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HCV can activate B cells via CD81 engagement: a molecular mechanism for B cell autoreactivity and cryoglobulinemia in HCV infection
252A
AASLD ABSTRACTS
HEPATOLOGY October 2001
313
314
HCV CAN ACTIVATE B CELLS VIA CD81 ENGAGEMENT: A MOLECUEAR MECHANISM FOR B CELL A U T O R E A C T M T Y A N D CRYOGLOBULINEMIA IN HCV INFECTION. Domenico Rosa, Giulietta Saletti, IRIS Re-
HEPATOCYTES EXPRESSING HCV STRUCTURAL PROTEINS ENHANCE APOPTOSIS OF ACTIVATED T CELLS IN VIVO. Lni Huang,
search Center, Siena Italy; Gabriele Pozzato, Pordenone General Hospital, Pordenone Ita!y; Sergio Abrignani, Chiron Corporation, Emeryvile, CA Although HCV tropism is not well defined, progress has been made studying the specific binding of recombinant HCV envelope glycoproteins to human cell surface proteins and assessing interaction of these surface Bgands with HCV virions. For instance, the major extracellular loop of the tetraspanin CD81 binds with high affinity the recombinant E2 protein as well as HCV particles as demonstrated by quantitative PCR. CD81 is a promiscuous molecule participating in different molecular complexes on various cell types, a fact that may influence its capacity to deliver signals to target cells. On B-lymphocytes, CD81 associates with CD21 and CD19, two molecules which, when appropriately engaged, lower the B cell activation threshold. Interestingly, chronic HCV infection is frequently marked by- the presence of antoreactive antibodies, and there is a tight association with a B lymphocyte proliferative disorder, mixed cryoglobulinemia. We therefore investigated the effects of CD81 engagement on human B lymphocyte activation in vitro. We have found that binding of recombinant HCV envelope glycoproteins or anti-CD81 antibodies to CD81 delivers a very modest costimulation signal to B cells that have been exposed to suboptimal activation stimuli (i.e., low concentrations of anti-IgM mAb). However, we have found that co-engagement of CD81 by HCV E2 and anti-CD81 mAbs (selected for their inability to interfere with E2-CD81 interaction) delivers a true and complete activation signal to B ceils in vitro, as demonstrated by cell proliferation and antibody production. This is surprising, as CD81 had not been considered an activation molecule previously. Moreover, we have found that B cells activated via CD81 engagement display a peculiar activated phenotype, as compared to B cells activated by anti-IgM mAb. Importantly, we have observed the same phenotype on B ceils taken from the peripheral blood of patients with HCV infection with or without cryoglobulinemia. We have demonstrated that engagement of CD81 on B lymphocytes in vitro can directly activate B cells and that these B cells display a phenotype similar to that found on B cells from patients with HCV but not with HBV infection. It is therefore possible that, in HCV infection, B cells would be abnormally activated due to this interaction between the virus (mimicked by E2 and anti-CD81 mAb in vitro) and the CD 19/CD21/CD81 complex, which would in turn facilitate production of the autoantibodies and rheumatoid factor commonly found in HCV infection. B cell activation involving the interaction of HCV with CD81 along with other genetic and environmental factors may be responsible for the development of HCV-associated cryoglobulinemia. In conclusion, our work suggests that HCV-associated cryoglobulinemia may be a direct consequence of the binding of HCV to CD81 on B cells. This offers a unique perspective into the molecular mechanism of this extra-hepatic manifestation of chronic HCV infection.
