Ralston, Nicholas V.C.

Candida albicans is an increasingly important fungal pathogen. Alveolar macrophages respond to fungal components such as zymosan by releasing arachidonic acid (AA) and AA metabolites. However, few studies hypothesized that macrophages respond to C. albicans by releasing AA and generating AA metabolites as a consequence of interaction of mannose and beta-glucan receptors with fungal cell wall components. [14C]AA-labeled rabbit alveolar macrophages released AA following stimulation with either live or heat-killed C. albicans. High-pressure liquid chromatography analysis revealed that 55% of the AA released was metabolized via cyclooxygenase and lipoxygenase pathways. The metabolites consisted of prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha, thromboxane B2, and leukotrienes B4 and D4. We further examined the roles of alpha-mannan and beta-glucan components of C. albicans in mediating these alterations of eicosanoid metabolism. Prior work in our laboratory has shown that soluble alpha-mannan and beta-glucan inhibit macrophage mannose and beta-glucan receptors, respectively. Incubation of alveolar macrophages with soluble alpha-mannan derived from C. albicans (1 mg/ml) resulted in 49.8% +/- 2.6% inhibition of macrophage AA release during stimulation with intact C. albicans (P = 0.0001 versus control). Macrophage AA release in response to C. albicans was also inhibited to a significant but lesser degree by soluble beta-glucan (36.2% +/- 1.3%; P = 0.008 versus control). These results indicate that C. albicans stimulates macrophage AA metabolism and that these effects are partly mediated by alpha-mannan and beta-glucan constituents of the fungus.

The role of Ca2+ was investigated in the response of alveolar macrophages to cotton tannin, an agent implicated in the lung disease byssinosis in textile mill workers. A physiological concentration of extracellular Ca2+ was found to be required for tannin-mediated release of radiolabeled arachidonic acid (AA). Flow cytometry using indo 1 indicated that tannin caused a rapid and dose-dependent Ca2+ increase in macrophages that also required extracellular Ca2+. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid virtually abolished the Ca2+ influx mediated by tannin but had little effect on intracellular Ca2+ release induced by thapsigargin, N-formylmethionyl-leucylphenylalanine, or thimerosal. A mechanism for extracellular Ca2+ influx was demonstrated by rapid Mn2+ quenching of indo 1 by tannin. Verapamil inhibited tannin-mediated Ca2+ influx and AA release, but the effective concentration was 100 microM. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid chelated all Ca2+ in the cells and effectively abolished the tannin response. Exposure to tannin was not associated with cytotoxicity, as judged by 51Cr release. The data suggest that tannin induces Ca2+ influx in alveolar macrophages, which represents an important prerequisite for a cell-signaling pathway resulting in the accumulation of free AA.

AbstractPrevious studies with macrophages that had been prelabeled with [14C]arachidonic acid (20∶4) have shown that condensed tannin is a potent agonist for the release of arachidonic acid. However, it has not been demonstrated that the percentage release of [14C]20∶4 accurately reflects the metabolic activity of the endogenous 20∶4 pool. In order to measure the 20∶4 mass release relative to the total cellular 20∶4 pool, the free fatty acids of freshly isolated alveolar macrophages were derivatized with a fluorescent reagent, and then separated and quantified by high‐performance liquid chromatography. The amounts of esterified fatty acids were measured by gas chromatography of the methyl esters. Free fatty acid levels were compared to those of the total esterified plus unesterified fatty acids to determine the actual percentage released of each fatty acid. Tannin‐stimulated release of 20∶4 mass reflected that previously reported for the release of [14C]20∶4 label but at a slower rate and at a much lower percentage indicating that [14C]20∶4 had been incorporated into part of a more reactive pool. The specificity of the fatty acid release induced by tannin and β‐1,3‐glucan, a known agonist for 20∶4 release, was also examined. Both agonists promoted an increase in the levels of free 20∶4 and of other fatty acids. A comparison of the absolute increases of each of the fatty acids indicated that tannin caused a preferential increase in the mass of free 20∶4, whereas β‐1,3‐glucan evoked a selective increase in the mass of 16∶0.