Complement and complement receptors

Complement and complement receptors C.D. Ross Division

of Rheumatology-lmmunology

University

of North

Carolina

Current

Opinion

and the Arthritis

at Chapel

in Immunology 1989,

Introduction

Il-interleukin;

mAb-monoclonal RCA-regulator

Abbreviations DNA; EBV-Epstein-Barr

LFA-lymphocyte

antibodies;

of complement

@

SCR-short

virus;

function-associated

MAC-membrane

activation;

2:5M2

Most normal individuals have two C4 alleles and two farms of the C4 protein (C4A and C4B) in their blood. A major focus of research on C4 has been the elucida tion of the function of these two C4 gene products in immune complex processing in patients with systemic lupus erythematosus (XE). It is generally agreed that the C4A gene product binds more eIlìciently to protein antigen-immune complexes than the C4B gene product does, resulting in enhanced immune complex solubilization and opsonization for clearance. This apparently explains why individuals who only have the C4B gene prod uct (i.e. are C4A null or C4A*QO) have a higher statistical risk for onset of autoimmune disease. In the last year, C4A deficiency was shown to be a determinant for SLE among four ethnic groups (Caucasoid, Chinese, Japanese and Black Americans) [101. Zn vitro studies showed that the purilied C4A3 variant bound to IgG four times more eflìciently than the puritïed C4Bl variant [ 111, and this made the C4A3 much more efficicent in prevention of immune complex precipitation [ 121. One hypothesis is that inefficient clearance of naturally occurring immune’ complexes by C leads to the sequestration of complexes at peripheral sites, and that the inflammation resulting from immune complex activation of neutrophils releases autoantigens that promote formation of autoantibodies (Fig. 1).

An excellent review on classical pathway methodology was written by Gigli and Tausk [4]. Research on Clq has centered on Clq receptors (sec below) and the mechanism of Clq interaction with immune complexes that leads to activation of Clr and Cls. Duncan and Winter [5] have used site-directed mutagenesis of a mouse immunoglobulin (Ig)G2t, to localize the Clq-binding site on IgG to three side chains of the CH2 domain containing the residues Glu318, Lys320 and Lys322. These residues were conserved within ether antibody isotypes, and a peptide mimic of this sequence inhibited C lysis. Al though the binding and activation of C 1 has been thought to be the critical function of IgG in C activation, studies that compared Ig isotypes revealed differences in regu lation of both Cl and C4 activation [ 61. Human IgG1, known to be more efficient in C lysis than IgG3, was less eflìcient than IgGs in Iìxing Clq, but more efficient than IgG3 in presenting activated Cls to C4. IgG1 produced 10.fold more C4 activation compared with IgG3. Using

lg-immunoglobulin;

USA

Volanakis and colleagues [9] have produced a hemolytically active recombinant C2 from a fuk-length cDNA clone isolated from a human liver cDNA library in hgtll. The C2 cDNA was cloned into the expression vector p91023(B) and used to transfect COS cells. Although the messenger RNA(mIXNA)produced by the vector construct was longer than the C2 mRNA identified in human liver, the recombinant C2 had the same molecular weight, antigenicity and hemolytic activity as plasma C2.

Classica1 pathway

cDNA_complementary

Research Center, Carolina,

monoclonal antibodies (mAb) to Cls, the binding site for C4 has been localized to the y-domain of Cls [7]. Analysis of complementary DNA (cDNA) clones of Clr and Cls demonstrated 40.5% homology, and Southern blotting indicated that the genes for Clr and Cls were arranged in a close ‘tail-to-tail orientation [8].

Complement (C) research has diversi&d with the recog nition that the C system includes more than 30 distinct plasma and membrane proteins. The functions of the individual components also overlap with other systems, such as coagulation, cellular adhesion function and the immune response. Because the plasma C proteins have largely been defined, the emphasis of research has shifted to the membtane components of C and to the more precise determination of the role of C in the immune response and autoimmune disease. Approximately half of tbe current papers in C research are devoted to C receptors. This review covers recent literature on: (1) the classical pathway; (2) the alternative pathway; (3) the terminal component pathway, and (4) C receptors. Several excellent reviews on all aspects of C were published in the last year [l-31.

C-complement;

Hill, North

attack complex; consensus

HAF-hereditary

angioedema;

antigen; LHR-long

homologous

mRNA-messenger

repeat; SLB-systemic

Current ScienceLtd ISSN0952-7915

repeat;

RNA;

lupus erythematosus.

Complement and complement receptorsRoss

Immune complexes circulate in plasma

Fig.1. Hypothetical mechanism for the generation of circulating autoantibody immune complexes in patients with deficiencies of components of the classica1 pathway of complement CC). In this scheme, it is proposed that immune complexes (IC) generated by normal responses to pathogens (e.g. bacteria) would be cleared inefficiently because of poor C fixation via the classical pathway. This would allow these IC to be sequestered at peripheral skes rather than being bound to red blood cell (RBC) CR, for clearance by normal mechanisms involving the macrophage phagocytic system in the liver. Such IC trapped in peripheral sites would produce inflammation, resulting in tissue damage that would release autoantigens (e.g. DNA, which is believed to be of major importante in patients with systemic lupus erythematosus), cytokines, and an inhibitor of DNAse (actin) that might otherwise be available to solubilize DNA autoantigens. Such DNA might then be recognized as an autoantigen and so stimulate formation of autoantibodies to DNA. Adapted with permission from a presentation by Professor P.J. Lachmann to the Federation of American Societies of Experimental Biology, New Orleans, Louisiana, March 1989.

Progress has also been made in understanding the mode of action of the Cl inhibitor and the consequences of its deficiency in the disease hereditary angioedema (HAE; for review, see [13]). At its reactive center, the Cl irhibitor contains an Arg444 that acts as the ‘bait’ for irreversible inactivation of esterases such as Cls. The inactive Cl inhibitor from a patient with type II HAE was found to have a point mutation in which the Arg444 had been changed to %444 [ 141. Longterm prophyfaxis of HAP is usually accomplished with the androgen Danazol, but in two patients where this was not tolerated, long-term therapy with purifìed Cl inhibitor concentrate was effeclive [15]. The possibility of obtaining a recombinant Cl inhibitor that could potentially be used for such therapy

was shown by the generation of a functional Cl inhibitor in transfected COS cells [ 161.

The alternative

pathway

One of the major remaining questions about the alternative pathway is the biochemical mechanism used for recognition of activating surfaces such as bacterial cell wak. Fixation of C3b onto soluble polysaccharides indicated that the type and size of the polysaccharides asso ciated with fïxed C3b are major factors in regulating the accessibility of the fixed C3b to the control protein fac-

51

52

Innate immunity

tor H [ 17,181. Maximum inhibition of factor H function (permitting activation of the alternative pathway) was estimated to occur at a length of 16 saccharide units. The possibility of altemative pathway regulation by host cel& secreted factor H or factor 1 was suggested by the iïnding of factor H synthesis by skin fibroblasts [ 191, and synthesis of both factors H and 1 by umbilical vein endotbelial cells [20]. Generation of the C5 convertase of the altemative pathway has been known to involve a cluster of iïxed C3b molecules that present the C5 substrate to a C3b,Bb enzyme complex. Kinoshita et al. [21] reported that the essential part of this C3b cluster is a covalently linked pair of C3b molecules that form a high-aifinity binding site for c5. Properdin, first described by Pillemer in 1954 (Science 1954, 120:28@-285) as the initiating agent of the alternative pathway, is now recognized as an Important regulator of the pathway. Properdin binds to C3b,Bb, stabilizing this complex, the C3/C5 convertase of the path way. Properdin was found to bind only to fixed C3b and not to fluid C3b, thereby restricting C to activating surfaces [22]. Properdin also protected fìxed C3b from cleavage by factor 1. Analysis of properdin synthesis by HL-60 cells showed that glycosylation and polymerization were early intracellular events that might control anomalous aggregation of subunits [ 231. Sequence analysis of a murine properdin cDNA clone revealed that properdin is composed primarily of six repeating motifs of - 60 amino acids. Similar sequence mot& have been obsetved in thrombospondin, the circumsporozoite protein of malaria parasites, and regions of the membrane attack complex (MAC) of C [ 241. The similarity of properdin to parasite surface molecules suggests that there may be a mechanism by which parasites avoid host defenses mediated by C.

