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C-Reactive Protein (CRP) Induced Signaling in Neutrophils
DISSERTATION
Presented in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University
By
Maoyen Chi, B.S., M.S.
The Ohio State University
2001
Dissertation Committee: Approved by Professor Richard F. Mortensen (Advisor) Professor Neil Baker Associate Professor Joseph Krzycki ^ A d^ser Professor Pravin Kaumaya Department of Microbiology UMI Number: 3031185
UMI
UMI Microform 3031185 Copyright 2002 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition Is protected against unauthorized copying under Title 17, United States Code.
Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. Ml 48106-1346 ABSTRACT
C-reactive protein (CRP) is the prototype of the acute phase reactants in
humans and its concentration is used as a measure of inflammatory and infectious
diseases. It circulates as a stable, highly conserved single pentamer of noncovalently associated identical subunits arranged in a flat pentameric disk.
The rapid and pronounced upregulation of CRP gene expression in liver hepatocytes by inflammatory cytokines, as well as the conservation of CRP both in the invertebrates and throughout the vertebrates suggests that CRP may have a role in both innate host resistance and the regulation of inflammatory reactions.
CRP exerts many of the effector activities associated with specific antibody. CRP and CRP-complexes are ligands for specific CRP-receptors (CRP-R) that have been reported to activate cells of the monocytic lineage. Recent studies have revealed that the predominant CRP-R on both human monocytes and neutrophils
(PMN) is the 40 kDa IgG FcyRII. The signaling pathways initiated by binding of
CRP to FcyRII have not yet been examined.
Previously, CRP was shown by this laboratory to inhibit both chemotaxis and the respiratory burst induced by unrelated ligands in PMN. Therefore. I
n wished to determine what signaling events were induced by CRP via FcyRII. In the experiments reported herein, I show that acute phase concentrations of CRP induce two related signaling pathways. The first is a positive signaling pathway in which CRP induces the tyrosine phosphorylation of the ITAM of FcyRIIa, Syk and PLCy2. It promotes PLCy2 and PI-3K translocation to the membrane. The second is a negative signaling pathway in which CRP induces tyrosine phosphorylation of SHIP in a dose-dependent manner and thus promotes the
SHIP-Shc interaction. CRP was also found to inhibit fMLP-induced membrane localization of PLCy2. Based on the kinetics of both positive and negative signaling, I propose a model in which CRP activates PMN by inducing a positive signal in the early stage of infection. In a later stage, CRP induces an inhibitory negative signal to modulate the earlier activation. The overall effect of CRP on
FcyRII-mediated signaling is one of regulating the response of phagocytic leukocytes to inflammatory agonists.
Ill ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. Richard Mortensen for his guidance throughout my graduate training. I would also like to thank the members of my dissertation committee. Dr. Joeseph Krzycki, Dr. Neil Baker and Dr. Pravin
Kaumaya for their comments and feedback on my work. I would especially like to thank Dr. Susheela Tridandapani and Dr. Mark Coggeshell for their generosity in providing me with reagents and more importantly their useful and enlightening discusscions.
IV To my son, wife and my family. VITA
February 8, 1965 ...... Bom in Suzhou, China
1983 ...... Dipolma of Pharmacy The Health Sciences School of Suzhou Medical College
1983-1992 ...... Pharmacist/Researcher Guanji Hospital, Suzhou, China
1994 ...... B.S., Biochemistry Washington State University, Pullman, WA, 99164
1995-1999 ...... GraduateTeaching Assistant Department of Microbiology The Ohio State University, Columbus, OH 43210
1995-Present ...... Graduate Research Assistant Department of Microbiology The Ohio State University, Columbus, OH 43210
VI PUBLICATIONS
1. Maoyen Chi, Susheela Tridandapani, Wanjian Zhong, Mark Coggeshall and Richard F. Mortensen (2001) C-Reactive Protein (CRP) Induces Signaling through IgG Fc-Receptor (Fc R) Ha on HL-60 Granulocytes, J. Immunol. 167: in press.
2. Maoyen Chi, Susheela Tridandapani, Wanjian Zhong, Mark Coggeshall and Richard F. Mortensen (2001) C-Reactive Protein (CRP) Induces Negative Signaling through SHIP in HL-60 Granulocytes, J. Immunol. (In preparation).
ABSTRACTS 1. Signaling via Leukocyte CRP-R:Inhibition of Activation Response. Interaction of Innate and Acquired Immunity, Society for Leukocyte Biology 34th Annual Meeting, Cambridge, Massachusetts. Oct., 2000
FIELDS OF STUDY Major Field: Microbiology/Immunology Minor Field: Computer and Information Science.
VII TABLE OF CONTENTS
Page Abstract ...... II
Acknowledgements ...... IV
Vita...... VI
List of Figures ...... XI
List of Abbreviations ...... XIII
Chapters:
1. Background ...... 1
I. C-Reactive Protein ...... I
A. Introduction ...... I
B. CRP Gene Structure and Expression ...... 5
C. Induction and Synthesis of C R P ...... 8
D. Protein Structure of CRP ...... 9
E. Biological Activities of CRP ...... 11
1. CRP Binding Activity ...... 11 2. Complement Activation ...... 12 3. Opsonization and Phagocytosis ...... 13
vni 4. Effect of CRP on Leukocyte Activities ...... 14 5. Biological Activities of CRP Related to Inflammation.. 16 6. CRP associated diseases ...... 18
n. IgG Fc Receptors ...... 20
A. Introduction ...... 20
B. Fey Receptor classification and structure ...... 20
C. FcyR Signaling ...... 23
1. Overview ...... 23 2. Syk Kinase ...... 24 3. Phospho 1 ypase Cy ...... 25 4. Phosphoinositidyi-3-Kinase ...... 26 5. Negative Signaling via FcyR ...... 28
D. CRP and FcyRs ...... 30
2. CRP Induces Positive Signaling Through IgG Fc-Receptor II on HL-60 Cells ...... 38
A. Introduction ...... 38
B. Materials and Methods ...... 41
1. Reagents and Cells ...... 41 2. Purification of CRP and SAP ...... 41 3. Membrane Localization of PLCy ...... 42 4. Granulocyte Activation and Immunoadsorption ...... 43 5. Tyrosine-phosphory 1 ation Analysis ...... 44 6. Immuno blot analysis ...... 45
C. Results...... 46
1. Effect of CRP on phosphorylation of FcyRIIa ...... 46 2. Effect of CRP on Syk Phosphorylation ...... 47 IX 3. Effect of CRP on the association of FcyRIIa and Syk 48 4. Effect of CRP on PLCy2 Phosphorylation and Membrane Localization ...... 49 5. Effect of CRP on PI-3K membrane translocation ...... 49
D. Discussion ...... 51
3. CRP Induces Negative Signaling Through SHIP-Shc on HL-60 Cells ...... 66
A. Introduction ...... 66
B. Materials and Methods ...... 69
1. Reagents and Cells ...... 69 2. Purification of CRP and SAP ...... 69 3. Membrane Localization of PLCy ...... 70 4. Granulocyte Activation and Immunoadsorption ...... 71 5. Tyrosine-phosphorylation Analysis ...... 72 6. Immuno blot analysis ...... 73
C. Results...... 74 1. Effect of CRP on phosphorylation of FcyRIIb ...... 74 2. Effect of CRP on SHIP Tyrosine Phosphorylation ...... 75 3. Effects of CRP on SHIP-Shc Interaction ...... 76 4. Kinetics of SHIP tyrosine phosphorylation ...... 76 5. Effects of CRP on PLCy2 membrane translocation induced by fMLP ...... 77
D. Discussion ...... 79
Summary...... 90
References ...... 93 LIST OF FIGURES
Figure Page
1. Overview of CRP Structure ...... 34
2. Interaction between CRP and Phosphocoline ...... 35
3. Receptor Binding face of CRP ...... 36
4. Classification of Fey Receptors ...... 37
5. Effect of CRP on Tyrosine Phosphorylation of
FcyRHa in HL-60 (G) Cells ...... 58
5a. Effect of CRP on Tyrosine Phosphorylation of
FcyRfla in HL-60 (G) Cells ...... 59
6. Kinetics of Tyrosine Phosphorylation of FcyRIIa
in Response to CRP ...... 60
7. Effect of CRP on Tyrosine Phosphorylation
of Syk in HL-60 (G) Cells ...... 61
8. Kinetics of Tyrosine Phosphorylation of Syk in Response to CRP... .62
9. Effect of CRP on the interaction between FcyRIIa and Syk ...... 63
10. Effect of CRP on Tyrosine Phosphorylation of
PLCy2 in HL-60 (G) Cells ...... 64
11. Effect of CRP on PLCy2 and PI-3K Membrane Translocation ...... 65
XI 12. Effect of CRP on Tyrosine Phosphorylation of FcyRIIb in HL-60 (G) Cells ...... 84
13. Effect of CRP on Tyrosine Phosphorylation of SHIP in HL-60 (G) Cells ...... 85
14. Effect of CRP on SHIP-Shc Interaction in HL-60 (G) Cells ...... 86
15. Kinetics of Tyrosine Phosphorylation of SHIP in Response to CRP ...... 87
16. Effect of CRP on PLCy2 Membrane Translocation
induced by fMLP ...... 88
17. CRP induced signaling model in PMN ...... 89
XII LIST OF ABBREVIATIONS
Ab Antibody APP Acute phase protein BCR B cell receptor C5a Complement 5 fragment a C/EBP CAT box enhencer binding protein CPS C-polysaccharide CRP C-reactive protein CRP-R C-reactive protein receptor DAG Diacylglycerol DMSO Dimethyl sulfoxide EBSS Earle’s balanced salt solution FcyR Fc gamma receptor fMLP Formyl-Met-Leu-Phe GDP Guanosine diposphate GTP Guanosine triposphate Ig Immunoglobulin IL-1 Interleukin I IL-6 Interleukin 6 IP3 Inositol trisphosphate ITAM Immunoreceptor tyrosine-based activation motif ITIM Immunoreceptor tyrosine-based inhibition motif LPS Li po polysaccharide mAB Monoclonal antibody MAPK Mitogen activated protein kinase PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PC Phosphocoline PH Plekstrin-homology PI-3K Phosphatidylinocitol 3-kinase PKC Protein kinase C PLCy Phospholipase C gamma PMA Phobol myristate acetate PMN Polymorphnuclear neutrophils xin PTB Phosphotyrosine-binding domain PTK Protein tyrosine kinase SAP Serum amyloid P component SDS Sodium dodecyl sulfate SH2 Src homology domain 2 SH3 Src homology domain 3 SHIP SH2 domain containing inositol 5-phosphatase TBS Tris-buffered saline TNF-a Tumor necrosis factor a Tyr-P Tyrosine phosphorylation
XIV CHAPTER 1
BACKGROUND
I. C-Reactive Protein
A. Introduction
The acute-phase response of inflammation is the immediate systemic response to tissue injury or bacterial infection and is considered part of “innate immunity”. Kushner (1) recently defined the acute phase response as a series of metabolic changes that results in the dramatic reorchestration of the synthesis and secretion of a group of plasma proteins, known as acute-phase proteins (APP). by liver hepatocytes in response to circulating inflammatory cytokines. .APP encompasses a broad range of proteins, including complement proteins (C2 through C9), protease inhibitors (otl-antitrypsin, o2-antiplasmin), coagulation proteins (fibrinogen), metal-binding proteins (ceruloplasmin), and certain response specific proteins (lipopolysaccharide-binding protein). Human C- reactive protein (CRP), named for its capability to bind to the C-polysaccharide
(CPS) of the pneumococcal cell wall, along with serum amyloid A (SAA) protein, are the two most pronounced APPs in humans. CRP was initially discovered at the Rockfeller Institute in 1930 by Avery and Francis as a precipitin of CPS (2). It is widely present in the
blood of patients with acute pneumonia. Unlike Immunoglobulin (Ig) antibodies,
CRP is present at the earliest stages of infection. Upon infection, its plasma
concentration increases quickly up to as much as a 1000-fold within 24 hours compared to normal (1) and rapidly decreases with the resolution of inflammation
(3, 4. 7). Thus, CRP is used as a clinical marker for acute inflammation in many diseases such as rheumatoid arthritis and the vasculitides. In fact, the increased synthesis of CRP by hepatocytes is used to monitor the presence of subclinical infection and evaluate the effects of treatment with anti-inflammatory drugs.
It should be pointed out that the pattern of APP varies among different species. Although CRP is the major APP in humans and where its concentration various dramatically during inflammation; whereas the level of the serum amyloid
P component (SAP), a structurally related protein, remains virtually unchanged.
In contrast to humans, the APP in mouse display the opposite pattern where mouse CRP only increases 2 to 3-fold, but mouse SAP can increase up to 50-fold with the kinetics similar to that of human CRP (5-7). The production of CRP is induced by a small number of “pro-” inflammatory cytokines, such as interlukin-I
(IL-1), IL-6 and tumor necrosis factor a (TNFa) (8, 9). Among these cytokines,
IL-6 is the most potent stimulus for the new synthesis of CRP by the liver. Characterized in all vertebrate species by its Ca'^-dependent ligand
binding to the phosphocoline (PC) moiety of the pneumococcal CPS. CRP is a
member of the conserved and phylogeneticily ancient superfamily of pentameric
proteins officially named as pentraxins. These proteins are highly conserved and
appear prior to the development of the adaptive or clonal immune response.
Pentraxins have been found in virtually all mammals and birds, amphibians and marine teleosts. The most primitive creature in which CRP has been found is the arachnid Limiiluspolyphemus (horseshoe crab) where it is better known as limulin
(10). CRP is a stable, cyclic pentameric protein consisting of five identical, non covalently linked subunits, each subunit contains 206 amino acids in a single poplypeptide chain and assembles into a flat pentameric disk (11).
Mediated through its PC-binding sites, CRP binds to a broad range of multivalent ligands that include damaged cells, certain bacteria and chromatin
(12). In addition to PC-containing ligands, CRP has also been reported to exhibit significant calcium-dependent and PC-inhibitable binding to eukaryotic cell membrane constituents. It also binds certain nuclear constituents that do not contain the PC moiety, such as small ribonucleoprotein particles (snRPs) (12).
