Immunological Investigations, Early Online: 1–11, 2014 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.932378
Anti-C1q in chronic hepatitis C Virus genotype IV infection: association with autoimmune rheumatologic manifestations
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Samia H. Fadda,1 Iman H. Bassyouni,1 Ahmed Hamdy,2 Nermeen A. Foad,2 and Iman E. Wali3 1
Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Egypt, 2 Department of Rheumatology and Rehabilitation, Faculty of Medicine, El-Fayoum University, Egypt, 3 Department of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Egypt A growing body of evidence suggests that anti-complement-1q (anti-C1q) antibodies are elevated in a variety of autoimmune disease. Therefore, we investigated their prevalence and clinical significance in plasma of patients with hepatitis C virus (HCV) genotype IV in the presence and absence of autoimmune extra hepatic manifestations in comparison to normal healthy individuals. Plasma Anti-C1q Abs levels were assessed by an Enzyme Linked Immunosorbant Assay in 91 chronic HCV-infected patients (51 with and 40 without autoimmune rheumatic manifestations) and 40 healthy volunteers matched for age and gender. Epidemiological, clinical, immunochemical and virological data were prospectively collected. Positive Anti-C1q antibodies were more frequent among HCV patients with extra-hepatic autoimmune involvement, than those without and healthy control subjects. No significant correlations were found between Anti-C1q levels with either the liver activity or the fibrosis scores. In HCV-patients with autoimmune involvements, plasma Anti-C1q levels were significantly higher in patients with positive cryoglobulin, and in those with lymphoma than in those without. These results were confirmed by multivariate analysis. Further large scale longitudinal studies are required to assess and clarify the significance and the pathogenic role of anti-C1q antibodies among HCV infected patients with positive cryoglobulinaemia and lymphoma. Keywords Anti-C1q antibodies, cryoglobulinaemia, hepatitis C virus, lymphoma
INTRODUCTION Infection with hepatitis C virus (HCV) remains a severe life-threatening medical and public health problem worldwide. Egypt has the highest
Correspondence: Dr. Iman H Bassyouni, Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Cairo 12613, Egypt. Rheumatology & Rehabilitation Department, El-Kasr El-Aini Hospital, Cairo, Egypt. E-mail: [email protected] or [email protected]
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prevalence of HCV infection in the world, averaging 15–25% in rural communities (Esmat et al., 2012; Zaki et al., 2010). HCV has emerged as a major cause not only of liver disease, but also of numerous extrahepatic autoimmune conditions (Jacobson et al., 2010). Immunopathological manifestations range from production of autoantibodies to overt autoimmune disease, and to immune-complex-mediated disorders, including, glomerulonephritis, vasculitis, cryoglobulinaemia as well as to the development of a secondary lymphoma (Vergani & Mieli-Vergani, 2013). It has been well established that chronic HCV infection plays important roles in the production of nonorgan-specific autoantibodies (NOSA), as well as organ-specific autoantibodies such as thyroid autoantibodies (Himoto & Masaki, 2012). There is substantial evidence that the immune complexes found within affected vessels are composed of HCV particles bound to anti-HCV antibodies, which locally activate the classical complement pathway (Qin & Gao, 2006). The complement (C) system is a set of biochemical pathways that removes pathogen components from an organism as part of the innate and acquired immunity programs (Trouw & Daha, 2011). Activation of the complement system triggers a wide range of cellular responses ranging from apoptosis (cell death) to opsonization (antigen/antibody binding) (Potlukova & Kralikov, 2008; Ricklin et al., 2010). C1q, the first component of the classical pathway of complement system, plays a significant role in the clearance of immune complexes and apoptotic bodies. Disruption of this process may lead to development of autoimmunity. C1q binds immune complexes by its 6 globular domains while it binds a variety of other ‘‘nonimmune’’ activators of the complement system, like C-reactive protein by its collagen-like region (CLR) (Kallenberg, 2008). C1q along with its physiological role in maintenance of homeostasis is involved in pathological conditions associated with the generation of anti-C1q autoantibodies in which CLR of C1q is the target of anti-C1q antibodies (Stoyanova et al., 2011). The liver is the major site for biosynthesis of 80–90% plasma complement components, and expresses a variety of complement receptors. The complement and complement receptors appear to be involved in liver injury and repair. However, the underlying mechanisms remain poorly understood (Qin & Gao, 2006). Anti-C1q antibodies are highly prevalent in prototypical autoimmune disease, systemic lupus erythematosus (SLE), exhibiting strong correlation with lupus nephritis (Hu et al., 2013; Mahler et al., 2013; Trouw et al., 2004; Zhang et al., 2011). These antibodies are also detected in hypocomplementemic urticarial vasculitis, rheumatoid vasculitis, IgA nephropathy, anti-glomerular basement membrane (anti-GBM) nephropathy, and HIV infection (Mahler et al., 2013). Similarly, autoantibodies are frequently found in patients with viral infections including Epstein-Bar (EB) virus, measles virus, and herpes simplex virus before the discovery of hepatitis C virus (HCV) (Himoto & Masaki, 2012). A growing body of evidence suggests that the complement system is also involved in the pathogenesis of a variety of liver disorders (Qin & Gao, 2006). Therefore, we investigated the prevalence and clinical significance of anti-C1q Abs in plasma of patients with HCV genotype IV in the presence and absence
Anti-C1q in HCV-associated rheumatologic manifestations
of autoimmune extra hepatic manifestations in comparison to normal healthy individuals.
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PATIENTS AND METHODS Patients’ assessment Fifty-one chronic HCV patients with rheumatological autoimmune manifestations were selected from the Department of Rheumatology, Cairo University hospitals. Inclusion criteria for HCV patients were (a) serum positive tests for anti-HCV antibodies and HCV-RNA; (b) absence of liver cirrhosis, concomitant HBV, HIV infections and hepatocellular carcinoma (through histology, laboratory evidence of liver failure and/or ultrasound-proven portal hypertension); and (c) exclusion of concomitant autoimmune diseases (rheumatoid arthritis, connective tissue diseases, etc.) HCV patients who received interferon therapy were excluded from the study. The recorded data of rheumatologic manifestations for every patient was as follows, arthralgia; arthritis; vasculitis, sicca manifestations; pulmonary; renal; haematological, lymphoproliferative disease, and neurological (peripheral and/or central nervous system manifestations). Each of the 51 HCV patients with autoimmune rheumatologic manifestations included in this study was matched by gender and age with HCV patients without such manifestations. Only patients without liver cirrhosis, hepatocellular carcinoma, and concomitant autoimmune diseases were included. All chronic HCV participants were subjected to careful historical interview and complete clinical examination. Demographic, clinical, immunochemical, and virological parameters were collected for each patient. Forty healthy subjects, sero-negative for HCV, matched for age and gender fulfilling the same exclusion criteria applied for HCV patients, were included as a healthy control group. The study was approved by our institutional ethics committee and informed consent was obtained from all patients. Laboratory and immunological investigations Virological studies Anti-HCV antibodies and HCV RNA were determined on serum clotted and centrifuged at 37 C and stored at 70 C, respectively. HCV antibody assayed using 3rd-generation enzyme linked immunosorbent assay (ELISA) (Abbott-Murex Biotech, Dartford, UK) and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) (Promega Co., MA, USA) Genotyping was performed using Murex HCV Serotyping ELISA Kits (Abbott-Murex Biotech, Dartford, UK). Hepatitis B surface antigens (HBsAg) (Sorin Biomedica Co., Italy) were assessed using commercially available kits. HIV antibody was assayed using 3rd generation Genscreen HIV 1/2 version 2 ELISA Kit (Bio-Rad Laboratories, France). Rheumatoid factor (RF) detection RF was assessed by the latex fixation method. A suspension of uniform polystyrene particles sensitized in glycine buffer with heat altered human IgG (BD Diagnostic Systems, Sparks, Maryland, USA) was incubated
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with progressive dilutions of human sera in microtitre wells. After incubation, the plates were inspected for observable agglutination.
