- Email: [email protected]
Anti-C-reactive protein antibodies in chronic hepatitis C infection: Correlation with severity and autoimmunity
Human Immunology (2007) 68, 844 – 848
Anti-C-reactive protein antibodies in chronic hepatitis C infection: Correlation with severity and autoimmunity Aharon Kessela,*, Ghadir Eliasa, Elsa Pavlotzkya, Eli Zuckermanb, Itzhak Rosnerc, Elias Toubia a b c
Division of Allergy and Clinical Immunology, Bnai Zion Medical Center, Faculty of Medicine, Technion, Haifa, Israel Department of Medicine A, Hepatology Unit, Bnai Zion Medical Center, Faculty of Medicine, Technion, Haifa, Israel Rheumatology Department, Bnai Zion Medical Center, Faculty of Medicine Technion, Haifa, Israel
Received 9 June 2007; received in revised form 10 June 2007; accepted 10 June 2007
KEYWORDS Anti-CRP antibodies; HCV; Cryoglobulinemia; RF
Summary The aim of this work was to detect circulating anti-C-reactive protein (CRP) antibodies in serum samples of patients with chronic hepatitis C virus (HCV) and to investigate a possible association with other autoimmune manifestations. A total of 94 patients with chronic HCV infections and 108 healthy controls were enrolled. All patients underwent a baseline evaluation: immunological assessment of cryoglobulin,antinuclear antibodies (ANA), rheumatoid factor (RF), anticardiolipin (aCLA), and anti-CRP antibodies. Patients with HCV underwent a liver biopsy scored according to the modified Knodell score. Anti-CRP antibodies were detected in 17% of HCV patients compared with 6.4% of the healthy controls (p ⬍ 0.025). When HCV patients positive for anti-CRP antibodies were compared with patients who were negative for anti-CRP antibodies, the prevalence of positive RF was significantly higher, 50% versus 17.9% (p ⱕ 0.05). Cryoglobulinemia was also significantly more frequent in patients who were positive for anti-CRP antibodies, 75% versus 32%, p ⱕ 0.01. ANA and aCLA did not differ significantly between the two groups. The presence of anti-CRP antibodies was associated with greater liver disease severity (histology activity index, 9 ⫾ 3.3 versus 6 ⫾ 2.9, p ⫽ 0.01). An increased prevalence of anti-CRP antibodies was manifested in HCV-infected patients. The presence of antiCRP antibodies correlated with the presence RF, cryoglobulinemia, and severity of liver disease. © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Introduction Hepatitis C virus (HCV) infection is one of the most common causes of chronic liver disease worldwide, affecting between 0.5% and 2% of the population of the Western world * Corresponding author. Fax: 97248359659. E-mail address: [email protected] (A. Kessel).
[1]. The majority of HCV-infected individuals do not resolve their infection, leading to the development of chronic hepatitis. The additional lymphotropism of HCV may be responsible, at least in part, for multiple immune-mediated extrahepatic manifestations of HCV infection, such as mixed cryoglobulinemic vasculitis and Sjögren-like syndrome, as well as the production of autoantibodies against a variety of self-antigens such as antinuclear (ANA), antiextractable nu-
0198-8859/$ -see front matter © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2007.06.008
845
Anti-CRP antibodies in chronic hepatitis C
ABBREVIATIONS aCLA ANA CRP ELISA ENA HAI HCV RF SLE SLEDAI
anticardiolipin antibodies antinuclear antibodies C-reactive protein enzyme-linked immunosorbent assay extractable nuclear antigen histology activity index hepatitis C virus rheumatoid factor systemic lupus erythematosus systemic lupus erythematosus activity index
clear antigen (ENA), anticardiolipin (aCLA), and anti-C1q antibodies [2–11]. C reactive protein (CRP) is one of the acute-phase proteins increased during systemic inflammation. It is produced primarily in the liver in response to infection, noninfectious systemic inflammation, and trauma. CRP may be particularly important in the protection against certain invasive bacterial mucosal pathogens, such as pneumococcus, meningococcus, and some H. influenzae strains, in the period preceding emergence of antibody-mediated opsonizing or batericidal activity [12]. In contrast to the importance of CRP in bacterial infection, its role in viral infection is still obscure. In chronic hepatitis C, levels of some acute-phase protein, such as C5b-9 levels, CH50, specific C4 activity, and CRP were lower than controls. Interestingly, some of these changes were reversible in patients who responded to HCV treatment [13–15]. In 1985 Robey et al. described autoantibodies against CRP in a patient with systemic lupus erythematosus (SLE ) [16]. Sjowall et al. detected antibodies to CRP in 36% of SLE patients, but no significant correlation between serum CRP levels and anti-CRP antibodies was present [17]. In other studies, anti-CRP antibodies were present in 78% and 23% of patients with SLE [18,19], with the difference frequency of anti-CRP in these studies probably a result of different detection methodologies used. Anti-CRP antibodies were also detected in the serum of patients with rheumatoid arthritis, primary biliary cirrhosis, and scleroderma [18]. The objective of the present study was to analyze circulating anti-CRP antibodies (Abs) in serum samples of patients with chronic HCV and to reveal a possible association between anti-CRP Abs and other autoimmune manifestations in these patients.