Brigham and Women's Hosp, Boston, MA; Nina Fowler, Beth Israel Deaconess Medical Ctr, Boston, MA; Wajahat Z Mehal, Yale University, New Haven, CT; Emmet Schmidt, Massachusetts General Hospital, Boston, MA; Nicholas Crispe, University of Rochester Medical Center, Rochester, NY; Margaret J Koziel, Beth Israel Deaconess Medical Ctr, Boston, MA Background: The majority of patients infected by HCV develop persistent infection. The mechanism underlying failure of HCV clearance in most patients is not understood. Using hepatocytes from HCV transgenic (Tg+) mice expressing HCV El, E2 and core proteins in the liver, we have shown previously that HCV Tg+ hepatocytes enhance apoptosis of polydonally activated T cells when they are co-cultured in vitro. This process appears to be mediated by increased expression of Fas ligand in HCV Tg+ hepatocytes. Based on these observations, we have postulated that infection of hepatocytes by HCV may enhance apoptosis of activated T cells in the liver, leading to attenuation of HCV-speciflc T cell responses and failure of viral clearance. Methods: We adoptively transferred CSFE-labeled B6-->Balb/C alloreactive T cells directly into the portal veins of HCV Tg+ mice expressing HCV structural proteins in hepatocytes(in Balb/C background). 15 hours after transfer, intrahepatic lymphocytes were isolated, and apoptosis of CSFE-labeled donor ceils was studied by FACS analysis after staining with Annexin V and Ph Liver histology was assessed, and ALT determined to see whether there was any associated liver injury. To study whether enhanced apoptosis and liver injury were mediated by Fas - FasL interaction, blocking experiments were performed by concomitant injection of an anti-FasL antibody. Results: We have found that apoptosis of donor T cells was significantly increased in the livers of HCV Tg+ mice 15 hours after adoptive transfer (26.9 + 10.5% in HCV Tg+ mice vs 13.6-+5.0% in HCV Tg- mice; P=0.002 by Mann-Whitney U test). In addition, liver injury was more profound in HCV Tg+ mice as reflected by significantly higher ALT (164-+75 in HCV Tg+ mice vs 85-+24 in HCV Tg- mice; P=0.03) and liver necrosis (P value=0.004 by Fisher's exact test). In preliminary experiments, concomitant injection of an anti-FasL antibody not only inhibited apoptosis of donor T cells but also prevented liver necrosis, suggesting that both enhanced T-cell apoptosis and liver injury are mediated by Fas - FasL interaction. Conclusion: We have extended our previous in vitro observations, and have provided evidence that hepatocytes expressing HCV proteins enhance apoptosis of activated T cells in vivo, which may contribute to HCV persistence. HCVspecific T cells would be activated in vivo and may undergo enhanced apoptosis upon exposure to HCV-infected hepatocytes, thus serving to attenuate an effective immune response. Increased expression of FasL induced by HCV infection may also sensitize hepatocytes to other injuries, which may be important in the pathogenesis of liver damage associated with HCV infection.
315
316
THE CHEMOKINE CXCL16 A N D ITS RECEPTOR CXCR6 ARE IMPORT A N T FOR P O S I T I O N I N G OF T CELLS W I T H I N THE LIVER. Heydtmann
HCV-SPECIFIC T CELL RESPONSES FROM ORAL LICHEN PLANUS DERIVED LYMPHOMONONUCLEAR CELLS. Pilli Massimo, Azienda Hosp di
Mathis, Liver Research Labs, Birmingham Uk; Stephan H~bscher, Dept of Pathology, Birmingham Uk; Mike Briskin, Millenium lnc, Boston, MA; David H Adams, Liver Research Labs, Birmingham Uk
Parma, Parma Italy; Paolo Vescovi, Maddalena Manfredi, Univ di Parma, Parma Italy; Amalia Penna, Albertina Cavalli, Cristina Cavallo, Alessandro Zerbini, Tiziana Giuberti, Carlo Ferrari, Gabriele Missale, Azienda Hosp di Parma, Parma Italy
Background & Aims: The recruitment and positioning of lymphocytes in the liver in hepatitis C is critical for viral clearance and the development of chronic inflammation. Chemokines, chemotactic cytokines that attract effector cells to sites of inflammation are critical in this process. The novel chemokine CXCL16 is unique amongst the main chemokine families in that it exists in a transmembrane as well as a soluble form. This provides a potential mechanism for localizing it to particular structures. We studied the role of this chemokine and its specific receptor CXCR6 in lymphocyte recruitment and retention in the liver in HCV. Materials & Methods: Lymphocytes were isolated from liver removed at transplantation and from paired peripheral blood samples. Primary cultures of cholangiocytes, hepatocytes