Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader

Peroxynitrite formation from macrophage-derived nitric oxide

Phagocytes, in particular macrophages and PMN, are now recognized as major components of inflammatory and immunologic reactions in the lung. Normally, macrophages represent the majority of phagocytes in the lower respiratory tract. These lung macrophages are morphologically and functionally heterogenous and include alveolar, interstitial, intravascular, and airway macrophages, each with characteristic morphologic and functional features. Through the presence of surface receptors for numerous ligands and through their large number of secretory products, lung macrophages can respond to environmental factors and account for most of the clearance of microparticles and microorganisms in the distal airways and the alveolar spaces. In addition, macrophages also play an important role in inflammatory processes through the release of oxygen radicals and proteolytic enzymes. Through the release of several cytokines, i.e., growth-promoting and inhibiting factors, lung macrophages may also influence both matrix damage and repair processes. Macrophages can also contribute to the alveolitis by recruitment of inflammatory and immune cells. This latter contribution is best demonstrated in migration movement of PMN. The normal distal airways generally contain a small number of PMN, but the pulmonary vascular bed represents a large reservoir of PMN. Some of them are in intimate contact with the endothelium, forming the so-called marginating pool of PMN. Because the capillary lumen is separated only from the alveolar space by a monolayer of endothelial and epithelial cells on each side of a thin interstitial matrix, it is likely that some inhibitory mechanism exists to prevent PMN from migrating towards the alveolar space. Such inhibitors of PMN migration are present both in serum and in the alveolar space, some being released by alveolar macrophages. However, alveolar macrophages can also secrete factors called chemotaxins that attract PMN to the airways, and this supports a central role for alveolar macrophages in the regulation of PMN traffic in the lungs. Thus, secretory products of alveolar macrophages are part of the regulatory mechanisms of PMN mobility and adherence that appears to be crucial in the initiation of some inflammatory reactions. The contribution of phagocytes to the defense against infection and tumor has been documented mostly in vitro. Thus, both oxygen radicals, in particular hydroxyl radicals and proteases such as lysozyme, are potent bactericidal agents. That phagocytes are also important defenders of the lungs in vivo is best supported by the observations in immunodeficient patients and animal models.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophages and polymorphonuclear neutrophils in lung defense and injury

Publisher Summary This chapter discusses the nature of endotoxins and their interactions with cells of the immune system. The direct interaction of endotoxin with B lymphocytes leading to the formation of antibodies to endotoxin as well as a spectrum of other immunoglobulin molecules is discussed. In addition, the interactions of endotoxins with T cells and macrophages and the associated immunoregulatory effects are discussed. The central role of the lipid A portion of the endotoxin molecule in cellular activation and the molecular events occurring at the cell surface in the course of stimulation are presented. The potential of endotoxins as therapeutic manipulators of the immune response in man is also evaluated. Major direct interactions of endotoxins with B lymphocytes have been documented, leading to synthesis and secretion of antibody directed not only against antigenic determinants on the endotoxin molecules themselves, but also with specificities characteristic of the complete repertoire of variable region gene products. Knowledge of the immunobiology of endotoxins has prompted investigations in their therapeutic uses in man as both antigenically distinct and cross-reactive immunogens in protection against gram-negative infections.

Bacterial endotoxins and host immune responses

Arachidonate and other unsaturated long-chain fatty acids were found to activate protein kinase C from human neutrophils. Kinase activation by arachidonate required calcium and was enhanced by diolein but did not require exogenous phosphatidylserine. Submaximal levels of arachidonate also enhanced the affinity of the kinase for calcium during activation by phosphatidylserine. Thus the release of arachidonate, which is triggered in many cell types by ligand-receptor interactions, could play a second messenger role in the regulation of cellular function by activation of protein kinase C.

http://science.sciencemag.org/content/sci/224/4649/622.full.pdf

A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase.

We studied the capacity of cultured mouse peritoneal macrophages to generate superoxide anion (O(2-)), the initial product of conversion of oxygen to microbicidal species, during phagocytosis of opsonized zymosan or upon contact with the membrane-active agent phorbel myristate acetate (PMA). Macrophages from mice infected with Bacille Calmette-Guerin (BCG) or injected intraperitoneally with thioglycollate broth or endotoxin, released up to 12 times more O(2-) than did resident peritoneal macrophages, depending upon the cell type and whether the stimulus was zymosan or PMA. There was little if any O(2-) release from resting (unstimulated) macrophages. The density of cells on culture dishes was an important variable since crowding of the dish markedly reduced the efficiency of O(2-) production. The enhanced O(2-) release of chemically elicited and infection-activated macrophages was noted after stimulation with a wide range of concentrations of PMA and zymosan, at all time points studied (up to 120 min), and with cells maintained for 140 rain to 16 days in culture. The O(2-) response of resident cells improved twofold to zymosan and ninefold to PMA during the first 3 days in culture. The capacity to release O~ appears to be limited to actively phagocytic cell types: murine macrophage-like tumor lines and cultured human monocytes released O(2-) when stimulated by PMA or zymosan, fibroblast and endothelial lines and embryo-derived cells did not. Activity of superoxide dismutase, which removes O(2-), was not detectable in culture supernates of any cell type, and thus, differences in detectable O(2-) could not be attributed to variations in the release of this enzyme. We conclude that the phagocytosis- associated respiratory burst is significantly enhanced in mononuclear phagocytes obtained ai~r chemical inflammation or BCG infection. Increased capacity to generate O(2-) and other oxygen radicals during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of activated macrophages.

https://rupress.org/jem/article-pdf/148/1/115/1089288/115.pdf

CA Godzik

Papers

Increased superoxide anion production by immunologically activated and chemically elicited macrophages.