cr1 and the cell membrane proteins that bind c3 and c4

Complement I

Immunol. Res. 6. 192-209 (1987)

II

�9 1987 S Karger AG, Basel 0257-277X/87/0063-019252.75/0

CR] and the Cell Membrane Proteins That Bind C3 and C4 A Basic and Clinical Review t

James G. Wilson, Nicolaos A. Andriopoulos, Douglas T. Fearon

Department of Rheumatology and Immunology, Brigham and Women's Hospital, and Departmen~ of Medicine, Harvard Medical School, Boston, Mass. USA; Department of Medicine, Division of Rheumatotogy, Hellenic Airforce and V.A. General Hospital, Athens, Greece; Department of Medicine, Vetcrans Administration Medical Center, Jackson, Miss. USA

Introduction

The human C3b/C4b receptor (CD35), termed complement receptor type 1 or CR 1, is a polymorphic glycoprotein [1-5], present on a variety of cell types, [1, 6-t0] that mediates binding between these cell types and particles or immune complexes that have activated complement and bear C3b and/or C4b. CR t participates in phagocytic reactions [11-17], in the clearance of immune complexes from the circulation [18], and perhaps in the regulation of immunoglobulin production by B lymphocytes [19]. The finding of functional [20, 21] and quantitative [9, 10, 22-27] deficiencies of CRl on erythrocytes [20-26], leukocytes [27] and glomerular podocytes [9, 19] of patients with systemic lupus e~thematosus (SLE) has suggested a role for abnormalities of CR1 in the pathogenesis of this disease. This suggestion, and the observation of genetically determined polymorphisms affecting the structure [1-5] and quantitative expression [23,

l Supported by grants AI 22833 and AM 36797 from the National Institutes of Health.

24, 26] of CR1 on cells of normal individuals prompted efforts to clone the CR 1 gene. The recent isolation and analysis of CRI cDNA [28, 29] and genomic [30] clones have provided an evolutionary model for the development of the CR1 structural altotypes [30] and have demonstrated that a polymorphic element linked to the CR I gene regulates the quantitative expression of CRI on erythrocytes [31]. This review will examine the cell and molecular biology of CR1, discuss the other known membrane proteins that bind fragments of C3 and C4, and consider the potential role of these complement-binding membrane proteins in inflammatory processes.

C3 and C3 Receptors

Cleavage of C3 by the convertases of the classical or alternative complement pathway [32] releases the anaphylotoxic fragment C3a [33] and exposes an internal thioester bond in the a-chain of C3b [34] that can bind to particles or immune complexes by a trans- acylation reaction [35] (fig. 1). Factor I-

Membrane Proteins That Bind C3 and C4 t93

mediated inactivation of C3b requires either factor H or CR1 as a cofactor [36, 37] and involves cleavage at two sites in the ct-chain, yielding the small fragment C3f and the he- molytically inactive iC3b fragment [38-41]. Cleavage by factor I at a third site in the a- chain requires CR 1 as a cofactor and releases the C3c fragment [42-44]. The C3d,g that remains bound to the activating surface or complex may undergo an additional cleavage by trypsin-like enzymes that remove C3g, leaving C3d as the residual bound fragment [45-48].

Six distinct membrane proteins in addi- tion to CR1 have been identified as having affinity for fragments of C3. The apparent Mrs, ligand specificities, and cellular distribution of these proteins are shown in table I.

CR2 (CD21, formerly termed the C3d receptor) is expressed on B lymphocytes [49- 52] and follicular dendritic cells [7], has affinity for the C3d region of iC3b, C3d,g and C3d [50, 52], and has further been identified as the B-cell receptor for the Epstein-Barr virus [53-56]. The biologic consequences of interaction between CR2 and C3d or C3d,g have not been established. However, the findings that cross-linked C3d,g can provide a necessary signal for the transition of activated, synchronized murine B cells from GI to S phase [57], and that incubation of B- cell-enriched human mononuclear cells with polyclonal anti-CR2 enhanced their proliferation in response to soluble products o f activated T cells [58], suggest that CR2 may reg- ulate B-cell growth. The additional observation that CR2 becomes phosphorylated upon treatment of human B cells with the tumor- promoting agent, phorbol myrislate acetate (PMA) [59], or after cross-linking of the membrane immunoglobulin of these cells [60] demonstrates the capacity of CR2 to

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Fig. 1. The proteolytic cleavage of C3. The hatched, thickened lines represent peptide chains. The position of the internal thioester that can form a covalent bond with an adjacent molecule is indicated in the or-chain. The intra-chain and inter-chain disulfide bonds are shown. * = Cleavage site for the classical or alternative pathway C3 convertase; l, 2, 3 = sites tbr sequential cleavage reactions mediated by factor I with H or CR1 as a cofactor: T = site for cleavage by twpsin-like enzymes that release C3g.

undergo a rapid chemical alteration through which receptor-mediated st imulation might be transduced to other cellular constituents.

CR3 (CDl lb ) , p150,95 ( C D I l c ) and a third protein, LFA- 1 (CD 11 a), are expressed

194 Wilson/A ndriopoulos/Fcaron

Table I. Membrane proteins that bind C3 and C4

Receptor Mr Ligands Cellular distribution

CR1 210,000-290,000 C3b, iC3b, C4b erythrocyte, neutrophil, monocyte, B lymphocyte, some T lymphocytes, eosinophil, mast cell, follicular dendritic cell, glomerular podocyte

CR2 t45,000 iC3b, C3d,g, C3d, B lymphocyte, follicular dendritic cell Epstein-Barr virus

CR3 ct 165,000 iC3b neutrophil, monocyte, large granular lymphocyte, 13 95,000 a follicular dendritic cell

CR4 9 C3d,g, iC3b, C3b neutrophil, platelet, B lymphocyte

gp45-70 45,000-70,000 C3b, iC3b, C4b neutrophil, monocyte, B lymphocyte, T lymphocyte

p150,95 ct 150,000 iC3b, ?C3d neutrophil, monocyte, large granular lymphocyte 13 95,000 ~

DAF 70,000 C3b, C4b erythrocyte, platelet, neutrophil, monocyte, B lymphocyte, T lymphocyte, endothelial cell

Apparently identical ~3 subunits as indicated by their immunoprecipitation with the monoclonaf antibody, anti-TSl/l 8 [61].

