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Murine polymorphonuclear leukocytes (PMN) are stimulated to produce hydrogen peroxide (H2O2) by some unopsonized fungi, including Coccidioides immitis spherules, Candida albicans blastospores, and Saccharomyces sp. blastospores (zymosan). In this communication we have examined the basis for this stimulation by studying the effects of mannan and its primary constituent, D-mannose, on PMN function in vitro, since this polysaccharide is a significant component in many fungal cell walls. Our results show that mannan stimulated H2O2 production and iodination by PMN and enhanced H2O2 production by cells stimulated with zymosan. Moreover, the amount of H2O2 released by PMN was directly related to the concentration of mannan, and mannan obtained commercially or prepared in our laboratory worked equally well. Mannose, on the other hand, inhibited H2O2 release from PMN stimulated with zymosan or mannan and reduced oxygen consumption, carbon dioxide production, superoxide release, and iodination by these cells as well. With respect to specificity of inhibition, 18 different monosaccharides were examined by using 2 different assays for H2O2 release; and only 2-deoxy-D-glucose, a potent glycolytic inhibitor, reduced H2O2 release from zymosan-stimulated PMN. Furthermore, H2O2 release from cells stimulated with phorbol myristate acetate or opsonized Sephadex was not inhibited by mannose whereas H2O2 release was reduced when PMN were stimulated with C. albicans blastospores or C. immitis spherules in the presence of this sugar. From these data we propose that initiation of PMN oxidative metabolic burst in response to some unopsonized fungi occurs through a mannose-specific mechanism.

Spleen cells from mice immunized with a variety of antigens and incubated in vitro with killed spherules of Coccidioides immitis lyse six to eight times more autologous murine erythrocytes than normal spleen cells and spherules. Cellular and biochemical events in this phenomenon were investigated to ascertain its significance. Kinetic studies suggested that hemolysis results from the activation of some immune cells by spherules. The capacity of spherules to activate these cells is rather unusual because, of the inert particles tested, only zymosan A and crude chitin demonstrated comparable activity. Furthermore, although the hemolytic phenomenon occurred in serum-free medium, more lysis was effected by immune cells and opsonized spherules or zymosan A than by immune cells and untreated fungal particles. Sheep, chicken, and human erythrocytes were not lysed in the hemolytic phenomenon; however, hemoglobin in chicken and sheep erythrocytes was oxidized. Both the murine erythrocyte lysis and oxidation of ovine hemoglobin correlated with the reduction of Nitro Blue Tetrazolium by immune cells adherent to spherules, and both phenomena appeared to be mediated by H2O2 released into the medium by activated cells. Spleen cells reactive with spherules could not be depleted by treatment with iron carbonyl, antiimmunoglobulin plus complement, or anti-brain-associated theta plus complement, but they were partially or completely depleted after rosette formation with erythrocytes coated with antibody or murine complement. Using light and electron microscopy, we noted that immune spleens contained more neutrophils than normal spleens, that these neutrophils reduced Nitro Blue Tetrazolium after stimulation with phorbol myristate acetate, and that they were the most prevalent cell type adherent to spherules after incubation in vitro.

This investigation analyzed the effect of labor on the phagocytic activity of neutrophils in women with uncomplicated term pregnancies. Nineteen healthy women who were not pregnant and did not use oral contraceptives or glucocorticoids served as controls. Peripheral venous blood samples were collected from 15 study patients who were in the active phase of labor (5-10 cm of dilation). Neutrophil phagocytic function was evaluated with the radioiodine fixation test. Assays were conducted utilizing both pooled homologous serum and autologous serum. There was no statistically significant difference in the neutrophil phagocytic function of laboring patients and controls. In addition, there was no evidence that serum from pregnant women exerted a depressant effect on phagocytosis.

The response of human polymorphonuclear leukocytes (PMN) to blastospores and pseudo-hyphae of the opportunistic fungus Candida albicans has been studied in vitro and in vivo. Of the fungicidal mechanisms elucidated thus far, the myeloperoxidase-hydrogen peroxide-halide system appears to be most effective against cells of this fungus. In our studies on the interaction between murine PMN and blastospores, we assayed the release of H2O2 by PMN incubated with viable or killed, unopsonized or opsonized blastospores by using two assay systems, lysis of murine erythrocytes and oxidation of scopoletin. Our results showed that PMN released increasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized killed blastospores, but released decreasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized viable blastospores. The oxidative metabolic burst by PMN in the presence of viable or killed blastospores was also measured by using reduction of nitroblue tetrazolium and chemiluminescence. Viable blastospores stimulated a stronger metabolic burst than killed blastospores, suggesting that PMN respond to live blastospores more vigorously than killed blastospores; however, live blastospores appear to alter or inhibit the release of H2O2 by PMN.

Iodide fixation by murine polymorphonuclear leukocytes (PMN) incubated with viable Candida albicans blastoconidia increases directly with yeast cell concentration up to about 3 x 10(6) cells per ml, but above this concentration bound activity declines dramatically. To understand the basis for this decline, we examined the oxidative metabolism of fungi and stimulated PMN and found some remarkable similarities between these cell types. Both produced 14CO2 when incubated with [1-14C]glucose, both reduced cytochrome c, and both fixed radiolabeled iodide, although the fungi required exogenous lactoperoxidase. In dose-response experiments, iodination by fungi with lactoperoxidase was identical to that with PMN, i.e., the maximum bound activity occurred in cultures with 10(6) to 3 x 10(6) blastoconidia per ml. Iodination by fungi with lactoperoxidase was reduced when blastoconidia were incubated at 25 degrees C or in the presence of catalase and the metabolic inhibitors rotenone, antimycin A, and 2-deoxyglucose. Results from assays for oxidation of scopoletin and o-dianisidine showed that 10(6) blastoconidia in 1.0 ml of medium released 0.5 to 0.7 nmol of H2O2 after 1 h, but 3 X 10(6) and 10(7) cells released significantly less H2O2. These results suggest that iodide fixation by PMN and low numbers of fungal cells may reflect a cooperative effort, with fungi generating some H2O2 that reacts with the myeloperoxidase released from the PMN. With high concentrations of blastoconidia, H2O2 activity appeared to be specifically inhibited, possibly to protect fungal cells from damage.