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.

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.

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.

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.

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.

PCT-233, a novel modulator of pro- and anti-inflammatory cytokine production.

Plant extracts have been implicated in various immunoregulatory effects that are poorly understood. Thus, we investigated the modulatory activity of PureCell Complex (PCT)-233, an active molecular complex from mesophyll tissue of Spinacia oleacea on the inflammatory process. Alveolar macrophages (AM) were treated with PCT-233 and/or budesonide, a well-known anti-inflammatory agent, before or after being stimulated with lipopolysaccharides (LPS). Pro- and anti-inflammatory cytokine production, tumour necrosis factor (TNF) and interleukin (IL)-10, respectively, were measured in cell-free supernatants at different times after the treatment. PCT-233 increased unstimulated AM release of both TNF and IL-10, whereas heat- and light-inactivated PCT-233 stimulated only the release of TNF without affecting IL-10 production, suggesting that different mechanisms are involved in the modulation of TNF and IL-10 release by PCT-233. The presence of LPS did not modify PCT-233-stimulated TNF production, but the ratio TNF/IL-10 production by LPS-stimulated AM was reduced significantly in the presence of PCT-233. Pretreatment of AM with PCT-233 and budesonide before LPS stimulation reduced TNF production at both protein and mRNA levels, whereas IL-10 production was increased. Moreover, TNF/IL-10 ratio was reduced further with the combination PCT-233/budesonide. Interestingly, AM treatment with PCT-233 and budesonide 18 h after LPS stimulation did not modulate TNF release significantly but it did increase IL-10 production, and a synergistic effect was observed with the combination PCT-233/budesonide. These exciting data suggest that PCT-233 possesses some anti-inflammatory properties, even when added during the inflammatory process, and could potentiate the effect of other anti-inflammatory agents.

Immunomodulatory effects of the tobacco-specific carcinogen, NNK, on alveolar macrophages.

Lung cancer is strongly associated with cigarette smoking. More than 20 lung carcinogens have been identified in cigarette smoke and one of the most abundant is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). We hypothesized that NNK modulates alveolar macrophage (AM) mediator production, thus contributing to carcinogenesis. An AM cell line, NR8383, was treated with [3H]NNK and lipopolysaccharide (LPS), and NNK metabolites released in supernatants were analysed by high-performance liquid chromatography (HPLC). NNK was metabolized by carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol (NNAL) or activated by alpha-carbon hydroxylation. AMs were also treated with NNK (100-1000 micro M), with and without LPS, for different periods of time (6-72 h), and mediators released in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) or the Griess reaction. NNK inhibited (in a concentration-dependent manner) AM production of tumour necrosis factor (TNF), macrophage inflammatory protein-1alpha (MIP-1alpha), interleukin (IL)-12 and nitric oxide (NO), whereas IL-10 production was increased. Cyclooxygenase inhibitors - NS-398 and indomethacin - and anti-prostaglandin E2 (anti-PGE2) antibody abrogated the NNK-inhibitory effect on MIP-1alpha production by AM. NNK stimulated the release of PGE2, and exogenous PGE2 inhibited AM MIP-1alpha production, suggesting that the NNK immunomodulatory effect may be mediated by PGE2 production. Thus, in addition to its carcinogenic effects, NNK may contribute to the lung immunosuppression observed in tobacco smokers.

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.

Redundancy or cell-type-specific regulation? Tumour necrosis factor in alveolar macrophages and mast cells.

Tumour necrosis factor (TNF) is an important inflammatory cytokine produced by several cell types. To test the hypothesis that there is cell-type-specific regulation and not redundancy of TNF production, we investigated its production by alveolar macrophages (AM) and peritoneal mast cells (PMC). Cell lysates of freshly isolated AM and PMC contained 9 +/- 3 pg and 57 +/- 17 pg of TNF/10(6) cells, respectively. Furthermore, unstimulated PMC expressed 4 x 10(3)-fold more attomols of TNF mRNA/microg total RNA compared with AM. These data may explain in part the greater TNF-dependent cytotoxicity of PMC. Furthermore, fixed PMC showed significantly higher TNF-dependent cytotoxic activity than AM (sevenfold), suggesting that PMC express more membrane TNF than AM. Although AM and PMC contain different amounts of TNF, antigen stimulation caused a similar release of TNF from sensitized rats. Interferon (IFN)-gamma, respectively, stimulated and inhibited AM and PMC TNF-dependent cytotoxicity whereas lipopolysaccharide (LPS) significantly stimulated TNF-dependent cytotoxicity in both cell types. However, TNF released (AM 400-fold and PMC threefold) and TNF mRNA expression, as measured by competitive reverse transcription-polymerase chain reaction (AM 7 x 10(3)-fold and PMC twofold), were considerably greater in LPS-stimulated AM than PMC. Our data indicate that TNF is differentially expressed in these two cell types and that its production is dependent on the nature of the stimulus. These data provide vital basis in experimental approaches aimed at modulating the effect of TNF in airway disease conditions involving both AM and mast cells.

Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: stimulation of IL-10 production.

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10-5-10-7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nomega-nitro-l -arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10-5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1. 7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nomega-nitro-l -arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.

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.

Rat mucosal mast cells: the cultured bone marrow-derived mast cell is biochemically and functionally analogous to its counterpart in vivo.

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.

Mechanisms of macrophage stimulation through CD8: macrophage CD8alpha and CD8beta induce nitric oxide production and associated killing of the parasite Leishmania major.

Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8alpha (OX8, 5 microg/ml) or CD8beta (341, 10 microg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8alpha and CD8beta stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 microg/ml), PP1 (src family kinase inhibitor, 5 microg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 microg/ml), and Ro 31-8220 (PKC inhibitor, 1 microM) significantly inhibited anti-CD8alpha- and anti-CD8beta-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8alpha stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the beta-chain of macrophage CD8, in addition to the alpha-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.

TGF-beta1 inhibits the release of histamine and tumor necrosis factor-alpha from mast cells through an autocrine pathway.

Transforming growth factor beta1 (TGF-beta1) is a member of a gene superfamily involved in the regulation of cell growth and differentiation, tissue repair, fibrosis, and inflammatory responses. Given the role of the mast cell (MC) in inflammation and fibrosis, the effect of TGF-beta1 on MC mediator release was studied. In vitro treatment of rat peritoneal MC (PMC) with TGF-beta1 (10(-10) M) for 20 h followed by washes inhibited (23%) antigen stimulated histamine release. Similar pretreatment of PMC with TGF-beta1 (10(-10) M) inhibited (27%) tumor necrosis factor-alpha (TNF-alpha) dependent cytotoxicity and reduced (31%) mRNA levels of TNF-alpha, but did not inhibit nitric oxide (NO) release. By contrast, the presence of TGF-beta1 throughout the cytotoxic assay, but without pretreatment of PMC did not modulate TNF-alpha release. At least 2 h pretreatment with TGF-beta1 was required to inhibit MC TNF-alpha-dependent cytotoxicity. This inhibitory effect of TGF-beta1 was abrogated by antibody to TGF-beta1. Interestingly, the treatment of PMC with anti-TGF-beta1 antibody alone significantly increased the release of histamine and TNF-alpha. Furthermore, freshly isolated rat PMC (10(7)) contained 35 +/- 7 pg latent TGF-beta1 and 51 +/- 9 pg was spontaneously released within 30 min of culture. However, stimulation of PMC with antigen inhibited the spontaneous release of TGF-beta1 by 43%. The duration of pretreatment with TGF-beta1 required to inhibit MC TNF-alpha release was similar to that required for downregulation of MC TNF-alpha-dependent cytotoxicity by IFN-gamma. TGF-beta1 and IFN-gamma had an additive inhibition on TNF-alpha release by PMC. This inhibitory effect was abrogated and TNF-alpha-dependent cytotoxicity was enhanced by the addition of anti-TGF-beta1 antibody, but not by anti-IFN-gamma. These results suggest MC mediator release is regulated by TGF-beta1 in an autocrine manner.

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.

Stem cell factor potentiates histamine secretion by multiple mechanisms, but does not affect tumour necrosis factor-alpha release from rat mast cells.

The effect of stem cell factor (SCF) on histamine and tumour necrosis factor-alpha (TNF-alpha) release from rat peritoneal mast cells (PMC) was determined and the intracellular pathways involved in the potentiation of histamine secretion were investigated. The effects of SCF (2-100 ng/ml) were examined following both short-term (0 and 20 min) and long-term (up to 24hr) preincubations with SCF. Pretreatment of PMC with SCF for 0 min (concurrent) or 20 min did not induce histamine secretion directly, but significantly increased antigen (Ag)-induced histamine secretion. SCF potentiated Ag-induced intracellular Ca2+ increase and calcium ionophore A23187-induced histamine secretion. Pertussis toxin (PT) inhibited SCF-induced potentiation of IgE-dependent histamine secretion, indicating that PT-sensitive G-proteins are involved in the immediate effects of SCF. In long-term incubation experiments, SCF pretreatment for 18-24 hr significantly enhanced Ag-induced histamine secretion, but did not affect Ag-induced intracellular Ca2+ levels. The effects of long-term incubation with SCF, but not the short-term effects, were blocked by cycloheximide. Interestingly, spontaneous and Ag-induced TNF-alpha release from rat PMC were not affected by pretreatment with SCF (2-500 ng/ml) for 1 to 24 hr. Thus, through immediate and delayed mechanisms, SCF potentiates histamine release from PMC, but has not effect on TNF-alpha release. The regulation of MC by SCF may be important in allergic and other inflammatory diseases.

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.