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 splenocytes 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 extent of DNA fragmentation, the hallmark of apoptosis, determined by agarose gel electrophoresis and the amount of fragmented DNA measured by ELISA method showed that the extent of apoptosis was clearly increased in splenocytes cultured in the presence of MS. Under the scanning and transmission electron microscopic observations, the large portion of splenocytes 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 proliferation by mitogens was strongly suppressed. When analyzed by surface immunolabelling flow cytometry, the subsets of lymphocytes (B, T, CD4 + T and CD8 ± T cells) were affected in a global non-selective manner. As determined by ultrafiltration, the molecular weights of apoptosis-facilitating factors present in MS appeared to be greater than 10 kDa. Upon fractionation with Sephadex G-200, the apoptotic factors were separated into 2 fractions. In summary, results obtained in the present study indicate that some unidentified endogeneous macromolecules present in MS may produce the stimulatory effect on the apoptosis and cause immunosuppression of splenocytes under culture.