C5 and the terminal

components

of

complement There were several excellent reviews in the last year on C5, C9, and the h4AC of C [2%28]. Sequence analysis of cDNA clones representing the entire C5 molecule indi cated that human C5 is 80% homologous with murine C5 [29]. C5 has extensive homology with C3, C4 and a2macroglobulin, but C5 lacks the intemal thioester bond characteristic of these other proteins. The homology between C9 and the cytotoxic T cell effec tor molecule perforin, initially revealed at the protein Sequence kevel, has now been coniïrmed at the cDNA leve1 by two laboratories [30,31]. In addition to their homologous structural features, both proteins function similady as potent tumoricidal molecules via generation of membrane-penetrating pores in target cell membranes. Northem blot analysis of mRNA indicated that perforin expression is restricted to cytotoxic T cells. Assembly of the MAC was found to be an inefficient process [32] and once assembled, the membrane-derived

MAC was an inhibitor of C activation by both the clas sical and altemative pathways [33]. The poot- efftciency of h4AC assembly resulted both from instability of the C5 convertase and a lack of incorporation of some Auidphase C5b into membrane MAC. The inhibition of C activation produced by excess unincorporated MAC may represent a form of C regulation. Micro-organisms have evolved several mechanisms for avoiding destruction by C (for review, see 1341). Well known mechanisms include inhibition of C activation, proteolytic destruction of fixed C, and masking of C-sensitive membrane components by capsular polysaccharides. Iida et al. [35] examined the mechanism of C resistance in Tqpanmoma cruzi. Although the infective amastigote stage of the organism was found to be a potent activator of the altemative pathway, MAC insertion and cytotoxicity were low. Amastigotes were shown to express a membrane protein that prevented insertion of assembled MAC complexes in a manner suggestive of the host cell C8binding protein or homologous restriction factor.

Complement

receptors

Several important discoveries were made about membrane C3 receptors. As many as six types of membrane C3 receptors are thought to exist (Table 1; Fig. 2). Iambris [36] bas reviewed the multifunctional role of C3 and the various receptors that react with C3. Using peptides derived from the C3 sequence, as well as peptidespecific mAb, Becherer and Lambris localized the binding sites in C3 for the C3b receptor, CR, [37], properdin [ 381 and factor H [39]. The binding site for CR2 in the C3d region had been mapped previously using similar techniques (Iarnbris et al., Proc Nat1 Acud Sci USA 1985, 82:423M239). Despite all the newly acquired data about C3 and its specific receptors, major questions about the cell biology of C3 receptors remain, and the mechanism by which C3 contributes to antigen recognition in the immune response is unknown. O’Neil et al. have reported further studies of the immune abnormalities of a colony of C3deficient dogs [ 401. As had been noted earlier with C3-deficient patients and CS-depleted animals, there was a deficiency in the primary immune response to T cell-dependent antigens. Although antibody hels increased with higher antigen doses and secondaty immmization, antib@ levels of 1gG did not come up to these of normal control dogs or litter mates that were heterozygous for ~3 deficiency. One possible mechanism of C3 function in the immune response is through presentation of antigens to T cells by macrophages or B ceR.s. In addition to pretious studies that also suggested t.h possibility, in vitr-c experiments were devised to show directly that fixed C3b or C4b antigens enhanced the effiCienCy of antigen-presenting cell function in activaton of T cell clones [ 411.

ComDlement

C3a receptor

CRl,H,B

CR2

Conglutinin

H

and complement

recetiors

Ross

CR3 P

cechain

\

COOH

H2N

B-chain

CS

CHO

Fig. 2. Schematic representation of the complement (C) 3 molecule, showing sites of enzymatic cleavage and regions of attachment to C receptors and other C components. Sites of C3 cleavage by C3 convertases, factor I (0, elastase (E), and trypsin fTJ are indicated. The sites for CR, 1371,CR,, CR, 167,691, C3a receptor, factor H (H), properdin CP),C5, factor B (B), and conglutinin are shown. The position of the residues corresponding to these sites is based on the oredicted amino acid sequence of C3 after subtracting the signal peptide sequence. Adapted from [361

CR1

CR1 (CD35; the C receptor speciíïc for C3b, C4b, and iC3b) is one of four receptors for bound fragments of C3. The extemal membrane domain of CR, consists of four or Iìve long homologous repeat (DIR) units, each of which contains seven short consensus repeat (KR) sequences of -61 amino acids [42]. The repeating SCR motif is characteristic of a large family of C regulatoty proteins that map to chromosome 1 in what has been termed the ‘regulator of complement activation’ (RCA) lotus [43,44]. Through deletion mutagenesis, CR1 transfectants were generated in COS cells that expres& different combinations of the four DIRs in recombinant membrane CR1 molecules. This allowed mapping of the C4b- and C3b-binding sites in CR1 to two adjacent IIIR at the terminus of the extemal membrane domain (Fig. 3) [45]. Considerably less is known about the murine homologs of human CR1 and CR2. Rat mAb to murine B cell CR, were described [46] which allowed studies of the structure and distribution of CR1 on different mouse cell types. Surprisingly, mouse platelets did not react with a mAb to CRr, suggesting mat the well known C3b receptor activity of mouse platelets (immune adherente) is mediated by a C receptor that is unrelated to CR,. The CR1 of primate erythrocytes and its function in im mune complex ckarance remains a leading area of research. Erythrocytes represent the major mechanism for sequestration and transport of circulating immune complexes to macrophages in the Ever. Two laboratories re-

ported that the CR1 of erythrocytes was arranged in large clusters that greatly increased the binding efficiency of immune complexes and compensated for the lower numbers of receptors per cell compared with blood leukocytes [47,48]. Zn vitro studies of C3b fixation to, and redcell binding of, DNtz/anti-DNAcomplexes showed that relatively large double-stranded DNA complexes were required for C3b opsonization and uptake by red cells 1491. DNA/anti-DNAcomplexes that bound to red cells characteristically bore one IgG molecule per 20-30 base pairs and less than one C3b molecule per IgG. C3b molecules were iïxed to IgG and not to the DNA in these complexes. Schifferli et al. [50] examined erythrocyte CR,dependent mechanisms of clearance in patients and normal volunteers injected with radiolabeled immune complexes of tetanus toxoid/anti-tetanus toxoid. Kinetic analyses revealed two different phases of clearance in patients who had either diminished C opsonizing activity in their plasma or low numbers of CR1 on their erythrocytes. The first rapid phase of ckara.nce, observed only in patients and not In normal volunteers, is thought to represent the trapping of free immune complexes in the capillary beds of peripheral organs. The second, slower, phase of clearance in patients probably represented the transport of erythrocyte-bound complexes to macrophages. Normal individuals or patients with normal C and erythrocyte CR1 levels showed only thk slower rype of clearance kinetics. Reductions in erythrocyte CR, had been shown previously to occur in SLE patients who had evidente of an ongoing process of C activation that was characterized

&Jio*k,ure,

and cel1typedistribution

s-re,

of complement

receptors.