on neutrophils, monocytes and large granular lymphocytes and are related in having an identical 13-chain that is paired noncova- lently with their three distinct s-chains [61 ]. CR3 was initially defined functionally by its capacity to mediate adherence between iC3b-bearing particles and monocytes or neutrophils [62]. Subsequent studies demonstrated its participation in the phagocytosis of iC3b-bearing particles by cultured monocytes [15, 17]. Its molecular identity was established by the ability of monoclonal ant i -Mac-l /OKM1 antibodies to inhibit the formation of iC3b-dependent rosettes [63], and to adsorb a monocyte protein that could bind specifically to iC3b-bearing sheep erythrocytes [64]. More recently, p150,95 has also been found to have affinity for iC3b [65], and indirect data suggest that this pro-

tein may mediate binding between cultured human monocytes and particles bearing C3d [66, 67]. A number of patients have been identified in whom inherited deficiencies of CR3, p150,95 and LFA-1 have been associated with delayed umbilical cord separation, recurrent infections, progressive periodonti- tis and persistent leukocytosis [68-721. Neu- trophils from these patients not only have an impaired ability to bind iC3b-bearing sheep erythrocytes and to ingest complement-opsonized droplets of paraffin oil, but also have a more general defect of adhesion-dependent functions, including adherence to surfaces, spreading, orientation in chemotactic gra- dients, aggregation, and antibody-dependent cellular cytotoxicity. It is unclear which components of the clinical syndrome are attrib- utable to defective binding of complement-

Membrane Proteins That Bind C3 and C4 195

derived ligands, and which are caused by the more general defects of adhesion-dependent function.

CR4 has been defined by studies of the competitive binding of soluble C3d,g, iC3b and C3b to neutrophils, platelets and B lymphocytes; it is distinct from other C3-binding proteins in its specificity and its cellular distribution [73, 74]. The glycoprotein(s) designated gp45-70 (also termed membrane cofactor protein or MCP) [75, 76] is present in detergent lysates of neutrophils, monocytes, and B and T lymphocytes, and binds specifically to Sepharose beads bearing C3b, iC3b or C4b, but not C3d, suggesting that its affinity is to a region in the C3c fragment [75]. Proteins of a similar Mr and having affinity for C3b have been identified in rabbit alveolar macrophages [77] and in murine erythrocytes, lymphocytes, macrophages and cultured fibroblasts [78]. The murine protein, termed p65, cross-reacted with rabbit polyclonal antibody to human CR1. CR4 and gp45-70 do not mediate rosette formation between the cells on which they reside and particles beating their respective ligands [73-76]. The biologic functions of these proteins are unknown, although the capacity of gp45-70 to serve as a cofactor for the factor- I-mediated cleavage of C3b and C4b suggests that it may have a role in preventing complement-mediated damage to host cell membranes [76].

Decay-accelerating factor (DAF) is present on erythrocytes [79], platelets, neutrophils, monocytes, B and T lymphocytes [80, 81] and endothelial ceils [82], and was initially identified based on its capacity to pro- mote decay of the classical and alternative pathway C3 convertases [79]. Efforts to demonstrate directly that DAF, like factor H, C4-binding protein (C4bp) and CR1, me-

diates its decay function by binding to C3b and C4b have been unsuccessful [83]. How- ever, when erythrocytes bearing C3b or C4b and having DAF either endogenously present (human erythrocytes) or experimen- tally incorporated (sheep erythrocytes) into their membranes were treated with the cross- linking reagent dithio-bis-(succinimidyl pro- pionate), DAF was specifically cross-linked to C3b and C4b, indicating that it had been spatially associated with these proteins in the cell membrane [83]. Moreover, recent analysis of DAF cDNA clones has shown the presence of repeating sequences homologous to a consensus repeat found in several other proteins having C3/C4-binding function [84] (see below). A critical role of DAF in protect- ing host cells against damage by complement is illustrated by the disease paroxysmal nocturnal hemoglobinuria, in which the absence of DAF from a population of erythrocytes [85, 68], granulocytes, monocytes and platelets [81, 87] is associated with complement- mediated hemolysis and pathologic intravas- cular thrombosis.

Cell Biology of CR1

CR1 has affinity for C3b, iC3b and C4b [I, 88-91], and is present on erythrocytes, neutrophils, monocytes/macrophages, B lymphocytes, some T lymphocytes, eosinophils, mast cells, follicular dendritic cells, glomerular podocytes, and in soluble form in plasma [1, 6-10, 92-94]. Four altotypes of CR 1 have been observed that differ in MT by more than 100,000 but appear not to differ at their ligand binding site [2-5, 95]. The two most common allotypes, termed F and S, have apparent molecular weights of 250,000 and 290,000, and gene frequencies

196 WilsonJAndriopoulos/Fearon

in Caucasian populations of approximately 80 and 20%, respectively [2-4]. A rarer allotype designated F" has an apparent Mr of 210,000 [4, 31], and a fourth allotype that is larger than the S form has also been reported [5]. Mr differences among the four allotypes persist after treatment with endoglycosidase F, indicating that they are not the result of differential glycosylation [3-5], and biosyn- thetic studies have supported this conclusion [96]. The occurrence of the same allotypic forms of CRI on the erythrocytes, neutrophils and monocytes of each individual [3-5, 97] indicates that CR1 of these three cell types is a product of the same gene. How- ever, the observation that neutrophils [97] and T lymphocytes [6] have CRI with an apparent molecular weight that is slightly larger than that of other peripheral blood cells of the same donor suggests the occurrence of either cell-specific differential splicing or post-translational modification.

The function of CR 1 depends on the cell type on which it resides. CR 1 oferythrocytes participate in the clearance [ 18] and processing [43] of immune complexes, while those of monocytes and granulocytes mediate or potentiate phagocytic and endocytic reactions [l 1-17, 98]. Although the function of CRI of B lymphocytes has not been established, treatment of mononuclear cells with polyclonal anti-CR 1 has been shown to en- hance the production of immunoglobulin in response to sub-optimal doses of pokeweed mitogen [19]. The functions of CR1 of T lymphocytes, follicular dendritic cells and glomerular podocytes and of soluble CR 1 in plasma are unknown.

CR 1 of unstimulated monocytes and neutrophils resides in two pools; 10-20% is expressed in clusters on the cell surface [98, 99], and the remainder exists in an intracel-

lular compartment from which it can be mo- bilized by chemotactic peptides or mechani- cal stimuli [99, 100]. Cross-linking of CR1 on the cell surface either with polyclonal antibody or indirectly with C3b and polyclonal anti-C3 induces adsorptive endocytosis of the receptor-ligand complexes through clathrin-coated pits and vesicles [98, 101].

