Pneumocystis cell wall beta-glucans induce dendritic cell costimulatory molecule expression and inflammatory activation through a Fas-Fas ligand mechanism.

Respiratory failure during Pneumocystis pneumonia is mainly a consequence of exaggerated inflammatory responses to the organism. Dendritic cells (DCs) are the most potent APCs in the lung and are key to the regulation of innate and adaptive immune responses. However, their participation in the inflammatory response directed against Pneumocystis infection has not been fully elucidated. Therefore, we studied the role of Pneumocystis carinii, as well as Saccharomyces cerevisiae, cell wall-derived beta-glucans, in DC costimulatory molecule expression. We further studied the impact of beta-glucans on subsequent T cell activation. Because cytokine secretion by DCs has recently been shown to be regulated by Fas ligand (FasL), its role in beta-glucan activation of DCs was also investigated. beta-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by beta-glucans elicits T cell activation and polarization into a Th1 patterned response, but with the conspicuous absence of IL-12. These observations differed from LPS-driven T cell polarization, suggesting that beta-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1beta and TNF-alpha secretion by beta-glucan-stimulated DCs was partially regulated by Fas-FasL. This suggests that dysregulation of FasL could further enhance exuberant and prolonged cytokine production by DCs following DC-T cell interactions, further promoting lung inflammation typical of Pneumocystis pneumonia.

Surfactant protein D enhances Pneumocystis infection in immune-suppressed mice.

To further determine the role of surfactant protein (SP)-D in the pathogenesis of Pneumocystis pneumonia, a mouse model of transgenic overexpression (OE) of SP-D was studied. These animals produce roughly 30- to 50-fold greater SP-D than their wild-type (WT) counterparts but show no other differences in lung morphology and function. Animals in both the SP-D OE and WT groups were depleted of CD4 lymphocytes with weekly injections of GK1.5 antibody, before Pneumocystis inoculation, and throughout the subsequent infection period. At various time points, mice were killed and analyzed for inflammatory parameters and organism burden. Proinflammatory cytokines in bronchoalveolar lavage fluid were elevated throughout the period of infection, with OE animals exhibiting significantly higher levels of TNF-alpha and macrophage inflammatory protein-2 compared with WT controls. The total number of cells in the lavage fluid was also increased significantly only in the OE group, whereas the cell differential composition demonstrated lymphocyte and eosinophil infiltration in both groups of animals. Significantly, the organism burden was markedly higher in the SP-D OE animals, whereas the WT mice demonstrated little alteration in organism number over the course of infection. These results further indicate that SP-D facilitates the development of Pneumocystis infection and related lung inflammation in an immunosuppressed mouse model.

Surfactant protein D-mediated aggregation of Pneumocystis carinii impairs phagocytosis by alveolar macrophages.

Pneumocystis carinii remains an important and potentially fatal cause of opportunistic pneumonia. Animal studies reveal that substantial quantities of surfactant protein D (SP-D) accumulate in the airspaces during P. carinii pneumonia and are particularly abundant in aggregates of organisms. Due to the multimeric structure of SP-D, we hypothesized that SP-D mediates aggregation of the organism. From previous clinical studies it is known that aggregated organisms are conspicuous in sections of lung tissue and bronchoalveolar lavage (BAL) fluids of humans with active P. carinii pneumonia. Herein, we observe that SP-D levels increased at least fourfold in BAL fluids of patients with P. carinii pneumonia. Next, a spectrophotometric sedimentation assay was developed to assess the aggregation of P. carinii in vitro by SP-D. P. carinii organisms were first stripped with glutathione to remove bound SP-D and subsequently incubated in the presence of SP-D and 2 mM calcium. P. carinii incubated with natural SP-D (10 micro g/ml) containing dodecamers and higher-order forms exhibited aggregation and enhanced sedimentation compared to that of glutathione-stripped P. carinii. Aggregation was also enhanced by the concentrated supernatant of rat BAL fluid, and this effect was abolished by the selective removal of SP-D from the lavage fluid. P. carinii aggregation was reduced by maltose, mannose, and EDTA, consistent with the role of the SP-D C-type lectin domain (CRD) in the aggregation event. Comparisons of different molecular forms of SP-D showed that dodecamers-but not trimeric subunits-mediate optimal aggregation of P. carinii. Aggregation of P. carinii by SP-D was shown to be responsible for the impaired phagocytosis of the organisms by alveolar macrophages. Thus, SP-D-mediated aggregation of P. carinii may represent one means by which the organism avoids elimination by the host.

Pneumocystis carinii cell wall beta-glucan induces release of macrophage inflammatory protein-2 from alveolar epithelial cells via a lactosylceramide-mediated mechanism.

Infiltration of the lungs with neutrophils promotes respiratory failure during severe Pneumocystis carinii (PC) pneumonia. Recent studies have shown that alveolar epithelial cells (AECs), in addition to promoting PC attachment, also participate in lung inflammation by the release of cytokines and chemokines. Herein, we demonstrate that a PC beta-glucan rich cell wall isolate (PCBG) stimulates the release of macrophage inflammatory protein-2 (MIP-2) from isolated AECs through a lactosylceramide-dependent mechanism. The results demonstrate that MIP-2 mRNA and protein production is significantly increased at both early and late time points after PCBG challenge. Although CD11b/CD18 (Mac-1, CR3) is the most widely studied beta-glucan receptor, we demonstrate that CD11b/CD18 is not present on AECs. This study instead demonstrates that preincubation of AECs with an antibody directed against the membrane glycosphingolipid lactosylceramide (CDw17) results in a significant decrease in MIP-2 secretion. Preincubation of the anti-CDw17 antibody with solubilized lactosylceramide reverses this effect. Furthermore, incubation of AECs with inhibitors of glycosphingolipid biosynthesis, including N-butyldeoxyno jirimycin and d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl, also results in a significant decrease in AEC MIP-2 production following challenge with PCBG. These data demonstrate that PC beta-glucan induces significant production of MIP-2 from AECs and that CDw17 participates in the glucan-induced inflammatory signaling in lung epithelial cells during PC infection.