Vuk-Pavlović, Zvezdana

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 Glycosphingolipid (GSL) Lactosyl Ceramide (LacCer) is thought to be involved in Pneumocystis interaction with the host. Our previous results show that treatment with glycosphingolipid synthesis inhibitor PDMP reduces the fungus lung burden as well as symptoms of inflammation in mice with Pneumocystis pneumonia (PCP). Experiments were set up to resolve whether anti-inflammatory effects were due to the specific mechanisms involved in innate immune responses to Pneumocystis, or to some general anti-inflammatory property of PDMP. Animals were infected with live organisms, or challenged with either beta-glucan (the pro-inflammatory component of Pneumocystis cell wall) or LPS (inducer of lung inflammation unrelated to Pneumocystis). At the experimental endpoint the following inflammatory parameters were assessed: leukocyte recruitment to the lungs, production of pro-inflammatory cytokines and chemokines, histopathology and lung damage. Results show that glucan-induced acute inflammation partially mimics the inflammation caused by the whole organism, and PDMP suppresses some of the inflammatory parameters affected in PCP. In contrast, although LPS induces comparable intensity of inflammation as live Pneumocystis or glucan, PDMP does not suppress any inflammatory parameters measured in this group. These results suggest that the mechanism of PDMP action is related to the features specific to Pneumocystis and host recognition of the microorganism. Further studies will attempt to determine the role of LacCer in these processes. Supported by NIH-RO1-HL62150 and Mayo Clinic

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

Abstract Monocyte maturation to macrophages and transformation into epithelioid granuloma cells in some granulomatous diseases are accompanied by the induction of membrane-bound angiotensin-converting enzyme (ACE). The physiologic and pathophysiologic roles of ACE generated in these processes are not known. The pattern and the mechanism of ACE induction in human monocytes are also not well understood. Dexamethasone is one of the agents reported to induce elevated ACE activity in human monocytes, and therefore a suitable tool for studying the phenomenon. This study shows that dexamethasone augments monocyte ACE in a Diphasic dose-dependent manner with maximum effect at 10-5 M concentration. Although it enhances the level of ACE activity, dexamethasone doss not alter the time course for ACE induction from that found in unstimulated monocytes. The ACE activity of monocytes cultivated in 10 nM dexamethasone and then exposed to 10-3 M diazosulfanilic acid (DASA) is reduced approximately by 80% in comparison with cells not treated with DASA, demonstrating that dexamethasone-induced ACE is an ectoenzyme. Dexamethasone does not increase the activity of other monocyte ectoenzymes: γ-glutamyltransferase, alkaline phosphodiesterase-I, and leucine aminopeptktase, showing that dexamethasone induction of ACE is a specific, rather than generalized, effect on plasma membrane enzymes. It is suggested that the increase in ACE activity is due to the increased rate of enzyme synthesis.

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.

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 Fitness of cell-mediated immunity is thought to depend on TCR diversity; however, this concept has not been tested formally. We tested the concept using JH−/− mice that lack B cells and have TCR Vβ diversity <1% that of wild-type mice and quasimonoclonal (QM) mice with oligoclonal B cells and TCR Vβ diversity 7% that of wild-type mice. Despite having a TCR repertoire contracted >99% and defective lymphoid organogenesis, JH−/− mice rejected H-Y-incompatible skin grafts as rapidly as wild-type mice. JH−/− mice exhibited T cell priming by peptide and delayed-type hypersensitivity, although these responses were less than normal owing either to TCR repertoire contraction or defective lymphoid organogenesis. QM mice with TCR diversity contracted >90%, and normal lymphoid organs rejected H-Y incompatible skin grafts as rapidly as wild type mice and exhibited normal T cell priming and normal delayed-type hypersensitivity reactions. QM mice also resisted Pneumocystis murina like wild-type mice. Thus, cell-mediated immunity can function normally despite contractions of TCR diversity >90% and possibly >99%.

