Feb 28, 1983 - GARY A. KORETZKY*, RONALD P. DANIELEt, WARNER C. GREENEt, AND PETER C. NOWELL*§. Departments of *Pathology and Laboratory ...
Proc. NatL Acad. Sci. USA Vol. 80, pp. 3444-3447, June 1983
Immunology
Evidence for an interleukin-independent pathway for human lymphocyte activation (T-cell growth factor/mitogen/A23187/calcium ionophore/immune response)
GARY A. KORETZKY*, RONALD P. DANIELEt, WARNER C. GREENEt, AND PETER C. NOWELL*§ Departments of *Pathology and Laboratory Medicine and of tMedicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and tNational Cancer Institute, Bethesda, Maryland 20205 Contributed by Peter C. Nouwell, February 28, 1983
receptor (14-17) does not inhibit cultures stimulated by A23187, although this antibody is a potent inhibitor of activation induced by other mitogens or antigens. In addition, although exogenous IL 2 greatly augments the proliferative response of human lymphocytes stimulated by phytohemagglutinin (PHA) or TPA, added IL 2 had no effect on proliferation after stimulation by A23187. These observations indicate that the mitogenic action of the ionophore may not only be independent of IL 1 but may also have an IL 2-independent component.
ABSTRACT Though lectin mitogen stimulation of T-cell proliferation is an interleukin 1- (IL 1), interleukin 2- (IL 2) dependent process, the calcium ionophore A23187 may be able to initiate T-lymphocyte proliferation by an additional pathway. That the action of A23187 is IL 1 independent was demonstrated by its ability to stimulate monocyte-depleted cells without the addition of exogenous IL 1. The IL 2 independence of A23187 was indicated by (i) the inability of exogenous IL 2 to augment A23187-induced proliferation and (ii) the inability of the monoclonal antibody anti-Tac (with specificity for the human IL 2 receptor) to inhibit proliferation mediated by A23187. By contrast, in cultures stimulated with the lectin mitogen phytohemagglutinin, additional IL 2 considerably increased proliferation, whereas anti-Tac routinely caused 60-90% inhibition. Although the ionophore caused some IL 2 production and resulted in IL 2 receptor expression, quantitative studies showed that our results could not be explained by excessive amounts of endogenous IL 2 interfering with the blockdng action of the antibody. Therefore, these data suggest that the action of A23187 as a human lymphocyte mitogen may be the result of at least two pathways-one dependent on the interleuidn-cell interactions and the other independent of these mediators.
MATERIALS AND METHODS Cell Preparation. Peripheral blood mononuclear cells (PBM) were obtained by Ficoll-Hypaque separation of heparinized venous blood drawn from healthy human volunteers. For macrophage depletion, PBM were resuspended at 2 X 106 ml in RPMI 1640 with 10% human serum and were incubated in T150 flasks (Coming). After at least 1 hr of incubation at 37°C in 5% CO2 in air, the nonadherent cells were gently poured from the flasks, centrifuged at 400 x g, and resuspended in minimal essential medium. The cells were then passed through a 25-ml G10 column previously washed with minimal essential medium, neomycin/kanamycin [1% (vol/vol)], 2 mM L-glutamine, and 5% serum for at least 2 void vol. The first 15 ml of effluent were designated MDC, which were routinely
