Pneumocystis carinii attaches to alveolar epithelial cells during the development of pneumonia. Adhesive proteins found within the alveolar space have been proposed to mediate P. carinii adherence to lung cells. Vitronectin (Vn), a 75-kDa glycoprotein present in the lower respiratory tract, has substantial cell-adhesive properties and might participate in the host-parasite interaction during P. carinii pneumonia. To address whether Vn binds to P. carinii, we studied the interaction of radiolabeled Vn with purified P. carinii organisms. Vn bound to P. carinii, occupying an estimated 5.47 x 105 binding sites per organism, with an affinity constant, Kd, of 4.24 x 10-7 M. Interestingly, the interaction of Vn with P. carinii was not mediated through the Arg-Gly-Asp cell-adhesive domain of Vn. Addition of Arg-Gly-Asp-Ser (RGDS) peptides did not inhibit binding. In contrast, Vn binding to P. carinii was substantially inhibited by the addition of heparin or by * Corresponding author. significance of Vn-microbe interactions in the pathogenesis of infection has not yet been established. Although P. carinii organisms encounter substantial concentrations of Vn in both normal and diseased lung, the role of Vn in P. carinii pneumonia has not been fully studied. Accordingly, this investigation was undertaken to determine whether Vn interacts with purified P. carinii and to determine which regions of Vn interact with the organism. To also determine the potential role of Vn in mediating P. carinii attachment to host epithelial cells, we assessed the impact of Vn in mediating P. carinii attachment to cultured A549 lung cells. MATERIALS AND METHODS Materials. All organic chemicals were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, Mo.) or Fischer Scientific Co. (Pittsburgh, Pa.) unless otherwise specified. Iodo-Beads were obtained from Pierce Chemical Co., Rockford, Ill.; fetal bovine serum was from GIBCO, Grand Island, N.Y.; and carrier-free Na'251 and Rainbow
Pneumocystis carinii is an opportunistic organism that causes severe lung injury in immunocompromised hosts. Macrophage responses to P. carinii are poorly defined. Arachidonic acid (AA) and its metabolites are potent mediators of inflammation and have been implicated in host response to microorganisms. We therefore examined the production of eicosanoids from rat and rabbit alveolar macrophages stimulated with purified P. carinii. [14C]AA-labeled rabbit macrophages released 8.50 +/- 1.33% of the incorporated [14C]AA after 90 min in response to P. carinii (P = 0.0001 compared with unstimulated controls). In contrast, a similar number of rat alveolar macrophages exhibited a smaller but significant response to P. carinii, releasing 3.84 +/- 1.54% of their [14C]AA after 90 min (P = 0.001 compared with control). We further determined that P. carinii stimulated substantial production of prostaglandin E2 and concurrently a small amount of leukotriene B4 release from alveolar macrophages. To further investigate whether serum opsonization of P. carinii enhances these alterations in AA metabolism, we assessed the effect of P. carinii immune serum on P. carinii-induced AA release. P. carinii opsonized with this antiserum caused significantly greater AA release from rat alveolar macrophages than either unopsonized P. carinii or organisms opsonized with nonimmune serum. Previous studies suggest that P. carinii interacts with macrophage beta-glucan and mannose receptors. However, incubation of macrophages with P. carinii in the presence of either soluble beta-glucan or alpha-mannan failed to alter the release of AA from macrophages in response to P. carinii. Macrophage release of eicosanoids represents a potentially important host inflammatory response to P. carinii infection.
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 have evaluated the effect of whole fungi on macrophage eicosanoid metabolism. We hypothesized that macrophages respond to C. albicans by releasing AA and generating AA metabolites as a consequence of interaction of mannose and I-glucan receptors with fungal cell wall components. ['4C]AA-labeled rabbit alveolar macrophages released AA following stimulation with either live or heat-killed C. albicans. Highpressure 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., 6-ketoprostaglandin F1,, thromboxane B2, and leukotrienes B4 and D4. We further examined the roles of a-mannan and I-glucan components of C. albicans in mediating these alterations of eicosanoid metabolism. Prior work in our laboratory has shown that soluble a-mannan and ,-glucan inhibit macrophage mannose and j-glucan receptors, respectively. Incubation of alveolar macrophages with soluble a-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 I-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 ot-mannan and j-glucan constituents of the fungus.
Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [14C]-AA labeled monocytes released 8.9 +/- 2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 16:1) (P = 0.0002). Prior studies indicate that soluble alpha-mannans and beta-glucans antagonize mannose and beta-glucan receptors, respectively. Preincubation of monocytes with alpha-mannan (100 micrograms/ml) caused 45.8 +/- 5.7% inhibition of [14C]-AA release, whereas beta-glucan (100 micrograms/ml) yielded 43.7 +/- 6.0% inhibition (P < 0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, alpha-mannan or beta-glucan failed to inhibit IL-1 beta release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by alpha-mannan and beta-glucan components of the fungus.
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