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The studies presented indicate that sensitized guinea pig lymph node lymphocytes release on exposure to antigen a soluble mediator of 35 to 70,000 daltons, which induces proliferation of nonimmune oil-induced, monocyte-derived peritoneal and noninduced alveolar macrophages. Cell counts and nuclear labeling experiments indicate that this proliferation can be effectively assayed under conditions of monolayer culture by tritiated thymidine incorporation. The magnitude and the kinetics of the proliferative response of macrophages in this system are dependent upon the concentration and the time of addition of the soluble mediator. Influences that operate to limit proliferation in this system appear to be high macrophage density, the production of prostaglandin by macrophages, and other mediators present in the lymphokine preparation, such as MIF. We have tentatively termed the active soluble mediator macrophage mitogenic factor (MMF); however, its similarity to a fibroblast-produced macrophage growth factor (MGF) might indicate that, like the interferons, a term like Type II MGF would also be appropriate. The biology of this soluble mediator would appear relevant in terms of the marked proliferation and activation of macrophages known to occur during states of intense T lymphocyte-mediated immunity, as occurs during infection with facultative intracellular bacterial pathogens.

The results of 47 experiments indicate that the lectin mitogens, PHA and Con A, increase cyclic GMP (cGMP) levels in human peripheral blood lymphocytes and their incubation medium 2- to 9-fold within 10 min of stimulation. At concentrations supraoptimal for the induction of proliferation, Con A, but not PHA, induces progressive increases in cAMP and lesser increases in cGMP. Succinylated Con A (S-Con A) does not exhibit inhibition of proliferation up to concentrations of 250 µg/ml and correspondingly does not produce the cAMP increases observed with native Con A. PHA and both forms of Con A induce modest increases in cGMP in the absence of calcium and no increases in the presence of EGTA. After preexposure to Con A in the absence of calcium, the addition of calcium produces increases in cGMP. The data are consistent with the interpretation that mitogen-induced increases in lymphocyte cGMP are totally Ca2+-dependent, yet only in part dependent on extra-cellular Ca2+. The interpretation is confirmed with a divalent cation ionophore A23187, which was shown to induce lymphocyte proliferation in close association with Ca2+-dependent increases in cGMP and at higher concentrations to inhibit the mitogenic effects in association with Mg2+-dependent increases in cAMP. Dose-response curves for each agent showed that this capacity to increase cGMP levels was correlated with the optimal mitogenic concentration as determined by assay of DNA synthesis. The data are consistent with the postulate that cGMP participates in the positive aspects of mitogen action and that cAMP participates in the inhibitory aspects. It is concluded that coparticipation of cGMP and Ca2+ as essential mediators of the mitogenic signal is a sound hypothesis.

The action of the purified thymic factor, thymopoietin, on populations of post-thymic lymphocytes has been studied. Thymopoietin, at concentrations as low as 1.5 ng/ml, uniquely enhanced the proliferative response of peripheral T cells from lymph node and spleen to allogeneic stimulation. Enhancement of the allogeneic response (MLR) was not produced by several polypeptide hormones, including insulin, ACTH, HCG, or Ubiquitin. Treatment of spleen cells with anti-Thy-1 antiserum almost completely abolished the MLR. Thymopoietin's stimulatory effects could not reverse this. Thymopoietin treatment of Thy-1+-enriched spleen cell populations enhanced the MLR even when thymopoietin was removed as early as 2 min after incubation with responding cells. The interaction of thymopoietin with peripheral Thy-1+ cell populations produced a rapid and transient rise in cyclic GMP levels and slightly decreased cyclic AMP levels. These results suggest that thymopoietin interacts with one or more Thy-1+ subpopulations and that this interaction involves early changes in cyclic nucleotide metabolism.