FACILITATION OF APOPTOSIS BY AUTOLOGOUS SERUM AND RELATED IMMUNOSUPPRESSION IN THE SPLENOCYTE CULTURE

The addition of adult mouse serum (MS) to the culture of mouse splenoc ytes resulted in an accelerated decrease of viable cell number during initial 24 h of culture as determined by the trypan blue dye exclusion test and the propidium iodide staining method. Furthermore, the exten t of DNA fragmentation, the hallmark of apoptosis, determined by agaro se gel electrophoresis and the amount of fragmented DNA measured by EL ISA method showed that the extent of apoptosis was clearly increased i n splenocytes cultured in the presence of MS. Under the scanning and t ransmission electron microscopic observations, the large portion of sp lenocytes showed morphological characteristics of apoptotic cells such as apoptotic body, condensed chromatin and shrunken appearance. With the accelerated rate of apoptosis, the immunocompetence of splenocytes such as the antibody production, natural killer cell activity, and pr oliferation by mitogens was strongly suppressed. When analyzed by surf ace immunolabelling flow cytometry, the subsets of lymphocytes (B, T, CD4 + T and CD8 + T cells) were affected in a global non-selective man ner. As determined by ultrafiltration, the molecular weights of apopto sis-facilitating factors present in MS appeared to be greater than 10 kDa. Upon fractionation with Sephadex G-200, the apoptotic factors wer e separated into 2 fractions. In summary, results obtained in the pres ent study indicate that some unidentified endogeneous macromolecules p resent in MS may produce the stimulatory effect on the apoptosis and c ause immunosuppression of splenocytes under culture.