(First of Two Parts) THE part played by phagocytes in defense against invading pathogens has been recognized since 1883. In that year, Metschnikoff, a Russian zoologist, reported that foreign particles injected into metazoans (in Metschnikoff's experiments, starfish larvae) were taken up by a population of "wandering mesodermal cells" that resided in interstitial tissues.1 He postulated a crucial role in host defense for these wandering cells, which he named "phagocytes." Since Metschnikoff's discovery, hundreds of scientists studying dozens of species have reported thousands of studies on these cells, perhaps the most widely recognized of which are those of the eminent English . . .

Oxygen-dependent microbial killing by phagocytes (second of two parts).

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

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

Using a specific and sensitive fluorometric assay, the H2O2 release from as few as 2 X 10(5) mouse peritoneal macrophages could be detected continuously and quantitated. It is emphasized that the assay measured H2O2 release, not production. Induction of H2O2 release required sequential application of two stimuli: the administration of an activating agent to the mice from 4 days to 10 wk before all harvest, and the exposure of the cells in vitro to a triggering agent. BCG was most effective as an activating agent, resulting in peritoneal macrophages which could be triggered to release H2O2 almost as copiously (8 nmol/10(6) macrophages per 5 min) as mouse peritoneal PMN (9 NMOL/10(6) PMN per 5 min). Casein and C. parvum could also serve as activators, but thioglycollate and FCS were ineffective after single injections. PMA was a potent triggering agent, resulting in a maximal rate of H2O2 release after a latency of about 40 s for cells in suspension. Other triggering agents included the ionophore A23187, concanavalin A in the presence of cytochalasin B, and phagocytosis. H2O2 release could be attributed to macrophages and PMN in peritoneal cell suspensions or in preparations of adherent peritoneal cells, but not to lymphocytes. Indirect evidence suggested that the H2O2 detected was formed from superoxide anion. These observations appear to justify renewed interest in the idea that H2O2 may be important in macrohpage antimicrobial and antitumor mechanisms.

https://rupress.org/jem/article-pdf/146/6/1648/1402769/1648.pdf

Hydrogen peroxide release from mouse peritoneal macrophages: dependence on sequential activation and triggering.

TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 29,79-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 59-upstream region, the CArG element in the 59 flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.

/pdf/production-of-hydrogen-peroxide-by-transforming-growth-jh6q7zmj8v.pdf

Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells.

Stimulation of guinea pig granolocytes by digitonin results in superoxide (O-2) generation. A continuous assay shows that there is a lag between the addition of digitonin and the onset of O-2 production. The rate of activation of the O-2 generating system is dependent upon the concentration of digitonin and the temperature. The final linear rate of O-2 production is affected by the concentration of digitonin, temperature, pH, and the presence of exogenous reduced pyridine nucleotides. Thus, factors which alter either the activation process or the activity of the O-2 generating system can affect O-2 production by stimulated granolocytes.

/pdf/superoxide-generation-by-digitonin-stimulated-guinea-pig-1ib9810fcf.pdf

Superoxide generation by digitonin-stimulated guinea pig granulocytes. A basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system.

Recent studies indicate that oxygen radicals such as superoxide or singlet oxygen may be important in the functional activity of human granulocytes. We have examined the possible importance of these radicals in the functional capacity of human blood monocytes. Monocytes, like granulocytes, generate chemiluminescence during phagocytosis. Chemiluminescence is impaired 50-90% by superoxide dismutase, an enzyme which enhances the dismutation of superoxide to hydrogen peroxide. These results indicate that superoxide is related to the chemiluminescence generated by monocytes. Superoxide dismutase in a concentration which impaired chemiluminescence also impaired the staphylococcal killing by monocytes. Hexose monophosphate shunt activity and hydrogen peroxide production by granulocytes and monocytes were also evaluated. The oxidation of [1-14C]glucose was used as a measure of hexose monophosphate shunt activity and the oxidation of [14C]formate as an estimation of hydrogen peroxide production. The oxidation of both substrates by monocytes was increased during phagocytosis but, in contrast to results in granulocytes, was not further increased by the addition of superoxide dismutase. These data indicate that superoxide may be important in bactericidal activity of human monocytes. Our results also suggest that the metabolism of oxygen radicals in monocytes and granulocytes may be different.

/pdf/a-comparison-of-the-metabolic-response-to-phagocytosis-in-1t49pv7pp8.pdf

A L Sagone

Papers

A comparison of the metabolic response to phagocytosis in human granulocytes and monocytes.