Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4+ T cells

Complement activation is traditionally thought to occur in the extracellular space. However, it has been suggested that complement proteins are activated and function at additional locations. T cells contain intracellular stores of C3 and C5 that can be cleaved into C3a and C5a and bind to intracellular receptors, which have been shown to be of vital importance for the differentiation and function of these cells. However, whether the origin of the complement proteins located within T cells is derived from endogenous produced complement or from an uptake dependent mechanism is unknown.The presence of intracellular C3 in T cells from normal donors was investigated by fluorescence microscopy and flow cytometry. Moreover, mRNA expression levels of several genes encoding for complement proteins with primary focus on C3, C3aR, C5 and C5aR1 during resting state and upon activation of CD4⁺ T cells were investigated by a quantitative PCR technique. Furthermore, the gene expression level was evaluated at different time points.We confirmed the presence of intracellular C3 protein in normal T-cells. However, we could not see any increase in mRNA levels using any activation strategy tested. On the contrary, we observed a slight increase in C3 and C5aR1 mRNA only in the non-activated T-cells compared to the activated T cells, and a decrease in the activated T-cells at different incubation time points.Our results show that there is a baseline intracellular expression of the complement C3, C5, C3aR and C5aR1 genes in normal CD4⁺ T cells, but that expression is not increased during T-cell activation, but rather down regulated. Thus, the pool of intracellular complement in CD4⁺ T cells may either be due to accumulated complement due low-grade expression or arise from the circulation from an uptake dependent mechanism, but these possibilities are not mutually exclusive.     

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis

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Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.     

C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cells

The 155-kDa complement regulator factor H (FH) is the predominant soluble regulatory protein of the complement system. It acts as a cofactor for the factor I-mediated conversion of the component C3b to iC3b, competes with factor B for a binding site on C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. The primary site of synthesis is the liver, i.e. FH-specific mRNA and protein were identified in both hepatocytes (HC) and Kupffer cells (KC). Previous studies in rat primary HC and KC had shown that the proinflammatory cytokine IFN-gamma influences the balance between activation and inhibition of the complement system through up-regulation of the inhibitory FH. In this study we show that C5a, as a product of complement activation, stimulates the expression of FH-specific mRNA and protein in KC and thus induces a negative feedback. Quantitative-competitive RT-PCR showed an approximate threefold C5a-induced up-regulation of FH. ELISA analyses revealed a corresponding increase in FH protein in the supernatants of KC. The up-regulation of FH was completely inhibited by the C5a-blocking monoclonal antibody 6-9F. Furthermore, an involvement of LPS and IFN-gamma was excluded, which strongly indicates a direct effect of C5a on the expression of FH in KC.     

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.     

Cells carrying C5b-9 complement complexes in human atherosclerotic wall

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.     

Distribution of exogenous complement factor H in mice in vivo

Factor H is an important regulator of complement activation in plasma and on cell surfaces in both humans and mice. If FH function is compromised, inappropriate complement activation on self‐surfaces can have disastrous effects as seen in the kidney diseases atypical haemolytic uremic syndrome (aHUS ) and C3 glomerulopathy. As FH constructs have been proposed to be used in treatment for these diseases, we studied the distribution of exogenous FH fragments in mice. Full‐length mFH , mFH 1‐5 and mFH 18‐20 fragments were radiolabelled, and their distribution was examined in WT , FH ^−/− and FH ^−/−C3^−/− mice in vivo. Whole body scintigraphy revealed accumulation of radioactivity in the abdominal part of the mice, but also to the thyroid gland and urinary bladder. At organ level in WT mice, some full‐length FH accumulated in internal organs, but most of it remained in the circulation. Both of the mFH fragments accumulated in the kidneys and were excreted in urine. For mFH 1‐5, urinary secretion is the likely cause for the accumulation. Concentration of mFH 18‐20 to kidneys was slower, and at tissue level, mFH 18‐20 was localized at the proximal tubuli in WT and FH ^−/−C3^−/− mice. No C3‐independent binding to glomeruli was detected. In conclusion, these results show that glomerular glycosaminoglycans and sialic acids alone do not collect FH in kidneys. Deposition of C3 fragments is also needed, which implies that in aHUS , the problem is in simultaneous recognition of C3 fragments and glycosaminoglycans or sialic acids by FH , not just the inability of FH to recognize glomerular endothelium as such.     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. I. Isolation and characterization of factor H: the serum cofactor for the C3b/C4b inactivator (factor I)

