Effect of antioxidants on apoptosis and cytokine release in fetal rat Type II pneumocytes exposed to hyperoxia and nitric oxide

The response of the fetal rat Type II pneumocyte (FTIIP), the stem cell of the alveolar epithelium, to hyperoxia would be helpful to understand the effects of oxygen-induced injury to the developing lung. Our goals were to evaluate the effect of antioxidants (AO) on apoptosis and release of cytokines in freshly isolated FTIIP (day-19) in the presence of 95% O2 and/or nitric oxide (NO). There was increased apoptosis in FTIIP exposed to hyperoxia alone and in combination with NO; this was significantly attenuated (p < 0.01) in the presence of 3 AO, namely grape seed proanthocyanidin extract (GSPE), superoxide dismutase (SOD) and catalase. The anti-inflammatory cytokine IL-10 has been shown to have a role in ameliorating tissue damage owing to persistent inflammation. The release of IL-10 was significantly decreased (p < 0.01) in the presence of GSPE and catalase, compared to control. Addition of SOD led to increased IL-10 compared to GSPE or catalase (p < 0.01) or the combination of GSPE + SOD + catalase (p < or = 0.01). Thus, in our in vitro model of hyperoxic and NO mediated injury to FTIIP, protection from apoptotic cell death with the addition of AO was associated with varying levels of IL-10 release. Our data suggest that the use of SOD and/or IL-10 may decrease hyperoxic lung injury by decreasing apoptosis. Further studies are needed to understand the mode of protection from catalase and GSPE.     

Nitric oxide augments release of chemokines from monocytic U937 cells: modulation by anti-inflammatory pathways

Nitric oxide (NO) appears to act as an inflammatory mediator on monocytic cells. Exogenous NO augmented release of chemokines from human promonocytic U937 cells and peripheral blood mononuclear cells. Pharmacological strategies aiming at modulation of NO-induced release of interleukin-8 (IL-8) were investigated in U937 cells in detail. Release of IL-8 was down-regulated by transforming growth factor beta2 (TGF-beta2), by the protein tyrosine-kinase inhibitor genistein, and via rises in intracellular cyclic AMP, generated by prostaglandin E(2), rolipram, pentoxifylline, forskolin, or dibutyryl-cyclic AMP. In addition, incubation with the synthetic glucocorticoid dexamethasone or suppression of activity of p38 mitogen-activated protein (MAP) kinases by SB-203580 modulated release of IL-8. Activation of p38 MAP kinases was confirmed by the demonstration of an augmented appearance of phosphorylated p38 in the presence of NO. The present data suggest that exposure to exogenous NO resembles activation of U937 cells by proinflammatory stimuli. The anti-inflammatory cytokine TGF-beta2, as well as anti-inflammatory or immunosuppressive agents such as genistein, pentoxifylline, rolipram, dexamethasone, and SB-203580 modulate inflammatory, chemokine-inducing actions of NO.     

Dexamethasone protects against high-dose endotoxin without loss of tolerance to oxygen

Endotoxin (500 micrograms/kg)-treated rats are very tolerant to hyperoxia (greater than 95% O2, 1 ATA). We have now attempted to determine if dexamethasone given to rats 1 h before a usually lethal dose of endotoxin would diminish endotoxin's lethality without substantially abrogating its capacity to confer tolerance to hyperoxia. Endotoxin (20 mg/kg) given alone killed 70-80% of air- or O2-breathing rats within 24 h; dexamethasone (0.6 mg) given 1 h before endotoxin decreased mortality at 24 h to 10-15%. About 90% of the rats that were alive 24 h after receiving dexamethasone plus endotoxin (20 mg/kg) survived 72 h of hyperoxia. Dexamethasone plus endotoxin (10 mg/kg) provided as much protection against pulmonary edema resulting from 72 h of hyperoxia as did 500 micrograms/kg endotoxin alone. Tolerance to hyperoxia produced by dexamethasone plus high-dose endotoxin was accompanied by a rise in the activity in the lung of antioxidant enzymes. We conclude that dexamethasone protects rats against the lethal effects of high doses of endotoxin without interfering with endotoxin's capacity to engender tolerance to hyperoxia.     