Production of a limited degree of receptor cross-linking with either monoc/onal anti- CR1 or dimeric C3b resulted in the attach- ment of CR1 to the cytoskeleton [102] and its redistribution into caps and patches that were associated with subptasmalemmal ac- cumulations of myosin [ 103]. This redistribution was inhibitable by cytochalasin D or chlorpromazine, and was accompanied by redistribution of IgG Fc receptors (FcR) to the same areas of the plasma membrane as CR1 [103]. CR1, CR3 and FcR that were immunoadsorbed from solubilized membranes of neutrophils were capable of binding ~25I-actin in amounts exceeding those bound by major histocompatibility complex class I of neutrophils or CR I of erythrocytes [I02]. These observations and the finding that phagocytic reactions of monocytes and granulocytes are inhibitable by cytochalasins B and D [104, 105] suggest that phagocytosis mediated by CR1, CR3 and FcR may be dependent on their direct or indirect interaction with cytoskeletal actin.

CR 1 and CR3 of resting myelomonocytic cells are able to bind ligand-coated particles but cannot mediate phagocytosis [14, 15]. The first demonstration that complement receptors could acquire phagocytic capability upon cellular activation was provided by studies in which murine peritoneal macrophages were treated with the soluble products of activated T cells [13]. It was shown subsequently that human monocytes and


neutrophils that had been incubated with PMA were capable of CRI- and CR3-mediated phagocytosis [15] and exhibited ligand-independent internalization of CR1 [106]. Three proteins of the extracellular ma- trix, fibronectin [16], serum amyloid P [17] and laminin [107] have also been shown to activate CR1 and CR3; thus, the acquisition of a capacity for complement-mediated phagocytosis may occur physiologically as cells migrate to the extravascular space.

The finding that CR1 and CR3 could be activated by PMA suggested a role for protein phosphorylation by protein kinase C [108] in altering the functional capacity of these receptors. Assessment of the incorpo- ration of 32po4 into CR1, CR3, and FcR of neutrophils showed that none of these proteins was constitutively phosphorylated. Stimulation of the cells with PMA induced rapid phosphorylation of CRI, but not of CR3 or FcR [59]. CRI of two other phagocytic cell types, monocytes and eosinophils, was also phosphorylated in response to treatment of the cells with PMA, but CR 1 of the non-phagovytic cell types, erythrocytes, B lymphocytes, and B lymphoblastoid cells was not phosphorylated [59]. Treatment of neutrophils with the chemotactic peptide N - formyl - methionyl - leucyl- phenylalanine (FMLP) also resulted in rapid phosphorylation of CR1 [59]. FMLP in the presence of fibronectin has been reported to induce expression of phagocytic activity by CRI [109]; thus two agents that can activate CR1 for phagocytosis also stimulate its phosphorylation. The lack of phosphorylation of CR3 in PMA-treated cells, despite its reported activation by this agent, suggests that the interactions with other cellular proteins that are required for phagocytosis may differ for CR1 and CR3.

Erythrocytes express approximately 500 CR1 molecules per cell [22, 23], 50- to 100- fold fewer than are present on monocytes, neutrophils and B lymphocytes [1]. How- ever, 85-90% of CR1 in the circulation resides on erythrocytes because of the predom- inance of this cell type in blood [1]. Serum- opsonized immune complexes bind rapidly to CR1 of erythrocytes [110, 111] and are subsequently released after C3b in the complexes is cleaved to C3d,g by factor I, with CRI as a cofactor [43, 111]. Kinetics of the binding and release reactions are dependent on both the nature of the immune complexes and the number of CR1 on the erythrocytes [111, 112]. Preformed immune complexes that were injected intravenously into pri- mates were bound by CRI of erythrocytes and deposited in the organs of the reticulo- endothelial system, primarily the liver [18]. Analysis of clearance studies employing IgG- or IgM-sensitized erythrocytes has indicated that complement receptors of reticuloendo- thelial cells mediate both a sequestration phase and a subsequent phagocytic phase in the clearance of ligand-bearing particles from the circulation [ 113, 114]. Similarly, immune complexes that are released from CR1 of erythrocytes by factor I may be bound and subsequently ingested by reticu- loendothelial phagocytes.

Normal individuals may differ from each other by as much as tenfold in the number of CR 1 on their erythrocytes, but for each individual this number represents a relatively stable characteristic [23]. The existence of a genetic mechanism regulating the number of CR1 on erythrocytes was initially indicated by population studies and pedigree analyses [23, 24, 26], and it was proposed that this number was regulated by at least two autosomal, codominant alleles [23]. Analyses of

198 Wilson/A ndriopoulos/Fearon

the CRI gene have now validated and ex- panded this model [31] (see below) and have thus established the existence of genetic differences among individuals that may in- fluence their relative capacities to clear and process immune complexes.

Molecular Biology of CRI

Approximately 80% of the coding sequence of CR1 has now been obtained by analysis of overlapping cDNA clones [28, 29]. Its structure is characterized by the occurrence of at least four tandem, direct, long homologous repeats (LHRs) of 450 amino acids, that are 70-95 % identical in their nucleotide and amino acid sequences [29]. Each LHR is comprised of seven repeated sequences of 60-65 amino acids, termed short consensus repeats (SCRs). Contained in each SCR are 10-15 conserved positions that also occur in other C3/C4-binding proteins including factor H, C4bp, C2, factor B, CR2 and DAF [84, 115-119] and in proteins not known to have this function, including the IL-2 receptor, t32-glycoprotein 1, Clr, haptoglobin a-chain and factor XIIIb [120- 124]. At the carboxyterminus of the receptor is a typical membrane-spanning region of 25 hydrophobic amino acids followed by four positively charged residues and a cytoplasmic tail of 39 residues [29]. The conserva- tion of four half-cystines per SCR and of amino acids such as proline, glycine and asparagine that are frequently present in /3- turns have ted to the proposal, by analogy to the structure of [3z-glycoprotein 1 [ 121], that disulfide linkages between the first and third and the second and fourth half-cystines create a triple-loop structure within each SCR. A series of such structures would be

predicted to form a semi-rigid, elongated molecule that could extend from the membrane of cells bearing CR 1 (fig. 2) and allow their interaction with C3b bound at relatively inaccessible sites.

An intervening sequence probe derived from a CR1 genomic clone hybridizes to at least three separate restriction fragments in Southern blots of DNA from normal individuals [30]. This observation suggests that the LHRs have evolved by a series of events involving duplication of large genomic segments containing exons encoding the con- stituent SCRs of an LHR. The analysis of restriction fragment length polymorphisms (RFLPs) in Southern blots of DNA from individuals having different structural allotypes of CR 1 indicates that these may be the products of alleles that differ in the number of LHRs that they encode [30].

