Predominance of type 1 (Th1) cytokine production in the liver of patients with HCV-associated mixed cryoglobulinemia vasculitis

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1 Journal of Hepatology 41 (2004) Predominance of type 1 (Th1) cytokine production in the liver of patients with HCV-associated mixed cryoglobulinemia vasculitis David Saadoun 1,2, Olivier Boyer 2,Hélène Trébeden-Nègre 2, Nicolas Limal 1, Véronique Bon-Durand 2, Marita Andreu 1, David Klatzmann 2, Jean Charles Piette 1, Patrice Cacoub 1,2, * 1 Service de Médecine Interne, Hôpital Pitié-Salpêtrière, 47 bd de l hôpital, Paris, France 2 Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, CNRS UMR 7087, Hôpital Pitié-Salpêtrière, Paris, France See Editorial, pages Background/Aims: Patients with hepatitis C virus (HCV) mixed cryoglobulinemia (MC) vasculitis have a higher mortality rate and more frequent incidence of cirrhosis than their cryoglobulin-negative counterparts. To compare the cytokine profile of liver-infiltrating T cells in HCV-infected patients with or without MC vasculitis. Methods: Hepatic biopsy specimens were obtained from HCV infected patients with and without MC vasculitis. Using intracellular staining and flow cytometry, we assessed the ability of freshly isolated liver T cells from these biopsies to produce IFN-g, TNF-a, IL-2, IL-4, and IL-10 in response to stimulation with PMA and ionomycin. Results: HCV-MC vasculitis patients compared to HCV-MC negative controls have an enhanced hepatic T cells production of Th1-type cytokines [i.e. TNF-a(30.3G13% vs. 15.5G5%, PZ0.01), IL-2 (20.2G9% vs. 10G4%, PZ 0.01) and IFN-g (22.2G11% vs. 9.4G4%, PZ0.008)], whereas IL-10, a representative Th2-type cytokine, was significantly lower (7.2G4% vs. 17G7%, PZ0.01). Conclusions: T cell from the liver of HCV-MC vasculitis patients display a significantly augmented liver Th1 profile compared to MC-negative controls. This enhanced production of type-1 cytokines may account for a more severe course of liver disease. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Mixed cryoglobulinemia vasculitis; Hepatitis C virus; Cytokine; Liver fibrosis 1. Introduction The syndrome of mixed cryoglobulinemia (MC) is characterized by the clinical triad of purpura, arthralgia, and asthenia. Cryoglobulins are composed of different immunoglobulins, of which the IgM rheumatoid factor (RF) component is monoclonal in type II MC and polyclonal in type III MC [1]. The clinical manifestations of MC result Received 2 April 2004; received in revised form 16 June 2004; accepted 9 August 2004; available online 27 August 2004 * Corresponding author. Address: Department of Internal Medicine, Hôpital Pitié-Salpétrière, 47 bd de l hôpital, Paris, France. Tel.: C ; fax: C address: patrice.cacoub@psl.ap-hop-paris.fr (P. Cacoub). from an immune complex-type vasculitis (MC vasculitis) with deposition of immunoglobulins and complement in vasculitis lesions [2]. Although 60 90% of patients who are chronically infected with the hepatitis C virus (HCV) have cryoglobulin present in their serum, only a small proportion will develop a clinically symptomatic vasculitis [3]. Patients with HCV-MC vasculitis have a higher mortality rate and more frequent incidence of cirrhosis than their cryoglobulin-negative counterparts [4]. HCV has been recognized as the major etiologic factor of MC, possibly through B cell clonal proliferation stimulated by HCV antigens [5]. The liver histology of HCV-infected patients with MC shows a combination of portal and/or lobular inflammatory cell infiltration frequently associated /$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 1032 D. Saadoun et al. / Journal of Hepatology 41 (2004) with lymphoid nodules resembling secondary lymphoid organs [6]. Immunohistochemical characterization has demonstrated that they consist mainly of B cells surrounded by a T cell zone [7]. These follicle-like structures often display a well-formed germinal center