Anick Langlois

Crucial implication of protein kinase C (PKC)‐δ, PKC‐ζ, ERK‐1/2, and p38 MAPK in migration of human asthmatic eosinophils

Asthma is associated with an eosinophil infiltration into the bronchial mucosa. 5‐Oxo‐6,8,11,14(E,Z,Z,Z)‐eicosatetraenoic acid (5‐oxo‐ETE), a potent eosinophil chemotactic factor, activates cell motility, adherence, and proteolysis, notably, by promoting CD11b expression, matrix metalloproteinase (MMP)‐9 secretion, and plasmin generation. We investigated the intracellular signaling pathways implicated in these various steps by using different, selective inhibitors. Human eosinophil migration through a reconstituted basement membrane in response to 5‐oxo‐ETE was greatly inhibited (≥72%) by the protein kinase C (PKC)‐δ, PKC‐ζ, ERK‐1/2, and p38 inhibitors. Our findings indicate that PKC‐δ mediates cell motility, CD11b expression, and MMP‐9 granule release. PKC‐ζ is also largely involved in eosinophil migration, although its specific targets remain undefined. ERK‐1/2 and p38 modulate CD11b expression; ERK‐1/2 is also involved in long‐term MMP‐9 secretion and p38 in the plasmin activation system. We demonstrated the crucial implication of PKC‐δ, PKC‐ζ, ERK‐1/2, and p38 in human blood eosinophil migration through extracellular matrix components. Targeting specific pathways may have therapeutic potential for the treatment of allergic airway inflammation.

Modulation of eosinophil activation in vitro by a nicotinic receptor agonist

Nicotinic receptor agonists decreased the infiltration of eosinophils into the lung and airways in a mouse model of asthma. To better understand the mechanisms implicated in this anti‐inflammatory phenomenon, the expression of nicotinic acetylcholine receptors (nAChRs) and the effect of dimethylphenylpiperazinium (DMPP), a nonselective nAChR agonist, on human blood eosinophils were studied. The expression of α‐3, ‐4, and ‐7 nAChR subunits on human blood eosinophils was measured by cell ELISA and immunocytochemistry. mRNA expression for all three subunits was evaluated by quantitative RT‐PCR. The effect of DMPP on leukotriene C4 (LTC4) and matrix metalloproteinase‐9 (MMP‐9) production, eosinophil migration, and intracellular calcium mobilization was measured. The results show that the α‐3, ‐4, and ‐7 nAChR subunits and mRNAs are expressed by blood eosinophils. In vitro treatment of these cells with various concentrations of DMPP reduced platelet‐activating factor (PAF)‐induced LTC4 production significantly. DMPP (160 μM) decreased eotaxin, and 5‐oxo‐6,8,11,14‐eicosatetranoic acid induced eosinophil migration through Matrigel by 40.9% and 55.5%, respectively. This effect was reversed by the nAChR antagonist mecamylamine. In addition, DMPP reduced MMP‐9 release and the inositol 1,4,5‐triphosphate‐dependent intracellular calcium increase provoked by PAF. Taken together, these results indicate that functional nAChRs are expressed on eosinophils and that nAChR agonists down‐regulate eosinophil function in vitro. These anti‐inflammatory effects could be of interest in the treatment of allergic asthma.

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5‐oxo‐eicosatetraenoate‐induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells.

Introduction Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with TH2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT1 and CysLT2 receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the TH2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells. Methods A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD4. The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively. Results IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD4 weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD4 had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT1 but not CysLT2. CysLT1 expression was increased by IL-13 but not by IL-4 and/or LTD4. Importantly, the upregulation of CysLT1 by IL-13 preceded eotaxin-3 release. Conclusions These results demonstrate a stepwise cooperation between IL-13 and LTD4. IL-13 upregulates CysLT1 expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.

Pulmonary CD103 expression regulates airway inflammation in asthma

Although CD103(+) cells recently emerged as key regulatory cells in the gut, the role of CD103 ubiquitous expression in the lung and development of allergic airway disease has never been studied. To answer this important question, we evaluated the response of Cd103(-/-) mice in two separate well-described mouse models of asthma (ovalbumin and house dust mite extract). Pulmonary inflammation was assessed by analysis of bronchoalveolar lavage content, histology, and cytokine response. CD103 expression was analyzed on lung dendritic cells and T cell subsets by flow cytometry. Cd103(-/-) mice exposed to antigens developed exacerbated lung inflammation, characterized by increased eosinophilic infiltration, severe tissue inflammation, and altered cytokine response. In wild-type mice exposed to house dust mite, CD103(+) dendritic cells are increased in the lung and an important subset of CD4(+) T cells, CD8(+) T cells, and T regulatory cells express CD103. Importantly, Cd103(-/-) mice presented a deficiency in the resolution phase of inflammation, which supports an important role for this molecule in the control of inflammation severity. These results suggest an important role for CD103 in the control of airway inflammation in asthma.

Lung CD200 Receptor Activation Abrogates Airway Hyperresponsiveness in Experimental Asthma

In allergic asthma, homeostatic pathways are dysregulated, which leads to an immune response toward normally innocuous antigens. The CD200-CD200 receptor pathway is a central regulator of inflammation, and CD200 expression was recently found to be down-regulated in circulating leukocytes of patients with asthma. Given the antiinflammatory properties of CD200, we investigated whether local delivery of recombinant CD200 (rCD200) could reinstate lung homeostasis in an experimental model of asthma. Brown Norway rats were sensitized with ovalbumin (OVA) and alum. rCD200 was intratracheally administered 24 hours before OVA challenge, and airway responsiveness to methacholine was measured 24 hours after the allergen challenge. Inflammation was also assessed by measuring cell recruitment and cytokine levels in bronchoalveolar lavages, as well as lung and draining lymph node accumulation of dendritic cells (DCs) and T cells. In sensitized rats, rCD200 abolished airway hyperresponsiveness, whereas the sham treatment had no effect. In addition, rCD200 strongly reduced OVA-induced lung accumulation of myeloid DCs, CD4(+) T cells, and T helper type 2 cells. This was associated with a strong reduction of OVA-induced IL-13 level and with an increase of IL-10 in supernatants of bronchoalveolar lavages. Lung eosinophilia and draining lymph node accumulation of myeloid DCs and T cells were not affected by rCD200. Overall, these data reveal that rCD200 can inhibit airway hyperresponsiveness in a model of asthma by a multistep mechanism associated with local alterations of the T cell response and the cytokine milieu.

