Alveolar macrophages from allergic lungs are not committed to a pro-allergic response and can reduce airway hyperresponsiveness following ex vivo culture.

BACKGROUND: We already demonstrated that adoptive transfer of alveolar macrophages (AMs) from non-allergic rats into AM-depleted allergic rats prevents airway hyperresponsiveness (AHR). We also showed that AMs from non-sensitized, but not from sensitized, allergy-prone rats can prevent AHR following allergen challenge in sensitized allergic animals, establishing the importance of rat immunological status on the modulation of AM functions and suggesting that an allergic lung environment alters AM functions. OBJECTIVE: We investigated how the activation of allergic AMs can be modulated to reinstitute them with their capacity to reduce AHR. METHODS: AMs from sensitized Brown Norway rats were cultured ex vivo for up to 18 h in culture media to deprogram them from the influence of the allergic lung before being reintroduced into the lung of AM-depleted sensitized recipient. AHR and cytokines in bronchoalveolar lavage (BAL) were measured following allergen challenge. AMs stimulated ex vivo with Bacillus Calmette-Guerin (BCG) were used as positive controls as BCG induces a T-helper type 1 activation in AMs. RESULTS: AMs ex vivo cultured for 4-18 h reduced AHR to normal level. Interestingly, pro-allergic functions of AMs were dampened by 18 h culture and they reduced AHR even after spending 48 h in an allergic lung microenvironment. Furthermore, transfer of cultured AMs caused an increase in the levels of IFN-gamma and IL-12 in BAL when compared with their ovalbumin control. After 18 h of ex vivo culture, AMs expressed reduced levels of TNF, IL-1alpha, IL-6, and Arginase-2 mRNAs compared with freshly isolated AMs, suggesting that ex vivo culture exempted AMs from lung stimuli that affected their functions. CONCLUSIONS: There is a significant crosstalk between lung microenvironment and AMs, affecting their functions. It is also the first report showing that sensitized AMs can be modulated ex vivo to reduce lung pro-allergic environment, opening the way to therapies targetting AMs.

A sphingosine-1-phosphate receptor 1 agonist inhibits tertiary lymphoid tissue reactivation and hypersensitivity in the lung.

Hypersensitivity pneumonitis is characterized by pulmonary accumulation of B-cell-rich tertiary lymphoid tissues (TLTs), which are alleged sites of amplification for antigen-specific responses. The sphingosine-1-phosphate receptor 1 (S1P1) regulates key mechanisms underlying lymphoid tissue biology and its chemical modulation causes lymphocyte retention in lymph nodes. Given the putative immunopathogenic impact of lymphocyte accumulation in TLTs, we investigated whether or not chemical modulation of S1P1 caused lymphocyte retention within TLTs in a model of hypersensitivity pneumonitis. Mice were exposed subchronically to Methanosphaera stadtmanae (MSS) in order to induce an hypersensitivity pneumonitis-like disease. MSS exposure induced B-cell-rich TLTs surrounded by S1P1-positive microvessels. Upon MSS rechallenge, the S1P1 agonist RP001 prevented the pulmonary increase of CXCL13, a chief regulator of B-cell recruitment in lymphoid tissues. This was associated with a complete inhibition of MSS rechallenge-induced TLT enlargement and with a 2.3-fold reduction of MSS-specific antibody titers in the lung. Interference with TLT reactivation was associated with a 77% reduction of neutrophil accumulation and with full inhibition of protein-rich leakage in the airways. Thus, an S1P1 agonist hinders TLT enlargement upon antigenic rechallenge and inhibits key pathognomonic features of experimental hypersensitivity pneumonitis.

Serotonin modulates the cytokine network in the lung: involvement of prostaglandin E2.