Cytokines in tolerance to hyperoxia-induced injury in the developing and adult lung

Cytokines are peptides that are produced by virtually every nucleated cell type in the body, possess overlapping biological activities, exert different effects at different concentrations, can either synergize or antagonize the effects of other cytokines, are regulated in a complex manner, and function via cytokine cascades. Hyperoxia-induced acute lung injury (HALI) is characterized by an influx of inflammatory cells, increased pulmonary permeability, and endothelial and epithelial cell injury/death. Some of these effects are orchestrated by cytokines. There are significant differences in the response of the developing versus the adult lung to hyperoxia. We review here cytokines (and select growth factors) that are involved in tolerance toward HALI in animal models. Increased cytokine expression and release have a cascade effect in HALI. IL-1 precedes the increase in IL-6 and CINC-1/IL-8 and this seems to predate the influx of inflammatory cells. Inflammatory cells in the alveolar space amplify the lung damage. Other cytokines that are primarily involved in this inflammatory response include IFN-gamma, MCP-1, and MIP-2. Certain cytokines (and growth factors) seem to ameliorate HALI by affecting cell death pathways. These include GM-CSF, KGF, IL-11, IL-13, and VEGF. There are significant differences in the type and temporal sequence of cytokine expression and release in the adult and newborn lung in response to hyperoxia. The newborn lung is greatly resistant to hyperoxia compared to the adult. The delayed increase in lung IL-1 and IL-6 in the newborn could induce protective factors that would help in the resolution of hyperoxia-induced injury. Designing a therapeutic approach to counteract oxygen toxicity in the adult and immature lung first needs understanding of the unique responses in each scenario.     

Transient injury to rat lung mitochondrial DNA after exposure to hyperoxia and inhaled nitric oxide

The effect of hyperoxia alone and in combination with inhaled nitric oxide (NO) on the integrity of lung mitochondrial DNA (mtDNA) in vivo was evaluated in Fischer 344 rats. PCR amplification of lung mtDNA using two sets of primers spanning 10.1 kb of the mtDNA revealed that inhalation of 20 ppm of NO in conjunction with hyperoxia (>95% O2) reduced the amplification of mtDNA templates by 10 +/- 1% and 26 +/- 3% after 24 h of exposure. The ability of mtDNA to amplify was not compromised in rats exposed to 80% O2, even in the presence of 20 ppm of inhaled NO. Surprisingly, exposure to >95% O2 alone for either 24 or 48 h did not compromise the integrity of mtDNA templates compared with air-exposed controls, despite evidence of genomic DNA injury. Interestingly, inhaling NO alone for 48 h increased mtDNA amplification by 12 +/- 2% to 21 +/- 7%. Injury to the lung mtDNA after exposure to >95% O2 plus 20 ppm of NO was transient as rats allowed to recover in room air after exposure displayed increased amplification, with levels exceeding controls by 20 +/- 3% to 29 +/- 4%. Increased amplification was not due to cellular proliferation or increased mitochondrial number. Moreover, the ratio of pulmonary mtDNA to genomic DNA remained the same between treatment groups. The results indicate that hyperoxia fails to induce significant injury to mtDNA, and whereas inhalation of NO with hyperoxia results in mtDNA damage, the lesions are rapidly repaired during recovery.     

Cytokines increase rat lung antioxidant enzymes during exposure to hyperoxia

Pretreatment with the combination of tumor necrosis factor/cachectin (TNF/C) and interleukin 1 (IL-1) increased glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD) activities in lungs of rats continuously exposed to hyperoxia for 72 h, a time when all untreated rats had already died. Pretreatment with TNF/C and IL-1 also increased, albeit slightly, lung G6PDH and GR activities of rats exposed to hyperoxia for 4 or 16 h. By comparison, no differences occurred in lung antioxidant enzyme activities of TNF/C and IL-1- or saline-pretreated rats exposed to hyperoxia for 36 or 52 h; the latter is a time just before untreated rats began to succumb during exposure to hyperoxia. The results raise the possibility that TNF/C and IL-1 treatment can increase lung antioxidant enzyme activities and that increased lung antioxidant enzymes may contribute to the increased survival of TNF/C and IL-1-pretreated rats in hyperoxia for greater than 72 h.     

