Antibody CR1-2B11 recognizes a non-polymorphic epitope of human CR1 (CD35).

Measurement of erythrocyte [red blood cells (RBC)] complement receptor type 1 (CR1, CD35) has the potential to serve as a sensitive assessment of complement activation and immune complex clearance. All previously reported monoclonal antibodies (MoAb) to the extracellular region of CR1 recognize epitopes within the long homologous repeats (LHR) of CR1 and the epitopes for the most frequently used MoAbs are repeated at least twice per CR1 molecule. Furthermore, CR1 exhibits structural polymorphism characterized by a variable number of LHR per molecule. Thus, accurate enumeration of cell surface CR1 using currently available MoAb would require that the results be corrected for the number of antibody epitopes per CR1 molecule encoded by each individual's alleles. To obtain a MoAb to a non-polymorphic epitope on human CR1, hybridomas were generated from mice immunized with recombinant soluble CR1 (sCR1) and MoAb were screened for those that recognized the full-length extracellular domain but failed to bind to all four recombinant LHR fragments. A single antibody, CR1-2B11, was identified and was found to recognize an epitope located wholly within SCR29-30 of CR1, NH2-terminal to an elastase cleavage site. Like other CR1 MoAb, the CR1-2B11 epitope expression decreased on old erythrocytes compared to younger cells and CR1-2B11 did not identify a CR1 'stump' on RBC. Importantly, CR1-2B11 immunofluorescence did not change with storage or handling of RBC, unlike the apparent decrease in immunofluorescence observed with other MoAb. CR1-2B11 should be useful for the accurate enumeration of RBC CR1.

A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1).

Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.

Complement receptor 1/CD35 is a receptor for mannan-binding lectin.

Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1-MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL-immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.

C5a-stimulated human neutrophils use a subset of beta2 integrins to support the adhesion-dependent phase of superoxide production.

Isolated human polymorphonuclear neutrophils (PMN) responded to human C5a with an immediate, transient release of superoxide lasting from 0.5 to 5 min. This was followed by a second release of superoxide, which began at 10 min after addition of C5a, was sustained for more than 30 min, and required ICAM-1 immobilized in the wells. F(ab')2 monoclonal antibody (mAb) preparations were used to dissect the role of individual beta2 integrins and to avoid the confounding effects of ligating Fc receptors. Anti-CD18 mAb treatment of the PMN had no effect on the immediate first phase but completely inhibited the second, adhesion-dependent phase of superoxide production. Anti-CR3 mAb only inhibited the adhesion phase of superoxide production partially, implying that other beta2 integrins were involved. A mixture of anti-CD11a, anti-CD11b, and anti-CD11c was not able to block superoxide production completely, suggesting a role for alphad/beta2. Surprisingly, blocking anti-LFA-1 mAb had no effect on superoxide production. Consistent with this observation, immobilized, purified ICAM-2, a specific counter-receptor for LFA-1, did not support the adhesion-dependent phase of-superoxide production. Thus, PMN treated with C5a used signals via CR3, P150/95, and alphad/beta2, but not LFA-1, to support superoxide production. LFA-1 has been shown by others to mediate most of the adhesion necessary for transendothelial migration in vivo. The inability of LFA-1 ligation to stimulate superoxide production may be an important means of preventing blood-vessel damage when PMN migrate across the endothelium.

Intercellular adhesion molecule 1 and beta2 integrins in C1q-stimulated superoxide production by human neutrophils: an example of a general regulatory mechanism governing acute inflammation.