Factor H, purified from mouse EDTA-plasma using a 4-step procedure, consists of a single polypeptide chain of Mr 150,000 on SDS-PAGE. Mouse H (Hmo) was required for the cleavage of fluid-phase mouse C3b by mouse I (Imo). The final product of degradation of fluid-phase mouse C3b was iC3b, consisting of fragments of the alpha'-chain (alpha'-70, alpha'-43) linked by disulfide bonds to an intact beta-chain. Imo alone was capable of cleavage of membrane-bound mouse C3b and of generating iC3b. The addition of Hmo nevertheless had an enhancing effect on Imo activity, but cleavage did not proceed beyond iC3b. These observations suggest that one important function of Hmo is to permit the inactivation of fluid-phase C3b, and to inhibit irreversibly its activity. The concentration of H in the plasma of male and female BALB/c mice was not significantly different. Among different inbred strains of mice, large differences were observed in the plasma levels of H, and plasma H levels were positively correlated with the plasma levels of C3. This observation, taken together with the well known role of H in the control of the activation of the alternative pathway, suggests that the turnover of C3 is controlled to some extent by H.     

Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice

Complement activation has a deep pathogenic influence in immunoglobulin (Ig)A nephropathy (IgAN). C3a and C5a, small cleavage fragments generated by complement activation, are key mediators of inflammation. The fragments exert broad proinflammatory effects by binding to specific receptors (C3aR and C5aR, respectively). However, no studies thus far have investigated the effects of C3a, C5a and their receptors on IgAN. We observed that C3aR and C5aR antagonists repressed IgA‐induced cell proliferation and interleukin (IL)‐6 and monocyte chemotactic protein 1 (MCP‐1) production in cultured human mesangial cells (HMCs). Furthermore, an IgAN mouse model induced by Sendai virus infection was employed to investigate the effects of C3aR and C5aR on IgAN in vivo for the first time. Wild‐type (WT) and several knock‐out mouse strains (C3aR^–/– or C5aR^–/–) were immunized intranasally with increasing doses of inactivated virus for 14 weeks and were subjected to two intravenous viral challenges during the time‐period indicated. In the Sendai virus‐induced IgAN model, C3aR/C5aR‐deficient mice had significantly reduced proteinuria, lower renal IgA and C3 deposition, less histological damage and reduced mesangial proliferation compared with WT mice. Both C3aR deficiency and C5aR deficiency, especially C3aR deficiency, inhibited renal tumour necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β, IL‐1β, IL‐6 and MCP‐1 expression significantly. However, C3aR/C5aR‐deficient and WT mice with IgAN did not differ with respect to their blood urea nitrogen (BUN) and serum creatinine levels. Our findings provide further support for the idea that C3aR and C5aR are crucially important in IgAN, and suggest that pharmaceutically targeting C3aR/C5aR may hold promise for the treatment of IgAN.     

Serum properdin consumption as a biomarker of C5 convertase dysregulation in C3 glomerulopathy

Properdin (P) stabilizes the alternative pathway (AP) convertases, being the only known positive regulator of the complement system. In addition, P is a pattern recognition molecule able to initiate directly the AP on non‐self surfaces. Although P deficiencies have long been known to be associated with Neisseria infections and P is often found deposited at sites of AP activation and tissue injury, the potential role of P in the pathogenesis of complement dysregulation‐associated disorders has not been studied extensively. Serum P levels were measured in 49 patients with histological and clinical evidence of C3 glomerulopathy (C3G). Patients were divided into two groups according to the presence or absence of C3 nephritic factor (C3NeF), an autoantibody that stabilizes the AP C3 convertase. The presence of this autoantibody results in a significant reduction in circulating C3 (P < 0·001) and C5 levels (P < 0·05), but does not alter factor B, P and sC5b‐9 levels. Interestingly, in our cohort, serum P levels were low in 17 of the 32 C3NeF‐negative patients. This group exhibited significant reduction of C3 (P < 0·001) and C5 (P < 0·001) and increase of sC5b‐9 (P < 0·001) plasma levels compared to the control group. Also, P consumption was correlated significantly with C3 (r = 0·798, P = 0·0001), C5 (r = 0·806, P < 0·0001), sC5b‐9 (r = ?0·683, P = 0·043) and a higher degree of proteinuria (r = ?0·862, P = 0·013). These results illustrate further the heterogeneity among C3G patients and suggest that P serum levels could be a reliable clinical biomarker to identify patients with underlying surface AP C5 convertase dysregulation.     