Genetic regulation of the quantitative expression of CR1 on erythrocytes has been proven by the demonstration of an RFLP that correlates with high, intermediate and low CRI number [31]. Normal individuals who are homozygous for either of two allelic HindIII fragments of 7.4 and 6.9 kb express high and low numbers, respectively, of CR l on erythrocytes; individuals who are heterozygous for these alleles have intermediate numbers of receptors. The controlling element that is identified by this RFLP regulates the expression of CR1 on erythrocytes by a cis-acting mechanism, and does not affect total CR 1 expression by leukocytes.

Evidence indicates that genetic elements other than that associated with the HindIII RFLP also participate in regulating the quantitative expression of CR 1 on erythrocytes. A three- to fourfold range of numbers of CR1 on erythrocytes is observed both among individuals who are homozygous for the 7.4-kb


LHR-A " L H R - B LHR-C LHR-D ~ "

Fig. 2. Diagram of the proposed structure of CR I. The COOH-terminal cytoplasmic tail and transmem- brane region are shown. Thirty SCRs extend from the cell membrane. The LHRs are indicated by brackets. An enlargement of a single SCR is shown in the inset to illustrate the triple loop structure. Reproduced from the "Journal of Experimental Medicine', 1987, vol. 165, p. 1107, by copyright permission of the Rockefeller Uni- versity Press.

form of the RFLP and among those who are heterozygous [31]. However, the relative amounts of CRl expressed on erythrocytes of individuals within these groups are stable over time [125], suggesting that the intra- group differences among individuals are genetically determined, rather than resulting from fluctuation about a genetically determined mean. This conclusion is supported by studies in which the known linkage between the genes for CR1, factor H and C4bp [t26] was exploited by 'haplotyping' individuals with respect to polymorphisms of these proteins [127]. Analysis of the segregation of these haplotypes in families, in conjunction with quantitation of CR1 on erythrocytes, suggested the existence either of more than two alleles at a single locus or of combinations of alleles at different loci determining the number of CR1 on erythrocytes. These pu- tative alleles, like those identified by the HindIII RFLP, were cis-acting, controlling expression only of the CR 1 structural allotype to which they were linked.

Finally, in situ hybridization studies and analysis of somatic cell hybrids using CRI cDNA probes have indicated that the linkage group comprised of the genes for CR 1, factor H, C4bp [126] and CR2 [128] is located on the long arm of chromosome 1, band q32 [128]. The application of techniques for the analysis of large chromosomal fragments [129] should allow determination of the or- der of these genes on the chromosome.

Abnormalities of CR1 in SLE

Patients with SLE have decreased numbers of CRI on their erythrocytes [20-26], B lymphoc)tes [27] and neutrophils [27], and CRI antigen is absent from glomerular podocytes affected by diffuse proliferative glomerulonephritis of SLE [9, I0]. In addi- tion, clearance studies utilizing radiolabeled, autologous erythrocytes that were sensitized with IgG anti-Rh(D) indicate that reticulo- endothelial phagocytes of SLE patients have

200 W ilson/ Andriopoulos/ Fearon

impaired function not only of FcRs [130, 131] but also of another, complement-dependent receptor, perhaps CR 1 [ 132, 133]. A relative deficiency of CR2 on B lymphocytes that correlates with that of CRI is also seen among patients with SLE [27].

Evidence that the number of CR1 on erythrocytes can be altered secondarily in SLE includes the temporal variation of this number in patients with active disease [21, 22, 25], its direct correlation with serum concentrations of C4 and inverse correlation with those of immune complexes [22], and the progressive loss of CR1 from erythrocytes that were transfused into a patient with active SEE [134]. An inverse correlation in the numbers of molecules of CR1 and C3d,g on erythrocytes of patients with SLE or complement-mediated hemolytic anemias suggests that CR1 loss may be a consequence of complementactivation near the cell surface [25]. An additional mechanism of CR I loss may be illustrated by a patient with active SLE in whom high titers of an autoantibody to CR1 were associated with absence of CRl from erythrocytes and its marked reduction on B lymphocytes and neutrophils [135]. De- creased titers of the autoantibody coincided with the patient's clinical improvement and were followed by a partial reversal of her receptor abnormalities.

The finding of decreased numbers o fCRl on erythrocytes among both patients with SLE and their healthy relatives in populations in Boston [23] and Tok~s [20, 26] has suggested that inheritance of low numbers of CRI on erythrocytes may represent a predis- posing factor for the development of SEE. However, the relatives of SLE patients in Cambridge, UK, were found not to differ from normal in their mean number of CR1 on erythrocytes [24], and these disparate re-

suits have prompted a careful reanalysis of the population in Boston [125]. In the more recent study, the mean number of CR1 on erythrocytes for 93 first-degree relatives of SLE patients in 28 kinships was 83 % of the normal mean, and the difference between these means was highly significant (p < 0.002). During 1-4 years, the number of CRI on erythrocytes was as stable a characteristic for patients with SLE as for their healthy relatives and normal individuals; thus any secondary processes affecting this number were either constant or of small magnitude. Patients differed from normal in the occurrence of the alleles identified by the CR1 HindIII RFLP, with fewer patients be- ing homozygous for the 7.4-kb fragment that is associated with high numbers of CR1 on erythrocytes. However, this finding did not wholly account for the relative CRI deficiencies of the patients and their family mem- bers. Rather, when normal individuals, patients and relatives of the patients were grouped according to their genotype for the HindlII RFLP, both patients and their relatives had lower mean numbers of CR1 on erythrocytes than did normal individuals of the same genotype. It is possible that the decreased numbers of CR1 on erythrocytes among the patients and their relatives were acquired. However, the temporal stability of these numbers and the lack of other apparent abnormalities among the relatives favor the interpretation that the decreased numbers of CRI on their erythrocytes were determined by genetic element(s) whose effects are addi- tive with those of the element identified by the HindlII RFLP.

To investigate whether geographically separated populations might differ in the occurrence of genes affecting CRI expression, the binding to e~'throcytes of the mono-


Fig. 3, Comparison of the number of CRI on erythrocytes (CRI/E) for patients with SLE with the mean number of CRI/E for each patienfs relatives. Patients and their relatives from Athens (A) and Bos- ton (B) were analyzed sepa- rately.

g-.

