B. Fleischer

Cyclooxygenase-2 inhibition induces apoptosis signaling via death receptors and mitochondria in hepatocellular carcinoma.

Inhibition of cyclooxygenase (COX)-2 elicits chemopreventive and therapeutic effects in solid tumors that are coupled with the induction of apoptosis in tumor cells. We investigated the mechanisms by which COX-2 inhibition induces apoptosis in hepatocellular carcinoma (HCC) cells. COX-2 inhibition triggered expression of the CD95, tumor necrosis factor (TNF)-R, and TNF-related apoptosis-inducing ligand (TRAIL)-R1 and TRAIL-R2 death receptors. Addition of the respective specific ligands further increased apoptosis, indicating that COX-2 inhibition induced the expression of functional death receptors. Overexpression of a dominant-negative Fas-associated death domain mutant reduced COX-2 inhibitor-mediated apoptosis. Furthermore, our findings showed a link between COX-2 inhibition and the mitochondrial apoptosis pathway. COX-2 inhibition led to a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, followed by translocation of Bax to mitochondria and cytochrome c release from mitochondria. Consequently, overexpression of Mcl-1 led to inhibition of COX-2 inhibitor-mediated apoptosis. Furthermore, blocking endogenous Mcl-1 function using a small-interfering RNA approach enhanced COX-2 inhibitor-mediated apoptosis. It is of clinical importance that celecoxib acted synergistically with chemotherapeutic drugs in the induction of apoptosis in HCC cells. The clinical relevance of these results is further substantiated by the finding that COX-2 inhibitors did not sensitize primary human hepatocytes toward chemotherapy-induced apoptosis. In conclusion, COX-2 inhibition engages different apoptosis pathways in HCC cells stimulating death receptor signaling, activation of caspases, and apoptosis originating from mitochondria.

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

BackgroundHepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro.MethodsRNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively.ResultsHere we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition.ConclusionOur data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies.

Clonal analysis of liver-infiltrating T cells in patients with LKM-1 antibody-positive autoimmune chronic active hepatitis

Autoantibodies against microsomal antigen of liver and kidney (LKM‐1) are diagnostic markers for a subgroup of autoimmune chronic active hepatitis (AI‐CAH). Cytochrome P4S0dbl, now classified as cytochrome P450 IID6, is the major antigen of LKM‐1 antibodies. Immunohistological studies suggest that hepatic injury in AI‐CAH is mediated by liver‐infiltrating T cells. In the present study the specificity and function of liver‐infiltrating T cells was analysed at the clonal level. Phenotypical characterization of 189 T cell clones isolated from four liver biopsies of LKM‐1 antibody‐positive patients showed an enrichment of CD4+CD8‐ T cells. Five CD4+CD8‐ T cell clones proliferated specifically in the presence of recombinant human LKM‐1 antigen (rLKM). This reaction was restricted to autologous antigen‐presenting cells and to HLA class II molecules. In order to see whether rLKM was also recognized by peripheral blood T lymphocytes (PBL) we tested the proliferative response of PBL from several individuals. PBL from three of the four patients with LKM‐1 antibody‐positive AI‐CAH proliferated to rLKM, whereas no response was seen with PBL from patients with LKM‐1 antibody‐negative chronic liver diseases and from healthy blood donors. These data demonstrate that the LKM‐1 antigen is recognized by liver‐infiltrating T cells in LKM‐1 antibody‐positive AI‐CAH. For further functional characterization, liver‐derived T cell clones were tested for their cytotoxic activity. In the presence of phytohacmagglutinin 24 out of 26 CD4‐CD8+ but also 20 out of 63 CD4+CD8‐ T cell clones lysed autologous as well as allogenic EBV‐transformed B cell lines or K562 cells. Five CD4‐CD8+ T cell clones lysed autologous but not allogenic B cell lines spontaneously in a HLA class I‐restricted manner. Although the antigen specificity of these clones is still unknown the data show the presence of autoreactive T cells at the site of inflammation that could contribute in the pathogenesis of AI‐CAH.

