The effect of nitric oxide (NO) on growth and major histocompatibility complex(MHC)-unrestricted cytotoxicity of interleukin(IL)-2-cultivated rat spleen nonadherent mononuclear cells was examined. NO donor sodium nitroprusside (SNP) at relatively low concentrations increased magnitude, as well as duration of IL-2-induced proliferative response of nonadherent splenocytes. SNP effect depended completely on released NO, because it was prevented by NO scavenger haemoglobin, but not mimicked by expired SNP solution, unable to generate NO, or ferricyanide, a second breakdown product of SNP. Other NO donors - SIN-1, SNAP and GSNO failed to exert SNP-like growth-enhancing action, probably as a consequence of rapid NO generation, compared to sustained NO release by SNP. All NO-releasing chemicals at high concentration blocked IL-2-induced proliferation. Growth-promoting effect of SNP-derived NO was independent of guanilat cyclase activation, because dibutyryl cGMP did not affect IL-2-triggered splenocyte proliferation. Macrophage NO acted in a manner similar to SNP; at low concentrations it promoted IL-2-induced splenocyte growth, however higher amounts were suppressive. Cytotoxicity of IL-2-activated splenocytes against NK-sensitive K562 cell line was significantly increased when SNP was present during cultivation with IL-2. Proportion of NKR-P1+ and CD25+ cells, as well as per cell expression of these important activation molecules were increased upon SNP treatment, suggesting possible mechanism for the observed NO action.

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) activity in murine L929 fibroblasts were investigated. IFN-g-induced NO production in L929 cells was mediated through an iNOS-dependent L-arginine-NO pathway, since it was abrogated by a selective inhibitor of iNOS, aminoguanidine. CsA applied simultaneously with IFN-g caused a dose-dependent reduction of NO synthesis in L929 cells. However, CsA did not influence the enzymatic activity of iNOS, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-g and any further induction was blocked by the protein-synthesis inhibitor cycloheximide. IFN-g-triggered expression of mRNA for interferon regulatory factor-1 was not reduced by CsA-treatment, suggesting that this iNOS transcription factor is not a target in CsA-mediated inhibition of NO synthesis. Finally, FK506 was not able to mimic the inhibitory effect of CsA on NO production in L929 cells, indicating the calcineurin-independent mechanism of CsA action. These results indicate that CsA suppresses NO synthesis in L929 cells independent of calcineurin inhibition, and interfering with intracellular pathways involved in the iNOS induction, rather than inhibiting its enzymatic activity.

Cells expressing markers of both natural killer and T lymphocytes (NK T cells) in humans and mice express a restricted T-cell receptor (TCR) repertoire, are of CD4- CD8- or CD4+ CD8- phenotype, and upon anti-CD3 stimulation secrete large amounts of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). NK T cells may be the primary source of IL-4-promoting T helper type 2 (Th2) responses and/or they might be involved in regulating the balance between Th1- and Th2-type immune responses, and may consequently affect susceptibility to autoimmune diseases associated with a skewed Th phenotype. We show that rat NK T cells selectively proliferate to IL-2, and use this fact to analyse cytokine production by NK T cells in two rat strains differentially susceptible to Th1- or Th2-type autoimmune diseases. Analysis by reverse transcription-polymerase chain reaction revealed that, in contrast to mouse, rat NK T cells secrete exclusively IFN-gamma and not IL-4 after anti-CD3 stimulation, and use a wider TCR-Vbeta repertoire, suggesting that rat NK T cells are not essential for the development of Th2-type CD4+ T-cell responses.