C3 nephritic factor protects bound C3bBb from cleavage by factor I and human erythrocytes

C3 nephritic factor is an autoantibody to the alternative-pathway C3 convertase (C3bBb) which increases the half-life of the convertase both in the presence and absence of serum regulatory proteins. Human erythrocytes contain membrane proteins which also can regulate C3bBb. One of these proteins, the C3b/C4b receptor (CR1), plays an important role in the processing of soluble immune complexes. C3b which is fixed to immune complexes binds to CR1 and is cleaved by factor I to C3c and C3dg. We have tested the effectiveness of the nephritic factor in protecting bound C3b from cleavage by factor I and human erythrocytes. Sheep erythrocyte intermediates EAC1423b were prepared using 125I-labeled C3 and incubated with factors B and D in the presence and absence of nephritic factor. Breakdown of C3b was measured by release of 125I-C3c following incubation with human erythrocytes and factor I. Purified IgG from two patients with nephritic factor prevented C3c release in a dose-dependent manner. Normal human IgG was ineffective as was nephritic factor in the absence of factor B. Factor P also inhibited the release of C3c in the presence of factor B with equivalent activity at approx. 20-fold higher concns than nephritic factor. These results indicate that nephritic factor can impair human erythrocyte dependent degradation of C3b in alternative-pathway-activating immune complexes.     

Consumption of C4b-binding protein (C4BP) during in vivo activation of the classical complement pathway.

C4BP has a central role in regulating the classical complement (C') pathway, but it is still uncertain whether or not it is consumed during in vivo complement activation. Attempts to demonstrate changes in C4BP plasma levels in systemic lupus erythematosus and essential mixed cryoglobulinaemia have failed, probably due to up-regulation of this protein during the inflammatory reaction. We have studied one patient with severe post-transfusion complement-mediated anaphylaxis (CMA), and 67 patients with hereditary C1 inhibitor deficiency (hereditary angioedema (HAE)). The first of these two conditions is characterized by the absence of systemic inflammatory reaction and the second by acute and chronic activation of the C' classical pathway. C4BP, C4BP-C4b complex, and soluble terminal C' complex (sC5b-9) were measured in the patients' plasmas by ELISA techniques and C3a and C4a by radioimmunoassays. In CMA, 15 min after the transfusion, there was a massive C' activation, with increases in C4a, C3a, sC5b-9, C4BP-C4b complexes and decreases in C4, C3 and C4BP. All parameters reverted to preinfusion values within 24 h. Depletion of C4 was correlated with that of C4BP. In patients with HAE, the median value of C4BP (83% range 54-165) was significantly lower (P < 0.0001) than in normal controls (99% range 70-159), with no difference between patients in remission or during acute attacks. C4BP-C4b complexes could not be detected in HAE patients. The results of this study indicate that C4BP is consumed in vivo during acute, and possibly during chronic activation of the C' classical pathway, and that this protein, after interaction with C4b, not longer circulates in plasma.     

Analysis of human C4A and C4B binding to an immune complex in serum.

Previous studies using isolated complement proteins have shown that more C4A than C4B binds to certain types of immune complexes. However, the in vivo binding of the C4 isoforms to an immune complex has not been investigated in detail and may differ from events when measured with the isolated proteins. We report here the binding of C4A and C4B to an immune complex of bovine serum albumin (BSA) anti-BSA as it occurs in serum. We found that when using the isolated C4 proteins more C4A than C4B bound to the complex, but in serum similar amounts of C4A and C4B were found to bind. Furthermore, these results were not explainable by a difference in activity between isoforms. In an attempt to explain these results a number of unexpected observations were noted. First C4A, but not C4B, bound specifically to a yet unidentified 38-kD serum protein. Second, when both covalent and non-covalent binding was assessed, we found that as serum concentration increased there followed a concomitant decrease in covalent binding and C4B was more affected than C4A. The potential biological significance of these findings is discussed.     

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.     

