Evidence for a cyclic guanosine monophosphate-dependent, carbon monoxide-mediated, signaling system in the regulation of TNF-alpha production by human pulmonary macrophages
J. Arias-Diaz, E. Vara, C. Garcia, N. Villa and J. L. Balibrea
Department of Biochemistry (Facultad de Medicina), Universidad Complutense, Madrid, Spain.
BACKGROUND AND OBJECTIVES: The second messenger cyclic guanosine
3',5'-monophosphate (cGMP) seems to be implicated in the release of tumor
necrosis factor alpha (TNF-alpha) by activated macrophages. There is
controversy regarding the potential of human macrophages to produce nitric
oxide (NO). Since guanylate cyclase can be activated also by carbon
monoxide (CO) and this gas may be formed endogenously, we examined the
ability of human pulmonary macrophages to produce CO in the presence of
lipopolysaccharide (LPS) or LPS+interferon gamma (IFN-gamma). In addition,
the source and the relative contribution of this molecule to the
LPS-induced increase in cell cGMP content and TNF-alpha release were
explored. DESIGN: Interstitial macrophages were obtained from multiple
organ donor lungs by enzymatic digestion. After 24-hour preculture,
purified macrophages were cultured for 24 hours in the presence or absence
of LPS, LPS+IFN-gamma, CO (250 and 500 mumol/L), sodium nitroprusside,
8-Br-cGMP, hemoglobin, methylene blue, zinc-protoporphyrin IX, hemin,
S-adenosylmethionine, deferoxamine mesylate, or combinations. The cGMP
content of the cells and TNF-alpha, CO, and NO release to the medium were
determined. RESULTS: In the presence of LPS, TNF-alpha production was not
accompanied by any detectable increase in the NO release to the medium.
However, an increase in medium CO concentration (mean +/- SEM) (5.81 +/-
0.20 vs 3.74 +/- 0.08 pmol/microgram protein; n = 11; P < .01) and cell
cGMP content (0.273 +/- 0.021 vs 0.138 +/- 0.019 pmol/microgram protein; n
= 10; P < .01) was observed. These changes were more pronounced in the
presence of LPS+IFN-gamma. Release of TNF-alpha also was induced by both
sodium nitroprusside and 8-Br-cGMP. In contrast, methylene blue, a
guanylate cyclase inhibitor, inhibited LPS-, LPS+IFN-gamma-, and sodium
nitroprusside-induced TNF-alpha production and cGMP increase; hemoglobin,
which traps CO, had a similar effect. CONCLUSION: Intracellular cGMP
increase, secondary to an endogenous production of CO, participates in the
release of TNF-alpha by activated human pulmonary macrophages.