Abstract Inhalation of cotton mill dust leads to the development of the occupational lung disease byssinosis in a portion of the exposed workers. Condensed tannins present in the dust have biologic activities consistent with the hypothesis that they are one of the etiologic agents for the disease. Inhalation of either cotton dust or tannin provokes an acute inflammatory response characterized by the influx of neutrophils into the airways. The secretion of a low-molecular-weight, lipid neutrophil chemotactic factor from the alveolar macrophages in response to tannin stimulation appears to be important in this inflammatory process. In these studies, the effect of tannin on the ability of alveolar macrophages and neutrophils to produce hydrogen peroxide was examined. Low concentrations of tannin itself induced a modest production of hydrogen peroxide from conditioned rabbit alveolar macrophages, while higher concentrations failed to induce peroxide production. In the presence of an independent stimulator of peroxide production (concanavalin A), tannin inhibited peroxide production at all concentrations examined. Aqueous extracts of cotton mill dust (CDE) had an identical effect on peroxide production in a manner that indicated that the tannin present in the dust was responsible for the effect. Like its direct effect on macrophage peroxide production, tannin induced modest peroxide production in human neutrophils. However, unlike its effect on macrophages, tannin enhanced the peroxide production induced by the presence of an independent stimulator (phorbol myristate acetate). CDE had a similar effect on peroxide production, but the dose-response curves suggested that only the high-molecular-weight polymers of tannin present in the CDE were able to enhance peroxide production. These results indicate that inhaled tannin would decrease the ability of resident alveolar macrophages to inactivate inhaled, potentially harmful cotton dust particles but would enhance the host-defense function of the recruited neutrophils. This difference in the action of tannin on neutrophils and macrophages may be of importance in the pathogenesis of byssinosis.

Abstract The condensed tannin present in cotton mill dust profoundly alters the functional capabilities of resident alveolar macrophages. Previous studies from this laboratory have shown that in vitro exposure of rabbit resident alveolar macrophages to condensed tannin significantly inhibits the ability of these cells to produce reactive oxygen intermediates or to ingest particles. In the present study, we demonstrate that condensed tannin also alters arachidonic acid (C20:4) metabolism in these cells. Exposure of rabbit resident alveolar macrophages to condensed tannin results in the time- and dose-dependent release of C20:4 from the membrane phospholipids. The release of C20:4 occurred only at tannin concentrations > 25 µg/ml and was maximal 90 min after the onset of exposure. The EC50 for release was 75 µg/ml. Exposure to 100 µg/ml tannin resulted in the release of 20 ± 3% of the [14C]C20:4 incorporated in the cell membrane. In comparison, exposure to 160 µg/ml zymosan resulted in the release of 14 ± 4% of the [14C]C20:4. For both tannin and zymosan, phosphatidylcholine and phosphatidylinositol were the principal sources of the released C20:4. Approximately 63% of the C20:4 released after zymosan stimulation was further metabolized, mainly via the cyclooxygenase pathway. The major metabolites were 6-ketoprostaglandin F1α, prostaglandin F2α, and prostaglandin E2. In contrast, only 24% of the C20:4 released by tannin was subsequently further metabolized. The metabolites formed were essentially evenly distributed between products of the cyclooxygenase pathway and the lipoxygenase pathway. Exposure of alveolar macrophages to 50 µg/ml tannin for 30 min reduced the ability of the cells to subsequently incorporate C20:4 by 50 to 70%. In contrast, exposure of the cells to 160 mg/ml zymosan for 30 min had only a minimal effect on the subsequent ability of these cells to incorporate C20:4. These results indicate that tannin promotes C20:4 release, at least in part, by inhibiting its reacylation back into phospholipids, a mechanism that differs from that of zymosan.

Abstract Pneumocystis carinii continues to cause severe pneumonia in immunocompromised patients. Surfactant protein D (SP-D), a lung collectin, markedly accumulates during P. carinii pneumonia and binds to glycoprotein A (gpA) on the surface of P. carinii, thereby enhancing interactions with alveolar macrophages. Herein, we report the structural basis of the interaction of SP-D with gpA. We demonstrate that natural SP-D binds to purified gpA in the presence of 2 mM calcium in a saturable, concentration-dependent manner, which is abolished by 10 mM ethylenediaminetetraacetic acid. Increasing concentrations of calcium under otherwise cation-free conditions significantly enhance SP-D binding to gpA, whereas manganese and magnesium cations have minimal effect. Maximal SP-D binding occurs at pH 7.4, with significant inhibition at pH 4. SP-D binding to gpA is also competitively inhibited by maltose > glucose > mannose > N-acetyl-glucosamine. Comparison of the binding of various natural and recombinant forms of SP-D to gpA reveals that the number of carbohydrate recognition domains (CRDs) in a given SP-D form determines the relative extent of binding to gpA. Maximal binding is observed with natural SP-D (dodecamers and higher order SP-D complexes) followed by recombinant dodecamers. In contrast, recombinant full-length trimers exhibit substantially less binding, which is similar to that observed with a recombinant truncated molecule consisting of the CRD and neck regions, and containing trimers of this portion of the molecule. Taken together, these findings strongly indicate that the CRD of SP-D mediates interaction with P. carinii gpA through its attached oligosaccharides and that the extent of SP-D binding to P. carinii is greatest with dodecamers and higher order forms of SP-D.