OBJECTIVE: To investigate the role of intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins in the production of superoxide (O2-) by C1q-stimulated human polymorphonuclear leukocytes (PMN). METHODS: PMN were pretreated with F(ab')2 fragments of monoclonal antibodies (mAb) that blocked or did not block beta2 integrin-mediated adhesion. The cells were added to wells coated with C1q, and the production of O2- was monitored kinetically as a color change due to reduction of cytochrome c. In some experiments, C1q was co-immobilized with purified ICAM-1. RESULTS: Blocking mAb to the shared beta2 integrin subunit, CD18, completely inhibited the O2- response triggered by immobilized C1q, while blocking mAb to the alpha subunits of the beta2 integrins each partially blocked the O2- response. PMN treated with C1q were found to activate the beta2 integrins lymphocyte function-associated antigen 1 and CR3 for binding to ICAM-1. Co-immobilization of ICAM-1 with C1q cooperatively triggered O2- production by PMN. CONCLUSION: beta2 integrin binding to an ICAM provided an essential costimulatory signal for O2-production triggered by C1q in PMN. Our findings suggest a model for PMN activation in which 2 stimuli are required for O2- production: a first signal that also activates PMN beta2 integrins, followed by a second, beta2 integrin-mediated signal, which occurs physiologically upon PMN binding to ICAM-1. The requirement for this dual signal for PMN generation of O2- would serve as a regulatory mechanism to limit the production of O2- to a tissue environment where C1q, or some other stimulus, is colocalized with stromal cells bearing up-regulated ICAM-1. This mechanism may explain why all tissues can express ICAM-1 and may explain in part why inhibitors of tumor necrosis factor alpha, a major physiologic stimulus of ICAM-1 up-regulation, are potent antiinflammatory agents.

C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.

Phylogenetic analysis of the homologous proteins of the terminal complement complex supports the emergence of C6 and C7 followed by C8 and C9.

The plasma complement system comprises several activation pathways that share a common terminal route involving the assembly of the terminal complement complex (TCC), formed by C5b-C9. The order of emergence of the homologous components of TCC (C6, C7, C8alpha, C8beta, and C9) has been determined by phylogenetic analyses of their amino acid sequences. Using all the sequence data available for C6-C9 proteins, as well as for perforins, the results suggested that these TCC components originated from a single ancestral gene and that C6 and C7 were the earliest to emerge. Our evidence supports the notion that the ancestral gene had a complex modular composition. A series of gene duplications in combination with a tendency to lose modules resulted in successive complement proteins with decreasing modular complexity. C9 and perforin apparently are the result of different selective conditions to acquire pore-forming function. Thus C9 and perforin are examples of evolutionary parallelism.

Role of complement component C1q in the IgG-independent opsonophagocytosis of group B streptococcus.

We investigated the role of complement component C1q in the IgG-independent opsonophagocytosis of type III group B Streptococcus (GBS) by peripheral blood leukocytes. We report that C1q binds to type III GBS both in normal human serum deficient in IgG specific for type III capsular polysaccharide and in a low-ionic strength buffer. The dissociation constant Kd ranged from 2.0 to 5.5 nM, and the number of binding sites Bmax ranged from 630 to 1360 molecules of C1q per bacterium (CFU). An acapsular mutant strain of GBS bound C1q even better than the wild type, indicating that the polysaccharide capsule is not the receptor for C1q. In serum, binding of C1q to GBS was associated with activation of the classical complement pathway. However, normal human serum retained significant opsonic activity after complete depletion of C1q, suggesting that the serum contains a molecule that is able to replace C1q in opsonization and/or complement activation. Mannan-binding lectin, known to share some functions with C1q, appeared not to be involved, since its depletion from serum had little effect on opsonic activity. Excess soluble C1q or its collagen-like fragment inhibited phagocytosis mediated by normal human serum, suggesting that C1q may compete with other opsonins for binding to receptor(s) on phagocytes. We conclude that, although C1q binds directly to GBS, C1q binding is neither necessary nor sufficient for IgG-independent opsonophagocytosis. The results raise the possibility that additional unknown serum factor(s) may contribute to opsonization of GBS directly or via a novel mechanism of complement activation.

In vitro and in vivo responses of murine granulocytes to human complement-derived, haemolytically inactive C5b67 (iC5b67).

