P. de Nadai

Antihistamines as anti-inflammatory agents

Histamine is an important chemical mediator of inflammation in allergic rhinitis, cutaneous allergic reactions and asthma. In the airways, it induces vasodilation of the postcapillary venules, with extravasation of fluid, protein and cells leading to mucosal oedema. It produces bronchoconstriction by direct stimulation of H1 receptors on smooth muscle and by indirect stimulation of parasympathetic reflexes. Airway hyper-responsiveness to histamine is a hallmark of asthma, and histamine is released from the airway mast cell granules. Increased plasma concentrations of histamine have been detected during both the earlyand late-phase reactions following bronchial allergen challenge [1]. The level of histamine in bronchoalveolar lavage fluid from asthmatics has been found to correlate with airway hyper-reactivity and with asthma severity [2]. Histamine also has significant immune pro-inflammatory properties mediated by numerous cell types, including T cells, epithelial and endothelial cells and dendritic cells. For example, histamine can induce the expression of adhesion molecules on endothelial cells, such as E-selectin or ICAM-1 [3], trigger the production of IL-6 and IL-8 by endothelial cells [4] and up-regulate costimulatory molecules on dendritic cells [5]. Because histamine influences cell types that govern immune and inflammatory reactions, the therapeutic potential of H1-antihistamines in asthma has generated a great deal of interest. H1-antihistamines have been widely and successfully used in the treatment of allergic rhinitis and urticaria; however, their possible use in the treatment of asthma has been a matter of debate for about 20 years. The first-generation H1-antihistamines (i.e. chlorpheniramine, mequitazine, promethazine, etc.) have marked sedative effects, due to blood–brain barrier crossing. The so-called second-generation antihistamines (i.e. loratadine, cetirizine, terfenadine, ebastine, etc.) were engineered to be highly selective for the H1 receptor and are characterized by minimal sedative effects, rapid onset of action and long halflife. However, they still have dose-related adverse effects at high doses, in particular, sedative and anticholinergic effects. To address these therapeutic issues, third-generation antihistamines have been recently developed, which are either active metabolites (i.e. desloratadine, fexofenadine) or enantiomers (levocetirizine) of second-generation compounds. They exhibit more potent H1-receptor antagonist and antiinflammatory activity than their parent compounds. The anti-allergic properties of antihistamines usually refer to their ability to inhibit mast cell and basophil activity linked to the early-phase reaction. Such activity includes the inhibition of classical preformed mediators, such as histamine and tryptase, and has been demonstrated for available antihistamines [6–9]. More recently, desloratadine was demonstrated to inhibit basophil cytokines such as IL-4 and IL-13 [10]. The importance of these cytokines in B cell activation and IgE synthesis would predict an interesting effect on IgE-mediated allergic reactions. The late-phase reaction is characterized by chemotaxis of inflammatory cells, including eosinophils and Th2 cells through the endothelial cell layer, followed by activation, differentiation and release of various mediators. New-generation antihistamines have shown inhibitory effects on the expression of adhesion molecules such as ICAM-1 on epithelial cells [11, 12] and P-selectin on endothelial cells [13], on eosinophil chemotaxis and activation both in vitro [14–17] and in vivo [18, 19], on inflammatory cytokine production such as IL-6 or GM-CSF [13, 20] and on chemokine release such as IL-8, RANTES and eotaxin [13, 17, 21, 22]. In addition, all the H1-antihistamines tested to date inhibit the constitutive H1-receptor-mediated NF-kB transcription factor activation [23, 24], probably explaining their effects on cytokine production. Few studies have evaluated the effects of antihistamines on Th2 lymphocytes, cells believed to orchestrate the allergic reaction [25, 26]. In these studies, terfenadine and desloratadine were found to inhibit Th2 cytokine production by peripheral blood T cells. In this issue of the journal, Nori et al. [27] have evaluated the effect of the second-generation H1-antihistamine ebastine on the production of Th2-type cytokines. Using T cells derived from healthy non-atopic volunteers, they showed that ebastine inhibited the secretion in vitro of IL-4 and IL-5, but not IL-2 and IFN-g. This effect was not observed with the first-generation antihistamine ketotifen. Furthermore, there was also an inhibitory effect on T cell proliferation and macrophage-derived pro-inflammatory cytokines such as IL-6 and TNF-a. However, these data were obtained with cells from non-atopic donors stimulated with non-specific stimuli. Furthermore, these effects were observed with high concentrations of ebastine that would be achieved in vivo only with doses administered at several times the therapeutic dose. It would be interesting to assess the effect of H1-antihistamines on allergen-stimulated cells derived from allergic patients. Future work might also evaluate the effect on the cytokine profile of the allergic reaction of ebastine administered orally to allergic patients at therapeutic doses. The clinical efficacy of second-generation H1-antihistamines in allergic rhinitis is well documented [28]. In asthma, the clinical evaluation of these new H1-antihistamines has been generally disappointing (for review see [29–31]). A metaanalysis of 19 double-blind, randomized, placebo-controlled studies of antihistamine in persistent asthma showed that the beneficial effects were outweighed by adverse effects [32]. However, some studies have reported beneficial effects. The first evidence for a potential benefit of new-generation Clin Exp Allergy 2003; 33:1476–1478