Many biological activities of CRP are associated with its specific IgG-like binding activities. It has been reported that CRP exerts a wide range of biological effects including activation of macrophages, the classical complement (C) pathway and platelets, opsonization of particles, induction of cytokine secretion and protection from microbial infection and the development of autoimmunity.
CRP was found selectively deposited at sites of inflammation where it accumulates (Reviewed in 14). Similar to antibodies, CRP binds and opsonizes some infectious pathogens and damaged host cells for phagocytosis via specific membrane receptors on leukocytes. CRP peptides generated by surface proteases on neutrophils at inflammation sites were found to modulate activities of leukocytes (14). CRP is a ligand for specific receptors on leukocytes, activating monocytes/macrophages and enhancing superoxide production by macrophages
(13), but inhibiting the chemotactic activity and the respiratory burst of neutrophils (PMN) (14. 15). CRP expressed as a transgene in mice has been shown to contribute to innate host protection by activating complement and mediating phagocytosis (16). CRP also has been shown to inhibit PMN infiltration of inflamed lungs (17), as well as septic shock in a transgenic mouse model (18).
Based on monocyte/macrophage opsonophagocytosis studies and ligand- binding data, leukocyte CRP receptors (CRP-R) were originally thought to be related to IgG Fc receptors (FcR) (19-21); however, more recent studies have revealed that the predominant CRP-R on both human monocytes and PMN is the 40 kDa IgG FcyRII (20-25). The signaling pathways trigged by binding of CRP to
FcyRII have not yet been determined.
B. CRP Gene Structure and Expression
The human CRP gene is a single copy gene located at q21-23 of chromosome I, a syntenic region that contains many genes that play immune and inflammation-related roles (26, 27). These genes include both the high-affinity and low-affinity Fc receptors (FcRs) for IgE and IgG (28-30) and both genes for the blood pentraxin family. In humans, CRP gene consists of two exons separated by an intron of 278 base pair (bp). The first exon encodes a leader peptide plus the first two amino acids of the mature protein product; the second exon encodes the remaining 204 amino acids (31). The transcribed portion of the CRP gene includes a long 3’-untranslated region (UTR), two heat-shock consensus sequences in the 5’-UTR and a long (GT)„ repeat region in the intron that might be involved in the regulation of gene expression (31).
The promoter of the human CRP gene contains the classical TATA box sequence (TAT A A AT) and the CAT box sequence (CAAT) at -81 and -29 base pairs upstream, respectively, of the mature mRNA cap site (31). There are two acute phase response elements (APRE), APREl and APRE2, that govern the IL-6 activation and contain the CAT box enhencer binding protein, or C/EBP, consensus sequences TG(G/A)AA from -94 to -50 and -137 to -106 (8). APRE I
bears the binding site for the liver-specific transcription factor, hepatocyte nuclear
factor I (HNFl), while APRE2 contains both an HNFl binding site (P site) and
an additional binding site (a site) for the transcription factor, NF-IL-6, which now
is called signal transducer and activator of transcription factor 3 (STAT 3). (32).
In vitro studies of human CRP gene expression using hepatoma cells have shown
that IL-6 dramatically induces CRP gene transcription (33-35). When IL-6 is combined with IL-ip, there is a synergistic transcriptional response (35. 36). The two IL-6 responsive elements, which are responsible for the cytokine-induced expression of human CRP, are proximal to the transcription initiation site. ST AT
3 is activated by EL-6 and phosphorylated in a PKC-dependent manner at its Ser
105 position (37). The STAT family members play a major role in mediating IL-
6-induced transcription. In human CRP gene there is a binding site for STAT 3 within the first 157 bp of the promotor (39, 40), which contains the TT(N) 4 /5 AA sequences that are capable of binding STAT proteins. Induction of human CRP expression by STAT 3 occurs via a cis-acting element centered at the minimal promoter region (39). The binding of IL-6 to its receptor complex leads to the phosphorylation of Janus kinases or JAK, which rapidly (15-60 min) phosphorylates STATS and then induces its dimerization and translocation to the nucleus (37). Activated STATS then binds to the specific response elements in the promoter regions of cytokine responsive genes. This laboratory has demonstrated
a consensus STAT 3 binding sequence that is functional in the promoter region of
the mouse CRP gene (32).
IL-1 also plays a regulatory role in CRP gene transcription. There is an IL-
1-responsive sequence between -42 and +15, which has been identified in the
human CRP gene that is believed to act as a transcription modulator (8). [L-I
regulated transcription of the human CRP gene is via the “acute box”, a c/x-acting sequence motif in the 5’UTR of ferritin mRNAs (41). Recently using transactivation assays, Kushner group (42) reported that transiently transfected
Hep 3B cells that over-expressed the Rel family protein p50, one of the heterodimer p65/p50 of the NF-k B transcription factors, displayed a remarkable increase in CRP gene transcription. In the presence of the over-expressed p50,
CRP expression was enhanced to the greatest extent in response to a combination of IL-1 P and IL-6. In contrast, over-expression of p65 abolishes the effects on
CRP induction by p50. In conclusion, these studies on CRP gene expression indicated that complex interplay of transcription factors act differently with acute phase genes vs. Ig genes. c. Induction and Synthesis of CRP
The dramatic increase in the plasma concentration of CRP is detectable as
early as four hours after tissue injury or infection and reaches a maximum level
within 24 to 48 hours (43). The rapid de novo synthesis and secretion of CRP by
liver hepatocytes accounts for almost all of its increasing in blood level. The fact
that a remote injury triggers quick elevation of CRP by hepatocytes implies a sensitive humoral mechanism for induction of the acute phase response. Indeed, several reports have shown that induction of CRP synthesis is mediated by various inflammatory cytokines such as DL-I and CL-6, but not tumor necrosis factor-a (TNF-a). IL-6 is the potent cytokine that induces CRP production.
Several investigators have identified the IL-6-responsive elements (8. 9. 45), along with a trans-acting IL-6-inducible nuclear protein that binds to the IL-6- responsive consensus element, at the 5’-flanking region of the CRP gene (38).
Samols, et ai. (44) have reported a novel mechanism at the post-transcriptional level for the rapid release of sequestered CRP by rabbit hepatocyts. They found that rabbit CRP mRNA could form a stable intramolecular duplex, which interferes with its in vitro translation capability, indicating that the concentration of translatable CRP mRNA was dramatically induced during the acute-phase response (44). In addition to IL-6 and IL-1, it has been reported that transforming growth factor P-1, interferon-y and the combination of all theses cytokines could
affect the regulation of CRP synthesis (46-48).
D. Protein Structure of CRP
CRP circulates as a pentamer of noncovalently associated subunits with
identical amino acid sequences, each consisting of a single polypeptide of 206
amino acids (49). The pentameric tertiary structure consists of a flat annular disk
of the subunits (Figl). The resolution of the crystal structure of CRP revealed
that each of the subunit has a flattened jellyroll appearance similar to that of Con
A, which consists 14 antiparallel 3-strands arranged into two 3-sheets with two
Ca^^-ions ligated into close proximity to each other contributing to the single PC-
binding site on one face of each subunit (50-52) (Fig2). All five of the lectin-like
PC-binding sites are located on the same "recognition-face" of the pentamer (53); whereas, the opposite face of the pentamer that contains a short a-helix is the
"effector-face" since it has the sites for Clq and leukocyte CRP-receptor (CRP-R) interactions (53-54) (Fig3). The crystallography study also showed that the first calcium-ligation site is mediated by residues of Asp 60, Asn 61, Glu 138, Aspl40 and the main peptide chain carbonyl group of Gin 139 (55). The second calcium- binding site is coordinated by Glu 138, Asp 140, Gin 150 and Glu 147. The more complete coordination of the second calcium-binding site suggests that this site is important for ligand binding (55). An additional crystallography study of CRP-PC
complex indicated that the PC-binding site on CRP is mediated by a phosphate-
calcium interaction, in which the phosphate binding at the calcium site is coordinated by a hydrophobic interaction to the three methyl groups of choline and the exposed face of a hydrophobic pocket formed by Phe 66, Leu 64 and Thr
76 (56) (Fig. 2). The side chains of Ser 68, Ser 74 and Glu 81 are all positioned at the opposite end of this hydrophobic pocket vs. the calcium-binding sites with the reorientation of the Glu 81 side chain optimizes the interaction with the choline nitrogen (56). By the short a-helix (residues 168-176) on the “effector-face”, there is also a deep, extended cleft, suggesting that this site could be involved in a ligand binding interaction (53-54) (Fig. 3). The walls and rim of this cleft are formed on one side by parts of the N and C termini and on the other side by the
177-182 loop and the C-terminal end of the a-helix. The cleft starts roughly at the center of the protein and extends to the central pore of each subunit of CRP.
Recent site-directed mutagenesis studies clearly shown that the actual Clq binding site is located at the pocket of the open end of the cleft (57). Aspll2, which is located at the relatively open end of the cleft towards the center of the pentamer, along with Tyrl75 originating from the helix, are the two important contacting residues for Clq binding, but the adjacent Lys 114 also interferes with the Clq binding, suggesting a possible role of the residue in the regulation of
10 CRP-Clq interactions (57). Because complement activation and opsonization by
CRP requires binding of CRP to an appropriate muitivalent ligand at the PC-
binding site, this cleft, which is on the opposite face from the "recognition-face"
for PC-binding, is most likely involved in Clq and FcyR. interaction and could be involved in CRP-leukocyte interactions. Thus, the structural features of CRP are consistent with a multifunctional blood protein that e.xerts its activities during the earliest stages of nonspecific or innate host response.
E. Biological Activities of CRP
I. CRP Binding Activity
The physiological functions of CRP are closely associated with its versatile recognition and binding activities for a wide range of biological molecules that contain the PC moiety or monophosphate esters. In addition to the
PC-containing ligands, CRP also exhibits significant calcium-dependent, PC- inhibitable specific binding to several eukaryotic cell constituents. CRP has been reported to bind chromatin (58) and certain histones such as H i, H2A and H2B
(59) under non-physiological conditions, to snRNPs (60-61) under physiological conditions (61), and to galactose-containing polysaccharides (62). The earliest binding specificity discovered for CRP was that it precipitated the pneumococcal
CPS in a manner analogous to anti-CPS antibodies (63-64). Subsequent studies revealed that a specific calcium-dependent, PC binding site plays a central role in 11 this interaction (65). The ubiquitous presence of PC within cells of bacteria, fungi,
parasites and membrane phospholipids such as lecithin and sphingomyelin in
eukaryotic cells suggests a potentially important role for CRP in the clearance of
exogenous infectious agents and endogenous damaged cells ( 6 6 ). The lectin-like
binding reactivity of CRP directed toward the PC-moiety serves as the defining
activity for CRP in all species.
2. Complement activation
The capability of CRP to activate the C pathway is well-documented (67-
84). Ligand-complexed CRP that is capable of activating C pathway was initially reported by Kaplan and Volanakis (67), as well as Siegal, et al. ( 6 8 ). The latter study showed that CPS complexed CRP could bind to human Clq and induce the full activation of the classic C pathway resulting in hemolysis (7). Later studies indicated that CRP complexed with phosphotidylcholine-containing liposomes, certain polycations, and nuclear DNA also activated the classical C pathway (69,
80). The effects of CRP on the alternative C pathway were not well defined until recently even though ligand-complexed CRP was reported to inhibit the activation of the alternative pathway in vitro (71). Since ligand-complexed CRP efficiently activates the classical human C pathway, this process can lead to the uptake of the
CRP-bound complexes by phagocytic cells. The earliest studies reported that CRP
12 induced C activation is restricted to C l, C4, C2 and C3 with little consumption of
C5-9 (72, 73). More recently, it was shown that CRP binds directly to the C regulatory protein, factor H (74), which in turn inhibits both the alternative C pathway and the newly discovered mannan-binding lectin (MBL) C pathway (74).
As a result of this binding, C3 and C5 convertase activities, as well as the deposition and generation of C5b-9 in the alternative pathway are reduced: the deposition of C3b in the MBL C pathway is also reduced (74).
3. Opsonization and Phagocytosis
Another important IgG-like binding activity of CRP is its binding to phagocytic leukocytes that result in the modulation of their phagocytic behavior.
More than fifty years ago, George Lofstrom (85) discovered that human acuie- phase serum containing CRP provided transient protection to mice against pneumococcal infection. Subsequent studies by others demonstrated that the phagocytic rate of leukocytes could be increased by partially purified CRP ( 8 6 ,
87). A more definitive study by Mortensen, et al. ( 8 8 ) using purified CRP bound to CPS-coated erythrocytes showed that CRP was required for ingestion by mouse macrophages and human monocytes, i.e. CRP was directly opsonic. Later in vivo experiments using mice challenged with pneumococci demonstrated that the protective effects of CRP were attributable to both its opsonic and complement
13 activation properties (89, 90). Szalai, et al. (91, 92) reported that human CRP transgenic mice are resistant to a lethal dose of Streptococcus pneumoniae and
Salmonella enterica serovar Typhimurium. In the latter study, it was found that transgenic mice infected with the virulent Typhimurium strain lived longer and had significant lower mortality than the non-iransgenic mice (92). In the CRP transgenic mouse, the blood clearance of salmonella was more rapid (0 - 4 h) and the number of living bacteria in the liver and spleen was less than the control after
7 days of challenge. Recently, Lysenko, et al. (93) showed that CRP mediates killing through the activation of complement when bound to Haemophilus influenzae. All of these reports clearly suggest that CRP plays an important role in early host defense against infections by accelerating the clearance of pathogens and perhaps enhancing the humoral branch of the immunity during later stage of infection.
4. Effect of CRP on Leukocyte Activities
Early observation of the “anti-inflammotory” effects of CRP came from the experiments in which transgenic mice expressing rabbit CRP were protected against PMN-infiltrating alveolitis under non-inflammatory conditions (94).
