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.

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␤-Glucans are polymers of D-glucose which represent major structural components of fungal cell walls. It was shown previously that fungi interact with macrophages through ␤-glucan receptors, thereby inducing release of tumor necrosis factor alpha (TNF-␣). Additional studies demonstrated that vitronectin, a host adhesive glycoprotein, binds to fungi and enhances macrophage recognition of these organisms. Since vitronectin contains a carbohydrate-binding region, we postulated that vitronectin binds fungal ␤-glucans and subsequently augments macrophage TNF-␣ release in response to this fungal component. To study this, we first determined the release of TNF-␣ from alveolar macrophages stimulated with fungal ␤-glucan. Maximal TNF-␣ release occurred with moderate concentrations of ␤-glucan (100 to 200 g/ml), whereas higher concentrations of ␤-glucan (>500 g/ml) caused apparent suppression of the TNF-␣ activity released. This suppression of TNF-␣ activity by high concentrations of ␤-glucan was mediated by the particulate ␤-glucan binding soluble TNF-␣, through the lectin-binding domain of the cytokine, rendering the TNF-␣ less available for measurement. Next, we assessed the interaction of vitronectin with ␤-glucan. Binding of 125 I-vitronectin to particulate fungal ␤-glucan was dose dependent and specifically inhibitable by unlabeled vitronectin. Furthermore, treatment of ␤-glucan with vitronectin substantially augmented macrophage TNF-␣ release in response to this fungal component. These findings demonstrate that fungal ␤-glucan can directly modulate TNF-␣ release from macrophages. Further, these studies indicate that the host adhesive glycoprotein vitronectin specifically binds ␤-glucan and augments macrophage cytokine release in response to this fungal element.