Standing, Joseph E.

Abstract Pneumocystis pneumonia is caused, at least partly, by inflammatory action of β-glucan, the major component of Pneumocystis cell wall. Glucan induces release of pro-inflammatory cytokines in alveolar macrophages and lung epithelial cells. While macrophages recognize glucan mainly by dectin receptors, epithelial cells utilize membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer) as glucan-binding molecule. The use of GSL inhibitors in vitro causes reduced NF-κB activation and inhibited IL-8 release in epithelial cells. To assess the impact of LacCer in lung inflammation, we treated Pneumocystis-infected mice with GSL inhibitor PDMP. The animals were immunosuppressed by GK 1.5 antibody, infected with Pneumocystis by intratracheal injection and, starting at 8 weeks post infection, treated with either 150 mg PDMP/kg of body weight - dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). The analysis of sacrificed animals showed that PDMP-treated mice had fewer leukocytes in the bronchoalveolar lavage fluid (BALF) than saline-treated controls with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP-2 and TNF-α (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q-PCR, showed a trend of decreased Pneumocystis-specific DNA copies in PDMP-treated animals. These results suggest that PDMP might have an impact on inflammatory response to Pneumocystis. Other GSL inhibitors are yet to be tested in similar in vivo experiments. This might lead to new options in treatment of Pneumocystis pneumonia. Supported by NIH RO1 HL62150 and Mayo

Abstract Alveolar macrophages (AMS) initiate inflammation during Pneumocystis carinii pneumonia by releasing cytokines including TNF-alpha. Recent studies suggest that macrophage responses to P. carinii are enhanced by serum opsonization, but the mechanisms of enhancement are not well defined. To determine whether macrophage release of TNF-alpha in response to P. carinii was augmented by immune opsonization, alveolar macrophages obtained from rabbits were cultured with P. carinii that had been opsonized with either nonimmune rabbit serum, immune serum generated against P. carinii, or an affinity-purified polyclonal Ab recognizing the major P. carinii surface Ag gp120. Each experiment also included organisms maintained in media alone (nonopsonized P. carinii). Opsonization of P. carinii with immune serum or gp120 Ab significantly enhanced macrophage TNF-alpha release. Interestingly, however, opsonization with nonimmune serum also increased TNF-alpha response to the organism. Because P. carinii is known to interact with the adhesive glycoproteins, vitronectin (VN) and fibronectin (FN), we hypothesized that they might also augment TNF-alpha release. Opsonization of P. carinii with VN or FN resulted in significant potentiation of macrophage TNF-alpha liberation. We further determined that VN and FN were present in increased quantities in the lower respiratory tract of patients with P. carinii pneumonia compared with normal volunteers. Additionally, VN and FN were demonstrated on the surface of freshly isolated P. carinii organisms by immunoblot analysis. Our study suggests that immune and nonimmune opsonins contribute to host defenses during P. carinii pneumonia by enhancing regional TNF-alpha release in response to the organism.

Abstract Recent studies suggest that TNF-alpha plays a central role in host defenses during Pneumocystis carinii pneumonia. To determine whether P. carinii directly stimulates TNF-alpha secretion, rat alveolar macrophages were cultured in the presence of purified P. carinii. Whereas unstimulated alveolar macrophages released only 13.0 +/- 2.7 pg/ml of TNF-alpha into the medium, macrophages stimulated with P. carinii released 108.2 +/- 20.4 pg/ml of TNF-alpha after overnight culture (p = 0.0001). Maximal TNF-alpha release was observed after 8 h of incubation and required a P. carinii: macrophage ratio of at least 2.5:1. Autoclaved P. carinii were also able to trigger TNF-alpha release from macrophages albeit at a reduced level. In view of recent evidence that P. carinii is phylogenetically related to the fungi and contains a beta-glucan-rich cell wall, we hypothesized that TNF-alpha release might in part be mediated by this cell wall component. Preincubation of macrophages with particulate beta-glucan derived from Baker’s yeast resulted in complete inhibition of TNF-alpha release in response to P. carinii. In addition, digestion of P. carinii with zymolyase, a preparation containing predominantly beta-glucanase activity, substantially reduced the ability of P. carinii to cause TNF-alpha release from macrophages. In a similar manner, macrophages incubated with P. carinii in the presence of laminariheptaose, an oligosaccharide that binds to macrophage beta-glucan receptors, also displayed decreased TNF-alpha release. Interestingly, TNF-alpha release may not be completely linked to the adherence of the organism to macrophages. Particulate beta-glucan significantly reduced P. carinii adherence to macrophages and also impaired TNF-alpha release. However, yeast mannan also significantly reduced P. carinii adherence to macrophages and also impaired TNF-alpha release. However, yeast mannan also significantly reduced P. carinii adherence but had no effect on TNF-alpha release. These data demonstrate that P. carinii can directly stimulate the secretion of TNF-alpha from alveolar macrophages and that this effect is largely mediated by beta-glucan components of the P. carinii cell wall.