Serotonin, well known for its role in depression, has been shown to modulate immune responses. Interestingly, the plasma level of serotonin is increased in symptomatic asthmatic patients and the use of anti-depressants, known to reduce serotonin levels, provokes a decrease in asthma symptoms and an increase in pulmonary function. Thus, we tested the hypothesis that serotonin affects alveolar macrophage (AM) cytokine production, altering the cytokine network in the lung and contributing to asthma pathogenesis. AMs were treated with different concentrations of serotonin (10(-11)-10(-9) M) or 5-HT(1) and 5-HT(2) receptor agonists for 2 h prior stimulation. T helper 1 (Th1) and Th2 cytokines, prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) were measured in cell-free supernatants. Serotonin significantly inhibited the production of tumour necrosis factor (TNF) and interleukin (IL)-12, whereas IL-10, NO and PGE(2) production were increased. These immunomodulatory effects of serotonin were mimicked by 5-HT(2) receptor agonist but were not abrogated by 5-HT(2) receptor antagonist, suggesting the implication of other 5-HT receptors. Inhibitors of cyclooxygenase and antibody to PGE(2) abrogated the inhibitory and stimulatory effect of serotonin on TNF and IL-10 production, respectively, whereas NO synthase inhibitor eliminated serotonin-stimulated IL-10 increase. Furthermore, PGE(2) significantly increased AM IL-10 and NO production. These results suggest that serotonin alters the cytokine network in the lung through the production of PGE(2). The reduction of Th1-type cytokine by serotonin may contribute to asthma pathogenesis.

Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds.

1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.5. Neither PF-5901 nor Wy-50,295 (1 or 10 microM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation.6. These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties.

Dysregulation of alveolar macrophages unleashes dendritic cell-mediated mechanisms of allergic airway inflammation.

Allergic asthma is a chronic inflammatory disorder characterized by eosinophilia and T helper type 2 (Th2) cell activation. However, little information is available on the mechanisms leading to this pathology. We previously showed that alveolar macrophages (AM) from rats with experimental asthma lose their ability to prevent asthma symptoms. To understand the implication of AM in lung immunity, we investigated the influence of AM sensitization status on lung dendritic cell (DC) activation induced by allergen challenge in vivo. Rat sensitized to ovalbumin developed airway inflammation (eosinophils and Th2 cells) and demonstrated myeloid DC (mDC) activation following allergen exposure. The replacement of AM of sensitized animals by AM from naive animals did not affect allergen-triggered eosinophilia but completely abolished lung mDC allergen capture and migration to the lymph nodes, as well as Th2 cell polarization. Moreover, immunosuppressive functions of naive AM occurred in conjunction with low engulfment of allergens but without variation of major histocompatibility complex II and CD23 expression. Interestingly, sensitized AM that were withdrawn from the inflammatory environment regained their immunosuppressive functions when transferred to sensitized rats. Thus, these are the first in vivo evidences showing that dysregulation of AM functions is sufficient to induce DC-triggered allergic response.

Interleukin-4 production by human alveolar macrophages.

BACKGROUND: IL-4 is a key factor for T helper type 2 (Th2) differentiation and Ig class switching to IgE and IgG(4) during the development of immune responses. IL-4 is produced by T cells, mast cells, basophils, and eosinophils. However, there is also evidence suggesting that rat alveolar macrophages (AMs) produce IL-4. OBJECTIVE: Given the importance of AMs and Th2-related diseases in the lung, we investigated the production of IL-4 by human AMs. METHODS: Human AMs were isolated from bronchoalveolar lavage, purified, and IL-4 production was investigated at mRNA and protein levels using real-time PCR, flow cytometry, immunocytochemistry, and ELISA. The presence of IL-4 was investigated in subjects with asthma or asymptomatic airway hyper-responsiveness, and in normal non-smokers. RESULTS: IL-4 and IL-4delta2 (a splice variant found in other IL-4 producing cells) mRNAs were found in all these subjects, but IL-4 expression could not be correlated with a particular disease. Protein production was verified by immunocytochemistry and flow cytometry analysis demonstrating, respectively, up to 69% and 59% positive AMs, regardless of the subject condition. Furthermore, phorbol-12-myristate-13-acetate and calcium ionophore stimulated the release of IL-4 after 48 h treatment in the presence of anti-IL-4 receptor antibody. CONCLUSION: Our results show for the first time that IL-4 and IL-4delta2 mRNA are expressed and IL-4 protein produced and released by human AMs, suggesting a contribution of these cells in the modulation of Th2 immune response.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a component of tobacco smoke, modulates mediator release from human bronchial and alveolar epithelial cells.

