FLOW CYTOMETRIC DETECTION OF ACTIVATION-INDUCED CELL-DEATH (APOPTOSIS) IN PERIPHERAL-BLOOD LYMPHOCYTE SUBPOPULATIONS FROM HEALTHY CATS

Human peripheral blood lymphocytes (PBLs) from healthy individuals are resistant to in vitro-induced apoptosis, but activated human lymphocy tes can readily undergo apoptosis. The activation of human lymphocytes is accompanied by the upregulation of a cell surface antigen, the maj or histocompatibility complex (MHC) class II-antigen. Only a minority of PBLs are usually MHC class II-antigen-positive in healthy humans. I n contrast, in healthy cats the majority of feline PBLs are MHC class II-antigen-positive. We have, therefore, investigated the sensitivity of feline peripheral blood lymphocytes obtained from specified pathoge n free (SPF) cats to the induction of apoptosis. Feline PBLs from SPF cats (n = 16) and human PBLs from healthy donors (n = 2) were isolated . After short-term culture, cells were examined for the presence of fr agmented DNA as a result of apoptosis by a DNA agarose gel electrophor esis method and for the presence of DNA double strand breaks by in sit u 3' end labeling. In addition, relative DNA content per cell was flow cytometrically determined using propidium iodide (PI) or 7-actinomyci n-D (7-AAD) and apoptotic cells were identified on the basis of a redu ced DNA content. Cell surface antigens and cellular DNA were analyzed simultaneously by dual-color flow cytometric analyses in order to stud y lymphocyte subsets. Single-and dual-color analysis revealed that, in contrast to human lymphocytes, feline lymphocytes rapidly underwent a poptosis when cultured overnight in medium. Furthermore, the majority of apoptotic cells was found within the MHC II-positive cell subset.