These same transgenic mice were also protected from endotoxin shock induced by lipopolysaccharide (LPS) and platelet-activating factor (PAF), however CRP did
14 not neutralize the PC-bearing PAF (95, 96). Buchta, el al. (97) observed that CRP
inhibited the superoxide (O'z) production and the secretion of vitamin 812
binding protein stimulated by phorbol myristate acetate (PMA). A subsequent
study demonstrated that CRP not only decreased the activation effects of PMA on
0 ~ 2 production, but also inhibited the 0 "% production stimulated by the bacterial
chemotactic peptide, formyl-methionine-leucine-phenylalanine (fMLP) (98). In
this study, CRP was also found to significantly reduce phosphorylation trigged by
PMA or fMLP (98). These data suggested that CRP could regulate the neutrophil
activities in a negative feedback manner. Later studies found that CRP inhibited
chemotoxis induced by fMLP by blocking the signal transduction pathway of p38
mitogen-activated protein kinase (p38 MAPK), which would ultimately lead to
the loss of actin polymerization and cell motility (99). Since CRP selectively accumulates at inflammatory sites where EL - 8 is produced, this laboratory examined the effects of CRP on the responsiveness of PMN to EL - 8 and fMLP, and found that purified human CRP inhibited the chemotactic response of PMN to
EL- 8 and fMLP (15). Furthermore mAh that was generated against the leukocyte
CRP receptor (CRP-R) also inhibited the chemotactic response. A synthetic CRP peptide (residues 27-38) that binds to the CRP-R had weak chemotactic activity, but two other CRP synthetic peptides (residues 174-185 and 191-205) inhibited chemotaxis of PMNs to both EL - 8 and fMLP (15). Zhong, et al. (15) found that
15 CRP inhibited PMA-induced O 2 production more efficiently than fMLP-triggered response. Upon closer examination, CRP inhibited the protein kinase C (PKC)- dependent assembly of the NADPH oxidase complex at the level of both phosphorylation and translocation of PKC-beta2 to the membrane at an acute phase threshold concentration of approximately 50 pg/mL of CRP. Translocation to the membrane and serine-phosphorylation of the major cytosolic p47-phox component of the NADPH oxidase complex was inhibited by CRP. CRP also inhibited membrane localization of activated Rac2, the low m.w. G protein
(GTP’ase) regulator for the assembly of the oxidase components in activated neutrophils. These results suggest a role of CRP as a regulator of leukocyte infiltration at inflammatory sites. By contrast, the activation effects of CRP to monocyte/macrophage cells lineage ( 1 0 0 ) suggest different regulation of these two leukocyte populations at the level of signaling.
5. Biological Activities of CRP Related to Inflammation
CRP was first reported to have tumoricidal activity in 1982 by Barta
(101). Using a metastatic tumor model C57BL76 mice, liposome-containing CRP significantly reduced the size and number of métastasés (101). A follow-up study showed that CRP incorporated into multilamellar vesicles activated macrophages that in turn displayed nonspecific tumoricidal activity against the syngeneic
16 T241 fibrosarcoma, B-16 melanoma cells and allogeneic Sarcoma I cells (102).
The same group later reported that only one of several synthetic peptides derived from CRP could mimic the antitumor effects of native CRP by activating both monocytes and alveolar macrophages in mice (103). In 1986, Zahedi and
Mortensen (104) also reported that purified CRP activated inflammatory mouse macrophages in vitro to a tumoricidal state and that the tumoricidal activation by
CRP was not due to any contaminating lipopolysaccharide, but rather that CRP at
10 fxg/ml induced significant tumoricidal capacity in resident macrophages (104).
CRP also exhibited protective effects against septic shock. Using transgenic mice expressing high levels of CRP, Samol’s group observed that the mice were remarkably protected from a lethal challenge of LPS (18). This group also observed similar protection with challenges from PAF and the combination of TNF-a plus IL-ip, but not with TNF-a alone, although PAF was able to bind
CRP. The mechanism by which CRP provides protection probably does not involve sequestration of PAF (18). The biologically inactive precursor of PAF, lyso-PAF, also bound CRP but did not render the transgenic mice sensitive to
PAF when CRP-expressing animals were simultaneously challenged with PAF and an excess of lyso-PAF (18). These results suggest that CRP’s protective effect against septic shock in vivo may involve negative signaling pathways in leukocytes.
17 CRP also was reported to enhance cytokine production by monocytes.
Using purified LPS-free CRP, Pue, et al. (105) found that human PBMC and lung
macrophages secret low level of IL-ip in response to either CRP (250 mg/ml) or
LPS (100 ng/ml). However, when CRP and LPS were combined, the secretion of
EL-ip by PBMC was increased in a synergistic manner that disappeared if CRP
was immunodepleted. CRP alone did not stimulate lung macrophages to produce
IL-ip or IL-lra. When combined with LPS, CRP Inhibited EL-ip and IL-lra release induced by LPS. These data suggest that acute phase levels of CRP may have divergent effects depending on the target population. CRP may on balance be largely pro-inflammatory to blood monocytes responding to LPS.
6 . CRP associated diseases
The majority of inflammatory leukocytes infiltrating the arterial wall in early atherogenesis are monocytes (106). By contrast, very few neutrophils are present in the lesion, unlike any other inflammatory site. Since CRP is the prototype acute-phase reactant in humans, the association of CRP with coronary artery disease has been explored in terms of a risk factor, prognostic marker, and now as a potential contributor to atherogenesis. A modest elevation of CRP blood levels is observed to be associated with patients with severe unstable angina
(107). This CRP level elevation also predicts an increased risk for myocardial
18 infarction (MI) and mortality in both MI and stroke patients (108). The concentration of CRP also correlates with other risk factor of coronary disease in hyperlipidémie patients and in normal subjects (106, 108).
Substantial new evidence has suggested that CRP may contribute to inflammation in atheroma and also may be actively involved in early atherogenesis. CRP displays Ca'^-dependent in vitro binding to LDL and VLDL
(109) and activates the complement system (74). Native CRP is found deposited at human atherosclerotic lesion site in human coronary arteries along with the terminal complement components C5b-9 (106, 108). CRP is also found co localized with foam cells in fatty streaks, suggesting an interaction of CRP with monocytes entering the early atherosclerolic lesions (110, 111). However, the pathological implication of these interactions are not understood yet, but of considerable interest to the heart disease research community.
19 II. IgG Fc Receptors
A. Introduction
Membrane receptors for the Fc domain (FcR) of Ig antibody molecules are
expressed on many hematopoietic cells and exist for each of the following Ig
isotypes IgG (FcyR), IgE (Fee) and IgA (FcoR). These FcRs all belong to the
immunoglobulin gene superfamily (112, 113). All of these receptors have a single
Fc-binding polypeptide chain (a-chain) that is folded into either two or three Ig domains in the extracellular portion of the receptor. Some of these receptors are also comprised of associated polypeptides needed to mediate signal transduction.
Cross-linking of these FcR with bivalent or muitivalent ligand links the humoral and cellular branches of immunity (115, 118). Among the three type of FcR, the
FcyR for IgG is the most widely distributed, extensively studied and the most relevant to the study with CRP reported herein.
B. Fey Receptor classification and structure
Human FcyRs are divided into three subclass, FcyRI, FcyRII, FcyRIII based on their relative affinity for IgG, gene structure, molecular size, expression pattern and recognition by specific monoclonal antibodies (114-116). All the three subclasses are members of the Ig gene superfamily since the extracellular domains that mediate the specific IgG binding share extensive sequence and
20 structural homology with each other and the Ig themselves. Most of the structural
differences are located on the cytoplasmic domains, suggesting their involvment
in different signal transduction mechanisms. (115, 117). FcyRI and FcyRIII are
oligomers that contains the a chain that bears the IgG binding domain, and are
associated with the dimers of the common y chain and Ç chains that are used by several immune receptors for signaling (Fig. 3) (118). FcyRII is a monomer and like FcyRin, it binds to immune complexes with low affinity, whereas FcyRI is classified as a high affinity receptor capable of binding monomers of IgG (115).
The genes A, B and C that encode these receptors map to chromosome 1 at q2I-
23, close to the same region where CRP gene is located (rev in 115). FcyRI and
FcyRII are encoded by three distinct genes: A, B and C, while FcyRIII is encoded by two genes A and B (115).
FcvRI. Human FcyRI (CD64) is a 72-kDa sialoglycoprotein. Its extracellular portion contains three Ig-like domains that are responsible for the high-affinity binding to IgG monomer (Fig.3). This receptor is expressed on macrophages, monocytes and interferon-y stimulated neutrophils.
FcyRII. Human FcyRII (CD32) is a 40-kDa sialoglycoprotein with a single membrane spanning polypeptide chain that has an extracellular portion that contains only two extracellular Ig-like domains (Fig.2). It binds only to muitivalent ligands or multimers of IgG with low affinity. FcyRII has three
21 isoforms: FcyRIIA, FcyRUB and FcyRIIC, which are the alternate splicing
products of its three genes. FcyRIIA bears the immunoreceptor tyrosine-based
activation motif (FTAM) in its cytoplasmic tail (Fig. 3). The ITAM motif contains
two pairs of tyrosine and leucine residues within the consensus sequence D/E-X7-
D/E-X2 -Y-X-X-L-X7 -Y-X-X-L, where X denotes any amino acid (115). This
sequence is both sufficient and necessary for signal transduction (115. 118).
FcyRIIA is expressed mainly by monocytes, macrophages and granulocytes and it
is the only FcyR found in platelets. FcyRIIB, on the other hand contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplamic domain
and is the only FcyR that has an intrinsic ITIM motif (119, 120). Contrast to
FT AM that contains two Y-X-X-L sequences, the consensus amino acid sequence
of the ITIM is a single (He/Val/Leu/Ser)-X-Y-X-X-(Leu/val), with X representing
any amino acid. FcyRIIB is predominantly expressed in T and B cells (119, 120).
FcyRIIC is the result of unequal cross-over between FcyRIIA and FcyRIIB, which
forms a hybrid that contains the extracellular and transmembrane domains of
FcyRIIB and the tail of FcyRIIA (121).
FcvRITT . Human FcyRIII (CD 16) is also a low affinity IgG receptor that binds only to IgG-containing complexes or IgG multimers. It has two isoforms,
FcyRHIA and FcyRIilB (Fig. 3) FcyRHIA is a receptor with a transmembrane portion and a short cytoplasmic tail. It is mostly expressed in natural killer (NK) on cells and macrophages, whereas FcyRIIIB (CD 16) is a
glycosylphosphatidylinositol (GPI)-anchored receptor that lacks the
transmembrane segment and cytoplamic tail (122). FcyRIIIB probably associates
with other receptors or co-receptors to transmit signal and to function.
Both FcyRI and FcyRIII exist as mutimeric complexes. In both of the FcyR
classes, the ot-chains that contain the IgG binding domain are associated with
heterodimers of homologous disulfide-linked y or ^ chain that contain the ITAM
motif (Fig.I). By contrast, the two FcyRII receptor subclasses exist only as
monomers with the important structural difference in their cytoplasmic domains
as the ITAM motif (FcyRIIa) or the ITIM (FcyRIIb) (118).
C. FcyR Signaling
1. Overview
Although FcyR structures are well established, the precise physiological
functions and roles played by each of the FcyR classes are still being defined. The present state of knowledge suggests that each of the receptors mediate distinct signal transduction pathways and consequently different biological activities (123,
124). It is still unknown whether a particular FcyR isoform or class mediates a unique effector function. However, one function shared by all three classes is the ability to mediate phagocytosis (125). 23 A general summary of the activation signaling events initiated by FcyRs
are as follows: Receptors are initially activated by clustering them with immune
complexes or IgG-opsonized particles that bring the FcyRs together in lipid rafts.
Both tyrosine residues in the ITAM motif of the cytoplasmic domains of these
receptors or FcyRI associated protein then become tyrosine phosphorylated (Tyr-
P) by probably Syk or Lyn family kinases (126). The phosphorylated tyrosines in
the ITAM motif serve as docking sites for protein tyrosine kinases (FTK) of the
Syk or Src families, or both. In turn, these PTKs undergo autophosphorylation
and become activated at these sites as a result of binding to the ITAM (127).
These activated PTKs then phosphorylate and activate a series of substrates including PTK Syk or Lyn, phospholipase C (PLC)y; phosphoinositol-3-kinase
(PI-3K) and the p38 mitogen-activated protein kinase (MAPK). The most relevant properties for each of these pathway components are described below.
2. Syk Kinase
The Syk family of PTKs are expressed by all hematopoietic cells and are essential for signal transduction for many cell surface immune receptors (128).
Syk has tandem Src homology domain 2 (SH2) domains in the N-terminal half of the molecule (rev 199) that is involved in association with the Tyr-P in the ITAM motif after receptor aggregation. Tyr-P of Syk and the consequent activation of
24 Syk enzymatic activity is one of the most rapid and earliest events after receptor
ligation (130, 131). The binding of Syk to the Tyr-P in ITAM results in a
conformational change and consequently increases its kinase activity (131. 132),
although the Src-family kinase can also phosphorylate and activate Syk (132). In
vivo, Tyr-P of Syk parallels the increase of its kinase activity. Thus, a possible pathway for immune receptor signaling is the binding of the SH2 domains of Syk to Tyr-P in ITAM followed by either auto-phosphorylation or Lyn-dependent phosphorylation of Syk, both events can contribute to the activation of Syk (132.
133)
3. Phospholypase Cy (PLCy)
PLC activation is one of the most common transmembrane signaling events elicited by receptors that regulate many cellular processes, including proliferation, differentiation, metabolism, secretion, contraction and sensory perception (rev in 139, 140). There are ten mammalian PLC isozymes identified to date that are all single polypeptides and can be divided into three classes, PLC-
P (contains 4 isoforms), PLC-y (2 isoforms) and PLC-5 (4 isoforms) (139-143).
PLC-y is the major isozyme that is tyrosine phosphorylated and activated by receptor PTK or nonreceptor PTK in response to ligation or cross-linking of membrane FcRs in different cell types ( 140). All three PLC isozymes catalyze
25 the hydrolysis of inositol-containing phospholipids, primarily, the
phosphatidylinositol-4,5-bisphosphate (PtdIns( 4 ,5 )P2 ). PLCy contains a variety of
protein-protein interaction domains specifically, the SH2 that permits Tyr-P
binding, the Src homology domain 3 (SH3), and also a single pleckstrin homology
(PH) domain that is responsible for protein-lipid interactions (R e v in
Activation of PLCy promotes its translocation from cytosol to the inner part of membrane where it "docks" via its PH domain to its major substrate PIP 2 , located on the inner lipid leaflet of the membrane. Upon activation, PLCy cleaves
PtdIns( 4 ,5 )P2 into two potent secondary lipid messengers; 1,2-diacylglycerol
(DAG) and inositol-1,4,5-triphophate (IP3). These two low m.w. messengers are well-documented activators of protein kinase C (PKC), and mobilize intracellular stores of Ca2+ via intracellular IP3 receptor (IP3R), respectively (142). Thus.
