CERTIFICATE OF ORIGINALITY
I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text. I also declare that the intellectual content of this thesis is the product of my own work, even though I may have received assistance from others on style, presentation and language expression.
(Signee THE KINETICS OF C9, VITRONECTIN AND THE
TERMINAL COMPLEMENT COMPLEX
by
James David Greenstein
Department of Nephrology, Prince Henry Hospital, Sydney, Australia
and
Faculty of Medicine, University of New South Wales, Sydney, Australia
Submitted/or the degree ofDoctor ofPhilosophy
1997 u i'J s vv 1 4 MAY 1998
LlBHARY A n
ABSTRACT
The primary pathways of complement activation converge to produce the terminal complement complex (TCC). Assembly of the TCC on a cell surface may result in the formation of a cytolytic pore, or membrane attack complex (MAC, i.e. C5b- 9(m)), composed of the five terminal complement components. Alternatively, complement activation in the plasma produces the soluble form, SC5b-9, which by the inclusion of regulatory proteins, vitronectin (S-protein) and clusterin, is rendered unable to disrupt cells. Activation of the complement system occurs in several autoimmune diseases although the role of the terminal complex in their pathogenesis remains uncertain. Measurement of its concentration in the plasma, or that of its components, provides limited information about terminal complement pathway activity. Specifically, the serum concentration of C9, a major component of the TCC, is usually in the normal range despite the presence of circulating immune complexes and evidence of accelerated consumption of the early complement components. Similarly, the plasma SC5b-9 concentration may be misleading due to the potential for in vitro complement activation in inadequately handled blood samples. To clarify this issue, kinetic studies of C9, its regulatory protein vitronectin and the SC5b-9 complex were performed in vivo. Their metabolism was investigated in experimental animals and C9 was also studied in eight normal human subjects and nine patients with autoimmune disease, including seven with systemic lupus erythematosus, one with mesangial lgA nephropathy and another with mixed essential cryoglobulinaemia. The fractional catabolic rate (FCR), plasma half-life (T112), compartmental distribution (i.e. extravascular/intravascular ratio, [EVIIV]) and indirectly, the plasma production rate, were determined. The relationship between these parameters and conventional, static measurements of complement and immunological activity was also addressed.
C9 was prepared from the plasma of a healthy human donor by established column chromatographic techniques and was radiolabelled with 1251. It retained full haemolytic activity and was able to be incorporated into human and rabbit SC5b-9, both spontaneously, and following activation with cobra venom factor (CVF) in vitro. Abstract III
Following intravenous administration to normal adult New Zealand white rabbits, 125I
C9 disappeared from the plasma with a final T112 of 25.2 ± 3.7 h, (mean± SD). The FCR was 5.99 ± 0.69 %/hand the EVIIV ratio was 0.72 ± 0.21. The distribution of 125I C9 among body tissues was similar to that observed for 1311-rabbit serum albumin (RSA) administered as a control, with a concentration of less than 50 % that of plasma in all organs tested (i.e. liver, spleen, kidney, lung, cardiac and skeletal muscle). Activation of complement in vivo by intravenous injection of CVF, resulted in the rapid disappearance of 125I-C9 fi:om the plasma and accumulation of protein-bound radioactivity in the spleen (exceeding the plasma concentration) and the liver. By contrast, the metabolism and distribution of 1311-RSA was unaffected by CVF, suggesting that these results were due to specific organ uptake of the TCC. This was confirmed by direct injection of 125I-SC5b-9. The complex disappeared rapidly fi:om the plasma, falling by 50% in 0.68 (0.55- 0.77) h, (median [range]), with less than 15% of the injected dose remaining after 4 h. Accumulation of protein-bound radiolabel was again noted in the spleen and liver, which contained up to 15 and 9 times respectively, the plasma concentration.
In healthy human subjects, the metabolic characteristics of highly purified, 125 sterile I-C9 were FCR: 2.92 ± 0.36 %/h, T112 : 42.5 ± 6.7 hand EV/IV: 0.56 ± 0.12.
Patients with reduced total serum complement haemolytic activity (i.e. CH50 < 68 % of n01mal human serum [NHS]) had a significantly higher FCR (3.57 ± 0.67 %/h) and shorter T112 (33.5 ± 6.8 h) than the control group (both p < 0.05). The plasma C9 production rate (calculated indirectly) was also greater in patients (0.11 ± 0.05 mg/kg per h) compared with control subjects (0.07 ± 0.03 mg/kg per h), (p < 0.05) and was associated with a higher serum C9 concentration (76 ± 13 mg/L vs 61 ± 14 mg/L, p < 0.05). By contrast, the serum C9 concentration was not correlated with its FCR. The plasma concentration of SC5b-9 was also higher in patients (515 [300 - 1879] j.lg/L) than in normal subjects (313 [229 - 402] j.lg/L), (p < 0.01) and showed a positive correlation with the FCR of C9 (r = 0.61, p < 0.01). The level of circulating immune complexes, but not the serum C3 or C4 concentration, also correlated positively with the
C9-FCR (r = 0.5, p < 0.05). Abstract IV
The metabolism of native and phosphorylated human vitronectin was examined m rabbits to establish the effects of phosphorylation. Selectively phosphorylated plasma, in which < 1 % of vitronectin was present as the 32P form, was administered intravenously and the disappearance of labelled molecules and antigenically detectable vitronectin (i.e. by enzyme-linked immunosorbant assay, [ELISA]) was studied 32 simultaneously. The plasma T112 of P-vitronectin was 8.9 ± 0.5 h which did not differ significantly :from that of the total vitronectin pool (8.0 ± 1.3 h). By contrast, 32P vitronectin had a significantly lower FCR than that of antigenically detectable molecules (10.85 ± 0.71 %/h vs 18.77 ± 1.57 %/h, p < 0.005) and a much smaller EVIIV (0.28 [0.15 - 0.36] vs 1.00 [0.48 - 1.60], p < 0.05). Complement activation with CVF generally produced only small and variable effects on the metabolism of antigenically detectable vitronectin. However, the rate of disappearance of 32P-vitronectin :from the plasma of the same animals was markedly accelerated in all cases following CVF administration.
These results demonstrate the complexity of C9, vitronectin and TCC metabolism in the normal state and during pathological complement activation. In particular, it was shown that C9 is rapidly metabolised in normal humans and that hypercatabolism occurs in patients with autoimmune disease and complement activation, despite the presence of normal or elevated serum C9 levels. The kinetic factors responsible for this have also been defmed. This data may be of relevance to future therapeutic strategies aimed at regulation of the terminal complement pathway. v
DECLARATION
"I hereby declare that this submission is my own work and that, to the best of my lmowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgment is made in the text."
L___ _j James David Greenstein VI
ACKNOWLEDGMENTS
A great deal of thanks goes to my supervisor A/Professor John Charlesworth for steering my path into Nephrology and for providing opportunities, help and inspiration during the course of this research. I am also indebted to A/Professor Bruce Pussell for his help and encouragement along the way and Professor Graham Macdonald for allowing this work to proceed in his department. I am very grateful to Dr Carol Morris for her assistance during the establishment phase of this work. Much thanks is also owed to Dr Philip Pealce for his encouragement and simply invaluable practical advice and assistance.
To the staff of the renal laboratories at Prince Henry Hospital I owe a debt of thanks for the cheery feel of the place. In particular, I am grateful toMs Sue O'Grady and Mr Tom Tzilopoulos for their technical assistance with immunological assays. I am also indebted to Drs Grant Luxton and Julie Wessels for their considerable contribution to the animal studies. Further, I acknowledge with thanks the staff of the Immunopathology Laboratory at Prince Henry Hospital, the Microbiology, Biochemistry and Haematology Laboratories of Prince of Wales Hospital, and the Prince Henry Hospital animal house for their assistance.
My appreciation to Professor Dennis Wakefield and Drs Malcolm Robertson and Frank Maccioni for allowing their patients to be studied, and also Sr Sue Champion and the many registrars, residents and nursing staff who were both subjects and assistants.
This work was conducted with the financial support of the National Health and Medical Research Council of Australia and the Australian Kidney Foundation for which I am grateful.
Much thanks and love to my mother and father who were always there and always believed. Finally, I dedicate this thesis to my wife Vicky and our daughters Emma, Jessica and Sarah, without whose love I would scarcely have bothered at all. VII
TABLE OF CONTENTS
SECTION PAGE
Abstract II Declaration v Aclmowledgments VI Index VII List of tables X List of figures XI Abbreviations XIV Symbols and units XVII
1.0 INTRODUCTION 1
1.1 The complement system and its activation 2 1.2 Components of the terminal complement pathway 11 1.3 Assembly and function of the TCC 28 1.4 Terminal complement complexes in disease 52 1.5 Metabolic studies of complement proteins 71 1.6 Aims of this thesis 83 1.7 General hypothesis 84
2.0 METHODS 85
2.1 Purification and preparation of proteins 86 2.2 Techniques for protein analysis 89 2.3 Radio labelling of proteins 92 2.4 Complement-dependent haemolytic assays 96 2.5 Activation of 125I-C9 and production of 125I-SC5b-9 in vitro 99 2.6 Radioimmunoassay of 125I-SC5b-9 100 2.7 Metabolic studies in experimental animals 103 Contents VIII
SECTION PAGE
2.8 Protocol for the study of C9 metabolism in human subjects 108 2.9 Analysis of metabolic data 116 2.10 Quantitation of blood proteins 120 2.11 Mathematical and statistical analysis 122
3.0 RESULTS 123
Purification and preparation of proteins 124
3.1 The purity and functional activity of C9 125 3.2 Molecular integrity and function of phosphorylated vitronectin 133
Studies in vitro 135
3.3 Activation of C9 in vitro 136 3.4 Antigenic and physical properties of the soluble TCC 142
Studies in experimental animals 149
3.5 The metabolism of C9 in normal rabbits 150 3.6 C9 metabolism in rabbits during complement activation 158 3.7 The metabolism of SC5b-9 in normal rabbits 166 3.8 The metabolism ofvitronectin and 32P-vitronectin normal rabbits 170 3.9 Vitronectin metabolism in rabbits during complement activation 175
Studies in human subjects 179
3.10 The metabolism of C9 in normal human subjects 180 3.11 C9 metabolism in patients with autoimmune disease 187 3.12 Correlations between C9 metabolic data and other immunological measurements 195 Contents IX
SECTION PAGE
4.0 DISCUSSION 205
4.1 The behaviour of C9 and SC5b-9 in vitro 206 4.2 The metabolism of C9 and SC5b-9 in experimental animals 211
4.3 The kinetics ofvitronectin and Ser378-P04-vitronectin in vivo 216 4.4 The metabolism of C9 in humans 219 4.5 Summary and concluding remarks 224
APPENDICES 227
1. Buffers required for the purification of C9 228 2. Polyclonal anti-sera used in radial immunodiffusion 231 3. Buffers used for polyacrylamide gel electrophoresis 232 4. Patient information sheet 233 5. Consent form for human metabolic studies 234 6. Calculation of the mean plasma (protein-bound) radioactivity 235 7. Derivation of the formula for whole body C9 content 236 8. Suppliers of materials and equipment used in this thesis 237
PERSONAL BIBLIOGRAPHY AND REFERENCES 239
Publications and presentations arising from this work 240 General references 241 X
LIST OF TABLES
TABLE TITLE PAGE
1. Metabolic effects of sub-lethal MAC deposition 41 2. Conditions associated with elevated levels of TCC in body fluids 53 3. Diseases associated with MAC tissue deposition 55 4. Studies reporting C9 neoantigens in renal tissue 57 5. Reports of congenital deficiency of the terminal complement pathway components and disease associations 68 6. The metabolic characteristics of early complement components in normal experimental animals 76 7. Studies of the metabolism of early complement components in healthy human subjects 78 8. Reports of the metabolism of the terminal complement complex and its components in experimental animals and human subjects 81 9. Characteristics of the murine monoclonal anti-human vitronectin antibodies used in this thesis 101 10. Physical data : patients and controls 109 11. Clinical data : patients with autoimmune disease 110 12. Renal function in patients 112 13. Patients' immunological data 113 14. 1-laematological parameters in patients with autoimmune disease 114 15. Recovery of C9 and total protein at stages of C9 purification 127 16. Comparison of the high MW serum fractions produced spontaneously and by CVF activation of 1251-C9-supplemented NHS 148 17. C9 metabolic data in normal New Zealand white rabbits 152 18. C9 metabolism in normal human subjects 183 19. C9 and SC5b-9 levels in normal human subjects 186 20. C9 metabolism in patients 190 21. C9 and SC5b-9 levels in patients 194 XI
LIST OF FIGURES
FIGURE TITLE PAGE
1. Overview of the complement cascade 3 2. The classical and lectin pathways of complement activation 5 3. The alternative pathway of complement activation 8 4. Model of C9 polymerisation 16 5. The domain structure of vitronectin 21 6. Diagrams of the classical and alternative C5 converting enzymes 29 7. The terminal complement pathway 31 8. Formation of the membrane attack complex 34 9. Inhibition of MAC assembly on homologous cells by CD 59 44 10. Compartmental models of protein kinetics 72 11. Purification of C9 by column chromatography 126 12. SDS-polyacrylamide gel electrophoresis of purified C9 128 13. Autoradiography of purified 125I-C9 129 14. FPLC Superose 12 gel filtration of purified 125I-C9 130 15. The haemolytic activity of purified C9 132 16. FPLC Superose 12 gel filtration of 32P-vitronectin 134 17. Activation of 1251-C9 in normal human serum 137 18. Activation of human 1251-C9 in rabbit serum 138 19. FPLC Superose 12 gel filtration of 1251-C9 incubated in NHS at 37°C 139 20. Incorporation of 1251-C9 monomer into a high MW form during prolonged incubation in NHS 141 21. Radioimmunoassay for C5 and C9 neoantigens in the TCC 143 22. Radioimmunoassay for vitronectin epitopes in the TCC 144 23. Heparin-Sepharose chromatography of 1251-C9 monomer and 1251- C9. -contammg. . comp 1exes 146 24. Stability ofthe TCC in lMNaCl 147 List offigures XII
FIGURE TITLE PAGE
125 25. Typical plasma and whole body 1-C9 disappearance curves in a normal rabbit 151 26. FPLC gel filtration of plasma samples from a normal rabbit gtven. 1251 -C9 154 125 131 27. Organ distribution of 1-C9 and 1-RSA in a normal rabbit 155 125 28. Uptake of 1-C9 by blood cells in a normal rabbit 157 125 29. Plasma 1-C9 disappearance curves in rabbits administered CVF 159 30. The effect of CVF on the FPLC gel filtration of plasma in rabbits gtven. 1251 -C9 160 125 31. Plasma disappearance curves for monomeric and complexed 1-C9 161 32. Plasma disappearance curves for free and protein-bound radioactivity 125 in a rabbit given 1-C9 followed by .CVF 163 125 131 33. Organ distribution of 1-C9 and 1-RSA during complement activation 164 125 34. Uptake of 1-C9 by circulating blood cells following CVF 165 131 125 35. The plasma disappearance curves for 1-C9 and 1-SCSb-9 administered concurrently to two rabbits 167 36. Organ uptake of 1251-SCSb-9 169 37. Plasma vitronectin disappearance curves in normal rabbits 171 38. Metabolic parameters of vitronectin in laboratory animals 172 39. FPLC Superose gel filtration of plasma from a normal rabbit 32 following injection of P-vitronectin 174 40. The disappearance of antigenically detectable vitronectin from the plasma of rabbits during complement activation 176 32 41. The disappearance of P-vitronectin from the plasma of rabbits during complement activation 177 32 42. FPLC gel filtration of plasma P-vitronectin following administration of CVF 178 List offigures XIII
FIGURE TITLE PAGE
43. The range of plasma 125I-C9 disappearance curves in healthy human subjects 181 44. The disappearance of whole body and plasma protein-bound radioactivity in a typical normal 125 I-C9 human turnover study 182 45. The daily FCR of C9 in normal human subjects 184 46. The metabolism of 125I-C9 in human subjects with pathological complement activation 188 47. The daily FCR of C9 in patients 189 48. Metabolic parameters of C9 in normal subjects and patients
with autoimmune disease and low CH50 192 49. The FCR of C9 calculated by Mathews' analysis compared with the metabolic clearance method 196 50. Plasma volume vs the difference between the C9-FCR calculated by Mathews' and the metabolic clearance methods 197
51. The relationship between the C9-FCR and its plasma T112 198 52. The relationship between the C9 plasma production rate, serum C9 concentration and whole body C9 content 199 53. C9 plasma production rate vs C9-FCR 200 54. C9-FCR vs the level of circulating immune complexes 202
55. The relationship between plasma T112 and C9-FCR and the CH50 203 56. Plasma SC5b-9 concentration vs C9 catabolic rate 204 XIV
ABBREVIATIONS
AA Arachidonic acid Ab Antibody ANA Anti-nuclear antibody AT III Anti-thrombin III BSA Bovine serum albumin Cl-Inh C 1 esterase inhibitor C3Nef C3 nephritic factor C4-, C8- & C9bp C4-, C8- & C9-binding proteins C5b-9(m) Membrane-bound CSb-9, i.e. the MAC CFD Complement fixation diluent CHso Total serum complement haemolytic activity CIC Circulating immune complex CRI Complement receptor type I. CRF Chronic renal failure CSF Cerebrospinal fluid CVF Cobra venom factor DAF Decay accelerating factor DEAE- Diethylaminoethyl-
dH20 Distilled H20 dsDNA Double stranded deoxyribonucleic acid EA Antibody-coated erythrocyte EAct-7 & EAct-s Complement intermediate assembled on EA cells EACA s-aminocaproic acid EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor ELISA Enzyme-linked immuno-sorbant assay EV Extravascular compartment EVIIV Extravascular/intravascular distribution ratio Abbreviations XV
FPLC Fine performance liquid chromatography GBM Glomerular basement membrane GFR Glomerular filtration rate GN Glomerulonephritis GPI Glycosylphosphatidylinositol Hb Haemoglobin HP Hemopexin HRF Homologous restriction factor HSA Human serum albumin Ig Immunoglobulin IgAN IgA nephropathy IL-l Interleukin 1 IP1,2&3 Inositolmono-, di- & triphosphate IV Intravascular compartment IVI Intravenous injection LDL Low density lipoprotein LTB Leukotriene B mAb Monoclonal antibody MAC Membrane attack complex of complement MASP Mannan-binding protein-associated serine protease MBP Mannan-binding protein MCP Membrane cofactor protein MEC Mixed essential cryoglobulinaemia MCGN Mesangiocapillary glomerulonephritis MW Molecular weight NHS Normal human serum NRS Normal rabbit serum OD Optical density PAGE Polyacrylamide gel electrophoresis PAI-l Plasminogen activator inhibitor type 1 PBS Phosphate-buffered saline Abbreviations XVI
PEG Polyethyleneglycol Pg Prostaglandin PHN Passive Heymann nephritis PKA cAMP-dependent protein kinase A PLA&PLC Phospholipase A & C PMSF Phenylmethylsulphonylfluoride PMNL Polymorphonuclear leucocyte PNH Paroxysmal nocturnal haemoglobinuria PPR Plasma production rate Ra/R2 Enterobacterial complement activating proteins RIA Radioimmunoassay ROM Reactive oxygen metabolites RSA Rabbit serum albumin RT Room temperature SC5b-9 The soluble terminal complement complex SDS Sodiumdodecylsulphate SGP-2 Sulphated glycoprotein 2 SLE Systemic lupus erythematosus SM Somatomedin T-AT III Thrombin-antithrombin III complex
Tll2 Half-life TCA Trichloroacetic acid TEMED N,N,N' ,N'-Tetramethylethylenediamine TCC Terminal complement complex TRJS Tris[hydroxymethyl]aminomethane TX Thromboxane Uprotein Urinary protein vol. Volume WBC9 Whole body C9 content WHO type World Health Organisation classification of lupus nephritis XVII
SYMBOLS AND UNITS
A An gstrom, 1.e.. 10"10 m A Ampere b Base, i.e. unit of nucleic acid length Ci Curie cpm Counts per minute D Dalton g Gram g x acceleration due to gravity h Hour IU International units L Litre m Metre M Mole/L mm Minute mol Mole p Statistical probability r Correlation coefficient rpm Revolutions per minute S/cm Electrical conductivity, i.e. Siemens/em s Sedimentation units s Second v Volts yr Year [x] Concentration of "x" 1
Section 1
INTRODUCTION 2
1.1 THE COMPLEMENT SYSTEM AND ITS ACTIVATION
GENERAL OVERVIEW
Towards the end of the nineteenth century the heat-labile bactericidal property of serum was first described and attributed to a factor, "alexin" (i.e. unnamed) [Buchner, 1889]. Subsequent studies by Bordet and others indicated that this factor was also involved in the haemolytic activity of immune serum [Bordet, 1895 & 1898]. Consequently, the term Complement was adopted as this action was deemed to be complementary to that of antibody.
The complement system is composed of 11 primary components and numerous soluble and membrane-bound regulatory proteins and receptors. Activation of complement proceeds by two main mechanisms : the classical pathway, typically initiated by the binding of immune complexes to the first complement component (i.e. C1), and the alternative pathway (see Fig. 1, page 3). Both of these mechanisms lead to the generation of a complexed serine protease capable of cleaving the third complement component (i.e. C3). The ultimate result of C3 activation is the recruitment of components C5, C6, C7, C8 and C9 (i.e. the terminal complement pathway), and generation of the terminal complement complex (TCC). Complement activation on a cell surface leads to the formation of the membrane attack complex (MAC or C5b-9(m)) with cytolytic and cell modulatory capabilities. Alternatively, activation in the plasma results in the assembly of a TCC (i.e. SCSb-9) which, by the inclusion of the regulatory proteins vitronectin and clusterin, is rendered soluble and unable to disrupt cell membranes.
The complement system is an important effector of the humoral immune mechanism through the generation of cytolytic pores and the antimicrobial activities of several intermediates and by-products of the active pathways. However, it has also become evident that complement is an important mediator of inflammation and that 3
Fig. 1. Overview of the complement cascade
The diagram below shows the major components of the complement system. The regulatory proteins and biologically active, soluble intermediates have been omitted for clarity but are discussed in later sections. The curved arrow shows the C3 feedback loop.
Activation pathways
Classical activation Alternative activation pathway pathway Cl, C2, C4 C3, Factor B, Factor D
Common pathway C3
Terminal pathways C5, C6, C7, C8, C9
SC5b-9 C5b-9(m) Soluble terminal complex Membrane attack complex Section 1.1 The complement system - overview 4
abnormal quantities of its components, or their dysregulation, may be associated with autoimmune disease such as glomerulonephritis (GN) and systemic lupus erythematosus (SLE).
More recently, interest has focused on the possible pathological role of the terminal complement pathway. Several investigators have reported the presence of components of the MAC at sites of inflammation in patients with autoimmune conditions, and elevated levels of SC5b-9 in their plasma. Nevertheless, the relationship between the TCC and tissue damage remains unclear.
This thesis is concerned mainly with the biology and metabolism of the TCC and, in particular, C9 and its inhibitor vitronectin. The following introductory sections review aspects of complement activation, the terminal complement complex, its components, and their putative role in disease.
ACTIVATION OF COMPLEMENT BY THE CLASSICAL PATHWAY
In Bordet's original studies, the haemolytic activity of sensitised serum was dependent on both a heat labile factor, i.e. complement, and a relatively heat stable component, now known to be immunoglobulin. Activation of the complement cascade by the binding of complexed antibody to Cl leads to the step-wise recruitment of additional components which constitute the "Classical pathway" (see Fig. 2, page 5).
Cl consists of three major subunits, Clq (molecular weight [MW] 462 kD), Clr (MW 83 kD) and Cis (MW 83 kD) [Law & Reid, 1995], which are maintained in 2 complexed form as Clqr2s2 [Sim & Reid, 1991] in the presence of Ca + ions [Lepow et al., 1963]. In humans and higher vertebrates, immunoglobulins, particularly of the lgG
(especially lgG1 & lgG3) and lgM classes, are able to bind via their Fe region to Clq following attachment to antigen. The large C 1q subunit is a hexamer comprising six filamentous collagen-like triple helices, each terminating in a globular, non-collagenous region. Some activators of the classical pathway, such as immune complexes, exert 5
Fig. 2. The classical and lectin pathways of complement activation
The classical pathway may be activated by a wide variety of soluble and membrane bound factors of which only a few are shown below. Inhibitory and regulatory effects are shown as dotted lines.
Ca2+ 1 Immune complexes
Clq-rrs2 * 2
MBP-polysaccharide ----C-a--++ L------_Cl-Inh
Clq-rrs2
... C4b breakdown by Factor I
C3 convertase ofthe classical pathway C4b2a ,.. · · · · · - --- · C4bp, DAF
I \ ~ ~ Convertase ...... ~.. dissociation and C4b ~ ~ .,.. -- destruction by Factor I
C3 ---+ C3b + C3a
Abbreviations
DAF decay accelerating factor, C4bp C4-binding protein, Cl-Inh Cl inhibitor, CRl complement receptor I, MASP mannan-binding protein-associated serine protease, MBP mannan-binding protein, MCP membrane cofactor protein, Ra/R2 enterobacterial polysaccharides.
*By convention, a bar over the component's name denotes the active form [see Harrison & Lachmann, 1986]. Section 1.1 The complement system - overview 6
their effect by binding to the globular regions of C1q [Burton & Woof, 1992], while others, including C-reactive protein [Jiang et al., 1991] and the lipopolysaccharides of certain strains of E.coli [Zohair et al., 1989], bind to the non-globular regions. Subcellular proteins [Rossen et al, 1988], including mitochondrial cardiolipin [Kovacsovics, 1985], are also capable of activating the classical pathway although their mechanisms are less clear. Coupling of ligands to C1q leads to a change in its conformation and triggers the auto-activation of the C1r subunits [Law & Reid, 1995]. In turn, the C1r molecules enzymatically cleave C1s (i.e. C1 esterase) in a calcium requiring reaction [Dodds et al., 1978, Law & Reid, 1995], leading to the activation of the Cl complex. Alternatively, C1 may be activated directly by the action of trypsin like enzymes [Cooper, 1982].
Activated C1 (i.e. C1) cleaves the fourth complement component, C4 (MW 205 kD [Law & Reid, 1995]), into a large portion, C4b, and a small fragment, C4a. C4 contains a reactive ~-cysteinyl-y-glutamyl thiolester bond in its a. chain [Sottrup-Jensen et al., 1985]. Following activation, this thiolester becomes unstable and may either be hydrolysed or bind covalently to a circulating immune complex (CIC), cell wall, or other suitable nucleophilic surface by an amide linkage [Law & Reid, 1995]. CT similarly activates C2 (MW 102 kD [Law & Reid, 1995]), splitting it into a large C terminal piece, C2a and a small, soluble N-terminal fragment, C2b. C4b acts, in effect, as a receptor for C2a with which it combines to form the hetero-dimer C4b2a. This complex contains a catalytic site within the C2a subunit which cleaves and activates the third complement component and constitutes the C3 converting enzyme (i.e. C3 convertase) of the classical pathway. Two other plasma proteins, i.e. mannan-binding protein (MBP) and mannan-binding protein-associated serine protease (MASP), in combination can also activate C4 and C2 directly after binding to certain enterobacterial polysaccharides [Law & Reid, 1995]. This is referred to as the lectin pathway (see Fig. 2, page 5). Section 1.1 The complement system - overview 7
Regulation of the classical activation pathway
The early phase of classical pathway activation is regulated by C1 esterase inhibitor (C1-Inh), an aTglobulin (MW 110 kD [Law & Reid, 1995]) which antagonises C1 by dissociating C1r and C1s from the complex [Sim & Reid, 1991]. Deficiency of this protein is associated with unrestrained complement activation and the life threatening condition, angioedema. C4-binding protein (C4bp), (MW 500 kD [Law & Reid, 1995]) also regulates the classical pathway C3 convertase by binding to C4 and promoting its dissociation from the complex. The membrane protein, decay accelerating factor (DAF), performs a similar function. C4bp is also a cofactor for Factor !-mediated catabolism of soluble C4, while membrane cofactor protein (MCP) and complement receptor 1 (CR1) serve this function when C4 is membrane-bound [Cooper, 1988]. These mechanisms inhibit the assembly of complement intermediates on autologous cells (see page 9).
THE ALTERNATIVE PATHWAY OF COMPLEMENT ACTIVATION
The alternative pathway is an ancient biological mechanism with ancestral components evident in some invertebrates [Parries & Atkinson, 1991]. It is largely independent of immunoglobulin and therefore has less specific triggers than the classical system (see Fig. 3, page 8). While some IgA-, IgE- and a limited range oflgG containing complexes may initiate the process, alternative pathway activation most often accompanies exposure to bacterial polysaccharides, lipopolysaccharides, certain viruses and virus-infected cells, or trypsin-like enzymes [Cooper, 1982, Reid, 1983, Cooper & Nemerow, 1983]. The central theme of the alternative pathway involves the slow, continuous generation of C3b in the plasma by the action of physiological C3- cleaving enzymes such as trypsin, thrombin and plasmin. Spontaneous hydrolysis of the thiolester bond within native C3 also occurs producing C3(H20) [Fearon & Austin, 1975a, Fishelson et al., 1984]. This latter reaction induces a C3b-like conformational change in native C3 [Pangburn & Muller-Eberhard, 1984, Lachmann & Hughes-Jones, 1984, Kinoshita, 1991]. Alternatively, the thiolester bond may react with hydroxyl or 8
Fig. 3. The alternative pathway of complement activation
Spontaneous activation of C3 and regulation of a positive feedback loop underlies the alternative complement pathway. Regulatory effects are shown as dotted lines.
Spontaneous hydrolysis of C3 activation by a suiface C3 in solution bound nucleoplzile (R-OH)
C3 + H20 --+ C3(H20) C3 + R-OH __.. C3(R-OH) t--- FactorB ---J Factor D -----+ C3(R-OH)B
t-Ba
C3(R-OH)Bb
~~------FactorH,CRl, MCP
C3 ----;. \ .. ~. C3b catabolism by Tryptic enzymes/ • -- --~~> Factor I
Properdin (P) and --"-- ---- FactorB stabilising surfaces C3bBP .._ Factor D
CRl, DAF · · · · · · · · ·~~> C3bBbP ····--+-dissociation & C3 catabolism by Factor I Stable C3 convertase oftlze alternative pathway Section 1.1 The complement system - overview 9
amino groups on a surface resulting in a covalently bound C3b-like C3. Both C3b and C3b-like C3 combine with the plasma protein, Factor B (MW 92 k:O [Law & Reid, 1995]), which is subsequently cleaved by the serine protease, Factor D (MW 24 k:O [Law & Reid, 1995]), liberating a soluble low MW fragment, Ba, and leaving the larger portion, Bb, attached to the complex [Lesavre et al., 1978]. The resulting species,
C3(H20)Bb and C3bBb, constitute the C3 convertases of the alternative pathway [Kinoshita, 1991]. They are Mg2+-containing metaloenzymes but may include other divalent cations [Fishelson et al., 1984]. Bb contains a catalytic site and performs a similar role to C2a in the classical pathway with which it bears structural similarity.
Most of the newly generated C3b is rapidly degraded in the plasma, or on an unfavourable surface, by the actions of the regulatory protein, Factor I (previously termed C3b inactivator), (MW 88 kD [Law & Reid, 1995]), and its co-enzyme, Factor H (previously termed P1H), (MW 150 kD [Law & Reid, 1995]). However, should a stabilising surface or molecule be available to which to bind, such as a bacterial cell wall or circulating immune complex, its half-life (Tw) is prolonged. Czop et al., (1978) suggested that surfaces which were deficient in sialic acid were the most effective for C3 activation. The plasma protein, Properdin (MW 220 kD [Law & Reid, 1995]) is also able to stabilise the convertase forming the complex, C3bBbP [Fearon et al., 1975b]. An amplification feed-back loop is established in which C3b contributes to the production of more C3 convertase [Pangburn & Muller-Eberhard, 1984, Lachmann & Hughes-Jones, 1984]. Large amounts of C3 are then split and accumulate on the activation surface, or are released into the fluid phase. C3b generated by the classical complement pathway may also be amplified via this process.
Regulation of the alternative pathway
Several mechanisms regulate the function of the alternative pathway. Factor H binds to soluble C3b leading to its destruction by Factor I [Cooper, 1988]. By contrast, surface-bound C3b exhibits relatively fewer Factor H attachment sites, which favours the binding and activation of Factor B [Kazatchkine et al., 1979]. However, deposition Section 1.1 The complement system - overview 10
of C3b on autologous cells is restricted. In this situation CR1 and MCP act as cofactors for Factor !-mediated catabolism of surface-bound C3b and promote dissociation of the alternative pathway C3 convertase in a manner analogous to the classical pathway counterpart [Cooper, 1988].
Dysregulation of the alternative pathway occurs in some patients with type II mesangiocapillary glomerulonephritis (MCGN) and partial lipodystrophy [Sissons et al., 1977]. The serum of these individuals contains an abnormal immunoglobulin which stabilises the C3 convertase [Davis et al., 1977, Scott et al., 1978]. This antibody (i.e. C3 nephritic factor [C3 NeF]) is most often directed against neoantigens of the C3bBb complex [Couser et al., 1985, Strife et al., 1990] and results in accelerated activation of C3 and profound reduction in the serum C3 concentration.
Cobra venom factor
An extract from the venom of the cobra Naja naja kouthia is capable of strongly activating the alternative pathway both in vivo and in vitro [Vogt, 1982, Vogel et al., 1984]. Cobra venom factor (CVF) is the reptilian analogue of mammalian C3b [Vogel et al., 1984, Couser et al., 1985]. It is able to bind to Factor B, which is then susceptible to cleavage and activation by Factor D. However, the resulting C3 convertase, CVFBb, is resistant to regulation by Factors I and H, and is therefore more potent than the homologous complex. CVF derived from Naja naja sp. also produces a very efficient C5 converting enzyme which rapidly depletes the late complement components from the circulation through exhaustive consumption [Von Zabern et al., 1980], (see Section 1.3, page 28). Several other constituents of cobra venom are also known to interact with complement through a variety of mechanisms [Vogt, 1982]. 11
1.2 COMPONENTS OF THE TERMINAL COMPLEMENT PATHWAY
The terminal complement pathway includes components C5 through to C9, the soluble regulatory proteins vitronectin and clusterin and the membrane-bound inhibitors CD59 and homologous restriction factor (HRF).
COMPLEMENT COMPONENTS
There is considerable homology between the complement proteins of the terminal pathway. In particular, C6, C7, and the a and f3 chains of C8 and C9 are structurally and antigenically closely related [Muller-Eberhard, 1986]. The genes for C7 [DiScipio et al., 1988], C8a [Howard et al. 1987a], C8f3 [Howard et al. 1987b] and C9 [DiScipio et al., 1984] have recently been identified and their nucleotide and corresponding amino acid sequences determined. These proteins share between 21 % and 25% homology on alignment. They have in common (from theN-terminal to the C-terminal) : (i) a thrombospondin module, (ii) a cysteine-rich region homologous with the low density lipoprotein (LDL) receptor type A, (iii) a MAC-specific domain, (iv) a lytic domain and (v) a C-terminal region homologous with the epidermal growth factor like (EGF) repeats of the LDL receptor type B [Law & Reid, 1995]. Perforin, the membrane damaging protein released from activated T cells, also has a central lytic domain and a C-terminal EGF-like region [Lichtenheld et al., 1988], suggesting a common ancestry with the terminal complement proteins.
Production of the terminal complement components
The liver is the major source of the terminal complement proteins with the exception of C7 [Hobart & Lachmann, 1977, Geng, 1986, Wurzner et al., 1994, Brauer Section 1.2 Components ofthe terminal pathway 12
et al., 1995]. In each case, extrahepatic synthesis has also been demonstrated. Human bone marrow cells produce C7 [Naughton et al., 1996], fibroblasts: C5 to C9 [Garred et al., 1990], macrophages: C5 [Werb, 1982], human peritoneal macrophages: C5 to C9 [Hetland & Bungum, 1988], alveolar macrophages: C6 to C8 [Pettersen et al., 1987] and C9 [Pettersen et al., 1988], polymorphonuclear leucocytes (PMNL): C6 and C7 [Hogasen et al., 1995] and glial/glioma cells elaborate C5 [Gasque et al., 1993] and C6 to C8 [Gasque et al., 1995]. It has been suggested that extrahepatic production of complement components may contribute to local inflammatory reactions [Brauer et al., 1995], although its relative contribution to plasma and tissue pools remains uncertain.
C5
The fifth component of complement is a 13 1 globulin [Cooper, 1988] with a molecular weight of 185 kD [Nilsson et al., 1972]. Its mean serum concentration is approximately 70 mg/L [Cooper, 1988, Law & Reid, 1995]. C5 is encoded at chromosome location 9q 32 - 34 [Law & Reid, 1995] and is synthesised as a single chain i.e. Pro-C5. A short basic tetrapeptide is subsequently removed to yield a disulphide-linked dimer with a C-terminal a chain ofMW 115 kD, and an N-terminall3 chain of 75 kD [Reid, 1988, Law & Reid, 1995]. C5 shares considerable homology with C3 and C4 based on eDNA data [Sottrup-Jensen, 1985], although notably, C5 lacks the intramolecular thiolester of C3 and C4. Consequently, neither C5 nor the TCC form covalent linkages (upon activation), that are characteristic of the earlier complement components [Reid, 1988]. C5 is degraded by plasmin, thrombin, kallikrein and other plasma proteases [DiScipio, 1992].
C6
C6 is a single chain glycoprotein [Podack et al., 1979] of MW 120 kD [Law &
Reid, 1995] and 132 electrophoretic mobility [Cooper, 1988]. Its mean plasma Section 1.2 Components ofthe terminal pathway 13
concentration is approximately 60 mg/L [Cooper, 1988, Law & Reid, 1995]. C6 is encoded on chromosome 5q [Morgan & Walport, 1991, Law & Reid, 1995].
C7
C7 is a single chain ~2 globulin [Podack et al., 1973] of MW approximately 110 kD [Law & Reid, 1995] which is encoded near the C6 locus on chromosome 5q [Morgan & Walport, 1991, Law & Reid, 1995]. It has a serum concentration of approximately 50- 60 mg/L [Cooper, 1988, Law & Reid, 1995].
C8
C8 consists of three chains: C8a (MW 64 kD), C8~ (MW 64 kD) and C8y (MW 22 kD) with a total MW of 150 kD [Law & Reid, 1995]. Native C8 has a spherical shape [Esser et al., 1993]. The a andy chains are united by a disulfide linkage (i.e. as
an ay hetero-dimer), while the ~ chain is held non-covalently [Ng et al., 1987]. Each
subunit is encoded by a single gene [Ng et al., 1987]. The C8a and C8~ genes lie in close proximity at chromosome location 1p34 [Michelotti et al., 1995, Law & Reid, 1995] while they chain gene is located on 9q [Law & Reid, 1995]. Although C8a and
C8~ are necessary for the lytic activity of the MAC, C8y, which is structurally dissimilar to the other subunits, is not required and its function remains unknown [Haefliger et al., 1987]. C8 has a serum concentration of approximately 50- 60 mg/L [Law & Reid, 1995].
C9
C9 is a glycoprotein of MW 71 kD [Law & Reid, 1995] and a-globulin electrophoretic mobility [Cooper, 1988]. It is encoded by a gene at chromosome Section 1.2 Components ofthe terminal pathway 14 location 5pl3 [Law & Reid, 1995] which includes 12 exons and spans at least 80 kb of DNA [Marazziti et al., 1987]. The complete nucleotide sequence of the C9 gene has been established using a eDNA library from human liver [DiScipio et al., 1984], and the corresponding peptide sequence consists of 53 7 amino acids in a single chain with a calculated MW of 60.7 kD. Post-translational glycosylation yields a final carbohydrate content of 7.8% by weight including 3 moles of glucosamine, 17.6 moles of neutral hexose and 7.4 moles of sialic acid per mole of C9 [Biesecker & Muller-Eberhard, 1980].
C9 has a concentration of 58 ± 8 mg/L in the serum [Biesecker and Muller Eberhard, 1980] and a similar plasma level (i.e. approximately 60 mg/L [Oleesky et al., 1986, Law & Reid, 1995]). However, C9 behaves as an acute-phase reactant and may have a considerably increased serum concentration during physiological stress [Charlesworth et al., 1979a, Oleesky et al., 1986, Adinolfi & Lehner, 1988]. For example, Lassiter et al., (1992) reported that the serum C9 concentrations in new mothers was 260 ± 47 mg/L compared with 42 mg/L in their neonates. Wyatt et al., (1981) also found a relatively high level of C9 (and C5 and C1-Inh) in the serum of smokers and proposed that this may be due to on-going low grade inflammation associated with complement activation. C9 is metabolised by several plasma enzymes including thrombin [Dankert & Esser, 1985], and trypsin [Dankert et al., 1985].
Functional domains of C9
Limited proteolysis by a-thrombin cleaves C9 between residues 244 and 245, into two putative domains termed C9a (N-terminus) and C9b (C-terminus). Although physiological activation of C9 does not involve such a reaction, proteolysis by plasmin, thrombin and other enzymes may be important to its catabolism and can frustrate attempts at purification. C9 exhibits amphiphilic properties typical of other integral membrane proteins [Singer, 1974, DiScipio et al., 1984], and its domains differ in their physical characteristics. For example, C9a contains a high proportion of acidic residues while C9b includes a higher fraction of hydrophobic amino acids [DiScipio et al., 1984]. Section 1.2 Components ofthe terminal pathway 15
Furthermore, Ishida et al., (1982) observed that phospholipid binding sites were present in the C9b domain. In particular, two helices in a segment including residues 293 - 334 have been proposed as sites of interaction between C9 and membrane lipids [Dupuis et al., 1993].