•

"S

. i':""

b

" . - ' i

~ 2 3 4 5 6 7 , 2 ~ - " ~ 5 ~ 7

CR1/E of Pro~and (x lO -~) CR1/E o~ Proband {'xlO-~l

clonal ant i -CRl antibody, Yz-I [106], was assessed among healthy individuals in Bos- ton and among healthy donors, patients with SLE and relatives of the patients in Athens, Greece. All assays were performed in Bos- ton, using erythrocytes from EDTA-antico- adulated whole blood that had been drawn less than 120h previously and stored at 4 ~ The number of CR1 on erythrocytes stored in this manner was found to be stable for at least 12 days. The mean number of CR 1 per erythrocytes for healthy individuals in Athens, 452 + 16 (SEM; n = 135), was significantly lower than that of the Boston population. 553 _+ 21 (n --- 100; p < 0.0005; Student 's t test). Patients in Greece had fewer CRI per erythrocytes, 257 + 31 (n = 19), than did healthy individuals (p < 0.0002), but the mean number of CRI per erythrocytes for 60 first-degree relatives of 18 of the patients, 445 +_ 24, did not differ from the mean for the normal population. To examine the combined effects of genetic regulation and pathologic processes related to SLE on CRI expression in the patients, the number of CR 1 per eD'throcytes for each

patient was compared to the mean number of CR1 per erythrocytes for the pat ient 's f~rst-degree relatives (fig. 3A). A significant correlation between these numbers (Spear- man correlation coefficient = 0.641; p < 0.005) indicated that genetic regulation of CRI expression was evident among the patients. Comparable results were obtained when patients and their relatives included in an earlier study of a Boston populat ion [125] were analyzed in the same fashion (fig. 3B; correlation coefficient = 0.404; p < 0.04). The findings that almost all patients in Ath- ens had fewer CR1 per erythrocytes than did their relatives and that the mean for all of the relatives did not differ f rom normal are not consistent with an autosomal codominant mechanism for inheritance of low numbers of CR1 on erythrocytes among the patients in this population. Thus, low CRI on erythrocytes of Greek patients may result from a recessive abnormali ty or may be acquired. The observat ion that the mean number of CR 1 on erythroc~tes for the normal population in Athens was approximately 20% lower than that in Boston demonstrates the exis-

202 Wilson/Andriopoulos/Fearon

tence of major differences between separate populations with respect to genetic factors affecting CR1 expression, and indicates the need for investigation of such factors at the molecular level.

Previous studies have established an association between inherited abnormali t ies o f the components of the classical complement pathway, C 1 q, C 1 r, C ls, C4, C2 and C3, and the occurrence o f a u t o i m m u n e disease [136]. Deposit ion of C3 on immune complexes is dependent on the normal activation of this pathway; thus a potential common effect o f deficiency of one of these proteins or o f a relative deficiency of CR1 on erythrocytes would be impaired processing of immune complexes. Such an impairment could result not only in the deposition of these complexes at peripheral tissue sites, but also in a failure of normal interaction between the complexes and cells o f the reticuloendotheliat system. The critical interactions affected could include C R I - and CR3-dependent phagocytosis by macrophages, antigen pre- sentation by macrophages and follicular dendritic cells, and perhaps regulation of the growth and matura t ion of B cells through CRI and CR2.

Conclusion

The covalent binding of C3 and C4 to particles or immune complexes allows their subsequent interaction with cell membrane proteins having affinity for portions of the C3 and C4 molecules. CR1 is a polymorphic glycoprotein comprised of a single peptide chain that has affinity for C3b, iC3b and C4b. CR1 participates in the clearance and processing of circulating immune complexes, in the phagocytosis o f C3b and C4b-

bearing particles, and as a cofactor for the cleavage of C3b by factor I. The latter function at tenuates complement act ivat ion near host cells and generates iC3b and C3d,g that can bind to any o f at least five additional membrane proteins. Inheri ted and acquired abnormali t ies of CR 1 may participate in the pathogenesis o f SLE by altering the clearance and processing of circulating immune complexes and perhaps also by affecting the regulation of B lymphocyte growth and maturation.

References

1 Fearon, D.T.: Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte. B lymphocyte, and monocyte. J. exp. Med. 152: 20- 30 (1980).

2 Dykman, T.R.; Cole, J.L.; Iida, K.; Atkinson. J.P.: Polymorphism of human eryIhrocyte C3b/C4b receptor. Proc. natn. Acad. Sci. USA 80: 1698-1702 (1983).

3 Wong, W.W.; Wilson, J.G.; Fearon, D.T.: Genetic regulation of a structural polymorphism of human C3b receptor. J. clin. Invest. 7 2 : 6 8 5 - 6 9 3 (1983).

4 Dykman, T.R.; Hatch, J.A.; Atkinson, J.P.: Poly- morphism of the human C3b/C4b receptor. Iden- tification of a third allele and analysis of receptor phenotypes in families and patients with systemic lupus erythematosus. J. exp. Med. 159:691-703 (1984).

5 Dykman, T.R.; Hatch, J.A.; Aqua, M.S.; Atkin- son, J.P.: Polymorphism of the C3b/C4b receptor (CR1): characterization of a fourth allele. J. Im- mun. 134:1787-1789 (1985).

6 Wilson, J.G.; Tedder, T.F.: Fearon, D.T.: Charac- terization of human T lymphocytes that express the C3b receptor. J. Immun. 131." 684-689 (1983).

7 Reynes, M.; Aubert, J.P.; Cohen, J.H.M.; Au- douin, J.; Tricotet, V.; Diebold, J.: Kazatchkine, M.D.: Human follicular dendritic cells express CRI, CR2 and CR3 complement receptor anti- gens. J. lmmun. 135:2687-2694 (1985).


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9 Kazatchkine, M.D.: Fearon, D.T.; Appay, M.D.; Mandet, C.; Bariety, J.: hnmunohistochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal diseases. J. clin. Invest. 69:900-912 (1982).

10 Emancipator, S.N.; lida, K.; Nussenwzeig, V.; Gallo, G.R.: Monoclonal antibodies to human complement receptor (CRI) detect defects in glomerular diseases. Clin. Immunol. Immunopathol. 27." 170-175 (1983).

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13 Griffin, LAx Griffin, F.M.: Augmentation ofmac- rophage complement receptor function in vitro. I. Characterization of the cellular interactions required for the generation ofa T lymphocyte product that enhances macrophage complement receptor function. J. exp. Med. I50:653-675 (1979).

14 Newman, S.L.: Musson, R.A.; Henson, P.M.: De- velopment of functional complement receptors during in vitro maturation of human monocytes into macrophages. J. Immun. I25. 2236-2244 (1980).

15 Wright, S.D.: Silverstein, S.C.: Tumor-promoting phorbol esters stimulate C3b and C3b" receptor- mediated phagocytosis in cultured human monocytes, J. exp. Med. 156:1149-1164 (1982).

