Cellular receptors for fragments of the third component of complement

Many of the biological effects of the complement system are mediated by cellular receptors which can bind the fragments of C3 that are covalently attached to activators of complement. These receptors, which are termed CR1, CR2 and CR3, reside on lymphocytes, myelomonocytic cells, erythrocytes and renal podocytes. Progress in understanding their roles in im munological reactions has recently accelerated, as summarized in this review by Douglas Fearon, because of the molecular identification of these membrane proteins.     

Lipopolysaccharides as complement inhibitors by complex formation with the purified third complement component (C3)

Lipopolysaccharides (LPS) from different bacteria in smooth or rough form (Y. enterocolitica, Y. pseudotuberculosis, E. coli, S. typhimurium, S. marcescens) strongly inhibited hemolytic C3 in incubation mixtures with purified C3. LPS from a core deficient mutant was still reactive, whereas lipid A no longer affected C3 activity. The physical state of LPS was critical for its effect on C3. Strand-like LPS structures formed by Ca++-induced aggregation of solubilized LPS, as shown by electron microscopy, demonstrated the highest reactivity with C3. Inhibition of hemolytic C3 was found to be due to complex formation between LPS and C3 by a hydrophobic reaction. The binding capacity of 1 microgram LPS-R and LPS-S was as high as 125 ng C3 and 56 ng C3, respectively. The C3b fragment required different reaction conditions for maximal binding. The strong binding capacity of LPS for the complement component C3 raises the possibility that LPS act as inhibitors of complement by interruption of the reaction cascade at local infectious sites with gram-negative bacteria.     

The multifunctional role of C3, the third component of complement

The third component of complement, C3 is an important participant in immune surveillance and immune response pathways. It is probably one of the most versatile and multifunctional molecules known, interacting with numerous serum proteins, cell surface molecules and foreign proteins. Here, John D. Lambris reviews the multiple interactions of C3 emphasizing recent work on these interactions and addresses some of the unanswered questions and controversies.     

Synthesis and regulation of the fourth component of complement (C4) in the human monocytic cell line U937: comparison with that of the third component of complement (C3).

Production of the fourth component of complement (C4) by the human monocytic cell line U937 and its regulation were investigated in comparison with the production of the third component of complement (C3) in a cell culture system. Although no detectable C4 was produced by U937 without stimulation, U937 was induced by recombinant interferon-gamma (IFN-gamma) to synthesize C4 in a dose- and time-dependent fashion. The production of C4 was reversibly inhibited by cycloheximide, indicating that it resulted from de novo synthesis. The C4 synthesized by U937 cells was functionally active as assessed by haemolytic assay. SDS-PAGE following biosynthetic labelling showed that subunit structure of C4 synthesized by U937 cells was identical with that of plasma C4 but that molecular weight of alpha-chain was greater than that of plasma C4. We compared the regulation of C4 synthesis with that of C3 synthesis. Although C3 synthesis by U937 cells was enhanced by IFN-gamma, lipopolysaccharide (LPS) and phorbol myristate acetate (PMA), C4 synthesis was induced only by IFN-gamma. LPS and IFN-gamma induced a synergistic increase in C3 synthesis by U937 cells. U937 cells incubated with LPS and IFN-gamma synthesized a greater amount of C4 than those incubated with IFN-gamma alone. Thus it was demonstrated that the synthesis of C3 and C4 was independently regulated. This study shows that the U937 cell line provides a useful model for studies on the synthesis of complement proteins and on the regulation of complement production.     

Characterization of tryptic fragments of human complement factor C3

C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6M guanidinium hydrochloride. No low mol. wt (Mr) fragments were identified. The polypeptide chains were characterized with regard to Mr, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the beta-chain (Mr 64,000) and two from the alpha'-chain (Mr 40,000 and 23,000). The beta-chain fragment was derived from the C-terminal part of the chain, and the 23,000-Mr component constituted the amino terminal end of the alpha-chain. The 40,000-Mr fragment emanated from the C-terminal end of the alpha-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. natn. Acad. Sci. U.S.A. 77, 5764-5768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.     