Subjects and methods Ninety-four consecutive patients suffering from chronic HCV infection, followed at the Liver Clinic of the Bnai Zion Medical Center (51 females and 43 males, mean age 53 ⫾ 22.9 years (range 19 to 82 years), were enrolled in the study. All patients were positive for anti-HCV antibodies, had detectable HCV RNA, were untreated at the time of survey, and had not previously received any antiviral treatments. The patients underwent a liver biopsy with stage of fibrosis and grade of activity determined (histology activity index (HAI), Knodell score). The Knodell score is composed of the summa-
tion of four individual scores representing periportal or bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation, and fibrosis. The score ranges from 0 to 22. Patients who had positive serological tests for HIV or hepatitis B surface antigen were excluded. All patients underwent a baseline evaluation including screening for rheumatoid factor (RF), cryoglobulins, and a panel of autoantibodies including ANA, aCLA, and anti-CRP antibodies. A control group, matched for age and sex, consisting of 108 individual members of the hospital staff was evaluated for the presence of anti-CRP. The staff routinely undergoes blood studies to exclude systemic and infectious diseases. The exclusion criteria for controls were liver disease, collagen or autoimmune diseases, chronic diseases, malignancy, and any other concurrent or previous illness known to affect liver function. The study was approved by the hospital’s ethics committee.
Autoantibody detection Antibodies to CRP were determined by an enzyme-linked immunosorbent assay (ELISA) protocol described by Rosenau and Schur [19]. Human CRP (Sigma C-4063) was diluted to 10 g/ml in phosphate-buffered saline (PBS) and coated onto Maxisorp ELISA plates (Nunc, Roskilde, Denmark) at room temperature overnight. The plates were washed with PBS-Tween (PBST) and blocked with blocking buffer (1% bovine serum albumin (BSA) in PBST) for 1 hour. The blocking solution was removed and 1:50 diluted plasma samples in duplicate were added into wells coated with antigen and duplicates were added into wells coated only with blocking buffer to control for unspecific binding. After 3 hours the plate was washed and goat anti-human IgG alkaline phosphatase (Sigma, St. Louis, MO, A-3150; diluted 1:1000) was added to each well. After 1 hour and another wash the assay was developed using 1 mg/ml p-nitrophenyl phosphate (Sigma N2765) in glycine buffer and the absorbance was read at 450 nm with a reference of 630 nm. Eighty blood donors were tested by this method to determine the mean OD ⫾ 2 SD for healthy individuals. Levels of anti-CRP above 2 SD were considered positive. The detection of antinuclear antibodies in the patients’ serum was assayed by indirect immunofluorescence using HEp-2 cells (Alphadia, Wavre, Belgium). Serum was considered positive for HEp-2 when immunofluorescence staining was observed at a dilution titer of ⱖ1:40. Rheumatoid factor was assayed by the 2-minute hemagglutination slide test (Wampole Laboratories, Princeton, NJ). Serum was considered positive for RF when agglutination was observed in a dilution titer of ⱖ1:10. Anticardiolipin antibodies were analyzed using a commercial ELISA kit (Genesis Diagnostics, Cambridgeshire, UK). The cutoff for a positive test was IgG ⱖ 15 unit/ml and IgM ⱖ 10 unit/ml. Cryoglobulin detection and analysis. After an overnight fast, patients’ venous blood samples were collected into prewarmed tubes and were allowed to clot at 37°C. After centrifugation and separation, the sera were incubated at 4°C for 3 days. The cryocrit was determined by centrifugation of the cooled serum in hematocrit tubes at 4°C. The cryoprecipitates were further analyzed and characterized by immunofixation electrophoresis (Immunofix kit; Helena Laboratories, Beaumont, TX). Anti-HCV antibody was detected by ELISA (Abbot Laboratories, North Chicago, IL), with HCV RNA detected by polymerase chain reaction (PCR) assay (Amplicor Roche Molecular Systems, Somerville, NJ).
Statistical analysis For comparisons of qualitative values a 2 test was used, whereas an unpaired Student t-test was used for quantitative values. Two-
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A. Kessel et al.
tailed p values of 0.05 or less were considered statistically significant.