Specificity

Available

Clq-R

Clq

collagen ‘tail

IWDI

CR, (CD35)

C3b > C4b > iC3b

44D,

Receptor

type

mAb

57F, C3To5,

Ell,

Yaz-1, J3B11,

J3D3

CR2 (CD21)

C3d region of iC3b = C3dg > C3d > C3b gp350 of Epstein-Barr

CR3 (CD1 1 blCD18)

Anti-B2,

HB-5, OKB7,

iC3b (RCD),

CD11 b-specific mAb: Anti-Mac-l

fl-glucan,

Anti-Leu

lipopolysaccharide,

fibrinogen, tfistoplasma

endothelial

cells,collagen

(?),

MN-41,

15, Anti-Mol,

(M1/70), OKM9,

OKMIO

capsulatum

iC3b, C3dgW, endothelial

(p150,95)

Histoplasma

capsulatum

‘35

C3d portion

of fluid-phase

FH-R

Factor

CSa-R

C5atC5adesArg

NO mAb reported

C3alC4a-R

C3a, C4a

NO mAb reported

antibodies;

OKMI,

44, 903, 904, 60.1, Vim-12

CR, (CDllc/CD18)

mAb, monoclonal

AB-1, BL13

virus

cells, collagen (?),

CDllc-specific Ki-Ml,

iC3b, C3dg, C3d

M5,

NO mAb reported

H, c$z form

R, receptor.

mAb. Anti-Leu

3.9, L29, KB23, Bu-15

NO mAb reported

The leukocyte

antigen

by frxation of C3 fragments to their erythrocytes (Ross et aC, J Immunoll985, 135:2005-2014). Whether this C3 íïxation resulted from transport of immune complexes or autoantibodies to erythrocytes was not established. Hammond et al [51] have now presented evidente that elevated levels of autoantibodies to cardiolipin are highly correlated with diminished levels of erythrocyte CR, in patients with SLE. Although patients with IgM antkardiolipin antibodies had 6xed C3 and low levels of CR1 on their erythrocytes, other patients with IgG anticardiolipin

workshop

name is given in brackets,

where it exists.

mtibdies occasionally exhibited 10~ CR, levels without detectable levels of red cell-fïxed C3.

CR* Important advances have been made in our understanding of the B cell C3d receptor, CR2 (CD21), that also serves as the receptor for Epstein-Barr virus (EBV). &-

Complement

and complement

receptors

Ross

Table 1. (continued) Cel1 type distribution

Structure Single-chain

glycoprotein,

65 kD

B lymphocytes

-

Neutrophils

high

-

Monocytes

-

Fibroblasts Platelets Single-chain

glycoprotein,

Tissue

very high low

Monocytes,

four allotypes:

A, 190 kD; B, 220 kD; C, 260 kD; D, 160 kD

high

-

very high

neutrophils

macrophages

B cells, eosinophils Kidney podocytes T cells

(-

Single-chain

glycoprotein,

14&145

-

Lymph

non-covalently

165 kD a-chain 95 kD P-chain

linked glycoprotein

(CDllb

chains:

antigen)

Monocytes Tissue

(CD18 antigen)

non-covalently

150 kD

Neutrophils

linked glycoprotein

a-chain(CD11 c

95 kD p-chain (CD18

chains:

antigen)

antigen)

Cytotoxic

Two

or more glycoprotein

subunits

of 50 kD;

total size unknown

47 kD protein-binding of 150-200

unit within

an oligomer

kD

Unknown

-

cells -

epithelial

cells -

very high low

high

-

-

low

high

-

high

-

-

low

high

low

T cells, natura1 killer cells, eosinophils -

Platelets

low

-

-

low

Neutrophils

low

B lymphocytes

-

Monocytes

-

high

Neutrophils

-

high

Mast

dendritic

pharyngeal

macrophages

Neutrophils

Unknown

high

T cells, natura1 killer cells, eosinophils

Monocytes Tissue

-

macrophages

Cytotoxic Two

low

node follicular

Thymocytes, Two

high high

low

B lymphocytes

kD

high

low

-

20%) -

Erythrocytes

-

-

high

cells

Neutrophils

-

high

Monocytes

-

high

Mast cells Eosinophils

-

high

Monocytes

-

high

Neutrophils

-

low

Platelets

very low (?)

-

High and low refer to levels of expression.

quence analysis of CR2 cDNA clones has indicated that there is a high degree of homology with CR, [52], and that CR1 and CR2 genes are linked within the RCA locus on chromosome 1 [46]. Expression of recombinant CR2 in mouse L cells was accomplished [ 531. The transfected L cells expres& C3d receptor activity and also EBV receptor activity, and were infected productivelywith EBV. This indicated that CR2 was the sole factor responsible for the tissue tropism of EBV. A rabbit antiserum to human CR2 cross-reacted with murine CR2 and allowed

the identification of a murine CR2 cDNA clone. Sequence analysis of the murine CR2 cDNA revealed 79% sequence homologywith human CR2 [ 541. However, murine B cells that expres& CR2 and C3d receptor activity did not bind EBV and were not susceptible to EBV infection. Important questions remain about me role of CR2 in the immune response. Although absent from mature T cells, small amounts of CR2 have been detected on thymocytes [55]. Ligands for CR2 have been shown to function as

55

56

Innate immunity

C4b

C3b

(C3b)

(C4b)

LHR-D

LHR-C

LHR-B

LHR-A

C3b (C4b)

Primary

TM-CYTO

binding sites

Secondary

binding sites

Fig.3. Schematic model depicting the types of short consensus repeat (SCR) making up each long homologous repeat (LHR) of comple-

ment receptor type 1 (CR,), and the predicted sites determining the specifìcities of the receptor for C3b and C4b. The sites of secondary binding specifïcities of these are indicated by the parentheses. TM-CYTO is the transmembrane-cytoplasmic domain of CR,. Adapted from [451. C, complement.

B cell growth factors, and B lymphoblastoid lines have been maintained in serum-free media supplemented with purilïed C3 or anti-CR2 mAb [56]. The normal ligand(s) for CR2 are unknown, but important diíferences in B cell responses have been noted between monovalent ligands such as fluid-phase C3d and multivalent liga& such as C3dgSepharose or F(ab’)L anti-CRz. Monovalent CR2 ligands suppress B cell activation, whereas multivalent ligands function as B cell growth factors that can augment stimulation by suboptimal doses of anti-IgM [57-591.

CR3 C%, the iC3b receptor or CDllb/CDlS, is a member of the CD18 family that shares a common fi-subunit (~~18) connected non-covalently to one of three cr-subunit types: CDlla (lymphocyte function-associated antigen, LFA, -l), CDllb (CR& and CDllc (CRq or p150,95). In the past year, several laboratories reported sequence data for the CR3 u-subunit (CD11 b) cDNA clones that showed that the cc-subunit, as well as the l3-subunit reported earlier, was a member of the integrin gene superfamily of adhesion-promoting surface molecules [ 6@-64]. It seems appropriate that CR3 should be viewed as both an iC3b receptor with primary responsibility for medi ating ingestion of C3-opsonized micro-organisms and as an adhesion-promoting receptor whose function is totally independent of fìxed C3.

C& appears to have multiple ligand specilïcities and functions, not all of which may be expres& by each in dividual membrane molecule. For example, the surface CR3 on resting neutrophils appears to be involved in the homotypic aggregation response to chemotactic fac-

tor (formyl-methionine-leucine-phenylalanine tripeptide or C5a), whereas the newly expressed membrane CR3 derived from cytoplasmic granules apparently bas no role in this reaction [65]. Other evidente for the existente of different classes of surface CR3 molecules has come from studies of the role of CR3 in phagmc and release reactions. Antibodies to C& have been found to inhibit a variety of neutrophil responses thought to involve other receptors with distinct ligand specifìcities, such as CR1 or FC receptors. An immobile subset of CR3 that cannot be modulated by cultivation of cells on anti-C%-coated culture surfaces is apparently involved [ 661. This immobile C& subset is presumably linked to the cytoskeleton and may function to transduce cellular activation events initiated by ligation of other receptor types that are not linked to the cytoskeleton (e.g. neutrophil FcRIII that is linked exclusively to the cell surface via a phosphatidylinositol anchor). Among the important new ligand specifìcities that have come to be recognized for C& are an RGD (arginineglycine-aspartate) sequence in iC3b and a separate distinct binding site for bacterial lipopolysaccharide (LF’S; endotoxin) [67]. It has also been claimed by Wright and colleagues [68-701 that the RGD specifïcity allows CQ to bind to fibrinogen and a cellsurface protein of Lez&muniu (gp63). If confïrmed, this would indicate that there is a further similarity between CR3 and other integrin molecules, such as the fibroblast fibronectin receptor, and the platelet fibrinogen receptor (gpIIb/IIIa) that have recognized specticities for RGD sites in fibronectin and fibrinogen. However, the RGD specifìcity of CR3 for fibrinogen has been disputed by Altieri et al. [ 711, and the RGD specificity of C& for LeAbmunM has been disputed by groups in two additional laboratories 172,731.

