C1q,a subunit of the first component of complement,enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37°C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37°C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 ± 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 ± 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 μg/ml of C1q significantly increased GMC-apoptosis up to 39.4 ± 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab′)₂-ER4G (F(ab′)₂-ER4G-GMC), while ER14-GMC or F(ab′)₂-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse κ-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.     

Spontaneous B cell hyperactivity in autoimmune-prone MRL mice

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.     

Defective prevention of immune precipitation in autoimmune diseases is independent of C4A*Q0

Increased prevalence of C4 null alleles is a common feature of autoimmune diseases. We have shown previously that complement-dependent prevention of immune precipitation (PIP) is defective in patients with systemic lupus erythematosus (SLE), and correlated this defect with C4A*Q0 and low levels of the C4A isotype. To further clarify the role of C4A in the aetiology of SLE, we now extend our studies to other diseases which have been associated with C4A*Q0. The frequency of C4A*Q0 was increased in Icelandic patients with coeliac disease (0.50; P < 0.001), Grave's disease (0.30; P = 0.002) and insulin-dependent diabetes mellitus (0.23; P = 0.04) and in British patients with dermatitis herpetiformis (0.42; P = 0.002) and this was reflected in low levels of C4A. In spite of this, PIP was normal in these patients, and in marked contrast to our previous observations on connective tissue diseases, PIP measurements in these patient groups correlated more strongly with levels of C4B (r = 0.51, P = 0.0000004) than C4A. Patients with increased levels of anti-C1q antibodies had significantly lower PIP than patients without such antibodies (P < 0.01) and a negative association of PIP with anti-C1q antibodies was also reflected in an increased prevalence (P = 0.006) and levels (P = 0.006) of anti-C1q antibodies in patients with subnormal PIP, as well as a negative correlation between PIP and anti-C1q antibodies (r = - 0.25, P = 0.02). These results show that the PIP defect cannot be explained by low levels of C4A alone and suggest that measurements of anti-C1q antibodies may be useful in future studies on the molecular cause of the PIP defect in autoimmune connective tissue disease.     

Antibody-mediated demyelination in experimental allergic encephalomyelitis is independent of complement membrane attack complex formation.

The effects of decomplementation by cobra venom factor (CVF) on the pathogenesis of inflammation and demyelination in experimental allergic encephalomyelitis (EAE) and acute antibody-mediated demyelinating EAE (ADEAE) have been quantified histologically and immunocytochemically. In rats immunized with 50 micrograms of myelin basic protein in Freund's complete adjuvant containing 100 micrograms heat-killed Mycobacterium tuberculosis H37Ra, clinical signs of EAE were completely suppressed by two injections of CVF given 9 and 12 days post-immunization. Suppression of clinical disease was associated with a dramatic reduction in peri-vascular inflammation in the CNS, although immunohistochemical staining identified small numbers of infiltrating T cells and macrophages. In contrast, CVF treatment had no significant effect on the clinical severity of ADEAE and although C9 deposition within the CNS was virtually abolished, there was no statistically significant decrease in the extent of demyelination or inflammation. These observations indicate that in the absence of complement components C3 and C5 an antibody-dependent cell-mediated cytotoxic response plays an important role in the pathogenesis of antibody-mediated demyelination. The major role of the complement cascade in EAE appears to be the generation of pro-inflammatory factors that enhance the inflammatory response within the CNS in animals facing a mild encephalitogenic challenge.     

Activation-induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals

T cell apoptosis has been proposed as an Important contributorto the functional defects and depletion of T cells in HIV-lnfectedIndividuals. However, the mechanisms Involved in this apoptosishave not been elucidated. We recently showed that peripheralblood T cells from HIV-infected individuals are especially susceptibleto Fas antigen-induced apoptosis. In this study we examine therole of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR)and CD30, receptors known to be involved In T cell activation-inducedcell death (AICD), in the spontaneous and activation (anti-CD3)-lnducedapoptosis of peripheral blood T cells from asymptomatic HIV-infectedindividuals. We report here that spontaneous and activation-InducedT cell apoptosis cannot be inhibited by reagents that blockinteractions of Fas, CTLA-4, p55 and p75 TNFR and CD30 withtheir respective ligands. We also show that IL-12, IFN-, IL-4and IL-10 cannot modify spontaneous, activation- and anti-Fas-inducedapoptosis. Anti-Fas preferentially Induced CD4⁺ T cell apoptosiswhereas AICD induced apoptosis equally In CD4⁺ and CD8⁺ T cells.We conclude that T cell AICD In HIV infection Is not mediatedby Fas, thus Indicating that Fas-Induced and activation-InducedT cell apoptosis are independent mechanisms of apoptosis whichmay play different roles in the pathogenesls of HIV infection.     