Morphology of monomeric C9
Electron microscopy has revealed monomeric C9 to be globular ellipsoid in shape [DiScipio, 1993]. DiScipio (1993) also found that the physical properties of C9 (e.g. its frictional rate) were consistent with a heart, or globular ellipsoid form by comparison with other molecules of known tertiary structure. High flux neutron and synchrotron X-ray scattering studies have indicated more precisely that native C9 has a folded, or "V" shaped structure, subtending an angle of about 10° between its arms [Smith et al., 1992]. Tschopp et al., (1988) have suggested that the cysteine-rich region (i.e. residues 101 - 111) located in the C9a domain, and the membrane-binding site of C9b (i.e. residues 245 - 34 7) are concealed in this conformation, maintaining the molecule's solubility in plasma (see Fig. 4, page 16).
The length of C9 has been estimated at 110- 120 A (11 - 12 nm) by neutron and electron scattering experiments. However, DiScipio (1993) considered that this was an overestimation compared with measurements based on physical and electron microscopic analysis, finding dimensions of 77 A (7.7 nm) x 70 A (7.0 nm) x 52 A (5.2 nm). Similarly, Podack and Tschopp (1982) proposed a length of 80 A (8 nm), and a width of 55 A(5.5 nm) for the C9 monomer.
Polymerisation of C9
C9 monomers undergo spontaneous, circular polymerisation in solution and on lipid membranes, forming a tubular structure which resembles the MAC [Biesecker et 16
Fig. 4. Model of C9 polymerisation
In its native form the membrane binding sites of C9 are concealed. Activation allows both membrane insertion and polymerisation to proceed. Regulatory/inhibitory effects are shown below by dotted lines.
C9 residues 10 1 - Ill conserved cysteine-rich domain
Folded soluble C9
+ Thrombin-sensitive hinge
C5b-8*, divalent cations
C9 unfolding and membrane insertion 4!' 4!' • 4!' • Lipid bilayer
Vitronectin heparin-binding residues 348-360, ...... ------."' Protamine
Polymerisation of C9
• TCC intermediate (see Section 1.3, page 33). (Adapted from Tschopp, et al., 1988) Section 1.2 Components ofthe terminal pathway 17
al., 1993]. The rate of this reaction is temperature dependent requiring several days at 37°C, or as little as 2 hat 46- 56°C [Podack & Tschopp, 1982]. There is a requirement for metal ions and poly-C9 formation is abolished by EDTA [Tschopp, 1984a]. 2 2 2 Divalent cations such as ci+, Cd +, Cu + and Zn + in particular, are the most efficacious. Thielens et al., (1988) showed that native C9 is a metaloprotein containing 2 1mole of Ca + per mole of C9. Removal of calcium (i.e. with EDTA) leads to instability, thermal lability, and loss ofhaemolytic activity. In vitro, the introduction of calcium ions may produce a three-fold increase in the affinity constant for C9 self association. This is further enhanced by a low ionic strength buffer [DiScipio, 1991].
Polymerisation of C9 involves the assembly of a cylindrical conduit. Podack & Tschopp (1982) found that poly-C9 cylinders had an internal diameter of 110 A (11 nm) and length of 160 A (16 nm). A wider torus at one end had internal and external diameters of 110 A (11 nm) and 220 A (22 nm) respectively. More recently, Biesecker et al., (1993) used cryo-electron microscopy to calculate the dimensions of poly-C9 particles. They found a dense inner ring of diameter 113 A(11.3 nm) to 181 A (18.1 nm) which was surrounded by a concentric outer rim extending to 254 A (25.4 nm) in diameter. The tail ofpoly-C9, i.e. opposite the torus end, is organised into two distinct globular domains each of 3 nm diameter [Tschopp, 1984b]. This structure is similar to that seen in C5b-7(m) (see Section 1.3, page 32). Poly-C9 tubules have an average MW of 1,050 kD although there is considerable heterogeneity [Tschopp, 1984b]. Tschopp et al., (1984b) reported that between 12 and 18 C9 units may participate in each poly-C9 complex, with approximately 50 - 75 % of such structures containing 14 to 16 molecules. Similarly, Podack & Tschopp (1982) noted that poly-C9 had a dodecameric appearance, while Biesecker et al (1993) found a 13-fold rotational symmetry in their slightly larger particles. As the approximate dimensions of monomeric C9 are only 80 A (8 nm) x 55 A (5.5 nm), [Podack & Tschopp, 1982], it has been proposed that the C9 monomer unfolds during polymerisation. This exposes the membrane-binding region of C9b and also sites in the C9a domain which allow polymerisation [Tschopp et al., 1988], (See Fig. 4, page 16). Laine and Esser (1989) have also identified refolding C9 molecules during membrane insertion. Examination with circular dichroism spectroscopy has confirmed that polymerisation is accompanied by a change in the Section 1.2 Components ofthe terminal pathway 18
conformation of C9. This is reflected in an increase in the proportion of the molecular
secondary structure present as ~-pleated sheets from 32- 38 % [Tschopp et al., 1982]. Moreover, the same phenomenon was observed when C9 was added to vesicles coated with C5b-8 and Podack et al., (1982?) have noted that circular polymerisation of C9 is induced by the C5b-8 assembly.
Poly-C9 forms transmembrane channels in lipid vesicles upon exposure of its hydrophobic domains [Tschopp et al., 1982], however in solution poly-C9 tubules form large aggregates, associating along a 40 A (4 nm) length of their tails. While these aggregates are dissociable in detergents, the poly-C9 complexes themselves are stable to all but strong reducing agents. This is due to internal disulphide linkages [Yamamoto & Migita, 1983].
Polymerisation of C9 exposes neoantigens which are not found on the native monomer but are also expressed on the TCC [Mollnes et al., 1985b]. This provides further evidence for the presence of C9 polymers within the terminal complex and is discussed more fully in Section 1.3, page 28. Of note, Dalmasso et al., (1989) reported that purified, monomeric C9 may also contain a neoantigen, not expressed by the serum protein. Denatured, monomeric C9 has similarly been shown to react with some monoclonal anti-C9 neoantigen antibodies (e.g. MCbC5 [Mollnes et al., 1985b]) emphasising the importance of meticulous purification and storage of this labile protein.
REGULATORY COMPONENTS OF THE TERMINAL COMPLEMENT PATHWAY
Vitronectin
Vitronectin is a multifunctional, adhesive glycoprotein ofMW 75 kD [Tomasini & Mosher, 1991] and a electrophoretic mobility [Cooper, 1988]. It is present in the Section 1.2 Components ofthe terminal pathway 19
plasma (and serum [Preissner, 1991]) at a concentration of approximately 200 - 400 mg/L [Tomasini & Mosher, 1991] and is found in a wide variety of body fluids and tissues. Vitronectin supports cell adhesion and has been referred to as serum spreading factor [Barnes & Silnutzer, 1983]. It also has considerable homology (if not identity) with the serum protein, epibolin, which promotes the spreading of epidermal cells [Stenn, 1981]. However, it is biochemically and antigenically distinct from the adhesive protein, fibronectin [Hayman et al., 1982]. In 1985, Suzuki et. al., analysed the vitronectin gene using eDNA clones and deduced its primary molecular sequence. It became apparent that vitronectin was identical with a previously described molecule termed S-protein (i.e. site-specific protein) which is a constituent of SC5b-9. [Tomasini & Mosher, 1986, Jenne & Stanley, 1985]. However, S-protein (vitronectin is the more common terminology) bears no similarity to the vitamin K-dependent regulatory protein of the coagulation pathway, protein S. The MW of vitronectin, calculated from the primary peptide sequence, is 52,371 D [Jenne & Stanley, 1985] indicating that post translational glycosylation, sulphation and phosphorylation contribute 25 - 30 % of it's weight.
Production and distribution ofvitronectin
Several tissues and cell types are known to contain or secrete vitronectin. The major source of plasma vitronectin is the liver and cirrhosis is associated with reduced plasma vitronectin concentration [Kemkes-Matthes et al., 1987]. Platelets [Parker et al., 1989], monocytes/macrophages [Hetland et al., 1989] and megakaryocytes [Kanz et al., 1988] all contain histochemically identifiable vitronectin. Approximately 1 % of the protein released by activated platelets consists of disulfide-linked multimeric and complexed vitronectin [Preissner et al., 1989a, Tomasini & Mosher, 1991]. It is also synthesised and secreted by several cell lines in vitro, including the Hep G2 hepatoma [Barnes & Reing, 1985], mesothelial cells, arterial smooth muscle cells [Preissner, 1991], a human testicular teratoma [Cooper & Pera, 1988], a glioblastoma cell line [Gladson et al., 1990 & 1991] and normal cultured fibroblasts [Hayman et al., 1983]. Section 1.2 Components ofthe terminal pathway 20
Vitronectin has been identified in a variety of body fluids and tissues. In addition to plasma, it occurs in amniotic fluid [Shaffer et al., 1984], urine [Preissner, 1 1991] and tears • Histochemical studies have also located vitronectin in the normal kidney, portal triads ofthe liver, embryonic lung, skeletal muscle, and capsular visceral surfaces [Hayman et al., 1983]. In the skin, vitronectin has been found in association with elastic elements of the dermis in an age-dependent manner, showing a sharp rise during puberty [Dahlback et al., 1989a]. However, it is not increased in sun-exposed areas, in contrast to C9 neoantigen with which it is often co-localised.
Vitronectin domain structure and function
The vitronectin gene is located at chromosome position 17 q 11 [Law & Reid, 1995], and includes eight exons and seven introns. It is 4.5- 5 kb in length from which a 1.7 kb transcript is produced [Jenne & Stanley, 1987], (see Fig. 5, page 21). Exon 1 is a leader region which remains mostly untranslated, contributing only the first 2 N terminus amino acids of the 459 residue polypeptide. Exon 2 (i.e. residues 3 - 42) encodes most of the sequence of somatomedin B (SM B) which forms the first 44 N terminus peptides of the mature molecule [Barnes et al., 1984, Suzuki et al., 1984 & 1985]. There is uncertainty over the significance of tl1is region to the function of vitronectin and over the function of free SM B itself. Unlike somatomedins A and C, somatomedin B is now thought to be devoid of intrinsic insulin-like growth promoting properties, which have been attributed to contamination with epidermal growth factor in earlier isolates [Heldin et al., 1981]. It is nevertheless possible that vitronectin is the precursor molecule from which SM B is cleaved although vitronectin, free of its 5kD N terminus, has not been identified in the plasma. The SM B region includes 8 of the 14 cysteine resides present in vitronectin [Suzuki et al., 1984] and inhibits the action of tryptic enzymes [Fryklund & Sievertsson, 1978]. '
1 Personal communication regarding vitronectin in tears from Dr C Morris, lately Senior Scientific Officer, The Renal Immunology Laboratory, Prince Henry Hospital, Sydney. 21
Fig. 5. The domain structure ofvitronectin
The vitronectin gene and protein domains are shown (a). The double ended arrows identify ligand binding and other functional sites. A graphic model of the conformational transition ofvitronectin during activation is shown in (b).
(a) Gene I II III IV V VI VII VIII introns 5' 3'
NH2 COOH
H
...... ~ ~ Integrins Crosslinldng site heparin, C9, Perforin, P AI -1 and plasminogen • • +~-+ P AI -1 Collagen
(b) Heparin, Thrombin-antithrombin, C5b-7, denaturants
SMB
cs
Abbreviations and symbols
CS connecting segment, H hinge region, HP hemopexin-like repeats, P proteolysis-sensitive site, PAI-l plasminogen activator inhibitor type 1, <( glycosylation site.
(Adapted from Preissner, 1991) Section 1.2 Components ofthe terminal pathway 22
The third exon encodes for a large peptide sequence (i.e. the connecting segment) which includes four functional regions. (i) The peptide triplet, Arg45-Gly46- Asp47 (RGD) which mediates the attachment ofvitronectin to cells through interaction with several integrin receptors [Ruoslahti & Pierschbacher, 1986, Cherney et al., 1993]. This sequence is shared with many other adhesive proteins including fibronectin, fibrinogen, Von Willebrand factor, thrombospondin, laminin and collagen. Vitronectin binding integrins are widely distributed and occur on megakaryocytes, platelets, endothelial cells, and several neoplastic cell lines. They include integrin types anb-f33, amb-f33, av-f3b av-f33 and av-f3s [Preissner, 1991, Cherney et al., 1993]. (ii) Residues 48 - 130 comprise a highly acidic region. It includes Tyr56 and Tyr59 which may be sulphated by the enzyme, tyrosylprotein sulfotransferase, found in the Golgi membrane. This region may be important in neutralising the cationic groups of the carboxyl terminus of vitronectin and so maintain the inactive molecule in a closed conformation [Jenne et al., 1989]. (iii) Gln93 is a potential site for cross-linking by Factor XIIIa!transglutaminase [Sane et al., 1988], and may be involved in the formation of vitronectin polymers. (iv) It has been proposed that a binding site for collagen is present in the vicinity of the cross-linking region of the connecting segment [Gebb et al., 1986, Izumi et al., 1988]. Cross-linking between vitronectin and collagen in the extracellular matrix could therefore occur in this region.
Exons III to VIII encode for 2 domains which are homologous with the haem binding plasma protein, hemopexin, and are found between residues 132-268 and 269- 459. A heparin-binding domain is located within the second hemopexin region [Preissner, 1991]. It contains a 40 amino acid, highly basic segment [Cardin et al., 1989] which binds to several sulphated polysaccharides including fucoidan and dextran sulfate [Tomasini & Mosher, 1991] Evidence suggests that this site may also be involved in the inhibition ofTCC assembly [Tschopp et al., 1988, Hogasen et al., 1992] although other regions may also have a role. This is discussed more fully in Section 1.3, pages 45 & 47. Section 1.2 Components ofthe terminal pathway 23
Conformational lability of vitronectin
In the native conformation, it is probable that the cationic heparin-binding region lies in proximity to vitronectin's acidic N-terminus, folded about a putative hinge at Pro268 [Hunt et al., 1987, Preissner, 1991]. A variety of ligands may induce native vitronectin to unfold. Denaturation, alkylation, reduction, binding to surfaces, a. thrombin, thrombin-antithrombin III complex (T-AT III) and heparin itself can induce a conformational change. This may result in increased affinity for heparin, y-thrombin, T
AT III and ~-endorphin by exposing encrypted sites [Barnes et al., 1985, Hayashi et al., 1985, Preissner et al., 1987, Preissner, 1991]. Monoclonal antibodies have been raised against the activated, unfolded form of vitronectin (e.g. 8E6 [Hayman et al., 1993]). These similarly exhibit greater affinity for vitronectin following complexing with ligands such as thrombin and T-AT III [Tomasini & Mosher, 1988, Tomasini et al., 1989]. Coagulation also changes the proportion ofvitronectin that binds to heparin. In plasma, only 2- 5% of the protein is in a heparin-binding form, whereas this is enriched three to four fold in serum [Izmni et al., 1989]. Furthermore, heparin-binding vitronectin appears to be in the form of aggregates, presenting a larger Stokes radius than the non-heparin-binding conformer [Izumi et al., 1989]. Coagulation also leads to the formation of a ternary complex between vitronectin and T-AT III [Ill & Ruoslahti 1985, Jenne et al., 1985].
Vitronectin polymorphisms
Vitronectin occurs in the plasma as single (MW 75 kD) and two chain forms (MW 65 kD + 10 kD), the latter being held together by sulfhydryl linkages [Akama et al., 1986]. This can be demonstrated by polyacrylamide gel electrophoresis (PAGE) performed under reducing conditions. Individuals may have one of three phenotypes in their plasma: type 1-1, which is enriched in single chain molecules, type 1-2, which has an equal mixture of single and double chain forms and type 2-2, in which mainly double chain vitronectin is found [Kubota et al., 1988]. The proportion of each type in the Section 1.2 Components ofthe terminal pathway 24
Caucasian population is consistent with that predicted by a Hardy-Weinberg equilibrium of two co-dominant alleles [Conlan et al., 1988]. It is due to a polymorphism at residue 3 81 which can be either methionine or threonine. This, in turn, determines the sensitivity of the peptide bond between Arg397 and Ala380 to proteolysis, with Thr381 promoting splitting [Kubota et al., 1990]. The responsible enzyme remains unknown although trypsin performs this ftmction in vitro [Podack & Muller-Eberhard, 1979]. The population frequency of each vitronectin phenotype is independent of age, sex and ABO blood group [Kubota et al., 1988] but varies with ethnicity. For example, a higher frequency of the single chain form in Japanese and Chinese populations has been reported [Kubota et al., 1988, Sun & Mosher, 1989]. However, this has not been associated with a propensity to disease.
There is little evidence for ftmctional differences between the single and two chain forms of vitronectin, although the latter has been reported to have a higher affinity for immobilised heparin [Sane et al., 1990], and Izumi et al., (1989) similarly found that heparin-binding multimeric vitronectin was enriched in the 65 kD form. Furthermore, a cAMP-dependent protein kinase released from thrombin-activated platelets preferentially phosphorylates the 75 kD polypeptide [K.orc-Grodzicki et al., 1988] as discussed below.
Phosphorylated vitronectin
Korc-Grodzicki et al., (1988 & 1990) and Shaltiel et al., (1993) have demonstrated that thrombin-activated platelets release a cAMP-dependent protein kinase (i.e. protein kinase A [PKA]) which selectively phosphorylates vitronectin. This enzyme transfers a P04 group to Ser378 [Korc-Grodzicki et al., 1990] which lies within a typical phosphorylation consensus sequence, i.e. Arg-Arg-Pro-Ser [Edelman et al., 1987], at the carboxyl terminus of the heparin-binding domain. The preferred substrate of PKA is non-cleaved vitronectin although incubation with heparin exposes the critical residues for modification in the two-chain form [Chain et al., 1990]. The pure catalytic subunit of PKA, derived from rabbit skeletal muscle, ftmctions similarly to the platelet- Section 1.2 Components ofthe terminal pathway 25
derived enzyme. Alternative phosphorylation loci may also be present, as vitronectin produced by HepG2 cells binds phosphate at sites distinct from Ser3 78 [McGuire et al., 1988].
McGuire et al., (1988) suggested that vitronectin could be phosphorylated in vivo by PKA and ATP released from injured cells or by platelets during haemostasis. Phosphorylated vitronectin has been shown to have decreased affinity for PAI-l [Shaltiel et al., 1993], although its physiological relevance remains unclear.
Clusterin
Clusterin was first isolated from the seminal fluid of the ram [Fritz et al., 1983] and the rat. In vitro it produces clustering of Sertoli cells from immature rats, haemagglutination [Fritz et al., 1983] and inhibition of C5b6-initiated haemolysis [O'Bryan et al., 1990]. Ram seminal clusterin was found to be homologous with sulphated glycoprotein 2 (SGP-2) which is the major protein secreted by rat testicular Sertoli cells [Tsuruta et al., 1990]. Subsequently, clusterin was identified in human semen and a correlation was found between its concentration (i.e. 2 - 15 g/L [Law & Reid, 1995]) and sperm fertility in vitro [O'Bryan et al., 1990]. Sequence analysis has shown that seminal clusterin is highly homologous with the serum protein, SP-40,40 [Kirszbaum et al., 1989, Tsuruta et al., 1990], an apolipoprotein (MW 80 kD), comprised of two non-identical peptide chains (i.e. a and f3) each of MW 40 kD [Murphy et al., 1988]. Its gene is located at chromosome position 8p21 [Purrello et al., 1991, Law & Reid, 1995]. Both chains are encoded on a single open reading frame contained on one mRNA molecule indicating that post-synthetic proteolysis is required to produce the mature protein [Kirszbaum et al., 1989]. A short segment of 23 amino acids (including cysteine) in the f3 chain of clusterin shows homology with comparable regions in C7, C8 and C9, but it otherwise bears little resemblance to the complement proteins [Kirszbaum et al., 1989]. The major site of clusterin production appears to be the liver [Hogasen et al., 1996] and it is present in the serum at a concentration of 35 to
105 ~g/mL [Murphy et al., 1988]. Section 1.2 Components ofthe terminal pathway 26
Clusterin derived from both serum and semen is an inhibitor of the TCC [O'Bryan et al., 1990]. It has been purified from SC5b-9 and termed complement lysis inhibitor [Jenne & Tschopp, 1989]. Its mechanism of complement inhibition is discussed in Section 1.3, pages 45 & 47.
Membrane-bound inhibitors of the terminal complement pathway
CD 59
CD59 was first characterised and cloned by Sugita et al., (1989). This protein inhibits the formation of MAC channels and was termed MACIF although it has been referred to by several other names including HRF20, protectin, MIRL and :MEM-43-Ag [Law & Reid, 1995]. It is a relatively small protein (MW 20 kD [Law & Reid, 1995]) containing only 103 amino acid residues [Lachmann, 1991] and its gene is located at chromosome position 11p13 [Law & Reid, 1995]. CD59 has been demonstrated in a wide range of tissues including skin, liver, kidney, lung, pancreas, salivary gland, placenta and the central nervous system [Lachmann, 1991]. Its expression on cultured human mesangial cells is induced by complement activation on the cell surface. As this phenomenon is abolished in C5 or C8 deficient sera, a role for the TCC is indicated [Shibata et al., 1991].
HRF
HRF has a MW of approximately 65 kD [Law & Reid, 1995] and is synonymous with C8-binding protein (C8bp), [Schonermark et al., 1986], C9-binding protein (C9bp) and MAC-inhibiting protein (MIP), [Watts et al., 1990]. Young et al., (1990) presented evidence that HRF is immunologically related to C8, C9 and perforin and may be a product of the complement supergene family. In common with other cell-bound complement regulators including CD59 [Lachmann, 1991] and DAF [Davitz et al., 1986], HRF is bound to the cell membrane via a glycolipid anchor, i.e. a glycosyl- Section 1.2 Components ofthe terminal pathway 27 phosphatidylinositol (GPI) linkage [Law & Reid, 1995] and can be cleaved from membranes by purified phosphatidylinositol-specific phospholipase C [Hansch et al., 1988b]. HRF has been identified on many cell types including erythrocytes, platelets, monocytes and lymphocytes [Blaas et al., 1988, Martin et al., 1988]. Glomerular mesangial cells contain HRF in their cytoplasm and on their surface. Deposition of MAC on cultured mesangial cells results in a transient, protein synthesis-independent increased expression ofHRF [Schieren et al., 1994].
The mechanisms by which CD59 and HRF inhibit the assembly of MAC on autologous cells are discussed in Section 1.3, page 42. 28
1.3 ASSEMBLY AND FUNCTION OF THE TCC
Activation of C5
Most of the C3b that is generated by the classical and alternative activation pathways is rapidly degraded. However, molecules that evade catabolism may become covalently linked to a variety of structures including prokaryotic cells and circulating immune complexes. Deposition of C3b on autologous cells is effectively inhibited by surface-bound regulatory mechanisms, although large quantities may accumulate on target surfaces forming a C3b cluster [Kinoshita, 1991]. When a C3b molecule becomes bound to a C3 convertase, the substrate specificity of the latter is modified and the resulting trimolecular complexes, C4b2a3b and C3bBb3b, become the C5 converting enzymes of the classical and alternative pathways, respectively [Daha et al., 1976, Kinoshita, 1991], (see Fig. 6, page 29). In the classical pathway C5 convertase, the C3b molecule is esterified to a single serine residue (i.e. Ser1217) in the a. chain of C4b, and both C3b and C4b are necessary for effective C5 activation [Law & Reid, 1995]. The requirement for functional C4b has been demonstrated by the study of convertases assembled with the defective C4 allotype, C4A6. This molecule has a single arginine to tryptophan substitution at residue 458 of its ~ chain. The resulting C4b2a3b complex neither binds to, nor cleaves C5, suggesting that the C5-binding site may reside within C4b [Ebanks et al., 1992]. As for the classical C3 converting enzyme, the catalytic site of the C5 convertase lies in the C2a subunit. In the alternative pathway, a similar reaction is catalysed by the Bb component of the complex.
Activation of C5 involves the cleaving of a single bond in its a. chain, releasing the first 74 amino acids (i.e. the anaphylatoxic fragment, C5a) from the larger portion,
C5b [Law & Reid, 1995]. However, this process is relatively inefficient and it has been estimated that between 60 and 200 molecules of C3 are split for every cleaved C5 [Bhakdi, 1988]. This has been demonstrated in studies of C5 catabolism in patients 29
Fig. 6. Diagrams of the classical and alternative CS converting enzymes
The classical (a) and alternative pathway (b) C5 convertases are derived from their respective C3 converting enzymes by the addition of a C3b molecule. C2a and Factor B contain the catalytic site.
(a)
C3b
Surface or immune ~~ complex -x-c-~ ~
C5 C5b+ C5a
(b)
C3b
Surface or ~~ immune -0-C-~ complex G
C5 CSb+ CSa
(Adapted from Law & Reid, 1995) Section 1.3 TCC: assembly & jUnction 30
with type II MCGN and circulating C3 Nef. Many of these individuals have a normal rate of C5 consumption and normal serum C5 levels, despite profoundly increased C3 catabolic rate. Furthermore, only some patients with C3 Nef have elevated plasma SC5b-9levels [Mollnes et al., 1986b], (see Section 1.4, page 54). These results indicate the inefficiency of formation of the soluble C5 convertase and activation of its substrate [Sissons et al., 1977].
Activation of C5 may also proceed by complement-independent mechanisms. These include plasma enzymes and also products of the myeloperoxidase-halide system of activated neutrophils (i.e. hypochlorite and taurine chloramine). In the latter case, C5 is not cleaved but is modified by oxidation of its methionine residues to methionine sulphoxide [Vogt & Hesse, 1994]. The resulting intact, but active molecule, is able to interact with components C6 to C9, to form a terminal complex. Tllis process may have relevance to inflammatory diseases in which leucocytic infiltration is prominent such as vasculitis and necrotising forms of glomerulonephritis.
Assembly of CSb-7
C5 activation is the final enzymatic step in the complement cascade. Thereafter, assembly of the TCC proceeds through spontaneous, non-covalent association of its components (see Fig. 7, page 31). C5b contains a metastable binding site for the sixth component (C6) and forms the nidus for TCC construction [Muller-Eberhard, 1986]. C5b6 is a relatively stable hetero-dimer [Yamamoto & Gewurz, 1978], in which C6 interacts with determinants in the a.-chain of C5b [DiScipio, 1992]. The efficiency of this reaction appears to be enhanced when complement activation and, in particular the generation of C5b, talces place on a surface rather than in solution [Bhakdi et al., 1988].
C5b6 has some affinity for lipid surfaces with which it may bind reversibly [Bhakdi et al., 1990] although this is relatively weak in comparison to that of the later intermediates of the terminal pathway. Soluble C5b6 may also bind to the seventh 31
Fig. 7. The terminal complement pathway
The terminal complement pathway is initiated with the proteolytic activation of CS by converting enzymes. Thereafter, assembly of the terminal complexes proceeds through non-enzymatic mechanisms. Regulatory effects are shown below by dotted lines.
CS ------. C5b + C5a
C5b6
CSb-7 ,...... -- vitronectin , 1 , .. - clusterin I I
Membrane insertion Soluble complex C5b-7(m) * SCSb-7
C5b-8(m) * SCSb-8 HRF ______,.. CD59 ----- · .,..
C5b-9(m) * SC5b-9 Membrane attack complex Soluble terminal complex
* "(m)" has been used by some authors to denote membrane-bound terminal complexes (see Bhakdi & Tranum-Jensen, 1984, Mollnes & Harboe, 1987, Bhakdi, 1988). #multiple C9 molecules may be incorporated (see page 33). Section 1.3 TCC: assembly & function 32 complement component (C7) to form the trimolecular complex, C5b-7. As for C6, C7 is held in this structure by association with the a chain of C5b [DiScipio, 1992]. However, while C5b6 remains loosely bound to the C3b subunit of the C5 convertase, C5b-7 is liberated into the fluid phase [Law & Reid, 1995]. This intermediate represents a branch point in the assembly of the TCC from which there are two main paths. Firstly, if a suitable lipid layer is available, C5b-7 can spontaneously insert into the membrane leading ultimately to the construction of the MAC. Alternatively, C5b-7 which remains free, rapidly forfeits its ability to interact with cell membranes due to the absorption of the regulatory plasma proteins, vitronectin and clusterin, producing the non-lytic soluble complex, SC5b-9.
THE MEMBRANE ATTACK COMPLEX
Assembly of the MAC and intermediate complexes
Assembly of the MAC (i.e. C5b-9(m)) from soluble C5b-7, C8 and C9 proceeds by several sequential reactions (see Fig. 7, page 31). The membrane-bound complexes, C5b-7(m) and C5b-8(m), are stable intermediates of this process with distinct physical and chemical characteristics.
C5b-7(m)
Insertion of C5b-7 into a membrane involves interactions between the lipid layer and exposed hydrophobic sites, primarily on C6 and C7 [Law & Reid, 1995]. This reaction, producing C5b-7 (m), is essentially irreversible [Tschopp, 1984b]. Electron microscopy of complement-coated lipid vesicles has shown that C5b-7(m) has an extended structure of length 25 nm and is attached to the lipid via a stalk measuring 8 - 10 nm long and 2 - 3 nm wide [Tschopp, 1984b]. This stalk is grouped into three consecutive globular domains, similar to that which occur in the tails of poly-C9 Section 1.3 TCC: assembly & function 33
[Tschopp, 1984b], (see Section 1.2, page 17). The widened region of C5b-7(m), on the upper part of the stalk, often appears to branch into two domains, each 15 - 17 nm in length [Tschopp, 1984b].
C5b-8(m)
Addition of one molecule of the eighth component of complement (C8) to C5b-
7 (m) proceeds through interactions between C5b and the ~ chain of C8 [Law & Reid, 1995]. This is associated with widening, but not lengthening of the C5b-7(m) stalk and greatly reduces its tendency to self aggregation [Tschopp, 1984b].
C5b-9(m) (The MAC)
The final stage in assembly of the MAC involves the accumulation of a variable number of C9 molecules (i.e. up to 18) by C5b-8(m) [Kinoshita, 1991]. It has been proposed that native C9 interacts with two distinct binding sites on the C8a-subunit of C5b-8(m). Binding to Site 1 is reversible while binding to Site 2 is accompanied by a conformational change in C9 leading to membrane insertion and ultimately, polymerisation and formation of a lytic pore [Lehto & Meri, 1993, Law & Reid, 1995], (see Fig. 8, page 34).
Structure and function of the MAC
Complement produces damage to cell membranes through its five terminal components acting in concert, as the MAC [Thompson & Lachmann, 1970, Lachmann & Thompson, 1970, Bhakdi & Tranum-Jensen, 1983a]. While its primary function is in anti-microbial defence, in recent years there has been considerable debate over the 34
Fig. 8. Formation of the membrane attack complex
Incorporation of C9 into the MAC initially involves a reversible interaction between the C9 monomer and Site 1, then irreversible binding to Site 2 on C5b-8(m). Polymerisation of C9 may then proceed.
multiple C9 molecules
C5b-8(m) l
Site 1
Site 2 Membrane
Reversible binding of C9 Irreversible elongation of C9 Polymerisation of C9 monomer to Site 1 of · with binding to Site 2 and MAC formation C5b-8(m)
(Adapted from Law & Reid, 1995) Section 1.3 TCC: assembly &function 35
mechanisms by which the MAC effects cytolysis or alterations to mammalian cellular processes.
Heterogeneity of terminal complexes
Membrane attack complexes deposited on erythrocytes in vitro vary in their C9 content according to the conditions which apply and, in particular, the amount of C9 available. Moreover, complexes of varying size may co-exist in proximity on lipid bilayers [Sims & Lauf, 1980, Boyle et al., 1981, Dalmasso & Benson, 1981]. At low serum concentrations, Bhakdi and Tranum-Jensen (1984) found that the C9 : C8 ratio was 2-3 : 1, i.e. consistent with a formula of (C5b-8)1 C92_3• However, complexes generated in an abundance of serum contained :from six to eight C9 molecules. Morgan et al., (1987) found a C9 : C8 molar ratio of 12 : 1 on neutrophils recovering from complement attack and in the membrane vesicles shed by exocytosis. Cyclic polymerised C9 may contain up to 18 C9 units [Tschopp, 1984b], (see Section 1.2, page 17) and Silversmith and Nelsestuen (1986) similarly found a maximum C9 : C5b-8 molar ratio of 16.2 ± 2.0 for complexes assembled on small phospholipid vesicles in vitro. By comparison, Bhakdi and Tranum-Jensen (1986) have shown that only one C9 molecule may be sufficient for the generation of a stable transmembrane pore. Moreover, C9 is not strictly required for the construction of a functional MAC as the tetramer, C5b-8, is able to lyse erythrocytes and some nucleated cells, albeit at a slower rate than the mature, C9-containing lesion [Stolfi, 1968, Podack & Muller-Eberhard, 1978, Biesecker, 1983, Podack & Tschopp, 1984, Law & Reid, 1995]. It is likely that many C5b-8 complexes do not acquire C9. Estimates of the proportion of membrane bound C8 molecules eliciting formation of the MAC in vitro, range from 1 in 8- 12, to 1 in 3 - 4 [Bhakdi & Tranum-Jensen, 1984]. Earlier reports of a dimeric MAC with the general formula (C5b-8)2 C9 12 [Biesecker et al., 1979, Podack & Muller-Eberhard, 1980], probably do not occur in most cases [Bhakdi & Tranum-Jensen, 1981, Tschopp, 1984b], although fusion of complexes has been proposed [Sims and Lauf, 1980]. Section 1.3 TCC: assembly &function 36
Measurements of the MW of the MAC have provided a range of results which is consistent with its variable C9 content. A MW of 1.0 to 1.3 x 106 D has been proposed for the more common, C9-unsaturated complexes [Bhakdi & Tranum-Jensen, 1981, Bhakdi et al., 1990], while the larger forms may weigh 1.5 - 1.6 x 106 D [Bhakdi & Tranum-Jensen, 1984]. The range of sedimentation rates reported for the MAC on sucrose density gradients is similarly broad, i.e. 25S- 40S [Bhakdi et al., 1990].
MAC-induced membrane permeability
Membrane attack complexes have been studied on natural cell membranes, unilamellar vesicles and planar lipid bilayers. The MAC has been found to form stable water-filled pores with slight selectivity for cations, but overall voltage insensitivity [Benz et al., 1986]. Several studies have also demonstrated leakage of intracellular macromolecules across complement-treated membranes including haemoglobin, ovalbumin, ribonuclease A, glucose-6-phosphate dehydrogenase and pancreatic trypsin inhibitor [Giavedoni et al., 1979, Ramm & Mayer, 1980, Dalmasso & Benson, 1981, Ramm et al., 1985, Malinski & Nelsestuen, 1989]. These observations indicate that a large breach in the membrane must be present. However, studies of the restricted diffusion of sucrose through MAC lesions have suggested a relatively small pore diameter of the order 16- 24 A (1.6- 2.4 nm) [Sims & Lauf, 1978 & 1980]. Ramm et al., (1982 & 1985) noted that the ability of MAC channels to conduct the passage of various molecules depended on the number of C9 units present in each complex, and hence, the channel size. Specifically, an average of one C9 molecule permitted the free movement of sucrose, two to three molecules allowed the passage of inulin and four C9 molecules were required for the permeability of ribonuclease A. In micelles bounded by a phospholipid monolayer, Malinski and Nelsestuen "(1989) similarly found that increasing the C9 content of MAC lesions was accompanied by progressive pore enlargement, with a maximal span of approximately 10 nm. Furthermore this was accompanied by the release of trapped macromolecules of increasing size up to, but not exceeding a diameter of 118 A(11.8 nm). By contrast, Sims and Lauf (1980) considered Section 1.3 TCC: assembly &function 37
that C5b-9 assemblies could fuse into larger units and that this contributed more to their functional heterogeneity than variations in their individual C9 content.
Morphology of the MAC
Membrane-damaging, pore-forming proteins occur in several situations in nature and include the cytolytic products of fungi, amoeba and certain parasites [Bhakdi & Tranum-Jensen, 1987]. Cytotoxic T lymphocytes also elaborate a pore-forming protein, perforin [Young & Cohn, 1987, Bhakdi & Tranum-Jensen, 1988], which exhibits some homology with domains in C6, C7, C8a., C8p and C9 [Lichtenheld et al., 1988]. However, the only component of the TCC which is capable of spontaneous polymerisation is C9 [Law & Reid, 1995], (see Section 1.2, page 15). Cyclic poly-C9 is similar in ultrastructure and dimensions to the MAC pore and it has been suggested that it makes up the major (or sole) part of its channel structure with the other components lying more peripherally [Muller-Eberhard, 1988]. A possible exception is the a.-chain of C8 which may also directly contribute to the pore. In support of this, Whitlow et al., (1985) studied C5b-9 lesions in erythrocytes and provided evidence that both C8 and C9 span the membrane and penetrate into the cytoplasm.
An issue of contention has been whether the MAC truly constitutes a water filled transmembrane pore (i.e. a lytic pore) [Mayer, 1972], or whether a functional conduit, or "leal(y patch" is produced by disturbance of the phospholipid molecules in the vicinity of the complex [Esser et al., 1979]. Induction of membranolytic enzymes, including phospholipases has also been proposed. For example, activation of arachidonic acid synthase and other enzymes leading to the mobilisation of membrane lipids has been noted. However, this appears to be a consequence of the transmembrane 2 flux of Ca + and other ions rather than a primary response [Salmon & Higgs, 1987, Morgan, 1989], (see page 40).
There is considerable evidence that the MAC lesion conforms to a true trans membrane pore. Bhakdi and Tranum-Jensen (1978) studied the morphology of C5b-9 Section 1.3 TCC: assembly & function 38 extracted from target membranes and reinserted into artificial lipid vesicles. They concluded that the MAC was a vertically oriented, hollow, cylindrical macromolecule with lipid binding regions enabling it to penetrate the membrane. Ultrastructural studies involving electron microscopy of freeze-fractured membranes also indicate that complexes containing a large number of C9 molecules resemble a hollow, water-filled protein cylinder which is, at least partly, embedded in the membrane [Podack et al., 1982a, Tranum-Jensen & Bhakdi, 1983], (see Fig. 8, page 34). McCloskey et al., (1989) studied C5b-8 and C5b-9 complexes assembled on synthetic phospholipid layers, using freeze etch electron microscopy. C5b-8 appeared as 12 nm particles on the external surface, which formed chain-like aggregates. By contrast, C5b-9 appeared as 27 nm rings on the external lipid surface. Upon splitting of the membrane C5b-9 partitioned with the outer leaflet leaving a hole of 17 nm diameter in the protoplasmic face. Tranum-Jensen et al., (1978) also examined the ultrastructure of C5b-9 assembled on sheep erythrocytes. They appeared as thin walled cylinders with an annulus on the extracellular side. The total height was 150 A (15 nm) to which the annulus contributed 30 A (3 nm). The internal diameter was uniform and measured 100 A (10 nm). The annulus had an external diameter of 200 A (20 nm) and the cylinders were oriented vertically within the membrane.
Esser (1991) has argued that the breach in membrane integrity produced by the MAC can be explained by a "leaky patch" model, rather than a rigid pore. He notes that lesions containing only three or four C9 molecules are fully cytotoxic although they may not exhibit a typical ring structure. It has been proposed therefore, that ring complexes consisting of more than 12 C9 monomers may be physiologically mlimportant [Muller-Eberhard, 1986, Esser, 1989-1990]. The leaky patch theory requires that local disarrangement of lipid molecules in the region of the MAC disturbs the membrane structure. Distortion of the lipid bilayer has been demonstrated by electron spin resonance spectroscopy [Esser et al., 1979] and more recently, by freeze fracture electron microscopy [McCloskey et al., 1989]. Approximately 5000 lipid molecules appeared to be influenced by each MAC lesion in these studies. Furthermore, proponents of the leaky patch hypothesis note that the MAC cannot be considered a true channel without satisfying certain physical requirements. These include (i) gating Section 1.3 TCC: assembly & jUnction 39
properties, (ii) agonist selectivity, (iii) turn on/turn off mechanisms, (iv) reflection co efficient, (v) diffusion behaviour and (vi) selective inhibition by chemical means or site directed mutagenesis. Many of these criteria have not been met. However, Podack et al., (1982a) have provided an intermediate perspective in this debate. They proposed that the MAC functions both by channel formation and lipid reorientation based on studies of the three dimensional structure of MAC on phospholipid vesicles.
Cellular responses to MAC deposition
MAC-induced cytolysis
Deposition of MAC on a cell surface results in disruption to its osmotic barrier and allows the passage of electrolytes and water across the membrane according to their electrochemical gradients and physical characteristics. The relatively high intracellular protein concentration results in cell swelling and ultimately lysis. It has also been shown that the osmotic pressure generated by extracellular macromolecules affords some protection from complement attack [Bhal(di & Tranum-Jensen, 1991]. A single MAC lesion containing very few or no C9 molecules may be sufficient to lyse metabolically-vulnerable cells such as erythrocytes. This has been referred to as the "one hit theory" [Mayer, 1972]. Kitamura and Inai (1974) studied, in detail, the reaction of C9 with cell-bound C 1-8. They found that guinea pig erythrocytes coated with C 1-8
(i.e. EAc1_8) could be lysed by only one molecule of either guinea pig or human C9, although human EAc1_8 cells appeared to require more than one C9 molecule for a critical lesion.