Results Anti-CRP antibodies were detected in 17% (16/94) of HCV patients compared with 6.4% (7/108) of healthy controls (p ⬍ 0.025). Among HCV patients, 50% (47/94) had at least one autoantibody ( ANA, RF, or aCLA). Specifically, 21.2% (20/94) had a positive ANA, 15.9% (15/94) had a positive aCLA, and 28.7% (27/94) had RF. A significantly higher frequency of anti-CRP antibodies was evident among HCV patients who had at least one autoantibody (ANA, RF, or aCLA) present compared with patients without autoimmune autoantibodies ( 27.9% versus 8.6%, p ⱕ 0.025). When patients with positive anti-CRP antibodies were compared with patients who were negative for anti-CRP antibodies, the prevalence of positive RF was significantly higher (50% versus 24.3%, p ⱕ 0.05). Cryoglobulinemia was also significantly more frequent in patients who were positive for anti-CRP antibodies (75% versus 32%, p ⱕ 0.01; Figure 1). Autoimmunity (the presence of ANA, RF, or aCLA) was significantly more frequent in patients who were positive for anti-CRP antibodies compared with patients without anti-CRP antibodies (75% versus 42%, p ⱕ 0.025; Figure 2). The presence of ANA and aCLA did not differ significantly between patients with positive or negative anti-CRP antibodies (Table 1). There was no significant difference between the ages and gender of patients with and without anti-CRP (Table1). On evaluation of liver histology, 18% of patients (9/50) had cirrhosis. The presence of anti-CRP antibodies was associated with more active liver disease (HAI 9 ⫾ 3.3 versus 6 ⫾ 2.9, p ⫽ 0.01). The presence of cirrhosis did not differ significantly between patients with positive or negative anti-CRP antibodies (20% versus 17.5%).
Discussion In the present study anti-CRP antibodies were observed in the serum of a significant proportion of patients with HCV
Figure 1. Cryoglobulinemia was significantly more frequent in patients with positive anti-CRP antibodies compared with patients with negative anti-CRP antibodies, 75% versus 32%, p ⱕ 0.01.
Figure 2. Among patients with positive anti-CRP antibodies the frequency of autoimmunity (the presence of ANA, RF, or aCLA) was statistically higher compared with patients without anti-CRP antibodies (75% versus 42%, p ⱕ 0.025).
infection. This finding correlated with the presence of autoantibodies detected in the sera of these patients [20]. It is not clear whether the presence of anti-CRP autoantibodies in HCV patients is an epiphenomenon, whether it is a part of nonspecific antibody production by virus activated B cells, or whether it actually reflects events of specific pathogenetic interest, just as the presence of other antibodies reflects active autoimmune pathologic processes in the context of HCV infection, i.e., vasculitis and arthritis. Consideration must be given to the putative additional significance of antiCRP antibodies in the context of HCV infection. The primary stimuli for CRP production are proinflammatory cytokines, especially interleukin (IL)-6, IL-1, and tumor necrosis factor ␣. CRP binds autologous ligands including modified plasma lipoproteins, damaged cell membranes, various phospholipids, small nuclear ribonucleoprotein particles, apoptotic cells, and extrinsic ligands, such as many glycan phospholipids and additional constituents of microorganisms, such as capsular and somatic components of bacteria, fungi, and parasites. Bound CRP then recognizes C1q and activates the classic complement cascade engaging C3 and the terminal membrane attack complex, C5-C9 [21]. CRP-mediated complement activation is initiated by binding of one molecule C1q to two adjacent molecules of CRP associated with a ligand [22]. In animal models CRP modulated inflammation. CRP influenced the course of autoimmune disease in lupus-prone (NZB ⫻ NZW) F1 mice by decreasing antigenic stimulation and enhancing clearance of nuclear antigens [23]. On the other hand, CRP was also protective in nephrotoxic nephritis, an immune complex nephritis model that does not involve autoantibodies [24]. The significance of anti-CRP antibodies in specific rheumatic diseases is not clear. However, Sjowall et al. demonstrated a positive correlation among SLE disease activity (SLE activity index [SLEDAI] score), anti-DNA antibodies, and serum levels of anti-CRP antibodies, respectively. Inverse correlations were demonstrated between anti-CRP antibody levels and C1q. Further-
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Anti-CRP antibodies in chronic hepatitis C Table 1
Comparative analysis of HCV-infected patients with or without anti-CRP autoantibodies.
Age (years ⫾ SD) Female/male ANA aCLA RF Cryoglobulin Histology activity disease (HAI ⫾ SD) Cirrhosis *
Anti-CRP positive (n ⫽ 16)
Anti-CRP negative (n ⫽ 78)
p value
57 ⫾ 14 8/8 6/16 2/16 8/16 9/12 9 ⫾ 3.3 (n ⫽ 9)* 2/10
53 ⫾ 16 46/32 14/78 13/78 19/78 15/47 6 ⫾ 2.9 (n ⫽ 41)* 7/41
NS NS NS NS ⱕ0.05 ⱕ0.01 ⱕ0.01 NS
Number of patients who underwent liver biopsy.
