Glutathione S-transferase M1 modulates allergen-induced NF-κB activation in asthmatic airway epithelium.

BACKGROUND: Glutathione S-transferase M1 (GSTM1) is a phase II enzyme and regulator of inflammatory signaling in airway epithelial cells. We have found upregulation of neutrophilic airway inflammation in atopic asthmatics expressing GSTM1 gene (GSTM1+) compared to GSTM1null asthmatics. We hypothesized that GSTM1 modulates NF-κB activation in bronchial epithelium in atopic asthmatics. We determined regulation of allergen-induced NF-κB activation in bronchial epithelium by GSTM1 in human atopic asthmatics in vivo. METHODS: Endobronchial biopsies and bronchoalveolar lavage fluid samples were collected from 13 GSTM1+ and 12 GSTM1null human atopic asthmatics at baseline and 24 h after segmental allergen challenge. A quantitative analysis of NF-κB activation in airway epithelium was accomplished using a polyclonal antibody against the phosphorylated p65 component of NF-κB. Elastase-positive neutrophils in the bronchial wall were quantified. RESULTS: Postallergen neutrophilia in airway subepithelium and epithelial lining fluid was greater in GSTM1+ compared to GSTM1null asthmatics. Airway eosinophilia was similar in GSTM1+ and GSTM1null asthmatics. Allergen-provoked NF-κB induction in bronchial epithelium was significantly greater in GSTM1+ compared to GSTM1null asthmatics. Activation of NF-κB activation in airway epithelial cells correlated with interleukin-8 concentrations and absolute neutrophil numbers in bronchoalveolar lavage fluid in GSTM1+ but not GSTM1null asthmatics. CONCLUSIONS: Allergen-induced neutrophilic airway inflammation in GSTM1+ asthmatics is associated with NF-κB activation in airway epithelial cells in vivo. These novel data provide a potential mechanism of the genomic link between GSTM1 polymorphism and airway neutrophilia in atopic asthma.

Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway.

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.

Characterization of a rat alveolar macrophage cell line that expresses a functional mannose receptor.

The mannose receptor is a single polypeptide transmembrane glycoprotein expressed on the surface of macrophages that binds and internalizes soluble and particulate ligands. Physiological ligands for this receptor are pathogens, such as mycobacteria, and extracellular acid hydrolases and peroxidases. Expression of the mannose receptor is tightly linked to the functional state of the macrophage: the receptor appears during differentiation, is increased by macrophage deactivating agents, and is reduced in the presence of macrophage activating agents. Studies of the mechanisms underlying these regulatory processes have been hampered by the lack of a stable cell line that expresses a functional and appropriately regulated mannose receptor. In this study we describe expression and modulation of the mannose receptor by the rat alveolar cell line NR8383. Similar amounts of the mannose receptor ligand horseradish peroxidase were internalized by both NR8383 cells and alveolar macrophages. In addition, NR8383 cells expressed immunoreactive mannose receptor protein and mannose receptor mRNA as detected by Northern analysis. Regulation studies showed that mannose receptor expression was regulated at the levels of activity, protein, and mRNA in NR8383 cells similarly to regulation in primary rat macrophages. In addition, NR8383 cells could be successfully transfected with a luciferase reporter gene, providing the transfectable, mannose receptor-positive macrophage cell line. These results support the hypothesis that NR8383 cells potentially represent the best current macrophage cell line for studying various aspects of macrophage function, and are particularly critical in studies of regulation of the mannose receptor, a key receptor in host defense and immune regulation.

Ingestion of Candida albicans down-regulates mannose receptor expression on rat macrophages.