Rhinovirus-16 Induced Release of IP-10 and IL-8 Is Augmented by Th2 Cytokines in a Pediatric Bronchial Epithelial Cell Model

Background

In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation.

Methods

Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID₅₀. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways.

Results

The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone.

Conclusion

Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Interactions of nitric oxide and oxygen in cytotoxicity: proliferation and antioxidant enzyme activities of endothelial cells in culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O2 in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-++ +ium-1,2-diolate) (DETA-NONOate) (100-500 microM), under normoxia (air), hypoxia (< 0.04% O2) or hyperoxia (88-94% O2). It was found that the dose dependency on NONOate was little affected by the ambient O2 concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H2O2 elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 microM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H2O2 removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.     

Mitogenic effect of endotoxin on lung and tolerance of rats to hyperoxia

Treatment of rats with endotoxin, as late as 24 h after beginning exposure to greater than 95 O2 at 1 atm, increases survival at 72 h from 20-30% to greater than 95% (J. Clin. Invest. 65: 1104, 1980), whereas treatment with corticosteroids reduces survival (Toxicol. Appl. Pharmacol. 47: 367, 1979). Since endotoxin is mitogenic to some cells and glucocorticosteroids decrease DNA synthesis by lung cells, we asked 1) is endotoxin mitogenic to the lung, and, if so, 2) is the mitogenic effect required for endotoxin to produce tolerance to hyperoxia? We found endotoxin administered in vivo does have a mitogenic effect on the lung as indicated by an increased rate of DNA synthesis by lung slices; dexamethasone blocked this effect. However, although dexamethasone given alone markedly diminished survival in hyperoxia, dexamethasone did not impair the protection conferred to rats by endotoxin against the edemogenicity and lethality of hyperoxia. Furthermore, dexamethasone did not diminish the rise of antioxidant enzyme activity in the lungs of endotoxin-treated O2-exposed rats. We conclude endotoxin can produce tolerance to hyperoxia even when its mitogenic action on the lung is substantially diminished.     

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.     

Influence of bisphosphonates on the production of pro-inflammatory cytokines by activated human articular chondrocytes

Bisphosphonates have anti-inflammatory effects in rheumatoid arthritis and chondroprotective effects in animal arthritis models but their influence on chondrocytes is not known. The aim of this study is to investigate whether bisphosphonates could influence the production of pro-inflammatory cytokines by activated chondrocytes. Therefore human articular cartilage explants were incubated at 48 h with clodronate, pamidronate or risedronate (10(-6) and 10(-8)mol/L), and dexamethasone (10(-8)mol/L). Subsequently, cultures were stimulated with IL-1, 10 ng/mL (n=6) or 1 ng/mL (n=10) for 48 h. Co-incubation was performed with or without bisphosphonates or dexamethasone. A flow cytometric microsphere-based immunoassay was used for the detection of the pro-inflammatory cytokines IL-6, IL-8, TNF-alpha, IL-1 and the regulatory cytokines IL-12p70 and IL-10 in the supernatants. Stimulation with IL-1 resulted in a dose dependent induction of IL-6 and IL-8, but no production of the other cytokines could be demonstrated. This production of IL-6 and IL-8 was neither inhibited nor enhanced by bisphosphonates. Only dexamethasone caused an inhibition of IL-6 production. In conclusion, there is no evidence on the level of articular cartilage cells that bisphosphonates would suppress or enhance IL-6 and IL-8 mediated joint destruction.     

Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury

Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratio was enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.     

Protection of lung tissue against ischemia/reperfusion injury by preconditioning with inhaled nitric oxide in an in situ pig model of normothermic pulmonary ischemia

