No abstract

Five different kinds of mouse lymphoma cells conditioned culture medium so that it became inhibitory to thiol-dependent cells but not to thiol-independent cells. Conditioning was rapid and appeared to be complete in 1-3 hr. The inhibitory effects could be reversed by concentrations of 2-mercaptoethanol higher than those usually required for growth promotion. Inhibitory factors were concentrated by ultrafiltration and chromatography on Sephadex G-10 which showed their apparent molecular weights to be 300-600. The factors were stable at 1000 and to proteases but were inactivated (after reduction) by iodoacetamide, indicating that they may be thiols or disulfides.Specific thiols and their corresponding disulfides promote the survival and replication in vitro of mouse lymphoid cells, a property which is retained in numerous lymphoma lines (1-4). Such substances, however, are either nonphysiological (particularly 2-mercaptoethanol which has been widely used in experimental systems) or are required at concentrations much greater than occur in vivo. On the other hand, thiol is not required by lymphoma cells when they are cocultivated with macrophages or other cells but, after separation, the supernatant medium does not continue to support growth (5, 6). The mechanism of these effects and their relationship to conditions in vio thus far have not been explained.We here present evidence that thiol-dependent lymphoma cells in vitro are subject to autoinhibition mediated through substances that they liberate into the medium. These have been partially characterized and appear to be thiols or disulfides. Growth promoting thiols (e.g., 2-mercaptoethanol) or cocultivated macrophages interrupt the autoinhibitory system, and these actions may therefore be significant for restoring cell proliferation. The experiments to be described were prompted by observations on the growth promoting effect of macrophages in cocultivation with lymphoma cells (6). Numerous attempts failed to show evidence that macrophages produced a stable conditioning of the medium, and an alternative explanation, that these cells might reverse an autoinhibition by L1210(V) cells, was therefore considered.In most of our experiments we used cells of the thiol-dependent lymphoma L1210(V), whose properties we compared with those of its thiol-independent variant L1210(A) (7). Three other cell lines were examined-EL4, RADA1, and ERLDand in these the same relationships between autoinhibition and thiol dependence were found. MATERIALS AND METHODSTumors. B6D2F1 mice were used to carry the following tumors in ascitic form: L1210(V), L1210(A), and EL4. ERLD (i) Supernatants were concentrated up to 20 times by using a UM05 or other ultrafiltration membrane (Amicon Co., Lexington, MA). Aliquots from these concentrated solutions were tested for their ability to support growth of the various cell lines in tissue culture.(ii) Supernatants were lyophilized to dryness and the residues were dissolved in water and chromatographed on Sephadex G-10.Measurement of Lymphoma Ce...

Sixteen 3 month old "nude" mice, 24 of their litter mates and 30 Swiss mice were injected subcutaneously with 0.1 ml suspension of the E variant of the encephalomyocarditis (EMC) virus. While the mortality rate of the litter mates and Swiss mice during 5-7 days after inoculation was more than 40%, none of the "nude" mice died during the experiment. The surviving animals were sacrificed at 24 h intervals from day one to seven days after injection. Brain suspensions assayed for the presence of the virus yielded significant titers at 24 h in all groups, which increased during 7 days. The litter mates and Swiss mice showed proliferation of lymphocytes and microglial cells in the perivascular areas of the brain during the fifth to the seventh day. The "nude" mice, on the other hand, displayed no perivascular lymphocytic infilteration during the same periods. Ultrastructurally, all groups showed aggregates of ribosomes in the cytoplasmic matrix on the third day, which became enlarged in size on the 5th day. At 7 days, both litter mates and Swiss mice showed an increased number of necrotic cells, while these changes were not observed in the "nude" mice. These findings suggest that the high mortality rate in immunologically normal mice was related to the efforts of T cells to eliminate virus-infected cells and to produce extensive necrosis, while T cell-depleted animals showed good survival rates.