PLCy is a key enzyme for propagating an activation signal in the phagocytic leukocytes.
4. Phosphoinositide 3-Kinase (PI 3-K)
PI 3-K phosphorylates the hydroxy group at position 3 of inositol ring to generate several different lipid messangers that are implicated in receptor- stimulatea signaling and in the regulation of membrane traffic. Activated PI-3K converts Ptdlns, PtdIns(4)P and PtdIns( 4 ,5 )P2 into PtdIns(3)P, PtdIns( 3 ,4 )P2 and
26 PtdIns( 3 ,4 ,5 )P3 respectively. These three lipid products in turn serve as
intracellular second messengers that activate downstream signaling events. PI-3K
is a heterodimeric protein that contains a regulatory subunit (p85) and a catalytic
subunit (pi 10) that are linked by a single covalent bond (145). Based on their
lipid substrate specificity and structure of the catalytic subunit of PI-3K, the enzyme has been categorized into three classes.
Class I: It phosphorylates Ptdlns, PtdIns(4)P and PtdIns(4.5)P:. However, its preferred substrate is Ptdln(4, 5)P]. This class has been further divided into
Class U and class Ig. The catalytic subunit of this class is a 110 kDa protein
(p 1 lOot, P, Ô or y) that interacts with SH2-domain containing proteins. The p85 regulatory subunit contains the SH2 domain that can bind to Tyr-P protein in the membrane, inducing an activation of the enzymatic activity in the pi 1 0 subunit
(146). This subunit also contains a SH3 domain and a breakpoint cluster region
(BCR)-homology domain (BH) for which the binding partner and biological role are not yet clear.
Class H: This is a 200-kDa enzyme. Its substrates are Ptdlns. PtdIns(4)P. but not Ptdlns( 4 ,5 )p 2 . The unique structural feature of this class is the carboxyl terminal C2 domain, which is responsible for its Ca'^-independent binding to lipids (146, 147).
27 Class HT: PI3-K is homologous to the yeast version of PI-3K. Its only
substrate is Ptdlns.
Class I PI-3K is the most often examined in leukocytes and therefore
most relevant to the study herein.
5. Negative Signaling via FcyR.
Among all the FcyRs, FcyRIIb is the only receptor that contains the ITIM
motif. The inhibitory pathway triggered by FcyR signaling are thought to be
mediated solely by the ITIM-containing FcyRIIb (Rev in 148. 149). The initial signaling events are similar to that of the activation pathway of FcyR signaling via an ITAM. Cross-linking of the FcyRIIb with another ITAM-bearing receptor induces the Tyr-P in the ITIM by the protein tyrosin kinase, Lyn (148. 149). The
Tyr-P on the ITIM of this receptor can in turn serve as a docking site for the SH2- domain containing inositol phosphatase (SHIP) (151-156). The consequence of the docking of SHIP to the receptor and its phosphorylation is the activation of this enzyme. The precise role of SHIP has not been fully established; however, it is thought that the phosphatase activity of SHIP hydrolizes PIP 3, which induces the dissociation of PH-domain-containing enzymes like PLCy from the membrane
(150. 154). To date, SHIP and FcyRIIb are the only proteins found to be tyrosine phosphorylated exclusively under negative signaling conditions in B cells when
28 the Fab fragment of a IgG ligand binds to the antigen receptor while the Fc part
bind to FcyRs. Coggeshall, et al. (155) reported that SHIP in B cells was recruited to the ITIM through its N-terminal SH2 domain. This SHIP-ITIM interaction triggers the subsequent binding of SHIP to She, an adaptor protein that contains a SH2 domain and a Tyr-P binding domatin (PTB). She is commonly associated with negative signaling after the dissociation of SHEP-ITIM due to the relatively high affinity between SHIP and She (155). Recently, Coggeshall’s group observed that clustering of either FcyRIIa or FcyRI is also effective in inducing SHIP phosphorylation and found that SHIP binds in vitro to a phosphorylated ITAM motif (155-157). This led to the proposal that SHIP inhibits
FcyR-mediated signal transduction by engaging ITAM motif-containing cytoplasmic domains of FcyRIIa and FcyRI-associated y-chain. These information strongly suggested that SHIP plays an essential role in mediating negative signaling.
29 D. CRP and FcyRs
Earlier investigators proposed the presence of specific CRP receptors on
leukocytes based on the functional activity of CRP-mediated phagocytosis ( 8 8 ,
158-160). Radioligand binding studies with CRP confirmed this speculation (161.
162). It was found that the binding of CRP to leukocytes and various human
leukocytic cell lines was calcium-dependent, rapid with a Tt / 2 < 0.5 min and had
an average affinity of 3 to 10 x 10~® M (Kd) for neutrophils and 1 x 10 M for
monocytes (19-21). Flow cytometry studies also confirmed that labeled CRP
specifically binds to 60% of human peripheral blood monocytes (163, 164) and
40% of the neutrophils (165).
Early observation of the inhibition of CRP binding to neutrophils by heat-
aggreated IgG led to the hypothesis that the CRP-receptor was somehow related
to IgG FcRs or possiblely IgG itself (161. 162). Other reports indicated that there
was no inhibition by monoclonal anti-FcyR antibodies (166). The initial findings on the interaction between CRP and leukcytes came from the observation that erythrocytes coated with C-polysaccharide (CPS) and reacted with CRP (E. CPS-
CRP), upon the addition of C or purified C components attached to both human B lymphocytes and peripheral blood monocytes ( 8 8 ). This interaction could also be reduced by blocking monocyte receptors with aggregated human y-globulin
(HGG), suggesting that this intereaction might be mediated by FcRs (164). Using
30 the same E.CPS-CRP model, Gewurz, et al. (219) found that heat-modified CRP
binds to PEL, T cells and B cells with a preferential overlap with IgG FcR-
bearing cells by microscopic visualization, indicating that CRP-binding cells
predominantly represent a subset of cells bearing FcR (2I9).The Mortensen
laboratory reported the isolation of two CRP-binding membrane proteins of about
40 kDa and 60 kDa from the human myelomonocytic cell line U-937 and that
these proteins appeared to be distinct from FcyRs (167, 168). More recent binding
studies have shown that the binding activity of CRP is associated with its ability
to activate phagocytic leukocytes. Both CRP and CRP-complexes are ligands for
specific CRP-receptors that have been reported to activate cells of the monocytic
lineage (14), but inhibit the activities of neutrophils (PMN) (15-19).
In 1995, Du Clos’s group observed the interaction of CRP with FcyRI in
COS-7 cells transfected with a cDNA encoding the receptor. Furthermore, CRP
that was mutated in the amino acid sequence homologous to the IgG sequence
proposed to interact with FcyRI failed to bind to transfected cells (190). This
study confirmed the binding of CRP to FcyRI and identified a site on CRP that is
essential for this binding. Several more recent studies from the same group have
further revealed that the predominant CRP-R on both human monocytes and PMN
is the 40 kDa IgG FcyRII (23-25). They found that COS-7 cells transfected with human FcyRUA cDNA bound CRP in a dose-dependent and saturable manner
31 consistent with receptor-mediated binding (24. 25). CRP bound to both
transfected cells and K-562 cells with similar kinetics, and in both cases binding
was inhibited by aggregated IgG (25). CRP also specifically precipitated
detergent solubilized FcyRI and FcyRII from the monocytic cell line. THP-1. In a
subsequent study by the same group, the binding of CRP to FcyRIIa on human
monocytes and neutrophils was shown to be allele-specific since CRP binding to
the FcyRUA R-I31 isoform from homozygotes on monocytes and neutrophils was
high, and the binding was inhibited by a R-specific monoclonal Ab (2.3). CRP
displayed much less binding to cells from FcyRIIA H-131 homozygotes (which
bind to Ig02 with high affinity). Furthermore, CRP binding studies using various
FcyR-deficient mice clearly shows that the binding of CRP to murine leukocytes
requires FcyR (22). CRP binding to leukocytes from y-chain-deficient and FcyRII-
deficient mice was reduced compared with binding to leukocytes from wild-type
mice or heterozygous control mice. Thus, the analysis of CRP binding to
macrophages, neutrophils, and lymphocytes provides direct evidence that
FcyRIIb I, FcyRIIb2, and FcyRI are the receptors for CRP on mouse leukocytes.
The most recent study from Du Clos’s group indicates that the phagocytic activity of the FcR y-chain deficient mouse bone marrow macrophages affects the ingestion of SAP opsonized zymosan particles, but not CRP coated particles (22).
CRP also displayed significant binding to peritoneal macrophages from y-chain
32 deficient mice. These results suggest that CRP and SAP have a different binding specificity for the FcyRs in mice in which CRP bind selectively to FcyRII whereas
SAP bind to FcyRI and FcyRIII. However, the phagocytosis activated by SAP is mediated by FcyRI and FcyRIII, not FcyRII. Taken together, these biochemical and genetic studies suggest that the major receptor for CRP on phagocytic cells is the FcyRII.
33 »
Fig. I OverView of CRP structure
34 Fig 2. Interaction between CRP and PC, showing on subunit of CRP
35 Fig. 3 Receptor binding face o f CRP
36 GPI-Anchor
a y/C a y/C A.B.C
Fig.4 Classification of Fcr receptors
37 CHAPTER 2
CRP Induces Positive Signaling Through IgG Fc- Receptor (FcyR) II on HL-60 Cells
A. Introduction
C-reactive protein (CRP) is the prototypical acute phase protein or reactant
in humans and therefore its blood level is used as an indicator of the presence and
severity of an inflammatory, as well as infectious diseases (I). CRP circulates as
a stable, single pentamer of five noncovalently associated identical protomers
arranged in a flat pentameric disk (53, 109). The recent resolution of crystal
structure of both CRP and CRP-phosphocholine (PC) complexes revealed that
each subunit has 14 antiparallel P-strands along a single polypeptide of 206 amino
acids that are arranged into two 3-sheets with two bound Ca'^-ions per subunit, both of which contribute to a single PC-binding site (50, 56). The lectin-like PC- binding sites are all on the same plane or the "recognition-face" of the pentamer; whereas, the opposite face of the pentamer is considered the "effector-face" that has the sites for binding to Clq (57) and perhaps specific for leukocyte CRP- receptors (CRP-R). The rapid and greatly amplified induction of CRP gene
38 expression in hépatocytes is driven by a synergistic interaction between EL-ip and
IL- 6 that induce several transcription factors (42. 57). The conserved biological
activities of CRP are consistent with a role as an effector of innate host resistance
and as a regulator of inflammation (22. 53. 169). This notion is supported by the
well-documented CRP activation of the classical complement (C) pathway
through C3, but inhibition of the amplification loop by recruiting and binding to
factor H (74) (14). In addition, CRP transgenes expressed in mice protect against certain bacterial pathogens by activating the C pathway, mediating their phagocytosis and inhibiting endotoxin (Rev in 12).
The most basic host protective activity shared by CRP and IgG Ab is the mediation of phagocytosis. The specific CRP-R characterized on cell lines corresponding to human phagocytic leukocytes possessed some of the properties of IgG Fc-receptors (FcyR), but were appeared distinct (Rev in 12. 14). Recent evidence clearly shows the major high affinity CRP-R on human monocytes and neutrophils (PMN) is the 40 kDa low affinity IgG FcR class, FcyRII, which has a higher affinity for CRP than IgG. (24, 25). However, CRP-mediated signaling via
FcyRII has not been explored. Since PMN are the most likely phagocytic leukocytes to interact with CRP or CRP-complex at an inflammatory sites (170), the signaling response of differentiated HL-60 granulocytes, a functional PMN- like cell line that possesses abundant FcyRII (171. 172), was examined in terms of
39 cell activation vs. IgG. In the experiments reported herein, I show that acute phase concentrations of CRP triggers the Tyr-P of the IT AM motif present in the cytoplasmic domain of FcyRIIa, activates Syk kinase and PLCy2 by inducing their
Tyr-P, and promotes membrane localization of PLCy2 and PI-3K. These observations provide an explanation for the CRP-mediated cellular activities of mediating phagocytosis.
40 B. Materials and methods
1. Cells and Reagents. The human promyelocytic cell line HL-60 was grown in
RPMI-I640 supplemented with 4% defined fetal bovine serum (FBS) and 6 % supplemented bovine calf serum (BCS) (HyClone, Logan, UT). To differentiate
HL-60 cells into granulocytes (G), ~4 xlO^ cells/ml were incubated with 1.2%
DMSO for 6 or 7 days until >90% of the cells reduced NBT dye and were capable of rapid reduction of ferricytochrome C in response to PMA (171. 173). Protein
G Sepharose*^ beads was purchased from Zymed (Burlingame, CA). .Affinity purified rabbit IgG anti-human PLCy2 (Cat#: sc-407) was purchased from Santa
Cruz Biotechnology. Biotin-labeled Goat-anti-Mouse IgG(y) and peroxidase labled streptavidin were purchased from KPL. Mouse monoclonal (mAb) anti-
FcRUa (IV.3) was gift from Dr. Susheela Tridandapani (Department of Internal
Medicine, Ohio State University). Anti-phosphotyrosine mAb mixture (Py20
(IgG2b), Py72.10.5, and 4G10 (IgG2b) in the ratio of 30:30:1), and rabbit anti- p85 were gifts from Dr. Mark Coggeshall (Oklahoma Medical Research
Foundation, Oklahoma city, OK, 73104).
2. Purification of CRP and SAP. CRP was purified as described elsewhere (15,
84). Briefly, serum SAP was removed from CRP-containing pleural or ascitic fluids by passage through a column of agarose beads. The eluted protein was then
41 passed through a 1 0 ml ( 2 0 mm diameter) column of p-aminophenyl-
phosphocholine (PC)-sepharose, washed extensively with TBS + 2mM Ca++ and
the bound protein eluted in TBS + 10 mM EDTA. A second round of affinity
purification on the PC-bearing matrix was used to remove trace amounts of other
proteins. The concentration of CRP or SAP was determined by ELISA or by RID
with sheep anti-human CRP. The protein was > 99% CRP or SAP based on reactivity in the competitive ELISA and by SDS PAGE. The concentration of endotoxin in the purified proteins was 0 . 1 -0 . 2 endotoxin units/mg protein
(Chromogenic Limulus assay, M. A. Bioprod., Walkersville, MD), corresponding to an LPS concentration of < 0.05 ng/mg of CRP or SAP.