Pneumocystis, an opportunistic fungus, causes pneumonia in immunocompromized hosts. Inflammation is induced, at least partly, by β‐eta‐glucan, the major component of Pneumocystis cell wall. Airspace‐lining epithelial cells recognize glucan by membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer). We treated Pneumocystis‐infected mice with two GSL inhibitors: PDMP and NB‐DNJ (Miglustat‐Actelion Pharmaceuticals). Starting at 8 weeks post infection, animals received either 150 mg PDMP or 1200 mg Miglustat/kg of body weight ‐ dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). Results show that PDMP‐treated mice had fewer leukocytes in bronchoalveolar lavage fluid (BALF) than saline‐treated controls, with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP‐2 and TNF‐alpha (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q‐PCR, showed decrease in PDMP‐treated animals. The anti‐inflammatory effects of Miglustat were less pronounced than PDMP effects. Neither inhibitor induced lung injury as measured by albumin leak into airspaces. Results suggest that GSL inhibitors might have an impact on inflammatory response to Pneumocystis, leading to new options in treatment of Pneumocystis pneumonia. Supported by NIH‐RO1‐HL62150 and Mayo Clinic

beta-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 beta-glucan receptors, thereby inducing release of tumor necrosis factor alpha (TNF-alpha). 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 beta-glucans and subsequently augments macrophage TNF-alpha release in response to this fungal component. To study this, we first determined the release of TNF-alpha from alveolar macrophages stimulated with fungal beta-glucan. Maximal TNF-alpha release occurred with moderate concentrations of beta-glucan (100 to 200 micrograms/ml), whereas higher concentrations of beta-glucan (> or = 500 micrograms/ml) caused apparent suppression of the TNF-alpha activity released. This suppression of TNF-alpha activity by high concentrations of beta-glucan was mediated by the particulate beta-glucan binding soluble TNF-alpha, through the lectin-binding domain of the cytokine, rendering the TNF-alpha less available for measurement. Next, we assessed the interaction of vitronectin with beta-glucan. Binding of 125I-vitronectin to particulate fungal beta-glucan was dose dependent and specifically inhibitable by unlabeled vitronectin. Furthermore, treatment of beta-glucan with vitronectin substantially augmented macrophage TNF-alpha release in response to this fungal component. These findings demonstrate that fungal beta-glucan can directly modulate TNF-alpha release from macrophages. Further, these studies indicate that the host adhesive glycoprotein vitronectin specifically binds beta-glucan and augments macrophage cytokine release in response to this fungal element.

Pneumocystis carinii causes life-threatening pneumonia in patients with impaired immunity. Recent studies suggest that alveolar macrophages interact with P. carinii through macrophage mannose receptors. However, the ligand(s) on P. carinii that is recognized by these receptors has not been fully defined. P. carinii contains a major mannose-rich surface antigen complex termed glycoprotein A (gpA). It was therefore hypothesized that gpA binds directly to macrophage mannose receptors and mediates organism attachment to these phagocytes. To assess this, gpA was purified from P. carinii by continuous-elution gel electrophoresis. 125I-labeled gpA bound to alveolar macrophages in a saturable fashion. In addition, gpA binding was substantially inhibited by both alpha-mannan and EDTA, further suggesting that gpA interacts with macrophage mannose receptors. Macrophage membrane proteins capable of binding to gpA were isolated with a gpA-Sepharose column. A 165-kDa membrane-associated protein was specifically eluted from the gpA-Sepharose column with EDTA (20 mM). This protein was identified as the macrophage mannose receptor by immunoprecipitation with a polyclonal anti-mannose receptor antiserum. To further investigate the role of gpA in P. carinii-macrophage interactions, 51Cr-labeled P. carinii cells were incubated with macrophages in the presence of increasing concentrations of soluble gpA, and organism attachment was quantified. Soluble gpA (2.5 mg/dl) competitively inhibited P. carinii attachment to alveolar macrophages by 51.3% +/- 3.7% (P = 0.01). Our findings demonstrate that gpA present on P. carinii interacts directly with mannose receptors, thereby mediating organism attachment to alveolar macrophages.

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 10(5) 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 digesting the organisms with heparitinase, suggesting that P. carinii may interact with the glycosaminoglycan-binding domain of Vn. To determine whether Vn might enhance P. carinii attachment to lung epithelial cells, we studied the binding of 51Cr-labeled P. carinii to cultured A549 lung cells. Addition of Vn resulted in significantly increased binding of P. carinii to A549 cells, whereas a neutralizing anti-Vn serum substantially reduced the binding of P. carinii to A549 cells. These data suggest that Vn binds to P. carinii and that Vn might provide an additional means by which P. carinii attaches to respiratory epithelial cells.

Abstract Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-α and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-α generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly β-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that β-glucan components of the P. carinii cell wall largely mediate TNF-α release. Furthermore, the soluble carbohydrate β-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble α-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-α release induced by the P. carinii cell wall fraction. P. carinii β-glucan-induced TNF-α release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii β-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate β-glucan receptors on the phagocyte.

Abstract 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 β-glucans, in DC costimulatory molecule expression. We further studied the impact of β-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 β-glucan activation of DCs was also investigated. β-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by β-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 β-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1β and TNF-α secretion by β-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.

Abstract 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 β-glucan and mannose receptors. However, incubation of macrophages with P. carinii in the presence of either soluble β-glucan or α-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.