Complement

CR&R, A neutrophil receptor activity for iC3b and C3dg fragments that had previously been designated as CQ now appears to represent at least two distinct surface molecules. CDllc or p150,95, the third member of the CD18 family, appears to be responsible for one of these activities and has been designated CQ, and accbrdingly, the second receptor activity, whose molecular str@ure is unknown, has been designated CRg [37]. CRq is present in small amounts on neutrophils and monqes, but is the predo minant type of C3 receptor ant3gen expressed on tissue macrophages that frequently lack detectable amounts of CR, and C% [74]. CQ has a divalent cation-dependent speciiïcity for fixed iC3b (and possibly also lïxed C3dg) on sheep erythrocytes (EC3bi or EC3dg) that closely resembles the specitïcity of C%. Sequence analysis of cDNA clones representing the asubunits of CR3 and CQ has indicated that there is approximately 87% homology [75] and linkage on chromosome 16 [76]. Features that distinguish CQ from the C3dgdimer receptor (CRs) are that CR5 activity is unaf fected by EDTA and expres& by platelets that lack detectable CDllc molecules (Vik and Fearon, J Immunol 1985, 134:2571-2579; Vil and Fearon, J Immunoll987, 138:254-258).

Clq and factor H receptors Attempts at isolation of the Clq receptor, Clq-R, of Raji cells by Clq affiity chromatography [ 77 J have revealed a structure (a 65 ka glycoprotein) that is similar to that reported originally by Ghebrehiwet et al. (I Exp Med 1980, 160:1375-1389). A rabbit antisera to the Raji cell ClqR immunoprecipitated similar molecules from Raji and U937 cells, suggesting that the Clq-R of lymphocytes and monocytes may be the same molecule. Other studies ?f phagocytic cell Clq-R have indicated that they function as opsonin reeeptors in a marmer analogous to the C3 receptors in their ability to augment the killing or phagocytosis of organisms coated with Clq [78,79]. A novel Clq-R has been identified on human umbilical vein endothelial cells that promotes the attachment of Clq-bearing immune complexes [80]. It was suggested that this endothelial cell Clq-R might have some role in mediating itn mune complex-associated vasculitis. Although there have been no recent structural studies of the factor H recep tor, M-R, Ripoche et al [Bl] have identilîed two farms of factor H, designated +1 and &, in normal plasma, by hydrophobic tinity chromatography, and found that only the 42 form was áble to bind to the M-R of Raji cells.

C5a receptor Much interest has focused on the neutrophil C5a receptor , CSa-R, because of the importante of this receptor

and complement

receptors

Ross

in mediating tissue injuv in inflammatoq diseases such as arthritis. Research on both recombinant C5a and the CSa-R has, as one of its goals, the development of therapeutic agents that will black CSa-media& inflammatoty reactions. Isolation of the neutrophil CSa-R in an active form was accomplished by solubilization of cells with digitonin and B-dodecyl maltoside [82]. The size of the isolated receptor, 52 kD, was the same as that previously estimated in experiments using photoaffinity probes. Sitedirected mutagenesis of recombinant C5a and studies of responses to the modilied recombinant C5a indicate that three C-terminal residues itlteract with the neutrophil CSa-R: Lys68, Leu72, and Arg74. In addition, amino acid substitutions in other parts of the C5a molecule indicated that there were two other discontinuous @ons of the C5a molecule that appear to act in concert to achieve full potency [83]. One of the functions thought to be mediated by the monocyte CSa-R is the release of,.interleukin (IL)-1. Arend et al. [84], however, present& evidente that highly purilïed C5a did not stimulate IL-1 release, and that C5a preparations that did stimulate IL-1 secretion contained a trace of contamination with LPS. Removal of LPS or incubation with the LPS inhibitor, polymyxin B, removed the IL-1-inducing activity from several dilferent types of C-fragment preparations including C5a, C3a, C3b, iC3b, C3d, Bb, and Ba.

Annotated references and recommended reading ??

00

Of interest Of outstanding

interest

1.

ATKINSON JP: Complement deficiency: predisposing factor to autoimmune syndromes. Am J Med 1988, 85:4%7. A review summarizing current evidente that inherited deficiencies of components of the classical pathway contribute to the onset of autoimmune disease. ??

CAMPBELL FQ IAw SKA, REIL)KBM, SIMRB: Structure, organization, and regulation of the complement genes. Annu Reu Immunol 1988, 6:161-196 A review summarizing current knowiedge of the molecular biology of complement system proteins. 2.

??*

Molecular organization and foncMUILER-EBERHARD HJ: tion of the complement system. Annu Ra, Bimbam 1988, 57:321-347. A review of aU aspects of complement system structure and function. 3.

??o

GIG~J 1, TAUSKFA: Cl, C4, and C2 components of the classical pathway of complement and regulatory proteins. Metb Enzymol1988, 162:626-638 A review of the methodology for isolation and functional analysis of classical pathway C components. 4.

??

DUNCAN AR, WINIER G: The binding site for Clq on IgG. Nature 1988, 332:73%739. An investigation, using sitedirected mutagenesis of murine IgGLb, of important residues in the CH, domain that are required for binding and activation of Clq. 5.

??e

6. .

BINDONCI, H&II G, BRÜGGEMANN M, WALDMANN H: Human monoclonal IgG isotypes differ in complement activating function at the leve1 of C4 as well as Clq. J Eqo Med 1988, 168:127-142.

57

58

Innate immunity A matched set of chimeric antibodies, each with an identical mouse V region linked to a different human Ig constant region, was used to show that IgGl antibodies were more efficient in C lysis than IgG3 antibodies because they were more efficíent in C4 activation, despite lower levels of Clq binding. 7.

M~?suMoTo M, NAGAW K, KITAMURA H, KURAMITS u S, NAGASAWA S, SEYAT: Probing the C4-binding site on Cls with monoclonal antibodies. Evidente for a C4/C4b-binding site on the ydomain. J Immunol 1989, 142:274+2750. The atrachment of C4 was shown to be fo the Pl region of the y-domain of Cls, using mAh to different parts of Cls and isolated Pl fragments. 0

8.

KUSUMO~ H, HIROSAWA S, WIER JP, HAGEN FS, KURACHIK: Human genes for complement components Clr and Cls in a close tail-to-tail arrangement. proc Nd Acad Sci USA 1988, 85:7307-7311. cDNA clones for Clr and Cls were used to probe human genomic DNA to show that the genes of Clr and Cls are located in a ‘ti-to-tail arrangement at a distance of 9.5 kilobases. 0

HORXJCHI T, b%ACON KJ, KIDD VJ, VOwruw~ JE: cDNA cloning and expression of human complement component C2. J Jmmund 1989, 142:210>2111. A fulLIer@ human C2 cDNA was cloned into the expression vector p91023(B) and used successfully to generate a fully active recombinant C2 by tmnsection into COS cells. 9.

??o

10.