and sinusoidal endothelial cells were established. Cell surface expression of CXCR6 and CXCL16 was carried out by flow cytometry and immunocytochemistry and lymphocyte adhesion to monolayers of human cholangiocytes was analysed in static adhesion assays in wich blocking antibodies against CXCR6 and CXCL16 were added. Results: A high proportion of liver-derived lymphocytes were CXCR6+ w h e ~ a r e d to peripheral blood. CXCR6 was expressed by the majority of CD8+ T cells and CD56+ cells in the liver compared with less than 50% in blood. A higher proportion of CXCR6+ T cells were detected in blood of HCV patients compared with healthy subjects. In the liver CXCR6+ lymphocytes were found in association with CXCL16+ stromal and parenchymal cells in inflammatory areas. In order to determine the cellular distribution of CXCL16 we stained cultured cholangiocytes, sinusoidal endothelial ceils and hepatocytes for CXCL16 expression and analsyed the subceflular distribution using confocal microscopy. The strongest staining was seen on epithelial cells with cholangiocytes in particular expressing high levels of membranous CXCL16 on cell surface villi. In order to determine whether the transmembrane CXCL16 can support adhesion directly we used antibodies against CXCL16 and CXCR6 in lymphocyte adhesion assays to monolayers of cholangiocytes. Compared to controls CXCL16 antibody reduced adhesion by 56.7 % and anti-CXCR6 by 49%. These results were similar to the levels of inhibition obtained by blocking CD18/ICAM-1. Summary: 1) A high proportion of liver infiltrating effector cells express CXCR6 2) In contrast to many other chemokines CXCL16 is concentrated on epithelial cells particularly cholangiocytes where it is expressed in a trans membrane form that supports lymphocyte adhesion. Conclusions: This is the first demonstration that CXC chemokines can directly support lymphocyte adhesion. The expression of CXCL16 by cholangiocytes suggests it might play a role in attracting and retaining effector cells to promote bile duct damage in inflammatory liver disease.
Hepatitis C virus infection is associated with a number of extra-hepatic manifestations, including several dermatologic diseases. Although the presence of positive and negative HCV-RNA strands has recently been demonstrated in oral mucosa of patients with oral Lichen planns, the direct pathogenetic role of HCV in the development of these lesions still remains controversial. METHODS: to assess whether HCV-specific T cells are present within the lesions and to characterize their antigen-specificity and functional features, T cells from mucosa biopsies of 9 HCV-positive patients with oral Lichen planus were isolated. Oral mucosa and peripheral blood-derived T cells were stained with activation and differentiation markers, tested with recombinant structural and non-structural HCV proteins and stained with HCV peptide/HLA-A2 and HLA-A3 tetramers. RESULTS: phenotypic analysis in 9 patients showed enrichment of DR+, CD69+ and CD45-RA- T cells in the oral biopsies compared to peripheral blood. HCV-specific CD4+ T cell lines were obtained in 4 patients from Lichen lesions but only in 2 of them from peripheral blood. Lichen- and peripheral blood-derived T cell lines were able to proliferate and to produce IFN-gamma upon stimulation with structural and non-structural HCV antigens and showed the same fine specificity. However, different clonal populations were present in oral mucosa and peripheral blood, as shown by TCR V/3 analysis of antigen-specific "17cells. Frequency of HCV-specific CD8+ T cells tested with 4 different HCV tetramers was higher in mucosa tissue than in the circulation in all patients tested. While peripheral blood-derived HCVspecific T cells could be expanded in-vitro by peptide stimulation, HCV-specific T-cells from Lichen biopsies failed to proliferate. CONCLUSIONS: the isolation of HCV-spedfic T ceils from Lichen lesions but not from the peripheral blood in two patients, the different VJ8 usage and the different frequency of HCV-specific T cells in the two compartments demonstrates that Lichen-derived T cells are not blood contaminants. For the first time HCV-specific T cell responses at the site of the lesions of an HCV-associated dermatologic disease have been demonstrated. Furthermore, the phenotypic and functional characteristic of HCV antigen-experienced T cells strongly suggests a role for HCVspecific T cell responses in the pathogenesis of oral Lichen planns in patients with chronic HCV infection.