more, in patients with active lupus nephritis anti-CRP positivity was manifested at flare [25]. C-reactive protein may mediate protection against autoimmune disease by enhancing clearance of nuclear antigens expressed on apoptotic cells. The presence of anti-CRP might disturb this process, thus increasing the exposure of autoantigens to the immune system and thereby promoting the development of autoantibodies. Specifically in the case of HCV infection, increased apoptosis of T and B (CD19⫹CD5⫺) lymphocytes in the presence of anti-CRP may prolong exposure of the nuclear antigens to (CD19⫹CD5⫹) B lymphocytes and thereby increase the possibility of autoantibody formation, consistent with the finding of the present study [26,27]. It should be emphasized that anti-CRP antibodies are directed against the monomeric (modified) form of CRP, i.e., epitopes that are hidden in the pentameric structure of CRP. Additionally, Motie et al. demonstrated that modified CRP binds immune complexes specifically and might therefore be involved in removal of immune complexes [28]. This observation may explicate our finding that HCV-infected patients with anti-CRP have higher levels of cryoglobulins, which are largely cold insoluble immune complexes. The finding that presence of anti-CRP is associated with more severe liver disease is consistent with previous reports in large prospective cohorts of HCV-infected patients, wherein markers of autoimmunity, particularly mixed cryoglobulins, correlated with extent of liver damage severity [29]. The effect of anti-CRP on HCV liver damage may be mediated via its interaction with complement. The role of complement system in enhancing neutralization of antibody-bound virus by several mechanisms, including direct lysis of enveloped viruses, has been demonstrated [30,31]. Meyer et al. specifically demonstrated the importance of the complement system in the clearance of antibodies to the HCV E2 glycoprotein, which play a central role in viral entrance to mammalian cells [32]. The presence of anti-CRP antibodies may possibly attenuate this process by inhibition of complement activation, leading to viral persistence. Thus, the association between anti-CRP positivity and liver disease activity in the present study is analogous to the correlation between SLEDAI score and levels of anti-CRP in serum of SLE patients. In conclusion, the increased prevalence of anti-CRP antibodies in HCV-infected patients was documented for the first time in the present study. The presence of anti-CRP antibodies is associated with the presence RF, cryoglobu-
linemia, and increased severity of liver disease in these patients.
References [1] Hoofnagle JH, Tralka TS. Management of hepatitis C virus. Proceedings of the National Institutes of Health Consensus Development Conference. Hepatology 1997;26(Suppl. 1):S1. [2] Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 1992;327: 1490. [3] Marcellin P, Descamps V, Marinot-Peignoux M, Larzul D, Xu L, Boyer N, et al. Cryoglobulinemia with vasculitis associated with hepatitis C virus infection. Gastroenterology 1993;104:272. [4] Misiani R, Bellavita P, Fenili D, Borelli G, Marchesi D, Massazza M, et al. Hepatitis C infection in patients with essential mixed cryoglobulinemia. Ann Intern Med 1992;117:573. [5] Haddad J, Deny P, Munz-Gotheil C, Ambrosini JC, Trinchet JC, Pateron D, et al. Lymphocytic sialadenitis of Sjogren’s syndrome associated with chronic hepatitis C virus liver disease. Lancet 1992;339:321. [6] Clifford BD, Donahue D, Smith L, Cable E, Luttig B, Manns M, et al. High prevalence of serological markers of autoimmunity in patients with chronic hepatitis C. Hepatology 1995;21:613. [7] Gumber SC, Chopra S. Hepatitis C: a multifaceted disease. Ann Intern Med 1995;123:615. [8] Hadziyannis SJ. The spectrum of extrahepatic manifestations in hepatitis C virus infection. J Viral Hepatol 1997;4:9. [9] Pawlotsky JM, Roudot-Thoraval F, Simmonds P, Mellor J, Ben Yahia MB, Andre C, et al. Extrahepatic manifestations in chronic hepatitis C and hepatitis C virus serotypes. Ann Intern Med 1995;122:169. [10] Prieto J, Yuste JR, Beloqui O, Civeira MP, Riezu JI, Aguirre B, et al. Anticardiolipin antibodies in chronic hepatitis C: implication of hepatitis C virus as the cause of the antiphospholipid syndrome. Hepatology 1996;23:199. [11] Saadoun D, Sadallah S, Trendelenburg M, Limal N, Sene D, Piette JC, et al. Anti-C1q antibodies in hepatitis C virus infection. Clin Exp Immunol 2006;145:308. [12] Weiser JN, Pan N, Mcgowan KL, Musher D, Martin A, Richards J. Phosphorylcholine on the lipopolysaccharide of Haemophilus influenza contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein. J Exp Med 1998;187:631. [13] Bíró L, Varga L, Pár A, Nemesánszky E, Csepregi A, Telegdy L, et al. Changes in the acute phase complement component and IL-6 levels in patients with chronic hepatitis C receiving interferon alpha-2b. Immunol Lett 2000;72:69.