Chemokines in cutaneous allergic reactions: the SCID model as a powerful tool to evaluate their interest as therapeutical targets

Abstract Chemokines are small chemotactic molecules actingthrough G-protein-coupled receptors. Their functions are very diverse in homeostatic as well as in inflammatory conditions. Recently, it has been shown that chemokines and their receptors also help control the tissue specificity of memory lymphocyte subsets, which could lead to their use as therapeutic targets in selective tissue diseases such as allergic cutaneous reactions. Currently available animal models are not relevant to the human situation, which is a reason as to why we have developed an in vivo model of humanized immunodeficient mice grafted with human skin and mononuclear cells. This model will allow us to evaluate, in a pre-human context, the bonafide effects of chemokines and to test them as potential therapeutic targets.

ILC2 and ILC3 contribute to house dust mite (HDM)-induced asthma in obese mice

Many epidemiological and clinical evidences suggest that obesity is an important risk factor for asthma, but the underlying mechanisms remain poorly understood. Besides adaptive immunity and adipokines, innate mechanisms have been involved in both diseases. Given the recent implication of group 2 innate lymphoid cells (ILC2) in asthma and adipose tissue homeostasis, and of ILC3 in obesity-associated airway hyperresponsiveness (AHR), the purpose of this study was to characterize the contribution of adaptive T helper cells versus ILC in a model of house dust mite (HDM)-induced asthma exacerbation by diet-induced obesity. Obesity was induced by a 60% high fat diet (HFD), and lean mice were fed with a low fat diet (LFD). Twelve weeks later, asthma was induced by intranasal administration of HDM. The cardinal features of experimental asthma were evaluated in combination with a flow cytometric analysis of the different adaptive or innate lymphoid population proportions and cytokine production. In lean mice, HDM induced the classical parameters of allergic asthma including increased airway hyperresponsiveness, IgE production, cell recruitment in the bronchoalveolar lavage with eosinophil, lymphocyte and neutrophil infiltrations. Histologic staining confirmed increased mucus accumulation in lungs of asthmatic mice. Feeding mice with HFD resulted in the exacerbation of some features of asthma including serum concentrations of IgE, recruitment of eosinophils in the bronchoalveolar lavage and airway hyperreactivity. Evaluation of the T cell cytokine profile in the lung protein extracts showed an exacerbation of the Th2 and Th17 profiles in obese mice compared to lean mice after HDM challenge. Moreover, the number of ILC2 and ILC3 NCR− cells was increased in HDM-challenged obese mice compared to HDM-challenged lean mice, with an increased intracellular expression of IL-13, IL-17A and IL-22. At the mRNA level, IL-33 mRNA was strongly increased in HDM-challenged obese mice as compared with HDM-challenged lean mice, suggesting that IL-33 might contribute to the induction of ILC2 in obese asthmatic mice. These results support the concept that obesity may exacerbate allergen-induced asthma by a mechanism involving in part ILC2 and ILC3.