AASLD ABSTRACTS
HEPATOLOGY October 2001
313
314
HCV CAN ACTIVATE B CELLS VIA CD81 ENGAGEMENT: A MOLECUEAR MECHANISM FOR B CELL A U T O R E A C T M T Y A N D CRYOGLOBULINEMIA IN HCV INFECTION. Domenico Rosa, Giulietta Saletti, IRIS Re-
HEPATOCYTES EXPRESSING HCV STRUCTURAL PROTEINS ENHANCE APOPTOSIS OF ACTIVATED T CELLS IN VIVO. Lni Huang,
search Center, Siena Italy; Gabriele Pozzato, Pordenone General Hospital, Pordenone Ita!y; Sergio Abrignani, Chiron Corporation, Emeryvile, CA Although HCV tropism is not well defined, progress has been made studying the specific binding of recombinant HCV envelope glycoproteins to human cell surface proteins and assessing interaction of these surface Bgands with HCV virions. For instance, the major extracellular loop of the tetraspanin CD81 binds with high affinity the recombinant E2 protein as well as HCV particles as demonstrated by quantitative PCR. CD81 is a promiscuous molecule participating in different molecular complexes on various cell types, a fact that may influence its capacity to deliver signals to target cells. On B-lymphocytes, CD81 associates with CD21 and CD19, two molecules which, when appropriately engaged, lower the B cell activation threshold. Interestingly, chronic HCV infection is frequently marked by- the presence of antoreactive antibodies, and there is a tight association with a B lymphocyte proliferative disorder, mixed cryoglobulinemia. We therefore investigated the effects of CD81 engagement on human B lymphocyte activation in vitro. We have found that binding of recombinant HCV envelope glycoproteins or anti-CD81 antibodies to CD81 delivers a very modest costimulation signal to B cells that have been exposed to suboptimal activation stimuli (i.e., low concentrations of anti-IgM mAb). However, we have found that co-engagement of CD81 by HCV E2 and anti-CD81 mAbs (selected for their inability to interfere with E2-CD81 interaction) delivers a true and complete activation signal to B ceils in vitro, as demonstrated by cell proliferation and antibody production. This is surprising, as CD81 had not been considered an activation molecule previously. Moreover, we have found that B cells activated via CD81 engagement display a peculiar activated phenotype, as compared to B cells activated by anti-IgM mAb. Importantly, we have observed the same phenotype on B ceils taken from the peripheral blood of patients with HCV infection with or without cryoglobulinemia. We have demonstrated that engagement of CD81 on B lymphocytes in vitro can directly activate B cells and that these B cells display a phenotype similar to that found on B cells from patients with HCV but not with HBV infection. It is therefore possible that, in HCV infection, B cells would be abnormally activated due to this interaction between the virus (mimicked by E2 and anti-CD81 mAb in vitro) and the CD 19/CD21/CD81 complex, which would in turn facilitate production of the autoantibodies and rheumatoid factor commonly found in HCV infection. B cell activation involving the interaction of HCV with CD81 along with other genetic and environmental factors may be responsible for the development of HCV-associated cryoglobulinemia. In conclusion, our work suggests that HCV-associated cryoglobulinemia may be a direct consequence of the binding of HCV to CD81 on B cells. This offers a unique perspective into the molecular mechanism of this extra-hepatic manifestation of chronic HCV infection.
