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 h eat treatment (42 degrees C, 30 minutes), and have found that hyperthe rmia increased the level of apoptosis to double that of spontaneous ap optosis in controls within Ci hours. Immunolabelling for Nuclear Mitot ic 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 dif fuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots. We then examined whether the heteroge neity in NuMA organization might be an indication of a differential re sponse of B and T lymphocytes to hyperthermia, and whether the presenc e 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 on ly 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 u nfractionated cultures, greater than or equal to 90% of nuclei that we re accumulating DNA strand breaks, as detected by TUNEL, exhibited NuM A 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 El cells, were spotty after heating, we conclude further that h yperthermia induces apoptosis preferentially in splenic T lymphocytes, The observation that the proportion of T cells was, on average, three fold greater in control than in heated samples after 24 hours in cultu re reinforces this conclusion.