and may act as true functional follicular structures [8]. The analysis of both B and T lymphocytes from the livers of HCV infected patients have provided particularly interesting data. The production of RF is associated with oligoclonal or monoclonal expansions of intrahepatic B cells [9]. The role of T cells at the site of lesions has been emphasized by Pham et al. [10]. Intrahepatic T cells have been shown to be clonally expanded. They express particular TCR Vb gene products, suggesting an HCV-driven intrahepatic immune response. An individual s Th1/2 differentiation is critical in influencing the liver disease progression and the course of HCV. Patients with severe liver disease, have enhanced expression of Type-1 cytokines [i.e. interferon (IFN)-g, interleukin (IL)-2] [11]. In contrast, the prevalent cytokine pattern of circulating virus-specific T cells from patients who recover spontaneously from acute hepatitis is Th1-like and a prevalent Th2 pattern of cytokine production is associated with viral persistence and chronic evolution [12,13]. Augmented Th1 cytokine production is also seen in patients that respond favorably to IFN-a therapy [14]. It has been suggested that HCV-MC patients are at higher risk of developing cirrhosis than HCV-cryoglobulin negative patients [3,4,15]. Recent data indicate that peripheral blood monocytes from HCV-related MC vasculitis patients produce higher level of Th1 polarizing cytokines than their cryoglobulin-negative counterparts [16]. However, to date, there is no available information on the cytokine profile of liver-infiltrating T cells in MC patients. Since the liver is the primary site of HCV infection, we investigated the cytokine profile of liver T cells from HCV-infected patients with and without MC vasculitis. Our data indicate that a type-1 cytokine pattern clearly predominates in hepatic T cells from HCV-MC vasculitis patients. 2. Patients and methods 2.1. Patients The study received approval from the institutional ethics committee and informed consent was obtained from all patients. Nineteen HCV-infected patients (median age 52.6 years) entered the study, seven of whom had symptomatic MC. All had histologically proven chronically active liver disease, and positive HCV antibodies and RNA. They were negative for hepatitis B surface antigen and human immunodeficiency virus antibodies. MC-positive patients had serum MC O0.05 g/l, on at least two occasions. Symptomatic MC was defined by serum MC associated with the triad of purpura arthralgia asthenia, with or without renal or neurologic involvement. None of the 19 patients had received antiviral or immunosuppressive treatment in at least the 6 months preceding their inclusion Virologic and immunologic serum markers HCV RNA and HCV genotype were done as previously described [17,18]. Cryoglobulins were isolated from the patients sera, purified, and characterized by immunoblotting at 37 8C as previously described [19]. In this study, patients were considered to have a significant amount of serum cryoglobulins if they had a minimum level of 0.05 g/l on two occasions. According to the classification of Brouet et al., all patients had either type II or type III MC, characterized by the presence of a monoclonal or polyclonal RF component, respectively [20] Histologic studies Liver biopsy specimens from the 19 chronic HCV-infected patients were evaluated according to the previously validated Metavir scoring system [21]. Our analysis focused on the inflammatory activity and the severity of fibrosis. Necroinflammatory activity was graded on a scale of 0 3 (0Zno activity; 1Zmild activity; 2Zmoderate activity; 3Zsevere activity), taking into account the severity of portal and lobular necroinflammatory lesions. Fibrosis was graded on a scale of 0 4 (0Zno fibrosis; 1Zstellate enlargement of the portal tract but without septa formation; 3Znumerous septa without cirrhosis; 4Zcirrhosis) Stimulation of cells and staining for intracellular