066 Implication de CCL18 dans le développement de l’asthme allergique

Introduction L’asthme allergique est caracterise par le recrutement au niveau pulmonaire de lymphocytes de type Th2, d’eosino-philes et de basophiles, grâce a un reseau complexe de chimiokines. CCL18 est une chimiokine exprimee majoritairement au niveau pulmonaire, par les cellules presentatrices d’antigene activees par des cytokines de type Th2, ce qui suggere une possible implication dans le developpement de l’asthme allergique. Methodes Des cellules mononucleees du sang peripherique de patient sains (S) ou asthmatiques et allergiques aux acariens ont ete mises en culture en presence de l’allergene majeur de Dermatopha-goides pteronyssinus (Derp 1). La production de transcrits CCL18 par ces cellules, ainsi que sa secretion a ete evalue apres contact avec l’allergene, en presence d’anticorps neutralisants anti-IL-4, anti-IL-13 ou anti-ILlO, mais egalement apres depletion des lymphocytes T ou des monocytes. La production de CCL18 par les cellules dendritiques de la lignee myeloide ou plasmacytoide en presence de Derp 1 a egalement ete testee. Le pouvoir chimiotaxique de CCL18 a enfin ete evalue vis-a-vis des lymphocytes de type Th2, et des basophiles. Resultats Les cellules de patients asthmatiques allergiques produisent du CCL18 48 a 72 heures apres contact avec l’allergene, mais pas les cellules de patients sains. Cette production est abolie en presence d’anticorps anti-IL-4 et anti-IL-13, ou apres depletion des lymphocytes T ou des monocytes. Une partie de la production de CCL18 semble directement issue des cellules dendritiques plasmacytoides, alors que l’autre partie semble provenir des monocytes actives par l’IL-4 et l’IL-13 produits par les lymphocytes. Enfin, de grandes quantites de CCL18 ont ete trouvees dans le lavage broncho-alveolaire de patients asthmatiques allergiques non traites. Fonctionnellement, CCL18 recrute les lymphocytes Th2 et les basophiles, mais pas les lymphocytes Thl ni les eosinophiles. CCL18 est egalement capable d’induire une liberation d’histamine par les basophiles. Conclusion Ces donnees mettent en evidence de nouvelles fonctions de CCL18, suggerant une implication forte de cette chimiokine dans le developpement de la reaction d’asthme allergique.

