NITRIC-OXIDE INHIBITS THE SECRETION OF T-HELPER-1-ASSOCIATED AND T-HELPER-2-ASSOCIATED CYTOKINES IN ACTIVATED HUMAN T-CELLS

Mechanisms regulating the balance of T-helper 1 (Th1) and T-helper 2 ( Th2) immune responses are of great interest as they may determine the outcome of allergic and infectious diseases. Recently, in mice, nitric oxide (NO), a powerful modulator of inflammation, has been reported t o preferentially down-regulate Th1-mediated immune responses. In the p resent study, we investigated the effect of NO on the production of Th 1- and Th2-associated cytokines by activated human T cells and human T -cell clones. Cytokine secretion was measured in the presence of the N O-donating agents 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acet ylpenicillamine (SNAP). Both NO-donors markedly inhibited the release of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-5, IL-10 and IL-4 by anti-CD3 activated T cells. A preferential inhibition of Th1- associated cytokines was not observed. Neither was nitrite found in th e supernatants of activated T cells, nor was specific mRNA for inducib le and constitutive NO synthase detectable, indicating that T cells th emselves did not contribute to the observed effect of the NO donors. C ostimulation with anti-CD28 monoclonal antibodies (mAb) prevented SIN- 1/SNAP-mediated down-regulation of cytokine production only in part. I n contrast, when T cells were stimulated by phorbol-ester and ionomyci n, they were refractory to SIN-1-induced inhibition of cytokine produc tion. When SIN-1 was added after the onset of anti-CD3 stimulation, th e inhibitory effect was found to be less pronounced, indicating that S IN-1 may interfere with early signal transduction events. The addition of superoxide dismutase (SOD) and catalase did not restore the effect s of SIN-1, demonstrating that the inhibition of cytokines was due to NO and not to oxygen intermediates. Furthermore, 8-Br-cGMP-mediated in crease of intracellular cGMP caused the same pattern of cytokine inhib ition as observed with SIN-1 and SNAP. Using a single cell assay, thes e agents were shown to reduce the frequency of IFN-gamma-producing T c ells, suggesting that not all T cells are susceptible to SIN-1/SNAP. H owever, cytokine production by purified T-cell subpopulations (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)) was equally impaired by NO donors. In conclusion, in contrast to the murine system, our results do not pr ovide evidence that NO preferentially inhibits Th1-cytokine secretion of activated human T cells in vitro.