NK Cell Proliferation and Cytokine Production Depends on the mTOR- and Calcineurin-Pathway but not p38 MAP-Kinase Pathway

Abstract

The role of NK cells during solid organ rejection is still nearly unexplored. Therefore we investigated the effect of routinely given immunosuppressants on the proliferation and cytokine production of fresh NK cells and NKL, a NK cell leukemia line. Methods: eFluor 670 stained NKL cells were treated with medium supplemented with DMSO, mTOR-inhibitor Sirolimus (Sir), calcineurin-inhibitors Tacrolimus (Tac) and Cyclosporin A (CsA) or MEK1/2- inhibitor U0126 (10 or 20 μM) in the presence or absence of IL-2. Supernatants were harvested at day 3 for the analysis of cytokines/chemokines by the Bioplex-technique and proliferative capacity was assessed by FACS-analyses at day 0 to day 3. Results: Sir, Tac and CsA inhibit the proliferation of NK and NKL cells in a dose-dependent manner. The anti-proliferative effect of the inhibitors can be partly recovered by IL-2. Sir causes the strongest anti-proliferative effect on NKL cells. Although CsA and Tac are both calcineurin-inhibitors, Tac shows a less inhibitory capacity regarding NKL proliferation. In contrast MEK1/2 inhibitor U0126 does not affect NKL proliferation in the presence of IL-2. Some of the cytokines/chemokines produced by NKL cells like GM-CSF or CCL5 are secreted in an IL-2- independent manner. Others like VEGF, CXCL8, IFN-g or IL-10 could only be detected in higher amounts in the presence of IL-2. Interestingly, the influence of these inhibitors on the cytokine/chemokine secretion does not show the same pattern as for NKL proliferation. The most potent inhibitor of NKL proliferation, Sir, only decreases VEGF-production, whereas CsA inhibits the secretion of VEGF, CXCL8 and IL-10. Surprisingly, MEK1/2 inhibitor U0126 induces a strong production of CXCL8, CCL5 and CCL2. Conclusions: Our results show that CNI as well as mTOR inhibitors effect proliferation of NKL cells which depends clearly on IL-2. Interestingly the responsiveness to IL-2 via mTOR inhibition plays a more critical role than the production of IL-2 itself since CsA and Tac do not inhibit NKL proliferation cells as strong as Sir. The MAP kinase pathway is not involved in this proliferation process in the presence of IL-2 as U0126 does not reduce the proliferation of NKL cells. Taken together, proliferation and cytokine/chemokine-production of NK cells are regulated differently by calcineurin and other pathways. Thus, immunosuppressants with high anti-proliferative capacity must not be automatically a good suppressor of cytokine production. Therefore, the efficacy of immunosuppressants should also be addressed with respect to cytokine and chemokine secretion.