Inhibition of cleavage of the third component of human complement (C3) by its small cleavage fragment, C3a: inhibition occurs with the classical-pathway, but not the alternative-pathway, C3 convertase

Activation of the third component of complement (C), C3, is central to the functioning of the C system in inflammation. Cleavage of C3 by the C3 convertases of both the classical and alternative pathways results in the formation of two split products, C3b and C3a. C3a inhibited cleavage of C3 by the classical-pathway C3 convertase. The inhibition varied in a concn-dependent relationship, with a concn of approximately 40 micrograms/ml yielding 50% inhibition. Removal of the carboxy terminal arginine from the C3a did not alter the inhibition. C3a did not inhibit cleavage of C3 by the alternative C pathway C3 convertase, or cleavage of C5 by C5 convertase. The C3-cleaving capacity of EAC142oxy that had been previously incubated with C3a could be recovered completely by washing the cells, indicating that the C3a binding to the EAC42oxy cell must have been reversed without having had an effect on the amount of C2 bound. Ribonuclease, a molecule of similar size and charge to C3a, did not affect C3 cleavage and C3a inhibition was not reduced by providing a surface for non-specific adsorption of the C3a, suggesting that the effect of C3a on C3 cleavage was not mediated by non-specific interaction with cell surfaces. C3a inhibited the C3-cleaving capacity of the fluid-phase enzyme, C42oxy, to the same degree as it inhibited the cell-bound enzyme, EAC42oxy, indicating that the C3a must interact with the C42 complex directly. Inhibition of C3 cleavage by C3a is the first demonstration of product inhibition of a complement enzyme. It may provide another control of C3 activation.     

Increased production of the third component of complement (C3) by monocytes from patients with systemic lupus erythematosus.

We measured in vitro C3 production by peripheral blood monocytes from patients with systemic lupus erythematosus (SLE), and found it to be significantly greater than that from normal controls. We also found that monocytes from SLE patients with active disease produced a markedly larger amount of C3 than those from SLE patients with inactive disease. Production of C3 by monocytes correlated with serum levels of anti-dsDNA antibodies and inversely correlated with serum C3 levels in SLE patients. Serial measurement of C3 in the culture supernatant from each SLE patient showed that C3 production by monocytes fell in parallel with a decrease of disease activity. The effect of corticosteroids was ruled out as there was no relation between the level of C3 production by monocytes and the dose of prednisolone. This seems to be the first study in which the C3 production was assayed at a cellular level in SLE patients, and this study suggests that the local C3 production is increased in SLE patients.     

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.     

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.     

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.     

Immunology: Investigations on the cell type responsible for the endometrial secretion of complement component 3 (C3)

It has been shown that and human endometria have the capacityto produce complement component 3 (C3). In rats, endometrialC3 is an oestrogen-dependent protein produced and secreted byglandular cells. The cell responsible for the synthesis andsecretion of human endometrial C3 has not been clearly defined.Our study was aimed at answering this question. Samples of endometriumobtained from hysterectomies were either immunostained for C3or digested with collagenase; then the stromal and glandularcells were separated and immunopurified (or not) with an antibodyto CD45 coupled to magnetic beads to eliminate the endometriallymphomyeloid cells. Cells were cultured for 2 weeks and C3measured in the medium by an in-house radioimmunoassay. Glandularas well as stromal cells stained positively for C3 and releasedC3 in vitro. The release of C3 from both cell types could beinhibited by cycloheximide. Epithelial cells produced significantlymore C3 than stromal cells, and endometrial C3 production washigher for both cell types when these were obtained from secretoryas compared to proliferative endometria. Lymphomyeloid cellswere possibly a source of C3 since after immunoadsorption ofthese cells, the remaining stromal or glandular cells producedsignificantly less C3. We conclude that endometrial stromal,glandular and lymphomyeloid cells all produce C3.     

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.     

C3研究进展

C3是3条补体激活途径的交汇点，系机体防御体系中的关键分子，是连接天然免疫和获得性免疫的桥梁之一，但其活化失调则会导致组织细胞损害，且其与病原体相互作用为微生物逃避补体攻击提供了可能。因此，深入了解C3的分子结构及其与其它蛋白、病原体、细胞的相互作用，对补体系统研究以及补体相关疾病和感染性疾病的治疗均有重要意义。     

The third complement factor (C3) and its in vivo cleavage products: interaction with lectins and precipitation with polyethylene glycol

Five molecular forms of C3 expressing D but not C epitopes were identified following in vivo activation of the complement system. Examination of concanavalin A (Con-A) reactivity in crossed immunoelectrophoresis revealed that native C3, C3c and the beta mobile form 4 of C3d were completely precipitated by 100 micrograms Con A/cm2. The alpha-1 mobile form 1 of C3d did not interact with Con A, whereas the alpha-2 mobile forms 2 and 3 were retarded in electrophoretic migration by Con A. Native C3, C3c, and forms 4 and 5 of C3d were precipitated by 12% (w/v) polyethylene glycol (PEG). Form 1 of C3d was soluble in these PEG concentrations, whereas forms 2 and 3 were partially precipitated.     