Murine polymorphonuclear leukocytes synthesize and secrete the third component and factor B of complement

Complement proteins in serum are synthesized mostly by hepatocytes and many other cell-types have also been shown to synthesize complement in various tissues. However, polymorphonuclear leukocytes (PMNs) have never been reported to secrete complement. This paper demonstrates the synthesis and secretion of C3 and factor B by murine peritoneal exudate PMNs elicited with OK432 (Streptococcus preparation). Using [35S]methionine incorporation and immunoprecipitation, C3 and factor B produced by PMN are found to be antigenically and physically identical to macrophage C3 and factor B. ELISA analysis reveals that culture supernatant of PMN--free of macrophage contamination--contains C3 antigen, and both flow cytometric analysis and immunoperoxidase staining also demonstrate the presence of intracellular C3 using special precautions to eliminate non-specific staining. The role of complement produced by PMN is currently unknown, but it is very important to take this new finding into consideration for further clarification of the roles of complement in extravascular inflammatory sites.     

Decreased synthesis of the third component of complement (C3) in hypocomplementenic systemic lupus erythematosus

Metabolism of the third component of complement (C3) was evaluated in eight normal subjects and nine patients with systemic lupus erythematosus (SLE). Six of the nine patients with SLE were hypocomplementemic; four of these had active renal disease. In the eight normals, the half-life survival (T 1/2) was 49–66 hr, the catabolic rate (Km) was 1·8 to 3·5% of plasma pool/hr, and the synthetic rate (Ks) 0·89–2·0 mg/kg/hr. Two SLE patients with normal C3 had a normal T 1/2, Km and Ks; one with increased C3 had a shortened T 1/2 and a high Km and Ks. Three patients with untreated SLE and depressed C3 had T 1/2 of 21 hr, 37 hr, and 69 hr, Km of 5·3%, 3·5% and 1·6% with Ks of 0·48, 0·95 and 0·23 mg/kg/hr, respectively. Three other patients with low C3 levels taking corticosteroids had normal Km but depressed Ks. Three patients were restudied after prednisone therapy. All had an increased C3 and Ks, two had an increase in T 1/2 and a decrease in Km, while one did not change T 1/2 or Km. In summary, five of six SLE patients with low C3 had a depressed Ks. The lowest value of C3 was generally associated with the lowest Ks. Depressed Ks occurred in patients with or without renal disease. These results indicate that static measurements of C3 do not quantify the degree of C3 utilization. The major determinant of the low C3 observed in this study of SLE was decreased synthesis of C3.     

Characterization of the third component of complement (C3) after activation by cigarette smoke

Activation of lung complement by tobacco smoke may be an important pathogenetic factor in the development of pulmonary emphysema in smokers. We previously showed that cigarette smoke can modify C3 and activate the alternative pathway of complement in vitro. However, the mechanism of C3 activation was not fully delineated in these earlier studies. In the present report, we show that smoke-treated C3 induces cleavage of the alternative pathway protein, Factor B, when added to serum containing Mg-EGTA. This effect of cigarette smoke is specific for C3 since smoke-treated C4, when added to Mg-EGTA-treated serum, fails to activate the alternative pathway and fails to induce Factor B cleavage. Smoke-modified C3 no longer binds significant amounts of [14C]methylamine (as does native C3), and relatively little [14C]methylamine is incorporated into its alpha-chain. Thus, prior internal thiolester bond cleavage appears to have occurred in C3 activated by cigarette smoke. Cigarette smoke components also induce formation of noncovalently associated, soluble C3 multimers, with a Mr ranging from 1 to 10 million. However, prior cleavage of the thiolester bond in C3 with methylamine prevents the subsequent formation of these smoke-induced aggregates. These data indicate that cigarette smoke activates the alternative pathway of complement by specifically modifying C3 and that these modifications include cleavage of the thiolester bond in C3 and formation of noncovalently linked C3 multimers.     

Leucocyte mobilizing factor: a new biological activity derived from the third component of complement

A simple method is described to assay for factors that mobilize leucocytes in the bone marrow. Guinea pig or rat femura were slowly perfused with Medium 199 and the cell concentration in the effluent was determined. Leucocyte mobilizing factor (LMF) was recognized by an increase in the effluent cell concentration following addition of the material to the perfusion medium. By reaction with C 142, the rabbit C3 molecule was cleaved, releasing a fragment with LMF activity. A purification procedure is described. The factor was devoid of chemotactic activity. It was negatively charged at pH 8.6. LMF may be involved in the pathogenesis of the leucocytosis associated with C dependent immune reactions in vivo.     