WA, PEREZ MC, ARMATlS PE: l?utial C4A deficiency is associated with susceptibility to systemic lupus erythematosus in black Americans. Ar&rU Rbeum 1988, 31:1171-1175. Among 59 black American patients with SIX, the incidence of C4A null genes was 32%, compared with 15.3% in a control black population. WILSON

??

ll. 0

KISHOREN, 5%~ D, SKANES VM, LEVINERP: The fluid-phase binding of human C4 and its genetic variants, C4A3 and C4B1, to immunoglobins. Mol Jmmund 1988, 25:81X319. In vitro studies of isolated C4A3 and C4Bl proteins showed that C4A3 bound preferentially to the heavy chain of Ig and associated protein antigens, promoting immune complex solubilization.

12. @

PAUL L, SKANESVM, MAYDEN J, LEVJNERP:

C4-mediated inbibition of immune precipitation and Merences in inbibitory action of genetic variant?+, C4A3 and C4Bl. Complement 1988, 5:11&119. C4A3 bound more e&ìciently and rapidly to immune complexes than C4B1, although both prevented immunoprecipitation and promoted the solubilization of preformed complexes. DAVISAE III Cl inbibitor and hereditary angioneurotic 13. 0 ederna Annu Rev Jmmwwll988, 6:595-628. A review of the molecular biology and fünctions of Cl inhibitor, as wefl as a review of genetic variants of the inhedted deficiency disease of Cl inhibitor, HAE. 14.

AUIAKKS, PE~IBERTONPA, ROSENFS, CAREU RW, LACHMANN PJ, HAIUUXINRA: Dysfunctional Cl-inhibitor(At), isolated &om a type JI hereditary-angio-oedema plasma, contains Pl ‘reactive centre’ (w-His) mutation. Biochem J 1988, 253:615-618. Sequence analysis of a dysfunctional Cl inhibitor protein from a patient with HAE demonstrated that an arginine residue thought to be necessary for Cl inhibitor function had been mutated to a histidine residue. ??

BORKK, WIIZKEG: Long-term prophyiaxis wîth Cl-inhibitor (Cl INH) concentrate in patients with recurrent angioedema caused by hereditary and acquired Cl-inbibitor deBciency. J AUetgy Clin Jmmunoll989, 83:677*2. 2 patients with HAF that could not tolerate, or did nof respond fo, standard Danazol therapy were shown to be treated effectivelywith a concentrate of puriIïed Cl inhibitor for a period of 1 year. 15. 0

16. ??o

Expression of fimctional human Cl inhibitor in COS cells. J Bid &em 1988, 263mnf%in9. A full-lengthhuman Cl inhibitor cDNA clone was transfected transiently into COS cells, resulting in the synthesis and secretion of a fully active Cl inhibitor molecule. EIDERING E, NUIJENSJH, HACK CE:

PANGBURN MK: Analysis of the mechanism of recognition in the complement altemative pathway using C3b-bound low molecular weight polysaccbaades. J Jmmunol 1989, 142:275+2765. A fluorometk assay was developed and used to analyse the ability of small C3b polysacchtide polymers to activate the altemative pathway by protection of the bound C3b from the regulatory protein factor H.

17.

??o

18. ??e

PANGBURN MK: Analy& of recognition in the altemative pathway of complement. Effect of polysaccharide size. J

Jmmund 1989, 142~2762770. The size requirement of polysac&uides covalently linked to C3b fqr activation of the altemative pathway by restriction of factor H revealed that a minimum length of 16 sacchadde units was required to interact with a domain on C3b located * 15 A from the thioester site of C3b. KATZY, STRUNKRC: Synthesis and regnlation of complement protein factor H in human skin fibroblasts. J Jmmunol 1988, 141:55+563. Skin fibroblasts were shown to synthesize factor H protein and ml@& and to increase levels of synthesis in response to interferon. 19. ??

20.

RIFWHE J, MITCHELL JA, ERDEI A, MADINC, MOFFAT~ B, MOKOENAT, GORDON S, SIM RB: Interferon y induces synthesis of complement altemative pathway proteins by human endotheu ceb in culture. J E35pMed 1988, 168:1917-1922. Endothelial cells were shown, by identiíïcation of both intrinsicaUy35s. labeled proteins and specifïc mIXNA,to synthesize and secrete altemative pathway factors B, H, and 1. me

21. 0

KINOSHITA T, TAKATA Y, KOZONO H, TAKEDA Y, HONG K, INOUE K: C5 convertase of the altemative complement

pathwayz covalent linkage between two C3b molecules within the trimolecuku complex enzyme. J Jmmurwll988, 1413895-3901. The C5 convertase was shown to con.& of a covalently linked dimer of 2 C3b molecules, which had increased alEnity for C5, that allowed eflìcient presentation of the C5 substrate to an adjaceht C3bBb enzyme complex. 22. 0

FARRIESTC, LACHMANN PJ, HARRISON RA: Analysis of the interactions between properdin, the tbird component of complement (C3), and its physiological activation products. BiLxbem J 1988, 252~47-54. Investigation of the interaction of properdin with different fluid-phase and fixed fmgments of C3 revealed that properdin bound preferentially to fixed C3b (or fixed C3b,Bb complexes) rather than any fluid-phase form of C3, and protected the C3b from cleavage by factor 1. FARIUES TC, ATKIN~~N JP: Biosyntbesis of properdin. J Jmmutwl 1989, 142:842-847. Properdin synthesis was examined in dimethylsulfoxide-dtiated HL-60 cells to determine the kinetics and squence of events leading to glycosylation and formation of the properdin polymers. 23. ??

24.

GCNJNDIS D, REID KBM: Properdin, the terminal complement components, tbrombospondin and tbe circurnsporozoite protein of malaria parasites contain similar sequence mot& Nature 1988, 335:82-85. Sequence analysis of mouse properdin cDNA showed -85% homology with human and rabbit properdin, and that most of the molecule consisted of 6 repeatingmotifs of -60 amino adds, resembling similar sequence mot& reported prevìously for the malaria surface protein thrombospondin. ??

and C3a, C4a, and C5a fkagments of the complement system. Meti Enzymd 1988, 162:57+625. A review of methods used to isolate nat& C3 and C5, as well as to generate the 3 anaphyiatoxins of the complement system. 25.

JANATOVA J: C3, C5 components

??

ESSERAF, SODEIZ JM: Membrane attack complex proteim C5b-6, C7, CS, and C9 of human complement. Met!wd Enzymd 1988, 162:551-578. A review of the biochemist~ and methodology required to iso& and functionalìy characterize the terminal components that make up the MAC.

26. 0

Complement

and complement

receptors Ross

27. 0

The binding site in C3b for properdin was identifïed through formation of inhibitoty synthetic peptides representing the C-terminus of the CLchain of C3.

DALMMWAP, FAK RJ, RARL: The pathobiology of the ter28. minal complement complexes. Cotn@ment 1989, 6:3648. * An ovetiew of data indicating a role for the MAC in the pathogenesis of various autoimmune, infectieus, and intlammatoty disease processes.

LUBRIS JD, AVIIAD, BECHERERJD, MULIER-EBERHARD HJ: A discontinuous factor H binding site in the third component of complement as delineated by synthetic peptides. J Bio1 Ckm 1988, 263:12147-12150. 2 discontinuous binding sites for factor H were mapped to the C3c and C3d regions of C3b by examining the binding of factor H or an anti-factor H idiotypic antibody to synthetic and cyanogen bromide peptides, derived from C3c and C3d.

STANIIY KK: Tbe molecular mechanism of complement C9 insertion and polymerisation in biological membranes. Curr Top Microbzbl Immunol 1988, l40:4%5. A review of C9 structure and functional events leading to C9 insertion and polymerixation in biological membranes.