cytokines Immediately after the biopsy, mg of liver tissue was placed in complete medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum; Gibco/BRL, Cergy Pontoise, France). Hepatic cells were obtained by mechanical dissociation. Freshly isolated hepatic mononuclear cells (MNC) were activated with PMA (50 ng/ml) and ionomycin (1 mg/ml) (Sigma, Saint-Quentin Fallavier, France) in the presence of GolgiPlug (1 ml/ml) (BD Pharmingen, le Pont de Claix, France) 6 hours at 37 8C in RPMI 1640 (Gibco/BRL). MNC were then centrifuged, washed with PBS (Sigma) and stained with APC-labeled anti-cd3 (SK7) (Becton-Dickinson, San Jose, CA), PC5-labeled anti-cd4 (13B8.2) (Coulter Immunotech, Marseille, France), and PerCP-labeled anti-cd8 (SK1) (Becton-Dickinson). APC-labeled mouse IgG1 (679.1Mc7) (Coulter Immunotech), PC5-labeled mouse IgG1 (679.1Mc7) (Coulter Immunotech), and PerCP-labeled mouse IgG1 (X40) (Becton- Dickinson) were used as isotypic controls. After centrifugation, cells were resuspended in 125 ml Cytofix/cytoperm (Becton-Dickinson) for 20 min at 4 8C and then washed twice with Perm/wash (Becton-Dickinson). Staining for the presence of intracellular cytokine was performed according to the technique described by Andersson et al. [22]. The following anti-cytokine monoclonal antibodies were used: FITC-labeled anti-ifn-g (4S.B3), antitumor necrosis factor-a (TNF-a, Mab11), anti-il-2 (MQ1-17H12), anti-il- 4 (MP4-25D2), and APC-labeled anti-il-10 from Becton-Dickinson. FITClabeled mouse IgG1 (X40), FITC-labeled rat IgG1 (R3-34), FITC-labeled rat IgG2a, k (R35-95), and APC-labeled rat IgG2a, k (R35-95) from Becton-Dickinson were used as isotypic controls. Stained cells were analyzed on a FACS-Calibur (Becton-Dickinson, San Jose, CA) Statistical analysis All data were expressed as meangsd. Population means were compared using the Mann Whitney U test. P values less than 0.05 were considered significant. 3. Results 3.1. Patients The main characteristics of the 19 HCV-infected patients are summarized in Table 1. Baseline characteristics of the 7

3 D. Saadoun et al. / Journal of Hepatology 41 (2004) Table 1 Main features of 19 HCV-infected patients including 7 patients with mixed cryoglobulinemia vasculitis Case Sex/age (years) Cryoglobulinemia-related symptoms Cryoglobulinemia type/level (g/l) ALT (ULN) HCV viremia (Mequiv/ml) HCV genotype MC vasculitis 1 M/39 Asthenia, II/ /3 2 F/48 Asthenia, purpura, II/ /1 arthralgia, polyneuropathy 3 F/57 Asthenia, II/ /2 4 M/56 Asthenia, II/ /4 arthralgia, nephropathy 5 M/42 Asthenia, II/ /1 6 F/71 Asthenia, purpura, II/ /2 poly- neuropathy 7 F/64 Asthenia, II/ /0 MC-negative controls 1 F/ /1 2 M/ /4 3 M/ /3 4 M/ /2 5 F/ /2 6 M/ /1 7 F/ /2 8 M/ /1 9 F/ /1 10 M/ /3 11 M/ /2 12 M/ /0 Metavir score (A/F) a M, male; F, female; ULN, times the upper limit of normal values; Mequiv/ml, million equivalent per milliliter; Type II are mixed cryoglobulins, composed with a monoclonal component, as described in Section 2. Monoclonal component of all patients was IgM kappa. a Metavir score: liver biopsy specimens were evaluated according to previously validated scoring system, graded on a scale of 0 3 for the inflammatory activity (A0 A3), and of 0 4 for the fibrosis (F0 F4). patients with MC vasculitis were as follows: sex ratio male to female 0.75, median age 53.8 (39 71) years, neuropathy present in 85% (nz6) and nephropathy in 14% (nz1). All patients had a type II, IgM kappa MC with a mean cryoglobulin level of 1.8 ( ) g/l. The mean duration of HCV infection was 16 (14 37) years and 19 (10 52) for MC patients and controls, respectively. The mean serum alanine aminotransferase (ALT) level was 3.4 ( ) times the upper limit of normal values and did not differ significantly from those of controls. Mean virologic data were similar in both groups, i.e. genotype (genotype 1 in 57% of MC patients vs. 75% in controls) and HCV viremia (mean viremia 1.2 Mequiv/ml in MC patients vs. 1.1 Mequiv/ml in