Importance of factors H and I for the adherence of C3b-coated erythrocytes to cells

The role of cell membrane-associated human factor H for the binding of cell-bound C3b to complement receptor-carrying (CR+) cells was investigated. Pretreatment of CR+ cells with antibodies to factor H inhibited the adherence of C3b-coated red cells to human tonsil lymphocytes (TL) and peripheral blood monocytes (M phi). The C3b receptor reactivity of human polymorphonuclear leucocytes (PMN) was not influenced and the one of Raji lymphoblastoid cells only slightly influenced; iC3b and C3d receptor reactivity was in no case affected. When diisopropylfluorophosphate (DFP) in a concentration of 0.1 mM was present during pretreatment of the CR+ cells with anti H, the antibodies gained the capacity to inhibit the adherence of C3b-coated erythrocytes to Raji cells; this effect was dose-dependent with respect to DFP. In contrast, there was no influence of DFP on the inhibition pattern of anti H in the case of TL and M phi. The adherence of C3b-coated erythrocytes to PMN remained unaffected by anti-H antibodies in the presence of DFP. Polyclonal as well as monoclonal antibodies directed against human factor I inhibited the binding of C3b cells to Raji cells but not to TL. Additionally, when anti I and anti H antibodies were both present, C3b receptor reactivity of Raji cells was inhibited to a larger extent than with either antibody alone; again, TL remained unaffected. Results obtained by washing the Raji cells before and after treatment with anti H and anti I suggest that the respective antibodies act on factor H primarily on the level of the cell membrane and on factor I in the fluid phase.     

C3 and C4 gene expression and interferon-gamma-mediated regulation in human glomerular mesangial cells.

The glomerular mesangial cell (GMC) plays a key role in the maintenance of glomerular structure and function and in the mediation of glomerular injury. To explore the potential of this cell to produce complement and react to local inflammatory signals, we studied the synthesis and regulation of the third and fourth components of complement in cultured human GMC. Using metabolic labelling and immunoprecipitation, we found that C3 and C4 polypeptide chains were synthesized and secreted by GMC. Interferon-gamma (IFN-gamma) led to an increase in C4 protein synthesis, but not C3 synthesis. There was a corresponding increase in C4 mRNA in IFN-gamma-activated cells, but no increase in C3 mRNA, as determined by semi-quantitative polymerase chain reaction (PCR) estimation. These results demonstrate that human GMC can synthesize C3 and C4 proteins, and that regulation of expression of the C4 gene is mediated by IFN-gamma. We hypothesize that GMC production of complement could influence the clearance of immune aggregates by the kidney and the mediation of glomerular injury.     

Allelic frequencies of HPA‐1 to 5 human platelet antigens in patients infected with hepatitis C virus

Studies have suggested that hepatitis C virus (HCV) may infect not only hepatocytes but may also be carried by platelets. Platelets express more than 20 polymorphic antigenic determinants on their surface, which are called human platelet antigens (HPA). To determine the allele frequency of the HPA‐1 to ‐5 in patients infected with HCV, blood samples were collected from 257 blood donors for the control group and from 191 patients infected with HCV. DNA was isolated and amplified for genes HPA‐1 to ‐4 using PCR Sequence Specific Primers (PCR‐SSP) and HPA‐5 using PCR‐Restriction Fragment Length Polymorphism (PCR‐RFLP). The allelic and genotypic frequency of HPA‐5a in patients infected with HCV was found to be significantly lower (P < 0.05) than in the controls, and HPA‐5b from patients infected with HCV was significantly higher (P < 0.05) than in controls. The increase in HPA‐5b allelic frequency in HCV infection may indicate a possible association between HCV infection and HPAs. J. Med. Virol. 81:757–759, 2009 © 2009 Wiley‐Liss, Inc.     

局灶节段性肾小球硬化症C4d表达的特点及意义

目的研究不同类型的原发性局灶节段性肾小球硬化症（FSGS）中C4d表达的特点及意义。方法采用免疫组化染色方法,并结合PASM—C4d套染和免疫组化双标染色方法,观察了97例FSGS中C4d在肾小球上沉积部位和强度,及其与病理类型之间的关系。结果88例（90．7％）FSGS的肾小球C4d阳性表达,C4d主要沉积在FSGS肾小球硬化病变处。细胞型、顶端型、塌陷型及非特殊型FSGS中均表现为硬化病变处血管袢C4d不均匀粗线状沉积,系膜区散在多少不等的颗粒状沉积;顶端型和非特殊型为节段沉积,细胞型和塌陷型为球性沉积。门部型FSGS主要在门部有C4d颗粒状沉积,未见血管袢沉积。结论补体经典途径激活参与了FSGS的发病机制;硬化病变处血管袢C4d不均匀粗线状沉积是细胞型、塌陷型、非特殊型和顶端型FSGS重要的免疫病理形态特点之一。     

