El-Bdaoui Haddad

The IL-6R α chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo

The cytokine IL-6 acts via a specific receptor complex that consists of the membrane-bound IL-6 receptor (mIL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). In this study, we investigated the role of IL-6R components in asthma. We observed increased levels of sIL-6R in the airways of patients with allergic asthma as compared to those in controls. In addition, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130-fraction constant led to suppression of Th2 cells in the lung. By contrast, blockade of mIL-6R induced local expansion of Foxp3-positive CD4+CD25+ Tregs with increased immunosuppressive capacities. CD4+CD25+ but not CD4+CD25- lung T cells selectively expressed the IL-6R alpha chain and showed IL-6-dependent STAT-3 phosphorylation. Finally, in an in vivo transfer model of asthma in immunodeficient Rag1 mice, CD4+CD25+ T cells isolated from anti-IL-6R antibody-treated mice exhibited marked immunosuppressive and antiinflammatory functions. IL-6 signaling therefore controls the balance between effector cells and Tregs in the lung by means of different receptor components. Furthermore, inhibition of IL-6 signaling emerges as a novel molecular approach for the treatment of allergic asthma.

Role of p38 MAP kinase in LPS‐induced airway inflammation in the rat

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED50=15.8u2003mgu2003kg−1) plasma levels of TNF‐α in rats challenged with LPS (1.5u2003mgu2003kg−1, i.p.). Aerosolized LPS induced a peak in TNF‐α levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2u2003h time point. Furthermore, the 4u2003h time point was associated with the peak in IL‐1β levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100u2003mgu2003kg−1), had no effect on peak TNF‐α levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100u2003mgu2003kg−1) significantly reduced both TNF‐α levels and neutrophilic inflammation. However, the BAL fluid from rats pre‐treated with either compound significantly inhibited TNF‐α release from cultured human monocytes 18u2003h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100u2003mgu2003kg−1) produced dose‐related inhibition of BAL IL‐1β levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4u2003h after LPS challenge. P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment. P38 kinase appears to be involved in the release of IL‐1β and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.

Differential Effects of Ebselen on Neutrophil Recruitment, Chemokine, and Inflammatory Mediator Expression in a Rat Model of Lipopolysaccharide-Induced Pulmonary Inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1–100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-α, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-α and IL-1β protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-α and IL-1β, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.

Functional characterization and biomarker identification in the Brown Norway model of allergic airway inflammation

The antigen‐induced inflammatory response in the Brown Norway rat is a model commonly used to assess the impact of novel compounds on airway eosinophilia. A detailed functional, cellular and molecular characterization of this model has not yet been performed within a single study. This information together with the temporal changes in this phenomenon should be known before this model can be used, with confidence, to elucidate the mechanisms of action of novel anti‐inflammatory drugs. Antigen challenge caused an accumulation of eosinophils in lung tissue 24 h after challenge. Accumulation of CD2+ T cells was not apparent until after 72 h. Interestingly, mRNA for the Th2 type cytokines interleukin (IL)‐4, IL‐5 and IL‐13 and eotaxin were elevated in lung tissue after challenge and the expression of IL‐13 and eotaxin protein increased at around 8–12 h. The temporal changes in both the biomarker production and the functional responses are important factors to consider in protocol design prior to initiating a compound screening program. A neutralising antibody (R73) against αβ‐TCR caused a significant reduction in T cell numbers accompanied by a significant suppression of eosinophil accumulation. Airway hyperreactivity (AHR) was not apparent in this specific Brown Norway model in sensitized animals after a single or multiple challenges although eosinophil influx was seen in the same animals. In conclusion, this is a convenient pre‐clinical model (incorporating the measurement of biomarkers and functional responses) for screening novel small molecule inhibitors and/or biotherapeutics targeted against T cell/eosinophil infiltration/activation.

Critical Role for T Cells in Sephadex-Induced Airway Inflammation: Pharmacological and Immunological Characterization and Molecular Biomarker Identification

Intratracheal instillation of Sephadex particles is a convenient model for assessing the impact of potential anti-inflammatory compounds on lung eosinophilia thought to be a key feature in asthma pathophysiology. However, the underlying cellular and molecular mechanisms involved are poorly understood. We have studied the time course of Sephadex-induced lung eosinophilia, changes in pulmonary T cell numbers, and gene and protein expression as well as the immunological and pharmacological modulation of these inflammatory indices in the Sprague Dawley rat. Sephadex increased T cell numbers (including CD4+ T cells) and evoked a pulmonary eosinophilia that was associated with an increase in gene/protein expression of the Th2-type cytokines IL-4, IL-5, and IL-13 and eotaxin in lung tissue. Sephadex instillation also induced airway hyperreactivity to acetylcholine and bradykinin. A neutralizing Ab (R73) against the αβ-TCR caused 54% depletion of total (CD2+) pulmonary T cells accompanied by a significant inhibition of IL-4, IL-13 and eotaxin gene expression together with suppression (65% inhibition) of eosinophils in lung tissue 24 h after Sephadex treatment. Sephadex-induced eosinophilia and Th2 cytokine gene and/or protein expression were sensitive to cyclosporin A and budesonide, compounds that inhibit T cell function, suggesting a pivotal role for T cells in orchestrating Sephadex-induced inflammation in this model.

Characterization of the effects of cannabinoids on guinea-pig tracheal smooth muscle tone: role in the modulation of acetylcholine release from parasympathetic nerves

We investigated the ability of the cannabinoid agonists CP55,940 (CB1/CB2) and anandamide (endogenous cannabinoid) to modulate electrical field stimulation (EFS)‐induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea‐pig trachea. We assessed whether modulation of transmitter release translated to an impact on functional responses by investigating the effect of these agents on contractile responses evoked by EFS and ACh. Furthermore, we evaluated the ability of these compounds to elicit bronchodilation in pre‐contracted guinea‐pig tracheal strips. CP55,940 and anandamide significantly inhibited EFS‐evoked ACh release (maximal inhibition of 35.1±2.9% and 33.4±6.4% at 1u2003μM, P<0.05, respectively). The CB1 receptor antagonist SR 141716A (1u2003μM), had no effect on ACh release and failed to reverse the inhibitory effect of CP55,940 (1u2003μM). Paradoxically, CP55,940 had no significant effect on EFS‐evoked cholinergic contractile responses. Furthermore, CP55,940 did not relax pre‐contracted tracheal strips or affect contractile responses to exogenous ACh. This lack of activity on smooth muscle tone is consistent with the fact that no detectable specific binding of [3H] CP55,940 was found in tracheal homogenates. These data suggest that cannabinoid agonists inhibit ACh release from cholinergic nerve terminals via activation of CB2 receptors but that this inhibitory action does not impact on functional responses such as cholinergic contraction.

Analysis of Gene Expression

Messenger (m)RNAs represent the information-carrying intermediates for protein synthesis. Northern blotting gives a snapshot of the relative abundance of an RNA transcript at a set time point by measuring the steady-state levels of an RNA transcript. The steady-state level of a particular mRNA species is determined by its rate of production, degradation, and transport from the nucleus to cytoplasm. Northern blotting allows investigation and quantification of changes in gene expression.