Antithrombin III inhibits lymphocyte proliferation, immunoglobulin production and mRNA expression of lymphocyte growth factors (IL-2, gamma-IFN and IL-4) in vitro

Background: Antithrombin III (AT-III) is a physiological inhibitor of throm bin and other serine proteases, and has antiinflammatory properties. Thromb in is known to enhance T lymphocyte activation in vitro and serine protease s can act as costimulators for lymphocyte proliferation and cytokine produc tion. We have previously shown that AT-III significantly inhibited allograf t rejection in a highly histoincompatible model of rat lung transplantation and in vitro cell proliferation in ConA-stimulated rat spleen cells. In th is study, we examined the involvement of cytokine gene expression in the ab ove inhibitory effect of AT-III. We also examined the effect of AT-III on s everal in vitro immune reactions in human peripheral blood mononuclear cell s (PBMCs). Methods: mRNA expression of cytokines/cytokine receptor importan t in lymphocyte activation was examined. Rat spleen cells were stimulated w ith Con-A with/without AT-III and submitted for reverse transcriptase-polym erase chain reaction (RT-PCR). To assess the effect of AT-III on human PBMC s, we examined the effects of AT-III on cell proliferation of human PBMCs s timulated in mixed lymphocyte reaction (MLR) (allogeneic stimulation), with OKT3 (T cell receptor activation) and with PHA (mitogenic stimulation). Th e effect of AT-III on PWM-stimulated immunoglobulin (Ig) production by huma n PBMCs was also examined. All experiments for cell proliferation were perf ormed in 10% serum and in serum-free (SF) media to determine whether AT-III exerted its effects through its interaction with thrombin in serum. Result s: mRNA expression of IL-2, gamma -IFN and IL-4 in ConA-stimulated rat sple en cells was nearly completely inhibited by AT-III at 15 IU/ml. mRNA levels for IL-6, IL-2R and TGF-betaI were not significantly affected by AT-Ill. A T-III showed a dose-dependent inhibition of cell proliferation in human PBN ICs. At 15 IU/ml, cell proliferation was inhibited by similar to 86%, simil ar to 81% and similar to 56% in the MLR-, OKT3- and PRA-stimulated PBMCs, r espectively in both serum and SF media. AT-III inhibited PWM-stimulated Ig production in a dose-dependent manner. IgG, IgM and IgA production was redu ced by similar to 60%, 80% and 70%, respectively in cultures incubated with 15 IU/ml AT-III. Conclusions: (1) Inhibition of IL-2, gamma -IFN and IL-4 mRNA expression might be responsible for inhibition of cell proliferation b y AT-III in ConA-stimulated rat spleen cells, (2) AT-III inhibits cell prol iferation in the MLR-, OKT3- and PHA-stimulated human PBMCs, and Ig product ion in PWM-stimulated human PBMCs, (3) The immune regulatory effects of AT- III are independent of its interaction with thrombin since similar levels o f suppression were seen in SIT media, and (4) These results suggest that AT -III has potent inhibitory effects on lymphocyte activation and cytokine pr oduction and may have potential applications as an immunomodulatory agent. (C) 2001 Elsevier Science B.V. All rights reserved.