848 [14] Dumestre-Perard C, Ponard D, Drouet C, Leroy V, Zarski JP, Colomb MG. Complement C4 monitoring in the follow-up of chronic hepatitis C treatment. Clin Exp Immunol 2002;127:131. [15] Kalabay L, Nemesanszky E, Csepregi A, Pusztay M, Dávid K, Horváth G, et al. Paradoxical alteration of acute-phase protein levels in patients with chronic hepatitis C treated with IFN-␣2b. Int Immunol 2004;16:51. [16] Robey FA, Jones KD, Steinberg AD. C-reactive protein mediates the solubilization of nuclear DNA by complement in vitro. J Exp Med 1985;161:1344. [17] Sjöwall C, Eriksson P, Almer S, Skogh T. Autoantibodies to C-reactive protein is a common finding in SLE, but not in primary Sjögren’s syndrome, rheumatoid arthritis or inflammatory bowel disease. J Autoimmun 2002;19:155. [18] Bell SA, Faust H, Schmid A, Meurer M. Autoantibodies to C-reactive protein (CRP) and other acute-phase proteins in systemic autoimmune diseases. Clin Exp Immunol 1998;113: 327. [19] Rosenau BJ, Schur PH. Antibodies to C reactive protein. Ann Rheum Dis 2006;65:674. [20] Cacoub P, Renou C, Rosenthal E, Cohen P, Loury I, LoustaudRatti V, et al. Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d’Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l’Hepatite C. Medicine 2000;79:47. [21] Pepys MB, Hirschfield GM. C-reactive protein: a critical update. J Clin Invest 2003;111:1805. [22] Sjowall C, Wettero J, Bengtsson T, Askendal A, Almroth G, Skogh T, et al. Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction. Biochem Biophys Res Commun 2007;5;352:251. [23] Du Clos TW, Zlock LT, Hicks PS, Mold C. Decreased autoantibody levels and enhanced survival of (NZB X NZW) F1 mice
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treated with C-reactive protein. Clin Immunol Immunopathol 1994;70:2. Rodriguez W, Mold C, Kataranovski M, Hutt J, Marnell LL, Du Clos TW. Reversal of ongoing proteinuria in autoimmune mice by treatment with C-reactive protein. Arthritis Rheum 2005; 52:642. Sjowall C, Bengtsson AA, Sturfelt G, Skogh T. Serum levels of autoantibodies against monomeric C-reactive protein are correlated with disease activity in systemic lupus erythematosus. Arthritis Res Ther 2004;6:R87. Toubi E, Kessel A, Goldstein L, Slobodin G, Sabo E, Shmuel Z, et al. Enhanced peripheral T cell apoptosis in chronic hepatitis c virus infection: association with liver disease severity. J Hepatology 2001;35:774. Toubi E, Kessel A, Peri R, Shmuel Z, Bamberger E, Sabo E, et al. Enhanced apoptosis of peripheral CD5neg B-lymphocytes from chronic HCV infected patients: reversal after anti-viral treatment. J Virol 2004;78:11379. Motie M, Brockmeier S, Potempa LA. Binding of model soluble immune complexes to modified C-reactive protein. J Immunol 1996;156:4435. Saadoun D, Asselah T, Resche-Rigon M, Charlotte F, Bedossa P, Valla D, et al. Cryoglobulinemia is associated with steatosis and fibrosis in chronic hepatitis C. Hepatology 2006;43:1337. Akahane Y, Miyazaki Y, Naitoh S, Takeda K, Tsuda F, Okamoto H, et al. Cold activation of complement for monotoring the response to interferon in patients with chronic hepatitis C. Am J Gastroenterol 1996;91:319. Cooper NR. Complement evasion strategies of microorganisms. Immunol Today 1991;12:327. Meyer K, Basu A, Przysiecki CT, Lagging LM, Di Bisceglie AM, Conley AJ, et al. Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein. J Virol 2002;76: 2150.
Anti-C-reactive protein antibodies in chronic hepatitis C infection: Correlation with severity and autoimmunity Aharon Kessela,*, Ghadir Eliasa, Elsa Pavlotzkya, Eli Zuckermanb, Itzhak Rosnerc, Elias Toubia a b c
Division of Allergy and Clinical Immunology, Bnai Zion Medical Center, Faculty of Medicine, Technion, Haifa, Israel Department of Medicine A, Hepatology Unit, Bnai Zion Medical Center, Faculty of Medicine, Technion, Haifa, Israel Rheumatology Department, Bnai Zion Medical Center, Faculty of Medicine Technion, Haifa, Israel
Received 9 June 2007; received in revised form 10 June 2007; accepted 10 June 2007
KEYWORDS Anti-CRP antibodies; HCV; Cryoglobulinemia; RF
Summary The aim of this work was to detect circulating anti-C-reactive protein (CRP) antibodies in serum samples of patients with chronic hepatitis C virus (HCV) and to investigate a possible association with other autoimmune manifestations. A total of 94 patients with chronic HCV infections and 108 healthy controls were enrolled. All patients underwent a baseline evaluation: immunological assessment of cryoglobulin,antinuclear antibodies (ANA), rheumatoid factor (RF), anticardiolipin (aCLA), and anti-CRP antibodies. Patients with HCV underwent a liver biopsy scored according to the modified Knodell score. Anti-CRP antibodies were detected in 17% of HCV patients compared with 6.4% of the healthy controls (p ⬍ 0.025). When HCV patients positive for anti-CRP antibodies were compared with patients who were negative for anti-CRP antibodies, the prevalence of positive RF was significantly higher, 50% versus 17.9% (p ⱕ 0.05). Cryoglobulinemia was also significantly more frequent in patients who were positive for anti-CRP antibodies, 75% versus 32%, p ⱕ 0.01. ANA and aCLA did not differ significantly between the two groups. The presence of anti-CRP antibodies was associated with greater liver disease severity (histology activity index, 9 ⫾ 3.3 versus 6 ⫾ 2.9, p ⫽ 0.01). An increased prevalence of anti-CRP antibodies was manifested in HCV-infected patients. The presence of antiCRP antibodies correlated with the presence RF, cryoglobulinemia, and severity of liver disease. © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Introduction Hepatitis C virus (HCV) infection is one of the most common causes of chronic liver disease worldwide, affecting between 0.5% and 2% of the population of the Western world * Corresponding author. Fax: 97248359659. E-mail address: [email protected] (A. Kessel).
