Splenic T lymphocytes die preferentially during heat-induced apoptosis: NuMA reorganization as a marker

We are investigating nuclear events during apoptosis in mouse splenic lymphocytes cultured immediately after isolation (controls) or after heat treatment (42 degreesC, 30 minutes), and have found that hyperthermia increased the level of apoptosis to double that of spontaneous apoptosis in controls within 6 hours. Immunolabelling for Nuclear Mitotic Apparatus Protein (NuMA) suggested that splenocytes were responding heterogeneously to the heat treatment. Whereas all nuclei in controls and about half of nuclei in heat-treated samples showed the usual diffuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots. We then examined whether the heterogeneity in NuMA organization might be an indication of a differential response of B and T lymphocytes to hyperthermia, and whether the presence of NuMA spots is related to the apoptotic process. NuMA labelling of heated fractionated splenocyte populations showed that 90% of nuclei in T-enriched cultures (less than or equal to 4% IgG+ cells), but only 25% of nuclei in B-enriched samples (less than or equal to 80% IgG+ cells), contained spots. As well, 2 hours after heat treatment of unfractionated cultures, greater than or equal to 90% of nuclei that were accumulating DNA strand breaks, as detected by TUNEL, exhibited NuMA spots. These data indicate that cells with NuMA spots are targetted for, or have initiated, the death program. Since most T cells, but few or no B cells, were spotty after heating, we conclude further that hyperthermia induces apoptosis preferentially in splenic T lymphocytes. The observation that the proportion of T cells was, on average, threefold greater in control than in heated samples after 24 hours in culture reinforces this conclusion.