Recovery from MAC attack
Activation of complement and deposition of the MAC on the surface of nucleated cells does not necessarily lead to cytolysis [Morgan, 1989]. Terminal Section 1.3 TCC: assembly &function 40
complexes can be shed from metabolically active cells by "capping" and vesiculation, and the T112 of MAC in this context is approximately 2 min [Morgan, 1997]. This process has been demonstrated in human platelets [Sims & Wiedmer, 1986], oligodendrocytes [Scolding et al., 1989], rat glomerular cells [Camussi et al., 1987] and others. Alternatively some cells, including neutrophils, process MAC both by exocytosis and endocytosis with subsequent internal degradation [Morgan et al., 1987].
Several interacting intracellular messengers may be responsible for the recovery of nucleated cells :from MAC attack [Morgan, 1989]. It has been suggested that ci+ influx through complement lesions may be the primary stimulus to membrane repair [Morgan, 1989]. Morgan and Campbell (1985) demonstrated that a transient rise in the intracellular ci+ level was required for rapid removal of the MAC :from the membrane of leucocytes following a sub-lethal attack. This involved both calcium influx and mobilisation of intracellular stores. The cytoplasmic concentration of cyclic AMP (cAMP) also rises following MAC deposition [Morgan, 1989] and this nucleotide has been shown to rescue cells (i.e. tumour cells) bearing lethal amounts of MAC [Boyle et al., 1976]. This suggests a role for guanine nucleotide-binding proteins (i.e. G proteins) and adenylate cyclase in the response to, and repair of MAC lesions [Morgan, 1989].
Metabolic effects of MAC deposition
A growing literature reports the effects of non-lethal doses of MAC on metabolically competent cells and, in particular, their production and release of active mediators such as oxygen radicals, activated enzymes and metabolites of arachidonic acid. This phenomenon has been demonstrated in leucocytes, platelets, glomerular cells, glial cells and others (See Table 1, page 41). Influx of ci+, facilitated by MAC lesions, may in many cases be the trigger for the elaboration of metabolic products in addition to membrane repair processes [Morgan, 1989]. However, an early response of platelets to MAC deposition appears to be an increase in membrane Na+ conductance leading to the loss of membrane polarity. Repolarisation involves active transport of both Na+ and K+ and requires extracellular calcium [Sims & Wiedmer, 1991]. 41
Table 1. Metabolic effects of sub-lethal MAC deposition
Cell type Species Effect Reference
Blood cells
PJVlNL Rat ROM & LTB4 production a, b PJVlNL Human ROM & LTB4 production c, d Monocytes Human ROM, IL-l & prostanoid release e Platelets Human Prothrombinase activation, a granule f, g, h secretion & TXB2 production
Glomerular cells
Epithelial cells Rat AA, PgE2, PgF2w TXA2 & TXB2 release, i,j PLC activation, IP2 & IP3 production Epithelial cells Human Collagen release k Mesangial cell Rat PgE2, IL-l-like factor release, l,m,n,o ROM production, phospholipase activation
Others
Peritoneal Rat AA, Pg~ & TXB2 production p macrophage Endothelial cells human& Release of growth factors q bovine 0 ligodendrocyte Rat LTB4 production r Glioma cells Rat AA mobilisation s Synoviocytes Human ROM, PgE2 production t, u Erythroleukaemic Human Synthesis of"large complement-induced v cells proteins" (i.e. L-CIP)
Abbreviations
AA arachidonic acid, DAG 1,2 diacylglycerol, IL-l interleukin 1, IP2 & IP3 inositol phosphates, LTB4 leukotriene B4, PgE2 prostaglandin E2, PgFza prostaglandin F2a, PLC phospholipase C, ROM reactive oxygen metabolites, TXA2 & TXB2 thromboxane A2 & B2•
References (a) Morgan, 1989, (b) Imagawa et al., 1987, (c) Morgan & Campbell, 1985 (d) Seeger et al., 1986, (e) Hansch et al., 1987, (f) Wiedmer et al., 1986, (g) Chang et al., 1993, (lz) Betz et al., 1987, (i) Cybulsky et al., 1989, (j) Hansch et al., 1988a, (k) Torbohm et al., 1990, (l) Adler et al., 1986, (m) Lovett et al., 1984, (u) Lovett et al., 1987, (o) Lianos & Zanglis, 1992, (p) Hansch et al., 1984, (q) Benzaquen et al., 1994, (r) Shirazi et al., 1987, (s) Shirazi et al., 1989, (t) Morgan et al., 1988, (11) Daniels et al., 1990, (v) Reiter & Fishelson, 1992.
(Adapted in part from Morgan, 1989) Section 1.3 TCC: assembly & JUnction 42
Deposition of C5b-8 has also been shown to have metabolic sequelae. Polymorphonuclear leucocytes release small amounts of prostaglandin and arachidonic acid following assembly of this complex on their surface, although C9 is required for a maximal response and, in particular, the secretion of leukotrienes (e.g. LTB 4) and other mediators [Imagawa, 1987]. Both C5b-8 and C5b-9 have also been shown to modulate the release of collagen from human glomerular epithelial cells in vitro. This may have a role in the development of sclerotic lesions in the kidney [Torbohm et al., 1990].
MAC deposition modulates the complement pathway by feedback inhibition. It inhibits the assembly of new C3 convertases and the cleavage of C5 by preformed converting enzymes [Bhakdi et al., 1988c]. Furthermore, sub-lytic doses of MAC appear to afford protection to metabolically competent cells from subsequent complement attack [Reiter et al., 1992]. The molecular basis of this remains unclear, however MAC assembly has been shown to up-regulate expression of the membrane bound complement inhibitors, DAF and HRF, in cultured human mesangial cells [Shibata et al., 1991, Schieren et al., 1994]. In the case of HRF, this is transient, independent of protein synthesis, and may involve mobilisation of an intracellular pool. C5b-9 deposition may similarly protect cells from subsequent attack by non complement pore-forming complexes (e.g. perforin), possibly through the elaboration of "large complement induced proteins" (L-CIP), [Reiter & Fishelson, 1992].
Recent evidence also suggests that non-lytic doses of MAC may have a mitogenic effect on cells, both directly [Halperin et al., 1993] and indirectly through the release of growth factors [Benzaquen et al., 1994]. It may also paly a role in regulating apoptosis [Morgan, 1997].
Inhibition of attack by homologous MAC
Many cells have a potent armoury of molecular mechanisms to evade attack by homologous MAC, i.e. "homologous restriction", or species-specific inhibition of complement attack. This may serve as an important inhibitor of reactive or bystander Section 1.3 TCC: assembly & function 43 cell lysis, described originally as "the complement-mediated lysis of un-sensitised cells", involving elements of the terminal pathway [Lachmann & Thompson, 1970, Thompson & Lachmann, 1970]. In particular, two integral membrane proteins, CD59 and HRF, have been shown to inhibit the actions of autologous MAC and reactive lysis (see Section 1.2, page 26). The mechanism of action of CD59 in particular, has been studied extensively. It binds to the ay subunit of C8 and the "b" domain of C9 during MAC assembly [Meri et al., 1990, Ninomiya & Sims, 1992] and inhibits the unfolding and membrane insertion of the first C9 unit (see Fig. 9, page 44). Consequently, further recruitment and polymerisation of C9 is also inhibited and the C8 : C9 ratio of the resulting complexes is 1:1 [Lachmann, 1991]. HRF has similarly been shown to restrict the number of bound C9 molecules to equimolar with respect to C7, and also to inhibit C5b-8-mediated cytolysis [Zalman & Muller-Eberhard, 1994]. The effect of homologous restriction is profound and it has been estimated that human erythrocytes may survive deposition of between 1,000 and 2,000 homologous terminal complexes without lysis [Bhakdi et al., 1990]. This is in contrast to the very few complexes required to lyse heterologous red cells.
HRF has also been found in the granules of cytotoxic lymphocytes, and it has been proposed that it may serve to protect target cells from antibody-dependent cell mediated cytolysis [Zalman et al., 1986]. However, Lachmann (1991) has noted that homologous restriction is not a feature of this process and the significance of HRF in this location remains unclear.
THE BIOLOGY OF SCSb-9
Assembly of SC5b-9 and its intermediates
The soluble complex, SC5b-9, is produced as a by-product of complement activation on target surfaces. Furthermore, greater quantities are generated when activation occurs on non-lipid structures such as bacterial cell walls [Bhakdi et al., 1990]. In this setting, nascent C5b-7 is less likely to encounter a suitable membrane 44
Fig. 9. Inhibition of MAC assembly on homologous cells by CD59
CD59 inhibits MAC formation on homologous cells by interfering with the interaction between C9 and Site 2 on C5b-8(m). The initial C9 monomer may still bind, however polymerisation and formation of a typical ring-like pore do not occur. (Compare with Fig. 8, page 34).
C9 monomers
C5b-8(m) ~
Site 1 +- CD59
Membrane
Binding of C9 monomer to Site 1 Failure ofconformationally on C5b-8(m) altered C9 to bind to Site 2 and inhibition ofMAC formation
(Adapted from Law & Reid, 1995) Section 1.3 TCC: assembly & function 45
with which to bind and form the MAC. Complement activation in the fluid-phase, i.e. by soluble immune complexes, also results in the production of SC5b-9. However, it has been estimated that only 3 - 20 % of C5b molecules produced by soluble C5 convertases ultimately become incorporated into the TCC [Bhakdi, 1988, Bhakdi et al., 1988a]. Several intermediate complexes involved in SC5b-9 assembly have been identified and correspond to those leading to MAC formation.
SCSb-7
C5b-7 which remains unbound, rapidly forfeits its ability to interact with cell membranes. This is due mainly to the non-covalent absorption of two plasma proteins, i.e. vitronectin [Podack et al., 1977, Jenne & Stanley, 1985], (see Section 1.2, page 18) and clusterin (SP-40,40) [Murphy et al., 1988], (see Section 1.2, page 25), forming the hydrophilic complex, SC5b-7. The resulting assembly has a globulin electrophoretic mobility and a MW of 668 kD [Podack et al., 1977].
The soluble inhibitors of the TCC are thought to occupy the exposed apolar, putative lipid-binding regions of C5b-7, thereby preserving its solubility in plasma. Specifically, a peptide sequence containing three intradomain disulfide bridges which is common to complement components C6 to C9 (and perforin), has been implicated by antibody inhibition studies [Tomasini & Mosher, 1991]. This domain is homologous to the LDL receptor [Sudhof et al., 1985] and contains a relatively large proportion of negatively charged residues [Tomasini & Mosher, 1991], (see Section 1.2, page 11).
Inhibition of CSb-7 membrane insertion by vitronectin and clusterin
Vitronectin binds to C5b-7 in a molar ratio of 1:1 [Podack et al., 1977] and effectively inhibits complement-mediated cytolysis with a dissociation constant (Ki) of approximately 40 1-Lg/mL [Podack et al., 1978]. It has been proposed that interactions Section 1.3 TCC: assembly &function 46 between vitronectin's heparin-binding domain, i.e. residues 348 - 360 and the highly conserved cysteine-rich region of the terminal complement components underlie this reaction [Tschopp et al., 1988, Hogasen et al., 1992]. However, recent evidence suggests that domains other than the heparin-binding site may be involved. Sheehan et al., (1995) demonstrated that cyanogen bromide digest fragments of vitronectin, devoid of heparin-binding activity, were also able to inhibit CSb-7 binding to immobilised, intact vitronectin
Clusterin inhibits the cytolytic potential of nascent CSb-7 at physiological plasma concentrations (i.e. approximately 50 J..Lg/mL) [Jenne & Tschopp, 1989] and renders it water soluble and inactive in a manner similar to vitronectin [Tschopp et al.,
1993]. In particular, clusterin binds to C7 and the ~-chain of C8 [Tschopp et al., 1993]. Murphy et al., (1989b) demonstrated that, on an equimolar basis, SP-40,40 (clusterin) was a more potent inhibitor of CSb-6-mediated haemolysis than vitronectin, however additional inhibition occurred when both proteins were present.
SC5b-8
One molecule of C8 combines with SCSb-7 to form SCSb-8. This intermediate has a nominal MW of 800 - 850 kD, a sedimentation coefficient of 19 - 20S and its effective molecular radius is approximately 10 nm [Bhakdi & Roth, 1981]. Each SCSb- 8 complex may contain two molecules ofvitronectin [Bhakdi & Roth, 1981].
SC5b-9
SCSb-9 is produced by the addition of one or more C9 units to SCSb-8. It has a MW of approximately 1000 kD and a sedimentation coefficient of 23 S [Kolb & Muller Eberhard, 1975a, Bhakdi et al., 1979, Bhakdi, 1988]. On average, each complex contains only two to three molecules of C9 [Kolb & Muller-Eberhard, 1975b, Bhakdi et Section 1.3 TCC: assembly & function 47
al., 1979, Bhakdi, 1988], that is, a lesser quantity than the MAC, although the latter exhibits considerable heterogeneity in this regard (see page 35). Bhakdi & Tranum Jensen (1983b) also found a similar subunit composition in rabbit SC5b-9.
Under the electron microscope, SC5b-9 appears wedge-shaped [Preissner et al., 1989b] or globular [Bhakdi & Roth, 1981]. It therefore lacks the characteristic ring structure of C9-saturated MAC [Bhakdi & Tranum-Jensen, 1987] but has, nonetheless, been described as a "masked cylinder" [Bhakdi et al., 1979]. While many studies have demonstrated the chemical stability of SC5b-9, Podack and Muller-Eberhard (1980) observed dissociation of vitronectin from the complex in 145 mM desoxycholate with re-emergence of its amphiphilic characteristics.
Vitronectin and clusterin inhibit C9 polymerisation within the TCC
It has been estimated that each mature SC5b-9 complex may contain two to three molecules ofvitronectin [Kolb & Muller-Eberhard, 1975b, Podack & Muller-Eberhard, 1980, Bhakdi & Roth, 1981, Bhakdi, 1988, Bhakdi et al., 1990]. Similarly, one to two molecules of clusterin may also be present [Bhakdi et al., 1990]. In addition to antagonising C5b-7 membrane insertion, there is evidence that both vitronectin and clusterin bind directly to C9 and inhibit its polymerisation within the TCC [Podack et al., 1984, Milis et al., 1993, Johnson et al., 1994]. This reaction appears to involve the heparin binding domain of vitronectin [Milis et al., 1993], while Tschopp et al., (1993) found that both clusterin subunits were equally potent inhibitors of MAC-associated haemolysis and zinc-induced polymerisation of C9. However, Milis et al., (1993) have suggested that vitronectin may exert most of its anti-complementary effect through the inhibition of C5b-7 membrane attachment, rather than antagonism of C9 directly.
The co-localisation of vitronectin with C9 neoantigens (found in SC5b-9, MAC and poly-C9) in tissues has also raised the possibility that vitronectin may be a regulator of tissue-bound MAC [Bhakdi et al., 1988b]. This is discussed further in Section 1.4, page 52. Section 1.3 TCC: assembly & function 48
Lipoproteins and inhibition of the TCC
Lipoproteins antagonise the formation of the MAC and may act in a similar manner to vitronectin [Lint et al., 1977, Podack et al., 1978]. In particular, LDL inhibits MAC assembly with a Ki of 34 J..Lg/mL [Podack et al., 1978]. Although the serum concentration of LDL is approximately 10 times that of vitronectin [Tomasini & Mosher, 1991], its role as a complement inhibitor in vivo remains ill defined. Notably, clusterin is also found in the plasma in association with lipoproteins and forms a high density complex with apolipoprotein A-I [Jenne et al., 1991].
Properties and putative functions of SC5b-9
As yet, no significant biological function in vivo has been confidently ascribed to SC5b-9. However, evidence from studies in vitro suggest possible roles in cell adhesion, intercellular matrix reactions and coagulation. Many of these functions may be mediated by the vitronectin subunit.
Cell binding
Biesecker (1990) demonstrated that L8 skeletal muscle-derived myoblasts adhered to plates coated with SC5b-9. Binding was strongly inhibited by pre-treatment of the coated plates with polyclonal anti-vitronectin antibody, while no inhibition occurred with anti-CS or anti-C8, and only slight inhibition with antibodies against C6, C7 and C9. Similarly, the fibronectin-derived peptide "GRGDSP", which includes the integrin-binding motif, inhibited L8 cell binding. Other groups have also noted the ability of SC5b-9 to promote cell spreading in vitro and therefore suggested that it may serve such a role in the tissues [Bhakdi, 1988]. Section 1.3 TCC: assembly & function 49
Heparin binding
Hogasen et al., (1992) found that SC5b-9 adhered to heparin-Sepharose. In this study, SC5b-9 was also shown to bind to the monoclonal antibody, 8E6, which recognises the conformationally altered, heparin-binding form of vitronectin. They proposed that the vitronectin heparin-binding site may be involved in the interactions between SC5b-9, cells and the intercellular matrix. Similarly, it has been shown that the binding of vitronectin-T-AT III complex to endothelial cells is dependent on the vitronectin heparin-binding site [de Boer et al., 1992].
Interactions with the coagulation pathways
AT III has been shown to associate with SC5b-9 in serum although the functional significance of this, if any, remains uncertain [K.olb & Kolb, 1983].
Interactions with P-endorphin
SC5b-9 expresses non-opiate binding sites for the C-terminus of P-endorphin [Schweigerer et al., 1983]. The significance of this is also uncertain, however the authors suggested that P-endorphin may modulate the immune response by this mechanism.
Contribution to tissue matrix TCC
The extent to which circulating SC5b-9 can be trapped by tissues, in the absence of in situ complement activation, remains unknown [Mollnes & Harboe, 1987]. Bhakdi (1988) considered that it was "primarily improbable" that SC5b-9 could diffuse from the plasma, through the endothelium, to find the connective matrix. However, its Section 1.3 TCC: assembly & function 50
behaviour at sites of endothelial damage is uncertain. Dalmasso et al., (1989) suggested that plasma SC5b-9 may contribute to tissue deposits and hypothesised that it could adhere to damaged structures and promote continued injury. Vitronectin may target SC5b-9 to sites of endothelial disruption through interactions involving its heparin binding domain and the sub-endothelial matrix [Bhakdi, 1988]. The frequently demonstrated coexistence of vitronectin and C9 neoantigens in inflamed tissue lends support to this proposal [Falk et al., 1985, Bariety et al., 1989], (see Section 1.4, page 52).
NEOANTIGENS OF THE TCC
Assembly of the TCC results in a conformational change in its components and exposure of hitherto cryptic epitopes, or neoantigens. Furthermore, there is reduced expression of determinants normally found on the native components [Kolb & Muller Eberhard, 1975a]. This phenomenon is not restricted to the terminal pathway and also occurs during activation of the early complement components such as C3, [Pettersen et al., 1988]. Kolb and Muller-Eberhard, (1975a) raised polyclonal antibodies against the soluble TCC by inoculation of rabbits with the purified complex. Anti-sera, specific for neoantigens, were obtained by absorption with fresh human serum. As the absorbed anti-sera precipitated the soluble TCC it was concluded that multiple neoepitopes must be present. They found that these antibodies recognised sites present on cell-bound C5b-7, C5b-8 and soluble C5b-9 (i.e. SC5b-9), but not the native components. Subsequently, Mollnes et al., (1985b) reported two murine monoclonal antibodies (mAb), i.e. MCaEll and MCbC5, which were specific for a neoantigen(s) ofpoly-C9, and recognised the soluble and tissue-bound TCC. However, MCaE11 (i.e. mAb aE11) proved to be superior in distinguishing polymeric from denatured monomeric C9 and has consequently been used widely in assays to detect and quantitate the TCC. Section 1.3 TCC: assembly & function 51
DETECTION AND QUANTITATION OF SC5b-9 AND THE MAC
The soluble TCC can be detected in human plasma using enzyme linked immunosorbent assays (ELISA) based on a combination of anti-CS and anti-C6 or anti C9 immunoglobulins [Mollnes et al., 1984]. However, a major problem of this approach is that plasma samples need to be fractionated initially to avoid interference from uncomplexed, native components. Recently, sensitive sandwich ELISAs have been developed which make use of monoclonal antibodies against neoantigens expressed by the TCC and, in particular, polymerised C9 [Falk et al., 1983, Mollnes et al., 1985a & b]. Monoclonal antibody aEll is currently in widespread use. These assays obviate the need to fractionate plasma but are still subject to several sources of error. In particular, Mollnes et al., (1985a & 1988a) have stressed the importance of drawing blood into ethylenediaminetetraacetic acid (EDTA) and keeping specimens cooled to :5: 4 oc to inhibit spontaneous complement activation in vitro, which may otherwise contribute to erroneously high SC5b-9 values [Hugo et al., 1987].
Early assays failed to detect the TCC in normal plasma [Falk et al., 1983]. However, it was subsequently found, albeit at a low level. Hugo et al., (1987) reported a concentration of < 100 - 200 ng/mL in healthy subjects and the normal range suggested by Behring Diagnostics (1990), which manufactures and distributes a frequently used SC5b-9 assay, is similarly low, i.e. 290 (200- 390) ng/mL, (median & (10- 90% range).
Immunohistochemical assays have also been established to detect the MAC in tissue deposits. Most often, these involve frozen tissue sections and similarly utilise monoclonal antibodies against C9 neoantigens with detection by fluorescein-conjugated or enzyme-linked second antibodies [Falk et al., 1983, Mollnes et al., 1985b, Mollnes & Harboe, 1987]. 52
1.4 TERMINAL COMPLEMENT COMPLEXES IN DISEASE
Several lines of evidence support a role for the terminal complement pathway in the pathogenesis of disease. For example, sensitive assays have revealed elevated levels of SC5b-9 in the plasma and serum of patients with a variety of disorders (see Table 2, page 53). However, there have also been some conflicting studies. Falk et al., (1985) found that patients with SLE had markedly elevated levels of SC5b-9 in their sera which returned to normal during clinical remission. Furthermore, the SC5b-9 concentration proved to be a more sensitive measure of disease activity than the levels of C3, C4 or total serum complement haemolytic activity (CH50). By contrast, Bhakdi (1988) found that a large group of untreated SLE patients did not have significantly elevated levels. Some of these discrepancies may relate to differences in specimen handling, such as the presence or absence of EDTA and the construction of the assay [Bhakdi et al., 1990]. Elevated levels of SC5b-9 have also been detected in several extravascular fluid compartments, including the cerebrospinal (CSF) and synovial fluids (see Table 2, page 53). It has been argued that quantitation of SC5b-9 at these sites could be of greater diagnostic relevance than plasma measurements since the complex may be removed more slowly [Bhakdi, 1988] and its presence may reflect local complement activation.
Deposits of MAC antigens have been detected in a wide variety of tissues from patients with both immunologically-mediated and non-immunological diseases. These include renal, neurological, dermatological, musculoskeletal, haematological, cardiovascular, endocrine, pulmonary and other disorders. Vitronectin has commonly been found in proximity to C9 neoantigens, and more recently, clusterin has also been identified in association with TCC deposits, particularly in the kidney [Eddy & Fritz, 1991, French et al., 1992]. Whether this results from the deposition of plasma SC5b-9 or association of the regulatory proteins with tissue MAC in situ, is not known, although Bhakdi et al., (1990) considered the latter scenario to be the more probable (see Section 1.3., page 49). Furthermore, it has been proposed that the presence of 53
Table 2. Conditions associated with elevated levels of TCC in body fluids
Fluid Disease Reference
Serum Active SLE a Guillain Barre Syndrome b Hashimoto's thyroiditis c Graves disease c Henoch-Schonlein purpura d
Plasma SLE e Membranous nephropathy e MCGNtypeii e,f Rheumatoid arthritis g,h Myocardial infarction i
Cerebrospinal fluid Multiple sclerosis j, k Infectious neurological disease j Radiculoneuritis j Guillain Barre syndrome k Cerebral SLE l Cerebral Sjogren's syndrome l
Synovial fluid Rheumatoid arthritis g,h Juvenile rheumatoid arthritis g
Urine Membranous nephropathy m
References
(a) Falk et al., 1985, (b) Koski et al., 1987, (c) Weetman et al., 1989, (d) Kawana & Nishiyama, 1992, (e) Horigome et al., 1987, (/) Mollnes et al., 1986b, (g) Mollnes et al., 1986a, (lz) Corvetta et al., 1992, (i) Mollnes et al., 1988b, OJ Mollnes et al., 1987, (k) Sanders et al., 1986b, (I) Sanders et al., 1987, (m) Schulze et al., 1991. Section 1.4 The TCC in disease 54
vitronectin and clusterin in the tissues indicates that the MAC may be in a haemolytically inactive form [Bhakdi, 1988, Murphy et al., 1989a], (see Section 1.3, page 45). However, only a relatively small proportion of tissue MAC appears to be associated with vitronectin (i.e.::;; 0.4 mole I mole C5b-9 [Bhakdi et al., 1988b]) and the significance of this requires clarification. A list of studies reporting tissue deposits of MAC is shown in Table 3, page 55.
THE ROLE OF THE TCC IN DISEASE PATHOGENESIS
In most cases, involvement of the TCC in the pathogenesis of tissue lesions is only implied by histochemical and serological studies. However, in some cases, there is direct evidence for its participation either in the disease itself, or an animal model thereof. Renal, neurological, musculoskeletal and dermatological diseases in particular, have undergone extensive scrutiny and provide insight into disease mechanisms.
THE TCC IN RENAL DISEASE
Several studies have demonstrated elevated levels of SC5b-9 in the plasma of patients with primary glomerulonephritis. Horigome et al., (1987) fotmd elevated TCC levels in eight of eleven patients with membranoproliferative GN (synonymous with MCGN) and one of six patients with membranous nephropathy. Mollnes et al., (1986b) similarly reported elevated plasma SC5b-9 levels in some patients with type II MCGN and partial lipodystrophy. In patients with lupus nephritis, Horigome et al., (1987) reported that the plasma TCC concentration was correlated positively with the circulating immune complex level (CIC) and negatively with the CH50, C3, C4 and C5 concentrations. 55
Table 3. Diseases associated with MAC tissue deposition
Organ I System Tissue Disease Reference
Kidney (See Table 4, page 57)
Nervous system Brain capillary endothelia Multiple sclerosis a Oligodendrocyte-derived Multiple sclerosis b membrane vesicles Substantia nigra Parkinson's disease c * Skeletal muscle Motor endplate Myasthenia gravis d Viable muscle fibres Myositis e Necrotic muscle fibres Myositis & m. dystrophy e,f microvasculature Dermatomyositis g
Joints Synovium Rheumatoid arthritis h,i Psoriatic arthritis i Synovial cell cytoplasm Rheumatoid arthritis j Osteoarthritis j Membrane vesicles Rheumatoid arthritis k
Sltin Dermal-epidermal zone SLE & discoid lupus # l, m Basement membrane Bullous pemphigoid m, n Dermal papillae Dermatitis herpetiformis # m, o Vessel walls Henoch-Schonlein purpura p Dermal elastic fibres Aging# q
Cardiovascular Necrotic myocardium & Myocardial infarction # r, s system peri-infarction zone Aorta: plaques & debris Arteriosclerosis # t, U, V, W
Endocrine Thyroid gland Autoimmlme X system thyroid disease
Lymphoid tissue Follicular dendritic cells Follicular hyperplasia# y
References
(a) Compston et al., 1989, (b) Scolding et al., 1989, (c) Yamada et al., 1992, (tl) Engel & Arahata, 1987, (e) Morgan et al., 1984b, (f) Engel & Biesecker, 1982 (g) Kissel et al., 1986, (II) Husby & Williams, 1985, (i) Sanders et al., 1986a, OJ Corvetta et al., 1992, (k) Morgan et al., 1988, (l) Biesecker et al., 1982, (m) Dahlback et al., 1989b, (11) Dahl et al., 1984, (o) Dahl et al., 1985, (p) Kawana & Nishiyama, 1992, (q) Dahlback et al., 1989a, (r) Schafer et al., 1986, (s) Rus et al., 1987, (t) Vlaicu et al., 1985, (u) Rus et al., 1986, (v) Niculescu et al., 1987, (w) Niculescu et al., 1989, (x) Weetman et al., 1989, (jJ) Halstensen et al., 1988.
* C7 and C9 detected but MAC neoantigens were not sought. #Co-localisation ofVitronectin and MAC. Section 1.4 The TCC in disease 56
Deposits of MAC in renal tissue have also been widely reported (see Table 4, page 57). Co-localisation of MAC and immune deposits occurs in lupus nephritis [Biesecker et al., 1981, Lai et al., 1989], idiopathic membranous nephropathy [Rauterberg et al., 1981, Lai et al., 1989], anti-glomerular basement membrane nephritis (anti-GBM disease) [Rauterberg et al., 1981], mesangial IgA nephropathy (IgAN) [Rauterberg et al., 1987], post streptococcal nephritis [Parra et al., 1984] and various other forms of glomerulonephritis including MCGN types I and II [Falk et al., 1983, Kazatchkine et al., 1983, Mariot et al., 1984, Hinglais et al., 1986, Falk et al., 1987, Dalmasso et al., 1989]. However, small deposits of MAC, in the absence of immunoglobulin or other complement components, have also been noted in many conditions including minimal change nephropathy, scleroderma, multiple myeloma, diabetes mellitus, hypertensive nephrosclerosis, crescentic nephritis and amyloidosis [Couser et al., 1985, Dalmasso et al., 1989]. In this case, the TCC was most often located along the tubular basement membrane, Bowman's capsule, the internal elastic lamina and media of blood vessels, and in juxtaglomerular and mesangial regions, particularly in areas of sclerosis. Normal adult renal tissue, but not that from newborn infants, may also contain small amounts of MAC [Falk et al., 1983, Dalmasso et al., 1989]. Specifically, fine, granular immunostaining of C9 neoantigens has been observed in apparently normal glomeruli and arterioles [Bariety et al., 1989].
The terminal pathway regulatory proteins have also been identified in renal tissue. Vitronectin has been found alone, or in association with C9 neoantigens in the lddneys of patients with lupus nephritis, post infectious nephritis, mesangial IgA nephropathy, membranous nephritis, MCGN, glomerulosclerosis and renal amyloidosis (i.e. type A amyloid) [Falk et al., 1987, Mollnes & Harboe, 1987, Dahlback et al., 1987, Bariety et al., 1989, Lai et al., 1989]. Falk et al., (1987) found that C9 neoantigen was accompanied by vitronectin in almost all cases. An exception was that when vitronectin was linearly deposited in relation to the glomerular basement membrane, C9 neoantigen was absent. They speculated that this reflected a primarily non-immunological process, as seen in diabetic nephropathy. Furthermore, Bariety et al., (1989) found that when vitronectin was absent from immune deposits, CSb-9 was similarly not detected in these 57
Table 4. Studies reporting C9 neoantigens in renal tissue
Site Disease Glomerular Mesangium Arteries & Tubulo- Reference capillaries arterioles interstitium
Normal kidney +* +* +* a, b, c, d, e Minimal change + + + b, c,f nephropathy Hypertension + + + a Nephrosclerosis + + + a, b, e Diabetes mellitus + + + a, b, c Amyloidosis In amyloid deposits (especially type A) c, e Multiple myeloma - c,f Obstructive + + + a, c uropathy Allograft rejection + + + b, c,f Interstitial + c,f nephritis IgAN + a, b, c, e, fg Membranous GN + + + + a, b, c, e, f h, i MCGNtypei + + + + a, b, c, e,f MCGNtypeii + + + a, b Anti-GBM disease + fh Post infectious GN + + + + b,j Lupus nephritis + + + a, b, c, e, WHO II, III, IV, V i, k, l, m Crescentic GN + + c,f i
References (a) Falk et al, 1983, (b) Falk et al, 1987, (c) Hinglais et al., 1986, (d) Kazatchkine et al., 1983, (e) Bariety et al., 1989, (f) Mariot et al., 1984, (g) Rauterberg et al., 1987, (lz) Rauterberg et al., 1981, (i) Lai et al., 1989, OJ Parra et al., 1984, (k) Biesecker et al., 1981 (I) Mollnes et al., 1985b, (m) Mollnes & Harboe, 1987. + C9 neoantigens present, - antigens absent. * Weak staining only. (Adapted from Faile et al., 1987 & Dalmasso et al., 1989) Section 1.4 The TCC in disease 58
deposits but was restricted to a fme granular mesangial distribution, as seen in normal kidneys. Clusterin is also often co-localised with the TCC in diseased renal tissues. It is usually accompanied by vitronectin and is rarely found in the absence of immunoglubulin [French et al., 1992]. In contrast to vitronectin [Bariety et al., 1989, Tomasini & Mosher, 1991], clusterin is seldom seen in healthy glomeruli [Murphy et al., 1988 & 1989a].
Physiological studies have indicated that the MAC may have a direct role in the pathogenesis of glomerulonephritis. Recently it has also been reported that the permeability of isolated glomeruli to albumin in vitro is increased following exposure to CSb-9 [Savin et al., 1994]. Furthermore, there is evidence that altered glomerular haemodynamics may be a direct consequence of terminal pathway attack. Several types of GN have been investigated as discussed below.
Membranous nephropathy and the Heymann model
Human membranous nephropathy is distinctive in that immunoglobulin deposits are largely confined to the sub-epithelial region and, despite the paucity of cellular infiltrate, it is associated with marked proteinuria. The Heymann nephritis rat model most closely resembles this disease and results from immunisation with a proximal tubular brush border extract, Fx1A [Heymann et al., 1959, Couser et al., 1976]. A more rapidly developing model, induced by injection of heterologous anti-Fx1A antibody, i.e. passive Heymann nephritis (PHN), produces a similar condition with sub-epithelial deposits and has been widely studied [Feenstra et al., 1975a & b]. Immune complexes fmm in situ [Couser et al., 1978, Van Damme et al., 1978] involving a 330 kD glycoprotein (gp330) produced by the epithelial cell [Kerjaschki & Farquhar, 1983, Kerjaschld, 1992]. Gp330 is located in the clathrin-coated pits on the "soles" of the podocyte processes. Its normal function is uncertain although it appears to be a receptor for plasminogen and is a member of a large family of structurally related glycoproteins which includes the enzyme maltase [Kerajaschld, 1992]. Terminal complement Section 1.4 The TCC in disease 59
components have been demonstrated in the glomerular capillary walls in the autologous and heterologous phases of PHN [Adler et al., 1984] and complement depletion with CVF (but not neutrophil depletion) significantly reduces or abolishes the proteinuria [Salant et al., 1980, Perkinson et al., 1985]. Moreover, when rats rendered C6-deficient by the administration of anti-C6 F(ab')2 fragments are given anti-Fx1A, the development of proteinuria is prevented despite antibody deposition [Baker et al., 1985 & 1989]. Similarly, Camussi et al., (1987) have shown a requirement for C6 in the mediation of anti-Fx1A-induced cytotoxicity to glomerular epithelial cells in culture. A model of C8-dependent proteinuria and MAC-induced glomerular epithelial cell toxicity has also been reported in the isolated, perfused rat lddney [Cybulsky et al., 1986].
The molecular pathology of Heymann nephritis has been studied in detail. Within 10 minutes of injection of polyclonal anti-gp330 into normal rats, immune deposits accumulate within the glomerular epithelial cells' coated pits [Kerjaschki, 1992]. The complexes are then shed from the cell membrane and become associated with, and immobilised by, the lamina rara externa of the GBM. MAC is also present in the sub-epithelial immune deposits in PHN [Adler et al., 1984, Perkinson et al., 1985] and human membranous nephropathy [Lai et al., 1989]. At this site, C5b-9 could interfere with the function of the slit pore diaphragm of the epithelial cell [Kanwar, 1984]. Damage to, or death of the this cell might then follow, although ultrastructural studies in experimental models have indicated that processing of complexes, rather than cytolysis, occurs [Hinglais et al., 1986]. In PHN, Kerjaschki et al (1989) have detected C5b-9 antigens inside the epithelial cell, within intracytoplasmic vesicles, and in association with membrane-bound vesicles in the urinary space. They proposed that deposited MAC may be endocytosed by the epithelial cell, transported across the cytoplasm, and excreted. Moreover, patients with membranous nephropathy [Schulze et al., 1991], and animals with PHN [Pruchno et al., 1989], have been found to excrete increased quantities of C5b-9 in their urine. The highest levels occur early in the disease, and in the most severe cases. It has also been suggested that renal epithelial cells may respond to MAC deposition by the activation of lipolytic and transmethylase enzymes leading to tl1e production of abnormal, leaky basement membrane. In support Section 1.4 The TCC in disease 60
of this proposal, is the observation that C6-deficient rabbits with serum sickness and sub-epithelial MAC deposits produce an abnormal glomerular heparan sulphate with reduced negative charge [Groggel et al., 1985a].
Studies of Heymann nephritis have provided strong evidence of a central role for the TCC in glomerular immunopathogenesis. However, more recent investigations of the active Heymann model have suggested that CD4+ and CD8+ T cells, and other effector mechanisms, may also be involved [Quiza et al., 1992, Penny et al., 1997].
Anti-GBM nephritis
Within 24 h of administration of nephrotoxic immunoglobulin, rats [Kuhn et al., 1977, Timmermans et al., 1990] and rabbits [Groggel et al., 1985b] develop significant proteinuria. This is associated with a prominent neutrophil infiltrate in glomeruli from 15 to 30 min following deposition of anti-GBM antibody, and is maximal at 2.5 to 4 h [Cochrane et al., 1965, Kuhn et al., 1977]. The degree of proteinuria is proportional to the magnitude of the infiltrate [Cochrane et al., 1965] and is reduced by neutrophil depletion [Cochrane et al., 1965, Henson, 1972, Groggel et al., 1985b]. However, the role of complement in this disease remains controversial. While some studies have shown a reduction in proteinuria (and neutrophil infiltrate) in experimental animals following complement depletion [Cochrane et al., 1965 & 1970], others have found to the contrary [Adler et al., 1984, Perkinson et al., 1985]. Previously, it was considered that complement contributed to capillary damage indirectly, by recruitment of inflammatory cells through the production of C5a and C3b-mediated immune adherence [Couser et al., 1985, Groggel et al., 1985b]. However, more recent studies have implied a direct pathogenic role for the MAC. In C6-deficient rabbits, which are unable to assemble the MAC yet can activate C5, Groggel et al., (1985b) found a significant reduction in heterologous-phase proteinuria compared with control animals. The quantity of bound anti-GBM immunoglobulin, number of infiltrating neutrophils and the degree of cellular proliferation were unaltered, however, these animals had better preservation of GFR. This last observation is particularly notable as complement Section 1.4 The TCC in disease 61 depletion with CVF has also been shown to reduce the deterioration in GFR observed in experimental anti-GBM disease [Blantz et al., 1978].
The mechanisms underlying MAC-induced glomerular dysfunction in anti-GBM nephritis are uncertain. However, direct interaction of the MAC with the GBM has been proposed. The GBM is composed mainly of protein and proteoglycan chains (i.e. collagen types IV and V, laminin, entactin etc, [Couser et al., 1985]) and lacks a cellular lipid bilayer. However, it can activate complement via the alternative pathway [Williams et al., 1984]. Furthermore, Kopp and Burrell (1982) demonstrated that MAC like structures form on antibody-coated alveolar basement membrane, which is biochemically similar to the GBM. Although some studies have failed to demonstrate the MAC in glomeruli of experimental animals with anti-GBM nephritis [Adler et al., 1984], it has been observed in human tissue [Rauterberg et al., 1981]. Thus, MAC insertion and accumulation within the GBM remain possible [Couser et al., 1985].
Mesangial lgA nephropathy
Neoantigens of the MAC are commonly found in the mesangium of patients with IgA nephropathy and tend to co-localise with the deposits of IgA, but not immunoglobulins of other classes [Rauterberg et al., 1987]. However, a report of three Japanese males with congenital C9 deficiency and biopsy-proven mesangial IgA nephropathy [Yoshioka et al., 1992] argues against the absolute requirement for MAC in the development of this disease. All three patients had presented at the age of eight or nine years with haematuria and proteinuria, detected on routine micro-urine screening at school. Renal biopsies showed mesangial proliferation, glomerular crescents (in one patient) and positive immunofluorescence for IgA, C3 and vitronectin. However, after three to nine years follow up, no patient had deterioration in renal function prompting speculation that deposition of MAC may be associated with disease progression. In accord with this, Eguchi et al., (1987) found a positive relationship between the quantity of C9 neoantigen deposited in glomeruli and the histological grading of IgA disease. Similarly, Tomino et al., (1987) found that severe glomerular lesions, including crescent Section 1.4 The TCC in disease 62 formation, were more common in lddneys with C9 and/or vitronectin deposition. They concluded that activation of the terminal complement pathway may be an exacerbating factor in IgA nephropathy. However, the molecular basis of these observations is uncertain. Rat mesangial cells release vasoactive prostanoids in response to MAC deposition [Lovett et al., 1984, Lianos & Zanglis, 1992], (see Section 1.3, page 40), which may lead to alterations in local glomerular haemodynamics and membrane damage, although the significance ofthis awaits clarification.
Haemodialysis
Activation of the terminal complement pathway occurs during haemodialysis. Deppisch et al., (1990) found that the greatest increase in plasma SC5b-9 levels occurred with Cupraphane dialysers and less with Hemophane and Polysulphone F6. This phenomenon has been used to study the behaviour of endogenously generated SC5b-9 in human subjects (see Section 1.5, page 82 & Deppisch et al., 1990).