[1]. The majority of HCV-infected individuals do not resolve their infection, leading to the development of chronic hepatitis. The additional lymphotropism of HCV may be responsible, at least in part, for multiple immune-mediated extrahepatic manifestations of HCV infection, such as mixed cryoglobulinemic vasculitis and Sjögren-like syndrome, as well as the production of autoantibodies against a variety of self-antigens such as antinuclear (ANA), antiextractable nu-
0198-8859/$ -see front matter © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2007.06.008
845
Anti-CRP antibodies in chronic hepatitis C
ABBREVIATIONS aCLA ANA CRP ELISA ENA HAI HCV RF SLE SLEDAI
anticardiolipin antibodies antinuclear antibodies C-reactive protein enzyme-linked immunosorbent assay extractable nuclear antigen histology activity index hepatitis C virus rheumatoid factor systemic lupus erythematosus systemic lupus erythematosus activity index
clear antigen (ENA), anticardiolipin (aCLA), and anti-C1q antibodies [2–11]. C reactive protein (CRP) is one of the acute-phase proteins increased during systemic inflammation. It is produced primarily in the liver in response to infection, noninfectious systemic inflammation, and trauma. CRP may be particularly important in the protection against certain invasive bacterial mucosal pathogens, such as pneumococcus, meningococcus, and some H. influenzae strains, in the period preceding emergence of antibody-mediated opsonizing or batericidal activity [12]. In contrast to the importance of CRP in bacterial infection, its role in viral infection is still obscure. In chronic hepatitis C, levels of some acute-phase protein, such as C5b-9 levels, CH50, specific C4 activity, and CRP were lower than controls. Interestingly, some of these changes were reversible in patients who responded to HCV treatment [13–15]. In 1985 Robey et al. described autoantibodies against CRP in a patient with systemic lupus erythematosus (SLE ) [16]. Sjowall et al. detected antibodies to CRP in 36% of SLE patients, but no significant correlation between serum CRP levels and anti-CRP antibodies was present [17]. In other studies, anti-CRP antibodies were present in 78% and 23% of patients with SLE [18,19], with the difference frequency of anti-CRP in these studies probably a result of different detection methodologies used. Anti-CRP antibodies were also detected in the serum of patients with rheumatoid arthritis, primary biliary cirrhosis, and scleroderma [18]. The objective of the present study was to analyze circulating anti-CRP antibodies (Abs) in serum samples of patients with chronic HCV and to reveal a possible association between anti-CRP Abs and other autoimmune manifestations in these patients.
Subjects and methods Ninety-four consecutive patients suffering from chronic HCV infection, followed at the Liver Clinic of the Bnai Zion Medical Center (51 females and 43 males, mean age 53 ⫾ 22.9 years (range 19 to 82 years), were enrolled in the study. All patients were positive for anti-HCV antibodies, had detectable HCV RNA, were untreated at the time of survey, and had not previously received any antiviral treatments. The patients underwent a liver biopsy with stage of fibrosis and grade of activity determined (histology activity index (HAI), Knodell score). The Knodell score is composed of the summa-
tion of four individual scores representing periportal or bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation, and fibrosis. The score ranges from 0 to 22. Patients who had positive serological tests for HIV or hepatitis B surface antigen were excluded. All patients underwent a baseline evaluation including screening for rheumatoid factor (RF), cryoglobulins, and a panel of autoantibodies including ANA, aCLA, and anti-CRP antibodies. A control group, matched for age and sex, consisting of 108 individual members of the hospital staff was evaluated for the presence of anti-CRP. The staff routinely undergoes blood studies to exclude systemic and infectious diseases. The exclusion criteria for controls were liver disease, collagen or autoimmune diseases, chronic diseases, malignancy, and any other concurrent or previous illness known to affect liver function. The study was approved by the hospital’s ethics committee.
