Human polymorphonuclear granulocytes in inflammation : studies with an emphasis on nitric oxide

University dissertation from Stockholm : Karolinska Institutet, Department of Medicine at Huddinge University Hospital

Abstract: The function of the polymorphonuclear neutrophil granulocyte (PMN) is of vital importance in inflammation. For the defense against infections PMNs are armored with different weapons such as chemotaxis, phagocytosis and production of cytotoxic substances. During the past decade, a new mediator and effector molecule, nitric oxide (NO), has been identified and, for PMNs production of NO, could constitute a way to further tailor the response according to the nature of the activating event. Nitric oxide release was studied by means of two highly sensitive and specific real-time methods, the oxyhemoglobin method and an electrochemical method. Human PMNs activated with the surface receptordependent agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophor, AMU and the direct stimulator of protein kinase C, phorbol myristate acetate (PMA), produced NO which was inhibited by a specific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA). The NO production induced by fMLP or A231987 was dependent on the presence of extracellular Ca2+ but this was not the case for PMA. The stimulatory effect of fMLP was almost completely inhibited by Bordetella pertussis toxin (PT). These results show a strong similarity with previously known pathways for other PMNs functions. Because arachidonate metabolites are potent mediators in inflammation, the effects of leukotriene B4 and the cysteinyl leukotrienes D4 and C4 were studied on the release of NO, in vitro, by PMNs. When activated with LTB4, LTD4 or LTC4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S12S-diHETE, PMNs produced NO in a stimulus- and concentration-dependent manner. Cysteinyl leukotrienes and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with PT, or chelation of cytosolic Ca2+ also efficiently inhibited this response. This effect differs from activation of the NADPH-oxidase for which only LTB4 is an activator. Both overproduction of O2- and/or NO radicals are important mediators of tissue injury in rheumatoid arthritis (RA). There were a high systemic NO production in patients with untreated RA and lower oxidative response in fMLP-stimulated PMNs, in vitro, compared to controls. When treated with a soluble tumor necrosis factor (TNF)-[alpha] receptor plasma nitrite and fMLP-induced PMN-derived O2- production both became similar to controls. In chronic granulomatous disease (CGD) treatment with gamma-interferon (IFN-[gamma]) is associated with reduced frequency of infections. In CGD patients an increased NO formation in fMLP-stimulated PMNs, in vitro, was detected after IFN-[gamma] administration with a maximal enhancement on day 3, concurrently with an increased bactericidal capacity. Thus, human PMNs possess the ability to produce NO after stimulation with various agonists. This could be an additional pathway for PMNs to exert or modulate functional responses in inflammation and infections.

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