16 Pommier, C.G.; Inada, S.; Fries, F.; Takahashi, T.; Frank, M.M.; Brown, E.J.: Plasma fibronectin enhances phagocytosis of opsonized particles by human peripheral blood monocytes. J. exp. Med. 157:1844-1854 (1983).

17 Wright, S.D.: Craigmyle, L.S.; Silverstein, S.C.: Fibronectin and serum amyloid P component stimulate C3b and C3bi-mcdiated phagocytosis in cultured human monocytes. J. exp. Med. 158: 1338-1343 (1983).

18 Cornacoff, J.B.; Hebert, L.A.; Smead, W.L.; Van Aman, M.E.; Birmingham, D.J.; Waxman, F.J.: Primate erythrocyte immune complex clearing mechanism. J. clin. Invest. 71:236-247 (1983).

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20 Miyakawa, Y.; Yamada, A.; Kosaka, K.; Tsuda, F.; Kosugi, E.; Mayumi, M.: Defective immune adherence (C3b) receptor on erythrocytes of patients with systemic lupus erythematosus. Lancet ii: 493-497 (1981).

21 Taylor, R.P.; Horgan, C.; Buschbacher, R.; Brun- ner, C.M.; Hess, C.E.; O'Brien, W.M.; Wanebo, H.J.: Decreased complement-mediated binding of antibody/3H-dsDNA immune complexes of the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hemato- logic malignancies. Arthritis Rheum. 26:736-744 (1983).

22 lida, K.; Mornaghi, R.; Nussenzweig, V.: Comple- ment receptor (CRt) deficiency in erythrocytes from patients with systemic lupus erythematosus. J. exp. IVied. 155:1427-1438 (1982).

23 Wilson, J.G.; Wong, W.W.; Schur, P.H.; Fearon, D.T.: Mode of inheritance of decreased C3b receptors on erythrocytes of patients with systemic lupus erythematous. New Engl. J. Med. 307. 981- 986 (1982).

24 Walport, M.J.; Ross, G.D." Mackworth-Young, C.; Watson, J.V.; Hogg, N_; Lachmann, P.J.: Family studies of er3'throcyte complement receptor type I levels: reduced levels in patients with SLE are acquired, not inherited, Clin. exp. Immu- not. 59:547-554 (1985).

25 Ross, G.D.; Yount, W.J.: Walport, M.J.; Winfield, J.B.; Parker, C.J.; Fuller, C.R.: Taylor, R.P.: Myones, B.L.; Lachmann, P.J.: Acquired loss of erythrocyte (E) CRI (C3b-receptor) in systemic lupus erythematosus and other diseases with au- toantibodies and/or complement activation. J. Immun. 135:2005-2014 (1984).

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27 Wilson, J.G.; Ratnoff, W.D.; Schur, P.H.: Fearon, D.T.: Decreased expression of the C3b/C4b receptor (CRI) and the C3d receptor (CR2) on B lymphocytes and of CR1 on neutrophils of patients with systemic lupus ervthematosus. Arthritis Rheum. 29:739-747 (1986).

204 Wilson/Andriopoulos/Fearon

28 Wong, W.W.; Klickstein, L.B.; Smith, J.A.: Wets, J.H.; Fearon, D.T.: Identification of a partial eDNA clone for the human receptor for complement fragments C3b/C4b. Proc. natn. Acad. Scl. USA 82:7711-7715 (1985).

29 Klickstein, L.B.; Wong, W.W.; Smith, J.A.; Weis, J.H.; Wilson, J.G.; Fearon, D.T.: Human C3b/C4b receptor (CR1): Demonstration of long homologous repeating domains that are composed of the short consensus repeats characteristic of C3/C4 binding proteins. J. exp. Med. 165: 1095- 1112(1987).

30 Wong. W.W.; Kennedy, C.A.; Bonaccio, E.T.; Wilson, J.G,; Ktickstein, L.B.; Weis, J.H,: Fearon, D.T.: Analysis of multiple restriction fragment length polymorphisms of the gene for the human complement receptor type I: duplication of genomic sequences occurs in association with a high molecular weight receptor allotype. J. exp. Med. I64:1531-1546 (1986).

31 Wilson, J.G.; Murphy. E.E.; Wong, W.W.; KJick- stein, L.B.; Wets, J.H.; Fearon, D.T.: Identifica- tion of a restriction fragment length polymorphism by a CRI eDNA that correlates with the number of CR1 on erythrocytes. J. exp. Med. 164: 50-59 (1986).

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43 Medof, M.E.; Prince, G.M.; Mold, C.: Release of soluble immune complexes from immune adherence receptors on human eLythrocytes is mediated by C3b inactivator independently of Igl H and is accompanied by generation of C3c. Proc. natn. Acad. Sci. USA 79:5047-5051 (1982).

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46 Law, S.K.; Fearon, D.T.; Levine, R.P.: Action of the C3b-inactivator on ceil:bound C3b. J. lmmun. 122:759-765 (1979).

47 Chaplin. H.; Monroe, M.C.: kachmann, P.J.: Fur- ther studies of the C3g component of the a2D fragment of human C3. Clin. exp. ImmunoL 51.- 639-646 (1982).

48 Davis, A.E., Ill; Harrison. RA.: Lachmann, PA.: Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d. g (a2D), and C3g. J. Immun. 132:1960-1966 (1984).

49 Eden, A.; Miller, G.W.: Nussenzweig, V.: Human lymphocytes bear membrane receptors for C3b and C3d. J. clin. Invest. 52:3229-3237 (1973).


50 Ross, G.D.; Polley, M.J.; Rabellino, E.M.; Grey. H.M.: Two different complement receptors on human lymphocytes; one specific for C3b and one specific for C3b inactivator-cleaved C3b. J. exp. Med. 138:798-811 (1973).

51 lida, K.; Nadler, L.; Nussenzweig. V.: Identifica- tion of the membrane receptor for the complement fragment C3d by means of a monoclonal antibody. J. exp. Med. 158:1021-1033 (1983).

52 Weis, J.J.: Tedder, T.F.: Fearon, D.T.: Identifica- tion of a 145,000 M~ membrane protein as the C3d receptor (CR2) of human B lymphocytes. Proe. nam. Acad. Sci. USA 81:881-885 (1984).

53 Fingeroth. J.D.; Weis, J.J.; Tedder, T.F.; Stromin- ger, J.L.; Biro, P.A.; Fearon, D.T.: Epstein-Barr virus receptor of human B lymphocytes is tile C3d receptor CR2. Proc, hath. Acad. Sci. USA 81. 4510-4514 (1984).