controls). Liver biopsy specimens often showed signs of chronic active hepatitis, but there were no significant differences in the distribution of necroinflammatory lesions or fibrosis between the groups. The mean Metavir A and Metavir F scores were 1.4 [0 3] vs. 1.2 [1 2] and 1.8 [0 4] vs. 1.8 [0 4] in MC positive and MC negative patients, respectively Characterization of cells collected from liver biopsy specimens Preparation of cells from mg liver specimens yielded 1! !10 6 MNC. A majority of hepatic MNC (56%) in HCV-MC vasculitis patients were of CD3 C phenotype, of which 24.7% expressed CD4 whereas 57.4% expressed CD8 surface molecules. Among MC-negative controls, the analysis of freshly

4 1034 D. Saadoun et al. / Journal of Hepatology 41 (2004) Table 2 Cytokine production by freshly isolated hepatic CD3 C, CD4 C and CD8 C T cells upon stimulation with PMA and ionomycin Cytokine CD3 C CD4 C CD8 C HCV-MC Ctrl HCV-MC Ctrl HCV-MC Ctrl TNF-a 60.4G12 * 45G G16 ** 15.5G5 41.9G11 * 27.5G10 IFN-g 43.1G14 ** 29.1G G11 ** 9.4G4 35.2G9 ** 21.2G8 IL-2 24G G7 20.2G9 * 10G4 20.4G8 * 9.4G6 IL-4 3G2 3.8G3 2G1 2.7G2 3G2 2.9G3 IL-10 15G3 ** 28G8 7.2G4 17G7 10G4 ** 27.3G14 * P%0.05, ** P%0.01 compared to controls. HCV, hepatitis C virus; MC, mixed cryoglobulinemia; Ctrl, controls. HCV MCZpatients with hepatitis C virus mixed cryoglobulinemia vasculitis. CtrlZpatients with chronic HCV infection without MC vasculitis. Intracytoplasmic cytokine production was determined by anti-cytokine monoclonal antibodies staining and flow cytometry, as described in Section 2. The numbers give the mean percentages GSD of gated CD3 C, CD4 C and CD8 C T cells that stained positive for each cytokine upon stimulation. isolated hepatic MNC revealed similar proportions (52% CD3 C cells including 26 and 54.2% of CD4 C and CD8 C cells, respectively). The differences in the proportions of CD3 C, CD4 C and CD8 C liver T cells between MC vasculitis patients compared to MC-negative controls were not significant Cytokine production of liver T cells activated by PMA and ionomycine Using intracellular staining and flow cytometry, we assessed the ability of freshly isolated liver T cells from chronic HCV-infected patients to produce type-1 (IFN-g, TNF-a, IL-2) and type-2 (IL-4, and IL-10) cytokines in response to stimulation with PMA and ionomycin for 6 h (Table 2 and Fig. 1). The proportion of hepatic CD3 C lymphocytes expressing TNF-a was significantly higher in those with MC vasculitis than in the cryoglobulin-negative controls (60.4G12% vs. 45G15%, PZ0.03). Similarly, the proportions of hepatic CD4 C and CD8 C T cells that produced TNF-a were significantly higher in the MC vasculitis group than in the control group (30.3G13% vs. 15.5G5% and 41.9G11% vs. 27.5G10%, PZ0.01 and PZ0.02, respectively). The proportion of hepatic CD3 C T cells from HCV-MC vasculitis patients expressing IFN-g was significantly higher than in those from controls (43.1G14% vs. 29.1G 11%, PZ0.04). When analyzed separately, IFN-g production by hepatic CD4 C and CD8 C lymphocytes was also greater in those with HCV-MC vasculitis than in the control group (22.2G11% vs. 9.4G4% and 35.2G9% vs. 21.2G 8%, PZ0.008 and PZ0.01, respectively). The proportions of hepatic CD4 C and CD8 C lymphocytes that produced IL-2 were higher in the HCV-MC vasculitis group than in the control group (20.2G9% vs. 10G4% and 20.4G8% vs. 9.4G6%, PZ0.01 and PZ0.02, respectively). In contrast to type-1 cytokines, IL-4 production by hepatic CD3 C, CD4 C and CD8 C cells from HCV-MC patients did not differ significantly from that of HCV controls. Furthermore, hepatic CD3 C lymphocytes in the HCV-MC vasculitis group showed a significantly lower IL-10 production compared with those of controls (15G3% vs. 28G8%, PZ0.004). Similarly, the proportions of hepatic CD4 C and CD8 C T cells that produced IL-10 Fig. 1. Production of interferon (IFN)-g, interleukin (IL)-2 and IL-10 by liver T cells from HCV-MC patient and control.