NEUROLOGICAL DISEASE
The TCC has frequently been detected in the CSF of patients with infectious diseases, radiculo-neuritis, multiple sclerosis [Sanders et al., 1986b, Mollnes et al., 1987], acute monophasic Guillain-Barre syndrome [Sanders et al., 1986b] and various other autoimmune conditions involving the central nervous system (including cerebral SLE and Sjogren's syndrome [Sanders et al., 1987]). However, plasma levels of SC5b-9 are often normal in primary neurological disease and Mollnes et al., (1987) found no positive correlation, overall, between the CSF and plasma TCC concentrations. This indicates the independence of systemic and central nervous system complement activation. Section 1.4 The TCC in disease 63
Multiple sclerosis
In patients with multiple sclerosis, Morgan et al., (1984a) found that the CSF C9 concentration was significantly reduced and proposed that this was due to local C9 consumption and generation of the MAC, which could mediate myelin damage. In support of this, the MAC has been detected on oligodendrocyte-derived membranes in the CSF of patients with multiple sclerosis and in experimental complement-mediated oligodendrocyte damage in vitro [Scolding et al., 1989]. The MAC has also been detected on cerebral capillary endothelium from patients with multiple sclerosis [Compston et al., 1989].
MUSCULOSKELETAL DISEASE
Myasthenia gravis
Myasthenia gravis, an autoimmune condition which is associated with loss of acetylcholine receptors from the motor neurone end plate, was the first disease in which the MAC was implicated in pathogenesis [Morgan, 1989]. TCC neoantigens have been demonstrated along the motor endplate [Engel & Arahata, 1987] and Sahashi et al., (1980) detected C9 on post-synaptic membranes, where structural damage was greatest. This group considered that assembly of the MAC contributed to loss of acetylcholine receptors, although complement-independent modulation by antibodies may also have been involved. Furthermore, a requirement for C6 has recently been reported in a rat model ofthis disease suggesting a critical role for the TCC [Morgan, 1989].
Myositis
The MAC has been detected in association with muscle fibres [Morgan et al., 1984b], and on the microvasculature [Kissel et al., 1986] in patients with myositis. Section 1.4 The TCC in disease 64
MAC also occurs on necrotic fibres in both inflammatory myopathies and muscular dystrophy [Engel & Biesecker, 1982, Morgan et al., 1984b].
Arthritis
Both normal and diseased synovial membranes express mRNA for several components of the terminal complement pathway, including C5, C6, C7, C9 and regulatory proteins including CD59 [Guc et al., 1993]. Several studies have also reported the presence of MAC antigens in the synovium [Husby & Williams, 1985, Sanders et al., 1986a, Corvetta et al., 1992] and elevated levels of TCC in the synovial fluid of patients with rheumatoid arthritis [Mollnes et al., 1986a, Corvetta et al., 1992]. One third of the rheumatoid patients studied by Mollnes et al., (1986a) also had elevated plasma SC5b-9 concentrations, although there was no correlation between the levels in these two compartments. Similarly, Corvetta et al., (1992) found no relationship between the plasma SC5b-9 levels and disease activity while the extent of synovial MAC deposition was related to the degree of inflammation. These observations suggest that systemic and intra-articular activation of the terminal complement pathways may be independently regulated. The MAC has also been detected in the synovial tissues of patients with psoriatic arthritis [Sanders et al., 1986a] and occasionally, in osteoarthritis [Corvetta et al., 1992].
DERMATOLOGICAL DISEASE
The MAC has been detected in the skin of patients with systemic and discoid lupus erythematosus [Biesecker et al., 1982], bullous pemphigoid [Dahl et al., 1984], Henoch-Schonlein purpura [Kawana & Nishiyama, 1992] and dermatitis herpetiformis [Dahl et al., 1985]. It tends to be localised in areas of immunoglobulin deposition and tissue damage, such as the basement membrane in pemphigoid. In Henoch-Schonlein purpura, Kawana & Nishiyama, (1992) reported the frequent deposition of MAC on skin Section 1.4 The TCC in disease 65
blood vessel walls and found that elevated serum SC5b-9 concentration correlated well with disease activity. Co-localisation of MAC antigens with vitronectin has also been demonstrated in the skin lesions of SLE and dermatitis herpetiformis, but not in pemphigoid [Dahl back et al., 1989b] or Henoch-Schonlein purpura [Kawana & Nishiyama, 1992]. Dahlback et al., (1989a) have also found age-related deposition of both vitronectin and the TCC on dermal elastic fibres.
OTHER CONDITIONS ASSOCIATED WITH TCC ASSEMBLY
Organ-specific autoimmune disease
MAC tissue deposits have been detected in autoimmune thyroid disease [Weetman et al., 1989]. Moreover, Oleesky et al., (1986) found that the serum C9 concentration was significantly elevated in patients with hyperthyroid Graves disease and that Graves' thyroid tissue reacted with monoclonal anti-C9 antibodies.
Xenograft rejection
There is evidence for the direct participation of the TCC in hyperacute xenograft rejection. Brauer et al., (1996) reported that specific depletion of C6 by antibodies (i.e. rat-anti-rat C6) had a significant effect on the survival of guinea pig cardiac xenografts in the Lewis rat. They proposed that C6 depletion may also be beneficial in patients undergoing rejection of allografts.
Haemolytic anaemia
Salama et al., (1987) found that the MAC may play a role in drug-induced immune haemolysis. Specifically, antibodies directed against the drug, Nomofensine, efficiently produced haemolysis which was related to the quantity of deposited C5b-9. Section 1.4 The TCC in disease 66
Test and Woolworth (1994) also reported that erythrocytes from patients with sickle cell disease showed increased binding of CSb-7 and C9, and were more sensitive to reactive lysis.
Non-immunological disease
MAC deposits are also seen in primarily non-immunological conditions, such as in the necrotic tissue within a region of myocardial infarction. Schafer et al., (1986) suggested that ischaemic myocardium has reduced ability to regulate local complement activation and found that MAC deposition was a "most sensitive tool" for detecting ischaemic myocardial lesions. Furthermore, release of intracellular constituents such as mitochondria may precipitate or maintain complement activation [Bhakdi, 1988]. MAC deposition on critically damaged tissue may accelerate cell death, augment inflammatory reactions and ultimately facilitate removal of debris and tissue repair [Bhakdi, 1988]. Similarly, arteriosclerotic blood vessels may contain deposits of MAC [Vlaicu et al., 1985, Rus et al., 1986, Niculescu et al., 1987 & 1989].
Cardiopulmonary bypass
Patients undergoing cardiopulmonary bypass have increased levels of SCSb-9 in their plasma [Salama et al., 1988, Videm et al., 1992]. Salama et al., (1988) found that plasma SCSb-9 levels rose three to eight fold immediately following commencement of cardiopulmonary bypass and that this level paralleled the degree of haemolysis. They also detected CSb-9-coated erythrocyte ghosts. Section 1.4 The TCC in disease 67
DEFICIENCY OF THE TERMINAL PATHWAY COMPONENTS
Complement proteins
Individuals who are congenitally deficient in one or more components of the terminal complement pathway have impaired ability to form the TCC (see Table 5, page 68). This may result from inheritance of null alleles or genes coding for a defective protein. The incidence varies with the components and according to the population. For example, C9 deficiency is unusual in Caucasians while the homozygous C9 null genotype has an incidence of almost 111000 in the Japanese population [Morgan & Walport, 1991]. While most individuals remain well, homozygotes commonly present after the age of ten years, with a median age of 17 years at the time of their first infection [Ross & Densen, 1984]. Typically, they develop recurrent episodes of meningococcal meningitis or gonococcal disease [Ross & Densen, 1984]. Septicaemia is commonly present at diagnosis and unusual serotypes are often isolated [Fijen et al., 1989]. Over 40 % of homozygotes are effected at some stage and relapse of partially treated infections is 10 times more common than in the normal population. Despite their increased susceptibility to menigococcal infection, individuals with late complement deficiencies also appear to be less likely to succumb to the illness than immuno-competent hosts [Ross & Densen, 1984]. This may be due, at least in part, to the attenuation of release of tumour necrosis factor from stimulated monocytes which is normally modulated by C5 and possibly the other late complement components [Barton & Warren, 1993].
Deficiency of regulatory factors
Deficiency of the membrane-bound regulatory proteins of the terminal complement pathway have also been described. Absence ofHRF and CD59 (and DAF) is associated with the rare disorder, paroxysmal nocturnal haemoglobinuria (PNH) [Lachmann, 1991]. Erythrocytes from patients with this condition are especially susceptible to attack by autologous early complement components and MAC, i.e. 68
Table 5. Reports of congenital deficiency of the terminal complement pathway components and disease associations
Factor Serum Disease Population Reference level (homozygote frequ.)
Serum components cs Absent Neisseria! infections, African-American a, b, c, rarely SLE, nephritis, d*, e Sjogrens syndrome
C6 Low, absent or MCGNtypei, Caucasian (1/60000) b, d*J f, dysfunctional meningococcal disease, & African-American g, h ± deficient C7 Raynaud' s phenomenon
C7 Low, absent or Proteinuria,. SLE, . Japanese (1125000) d*, g, h, dysfunctional memngococcaemm, & Sephardic Jews i,j,k,l, ± deficient C6 Raynaud' s phenomenon, m, n, o,p scleroderma, ankylosing spondylitis C8 Absent Meningococcal disease, Sephardic Jews b, h, n, p, SLE q C8j3 Absent Meningococcal disease, Caucasian h, m, r ±lowC8ay juvenile chronic arthritis, exanthem C8ay Absent African-American & d*, h, s ±low C8j3 Japanese
C9 Absent Meningococcal disease Japanese (111 000), h, t, U, V, SLE, IgAN, post- rarely Caucasian W,X streptococcal GN 50% Chronic neutropenia Japanese, 1 case y +absent C2
Cell-bound regulatory factors GPI linker PNH h,z CD59 PNH-like illness 1 case a a
References (a) Rosenfeld et al., 1976, (b) Cooper, 1982, (c) Wang et al., 1995, (d) Inai et al., 1989, (e) Schoonbrood et al., 1995 (/)Coleman et al., 1979, (g) Wurzner et al., 1991, (lz) Morgan & Walport, 1991, (i) Zeitz et al., 1981, OJ Loirat et al., 1980, (k) Nemerow et al., 1978 (I) Egan et al., 1994, (m) Platonov et al., 1993, (n) Schlesinger et al., 1990, (o) Ammann & Fudenberg, 1982, (p) Zimran et al., 1987, (q) Louaib et al., 1994, (r) Wulffraat et al., 1994, (s) Tedesco et al., 1990, (t) Nagata et al., 1989, (u) Yoshioka et al., 1992, (v) Maruyama et al., 1995, (w) Takeda et al., 1994, (x) Hironaka et al., 1993, (j!) Kaneko et al., 1993, (z) Nicholson-Weller, 1988, (aa) Yamshina et al., 1990. * This study reports complement deficiencies in healthy blood donors. Section 1.4 The TCC in disease 69 reactive lysis [Lachmann, 1991]. Mild leucopoenia and thrombotic episodes are also characteristic. Forman et al., (1984) reported 26 patients with PNH and a variety of clinical features including abdominal pain, haemolytic crisis, acute renal failure, thrombosis, sepsis and bone marrow hypoplasia. PNH has also been associated with non-lymphocytic leukaemias [Nicholson-Weller, 1988].
Attachment of HRF (i.e. C8bp) [Hansch et al., 1988b], CD 59 [Morgan & Walport, 1991] (and DAF [Davitz et al., 1986, Hansch et al., 1988b]) to the cell membrane is via a GPI-linkage (see section 1.2, page 26). Failure of post-translational synthesis or attachment of this lipid group appears to be the underlying defect ofPNH in most cases [Nicholson-Weller, 1988, Morgan & Walport, 1991]. In support of this, PNH cells also commonly lack several other GPI-linked membrane proteins including alkaline phosphatase [Beck & Valentine, 1951], lymphocyte function-associated antigen 3 (i.e. LFA-3) [Selvaraj et al., 1987] and the Fey receptor (III), (i.e. CD16) [Selvaraj et al., 1988].
In most cases, PNH is an acquired clonal disorder of haematopoietic cells [Morgan & Walport, 1991], resulting from a somatic mutation occurring, or manifesting, in the second or third decade of life [Nicholson-Weller, 1988]. However, a single individual has been reported with genetic deficiency of CD59 [Yamashina et al., 1990]. This patient suffered from a PNH-like illness associated with several episodes of haemolysis and two cerebral infarctions by the age of 22 years. His parents were cousins but did not show any symptoms ofPNH.
Genetic deficiency of vitronectin has not been described in humans or in other higher organisms [Bhakdi, 1988, Zheng et al., 1995]. This suggests that it may play a critical role in normal physiology. However, genetically-altered mice, unable to produce vitronectin, demonstrated normal development and fertility in a recent study by Zheng et al., (1995). This issue requires clarification. Section 1.4 The TCC in disease 70
Autoantibodies to C9 neoantigens in disease
Autoantibodies directed against complement neoantigens are well described. In a study by Strife et al., (1990), greater than 75% of patients with hypocomplementaemic MCGN had detectable autoantibodies against neoantigens of the C3 convertase. By analogy with immunoconglutinins, i.e. autoantibodies directed against bound C3 or C4, Kolb and Muller-Eberhard (1975a) proposed that antibodies against neoantigens on C5b-9 may arise during infection or chronic immunologic diseases involving complement activation. Dalmasso et al., (1985 & 1989) reported that low levels of anti C9 neoantigen antibodies were often found in normal subjects and that increased levels, including both IgM and IgG isotypes, occurred in patients with autoimmune, infectious and malignant disease. They were shown to react with linear aggregates of C9 but not with SC5b-9 or the MAC and therefore differed from monoclonal antibodies (e.g. mAb aE11) in their specificity. The importance of these antibodies to the pathogenesis of disease remains uncertain 71
1.5 METABOLIC STUDIES OF COMPLEMENT PROTEINS
Metabolic studies provide dynamic information about protein synthesis, catabolism and distribution that are un-obtainable from single, or even sequential measurements of component concentrations. In the case of complement proteins, such studies have also contributed to an understanding of pathway activation and regulation in health and disease.
THEORETICAL CONSIDERATIONS
Several mathematical models and techniques have been devised to analyse the metabolism of trace-radiolabelled proteins in vivo. These involve determination of the rate and pattern of disappearance of radioactivity from the plasma, or a combination of plasma and urinary excretion data, following an intravenous injection of radiolabelled protein. In general, all methods assume that the protein is distributed between a central intravascular (IV), i.e the plasma, and one or more extravascular (EV) compartments (see Fig. 10, page 72), between which molecules freely move. Moreover, Nosslin (1973) noted that irrespective of the system's true structure, it is possible to consider an "equivalent two compartment model" in which the individual EV pools are combined (see Fig. 1O(b ), page 72). The IV and EV spaces are also connected irreversibly to an excretory pool, to which the urine contributes the major portion. Interpretation of metabolic data requires the veracity of several general assumptions regardless of the mathematical model employed as outlined byNosslin (1973). These include:
1. "The metabolism of the labelled protein is identical with that of the native unlabelled protein".
The integrity and function of the protein must be intact in vitro and remain so following radiolabelling. Indication of the presence of denatured material in vivo 72
Fig. 10. Compartmental models of protein kinetics
In Mathews' model (a), a central IV space (i.e. celll) is connected reversibly with several EV compartments (i.e. cells 3 to n), and unidirectionally with an excretory pool (i.e. cel12). In Nosslin's "equivalent two compartment model" (b), the EV spaces are viewed as a single entity. In these diagrams "K" denotes reaction rate constants.
(a)
(Adapted from Mathews, 1957)
(b)
Kr IV Sum of all Pool EVPools K2
K3 K4
Excretion
(Adapted from Nosslin, 1973) Section 1.5 Complement metabolism 73
includes a very rapid early fall in plasma protein-bound radioactivity associated with the excessive release of free radio label.
2. "There is no reutilisation of the radioactive label".
It has been suggested that this requirement was not fulfilled in earlier studies using 358-, 15N- and 14C-labelled molecules [Berson & Yallow, 1957]. However, in the case of isotopes of iodine, urinary excretion is relatively prompt and reutilisation is minimal once thyroidal uptake has been blocked by prior treatment with stable iodide [Berson & Yallow, 1957]. The distribution of iodide within the body has been extensively studied and estimates of the size of the Iodide space range between 6 - 9 times the plasma volume in the rabbit [Zizza et al., 1959, Regoeczi, 1963] and 10- 12 litres in humans [Bianchi et al., 1973]. This includes the intravascular compartment and several organs and tissues. In particular, the skin, hair and intestinal tract, have been found to accumulate intravenously administered 1311. Uptake oflodide by the unblocked thyroid gland may also be considerable over time [Regoeczi, 1963]. Despite its relatively large volume of distribution, iodide liberated by catabolism of labelled protein is excreted rapidly and steadily with a T112 of 6 - 12 h [Zizza et al., 1959]. This is shorter than the plasma half-life of most complement proteins and is therefore, not rate limiting.
Zizza et al., (1959) found that 90.5 - 98.1 % of injected 131I was recoverable in the urine and faeces of rabbits after four days, with only 1.9 - 7.6 % excreted in the faeces. This group also investigated the nature of radio-iodinated protein breakdown products released following injection of 131I-albumin. While the great majority of urinary radioactivity was present as free iodide, between 7 % and 18 % included mono iodotyrosine, di-iodotyrosine and other small organo-iodides. However, these were found to share the same volume of distribution and kinetics as free iodide and, in particular, there was no evidence for their retention or recycling. Section 1.5 Complement metabolism 74
3. "The system is in a steady metabolic state during the study".
This is established by both clinical assessment and measurement of the plasma concentration of the specific protein under study on several occasions during the sampling period.
4. "Newly synthesised protein is distributed in the intravascular pool before entering any extravascular pool".
ANALYTICAL METHODS
The various approaches developed to analyse metabolic data have particular applications and limitations. In addition, some make specific assumptions not covered by the general statements outlined above. The method described by Mathews (1957), (see Section 2.9, page 117) allows calculation of the fractional catabolic rate (FCR) and extravascular I intravascular distribution ratio (EV!IV) by analysis of the plasma disappearance curves alone. It assumes that catabolism occurs in the intravascular compartment or in a compartment with which it is in rapid exchange. Notably, Mathews' approach is applicable in subjects with renal impairment and, as such, offers advantages over the excretion-dependent techniques including the metabolic clearance method of Berson and Yallow (1957), (see Section 2.9, page 118) and the integrated rate equations method of Nosslin (1973). However, it requires the protein to attain compartmental equilibration and for the plasma radioactivity disappearance curve to assume a final log-linear (i.e. mono-exponential) decay. Some studies have provided examples where this is not the case and these are not appropriate for Mathews' analysis [Charlesworth et al., 1974b]. Nonetheless, Reeve et al., (1982) considered that Mathews' technique was the one of choice for calculating EV/IV ratios whenever the FCR could be confidently determined. Section 1.5 Complement metabolism 75
The metabolic clearance method described by Berson and Yallow (1957) allows calculation of the FCR from the ratio of the excreted radioactivity to the mean protein bound plasma counts for a defined time interval. This technique relies on accurate urine collections and is not suitable where there is retention of unbound label, such as occurs in renal impairment. A tendency to underestimate the FCR is also seen if only early data are used [Reeve et al., 1982]. During this period, net movement of released label from the site(s) of protein catabolism to its wider distribution space may delay its excretion. Otherwise, this method offers simplicity, has no requirement for a particular pattern of plasma protein disappearance and is not dependent on complete protein equilibration. Providing the T112 of the protein under study does not approach the value for renal iodide clearance and there are reliable urine collections, this technique has several advantages over other methods [Reeve et al., 1982].
In this thesis, the methods of both Mathews, and Berson and Yallow have been used where possible. Section 2.9, page 116, outlines the procedures and formulae employed.
STUDIES OF COMPLEMENT METABOLISM IN VIVO
Complement metabolism in vivo was first investigated 30 years ago by Alper and Rosen (1967) who examined the behaviour of C3 in normal subjects and patients with acute glomerulonephritis, nephrotic syndrome, haemolytic anaemia, rheumatoid arthritis and hereditary angioneurotic oedema. Since then, many studies of complement metabolism have been reported. Published metabolic studies of the early human components in experimental animals are listed in Table 6, page 76, while Table 7, page 78, summarises the metabolic data in healthy human subjects.
Metabolic studies have provided insight into the mechanisms responsible for altered plasma/serum concentrations of the complement components in disease. For example, several investigators have noted that both reduced plasma production rate (PPR) and increased EV distribution contribute to reduced levels of individual 76
Table 6. The metabolic characteristics of early complement components in normal experimental animals
Protein Species FCR Reference
(h) (%/h)
HumanC3 Rabbit 35-40 2.4-2.8 a
Rabbit C3 Rabbit 29.3 4.3 b
Rabbit 40 2.7 c
Rabbit C3a Rabbit * * b
Rabbit C3b Rabbit * * b
Rabbit iC3b Rabbit * * b
HumanC3c Rabbit 24 4.5 a
HumanC3d Rabbit >50 a
Human Factor H Rabbit 30-45 0.39-1.45 d#
Data is presented as the mean or range.
References
(a) Charlesworth et al., 1974a, (b) Peake et al., 1991, (c) Manthei et al., 1984, (d) Charlesworth et al., 1979b.
* Metabolised too rapidly for reliable calculation of metabolic parameters. # Factor H is referred to as ~ 1H in this article. Section 1.5 Complement metabolism 77
components during pathological complement activation. In their study of C3 metabolism, Alper and Rosen (1967) found this to be the case. Charlesworth et al., (1974b) also found that a combination of hypercatabolism, reduced production and altered compartmental distribution of C3 occurred simultaneously in patients with reduced serum C3 levels. Sliwinski and Zvaifler (1972) concluded that depression of synthesis was the major contributor to reduced serum C3 concentration in patients with SLE. By contrast, a study of C1q metabolism in patients with acquired hypogammaglobulinaemia demonstrated that a marked increase in the EV distribution was the predominant cause of reduced serum C 1q concentrations [Kohler & Muller Eberhard, 1972]. The plasma production rate of C1q was normal in these subjects. Pussell (1982) similarly found that patients with hypogammaglobulinaemia and reduced serum C 1q levels had both increased EV distribution and FCR of C 1q, without a corresponding increase in the C 1q synthesis rate.
Normal, or elevated serum complement levels may also occur in inflammatory and infectious diseases. Several complement proteins have increased plasma production rate during physiological stress, i.e. acute phase reactivity. These include C1s, C3, C4 and, in particular, C9 and Factor B (Charlesworth et al., 1979a, Oleesky et al., 1986, Adinolfi & Lehner, 1988]. For example, in uncomplicated infectious mononucleosis, Charlesworth et al., (1982) found that increases in serum C3 concentration were associated with an increase in the C3 synthesis rate. They proposed that this allowed effective processing of immune complexes (i.e. complement-mediated solubilisation) and thereby minimised tissue damage. This was in contrast to SLE in which the early complement component levels are often reduced, immune complex solubilisation is impaired and tissue deposition of complexes occurs. Swaak et al., (1988) found that a group of patients with rheumatoid arthritis had hypercatabolism of C3, yet none had depressed serum C3 levels. They proposed that, in this case, accelerated C3 consumption was fully compensated for by increased synthesis. Charlesworth et al., (1989b) similarly found that both hypercatabolism of C3 and elevated C3 production rate occurred in patients with rheumatoid arthritis. Furthermore, patients with ankylosing spondylitis have also been shown to have moderately increased rates of C3 turnover despite elevated serum C3 levels [Brinch et al., 1982b]. 78
Table 7. Studies of the metabolism of early complement components in healthy human subjects
Protein Tvz FCR EVIIV PPR Reference
(h) (%/h) (mglkg per h)
Clq 37 2.84 0.5 0.178 a
C3 2.35 0.9 1.38 b 2.12 1.16 c 1.83 0.83 d 2.5 1.5 e 71 1.66 0.35 0.81 f 1.71 0.69 g 74 1.54 0.45 0.76 h 56 1.61 0.39 0.55 i
C4 54 1.87 0.45 0.30 i 1.40 0.21 g
C4A3,Bl * 1.95 0.43 j C4A3,BO * 1.99 0.31 j
FactorB 66 1.98 1.07 0.18 f# 1.4- 1.9 0.15- 0.19 g
FactorH 76 1.32 0.52 0.37 kt
Data is presented as the mean or range.
References
(a) Pussell, 1982, (b) Alper & Rosen, 1967, (c) Petz et al., 1968, (d) Hunsicker et al., 1972, (e) Sliwinski & Zvaifler, 1972, (f) Charlesworth et al., 1974b, (g) Ruddy et al., 1975, (h) Sissons et al., 1977, (i) Charlesworth et al., 1989a, 0) Peake et al., 1989, (k) Charlesworth et al., 1979b.
* Allotypes of C4. #Factor B is referred to as the glycine-rich beta glycoprotein in this article. t Factor His referred to as j31H in this article. Section 1.5 Complement metabolism 79
The activity of inflammatory disease has been found to effect the metabolic behaviour of complement proteins. In a dual isotope study of C3 and C4 metabolism in SLE, Charlesworth et al., (1989a) noted that hypercatabolism of the fourth component occurred regardless of disease activity. However, only patients with active disease had significantly lower C4 plasma production rates than the control group. In a prospective study, Swaak et al., (1986) similarly noted that increased C3 consumption occurred in SLE patients irrespective of the disease activity. However, in the period preceding an exacerbation, impaired C3 synthesis was observed, in contrast to the stable disease phase. Studies of this type provide a basis for the observation that serum C3 and C4 levels are often reduced in active SLE [Milis et al., 1992].
Hypercatabolism of the early complement proteins has also been reported in several non-rheumatological, non-renal diseases. In particular, Brinch et al., (1980) found that patients with adult coeliac disease had elevated C3-FCR and that this was greatest in active, untreated cases. Patients with ulcerative colitis and associated hepatobiliary disease have also been found to have increased rates of C3 catabolism
[Brinch et al., 1982a]. ~
In some cases, the site of complement activation can be inferred from metabolic investigations. In their study of C3 metabolism in patients with post streptococcal glomerulonephritis, Endre et al., (1984) found that the FCR of C3 was markedly increased (i.e. 3.02 - 6.99 %/h) compared with the control group (1.56 - 2.12 %/h). Several patients had profoundly reduced serum C3 levels and correspondingly low C5 concentrations. The authors concluded that this was most consistent with the formation of a surface-bound C3/C5 convertase as previous metabolic studies have shown the relative inefficiency of the soluble converting enzymes [Sissons et al., 1977, discussed below].
Metabolic studies have also shed light on the molecular mechanisms and regulation of the complement cascade in vivo. For example, Carpenter et al., (1969) demonstrated hypercatabolism of C4 in patients with hereditary angioedema who lack the natural inhibitor of the C1 esterase. Several investigators have also noted the effects Section 1.5 Complement metabolism 80
of component depletion on the "down-stream" generation of active complement proteases. In their study of SLE patients, Charlesworth et al., (1989a) speculated that markedly reduced C4 production in active disease could ultimately limit the generation of the classical C3 convertase (i.e. C4b2a), and thereby reduce the C3 turnover rate. Moreover, in a simultaneous study of Factor B and C3 metabolism, the turnover rate of Factor B was generally increased in patients with C3 hypercatabolism except when significant hypocomplementaemia was present. In the latter case, Factor B metabolism was normal, or nearly so, and the authors suggested that this was due to a deficiency of C3b, restricting the activation of Factor B by Factor D [Charlesworth et al., 1974b].
Metabolic studies of the terminal complement pathway
There have been relatively few published reports of the metabolism of terminal complement components in either experimental animals or humans (see Table 8, page 81 ). In 1977 Sissons et al., studied the metabolism of C5 and its relationship to C3 turnover in normal subjects and patients with pathological complement activation. In patients with SLE they foWld that the FCR and EV /IV distribution of both C3 and C5 were increased although, in general, the catabolic rate of C5 was less affected than that of C3. Moreover, in two patients with C3 Nef, C5 turnover was normal despite markedly increased C3 catabolism. They suggested that little or no C5 convertase was generated by the fluid-phase alternative pathway in these cases [Sissons et al., 1977]. The metabolism of radio labelled C5 :fragments, C5a and C5a des arginine, has also been investigated in experimental animals. Fifty percent of the injected dose was removed from the circulation within two minutes. Highly vascular organs including the lWlgs, spleen, liver and kidneys were largely responsible for this rapid clearance [Webster et al., 1982].
A study of the metabolism of 125I-labelled C8 and C9 in the rat was reported (in abstract) by Dalmasso and Falk (1987). They foWld that both proteins were very rapidly 81
Table 8. Reports of the metabolism of the terminal complement complex and its components in experimental animals and human subjects
Experimental animals
Component Species Tvz Reference
HumanCSa Rabbit <2min a HumanCSa Rabbit <2min a (des arginine) RatC8 Rat 22h b RatC9 Rat 12.5 h b Rat SCSb-9 Rat 2-3 h b Rabbit SCSb-9 Rabbit 30-50 min c C6D Rabbit* 30-50 min c Human SCSb-9 Rabbit "Virtually the same as c for rabbit SC5b-9" #
Normal human subjects
Component Tvz FCR EV/IV PPR Reference
(%/h) (mg/kg per h) cs 1.6-2.2 0.06-0.11 d 63 h 1.74 0.44 0.09 e SCSb-9 42-58 min It
Data is presented as mean or range.
References
(a) Webster et al., 1982, (b) Dalmasso & Falk., 1987, (c) Hugo et al., 1989, (d) Ruddy et al., 1975, (e) Sissons et al., 1977, (f) Deppisch et al., 1990.
* C6-deficient rabbits. # Stated in this reference but results not shown. t In this study SC5b-9 was produced in vivo during haemodialysis. Section 1.5 Complement metabolism 82
eliminated from the plasma with a final T112 of22 h for C8 and 12.5 h for C9. However,
2/3 of the radioactivity disappeared from the plasma in the early phase with a T112 of approximately 3 h in each case.
A small number of studies have examined the behaviour of SC5b-9 in laboratory animals. In the rat, Dalmasso and Falk (1987) found that this complex was rapidly removed from the plasma with a T112 of only 2- 3 h. In 1989, Hugo et al., reported the metabolism of SC5b-9 in normal and C6-deficient rabbits. They administered inulin activated rabbit serum intravenously and monitored plasma levels with a sandwich ELISA based on mono- and polyclonal antibodies against the terminal complex. The
T112 was 30 - 50 min with a return to basal levels after 2 - 3 h. No differences were observed between the clearance rates of homologous and human SC5b-9. The complex was removed even more rapidly from the plasma of C6-deficient animals. The authors estimated that normal basal plasma SC5b-9 concentrations were maintained by the spontaneous turnover of terminal pathway components at a rate of approximately 0.2 %/h.
Deppisch et. al., (1990) investigated the kinetics of SC5b-9 in patients undergoing haemodialysis, taking advantage of the complement activating properties of dialysis membranes . They found that the greatest increase in plasma levels occurred with Cupraphan dialysers and less with Hemophan and Polysulphone F6. From its behaviour following cessation of dialysis, they estimated the plasma T112 of SC5b-9 to be 48 (42- 58) min. 83
1.6 AIMS OF THIS STUDY
The terminal complement complex has been implicated in the pathogenesis of autoimmune disease. However, measurement of its concentration in the plasma or that of its components provides limited information about terminal pathway activity. The broad intention of this thesis was to study the dynamic characteristics of the terminal complement pathway in vivo. Specifically, the aims were:
1. Preparation of sterile, fully functional, radio labelled C9 suitable for studies in both laboratory animals and human subjects.
2. To define the metabolic characteristics of C9 in experimental animals. In particular, to determine its plasma half-life, fractional catabolic rate, plasma production rate and compartmental distribution in the normal state and during complement activation.
3. To examine the metabolism of SC5b-9 in laboratory animals, especially with regard to its rate of elimination from the plasma and organs of sequestration.
4. To study the metabolism of C9 in normal human subjects and patients with autoimmune disease. In particular, to clarify the relationship between the kinetic parameters of C9 and the plasma/serum levels of complement components, terminal complexes and other indices of immunological activity.
5. Investigation of the metabolism of vitronectin in experimental animals during basal complement activity and following activation. Phosphorylated vitronectin was also studied to determine whether this form differed in its metabolic characteristics or recruitment by the complement cascade. 84
1. 7 GENERAL HYPOTHESIS
This study examined the hypothesis that complement activation in experimental animals and patients with autoimmune disease is associated with altered metabolism of the terminal complement complex and its constituents. This is reflected in increased fractional catabolic rate and altered compartmental distribution of its components, C9 and its inhibitor, vitronectin. 85
Section 2
METHODS 86
2.1 PURIFICATION AND PREPARATION OF PROTEINS
PURIFICATION OF HUMAN C9
A list of reagents, their manufacturers and buffer formulae used in the purification of C9 may be found in Appendix 1, page 228, and Appendix 2, page 231, lists the anti-sera used in radial immunodiffusion. Appendix 8, page 237, includes the addresses of suppliers of materials and equipment used in these studies.
General procedures
C9 was prepared from plasma by a modification of the method described by Biesecker and Muller-Eberhard, (1980). All steps were performed at 4°C, except where noted otherwise. 200 mL of blood was drawn from a healthy donor, and combined with 8 mL of 86 mM EDTA. The plasma was separated by centrifugation at 1200 g, for 5 mm at room temperature (RT), and solid benzamidine and phenylmethylsulphonylfluoride (PMSF), (20 mg/mL in methanol), were added to 100 mL of plasma to give final concentrations of 25 mM and 0.6 mM respectively. The plasma was added to twice its volume of 21 % polyethyleneglycol 4000 (PEG 4000) containing K2HP04 (25 mM), NaCl (90 mM), Na2EDTA (10 mM), benzamidine (25 mM), PMSF (0.5 mM) and chloramphenicol (25mg/L) at pH 7.0, and incubated at 4°C with gentle stirring for 30 min. The precipitate was removed by centrifugation (3000 g, 10°C, 30 min) and solid PEG 4000 was added to the supernatant to produce a final concentration of21 %(i.e. an additional14 g/100 mL). Following further incubation at 4°C for 30 min with gentle stirring, the mixture was centrifuged (3000 g, 10°C, 30 min) and the supernatant discarded. The precipitate was re-dissolved in 20- 30 mL of buffer containing K2HP04 (100 mM), NaCl (150 mM), Na2EDTA (5 mM), benzamidine (25 mM) and chloramphenicol (25 mg/L) at pH 7.0, loaded onto a lysine-Sepharose column (1.6 x 28 em), (Amrad Pharmacia, Melbourne, Vic, Australia) and washed with the same buffer at a rate of 50 mL/h until there was no further elution Section 2.1 Protein purification 87
of protein. The eluent was diluted with four times its volume in a buffer containing benzamidine (25 mM), Na2EDTA (5 mM), s-aminocaproic acid (EACA), (30 mM) and chloramphenicol (25 giL) at pH 7.0, and solid NaCl (70 mMIL) was added. The solution was loaded onto a DEAE-A50-Sephadex column (5 x 25 em, Amrad
Pharmacia) and washed with starting buffer containing K2HP04 (25 mM), NaCl (100 mM), Na2EDTA (5 mM), benzamidine (25 mM), EACA (30 mM) and chloramphenicol (25mg/L) at pH 7.0, until the eluent attained a constant baseline level with the Biorad protein assay (Biorad, Sydney, NSW, Australia), (see Section 2.2, page 89). The column was eluted with a linear NaCl gradient from 100 mM (conductivity: 9.5 mS/cm) to 400 mM (conductivity: 33 mS/cm) at pH 7.0, at a flow rate of 50 mL/h and 10 mL fractions were collected. C9-containing fractions which were free of the plasma protein ceruloplasmin, as determined by radial immunodiffusion [Mancini et al., 1965], (see
Section 2.10, page 120), were dialysed against K2HP04 buffer (25mM) containing KCL (1 OOmM) at pH 7.5 and loaded onto an hydroxylapatite column (2.5 x 16.5 em, Biorad).
This column was washed with 80 mM phosphate starting buffer made from K2HP04 (80 mM), NaH2P04 (80 mM) and KCl (100 mM) at pH 7.7, until the eluent yielded a stable baseline level with the Pierce protein assay (Pierce, Rockford, 11, USA), (see Section 2.2, page 89). A linear phosphate gradient from 80 mM (conductivity: 15 mS/cm) to 400 mM (conductivity: 38 mS/cm) was applied to the column at a rate of 40 mL/h and 5 mL fractions were collected. C9-containing fractions (determined by radial immunodiffusion) were concentrated using a Centricon microconcentrator (Amicon,
Beverly, MA, USA), frozen and stored at -70°C, in 50 ~-tL aliquots in Cryo-tubes (Nunc, Roskilde, Denmark).
Procedures and precautions observed in the purification of C9 for metabolic studies in humans
C9 used in human metabolic studies was purified from the plasma of a healthy donor who was negative for hepatitis B surface antigen and antibodies against the human immunodeficiency virus types 1 and 2 and hepatitis C virus. All buffers were made from sterile, pyrogen-free water (Baxter, Old Toongabie, NSW, Australia) and all Section 2.1 Protein purification 88
implements and containers coming into contact with these buffers or C9-containing solutions, were steam sterilised or exposed to 0.1 M NaOH and 80 % ethanol (Ajax Chemicals, Sydney, NSW, Australia). Sepharose columns were treated with 0.1 M
NaOH and 20 % ethanol prior to use. Purified C9 was stored in 50 ~-tL aliquots in sterile, pyrogen-free Cryo-tubes (Nunc), and thawed once, immediately prior to use.
Biological safety testing of C9 and 125I-C9
Aliquots of purified C9 and 125I-C9 were cultured for aerobic, anaerobic and fungal organisms and a large quantity, equivalent to 100 times the human dose (with respect to body weight), was administered intravenously to rabbits and their rectal temperature measured at 15 min intervals for 3h to assess pyrogenicity. Microbiological 1 cultures and pyrogen tests were negative • Immediately prior to administration, the radio-labelled protein was passed through a sterile, pyrogen free, low protein-binding
0.2 ~-tm filter (Gelman, Sydney, NSW, Australia).
PREPARATION OF VITRONECTIN
Vitronectin used for metabolic studies in vivo was not purified, but rather selectively phosphorylated in human plasma using a 3,5 cAMP-dependent kinase from rabbit muscle (Sigma, Sydney, NSW, Australia), as described in Section 2.3, page 93. The donor plasma contained both single and two chain forms (i.e. type 1-2). Vitronectin used for studies in vitro was purified by heparin-affinity chromatography (Amrad Pharmacia) following treatment with 8 M urea as described by Hayashi (1993), although no reducing agent was added. During its purification, the elution pattern of vitronectin was monitored by radial immunodiffusion in agarose gels containing polyclonal sheep 2 anti-human vitronectin antibody (The Binding Site, Birmingham, UK) .
1 I acknowledge the assistance of the Microbiology Department, Prince of Wales Hospital, Sydney. 2 I acknowledge the contribution of Dr Philip Peake, (Principal Scientific Officer, The Renal Immunology Laboratory, Prince Henry Hospital, Sydney), to the preparation ofvitronectin used in this study. 89
2.2 TECHNIQUES FOR PROTEIN ANALYSIS
MEASUREMENT OF TOTAL PROTEIN CONCENTRATION
The Pierce Assay
50 mL of Pierce BCA protein assay reagent A, containing Na2C03, NaHC03, Bicinchoninic acid and sodium tartrate in 0.2N NaOH, was added to 1 mL of reagent B,
containing 4 % CuS04, to make a stock solution according to the manufacturer's
instructions (Pierce). 10 ~-tL aliquots of each sample were transferred to the wells of a microtitre plate (Bio-tek, CSL Biosciences, Melbourne, Vic, Australia), to which 200
~-tL of Pierce stock solution was added. The plates were incubated at 37°C, for 30 min and the optical density (OD) was measured at 540 nm using a Ceres-900 microtitre plate reader (Bio-tek). A standard curve was constructed using known dilutions of bovine serum albumin (BSA), (Sigma) in the sample buffer and the protein concentration was determined by reference to the standard curve. This technique was not used for buffers with a variable or high phosphate concentration, which can interfere with the colour generating reaction.
The Biorad Assay
Standard assay
The Biorad protein assay concentrate (Biorad) was diluted in 4 volumes of
dH20, and 1 volume of each test sample was added to 5 volumes of the diluted dye and allowed to incubate for at least 5 min at RT. The OD at 620 nm was measured using a Ceres-900 microtitre plate reader (Bio-tek), and the protein concentration determined with respect to a dilution series of BSA, after subtracting the absorbance in the blank (i.e. buffer only) sample. Section 2.2 Techniques for protein analysis 90
Biorad micro assay
0.2 mL of the Biorad protein assay concentrate was added to 0.8 mL of sample in a Ceres-900 microtitre plate (Bio-tek). After incubation for 5 min at RT the OD was read at 620 nm using a microtitre plate reader (Bio-tek), and compared with a dilution series ofBSA as described above.
POLYACRYLAMIDE GEL ELECTROPHORESIS
The formulae of buffers and suppliers of ingredients used in polyacrylamide gel electrophoresis (PAGE) are shown in Appendix 3, page 232.
PAGE was performed using a discontinuous buffer system [Laemmli, 1970] and the Mini-Protean II dual slab cell (Biorad). The separating gel contained 10% polyacrylamide and the buffers included sodiumdodecylsulphate (SDS) and 13-2- mercaptoethanol unless noted otherwise. Standard molecular weight markers, from 29 - 205 kD, were also included (Amrad Pharmacia). A power source (Biorad) supplied a constant potential of 200 V, and the duration was determined by the progress of a bromophenol blue tracker dye. Gels were stained in 0.5 % Coomassie Brilliant Blue (Sigma) and dried in a heated gel drier (Amrad Pharmacia).