3. Membrane Localization of PLCv. HL-60 (G) cells at lO’ in 100 ul of EBSS buffer (pH 7.4) were incubated on ice for 30 min and then at 37°C for 10 min before stimulation with various concentrations of purified human CRP. Reactions were stopped by sonicating the cells on ice for 1 min and centrifuging at 800x g at
4°C for 10 min to remove nuclei. The supernatants were mixed with a modified extraction buffer of: Tris (50 mM), pH 7.5, EGTA (2 mM), sodium ortho vanadate (10 mM), leupeptin 1 pg/ml, aprotinin 0.2 pg/ml, and fresh PMSF (2 mM); and then centrifuged at 60,000 rpm, 4°C (Beckman, TLA-100 rotor) for 30 min. The membrane pellet was washed once by resonicating in the extraction
42 buffer and centrifuging under the same conditions to remove any traces amount of
cytosolic proteins. The membrane preparations were stored at -70 °C and the
protein concentration measured by the BCA method using BSA as the standard
(Pierce Chemical, Rockford, IL). An equal amount of protein per lane for each
sample was separated by SDS-PAGE and then transferred to nitrocellulose
membranes (Micron) at 60 V. The membrane was saturated in 5% low fat
powdered milk in TBS containing 0.1% Tween-20, incubated overnight with one
of the antibody probes, washed, and then incubated with a HRP-labeled secondary
reagent and the reaction was developed with chemoluminescent reagents
(Kirkegaard-Perry Labs, Gaithersburg, MD). Rabbit anti-human PLCy2 (1:1,000)
was used as the primary Ab. HRP-conjugated anti-rabbit-IgG( 1:2,500) (KPL) or
protein G-HRP (1:4,000, Zymed) were used as secondary reagents.
4. Granulocyte Activation and Immunoadsorption. Differentiated HL-60 cells
were washed twice and then suspended in HBSS containing lOmM HEPES (pH
7.4). Cells (10 X 10® / sample) were incubated at4°C for 30 min and then at 37°C
for 10 min. After that cells were treated with various concentrations of purified human CRP (5 ug/ml - 200ug/ml) for 10 min. In cells treated with both reagents, the procedures were the same except that CRP was incubated first followed by fMLP. In positive control, cells were stimulated with 10 ul of 3 mM sodium
43 orthovanadate solution (1 ul of 33tnM sodium ortho vanadate, lu! of 30% H 2 O2 ,
98 ul of (IH 2 O) for 3 min at 37°C. Cells were then lysed in Triton lysis buffer
(TLB; PBS, lOmM HEPES, lOmM EDTA, and 1% Triton X-lOO, pH 7.4,
supplement with 3 mM sodium orthovanadate, 20 ug/ml aprotinin, 40 ug/ml
leupeptin and 2ug/ml pepstatin A. Insoluble material was removed by centrifugation at 16,000 x g for 1 0 min, and the supemant was immunoadsorbed overnight at 4°C with specific Ab (2ug/ml) mixed with 10 ul of recombinant protein G-Sephorose (Zymed) that were pre-incubated for two hours at room temperature or 4°C overnight and unbound Ab was washed away by ice-cold PBS buffer for three repeats. Following immunoadsorption, unbound proteins were removed with four time washes of TLB plus 1 mM sodium orthovanadate and subject to Westem blot analysis as described below.
5. Tvrosine-phosphorvlation Analysis. Detection of tyrosine-phosphorylation was accomplished by immunoprécipitation of the FcyRJIa, PLCy2, p85 and SHIP from whole cell lysates using 1 ul of each of the following antibodies: mAb anti-
FcyRIIa, rabbit IgG anti-human PLCy2, rabbit anti-p85 or rabbit anti-SHIP and protein G-Sepharose beads (10 pi). The beads were washed 3 times in Dulbecco’s
PBS (D-PBS) plus sodium ortho-vanadate (10 mM) before separation by SDS-
PAGE and immunoblotting. Phosphotyrosine in the proteins was detected using
44 anti-phosphotyrosine cock tail and biotin-labeled Goat-anti-Mouse IgG(y) and peroxidase labled streptavidin. The amount of proteins examined was detected on the same blots by reprobing using the goat Abs (1:2,000) followed by protein G-
HRP (1:4,000). The chemoluminescence intensity was detected and quantified by using a Lumi-Imager™ from Mannheim-Boehringer.
6 . Immuno blot analysis. After elution of adsorbed proteins from immobilized
Abs by boiling for 1 min in SDS sample buffer containing 5% 2-ME, the immunoadsorbed proteins along with Rainbow protein m.w. markers (Amersham) were separated by SDS-PAGE and were transferred electrophoreticlly to nitrocellulose membranes (Micron) at 60 V. Membranes were incubated for 30 min in 5% nonfat milk TBS. Blots were then incubated sequentially with the immunoblotting Ab and peroxidase-conjugated anti-rabbit Ab or biotin-labeled anti-mouse Ab then peroxidase-labeled streptavidin for Ih each at room temperature with three 15 min washes buffer (lOmM Tris-HCL, pH 7.5, 150mM
NaCL, and 0.1% Tween-20) after each step. Bound Abs was visualized by using
Lumi-Imager™ from Mannheim-Boehringer or autograph on films (Kodak).
When film was used, the image was scanned and the chemoluminescence intensity was quantified by using a Lumi-Imager™ from Mannheim-Boehringer.
45 c . Results
1. Effect of CRP on phosphorylation of FcvRIIa. Since the FcyRII class accounts
for most of the high affinity CRP-binding to human monocytes and PMN (23), we examined whether CRP could initiate signaling events via this receptor by measuring the Tyr-P of FcyRIIa. Immunoprécipitation of the FcyRII by the class specific mAb IV.3 subsequent to binding of human CRP to differentiated HL-60 cells should reveal whether CRP directly signals via FcyRIIa since it is the only human FcyR class with an intrinsic ITAM motif in its cytoplasmic region (1 18).
When CRP was allowed to bind to HL-60 cells in an aggregated, but soluble form, tyrosine phosphorylation of FcyRIIa occurred in a dose-dependent fashion with as little as 10 ug/ml of CRP (Fig. 5, 5a). Maximum levels of a signaling were consistently observed at acute phase levels of CRP of 100-200 ug/ml. Purified
CRP that had not been deliberately aggregated induced a similar level of Tyr-P of
FcyRIIa (Fig.5). Heat-aggregated, soluble human IgGi at the same concentration triggered more tyrosine phosphorylation of FcyRIIa as that of CRP (Fig.5). Thus, the range of effective molar concentrations for CRP (120 kDa ) for FcyRII signaling is similar to that of human IgG. The kinetics of FcyRIIa tyrosine phosphorylation was followed over the interval of 0.5 to 12 min with aggregated
CRP (100 pig/ml); the signal appeared as early as 1 min and reached a maxium at
1.5 min (Fig. 6 ). When either aggregated or monomeric CRP was allowed to bind 46 to HL-60 (G) cells prior to the addition of aggregated IgG, the signal intensity
was not altered (data not shown). The 40kDa appears to be diffused since it
corresponding to a glycoprotein (See figures 5 and 6 ). The results indicate that
FcyRIIa Tyr-P and activation is a rapid and early event triggered by clustering of
the receptor for CRP.
2. Effect of CRP on Svk Phosphorvlation. Src gene family kinases are activated
during the earliest stage of FcyR signaling and phagocytosis, although the
mechanism of activation of these tyrosine kinases by FcyRIIa is not yet fully
understood (174). The Tyr-P of the IT AM on FcyRIIa permits the recruitment of
the SH2-domain containing 72 kDa Syk kinase that is present exclusively in the cytoplasm of leukocytes (175, 176). Binding of Syk to a diphosphorylated IT.AM
results in a conformational change that increases its kinase activity ( 178). In viva assays also showed that the Tyr-P of Syk correlates with its kinase activity (227).
Syk, but not Zap70, another related kinase, is required for FcyR to mediate phagocytosis (179, 180). Syk is also required for FTAM-dependent activation of actin assembly and subsequent FcyRIIa-mediated phagocytosis (176). Thus, we assessed its activation in response to CRP. Tyr-P of Syk in HL-60 (G) cells was triggered by incubation with aggregated CRP (Fig. 7). The Tyr-P signal observed with acute phase concentrations of aggregated CRP (>I00 pg/ml) was similar to
47 that induced by aggregated human IgG when similar amounts of Syk protein were
compared (Fig. 7). There was no significant difference in the intensity of the
signal of phosphorylated Syk when unaggregated CRP (>100 ug/ml) was
compared to heat aggregated CRP. In a separate kinetic experiment, the P-Tyr of
Syk was detected as soon as I min and reached maxium levels at 2 min after CRP
stimulation (Fig. 8 ). This result correlates with the time course study on Tyr-P of
FcyRIIa (Fig. 6 ), and indicates that the activation of both FcyRIIa and Syk are
early and rapid events upon receptor clustering by CRP.
3. Effect of CRP on the association of FcvRIIa and Svk. Since Syk contains N-
terminal tandem SH2 domains and that domain binds to Tyr-P (177), the binding
of Syk to the Tyr-P in the ITAM of FcyRIIa is thought to induce a conformational
change that increases Syk kinase activity (16-17, 178). Since CRP induced the
Tyr-P of FcyRIIa and Syk, we tested the effects of CRP on the binding interaction
between FcyRIIa and Syk. HL-60 (G) cells were stimulated with concentrations of
aggregated CRP followed by immunoprécipitation of FcyRIIa and then probed with either anti-Tyr-P or anti-Syk. The co-precipitating Syk became Tyr-P in response to both CRP and aggregated IgG (Fig.9). This result is consistent with the previous finding that CRP induces the Tyr-P of Syk. The study also demonstrated that Syk is coimmunoprecipitated with the FcyRIIa even in cells
48 that were not treated with CRP (Fig.9, lane 1). Therefore, the result is not conclusive whether the association of FcyRIIa with Syk is CRP dependent.
4. Effect of CRP on PLCv2 Phosphorylation and Membrane Localization. A downstream signaling event following activation of FcyRIIa and Syk is the phosphorylation and recruitment to the membrane of PLCy (181), which in turn generates the soluble lipid second messengers, IP 3 and DAG, respectively (140).
The major PLC isoform in PMN and HL-60 (G) is the PLCy2 (140). Therefore,
HL-60 (G) cells incubated with increasing concentrations of CRP were used to isolate membrane bound phosphorylated PLCy2. When CRP was used at the concentration of as low as 10 pg/ml, Tyr-P of PLCy2 is clearly induced (Fig. 10).
Translocation of PLCy2 from the cytosol to the membrane was also readily detected in response to aggregated CRP (Fig. 11, panel A). These results are consistent with activation of PLCy in response to CRP.
5. Effect of CRP on PI-3K membrane translocation. PI-3K plays a pivot role in
FcyRIIa signaling in leukocytes since it generates lipid secondary messangers required for phagocytosis (182). The p85 is the regulatory subunit of PI-3K. Upon activation, PI-3K mobilizes from the cytosol to the membrane and produces various lipid secondary messengers, an event associated with FcyRIIa signaling
49 (183). Therefore to study the effects of CRP on PI-3K activation, cellular membrane was purified and transferred to a NC membrane. The presence of PI-
3K was examined by immunoblotting the membrane with an Ab to the p85 regulatory subunit of PI-3K. HL-60 cells were treated with various concentrations of CRP or IgG. Exposure of the cells to acute phase concentrations of CRP induced translocation to the membrane of PI-3K (Fig. 11, panel B). The amount of p85 subunit is increased in cells stimulated with 100-200 ug/ml of CRP when compared with the untreated cells (Fig. 11. panel B). IgG induced the same effect of PI-3K membrane mobilization. This study shows that CRP increases the membrane translocation of PI-3 kinase in differentiated HL-60 cells.
50 D. Discussion
Activation of neutrophils and macrophages is the key cellular event for
effecting innate host resistance. Since the acute phase reactant CRP Is considered
a link between the early systemic host response and subsequent specific immunity
(12, 14. 169), the signaling mechanism whereby CRP activates PMN is critical for understanding the opsonic activity and antimicrobial role of CRP. Therefore, the focus of this study was to determine whether CRP functions in a manner analogue to IgG Ab in terms of signaling via FcyRIIa. The distribution of this human FcR class on almost all hematopoietically-derived cells (172) corresponds exactly with the distribution for specific leukocyte CRP-R (8.14). In addition, the FcyRII class has recently been shown by DuClos and his colleagues to serve as the major functional leukocyte CRP-R (23-25). One of the unique properties of FcyRIIa is that the ITAM required for propagating the initial steps of signaling is present within the cytoplasmic domain of the receptor’s a-chain itself and not on an associated protein ( 149). The major new findings in this study are that the CRP ligand, when presented either as a monomer or in a multimeric complex, not only triggers the Tyr-P of the FcyRIIa ITAM, but also propagates the signal as indicated by the Tyr-P of the receptor-associated Syk kinase (176) and the downstream effector PLCy2 (140). CRP-induced FcyRIIa clustering also induced the translocation to the membrane of both PLCy2 and PI-3K, events required for
51 phagocytosis (140, 182). These events triggered by CRP occurred with the same kinetics as IgG immune complexes, indicating that CRP is as capable as IgG Ab of mediating phagocytosis and subsequent anti-microbial activities in PMNs.
Overall, the findings are consistent with the proposed link between CRP and its receptor in augmenting innate host resistance (12, 169). Almost all of the biological activities ascribed to CRP, especially its ability to opsonize microbial pathogens and activate the early steps of the classical C pathway, are consistent with a role in host protection (14, 53, 74). The first studies of CRP-mediated anti microbial host resistance used passively infused human CRP in mice, which were followed later by experiments using transgenic mice expressing human or rabbit
CRP genes to document CRP-dependent protection (rev. 12,14).