Autoreactive liver-infiltrating T cells in primary biliary cirrhosis recognize inner mitochondrial epitopes and the pyruvate dehydrogenase complex

Primary biliary cirrhosis (PBC) is characterized by lymphoid infiltrates in the portal tracts of the liver and the occurrence of antimitochondrial autoantibodies in serum directed against components of the pyruvate dehydrogenase complex and the other alpha-keto acid dehydrogenase complexes. These enzymes are located on the inner mitochondrial membrane. The destruction of the biliary tract in PBC is thought to be mediated by autoreactive liver-infiltrating T cells exerting cytotoxic activity or releasing certain lymphokines. In this study the reactivity of liver infiltrating T cells was shown to a bovine pyruvate dehydrogenase complex (PDH), a purified E2 subunit (PDH-E2) and a crude preparation of human liver mitoplasts (HLM), i.e. mitochondria depleted of their outer membranes. Peripheral blood lymphocytes (PBL) from 11 of 15 patients (73.3%) with PBC showed a HLA class II-restricted proliferative response to the PDH complex whereas PBL from patients with chronic viral hepatitis, autoimmune hepatitis or extrahepatic cholestatic icterus (n = 20) and healthy controls (n = 5) did not. In addition 13 of 15 PBL from patients with PBC (86.6%) and three of nine PBL from patients with autoimmune hepatitis (33.3%) reacted with the crude HLM preparation whereas no reactivity was found with PBL from eight patients with chronic viral hepatitis, three patients with extrahepatic cholestasis or five healthy controls. Clonal analysis of 115 liver-infiltrating T cells derived from two diagnostic liver biopsies of patients with PBC revealed a predominance of activated CD4+CD8- T helper cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera orpaticnts with autoimmune liver disorders. Live‐nfiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (A‐AH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclcar cells (PBMC) from patients with A‐AH or PBC but not chronic viral hepatitis secreted ant‐SGPR antibodies in vitro. In this study we characterized the influence of live‐nfiltrating T cells on the secretion of ASGP‐pecific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with A‐AH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibod‐ecrcting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supematants and showed a proliferative response to ASGPR. T cel‐epleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+ CD8− live‐nfiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous ant‐SGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8− and two CD4−CD8+ T cell clones no‐esponding to ASGPR provided this help for antibody secretion. Ant‐SGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclona‐ctivating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGP‐pecific antibodies. This help can be provided by ASGP‐esponsive T helper cells via cellular interactions.

Analysis of the in vitro cytokine production by liver-infiltrating T cells of patients with autoimmune hepatitis.

The pathogenic mechanisms underlying the development of autoimmune hepatitis (AIH) are still unclear. Since AIH is associated with the presence of various autoantibodics and certain HLA subtypes, it is likely that T and B cells play a major role in this disease. In this study we have determined the functional capacities of in vivo preactivated liver‐infiltrating T cells (LTC) from patients with AIH. As controls we used LTC from patients with non‐autoimmune hepatitis (non‐AIH). Our results show that preactivated LTC from patients with AIH predominantly (190/255 clones) reside in the CD4+ population, whereas LTC in non‐AIH are dominated by the CD8+ phenotype (148/254 clones). In view of this finding we have investigated the cytokine secretion patterns of 102 randomly chosen CD4+ T cell clones from six patients with AIH. As controls we have used 58 CD4+ LTC from 11 patients with non‐AIH. All clones were stimulated by lectin and irradiated accessory cells and subsequent cytokine production was evaluated. LTC from patients with AIH have a lower interfcron‐gamma (IFN‐γ)/IL‐4 ratio compared with LTC from non‐AIH. Although clones from some patients with AIH produced very high amounts of IL‐4 in vitro, this was not a constant finding. These results show that in two preactivated LTC from patients with AIH are mostly CD4+ T cells that produce more IL‐4 than IFN‐γ. In contrast. LTC from patients with non‐AIH are dominated by CD8+ and CD4+ T cells that produce significantly less IL‐4 than IFN‐γ. Thus, liver‐infiltrating T cells from patients with AIH and non‐AIH belong to different functional T cell subsets. This may have implications for the regulation of humoral and cellular immune responses in inflammatory liver disease.