Respiratory epithelial cells are known to contribute to immune responses through the release of mediators. The aim of this study was to characterize the immunomodulatory effects of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco carcinogen, on respiratory epithelial cells and to compare two metabolic pathways, alpha-methylhydroxylation and alpha-methylenehydroxylation, involved in these effects using selective precursors, 4-(acetoxy-methylnitrosamino)-1-(3-pyridil)-1-butanone (NNKOAc) and N-nitroso (acetoxymethyl) methylamine (NDMAOAc), respectively. Human bronchial and alveolar epithelial cell lines, BEAS-2B and A549, respectively, were treated with NNK, NNKOAc and NDMAOAc for 24 h with and without tumour necrosis factor (TNF) and mediators released in cell-free supernatants were measured by enzyme-linked immunosorbent assay (ELISA). NNK significantly inhibited interleukin (IL)-8, IL-6 and monocyte chemoattractant protein-1 (MCP-1) production in both cell types. Similar results were observed with primary bronchial and alveolar epithelial cells. Although NNK increased prostaglandin E(2) (PGE(2)) production by A549 cells, its immunomodulatory effects were not mediated by PGE(2) according to the results with cyclo-oxygenase inhibitors. NNKOAc mimicked NNK effects, whereas NDMAOAc significantly inhibited IL-8 production in BEAS-2B cells and MCP-1 in both cell types. These results demonstrate that NNK and its reactive metabolites have immunosuppressive effects on respiratory epithelial cells, which could contribute to the increased respiratory infections observed in smokers and the development and/or the progression of lung cancer.

Histamine inhibits tumor necrosis factor alpha release by mast cells through H2 and H3 receptors.

Histamine was one of the first inflammatory mediators thought to be important in the pathophysiology of asthma, but it is not now thought to be a mediator with primary importance in airway constriction. However, histamine has several effects that may be relevant. One of these effects, its immunoregulatory role, has been largely ignored in asthma. Thus, because mast cells (MC) are an important source of histamine and cytokines, the modulation by histamine of the release of one cytokine, tumor necrosis factor alpha (TNF alpha), was investigated. Rat peritoneal MC (PMC) were pretreated with different concentrations of histamine (10(-14) to 10(-4) M) for 2 h before being tested for their TNF alpha-dependent cytotoxicity. A concentration-dependent inhibition of cytotoxicity was observed from 21% at 10(-12) M to 38% at 10(-4) M, reaching a plateau at 10(-8) M. At least 1 h pretreatment with histamine or its presence throughout the cytotoxic assay was required for the inhibitory effect of histamine. This inhibition was abrogated by indomethacin or anti-PGE2, suggesting that PGE2 may be an important mediator in the inhibition of TNF alpha by histamine. To investigate the type of histamine receptor implicated in this effect, PMC were treated for 20 min with H1 (clemastine and diphenhydramine), H2 (ranitidine and cimetidine), or H3 (thioperamide) receptor antagonists before the addition of histamine. H2 or H3 antagonists abrogated the inhibitory effect of histamine on PMC TNF alpha-dependent cytotoxicity. Furthermore, blockage of H2 receptors with ranitidine increased the release of TNF alpha from PMC stimulated with antigen, suggesting that histamine released by MC within 10 min of antigen stimulation downregulates the subsequent release of TNF alpha from the same MC population. These results suggest that histamine may act as an autocrine regulator of cytokine release by MC and thus modulate inflammatory responses in allergic asthma.