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Cryoglobulin detection Venous blood was taken from fasting patients at 37 C, allowed to clot, and then separated by centrifugation. After centrifugation, the supernatant was removed from the serum, incubated at 4 C for 8 days, and examined daily for cryoprecipitation. Plasma Anti-C1q Abs levels determination Blood samples were collected into pyrogen free, EDTA- tubes, allowed to clot at 4 C for 1 h, and then centrifuged at 2000 g for 10 min. The plasma obtained was divided, and then aliquots were stored at 70 C until assayed on the same day to eliminate day to day interassay variability. A commercial ELISA kit (ORGENTEC Diagnostika GmbH, Mainz, Germany) was used for assaying Anti-C1q ab levels. In this assay a 96-well plate was coated with human C1q which was then used as the antigen to which the Anti-C1q autoantibodies can bind. In brief, each plasma sample was diluted in a highsalt buffer in order to prevent the binding of immune complexes then transferred to the wells of the ELISA plate and assayed according to the manufacturer’s instructions. The optical densities were measured at 450 nm converted into units (U/ml) by being plotted against the autoantibody concentration of the standards given by the manufacturer. The lower detection limit for the Anti-C1q test was determined at 0.5 U/ml. Values greater than 10 U/ml were considered positive for Anti-C1q antibodies as recommended by the manufacturer’s protocol. Statistical analysis The Statistical Package for Social Sciences (SPSS) version 15 (LEAD Technology Inc., Charlotte, NC, USA) was used to analyze the data. Continuous variables were summarized through the median (range) and categorical variables using absolute values and percentages. Mann–Whitney U-test was used to compare two independent groups and Kruskal–Wallis test was adopted for comparing more than two groups. Associations between categorical groups were tested using the Chi-square test ( 2) with Yates correction or Fisher’s exact test as appropriate. Spearman’s rank correlation test was used as a measure of association of quantitative variables. For those variables that were significantly influencing Plasma Anti-C1q levels (p50.05), a standard linear multiple regression analysis for Anti-C1q levels as dependent variable was performed. For the regression model, adjusted R2, Beta, b-value and standard error b were recorded.
RESULTS Patients characteristics The demographic, clinical, virological and laboratory parameters of the HCV-infected patients with and without autoimmune rheumatologic involvement are reported in Table 1. ESR were significantly higher and platelet count were lower in patients with rheumatologic manifestations (p = 0.025 and
Anti-C1q in HCV-associated rheumatologic manifestations
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Table 1. Clinical, demographic and laboratory characteristics in HCV patients with and without rheumatologic involvement.
Variable
HCV with rheumatological involvement N = 51
HCV without rheumatological involvement N = 40
Age (years) Female/Male ESR (mm/h) Hemoglobin (g/dl) Leucocytes (103/ml) Platelets (103/ml)) C3 (mg/dl) C4 (mg/dl) Creatinine (mg/dl) ALT (u/L) AST(u/L) Serum albumin (g/dl) HCV-RNA* 105 (IU/mL) Metavir Activity score A0 A1 A2 Metavir fibrosis score F0 F1 F2 F3
50 (19–65) 38/13 40 (10.00–150.00) 11.50 (6.30–15.00) 5.20 (2.40–61.00) 214 (8.00–890.00) 142.2 (21.45–309.14) 21.71 (4.68–74.49) 0.80 (0.30–5.30) 40.00 (5.00–173.00) 40.00 (9.00–125.00) 3.60 (1.50–4.50) 0.76 (0.00–16.61) No (%) 4 (7.84%) 19 (37.25%) 28 (54.9%) No (%) 2 (3.92%) 22 (43.14%) 13 (25.49%) 14 (27.45%)
50.50 (20–65) 26/14 36 (15.00–68.00) 12.00 (8.80–15.80) 5.30 (1.62–9.90) 244 (10.00–435.00) 118.79 (21.45–287.17) 24.88 (4.68–118.63) 0.90 (0.40–3.00) 36.00 (18.30–304.00) 37.00 (13.50–424.00) 3.65 (2.20–5.00) 0.09 (0.01–45.66) No (%) 5 (12.5%) 18 (45%) 17 (42.5%) No (%) 0 (0%) 17 (42.5%) 16 (40%) 7 (17.5%)
p Value 0.644 0.361 0.025* 0.084 0.083 0.045* 0.179 0.267 0.423 0.383 0.234 0.090 0.295 0.224
0.934
Continuous variables: Median (range); categorical variables: absolute value (%).Mann– Whitney U-test was used to compare continuous variables & 2 or Fisher exact test were used to compare frequencies; AST, aspartate transaminase; ALT, Alanine transaminase; *p50.05.
0.045, respectively). However, no statistically significant differences were found among all the other mentioned variables in the table between HCV patients with autoimmune rheumatologic involvement compared to those without these manifestations. All our patients had HCV genotype IV and none of them had either cirrhosis, history of alcohol consumption, HBV, or HIV. Anti-C1q Abs levels in studied population Anti-C1q antibodies (values greater than 10 U/ml) were detected in 16 HCV patients with extra-hepatic autoimmune involvement (31.37%), 6 HCV patients without such involvement (15%), and in only 3 healthy control subjects (7.5%) with a high statistical significant difference (p50.001). The median plasma Anti-C1q levels were higher in HCV patients with autoimmune rheumatologic manifestations (5.74 U/ml; range 1.01–52.27) than their levels in HCV patients without such manifestations (3.42 U/ml; range 0.16–58.24) and in the healthy control group (4.02 U/ml; range 0.60–27.50, Kruskal–Wallis test p = 0.038). Using the Mann–Whitney U tests, it was shown that Anti-C1q levels exhibited a statistical significant difference in HCV patients with autoimmune rheumatologic manifestations when compared to HCV patients without such
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Figure 1. Plasma Anti-C1q levels in HCV patients with and without rheumatic involvement and Healthy controls. The lines inside the boxes indicate the median; the outer borders of the boxes indicate 25th and 75th percentiles; the bars extending from the boxes indicate the 10th and 90th percentiles; the symbols above the bars represent the outliers.