Association of human platelet antigens polymorphisms with susceptibility to hepatitis C virus infection in Chinese population

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. Previous studies have identified a number of single nucleotide polymorphisms that are associated with HCV infection. Human platelet antigens (HPAs) polymorphisms play an important role in several diseases. Here, we demonstrated the association of the HPA‐2, HPA‐3, HPA‐5 and HPA‐15 polymorphisms with susceptibility to HCV infection in Chinese population. Overall, 118 patients with HCV and 167 controls were genotyped for HPAs. There were no significant differences in the allele and genotype frequency distribution for the HPA‐3, HPA‐5 and HPA‐15 systems between the patients with chronic HCV infection and the healthy controls (p > .05). However, the genotype frequency of HPA‐2aa was significantly lower, while HPA‐2ab/bb was significantly higher in patients than that in the controls (p = .006). The allele frequency of HPA‐2a in patients was significantly lower than that in the control group (p = .005). In contrast, HPA‐2b in patients was significantly higher than that in the control group (p = .005). We conclude that HPA‐2 polymorphism is associated with susceptibility to HCV infection, and individuals carrying the HPA‐2b allele may have a higher risk of HCV infection compared with individuals carrying HPA‐2a.     

Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies

Detection of peritubular capillary (PTC) C4d deposition in tissue sections of renal allograft biopsies became an important aid in the diagnosis of antibody-mediated rejection. Pathologists in many major transplant centers now routinely stain renal allograft biopsies for C4d. Currently, there are 3 commercially available antibodies. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections. A polyclonal antibody is used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system. The goal of our study was to compare these antibodies and methodologies in our renal allograft biopsy material. Twenty renal allograft biopsies with diffuse or focal PTC C4d staining, using immunofluorescence methods on frozen sections, were selected for this study. These biopsies were tested with the 3 commercially available anti-C4d antibodies (Biogenesis, Brentwood, Calif, cat no. 222-8004; Quidel Corporation, Santa Clara, Calif, cat no. A213; and ALPCO Diagnostic, Windham, NH, cat no. 004-BI-RC4D). Both monoclonal antibodies (Biogenesis and Quidel) were tested with a 3- and a 2-step indirect IF method on frozen sections. The polyclonal antibody (ALPCO) was applied to formalin-fixed paraffin sections using immunoperoxidase methodology. In selected cases, the polyclonal antibody was tested on frozen sections with a 3-step indirect IF method. To exclude possible false-negative staining with the IF method, we selected 10 additional biopsies that showed PTC margination of inflammatory cells, but were C4d-negative or only focally positive, and tested them with the ALPCO antibody on paraffin sections. We have found that all methodologies and antibodies tested provided adequate results with only minor differences between them. Perhaps the most sensitive method is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. We prefer the 2-step indirect IF method with the Quidel monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost. The advantages and disadvantages of the individual methodologies are discussed.     

Different HLA antigen associations for the functionally active and inactive products of the complement C4F1 allele

The genetic polymorphism of the fourth component of human complement. C4, was studied in 945 unrelated Caucasian individuals. A third allele of the C4F (Rodgers) locus, termed C4F₁ was demonstrated. This allele is characterized using immunofixation electrophoresis, by the presence of an additional fast-moving anodal band of C4 which distinguishes it clearly from the common C4F variant. The allelic frequencies fit the Hardy-Weinberg equilibrium assuming three alleles at the C4F locus: C4F, C4f⁰, and C4F₁. The functional activity of the C4F variants was investigated using a specific hemolytic overlay technique for C4. It was found that in almost all individuals (75 out of 78), the C4F₁ allele codes for a functionally inactive C4 product only when it occurs on an HLA-B17 positive haplotype but that the same allele codes for a functionally active fast variant of C4 when it occurs on an HLA-B37 positive haplotype (18 out of 18). Very strong genetic linkage disequilibrium was observed for the C4F₁ allele with HLA-B17 and B37. The active and inactive C4F₁ variant also has marked nonrandom gametic association to different alleles of the of locus and to HLA-C locus determinants. No further variants of the C4S (Chido) locus have been identified so far. Rodgers (Rg) typing by the plasma inhibition test of anti-Rg antiserum has shown that plasma from individuals homozygous for the C4F₁ allele is only able to partially inhibit anti-Rg whereas all C4F positive individuals totally inhibited the reaction.     