Inhibition of mast cell-mediated cytotoxicity by IFN-alpha/beta and -gamma.

Although IFN enhance the cytotoxic activity of NK cells, K cells, and monocytes, IFN-alpha/beta and IFN-gamma did not stimulate the cytotoxic activity of rat peritoneal mast cells (PMC), but had an inhibitory effect. Preincubation for 2 h with 100 and 200 U/ml of IFN-gamma and IFN-alpha/beta, respectively, inhibited PMC cytotoxicity against WEHI-164 target cells. Lower concentrations of IFN-gamma (12.5 U/ml) and IFN-alpha/beta (25 U/ml) inhibited cytotoxicity of PMC after 8 h preincubation. The inhibitory effect of IFN was concentration and time dependent. In contrast to cytotoxicity, the release of histamine by PMC was not stimulated by the target cells WEHI-164 and there was no correlation between histamine release and cytotoxic activity of PMC. Specific antibody to subclasses of IFN prevented the inhibition of PMC cytotoxic activity, but preincubation with antibodies to the alternate subclass of IFN did not affect the observed inhibition. Moreover, the presence of both subclasses of IFN showed an additive inhibition of PMC cytotoxicity. The cytotoxic activity of PMC can be completely inhibited by the addition of anti-TNF during the assay. At high concentrations (400 U/ml), IFN inhibited the release of TNF from PMC. In the presence of RNA or protein synthesis inhibitors, IFN did not inhibit cytotoxicity of PMC further. We postulate that IFN may alter gene expression in mast cells in a manner that down-regulates their functions.

Alveolar macrophages of allergic resistant and susceptible strains of rats show distinct cytokine profiles.

Brown Norway rats are widely used as a model of asthma, whereas Sprague Dawley rats do not develop allergic reactions under the same conditions. Given the importance of alveolar macrophages (AM) in down-regulating cellular immune responses in the lung, we postulated that the different susceptibilities in the development of airway allergic reactions in these rat strains may be related to functional differences in their AM. We investigated the production of important mediators in asthma, namely tumour necrosis factor (TNF), interleukin-10 (IL-10), IL-12, IL-13, nitric oxide (NO) and macrophage inflammatory protein-1alpha (MIP-1alpha), by AM of unsensitized Sprague Dawley and Brown Norway rats. AM were purified by adherence and stimulated with OX8 (anti-CD8 antibody) or LPS. OX8 stimulation significantly increased the release of TNF, IL-10 and NO in both strains of rats, whereas MIP-1alpha and IL-12 release were increased in Brown Norway rats only. Interestingly, stimulated AM from Sprague Dawley rats released significantly more TNF and less IL-10, IL-12, IL-13, MIP-1alpha and NO compared with AM from Brown Norway rats. These differences were also observed at the mRNA level, except for TNF. Thus, AM from Brown Norway and Sprague Dawley rats are functionally different. Furthermore, LPS- and OX8-stimulated AM from Brown Norway rats produce more Th2 type cytokines (IL-10 and IL-13) than AM from Sprague Dawley rats, suggesting that these cells may play an important role in creating a cytokine milieu that may favour the development of allergic reactions.

Priming of alveolar macrophages by leukotriene D(4): potentiation of inflammation.