manifestations and the healthy control groups (p = 0.012 and 0.029, respectively). On the other hand, there was no significant difference in Anti-C1q levels between patients with HCV without autoimmune manifestations vs healthy controls (p = 0.593). Figure 1 demonstrates the Anti-C1q levels in the different groups under study. Using Spearman rank correlation analysis (Table 2), Anti-C1q antibodies were found to be negatively correlated with C4 (r = 0.215, p = 0.040) and positively correlated with HCV RNA viral Load (r = 0.228, p = 0.029). On the other hand, age, serum albumin, aminotransferases and C3 didn’t reveal any statistical significant correlation with Anti-C1q levels. Furthermore, no significant correlations were found between Anti-C1q levels with either the METAVIR activity or the fibrosis scores (p40.05). Association of plasma Anti-C1q ab levels with clinical and laboratory parameters in HCV-associated rheumatologic involvements HCV-associated rheumatologic involvements are presented in Table 3. Articular involvement was the most common clinical feature reported by this group of patients (53%) followed by vasculitis in 17 patients (33.3%). Further analysis was performed in order to compare the levels of plasma Anti-C1q ab in the presence and absence of some clinical manifestations (Table 3). As illustrated through the table, plasma Anti-C1q levels were significantly higher in patients with positive cryoglobulin (p = 0.001), and in those with
Anti-C1q in HCV-associated rheumatologic manifestations Table 2. Correlations of Anti-C1q antibodies level with laboratory parameters and liver scores of activity and fibrosis in HCV infected patients.
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Parameter
r
ESR (mm/1st hr) Hemoglobin (g/dl) Leucocytes (103/ml) Platelets (103/ml)) C3 (mg/dl) C4 (mg/dl) ALT (u/L) AST(u/L) Serum albumin (g/dl) Creatinine (mg/dl) HCV-RNA* 105(IU/mL) Metavir activity score Metavir fibrosis score
p
0.082 0.131 0.023 0.141 0.054 0.215 0.019 0.029 0.038 0.054 0.228 0.134 0.055
0.438 0.217 0.829 0.182 0.610 0.040* 0.855 0.788 0.722 0.610 0.029* 0.205 0.603
AST, aspartate transaminase; ALT, Alanine transaminase; C, complement; *p50.05.
Table 3. Comparison of the plasma anti-C1q levels in HCV patients with autoimmune involvement in the presence and absence of some clinical manifestations and autoantibodies. Patients Affected Clinical feature
No.
Arthritis Vasculitis Skin rash Neuropathy Nephritis Hematological Sjogren Syndrome Lymphoma RF +ve ANA +ve Cryoglobulin +ve
27 17 14 16 5 13 5 5 23 18 16
Median (range) 5.37 6.42 5.87 4.98 6.05 6.43 3.60 14.3 6.5 4.90 11.70
(1.01–20.95) (1.01–20.95) (2.82–20.95) (1.01–20.95) (4.47–19.67) (2.53–52.27) (1.01–52.27) (12.35–52.27) (1.01–45.96) (2.55–16.16) (2.53–45.96)
Patients Unaffected No.
Median (range)
p Value
24 34 37 35 46 38 46 46 28 33 35
6.03 (1.06–52.27) 5.32 (1.06–52.27) 5.37(1.01–52.27) 6.01 (1.06–52.27) 5.31 (1.01–52.27) 5.12 (1.01–20.95 5.87 (1.01–52.27) 5.19 (1.01–45.96) 4.50(1.06–52.27) 6.00 (1.01–52.27) 3.75 (1.01–52.27)
0.85 0.83 0.90 0.53 0.33 0.13 0.41 0.002* 0.056 0.844 0.001*
Data are Median (range),*p50.05. Mann–Whitney U-test was used; *p50.05; ANA, antinuclear antibodies; RF, rheumatoid factor.
lymphoma (p = 0.002) than in those without. On the other hand, we did not find significant differences in Anti-C1q levels in relation to other organ involvement or with positive RF or ANA. To confirm these results, we performed a multivariate analysis to estimate the independent association of each covariate with Anti-C1q as the dependent variable against the variables that yielded p values 50.05 according to the univariate analysis among the group of patients with autoimmune rheumatic involvement. The model revealed that the presence of lymphoma had the highest impact (b = 15.36, Beta = 0.470, p50.0001) followed by the presence of cryoglobulins
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(b = 7.637, Beta = 0.364, p = 0.004). On the other hand, C4 levels (b = 0.117, Beta = 0.169, p = 0.161) was not significant in this model (b = 0.742, Beta = 0.038, p = 0.835). The coefficient of determination (R2) for the generated model was 0.352 implying that these two factors explain almost 35% of the variability in plasma Anti-C1q levels among HCV patients with autoimmune extra hepatic involvement.
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DISCUSSION Chronic HCV infection frequently leads to autoimmune response including the production of autoantibodies and the coincidence of autoimmune diseases (Himoto & Masaki, 2012). Recently, anti-C1q antibodies are demonstrated to be pathogenic in the target organ damage of many autoimmune diseases, by facilitating C1q deposition and enhancing complement activation via classical pathway (Hu et al., 2013; Potlukova & Kralikov, 2008). The anti-C1q antibodies have been shown to be associated strongly with immune complex diseases, most prominently with hypocomplementaemic urticarial vasculitis syndrome, systemic lupus erythmatosus, Behcet’s disease, and severe rheumatoid arthritis (Bassyouni et al., 2014; El-Hewala et al., 2011; Yang et al., 2012). In this study, we showed that anti-C1q antibodies was found in 31.37% of the chronically HCV-infected patients with autoimmune extrahepatic manifestations compared to 15% in chronically HCV-infected patients without these manifestations and 7.5% of the healthy control. Our study shows that prevalence of anti-C1q was comparable with that previously reported in Caucasian populations. Saadoun and coworkers (2006) have detected anti-C1q antibodies in 26% of their HCV patients compared to 10% of healthy donors. Similarly, Anti-Clq antibodies have been detected in 38% of HCV patients compared with 2% of healthy controls in another study (Lienesch et al., 2006). This increased production of anti-C1q antibodies might be a part of the widerange, exaggerated autoantibody produced against autoantigens, characteristic of HCV. The production of these antibodies is indicative of a heightened immune reactive state in which epitope spread and molecular mimicry have resulted in multiple serological reactivities, and concurrent immune disorders (Bianchi et al., 2007). Approximately 25–30% of patients with chronic HCV are positive for non-organ specific auto-antibodies, i.e. antinuclear antibodies, smooth muscle antibodies, and liver-kidney microsomal antibody type 1 (Indolfi et al., 2012). It has been reported that HCV-encoded core protein interacts directly with the receptor for the globular domain of C1q protein (gC1q-R) representing an efficient way to affect the host T- and B-cell immunity (Stoyanova et al., 2011). Herein, we further analyzed association of anti-C1q antibodies levels with the presence and absence of different autoimmune extrahepatic involvements. Cryoglobulins were found among 31.4% those HCV patients. HCV infection is one of the major causes of mixed cryoglobulinaemia (Lauletta et al., 2012). Anti-C1q antibodies associated significantly with the presence of cryoglobulins in our HCV patients. Indeed, the IgM–IgG complexes as found in MC are good receptors for C1q. Furthermore, it has been reported that C1q protein and C1q binding activity are enhanced in the cryoprecipitates of HCV-infected patients.