AUTORADIOGRAPHY
Hyperfilm-MP (Amersham, Sydney, NSW, Australia) was applied to the dried polyacrylamide gel and placed into a sealed cassette which contained an intensifying screen (Dupont, Sydney, NSW, Australia). Approximately 105 cpm of 125!-labelled protein was exposed for 2 hat RT, while for 32 P-labelled proteins, 104 cpm was exposed for 12 h at -70°C. The film was developed in GBX developer (Kodak, Coburg, Vic, Australia) and fixed in GBX fixer, as described by the manufacturer (Kodak). Section 2.2 Techniques for protein analysis 91
FPLC GEL FILTRATION CHROMATOGRAPHY
Gel filtration of proteins was performed with a Fine Performance Liquid Chromatography (FPLC) Superose 12 column (Amrad Pharmacia). It measured 1 x 30 em with an exclusion volume of approximately 8 mL for molecules of MW above 2 x 106 D. 200 J.!L samples were applied to the column and the flow rate adjusted to 0.5 mL/min. 500 J..LL fractions were collected. For iodinated proteins, the elution buffer was phosphate-buffered saline (PBS), (Oxoid, Melbourne, Vic, Australia), containing ' 0.1 % stable Kl to reduce interactions between free and protein-bound iodide and the column matrix. Otherwise, PBS alone was used. The column was calibrated using standard MW markers (Sigma)
HEPARIN-SEPHAROSE CHROMATOGRAPHY OF C9-CONTAINING SERUM FRACTIONS
CL-6B heparin-Sepharose (Amrad Pharmacia) was prepared by swelling and washing 4g of the dried powder in dH20, equilibrating in PBS and packing into a glass column measuring 1.5 x 8 em (i.e. approximately 14 mL). This was subsequently equilibrated in starting buffer containing 15 mM K2HP04, brought to pH 7.0 with HCl. 100 J.!L of sample (in PBS I 0.1 % KI) was diluted with 150 J..LL of starting buffer and 200 J..LL loaded onto the column, washed in 2 column volumes of starting buffer and a linear salt gradient applied from 0- 1000 mM NaCl in 15 mM K2HP04• The flow rate was 0.5 mL/min.
Techniques used in the analysis and quantitation of blood proteins are described in Section 2.10, page 120. 92
2.3 RADIOLABELLING OF PROTEINS
THE LACTOPEROXIDASE TECHNIQUE OF IODINATION
C9
This method was based on that described by Marchalonis, (1969). To 50 J-LL of freshly thawed, purified C9 (approximately 300 J-Lg/mL), was added 7 J-LL of lactoperoxidase (Sigma), (diluted 1/50 in PBS), 2J-LL ofNa1251 (ANSTO, Sydney, NSW, 5 Australia) and 5J..LL of H20 2 (1/10 dilution of a 30 % stock solution in PBS). The reactants were incubated, on ice, for 15 min with gentle agitation every 5 min. Iodination was stopped by the addition of 20 J-LL of a solution containing 0.6 %non
radioactive KI and 0.14 % NaN3 in PBS, and left on ice for a further 5 min. The mixture was loaded onto a pre-blocked (i.e. with 1 % BSA in PBS) Sepharose G25 (PD- 10) column (Amrad Pharmacia) with a gel bed volume of approximately 8 mL, and eluted with 1% BSA in PBS. 0.5 mL fractions (i.e. 10 drops) were collected and the radioactivity in 5 J..LL aliquots of these fractions was measured in a multiwell, NE 1600 gamma counter (Nuclear Enterprises, Edinburgh, Scotland). Two peaks of radioactivity were observed, i.e. an early peak containing the radiolabelled protein and a later peak consisting of free 1251. Typically, 20 - 60 % of eluted radioactivity was protein-bound. This method was used to iodinate proteins for studies in experimental animals
Rabbit serum albumin
Rabbit serum albumin (RSA), (Sigma) was dissolved in PBS to give a 1 mg/mL solution. 100 J..LL of RSA was added to 2J..LL of Na1311 (Amersham) and radiolabelled, using the lactoperoxidase technique as described above. 1311 radioactivity was counted in a Packard Minaxi, single well gamma counter (Packard, Fivedock, NSW, Australia). Section 2.3 Protein radio/abe/ling 93
THE IODOBEAD TECHNIQUE OF IODINATION 1
This teclmique was a modification of that described by Markwell, (1982). One Iodobead (Pierce) was washed in PBS and incubated with 50 f..LL of PBS and 2 f..LL ofNa 1251 (i.e approximately 200 f..LCi) for 5 minutes at RT. 50 f..LL of purified C9 was added and the reactants were incubated for a further 10 min at RT with occasional agitation. The solution was removed from the Iodobead and added to 20 f..LL of stable KI (6 mg/mL) for a further 5 min at RT. 100 f..LL of 0.1 %human serum albumin (HSA), (NSW Red Cross Blood Transfusion Service, Sydney, NSW, Australia) or BSA for non human studies, was added and the solution promptly transferred to an 8 mL Sepharose G25 column for separation of free from bound Iodide. This column was pre-blocked with 3 mL of 1% HSA (or BSA as appropriate), then washed with a further 100 mL of PBS. The column was eluted with 12 mL of PBS and 0.5 mL fractions were collected and counted as described above. The first elution peak, containing 1251-C9, was stored at 4°C for no longer than 3 h before use in metabolic studies in vivo, or up to 3 days prior to use in vitro. The specific activity of 1251-C9 was approximately 2.5 f..LCilf..Lg. This was the only method used to iodinate proteins for metabolic studies in human subjects.
PHOSPHORYLATION OF VITRONECTIN
Selective trace phosphorylation of plasma vitronectin
Vitronectin was selectively trace phosphorylated using a 3,5 cAMP-dependent protein kinase from rabbit muscle (Sigma) according to the method of Korc-Grodzicki et al., (1990). Fresh EDTA human plasma was equilibrated with 50 mM Hepes buffer at pH 7.5 containing 10 mM Mg(CH3C00)2, using a G25 Sephadex column. 10 f..LL of 1mM cAMP (Sigma), 20 f..LL of protein kinase (1mg/mL) and 10 f..LL (80 f..LCi) ofy-32P ATP (approximately 10"8 M), (Amersham), were added to 2.5 mL of the equilibrated
1 "Iodobeads" are non-porous polystyrene spheres (2.8 mm diameter) coated with the oxidant, N-chloro benzenesulfonamide (i.e. Chloramine-B). Section 2.3 Protein radiolabelling 94
plasma and incubated for 2 hat 37°C. The unreacted y-32P-ATP was separated from the labelled vitronectin by two passages through a G25 Sephadex column. 32P-vitronectin was quantitated by measurement of Cerenkov radiation in a liquid scintillation Packard 300C counter.
Quantitative phosphorylation of vitronectin
Purified vitronectin and pooled normal human serum [NHS] were quantitatively phosphorylated as described above, except that 1OJ..LL of 20 rnM stable ATP (containing 2 1 80 J..LCi y_3 P-ATP) was added to the mixture and 50 J..LL of protein kinase was used . Typically, quantitative labelling leads to the incorporation of a molar ratio of phosphate : vitronectin of 0.25 : 1 [K.orc-Grodzicki et al., 1990]. Purified vitronectin or NHS incubated in the absence of protein kinase served as a control.
MEASUREMENT OF FREE RADIOACTIVITY
Iodinated proteins
20 J..LL of radiolabelled protein was diluted with 480 J..LL of PBS containing 1 % BSA, and the total radioactivity was counted in a multiwell, NE 1600 gamma counter (Nuclear Enterprises). 1.5 mL of 24% TCA was added and the resulting precipitate removed by centrifugation at 600 g, for 20 min at RT and filtration through nylon wool. A 0.5 mL aliquot of :filtrate (containing unbound 1251) was counted in a gamma counter and the fraction of free radioactivity calculated, allowing for dilution, as shown below.
Unbound radioactivity (cpm) x 4 Free radioactivity (%) = X 100 Total radioactivity ( cpm)
1 I acknowledge the contribution of Dr Philip Peake, (Principal Scientific Officer, The Renal Immunology Laboratory, Prince Henry Hospital, Sydney), to the radiolabelling ofvitronectin used in this thesis. Section 2.3 Protein radio/abe/ling 95
Phosphorylated vitronectin
Free radioactivity in solutions of 32P-vitronectin was measured as described for iodinated proteins, except that the bound protein was precipitated in an equal volume of 24 % TCA and the Cerenkov radiation was counted in a liquid scintillation counter, as described above. 96
2.4 COMPLEMENT-DEPENDENT HAEMOLYTIC ASSAYS
Haemolytic assays were used for the measurement of (i) the CH50, (ii) the specific haemolytic activity of purified C9 and 125I-C9, and (iii) the ability of preparations of 32P-vitronectin to inhibit lytic pore formation in vitro. The first assay used antibody-sensitised sheep erythrocytes (EA) as target cells while the others used the cell-bound complement intermediates, EAc1_7 or EAc1_8, as substrates for C9- dependent haemolytic reactions.
PRODUCTION OF ANTIBODY-SENSITISED SHEEP ERYTHROCYTES
5 mL of sheep erythrocytes in Alsevers Solution (Armadeus, Brooklyn, Vic, Australia) was washed in complement fixation diluent (CFD), (Oxoid) and the concentration adjusted to 109 cells/m.L. 10 mL of rabbit anti-sheep haemolysin (Hunter Antisera, Newcastle, NSW, Australia), diluted 1/2000 in CFD was added, dropwise, to 10 mL of erythrocytes and incubated for 15 min at RT with gentle stirring. The resulting EA cells were washed (x3) in CFD and stored at 4°C for no longer than 48 h before use.
Measurement of the CH50
Aliquots of NHS and test sera were serially diluted (i.e. 1/50 - 1/320) in CFD. 200 )..LL of each dilution and 100 )..LL ofEA cells (5 x 108 cells/m.L) were introduced into test tubes and gently mixed. The tubes were incubated in a 37°C water bath for 10 min then quenched with 2 mL of cold CFD, separated by centrifugation (400 g for 5 min at RT), and the OD of the supernatant was measured at 415nm using a Ceres-900 microtitre plate reader (Bio-tek). Haemolytic dose-response curves were constructed and the CH50 was calculated, as a proportion ofNHS, by comparing the linear regions of the test sera and NHS haemolytic curves. 2.4 Haemolytic assays 97
PRODUCTION OF EAc1-7 AND EAc1-s
5 mL of EA cells was washed in CFD (x3) and the concentration adjusted to 5x108 cells/mL. 600 J..LL of undiluted C9-deficient serum (Sigma) was incubated with
150 J..LL ofEA at 37°C for 15 min with occasional gentle agitation. The resulting EAc1_8 cells (108 cells/mL ) were washed in PBS (x3) and used immediately in haemolytic
assays to minimise the amount of spontaneous lysis. EAc1_7 cells were made similarly, using C8-deficient sera (Sigma).
C9-dependent haemolytic assay
A C9-dependent assay was developed to assess the haemolytic activity of purified and radiolabelled C9. Briefly, 15 J..LL of EAc1_8 cells was added to 2- 200J..LL aliquots of C9, 1251-C9, or NHS as a control. Each dilution was made up to 400 J..LL with CFD and incubated at 37°C for 30 min. Background haemolysis was determined by
incubation of 15 J..LL of EAc1_8 cells with 375 J..LL of CFD alone, while maximal haemolysis was measured after addition of 375 J..LL of dH20, instead of CFD. Following incubation, the cells were centrifuged at 400 g for 10 min, and 200 J..LL aliquots of each supernatant were transferred to a microtitre plate and the OD at 415 nm was measured using a Ceres-900 microtitre plate reader (Bio-tek). The fractional haemolysis was calculated for each sample as shown below.
OD415nm (sample)- OD415nm (background) Haemolysis (%) = xlOO OD415 nm (maximal)
Results were corrected for the relative C9 concentration of each solution as measured by radial immunodiffusion in agarose gels (see Appendix 2, page 231). Haemolytic dose-response curves were constructed and the specific haemolytic activities of C9 and 1251-C9 were determined with respect to NHS by comparison of the linear regions of their haemolytic curves. 2. 4 Haemolytic assays 98
Complement inhibition by 32P-vitronectin
The effect of phosphorylated vitronectin on classical pathway-mediated haemolysis was tested by measuring the CH50 in control and quantitatively phosphorylated sera. The capacity of purified phosphorylated vitronectin to inhibit C9- dependent lysis of complement-coated cells was also determined as described by Milis et al., (1993). This assay quantitated the lysis ofEAc;1_7 cells in the presence of C8 (0.65 nmol/L), (Sigma), limiting C9 (1.4 nmol/L), (Sigma) and varying amounts of vitronectin (i.e. 2 - 15 ~-tmol!L) during a 30 min incubation at 37°C. Haemolysis was measured photometrically as previously described 1 and the inhibition due to phosphorylated and non-phosphorylated vitronectin preparations was determined.
1 I acknowledge the contribution of Dr Philip Peake, (Principal Scientific Officer, The Renal Immunology Laboratory, Prince Henry Hospital, Sydney), to the conduct of this assay. 99
2.5 ACTIVATION OF 1251-C9 AND PRODUCTION OF 1251-SCSb-9 IN VITRO
50 11L of purified 1251-human C9 (i.e. approximately 1 11g) was added to 50 11L of fresh NHS, 100 11L of CFD and 8 11L (i.e. 2 11g) of purified CVF. For the activation of 1251-human C9 in rabbit serum, normal rabbit serum (NRS) was substituted for NHS, and in both cases, PBS replaced CVF in control incubations. Following incubation for 3 h at 3 7°C, 200 J.LL of each solution was loaded onto an FPLC Superose 12 gel filtration column and eluted with PBS containing 0.1 % Kl. The high MW SC5b-9-containing fractions (i.e. approximately 1,000 kD) and the monomeric C9-containing fractions were collected and stored at 4°C for no longer than 18 h prior to use in vivo. Fresh NRS supplemented with 1251-C9 was used to produce 1251-SC5b-9 for metabolic studies in rabbits, while 125I-C9-supplemented NHS provided 1251-SC5b-9 for radioimmunoassays and studies in vitro.
PROLONGED INCUBATION OF 1251-C9 IN NHS
For studies involving prolonged incubation of 1251-C9 in NHS (i.e. for 3 days), 50 11L of pure 1251-C9 monomer, fractionated by FPLC gel filtration on Superose 12, was added to 1 mL of fresh NHS in a polystyrene tube and left in an incubator set to 37°C for up to 3 days, with occasional agitation. At approximately 1 h, 12 h, 1, 2, and 3 days of incubation, a 200 11L aliquot was removed for analysis by FPLC Superose 12 gel filtration, and a further 20 11L for precipitation with 24 % TCA and determination of the free iodide content as described in Section 2.3, page 94. 100
2.6 RADIOIMMUNOASSAY OF 1251-SCSb-9
A radioimmunoassay (RIA) using antibody-coated tubes was developed to investigate the components of the soluble TCC produced in vitro. Specifically, C5, C9 neoantigens and vitronectin epitopes were sought. For vitronectin, a panel of four murine monoclonal antibodies was employed. Each of these antibodies (i.e. mAb 2.11, 13.9, 19.16 and 24.4) had previously been shown to interfere with particular biological functions ofvitronectin as summarised in Table 9, page 101.
PREPARATION OF ANTIBODY-COATED TUBES
Protein-binding polystyrene tubes (Nunc) were coated with either (i) affinity purified polyclonal anti-human C5 globulin (Dako, Glostrup, Denmark), (ii) monoclonal mouse anti-human vitronectin (see Table 9, page 101) or (iii) polyclonal goat anti mouse lgG-Fc (Jackson Immunosearch, West Grove, PA, USA). Each antibody was initially diluted to 25 J-Lg/mL in 200 mM carbonate/bicarbonate coating buffer (pH 9.6) and 200 J-LL aliquots were transferred to the polystyrene tubes and left for 2 hat RT. After decanting, non-specific binding sites were blocked with 500 J-LL of PBS containing 0.5% Tween 20, 1% BSA and 5% lactose, for 30 min at 37°C. The tubes coated with goat anti-mouse lgG-Fc were subsequently incubated with monoclonal anti-C9 neoantigen antibody, i.e. mAb aE11 (Dako) at a concentration of 5 J-Lg/mL in PBS for a further 30 min at RT. Tubes treated with blocking solution only were used as controls.
PROTOCOL FOR THE RIA
1251-SC5b-9 was produced by the action of CVF on NHS supplemented with 125I-C9 according to the method described in section 2.5, page 99. Two other C9- containing solutions were also tested: (i) the high MW (i.e. approximately 1000 kD) serum fraction generated spontaneously, i.e. in the absence of CVF, by incubation of Table 9. Characteristics of the murine monoclonal anti-human vitronectin antibodies used in this thesis
Interference with vitronectin's biological activity
Clone Clone Immuno Cell Inhibition of Heparin Collagen Reference code number globulin binding TCC binding binding subclass formation
2.11 HV2 + a
13.9 HV14 ++ * b
19.16 HV22 ++ * b
24.4 HV23 +++ a
References (a) Morris et al., (1994), (b) Morris et al., (1992). - No significant effect,+,++,+++ increasing strength of effect. *Not tested. Section 2.6 Radioimmunoassay of125J-SC5b-9 102
NHS with 1251-C9 at 3 7°C for 3 h, and (ii) monomeric purified 1251-C9. 200 J.LL of each solution was incubated in the coated tubes for 3h at RT, with gentle shaking. The initial radioactivity was counted in a LKB Wallac 1261 Multi gamma counter (Linbrook, Thornleigh, NSW, Australia), the tubes were then washed four times in PBS containing 0.05% Tween 20, and the residual (i.e. bound) radioactivity was counted and expressed as a percentage of the initial counts. 103
2.7 METABOLIC STUDIES IN EXPERIMENTAL ANIMALS
Adult male New Zealand white rabbits weighing 2.5 - 4.5 kg were used in all studies. The animals which received proteins labelled with 1251 or 1311 were pre-treated with KI-supplemented drinking water (100 mg/L) for three days prior to, and throughout the study period, to saturate the physiological iodide space [see Charlesworth et al., 1974a]. Their diet was standard rabbit pellets (Doust & Rabbidge, Concord, NSW, Australia). Rabbits which were given phosphorylated proteins received normal water 1 and diet • The animals were housed in metabolic cages (Mascot Wireworks, Enfield, NSW, Australia) during the sampling period following which a lethal intravenous (lVI) dose of pentobarbitone was administered. These studies had the approval of The Animal Care and Ethics Committee of The University ofNew South Wales.
THE METABOLISM OF 1251-C9 IN RABBITS
Purified human C9 was thawed at 37°C, labelled with 1251 and passed through a 0.2 1-1m filter (Gelman) immediately prior to administration (see Section 2.3, page 92). Freshly prepared rabbit serum albumin was labelled with 1311 as described in Section 2.3, page 92. The animals received an injection of radiolabelled protein via a marginal ear vein and blood samples were drawn from a marginal vein on the contralateral ear at 10 min, 3, 6, and 12h, then 2 or 3 times daily until s; 8% of the initial protein-bound radioactivity remained in the plasma.
Protocols for metabolic studies of 1251-C9 in rabbits
1. Five normal rabbits received 5 - 10 1-1Ci of 1251-C9.
1 I acknowledge the assistance of the staff of the Animal House, Prince Henry Hospital, Sydney. Section2.7 Protocols for animal studies 104
2. Three rabbits received 5- 10 1-LCi of 1251-C9 followed by 50 1-Lg/kg of purified CVF
at either 10 min (n = 2) or at 24 h (n = 1). Control animals received an equivalent volume of isotonic saline (i.e. approximately 1.5 mL) instead of CVF.
3. Two rabbits were given approximately 10 1-LCi of 1251-C9 concurrently with approximately 5!-LCi of 1311-RSA, followed by 200 1-Lglkg of CVF at 10 min. Control animals received isotonic saline in place of CVF. These animals were killed at 6h and samples of their plasma, liver, spleen, kidneys, lungs, myocardium and skeletal muscle were obtained for counting of radioactivity (see page 105).
THE METABOLISM OF 1251-SC5b-9
Aliquots of freshly thawed human C9 were labelled with either 1251 or 1311 on the day prior to the study. 1251-SCSb-9 was prepared as described in section 2.5, page 99, and kept for no longer than 18 h at 4 oc before administration. Radio labelled proteins were injected via a marginal ear vein and 0.5 mL blood samples were drawn from a vein on the contralateral ear into EDTA at 10 min, 30 min, 1, 2, and 4 h. Three rabbits received 0.5 - 1 1-LCi of 1251-SCSb-9 and two of these animals were given a concurrent dose of 0.5 - 1 1-LCi of 1311-C9 which had been pre-incubated in a similar fashion to the 1251-SCSb-9, except for the omission of CVF. These two animals were killed at 4 hand their organs and tissues harvested as described below.
Collection and analysis of samples from studies of iodinated proteins
Plasma samples
Blood samples were drawn into Vacutainer tubes containing solid EDTA (Greiner labortechnik, Austria) and promptly separated by centrifugation at 400 g for 5 min at RT. The following aliquots were analysed : (i) 200 I-LL for FPLC gel filtration on Section2.7 Protocols for animal studies 105
Superose 12 matrix, (ii) 0.5 mL for counting of total radioactivity in multiple or single well gamma counters, (iii) 0.5 mL for precipitation with 1.5 mL of24% TCA; this was then centrifuged at 600 g for 20 min, the supernatant filtered through nylon wool and counted in a gamma counter to quantitate free isotope, and (iv) 10 ~-tL for measurement of the C3 concentration by radialimmunodiffusion in agarose gels as described in Section 2.1 0, page 120.
Blood cells
Following separation from plasma, the packed cells were washed seven times in PBS. 0.5 mL of cells was taken from the centre of the pellet for counting of total radioactivity. A further 0.5 mL aliquot was added to an equivalent volume of 24 % 1 TCA • The precipitate was removed by centrifugation at 600 g for 20 min at RT and filtration through nylon wool, and the supernatant counted in a Linbrook LK.B Wallac 1261 gamma counter to assess free radioactivity.
Tissues
Immediately following a lethal intravenous dose of pentobarbitone, the liver, spleen, kidneys, lungs, heart and samples of skeletal muscle (from the quadriceps femoris) were removed, washed in saline, cleared of adherent fat and a portion of each tissue weighed and placed into polystyrene tubes for counting of total radioactivity. A further sample of each tissue was homogenised with an Omni 2000 homogeniser (Omni International, Waterbury, CT, USA) in ice cold PBS containing 25 mM benzamidine,
0.5 mM PMSF and 4.4 mM Na2EDTA. A 0.5 mL aliquot of organ homogenate was added to 1.5 mL of 24 % TCA, centrifuged at 600 g for 20 min at RT and the supernatant (containing free isotope) was filtered through nylon wool and counted. All specimens obtained from dual isotope studies were counted in a single well Packard Minaxi auto gamma 5000 series gamma counter, allowing for a pre-determined 16 %
1 This procedure both lysed the cells and precipitated their proteins. Section2.7 Protocols for animal studies 106 crossover of 1311 rad' wac t' 1v1 'ty m. t o th e 1251 c h anne 1. Th e re1 atiVe . tissue . rad' wactlVIty . . was calculated with respect to the plasma as shown below.
Bound tissue radioactivity ( cpm/g) Relative tissue = radioactivity Bound plasma radioactivity (cpm/mL)
Urine
Urine was collected throughout the study period and was tested for protein bound radioactivity by precipitation with 24% TCA using BSA as a carrier. 0.5 mL of urine was added to 0.5 mL of 1 % BSA in PBS and 1 mL of 24 % TCA. The mixture was vigorously shaken and separated by centrifugation at 600 g for 20 min at RT and filtration through nylon wool. The supernatant, containing unbound radioactivity, was counted in a multiwell Nuclear Enterprises NE 1600 gamma counter. Urine was not obtained in studies of 1251-SC5b-9 metabolism due to their very short duration.
THE METABOLISM OF 32P-VITRONECTIN IN EXPERIMENTAL ANIMALS
Eight animals received 3 mL of human plasma containing approximately 106 cpm of trace-phosphorylated , vitronectin (i.e. approximately 0.5% of vitronectin molecules) via a marginal ear vein. The protein concentration of this material was approximately 10% of the original plasma. Four of these animals were given 0.25 mg of purified CVF intravenously at 10 min, while four control rabbits received an equivalent volume (i.e. 1 mL) of PBS. Blood samples, drawn into Vacutainer tubes containing a small quantity of solid EDTA, were talcen from a marginal vein on the contralateral ear at 10 min, 2, 3 and 6 h, and subsequently 3 to 4 times daily until < 8% of the injected protein-bound radioactivity remained. The labelled preparations used in these experiments were less than three days old and thawed once only prior to use. Section 2.7 Protocols for animal studies 107
Analysis of plasma samples from studies of 32P-vitronectin
Aliquots of plasma were taken for the following measurements : (i) 0.5 mL for measurement of total radioactivity (see below), (ii) 0.5 mL for precipitation with an equal volume of 24 % TCA for determination of free radioactivity as described for iodinated proteins, (iii) 50 J.!L for determination of total human vitronectin by ELISA as described below, and (iv) 200 J.!L for analysis by FPLC Superose 12 gel filtration.
Quantitation of radioactivity
Phosphorylated vitronectin was quantitated by measurement of Cerenkov radiation in a liquid scintillation, Packard 300C counter.
ELISA for total vitronectin
An ELISA was established in our laboratory to quantitate the total human 1 vitronectin in rabbit serum during metabolic studies • Briefly, 20 J.!L of sample was mixed with an equal volume of 20 mM phosphate buffer (pH 6. 7). The mixture was diluted to 1/20 by the addition of 1% skim milk powder (Carnation, Sydney, NSW, Australia) in PBS. 50 J.!L samples were introduced into the wells of a micro-titre plate which had been coated with anti-vitronectin mAb 24.4 (0.5 mg/mL) in PBS (see Table 9, page 101 ), and blocked with 1% skim milk powder. Vitronectin was detected by the addition of suitably diluted polyclonal sheep anti-human vitronectin antibody (The Binding Site), followed by donkey anti-sheep IgG conjugated with alkaline phosphatase (Sigma). P-nitrophenol phosphate substrate (1 mg/mL), (Sigma) was added and the OD read at 405 nm using a Bio-tek Ceres 900 plate reader. The results, in duplicate, were calibrated against serial dilutions of human plasma in rabbit plasma, which was similarly treated.
1 I acknowledge the contribution of Dr Philip Peake (Principal Scientific Officer, The Renal Immunology Laboratory, Prince Henry Hospital, Sydney) to the development of this assay. 108
2.8 PROTOCOL FOR THE STUDY OF C9 METABOLISM IN HUMAN SUBJECTS
SUBJECTS
Nine patients and eight healthy control subjects were studied. The groups were matched for age but not for sex. Their physical data are summarised in Table 10, page 109. Informed consent was obtained from all participants prior to commencement of the study (see Appendices 4 & 5, pages 233 & 234 for information & consent forms) and subjects were free to withdraw at any time although none did so. All pre-menopausal female subjects had a negative blood or urine pregnancy test (i.e. 13-HCG assay) within four days of commencement and a history of adverse reactions to iodine was sought.
Normal subjects
The control group was drawn from healthy laboratory, medical and nursing staff. Two females and six males were studied (age [mean± SD]: 34 ± 7 yr).
Patients
Nine patients (aged 35 ± 11 yr), including seven with SLE and one each with mesangial lgAN and mixed essential cryoglobulinaemia (MEC) were studied. Eight were female and one, with IgAN, was male. Seven had biopsy proven glomerulonephritis. Table 11, page 110, lists their major clinical characteristics and pharmacological therapy. The patients with SLE fulfilled the diagnostic criteria of the American Rheumatism Association [Tan et al., 1982]. Three patients had chronic renal impairment (CRF) and a serum creatinine concentration greater than 0.11 mmol/L, 109
Table 10. Physical data : patients and controls
Subject Diagnosis Age Sex Weight
(yr) (kg)
Patients
1. SLE 19 F 50.0 2. IgA 34 M 103.0 3. SLE 24 F 53.5 4. MEC 48 F 56.0 5. SLE 38 F 82.0 6. SLE 36 F 71.0 7. SLE 26 F 51.5 8. SLE 53 F 48.5 9. SLE 41 F 43.0
Mean±SD 35±11 62.1 ± 19.6
Controls
1. 33 M 105.0 2. 44 M 92.0 3. 29 M 69.0 4. 28 M 57.1 5. 27 M 85.0 6. 32 F 84.0 7. 40 F 59.5 8. 42 F 69.0
Mean±SD 34±7 77.6 ± 16.7 Table 11. Clinical data : patients with autoimmune disease
Subject Diagnosis Clinical features Pharmacological therapy & daily dose *
2 1. SLE GN (WHO type IV), CRF, Prednisolone 25 mg, cyclophosphamide 0.75 g/m IVI per proteinuria, hypertension month, amlodipine, prazosin, ranitidine
2. IgAN GN,CRF Atenolol 50 mg
3. SLE GN, proteinuria Prednisolone 10 mg, azathioprine 100 mg, frusemide 40 mg, ranitidine 150 mg
4. MEC Raynaud' s phenomenon Prednisolone 15 mg, enalapril 15 mg, allopurinol, carbamazepine
5. SLE Inactive disease Prednisolone 12.5 mg, thalidomide 100 mg, coumadin, aspirin
6. SLE GN (WHO type IV), Prednisolone 12.5 mg, cyclophosphamide 0.75 g/m2 IVI per proteinuria month
7. SLE GN, CRF Cyclosporine 50 mg, felodipine 5 mg, ranitidine 300 mg, ferrous sulphate, sodium bicarbonate
8. SLE Inactive disease Hydroxychloroquine sulphate
9. SLE Pulmonary haemorrhage, Prednisolone 100 mg, ranitidine 300 mg seizures, proteinuria
* Daily dose unless otherwise noted. Section 2.8 Protocol for human studies 111
although none required dialysis. Tables 12 - 14, pages 112 - 114, show the patients' renal function, immunological and haematological profiles respectively. Patients 1 and 9 were in hospital at the time of the study, while the remaining subjects were outpatients.
Medications
Seven of the nine patients were receiving immunosuppressive medications at the time of study. Six were taking oral corticosteroids on a regular basis. Four were receiving::::; 15 mg/day of prednisolone while patients 1 and 9 were taking 25 mg/day and 100 mg/day respectively. Two patients with SLE (i.e. patients 1 & 6) had received 2 intravenous cyclophosphamide (0.75 g/m ) for diffuse proliferative glomerulonephritis in the 4 months prior to the study, while patient 3 was taking azathioprine (100 mg/day) and patient 7 was receiving a small dose of cyclosporine (50 mg/day, i.e. 1 mg/kg per day). Several patients were also taking antihypertensive and other medications as summarised in Table 11, page 110.
EXPERIMENTAL PROTOCOL
All subjects received oral potassium Iodide, 60 mg thrice daily, for 3 days prior to injection and continuously throughout the period of sampling (i.e. for a total of 7 days) to block thyroid iodide uptake and saturate the physiological iodide space [see
Charlesworth et al., 1974b & 1979b, Peake et al., 1989]. 5 ~-tCi of freshly labelled and filtered 125I-C9 (in 2- 3 mL of sterile normal saline [Baxter]) was administered via an antecubital vein following the collection of 5 mL of clotted and EDTA blood samples for the determination of baseline serology. At 15 min, a sample of blood was drawn into EDTA (i.e. at T1) from the contralateral arm or another vein on the ipsilateral arm to avoid possible contamination with radioactivity remaining at the injection site. Subsequent blood samples were similarly drawn from a vein other than that used for the injection. 5 mL blood samples were taken at 3 and 6 h, then twice daily for 2 days and Table 12. Renal function in patients
Subject Diagnosis Serum creatinine * Creatinine clearance # Urinary protein t
(mmol/L) (mL/s) (g/24h)
1. SLE 0.19 1.0 3.2 2. IgAN 0.23 (1.0) 3. SLE 0.07 2.2 4.1 4. MEC 0.07 (1.3) 5. SLE 0.07 (2.1) 6. SLE 0.07 1.9 7.8 7. SLE 0.17 0.6 8. SLE 0.06 (1.2) 9. SLE 0.06 (1.2) 0.7
Laboratory normal range 0.06-0.11 1.2-2.7 <0.15
*I acknowledge the assistance of the Clinical Chemistry Department, Prince of Wales Hospital, Sydney, for measurement of the serum creatinine concentration. # Creatinine clearance was calculated from 24 h urine to plasma ratios, except for those results in parenthesis which were estimated by the method of Cockcroft & Gault, (1976), as 24 h urinary creatinine measurements were not available. t Results are for patients with proteinuria. Table 13. Patients' immunological data
Subject Diagnosis ANA * dsDNA * CIC CHso Serum C3 Serum C4 binding
(Titre) (IUimL) # (%) (%) (giL) (giL)
1. SLE 1/320 13 25 57 0.60 0.26 2. IgAN 17 48 1.11 0.38 3. SLE 1/320 10 33 34 0.56 0.17 4. MEC 1/2560 94 9 0.68 <0.04 5. SLE 1/320 33 48 31 0.68 0.17 6. SLE 1/1280 12 77 9 0.39 0.10 7. SLE 1/320 24 24 72 0.73 0.40 8. SLE 39 128 0.81 0.31 9. SLE 1/320 2 13 33 0.67 0.04
Laboratory normal 0-7 0-16 68-138 0.65-1.26 0.17-0.46 range
* I acknowledge the assistance of the Immunopathology Laboratory, Prince Henry Hospital, Sydney, for quantitation of the anti-nuclear antibody titre (ANA) and double strand DNA ( ds DNA) binding. # International Units/mL. Table 14. Haematological parameters in patients with autoimmune disease
Subject Diagnosis Haemoglobin * Leucocytes * Platelets*
(giL) (x J09 IL) (x Jo9 IL)
1. SLE 130 5.94 170 2. IgAN 153 7.76 232 3. SLE 120 7.16 313 4. MEC 105 7.53 382 5. SLE 138 4.10 195 6. SLE 123 5.70 366 7. SLE 67 4.80 366 8. SLE 143 4.75 235 9. SLE 108 9.26 271
Laboratory normal range 115-165 3.0 -10.0 150-400
*I acknowledge the assistance of the Department ofHaematology, Prince of Wales Hospital, Sydney, for the quantitation ofhaematological indices. Section 2.8 Protocol for human studies 115 daily for a further 2 days yielding a total of 8 samples. Blood was collected into Vacutainer tubes (Greiner Labortechnik) containing a small amount of solid EDTA. Plasma was separated by centrifugation at 400 g for 5 min at RT, within 10 minutes of collection or, if a delay was unavoidable (e.g due to travelling time), the blood was cooled immediately following collection and transported on ice. No delay exceeded 90 min. A 1 mL aliquot of plasma was removed for counting of total radioactivity and a further 1 mL for precipitation with an equal volume of 24 % TCA to quantitate the free radioactivity as described for animal studies (see Section 2.7, page 104). The remainder was frozen promptly and stored at -20°C pending serological and complement measurements (see Section 2.10, page 120). Complete daily urine collections were obtained from all subjects for each of the 4 days of testing. Free urinary radioactivity was assessed by precipitation in 24 % TCA using 1 % BSA in PBS as a carrier (see Section 2.7, page 106). All samples were quantitated using a Packard Minaxi auto gamma 5000 series gamma counter.
This protocol was in accordance with the guidelines ofthe National Health and Medical Research Council of Australia (1991) and had the approval of the Research Ethics Committee of the Eastern Sydney Area Health Service. 116
2.9 ANALYSIS OF METABOLIC DATA
A review of the theory and limitations of metabolic studies in vivo may be found in Section 1.5, page 71.
CALCULATION OF THE INITIAL PROTEIN-BOUND PLASMA RADIOACTIVITY
Following intravenous injection of a measured quantity of radio labelled protein, the protein-bound plasma radioactivity at time zero (i.e. T0 ) was determined assuming that 97% of the injected dose remained in the plasma at 10 min for rabbits, and 15 min 125 for human subjects (i.e. at T1) [see Pussell et al., 1985]. For I-SC5b-9 this assumption was inappropriate due to its very rapid clearance from the circulation. In 125 this case the plasma T0 level was calculated by dividing the injected I-SC5b-9 dose by the plasma volume determined from the dilution of concurrently administered 131I-C9 (see below)
CALCULATION OF THE PLASMA VOLUME
The plasma volume was calculated from the initial dilution of injected 125I-C9, 131I-C9 and 32P-vitronectin, and expressed with respect to body weight as shown below [see Reeve et al., 1982].
Protein-bound injected dose (cpm) Plasma volume (mL/kg) = Protein-bound plasma x Weight (kg) radioactivity at T0 (cpm/mL) Section2.9 Analysis ofmetabolic data 117
CALCULATION OF WHOLE BODY RADIOACTIVITY
The residual whole body radioactivity was calculated by subtracting the cummulative urinary counts from the injected dose [see Charlesworth et al., 1974b].
CONSTRUCTION OF PLASMA DISAPPEARANCE CURVES
Plasma protein-bound radioactivity was plotted, with respect to time (T), as a
percentage of the protein-bound radioactivity at T0, using a semi-logarithmic grid.
MATHEWS' METHOD OF PLASMA CURVE ANALYSIS
By applying a line of best fit, the final log-linear phase of the plasma curve
could be described with respect to time, i.e. f(T), with ordinate intercept, C1 and rate constant, br, as shown 1 .
f(1) = Cl e -bt T
This equation was subtracted from the original curve to give a second
exponential equation of the same type, with ordinate intercept, C2 and rate constant, b2. br, Cr, b2 and C2 were determined by computer-assisted graphics (CA Cricket Graph), (Computer Associates, Sydney, NSW, Australia).
CALCULATION OF THE FCR BY MATHEWS' METHOD
2 The FCR was calculated as a % of the plasma pool/h as shown below .
10000 FCR(%/h) =
1 Equations were initially derived in base 10 then converted to base e by recalculating b as Loge 1Ob. 2 Mathews' original equation for FCR has been multiplied 10000 fold as in this thesis the values for C 1 and C2 were derived as percentages, and the FCR was also expressed as a percentage. Section2.9 Analysis ofmetabolic data 118
CALCULATION OF THE EV/IV BY MATHEWS' METHOD
EVIIV =
CALCULATION OF THE PLASMA T112
The plasma T112 was determined from the slope of the final exponential phase of the plasma disappearance curves as shown below [see Mathews, 1957].
loge 0.5 -0.69
CALCULATION OF FCR BY THE METABOLIC CLEARANCE METHOD
The metabolic clearance [Berson & Yallow, 1957] was determined for subjects with normal serum creatinine concentration (i.e. 0.06 - 0.11 mmol/L) and normal creatinine clearance (i.e. 1.2 - 2. 7 mL/min). It was calculated for each timed period of urine collection (jj,T) from the ratio of the urinary radioactivity to the mean, protein 1 bound plasma radioactivity and converted to a percentage as shown below •
Urinary radioactivity (cpm) Metabolic clearance (%/h) = ------X 100 Mean protein-bound x jj,T (h) plasma radioactivity (cpm)
The mean metabolic clearance was calculated by taking the average of several days, with the exclusion of the first 24 h period, i.e. prior to compartmental equilibration (see Section 1.5, page 71).
1 The calculation of mean protein-bound plasma radioactivity is shown in Appendix 6, page 235. Section2.9 Analysis ofmetabolic data 119
CALCULATION OF THE PLASMA C9 PRODUCTION RATE
The PPR of C9 was calculated assuming that, in a steady state, the total amount of protein leaving the system through catabolism is equal to that entering, i.e. by de novo synthesis and I or release from stores 1 [see Charlesworth et al., 1989a].
FCR (%/h) x serum [C9] (mg/mL) x plasma vol. (mL/kg) PPR (mg/kg per h) = 100
CALCULATION OF THE WHOLE BODY C9 CONTENT
The whole body C9 content (WB C9), i.e. the sum of the C9 mass in all extravascular and the intravascular compartments, was determined for human subjects 2 as shown •
WB C9 (mg/kg) = Plasma vol.(mL/kg) x serum [C9] (mglmL) x (EVIIV + 1)
CALCULATION OF THE VOLUME OF DISTRIBUTION OF C9
The volume of distribution of C9 was taken as that volume in which the whole body C9 content would appear to be distributed at steady state, if its concentration throughout that volume was the same as in the serum [see Mayer et al., 1980]. This was determined in human subjects as shown below.
WB C9 (mg/kg) C9 volume of distribution (mL/kg) = Serum [C9] (mg/mL)
1 The FCR used in this equation was derived by Mathews' analysis as this was obtainable in all subjects. 2 The derivation of the formula for whole body C9 content may be found in Appendix 7, page 236. 120
2.10 QUANTITATION OF BLOOD PROTEINS
COMPLEMENT COMPONENTS & CERULOPLASMIN
C3, C4, C9 and ceruloplasmin were quantitated by single radial immunodiffusion based on the method of Mancini et al., (1965). Polyclonal anti-sera were added to molten 1 % agarose (Sigma) in barbital buffer 1 and allowed to set at RT. 3 mm wells were punched into the gels and 5 or 10 J-LL aliquots of sample were introduced and incubated for approximately 24 hat RT in a humidified chamber. The gels were emersed in normal saline for 6 - 8 h to remove non-precipitated protein and then dehydrated in air at RT and stained in 0.5 % Coomassie brilliant blue (Sigma). Samples of known concentration, or dilutions ofNHS, were used to construct a standard curve. The diameter of the precipitin ring was measured with a jeweller's eye piece and the square of this value was used to determine the absolute, or relative quantity of each protein by reference to the standard curves. Appendix 2, page 231, lists the volumes and manufacturers of the anti-sera used.