5 D. Saadoun et al. / Journal of Hepatology 41 (2004) were lower in the HCV-MC vasculitis group than in the control group (7.2G4% vs. 17G7% and 10G4% vs. 27.8G 14%, PZ0.01 and PZ0.003, respectively). There was no correlation between cytokine levels and clinical (cryoglobulinemia-related symptoms) or laboratory (ALT level, HCV viremia and histologic Metavir score) findings in patients with HCV-MC vasculitis or in HCV controls (data not shown). 4. Discussion Many CD3 C T cells infiltrate the liver of patients with chronic HCV infection. Besides minor subsets such as NK-T cells, these CD3 C liver-infiltrating cells are mostly conventional CD4 C and CD8 C T lymphocytes which have the potential to secrete type-1 or type-2 cytokines [23]. To analyze the type of effector lymphocytes present in the liver of HCV-infected patients, we activated freshly isolated hepatic T cells and assayed their production of TNF-a, IFN-g, IL-2, IL-4, and IL-10 at the single-cell level by intracellular staining and flow cytometry. Our data provide the first direct demonstration of an increased production of type-1 cytokines (i.e. TNFa, IFN-g and IL-2) and a decreased production of type-2 cytokines (i.e. IL-4 and IL-10) by liver T cells of MC patients. Although, no correlation was found between cytokine levels and clinical or laboratory findings in HCV-MC patients and in controls this study was not design to answer this question. The low production of IL-4 in the two groups of HCV infected patients is consistent with a previous report showing that intrahepatic T cells from HCV-infected individuals produce IFN-g but not IL-4 [24]. These results are also in line with the previous observation that peripheral blood monocytes from HCV-related MC vasculitis patients produce greater level of Th1-polarizing cytokines (i.e. IL-12) than HCV cryoglobulin-negative controls [16]. The clinical distinction of chronic HCV-infected patients with and without symptomatic MC is thus also reflected at the cytokine level. Together, these data suggest a model of MC vasculitis pathogenesis involving activation of T cells by intrahepatic antigen recognition and induction of an immune reaction dominated by Th1 helper cells. During B cell differentiation, the presence of type-1 or type-2 cytokines determines the switch to different Ig isotypes. For instance, in mice a Th1 profile is associated with the preferential production of IgG2a while a Th2 profile favors IgG1 and IgE [25]. In human, type-1 cytokines support the production of IgG1 and IgG3 subclasses in contrast to type-2 cytokines that bias B cell responses to IgG4 and IgE [26 28]. In patients with HCV-MC, it is presumable that B cell clonal expansion takes place in the liver where lymphoid aggregates are abundant and RF are produced [9]. The primary role of HCV itself in cryoglobulin production is mainly suggested by the selective concentration of the virus in cryoglobulins [5]. The type of IgG in cryoprecipitate may reflect the mechanisms that underlie the production of IgG anti-hcv and IgM RF, and the formation of MC. Indeed, in HCVassociated MC type II and MC type III cryoprecipitates, IgG are predominantly IgG1 and IgG3 [29]. Chronic antigenic stimulation can bias T helper cell differentiation toward the Th1 subset. The production of IFN-g and IL-2 can therefore activate B cells to produce strongly opsonizing antibodies belonging to certain IgG subclasses (IgG1 and IgG3) [30]. These data are consistent with our findings indicating a greater liver Th1 differentiation in MC vasculitis patients. This lends further support to the hypothesis that an HCV-driven intrahepatic immune response has an important role in the pathogenesis of HCV-MC. An imbalance of type-1 and type-2 cytokine production seems to be implicated in the liver disease progression of HCV infected patients. Patients with severe liver disease, i.e. cirrhosis, have enhanced expression of both IFN-g and IL-2 as compared to healthy controls and those with mild liver disease [11]. In contrast, HCV negative controls and HCV positive patients with mild inflammation showed higher expression of IL-10 compared with patients having overt liver disease. IL-10 has the ability to regulate the production of inflammatory cytokines and to modulate liver matrix remodeling by reducing collagen type 1 and enhancing metalloproteinase gene expression [31]. IL-10 deficient mice are susceptible to hepatic inflammation induced by chemical compounds [32]. These findings suggest that IL-10 deficiency may contribute to hepatic inflammation and fibrosis in HCV-MC vasculitis patients. Intrahepatic messenger RNA levels of IFN-g and IL-2 correlate with liver fibrosis and portal inflammation, suggesting that type-1 cytokines might play a role in mediating hepatocellular damage. The present observation of increased hepatic T cells production of type-1 cytokines in HCV-MC patients is therefore consistent with the fact that such patients are at higher risk of developing cirrhosis than HCV-cryoglobulin negative patients [3,15]. Indeed, a recent meta-analysis of 19 studies on a total of 2323 patients with chronic HCV found a highly significant association between cirrhosis and cryoglobulinemia after adjustment for age, gender, and estimated duration of disease [4]. However, one italian study with heterogen cryoglobulinemic population found opposite data indicating a decrease prevalence of cirrhosis in HCV-MC patients [33]. In patients presenting with HCV-MC vasculitis, IFN-a and ribavirin may exert their effects via inhibition of viral replication [34,35] rather than by complete eradication of the virus, or by an anti-inflammatory action [36 38]. The Th1/Th2 differentiation is critical in influencing liver disease progression in HCV-infected patients. In addition, it can bias the B cell antibody production toward IgG1 and

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