Haemolytically inactive C5b67 (iC5b67), which was made from purified human components and decayed to a haemolytically inactive form, was evaluated as an agonist for murine leucocytes both in vitro and in vivo. In an in vitro assay, iC5b67 stimulated chemotaxis for both neutrophils purified from mouse bone marrow and splenic eosinophils of IL-5 transgenic mice. The stimulation was dose-dependent, with high dose inhibition. As with human neutrophils, iC5b67 also failed to up-regulate CR3 (CD11b/CD18) expression and to stimulate superoxide generation in murine bone marrow neutrophils, in vitro. In vivo, iC5b67 elicited an inflammatory response in a mouse model of pleuritis. A marked infiltration of neutrophils, which peaked at 4 h, was followed by an infiltration of eosinophils and mononuclear leucocytes. This inflammatory response was dose- and time-dependent. However, the protein concentration in the pleural wash fluid did not increase, indicating that iC5b67 did not induce a capillary leak. Although the infiltration of neutrophils could not be reproduced by pure C7 or human serum albumin (HSA), C5b6 did induce an influx of neutrophils. We were able to document the existence of C7, both antigenically and functionally, in pleural washes of normal mice, making it likely that the activity of C5b6 resulted from the in situ formation of C5b67 and iC5b67. The mouse model of pleuritis promises to be a useful in vivo system in which to evaluate the pro- and anti-inflammatory effects of iC5b67 that have been noted in vitro.

C1q-binding proteins and C1q receptors.

Aggregated or immobilized complement C1q induces cellular responses in many different cell types. C1q-induced cellular responses may be involved in host defense and in protection against autoimmunity because C1q-deficient humans have infectious complications and a very high incidence of autoimmune disease. The search for the C1q receptor(s), which has been ongoing for 25 years, has led recently to the recognition that proteins identified as binding to C1q may be divided into two groups: C1q-binding molecules that are normally intracellular; and cell surface C1q receptors.

Complement receptor type 1 (CR1, CD35) is a receptor for C1q.

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

Increased susceptibility to endotoxin shock in complement C3- and C4-deficient mice is corrected by C1 inhibitor replacement.

Endotoxin shock is a life-threatening syndrome associated with a Gram-negative infection and mediated by a systemic inflammatory response. As a major effector of inflammation, the complement system has been implicated in both the pathogenesis and the protection from endotoxin shock. To clarify the role of complement in endotoxin shock, we have used mice totally deficient in either complement component C3 or C4. We found that both the C3- and C4-deficient mice were significantly more sensitive to endotoxin than wild-type controls. The endotoxin-challenged complement-deficient mice failed to clear endotoxin efficiently from the circulation and this led to excess consumption of C1 inhibitor protein (C1 INH), a major regulator of both complement and the contact system of blood coagulation. Replacement of C1 INH rescued the endotoxin-challenged complement-deficient mice from shock and death. These findings suggest a novel therapy for treatment of endotoxemia with C1 INH protein.

Serum amyloid P component forms a stable complex with human C5b6.

A 30,000 m.w. protein bound tightly to C5b6, which was formed by activating C7-depleted human serum with zymosan. The protein remained bound to the C5b6 complex during the isolation procedure for C5b6, including chromatography on lysine-Sepharose and an anion exchange resin. Following electrophoresis and electroblotting of the C5b6 complex to a polyvinylidene difluoride transfer membrane, the 30,000 m.w. protein was microsequenced. The 24 N-terminal amino acid sequence was determined and showed identity of the 30,000 m.w. protein with the serum amyloid P (SAP) component. The C5b6-SAP complex did not dissociate in the presence of 10 mM EDTA, which distinguishes SAP-C5b6 binding from SAP's usual Ca2+-dependent binding to other molecules. SAP, which was isolated from serum by chromatography, was able to bind to preformed C5b6, which had been assembled and isolated from purified components. Functionally, the C5b6-SAP could bind C7, and the resulting C5b67-SAP complex had only moderately lower specific hemolytic activity than that of C5b67. In addition, hemolytically inactive C5b67-SAP, such as hemolytically inactive C5b67, was chemotactically active for neutrophils, while isolated SAP had no effect on cell mobility. Because SAP reacts with other serum proteins and with cells, it is likely that the addition of SAP to terminal complement complexes may affect the fate of these complexes.