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Anti-C1q in HCV-associated rheumatologic manifestations
The wide expression of C1q receptor on the surface of blood and endothelial cells enhance their specific binding to immune complexes containing HCV core protein which may represent an important pathogenetic mechanism in the cryoglobulin-related pathway (Lauletta et al., 2012). The formation of neoantigens within the C1q molecule after the binding of C1s and C1r to C1q has been documented. The presence of C1q-cryoglobulin complexes enhance the generation of autoantibodies against C1q, anti-C1q antibodies are directed against a neoepitope that is expressed in bound form of C1q (Saadoun et al., 2006). Among the numerous extrahepatic manifestations of HCV, the interaction of the virus with B-cells is a major concern of chronic HCV infection which might undergo an oncogenic event giving survival advantage to a subclonal population, resulting in lymphoproliferative disorder (Bachy et al., 2010; Yao et al., 2011). Lymphoma was present in 9.8% of our HCV patients with auto immune manifestations. An important result of our study was the association of anti-C1q antibodies with lymphoma. Cytokines and growth factors produced by chronic infection are suspected to play a key role in B-cell transformation. Growing body of evidence has clarified the role of B-cell activating factor (BAFF) in regulating B-cell survival, proliferation, and differentiation into immunoglobulin-secreting cells (Lauletta et al., 2012). BAFF deregulation has been demonstrated in HCV infection as well as B-cell lymphoproliferative disorders (De Re et al., 2012; Se`ne et al., 2007; Sun et al., 2008), which might explain the enhancement of autoantibodies production including anti-C1q in lymphoma in case of HCV infection. In this study, we did not find a significant association between anti-C1q antibodies with other extra hepatic autoimmune manifestations. HCV infection is a risk factor for proteinuria and/or impaired renal function (Morales et al., 2012). Although Anti-C1q antibodies have been best described in patients with lupus nephritis (Akhter et al., 2011); however, there was no association between anti-C1q with renal involvement in our HCV patients. Several mechanisms other than immune complex deposition in the glomeruli have been found in the pathogenesis of renal affection associated with chronic HCV infection (Morales et al., 2012). Direct mesangial injury by HCV infection has been documented (Barsoum, 2007). HCV RNA and related proteins have been found in mesangial cells, tubular epithelial cells, and endothelial cells of glomerular and tubular capillaries. Recently, it has been described that toll-like receptor-3 (TLRs), primary proteins expressed on immune and nonimmune cells as key components of the innate immune system, could have a pathogenic role establishing a link between viral infection and glomerulonephritis (Morales et al., 2012). Another possible mechanism underlying HCV-related kidney injury is nonimmunologically mediated. HCV-positive patients have elevated levels of fasting serum insulin and insulin resistance, and higher prevalence of diabetes (Sarafidis & Ruilope, 2006). Anti-C1q antibodies negatively correlated with C4 in our studied HCV patients. Most HCV–MC patients showed decreased levels of the early complement components C1, C4 and C2, whereas C3 levels fluctuated with the disease course. Experiments performed to define the mechanism
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responsible for this complement profile showed that activation of the early complement components in serum was due to the activation of the classical pathway by mixed cryoglobulins (Saadoun et al., 2006). Herein, no significant correlations were found between anti-C1q antibodies levels with histopathological grading of liver biopsy assessed by METAVIR activity and fibrosis scores as reported in a previous study (Saadoun et al., 2006). In conclusion, this study has demonstrated an increased prevalence of antiC1q antibodies in HCV infected patients (type IV) with autoimmune extrahepatic manifestations especially patients with positive cryoglobulinaemia and lymphoma. Anti-C1q autoantibodies could be a potential candidate for a biomarker panel for lymphoma diagnosis. Indeed, by using a cross-sectional study design we cannot rule out that excess circulating Anti-C1q is a consequence rather than a cause of those associations. We also couldn’t determine if the up-regulation of anti-C1q in our HCV patients with autoimmune involvement is pathogenic, an epiphenomenon of aberrant tissue damage, or compensatory to an uncontrolled immune response. Furthermore, the autoimmune manifestations in our HCV group are highly heterogeneous with a small number of patients. Further prospective large scale, longitudinal studies are needed to elucidate their precise pathogenic role and their clinical relevance in chronic HCVinfected patients.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Anti-C1q in HCV-associated rheumatologic manifestations
Hu SY, Jia XY, Yang XW, et al. (2013). Glomerular C1q deposition and serum anti-C1q antibodies in anti-glomerular basement membrane disease. BMC Immunol, 21, 14–42. Indolfi G, Bartolini E, Olivito B, et al. (2012). Autoimmunity and extrahepatic manifestations in treatment-naı¨ve children with chronic hepatitis C virus infection. Clin Dev Immunol, 2012, 785627. Jacobson IM, Cacoub P, Dal Maso L, et al. (2010). Manifestations of chronic hepatitis C virus infection beyond the liver. Clin Gastroenterol Hepatol, 8, 1017–29. Kallenberg CG. (2008). Anti-C1q autoantibodies. Autoimmun Rev, 7, 612–5. Lauletta G, Russi S, Conteduca V, Sansonno L. (2012). Hepatitis C virus infection and mixed cryoglobulinemia. Clin Dev Immunol, 2012, 502156. Lienesch DW, Sherman KE, Metzger A, Shen GQ. (2006). Anti-Clq antibodies in patients with chronic hepatitis C infection. Clin Exp Rheumatol, 24, 183–5. Mahler M, van Schaarenburg RA, Trouw LA. (2013). Anti-C1q autoantibodies, novel tests, and clinical consequences. Front Immunol, 14, 117. Morales JM, Kamar N, Rostaing L. (2012). Hepatitis C and renal disease: Epidemiology, diagnosis, pathogenesis and therapy. Contrib Nephrol, 176, 10–23. Potlukova E, Kralikov P. (2008). Complement component C1q and Anti-C1q antibodies in theory and in clinical practice. Scand J Immunol, 67, 423–430. Qin X, Gao B. (2006). The complement system in liver diseases. Cell Mol Immunol, 3, 333–40. Ricklin D, Hajishengallis G, Yang K, Lambris JD. (2010). Complement: A key system for immune surveillance and homeostasis. Nat Immunol, 11, 785–97. Saadoun D, Sadallah S, Trendelenburg M, et al. (2006). Anti-C1q antibodies in hepatitis C virus infection. Clin Exp Immunol, 145, 308–12. Sarafidis PA, Ruilope LM. (2006). Insulin resistance, hyperinsulinemia, and renal injury: Mechanisms and implications. Am J Nephrol, 26, 232–44. Se`ne D, Limal N, Ghillani-Dalbin P, et al. (2007). Hepatitis C virus-associated B-cell proliferation—The role of serum B lymphocyte stimulator (BLyS/BAFF). Rheumatology (Oxford), 46, 65–9. Stoyanova V, Petrova S, Tchorbadjieva M, et al. (2011). New insight into the autoimmunogenicity of the complement protein C1q. Mol Immunol, 48, 678–82. Sun J, Lin Z, Feng J, et al. (2008). BAFF-targeting therapy, a promising