MM, BARNLJMSR NOACKD, TACKBF: Molecular analysis of human complement compo nent C5: localization of the structural gene to chromosome 9. Biocbemistry 1988, 27:14741482. The isolation of a C5 cDNA clone representing a segment of the C5 molecule extending from the 3’untranshued region through 58% of the 8.chain permitted a nearly complete analysis of C5 primary strucmre. 29. 00

WETSELRA, IEMONSRS, IE Bwu

SHINKAIY, TAKIO K, OKUMLIRAK: (Letter to the Bditor) Homology of perforin to the ninth component of complement (C9). Nature 1988, 334:525-527. Sequence analysis of the CDNAclone of mouse perforin showed extensive homology with C9 in functionally conserved regions. 30. ??*

LOWREYD, AEBISCHERT, OISEN K, LKXTENHEIDM, RUPP F, HENGARTNER H, PODACKER Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homologe to complement component C9. Proc Nat1 Acad Sci USA 1989, 86:247-251. Sequence analysis of murine perforin cDNA showed extensive regions of homology with certain functional domains of C9, C8a, C88, and C7 and northem blotting demonsttated restricted expression of perforin mRNA in cytotoxic T celb.

31. oo

BHAKDIS, FASSBENDER W, HUGO F, CARRENOM-P, BERSTECHER C, MlT P, KAiXTCHKlNEMD: Relative inefficiency of terminal complement activation. J Itnmund 1988, 141:3117-3122. Formation of the terminal component complex was found to be inefficient because of a low rate of formation and rapid rate of decay of the C5 convertase, followed by poor incorporation of newly fonned fluid-phase C5b into membrane complexes.

32. 0

33.

BHAKLXS, MAILLET F, MUHLYM, KA~ATCHKINE MD:

The cy-

tolytic C5b-9 complement complex: feedback inhibition of complement activation. Proc Nat1 Acad Sci US.4 1988, 85:1912-1916. Bound and fluid-phase MAC, C5b-9(m), were shown to inhibit C activa tion through inhibition of both C3 convertase formation and utilization of c5. ??

JOINER KA Complement evasion by bacteria and parasites. 34. 0 Annu Reu Microbabl 1988, 42:201-230. A review of the C system and its various mechanisms for cytotoxicity and opsonixation of bacteria and parasites, as weIJ as the mechanisms evolved by these organisms to aade C. IJDAK, WHITUIW MB, NUSSENZWEIG V: Amastigotes of Tv pbosoma wuzi escape destruction by the terminal complement components. J Eq Med 1989, 169881892. Amastigotes were shown to express an unidentibsd membrane protein that prevented the insettion of C MAC in a marmer resembling host celi membrane CS-binding protein or homologous restriction factor.

35. 0

LAMBtcsJD: The multiRmctional role of C3, the third component of complement. Immunol Tbaízy 1988, 9387-392. A rev+ew of the structure, binding sites, and functions of C3 and C3 receptors. 36. ??

BECHERER JD, IAMBtusJD: Identitïcation of the C3b receptorbinding domain in third component of complement. J Biel C&m 1988, 2631458614591. A region at the N-terminus of the a-chain of C3 was shown to farm the binding site for the C3b receptor, CR,. 37. .*

38. 0

DAOUDAXIME, BECHERERJD, IAMBRIsJD: A 34-amino acid peptide of the third component of complement mediates properdin binding. / Immunol 1988, 140:1577-1580.

39. 0

O’NEIL KM, OCHS HD, HEUER SR, CORK IC, MORRIS JM, WINK!XISTEIN JA Role of C3 in humoral immunity: defective antibody production in C3deficient dogs. J Immunoll988, 140:193!Sl%5. C3-deEcient dogs exhibited defective pdmary and secondary immune responses to T cell-dependent antigens that were not normalized by increased antigen doses. 40. om

ARVIEUXJ, YSSELHz COLOMBMG: Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones. Immunobgv 1988, 65:229-236. Stimulationof tetanus toxoid-specifìc T cell clones was enhanced when the tetanus toxid antigen was coupled to either C3b or C4b for presentation by B lymphoblastoid ceUs on which the antigen was associated with either CR1 or CR*.

41. uu

WONGWW, CAHIUJM, ROSENMD, KENNEDYCA, BONACCIO ET, MORRISMJ, Wnso~ JG, KLKXTEIN LB, FEARONDT: Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and identitîcation of a new CRl-Eke allele. J E3qbMed 1989, 169H7-861. Sequence analysis of cDNA clones and genomic DNA encoding the entire CR, molecule demonstrated that the low and high molecular weight allotypes of CR1 were made up of 4 of 5 LHRs, 1 of which was duplicated in the higher molecular weight allotype.

42. ??*

RFY-Cis J, RUBINUEINP, RODRIGUEZDE CORDOBAS: A physical map of the buman regulator of complement ac&tion gene cluster linking the complement genes (CRl, CR2, DAF1 and C4BP. J EqO Med 1988, 16ïYiS669. The genes encoding seveml C regulatory proteins and 2 types of C re ceptors were mapped to a lotus on chromosome 1, termed the RCA

43. uo

CARROILMC, ALZOT EM, KKZMANPJ, KIXKST!XN LB, SMITH JA, FEARONDT: Organixation of the genes encoding complement receptors type 1 and 2, decay-accelerating factor, and C4-binding protein in the RCA lotus on human cbromosome 1. JE@ Med 1988, 16~1271-1280. The arrangement of genes encoding 2 C receptors and 2 regulatory proteins of the C system was mapped within the lotus of chromosome 1 tenncd the RCA.

44. 00

KUCK.?TEIN LB, BARTOWTJ, MIIJXIC V, RABSONLD, SMtTHJA, FEARONDT: Identification of distinct C3b and C4b recognition sites in the human C3bK4b receptor (CRl, CD35) by deletion mutagenesis. J E@ Med 1988, 168:169+1718. Transfection of COS cells with recombinant CR, cDNA constructed to be deEcient in 1 of the 4 LHR of CR, allowed mapping of the C4b- and C3b-binding sites of CR, to 2 adjacent LHRs at the end of the external membrane domah. 45. ??e

KIN~XHITA T, TAKEDAJ, HONG K, KOZONOH, SAKAIH, INOUE K: Monoclonal antibodies to mouse complement receptor type 1 (CRl): their use in a distribution study showing that mouse eqthroqtes and platelets are CRl-negative. J Immutwll988, 1403066-3072. A rat anti-CR, mAb was generated from CR1 isolated from murine splenic B cells and used to e xamfne the expression of CR1 on different mouse ceU types.

46. 00

47. ??

Direct eviPACCAUDJ-P, CARPENIIERJ-l, S~HIFFERLIJA dence for the clustered nature of complement receptors type 1 on the eq&rocyte membrane. J Immunol 1988, 141:3~3894.

59

60

Innate immunity Localizauon of CR1 OIIerythrocyte membranes by immunofluorescence mi~roxopy and immunogold electron microscopy demonstrated a clustered distribution that may explain the high efficiency of immune complex uptake by red cells that occurs despite low overall numbers of CR, per teil. J, KAZATCHKINE m: DistribUtiOfl in CluSterS Of CHEVA,.,ER complement receptor type one (CRl) on human erythroqtes. J Immunol 1989, 142:2031-2036. Electron mic~~scopy e xamination of freeze-fracture replicas of erythrocytes on which CR1 was labeled tith colloidal gold-labeled antibody indicated that the majorily of surface CR1 was located in large clusters.

48. 0

EDBERG JC, Tosrc L, WRIGHT EL, SUTHEIUAND WM, TA~OR RP: Quantitative analyses of the relationsbip between C3 consumption, C3b Capture, and immune adherente of complement-fïxing antibody DNA/lmmune complexes. J Irn~ murw1 1988, 141:425-8-4265. merent sized complexes of DNA and human IgG anti-double-stranded DNAwere prepared and arAysed for C activation, numbers of IgG and C3 molecules bound per complex, and the efficiency of complex uptake by eq&rocytes via CR,.

