Granulocyte-macrophage colony-stimulating factor and interleukin-3 potentiate interferon-gamma-mediated endothelin production by human monocytes: role of protein kinase C.

Monocytic cells have been shown to produce endothelin, a potent vasoconstrictor molecule with immune modulating properties. The signalling mechanisms involved in this response are presently unclear. Monocytes are also believed to play an important role in inflammatory bowel disease (IBD). The objective of this study was to characterize the role of various cytokines, bacterial lipopolysaccharide (LPS) and colony-stimulating factors on the production of endothelin (ET) by freshly isolated human monocytes. Compelling circumstantial evidence exists for the conditions being investigated occurring in inflamed bowel mucosa to where monocytes migrate. Whereas LPS stimulated the release of 7 pg ET/2x106 cells in 40 hr, interferon-gamma (IFN-gamma) stimulated 45 pg ET/2x106 cells in 40 hr. There was an additive response when the two stimuli were employed together. Significantly the addition of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) effected a two- to threefold, dose-dependent increase in the production of ET. Production of endothelin was reproducibly blocked by the addition of the protein kinase C (PKC) inhibitors staurosporine and H7, as well as by the protein synthesis inhibitor cycloheximide. Assessment of the activities of the alpha and beta isoforms of conventional protein kinase C (PKC), as determined by MonoQ column fractionated calcium and lipid activatible phosphotransferase activity towards myelin basic protein (MBP) revealed an additive effect of using LPS, IFN-gamma and GM-CSF, which was even greater than that demonstrated for phorbol myristate acetate (PMA). Additionally the secretion of ET by monocytes from Crohn's disease patients (in remission) was analysed and compared with an age-matched control group. There was no significant difference between the two. These results: (1) demonstrate an important synergistic role for GM-CSF and IL-3 in the predominantly IFN-gamma-mediated ET production by normal human monocytes; (2) indicate a possible role for the protein kinase C signalling pathway in this response; and (3) argue against a primary abnormality of ET production in peripheral monocytes from patients with Crohn's disease.