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-γδ+ lymphocytes

T lymphocyte responses to heterologous or self 65‐kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65‐kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65‐kD hsp, tetanus toxoid (TT), heat‐killed or live Yersinia enterocotitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR‐γδ+ lymphocytes in the 65‐kD hsp‐stimulatcd SF‐derived lines. This expansion of TCR‐γδ+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR‐γδ+ cells was also shown after SFMC stimulation with live, but not with heat‐killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR‐γδ+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65‐kD hsp. Out of a panel of TCR‐γδ+ T lymphocyte clones (TLC) derived from a human 65‐kD hsp‐stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR‐αβ+ TLC responded vigorously to the human 65‐kD hsp and in some cases also cross‐recognized the mycobacterial hsp homologue and/or heat‐killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR‐γδ+ cells in response to 65‐kD hsp or live bacteria.

A sensitive and specific bioassay for the detection of human interleukin-10

Interleukin-10 (IL-10) is a novel cytokine that is produced by T cells, macrophages, B cells and keratinocytes. It has been shown to inhibit cytokine production and proliferation by T cells when macrophages are used as accessory or antigen presenting cells. Monokine production by macrophages is effectively downregulated by IL-10 and it can be used as a growth factor by CD4, CD8 and gamma/delta positive T cells as well as mast cells and B cells. It is because of these pleiotropic immunoregulatory effects that the detection of IL-10 in the supernatants of T cells, B cells, macrophages and other cells is important for many scientific questions. Here we describe a simple and sensitive bioassay specific for human IL-10 using the IL-10 dependent growth of the mouse mast cell line D36. Our data show that this assay is not crossreactive with hIL-1 beta, hIL-2, hIL-3, hIL-4, hIL-5, hIL-6, hIL-9, hIL-12, hGM-CSF and hTNF-alpha and that it can be completely blocked by an antibody against human IL-10. The hIL-10 induced growth of the D36 cell line is dependent on the presence of mIL-4. Human IL-10 can be measured in a concentration range from approximately 10 U/ml to 0.05 U/ml. This assay is only of limited use for the measurement of IL-10 in human blood samples since it is inhibited by the presence of human serum.

Hepatitis C virus antibody secretion in vitro by peripheral blood lymphocytes

A recombinant polypeptide corresponding to a virus-specific cDNA clone (c100-3) serves as the antigen for a hepatitis C virus (HCV) antibody assay. Previous investigations have shown an 80% prevalence of HCV antibodies in sera of patients suffering from post-transfusional chronic hepatitis non-A, non-B, but positive results were also obtained for 30 to 70% of sera from patients with chronic hepatitis B or autoimmune hepatitis. In this study we show that HCV antibodies are secreted by peripheral blood lymphocytes (PBL) in vitro. PBL from 12/35 patients with chronic non-A, non-B hepatitis and 1/6 patients with chronic active hepatitis B spontaneously secreted HCV antibodies in cell culture supernatants. The results were confirmed by neutralisation assay and ELISAs using recombinant and synthetic polypeptides derived from the c100-3 antigen and from the HCV core antigen. Two patients suffering from non-A, non-B hepatitis were negative for HCV antibodies in serum, but their PBL produced HCV c100-3 antibodies in vitro. PBL from patients suffering from autoimmune chronic hepatitis, primary biliary cirrhosis, toxic-liver injury and healthy blood donors did not produce antibodies to HCV c100 antigen irrespective of HCV antibody test results in their sera. Polyclonal B cell activation or mitogenic stimulation of T helper cells led to increased immunoglobulin synthesis by PBL in vitro, but did not lead to enhancement of specific HCV antibody production. In addition, HCV antibody production was not induced by these stimulation procedures in control lymphocytes. This spontaneous HCV antibody production in vitro suggests persistent antigenic stimulation of the B cells in vivo.