The role of macrophages in the activation of T lymphocytes by concanavalin A. III. Macrophage-independent activation.

Abstract

AbstractLymphocytes from the mesenteric lymph nodes of Lewis rats were depleted of macrophages by absorption for 60 min at 37°C on 0.1‐mm glass beads in the presence of 30% fetal calf serum. Lymphocytes which passed the column (total recovery 30‐50%) contained less than 0.2% macrophages, as assessed by neutral red ingestion or nonspecific esterase staining. At cell densities which allowed optimal proliferation of concanavalin A (Con A)‐stimulated, untreated lymphocytes (2 × 106 cells/ml), macrophage‐depleted lymphocytes were completely incapable of initiating DNA synthesis. The proliferative response to Con A was fully restored by adding back macrophages obtained from the peritoneal cavity. The help of macrophages was optimal at concentrations of 0.8 × 105 cells/ml, which corresponds to about 4% of lymphocytes. Restitution of the mitotic response of macrophage‐depleted lymphocytes was also achieved by raising cell densities to 5 × 106/ml. At this cell density, addition of macrophages was without any effect. Early events in lymphocyte activation, such as the increase of the incorporation of 14C‐oleate into membrane phospholipids after 4 h stimulation, was identical in the presence or absence of macrophages independent of the cell density ranging between 1 × 106 and 10 × 106 cells/ml. Similarly, the Con A‐stimulated early increase in the incorporation of [3H]uridine into RNA was indistinguishable in macrophage‐containing or ‐depleted lymphocytes. At low cell densities, only macrophage‐containing lymphocytes proceeded to increase RNA synthesis, until S‐phase, whereas in the absence of macrophages RNA synthesis approached background values after 40 h of Con A stimulation. Macrophage‐depleted lymphocytes, too, proceeded with an increase in RNA synthesis, when stimulated at high cell densities. The data suggest that macrophages are not required for the initiation of lymphocyte activation. Their role in supporting mitosis may be the contribution of a second signal close to the G1/S boundary extending the size of the proliferating population.