Korean J Hematol.  1997 Aug;32(2):234-247.

Nitric Oxide Synthesis in Murine Peritoneal Macrophages by Antithrombin III

• Kwak JY
• Park SY
• Lee HS
• Sohn MH
• Yim CY

Abstract

BACKGROUND: During the development of inflammatory responses, various cytokines including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) which activate macrophages to synthesize nitric oxide (NO) and hemostatic cascades are secreted. However, effects of various factors involved in hemostasis on NO synthesis have been little known. In this report, we studied the effects of antithrombin III (AT-III) on NO synthesis by thioglycollate-elicited peritoneal macrophages of BALB/c mouse.
METHODS
Macrophages were obtained from BALB/c mice injected with 3% thioglycollate and cultured with or without various reagents. Nitrite concentration was measured using a modification of Griess reaction. Immunoblot analysis for inducible nitric oxide synthase (iNOS), AT-III receptor binding study and assay of TNF secretion were performed.
RESULTS
AT-III alone did not induce nitrite synthesis by macrophages. However, AT-III induced nitrite synthesis in the presence of IFN-gamma in a dose dependant manner. iNOS was also expressed in macrophages by the treatment with IFN-gamma/AT-III. Addition of 0.5mM NG-monomethyl-L-arginine markedly inhibited IFN-gamma/AT-III-induced nirtite synthesis. Treatment with 5microgram/mL polymyxin B, which inactivates LPS, did not inhibit IFN-gamma/AT- III-induced nitrite synthesis, excluding inadvertent endotoxin contamination. Addition of rabbit anti-human AT-III polyclonal IgG antibodies, but not control rabbit IgG, inhibited IFN-gamma/ AT-III-induced nitrite production. Treatment of macrophages with heparin did not augment, but inhibited both IFN-gamma/AT-III- and IFN-gamma/LPS-induced nitrite synthesis.
CONCLUSION
Our results indicated that AT-III acted as a modulator of macrophage activation for L-arginine dependent NO synthesis and that AT-III might be an important molecule in the regulation of immune responses by macrophage activation to induce NO synthesis as well as in the regulation of hemostasis. Heparin was not a cofactor in the AT-III-induced NO synthesis.

Keyword

Antithrombin III; Nitric oxide; Macrophage; Heparin

MeSH Terms

Animals
Antibodies
Antithrombin III*
Arginine
Cytokines
Hemostasis
Heparin
Immunoglobulin G
Indicators and Reagents
Interferon-gamma
Interleukin-1
Macrophage Activation
Macrophages
Macrophages, Peritoneal*
Mice
Nitric Oxide Synthase Type II
Nitric Oxide*
omega-N-Methylarginine
Polymyxin B
Tumor Necrosis Factor-alpha
Antibodies
Antithrombin III
Arginine
Cytokines
Heparin
Immunoglobulin G
Indicators and Reagents
Interferon-gamma
Interleukin-1
Nitric Oxide
Nitric Oxide Synthase Type II
Polymyxin B
Tumor Necrosis Factor-alpha
omega-N-Methylarginine