strategy for treating autoimmune diseases. Eur J Pharmacol, 597, 1–5. Trouw LA, Daha MR. (2011). Role of complement in innate immunity and host defense. Immunol Lett, 138, 35–7. Trouw LA, Groeneveld TW, Seelen MA, et al. (2004). Anti-C1q autoantibodies deposit in glomeruli are only pathogenic in combination with glomerular C1q-containing immune complexes. J Clin Invest, 114, 679–88. Vergani D, Mieli-Vergani G. (2013). Autoimmune manifestations in viral hepatitis. Semin Immunopathol, 35, 73–85. Yang XW, Tan Y, Yu F, Zhao MH. (2012). Combination of anti-C1q and anti-dsDNA antibodies is associated with higher renal disease activity and predicts renal prognosis of patients with lupus nephritis. Nephrol Dial Transplant, 27, 3552–9. Yao ZQ, Ni L, Zhang Y, et al. (2011). Differential regulation of T and B lymphocytes by PD-1 and SOCS-1 signaling in hepatitis C virus-associated non-Hodgkin’s lymphoma. Immunol Invest, 40, 243–64. Zaki Mel S, Saudy N, El Diasty A. (2010). Study of nitric oxide in patients with chronic hepatitis C genotype 4: Relationship to viremia and response to antiviral therapy. Immunol Invest, 39, 598–610. Zhang CQ, Ren L, Gao F, et al. (2011). Anti-C1q antibodies are associated with systemic lupus erythematosus disease activity and lupus nephritis in northeast of China. Clin Rheumatol, 30, 967–73.
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Anti-C1q in chronic hepatitis C Virus genotype IV infection: association with autoimmune rheumatologic manifestations
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Samia H. Fadda,1 Iman H. Bassyouni,1 Ahmed Hamdy,2 Nermeen A. Foad,2 and Iman E. Wali3 1
Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Egypt, 2 Department of Rheumatology and Rehabilitation, Faculty of Medicine, El-Fayoum University, Egypt, 3 Department of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Egypt A growing body of evidence suggests that anti-complement-1q (anti-C1q) antibodies are elevated in a variety of autoimmune disease. Therefore, we investigated their prevalence and clinical significance in plasma of patients with hepatitis C virus (HCV) genotype IV in the presence and absence of autoimmune extra hepatic manifestations in comparison to normal healthy individuals. Plasma Anti-C1q Abs levels were assessed by an Enzyme Linked Immunosorbant Assay in 91 chronic HCV-infected patients (51 with and 40 without autoimmune rheumatic manifestations) and 40 healthy volunteers matched for age and gender. Epidemiological, clinical, immunochemical and virological data were prospectively collected. Positive Anti-C1q antibodies were more frequent among HCV patients with extra-hepatic autoimmune involvement, than those without and healthy control subjects. No significant correlations were found between Anti-C1q levels with either the liver activity or the fibrosis scores. In HCV-patients with autoimmune involvements, plasma Anti-C1q levels were significantly higher in patients with positive cryoglobulin, and in those with lymphoma than in those without. These results were confirmed by multivariate analysis. Further large scale longitudinal studies are required to assess and clarify the significance and the pathogenic role of anti-C1q antibodies among HCV infected patients with positive cryoglobulinaemia and lymphoma. Keywords Anti-C1q antibodies, cryoglobulinaemia, hepatitis C virus, lymphoma
INTRODUCTION Infection with hepatitis C virus (HCV) remains a severe life-threatening medical and public health problem worldwide. Egypt has the highest
Correspondence: Dr. Iman H Bassyouni, Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Cairo 12613, Egypt. Rheumatology & Rehabilitation Department, El-Kasr El-Aini Hospital, Cairo, Egypt. E-mail: [email protected] or [email protected]
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prevalence of HCV infection in the world, averaging 15–25% in rural communities (Esmat et al., 2012; Zaki et al., 2010). HCV has emerged as a major cause not only of liver disease, but also of numerous extrahepatic autoimmune conditions (Jacobson et al., 2010). Immunopathological manifestations range from production of autoantibodies to overt autoimmune disease, and to immune-complex-mediated disorders, including, glomerulonephritis, vasculitis, cryoglobulinaemia as well as to the development of a secondary lymphoma (Vergani & Mieli-Vergani, 2013). It has been well established that chronic HCV infection plays important roles in the production of nonorgan-specific autoantibodies (NOSA), as well as organ-specific autoantibodies such as thyroid autoantibodies (Himoto & Masaki, 2012). There is substantial evidence that the immune complexes found within affected vessels are composed of HCV particles bound to anti-HCV antibodies, which locally activate the classical complement pathway (Qin & Gao, 2006). The complement (C) system is a set of biochemical pathways that removes pathogen components from an organism as part of the innate and acquired immunity programs (Trouw & Daha, 2011). Activation of the complement system triggers a wide range of cellular responses ranging from apoptosis (cell death) to opsonization (antigen/antibody binding) (Potlukova & Kralikov, 2008; Ricklin et al., 2010). C1q, the first component of the classical pathway of complement system, plays a significant role in the clearance of immune complexes and apoptotic bodies. Disruption of this process may lead to development of autoimmunity. C1q binds immune complexes by its 6 globular domains while it binds a variety of other ‘‘nonimmune’’ activators of the complement system, like C-reactive protein by its collagen-like region (CLR) (Kallenberg, 2008). C1q along with its physiological role in maintenance of homeostasis is involved in pathological conditions associated with the generation of anti-C1q autoantibodies in which CLR of C1q is the target of anti-C1q antibodies (Stoyanova et al., 2011). The liver is the major site for biosynthesis of 80–90% plasma complement components, and expresses a variety of complement receptors. The complement and complement receptors appear to be involved in liver injury and repair. However, the underlying mechanisms remain poorly understood (Qin & Gao, 2006). Anti-C1q antibodies are highly prevalent in prototypical autoimmune disease, systemic lupus erythematosus (SLE), exhibiting strong correlation with lupus nephritis (Hu et al., 2013; Mahler et al., 2013; Trouw et al., 2004; Zhang et al., 2011). These antibodies are also detected in hypocomplementemic urticarial vasculitis, rheumatoid vasculitis, IgA nephropathy, anti-glomerular basement membrane (anti-GBM) nephropathy, and HIV infection (Mahler et al., 2013). Similarly, autoantibodies are frequently found in patients with viral infections including Epstein-Bar (EB) virus, measles virus, and herpes simplex virus before the discovery of hepatitis C virus (HCV) (Himoto & Masaki, 2012). A growing body of evidence suggests that the complement system is also involved in the pathogenesis of a variety of liver disorders (Qin & Gao, 2006). Therefore, we investigated the prevalence and clinical significance of anti-C1q Abs in plasma of patients with HCV genotype IV in the presence and absence
Anti-C1q in HCV-associated rheumatologic manifestations
of autoimmune extra hepatic manifestations in comparison to normal healthy individuals.