Brigham and Women's Hosp, Boston, MA; Nina Fowler, Beth Israel Deaconess Medical Ctr, Boston, MA; Wajahat Z Mehal, Yale University, New Haven, CT; Emmet Schmidt, Massachusetts General Hospital, Boston, MA; Nicholas Crispe, University of Rochester Medical Center, Rochester, NY; Margaret J Koziel, Beth Israel Deaconess Medical Ctr, Boston, MA Background: The majority of patients infected by HCV develop persistent infection. The mechanism underlying failure of HCV clearance in most patients is not understood. Using hepatocytes from HCV transgenic (Tg+) mice expressing HCV El, E2 and core proteins in the liver, we have shown previously that HCV Tg+ hepatocytes enhance apoptosis of polydonally activated T cells when they are co-cultured in vitro. This process appears to be mediated by increased expression of Fas ligand in HCV Tg+ hepatocytes. Based on these observations, we have postulated that infection of hepatocytes by HCV may enhance apoptosis of activated T cells in the liver, leading to attenuation of HCV-speciflc T cell responses and failure of viral clearance. Methods: We adoptively transferred CSFE-labeled B6-->Balb/C alloreactive T cells directly into the portal veins of HCV Tg+ mice expressing HCV structural proteins in hepatocytes(in Balb/C background). 15 hours after transfer, intrahepatic lymphocytes were isolated, and apoptosis of CSFE-labeled donor ceils was studied by FACS analysis after staining with Annexin V and Ph Liver histology was assessed, and ALT determined to see whether there was any associated liver injury. To study whether enhanced apoptosis and liver injury were mediated by Fas - FasL interaction, blocking experiments were performed by concomitant injection of an anti-FasL antibody. Results: We have found that apoptosis of donor T cells was significantly increased in the livers of HCV Tg+ mice 15 hours after adoptive transfer (26.9 + 10.5% in HCV Tg+ mice vs 13.6-+5.0% in HCV Tg- mice; P=0.002 by Mann-Whitney U test). In addition, liver injury was more profound in HCV Tg+ mice as reflected by significantly higher ALT (164-+75 in HCV Tg+ mice vs 85-+24 in HCV Tg- mice; P=0.03) and liver necrosis (P value=0.004 by Fisher's exact test). In preliminary experiments, concomitant injection of an anti-FasL antibody not only inhibited apoptosis of donor T cells but also prevented liver necrosis, suggesting that both enhanced T-cell apoptosis and liver injury are mediated by Fas - FasL interaction. Conclusion: We have extended our previous in vitro observations, and have provided evidence that hepatocytes expressing HCV proteins enhance apoptosis of activated T cells in vivo, which may contribute to HCV persistence. HCVspecific T cells would be activated in vivo and may undergo enhanced apoptosis upon exposure to HCV-infected hepatocytes, thus serving to attenuate an effective immune response. Increased expression of FasL induced by HCV infection may also sensitize hepatocytes to other injuries, which may be important in the pathogenesis of liver damage associated with HCV infection.
315
316
THE CHEMOKINE CXCL16 A N D ITS RECEPTOR CXCR6 ARE IMPORT A N T FOR P O S I T I O N I N G OF T CELLS W I T H I N THE LIVER. Heydtmann
HCV-SPECIFIC T CELL RESPONSES FROM ORAL LICHEN PLANUS DERIVED LYMPHOMONONUCLEAR CELLS. Pilli Massimo, Azienda Hosp di
Mathis, Liver Research Labs, Birmingham Uk; Stephan H~bscher, Dept of Pathology, Birmingham Uk; Mike Briskin, Millenium lnc, Boston, MA; David H Adams, Liver Research Labs, Birmingham Uk
Parma, Parma Italy; Paolo Vescovi, Maddalena Manfredi, Univ di Parma, Parma Italy; Amalia Penna, Albertina Cavalli, Cristina Cavallo, Alessandro Zerbini, Tiziana Giuberti, Carlo Ferrari, Gabriele Missale, Azienda Hosp di Parma, Parma Italy
Background & Aims: The recruitment and positioning of lymphocytes in the liver in hepatitis C is critical for viral clearance and the development of chronic inflammation. Chemokines, chemotactic cytokines that attract effector cells to sites of inflammation are critical in this process. The novel chemokine CXCL16 is unique amongst the main chemokine families in that it exists in a transmembrane as well as a soluble form. This provides a potential mechanism for localizing it to particular structures. We studied the role of this chemokine and its specific receptor CXCR6 in lymphocyte recruitment and retention in the liver in HCV. Materials & Methods: Lymphocytes were isolated from liver removed at transplantation and from paired peripheral blood samples. Primary cultures of cholangiocytes, hepatocytes