LPS-induced murine abortions require C5 but not C3, and are prevented by upregulating expression of the CD200 tolerance signaling molecule

PROBLEM: Lipopolysaccharide (LPS) acts via tlr4 to promote Th1 cytokine secretion and abortions. LPS is an essential co-factor in spontaneous abortion in the CBA x DBA/2 model and in stress-triggered abortions. In the CBA x DBA/2 model, C3a, C5a, and fgl2 prothrombinase participate in triggering inflammation that terminates embryo viability. As fgl2 prothrombinase (via thrombin) can generate C5a, it was predicted that LPS-driven abortions (which require fgl2) would be independent of C3. CD200Fc can prevent abortions in the CBA x DBA/2 model, but an action through Fc could not be excluded. METHOD OF STUDY: C3(-/-) and C5(-/-) knock-out mice on a B6 background were syngeneically mated and Salmonella enteritidis LPS was administered i.p. on day 6.5 or pregnancy along with 2 mg progesterone in sesame oil s.c. The total number of implants and the number of resorbing embryos were counted on day 13.5 of pregnancy. CD200-rtTA double transgenic homozygous males (B6 background) mated with B6(+/+) females were similarly treated. To up-regulate CD200 expression in embryonic trophoblasts, doxycycline was added to the drinking water from the time of mating. RESULTS: The LPS boosted the abortion rate from 15.5% (control) to 42.0% in C3(-/-) mice (chi(2) = 9.28, P < 0.005). In C5(-/-) mice, there was no increase in abortion rate with LPS compared to progesterone-treated controls (22.8%versus 26.3%, P = NS). LPS-treated transgenic mice given LPS + progesterone had a 42.5% abortion rate, but when the mice were given doxycycline to induce expression of CD200 by the embryo, the abortion rate was only 8.3% (chi(2) = 14.40, P < 0.005, Fisher's exact test P = 0.00007). CONCLUSION: C5, but not C3, appears necessary for LPS-driven abortions. Up-regulation of CD200 can prevent LPS-driven abortions, possibly by altering dendritic cells to promote Treg cell development or by a direct suppressive action on macrophages and mast cells that also express CD200 receptors.     

Inhibition of interleukin 3 function by a fragment of the third component of complement

A C3d-like (C3d-1) fragment of 33 kDa was isolated and its biological activity studied. The fragment was generated from guinea pig C3b by porcine pancreas kallikrein and purified by fast protein liquid chromatography. The C3d-like fragment inhibited interleukin (IL) 2-dependent T lymphocyte proliferation. The suppressive activity of the described C3d-1 fragment was not restricted to lymphocytes as targets but inhibited in addition the proliferation of a nonlymphocyte mast cell line which was strictly IL3-dependent in its proliferative capacity. Kinetic studies implied early stages of cellular proliferation to be influenced. Furthermore, the C3d-1 fragment was not only an inhibitor of cellular proliferation but was also a potent inducer of leukocytosis.     

An NcoI polymorphism in the human complement component 7 (C7) gene

A novel polymorphic site has been found in the 3′ untranslated region (UTR) of the human complement component 7 (C7) gene. The polymorphic site at 14-bp downstream from the TAG stop codon was either C or A (Nco I-digested), with allele frequencies of 0.660 and 0.340. This NcoI polymorphism would be useful to perform a DNA marker haplotype study in patients with deficiencies of the complement genes, such as C6, C7, C9, which are located closely on chromosome 5p13. Received: March 25, 1999 / Accepted: April 3, 1999     

Secretion of the C3 component of complement by peritoneal cells cultured with encapsulated Cryptococcus neoformans.

Two isolates of Cryptococcus neoformans were identified as being widely divergent in pathogenic potential after intratracheal infection of mice. These isolates differed in their ability to upregulate capsule synthesis when grown under tissue culture conditions, and this property correlated with virulence. We postulated that differential capsule synthesis may cause differential stimulation of macrophages to produce products such as complement components. To test this hypothesis, heat-killed yeast cells were incubated with normal mouse peritoneal cells (PC) before the level of C3 secreted was determined. Cryptococcal stimulants were grown on mycological agar, which does not promote capsule synthesis, or in RPMI 1640 at 37 degrees C in an atmosphere of 5% CO2, which stimulates capsule synthesis, to determine the role that the capsule plays in the induction of C3 secretion. C3 levels were elevated in cultures containing cryptococci grown in RPMI 1640 at 37 degrees C in an atmosphere of 5% CO2, and the level of C3 detected was correlated with the amount of capsule expressed by the yeast cell stimulant. Nonencapsulated mutants of C. neoformans did not stimulate C3 secretion. Purified capsular polysaccharide (glucuronoxylomannan [GXM]) also stimulated the PC to secrete C3. Two signals were required before GXM stimulated C3 secretion. The second signal was identified as endotoxin present in small amounts (0.06 ng per ml) in tissue medium. Endotoxin may provide a priming stimulus for PC to express receptors or other cytokines needed for effective stimulation of C3. These experiments show that enhancement of C3 secretion by C. neoformans is due to GXM and is correlated with the virulence of the cryptococcal isolate.     

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.     

The physiological breakdown of the third component of human complement

The C3b INA-dependent breakdown of fluid phase C3b has been shown to have an absolute requirement for a second factor. This factor is contained in catalytic amounts (with respect to C3b) of highly purified β1H. β1H alone does not cause proteolysis of C3b. In the presence of C3b INA and β1H, proteolysis of the larger3 polypeptide chain (116K) of C3b occurs. Initially, a single scission gives two chains of 68K and 46K. A subsequent split of the 46K chain yields a smaller product of 43K. All of these chains remain covalently bonded to the β chain of C3b. These initial reactions are the same whether purified components or whether low concentrations of serum as a source of C3b INA and β1H are used. Pre-treatment of C3b, C3b INA and β1H with DFP has no effect on these events.Further protelysis of the 68K. chain requires a DFP-sensitive protease and leads to the formation of an additional high molecular weight breakdown product of C3. This has the same polypeptide chain composition as the high molecular weight breakdown product of C3 purified from aged serum, and is therefore identified as C3c.     

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.