Cysteinyl leukotrienes (LTs), including LTC(4), LTD(4), and LTE(4), are well known to induce bronchoconstriction and increase bronchial hyperreactivity, mucus secretion, and vascular permeability. Interestingly, alveolar macrophages (AMs) express LTD(4) high-affinity receptor. These cells represent a major source of inflammatory mediators implicated in the pathophysiology of asthma. Thus, we investigated the immunomodulatory effects of LTD(4) on the production of inflammatory mediators such as macrophage inflammatory protein (MIP)- 1alpha, tumor necrosis factor (TNF), and nitric oxide (NO) by AMs. NR8383 cells, an AM cell line, were pretreated with LTD(4) (10(-11) M) for different periods of time and stimulated or not with lipopolysaccharide (LPS) for 2 h. Although LTD(4) treatment did not modulate the release of MIP-1alpha and TNF, this treatment (6 h) significantly increased the release of these mediators when AMs were further stimulated with LPS (increases of 47 and 21%, respectively). Further, LTD(4) pretreatment increased messenger RNA (mRNA) levels of MIP-1alpha and TNF. These effects of LTD(4) were abrogated by the presence of a LTD(4) receptor antagonist, Verlukast (MK-679), showing the specificity of LTD(4). Interestingly, LTD(4) treatment significantly increased the release of NO by LPS-stimulated AMs without modulating mRNA levels of the inducible NO synthase. Our data suggest that LTD(4) primes AMs to release more MIP-1alpha, TNF, and NO after stimulation. Thus, in addition to its potent bronchoconstrictor effect, LTD(4) may participate in the inflammatory process seen in asthma by potentiating the production of proinflammatory mediators by AMs during immunologic stimuli.

IFN-gamma potentiates the release of TNF-alpha and MIP-1alpha by alveolar macrophages during allergic reactions.

Viral infections play an important role in the exacerbation of asthma. The production of interferons (IFNs) is well known to limit viral spread, but IFN-gamma can also prime alveolar macrophages to release more inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha). Given the importance of these cytokines, we have investigated the effect of IFN-gamma on their release by alveolar macrophages during stimulation by immunoglobulin (Ig)E/anti-IgE. Alveolar macrophages from normal or Nippostrongylus brasiliensis-infected rats, the latter having increased numbers of low-affinity receptors for IgE (Fcepsilon RII) on their alveolar macrophages, were treated with IgE for 2 h and stimulated with anti-IgE for 18 h. The increase of TNF-alpha release (153 +/- 48 pg/10(6) cells) by IgE/anti-IgE occurred only with alveolar macrophages from infected rats. The messenger RNA level for TNF-alpha in rat alveolar macrophages was also increased by stimulation with IgE/anti-IgE. Treatment with IFN-gamma prior to stimulation with IgE/anti-IgE showed a time- and concentration-dependent increase of TNF-alpha release. Interestingly, IgE/anti-IgE treatment did not stimulate the release of MIP-1alpha (15 +/- 5 pg/10(6) cells), but IFN-gamma treatment alone and with IgE /anti-IgE significantly increased and potentiated MIP-1alpha release (98 +/- 40 pg/10(6) cells) by alveolar macrophages, respectively. These results suggest that IFN-gamma produced at times such as during viral infections primes alveolar macrophages for enhanced release of inflammatory mediators during allergic reactions, thereby contributing to the inflammatory process.

Anti-inflammatory effect of beta 2-agonists: inhibition of TNF-alpha release from human mast cells.

Beta 2-agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D2 from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-alpha (TNF-alpha), there is no information about their regulation by beta 2-agonists. Thus given the importance of TNF-alpha in inflammation and the widespread use of beta 2-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) beta 2-agonists on the secretion of TNF-alpha from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-alpha (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-alpha-sensitive cell line, WEHI-164, with an IC50 of 71, 50, and 29 nmol/L, respectively. Specificity for beta-adrenergic receptors was shown with propranolol. The inhibitory effect of beta 2-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-alpha release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, beta 2-agonists did not show tachyphylaxis for the inhibition of TNF-alpha release. Thus selective beta2-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-alpha from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of beta-agonists may be important in their mode of action in the treatment of allergic diseases.

Inhibitory effects of sulfasalazine and its metabolites on histamine release and TNF-alpha production by mast cells.