ELISA ASSAY FOR PLASMA SC5b-9
Blood samples were collected and processed as described in section 2.8, page 111, and according to the recommendations of Mollnes et al., (1988a). The plasma SC5b-9 concentration was measured using a proprietary ELISA (Behring Diagnostics, Kingsgrove, NSW, Australia). This method utilised mAb aEll-coated tubes as the capture system, and peroxidase-conjugated polyclonal anti-CS antibody to detect the bound complex. Briefly, 100 J-LL of each sample was introduced into the coated tubes and incubated for 30 min in a 37°C water bath with occasional agitation. After
1 Barbital buffer : 41.2 g Na Barbital+ 8.0 g Barbital made to 2000 mL with dH20 (pH 8.6) to produce a (5x) stock solution. Section 2.10 Quantitation ofspecific blood proteins 121 washing in phosphate/Tween buffer, 200 J.LL of anti-CS peroxidase-conjugated stock solution was added and incubated for a further 30 min at 37°C. The tubes were washed, and 200 J.LL of a-phenylenediamine dihydrochloride chromogenic substrate 1 was added and incubated for a further 30 min at RT in the dark. The reaction was stopped by the addition of 1 mL of 1M H2S04 and sample aliquots were transferred to a micro-titre plate for measurement of the OD at 490 nm using a Bio-tek Ceres 900 plate reader. SCSb-9 samples of known concentration, supplied with the assay kit, allowed construction of a standard curve.
THE C1 q-BINDING ASSAY FOR CIRCULATING IMMUNE COMPLEXES
This assay was described by Zfibler et al., (1976). Briefly, a 100 J.LL aliquot of serum was incubated with 0.2 M Na2EDTA/Na4EDTA (pH 7.4) at 3rC for 30 min. 125I-C1q stock solution was centrifuged at 30000 rpm for 60 min at 4°C to remove aggregates, and 20 J.LL of the supernatant was added to the serum/buffer solution and incubated for 30 min at 4°C. 1 mL of 3% PEG 6000 in 0.1 M borate buffer (pH 8.3) and 0.05% Tween 20 was introduced and incubated for a further 2 hat 4°C. Following centrifugation at 3000 g, the supernatant was removed and the radioactivity in the pellet counted using a Linbrook LKB Wallac 1261 gamma counter. The results were expressed as a percentage of the total precipitable protein, i.e. with 24% TCA. Aggregated human globulin served as the positive control.
MEASUREMENT OF ANTI-NUCLEAR ANTIBODIES
The serum anti-nuclear antibody titre was measured using Hep-2 cells in a proprietary assay (Sanofi Pasteur, Sydney, NSW, Australia) and the ds DNA binding 2 was determined by RIA (Johnson & Johnson, Sydney, NSW, Australia) •
1 a-phenylenediamine dihydrochloride in citrate/phosphate buffer/H20 2• 2 I acknowledge the staff of the Immunopathology Laboratory of Prince Henry Hospital, Sydney, for their assistance with the quantitation of anti-nuclear antibodies. 122
2.11 MATHEMATICAL AND STATISTICAL ANALYSIS
Data was stored and results were calculated using the QBasic programming environment (Microsoft, Redmond, WA, USA), and CA Cricket Graph (Computer Associates) was used to analyse graphical data.
Statistical analysis was performed using an Apple Macintosh desktop computer (Cupertino, CA, USA) and the INSTAT statistical package version 2 (Graphpad Software, San Diego, CA, USA). In general, data conforming to a normal distribution was expressed as the mean ± standard deviation (mean ± SD) and the statistical difference between such groups was tested using a two tailed Student's t-test. An F-test was initially applied to establish that the SDs of each data set were not significantly different, else the t-test was inappropriate. When this was the case, and when the data appeared not to be normally distributed, it was expressed as the median and range (median [range]) and a non-parametric, Mann Whitney U-test was applied. A statistical probability (p) < 0.05 was accepted as significant. Correlation coefficients (r) were derived by linear regression analysis. 123
Section 3
RESULTS 124
Purification and preparation of proteins 125
3.1 THE PURITY AND FUNCTIONAL ACTIVITY OF C9
C9 PURIFICATION
The critical steps in obtaining a highly pure preparation of C9 proved to be chromatography on DEAE-A50-Sephadex and hydroxylapatite matrices. Although many proteins were present in the C9-containing fractions eluted from the DEAE-A50- Sephadex column, only those which were free from ceruloplasmin were collected (as shown in Fig. 11(a), page 126), to avoid contamination of the final preparation. Finally, C9 was eluted cleanly from an hydroxylapatite column (see Fig. 11 (b), page 126).
Samples were obtained at several stages during a typical C9 purification and the relative C9 content and total protein concentration were measured. Table 15, page 127, summarises these results. Overall, the C9 yield was 5. 7 % with a fmal purification of 850 fold.
Reducing SDS-PAGE of C9-containing fractions stained with 0.5 % Coomassie Brilliant Blue revealed only one band of approximate MW 70 kD (see Fig. 12, page 128). Similarly, auto-radiography of 125I-C9 showed only one labelled band corresponding with the stained gel (see Fig. 13, page 129). Analysis of native (i.e. non reduced) 125I-C9 by FPLC gel filtration on Superose 12 demonstrated that in excess of 90% of the protein-bound radioactivity resided in a single peak consistent with C9. A small peak representing< 5 % of protein-bound radioactivity, was present in the high MW region, i.e. approximately 1000 kD, while a smaller peak or shoulder (i.e.< 4 %), was seen in the 200- 300 kD zone (see Fig. 14, page 130). Free 1251 was also present in the appropriate low MW region (not shown in Fig. 14). The proportion of free 1251 estimated from the areas under the gel filtration curve was in close agreement with the calculations based on precipitation with TCA, i.e. 1 - 4 %. 126
Fig. 11. Purification of C9 by column chromatography
Protein elution from DEAE-A50-Sephadex (a) and hydroxylapatite (b) columns in NaCl and phosphate gradients respectively. The dotted lines show buffer conductivity and the arrows show C9 and ceruloplasmin-containing fractions. C9 .. ... Ceruloplasmin.. ... (a) 400 40
300 30 .•... -····---·--···········" 200 ·········· ····· 20 ..-.. ········· ~ ~ , .• ---·-··--··--..------bS) t::d ::i 100 10 '-" ::::;= ~ 0 .., ...... = ~ (') ...... 1-< 0 0 0 ~ ~ = =t.l .....=(') 0 .... t.l= ....< ..... C9 ... q ...... =~ .. ..-.. 0 1-< 40 9 ~ 00. (') ...... ~ _ -9 - _..----·~~-····"''~·-" '-" 0 20 H 30
15 ,._..,.----·/""''"''"'"""/"'~~/·" 20 10
10 5
0 0 25 50 75 100 Fraction number Table 15. Recovery of C9 and total protein at stages of C9 purification
Protein recovery Stage Volume Total protein Total C9 C9 purification concentration
(mL) (giL) (% initial plasma) (fold)
Plasma 105.0 68.0 100 100 1.0
Post 21% PEG 4000 24.0 59.0 19.8 61.0 3.1
Post lysine-Sepharose 114.0 11.0 17.5 59.0 3.4 chromatography
Post DEAE-Sephadex 74.5 0.10 0.10 10.0 100 chromatography
Purified C9 1.4 0.34 0.0067 5.7 851
125 Fig. 14. FPLC Superose 12 gel filtration of purified I-C9. The arrows show MW markers in kD.
Blue dextran 660 66 12.3 ~ ~ ~ ~ ...... -...... 0.6 t.J ~ -Q .... 0.5 "'0 ~ 1-< "'0 0.4
=Q ,.Q= ....I 0.3 =~ Q -1-< =.. 0.2 ~ Q 0.1 ....=Q t.J -~ 1-< ~ 0 10 20 30 40 Column fractions ...... CH Q Section 3.1 C9 purity & activity 131
C9 HAEMOLYTIC ACTIVITY
The haemolytic response curve for the reaction of EAc1_8 cells with C9 was linear at low C9 concentrations (i.e.< 3 ng/mL), but began to plateau at 25- 30% of the 125 maximal haemolysis produced by dH20. Purified C9 and I-C9 retained full haemolytic activity with respect to NHS and their haemolytic dose-response curves were essentially superimposed over the linear phase (see Fig. 15, page 132).
Background haemolysis (i.e. due to EAc1_8 alone), was approximately 15 % and was subtracted from the results.
INCORPORATION OF PURIFIED 1251-C9 INTO 1251 SCSb-9 IN VITRO
Purified 1251-C9 was shown to interact in vitro with the other components of the terminal complement pathways of both rabbits and humans to generate a high MW complex consistent with SC5b-9. This is reviewed in Section 3.3, page 136.
STABILITY OF C9 DURING STORAGE
Purified C9, stored for three months at -70°C, retained full haemolytic activity following radiolabelling. Similarly, there was no evidence of aggregation or fragmentation of 125I-C9 on FPLC Superose analysis following storage at 4°C for one week. 132
Fig. 15. The haemolytic activity of purified C9
e 125I-C9
--h:-- Unlabelled purified C9
-o- Normal human serum
50
40
_.-._ -~ .....e ~ ~ e 30 ~ '-' .....Cl.l ;;.....Cl.l 0 -e 20 d) =~
10
0
0 5 10 15 20 25
C9 concentration (ng/mL) 133
3.2 MOLECULAR INTEGRITY AND FUNCTION OF PHOSPHORYLATED VITRONECTIN
SELECTIVE PHOSPHORYLATION OF VITRONECTIN
Typically, trace phosphorylation of human plasma with cAMP-dependent kinase and y-32P-ATP resulted in the labelling of approximately 0.5 % ofvitronectin molecules. (By comparison, quantitative phosphorylation of vitronectin leads to the incorporation of a molar ratio of phosphate: vitronectin of 0.25 : 1 [see Section 2.3, page 94 & Korc Grodzicki et al., 1990]). Autoradiography of phosphorylated plasma on reduced SDS PAGE showed a single labelled band of MW 75 kD. Non-denaturing FPLC gel filtration demonstrated that both antigenically-detected and 32P-vitronectin were largely monomeric, although approximately 4 % of molecules were present in the 1000 kD region. Fig. 16, page 134, shows an FPLC gel filtration of NHS following selective phosphorylation of vitronectin. In this preparation, approximately 10 % of protein bound radioactivity was in the high MW region.
Vitronectin purified by heparin-affinity chromatography migrated as a doublet on reduced SDS-PAGE with Coomassie Blue-stained bands of MW 75 kD and 65 kD. The purity of this preparation, estimated from stained gels, appeared to be in> 95 %.
THE EFFECT OF VITRONECTIN PHOSPHORYLATION ON COMPLEMENT FUNCTION IN VITRO
Complement-mediated haemolysis was unaffected by quantitative phosphorylation of serum vitronectin. No significant difference in the CH50 was observed between phosphorylated sera and sera incubated in the absence of protein kinase. Similarly, purified vitronectin retained unaltered ability to inhibit C8/C9- dependent lysis ofEAc1•7 cells following quantitative phosphorylation. 134
Fig. 16. FPLC Superose 12 gel filtration of 3 2P-vitronectin
Normal human serum in which the vit:ronectin was selectively labelled by incubation with 32P-ATP and cAMP-dependent protein kinase. The arrows show MW markers in kD.
Blue dextran 6 6 0 66 ! ! 4000
3000 s ~ ...._.~ ...... ~ ...... j;l- 2000 ~ .....=0 "CC ~=
1000
0
10 15 20 25 30 35
Fraction number 135
Studies in vitro 136
3.3 ACTIVATION OF 1251-C9 IN VITRO
INCUBATION WITH CVF
After 3 h of incubation in NHS in the presence of CVF at 37°C, 90 - 95 % of labelled C9 became incorporated into a high MW fraction (i.e. approximately 1000 kD) as shown by FPLC gel filtration on Superose 12 (see Fig. 17(b), page 137). By comparison, CVF -activated NRS, incubated under similar conditions, yielded a high MW peak representing approximately 30% of total radioactivity (see Fig. 18(b), page 138). In both cases, there was also a modest rise in the minor peak of radioactivity in the 200- 300 kD region.
CONTROL STUDIES
Incubation of 125I -C9-supplemented NHS at 37°C in the absence of CVF (i.e. with an equivalent volume of PBS), produced a small increment in high MW radioactivity representing approximately 10 % of the total counts (see Fig. 17 (c), page 137). This was in excess of the corresponding high MW peak present, as a contaminant, in purified C9 (compare with Fig. 17(a), page 137). The corresponding control incubation with NRS produced a smaller rise in high MW radioactivity, i.e.< 5% of the total protein-bound counts (see Fig. 18(c), page 138).
Prolonged incubation of 1251-C9 in NHS
Incubation of 125 I-C9 with fresh human serum at 37°C for up to 65 h, produced a progressive shift of radioactivity from the C9 monomeric peak to the high MW region as shown in Fig. 19, page 139. This conversion was most rapid in the first 12 h and slowed considerably by 46 h at which time each peak contributed almost equally to the 137
Fig. 17. Activation of 125I-C9 in normal human serum
FPLC gel filtration of purified 125I-C9 (a) and 125I-C9 incubated for 3 h in NHS in the presence (b) and absence (c) of CVF. The arrows show MW markers in kD.
Blue dextran 660 12.3 + + + 0.6
0.4
0.2
...... 0 ...... I> 0 ~ ~ .....0 0.6 "'CC ~ 1-< "'CC = 0.4 0 ,.Q= .....=I ....Q,) 0 1-< 0.2 ~
~ 0
=0 ...... 0 ~ ~ 1-< ~ 0.6
0.4
0.2
0 10 20 30 40 Fraction number 138
Fig. 18. Activation of human 12 5 I-C9 in rabbit serum
FPLC gel filtration of purified 125I-C9 (a) and 125I-C9 incubated for 3 h in NRS in the presence (b) and absence (c) of CVF. The arrows show MW markers in kD.
Blue dextran 660 66 12.3 + + + + 0.6
0.4
0.2
.....1>...... ;;...... 0 Col d .....0 "0 0.6 d 1-o "0
0= 0.4 ,.Q= .....I .....~= 0 1-o ~ 0.2
~ 0
...... =0 Col 0 d 1-o ~ 0.6
10 20 30 40 Fraction number Fig. 19. FPLC Superose 12 gel filtration of 12 5 I -C9 incubated in NHS at 37° C Samples taken at 1 h (a), 13 h (b), 46.7 h (c) and 64.7 h (d). The arrows show MW markers in kD.
Blue dextran 660 66 Free iodide
0.3 ++ + + (a) (b)
_e. 0.2 .....:;...... C.,) e':l .....0 0.1 "C e':l ~ -.....e':l 0 .....0 0.3 'S (c) (d)
=0 ...... C.,) 0.2 e':l ~ ~ 0.1
0 0 20 40 60 70 0 20 40 60 70
...... Fraction number (N \C Section 3.3 Activation of125 I-C9 in vitro 140 total protein-bound radioactivity (see Fig. 20, page 141). The proportion of free 1251 remained less than 3% throughout the experiment and there was no evidence of fragmentation of the protein. 141
Fig. 20. Incorporation of 125I-C9 monomer into a high MW form during prolonged incubation in NHS
Changes in the proportion of 125I-C9 monomer and the high MW (i.e. approximately 1000 kD), 125I-C9-containing serum fraction during prolonged incubation of 125I-C9 in NHS at 37°C. This figure was derived from the area under the curves in Fig. 19, page 139 and the time zero sample.
• Monomeric 125 I-C9
-o- High MW 125I-C9-containing serum fraction
-o- Free 1251
100
75 .....;;...... ;;...... ~ ~ .....Q "CC ~ ""' 50 -.....~ .....Q ~ Q ~ 25
0 0 25 50 75
Time (h) 142
3.4 ANTIGENIC AND PHYSICAL PROPERTIES OF THE SOLUBLE TCC
RADIOIMMUNOASSAY FOR SC5b-9 COMPONENTS
A radioimmunoassay was used to investigate the presence of components of 1251- SCSb-9 produced in vitro. Specifically, C9 neoantigens, CS and four epitopes of vitronectin were sought. The high MW fractions (i.e. approximately 1000 kD), produced spontaneously in control sera and by CVF-activation of 1251-C9-supplemented NHS were studied (see Section 3.3, page 136). As a further control, the high MW contaminant, present in small quantity in purified 1251-C9 was also tested.
Binding to antibodies against C9 neoantigens and C5
The high MW fractions of both CVF-activated and control sera bound to anti-CS and mAb aE11 (see Fig. 21, page 143). Approximately 11 % of CVF-activated high MW radioactivity was retained by anti-CS, and 39% by mAb aE11-coated tubes. The corresponding values for the spontaneous material were 18 % and 50 % respectively. By contrast, monomeric 1251-C9 showed no significant affinity for either of these antibodies, with only 0.1 % and 0.4 % of counts retained by anti-CS and mAb aE11 respectively.
Binding to monoclonal anti-vitronectin antibodies
The spontaneous high MW serum fraction bound significantly to all four anti vitronectin antibodies with 32 - 74% of counts retained (see Fig. 22, page 144). The CVF-activated sera also bound to anti-vitronectin mAbs 2.11 and 13.9, with 35 %and 143
Fig. 21. Radioimmunoassay for CS and C9 neoantigens in the TCC
Radioimmunoassay for the presence of complexed C5 and C9 neoantigens in the high MW serum fractions (i.e. approximately 1000 kD), produced spontaneously and by CVF activation of 125I -C9-supplemented NHS. Results obtained with 125I-C9 monomer are shown for comparison.
• Anti-C9 neoantigen (mAb aEll)
Anti-CS
,...... 80 fl.l
==0 ~ .....co= .....-...... 60 .....= ~ 0 ~..._, .e- 40 ·~ ...... ~ co= .....0 "'CC co= ~ 20 "'CC ~ ...... =co= ~ ~ 0
Monomeric C9 Spontaneous CVF-activated (High MW serum fractions)
125I-C9-containing fractions 144
Fig. 22. Radioimmunoassay for vitronectin epitopes in the TCC
Radioimmunoassay for the presence of complexed vitronectin epitopes in the high MW serum fractions (i.e. approximate!!, 1000 kD), produced spontaneously and by CVF activation of 12 I-C9-supplemented NHS. Results obtained with 125I-C9 monomer are included for comparison.
5I Anti-vitronectin mAb 24.4 1m Anti-vitronectin mAb 19.16 • Anti-vitronectin mAb 13.9
Anti-vitronectin mAb 2.11
,-... 80 til -= 0 =CJ ....~ -.... 60 ....-= ~ 0 ~ '-" .....0 40 ....;;.. CJ -~ ....0 "'t:: ~ ~ 20 "'t:: ....=~ ~ ~ ~-
Monomeric C9 Spontaneous CVF -activated (High MW serum fractions)
12 5 I-C9-containing fractions Section 3.4 Properties ofthe soluble TCC 145
69% of counts retained respectively. However, less than 0.5% of counts were retained by anti-vitronectin mAbs 19.6 and 24.4. Monomeric 125I-C9 bound only minimally to the anti-vitronectin mAbs, with < 0.1 % of counts retained. Similarly, < 0.1 % of radioactivity was retained by the control blocked tubes.
The high MW contaminant of purified 125I-C9 showed minimal binding (i.e. < 0.5 %) to all antibodies with the exception of mAb aE11 which retained approximately 5% of initial radioactivity.
HEPARIN-SEPHAROSE CHROMATOGRAPHY
The behaviour of CVF-activated and the spontaneous high MW fraction of 125I C9-supplemented NHS was investigated using heparin-Sepharose chromatography. Purified monomeric 125I-C9 was also studied as a control. Fig. 23, page 146, demonstrates that all three solutions bound to heparin-Sepharose in low ionic strength buffer. However, while the 125I-C9 monomer was completely eluted relatively early in the NaCl gradient, i.e. from 0.3 to 0.6 M, both of the high MW 125I-C9-containing complexes were released over a broader ionic range and with similar profiles.
STABILITY OF THE SOLUBLE TCC IN HIGH SALT CONCENTRATION
The spontaneous and CVF -activated high MW fractions of 1251-C9- supplemented NHS were studied by FPLC Superose 12 gel filtration after equilibration of each solution, and the column, in 1 M NaCL Only one significant peak of radioactivity was seen in the high MW region (i.e. approximately 1000 kD) and neither sample showed evidence of fragmentation or release of free iodide (see Fig. 24, page 14 7). Table 16, page 148, summarises the physical and antigenic characteristics of the spontaneous and CVF -activated high MW complexes. Fig. 23. Heparin-Sepharose chromatography of 125I-C9 monomer and 125I-C9-containing complexes
Heparin-Sepharose chromatography of 125I-C9 monomer and the high MW fractions (i.e. approximately 1000 kD) produced spontaneously and from CVF activation of125I-C9-supplemented NHS. The dotted line shows the applied NaCl gradient.
0.2 1 ,,,'' --o-- Monomeric 125I-C9 , ....>. ,,,,," .... Spontaneous High MW j;o- • / ...... serum fraction / ~ , > ~ 0.15 / 0.75 ~ ....Q ~ CVF-activated high MW ,/ ~..... "'t:l serum fraction ,/ -~ ~ ~ 1-< ,, / z "'t:l / ~ ~ , .... (j ~ ~ 0.1 0.5 •...-:. (Jel- .... '"'S = , ~ ~ ~ 0 , ..... ,,," ~ =0 = ...... ,, ..-..- ~ 0.05 , 0.25 ~ ~ / "---' 1-< ,/ ~ , ,, / / ,/ 0 0 0 20 40 60 80
~ Column fractions ol:::o =-- 147
Fig. 24. Stability of the TCC in 1M NaCI
FPLC gel filtration of the high MW fractions (i.e. approximately 1000 kD), produced spontaneously (a) and by CVF activation of1 25I-C9-supplemented NHS (b). The arrows show MW markers in kD.
Blue dextran 6 6 12.3 0.24 Free Iodide
0.5 t t t (a)
0.4
0.3
0.2 ....._e. lio- ...... ~ 0.1 c:l .....0 '"1:1 c:l ~ 0 -...... c:l 0 ..... 0.5 ~ 0 (b)
=0 ...... 0.4 ~ c:l ~ ~ 0.3
0.2
0.1
0
0 20 40 60 80
Fraction number 148
Table 16. Comparison of the high MW serum fractions produced spontaneously and by CVF activation of 1251-C9-supplemented NHS
Property Spontaneous high CVF-activated high MW serum fraction MW serum fraction
Physical properties
Molecular weight ~ 1000 kD ~ lOOOkD
Stability in 1M NaCl + +
Heparin binding + +
Antigenic properties
Binds anti-CS Ab + +
Binds mAb aE11 + +
Binds anti-Vn mAb 2.11 * + +
Binds anti-Vn mAb 13.9* + +
Binds anti-Vn mAb 19.6* +
Binds anti-Vn mAb 24.4* +
+ Property is present. - Property is absent. * See Table 9, page 101, for a description of these antibodies. 149
Studies in experimental animals 150
3.5 THE METABOLISM OF C9 IN NORMAL RABBITS
None of the five animals showed any ill effects during the sampling period of these studies. However, one rabbit was temporarily oliguric during the investigation and was excluded from calculations involving urinary data.
PLASMA 1251-C9 AND WHOLE BODY RADIOACTIVITY DISAPPEARANCE CURVES
Following injection of 125I-C9, protein-bound plasma radioactivity decayed in two phases. An early rapid decline in the first 24 h was followed by a slower, log-linear disappearance, with less than 10 % of the injected radioactivity remaining after 72 h. The whole body radioactivity fell more slowly than the plasma protein-bound counts, particularly in the first 24 h (see Fig. 25, page 151). Free iodide was released at a steady rate throughout the turnover period remaining< 5 %of the total plasma radioactivity.
METABOLIC PARAMETERS
The plasma T112 of C9 was 25.2 ± 3.7 h (mean± SD), the FCR was 5.99 ± 0.69 %/h, derived by Mathews' plasma curve analysis, and 5.47 ± 0.77 %/h calculated by the metabolic clearance technique. The difference between these two measurements of FCR was not statistically significant. The EVIIV distribution ratio was 0.72 ± 0.21. These data are summarised in Table 17, page 152.
THE PLASMA VOLUME
The plasma volume was 35.0 ± 3.0 mL/kg. 151
Fig. 25. Typical plasma and whole body 12 5 I -C9 disappearance curves in a normal rabbit
-o- Residual whole body radioactivity
• Plasma protein-bound radioactivity
100
.-... ~ (11 0 "t:l "t:l ...... ~ ~ ~ .i:i..... ~ 10 '-" .....t> ;> ...... ~ Cd .....0 "t:l Cd ~
1
0 20 40 60 80
Time (h) Table 17. C9 metabolic data in normal New Zealand white rabbits
FCR Animal Weight Plasma Plasma curve Metabolic EVIIV volume analysis clearance
(kg) (mL/kg) (h) (%/h)
1. 3.435 34.4 25.9 5.72 4.59 0.75 2. 3.498 30.3 27.6 5.27 5.50 0.57 3. 2.665 38.3 20.6 6.99 * 0.66 4. 3.472 35.3 29.5 5.58 5.31 1.08 5. 3.936 36.8 22.1 6.38 6.47 0.56
Mean±SD 3.401 ± 0.459 35.0 ±3.0 25.2 ± 3.7 5.99 ± 0.69 5.47 ± 0.77 0.72 ± 0.21
* Not calculated due to oliguria. Section 3.5 C9 metabolism in normal rabbits 153
EXCRETED RADIOACTIVITY
The unne was the major pathway for excretion of radioactivity with approximately 52 (49- 56)% (median [range]) ofthe injected dose recovered after 48h,
and greater than 83% after 8 days from non-oliguric animals (n = 4). TCA-precipitable urinary radioactivity was < 10 % of the total urine activity. Faecal radioactivity represented < 1 % of the urinary counts.
FPLC GEL FILTRATION CHROMATOGRAPHY OF PLASMA SAMPLES
The plasma from three normal rabbits was analysed by FPLC gel filtration chromatography on Superose 12. The results for samples taken from a rabbit at 10 min, 1, 8, and 21 h following injection of 1251-C9 are shown in Fig. 26, page 154. The major portion of radioactivity remained in the monomeric C9 region and, in particular, there was no evidence of accumulation of high MW labelled compounds or fragmentation of the molecule.
UPTAKE OF 1251-C9 AND 131 1-RSA BY TISSUES
The distribution of radioactivity to solid organs was measured in two normal 125 131 1 125 rabbits given 1-C9 and 1-RSA, and sacrificed at 6 h • Tissue protein-bound 1 (i.e. due to, or derived from 1251-C9) was present at less than 50% of the concentration of plasma protein-bound 1251 in all organs examined. Its distribution was similar to that of protein-bound 1311 (i.e. due to, or derived from 1311-RSA), (see Fig. 27, page 155). Calculation of the free organ radioactivity by subtraction of the bound from total organ counts, demonstrated accumulation of both iodide isotopes in the kidney.
1 Organ radioactivity was expressed as protein-bound (cpm/g fresh tissue)/(cpm/mL plasma). Fig. 26. FPLC gel filtration of plasma samples from a normal rabbit given 12 5I-C9 Samples drawn at 10 min (a), 1 h (b), 8 h (c) and 21 h (d) following injection. Arrows show MW markers in kD.
Blue dextran 660 66 15 t t t (a) (b) ,-... (f') 10 ~ ~
~ ~e 5 6 ~ C.l '-' 0 .....c ...... 15 C.l (c) (d) COd ....0 , 10 COd ~ 5
0 10 20 30 40 10 20 30 40
Fraction number Fig. 27. Organ distribution of 125I-C9 and 1311-RSA in a normal rabbit
Tissue protein-bound 125 I (•) and 131 I (t[;) expressed as (cpm per g fresh tissue) I (cpm per mL plasma) in a rabbit given both 125 I-C9 and131 I-RSA and killed at 6 h.
2 ...... ;;...... tJ ~ .....0 "'t:: 1.5 :...~ "'t::
=0 ,.Q 1 I ...... ~= 0:... ~ ~;;.. 0.5 ...... ~ -~ ~ 0
Plasma Liver Spleen Kidney Lung Heart Muscle Section 3.5 C9 metabolism in normal rabbits 156
UPTAKE OF 1251-C9 BY CIRCULATING CELLS
The radioactivity bound to circulating blood cells was measured in two normal rabbits. Washed, packed blood cells (i.e. mostly erythrocytes) contained less than 0.01% of the plasma radioactivity (see Fig. 28, page 157). 157
Fig. 28. Uptake of 125I-C9 by blood cells in a normal rabbit
Protein-bound radioactivity associated with plasma and washed, packed blood cells from a normal rabbit given 125 I-C9.
• Plasma --o- Cells
105
,.-.., ~ ~ 104 ~ ~ '-' ;;...... 103 ...... ~ ~ "= .....0 "'t:: 2 "= 10 ~
101
1 0 10 20 30 40 50 60
Time after injection (h) 158
3.6 C9 METABOLISM IN RABBITS DURING COMPLEMENT ACTIVATION
PLASMA 1251-C9 DISAPPEARANCE CURVES
Following the administration of CVF there was a marked increase in the rate of disappearance of protein-bound radioactivity from the plasma. This was most evident in the first 2 - 12 hand was seen in animals given CVF at 10 min and at 24 h following injection of 125I-C9. However, the final phase of 125I-C9 disappearance was parallel to that observed in normal rabbits (see Fig. 29, page 159). CVF administration was also associated with a marked fall in the serum C3 concentration of approximately 60 % by 6 hand 95 % at 20 h. This was not seen in animals given 125I-C9 alone. Metabolic parameters were not calculated from these studies because of loss of steady state conditions after injection ofCVF.
FPLC GEL FILTRATION OF PLASMA SAMPLES
FPLC gel filtration of plasma samples taken following CVF injection showed a new high MW peale of approximately 1000 kD, con~istent with SC5b-9. This was evident after only 1 hand rapidly declined to a minimal level at 21 h (see Fig. 30, page 160). An estimate of the absolute contributions of monomeric and complexed 125I-C9 was made by expressing the area under each peak as a proportion of the area under the whole curve and multiplying by the measured protein-bound radioactivity. This analysis showed that the appearance of high MW radioactivity occurred during the period of most rapid fall in the 125I-C9 monomer. Furthermore, the true rate of disappearance of monomeric 125I-C9 was faster than the total protein-bound radioactivity, particularly during the first 10 h following CVF injection (see Fig. 31, page 161). However, there was no evidence of fragmentation of the molecule into smaller, radio labelled species. 159
Fig. 29. Plasma 125 I-C9 disappearance curves in rabbits administered CVF
One rabbit was given CVF at 10 min following injection of 125I-C9 and the other at 25 h. Results obtained in a normal animal are included for comparison.
-o- Normal rabbit
• CVF injected at 10 min
• CVF injected at 25 h
100 ,.-... (!) l:ll Q "'d "'d (!) ....C) (!) ....'S' ~.._, .....0 ....~ C) 10 C':S ....Q "'d C':S ~ "'d
Q= ,.Q= ....I =(!) ....Q ~ ~ CVF CVF 1 0 20 40 60 80
Time (h) Fig. 30. The effect of CVF on the FPLC gel filtration of plasma in rabbits given 125I-C9 At 10 min, one animal received CVF (•), while the other received an equivalent volume of isotonic saline (D). Plasma samples were taken just before administration ofCVF or saline (a), 1 h (b), 8 h (c) and 2lh (d) after injection. Arrows show MW markers in kD.
Blue dextran 660 66 15 •• • (b) 10 ,-..., ~ ~ ~ 5 ~ ~ i 0 ~ ~ '-' 15 ~ ..... (c) (d) .....-I> ~ ~ 10 -.....0 "'0 ~ ~ 5
0 10 20 30 40 10 20 30 40
Fraction number 161
Fig. 31. Plasma disappearance curves for monomeric and complexed 12 ~I-C9
Plasma radioactivity disappearance curves for a rabbit given 125 I-C9 followed by CVF at 10 min. The 125J-C9 monomer and 125J-SC5b-9 curves were derived from the area under the gel filtration profile shown in Fig. 30, page 160, and the total protein-bound radioactivity.
-o- Monomeric 125I-C9 e 125 I-SC5b-9-containing fraction
A Total protein-bound radioactivity
,-... ~ 105 ~ ~ C.,) '-" ...... t> ...... ;;... ~ C.,) ~ ...... Q 104 '"t:l ~ ~
0 10 20 30 40 50 60
Time after injection (h) Section 3.6 C9 metabolism in rabbits during complement activation 162
RELEASE OF FREE 1251
Free 1251 remained low during the first 3h following CVF administration but continued to rise thereafter for approximately 24h (see Fig. 32, page 163). 1251 represented almost 40 % of the total plasma radioactivity in some animals.
TISSUE DISTRIBUTION OF 1251-C9 AND 131 1-RSA FOLLOWING CVF
Two rabbits were administered CVF at 10 min following injection of 1251-C9 and 1311-RSA and sacrificed at 6 h. These animals showed accumulation of protein-bound 1251-C9 in the spleen (up to 1.6 x the plasma concentration) and the liver (up to 0.7 x the plasma level), with minimal accumulation in the other organs. Administration of CVF had no appreciable effect on the catabolism or distribution of 1311-RSA (see Fig. 33, page 164).
UPTAKE OF 1251-C9 BY CIRCULATING BLOOD CELLS
Following injection of CVF there was a small but consistent rise in the radioactivity associated with washed, packed blood cells. This was maximal at 8 h and approached 10 %of the simultaneous plasma protein-bound radioactivity (see Fig. 34, page 165). 98 % of cell-associated radioactivity was removed by precipitation in 24 % TCA and filtration through nylon wool. 163
Fig. 32. Plasma disappearance curves for free and protein-bound radioactivity in a rabbit given 12 5I-C9 followed by CVF
CVF was given 10 min after administration of1251-C9.
• Free 1251
--o-- Monomeric 1251-C9
-o- 1251-SCSb-9-containing high MW fraction
,-.._ ~ ~ 105 ~ .._.,CJ t> .....;;...... CJ ~ .....Q "'CC ~ 104 ~
0 10 20 30 40 50 60
Time after injection (h) Fig. 33. Organ distribution of 12 5 I-C9 and 131 1-RSA during complement activation
Tissue protein-bound125 I (a) and 131 I (b), expressed as (cpm per g fresh tissue) I (cpm per mL fresh plasma), in two rabbits given 125I-C9 and 131 I-RSA and killed at 6 h. One rabbit received CVF at 10 min (•) while the other an equal volume of normal saline (t"i').
2 (a) 1.5 ...... » ...... > CJ ~ 1 .....Q '"C ~ 1-< 0.5 '"C
Q= 0 ,.Q= I ..... 2 ....~= Q (b) 1-< ~ 1.5 ~ ...... > ~ 1 -~ ~ 0.5
0 Plasma Liver Spleen Kidney Lung Heart Muscle 165
Fig. 34. Uptake of 12 5 I-C9 by circulating blood cells following CVF
Protein-bound radioactivity associated with packed blood cells and plasma in two rabbits given 125I-C9 followed, at 10 min, either by CVF (solid symbols) or isotonic saline (open symbols).
A Plasma (rabbit given CVF)
• Cells (rabbit given CVF) -t:r-- Plasma (rabbit given saline) --o-- Cells (rabbit given saline)
105
,.-., ~ 104 ~s ~ ~ '-" ...... 103 ...... ~ ~ .....0 "t:: 102 ~ ~
101
1 0 10 20 30 40 50 60
Time after injection (h) 166
3.7 THE METABOLISM OF SC5b-9 IN NORMAL RABBITS
1251-SCSb-9 PLASMA DISAPPEARANCE CURVES
Three rabbits received approximately 0.5 j.tCi of 125I-SC5b-9, two of which were given a similar concurrent dose of 131I-C9, which had been pre-incubated at 37°C for 3 h as a control. In each case 125I-SC5b-9 was cleared rapidly from the plasma, declining by 50% in 0.68 (0.55- 0.77) h, and with less than 15% of the injected dose remaining at 4 h (see Fig. 35, page 167). The rate of disappearance of 131I-C9 over the course of this study was similar to non-incubated 125! C9 (see Section 3.5, page 150). Free 125I was rapidly released and it exceeded 75 % of the total 1251-associated radioactivity in the plasma within 4 h of injection. This was in contrast to free 131I which accounted for less than 6 % of the plasma radioactivity due to this isotope and was liberated steadily throughout the study period.
PLASMA VOLUME
Calculation of the plasma volume based on the initial dilution of 125I-SC5b-9
gave a value of 53.0 (48.3 - 68.0) rnL/kg (n = 3). This was higher than that derived from the dilution of similarly prepared, concurrently administered 131I-C9 (i.e. 43.0 &
45.3 rnL/kg, [n = 2]).
EXCRETED RADIOACTIVITY
Due to the brevity of these studies, urinary radioactivity was not routinely measured. However, one animal had excreted 69.8 %of the injected dose by 20.5 h following administration of 1251-SC5b-9. 167
Fig. 35. The plasma disappearance curves for 131I-C9 and 12 5J-SC5b-9 administered concurrently to two rabbits
• rabbit 1 : 131 I-C9 • rabbit 1 : 125 I -SCSb-9 -o- rabbit 2: 131 I-C9
---o-- rabbit 2: 125 I-SCSb-9
100 ,.-..., Q,) l:ll 0 "'t:: "'t:: Q,) ....C.l Q,) ·s..... ~ '-" ...... e- ...... C.l 10 ~ .....0 "'t:: ~ ~ "'t::= 0 .Q= I .....= ....Q,) 0 ~ ~ 1 0 1 2 3 4 5
Time (h) Section 3.7 SC5b-9 metabolism in rabbits 168
UPTAKE OF 1251-SCSb-9 BY TISSUES
Protein-bound and free radioactivity were measured in the liver, spleen, kidney, lung, cardiac and skeletal muscle in two animals given 1251-SCSb-9 and 1311-C9, and sacrificed at 4 h. Marked uptake of protein-bound 1251 was observed in the spleen and liver up to 15 and 9 times the plasma concentration respectively (see Fig. 36, page 169). The other organs had concentrations which were similar to, or only slightly greater than the plasma. By comparison, the concentration of protein-bound 1311 was less than 50% of the plasma level in all organs tested and was similar in distribution to non-incubated 1251-C9 (see Fig. 27, page 155). The kidneys were again noted to accumulate large quantities of free, but not protein-bound radio label.
RADIOACTIVITY ASSOCIATED WITH CIRCULATING BLOOD CELLS
Radioactivity associated with multiply washed, packed blood cells remained insignificant, i.e.< 0.5% of total plasma counts. Fig. 36. Organ uptake of 12 5 I-SC5b-9
Tissue protein-bound 125 I (•) and 131 I c;~:) expressed as (cpm per g fresh tissue) I ( cpm per rnL plasma) in a rabbit given both 125 I -SC5b-9 and 131 I -C9 and killed at 4 h.
20 ...... ;;...... ~ .....=0 15 "0= "0""
0= ,.Q= 10 ....I .....=~ 0 ""~ ~ 5 ;;...... -=~ ~ 0
Plasma Liver Spleen Kidney Lung Heart Muscle 170
3.8 THE METABOLISM OF VITRONECTIN AND 32P-VITRONECTIN IN NORMAL RABBITS
PLASMA DISAPPEARANCE CURVES
Following injection of approximately 3 mL of trace-phosphorylated human plasma (containing< 1% 32P-vitronectin), antigenically-detected human vitronectin was eliminated very rapidly from the plasma with only 25 - 35 % of the dose remaining at 5h. The pattern of disappearance of 32P-vitronectin from the plasma of these animals was similar, although less rapid in the first 10 h. (see Fig. 37, page 171). Free 32P represented approximately 10 % of the total plasma radioactivity and was released at a steady rate throughout the study period.
METABOLIC PARAMETERS
The T112 of antigenically-detected vitronectin was 8.0 ± 1.3 h, the FCR was 32 18.77 ± 1.57 %/h, and the EVIIV was 1.00 (0.48- 1.60). The T112 of P-vitronectin, i.e. 8.9 ± 0.5 h, was similar to that of antigenically-detected molecules. However, the FCR of 32P-vitronectin, i.e. 10.85 ± 0.71 %/h, was significantly lower than that of the whole vitronectin pool (p < 0.005). Phosphorylated vitronectin also had a significantly smaller EVIIV distribution ratio, i.e. 0.28 [0.15- 0.36], compared with the ELISA-detected form (p < 0.05). These results are summarised in Fig. 38, page 172.
FPLC GEL FILTRATION OF PLASMA
FPLC gel filtration of plasma samples taken during a normal study revealed only one major peak of radioactivity, consistent with the molecular weight of monomeric 171
Fig. 37. Plasma vitronectin disappearance curves in normal rabbits
The range of disappearance curves for antigenically detectable vitronectin and 32P-vitronectin from the plasma of four rabbits. The ranges represent the mean± SD .
32p . . • -v1tronectm
!WI! Antigenically detectable vitronectin
100
e~ fll ~ -~ ..=Q.l .....-= bJ) ...... = =~ 10
Q.le
'"'Q.l fll 0 '"t:l ~ 0 ~
1
0 10 20 30 Time following injection (h) 172
Fig. 38. Metabolic parameters of vitronectin in laboratory animals
The Tl/2 (a), FCR (b) and EV/N (c) for antigenically-detected and 32 P-vitronectin in four normal rabbits. The figures represent the mean+ SD except for EV/N, where the median and range are shown.