补体C5b-9复合物促进树突状细胞成熟

探讨补体C5b-9复合物对树突状细胞成熟及免疫学功能的影响。rhGM-CSF+rhIL-4体外诱导单核细胞分化为不成熟树突状细胞,体外于不成熟树突状细胞表面用补体蛋白纯品组装CSb-9,37℃,5%CO_2温育96 h,流式分析细胞表型及抗原捕获能力;与同种异体CD4~+和CD8~+T细胞共培养检测其免疫刺激功能;ELISA检测细胞因子分泌。结果显示,亚溶解型C5b-9处理DC表面标志CD83、HLA、CD80、CD86、B7-H1、B7-H3、B7-H4以及BTLA等表达上调;DC分泌IL-12及TNF-α上调,抗原捕获能力降低;C5b-9处理DC刺激CD4~+T细胞活化及分泌IFN-γ、IL-2能力增强。结论:补体C5b-9复合物可以促进树突状细胞成熟,衔接天然免疫和特异性免疫。     

Evaluation of serum levels of C3 and C4 complement factors in patients with beta thalassemia major in Khuzestan Province,Southwest Iran

Background and Objectives: Thalassemia syndrome is the most common genetic disorder in the world and infection is the second cause of death in these patients. Measurement of serum C3 and C4 complement factors in serum was done in 60 patients with beta thalassemia major in comparison with 30 healthy subjects as control group.Materials and Methods: The serum level of C3 and C4 complement factors in 60 patients with beta thalassemia major who were randomly selected from among the patients referred to Shafa Hospital of Ahvaz was evaluated and compared with 30 samples from healthy individuals with no history of recent infectious or autoimmune diseases. It should be noted that single-radial-immunodiffusion assay was used in this study.Results: This study has shown a significant reduction in serum levels of C3 and C4 in patients compared to controls (P value < 0.05).Discussion: Decreased synthesis or increased consumption of complement factors in patients receiving multiple blood transfusions might lead to continuous contact between the immune system and various antigens, causing nonstop use of complement factors, recurrent infections, changes in parameters of the immune system due to iron overload as well as exposure to infectious factors such as HBV, HCV, HIV, and HTLV through blood transfusion.     

Localization of the terminal C5b-9 complement complex in the human aortic atherosclerotic wall

The terminal C5b-9 complement complex was investigated in 15 aortic, 2 femoral fibrous plaques and 5 fatty streaks aortic intimae using indirect immunofluorescence and immunoperoxidase. All the fibrous plaques presented C5b-9 deposit-like threads in the fibrous cap and masses in the amorphous areas of the plaque. The deposits were frequently associated with other immune-related proteins such as: IgG, IgA, IgM, Clq, C3c and C4 which were simultaneously investigated. Fatty streaks intimae presented no C5b-9 and complement component deposits. Whereas the demonstration of the complement components could merely reflect a non-specific trapping, the presence of assembled C5b-9 in the damaged tissue is more indicative of the involvement of complement activation in the progression of atherosclerotic lesions.     

Purification of human C5a des arg by immunoadsorbent and molecular sieve chromatography

Human C5a des arg was isolated from complement-activated serum by immunoadsorption followed by Sephadex G-75 chromatography. C5a des arg obtained by this 2-step procedure was shown to be immunologically identical to C5a des arg purified by a conventional multi-step method, homogeneous on SDS-polyacrylamide gels, and biologically active. Although this technique yields approximately the same amount of C5a des arg/liter of activated serum as that obtained by conventional methods, its simplicity and relative rapidity make it a practical alternative.     