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PATIENTS AND METHODS Patients’ assessment Fifty-one chronic HCV patients with rheumatological autoimmune manifestations were selected from the Department of Rheumatology, Cairo University hospitals. Inclusion criteria for HCV patients were (a) serum positive tests for anti-HCV antibodies and HCV-RNA; (b) absence of liver cirrhosis, concomitant HBV, HIV infections and hepatocellular carcinoma (through histology, laboratory evidence of liver failure and/or ultrasound-proven portal hypertension); and (c) exclusion of concomitant autoimmune diseases (rheumatoid arthritis, connective tissue diseases, etc.) HCV patients who received interferon therapy were excluded from the study. The recorded data of rheumatologic manifestations for every patient was as follows, arthralgia; arthritis; vasculitis, sicca manifestations; pulmonary; renal; haematological, lymphoproliferative disease, and neurological (peripheral and/or central nervous system manifestations). Each of the 51 HCV patients with autoimmune rheumatologic manifestations included in this study was matched by gender and age with HCV patients without such manifestations. Only patients without liver cirrhosis, hepatocellular carcinoma, and concomitant autoimmune diseases were included. All chronic HCV participants were subjected to careful historical interview and complete clinical examination. Demographic, clinical, immunochemical, and virological parameters were collected for each patient. Forty healthy subjects, sero-negative for HCV, matched for age and gender fulfilling the same exclusion criteria applied for HCV patients, were included as a healthy control group. The study was approved by our institutional ethics committee and informed consent was obtained from all patients. Laboratory and immunological investigations Virological studies Anti-HCV antibodies and HCV RNA were determined on serum clotted and centrifuged at 37 C and stored at 70 C, respectively. HCV antibody assayed using 3rd-generation enzyme linked immunosorbent assay (ELISA) (Abbott-Murex Biotech, Dartford, UK) and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) (Promega Co., MA, USA) Genotyping was performed using Murex HCV Serotyping ELISA Kits (Abbott-Murex Biotech, Dartford, UK). Hepatitis B surface antigens (HBsAg) (Sorin Biomedica Co., Italy) were assessed using commercially available kits. HIV antibody was assayed using 3rd generation Genscreen HIV 1/2 version 2 ELISA Kit (Bio-Rad Laboratories, France). Rheumatoid factor (RF) detection RF was assessed by the latex fixation method. A suspension of uniform polystyrene particles sensitized in glycine buffer with heat altered human IgG (BD Diagnostic Systems, Sparks, Maryland, USA) was incubated
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with progressive dilutions of human sera in microtitre wells. After incubation, the plates were inspected for observable agglutination.
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Cryoglobulin detection Venous blood was taken from fasting patients at 37 C, allowed to clot, and then separated by centrifugation. After centrifugation, the supernatant was removed from the serum, incubated at 4 C for 8 days, and examined daily for cryoprecipitation. Plasma Anti-C1q Abs levels determination Blood samples were collected into pyrogen free, EDTA- tubes, allowed to clot at 4 C for 1 h, and then centrifuged at 2000 g for 10 min. The plasma obtained was divided, and then aliquots were stored at 70 C until assayed on the same day to eliminate day to day interassay variability. A commercial ELISA kit (ORGENTEC Diagnostika GmbH, Mainz, Germany) was used for assaying Anti-C1q ab levels. In this assay a 96-well plate was coated with human C1q which was then used as the antigen to which the Anti-C1q autoantibodies can bind. In brief, each plasma sample was diluted in a highsalt buffer in order to prevent the binding of immune complexes then transferred to the wells of the ELISA plate and assayed according to the manufacturer’s instructions. The optical densities were measured at 450 nm converted into units (U/ml) by being plotted against the autoantibody concentration of the standards given by the manufacturer. The lower detection limit for the Anti-C1q test was determined at 0.5 U/ml. Values greater than 10 U/ml were considered positive for Anti-C1q antibodies as recommended by the manufacturer’s protocol. Statistical analysis The Statistical Package for Social Sciences (SPSS) version 15 (LEAD Technology Inc., Charlotte, NC, USA) was used to analyze the data. Continuous variables were summarized through the median (range) and categorical variables using absolute values and percentages. Mann–Whitney U-test was used to compare two independent groups and Kruskal–Wallis test was adopted for comparing more than two groups. Associations between categorical groups were tested using the Chi-square test ( 2) with Yates correction or Fisher’s exact test as appropriate. Spearman’s rank correlation test was used as a measure of association of quantitative variables. For those variables that were significantly influencing Plasma Anti-C1q levels (p50.05), a standard linear multiple regression analysis for Anti-C1q levels as dependent variable was performed. For the regression model, adjusted R2, Beta, b-value and standard error b were recorded.
RESULTS Patients characteristics The demographic, clinical, virological and laboratory parameters of the HCV-infected patients with and without autoimmune rheumatologic involvement are reported in Table 1. ESR were significantly higher and platelet count were lower in patients with rheumatologic manifestations (p = 0.025 and
Anti-C1q in HCV-associated rheumatologic manifestations
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Table 1. Clinical, demographic and laboratory characteristics in HCV patients with and without rheumatologic involvement.
Variable
HCV with rheumatological involvement N = 51
HCV without rheumatological involvement N = 40
Age (years) Female/Male ESR (mm/h) Hemoglobin (g/dl) Leucocytes (103/ml) Platelets (103/ml)) C3 (mg/dl) C4 (mg/dl) Creatinine (mg/dl) ALT (u/L) AST(u/L) Serum albumin (g/dl) HCV-RNA* 105 (IU/mL) Metavir Activity score A0 A1 A2 Metavir fibrosis score F0 F1 F2 F3
50 (19–65) 38/13 40 (10.00–150.00) 11.50 (6.30–15.00) 5.20 (2.40–61.00) 214 (8.00–890.00) 142.2 (21.45–309.14) 21.71 (4.68–74.49) 0.80 (0.30–5.30) 40.00 (5.00–173.00) 40.00 (9.00–125.00) 3.60 (1.50–4.50) 0.76 (0.00–16.61) No (%) 4 (7.84%) 19 (37.25%) 28 (54.9%) No (%) 2 (3.92%) 22 (43.14%) 13 (25.49%) 14 (27.45%)
50.50 (20–65) 26/14 36 (15.00–68.00) 12.00 (8.80–15.80) 5.30 (1.62–9.90) 244 (10.00–435.00) 118.79 (21.45–287.17) 24.88 (4.68–118.63) 0.90 (0.40–3.00) 36.00 (18.30–304.00) 37.00 (13.50–424.00) 3.65 (2.20–5.00) 0.09 (0.01–45.66) No (%) 5 (12.5%) 18 (45%) 17 (42.5%) No (%) 0 (0%) 17 (42.5%) 16 (40%) 7 (17.5%)
p Value 0.644 0.361 0.025* 0.084 0.083 0.045* 0.179 0.267 0.423 0.383 0.234 0.090 0.295 0.224
0.934
Continuous variables: Median (range); categorical variables: absolute value (%).Mann– Whitney U-test was used to compare continuous variables & 2 or Fisher exact test were used to compare frequencies; AST, aspartate transaminase; ALT, Alanine transaminase; *p50.05.