086 Un BCG produisant de L’IL-18 inhibe la réaction pulmonaire allergique dans un modèle murin

Introduction Le parallelisme observe entre l’augmentation de la prevalence des maladies allergiques et la diminution des infections dans les pays industrialises a conduit a l’emergence du concept que l’absence de stimulation bacterienne conduirait a un defaut de tolerance vis-a-vis d’antigenes de l’environnement. Afin d’augmenter les proprietes immuno-modulatrices du BCG {Mycobacterium bovis, bacille Calmette et Guerin), nous avons construit un BCG produisant de l’IL-18 (BCG-IL-18). L’IL-18 augmente la production d’IFN-γ, mais peut egalement augmenter la reponse Th2 dans certains modeles murins de reaction allergique. Le but de notre travail etait d’evaluer in vivo l’effet du BCG-IL-18, administre apres sensibilisation, sur la reaction allergique pulmonaire. Methodes Les souris ont ete sensibilisees par deux injections intraperitoneales d’ovalbumine (OVA) et d’alum aux jours JO et J10 suivies de 3 aerosols d’OVA (J20, 21 et 22). A J23, le BCG-IL-18, comparativement au BCG non recombinant, a ete administre par voie intratracheale. L’effet de cette injection a ete analyse apres provocation antigenique (J62-63) sur differents parametres de la reaction allergique pulmonaire. Resultats Les souris sensibilisees a l’OVA developpent une hyperreactivite bronchique (HRB) et presentent une inflammation pulmonaire caracterisee par une eosinophilie dans le lavage broncho-alveolaire (LBA), un infiltrat peribronchique et une production de mucus. Le BCG et le BCG-IL-18 inhibent l’HRB et l’afflux d’eosinophiles dans le LBA et la production de mucus, mais seule l’inhibition par le BCG-IL-18 est significative. La production d’IL-5 dans le LBA est diminuee significativement alors que celle d’IFN-γ augmente faiblement apres le traitement par le BCG-IL-18. Cependant, l’infiltrat pulmonaire persiste avec le traitement par le BCG et le BCG-IL-18, bien que sa nature n’ait pas ete determinee. Enfin les taux seriques d’IgE totales, IgE, IgGl et IgG2a specifiques d’OVA ne sont pas modifies par l’injection avec le BCG ou le BCG-IL-18. Conclusions Cette etude montre que le BCG-IL-18 peut inhiber une reaction pulmonaire allergique etablie et semble avoir un potentiel immuno-modulateur plus important que le BCG. Ce modele d’inhibition pourrait se reveler interessant pour etudier les mecanismes de regulation induits par un agent microbien immuno-modulateur.

072 Développement d’une méthode de purification de la CC chimiokine PARC/CCL18 et ses dérivés par passage sur gel de silice

Introduction Dans la reponse allergique, le processus inflammatoire joue un role deletere. Parmi les mediateurs de l’inflammation les chimiokines, dont le PARC/CCL18 fait partie, semblent impliquees dans de nombreuses pathologies. Il a ete montre que le PARC etait secrete non seulement des l’intrusion de l’allergene par les cellules dendritiques, mais aussi beaucoup plus tardivement (≈ 36-48H), ce qui pourrait l’inclure dans le processus perennisant la reponse allergique. Materiels et methodes Le PARC ainsi que ses derives sont produits par un systeme eucaryote (CHO DG44). Les cellules sont cultivees sans serum de veau foetal. L’ensemble des proteines est dose a l’aide du kit Protein assay de BioRad et dune lecture de l’absorbance. Le dosage du PARC est obtenu par un dosage ELISA sandwich. Pour une resolution plus importante, les solutions sont dosees par migration electrophoretique et revelation au nitrate d’argent. La silice utilisee pour les experiences nous a ete fournie par Millipore (Granular Silice, Si-500A, 90-130 um, Matrex®). Resultats L’incubation de surnageant de culture pure sur la silice ne permet pas la fixation des proteines sur la silice. Il a donc ete necessaire de definir les conditions permettant une adsorption quantitative. Nous avons reussi a adsorber 95 % du PARC et 80 % des proteines en tamponnant le surnageant en 0,5 M NaCl, Tris 20 mM, pH 8. En ce qui concerne l’elution des proteines, un gradient de concentration de 0,1 a 5 M NaCl n’a pas permis la desorption. Apres avoir teste de nombreux eluants, nous avons decouvert qu’une solution de PolyEthylene Glycol (PEG) a 20 % permettait d’eluer selectivement une majorite des proteines totales hormis le PARC. En outre, une solution KHCO3 1 M, NaCl 0,5 M, Tris 20 mM pH 8 permet de creer un differentiel d’elution entre les proteines totales et le PARC (20 % contre 80 %). Ces resultats nous ont suggere l’etablissement d’une sequence d’elution PEG-KHCO3. L’analyse avec le nitrate d’argent du second eluat nous donne un taux d’enrichissement proteique en PARC de l’ordre de 1 000. Conclusions Cette technique nous permet de concentrer considerablement le PARC, permettant par la suite d’optimiser une purification par adsorption desorption sur Heparine-Sepharose, et finalement obtenir des concentrations en PARC suffisamment elevees pour les manipulations en laboratoire.