Secretion and function of the third component of complement (C3) by murine leukocytes.

Our new finding of de novo synthesis and secretion of C3 by both murine peritoneal macrophages and polymorphonuclear leukocytes (PMN) was confirmed by the incorporation of [35S]methionine into C3 molecules and their complete inhibition by cycloheximide. The methods of secretion of C3 from these two types of cells were compared by examining the C3 contents in their culture supernatants. Completely different modes of secretion were observed, i.e. although macrophages synthesize and secrete C3 constitutively, PMN has a mechanism to store the already synthesized C3 in the cell and secrete it in response to stimuli. Protein kinase C (PKC) activators, e.g. 12-O-tetradecanoyl-phorbol 13-acetate, dioctanoyl glycerol, and mezerein, as well as calcium ionophore A23187 stimulate the secretion of C3 from PMN. These results suggest the involvement of PKC and the calmodulin pathway. A very sensitive method for measuring C3 activity was developed which enabled us to show for the first time that C3 secreted by PMN had opsonizing activity and that particles cultured with PMN were phagocytosed effectively.     

An inherited deficiency of the third component of complement, C3, in guinea pigs

Hereditary deficiency of the third component of complement, C3, is found very seldom in the human. C3 deficiency is associated with severe bacterial infections revealing the central role of C3 in complement activation via the classical or alternative pathway. We describe a new hereditary C3 deficiency in strain 2 guinea pigs. Serum from these animals had a markedly reduced lytic activity in a standard assay for complement-dependent, antibody-mediated cytotoxicity. In functional assays of individual components, the hemolytic activity of the components C4, C2, C5 and of factors B, D and H was in the normal range. The functional C3 titer, and similarly C3 antigenic activity in the serum of these C3-deficient animals (C3D) was on average only 5.7% of normal activity. Typing the animals with alloantisera or monoclonal antibodies to guinea pig Ia-antigens revealed that the C3D animals had the major histocompatibility complex-haplotype of inbred strain 2 guinea pigs (B.1, Ia.2,4). The C3 defect is not linked to the major histocompatibility complex and, in addition, is not linked to a C3a receptor deficiency. Macrophages and hepatocytes of the C3D animals have an unimpaired capacity for synthesis and secretion of C3 as measured by enzyme-linked immunosorbent assay. There was no indication for hypercatabolism of normal C3 by the animals as shown by plasma clearance of 125I-radiolabeled C3. Thrombocytes of the C3D animals responded normally to stimulation with purified C3a in an ATP-release assay without an indication for a desensitization in vivo. Possibly the fault resides in an enhanced susceptibility of their own C3 to proteolysis. However, C3 partially purified from the plasma of the C3D animals or secreted by hepatocytes exhibited no obvious structural differences to purified normal C3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis or in immunoblotting. The C3D serum had a reduced bactericidal activity compared to normal or to C4-deficient serum. Nevertheless, the animals are apparently healthy without an indication for increased frequency of bacterial infections. These guinea pigs provide an unique model for analysis of the biological functions of C3 in vivo and in vitro without the need for artificial C3-depletion procedures with all their known and unknown side-effects.     

Behavior of soluble human 125I-labeled C3b,the third component of complement,after binding to human cells

The behavior of ¹²⁵I-labeled C3b incubated with two C3b receptor-positive cells (human erythrocytes and the B lymphoblastoid Raji line), one C3b receptor-negative cell (T lymphoblastoid CEM line) and solubilized membranes from each cell was analyzed by sucrose density gradient (SDG) and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Whichever whole cell was tested, the unbound ¹²⁵I-labeled C3b recovered in the cell supernatant was not cleaved. When ¹²⁵I-labeled C3b was bound to whole cells or incubated with solubilized membranes, three different activities were detected: (a) nonspecific C3b polymerization, induced on the membrane of C3b receptor-positive or C3b receptor-negative cells; (b) specific C3b receptor activity solubilized only from the membrane of the two C3b receptor-positive cells and (c) C3b hydrolytic activity, inhibited by 5 x 10^?4 M phenyl methyl sulfonyl fluoride, only extracted from human erythrocyte membranes and carried by a molecule different from that of C3b receptor. C3b receptor activity solubilized from Raji and human erythrocyte membranes was detected by a 12 S peak complex formation on a 10-30 % SDG and characterized by an affinity constant of 2 x 10⁷ to 4 x 10⁷ mol^?1. Hydrolysis of labeled C3b (M_r = 175 000) by solubilized human erythrocyte membranes led to the formation of a split product of M_r = 35 000 consisting of two disulfide-linked polypeptide chains of M_r = 17 000. This is the first report of a breakdown of C3b on cell membranes different from the physiological breakdown described in the fluid phase.     