0.045, respectively). However, no statistically significant differences were found among all the other mentioned variables in the table between HCV patients with autoimmune rheumatologic involvement compared to those without these manifestations. All our patients had HCV genotype IV and none of them had either cirrhosis, history of alcohol consumption, HBV, or HIV. Anti-C1q Abs levels in studied population Anti-C1q antibodies (values greater than 10 U/ml) were detected in 16 HCV patients with extra-hepatic autoimmune involvement (31.37%), 6 HCV patients without such involvement (15%), and in only 3 healthy control subjects (7.5%) with a high statistical significant difference (p50.001). The median plasma Anti-C1q levels were higher in HCV patients with autoimmune rheumatologic manifestations (5.74 U/ml; range 1.01–52.27) than their levels in HCV patients without such manifestations (3.42 U/ml; range 0.16–58.24) and in the healthy control group (4.02 U/ml; range 0.60–27.50, Kruskal–Wallis test p = 0.038). Using the Mann–Whitney U tests, it was shown that Anti-C1q levels exhibited a statistical significant difference in HCV patients with autoimmune rheumatologic manifestations when compared to HCV patients without such
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Figure 1. Plasma Anti-C1q levels in HCV patients with and without rheumatic involvement and Healthy controls. The lines inside the boxes indicate the median; the outer borders of the boxes indicate 25th and 75th percentiles; the bars extending from the boxes indicate the 10th and 90th percentiles; the symbols above the bars represent the outliers.
manifestations and the healthy control groups (p = 0.012 and 0.029, respectively). On the other hand, there was no significant difference in Anti-C1q levels between patients with HCV without autoimmune manifestations vs healthy controls (p = 0.593). Figure 1 demonstrates the Anti-C1q levels in the different groups under study. Using Spearman rank correlation analysis (Table 2), Anti-C1q antibodies were found to be negatively correlated with C4 (r = 0.215, p = 0.040) and positively correlated with HCV RNA viral Load (r = 0.228, p = 0.029). On the other hand, age, serum albumin, aminotransferases and C3 didn’t reveal any statistical significant correlation with Anti-C1q levels. Furthermore, no significant correlations were found between Anti-C1q levels with either the METAVIR activity or the fibrosis scores (p40.05). Association of plasma Anti-C1q ab levels with clinical and laboratory parameters in HCV-associated rheumatologic involvements HCV-associated rheumatologic involvements are presented in Table 3. Articular involvement was the most common clinical feature reported by this group of patients (53%) followed by vasculitis in 17 patients (33.3%). Further analysis was performed in order to compare the levels of plasma Anti-C1q ab in the presence and absence of some clinical manifestations (Table 3). As illustrated through the table, plasma Anti-C1q levels were significantly higher in patients with positive cryoglobulin (p = 0.001), and in those with
Anti-C1q in HCV-associated rheumatologic manifestations Table 2. Correlations of Anti-C1q antibodies level with laboratory parameters and liver scores of activity and fibrosis in HCV infected patients.
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Parameter
r
ESR (mm/1st hr) Hemoglobin (g/dl) Leucocytes (103/ml) Platelets (103/ml)) C3 (mg/dl) C4 (mg/dl) ALT (u/L) AST(u/L) Serum albumin (g/dl) Creatinine (mg/dl) HCV-RNA* 105(IU/mL) Metavir activity score Metavir fibrosis score
p
0.082 0.131 0.023 0.141 0.054 0.215 0.019 0.029 0.038 0.054 0.228 0.134 0.055
0.438 0.217 0.829 0.182 0.610 0.040* 0.855 0.788 0.722 0.610 0.029* 0.205 0.603
AST, aspartate transaminase; ALT, Alanine transaminase; C, complement; *p50.05.
Table 3. Comparison of the plasma anti-C1q levels in HCV patients with autoimmune involvement in the presence and absence of some clinical manifestations and autoantibodies. Patients Affected Clinical feature
No.
Arthritis Vasculitis Skin rash Neuropathy Nephritis Hematological Sjogren Syndrome Lymphoma RF +ve ANA +ve Cryoglobulin +ve
27 17 14 16 5 13 5 5 23 18 16
Median (range) 5.37 6.42 5.87 4.98 6.05 6.43 3.60 14.3 6.5 4.90 11.70
(1.01–20.95) (1.01–20.95) (2.82–20.95) (1.01–20.95) (4.47–19.67) (2.53–52.27) (1.01–52.27) (12.35–52.27) (1.01–45.96) (2.55–16.16) (2.53–45.96)
Patients Unaffected No.
Median (range)
p Value
24 34 37 35 46 38 46 46 28 33 35
6.03 (1.06–52.27) 5.32 (1.06–52.27) 5.37(1.01–52.27) 6.01 (1.06–52.27) 5.31 (1.01–52.27) 5.12 (1.01–20.95 5.87 (1.01–52.27) 5.19 (1.01–45.96) 4.50(1.06–52.27) 6.00 (1.01–52.27) 3.75 (1.01–52.27)
0.85 0.83 0.90 0.53 0.33 0.13 0.41 0.002* 0.056 0.844 0.001*
Data are Median (range),*p50.05. Mann–Whitney U-test was used; *p50.05; ANA, antinuclear antibodies; RF, rheumatoid factor.
lymphoma (p = 0.002) than in those without. On the other hand, we did not find significant differences in Anti-C1q levels in relation to other organ involvement or with positive RF or ANA. To confirm these results, we performed a multivariate analysis to estimate the independent association of each covariate with Anti-C1q as the dependent variable against the variables that yielded p values 50.05 according to the univariate analysis among the group of patients with autoimmune rheumatic involvement. The model revealed that the presence of lymphoma had the highest impact (b = 15.36, Beta = 0.470, p50.0001) followed by the presence of cryoglobulins
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(b = 7.637, Beta = 0.364, p = 0.004). On the other hand, C4 levels (b = 0.117, Beta = 0.169, p = 0.161) was not significant in this model (b = 0.742, Beta = 0.038, p = 0.835). The coefficient of determination (R2) for the generated model was 0.352 implying that these two factors explain almost 35% of the variability in plasma Anti-C1q levels among HCV patients with autoimmune extra hepatic involvement.