High prevalence of hepatitis C virus subtypes 4c and 4d in Malaga (Spain): phylogenetic and epidemiological analyses

Hepatitis C virus (HCV) is a major aetiological agent of chronic hepatitis and it may lead to the development of liver cirrhosis and hepatocellular carcinoma. HCV has been classified into six clades as a result of high genetic variability. A commercial procedure to genotype HCV in 678 patients from Carlos Haya Regional University Hospital, Malaga was used to study the distribution of HCV genotypes in Malaga, southern Spain. A high prevalence of HCV-4 (10.2%) was found. This genotype is found more commonly in Egypt, Central Africa and the Middle East. The distribution of the different subtypes in the 69 patients with HCV-4 was as follows: 4.3% subtype 4e, 7.2% subtype 4a, 11.5% not subtypable, and 76.8% subtype 4c/4d. Of the 53 4c/4d patients, 69% were intravenous drug users and 31% non-intravenous drug users. In order to characterise further the HCV-4c/4d patients, sequences of the non-structural 5B gene (393 bp) were obtained from 36 HCV-4c/4d-infected untreated patients. Phylogenetic tree topologies distinguished clearly the two subtypes: 11 patients were infected by subtype 4c and 25 by 4d. This phylogenetic analysis, reinforced by the epidemiological characteristics, suggests the extension of the HCV-4c and -4d subtypes in the area of Malaga among both intravenous drug users and non-intravenous drug users.     

Conglutination and haemolysis of unsensitized human erythrocytes by bovine serum complement

Unsensitized human erythrocytes (E) were haemolyzed by bovine serum to a titre of 1:16-1:32. In the single dilution beyond the haemolytic endpoint, the cells were conglutinated. In dilutions in which haemolysis occurred, cells were conglutinated before being lyzed. With no or minimal haemolysis, conglutination to 1:32-1:64 occurred in tests using insulin-absorbed serum, serum heated at 50 degrees C for 30 min and in tests incubated at 4 degrees C for 30 min. In two-stage tests, EDTA and Mg2+-EGTA prevented bovine C sensitization of human E for conglutination by bovine serum heated at 56 degrees C for 30 min. EDTA prevented haemolysis, but haemolysis to 1:16-1:32 occurred with serum dilutions containing Mg2+-EGTA. Haemolytic activity was restored to serum heated at 50 degrees C by a factor B-containing fraction. Conglutination and haemolysis were blocked by heating serum at 56 degrees C for 30 min and were reduced to low titres by absorbing serum with zymosan. These results strongly suggest that the conglutination reaction involved the classical activation pathway whereas the haemolytic reaction involved the alternative activation pathway. Thus, with dilutions of untreated or treated bovine serum, two C-dependent reactions and the pathways involved can be demonstrated by using unsensitized human E as an indicator system.     

Yersinia pestis Ail recruitment of C4b‐binding protein leads to factor I‐mediated inactivation of covalently and noncovalently bound C4b

The outer membrane protein Ail of Yersinia pestis mediates several virulence functions, including serum resistance. Here, we demonstrate that Ail binds C4b‐binding protein (C4BP), the primary fluid‐phase regulator of the classical and lectin pathways. Non‐covalent binding of C4 and C4b to Ail was also observed. C4BP bound to Ail can act as a cofactor to the serine protease factor I (fI) in the cleavage of fluid‐phase C4b. Employing a panel of C4BP alpha‐chain mutants, we observed that the absence of complement control protein domain 6 and 8 reduced binding to Ail. Immunoblot analysis of normal human serum (NHS)‐treated bacteria revealed minimal C4b alpha’‐chain complexes with bacterial outer membrane targets. Addition of the anti‐C4BP monoclonal antibody MK104 to NHS restored C4b‐alpha’ chain target complexes, suggesting that C4b binds covalently to targets on the Y. pestis surface. C4b bound to Ail noncovalently was also cleaved in a C4BP and fI‐dependent manner, leaving the C4c fragment bound to Ail. MK104 also prevented the cleavage of noncovalently bound C4b. Collectively, these data suggest that when C4BP is bound to Ail, fI can cleave and inactivate C4b that has bound covalently to bacterial surface structures as well as C4b bound noncovalently to Ail.     