54 Nemerow, G.R.; Wolfert, R.; McNaughton, M.E.; Cooper, N.R.: Identification and characterization of the Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2). J. Virol. 55:347-351 (1985)_

55 Nadler, L.M.; Boyd, A.W.; Park, E.; Anderson, K.C.; Slaughenhoupt, B.; Thorley-Lawson, D.A.; Schlossman, S.F.: The B cell-restricted glyeopro- rein B2 is the receptor for Epstein-Barr virus; in Reinhe~, Hayncs, Nadler, Bernstein, Leukocyte typing It, pp. 509-518 (Springer, New York 1986).

56 Frade, R.: Barel, M.; Ehlin-Henriksson, B.; Klein, G.: gpl40, the C3d receptor of human B [ympho- cytes, is also the Epstein-Barr virus receptor. Proc. natn. Acad. Sci. USA 82:1490-1493 (1985).

57 Melchers, F.; Erdei, A.; Schulz, T.; Dierich, M.P.: Growth control of activated, synchronized murine B cells by the C3d fragment of human complement. Nature, Lond. 317:264-267 (1985).

58 Frade, R.; Crevon, M.C.; Barel, M.; Vazquez, A.; Krikorian, L.: Charriaut, C.; Galanaud, P.: En- hancement of human B cell proliferation by an antibody to the C3d receptor, the gp 140 molecule. Eur. J. Immunol. 15:73-76 (1985).

59 Changelian, P.S.; Fearon, D.T.: Tissue-specific phosphorylation of complement receptors CRI and CR2. J. exp. Med. 163:101-115 (1986).

60 Barel, M.; Vazquez, A.; Charriaut, C.; Aufredou, M.T.; Galanaud, P.; Frade, R.: gp 140, the C3d/EBV receptor (CR2), is phosphorylated upon

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61 Sanchez-Madrid, F.; Nagy, J.A.; Robbins, E.; Si- mon, P.; Springer, T.A.: A human leukocyte dig ferentiation antigen family with distinct or-subunits and a common !8-subunit: the lymphocyte function-associated antigen (LFA-I), the C3bi complement receptor (OKMI/Mac-I), and the p150,95 molecule. J. exp. Med. 158:1785-1803 (1983).

62 Ross, G.D.; Newman, S.L.; Lambris, J.DA Deve- ry-Pocius, .I.E.; Cain, J.A., Lachmann, P.J.: Gen- eration of three different fragments of bound C3b with purified factor I or serum. J. exp. Med. 158: 334-352 (1983).

63 BeIler, D.I.; Springer, T.A.; Schreiber, R.D.: Anti- Mac-I selectively inhibits the mouse and human type three complement receptor. J. exp. Med. 156: 1000-1009 (1982).

64 Wright, S.D.; Rao, P.E.; Van Voorhis, W.C.; Craigmyle, L.S.; Iida, K.; Talle, M.A.; Westberg, E.F; Goldstein, G.; Silverstein, S.C.: ldentifica- lion of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies. Proc. nam. Acad. Sci. USA 80:5699-5703 (1983).

65 Micklem, K.J.; Sire, R.B.; Isolation of complement-fragment-iC3b-binding proteins by affinity chromatography: the idenfication of p150,95 as an iC3b-binding protein. Biochem. J. 231: 233- 236 (1985).

66 lnada, S.; Brown, E.J.; Gaither, T.A.: Hammer, C.H.; Takahashi, T.; Frank, M.M.: C3d receptors are expressed on human monocytes alter in vitro cultivation. Proc. natn. Acad. Sci. USA 80:235 I- 2355 (1983).

67 Wright, S.D.; Licht, M.R.; Silverstein, S.C.: The receptor for C3d (CR2) is a homologue of CR3 and LFA-I (Abstract). Fed. Proc. 43:413 (1984).

68 Crowley, C.A.; Curnutte, J.T.; Rosin, R.E.; Andre- Schwartz, J.; Gallin, J.l.; Klempner, J.l.; Snyder- man, R.; Southwick. F.S.; Stossel, T.P.; Babior, B.M.: An inherited abnormality of neutrophil adhesion: its genetic transmission and its association with a missing protein. New Engl. J. Med. 302: 1163-1168 (1980).

69 Bowen, T.J.; Ochs, H.D.; Altman, L.C.; Price. T.H.; VanEpps. D.E.; Brautigan, D.L.; Rosin, R.E.; Perkins, W.D.; Babior, B.M.; Klebanoff, S.J.; Wedgwood. R.J.: Severe recurrent bacterial

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70 Arnaout, M.A.; Pitt. J.; Cohen, H.J.; Metamed, J.; Rosen, F.S.; Cotton, H.R.: Deficiency ofa granu- locyte-membrane glycoprotein (GPt 50) in a boy with recurrent bacterial infections. New EngL J. Med. 306:693-699 (1982).

71 Dana, N; Todd, R.F., llI; Pitt, J.; Springer, T.A.; Arnaout, M.A.: Deficiency of a surface membrane glyeoprotein (Mol) in man. J. clirl. Invest. 73: 153-159 (1984).

72 Anderson, D.C.; Schmalstieg, F.C.; Arnaout, M.A.; Kohl, S.; Tosi, M.F.; Dana, N.; Buffone, G.J.; Hughes, B J_; Brinkley, B.R.; Dickey, W.D.; Abramson, J.S.; Springer, T.; Boxer, L.A.; Hollers, J.M.; Smith, C.W.: Abnormalities of polymorphonuclear leukocyte function associated with a herit- able deficiency of high molecular weight surface glycopteins (GP138): common relationship to di- mirdshed cell adherence. J. din. Invest. 74: 536- 551 (198).

73 ViM D.P.; Fearorl, D.T.: Neutrophils express a receptor for iC3b, C3d.g, and C3d ~,l~at is distinct from CRt, CR2, and CR3. J. fmmun. 134: 257i- 2579 (I985).

74 Vik, D.P.; Fearon, D.T.: Cellular distribution of complement receptor type 4 (CR4): expression on human platelets J. Immun. 138: 254-258 (1987).

75 Cole, .I.L.; Housley, G.A., Jr.; Dykman, T.R.; MacDermott, RP.; Atkinson, J.P.: Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines. Proc. num. Acad. Sci. USA 82: 859- 863 (1985).

76 Seya, T.: Turner, J.R.; Atkinson. J.P.: Purification and characterization of a membrane protein (gp45-70) that is a cothctor for cleavage of C3b and C4b. J. exp. Med. 163:837-855 (1986).

