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 thrombin and other serine proteases, and has antiinflammatory properties. Thrombin is known to enhance T lymphocyte activation in vitro and serine proteases can act as costimulators for lymphocyte proliferation and cytokine production. We have previously shown that AT-III significantly inhibited allograft rejection in a highly histoincompatible model of rat lung transplantation and in vitro cell proliferation in ConA-stimulated rat spleen cells. In this study, we examined the involvement of cytokine gene expression in the above inhibitory effect of AT-III. We also examined the effect of AT-III on several in vitro immune reactions in human peripheral blood mononuclear cells (PBMCs).


METHODS
mRNA expression of cytokines/cytokine receptor important in lymphocyte activation was examined. Rat spleen cells were stimulated with Con-A with/without AT-III and submitted for reverse transcriptase-polymerase chain reaction (RT-PCR). To assess the effect of AT-III on human PBMCs, we examined the effects of AT-III on cell proliferation of human PBMCs stimulated in mixed lymphocyte reaction (MLR) (allogeneic stimulation), with OKT3 (T cell receptor activation) and with PHA (mitogenic stimulation). The effect of AT-III on PWM-stimulated immunoglobulin (Ig) production by human PBMCs was also examined. All experiments for cell proliferation were performed in 10% serum and in serum-free (SF) media to determine whether AT-III exerted its effects through its interaction with thrombin in serum.


RESULTS
mRNA expression of IL-2, gamma-IFN and IL-4 in ConA-stimulated rat spleen cells was nearly completely inhibited by AT-III at 15 IU/ml. mRNA levels for IL-6, IL-2R and TGF-beta1 were not significantly affected by AT-III. AT-III showed a dose-dependent inhibition of cell proliferation in human PBMCs. At 15 IU/ml, cell proliferation was inhibited by approximately 86%, approximately 81% and approximately 56% in the MLR-, OKT3- and PHA-stimulated PBMCs, respectively in both serum and SF media. AT-III inhibited PWM-stimulated Ig production in a dose-dependent manner. IgG, IgM and IgA production was reduced by approximately 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 by AT-III in ConA-stimulated rat spleen cells, (2) AT-III inhibits cell proliferation in the MLR-, OKT3- and PHA-stimulated human PBMCs, and Ig production in PWM-stimulated human PBMCs, (3) The immune regulatory effects of AT-III are independent of its interaction with thrombin since similar levels of suppression were seen in SF media, and (4) These results suggest that AT-III has potent inhibitory effects on lymphocyte activation and cytokine production and may have potential applications as an immunomodulatory agent.
Zuo
X J
XJ
Department of Pediatrics, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA 90048, USA.
Nicolaidou
E
E
Okada
Y
Y
Toyoda
M
M
Jordan
S C
SC
D000818:Animals; D000917:Antibody Formation / Q000187:drug effects*; D000990:Antithrombin III / Q000494:pharmacology*; D002478:Cells, Cultured / Q000187:drug effects; D003208:Concanavalin A / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis; D007074:Immunoglobulin G / Q000096:biosynthesis; D007075:Immunoglobulin M / Q000096:biosynthesis; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D015847:Interleukin-4 / Q000096:biosynthesis / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects*; D007959:Lymphocyte Culture Test, Mixed; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology
D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D012333:RNA, Messenger; D003208:Concanavalin A; D015847:Interleukin-4; D007371:Interferon-gamma; D000990:Antithrombin III