Véronique Turmel

Microbiological and Inflammatory Factors Associated with the Development of Pneumococcal Pneumonia

Pneumococcal pneumonia still is associated with a high mortality rate, despite appropriate antimicrobial therapy. Many gaps remain in the understanding of the pathogenesis of this deadly infection. The microbial and inflammatory events that characterize survival or death after intranasal inoculation of mice with an LD(50) inoculum of Streptococcus pneumoniae were investigated. Survival was associated with rapid bacterial clearance and low inflammation (surfactant and red blood cells in alveoli), but no neutrophil recruitment or lung tissue injury was noted. By contrast, death was preceded by strong bacterial growth that peaked 48 h after the infection and was associated with gradual increases in pulmonary levels of interleukin-6, macrophage inflammatory protein (MIP)-1alpha, MIP-2, monocyte chemoattractant protein-1, KC, and neutrophil recruitment. The injection of tumor necrosis factor-alpha or the addition of lipopolysaccharide or heat-killed S. pneumoniae to the inoculum enhanced early host response and survival. These observations may help develop appropriate markers of evolution of pneumonia, as well as new therapeutic strategies.

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.

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.

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, α‐methylhydroxylation and α‐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 E2 (PGE2) production by A549 cells, its immunomodulatory effects were not mediated by PGE2 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.

Alveolar macrophages reduce airway hyperresponsiveness and modulate cytokine levels

ABSTRACT The authors have recently demonstrated that alveolar macrophages (AMs) are important in protecting against early phase reactions and airway hyperresponsiveness following allergen challenge. To further understand the mechanisms involved, the authors investigated the capacity of AMs to modulate airway inflammation and cytokine levels in bronchoalveolar lavage (BAL). AMs from allergy-susceptible Brown Norway (BN) rats or allergy-resistant Sprague-Dawley (SD) rats were transferred into AM-depleted BN rats 24 hours prior to allergen challenge. Methacholine-induced airway hyperresponsiveness was examined 24 hours following ovalbumin challenge. Total cells, cell types, and cytokine levels (tumor necrosis factor [TNF], interleukin [IL]-4, IL-10, IL-12 and IL-13) in BAL were measured 24 hours after allergen challenge. The transfer of AMs from SD rats into AM-depleted BN rats 24 hours before allergen challenge eliminated methacholine-induced airway hyperresponsiveness, but did not modify the number and the type of inflammatory cells in BAL. Levels of IL-13 and TNF were significantly higher in BAL of BN rats compared with SD rats. Interestingly, IL-13 and TNF levels were significantly increased and inhibited, respectively, in BN rats that received AMs from SD rats compared with BN rats. Our data suggest that AM modulation of cytokine milieu is involved in the reduction of airway hyperresponsiveness.

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.

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.