77 Schneider, R.J.; Kulczycki, A., Jr.; Law, S.K.; At, kinson, J.P.: Isolation of a biologically active macrophage receptor for the Ihird component of complement. Nature, Land 290:789-792 (198 I).

78 Wang. W.W.; Fearon, D.T.: p65: a C3b-binding protein on routine cet[s that shares antigenic de- terminants with the human C3b receptor (CRI) and is distinct from murine C3b receptor. J. Im- mure 134:4048-4056 (1985).

79 Nichoison-WelIer. A.; Burg< J.; Fearon, D.T.: Weller, P.F.; Austen, K_F.: isolation of a human erythrocyte membrane glycoprotem with decay- accelerating activity for C3 corlverlases of ~he complement system. L Immun. 129. t84-t89 (1982).

80 Nicholson-Weller, A.; March, J.P.; Rosen, C.E.; Spicer, D.B.; Austen, K F.: Surface membrane expression by human blood teukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system. Blood 65:1237-1244 (t985).

81 Kinoshita, T.; Medal, M.E.; Silber, R.; Nussen- zweig, V.: Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J. exp. Med. 162." 75-92 (t985).

82 Asch, A.S.; Kinoshita, T.; Jaffe, E.A.; Nussen- zweig, V.: Decay accelerating factor is present on cultured human umbilical vein endothelial cells. J. exp. Meal. 163:221-226 (1986).

83 Kinoshita, T.; Medof, M.E.; Nussenzweig, V.: En- dogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes. J. Immnn. 136:3390-3395 (1986).

84 Caras, I.W.; Davitz, M_A.; Rhee, L.; Weddelt, G.; Martin, D.W., Jr.; Nussenzweig, V.: Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins. Nature, Land. 325:545-549 (1987).

85 Nicholson-Weller, A.; March, J.P.; Rosenfeld, S.I.; Austen, K.F.: Affected erythrocytes of patients with paroxysmal noctuiTml hemoglobirluria are deficient in the complement regulatory protein, decay accelerating factor_ Proc. natn. Acad. Sci. USA 80:5066-5070 (t983).

86 Pangburn, M.K.; Schre~ber, R.D.; Mtiller-Eber- hard, H.J.: Deficiency. of an erythrocyte membrane protein with complement regulatory, activity in paroxysmal nocturnal hemoglobinuria. Proc. natn. Acad. Sci. USA 80" 5430-5434 (t983).

87 Nieholson-Wetler, A.; Spicer, D.B.; Austen, K.F.: Deficiency of the complement regulatory protein, 'decay-accelerating factor', on membranes of granulocytes, monocytes, and plalelels in paroxysmal nocturnal hemogtobinuna. New Engl. 9. Meal. 312." I091-1097 (1985).

88 Nishioka, K.; Linscott, W.D.: Components of guinea pig complement I. Separation of a serum


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90 Cooper, N.,R.: Immune adherence by the fourth component of complement. Science 165:396-398 (1969)_

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92 Gupta, S.; Ross, G.D.; Good, R.A.; Siegal, F.P.: Surface markers of human eosinophils. Blood 48: 755-763 (1976).

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94 Yoon, S.H.; Fearon, D.T.: Characterization of a soluble form of the C3b/C4b receptor (CRI) in human plasma. J. Immun. 1 3 4 : 3 3 3 2 - 3 3 3 8 (1985).

95 Seya, T.; Holers, V.M.; Atkinson, J.P.: Purifica- tion and functionaI analysis of the polymo~hic variants of the C3b/C4b receptor (CR 1) and comparison with H, C4b-binding protein, (C4bp), and decay accelerating factor (DAF). J. Immun. 135: 2661-2667 (1985).

96 Lublin, D.M.; Gritt'ith, R.C.; Atkinson, J.P.: In- fluence of glycosylation on allelic and cell specific Mr variation, receptor processing and ligand binding of the human complement C3b/C4b receptor. J. biol. Chem. 26l: 5736-5741 (1986).

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98 Abrahamson, D.R.; Fearon, D.T.: Endocytosis of the C3b receptor of complement within coated pits in human polymorphonuclear leukocytes and monocytes. Lab. Invest. 48:162-168 (1983).

99 Fearon, D.T.: Collins, L.A.: Increased expression of C3b receptors on polymorphonuclear leukocytes induced by chemotactic lhctors and by purification prodecures. J. Immun. I30." 370-375 (1983).

100 Lee, J.; Hakim, R,M.; Fearon, D.T.: Increased expression of the C3b receptor by neutrophils and complement activation during haemodialy- sis. Clin. exp. Immunol. 56:205-214 (1984).

101 Fearon, D.T.; Kaneko, I.: Thomson, G.G.: Mem- brane distribution and adsorptive endocytosis by C3b recptors on human potymorphonuclear leukocytes. J. exp. Med. 153: 1615-1628 (1981).

102 Jack, R.M.; Ezzell, R.M.; Hartwig, J.; Fearon, D.T.: Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils. J. Immun. 137. 3996- 4003 (1986).

103 Jack, R.M.; Fearon, D.T.: Altered surface distribution of both C3b receptors and Fc receptors on neutrophits induced by anti-C3b receptor or ag- gregated IgG. J. Immun. 1 3 2 : 3 0 2 8 - 3 0 3 3 (1984).

104 Stossel, T.P.: Contractile proteins in phagocytosis: an example of cell surface-to-cytoplasm com- munication. Fed. Proc. 36:2181-2184 (1977).

105 Lin, D.C.; Tobin, K.D.; Grumet, M.; Lin, S.: Cytochalasins inhibit nuclei-induced actin poly- merization by blocking filament elongation. J. Cell Biol. 84:455-460 (1980).

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107 Bohnsack, J.F.; Kleinman, H.K.; Takahashi, T.; O'Shea, J.J.; Brown, E.J.: Connective tissue proteins and phagocytic cell function. Laminin enhances complement and Fc-mediated phagocytosis by cultured human macrophages. J. exp. Med. 161:912-923 (1985).

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112 Horgan, C.; Taylor. R.P.: Studies on the kinetics of binding of comNement-fixing dsDNA/anti- dsDNA immune complexes to the red blood cells of normal individuals and patients with systemic lupus erythematosus. Arthritis Rheum. 27. 320- 329 (t984).

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117 Campbell, R.D.: Bentley, D.R.: The structure and genetics of the C2 and factor B genes. Immu- nol. Rev. 87" I9-37 (1985).

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Dr. James G. Wilson Veterans Administration Medical Center 1500 E. Woodrow Wilson Drive Jackson, MS 392 I6 (USA)

cr1 and the cell membrane proteins that bind c3 and c4

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