Lymphocyte activation and cytokine production by Pisum sativum agglutinin (PSA) in vivo and in vitro

Mice spleen cells were incubated in vitro for 24 h with Pisum sativum agglutinin (PSA). The addition of these supernatants (SN) to macrophage cultures induced the production of nitric oxide (NO) by these cells in a dose-dependent manner. NO release was blocked in the presence of IFN gamma Antibodies and partially inhibited by TNF alpha Antibodies. The ability of PSA in inducing the production of IFN gamma and TNF alpha by spleen lymphocytes was confirmed assaying these cytokine levels in the SN. Spleen cells stimulated in vitro with PSA were highly activated showing an increased expression of the earlier activation marker, CD69, and a great proliferative response. On the other hand, spleen cells obtained from mice treated with PSA 24 h earlier, did not produce significant levels of IFN gamma or TNF alpha when incubated in vitro and showed a significantly lower proliferation rate when pulsed in vitro with PSA or Concanavalin A (ConA). The lower responsiveness to mitogens was also evident after 48 and 72 h after the treatment in vivo with the lectin. Nevertheless, the flow cytometric analysis of spleen lymphocytes obtained from PSA-treated Animals showed a high degree of activation in cells CD3+. There was a decrease in the expression of L-selectin and VLA-4, when compared to controls, in parallel with a significant increase in the expression of CD69 and CD122 (IL-2R) in lymphocytes recovered from PSA-injected Animals. The data point to evidence that PSA induces immunomodulatory effects, activating spleen lymphocytes in vivo, which become unresponsive to a second stimulation in vitro.