49. 0

SCHIFF!%UJA, NG YC, ESTREICHERJ, WALPORTMJ: The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the clrculation of humans complementand eq&roqte complement receptor ldependent mechanisms. J Immunol 1988, 140899-904. Isolated l*%labeled immune complexes of tetanus toxoid and human IgG anti-tetanus toxoid were injected into normal subjects and patients with deficiencies of C or erythrocyte CR, and examined for the kinetics of cleamnce from the circulation. 50. 00

HAMMOND A, RUDGEAC, IQUOU S, BOWCOCKSJ, WALPORT MJ: Reduced numbers of complement receptor type-1 on e+hroqtes are associated with increased levels of anticardiolipin antibodies - findings in patients with systemic lupus erythematosus and the antiphospholipid syndrome. Ar&riti Rbeum 1989, 32:259264. IQSS of e@rocyte CR1 in patients with SLE was shown to correlate with increased leve& of anticardlolipin autoantibodies that were shown to be associated with the surface of eqthrocytes. 51. ??

WEls JJ, TOOTHAKE R LE, SMITHJA, WEIS JH, FF.ARON DT: Struchne of the human B lymphocyte receptor for C3d and the Epstein-Barr virus and relatedness to other members of the family of C3K4 binding proteins. J Eq0 Med 1988, 167:1047-1066. CR* structure was determined from sequence analysis of overlapping cDNA clones; evidente for altemative RNA splicing was found that was consistent with CR2 containing 15 or 16 SCR units. 52. 00

m JM, HAYWARJISD, HICKEY JC, FEARON DT: Epstein- Barr virus (EBV) infection of murine L cells expressing recombinant human BBVK3d receptor. Proc Nd Acad Sci USA 1988, 85:9307-9311. Transfection of murine L cells with human CR2 cDNA permit& expres~ sion of CR2 antigens and C3d receptor activity, and allowed the L celIs to be infected with EBV. 53. ??o

FINGEROTH JD, BENEDICTMA, LEXY DN, STROMINGER JL: Identifîcation of murine complement receptor type 2. Proc Nut1 Acad Sci US4 1989, 86~242-246. Mouse B cell CR2 was identilïed through a rabbit anti-human CR2 that cross-reacted with mouse CR2 and a mouse CR2 cDNA clone was isolated by screening with a human CR2 cDNA 54. ??*

Tsom CD, IAMBRISJD: Expression of CR2/EBV receptors on hmnan thymocytes detected by monoclonal antihodies. Eur J Immunol 1988, 18:12W1302. CR2 which is a marker of mature B cells and is absent Erom mature T cells, was identifìed on thymocytes by Euorescence staining and immunoprecipitation with 3 mAb to CR2. 55.

~01 of a Burkitt lymphoma ceU line responsiveness to growth signals engaging the C3D/EBV receptor. Immunology 1988, 65:237-242. Further evidente that CR2 can function as a B cell groti factor receptor was obtained with the demonstration that purifìed C3 would allow the maintenance and proliferation of Raji B-type lymphoblastoid cells in a serum-free media.

BOHNSACK JF, COOPERNR CR2 ligands modulate human B 57. ??e cell activation. J Immunoll988, 141:256+2576. The function of CR2 in lymphocyte activation was explored using various soluble and particulate C3dg ligands, anti-CR2 mAb and co-stim~ ulating agents. A wide vadety of different ligands was examined, and monotient soluble ligands such as C3d fragments were shown fo suppress activation, whereas solid-phase or multivalent ligands were found to augment lymphocyte activation. CARTERRH, SPYCKERMO, NG YC, HOFFMANR, FEARONDT: Synergistic interaction between complement receptor type 2 (CD21) and membrane IgM on B lymphocytes. J Zmmunol 1988, 141:457-463. B cell proliferation was induced by combined stimulation with monoclonal anti-IgM and Sepharoseanti-CR2; B cell stimulation with CR2 ligands alone was nat observed. 58. ??e

~RNHARDTW, MELCHERSF: The role of C3 and its fragments in the control of S phase entry of activated mouse B lymphocytes via the complement receptor type 2. m Clin Immunogenet 1988, 5:115122. A review of earlier data from this laboratory (Nuture 1985, 317: 264267) which indicated that ligands for CR2 participate in the reg ulation of B cell activation in marmer similar to B cell growth factor-u derived from macrophages. 59. ??

60. ??e

CORBI AL, KISHIMOTOTK, MULERLJ, SPRINGER TA The human leukocyte adhesion glycoprotein Mac-l (complement receptor type 3, CDllb) a subunit. Cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B. J Bio1 Cbem 1988, 263:1240+12411. The sequence of the a-subunit of CR3 (Macl, CDllb) was determined from a ful&length cDNA clone and this revealed 87% homology with the a-subunit of CQ as well as evidente for membership of the integrin gene family. 61. ??e

PYTEIA R Amino acid sequence of the murine Mac-l a chain reveals homology with the integrin family and an additional domain related to von Willebrand factor. EMBO J 1988, 71371-1378. The amino acid sequence of the a-chain of murine C& (Mac-1) was determined from overlapping cDNA clones and shown to have a high degree of homology with members of the integrin gene family. 62.

ARNAOUTMA, REMOLD-O’DONNELL E, PIERCE MW, HARRISP, TENEN DG: Molecular cloning of the u subunit of human and guinea pig leukocyte adhesion glycoprotein Mol: chromosomal localization and homology to the cz subunits of integrins. Proc Nat1 Acad Sci USA 1988, 85:27762780. Cloning and sequencing of cDNA representing the a-subunits of guinea pig and human C& (Mol) indicated homology with members of the integrin gene family. ??

ARNAOUT MA, GUPTASK, PIERCEMW, TENENLX? Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mol (CR3). J Cel1 Bio1 1988, 106:2153-2158. Complete sequence analysis of a cDNA clone of human CQ a-subunit revealed the structure of transmembmne and cytoplasmic domains, 3 putative calcium-binding domains, and further evidente for membership of the integrin gene family.

63. ??

??

56. ??

PERNEGGERG, SCHUL?!TF, HOSP M, MYONESBI, PETZERAL, EIGENIZER A, BOCK G, WICK G, DIERICHMP: Cell cycle con-

64.

HICKSTEINDD, HICKEYMJ, OZOLSJ, BAKERDM, BACKAL, ROTH GJ: cDNA sequence for the uhí subunit of the human neutrophil adherente receptor indicates homology to integrin a subunits. Proc Nat1 Acad Sci USA 1989, 86257-261. Sequence analysis of a cDNA encoding the a-subunit of C% showed: a region of homology with von Wfflebrand factor, the transmembmne domain, and the cytoplasmic domain. ??

Complement BUYON JP, ABRAMSONSB, PHUIPSMR, SIADE SG, Ross GD, WEKQiANNG, WINCHESTER RJ: Dissociation between increased surface expression of Gp165/95 and homotypic neutrophil aggregation. J Immund 1988, 140:3156-3160. Treaunent of neutrophils with anti-CR3(Gp165/95) mAb wz shown to prevent neutrophil aggregation induced by formyl-methionine-leucinephenyiakmine tripeptide through blockade of a subset of membrane CR3 present on the surface of unstimuiated cells. 65. 0

GRAWAM II, GRESHAM HD, BROWN EJ: An immobse subset of plasma membrane CDllbKD18 (Mac-1) is i&olved in phagocytosis of targets recognized by multiple receptors. J Immunol 1989, 142:2352-2358. An immobiie subset of membrane C& (‘iael, CDllb/CD18) was demonstrated on phorbol-stimulated neutrophils and monmes that appeared to be involved in the phagocytosis of particles bound to the ceU surface, initiallyvia CR, or FC receptors. 66.

??e

WRIGHTSD, LEVINSM, JONG MTC, CHAD2, KABBASH’ LG: CR3 (CDllbKD18) expresses one binding site for Arg-Gly-Aspcontaining peptides and a second site for bacterial lipopolysaccharide. J E@ Med 1989, 169~175-184. Monocyte phagocytosis of Escherichia coliwas inhibited by soluble LPS or anti-CQ mAb that did not black the putative RGD-binding site. 67. ??e

68.