The frequency of infection and death due to various Candida species has increased steadily during the past decade, with mucocutaneous candidal infections as a common problem in the immunocompromised host. Mononuclear phagocytes are important in phagocytosis of this organism. In areas where there are low levels of opsonins, the macrophage-specific mannose receptor plays a dominant role in mediating Candida albicans ingestion. Following receptor-mediated infection, the host macrophage produces inflammatory cytokines and mediators that lead to ultimate killing of the invading Candida. Infection of macrophages by pathogens often leads to altered function that might effect their subsequent host defense properties. For example, function of both the complement receptor type 3 and the mannose receptor are down-regulated following exposure to pathogens or pathogen-derived products. In the current study, we have examined the down-regulation of mannose receptor expression following Candida infection and have investigated possible mechanisms that might be involved. Mannose receptor activity was decreased following 24 h postinfection with Candida. Both tumor necrosis factor and nitric oxide were produced during the infection, and inhibition of the these mediators partially blocked the effect on the receptor. Infection with Candida also inhibited the ability of dexamethasone to up-regulate mannose receptor expression. Finally, mannose receptor protein turnover was accelerated in Candida-infected macrophages. We conclude that Candida down-regulates one of the receptors involved in its internalization through a combination of production of modulatory molecules and enhanced receptor degradation. These results support the hypothesis that pathogens that infect macrophages have the ability to alter the phagocytic pathways available for subsequent host defense.

Purification of a cell-surface receptor for surfactant protein A.

In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens.

Dexamethasone blocks the interferon-gamma-mediated downregulation of the macrophage mannose receptor.

The macrophage mannose receptor is highly susceptible to modulation by a variety of proinflammatory and antiinflammatory agents. Previous studies have demonstrated that mannose receptor activity is dramatically increased in rat bone marrow-derived macrophages by dexamethasone treatment and decreased following incubation with interferon-gamma (IFN-gamma). Regulation by both agents occurs at least in part by alteration of mannose receptor mRNA levels. In the present study, we have investigated the ability of dexamethasone to block mannose receptor downregulation in rat marrow macrophages by IFN-gamma. Incubation of rat macrophages with IFN-gamma resulted in downregulation of mannose receptor activity, receptor synthesis, and mRNA levels, with no change in turnover rate of the receptor. IFN-gamma appeared to act at least partially through the generation of nitric oxide: inclusion of the nitric oxide inhibitor N-monomethyl arginine inhibited the IFN-gamma-induced effects on the mannose receptor by approximately 50%. When cells were coincubated with dexamethasone plus IFN-gamma, dexamethasone blocked the decrease in mannose receptor activity, synthesis, and mRNA. Additionally, dexamethasone inhibited nitric oxide production in response to IFN-gamma. These results suggest that mannose receptor expression is downregulated by IFN-gamma by at least two mechanisms: a decrease in mannose receptor mRNA levels and inhibition involving the production of nitric oxide. Dexamethasone has the capability of blocking the effect of IFN-gamma on mannose receptor expression through inhibition of IFN-gamma-mediated downregulation of mannose receptor transcription and/or inhibition of IFN-gamma-mediated induction of nitric oxide production.

Purification of type I and type II tumor necrosis factor receptors from human lung tissue.

Two receptors for tumor necrosis factor-alpha (TNF) were purified from detergent-solubilized human lung tissues by adsorption to TNF-Sepharose, followed by elution with low pH. By SDS-PAGE analysis, the two proteins had molecular weights of 75 and 55 kD. Using a soluble receptor assay, a binding affinity of approximately 1.2 nM was calculated for the isolated lung receptors. Each protein, isolated by electroelution from polyacrylamide gels, specifically bound TNF. Antibodies raised against the mixture of type I and II receptors bound specifically to both purified receptors by immunoblot analysis. Both the 75- and 55-kD receptors could be precipitated from 125I-surface-labeled or 35S-methionine-labeled U937 cells using TNF-Sepharose or anti-receptor antibodies. In addition, the anti-TNF receptor antibodies partially blocked binding of TNF to U937 cells and specifically immunoprecipitated 125I-TNF cross-linked to its receptors on U937 cells. These results demonstrate that both type I and II TNF receptors can be isolated from human lung tissue by ligand affinity chromatography, and that U937 cells express both TNF receptor types.

Paradoxical regulation by PGE-2 on release of neutrophil chemoattractants by rat bone marrow macrophages.

