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The Kinetics of C9, Vitronectin and the Terminal Complement

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The Kinetics of C9, Vitronectin and the Terminal Complement 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
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