Antigen-induced T cell death is regulated by CD4 expression

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.     

Complement classical pathway components are all important in clearance of apoptotic and secondary necrotic cells

Inherited deficiencies in components of the classical complement pathway are strong disease susceptibility factors for the development of systemic lupus erythematosus (SLE) and there is a hierarchy among deficiency states, the strongest association being with C1q deficiency. We investigated the relative importance of the different complement pathways regarding clearance of apoptotic cells. Phagocytosis of labelled apoptotic Jurkat cells by monocyte‐derived macrophages in the presence of sera from individuals with complement deficiencies was studied, as well as C3 deposition on apoptotic cells using flow cytometry. Sera from individuals deficient in C1q, C4, C2 or C3 all showed decreased phagocytosis. Mannose binding lectin (MBL) and the alternative pathway did not influence phagocytosis. Notably, the components of the complement classical pathway, including C1q, were equally important in clearance of apoptotic cells. This indicates that deposition of C3 fragments is of major significance; we therefore studied C3 deposition on apoptotic cells. Experiments with MBL‐deficient serum depleted of C1q or factor D confirmed the predominance of the classical pathway. At low dilution, sera deficient of C1q, C4 or C2 supported C3 fragment deposition demonstrating alternative pathway activation. In conclusion, we have found that complement‐mediated opsonization and phagocytosis of apoptotic cells, particularly those undergoing secondary necrosis, are dependent mainly upon an intact classical pathway. The alternative pathway is less important, but may play a role in some conditions. C1q was not more important than other classical pathway components, suggesting a role in additional pathogenetic processes in SLE other than clearance of apoptotic cells.     

Immunoglobulins and complement factor C4 in adult rhinosinusitis

We assessed whether complement and its factor C4 or abnormal immunoglobulin levels are associated with chronic or recurrent rhinosinusitis. We used multiple patient and control groups to obtain clinically meaningful data. Adult chronic or recurrent rhinosinusitis and acute purulent rhinosinusitis patients were compared with unselected adults and controls without previous rhinosinusitis. Associated clinical factors were reviewed. Levels of immunoglobulins, plasma C3, C4 and classical pathway haemolytic activity were analysed. C4 immunophenotyping was used to detect C4A and C4B deficiencies as null alleles. Complement was up-regulated in rhinosinusitis. C4A nulls and low IgA, IgG, IgG1, IgG2, IgG3 and IgG4 levels were all more common in chronic or recurrent rhinosinusitis patients than in unselected and healthy controls. We searched for relevant differences between the patient groups. According to stepwise logistic regression analysis, nasal polyposis [odds ratio (OR) 10.64, 95% confidence interval (CI) 2.5-45.7, P = 0.001], bronchial asthma (OR 8.87, 95% CI 2.3-34.9, P = 0.002), C4A null alleles (OR 5.84, 95% CI 1.4-24.9, P = 0.017) and low levels of IgG4 together with either IgG1 or IgG2 (OR 15.25, 95% CI 1.4-166.8, P = 0.026) were more common in chronic or recurrent rhinosinusitis than in acute rhinosinusitis patients. Isolated low IgG subclasses had limited value in patient assessment. C4A null alleles are associated with chronic or recurrent rhinosinusitis, potentially through their effect on immune defence and inflammation control. Multiple clinical and immunological parameters may need to be evaluated when searching for prognostic variables.     

A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site,is a selective inhibitor of the alternative complement pathway

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b α chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.     

Consumption of erythrocyte CR1 (CD35) is associated with protection against systemic lupus erythematosus renal flare

Erythrocyte complement receptor type one (E-CR1) is thought to protect against immune complex (IC) disease through interactions that lead to E-CR1 consumption, and low E-CR1 levels are characteristic of systemic lupus erythematosus (SLE). The purpose of this study was to test the hypothesis that E-CR1 consumption can predict or mark SLE flare. Recurrently active SLE patients [n = 43; 28 with past or present major renal manifestations (SLER) and 15 without (SLENR)], were evaluated every 2 months by detailed protocol testing (mean follow-up 22 months), including direct measurements of E-CR1 levels using a radioimmunoassay. In all patients, detectable E-CR1 levels fluctuated widely through acute periods of consumption and regeneration, preventing the use of any single value as a baseline. However, when individual chronic baseline values were used, determined as the mean of all E-CR1 values 4 months or more from a flare, a clear trend was observed. In 16 of 16 instances of non-renal flare in SLER patients, E-CR1 levels decreased at flare (mean decrease 34%, P < 0.0001). In contrast, no consistent difference was observed for flare in SLENR patients or for renal flare in SLER patients. Changes in E-CR1 levels did not correlate with plasma CR1 levels. In conclusion, single occurrences of E-CR1 consumption did not generally predict or mark SLE flare. However, compared to the average E-CR1 levels measured during no-flare intervals, E-CR1 consumption in SLER patients at flare was strongly associated with freedom from signs of renal involvement. We postulate that E-CR1 consumption reflects E-CR1 function that includes protecting against SLE nephritis.     