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DISCUSSION Chronic HCV infection frequently leads to autoimmune response including the production of autoantibodies and the coincidence of autoimmune diseases (Himoto & Masaki, 2012). Recently, anti-C1q antibodies are demonstrated to be pathogenic in the target organ damage of many autoimmune diseases, by facilitating C1q deposition and enhancing complement activation via classical pathway (Hu et al., 2013; Potlukova & Kralikov, 2008). The anti-C1q antibodies have been shown to be associated strongly with immune complex diseases, most prominently with hypocomplementaemic urticarial vasculitis syndrome, systemic lupus erythmatosus, Behcet’s disease, and severe rheumatoid arthritis (Bassyouni et al., 2014; El-Hewala et al., 2011; Yang et al., 2012). In this study, we showed that anti-C1q antibodies was found in 31.37% of the chronically HCV-infected patients with autoimmune extrahepatic manifestations compared to 15% in chronically HCV-infected patients without these manifestations and 7.5% of the healthy control. Our study shows that prevalence of anti-C1q was comparable with that previously reported in Caucasian populations. Saadoun and coworkers (2006) have detected anti-C1q antibodies in 26% of their HCV patients compared to 10% of healthy donors. Similarly, Anti-Clq antibodies have been detected in 38% of HCV patients compared with 2% of healthy controls in another study (Lienesch et al., 2006). This increased production of anti-C1q antibodies might be a part of the widerange, exaggerated autoantibody produced against autoantigens, characteristic of HCV. The production of these antibodies is indicative of a heightened immune reactive state in which epitope spread and molecular mimicry have resulted in multiple serological reactivities, and concurrent immune disorders (Bianchi et al., 2007). Approximately 25–30% of patients with chronic HCV are positive for non-organ specific auto-antibodies, i.e. antinuclear antibodies, smooth muscle antibodies, and liver-kidney microsomal antibody type 1 (Indolfi et al., 2012). It has been reported that HCV-encoded core protein interacts directly with the receptor for the globular domain of C1q protein (gC1q-R) representing an efficient way to affect the host T- and B-cell immunity (Stoyanova et al., 2011). Herein, we further analyzed association of anti-C1q antibodies levels with the presence and absence of different autoimmune extrahepatic involvements. Cryoglobulins were found among 31.4% those HCV patients. HCV infection is one of the major causes of mixed cryoglobulinaemia (Lauletta et al., 2012). Anti-C1q antibodies associated significantly with the presence of cryoglobulins in our HCV patients. Indeed, the IgM–IgG complexes as found in MC are good receptors for C1q. Furthermore, it has been reported that C1q protein and C1q binding activity are enhanced in the cryoprecipitates of HCV-infected patients.
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Anti-C1q in HCV-associated rheumatologic manifestations
The wide expression of C1q receptor on the surface of blood and endothelial cells enhance their specific binding to immune complexes containing HCV core protein which may represent an important pathogenetic mechanism in the cryoglobulin-related pathway (Lauletta et al., 2012). The formation of neoantigens within the C1q molecule after the binding of C1s and C1r to C1q has been documented. The presence of C1q-cryoglobulin complexes enhance the generation of autoantibodies against C1q, anti-C1q antibodies are directed against a neoepitope that is expressed in bound form of C1q (Saadoun et al., 2006). Among the numerous extrahepatic manifestations of HCV, the interaction of the virus with B-cells is a major concern of chronic HCV infection which might undergo an oncogenic event giving survival advantage to a subclonal population, resulting in lymphoproliferative disorder (Bachy et al., 2010; Yao et al., 2011). Lymphoma was present in 9.8% of our HCV patients with auto immune manifestations. An important result of our study was the association of anti-C1q antibodies with lymphoma. Cytokines and growth factors produced by chronic infection are suspected to play a key role in B-cell transformation. Growing body of evidence has clarified the role of B-cell activating factor (BAFF) in regulating B-cell survival, proliferation, and differentiation into immunoglobulin-secreting cells (Lauletta et al., 2012). BAFF deregulation has been demonstrated in HCV infection as well as B-cell lymphoproliferative disorders (De Re et al., 2012; Se`ne et al., 2007; Sun et al., 2008), which might explain the enhancement of autoantibodies production including anti-C1q in lymphoma in case of HCV infection. In this study, we did not find a significant association between anti-C1q antibodies with other extra hepatic autoimmune manifestations. HCV infection is a risk factor for proteinuria and/or impaired renal function (Morales et al., 2012). Although Anti-C1q antibodies have been best described in patients with lupus nephritis (Akhter et al., 2011); however, there was no association between anti-C1q with renal involvement in our HCV patients. Several mechanisms other than immune complex deposition in the glomeruli have been found in the pathogenesis of renal affection associated with chronic HCV infection (Morales et al., 2012). Direct mesangial injury by HCV infection has been documented (Barsoum, 2007). HCV RNA and related proteins have been found in mesangial cells, tubular epithelial cells, and endothelial cells of glomerular and tubular capillaries. Recently, it has been described that toll-like receptor-3 (TLRs), primary proteins expressed on immune and nonimmune cells as key components of the innate immune system, could have a pathogenic role establishing a link between viral infection and glomerulonephritis (Morales et al., 2012). Another possible mechanism underlying HCV-related kidney injury is nonimmunologically mediated. HCV-positive patients have elevated levels of fasting serum insulin and insulin resistance, and higher prevalence of diabetes (Sarafidis & Ruilope, 2006). Anti-C1q antibodies negatively correlated with C4 in our studied HCV patients. Most HCV–MC patients showed decreased levels of the early complement components C1, C4 and C2, whereas C3 levels fluctuated with the disease course. Experiments performed to define the mechanism
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responsible for this complement profile showed that activation of the early complement components in serum was due to the activation of the classical pathway by mixed cryoglobulins (Saadoun et al., 2006). Herein, no significant correlations were found between anti-C1q antibodies levels with histopathological grading of liver biopsy assessed by METAVIR activity and fibrosis scores as reported in a previous study (Saadoun et al., 2006). In conclusion, this study has demonstrated an increased prevalence of antiC1q antibodies in HCV infected patients (type IV) with autoimmune extrahepatic manifestations especially patients with positive cryoglobulinaemia and lymphoma. Anti-C1q autoantibodies could be a potential candidate for a biomarker panel for lymphoma diagnosis. Indeed, by using a cross-sectional study design we cannot rule out that excess circulating Anti-C1q is a consequence rather than a cause of those associations. We also couldn’t determine if the up-regulation of anti-C1q in our HCV patients with autoimmune involvement is pathogenic, an epiphenomenon of aberrant tissue damage, or compensatory to an uncontrolled immune response. Furthermore, the autoimmune manifestations in our HCV group are highly heterogeneous with a small number of patients. Further prospective large scale, longitudinal studies are needed to elucidate their precise pathogenic role and their clinical relevance in chronic HCVinfected patients.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Anti-C1q in HCV-associated rheumatologic manifestations
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