Yasuko Kawamoto

There is increasing evidence for the role of the Fas/Fas ligand interaction in the immunoregulation of T-cells. We studied the expression of the Fas ligand (FasL) in activated peripheral T-cells in vitro, and its relation to autonomous cell death by flow cytometry. Following the stimulation of lymph node T-cells with anti-CD3 and rIL2, the mRNA level of FasL increased more than four times during the first 2 days over the level before stimulation. The surface expression of FasL was observed on 27% of the population at day 2 after stimulation and increased to approximately 50% at day 3. Kinetic analysis by flow cytometry, however, indicated that all T-blasts transformed during activation did not express FasL. FasL expression became evident simultaneously with the termination of cell expansion. Since cells remained viable (> 90%) at day 3 as judged by trypan blue-exclusion, cell membranes expressing Fast were supposed to be still intact. Concomitantly with Fast-expression, spontaneous DNA fragmentation was observed. These observations support the idea that autonomous Fas/FasL interaction mediates apoptosis in activated peripheral T-cells as demonstrated in T-cell hybridoma or established T-cells. (C) 1997 Elsevier Science B.V.

Na-CBZ-L-lysine thiobenzyl ester (BLT)-specific proteases in cytoplasmic granules of intraepithelial lymphocytes in the murine intestine (iIEL) were characterized. BLT-specific proteases were isolated with the Sephacryl S-200 column chromatography, and the sample isolated contained a protein with a molecular weight of 58 kDa. The 58 kDa protein consisted of the homodimer of the 30 kDa subunits. The 58 kDa protease was detected by [&lt sup&gt 3&lt /sup&gt ] diisopropylfluorophosphate (DFP)-labeling, and also detectable by the immunoblotting using an antibody against the partial synthetic peptide of granzymce A. The cytoplasmic granules of iIEL were stained positively by an immunofluorescence with anti-granzyme A antibody. Therefore, it was suggested that the major BLT-specific proteases present in cytoplasmic granules of iIEL, might be granzyme A.

The natural killer (NK)-like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the participation of serine proteases in it were investigated, We monitored the cytotoxicity of iIEL with a sensitive cytotoxic assay using laser how cytometry. iIEL exhibited NK-like cytotoxicity on YAC-1 target cells, Benzamidine, a serine protease inhibitor, inhibited significantly both Na-CBZ-L-lysine thiobenzyl ester (BLT)-specific serine protease activity and iIEL-mediated NK-like cytotoxicity. These results suggest that BLT-specific serine proteases may participate in NK-like cytotoxicity of murine iIEL.