Effects of iodipamide on human C3 and factor B in vitro

The effects of iodipamide on C3 and factor B in normal human serum and in purified form have been examined by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Temperature-dependent changes in immunoelectrophoretic profile's have been observed; however, these are not the same as those obtained after treatment of normal human serum (NHS) with cobra venom factor Naja naja. Analyses of iodipamide-treated NHS and purified C3 and factor B by reducing SDS-PAGE indicate that no macromolecular changes have occurred in C3 and factor B that can be ascribed to proteolysis (i.e., activation). The changes observed in C3 and factor B, including loss of hemolytic activity, appear to be due to direct interactions between iodipamide and C3 and factor B. In the case of factor B, iodipamide treatment at 37° C induces aggregation, which is reversible upon reduction with β-mercaptoethanol.     

Binding and activation of C1 by cell bound IgG: Activation depends on cell surface hapten density

We have investigated the binding and activation of C1 by IgG-anti methotrexate antibody at cell surfaces. Under conditions where variation in cell surface hapten density had no effect on binding of IgG. the number of C1 (or its active form, C1) bound by the IgG was independent of hapten density. The ability of the C1 binding IgG complex to activate C1, however, was decreased with decreasing density of the hapten. The decreased ability to activate bound C1 was paralelled by decreased ability to activate the hemolytic sequence in whole complement. The results were interpreted to mean that binding of C1 was the result of aggregation (doublet formation) by IgG while activation of the bound C1 depended on changes induced in the IgG molecule by straddling hapten molecules at varying distances.     

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.     

Significance of C4d deposition in the follicular lymphoma and MALT lymphoma and their relationship with follicular dendritic cells

We evaluated the deposition of C4d in follicular lymphomas (FL) and extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma). Deposition of C4d was detected in 118 lymphoma tissues from patients with lymphoma and in 20 reactive hyperplasia lymphadens (RHL) using immunohistochemistical methods. FL, MALT lymphoma, and RHL were studied using double staining for CD35/C4d and Bcl-2/C4d. We studied 26 FL tissues, 19 of which showed C4d deposition. C4d deposition was detected around the follicular dendritic cells (FDCs) in the neoplastic follicles. There was no significant difference between the positive ratio of C4d and the grades of FL. We studied 12 MALT lymphoma tissues, six of which displayed C4d deposition. In these tissues, C4d deposition was detected in the peripheral region of partially colonized follicles in the form of an irregular ring, but was not found in the central region. C4d deposition was negative in completely colonized follicles. There was no C4d deposition in diffuse large B-cell lymphomas, mantle cell lymphomas, B-small lymphocytic lymphomas, T-lymphoblastic lymphomas, peripheral T-cell lymphomas, and anaplastic large cell lymphomas. C4d around the FDCs in the neoplastic follicles was a specific indicator for FL. C4d deposition in partially colonized follicles of MALT lymphoma was completely different from that in neoplastic follicles of FL, forming a key point for differential diagnosis.     

肾小球C4d沉积在慢性肾病早期诊断及预后的价值分析

目的 分析肾小球沉积C4d在慢性肾病中的价值,为临床提供可靠依据。方法 本次研究选自2014年1月～2017年1月医院肾内科肾脏病理检验患者194例,其中慢性肾脏病86例,急性肾损伤性疾病108例,均采用免疫组织化学检验肾小球C4d,进行沉积分型,慢性肾脏病对象划分为有效组（8周内随访蛋白尿得到控制）、对照组。对比慢性肾脏病、急性肾损伤患者肾小球C4d沉积情况,分析慢性肾脏病在C4d沉积表达中是否存在独立作用。分析肾小球C4d沉积是否在慢性病治疗中起到作用。结果 慢性肾脏病肾小球C4d阳性率、毛细血管袢区沉积伴系膜区沉积比重高于急性肾脏病对象,差异有统计学意义（P＜0.05）。肾小球毛细血管袢区C4d沉积成为慢性肾脏病疾病未获得控制的独立影响因素（P＜0.05）。结论 肾小球C4d沉积在早期诊断及判断预后具有价值,肾小球C4d毛细血管袢区沉积伴系膜区沉积慢性肾脏病风险较高,同时在疗效预测上有更高的价值。