During the 1940's. there were extensive studies on CRP that was immunochemicaly modified to determine if it interacted with other molecules or cells. It was found that under certain conditions such as treatment with acid, urea chelation or heating, pentemeric native-CRP dissociated into free subunits and underwent a spontaneous change in conformation, which resulted in the expression of a new conformation termed “neo-CRP”. “Neo-CRP” exhibits distinct antigenecity, electrophoretic mobility and ligand binding reactivity 1215 -
219). This form of CRP also displayed higher binding affinity to tissues than native CRP, which implied some fundamental biological functions of the **neo-
52 CRP”. In vivo studies had shown that heat aggregated neo-CRP was capable of
activating complement (67-69), opsonzation ( 8 8 . 166). and modulating the
responsiveness of lymphocytes (220. 221), NK cell (222, 223). macrophages
(224), PMNs (165) and platelets (225, 226). Using cytometric analysis and
bimodal staining pattern, Bray, et al. (218) revealed a distinct diagonal staining
pattern that was observed only when large granular lymphocytes were co-slained with anti-neo-CRP and either anti-CD 16 (anti-FcyRIII) or IV.3 (anti-FcyRll).
This finding suggested a 1:1 relation between anli-FcyR and deierniinanu recognized by anti-neo-CRP. It also demonstared that “neo-CRP" was physically associated with the FcyR present on large granular lymphocytes. It must he emphasized that there is no physiological equivalent of “neo-CRP”.
Furthermore, Gewurz, et al. (219) found that heat aggregated CRP bound to FcyR bearing cells. Zeller, et al. (189) has reported that the Fc/RIl-specillc mAb IV.3 significantly suppressed the activation of monocytes by heat- aggregated CRP. In addition, mAb IV.3 failed to reduce binding of aggregated
CRP to either monocytes or PMN, indicating that, although aggregated CRP does not bind to phagocytic cells at the IgG-binding determinant of FcyRII, CRP does alters aggregated IgG-induced cell activation through this receptor. Therefore, the use of heat-aggregated CRP is justified in the present receptor binding studies.
53 especially when one considers the long-standing dogma of a requirement for
dimerization of FcyR for signaling.
Since CRP is capable of binding to the three classes of FcyRs, it is
possible that the extensive Tyr-P of proteins in a receptor immunoprecipitaion
study induced by CRP could potentially come from more heterogeneous sources.
However, since the mAb IV.3 used in the immunoprécipitation studies here is
specific for only the FcyRII class, the observed Tyr-P protein bands in the
immunoprécipitation study are the result of the Tyr-P of the FcyRII. rather than
FcyR I or FcyRIII. In addition, since the expression of FcyRIIb in
neutrophils/monocytes is only one tenth of that of the FcyRIIa (118). the Tyr-P signal is predominantly from FcyRIIa, most likely from the ITAMs. However, this data does not rule out the possibility that CRP also induces the Tyr-P of Fr/RI and FcyRIII.
Tyr-P of Syk (178) and PLCy (139, 140) is required for the activation of their enzymatic activities. Furthermore, the degree of Tyr-P correlates with their activities (227). Therefore, the Tyr-P signal is an indication of the activation of both enzymes. Following the activation of FcyRIIa indicated by the Tyr-P of its
ITAMs, the immediate next event is the activation of Syk. The kinetic studies of
Tyr-P on both Fm/RIIa and Syk shown the same pattern of activation: a rapid Tyr-
P that quickly culminates after approximately 2 minutes of stimulation by CRP, 54 then the signal decays almost completely by 8 minutes (Pigs.6, 8). This identical
Tyr-P pattern between the FcyRIIa and Syk suggests a close interaction between
the two proteins. Indeed, the immunocoprecipitation of FcyRIIa and Syk clearly suggests a close protein-protein interaction in the membrane (Fig. 9). However,
based on result of this study, it is not conclusive whether the interaction between
Syk and FcyRIIa is induced by CRP treatment. In fact, the two proteins were coimmunoprecipitated even in cells without CRP treatment, suggesting that either
FcyRIIa and Syk are associated in the resting state or there is some unknown factor(s) that prompts their interaction in resting cells.
One issue raised by the findings herein is whether the signaling pathway initiated by CRP via FcyRIIa is identical to that initiated by IgG. The proximal signal transduction events appear to be qualitatively the same in terms of the critical components of the signaling pathway that are activated. Furthermore, the relative efficiency of activation as judged by the kinetics is very similar for CRP and IgG. This result might be anticipated if both agonists activate the initial kinase for phosphorylating the two Tyr within the tandem Y-X-X-I/L motifs of the ITAM of the receptor (149, 184). The initiating PTK for the FcyRs has so far only been identified as an Src family kinase (184, 185). Recent work from one of
Coggeshall’s group suggests that Syk, through its SH2-domains, binds directly to the ITAMs to serve as an adapter protein for the SH2 domains of the p85 subunit
55 of PI-3K resulting in concomittant recruitment of PI-3K (185). Since PI-3K is
essential for signaling and for propagating downstream events leading to
membrane movement and phagocytosis (182, 186), the documentation of its recruitment to the membrane, and presumably to the FcyRIIa, in response to CRP is evidence that CRP is capable of generating the intracellular mediators needed for phagocytosis. In earlier experiments using the same system, we demonstrated an increase in the kinase activity of P1-3K in membrane fractions in response to
CRP at levels >50 ug/ml (40). The activated PI-3K generates IP3, which promotes distal signaling events by binding to PH domains of many different enzymes, including PLCy2 in neutrophils (140. 182, 187). The localization of
PLCy2 to the membrane and mobilization of intracellular Ca-+ stores in response to CRP also represent crucial downstream events required for leukocyte activation and phagocytosis (188). Indirect evidence that CRP signalled via FcyRIIa on
PMN was first gathered by Zeller and her colleagues who found that CRP potentiated the aggregated IgG-induced activation of the respiratory burst of
PMNs (165), but that mAb IV.3 could, not block the potentiation induced by CRP
(189). This latter finding, as well as many earlier attempts to define the CRP-R with specific mAh reagents for FcyRs (Zhong 1997), suggests that the recognition sites on FcyRIIa for IgG and CRP are distinct. Since 93% of activated PMNs and virtually all HL-60(G) cells (15) bind aggregated CRP (165, 189), CRP may be
56 able to "prime" PMN for IgG-compIex activation through FcyRIIa when both are
present. The structural basis for the functional similarity between CRP and the Fc
of IgG has not yet been resolved other than the suggestion that the only shared.
accessible sequence of ^^^YLGGP of CRP is involved in binding to Fc RI (190).
Recently, the Tyr-175 residue was shown to be critical for the binding of Clq to
CRP and is part of the unusual extended deep cleft of the central pore of the
pentraxin (57). The pentagonal arrangement of the identical CRP subunits with
the effector face on the plane opposite the PC-binding face is compatible with
FcR clustering.
The specific finding of signaling of CRP via FcyRIIa in this report does
not preclude CRP-induced signaling via FcyRI (190), or even FcyRIII. which bind
CRP and/or SAP (191). The regulation of CRP-mediated activation of PMN is
likely to involve the use of negative signaling receptors such as FcyRIIb or may
use the ITAM of FcyRIIa to recruit phosphatases (148). The inflammatory milieu
where CRP accumulates may provide the critical density needed for eventually modulating the cell activation.
57 IP; Anti-FcyRIIa (IV’3 )
1 2 3 4 5 6 A: And-Tyr-P jq ^ IgGHCham 33K FcyRIIa
m m m
B: Bb-and-FcTRIU u m m ^ Anti-FcyRIIa AHo CRP (cgftnl); 0 10 too 200 HaCRP: 200 AlgO 100
Figure 5. EBeet of human CRP on the Tyr-P of FcRylla. HL-60 cells (10-13 x 10') vnre incviMted with 10-200 ug/ml of CRP et TIK for 3 min end then lysed. Cytosolic coOecdons weie then munnnoptecipiteted with 1 ugkemple of mAb nuose-entt- FcyRII (IV J) et overnight end seperete by SDS-PAGE (7J%) kibre transfer to NC membrane. The membrane wes first pttibed wüh enti-Tyr-P (A), end then le-piobed with rabbit-enti-Fc FcyRUe that a specific to the cytoplasmic domain of the receptoi(B). The signals were detected by chemoluminescene end euterediognphy. The chemoluminescence intensity wes queiSifiedby using e Lumi-Imager™ fiom Mannheim-Boehringer. The FcyRIIa -41 kOebend is indicated by the enow. A - Heat eggregtte
58 IP; a-Fc7Rnn (IV’3 ) Blot; a-P-i>T
50 K H C hniii F cy R IIn 35 K
HuCRP (ng/ml): 0 5 SO 200 0 50 fMLP (nM) : 0 0 0 0 100 100
F ^ u e Si. EXfett if human CRP on the tyniiae-photpliiiylatiin of tfie FcRtH DifTotentiated HL.SO caOs (10 z 10*) won mcubated with S to 200 Mg(inl of CRP at 37*0 for 10 mm anl then lysed. The cytosolic fraction was Ik n imraiinopnctpitated with 2 Mg o f moose-anti- FcRtU (mAh IV3) and immunoblotted with anti-P-Tyr. The moose IgS.v H-chain is shoom as a 35-60 kOa band and the m w matkeis on a 7 5% gel shown. The chenolmninescence intensity was qoaitiOed by using a Lnmi-lmaget™ Qom Mannheim-Boehiinget
59 IP; OrFc^RIIa (IV3 )
A; Amd-TyrP 1 2 3 4 5 6 7 50K- H Chain FcyRUa 35K-
B; Rb-anti-FcrRIU H Chun i î i ï a f t r ^ FcrRJI» Time(Mn): 0 0 5 1 1 5 3 (5 12 iCRPCaglml): 100 ------»
ncoxe *. #f Tti-P of FtRrlU in R ^ n e to CRP. H L ^ (O) ceRs w«is mciiwiwl (or düTemnl nuervob tnth 100 is/mlof «ggRgttedCBf pnoi to mununopmcipAoioa mth mAb 1V3 and pmoing (ho wpunled wnh ond-Tyr-P (A), foQowedbynfabit anti- FqfRIla(B). The chetnohnniiesceiica mtensitywasdetectetland qoanttftdbytsinga Lami.Iintgei™fiamMaBnheim-Boahtuigtr.
60 IP: Mo-anti-S'ylc
A: Antl-Tjr-P 1 2 3 4 75 K Syk 55 K H Chain B: And-Syk 75 K Syk 55 K = t H Chain A HuCRP (ugtol): 0 100 200 HuCRP: 200 A IgG (ug/ml) 100
Figure 7. Effect o f CRP on the T jr-P of S]4c in Hli-60 (G) cells. Cÿtosol pnpatatiotis fiotncells incubated with 100 oi 200 ug^tnl of aggregated CRP vs. eggiegited IgO (100 Ugitnl) were immonopiecipitated withanti-Syktno mAb and separated by SDS-PAOE before ptobing with anttXyr-P (A) and then with anti.SykmAb(B> The Svk band is •'>2 kOaasshowiL
61 IP; Mo-niiti-Svk
75 K A : A nii-T yr-P
Ig G H ■ III
75 K B: Aati^yk ^ ■*—IgG H
Tim (mm); 0 OJ 1 2 4 S 12 tu v m A HuCRP lOOiigtel ______ngunS. KiiKiictofTyT-PofSykhRiponsetg CRP. ThedmcomsaofTyr-PofSyk (72kO&) m the cyta9olofHL^(G) cells in teepome to aggregated CRP at lOOug/W. N*; VO^ was used as a positive contml IbrTyrf (A). Immunoblot ptobed with anti-Tyrf (B) Immunohlot of the same metdbrane reptobedwithanti-Syic.ThechemohuninesceiKe mtensitywasdetectedandquentifiedbyusmga Umu. Imager™ fio m Mansheim-Boehiingei.
62 IP: œ-FcYRIIa (I\’3)
A: Anti-Tyr-P 75 K 72 kD 50 K
Syk B: And-Syk
AHuCRP (ng^tnl): 0 10 100 200 SAP: 100 AIgG 100 N«3 V0 4 +
FiguieP. EBKttfCRPonthetetenctimbctMreeaSykaidFcRlb. HWO(0)celb (10x106 perlOOd) were incdb«ted with aggtegftted CRP, pmi&d human SAP or agpagated IgO. Call lysate» ware immmepiecipitated with anti-Fc RHa mAh IVS and the co- piecipitaad pntans sepazatad by SDSP AGE, blottad and pnbad with eiUi-P* V and subsequently with the anti Syk mo mAb. Blot is mpmsentath* of3 similar a xperimants.
