Elyse Y. Bissonnette

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 (Nω-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, Nω-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.

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 α‐carbon hydroxylation. AMs were also treated with NNK (100–1000 µM), 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‐1α (MIP‐1α), 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‐1α production by AM. NNK stimulated the release of PGE2, and exogenous PGE2 inhibited AM MIP‐1α 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.

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 IgG4 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.

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‐HT1 and 5‐HT2 receptor agonists for 2 h prior stimulation. T helper 1 (Th1) and Th2 cytokines, prostaglandin‐E2 (PGE2) 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 PGE2 production were increased. These immunomodulatory effects of serotonin were mimicked by 5‐HT2 receptor agonist but were not abrogated by 5‐HT2 receptor antagonist, suggesting the implication of other 5‐HT receptors. Inhibitors of cyclooxygenase and antibody to PGE2 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, PGE2 significantly increased AM IL‐10 and NO production. These results suggest that serotonin alters the cytokine network in the lung through the production of PGE2. The reduction of Th1‐type cytokine by serotonin may contribute to asthma pathogenesis.

Alveolar macrophages in allergic asthma: an expression signature characterized by heat shock protein pathways.

Abstract The implication of alveolar macrophages (AM) in asthma, a Th2 disease, has not been well characterized. Thus, the goal of this study is to better characterize AM phenotype of allergic asthmatic compared with normal subjects using genomic expression analyses. Microarray analyses were performed with AM isolated from bronchoalveolar lavage. Robust multiarray analysis (RMA) normalization and Smyths moderated t test were used to select differentially expressed genes. Fifty differentially expressed genes were identified. Nineteen have been classified in categories linked to stress or immune responses and among them; nine are part of the heat shock protein (HSP) family. Difference of expression for three (HSPD1, PRNP, SERPINH1) of the five selected genes were validated using real-time reverse transcription–polymerase chain reaction. Enzyme-linked immunosorbent assay was used to measure the protein level of heat shock protein 60 (HSP60), the protein encoded by HSPD1, and showed difference in AM protein level between allergic asthmatic and control subjects. In summary, this study suggests that HSP gene family, particularly HSP60, is involved in AM functions in a context of allergic asthma. These results also support the involvement of AM immune functions in the development of an allergic asthmatic response.

Endothelins regulate mediator production of rat tissue-cultured mucosal mast cells. Up-regulation of Th1 and inhibition of Th2 cytokines.

Mast cells have been shown to produce endothelin‐1 (ET‐1) and to express ET receptors. Thus, we postulated that ETs modulate mast cell mediator production in an autocrine manner. Rat tissue‐cultured mast cells (RCMC‐1) were incubated with exogenous ET‐1 or ET‐3, and β‐hexosaminidase release and TNF, IL‐4, IL‐10, IL‐12, IL‐13, macrophage inflammatory protein‐1α (MIP‐1α), and nitric oxide (NO) production were investigated. ET‐1 and ‐3 induced the release of β‐hexosaminidase and TNF and of mRNA expression. An antagonist of the ETB receptor subtype abrogated ET‐stimulated TNF release, although ETA and ETB receptors have been identified by immunocytochemistry. It is interesting that ET‐1 and ET‐3 inhibited (25–30%) mRNA expression of Th2‐type cytokines (IL‐4, IL‐10, and IL‐13) and increased IL‐12 release (39% and 41%, respectively) without affecting MIP‐1α and NO production. Thus, our data suggest that ETs may play an important role in modulating the cytokine network by regulating Th1/Th2 cytokine production by mast cells.

Regulation of normal and cystic fibrosis airway epithelial repair processes by TNF-α after injury.

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24-48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K(+) channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and K(ATP) currents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K(+) channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.

Alveolar Macrophage Subpopulations in Bronchoalveolar Lavage and Induced Sputum of Asthmatic and Control Subjects

Background. Alveolar macrophages (AM) are the most numerous immune cells in the airways and are involved in the immunological homeostasis of the lung. Intriguingly, their role in asthma remains unclear probably, in part, because of their heterogeneity. Objective. To characterize AM population from bronchoalveolar lavage (BAL) and induced sputum (IS) of asthmatic and normal subjects using specific biomarkers. Methods. Non-asthmatic non-allergic and allergic mild asthmatic subjects were recruited for this study. AM were obtained from BAL and IS and cytospins were prepared. Immunocytochemistry was performed for nine cellular markers (CD68, RFD7, CD14, CD11b, CD83, CD64, CD80, CD86, and FIZZ1). Results. Asthmatic subjects had more AM RFD7+ in BAL compared with IS, whereas control subjects had more AM RFD7+ in IS than in BAL. Consequently, there was an increased number of AM RFD7+ in BAL of asthmatic subjects compared with BAL of control subjects. AM CD11b+ was higher in BAL than in IS in both groups. The expression of FIZZ1, marker of macrophage alternative activation, was similar in asthmatic and normal subjects. Conclusion. The expression of cellular markers on AM differs according to their localization in the lung. Subpopulations of AM may contribute to the inflammatory profile observed in asthmatic subjects.

Coumarinic derivatives show anti-inflammatory effects on alveolar macrophages, but their anti-elastase activity is essential to reduce lung inflammation in vivo

We have previously demonstrated the potency of coumarinic derivatives to inhibit human leukocyte elastase. Given the anti-inflammatory activities of some coumarins, we investigated the capacity of our coumarinic derivatives to inhibit inflammation and whether their anti-elastase activity was essential for their anti-inflammatory functions. All compounds studied were coumarinic derivatives displaying differential anti-proteinase activity. Coumarinic derivatives 1, 2, and 3 efficiently inhibited human leukocyte elastase in vitro, whereas the coumarinic derivative 4 did not show inhibitory activity. The anti-inflammatory effect of these compounds and a coumarin control, scopoletin, on interleukin-6 (IL-6), tumor necrosis factor (TNF), and macrophage chemotactic protein-1 (MCP-1) release was studied using lipopolysaccharide (LPS)-stimulated alveolar macrophages. The in vivo effect of compound 2, that inhibits elastase, and compound 4, that does not show proteinase inhibition, was investigated using a mouse model of LPS-induced lung inflammation and elastase-induced acute lung injury. All investigated coumarinic derivatives, regardless of their anti-proteinase activity, significantly inhibited IL-6 and TNF production by LPS-stimulated alveolar macrophages. However, only compounds 2, 3, and 4 significantly reduced MCP-1 release. Compound 2 attenuated LPS-induced leukocyte recruitment in bronchoalveolar lavage, whereas no inhibition was observed with compound 4 devoid of elastase inhibitory capacity. Interestingly, MCP-1 level was reduced in bronchoalveolar lavage of compound 4 treated mice, whereas TNF and IL-6 levels were not modulated by coumarins. Furthermore, compound 2, but not 4, reduced elastase induced lung injury. Our data suggest that although coumarinic derivatives have anti-inflammatory properties, their anti-elastase activity is essential to reduce lung inflammation in vivo.

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.