Sulfasalazine is an effective treatment in some inflammatory diseases that exhibit mast cell (MC) hyperplasia. However, its effect on MC has been incompletely studied. We have established that sulfasalazine inhibits the release of histamine and TNF-alpha from MC. Sulfasalazine and its metabolites, 5-aminosalicylic acid (5-ASA) and to a lesser extent sulfapyridine, inhibited Ag-stimulated histamine release from rat peritoneal MC in a concentration-dependent manner with a 50% inhibitory concentration of 6 x 10(6)M, 8 x 10(-6)M, and 3 x 10(-4)M, respectively. Similar results were observed with sulfapyridine and 5-ASA on Ag-stimulated histamine release of another population of MC, namely rat intestinal mucosal MC, but sulfasalazine was markedly less potent than its metabolites. Interestingly, sulfasalazine and sulfapyridine, but not 5-ASA, inhibited Ag-stimulated TNF-alpha released by MC. Similar results were observed with MC-mediated cytotoxic activity in which sulfasalazine and sulfapyridine, but nor 5-ASA, inhibited MC TNF-alpha-dependent cytotoxicity in a concentration-dependent manner. The addition of sulfasalazine to MC, up to 12 h after the cytotoxic assay (16 h) had started, significantly inhibited cytotoxic activity, suggesting that sulfasalazine inhibited the cytotoxic mediator, TNF-alpha. Indeed, affinity studies demonstrated that sulfasalazine binds TNF-alpha. Furthermore, the inhibition of MC cytotoxicity by sulfasalazine appeared to require new protein synthesis. Pretreatment of MC with sulfasalazine also inhibited the release of TNF-alpha and reduced the levels of TNF-alpha mRNA. Thus, sulfasalazine inhibits MC-mediated, TNF-alpha-dependent cytotoxicity by multiple mechanisms: competitive inhibition of soluble TNF-alpha, reduction of levels of TNF-alpha mRNA, and inhibition of TNF-alpha release.

Regulation of mRNA levels of TNF-alpha and the alpha chain of the high-affinity receptor for IgE in mast cells by IFN-gamma and alpha/beta.

Rat mast cell lines (hybrid rat mast cells, HRMC, and rat cultured mast cells, RCMC) and mast cells from the rat body cavity were used to test the hypothesis that IFN-alpha/beta and IFN-gamma inhibit tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity by depressing the steady-state levels of mRNA for TNF-alpha. In vitro treatment of mast cells with IFN-gamma and IFN-alpha/beta depressed mRNA levels. By contrast, IFN pretreatment of mast cell lines induced an increase in levels of mRNA for the IFN-inducible gene, 2'5'-oligoadenylate synthetase and also for high-affinity IgE-receptor-alpha. The IFN-mediated regulation of mast cells may be an important mechanism in the control of inflammatory pathways characterized by Th1- and Th2-type responses.

Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.

TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10(-3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha-dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions.

Interferon and antiallergic drug regulation of histamine and tumor necrosis factor-alpha in rat mast cell subsets.

We have further characterized the heterogeneity of mast cells (MCs) by comparing the ability of rat peritoneal MCs (PMCs) and intestinal mucosal MCs (IMMCs) to produce tumor necrosis factor (TNF)-alpha and by investigating its regulation by interferon (IFN) and the antiallergic drugs nedocromil sodium (NED) and sodium cromoglycate (SCG). Although IMMCs store less TNF-alpha than PMCs, they produced comparable amounts of TNF-alpha in cytotoxic assays. Just as SCG and NED inhibit histamine secretion from PMCs but not IMMCs, IFN exhibited a similar differential effect on histamine release from these cells. However, SCG, NED, and IFN inhibit TNF-alpha-dependent cytotoxicity by both PMCs and IMMCs and reduce the steady-state levels of mRNA for TNF-alpha in PMCs. Thus, the modulation of MC mediator release depends upon the MC population and mediator studied. The inhibitory effect of SCG and NED on TNF-alpha release from MCs may explain some of their anti-inflammatory and therapeutic effects.