and sinusoidal endothelial cells were established. Cell surface expression of CXCR6 and CXCL16 was carried out by flow cytometry and immunocytochemistry and lymphocyte adhesion to monolayers of human cholangiocytes was analysed in static adhesion assays in wich blocking antibodies against CXCR6 and CXCL16 were added. Results: A high proportion of liver-derived lymphocytes were CXCR6+ w h e ~ a r e d to peripheral blood. CXCR6 was expressed by the majority of CD8+ T cells and CD56+ cells in the liver compared with less than 50% in blood. A higher proportion of CXCR6+ T cells were detected in blood of HCV patients compared with healthy subjects. In the liver CXCR6+ lymphocytes were found in association with CXCL16+ stromal and parenchymal cells in inflammatory areas. In order to determine the cellular distribution of CXCL16 we stained cultured cholangiocytes, sinusoidal endothelial ceils and hepatocytes for CXCL16 expression and analsyed the subceflular distribution using confocal microscopy. The strongest staining was seen on epithelial cells with cholangiocytes in particular expressing high levels of membranous CXCL16 on cell surface villi. In order to determine whether the transmembrane CXCL16 can support adhesion directly we used antibodies against CXCL16 and CXCR6 in lymphocyte adhesion assays to monolayers of cholangiocytes. Compared to controls CXCL16 antibody reduced adhesion by 56.7 % and anti-CXCR6 by 49%. These results were similar to the levels of inhibition obtained by blocking CD18/ICAM-1. Summary: 1) A high proportion of liver infiltrating effector cells express CXCR6 2) In contrast to many other chemokines CXCL16 is concentrated on epithelial cells particularly cholangiocytes where it is expressed in a trans membrane form that supports lymphocyte adhesion. Conclusions: This is the first demonstration that CXC chemokines can directly support lymphocyte adhesion. The expression of CXCL16 by cholangiocytes suggests it might play a role in attracting and retaining effector cells to promote bile duct damage in inflammatory liver disease.
Hepatitis C virus infection is associated with a number of extra-hepatic manifestations, including several dermatologic diseases. Although the presence of positive and negative HCV-RNA strands has recently been demonstrated in oral mucosa of patients with oral Lichen planns, the direct pathogenetic role of HCV in the development of these lesions still remains controversial. METHODS: to assess whether HCV-specific T cells are present within the lesions and to characterize their antigen-specificity and functional features, T cells from mucosa biopsies of 9 HCV-positive patients with oral Lichen planus were isolated. Oral mucosa and peripheral blood-derived T cells were stained with activation and differentiation markers, tested with recombinant structural and non-structural HCV proteins and stained with HCV peptide/HLA-A2 and HLA-A3 tetramers. RESULTS: phenotypic analysis in 9 patients showed enrichment of DR+, CD69+ and CD45-RA- T cells in the oral biopsies compared to peripheral blood. HCV-specific CD4+ T cell lines were obtained in 4 patients from Lichen lesions but only in 2 of them from peripheral blood. Lichen- and peripheral blood-derived T cell lines were able to proliferate and to produce IFN-gamma upon stimulation with structural and non-structural HCV antigens and showed the same fine specificity. However, different clonal populations were present in oral mucosa and peripheral blood, as shown by TCR V/3 analysis of antigen-specific "17cells. Frequency of HCV-specific CD8+ T cells tested with 4 different HCV tetramers was higher in mucosa tissue than in the circulation in all patients tested. While peripheral blood-derived HCVspecific T cells could be expanded in-vitro by peptide stimulation, HCV-specific T-cells from Lichen biopsies failed to proliferate. CONCLUSIONS: the isolation of HCV-spedfic T ceils from Lichen lesions but not from the peripheral blood in two patients, the different VJ8 usage and the different frequency of HCV-specific T cells in the two compartments demonstrates that Lichen-derived T cells are not blood contaminants. For the first time HCV-specific T cell responses at the site of the lesions of an HCV-associated dermatologic disease have been demonstrated. Furthermore, the phenotypic and functional characteristic of HCV antigen-experienced T cells strongly suggests a role for HCVspecific T cell responses in the pathogenesis of oral Lichen planns in patients with chronic HCV infection.