Apoptosis of cultured rat glomerular mesangial cells induced by IgG2a monoclonal anti-Thy-1 antibodies.
Anti-Thy-1 nephritis is a model of mesangial proliferative glomerulonephritis. It has been suggested that apoptosis, which is a counteracting regulatory mechanism against undesired cell proliferation, is involved in sequential histological changes in this model. In the present study, we investigated whether IgG2a anti-Thy-1 monoclonal antibody (ER4) or its F(ab')2 fragments are able to induce apoptosis of rat glomerular mesangial cells (GMC) in vitro. After co-culture with ER4 or its F(ab')2 fragments, apoptosis was assessed by morphological studies with Hoechst 33258 stain and FITC-annexin V. The latter detects the dislocation of negatively charged phospholipid, phosphatidylserine, from the inner to the outer leaflet of the membrane during apoptosis. This is a sensitive method for the detection of apoptosis. Under fluorescent microscopy, distinct nuclear condensation and positive reactivity with FITC-annexin V were observed in cells co-cultured with ER4 or its F(ab')2 fragments. The results obtained by FACS analysis with annexin V showed a direct correlation with the detection of apoptosis with the terminal deoxynucleotidyl transferase reaction (TDT). Up to 19% and 23% of rat GMC, which were co-cultured for 24 hours with 1 microgram/ml (0.5 microgram/l x 10(5) cells) of ER4 or its F(ab')2 fragments, were labeled by TDT, respectively. With annexin V, up to 34% and 31% cells displaying apoptosis were seen. The degree of apoptosis as measured by the annexin V method was dependent on the concentration of ER4 and time of incubation in the presence of ER4. Finally, apoptosis was confirmed by gel electrophoresis of DNA isolated from the cells co-cultured with each monoclonal antibody (MAb). DNA extracts from cells co-cultured with ER4 or its F(ab')2 fragments demonstrated typical internucleosomal DNA fragmentation. Medium alone, controls of anti-human C3bi receptor MAb (IB4) and anti-rat MHC class I MAb (OX18) showed neither nuclear changes nor significant labeling of the cells with the TDT reaction or with the annexin V. Taken together, these results demonstrate for the first time that anti-Thy-1 MAb is able to induce apoptosis of rat GMC in vitro. The Thy-1 antigen on rat GMC, therefore, seems to function as one of the molecules regulating cell death and thereby may determine the degree of mesangial alteration in Thy-1 nephritis.
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