EVALUATION OF GMI GANGLIOSIDE-MEDIATED APOPTOSIS IN FELINE THYMOCYTES

Cats with inherited GM1 gangliosidosis (GM1 mutant cats) have prematur e thymic involution characterized by decreased total thymocytes primar ily affecting the CD4(+) CD8(+) subpopulation. While GM1 mutant cats h ave increased cell surface GM1 gangliosides, as determined by cholera toxin B binding, on both thymocytes and peripheral lymph node cells on ly thymocytes show increased apoptosis. To determine if GM1 gangliosid es can increase the occurrence of apoptosis in feline thymocytes direc tly, we added exogenous GM1 ganglioside (GM1) to feline thymocyte prim ary cultures and compared the results to apoptotic changes seen in unt reated cells or in cells treated with dexamethasone (Dex), a known ind ucer of thymocyte apoptosis in other species. Incorporation of exogeno us GM1 into thymocyte cytoplasmic membranes was confirmed by flow cyto metric analyses of cholera toxin B labelling. Apoptosis in feline thym ocytes was analyzed by electron microscopy, spectrophotometric evaluat ion of DNA fragmentation, flow cytometric enumeration of apoptotic nuc lei, and gel electrophoretic analysis of degraded DNA, Alterations in percentages of thymocyte immunophenotype following GM1 incorporation w ere determined by flow cytometric analyses of labelled cell surface ma rkers for feline CD4 and CD8, Because in vitro addition of GM1 ganglio sides has been reported in other species to decrease surface expressio n of CM on both thymocytes and peripheral lymphocytes, we evaluated GM 1-associated downregulation of CD4 on the surface of feline thymocytes and peripheral lymph node cells by flow cytometry, Additionally, we c ompared the apoptotic response of the more mature peripheral lymph nod e cells to the less mature thymocytes, Our results indicate that incor poration of exogenous GM1 into feline thymocyte cell membranes produce s a dose-dependent increase of apoptotic cell death. Although, CD4 exp ression on both feline thymocyte and lymph node cell membranes was abr uptly decreased after introducing exogenous GM1, enhanced apoptotic de ath was observed only in thymocytes, not in lymph node cells at the sa me GM1 concentration. Enhancement of thymocyte apoptosis appears to be age-related since cells derived from cats <3 months of age were more vulnerable than those from cats >3 months of age. (C) 1998 Elsevier Sc ience B.V. All rights reserved.