63 IP; a-PLCyl
AjAntf-Tyr-P 1 2 3 4 5 6 160 K — » Î 145 kD
BiAmdPLCy:
A HuCRP (ugtol): 0 10 100 200 HuCRP: 200 A IgG 100
Hcue II. EBetlf of CRP oa PLC 2 Tyr-P. HL.60 (G) cello (10x106) were stimuleted with eggregeted CRP or eggtegated lÿl for 10 nun. Cell lysatee were imnuirtoprectpiteted with entt-PLC 3 at 4 C overnight arxl the precipitated proteins eepaiated by SEEPAGE (7 JV. gel) The blot was probed first with anti-P-Y (A) and then leprobed with arti-PLC 2 IgO Ab (B) ThePLC2buidis> 145 hOa as shown
64 A: Anti-nCfZ
160 K Anti-PLCy2 B: Anti- n-tK
75 K ■ Anti-p85
a Hu CRP (ugtol): 0 100 200 0 0 0 AHuIgG (u^ml) 0 0 0 10 100 200
Figwm ll.EBe 65 CHAPTER 3 CRP Induces Negative Signaling Through IgG Fc- Receptor (FcyR) H on HL-60 Cells A. Introduction The initial observations of the effects of CRP on PMNs usually reported an inhibitory effect of this protein on the physiological activities induced by agents such as fMLP, LPS, or PMA (194, 199). Mortensen, et al. (192) first reported that CRP inhibited the proliferative response of T-cells in both murine and human mixed lymphocyte reactions and thus inhibited the generation of cytolytic T-cells. At the same time, CRP was also discovered to inhibit the secretory response following platelet aggregation by heat aggregated human y- globulin and thrombin induced by poly-L-Lysine (193). CRP was shown to selectively inhibit alternative complement activation activated by the pneumococcal C-polysaccharide (71, 89). Marcelletti, et al. (195) reported that CRP inhibited the in vitro clonal proliferation of Granulocytes/Monocyte (G/M) stem cells and noticed that the inhibition was specific for G/M-stem cells committed to the monocyte lineage. In fact, G/M stem cells that possess Fc receptors at the time of CRP exposure were susceptible to inhibition of colony formation. This report first implied CRP’s inhibitory effects via FcR. In 1987, 66 Buchta, et al. (202) made the initial observation that CRP inhibited superoxide production and chemotaxis induced by PMA and con A, but it also enhanced neutrophil phagocytosis. To my knowledge, this was the first report showing that CRP exhibits both activation and inhibitory effects on leukocytes. The same group also reported that CRP reduced the extent of protein tyrosine phosphorylation induced by PMA and fMLP in a concentration-dependent fashion when all of the cellular proteins were assayed (301), clearly suggesting that the inhibitory effect of CRP was exerted at the signaling level. Later CRP was shown to directly inhibit macrophage migration and the ability of activating macrophages to release superoxide anion (0? ) (198). Since then, numerous groups have shown the inhibitory effect of CRP on neutrophil (PMN) activities. Kew, et al. (203) demonstrated that CRP inhibited random neutrophil movement and chemotaxis induced by C5a. Zhong, et al. (15) reported the inhibitory effect of CRP on several inflammatory activities of PMNs including chemotaxis and respiratory burst via inhibition of phosphorylation of protein kinase C (PKC) (14). The group also reported that CRP inhibited both the membrane translocation and serine- phosphorylation of p47-phox and Rac2, a low m.w. G protein regulator hence blocking the assembly of the NADPH-dependent oxidase in activated PMN. (15). Heuertz’s group demonstrated that CRP inhibits neutrophil chemotaxis in response to fMLP via inhibition of the p38MAP kinase activity that is required for 67 this response to all G-protein coupled receptors (GPCR) (200). This evidence strongly support a negative inhibitory role of CRP in modulating neutrophil activities activated by external effectors. In the previous chapter, I showed that CRP rapidly activates a series of positive signaling events upon receptor clustering. Although it may seem a paradox that a single molecule elicits dramatically opposite effects on the same target cell, such effects are consistent with a regulated signaling response. To reveal the inhibitory mechanism for PMN activities, the effects of CRP on several intracellular targets were examined. In this chapter, I show that CRP activates SHIP, a phosphatase that is involved in negative signaling by inducing its Tyr-P; CRP also promotes SHIP-Shc interaction, an event commonly observed after Tyr- P of SHIP in negative signaling. I also found that the activation of SHIP by CRP is more delayed when compared to its activation of both FcyRIIa and Syk. CRP also reduced the membrane translocation of PLCy2 induced by fMLP. 68 B. Material and methods 1. Cells and Reagents. The human promyelocytic cell line HL-60 was grown in RPMI-1640 supplemented with 4% defined FES and 6% supplemented bovine calf serum (HyClone, Logan, UT). To differentiate HL-60 cells into granulocytes (G), -4 X10^ cells/ml were incubated with 1.2% DMSO for 6 or 7 days. Protein G Sepharose"^ beads was purchased from Zymed (Burlingame, CA). Affinity purified rabbit IgG anti-human PLCy2 (Cat#: sc-407) was purchased from Santa Cruz Biotechnology. Biotin-labeled Goat-anti-Mouse IgG(Y) and peroxidase labled streptavidin were purchased from KPL. Anti-FcyRII cocktail (AT 10, KB61, IV.3), polycolonal rabbit anti- FcyRIIb (263) were gitts from Dr. Susheela Tridandapani (Department of Internal Medicine, Ohio State University) (38). Anti-phosphotyrosine mAb mixture (Py20 (IgG2b), Py72.10.5, and 4G10 (IgG2b) in the ratio of 30:30:1), rabbit anti-She and rabbit anti-SHIP were gifts from Dr. Mark Coggeshall (Oklahoma Medical Research Foundation, Oklahoma City, OK, 73401). CRP was purified from human ascites exactly as described by us earlier (24). 2. Purification of CRP and SAP Human CRP and SAP was purified as described elsewhere (ref). Briefly, SAP component was removed from CRP-containing pleiu-al or ascitic fluids by passage through a column of agarose beads (A-15m, 69 Bio-Rad). The eluted protein was then passed through a 10 mi (20 mm diameter) column of p-aminophenyl-phosphocholine (PC)-sepharose, washed extensively with TBS + 2mM Ca-H- and the bound protein eluted in TBS + 10 mM EDTA. A second round of affinity purification on the PC-bearing matrix was used to remove trace amounts of other proteins. The concentration of CRP or SAP was determined by ELISA or by RID with sheep anti-human CRP or sheep anti human SAP. The protein was > 99% CRP or SAP based on reactivity in the competitive ELISA and by SDS-PAGE. The concentration of endotoxin in the purified CRP or SAP was 0.1-0.2 endotoxin units/mg protein (Chromogenic Limulus assay, M. A. Bioprod., Walkersville, MD), corresponding to an LPS concentration of < 0.05 ng/mg of CRP. 3. Membrane Localization of PLCv. HL-60 (G) cells at 10^ in 100 ul of EBSS buffer (pH 7.4) were incubated on ice for 30 min and then at 37°C for 10 min before stimulation with various concentrations of purified human CRP. Reactions were stopped by sonicating the cells on ice for 1 min and centrifuging at 800x g at 4°C for 10 min to remove nuclei. The supernatants were mixed with a modified extraction buffer of: Tris (50 mM), pH 7.5, EOT A (2 mM), sodium ortho vanadate (10 mM), leupeptin 1 ng/ml, aprotinin 0.2 jag/ml, and fresh PMSF (2 mM); and then centrifuged at 60,000 rpm, 4'^C (Beckman, TLA-100 rotor) for 30 70 min. The membrane pellet was washed once by resonicating in the extraction buffer and centrifuging under the same conditions to remove any traces amount of cytosolic proteins. The membrane preparations were stored at -70 °C and the protein concentration measured by the BCA method using BSA as the standard (Pierce Chemical, Rockford, IL). An equal amount of protein per lane for each sample was separated by SDS-PAGE and then transferred to nitrocellulose membranes (Micron) at 60 V. The membrane was saturated in 5% low fat powdered milk in TBS containing 0.1% Tween-20, incubated overnight with one of the antibody probes, washed, and then incubated with a HRP-labeled secondary reagent and the reaction was developed with chemoluminescent reagents (Kirkegaard-Perry Labs, Gaithersburg, MD). Rabbit anti-human PLCy2 (1:1,000) was used as the primary Ab. HRP-conjugated anti-rabbit-IgG( 1:2,500) (KPL) or protein G-HRP (1:4,000, Zymed) were used as secondary reagents. 4. Granulocvte Activation and Immunoadsorption. Differentiated HL-60 cells were washed twice and then suspended in HBSS containing lOmM HEP ES (pH 7.4). Cells (10 X 10® / sample) were incubated at 4°C for 30 min and then at 37°C for 10 min. After that cells were treated with various concentrations of purified human CRP (5 ug/m - 200ug/ml) for 10 min. In cells treated with both reagents, the procedures were the same except that CRP was incubated first followed by 71 fMLP. In positive control, cells were stimulated with 10 ul of 3 mM sodium orthovanadate solution (I ul of 33mM sodium orthovanadate, lul of 30% H 2 O2 , 98 ul of CIH 2 O) for 3 min at 37°C. Cells were then lysed in Triton lysis buffer (TLB; PBS, lOmM HEPES, lOmM EDTA, and 1% Triton X-lOO, pH 7.4. supplement with 3 mM sodium orthovanadate, 20 ug/ml aprotinin, 40 ug/ml leupeptin and 2ug/ml pepstatin A. Insoluble material was removed by centrifugation at 16,000 x g for 10 min, and the supemant was immunoadsorbed overnight at 4°C with specific Ab (2ug/ml) mixted with 10 ul of recombinant protein G-Sephorose (Zymed) that were pre-incubated for two hours at room temperature or 4°C ovemight and unbound Ab was washed away by ice-cold PBS buffere for three repeats. Following immunoadsorption, unbound proteins were removed with four time washes of TLB plus 1 mM sodium orthovanadate and subject to Western blot analysis as described below. 5. Tvrosine-phosphorvlation Analvsis. Detection of tyrosine-phosphorylation was accomplished by immunoprécipitation of the FcRIIa, PLCy2, p85 and SHIP from whole cell lysates using 2 ul of each of the following antibodies: mAb anti- FcRIIa, rabbit IgG anti-human PLCy2, rabbit anti-p85 or rabbit anti-SHIP and protein G-Sepharose beads (10 pi). The beads were washed 3 times in Dulbecco’s PBS (D-PBS) plus sodium ortho-vanadate (10 mM) before separation by SDS- 72 PAGE and immunoblotting. Phosphotyrosine in the proteins was detected using anti-phosphotyrosine cock tail and biotin-labeled Goat-anti-Mouse IgG(y) and peroxidase labled streptavidin. The amount of proteins examined was detected on the same blots by reprobing using the goat Abs (1:2,000) followed by protein G- HRP (1:4,000). The chemoluminescence intensity was detected and quantified by using a Lumi-Imager™ from Mannheim-Boehringer. 6. Immuno blot analvsis After elution of adsorbed proteins from immobilized Abs by boiling for I min in SDS sample buffere containing 5% 2-ME, the immunoadsorbed proteins along with Rainbow protein m.w. markers (Amersham) were separated by SDS-PAGE and were transferred electrophoretically to nitrocellulose membranes (Micron) at 60 V. Membranes were incubated for 30 min in 5% nonfat milk TBS. Blots were then incubated sequentially with the immunoblotting Ab and peroxidase-conjugated anti-rabbit Ab or biotin-labeled anti-mouse Ab then peroxidase-labeled straptavidin for Ih each at room temperature with three 15 min washes buffer (lOmM Tris-HCL, pH 7.5, 150mM NaCL, and 0.1% Tween-20) after each step. Bound Abs were visualized by using Lumi-Imager™ from Mannheim-Boehringer or autograph on films (Kodak). In case of film, image was scanned and the chemoluminescence intensity was quantified by using a Lumi-Imager™ from Mannheim-Boehringer. 73 c. Results 1. Effects of CRP on Tvr-P of FcyRIIb. FcyRIIb is the only human FcyR that contains the cytoplasmic ITIM motif. Like FcyRUa, this receptor also bears two Ig-like domains on its extra cellular part that are extremely similar in sequence (118. 207), suggesting very similar binding behavior to the FcyRIIa. Since FcyRUa was found to bind to CRP with high affinity (25), it was reasonable to expect that CRP can also interact with FcyRIIb. Therefore, FcyRIIb is an ideal candidate to explore a possible mechanism for the negative signaling induced by CRP. Since the expression of FcyRIIb is only one tenth of that of the FcyRIIa more cells were needed to detect its presence (118). Thus, 30 x 10*^ HL-60 (G) cells that has been differentiated with 1.2% DMSO for 5-6 days were used for immunoprecipiation and then probed for the tyrosine phosphorylation of the receptor. There is no distinct band visible at 40 kDa or 62 kDa (Fig. 12). Since FcyRIIb is differentially glycosylated in different cells (155), it will migrate as a long smear between the ranges of 40 kDa to 62 kDa. Due to the dominant signal of the IgG heavy chain, the Tyr-P signal of FcyRIIb could be potentially shadowed. Therefore, to exclusively measure the Tyr-P of FcyRIIb, better methodology and reagents are required. 74 2. Effects of CRP on Tvr-P of SHIP. Bucha, et al. (201) were the first to report that CRP pretreament of human neutrophils stimulated with fMLP results in a significant reduction in the degree of phosphorylation of several intracellular proteins. A more recent study shows that CRP inhibits fMLP-induced p38 MAP kinase activity in a dose-dependent manner (204). This laboratory found that CRP inhibited both fMLP-induced chemotaxis and the fMLP-triggered oxidative burst. Together, these findings suggest that CRP can play a regulatory or inhibitory role on neutrophil activities. A recent SHIP mutagenesis study in T and B lymphocyte clear shows that SHIP'^’ B lymphocytes exhibited a prolonged Ca2+ influx and increase proliferation upon BCR-FcyRUB coligation, suggesting that SHIP was essential for FcyRUB-mediated negative signaling (205). The same study also found that SHIP could act as a negative regulator of MAPK signaling (205). Since SHIP has been reported to be recruited by both FcyRIIa and FcyRIIb and regulates signaling in B-cells (151-156), I tested whether CRP treatment of HL-60(G) cells induced the activation and recruitment of SHIP. In the initial studies of CRP treatment of these cells followed by immunoprécipitation of FcyRIIa, we observed the recruitment of SHIP to FcyRIIa as expected. CRP triggered a dose-response increase in Tyr-P of SHIP with the strongest Tyr-P signal induced by 100 to 200 ug/ml of aggregated CRP (Fig. 13). This result indicates that CRP is capable of inducing SHIP activation at acute phase concentrations. 