Prostaglandin E2 (PGE2) was shown to cause up to a 110% increase in the release into media of soluble chemoattractants for neutrophils by cultured rat bone marrow macrophages (RBMM) during a 16 hour incubation period. Coincubating with concentrations of PGE2 of 10 nM and below did not stimulate release of chemoattractants while concentrations between 10(2) and 10(4) nM increased the chemotactic activity of conditioned medium by 40% to 110% (p less than 0.05). In contrast to the effect of coincubating, pre-treatment with PGE2 for 2 and 4 hours was ineffective in stimulating the release of chemoattractants by RBMM. We also assessed whether PGE-2 modulated the release of chemoattractants by RBMM stimulated with endotoxin (LPS). LPS caused a four fold increase in the production of chemoattractants with a peak effect found at an LPS concentration of 1 microgram/ml. Coincubating with PGE2 in concentrations between 10(2) and 10(4) nM paradoxically decreased LPS-stimulated production of chemoattractants by up to 40% (p less than 0.05). Pre-treatment with PGE2 for 4 hours partially blocked LPS-stimulated release of chemotactic activity. These data indicate that PGE-2 has paradoxical effects on the production of chemoattractants by RBMM: being independently stimulatory but down regulating the effects of LPS. These findings suggest the possibility that the activation state of the RBMM may determine the effect of PGE2: quiescent RBMM can be stimulated by PGE2 but LPS-activated RBMM may be suppressed.

Modulation of mannose receptor activity by proteolysis.

Macrophages express a receptor on the cell surface that functions to clear glycoproteins from the extracellular milieu. The activity of this receptor is sensitive to treatment with trypsin. In inflammatory situations, macrophages are activated and exposed to increased levels of extracellular proteases. Under these conditions, mannose receptor activity on the macrophages is diminished. We therefore decided to study the effects of trypsin treatment on the structure and activity of cell-associated and purified receptor that might contribute to the activation-associated receptor down-regulation. Trypsin treatment (1 microgram/ml for 3 h) resulted in the production of a 140 kDa, trypsin-resistant fragment from both intact cells and isolated receptor. This fragment was no longer able to bind ligand. The remaining 35 kDa fragment apparently is further degraded into smaller fragments, since no evidence of this domain was found on Coomassie Blue-stained gels. The 140 kDa fragment retained immunoreactivity and contained at least a portion of the iodinated tyrosine residues following surface labelling with Na125I. Neither calcium nor ligand protected the receptor from proteolysis. In addition, prior treatment with oxidants did not increase the susceptibility of the receptor to trypsin digestion. We conclude from these results that the macrophage mannose receptor is clipped by the serine protease trypsin at the cell surface, resulting in the release and further degradation of the binding domain, and the production of a membrane-associated 140 kDa fragment. This trypsin-mediated down-regulation of receptor activity might be important in controlling glycoprotein clearance during inflammation.

Down-regulation of mannose receptor activity in macrophages after treatment with lipopolysaccharide and phorbol esters.

We have investigated the effects of LPS and PMA on the expression of functional mannose receptors in rat bone marrow-derived macrophages. After 48 h of treatment with LPS (10 ng/ml) and PMA (100 nM), mannose receptor activity was reduced by 70 to 80%. The effect of these agents on receptor activity was not reversible, and activity continued to decline after the agents were removed. Pretreatment of cells with dexamethasone was effective in blocking the LPS/PMA-induced down-regulation. Serine protease inhibitors did not block the reduction in receptor activity, suggesting that proteolysis is not involved in receptor down-regulation. LPS/PMA treatment did not increase turnover of the receptor. Ligand uptake studies showed that the total capacity of the uptake system was reduced by 80%, although the Kuptake was unaffected. Binding of 125I-mannose-BSA to intact macrophages showed a 70% decrease in surface receptor activity after treatment with LPS/PMA. LPS/PMA treatment had no effect on total receptor synthesis as quantitated by immunoprecipitation of metabolically labeled receptor. However, binding of metabolically labeled receptor to mannose-Sepharose, and binding of 125I-mannose-BSA to immunoprecipitated receptor revealed that intracellular plus surface binding sites were reduced to approximately 30% after LPS/PMA treatment. These results suggest that LPS/PMA treatment of macrophages results in an inactivation of mannose receptors with no effect on receptor turnover or biosynthesis.

