There was not an absolute elimination of either CD4+ or CD8+ T cells, consistent with observations that these mice do not have abnormalities in CD4+ or CD8+ cells compared to littermate controls (data not shown). of anti-viral reactions and facilitating viral persistence. test. (B) The relative increase of early apoptotic cells raises after co-culture with Tg hepatocytes. With this example, data for Tg hepatocytes were normalized to the appropriate Tg? control. Results for both CD4+ and CD8+ remain statistically significant at 4 and 15 h. (C) All annexin V+ cells, showing improved apoptosis of both CD4+ and CD8+ T cells at 15 h, and an increase in CD4+ AV+ cells at 4 h. (D) Relative increase in all AV+ cells. When all annexin V+ cells were analyzed (Fig. 2C), the variations remained statistically significant, with the exception of Dinoprost tromethamine the difference between CD8+ cells at 4 h. As expected, the absolute ideals for those AV+ increased over time. For example, in the case of CD4+ T cells, the median value of AV+ cells after 15 h of co-culture was 17.3% after co-culture with non-Tg hepatocytes and 28.5% after co-culture with HCV Tg hepatocytes. For CD8+ T cells, the median value of 30% of cells after co-culture for 15 h increased to 37% after co-culture with HCV Tg hepatocytes. Similarly, there is an increase in the relative degree of apoptosis if all AV+ cells are measured, with the exception of CD8+ cells at the early time point (Fig. 2D). The data suggest that CD8+ T cells at 4 h are primarily still in the early phases of apoptosis and have not progressed to later on stages. Non-activated T cells experienced very low levels of apoptosis, and there is no difference between Tg+ and Tg? for non-activated cells (data not shown). Taken collectively, the data suggest that the manifestation of HCV structural proteins in hepatocytes increases the apoptosis of triggered CD4+ and CD8+ T cells. Improved apoptosis of triggered T cells is definitely associated with upregulation of Fas ligand manifestation on HCV transgenic hepatocytes Activated T cells may be induced to undergo apoptosis by a variety of death-inducing molecules, including FasL (also known as CD95 ligand), TNF (Crispe, 1999), galectin-1 (Baum et al., 1995), and TNF related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand) (Jeremias et al., 1998). To study whether any of these death-inducing molecules is responsible for improved apoptosis of triggered T cells induced by hepatocytes expressing HCV proteins, we analyzed and compared the manifestation of these molecules between HCV Tg+ and non-Tg hepatocytes. We performed Western blotting analysis to study the manifestation of FasL, TRAIL, and galectin-1 using specific antibodies against these molecules. The level of FasL manifestation was improved in HCV Tg hepatocytes relative to their non-Tg counterparts Mouse Monoclonal to Rabbit IgG (Fig. 3A). The findings were related for total RNA using RT-PCR (Fig. 3B), as well as analysis of cell surface FASL manifestation using circulation cytometry (Fig. 3C). To measure the concentration of TNF in the supernatants of co-culture of hepatocytes and triggered T cells, we used ELISA. No statistically significant difference was observed between HCV Tg+ hepatocytes and non-Tg hepatocytes at either 4 or 15 h Dinoprost tromethamine of co-culture (data not shown). Manifestation of either Dinoprost tromethamine TRAIL or galectin-1 was not observed in Tg or non-Tg hepatocytes (data not demonstrated). After co-culture with HCV-expressing hepatocytes, there was no switch in FasL manifestation on lymphocytes (Fig. 3D). Open in a separate windows Fig. 3 Improved manifestation of Fas ligand in HCV transgenic hepatocytes expressing structural proteins. (A) Cell lysates (200 g) were subjected to Western blot analysis using specific polyclonal rabbit anti-mouse Fas ligand antibodies. The level.