Production of the third and fourth component of complement (C3, C4) by smooth muscle cells.

Production of the third and fourth components of complement (C3, C4) by smooth muscle cells was investigated by using normal human aortic smooth muscle cells (AoSMC), human smooth muscle cell line (G402) and vascular smooth muscle cells obtained from human umbilical cord vein (UVSMC). AoSMC spontaneously produced both C3 and C4 at 15 ng/10(6) cells/72 hr and 22 ng/10(6) cells/72 hr, respectively, and both were enhanced by interferon-gamma (IFN-gamma). Although phorbol 12-myristate 13-acetate (PMA) and tumour necrosis factor-alpha (TNF-alpha) enhanced C3 production, C4 production was reduced by these agents. On the other hand, G402 produced C4 but not C3 in a dose-dependent manner when cultured with IFN-gamma. UVSMC produced only a small amount of C3 and C4 compared with AoSMC or G402. C3 and C4 produced by AoSMC were confirmed to be identical with their human serum counterparts as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and measurement of haemolytic activity. Northern blotting analysis showed that the expression of mRNA of C3 and C4 was enhanced by TNF-alpha and IFN-gamma, respectively, in AoSMC. Our findings suggest the importance of smooth muscle cells as a source of components of complement in vascular diseases including vasculitis.     

Expression of the binding molecules for factor B and its fragment, Bb, of human complement on human monocytes after in vitro cultivation

We have examined the expression of the binding molecules for Bb of human complement on the surface of resting and cultured human monocytes. Flow cytometry using biotinylated anti-B antibodies and phycoerythrin-avidin showed that although resting monocytes did not bind Bb significantly, monocytes cultivated for 24 h in the presence of fetal calf serum were capable of binding with Bb and its precursor B, but not with Ba fragment. The Bb-binding molecules were pronase-sensitive, suggesting that membrane proteins are associated with the Bb-binding molecules. In addition to human monocytes, liquid paraffin-induced guinea pig inflammatory macrophages were also found to express Bb-binding molecules on their surface. This implies that Bb receptor-like molecules become expressed during activation and differentiation of monocytes, just as observed with the C3d-receptor of monocytes.     

Compstatin,a peptide inhibitor of complement,exhibits species-specific binding to complement component C3

Although activation of complement protein C3 is essential for the generation of normal inflammatory responses against pathogens, its unregulated activation during various pathological conditions leads to host cell damage. Previously we have identified a 13-residue cyclic peptide, Compstatin, that inhibits C3 activation. In this study, we have examined the species-specificity of Compstatin. Bimolecular interaction analysis using a real-time surface plasmon resonance-based assay showed that Compstatin exhibits exclusive specificity for primate C3s and does not bind either to C3s from lower mammalian species or to two structural homologs of C3, human C4 and C5. Furthermore, it showed that although the kinetics of binding of Compstatin to non-human primate C3s were distinctly different from those to human C3, like human C3 its mechanism of binding to non-human primate C3 was biphasic and did not follow a simple 1:1 interaction, suggesting that this binding mechanism could be important for its inhibitory activity. Analysis of Ala substitution analogs of Compstatin for their inhibitory activities against mouse and rat complement suggested that the lack of binding of Compstatin to mouse and rat C3s was not a result of sterically hindered access to the binding pocket due to individual bulky side chains or the presence of charge on the Compstatin molecule. These results suggest that Compstatin's exclusive specificity for primate C3s could be exploited for the development of species-specific complement inhibitors.