10 (a) Not significant
7.5 ..=..-.. '-' <'I 5 1""( ~- 2.5
0
25 (b) p < 0.005 20 ~ ~ 15 '-' u~ 10 ~ 5
• • • • •• I' •• ~ • 0
2 (c) p < 0.05
1.5 > ~ 1 ~ 0.5
0
32P-vitronectin Antigenically -detected vitronectin Section 3.8 Vitronectin metabolism in normal rabbits 173
vitronectin (see Fig. 39, page 174). Two smaller peaks were also evident with MW approximately 1000 kD and 200-300 kD.
PLASMA VOLUME
The plasma volume calculated from the initial dilution of 32P-vitronectin was
37.5 ± 1.8 mL/kg (n = 6) which compared closely with previous estimates of plasma volume in rabbits of similar size using the dilution of 1251 C9, i.e. 35.0 ± 3.0 mL/kg.
URINARY RADIOACTIVITY
Urinary Cerenkov radioactivity could not be accurately counted in these studies due to interference by precipitates in the urine. Clarification by acidification and dilution of the urine yielded higher, but inconsistent results. 174
Fig. 39. FPLC Superose gel filtration of plasma from a normal rabbit following injection of 3 2 P-vitronectin
Plasma from a rabbit drawn at approximately 2.5 h (a) and 6 h (b) following injection of 32P-vitronectin. The arrows show MW markers in kD.
Blue dextran 660 66 12.3 t t 0.4 (a)
0.3
...... ;;.... I> 0.2 ...... C) ~ ....0 't:l ~ ~ 0.1 't:l= 0 ,.Q= ....=I 0 ...... Q,l 0 ~ 0.4 ~ <:i-1 0
=0 ...... 0.3 C) ~ ~ ~ 0.2
0.1
0
10 15 20 25 30 35
Column fractions 175
3.9 VITRONECTIN METABOLISM IN RABBITS DURING COMPLEMENT ACTIVATION
PLASMA DISAPPEARANCE CURVES
The effect of CVF on the plasma disappearance rate of antigenically-detected vitronectin was variable and overall, the plasma curves were superimposed on those of normal rabbits (see Fig. 40, page 176). By contrast, the elimination of 32P-vitronectin from the plasma was more rapid in all animals given CVF, compared with the control group (see Fig. 41, page 177). Free 32Phosphate was also released more rapidly than in the control studies and represented up to 15 % of the total plasma counts.
FPLC GEL FILTRATION OF PLASMA SAMPLES
FPLC Superose 12 gel filtration chromatography of plasma samples taken from animals given 32P-vitronectin, and then CVF at 10 min, revealed accumulation of radioactivity in the high MW region (i.e. approximately 1000 kD) which was in excess of the small, similarly placed peale seen in control studies. This was evident from 2.5 h following injection and was accompanied by a relatively lower proportion ofvitronectin monomer (see Fig. 42, page 178). 176
Fig. 40. The disappearance of antigenically detectable vitronectin from the plasma of rabbits during complement activation
Four rabbits were given CVF at 10 min after injection of trace-phosphorylated vitronectin, and four normal animals served as controls. The ranges represent the mean± SD.
• Rabbits given CVF
Control animals
100
~ er:n ~ -~ cu ..=...... = ....t=l) ....= 10 ~= ecu ~ cu r:n 0 "C ~ 0 ~
1
0 10 20 30
Time following injection (h) 177
Fig. 41. The disappearance of 3 2P-vitronectin from the plasma of rabbits during complement activation
Four rabbits were given CVF at 10 min following injection and four normal animals served as controls. The ranges represent the mean± SD.
• Rabbits given CVF
~-,. ,...~ ~ ·i Control animals
100
c:l
arll c:l -~ Q,) ..cl...... s =~ ·s..... 10 ac:l Q,) 1-1 Q,) rll 0 "1:1 ~ 0 ~
CVF 1 0 10 20 30
Time following injection (h) 178
Fig. 42. FPLC gel filtration of plasma 3 2P-vitronectin following administration of CVF
Plasma from two rabbits drawn at approximately 2.5 h (a) and 6 h (b) following injection of 32P-vitronectin. At 10 min, one rabbit was given 0.25 mg of CVF (•), while the other was given normal saline (D). The arrows show MW markers in kD.
Blue dextran 660 66 12.3
0.4 • •(a)
0.3
;;...... 0.2 .....-i> ~ -~ .....0 '"t:l ~ 0.1 '"t:l"'"
=0 ..c I 0 ..... =Q,l -0 0.4 "'"Q.c ~ 0 .....=0 0.3 ~ -~ ~"'" 0.2
0.1
0
10 15 20 25 30 35
Column fractions 179
Studies in human subjects 180
3.10 THE METABOLISM OF C91N NORMAL HUMAN SUBJECTS
PLASMA 1251-C9 DISAPPEARANCE CURVES
Figure 43, page 181, shows the pattern of disappearance of protein-bound 125I C9 from the plasma of eight healthy subjects. In each case, a relatively rapid early decline was followed by a slower, persistent log-linear phase. Figure 44, page 182, shows the disappearance curves for both plasma 125I -C9 and the residual whole body radioactivity in a typical control subject. In general, the curve of elimination of whole body radioactivity ran parallel to that of protein-bound plasma counts following equilibration. However, in subjects with large plasma volumes (i.e. > 2500 mL) the whole body radioactivity tended to decline at a slower rate than the plasma. Free iodide was released at a steady rate throughout the study period and represented < 7 % of the total counts in each plasma sample.
METABOLIC PARAMETERS
The metabolic data for eight normal subjects are shown in Table 18, page 183.
Calculated by Mathews' analysis of the plasma curves, the T112 of C9 was 42.5 ± 6.7 h and the FCR was 2.92 ± 0.36 %/h. Figure 45, page 184, shows the FCR calculated daily by the metabolic clearance method for the normal group. This remained stable throughout the study period, with the exception of day 1 during which it was relatively low. The average metabolic clearance, excluding day 1, was 2.88 ± 0.49 %/h for normal subjects. The EVIIV was 0.56 ± 0.12, and plasma C9 was produced at a rate of 0.07 ± 0.03 mg/kg per h. 181
Fig. 43. The range of plasma 12 5 I -C9 disappearance curves in healthy human subjects
The shaded area represents the range of plasma disappearance curves for eight normal subjects.
100
,.-.._ ~ 1:1) Q "C .....~ -...... = ~ '-' .....;;...... :;...... ~ c:l .....Q "C c:l ~ "C= =Q ,.Q I .....= .....~ Q ~ ~ 10 sc:l 1:1) c:l -~
5
0 20 40 60 80
Time following injection (h) 182
Fig. 44. The disappearance of whole body and plasma protein-bound radioactivity in a typical normal 12 51-C9 human turnover study
The dotted line is an extrapolation of the final phase of the plasma disappearance curve used to determine the T112 and the ordinate intercept.
• Whole body radioactivity D Plasma protein-bound radioactivity
100
~ (/l 0 ...... "C ...... "C .... ~ ...... ~ ...... ~ ...... ';:;' ~ 0 ~ '-" .....n ...... ~ ~ .....0 "C ~ ~ 10
5
0 25 50 75 100
Time following injection (h) Table 18. C9 metabolism in normal human subjects
FCR Subject Plasma Plasma curve Metabolic EVIIV PPR volume analysis clearance
(mL/kg) (h) (%/h) (mg!kg per h)
1. 37.7 30.0 3.66 3.45 0.37 0.10 2. 47.5 38.7 2.66 2.14 0.45 0.10 3. 36.5 41.2 2.93 3.43 0.53 0.05 4. 28.8 49.8 2.83 3.30 0.69 0.04 5. 43.2 48.5 2.77 2.53 0.71 0.05 6. 36.9 40.7 3.21 2.72 0.59 0.07 7. 35.4 49.8 2.57 3.01 0.61 0.05 8. 45.9 41.7 2.72 2.44 0.53 0.10
Mean±SD 39.0±6.2 42.5 ± 6.7 2.92 ± 0.36 2.88 ± 0.49 0.56 ± 0.12 0.07 ± 0.03 184
Fig. 45. The daily FCR of C9 in normal human subjects
The mean FCR (+ SD) of C9 calculated by the metabolic clearance method for each 24 h period of urine collection.
5
4
1
0
1 2 3 4
Days Section 3.10 C9 metabolism in normal humans 185
C9 LEVELS AND VOLUME OF DISTRIBUTION
The serum C9 concentration was 61 ± 14 mg/L. This was measured on three occasions during the experimental period in each subject and varied by less than 8 % about the mean. The whole body C9 content was 3. 7 ± 1.1 mg/kg and this was distributed throughout a volume of60.6 ± 9.3 mL/kg (see Table 19, page 186).
PLASMA SC5b-9 LEVELS
The median plasma SC5b-9 level was 313 (range [229- 402]) J..Lg/L, (see Table 19, page 186.
PLASMA VOLUME
The plasma volume calculated for normal subjects, was 39.0 ± 6.2 mL/kg (see Table 18, page 183.
RADIOACTIVITY ASSOCIATED WITH CIRCULATING CELLS
Radioactivity associated with packed, washed blood cells was negligible (i.e. < 1% of the plasma counts).
URINARY RADIOACTIVITY
Control subjects excreted 63.8 ± 6.7 % of the injected dose by the end of the fourth day. TCA-precipitable radioactivity in the urine was 4.0 ± 2.0% of total counts. Table 19. C9 and SC5b-9 levels in normal human subjects
Subject Serum C9 Plasma SC5b-9 Whole body C9 C9volumeof content distribution
(mg/L) (pg/L) (mg/kg) (mL/kg)
1. 75 402 3.9 51.6 2. 76 355 5.2 68.9 3. 47 307 2.6 55.8 4. 47 309 2.3 48.7 5. 42 367 3.1 73.9 6. 61 318 3.6 58.7 7. 59 307 3.4 57.0 8. 77 229 5.4 70.2
Mean±SD or 61±14 313 (229- 402) 3.7 ± 1.1 60.6 ±9.3 median (range) 187
3.11 C9 METABOLISM IN PATIENTS WITH AUTOIMMUNE DISEASE
PLASMA 1251-C9 DISAPPEARANCE CURVES
The pattern of disappearance of 125I -C9 from patients' plasma was similar to that of control subjects. In particular, the terminal phase was log-linear with respect to time in all cases. However, patients with very active immunological disease eliminated C9 from the plasma more rapidly than the normal group. Figure 46, page 188, shows the plasma disappearance curves for two such patients : patient 6 with diffuse proliferative lupus nephritis and patient 9 who had SLE complicated by pulmonary haemorrhage and epileptic seizures. It should be emphasised that patient 9 was haemodynamically stable and had not been actively bleeding in the week prior to the study. Free Iodide was released at a steady rate and remained < 10 % of the plasma radioactivity in most cases, although there was a slightly higher proportion in patients with hypercatabolism of C9, i.e. up to 11 % in patients 6 and 9, and 20 % in patients with renal impairment (i.e patients 1, 2 and 7).
METABOLIC PARAMETERS
With the exception of day 1, the daily metabolic clearance of C9 in patients with normal serum creatinine levels and normal creatinine clearance (n = 6) exceeded that in the control group although this did not reach statistical significance (see Fig. 47, page 189). However, the average FCR ofC9, calculated by the metabolic clearance method, (3.58 ± 0.57 %/h) was significantly greater than that of healthy subjects (2.88 ± 0.49, p
< 0.05). Calculated by plasma curve analysis (n = 9), the FCR was 3.38 ± 0.70 %/h, the
T112 37.6 ± 10.2 h, and the EV/IV was 0.55 ± 0.19. These were not significantly different from the control group (see Table 20, page 190). 188
Fig. 46. The metabolism of 12 5 I-C9 in human subjects with pathological complement activation.
125I-C9 plasma disappearance curves for two patients with SLE and C9 hypercatabolism: patient 6 C•) with diffuse proliferative lupus nephritis and patient 9 C•) with recent pulmonary haemorrhage and grand-mal seizures. Both had reduced serum CHso and C4 concentration, while patient 6 also had low serum C3 and elevated circulating immune complexes. The normal range is shaded.
100
,-..,. Cl) C'll 0 "'0 "'0 .....Cl) C.l Cl) ...."S' ~ '-' ...... 1>...... i> C.l ~ ....0 "'0 ~ 1-o "'d
=0 ,.Q= ....I .....Cl)= 0 1-o 10 ~ s~ C'll ~ -~ 5 0 20 40 60 80
Time following injection (h) 189
Fig. 47. The daily FCR of C9 in patients
The day to day FCR of C9 calculated by the metabolic clearance method in six patients with autoimmune disease and normal serum creatinine levels. The results for eight control subjects are also included. The figure shows the mean and SD for each 24 h period of urine collection.
• Patients
mJ Control subjects
5
4
3 u~ :\ 2 u
1
0 1 2 3 4
Day Table 20. C9 metabolism in patients
FCR Patient Diagnosis Plasma Tvz Plasma curve Metabolic EVIIV PPR volume analysis clearance
(mL/kg) (h) (%/h) (mg/kg per h)
1. SLE 38.7 34.7 2.76 * 0.19 0.08 2. IgAN 25.0 36.9 3.51 * 0.68 0.06 3. SLE 42.7 42.7 2.80 3.78 0.54 0.07 4. MEC 54.1 38.9 3.45 3.18 0.67 0.16 5. SLE 33.4 31.7 3.66 2.91 0.60 0.10 6. SLE 50.1 25.8 4.57 4.40 0.49 0.18 7. SLE 43.7 48.3 2.64 * 0.55 0.11 8. SLE 42.1 55.5 2.76 3.19 0.86 0.07 9. SLE 56.7 24.1 4.24 3.99 0.37 0.20
Mean±SD 42.9 ± 10.0 37.6 ± 10.2 3.38 ± 0.70 3.58± 0.57 0.55 ± 0.19 0.11 ± 0.05
* not calculated due to renal impairment. Section 3.11 C9 metabolism in patients 191
Patients with low CH50
Patients with CH50 < 68 % of NHS (i.e. below the lower limit of the laboratory normal range, n = 7) had a C9-FCR that was significantly greater than the control group when calculated by both plasma curve analysis (3.57 ± 0.67 vs 2.92 ± 0.36 %/h, p <
0.05), and the metabolic clearance method (3.65 ± 0.60 [n = 5] vs 2.88 ± 0.49 %/h, p < 0.05), (see Fig. 48, page 192). This was associated with a significantly shorter plasma
C9-T112 compared with control subjects (33.5 ± 6.8 vs 42.5 ± 6.7 h, p < 0.05). As for the whole patient cohort, the EVIIV ratio ofthe low CH50 sub-group (0.51 ± 0.18) was not significantly different from that of the normal subjects.
PATIENTS WITH SLE
Patients with SLE and normal serum creatinine and creatinine clearance (n = 5) had significantly greater C9-FCR, calculated by the metabolic clearance method, than the control group (3.65 ± 0.60 vs 2.88 ± 0.49 %/h, p < 0.05). From plasma curve analysis, the FCR of C9 in all SLE patients (n = 7) was 3.35 ± 0.80 %/h, the T112 was 37.5 ± 11.7 h, and the EVIIV was 0.51 ± 0.21. These values were not significantly different from those of control subjects. However, SLE patients with low CH50 (n = 5) had a significantly shorter C9-T112 than that of the control group (31.8 ± 7.5 vs 42.5 ± 6.7 h, p < 0.05).
C9 LEVELS AND THE C9 VOLUME OF DISTRIBUTION
The plasma C9 production rate in patients (0.11 ± 0.05 mg/kg per h) was significantly higher than in the control group (0.07 ± 0.03 mg/kg per h, p < 0.05) and was accompanied by a significantly higher serum C9 concentration (7 6 ± 13 vs 61 ± 14 mg/L, p < 0.05). The volume of distribution of C9 (66.2 ± 16.2 mL/kg) and the whole body C9 content (5.1 ± 1.6 mg/kg) were similarly greater than those of the 192
Fig. 48. Metabolic parameters of C9 in normal subjects and patients with autoimmune disease and low CH5 0
The T112 (a), FCR (b) and EVIIV (c) of C9 in seven patients with CH50 < 68% and eight control subjects. The figures show the mean and SD.
50 (a) p < 0.05 40 ,-.. ...c= '-' ('l 30 1-l ~- I Q\ 20 u 10
0
5 (b) p < 0.05 ,-.. g_g"'t:: 4 ~~ '-'e 3 Ufll~- ~~ 2 Q\..c:l·~ u-~ ~ 1 '-' 0 Section 3.11 C9 metabolism in patients 193 control group (60.6 ± 9.3 mL/kg & 3.7 ± 1.1 mg/kg respectively), however, the differences fell just short of statistical significance (i.e. 0.05 < p < 0.06), (see Table 21, page 194).
PLASMA SC5b·9
The plasma SC5b-9 level was 515 (300 - 1879) and was significantly higher in patients (p < 0.01) than in normal subjects (313 [229- 402]), (see Table 21, page 194).
RADIOACTIVITY ASSOCIATED WITH CIRCULATING CELLS
Blood cell-associated radioactivity was negligible, amounting to less than 0.5 % of the plasma counts.
PLASMA VOLUME
The mean plasma volume calculated for patients was 42.9 ± 10.0 mL/kg, which was not significantly different from the control group (39.0 ± 6.2 mL/kg), (see Table 20, page 190).
URINARY RADIOACTIVITY
Patients excreted 57.5 ± 7.4 % (n = 9) of the injected dose by the end of the fourth day of urine collection. This figure was 60.1 ± 7.4% for the six patients with normal creatinine clearance, compared with 63.8 ± 6.7% for the control group. TeA precipitable radioactivity in patients' urine was 6.0. ± 2.0% of the total urinary counts which did not significantly differ from that of the control group, i.e. 4.0 ± 2.0 %. Table 21. C9 and SC5b-9 levels in patients
Subject Diagnosis Serum C9 Plasma SC5b-9 Whole body C9 C9volumeof content distribution
(mg/L) (pg/L) (mglkg) (mL/kg)
1. SLE 76 526 3.5 46.0 2. IgAN 71 387 3.0 42.0 3. SLE 56 300 3.7 65.8 4. MEC 85 515 7.7 90.3 5. SLE 84 482 4.5 53.4 6. SLE 77 1879 5.7 74.6 7. SLE 98 1058 6.6 67.7 8. SLE 57 384 4.5 78.3 9. SLE 82 704 6.4 77.7
Mean±SD or 76±13 515 (300 - 1879) 5.1 ± 1.6 66.2 ± 16.2 median (range) 195
3.12 CORRELATIONS BETWEEN C9 METABOLIC DATA AND OTHER IMMUNOLOGICAL MEASUREMENTS
FCRAND T112
The FCR ofC9 calculated by Mathews' plasma curve analysis, was significantly correlated with the metabolic clearance (r = 0.68, p < 0.01, n = 14) as shown in Fig. 49, page 196. However, the latter method tended to yield a lower result than plasma curve analysis in subjects with large plasma volumes. Specifically, a significant positive correlation (r = 0.66, p < 0.05) was found between the plasma volume and the difference between the FCR calculated by Mathews' method and that by the metabolic clearance technique (see Fig. 50, page 197).
The C9-FCR calculated for all subjects by Mathews' method, showed a highly significant negative correlation (r = -0.83, p < 0.0001) with the C9 plasma T112 (see Fig. 51, page 198).
THE SERUM C9 CONCENTRATION
The plasma C9 production rate showed a significant positive correlation with the serum C9 concentration (r = 0.75, p < 0.005) for the whole study group (see Fig. 52(a), page 199). By contrast, the FCR bore no significant relationship with the serum C9 level (not shown). However, the C9 plasma production rate was positively correlated with the C9-FCR (r = 0.74, p < 0.001), (see Fig. 53, page 200). 196
Fig. 49. The FCR of C9 calculated by Mathews' analysis, compared with the metabolic clearance method
Statistics are for the combined group of control subjects and patients with normal serum creatinine and creatinine clearance (n = 14).
• Patients r = 0.68 D Control subjects p < 0.01
5
,-..., ~ ~ • '--' "C 0 ~ 4 sQ,) • rJ:J !::: • Q,) ..=..... ~ D D ~ D ~ ,.Q • • u~ 3 D ~ I =-- • u D
D D
D 2 2 3 4 5
C9-FCR by the metabolic clearance method (%/h) 197
Fig. 50. Plasma volume vs the difference between the C9-FCR calculated by Mathews' and the metabolic clearance methods
Statistics are for the combined group of control subjects and patients with normal serum creatinine and creatinine clearance (n = 14).
r = 0.66
p < 0.05
1.5
,.-.. Cl) <:.1
~ 1-<= 1 ~~ u~ ~<:.I 0\= • uc,.Q .....~ 0.5 Cl) • • ~ '-" • •• • • l:ll • = 0 •...Cs=
,.-.. "t:: ..=Q -0.5 ...... • • uS~CI)
~-ll:ll 0\!:=: UCI) ..=..... -1 ~ • ~ '-"
-1.5 1000 2000 3000 4000 5000
Plasma volume (mL) 198
Fig. 51. The relationship between the C9-FCR and its plasma T 112
The statistics are for the combined study group (n = 17).
• Patients r = -0.83 D Control subjects p < 0.0001
5
4.5 • • 4 -"C ~] ~~ ---s oe ~. UCIJ 3.5 ~~ •• o 2 20 30 40 50 60 C9-T 112 (h) 199 Fig. 52. The relationship between the C9 plasma production rate, serum C9 concentration (a) and the whole body C9 content (b) Statistics are for the combined group of patients and control subjects (n = 17). • Patients D Control subjects (a) 100 ~ • ~ 90 '-"s =0 • ..... 80 • • ~ g -'"" • • ~ =Col 70 • =0 Col =--. D u 60 D s I = 50 ~ DO 00.'"" r = 0.75 p < 0.005 40 D (b) 8 • ~ bll 7 ~ • • '-"s 6 =--. • u B ...... 5 "C 0 ,.Q • • ~ 4 D 0 -..= D ~. ~ 3 De D r = 0.85 D p < 0.0001 2 0 0.05 0.1 0.15 0.2 0.25 C9 Plasma production rate (mg/kg per h) 200 Fig. 53. C9 plasma production rate vs C9-FCR The statistics are for the combined group of patients and control subjects (n = 17). • Patients r = 0.74 D Control subjects p < 0.001 0.25 ,.-,. ..d ~ 0.2 aJ • ~ l:lJ) • s~ • .....aJ 0.15 Cd ~ Q ...... = tJ "C= 0.1 •D:I D Q ~ ~ Cd s • D tf.l • Cd -~ 0.05 D D D • c=-, u D 0 2 2.5 3 3.5 4 4.5 5 C9-FCR (%/h) (Mathews' method) Section 3.12 Correlations between metabolic data 201 THE WHOLE BODY C9 CONTENT AND VOLUME OF DISTRIBUTION A highly significant correlation was found between the plasma C9 production rate and the whole body C9 content for all subjects (r = 0.85, p < 0001), (see Fig. 52(b), page 199). Similarly, the distribution volume of C9 showed a significant positive correlation with the plasma production rate (r = 0.59, p < 0.05), (not shown). SERUM C3, C4 AND CIC LEVELS The FCR of C9 was poorly correlated with the serum C3 and C4 concentrations but showed a significant positive relationship with the level of circulating immune complexes (r = 0.5, p < 0.05), (see Fig. 54, page 202). THE SERUM CH50 In the patient group (n = 9), the plasma C9-T112 showed a positive correlation with the CH50 (r = 0.77, p < 0.05), (see Fig. 55(a), page 203). While the C9-FCR calculated by Mathews' method, appeared negatively related to the CH50 (r = - 0.64), this did not quite attain statistical significance. (see Fig. 55 (b), page 203). THE PLASMA SCSb-9 CONCENTRATION The plasma concentration of SC5b-9 was positively correlated with the C9-FCR (r = 0.61, p < 0.01). The plasma SC5b-9 level and the C9 catabolic rate, expressed as 1 mg of C9 consumed per L of plasma per h , were similarly correlated (r = 0.64, p < 0.01) as shown in Fig. 56, page 204. The serum C9 concentration and the plasma SC5b- 9 level were also correlated (r = 0.45) but this was not of statistical significance. 1 Calculated from the product of the C9-FCR and the serum C9 concentration. 202 Fig. 54. C9-FCR vs the level of circulating immune complexes The statistics are for the combined patient and control groups (n = 17). • Patients r = 0.50 D Control subjects p < 0.05 5 • • -.. "t::: 4 -.,Q ~~ ~Q,j ._.e D • ~ ~Cll u~ • ~Q,j • ·..= =" .j.l UC'J D 6 3 D D D D e D • • D • 2 0 25 50 75 100 CIC (%) 203 Fig. 55. The relationship between plasma T 112 (a) and C9-FCR (b) and the CH5 0 The figures and statistics are for patients only (n = 9). (a) r = 0.77, p < 0.05 60 • 50 ...-. • 0 ("! ...... 1-1 40 • ~ I • u=" • • 30 • • • 20 (b) r = -0.64, Not significant 5 4.5 • ...-. "'C • ~~ 4 ~'S '-'El Ul"l.l~- • ~~ 3.5 •a.l • ="..C: • u-ed ~ '-' 3 • • • 2.5 • 0 50 100 150 CH50 (% NHS) 204 Fig. 56. Plasma SCSb-9 concentration vs C9 catabolic rate Statistics are for the combined patient and control groups (n = 17). • Patients r = 0.64 D Control subjects p < 0.01 2000 • ,-.., ~ bJ) ::i -'-" 1500 ...... =0 ~ .....~ Q,) (,j= 0 1000 • (,j= ="I ,.Q l() u 00. • e~ til 500 ~ • •• D -~ D IDC)• cP • D 0 0 1 2 3 4 C9 catabolic rate (mg/L per h) 205 Section 4 DISCUSSION 206 4.1 The behaviour of C9 and SC5b-9 in vitro This thesis describes studies of the metabolism of C9, vitronectin and SC5b-9 in experimental animals, and C9 in normal human subjects and patients with autoimmune disease. As a preliminary, these proteins and complexes were first examined in vitro to establish their molecular integrity and function. In particular, C9 was studied with regard to its haemolytic activity and recruitment by the terminal complement pathway and SC5b-9 was investigated for its antigenic characteristics. C9 was purified from human plasma with minimal contamination by extraneous proteins and in high yield (i.e. 5.7 %) consistent with previously published results [Biesecker & Muller-Eberhard, 1980]. The critical step in this process was fractionation by DEAE-A50-Sephadex chromatography. C9 has a high affinity for this matrix and was eluted after the major protein peak. However, it was important to collect only those C9-containing fractions which were free of the plasma protein ceruloplasmin, as it was not effectively separated from C9 by subsequent hydroxylapatite chromatography. This was achieved by testing eluted fractions for both C9 and ceruloplasmin by radial immunodiffusion. Several techniques were used to iodinate C9, both in preliminary studies and in experiments reported in this thesis. The chloramine-T method [McConahey & Dixon, 1966] proved unsatisfactory, resulting in a significant loss of specific haemolytic activity and behaviour in vivo that was indicative of denaturation. These results were not included in this thesis and the method was abandoned. Proteins used for studies in experimental animals and in vitro, were iodinated by the lactoperoxidase technique. By contrast, this yielded a fully functional, intact molecule and achieved a high incorporation of radioactive label, i.e. 30- 50%. A disadvantage ofthis method was the unavoidable contamination of the sample with bovine lactoperoxidase. However, only a small amount of enzyme was required and represented no greater than 2 % of the total protein. C9 prepared for human metabolic studies was iodinated using Section 4.1 Discussion 207 "Iodobeads" (Pierce), which are non-porous, polystyrene spheres coated with N-chloro benzenesulfonamide (i.e. Chloramine-B). This method offered significant advantages in that it required minimal manipulation of the protein and preserved its physical integrity and specific haemolytic activity. The iodination reaction was halted simply by removal of the bead from the reactants. Furthermore, neither the addition of a reducing agent nor metabolic inhibitor was required, in contrast to iodination by soluble chloramine-T and lactoperoxidase. In the current study, stable iodide was also introduced to quench any oxidant which may have become dislodged from the bead. Finally, a small quantity of HSA was added to stabilise the radiolabelled protein and to protect it from auto irradiation. To date, solid-phase iodination techniques have not been used in reported studies of protein metabolism in human subjects although they have several favourable characteristics and are well suited to this task. FPLC gel filtration chromatography of 125I-C9 on Superose 12 demonstrated that greater than 90 % of radioactivity was present as a single peak, consistent with the C9 monomer. However, the elution profile of 125I-C9 and that of other iodinated proteins, was slightly delayed and tended to underestimate the MW indicated by gel electrophoresis. This may have been due to interactions between the iodide group and the column matrix as it was minimised by the introduction of stable iodide to the buffer. Radioactive contamination of the column was also reduced in this manner. In addition to the major peak, purified 125I-C9 also contained a small amount of radioactivity in the high MW region (i.e. 1000 kD) and a smaller peak, or shoulder, at 200 to 300 kD. In the absence of visible contamination of either Coomassie blue-stained gels or autoradiographs, these high MW peaks probably represented trace quantities of multimeric C9 in an otherwise pure, monomeric preparation. This was further supported by a radioimmunoassay in which the 1000 kD species was significantly retained by tubes coated with antibodies against C9 neoantigens. The specific haemolytic activity of C9 was tested using EAc1_8 as target cells in a C9-dependent assay. The dose-response curves of purified C9, 125I-C9 and NHS were all linear at low C9 concentrations. Furthermore, the curves were superimposed during this phase indicating that the specific haemolytic activity of purified and radiolabelled Section 4.1 Discussion 208 C9 was fully preserved. This assay was also very sensitive and haemolysis was detected at C9 concentrations of less than 1 in 10,000 that of normal plasma, even after subtraction of background lysis. However, as the concentration of C9 was increased, and after attaining 25 - 30 % of maximal haemolysis, the curves tended to plateau. Beyond this point the haemolytic response was not sensitive to the availability of C9 and some other factor, possibly the number of C5b-8-bearing cells, was limiting the reaction. Fluid-phase activation of C9 was also investigated in vitro. In the presence of CVF, NHS promptly incorporated monomeric 125I -C9 into a high MW form of approximately 1000 kD. Following incubation at 37°C for 3 h, this fraction contained more than 90 % of the total radiolabelled protein. Furthermore, a radioimmunoassay demonstrated a complex containing C9 neoantigens, C5 and vitronectin, consistent with SC5b-9. However, an unexpected observation was that only two of a panel of four anti vitronectin m.Abs showed affinity for this complex. The non-binding antibodies were distinctive in that they had previously been shown to antagonise the attachment of vitronectin to cells. This suggests that the cell-binding site of vitronectin may be altered or concealed when incorporated into ~he TCC. Indirect support for this was provided by studies of the distribution of 125I-SC5b-9 in vivo which demonstrated that only the spleen and liver were sites of appreciable accumulation. If vitronectin's cell-binding sites had been available, the expected distribution of SC5b-9 would have been much larger, reflecting the widespread expression of vitronectin integrin receptors. A further teleological consideration is that vitronectin might defeat its primary purpose in the complement system of limiting damage to bystander cells if it were to avidly and indiscriminately mediate the adherence of terminal complexes to tissues. SC5b-9 has been reported to bind to cells in vitro by a process that was antagonised by anti vitronectin antibodies [Biesecker, 1990]. However, in that study SC5b-9 was immobilised on a solid phase which could have changed the conformation and thereby the availability of vitronectin's cell-binding epitopes. Several attempts were made in the current work to resolve this issue using cultured, human umbilical vein cells as Section 4.1 Discussion 209 targets for SC5b-9 in solution. Unfortunately, these were thwarted by technical problems but would be worthy of further effort. Incubation of 125I -C9-supplemented human serum in the absence of CVF was also associated with an increase in the proportion of high MW radioactivity, albeit much smaller than that following CVF-activation. Approximately 10- 15% of protein-bound radioactivity resided in the 1000 kD region after incubation at 37°C for 30 min. This "spontaneous" high MW species contained C9 neoantigens and complexed C5, i.e. characteristic of the TCC. However, in contrast to the CVF-activated form, it bound to all four anti-vitronectin mAbs including those which inhibited cell-binding. These results suggest the existence of two variants of the soluble TCC which, despite their antigenic differences, share several physical characteristics. Specifically, they were of similar MW and were stable in 1 M NaCI. They both bound to heparin-Sepharose and were eluted from the column over a similar broad range of NaCl concentration. This last observation is consistent with previous studies which have shown that complex formation is associated with exposure of vitronectin's heparin-binding site [Hogasen et al., 1992]. However, the structure of the spontaneous complex and the factors responsible for its production are unclear. One possibility is that it resulted from spontaneous complement activation with slow generation of the TCC [see Hugo et al., 1987]. Soluble immune complexes, foreign surfaces and activated enzymes may all stimulate complement activity in vitro. However, such a process would not account for the observed antigenic differences between the terminal complexes as they would both be the end products of complement pathway activation. Alternatively, assembly of the TCC or a TCC-like entity, in the absence of early pathway activation, may have been responsible. Kolb et al., (1973) observed interactions between soluble native C5 and C6, C5 and C7, C5 and C8, and C8 and C9. They noted that a C5-9 complex could be produced by the spontaneous combination of the individual components in solution. Furthermore, it was proposed that its morphology resembled a pyramid with uncleaved C5, C6, C7 and C8 at the comers and up to six C9 molecules bound to the C8 subunit [Kolb et al., 1972]. Esser et al., (1993) have also noted that C8 and C9 can associate, side by side, to form a globular heterodimer in physiological ionic strength buffer. A feature of these reactions is their reversibility. To examine tllis issue further in the Section 4.1 Discussion 210 current study, 1251-C9 was incubated in NHS at 37°C for up to 65 h. A progressive shift of radioactivity from the monomeric to high MW region was again noted. This was most rapid in the first 12 hand was slowed thereafter with the proportion of monomeric and complexed 1251-C9 approaching equivalence by 4 7 h and remaining essentially unchanged at 65 h. One explanation of this behaviour is that the reactants and products were attaining chemical equilibrium and implies that complexing of 125I-C9 was reversible. It is uncertain whether such a reaction would occur in vivo where both monomeric and complexed C9 are rapidly removed and metabolised. However, the spontaneous and reversible production ofpoly-C9, TCC, or a TCC-like complex in the plasma could influence the local kinetics and distribution of the components of the terminal complement pathway. Recent work in our laboratory has also demonstrated that free vitronectin may be heterogeneous with regards to its affinity for monoclonal antibodies in a similar manner to the TCC. Specifically, plasma vitronectin was found to associate only weakly with mAbs 19.16 and 24.4 (i.e. the cell-binding inhibitory antibodies), but bound strongly to mAbs 13.9 and 2.11. This pattern was similar to that observed for CVF-activated SC5b- 9. By contrast, vitronectin purified from serum, bound strongly to all four antibodies 1 and, in this regard, resembled the spontaneously-generated TCC • These observations provide indirect evidence for the regulation of cell-binding epitopes of free vitronectin, in addition to the complexed molecule within the TCC. It is therefore notable that Panetti and McKeown-Longo (1993a & b) found that conformationally-altered, but not native vitronectin, underwent endocytosis and catabolism by fibroblasts in an integrin receptor-mediated process. Further work is required to establish whether sub-types of the soluble TCC have particular functions and, if so, the manner by which their expression of epitopes influences their behaviour, distribution and metabolism. These studies emphasise the intricate physiology of the terminal complement pathway. The discussion which follows considers the kinetics of the TCC and its major components, in vivo in the normal state and during pathological complement activation. 1 Personal communication from Ms S O'Grady, Scientific Officer, The Renal Immunology Laboratory, Prince Henry Hospital, Sydney. 211 4.2 The metabolism of C9 and SC5b-9 in experimental animals There have been numerous studies of the metabolism of human and homologous complement proteins in laboratory animals. The rabbit is a particularly good subject, not only for its physical characteristics, but also for the similarity between the rabbit and human complement systems [Bhakdi & Tranum-Jensen, 1983b, Peake et al., 1991]. In the current study, incubation of human 125I-C9 with fresh rabbit serum in the presence of purified CVF in vitro, produced a shift of radioactivity from the monomeric to a high MW (i.e. approximately 1000 kD) serum fraction consistent with the generation of SC5b-9. After 3 h, approximately 30% of the protein-bound radioactivity resided in the high MW region. Although slower than the comparable reaction in human serum, this observation provides further evidence that human C9 can be utilised by the terminal complement pathway of the rabbit. The investigation of protein metabolism in experimental animals offers several opportunities, not available from studies in human subjects. Firstly, animals may be given a relatively large radioactive dose which improves the accuracy of quantitation. This is particularly important for proteins which are rapidly eliminated from the plasma, such as the complement components. Furthermore, animals may be sacrificed and the precise distribution of radiolabelled protein determined. The effects of controlled complement activation can also be assessed. In the current study, a highly purified form of CVF was used to stimulate the fluid-phase alternative pathway. Although the rate and extent of complement activation produced by CVF is generally far greater than that seen in human disease, it provides a stimulus sufficient for the accumulation and, therefore study of relatively short-lived species such as the TCC. Finally, animal studies provide a necessary screening test for the biological safety of preparations destined for study in human subjects. This is in addition to the rigorous in vitro tests of sterility previously described. Section 4.2 Discussion 212 In the current study, the disappearance of 125 I -C9 from the plasma of healthy rabbits occurred in two phases. In the first 12 - 18 h there was a rapid decline in protein-bound radioactivity followed by a slower decay. This pattern has been observed repeatedly in the past for complement and other proteins, and extensively analysed. The early phase is characterised by protein re-distribution in addition to catabolism, with a net movement of molecules from the intravascular to extravascular sites. It may also involve the rapid elimination of denatured material which can arise through improper handling during protein purification or exposure to harsh conditions during radiolabelling. For example, an early preparation of C9, not reported in this thesis, showed a significant decline (i.e. > 90 %) in haemolytic activity following trace iodination by the chloramine-T technique. Following injection into rabbits it disappeared very rapidly from the plasma, with less than 20 % of the injected dose remaining after 4 h. This was accompanied by a marked release of free iodide representing approximately 40 % of the total plasma activity. In the animal studies described in this thesis, C9 was labelled by the more gentle lactoperoxidase method and, after injection it released free iodide at a steady rate, amounting to < 5 % of the total plasma counts. Furthermore, estimates of the plasma volume based on isotope dilution (i.e. 35.0 ± 3.0 mL/kg) were similar to previous reports [see Regoeczi, 1963]. It is therefore unlikely that hypercatabolism of denatured molecules played a significant role in the present studies. Following compartmental equilibration, the 125I-C9 plasma disappearance curves attained a mono-exponential phase, from which the plasma T112 was derived. This persisted for the duration of sampling, i.e. until less than 8 % of the initial dose remained in the plasma. In all cases, the plasma curves were adequately described by the sum of only two exponential equations. The T112 of plasma C9 in rabbits was approximately 25 h. This was shorter than results reported previously for other human complement proteins in the rabbit, including C3 (35- 40 h [Charlesworth et al., 1974a]) and Factor H (30- 45 h [Charlesworth et al., 1979b]). However, to date there have been very few studies of the metabolism of terminal complement components in either humans or laboratory animals. In an abstract describing the metabolism of rat C8 and C9 in the rat, Dalmasso & Falk, (1987) Section 4.2 Discussion 213 reported the T112 of C8 to be 22 hand that of C9, 12.5 h. These values indicate very rapid elimination of both proteins from the plasma. However, the methodology employed was not described and metabolic studies in small animals may be fraught with technical difficulties. In the present study, the FCR of 125I-C9 (i.e. 5.99 %/h by Mathews' analysis) was also relatively high when compared with the behaviour of other human complement proteins in rabbits. In particular, it exceeded the FCR of C3 (2.4 - 2.8 %/h [Charlesworth et al., 1974a]) and Factor H (0.39 - 1.45 %/h [Charlesworth et al., 1979b]). Several factors may have contributed to the particularly rapid turnover of C9 in normal animals. Specifically, it is the only complement component which is capable of forming polymers, both spontaneously and in response to complement activation. Furthermore, it may combine spontaneously with the other proteins of the terminal complement pathway. By analogy with SC5b-9, it is likely that these macromolecular complexes would be rapidly eliminated from the plasma although, as previously discussed, they may also be unstable and dissociate spontaneously. FPLC-gel filtration chromatography allowed changes in the molecular weight of radio-labelled proteins to be studied in the course of their metabolism in vivo. In normal rabbits, C9 remained largely monomeric (i.e. > 90 % of the protein-bound plasma radioactivity) without evidence of accumulation of high molecular weight complexes or fragmentation. While this observation does not strictly discount either aggregation or disintegration from the catabolic process, it argues that any species thereby formed must have a very short plasma half-life. Treatment of animals with CVF following injection of 125I-C9 caused profound changes in the metabolic characteristics of C9. Most notable was a marked increase in its rate of disappearance from the plasma in the first 6 - 8 h. A concurrent rapid fall in the serum C3 concentration during this time also suggested that complement activation was occurring. In these animals, gel filtration of plasma demonstrated the early appearance (i.e. after only 1 hour) of a high MW, C9-containing species consistent with SC5b-9. However, as previously, there was no evidence of C9 fragmentation. Analysis of the area under the gel filtration curve showed that the rate of elimination of the C9 monomer during complement activation was even more rapid than suggested by gross Section 4.2 Discussion 214 measurement of the total plasma protein-bound radioactivity. Furthermore, generation of the high MW species occurred when the rate of disappearance of monomeric C9 was at its most rapid. Despite this, release of free iodide was initially small and rose to a maximum only after the decline of the high MW protein peale In their study of the metabolism of human C3 in rabbits, Charlesworth et al., (1974a) similarly found that while CVF increased the rate of C3 disappearance from the plasma, it was not accompanied by release of free iodide. They attributed this to extravasation of the protein or its breal(down products, rather than hypercatabolism per se. The current results also suggest that CVF does not directly accelerate C9 catabolism, but rather promotes its incorporation into high MW complexes (presumably SC5b-9), which are then rapidly catabolised. This was investigated directly by examining the in vivo 125 behaviour of I-SC5b-9 which proved to have a very short plasma T112• It was not possible to apply the same analytical techniques to derive the metabolic parameters of SC5b-9, as its turnover rate was so rapid. However, plasma levels fell by 50% in only 0.7 h, leaving less than 15 % of the injected dose after 4 h. While both extravascular sequestration and catabolism may have contributed, the great release of free iodide (i.e. > 60 % of total plasma activity at 5 h) argues strongly in favour of hypercatabolism. Rabbits given 125I-SC5b-9 also received 131I-C9 which had been pre-incubated in fresh rabbit serum for 3h at 37°C (in the absence of CVF) as a control for the production of SCSb-9. The rate of disappearance of 131I-C9 from the plasma of these animals was similar to non-incubated C9, indicating that the method of SC5b-9 generation in vitro was unlikely to have produced denaturation. These results are consistent with previous studies of both rabbit and human SC5b-9 in the rabbit by Hugo et al., (1989), who reported a plasma T112 of 30 - 50 min for both homologous and heterologous complexes. Several approaches were employed to examine the compartmental distribution of C9 in normal and complement-activated animals. The EV /IV ratio, calculated by analysis of the plasma curves, was 0.72 ± 0.21. This indicates that at equilibrium, approximately 40 % of the total C9 pool resided in the extravascular compartment. It should be emphasised that the term "extravascular" in this context refers to a functional, rather than anatomical entity and may involve, for example, binding sites on endothelial and circulating cells. However, in this study washed packed blood cells were Section 4.2 Discussion 215 demonstrated to retain only negligible quantities of protein-bound 1251 in the absence of complement activation and therefore did not contribute significantly to the extravascular distribution. The sequestration of protein-bound radio label by specific organs was also measured directly in rabbits during 125I-C9 turnover. In normal animals, the tissue levels were low and varied from 5 % of the plasma concentration in skeletal muscle to 50 % in the lungs after 6 h. This corresponded closely with the distribution of RSA administered as a control protein (i.e. not directly involved in the complement cascade [see Brinch et al., 1982a & b]). By contrast, 6 h after administration of CVF there was accumulation of protein-bound 1251 in the spleen and liver, while uptake by the kidneys, lungs, cardiac and skeletal muscle remained similar to control animals. As no effect of CVF on the distribution of RSA was observed, these results most likely represented specific organ uptake of SCSb-9, rather than changes in tissue blood flow or vascular permeability. Similarly, the spleen and liver were prominent sites of accumulation of protein-bound 1251 when 1251-SCSb-9 was administered directly. Although the fate of SCSb-9 sequestered by these organs was not investigated, it remains probable that the spleen and liver, or the reticuloendothelial elements therein, are sites of metabolism of the TCC and its components. Protein-bound radioactivity was also accumulated by circulating cells in rabbits given CVF following the administration of 125I-C9. As this was not observed in animals which received either 125I-C9 alone or 1251-SCSb-9, it probably resulted from the deposition of CSb-7 and subsequent assembly of the terminal complex. The presence of these cells in the blood after two days, by which time the plasma radioactivity had declined to < 5 % of the initial dose, indicates that the resulting MAC was neither rapidly processed nor appreciably haemolytic; the latter being consistent with the effect of homologous restriction mechanisms. However, these observations suggest that circulating cells may play a role in the sequestration and transport of terminal complement complexes, although further clarification is required. This study demonstrates that C9 is a widely distributed, rapidly metabolised protein. Complement activation leads to a change in the ratio of C9 : SCSb-9 in the plasma and, once formed, SCSb-9 is rapidly catabolised, probably by cells in the liver and spleen. 