Assessment of complement C4 gene copy number using the paralog ratio test

The complement C4 locus is in the class III region of the MHC, and exhibits copy number variation. Complement C4 null alleles have shown association with a number of diseases including systemic lupus erythematosus (SLE). However, most studies to date have used protein immunophenotyping and not direct interrogation of the genome to determine C4 null allele status. Moreover, a lack of accurate C4 gene copy number (GCN) estimation and tight linkage disequilibrium across the disease‐associated MHC haplotypes has confounded attempts to establish whether or not these associations are causal. We have therefore developed a high throughput paralog ratio test (PRT) in association with two restriction enzyme digest variant ratio tests (REDVRs) to determine total C4 GCN, C4A GCN, and C4B GCN. In the densely genotyped CEU cohort we show that this method is accurate and reproducible when compared to gold standard Southern blot copy number estimation with a discrepancy rate of 9%. We find a broad range of C4 GCNs in the CEU and the 1958 British Birth Cohort populations under study. In addition, SNP‐C4 CNV analyses show only moderate levels of correlation and therefore do not support the use of SNP genotypes as proxies for complement C4 GCN. Hum Mutat 31:1–9, 2010. © 2010 Wiley‐Liss, Inc.     

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.     

HLA typing of cultured amniotic cells for the prenatal diagnosis of complement C4 deficiency

It has recently been reported that HLA typing can be used for the prenatal diagnosis of the HLA-linked monogenetic disease, congenital adrenal hyperplasia (21-hydroxylase deficiency type). We describe here the use of HLA typing of cultured amniotic cells for the prenatal diagnosis of another HLA-linked disease, complement C4 deficiency. Although C4 is actually coded for by genes at two very closely linked loci, mapping near the HLA-B locus, absolute C4 deficiency behaves genetically like a monogenetic autosomal recessive disease linked to HLA. The HLA typing results for amniotic cells from a pregnancy at risk for C4 deficiency predicted that the fetus would share only one HLA haplotype with the affected child and would therefore be a clinically normal heterozygous carrier of the C4 deficiency alleles with half-normal levels of C4 activity. These predictions were confirmed after delivery.     

Identification of two new C4 alleles by DNA sequencing and evidence for a historical recombination of serologically defined C4A and C4B alleles

Nucleotide polymorphisms of the C4 genes were investigated by direct sequencing of seven different homozygous typing cells from the 10IHW panels. Two novel sequences were identified within the C4d region of the C4 genes. Our sequencing analyses extend previous findings suggesting that a recombination hot spot is likely to have occurred between codon positions 1157 and 1186 within the C4d region. The classification of electrophoretically defined C4A and C4B alleles can be further subtyped by sequencing. Because the central major histocompatibility complex region that carries various copies of the C4 gene has been associated with a range of disorders; further analysis at the sequence level within the C4 locus may provide informative genetic markers for the investigation of disease-associated polymorphisms.     

B cell abnormality and autoimmune disorders

Various abnormalities have been described in B cells from patients with systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and lupus-prone mice. Many of the abnormalities do not appear to be connected with the pathogenesis of the disease. However, various animal models developing lupus-like disease including both spontaneous mutants such as (NZB × NZW)F1 and MRL/lpr and mice generated by transgenic or knockout technology such as Bim-deficient and CD40L-transgenic mice show defect in apoptosis of mature B cells induced by ligation of the B cell antigen receptor (BCR). BCR-mediated apoptosis appears to be involved in deletion of self-reactive B cells. Thus, defect in BCR-mediated apoptosis is a widely observed B cell abnormality in lupus-prone mice and may play a role in the pathogenesis of systemic autoimmune diseases by abrogating deletion of self-reactive B cells.     

Increased frequency of complement C4 'null' alleles in recurrent spontaneous abortions.