75 3. Effect of CRP on SMP-Shc interaction. In FcyR mediated negative signaling in B cells, the phosphorylated SH-2 domain of SHIP serves as a docking site for recruiting the adaptor protein She (ref). Once the SHIP-Shc interaction is established, subsequent signaling events are triggered. Therefore, we also tested whether a SHIP-Shc interaction occurred in response to treatment of HL-60 (G) cells with CRP. The experiments of immunoprécipitation of SHIP followed by probing for She showed that a dose-response of She co-associated with SHIP in response to CRP treatments (Fig. 14), indicating that CRP can induce the interaction between SHIP and She. 4. Kinetics of SHIP tvrosine phosphorylation. In chapter 2 I described that CRP rapidly activated a series of intracellular activities in PMN and the maximum response illustrated the Tyr-P of FcyRIIa and Syk was maximal after I minute of treatment. Here I also found that CRP triggered the negative signaling by inducing the tyrosine phosphrylation of SHIP and promote the SHIP-Shc interaction after SHIP is tyrosine phosphorylated, two typical events in negative signaling. It appears conflicting that CRP as a prototype acute phase reactant possesses two seemingly opposite effects on the same target. To explain the above observations, I hypothesized that there would be a time lag of the negative inhibition effect of CRP after the protein triggers the positive activation 76 mechanism. Biologically, it also makes sense that if a ligand triggers an activation response, the magnitude of that response has to be carefully modulated. To test this hypothesis, a time course study of SHIP phosphorylation in HL-60 (G) induced by 100 ug/ml of aggregated CRP over the interval of 0 to 12 minutes was performed. The P-Tyr signal of SHIP increased with time and culminated at 7 minutes after CRP stimulation (Fig. 15). By contrast, the kinetics of FcyRIIa P-Tyr reached maximum level after only 1 minute of CRP exposure. Thus, the Tyr-P of SHIP is slower and later than the phosphorylation of the FcyRIIa, suggesting that there is a time lag for the regulatory signal to occur. 5. Effects of CRP on PLCv2 membrane translocation induced bv fMLP. The fMLP receptor on PMNs belongs to the trimeric GPCR family. It is well- established that the human PMN display a fMLP receptor-mediated signaling via a G-protein, PLC activation, IP3 generation, Ca2-t- mobilization and PKC activation (206-208). In addition, numerous studies have shown that fMLP induces superoxide production in neutrophils and macrophages mediated by this GPCR. Since this activity is of granulocytes, inhibitable by CRP treatment, the interaction between the CRP induced signaling pathway and the fMLP-triggered signaling pathway was explored. Since PLCy2 was activated by CRP treatment alone (Fig. 10, 11) and is also involved in the fMLP-receptor signaling, the 77 enzyme appeared to be an ideal candidate for testing the interaction between CRP and fMLP induced signaling. Differentiated HL-60 cells were incubated with various concentration of purified CRP prior to the exposure of the cells to fMLP. Isolated membrane proteins were separated by SDS-PAGE (7.5% gel) and then subjected to immunoblotting (Fig. 16). When CRP was used at an acute phase level of 200 ug/ml, along with IGOnM fMLP, translocation to the membrane of PLCy2 was readily detected (Fig. 16). Furthermore, a relative decrease in PLCy2 membrane translocation was observed in cells that were stimulated with both CRP and fMLP, with the extent of inhibition dependent on the concentration of CRP (Fig. 16). This experiment demonstrates that either CRP or fMLP promoted the membrane translocation of PLCy2, which is consistent with previous findings. However, pretreatment with CRP inhibited the activation of PLCy2 induced by fMLP. 78 D. Discussion A long-standing controversy exists whether CRP is a pro- or anti inflammatory protein. In chapter one, I described the finding that CRP induce positive signaling events that involve activation of multiple enzymatic activities. This activation effect of CRP was not previously appreciated, while CRP’s negative inhibitory activities were extensively documented (15. 97-99. 192-194). To my knowledge, the most detailed information on the inhibitory signaling mechanism of CRP at the molecular level are as follows: CRP inhibits the kinase activity of the p38 MAP (99) and PKC (15), CRP inhibits membrane translocation of the p47 subunit of the NADPH-dependent oxidase and Rac, a low m.w. G protein (15). Although these studies have revealed some information about the inhibitory mechanisms of CRP, there are still many critical parts of the picture that are missing such as which receptor is mediating the inhibitory function, which proteins are involved in the pathway, what is the sequence of those events. In the results above I found that CRP induced the Tyr-P of SHIP and promoted the SHIP-Shc binding. The later two events are associated with negative signaling. I also showed the kinetics of tyrosine phosphorylation of SHIP, which is much delayed relative to that for Tyr-P of Fcylla. Furthermore, CRP was also found to inhibit the membrane translocation of PLCy2 induced by fMLP. Due to the limit of the available antibody, I cannot make any conclusion on whether CRP 79 has effect on the Tyr-P of FcyRIIb. Better methodologies and reagents are needed to further pursue the study. Negative signaling involving SHIP and She was initially identified in B cells where surface Ag-receptors (BCR) and FcRs are co-clustered by IgG molecules in which the Fab portion of IgG Ab molecule binds to the BCR and the Fc portion binds to the FcR. The cross-linking of the two types of receptors triggers a series of events (151-153), which culminate in the inhibition of B cell activation (209, 210). Physiologically, this inhibitory effect represents a negative feedback of excess IgG that inhibit further Ab production by B cells. In contrast to the negative signaling pathway, the positive pathway is initiated by co-ligation of the BCR alone, which induces a series of biochemical responses that eventually lead to the B cell proliferation and differentiation into antibody producing cells (211). Two signaling molecules have been implicated in the negative signaling in modulation of immune receptor activation: SHIP, and another tyrosine phosphatase termed SH2-containing protein tyrosine phosphotase 1 (SHP-i). The inhibitory signaling of FcyRIIb is dependent on SHIP but not SHP-1 (211). SHIP is selectively tyrosine phosphorylated at the SH2 domain and associates initially with the rriM of FcyRIIb (155) and subsequently with the adapter protein She under negative signaling conditions in B cells (155). SHIP is a negative regulator 80 in multiple cell types including B cells (155, 215) mast cells (150) developing Xenopus oocytes (212) and cells responding to G-CSF (213) and M-CSF (214). The finding of tyrosine-phosphorylation of SHIP and the SHIP-Shc interaction induced by CRP in neutrophils strongly suggests that CRP is also capable of inducing negative signaling and that this negative signaling is mediated by the FcyRII-SHIP-Shc cascade. Most recently, using FcyRII knockout mice Coggeshall’s group reported that SHIP is sufficiently Tyr-P even in FcyRir/'(157) and SHIP is able to negatively regulate FcyRs-mediated phagocytosis via the IT AM of FcyRI/m, suggesting that the induction of its Tyr-P is not solely dependent on ITIM and ITAM can also transduce negative signal. In the report herein, the Tyr-P of SHIP induced by CRP could also be a result of tyrosine phosphorylated ITAM in FcyRIIa. However, more experiments involving gene knockout are required to support the notion. The fMLP-receptor belongs to the G-protein coupled receptor (GPCR) super family that is composed of seven transmembrane a-helixes. The trimeric G- protein: a, 3, and y subunits coupled with GDP and are associated with the cytoplasmic domain of the GPCR in resting state. Activation of the GPCR induces the dissociation of the trimers into p-y dimmer and the a monomer that binds to GTP. These proteins further interact with PLC and PKC to generate IPS, 81 arachidonic acid (AA), and influence the introcellular Ca-H- signaling. The inhibitory effect of CRP on fMLP-induced PLCy2 translocation to the membrane might be the net result of possible interactions between the FcyRIIb-SHIP-Shc signaling pathway and the GPCR signaling pathway in which the CRP-activated SHIP suppresses the GPCR signaling pathway. Zhong, et al. (15) reported that CRP inhibits superoxide production and chemotaxis induced by fMLP. This is consistent with the SHIP activation and SHIP-Shc complex formation. Therefore, I speculate that the inhibitory effects of CRP are mediated by SHIP and there are uncharacterized signaling events downstream of the SHIP-Shc interaction that eventually affect the assembly of NADPH-dependent oxidase and other cellular activities that are induced by fMLP. It seems to be a paradox that CRP possesses dual capability to induce both the positive and negative signaling. However, based on my kinetic studies, along with the studies on the positive and negative signaling of CRP, I am proposing a model (Fig. 17) of CRP in regulating the phagocytic activity of neutrophils at the signaling level by first rapidly activating positive signaling pathway, which involved a rapid Tyr-P of ITAM and Syk, then later, activates PLC and PI-3K that will generate secondary lipid messangers. After the activation effects reach some point, CRP attenuates these activities by inducing the SHIP-Shc mediated negative signaling pathway. In this model, SHIP can bind to the Tyr-P of ITAM 82 on FcyRIIa, but that does not exclude the possibility that it may bind to the Tyr-P of FcyRIIb. The net effect of this precisely regulated cellular mechanism is a controlled phagocytic activity of neutrophils in innate host defense. 83 IP: Anri-FcvRn cocktail (ATIO, 0 6 1 , IV3) A: Blat: uitt-TjrT-P 1 2 3 4 5 6 7 50K ■ [gOHChua 33 K . B: Blok AntiFrrRIlb (20) IgOHCkin AHoCRF (t]g6nl): 0 to 100200 HuSAP; too AlgO too tU jV O , Flguc 12. ESsctof ttunuaCItPon the Tyr-P of FcRfllh. HL.60 celt: (20-30 x t0‘) were incttbalMi witlt tO-200 pg/mtofCRP el OTC lord minend itieatywi Cytosolic collections were then nunuDopiecrpitated with tp g ^ p ls ofanti-FcyRn eoclctail(AT10, KBdt. IV J), et f C ovetniglrl end wparele bySDS-PAOE (73%) belore tienslerto NC menirene Ttie membrane was fiist probed withanti-Tyr-P (A), end then re-probed witli ratibit-entr-Fe FcyRIIb (263) (B). The signets were detected by chemolnninescene end eutoradiographjr. The Fciiuie -4) kDe bend is indicated by tlie arrow. A* Heat aggregate 84 IP: Auti-SHIP 160 K SHIP ABtet: AntUTji-P SHIP BBltfeAnd-SHIP ÙL HuORP (ug/ml): 0 10 100 200 A(ug/ml) 100 Figinc 13, EBéctofCItPoiitheT]rT-PofSHIPinRL'CI(Ocent. CjrtosaIpnp« 2atioM Qom ctSt mcnbated trith 10 or 200 pgtal of tggngUKl CRP mreimittqnopneipitatwi with 85 IP; Anci-Shc Blot: Aiiti-SHTP 1 2 3 4 5 6 160 K ^ _| - -►-SHIP AHuCRP(ugital): 0 10 100 200 HuSAP (u^ml): 100 AIgG (u^ml) 100 Flgun 14L Eaéct(fCRPoKtk>te 86 IP: a-SH IP A Bkt: o^P-Tyr 1 2 3 4 3 6N»yo. ItfOK . SHIP ■ " s B Bloc a-SHIP SHIP Time (min): 0 0.5 1 3 7 12 A HuCRP 100 Mgünl ______ Ficon 15. EBèetof CKPondieTTr-PofSHIPiiiHL-CO (G)ceUi. Cytosol pnpentiotis fiom esUtiacdrntedwitli 100 MgAnlofaggRgatMlCRP overindieatedtime inten'ab wen immnnopiecipitated withanti-SHIP and sepaiaied hy SDS-PAGE be&n pmbingwith and- Tyr-P (A) and then with and-SHIP (B). The SHIP band is -145 kDa as shown. The chemohiminescence intensity was detected and cjuantifiedby nsmg a Lnmi-Imager™ Oora Mannheiia-Boehhnger. 87 4 5 160 K PLCyi s AHuCRP(ug/mI):0 200 20 200 fMLP (nM> : 0 0 too 100 Flc«n W. E&(t *( CRP «a m iW a th n *f FLCyZ m 1l« mernhnneimdmceAhy l&nf. H L^(G ) a lb (20x106 per 200 nOtnniiictibated with lOOaMiMIP for 3 min tfter incitelion with aggngeledCRP for 10 mis and than cell nanfaraae* wen ptutOed by iiltiaantnOigitiBXL Aneqaal «noant of membrane proteia (60 to 80 n;) waanpanted by SDS-PAOE, Innafemd and pmbed wfh nbbtt-anli-hnmas PLCr2 (1:1G00). 88 + Activiition - Inhibition CRP ffiILP CD?- ^PKC Phagocytosis Inhibition Fig 17 CRP induced signaling in neutrophils 89 SUMMARY The acute-phase response is the early and immediate phase of systematic inflammation after tissue injury or bacterial infection. It is characterized by fever, increased vascular permeability, leukocytosis, and the radical change of the synthesis and rapidly increased secretion of plasma proteins by hepatocytes, which are known as acute-phase proteins or more commonly APRs. C-reactive protein (CRP) is the prototype acute-phase APR in humans. During acute phase response, its plasma concentration can be increased up to as much as 1000-fold compared to normal. CRP exists as a very stable and functional pentamer of identical noncovelently associated protomer, each subunit consisting of 206 amino acids and assembled as a flat pentamaric disk. CRP is capable of Ca^- dependent lectin-like binding to phosphocholine-containing substrates and other monophosphate esters with a single such binding site per subunit. Along with other APRs, CRP has been classified as an innate “recognition lectin”, not only because the three-dimensional structure of each of its five identical subunits is similar to ConA, but also because of its potential to couple nonspecific host response with specific immunity. CRP exerts many of the effector activities often associated with specific IgG antibodies. It serves as a ligand for a specific CRP receptor (CRP-R) on 90 leukocytes, activating monocytes/macrophages, but inhibiting the chemotactic activity and the respiratory burst of neutrophils (PMN). CRP expressed as a transgene in mice has been shown to contribute to innate host protection by activating complement and mediating phagocytosis. CRP also has been shown to inhibit PMN infiltration of inflamed lungs, as well as septic shock in a transgenic mouse model. Earlier, Leukocyte CRP-R was thought to be unique but containing characteristics that are related to IgG Fc-R. However, recent studies have identified that the 40 kDa IgG FcyRH is the predominant CRP-R in both human monocytes and PMN. Previous research in this laboratory revealed that CRP inhibits the superoxide (O2 ) production in neutrophil-like HL-60 granulocytes activated by the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and PMA by inhibiting the p47-phox protein, a critical component of the NADPH-dependent oxidase, and PKC membrane translocation, thus blocking the assembly of the active NADPH oxidase. This laboratory has also shown that CRP inhibited PKC activation, while activating phophoinosital-3 kinase (PI3-K) activity. However, the detailed CRP-R signaling pathway influenced by binding of CRP to FcyRH has not been studied. In this report, my study showed that acute phase concentration of CRP induces the positive signaling and negative signaling pathways in a sequential manner. In the positive signaling, CRP induced a series of activation event including tyrosine phosphorylation of the ITAM of FcyRIIa. Syk and PLCy2, promotion of the interaction between FcyRIIa and Syk as well as the translocation of PLCy2 and PI-3K to membrane. In negative signaling, CRP induces tyrosine phosphorylation of SHIP in a dose-response manner and it promotes the SHIP-interaction. CRP was also found inhibit fMLP-induced membrane localization of PLCy2. 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