216 4.3 The kinetics of vitronectin and Ser378-P04-vitronectin in vivo Vitronectin is difficult to purify in the native form because of its propensity for self-association and adherence to surfaces. Moreover, the methods commonly used for its purification often involve a denaturing step (such as exposure to 8M urea) prior to absorption on heparin-Sepharose [Yatohgo et al., 1988, Hayashi, 1993], or the use of chaotropes or buffers of extreme pH in immuno-affinity chromatography [Hodgetts & Morgan, 1989]. While vitronectin purified by these methods promotes cell spreading and inhibits complement, it is inappropriate for use in metabolic studies. However, these problems were largely overcome in the current study by investigating the behaviour of unpurified, human vitronectin in rabbits, detected by an anti-human vitronectin ELISA. The only chemical processing was specific trace labelling of vitronectin with 32P by cAMP-dependent protein kinase, to allow the simultaneous examination of both native (i.e. the great majority of molecules) and 32P-vitronectin. Autoradiography of phosphorylated plasma revealed only one labelled band, consistent (in MW) with intact vitronectin, while FPLC gel filtration showed that this was largely monomeric, with less than 5% present as high MW radioactivity. Following injection into rabbits, both native vitronectin and its phosphorylated form, were rapidly removed from the plasma with similar, short half-lives of 8 - 9 h. By comparison, the unrelated multifunctional glycoprotein, fibronectin, has also been shown to have a very short T112 of approximately 16h in the rabbit [Pussell et al., 1985]. However, the compartmental distributions of native and phosphorylated vitronectin were significantly different. Specifically, the native, ELISA-detected form was distributed equally between the intra- and extravascular spaces (i.e. median EV /IV of 1), whereas, approximately 80 % of the 32P-vitronectin was located in the plasma. Their fractional catabolic rates were also dissimilar, with that of native vitronectin being approximately 19 %/h, compared with 11 %/h for the phosphorylated molecule. Nevertheless, in both cases, the FCR was very high in comparison to complement proteins, and higher than that of fibronectin (i.e. 4.0 - 6.27 %/h [Pussell et al., 1985]). Several factors could have contributed to the observed differences between native and Section 4.3 Discussion 217 phosphorylated vitronectin. Firstly, phosphorylation may have selected a subset of molecules with particular characteristics. For example, only single chain (i.e. uncleaved) vitronectin is significantly phosphorylated under the conditions employed in these studies and, therefore, the results may have reflected differences in the behaviour of single and double chain molecules. Although there is little evidence to suggest that these variants function differently, this issue could be investigated further by studying the metabolism of vitronectin from a donor producing only the single chain form (i.e. phenotype 1-1). Secondly, phosphorylation may have denatured vitronectin molecules leading to altered metabolic characteristics. However, this seems unlikely in view of the minimal manipulation of material prior to injection. Furthermore, free 32P was released at a steady rate throughout the turnover period and estimates of the plasma volume based on protein-bound 32P dilution (i.e. 37.5 ± 1.8 mL/kg), were consistent with previously determined values [see Regoeczi, 1963]. These results support the molecular integrity ofvitronectin. A third, and more plausible explanation, is that phosphorylation altered the metabolism of vitronectin by a physiological process. The phosphorylation sensitive site at Ser378 lies within the heparin-binding domain, which is thought to be important in maintaining the folded conformation of native vitronectin, and is involved with binding to C9 and other ligands. Insertion of a phosphate. group into this highly basic region could therefore influence the molecule's conformation and metabolism. Complement activation in vivo also affected native and phosphorylated vitronectin differently. The rate of elimination of 32P-vitronectin was markedly increased in all animals given CVF, particularly within the first 10 h following injection. Furthermore, FPLC gel filtration demonstrated that a high MW (approximately 1000 kD), 32P-containing species was produced, consistent with 32P SC5b-9. However, the response of antigenically-detected vitronectin was variable and, in general, the plasma disappearance curves were superimposed over those of the control animals. These results suggest that phosphorylated vitronectin may be preferentially incorporated into the TCC. Recent work in our laboratory has also provided evidence, in vitro, that this may be the case: the fraction of 32P-vitronectin (in phosphorylated NHS) which became incorporated into the TCC following activation by CVF, was approximately twice that of antigenically-detected and 1251-labelled molecules [Peake & Charlesworth, 1995]. Section 4.3 Discussion 218 Despite evidence of enhanced participation of 32P-vitronectin in the terminal complement pathway, phosphorylation had no apparent effect on vitronectin's ability to inhibit complement-mediated haemolysis in vitro. Specifically, phosphorylated, purified vitronectin retained, unaltered, its ability to inhibit C8/C9-dependent lysis of EAc1_7 cells and quantitative phosphorylation had no significant effect on the CH50• This discrepancy may have been due, at least in part, to the existence of multiple complement inhibitory sites on vitronectin. For example, a 43 kD CNBr digest fragment has been identified which strongly binds to C5b-7 and inhibits CSb-8-induced C9 polymerisation, although at a much higher concentration than that required for SC5b-7 formation [Sheehan et al., 1995]. However, this region does not include the heparin-binding domain and therefore may be unaffected by phosphorylation. This issue could be clarified by examining the effect of phosphorylation on the complement inhibitory activity of heparin-binding and other vitronectin fragments. Vitronectin is an adhesive protein which occurs widely in the connective tissue matrices. The relatively large EV!IV ratio found for antigenically-detected molecules in the current study, is consistent with this distribution. However, the low EV !IV ratio of 32P-vitronectin suggests that this form is primarily an intravascular species and is therefore, well placed to influence fluid-phase reactions. This may be of particular relevance to complement activation at sites of inflammation within the microvasculature. For example, protein kinase A, released from activated platelets, could phosphorylate vitronectin and thereby facilitate the inactivation and removal of potentially cytotoxic terminal complement complexes. In this scenario, platelets and their products may play a central role in the regulation of the terminal complement pathway, although clarification is required. The function of phosphorylated vitronectin remains uncertain. However, the current study indicates that its compartmental distribution, catabolic rate and involvement with the terminal complement pathway differ from the unphosphorylated form. This may have consequences for its site and mode of action. 219 4.4 The metabolism of C9 in humans Accurate interpretation of metabolic data requires that the labelled protein functions in a manner identical with the native molecule. Furthermore, material administered to human subjects must be prepared under rigorous conditions to assure its sterility and biological safety. The preparations used in this study were shown to be sterile and fully functional in vitro, and analysis of in vivo metabolic data supported their molecular integrity. In particular, there was steady release of iodide throughout the sampling period and calculation of the urine/plasma radioactivity ratio yielded a stable value for FCR (i.e. the daily metabolic clearance) after the first 24h period. Furthermore, plasma volumes calculated from the initial isotope dilution were consistent with previously published values [Lentner, 1984]. By contrast, the presence of denatured protein can lead to a considerable overestimation of the plasma volume due to hypercatabolism of damaged molecules. The patients in this study were biochemically and haemodynamically stable, with minimal variation in their plasma C9 levels. None had significant haemorrhage during the investigation nor other means of protein loss. Moreover, the urinary protein bound radioactivity, as a proportion of the total excreted counts, was minimal and comparable for both healthy subjects and patients, including those with proteinuria. Consequently, no correction for this value was made in the calculation of metabolic parameters. The FCR of C9 in normal subjects (i.e. 2.92 ± 0.36 %/h) was greater than that reported previously for other complement proteins, including C3 (1.61 %/h [Charlesworth et al., 1989a]), C4 (1.87 %/h [Charlesworth et al., 1989a]), C5 (1.74 %/h [Sissons et al., 1977]) and the regulatory protein, Factor H (1.32 %/h [Charlesworth et al., 1979b]). However, it was similar to the FCR of C1q (2.84 %/h [Pussell, 1982]), and less than that of the non-complement, adhesive glycoprotein, fibronectin (4.81 %/h [Pussell et al., 1985]). The plasma T112 of C9 (i.e. 42.5 ± 6.7 h) reflected its high metabolic rate and was closely correlated (negatively) with the FCR. Accordingly, the Section 4.4 Discussion 220 C9-T112 was shorter than that determined for C3 (71h [Charlesworth et al., 1974b]), Factor B (66 h [Charlesworth et al., 1974b]) and Factor H (76 h [Charlesworth et al., 1979b]). By comparison, human C9 was metabolised in normal rabbits with a FCR of 5.99 ± 0.69 %/h. The increased metabolic rate in rabbits was probably due to the more rapid elimination of heterologous protein. In human subjects with normal serum creatinine concentration and creatinine clearance rate, a close correlation was found between the FCR calculated by Mathews' analysis, and the metabolic clearance method. While both are reliant on the plasma radioactivity, only the metabolic clearance approach requires excretory data and so these techniques are relatively independent. However, some differences between the results were noted. Specifically, in subjects with plasma volumes greater than 2500 mL the FCR, derived from the metabolic clearance, tended to be lower than that found by Mathews' analysis. This may have been due to the accumulation of free isotope in the physiological iodide space and consequent delay in the excretion of radioactivity during the early phase of the turnover study. Furthermore, the disappearance of whole body radioactivity in smaller subjects ran parallel to the plasma curve, while for heavier individuals, the whole body radioactivity declined at a slightly slower rate than the plasma. A mathematical correction for free body iodide is possible by measuring the size of the iodide space, although this generally requires the concurrent administration of a second iodide isotope and was not performed in this study [see Regoeczi, 1963, Charlesworth et al., 1974a]. However, to minimise error the average metabolic clearance was calculated with the exclusion of the first 24 hour period. The mean EVIIV ratio of C9 in normal humans (i.e. 0.56 ± 0.12) indicates that approximately 1/3 of the total C9 pool resided in an extravascular compartment, which is a higher proportion than that previously observed for C3 (EV/IV: 0.39 [Charlesworth et al., 1989a]) and C4 (EVIIV: 0.45 [Charlesworth et al., 1989a]), but comparable to Factor H (0.52 [Charlesworth et al., 1979b]). The loci of extravascular sequestration of C9 in humans were not determined, and the radioactivity associated with circulating blood cells was negligible. However, this extravascular pool may serve as an immediately available source ofC9 at sites oftissue damage. Section 4.4 Discussion 221 In the current study, seven patients showed abnormally low serum CH50, of which six also had raised levels of circulating immune complexes. For this group, the FCR of C9 was significantly greater, and the T112 shorter, than for normal subjects. By contrast, the EV /IV ratio of C9 in patients did not differ significantly from that of the control group. However, this does not imply that activated C9 and its complexes are not deposited in extravascular sites. To the contrary, rabbit studies have shown that SC5b-9 is rapidly sequestered by the liver and the spleen while the MAC is, by definition, tissue-bound. Nevertheless, only a portion of this complexed C9 may be available for exchange with the plasma pool and, therefore, is not reflected in the EV/IV ratio. The serum C9 concentration was higher in patients than control subjects and this value correlated with the C9 production rate, but not the FCR. This was in contrast to previous studies of C3 and C4 metabolism in SLE, where there was an inverse relationship between the FCR and the proteins' serum concentration [Charlesworth et al., 1989a]. Patients also had greater total body C9 content than the normal subjects. However, in the absence of altered compartmental distribution, plasma production became the major determinant of the serum C9 concentration, and the current study demonstrated the capacity of increased production to compensate for accelerated C9 removal during complement activation. The factors controlling this enhanced production rate were not examined but it is likely that acute phase reactivity plays a significant role [see Charlesworth et al., 1979a, Adinolfi & Lehner, 1988]. It should also be noted that the C9 synthetic rate was not calculated directly and that new protein entering the plasma compartment could have derived from a combination of newly synthesised protein and material released from intracellular stores. In the subgroup of patients with SLE, subjects with normal serum creatinine and creatinine clearance had a significantly greater C9-FCR, calculated by the metabolic clearance method, than the controls. In particular, this group contained two noteworthy subjects with active disease. A renal biopsy of patient 6 had initially shown membranous lupus nephritis (i.e. WHO type V), while a more recent biopsy revealed diffuse proliferative disease (i.e. WHO type IV). At the time of this study the patient had persistent proteinuria of 8 g/day. The other subject, patient 9, had a long history of Section 4.4 Discussion 222 SLE including renal disease, although her . renal function was normal during the investigation. This subject had experienced life-threatening pulmonary haemorrhage and cerebral seizures in the month prior to study which settled with immunosuppression, and for which no other cause was found. In each case, the CH50 was reduced and there were low levels of either, or both, C3 and C4. The C9-FCR was markedly elevated and the T112 was reduced, while the plasma C9 disappearance curves lay outside the normal range. The FCR of C9 was significantly correlated with the level of circulating immune complexes, while the T112 showed a positive correlation with the CH50• In contrast, the serum C3 and C4 levels were poorly correlated with the C9-FCR. These observations demonstrate that serum levels of the early complement components may poorly reflect the activity of the terminal pathway. There are several possible explanations for this. Specifically, activation of C5, especially in the fluid-phase, is known to be relatively inefficient, requiring 60 - 200 C3 molecules for each cleaved C5 [Bhakdi, 1988]. Furthermore, substrate exhaustion by excessive activation of the early pathways, could also contribute to defective formation of the C3 and C5-converting enzymes. The regulatory molecules vitronectin, clusterin and homologous restriction factors, may also modify the terminal pathway's response to complement activation. The plasma SC5b-9 concentration in the control group was consistent with the normal range suggested by Behring Diagnostics, the assay manufacturer. In patients, the plasma SC5b-9 level was significantly higher than in healthy subjects, and in contrast to the C9 monomer, was closely correlated with the rate of C9 catabolism. However, in one notable case (i.e. patient 7), a relatively low C9-FCR (i.e. 2.64 %/h) was accompanied by one of the highest levels of plasma SC5b-9 in the study population (i.e. 1058 J-Lg/L). This patient suffered from SLE and had been upwell during the previous year, with exacerbations of renal, haematological and cerebral disease. However, aside from stable chronic renal impairment (i.e. serum creatinine of 0.17 mM), she was well at the time of study. Her plasma C9 production rate was elevated (i.e. 0.11 mg/kg per h) and consequently, the serum C9 concentration was also very high (i.e. 98 mg/L). These results suggest that the increased plasma SC5b-9 level in this Section 4.4 Discussion 223 subject resulted :from a normal C9-FCR acting on a high serum concentration of C9 monomer, although defective clearance of the terminal complex cannot be discounted. It remains unclear whether such disparity between the rate of C9 production and its :fractional catabolism represents a physiological, or pathological state. However it is possible that some patients with a persistently elevated C9 production rate but normal C9 catabolism, could generate an excess of terminal complement complexes leading to continual, or recurrent tissue damage. Several patients in this study were receiving immunosuppressive and other pharmacological treatments. However, in most cases, only low or maintenance doses of corticosteroids (± azathioprine or cyclophosphamide) were used, with the exceptions of patients 1 and 9 who were taking 25 and 100 mg of prednisolone per day respectively. There have been no detailed studies of the effects of these agents on complement turnover. However, in high dosage, prednisolone induces whole body protein catabolism and accelerates amino acid oxidation, although it has little effect on estimates of protein synthesis [Haymond & Horber, 1992]. Sliwinski and Zvaifler (1972) found that treatment with corticosteroids had variable effects on C3 metabolism. Specifically, the FCR fell in some patients, while in others the synthesis rate was increased and the FCR remained unaltered. In general, immunosuppressive agents may ameliorate complement activity through reductions in autoantibody synthesis, and immune complex formation. However, until formal studies of the effects of these medications are performed, the issue will remain unresolved. This study demonstrated the high rate of C9 metabolism in both normal subjects and patients with autoimmune disease. It also clarified the metabolic basis for the maintenance of normal, or elevated C9 levels in the presence of pathological complement activation. In contrast to other complement proteins, the compartmental distribution of C9 was unchanged during disease, however there was a highly significant increase in its plasma production rate which fully compensated for hypercatabolism. 224 4.5 SUMMARY AND CONCLUDING REMARKS Complement components are among the most rapidly catabolised plasma proteins. This preswnably reflects not only their frequent involvement in normal immunological processes, but also the innate tendency of the activation mechanisms, particularly the alternative pathway, to be switched on. The current study demonstrated that C9 is also rapidly turned over in the circulation of both animal and hwnan subjects. Moreover, it was shown for the first time, that patients with autoimmune disease and pathological complement activation exhibit hypercatabolism of C9 despite having serum C9 concentrations in the normal or elevated range. This was associated with a marked increase in the plasma C9 production rate, without significant alteration in its compartmental distribution. The mechanisms which ultimately lead to deposition of the TCC and its persistence in the tissues remain uncertain. It is also unclear whether SCSb-9 formed in the plasma contributes to these deposits. In the current study the plasma concentration of SCSb-9 was shown to be positively correlated with the rate of C9 catabolism and, therefore, the turnover of the terminal complement pathway. However, the molecular mechanisms whereby SCSb-9 is removed from the circulation are unknown. It has been proposed that vitronectin may serve to guide SCSb-9 and other macromolecules to their sites of metabolism through its affinity for a variety of cellular and interstitial epitopes. This has been referred to as its "scavenger" role [Hogasen et al., 1992]. However, in this study, SCSb-9 was shown to accwnulate only in the spleen and the liver, despite the widespread distribution of vitronectin receptors. Monoclonal antibody binding studies in vitro provided a possible explanation for this observation, suggesting that vitronectin' s cell-binding sites may be concealed following its incorporation into the TCC. It is therefore possible that disturbed expression, or regulation of these cell binding regions could lead to abnormal, more diffuse distribution of SCSb-9. Co localisation of vitronectin with C9 neoantigens in inflamed tissues is consistent with this postulate. Although the potential of this complex to produce tissue damage remains unproven, this issue requires clarification. Section 4.5 Summary & Conclusions 225 The tissue distribution of C9 and SC5b-9 was studied in normal laboratory animals during fluid-phase complement activation. However, it was not possible to examine the distribution of these proteins in human subjects with complement activating diseases, despite its obvious relevance. Although the EV!IV ratio of C9 in patients was not significantly different to the control subjects, it is possible that the distribution of terminal complement proteins would be affected by pre-existing tissue lesions. For instance, SC5b-9 could deposit at sites of denuded endothelium through the interaction of vitronectin's heparin-binding domain and exposed polysaccharides in the interstitial matrix. It would be insightful to examine the metabolism and distribution of these proteins in animal models of human immunological disease such as Heymann, or nephrotoxic nephritis. Studies in animals with primarily non-immunological disease, such as atherosclerosis, could also reveal a secondary role for the TCC in the maintenance and progression of tissue damage. Several interactions between the coagulation cascade, platelets and the terminal complement pathway have been described, or proposed. However, the role of protein kinase A, which selectively phosphorylates vitronectin upon release from activated platelets, remains unclear. In the current study, phosphorylated vitronectin had markedly altered metabolic characteristics compared with the native form. Specifically, it had a smaller extravascular distribution and FCR. Activation of platelets, complement and other protein systems may occur simultaneously at sites of vascular damage raising the possibility that phosphorylated vitronectin could be an important participant in SC5b-9 assembly. Studies in vivo, presented in this thesis, and some recent work in our laboratory in vitro, have provided evidence that phosphorylated vitronectin may be preferentially incorporated into the TCC. These observations deserve further study, particularly with regard to the possible effects of phosphorylation on the expression ofvitronectin's cell-binding epitopes and the molecule's propensity to form polymers and macromolecular complexes. Therapy for autoimmune disease is, in the main, non-specific and directed at the earliest phases of the immune response. However, increasing evidence for the participation of the TCC in the development of tissue lesions raises the issue of Section 4.5 Summary & Conclusions 226 treatments directed towards modulation of the terminal complement pathway. This would have considerable theoretical advantage over measures to inhibit complement activation, and treatments such as corticosteroids and cytotoxic agents which cause profound, non-specific immunosuppression. In particular, inhibition of the complement cascade beyond the cleaving of C5 would preserve the anaphylatoxic and opsonic activities of the early pathways, while still inhibiting TCC assembly. However, this may be unhelpful in conditions where the anaphylatoxins are thought to be of pathological significance (such as anti-basement membrane nephritis). One possible approach would involve inhibition or depletion of terminal components by the use of monoclonal antibodies or Fab-fragments. However, results presented in this thesis suggest that C9 may be a poor target for such therapy. In particular, it is seldom likely to be the rate limiting step in TCC assembly and the increase in plasma production which accompanies hypercatabolism is most effective in maintaining normal or elevated circulating levels. The other TCC components, which have less marked acute-phase responses, may be more amenable to manipulation. Monoclonal antibodies directed against C5 are currently undergoing trial in humans. There is also potential for the administration of solubilised complement inhibitors such as CD59 and HRF, which act as antagonists of TCC assembly. Early pathway regulators such as soluble CRl have already demonstrated effectiveness as complement inhibitors in animal models and are being considered in human disease [Atkinson, 1997]. A further possibility involves the transgenic expression of these naturally occurring complement inhibitors in xenografts to reduce the risk of acute rejection. The ultimate efficacy of these regimens will depend on the half-life and stability of the agents and their ease and acceptability of administration. However, the prospect of more selective, and disease-specific immunotherapy is ample impetus to continuing efforts. The data presented in this thesis provides direct evidence of pathological activation of the terminal complement pathway in patients with autoimmune disease. Furthermore, the metabolic factors which regulate circulating levels of the C9 monomer, its inhibitor vitronectin and its soluble, complexed form, SC5b-9, have been defined and quantitated. These results provide further insight into the mechanisms underlying immunological disease and would be of relevance to therapeutic protocols aimed at manipulation of the terminal complement pathway. 227 APPENDICES 228 APPENDIX 1 BUFFERS REQUIRED FOR THE PURIFICATION OF C9 Ingredient Concentration Quantity Manufacturer EDTA, pH 7.2-7.6 Approx. 86 mM (a) 100mL Baxter 4.46g Ajax (b) 100mL Baxter 1.20 g Ajax Dropwise add solution (b) to (a) until pH 7.2- 7.6. [see Whaley, 1985] Initial plasma buffer Fresh plasma 100mL Benzamidine 25mM 0.391 g Sigma 1 PMSF/methanol 0.6mM 500 ~-tL Sigma/Ajax PEG 4000 21% buffer, pH 7.4 500mL dH20 500mL Baxter K2HP04 25mM 2.177 g Ajax NaCl 90mM 2.629 g Ajax Na2EDTA 10mM 1.861 g Ajax Benzamidine 25mM 1.957 g Sigma 2 PMSF/methanol 0.5mM 400 ~-tL Sigma/Ajax PEG4000 21% 105.0 g Ajax Chloramphenicol 25 mg/L 12.5 mg Parke Davis 1 Solution made by adding 100.0 mg of PMSF to SmL of ice cold methanol just prior to use. 2 Solution made by adding 43.0 mg ofPMSF to 400 IlL of ice cold methanol just prior to use. Appendix 1 229 Ingredient Concentration Quantity Manufacturer Post lysine-Sepharose buffer, pH 7.0 1000 mL dH20 1000 mL Baxter Benzamidine 25mM 3.915 g Sigma Na2EDTA 5mM 1.861 g Ajax EACA 30mM 3.936 g Sigma Chloramphenicol 25 mg/L 25.0mg Parke Davis DEAE-Sephadex starting buffer, pH 7.0 5000 mL dH20 5000mL Baxter K2HP04 20mM 17.42 g Ajax Na2EDTA 5mM 9.305 g Ajax Benzamidine 25mM 19.57 g Sigma EACA 30mM 19.68 g Sigma NaCL lOOmM 29.22 g Ajax Chloramphenicol 25 mg/L 125.0 mg Parke Davis DEAE-Sephadex final buffer 1500 mL DEAE-Sephadex column starting buffer for a 5 x 20 em column 1500mL NaCl 400mM 26.30 g Ajax Lysine column regeneration buffer, pH 7.5 500mL dH20 500mL Baxter EACA 200mM 13.12 g Sigma NaCl 1M 29.22 g Ajax K2HP04 50mM 4.354 g Ajax Hydroxylapatite equilibration buffer, pH 7.4 5000 mL dH20 5000mL Baxter K2HP04 25mM 21.76 g Ajax KCl lOOmM 37.27 g Ajax Appendix I 230 Ingredient Concentration Quantity Manufacturer Hydroxylapatite 80 mM phosphate starting buffer, pH 7.7 (a) 500mL dH20 500mL Baxter K2HP04 80mM 6.967 g Ajax KCl lOOmM 3.727 g Ajax (b) 500mL dH20 500mL Baxter NaH2P04 • 2H20 80mM 6.240 g Ajax KCl lOOmM 3.727 g Ajax Dropwise add solution (b) to (a) until pH 7.7 Hydroxylapatite 400 mM phosphate final buffer, pH 7. 7 (a) 500mL dH20 500mL Baxter K2HP04 400mM 34.83 g Ajax KCl lOOmM 3.727 g Ajax (b) 500mL dH20 500mL Baxter NaH2P04 • 2H20 400mM 31.20 g Ajax KCl lOOmM 3.727 g Ajax Dropwise add solution (b) to (a) until pH 7.7 231 APPENDIX 2 POLYCLONAL ANTI-SERA USED IN RADIAL IMMUNODIFFUSION Anti-serum Volume of anti Sample volume Manufacturer serum per 10 mL of agarose (Sigma) (j.tL) (j.tL) Anti-human C3 150 5 Cappell Anti-rabbit C3 200 5 Cappell Anti-human C4 100 5 Daleo Anti-human C9 50 10 Calbiochem Anti-human 200 10 Dako ceruloplasmin Anti-human 80 5 The Binding Site vitronectin 232 APPENDIX 3 BUFFERS USED FOR POLYACRYLAMIDE GEL ELECTROPHORESIS Ingredient Quantity Supplier 10% Separating gel 10mL dH20 4.02mL 1.5 M Tris-HCl, pH 8.8 2.50mL Sigma 10% (w/v) SDS stock solution 100 !-LL Biorad 30 % Acrylamide + N'N' -his-methylene- acrylamide (8 g/lOOmL) 3.33 mL Biorad 10% ammonium persulphate (fresh daily) 50 !-LL Biorad TEMED 5 !-LL Biorad Stacking gel 10mL dH20 6.10 mL 0.5 M Tris-HCl, pH 6.8 2.50mL Sigma 10% (w/v) SDS stock solution 100 !-LL Biorad 30 % Acrylamide + N'N' -his-methylene- acrylamide (8 g/1 00 mL) 1.30mL Biorad 10% ammonium persulphate (fresh daily) 50 !-LL Biorad TEMED 10 !-LL Biorad Running buffer (Sx concentrate) 1000 mL dH20 1000 mL Tris base 15.0 g Sigma Glycine 72.0 g Ajax SDS 5.00 g Biorad Reducing sample buffer 1 8mL dH20 4.00mL 0.5 M Tris-HCI, pH 6.8 1.00 mL Sigma Glycerol 0.80mL Ajax 10% (w/v) SDS 1.60 mL Biorad 2-(3-mercaptoethanol 0.20mL Merle 0.05 % (w/v) bromophenol blue 0.20mL BDH Chemicals 1 One volume of sample was diluted in at least four volumes of sample buffer. 233 APPENDIX4 PATIENT INFORMATION SHEET You are invited to participate in a study of a blood protein which, we believe contributes to the cause of your kidney disease. We hope to improve our understanding of how this protein behaves in the circulation before it deposits in the kidney and causes damage. You were selected as a possible participant in this study because of your particular type of"nephritis". If you decide to participate we will require to inject a very small amount of the protein into a vein in your arm. The protein has been highly purified and sterilised. It has been carefully checked for infection. A small amount of radioactivity has been linked to the protein so that we can follow its behaviour in your blood. The amount of radioactivity is similar to that used for many diagnostic tests in medicine. After the protein has been injected, we will need to take a I 0 mL blood sample from your arm twice on the first three days, and once each day for the next two days. We would also ask you to collect a 24 h urine sample for four days. You are asked to take iodide solution for three days before and during the test to prevent any lodgement of radioactivity in the thyroid gland. The main discomfort for you will be to have blood tests as well as the initial injection. Where possible, we will try to take these samples at the same time you are having routine tests for your kidney disease. We have performed over 200 such tests in our patients with various forms of kidney disease. None has suffered a side effect apart from the inconvenience of blood tests. Two other potential risks include infection at the time of injection and the effects of radioactivity. We emphasise that these risks are very small because of the careful methods of preparation of the protein. We cannot and do not hold out that you will receive any benefits from this study, although we hope to improve our understanding of your illness. You will not require to change your treatment in any way. Any information about you that is obtained in connection with this study will remain confidential and will be disclosed only with your written permission. However, the results of the study may be published or disclosed to other people in a way that will not identify you. Whether you take part in this study or not, it will not make any difference to the medical treatment you will receive in/from the Renal Unit at Prince Henry Hospital. If you decide to take part, you can still pull out at any time and this will not make any difference to your medical treatment either. If you have any questions at any time, Professor Charlesworth or Drs Pussell or Greenstein with be happy to answer them. You will be given a copy of this form to keep. 234 APPENDIX 5 CONSENT FORM FOR HUMAN METABOLIC STUDIES DECLARATION BY INVESTIGATOR I hereby certify that I have disclosed the risks that may be involved, in terms readily understood by the patient. Date Signature of investigator CONSENT BY PATIENT I hereby certify that I have read and understood all the information provided and agree to participate in the research proposal described above. Date Signature of patient Relationship to patient if patient is unable to consent Signature ofWitness: Nature of Witness: REVOCATION OF CONSENT BY PATIENT I hereby wish to WITHDRAW my consent to participate in the research proposal described above and understood that such withdrawal WILL NOT jeopardise any treatment or my relationship with the Hospital or any medical attendants. Date Signature The section for revocation of consent by the patient should be forwarded to NProf JA Charlesworth, The Renal Unit, Prince Henry Hospital, Sydney. 235 APPENDIX 6 CALCULATION OF MEAN PLASMA (PROTEIN-BOUND) RADIOACTIVITY Plasma disappearance curves can be resolved into the sum of two exponential equations of the form f(7) = C e -br , to give a general equation, F(7), for plasma 1 (protein-bound) radioactivity (PR) with respect to time (7) as shown below • (1) PR = F(1) = cl e -bt T + Cz e -b2 T where Cb C2, b1 and b2 are constants which can be resolved graphically, or arithmetically, as described in Section 2.9, page 117. If the time interval, T1 to T2, is divided into "n" segments each of ~T duration, then the mean plasma radioactivity over this interval is found as the average of the sum of the discrete PR levels in the limit as n approaches infinity as shown below. 7'2 T2 (2) Mean PR = LF(T) , but n = (T2 - T1) , ther~fore mean PR = LF(T) ~T TI 1'1 n ~T (Tz- T1) limn"""* oo lim ~T--* 0 The definite integral may then be calculated as shown. (3) MeanPR Substituting equ. (1) into equ. (3) and solving, yields equ. (4). -b2 Tt -bt Tz C ze C 1 e ----+---- (4) MeanPR= 1 Equations were initially derived in base 10 then converted to base e by recalculating b as Loge 1Ob. 236 APPENDIX 7 DERIVATION OF THE FORMULA FOR WHOLE BODY C9 CONTENT The WB C9 is the sum of the C9 contained in the EV and IV compartments, i.e. equ. (1). (1) WBC9 = EV +IV EV can be expressed as the product of the EV/IV ratio and the IV, i.e. equ. (2). (2) EV = EV x IV IV Substituting equ. (2) into equ. (1) yields equ. (3). (3) WB C9 = (EV x IV) + IV IV Rearranging the terms in equ. (3) gives equ. (4). (4) WB C9 = IV x (EV + 1) IV The EV/IV ratio is determined by metabolic analysis (see Section 2.9, page 118) and the IV is determined by equ. (5). (5) IV = serum [C9] x plasma vol. 1 Substituting equ. (5) into equ. (4) yields equ. (6). (6) WB C9 =(serum [C9] x plasma vol.) x (EVIIV + 1) 1 Assuming that the serum and plasma C9 concentrations are equivalent [see Biesecker & Muller Eberhard, 1980, Oleesky et al., 1986, Law & Reid, 1995]. 237 APPENDIX 8 SUPPLIERS OF MATERIALS AND EQUIPMENT USED IN THIS THESIS Manufacturer Supplier's address Ajax Chemicals Sydney, NSW, Australia Amersham Sydney, NSW, Australia Amicon Beverly, MA, USA Amrad Pharmacia Melbourne, VIC, Australia ANSTO Sydney, NSW, Australia Apple Macintosh Cupertino, CA, USA Armadeus Brooklyn, Vic, Australia Baxter Old Toongabie, NSW, Australia BDH Chemicals Epping, NSW, Australia Behring Diagnostics Kingsgrove, NSW, Australia Biorad Sydney, NSW, Australia Bio-tek (See CSL Biosciences) Calbiochem Sydney, NSW, Australia Computer Associates Sydney, NSW, Australia Cappell (See Organon Teknika) Carnation Sydney, NSW, Australia CSL Biosciences Melbourne, Vic, Australia Dako Glostrup, Denmark Doust & Rabbidge Concord, NSW, Australia Dupont Sydney, NSW, Australia Gelman Sydney, NSW, Australia Graphpad Software San Diego, CA, USA Greiner Labortechnik Austria HD Scientific Sydney, NSW, Australia Hettich (See HD Scientific) Appendix8 238 Manufacturer Supplier's address Hunter Antisera Newcastle, NSW, Australia Jackson Immunosearch West Grove, P A, USA Johnson & Johnson Sydney, NSW, Australia Kodak Coburg, Vic, Australia Linbrook Thornleigh, NSW, Australia Mascot Wireworks Enfield, NSW, Australia Merk Epping, NSW, Australia Microsoft Redmond, WA, USA NSW Red Cross Blood Transfusion Service Sydney, NSW, Australia Nuclear Enterprises Edinburgh, Scotland Nunc Roskilde, Denmark Omni International Waterbury, CT, USA Organon Teknika Ternhout, Belgium Oxoid Melbourne, Vic, Australia Packard Fivedock, NSW, Australia Parke Davis Springvale, Vic, Australia Pierce Rockford, IL, USA Sanofi Pasteur Sydney, NSW, Australia Shimadzu Japan Sigma Sydney, NSW, Australia Sorvall (See Dupont) The Binding Site Birmingham, UK 239 PERSONAL BIBLIOGRAPHY AND REFERENCES 240 PUBLICATIONS AND PRESENTATIONS ARISING FROM THIS WORK ARTICLES 1. Greenstein, J. D., P. W. Peake and J. A. Charlesworth. (1995) The kinetics and distribution of C9 and SC5b-9 in vivo: effects of complement activation. Clin Exp Immunol, 100: 40. 2. Greenstein, J.D., P. W. Peake and J. A. Charlesworth. (1996) The metabolism ofC9 in normal subjects and in patients with autoimmune disease. Clin Exp Immunol, 104: 160. 3. Peake, P. W., J. D. Greenstein, B. A. Pussell and J. A. Charlesworth. (1996) The behaviour of human vitronectin in vivo: effects of complement activation, conformation and phosphorylation. Clin Exp Immunol, 106: 416. PUBLISHED ABSTRACTS 1. Charlesworth J. A., J. D. Greenstein and P. W. Peake. (1995) The metabolic characteristics of C9 in normal subjects and patients with autoimmune disease. Nephrology, 1(5): 445. PRESENTATIONS OF PORTIONS OF THIS WORK 1. The Scientific Meeting of the Australian Society for Medical Research (NSW branch). Sydney, July, 1995. 2. The TOW Research Meeting of the Prince of Wales and Prince Henry Hospital group. Sydney, November, 1995. 3. The 6th Asian Pacific Congress ofNephrology. Hong Kong, December, 1995. 241 GENERAL REFERENCES Adinolfi, M. and T. Lehner. (1988) C9 and Factor B as acute phase proteins and their diagnostic and prognostic value in disease. Exp Clin Immunogenet, 5(2-3): 123. Adler, S., P. J. Baker, P. Pritzl and W. G. Couser. (1984) Detection of terminal complement components in experimental immune glomemlar injury. Kid Int, 26(6): 830. Adler, S., P. J. Baker, R. J. Johnson, R. F. Ochi, P. Pritzl and W. G. Couser. (1986) Complement membrane attack complex production of reactive oxygen metabolites by cultured rat mesangial cells. J Clin Invest, 77(3): 762. Akama, T., K. M. Yamada, N. Seno, I. Matsumoto, I. Kono, H. Kashiwaga, T. Funald and M. Hayashi. (1986) Immunological characterisation of human vitronectin and its binding to glycosaminoglycans. J Biochem, 100(5): 1343. Alper, C. A. and F. S. Rosen. (1967) Studies ofthe in vivo behaviour of human C3 in normal subjects and patients. J Clin Invest, 46: 2021. Ammann, A. J. and H. Fudenberg (1982). Immunodeficiency diseases. Basic & Clinical Immunology. 4th ed., Stites, D. P., J. D. Stobo, H. H. Fudenberg and J. V. Wells, Eds., Lange. Los Altos. 395. Atkinson, J. (1997) Inhibition of the complement system m clinical medicine. Nephrology, 3(supp 1): S24. Baker, P. J., R. F. Ochi, S. Adler, R. J. Johnson and W. G. Couser. (1985) C6 depletion abolishes proteinuria in experimental membranous nephropathy. Clin Res, 33(2): 475. Baker, P. J., R. F. Ochi, M. Schulze, R. J. Johnson, C. A. Campbell and W. G. Couser. (1989) Depletion of C6 prevents development of proteinuria in experimental membranous nephropathy in rats. Am J Pathol, 135(1): 185. Bariety, J., N. Hinglais, S. Bhakdi, C. Mandet, M. Rouchon and M. D. Kazatchkine. 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