APOPTOSIS OF CULTURED RAT GLOMERULAR MESANGIAL CELLS INDUCED BY IGG2AMONOCLONAL ANTI-THY-1 ANTIBODIES

Anti-Thy-1 nephritis is a model of mesangial proliferative glomerulone phritis. It has been suggested that apoptosis, which is a counteractin g regulatory mechanism against undesired cell proliferation, is involv ed in sequential histological changes in this model. In the present st udy, we investigated whether IgG2a anti-Thy-1 monoclonal antibody (ER4 ) or its F(ab')2 fragments are able to induce apoptosis of rat glomeru lar mesangial cells (GMC) in vitro. After coculture 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 disloca tion 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 mi croscopy, distinct nuclear condensation and positive reactivity with F ITC-annexin V were observed in cells co-cultured with ER4 or its F(ab' )2 fragments. The results obtained by FAGS analysis with annexin V sho wed a direct correlation with the detection of apoptosis with the term inal deoxynucleotidyl transferase reaction (TDT). Up to 19% and 23% of rat GMC, which were co-cultured for 24 hours with 1 mu g/ml (0.5 mu g /1 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 apo ptosis were seen. The degree of apoptosis as measured by the annexin V method pas dependent on the concentration of ER4 and time of incubati on in the presence of ER4. Finally, apoptosis was confirmed by gel ele ctrophoresis of DNA isolated from the cells co-cultured with each mono clonal antibody (MAb). DNA extracts from cells co-cultured with ER4 or its F(ab')2 fragments demonstrated typical internucleosomal DNA fragm entation. Medium alone, controls of anti-human C3bi receptor MAb (IB4) and anti-rat MHC class I MAb (OX18) showed neither nuclear changes no r significant labeling of the cells with the TDT reaction or with the annexin V. Taken together, these results demonstrate for the first tim e 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 degr ee of mesangial alteration in Thy-1 nephritis.