Complement C3-targeted therapy in C3 glomerulopathy,a prototype of complement-mediated kidney diseases

C3 glomerulopathy (C3G) is a rare and complex kidney disease that primarily affects young adults. Renal outcomes remain poor in the absence of specific treatment. C3G is driven by uncontrolled overactivation of the alternative complement pathway, which is mainly of acquired origin. Functional characterization of complement abnormalities (i.e., autoantibodies targeting complement components and variants in complement genes) identified in patients and experimental models of the disease improved the understanding of the disease, making C3G a prototype of complement-mediated diseases. The contribution of C3 convertase, as well as C5 convertase, in disease occurrence, phenotype, and severity is now well established, offering various potential therapeutic interventions. However, the lack of sufficient efficiency in anti-C5 therapy highlights the extreme complexity of the disease and the need for new therapeutic approaches based on C3 and C3 convertase axis inhibition. Here, we provide an overview of the complement activation mechanism involved in C3G and discuss therapeutic options based on complement inhibitors, with a specific focus on C3 inhibition.     

Influence of time,temperature and coagulation on the measurement of C3, C3 split products and C4

Quantitative and qualitative immunoelectrophoretic analyses of circulating C3, C3 split products and C4 were performed in matched EDTA plasma and serum obtained from 5 normal subjects and stored for up to 48 h at room temperature (18°C–22°C) and 4°C. Fluctuations in apparent levels of C3 were greater in serum than plasma stored at room temperature, a fall in levels seen by 24 h being followed by a significant increase. By contrast, levels of C3 did not alter if stored at 4°C. C4 levels in both EDTA plasma and serum remained unchanged for 24 h, a slight decrease being seen at 48 h. Levels of C4 remained constant if samples were stored at 4°C. Crossed immunoelectrophoresis revealed a significant progressive decrease in C3 levels and a simultaneous increase in C3c occuring after 4 h in serum and 8 h in EDTA plasma, stored at room temperature. In studies conducted at 4°C, similar but delayed fluctuations were seen. A progressive and significant increase in C3d levels was seen in both plasma and serum samples stored at room temperature, levels rising to 276% (plasma) and 308% (serum) of levels seen at zero time. At 4°C marginal increases in C3d levels only were observed. These results suggests that in vitro degradation of C3 and C4 are readily facilitated by temperature, time and coagulation, and that conditions of collection and storage of samples must be optimized for the accurate definition of activation of the complement cascade.     

Genetic detection of the silent allele (*Q0) in hereditary deficiencies of the human complement C6, C7, and C9 components

DNA polymorphisms (RFLPs) of the human complement component C6, C7, and C9 genes were studied in three C7-deficient (C7D) families, one C6-deficient (C6D) family, and one C9-deficient (C9D) family. The 3 loci are closely linked on human chromosome 5. The haplotypes carrying the “silent” allele (C7*Q0, C6*Q0, and C9*Q0) were defined in each family, allowing for the detection of carriers among asymptomatic relatives. This paper describes familial studies on a type of hereditary trait, characterized by recurrent Neisseria infections in individuals homozygous for “silent” alleles at the C6, C7, or C9 loci. © 1995 Wiley-Liss, Inc.     

Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease)

Introduction

Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology.

Subjects

Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded.

Results

Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls.

Conclusion

We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.     

Isolation and characterization of the C3b-binding entity of C3b-receptor from human erythrocytes

Applying 2 M KBr, membranes of E^hu were solubilized. By C3-affinity chromatography an activity could be isolated that inhibited the immune adherence reaction and C3b-dependent rosette formation. Since this material did not agglutinate EAC14^oxy 23b we termed it monovalent C3b receptor (mC3bR). PAGE and SDS-PAGE and staining with Coomassie brillant blue and PAS reagent revealed a single glycoprotein band with a mol. wt. of 55,000–60,000 daltons and an electrophoretic mobility comparable to ovalbumin. This mC3bR proved to be antigenetically related to gp 205 [17]. The potential of mC3bR to react with C3b-carrying particles was not destroyed by heat and trypsin treatment but by neuraminidase or periodic acid treatment suggesting that mC3bR reacted by its carbohydrate moiety with C3b. As by mC3bR, immune adherence could be inhibited by D-glucose and D-galactose but not by their optical antipodes, L-glucose and L-galactose.