Autoantibody detection Antibodies to CRP were determined by an enzyme-linked immunosorbent assay (ELISA) protocol described by Rosenau and Schur [19]. Human CRP (Sigma C-4063) was diluted to 10 g/ml in phosphate-buffered saline (PBS) and coated onto Maxisorp ELISA plates (Nunc, Roskilde, Denmark) at room temperature overnight. The plates were washed with PBS-Tween (PBST) and blocked with blocking buffer (1% bovine serum albumin (BSA) in PBST) for 1 hour. The blocking solution was removed and 1:50 diluted plasma samples in duplicate were added into wells coated with antigen and duplicates were added into wells coated only with blocking buffer to control for unspecific binding. After 3 hours the plate was washed and goat anti-human IgG alkaline phosphatase (Sigma, St. Louis, MO, A-3150; diluted 1:1000) was added to each well. After 1 hour and another wash the assay was developed using 1 mg/ml p-nitrophenyl phosphate (Sigma N2765) in glycine buffer and the absorbance was read at 450 nm with a reference of 630 nm. Eighty blood donors were tested by this method to determine the mean OD ⫾ 2 SD for healthy individuals. Levels of anti-CRP above 2 SD were considered positive. The detection of antinuclear antibodies in the patients’ serum was assayed by indirect immunofluorescence using HEp-2 cells (Alphadia, Wavre, Belgium). Serum was considered positive for HEp-2 when immunofluorescence staining was observed at a dilution titer of ⱖ1:40. Rheumatoid factor was assayed by the 2-minute hemagglutination slide test (Wampole Laboratories, Princeton, NJ). Serum was considered positive for RF when agglutination was observed in a dilution titer of ⱖ1:10. Anticardiolipin antibodies were analyzed using a commercial ELISA kit (Genesis Diagnostics, Cambridgeshire, UK). The cutoff for a positive test was IgG ⱖ 15 unit/ml and IgM ⱖ 10 unit/ml. Cryoglobulin detection and analysis. After an overnight fast, patients’ venous blood samples were collected into prewarmed tubes and were allowed to clot at 37°C. After centrifugation and separation, the sera were incubated at 4°C for 3 days. The cryocrit was determined by centrifugation of the cooled serum in hematocrit tubes at 4°C. The cryoprecipitates were further analyzed and characterized by immunofixation electrophoresis (Immunofix kit; Helena Laboratories, Beaumont, TX). Anti-HCV antibody was detected by ELISA (Abbot Laboratories, North Chicago, IL), with HCV RNA detected by polymerase chain reaction (PCR) assay (Amplicor Roche Molecular Systems, Somerville, NJ).
Statistical analysis For comparisons of qualitative values a 2 test was used, whereas an unpaired Student t-test was used for quantitative values. Two-
846
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tailed p values of 0.05 or less were considered statistically significant.
Results Anti-CRP antibodies were detected in 17% (16/94) of HCV patients compared with 6.4% (7/108) of healthy controls (p ⬍ 0.025). Among HCV patients, 50% (47/94) had at least one autoantibody ( ANA, RF, or aCLA). Specifically, 21.2% (20/94) had a positive ANA, 15.9% (15/94) had a positive aCLA, and 28.7% (27/94) had RF. A significantly higher frequency of anti-CRP antibodies was evident among HCV patients who had at least one autoantibody (ANA, RF, or aCLA) present compared with patients without autoimmune autoantibodies ( 27.9% versus 8.6%, p ⱕ 0.025). When patients with positive anti-CRP antibodies were compared with patients who were negative for anti-CRP antibodies, the prevalence of positive RF was significantly higher (50% versus 24.3%, p ⱕ 0.05). Cryoglobulinemia was also significantly more frequent in patients who were positive for anti-CRP antibodies (75% versus 32%, p ⱕ 0.01; Figure 1). Autoimmunity (the presence of ANA, RF, or aCLA) was significantly more frequent in patients who were positive for anti-CRP antibodies compared with patients without anti-CRP antibodies (75% versus 42%, p ⱕ 0.025; Figure 2). The presence of ANA and aCLA did not differ significantly between patients with positive or negative anti-CRP antibodies (Table 1). There was no significant difference between the ages and gender of patients with and without anti-CRP (Table1). On evaluation of liver histology, 18% of patients (9/50) had cirrhosis. The presence of anti-CRP antibodies was associated with more active liver disease (HAI 9 ⫾ 3.3 versus 6 ⫾ 2.9, p ⫽ 0.01). The presence of cirrhosis did not differ significantly between patients with positive or negative anti-CRP antibodies (20% versus 17.5%).
Discussion In the present study anti-CRP antibodies were observed in the serum of a significant proportion of patients with HCV
Figure 1. Cryoglobulinemia was significantly more frequent in patients with positive anti-CRP antibodies compared with patients with negative anti-CRP antibodies, 75% versus 32%, p ⱕ 0.01.