Effect of endotoxin-induced cell injury on 70-kD heat shock proteins in bovine lung endothelial cells.

Heat shock proteins (HSPs) have been remarkably conserved throughout evolution. It has been assumed that induction of HSPs remains a stereotypic response to injury, important for survival of eukaryotic cells during euthermic injury. However, there are few studies of this phenomenon in endothelial cells, and none in pulmonary endothelial cells. We studied the induction of synthesis of 70-kD proteins in bovine pulmonary artery endothelial cells (BPAECs) in response to heat shock and to euthermic injury induced by bacterial endotoxin. First, in response to heat, BPAECs showed rapid and reversible heat-induced synthesis of 70-kD proteins, readily detectable by one-dimensional SDS-PAGE of [35S]methionine-labeled BPAECs. Heat shock at 42 degrees C for 3 h or 43 degrees C for 2 h suppressed total protein synthesis by 30% (P less than 0.001) but an increased rate of synthesis of 70-kD protein continued, representing an increasing fraction of total protein synthesis. Heat-induced synthesis of 70-kD protein returned to baseline levels 8 h after heat shock. Northern analysis showed that mRNA for a protein homologous to a conserved amino acid sequence in the family of species-homologous 70-kD heat shock proteins (HSP 70) was induced by a 15-min incubation at 42 degrees C and remained detectably increased for 6 h. We next assessed whether euthermic injury by bacterial endotoxin (LPS) generated a similar response. LPS was cytotoxic by BPAECs as assessed morphologically, by release of 51Cr from prelabeled cells, and by a significant suppression of total protein synthesis (range, 35 to 70%; P less than 0.001). Despite cytotoxicity, LPS did not induce 70-kD protein at a level that could be detected by SDS-PAGE, and no increase in mRNA for HSP 70 was detected by Northern analysis. LPS-injured BPAECs remained "competent" to induce both 70-kD proteins and mRNA for HSP 70 in response to heat shock. We conclude that at least quantitatively, induction of HSP 70 by BPAECs is not a stereotypic response to injury but rather is at least relatively injury-specific. However, competence to induce HSP 70 appears to be extremely resilient: it is retained in dysfunctional BPAECs in the face of profound inhibition of global protein synthesis, suggesting an important homeostatic role.

Transfer of persulfide sulfur from thiocystine to rhodanese.

THiocystine (bis-[2-amino-2-carboxyethyl]trisulfide) is a natural substrate for rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1). Analogs of thiocystine were prepared by eliminating the carboxyl or amino group or by lengthening the carbon chain. Of these only homothiocystine (bis-[2-amino-2-carboxypropyl]trisulfide) had appreciable activity as a substrate. At pH 8.6, the optimum for rhodanese, transfer of sulfane sulfur to cyanide in the presence of rhodanese was nonspecific. Only the sulfane sulfur of 35S-labeled thiocystine was transferred to rhodanese. Thus, thiocystine and thiosulfate both produce a rhodanese persulfide as a stable intermediate in sulfur transfer.

Partial purification and properties of ovine liver Echinococcus granulosus protoscolices phospholgucose isomerase.

Echinococcus granulosus protoscolex is the actual larval stage of the cestode causing echinococcosis both in man and animals. In the present report, certain properties of phosphoglucose isomerase from the ovine liver E. granulosus protoscolices have been studied and compared with those of the hydatid cyst fluid and the healthy ovine liver enzymes. The protoscolices enzyme prepared in a manner similar to the hydatid cyst fluid and the ovine liver enzymes exhibited the following properties: (1) pH optimum of 8.2 (2) KM value of 0.23 mM, (3) the enzyme was inhibited in the presence of high concentrations of alpha-D-glucose 6-phosphate, (4) no detectable inhibition of the enzyme was observed in the presence of phosphate ion up to 4.1 mM, (5) the protoscolices enzyme was less thermostable as compared to the hydatid cyst fluid and the ovine liver enzymes, (6) the protoscolices enzyme had a lower Ki value (0.7 mM) as compared to either the hydatid cyst fluid (1.1 mM) or the ovine liver enzymes (4.6 mM) when 6-phosphogluconic acid was used as a competitive inhibitor.