Typings for major histocompatibility antigens HLA A, B, C and DR and for complement C4A, C4B and factor B were performed for 59 Finnish couples experiencing at least three consecutive recurrent spontaneous abortions (RSA). Forty-one of them were primary abortion (PA) couples with no children and 18 were secondary abortion (SA) couples who had one or two children before abortions. HLA sharing in A and B loci was slightly but significantly increased (15%, P less than 0.05) among RSA couples compared to the controls, as was DR sharing among SA couples compared to PA couples (50% versus 22%, P less than or equal to 0.05). The most interesting new finding, however, was the statistically significant increase of complement C4 functionally silent, i.e. C4 'null', alleles in RSA couples. C4 is a duplicated gene and its products differ in their functions in the complement cascade. C4A null alleles were equally increased in PA wives and in PA husbands (32%, P less than or equal to 0.05) compared to the controls (18%) and C4B null alleles in SA wives (56%, P less than or equal to 0.05) and in SA husbands (50%) compared to the controls (29%). Therefore, the offspring of RSA couples have a significantly increased risk of inheriting several null alleles. The majority, 95% (P less than 0.001) of PA couples and 83% of SA couples, had at least one C4A or C4B null in their phenotypes compared to 66% among Finnish controls.     

Murine glomerular mesangial cell uptake of apoptotic cells is inefficient and involves serum-mediated but complement-independent mechanisms

An increased number of apoptotic bodies have been detected in glomeruli of non-nephritic kidneys of C1q-deficient mice. In these mice an in vivo impaired uptake of apoptotic cells by peritoneal macrophages was also demonstrated. Here we investigated whether C1q plays a role in the in vitro clearance of apoptotic cells by glomerular mesangial cells. Phagocytosis was assessed using a novel flow cytometric assay that was validated by immunofluorescence studies. The uptake of apoptotic cells by mesangial cells, measured as percentage of mesangial cells ingesting apoptotic cells, was approximately 25%, 10% and 10% for a T cell lymphoma line (RMA), thymocytes and neutrophils, respectively. The uptake reached a plateau phase after 3 h, was specific for apoptotic cells and was mediated by serum but not by complement components C1q or C3. The phagocytosis of apoptotic cells was significantly inhibited by Arg-Gly-Asp-Ser (RGDS), a peptide capable of blocking the interaction of thrombospondin with CD36 or the vitronectin receptor. Pretreatment of the mesangial cells with dexamethasone (200 nm) but not with LPS increased the uptake markedly. These findings indicate that murine mesangial cells are capable of taking up syngeneic apoptotic cells, although much less efficiently than professional phagocytic cells. They also show that serum proteins other than complement components mediate the removal of apoptotic cells by murine mesangial cells in vitro.     

Increased frequency of the null allele at the complement C4b locus in autism.

Associations between C4 deficiency and autoimmune disorders have been found over the past several years. Since autism has several autoimmune features, the frequencies of null (no protein produced) alleles at the C4A and C4B loci were studied in 19 subjects with autism and their family members. The autistic subjects and their mothers had significantly increased phenotypic frequencies of the C4B null allele (58% in both the autistic subjects and mothers, compared with 27% in control subjects). The siblings of the autistic subjects also had an increased frequency of the C4B null allele, but this increase was not significant. The fathers had normal frequencies of this null allele. All family members had normal frequencies of the C4A null allele, all normal C4A and C4B alleles and all BF and C2 alleles.     

Cellular localization of complement C3 and C4 transcripts in intestinal specimens from patients with Crohn's disease

It has been suggested that the increase in C3 and C4 levels in jejunal perfusates of patients with Crohn's disease (CD) results from local intestinal synthesis of complement. The present study evaluated the expression of these complement genes in inflamed tissues from patients with CD. Surgically resected specimens from patients with CD and control tissue obtained from subjects with adenocarcinoma of the colon were evaluated for C3 and C4 gene expression by the use of 35S-labelled anti-sense RNA probes. All tissue samples, diseased and normal tissue, expressed C4 mRNA throughout in the intestinal epithelium. C3 mRNA was not detected in epithelial cells in histologically normal tissue, but in diseased specimens there was a focal distribution of C3 mRNA in epithelial cells of the crypts, but not in villous epithelium. Focal C3 gene expression correlated with crypt abscess formation and the presence of polymorphonuclear leucocytes in the lumen of the crypts. In addition, C3 mRNA was also found in macrophages of the submucosa. These macrophages were CD68+, fusiform with faint cytoplasm and morphologically different from the large rounded lamina propria macrophages, which do not express C3 mRNA. Multinucleated giant cells did not express either C3 or C4 genes. In addition to its presence in intestinal epithelium, C4 mRNA was also expressed in mast cells, which however did not express C3 mRNA. These observations identify cells in the intestinal wall expressing complement genes and support the hypothesis that there is local regulated production of complement in the intestine of patients with CD, and subsequent complement activation may contribute to the inflammatory process.