WRIGHTSD, WEKZ p, HUANGAJ, LE?ANSM, SIL~ERXEINSC, ~QIKEJD: Complement receptor type three (CD1 lWCD18) of human polymorphonuclear leukocytes recognizes fïbrinogen. Proc Natl Acad Sci CX4 1988, 85~77347738. A single binding site in CRJ for lïxed iC3b and fibrinogen was suggested by inhibition of neutrophil attachment to fibrinogen-coated surfaces or iC3b-coated erythmcytes by either fibrinogen peptides derived from the y-chain of fìbrinogen or anti-CRj. ??

RUSSELLDG, WRIGM SD: Complement receptor type 3 (CR3) binds to an Atg-Gíy-Asp-containing region of the major surface glycoprotein, gp63, of Leisbmania promastigotes. J E.Q Med 1988, 1681279292. A specificity of CQ for a similar RGD sequence contained in both iC3b and gp63 from mnia promastigotes was shown to mediate phagocytosis of i.e&mania. 69. ??e

70.

RUSSELL DG, TALW,%? -ROHANAP, ZELECHOWZIJ:

Antibod-

ies raised against synthetic peptides from the Arg-GlyAsp-containing region of the Leisbmania surface protein gp63 cross-react with human C3 and interfere with gp63-mediated binding to macrophages. InfKt Immun 1989, 57:630-632. The specilìcity of Ç% for common RGD-centered sequence in both iC3b and Leishmarìiu gp63 was indicated by the demonsuation of antibodies to gp63 that cross-reacted with iC3b and inhibited LeLrhmania binding to CQ. ??

71.

ALXIERI lX, BADER R, MANIWCCI PM, EDGINGTON TS: Oligospecifìcity of the celhdar adhesion receptor MAC-1 encompasses an inducible recognition specifïcity for fibmogen. J Cel1Biel 1988, 107:1893-1900. Monocyte binding to fibrinogen was inhibited by anti-Cg (OKMl) but not by RGD peptides derived from fibrinogen or another anti-CR3 (OKMlO) thought to be specilïc for the putative RGD-binding site of ??

CR3. 72. e

Wnso~ ME, PEARSONRD: Roles of CR3 and mannosc receptors in the attachment and ingestion of Lekbmania dotwvani by human mononuclear phagocytes. Infat Immun 1988, 56:36>369. Bvidence was gathered that both mannose receptors and C% paxticipated in the attachment of &&muniu to monocytes, but that the la-biiding site of CQ rather than the putative RGD-binding site was involved, because inhibition with OKMl was much strenger than inhibition by OKh410. 73. ??

Monoclonal antibodies that recognize distinct epitopes of the macrophage type three complement receptor diJTer in their ability to inhibit

COOPERA, ROSENH, BIACKWEUJM:

and complement

receptors Ross

binding of Leisbmania promastigotes harvested at different phases of their growth cycle. Immutadogy 1988, 65:511-514. Data are presented that L-&mzunia attachment to murine macrophages is mediated by the leain-binding site of CR3 rather than the putative RGD-binding site of C+. MYONE_~ BL, DAIZEU.JG, HOGGN, Ross GD: Neutrophu and monocyte cell surface p150Q5 has iC3b-receptor (CR& activity resembling CR+ J Clin Invest 1988, 82:m51. Membrane surface CDllc/CD18 (p150,95) was designated C& because it was shown to function as an iC3b receptor tith activity that was homologous fo the closely related surface molecule, CQ (CDllb/CD18). 74. ??e

75. ??e

CORBIAL, MULERLJ, O’CONNER K, IARSONRS, SPRINGER TA

cDNA cloning and complete prinsje structure of the a subunit of a leukocyte adhesion glycoprotein, p150,95. EMBO J 1988, 6~40234028. Sequence analysis of a cDNA clone representing the a-subunit of p150,95 (CQ) revealed 3 putative calciumbinding domains and homology with the integrin gene family.

CORBIAL, LARSON RS, KLWIMOTOTK, SPRINGER TA, MORTON CC: Chromosomal location of the genes encoding the leukocyte adhesion receptors LFA-1, Mac- 1 and ~15095. Identification of a gene cluster invohred in cell adhesion. J E_x$I Med 1988, 167:1597-1607. The genes for the a-subunits of the CD18 family were mapped to a region of the short arm of chromosome 16 between bands pll and ~13.1, whereas the gene for the common CD18 p-subunit was mapped to chromosome 21, band q22. 76. ??e

Md Immund 1988, 25:1067-1073. The synthesis and StIUCNre of the Clq receptors expressed by Rajicells and U937 cells were charactedzed and shown to be similar to the strucNre reported earlier by Ghebrehiwet (J Immunoll986, 137:61?3-624). 77.

ERLIEI A, REIDKBM: The Clq receptor.

??

Clq enhancement of antibodydependent granulocyte-mediated killing of nonphagocytosable targets in vitro. J Clin Invest 1988, 82:945-%9. Neutrophil IgGdependent killingof the microfilariae Diroyíkzriüimmitis wai enhanced by the addition of Clq. 78. ??e

HA~~ADAA, YOUNG J, CHMIELEWSKI RA, GREENEBM:

Clq enhances the phagocytosis of Cryptouxcus neofonnans blastospores by human monocytes. J Immunol 1988, 141:592-597. Cultivation of monocytes on Clq-coated surfaces enhanced the phagocytosis of Cryptoc0ccu.sneo*q with or without prior opsonization with IgG 79.

BOBAK DA, WASHBURN RG, FRANKMM:

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AMM, HIEMZXXA PS, KLU MOHAMAD N, DAHAMR, MILTENBURG VAN ES & VAN HINSBERGH VWM: The complement subcomponent Clq mediates binding of immune complexes and aggregates to endothelial cells in vitro. Eur J Immunol 1988,18:783787. The existente of an endothelial cell receptor for Clq was suggested by the finding of saturable binding of lz5I-Clq to endothelial cells that was blocked by the isolated collagen portion of Clq.

80. ??

81.

RIFQZHEJ, ERDEIA, GILBERTD, AL SAL[HIA, FONTAINEM, SAMRB: Two populations of complement factor H differ in their abiiity to bind to cell surläces. Bzkbem J 1988, 253:475-480. Plasma factor H was separated into 2 fractions, designated 91 and 42 on the basis of elution charactetistics in hydrophobic atlìnitychromatography; only the 42 farm was found to bind to Raji cell factor H receptors. ??

ROIUNSTE, SICUANOS, SPRINGER MS: Solubilization of the functional C5a receptor fkom human polymorphonuclear leukocytes. J Biel Gwm 1988, 263:52&526. The neutrophil C5a receptor of 52 kD was solubilized in an active form and shown to exist on the cell surface primarily as an oligometic farm estirnated to be 150-200kD by gel filtration. 82. ??

83. ??e

MOLUWN KW, MANDECKIW, ZUIDERWEGERP, FAIR L, FEY TA, KRAUSERA, CONWAYRG, MILLER r, EDALJI RP, SHAUCROSS MA, LANEB, Fox JL, GREERJ, CAKTERGW: Identifìcation of

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Innate immunity receptor-binding residues in the intlammatoxy complement protein C5a by site-directed mutagenesis. Proc Nat1 Acad Sci USA 1939, 86292-296. At least 3 discontinuous regions of C5a were identifïed by site-directed mutagenesis of recombinant C5a that appeared to be important for binding to neutrophil C5a receptors.

ARENDWP, MASWNIRJ, NIEMANN M, GICW PC: Absente of induction of IL-1 production in human monocytes by complement fragments. J ImmunoIl98!9, 142973-178. Purilkd preparations of C5a, C3a, C3b, iC3b, Bb and Ba fragments that had been depleted of endotoxin failed to induce monocyk secretion of IL-1. 84. 00