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

BackgroundIn vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.MethodsBALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.ResultsAAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4+ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.ConclusionAirway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.

Methanosphaera stadtmanae induces a type IV hypersensitivity response in a mouse model of airway inflammation

Despite improved awareness of work‐related diseases and preventive measures, many workers are still at high risk of developing occupational hypersensitivity airway diseases. This stems from a lack of knowledge of bioaerosol composition and their potential effects on human health. Recently, archaea species were identified in bioaerosols, raising the possibility that they play a major role in exposure‐related pathology. Specifically, Methanosphaera stadtmanae (MSS) and Methanobrevibacter smithii (MBS) are found in high concentrations in agricultural environments and respiratory exposure to crude extract demonstrates immunomodulatory activity in mice. Nevertheless, our knowledge of the specific impact of methanogens exposure on airway immunity and their potential to induce airway hypersensitivity responses in workers remains scant. Analysis of the lung mucosal response to methanogen crude extracts in mice demonstrated that MSS and MBS predominantly induced TH17 airway inflammation, typical of a type IV hypersensitivity response. Furthermore, the response to MSS was associated with antigen‐specific IgG1 and IgG2a production. However, despite the presence of eosinophils after MSS exposure, only a weak TH2 response and no airway hyperresponsiveness were observed. Finally, using eosinophil and mast cell‐deficient mice, we confirmed that these cells are dispensable for the TH17 response to MSS, although eosinophils likely contribute to the exacerbation of inflammatory processes induced by MSS crude extract exposure. We conclude that, as MSS induces a clear type IV hypersensitivity lung response, it has the potential to be harmful to workers frequently exposed to this methanogen, and that preventive measures should be taken to avoid chronic hypersensitivity disease development in workers.

CD34 Differentially Regulates Contractile and Noncontractile Elements of Airway Reactivity

&NA; Airway hyperresponsiveness (AHR), a major hallmark of asthma, results from alterations of contractile and noncontractile elements of airway reactivity. CD34 is a sialomucin that is expressed on various cells involved in asthma, such as eosinophils and airway smooth muscle precursors, highlighting its potential influence in AHR. To study the role of CD34 in regulating the contractile and noncontractile elements of AHR, AHR was induced by chronic exposure to house dust mite (HDM) antigen. To assess the role of CD34 on the contractile elements of AHR, airway reactivity and airway smooth muscle contractility in response to methacholine were measured. To assess CD34s role in regulating the noncontractile elements of AHR, a chimeric mouse model was used to determine the impact of CD34 expression on inflammatory versus microenvironmental cells in AHR development. Extracellular matrix production, mucus production, and mast cell degranulation were also measured. Whereas wild‐type mice developed AHR in response to HDM, a loss of airway reactivity was observed in Cd34−/− mice 24 hours after the last exposure to HDM compared with naive controls. This was reversed when airway reactivity was measured 1 week after the last HDM exposure. Additionally, mast cell degranulation and mucus production were altered in the absence of CD34 expression. Importantly, simultaneous expression of CD34 on cells originating from the hematopoietic compartment and the microenvironment was needed for expression of this phenotype. These results provide evidence that CD34 is required for AHR and airway reactivity maintenance in the early days after an inflammatory episode in asthma.

Hypersensitivity pneumonitis onset and severity is regulated by CD103 dendritic cell expression

Background Pulmonary dendritic cells drive lung responses to foreign antigens, including Saccharopolyspora rectivirgula, a causative agent of hypersensitivity pneumonitis. While the airway inflammatory mechanisms involved in hypersensitivity pneumonitis are well described, the mechanisms leading to the break in homeostasis and hypersensitivity pneumonitis onset are not well-described, and could involve CD103+ dendritic cells, which are found at baseline and during inflammatory responses in the lung. However, recent demonstration of the ability of CD103+ dendritic cells to induce inflammatory responses starkly contrasts with their classically described role as regulatory cells. These discrepancies may be attributable to the lack of current information on the importance of CD103 expression and modulation on these cells during inflammatory episodes. Methods To verify the importance of CD103 expression in the regulation of hypersensitivity pneumonitis, wild-type and Cd103-/- mice were exposed intranasally to S. rectivirgula and airway inflammation was quantified. Surface expression of CD103 in response to S. rectivirgula exposure was studied and cell transfers were used to determine the relative importance of CD103 expression on dendritic cells and T cells in regulating the inflammation in hypersensitivity pneumonitis. Results Cd103-/- mice developed an exacerbated inflammatory response as early as 18h following S. rectivirgula exposure. CD103 expression on dendritic cells was downregulated quickly following S. rectivirgula exposure, and cell transfers demonstrated that CD103 expression on dendritic cells specifically (and not T cells) regulates the onset and severity of this response. Conclusion All in all, we demonstrate that CD103 expression by dendritic cells, but not T cells, is crucial for homeostasis maintenance and the regulation of the TH17 airway inflammatory response in hypersensitivity pneumonitis.