Topical administration of nitric oxide (NO) by inhalation is currently used as therapy in various pulmonary diseases, but preconditioning with NO to ameliorate lung ischemia/reperfusion (I/R) injury has not been fully evaluated. In this study, we investigated the effects of NO inhalation on functional pulmonary parameters using an in situ porcine model of normothermic pulmonary ischemia. After left lateral thoracotomy, left lung ischemia was maintained for 90 min, followed by a 5h reperfusion period (group I, n = 7). In group II (n = 6), I/R was preceded by inhalation of NO (10 min, 15 ppm). Animals in group III (n = 7) underwent sham surgery without NO inhalation or ischemia. In order to evaluate the effects of NO preconditioning, lung functional and hemodynamic parameters were measured, and the zymosan-stimulated release of reactive oxygen species in arterial blood was determined. Animals in group I developed significant pulmonary I/R injury, including pulmonary hypertension, a decreased pO(2) level in pulmonary venous blood of the ischemic lung, and a significant increase of the stimulated release of reactive oxygen species. All these effects were prevented, or the onset (release of reactive oxygen species) was delayed, by NO inhalation. These results indicate that preconditioning by NO inhalation before lung ischemia is protective against I/R injury in the porcine lung.     

Interactions of nitric oxide and oxygen in cytotoxicity: Proliferation and antioxidant enzyme activities of endothelial cells in culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O₂ in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-1-ium-1,2-diolate) (DETA-NONOate) (100–500 μM), under normoxia (air), hypoxia (< 0.04% O₂) or hyperoxia (88–94% O₂). It was found that the dose dependency on NONOate was little affected by the ambient O₂ concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H₂O₂ elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 μM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24 h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H₂O₂ removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.     

Prophylactic effects of dexamethasone in lung injury caused by hyperoxia and hyperventilation.

To determine if prophylactic corticosteroids would prevent acute lung injury caused by hyperoxia and barotrauma, 29 piglets (1.2 +/- 0.3 kg, 1-2 days of age) were studied. Ten piglets were hyperventilated [arterial PCO2 (PaCO2) 15-20 Torr] with 100% O2 for 48 h and compared with 10 piglets treated with the identical management but given 0.7 mg/kg of dexamethasone at time 0 and every 12 h for the 48-h study. Six piglets were normally ventilated (PaCO2 40-45 Torr) for 48 h with 21% O2 as an additional control group. Pulmonary function and tracheal aspirates were examined at time 0 and every 24 h. Bronchoalveolar lavage was performed for surfactant analyses at the conclusion of the study. In animals treated with hyperoxia and hyperventilation, lung compliance decreased 32% and tracheal aspirate polymorphonuclear leukocyte (PMN) chemotactic activity increased by 51%, cell counts by 204%, number of PMNs by 277%, elastase activity by 111%, and albumin concentration by 328% over 48 h (P less than 0.05). In contrast, dexamethasone-treated piglets had increases in only tracheal aspirate albumin concentration (206%) over the 48-h study. All cellular and biochemical variables were lower in dexamethasone-treated compared with hyperoxic hyperventilated piglets. Room air normal ventilation controls had only a 108% increase in tracheal aspirate albumin concentration noted. Despite quantitative differences in surfactant among the three groups, activity was unaffected. Results indicate that hyperoxia and hyperventilation for 48 h causes significant inflammatory changes and acute lung injury and that prophylactic high-dose dexamethasone significantly ameliorates this lung damage.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Inhibition of LPS-induced iNOS and NO synthesis in primary rat microglial cells

Nitric oxide (NO) has been implicated in the etiopathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), and inhibition of NO synthesis has been proposed to be a possible mechanism of action of drugs to treat MS. In the present study, we investigated the inhibitory effect on NO synthesis of various steroids, cytokines and drugs used or proposed for the treatment of MS. As a model system, we used primary rat microglial cells which produce NO synthase and subsequently release NO upon stimulation with lipopolysaccharide (LPS). Among the substances tested, the glucocorticoids prednisone, hydrocortisone, dexamethasone and progesterone as well as transforming growth factor-beta (TGF-beta) dose-dependently inhibited LPS-induced nitric oxide synthase (iNOS) and NO synthesis. In contrast, COP-1, the phosphodiesterase inhibitors rolipram and pentoxifylline, the cytokines interleukin-10 (IL-10) and interferon-beta (IFN-beta) as well as the steroids beta-estradiol, testosterone, and dehydroepiandrosterone (DHEA) showed no inhibitory effect. Cholesterol slightly, but not significantly, increased LPS-induced nitric oxide synthesis. We conclude from the present study that with respect to treatment of MS, inhibition of NO synthesis may be an important mechanism of action of glucocorticoids and transforming growth factor-beta, but not of other drugs used or proposed to treat MS.