Signaling by hemolytically inactive C5b67, an agonist of polymorphonuclear leukocytes.

The hemolytically inactive complement component complex C5b67, designated iC5b67, can signal human polymorphonuclear leukocytes (PMN) both as a pertussis toxin-inhibitable agonist for chemotaxis and as an antagonist for C5a- and FMLP-stimulated chemotaxis and superoxide production. The signaling pathways utilized by iC5b67 have been further investigated. In contrast to mastoparan, iC5b67 failed to directly activate G proteins to stimulate inositol phosphate formation in COS cells that had been transfected with G alpha 16. In COS cells co-transfected with both G alpha 16 and the C5a receptor, iC5b67 could neither activate phospholipase C nor inhibit C5a receptor-mediated activation of phospholipase C. iC5b67 stimulated GTPase activity in a membrane-enriched fraction from PMN. These data support the hypothesis that iC5b67 signals through a unique receptor, likely G protein linked, but distinct from the C5a receptor. iC5b67 was able to mobilize intracellular stores to elicit increases in intracellular Ca2+. Based on the effects of herbimycin A, wortmannin, and chelerythrine on iC5b67-induced PMN chemotaxis, iC5b67 signaling involved activation of tyrosine and phosphatidylinositol 3-kinases, but not protein kinase C. Relevant to the capacity of iC5b67 to antagonize PMN superoxide production, iC5b67 induced rapid and sustained increases in intracellular cAMP, which others have shown can inhibit superoxide formation. Although iC5b67 antagonizes C5a and FMLP receptor-mediated superoxide generation, iC5b67 had no effect on PMA-induced superoxide formation. The distinct agonist and antagonist signaling pathways activated by iC5b67 in the PMN diverge soon after initial iC5b67 receptor-mediated transduction steps.

Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes.

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

Regulation of C1q receptor expression on human polymorphonuclear leukocytes.

The C1q receptor (C1qR) is expressed on a variety of cells, including polymorphonuclear leukocytes (PMN), in which stimulation by the C1qR leads to activation as measured by superoxide production. To investigate the expression and modulation of the C1qR on PMN, the binding of biotinylated C1q to PMN in suspension was measured by flow cytometry. Biotinylated C1q bound in a saturable and specific manner to PMN and the use of low ionic strength buffers enhanced binding. Covalent coupling of C1q to Sepharose beads allowed the affinity precipitation of a single 125-kDa band from surface iodinated PMN. The apparent molecular mass of the C1qR increased to 135 kDa upon reduction. Freshly isolated PMN had a uniform expression of C1qRs and phorbol myristate acetate induced a unimodal up-regulation of receptors. The inflammatory peptide FMLP rapidly up-regulated receptors by up to fivefold, and the high level of expression remained constant over 45 min. Taxol inhibited the FMLP induction of C1qR up-regulation, indicating that the ability to move the intracellular stores of C1qR depends on normal microtubule functioning. Thus, the C1qR is a constitutively expressed protein of the human PMN plasma membrane and it is rapidly up-regulated from a discrete intracellular pool of preformed molecules with the same kinetics as CR1 and CR3. It is likely that the C1qR is a component of the PMN complement receptor exocytic vesicle (CREV), in which CR1 and CR3 are also stored.

Structure and function of decay accelerating factor CD55.

Studies of decay-accelerating factor (DAF) function and structure are reviewed. DAF was first recognized as a species restricting factor operating at the level of C3/C5 activation. Cloning of the gene indicates that DAF has four short consensus repeats of the type characteristic of the regulators of complement activation gene cluster family. The third short consensus repeat is responsible for DAF's complement regulatory activity and signaling. DAF, like other glycophosphatidylinositol (GPI) anchored proteins, is associated with tyrosine kinases, and these kinases are probably the signaling devices. The details of how DAF's GPI anchor in the outer leaflet of plasma membrane connects with the tyrosine kinases on the inner leaflet are not known. Although DAF does not have an essential role in controlling hemolysis of erythrocytes, it does have important role in regulating the deposition of C3 on nucleated cells. The therapeutic potential of DAF is discussed.