Figure 2. Among patients with positive anti-CRP antibodies the frequency of autoimmunity (the presence of ANA, RF, or aCLA) was statistically higher compared with patients without anti-CRP antibodies (75% versus 42%, p ⱕ 0.025).
infection. This finding correlated with the presence of autoantibodies detected in the sera of these patients [20]. It is not clear whether the presence of anti-CRP autoantibodies in HCV patients is an epiphenomenon, whether it is a part of nonspecific antibody production by virus activated B cells, or whether it actually reflects events of specific pathogenetic interest, just as the presence of other antibodies reflects active autoimmune pathologic processes in the context of HCV infection, i.e., vasculitis and arthritis. Consideration must be given to the putative additional significance of antiCRP antibodies in the context of HCV infection. The primary stimuli for CRP production are proinflammatory cytokines, especially interleukin (IL)-6, IL-1, and tumor necrosis factor ␣. CRP binds autologous ligands including modified plasma lipoproteins, damaged cell membranes, various phospholipids, small nuclear ribonucleoprotein particles, apoptotic cells, and extrinsic ligands, such as many glycan phospholipids and additional constituents of microorganisms, such as capsular and somatic components of bacteria, fungi, and parasites. Bound CRP then recognizes C1q and activates the classic complement cascade engaging C3 and the terminal membrane attack complex, C5-C9 [21]. CRP-mediated complement activation is initiated by binding of one molecule C1q to two adjacent molecules of CRP associated with a ligand [22]. In animal models CRP modulated inflammation. CRP influenced the course of autoimmune disease in lupus-prone (NZB ⫻ NZW) F1 mice by decreasing antigenic stimulation and enhancing clearance of nuclear antigens [23]. On the other hand, CRP was also protective in nephrotoxic nephritis, an immune complex nephritis model that does not involve autoantibodies [24]. The significance of anti-CRP antibodies in specific rheumatic diseases is not clear. However, Sjowall et al. demonstrated a positive correlation among SLE disease activity (SLE activity index [SLEDAI] score), anti-DNA antibodies, and serum levels of anti-CRP antibodies, respectively. Inverse correlations were demonstrated between anti-CRP antibody levels and C1q. Further-
847
Anti-CRP antibodies in chronic hepatitis C Table 1
Comparative analysis of HCV-infected patients with or without anti-CRP autoantibodies.
Age (years ⫾ SD) Female/male ANA aCLA RF Cryoglobulin Histology activity disease (HAI ⫾ SD) Cirrhosis *
Anti-CRP positive (n ⫽ 16)
Anti-CRP negative (n ⫽ 78)
p value
57 ⫾ 14 8/8 6/16 2/16 8/16 9/12 9 ⫾ 3.3 (n ⫽ 9)* 2/10
53 ⫾ 16 46/32 14/78 13/78 19/78 15/47 6 ⫾ 2.9 (n ⫽ 41)* 7/41
NS NS NS NS ⱕ0.05 ⱕ0.01 ⱕ0.01 NS
Number of patients who underwent liver biopsy.
more, in patients with active lupus nephritis anti-CRP positivity was manifested at flare [25]. C-reactive protein may mediate protection against autoimmune disease by enhancing clearance of nuclear antigens expressed on apoptotic cells. The presence of anti-CRP might disturb this process, thus increasing the exposure of autoantigens to the immune system and thereby promoting the development of autoantibodies. Specifically in the case of HCV infection, increased apoptosis of T and B (CD19⫹CD5⫺) lymphocytes in the presence of anti-CRP may prolong exposure of the nuclear antigens to (CD19⫹CD5⫹) B lymphocytes and thereby increase the possibility of autoantibody formation, consistent with the finding of the present study [26,27]. It should be emphasized that anti-CRP antibodies are directed against the monomeric (modified) form of CRP, i.e., epitopes that are hidden in the pentameric structure of CRP. Additionally, Motie et al. demonstrated that modified CRP binds immune complexes specifically and might therefore be involved in removal of immune complexes [28]. This observation may explicate our finding that HCV-infected patients with anti-CRP have higher levels of cryoglobulins, which are largely cold insoluble immune complexes. The finding that presence of anti-CRP is associated with more severe liver disease is consistent with previous reports in large prospective cohorts of HCV-infected patients, wherein markers of autoimmunity, particularly mixed cryoglobulins, correlated with extent of liver damage severity [29]. The effect of anti-CRP on HCV liver damage may be mediated via its interaction with complement. The role of complement system in enhancing neutralization of antibody-bound virus by several mechanisms, including direct lysis of enveloped viruses, has been demonstrated [30,31]. Meyer et al. specifically demonstrated the importance of the complement system in the clearance of antibodies to the HCV E2 glycoprotein, which play a central role in viral entrance to mammalian cells [32]. The presence of anti-CRP antibodies may possibly attenuate this process by inhibition of complement activation, leading to viral persistence. Thus, the association between anti-CRP positivity and liver disease activity in the present study is analogous to the correlation between SLEDAI score and levels of anti-CRP in serum of SLE patients. In conclusion, the increased prevalence of anti-CRP antibodies in HCV-infected patients was documented for the first time in the present study. The presence of anti-CRP antibodies is associated with the presence RF, cryoglobu-
linemia, and increased severity of liver disease in these patients.
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