Nitric oxide inhibits the secretion of T‐helper 1‐ and T‐helper 2‐associated cytokines in activated human T cells‘pa

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Abstract
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 to preferentially down‐regulate Th1‐mediated immune responses. In the present study, we investigated the effect of NO on the production of Th1‐ and Th2‐associated cytokines by activated human T cells and human T‐cell clones. Cytokine secretion was measured in the presence of the NO‐donating agents 3‐morpholinosydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP). Both NO‐donors markedly inhibited the release of interferon‐γ (IFN‐γ), 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 the supernatants of activated T cells, nor was specific mRNA for inducible and constitutive NO synthase detectable, indicating that T cells themselves did not contribute to the observed effect of the NO donors. Costimulation with anti‐CD28 monoclonal antibodies (mAb) prevented SIN‐1/SNAP‐mediated down‐regulation of cytokine production only in part. In contrast, when T cells were stimulated by phorbol‐ester and ionomycin, they were refractory to SIN‐1‐induced inhibition of cytokine production. When SIN‐1 was added after the onset of anti‐CD3 stimulation, the inhibitory effect was found to be less pronounced, indicating that SIN‐1 may interfere with early signal transduction events. The addition of superoxide dismutase (SOD) and catalase did not restore the effects of SIN‐1, demonstrating that the inhibition of cytokines was due to NO and not to oxygen intermediates. Furthermore, 8‐Br‐cGMP‐mediated increase of intracellular cGMP caused the same pattern of cytokine inhibition as observed with SIN‐1 and SNAP. Using a single cell assay, these agents were shown to reduce the frequency of IFN‐γ‐producing T cells, suggesting that not all T cells are susceptible to SIN‐1/SNAP. However, 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 provide evidence that NO preferentially inhibits Th1‐cytokine secretion of activated human T cells in vitro.