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Background Type-17 inflammation characterizes psoriasis, a chronic skin disease. Because several inflammatory cytokines contribute to psoriasis pathogenesis, inhibiting the simultaneous production of these cytokines in TH17 cells may be beneficial in psoriasis. We found that Cav1.4, encoded by CACNA1F, was the only Cav1 calcium channel expressed in TH17 cells. Objective We sought to investigate the role of Cav1.4 expression in early TH17-activation events and effector functions, as well as its association with TH17 signature genes in lesional psoriatic (LP) skins. Methods Transcriptional gene signatures associated with CACNA1F expression were examined in LP skins by RT-PCR and in situ hybridization. Cav1 inhibitor and/or shRNA lentivectors were used to assess the contribution of Cav1.4 in TH17 activation and effector functions in a 3-dimensional skin reconstruction model. Results CACNA1F expression correlated with inflammatory cytokine expression that characterizes LP skins and was preferentially associated with RORC expression in CD4+ and CD4− cells from LP biopsies. Nicardipine, a Cav1 channel antagonist, markedly reduced inflammatory cytokine production by TH17 cells from blood or LP skin. This was associated with decreased TCR-induced early calcium events at cell membrane and proximal signaling events. The knockdown of Cav1.4 in TH17 cells impaired cytokine production. Finally, Cav1 inhibition reduced the expression of the keratinocyte genes characteristic of TH17-mediated psoriasis inflammation in human skin equivalents. Conclusions Cav1.4 channels promote TH17-cell functions both at the periphery and in inflammatory psoriatic skin.

International audience; Background Voltage-gated calcium (Ca(v)1) channels contribute to T-lymphocyte activation. Ca(v)1.2 and Ca(v)1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. Methods We generated mice deleted for Ca(v)1.2 in T cells or Ca(v)1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Ca(v)1.2 or Ca(v)1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Ca(v)1.2 and Ca(v)1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. Results We demonstrated non-redundant and synergistic functions of Ca(v)1.2 and Ca(v)1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Ca(v)1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Ca(v)1.2 and Ca(v)1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Ca(v)1.2 and Ca(v)1.3 must be co-expressed within the same CD4(+) T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Ca(v)1.2 and Ca(v)1.3, the expression of both channels by activated CD4(+) T cells from asthmatic children was associated with increased Th2-cytokine transcription. Conclusions Thus, Ca(v)1.2 and Ca(v)1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.

Voltage-gated calcium (CaWe generated mice deleted for CaWe demonstrated non-redundant and synergistic functions of CaThus, Ca

Type-17 inflammation characterizes psoriasis, a chronic skin disease. Because several inflammatory cytokines contribute to psoriasis pathogenesis, inhibiting the simultaneous production of these cytokines in TWe sought to investigate the role of CaTranscriptional gene signatures associated with CACNA1F expression were examined in LP skins by RT-PCR and in situ hybridization. CaCACNA1F expression correlated with inflammatory cytokine expression that characterizes LP skins and was preferentially associated with RORC expression in CD4Ca

Calcium is a second messenger essential, in all cells, for most cell functions. The spatio-temporal control of changes in intracellular calcium concentration is partly due to the activation of calcium channels. Voltage-operated calcium channels are present in excitable and non-excitable cells. If the mechanism of voltage-operated calcium channels is well known in excitable cells the Ca

Asthma and allergy: what about the differences between men and women? Allergic asthma is a chronic pulmonary disease characterized by bronchial hyper responsiveness, hyper production of mucus and remodeling of the airways. Asthma, which often begins before the age of 5, is the most common chronic disease in children and affects approximately 10% of the population in affluent societies. As it is the case for many allergic diseases, asthma affects men and women differently. In childhood, pathology is more common in boys, but this trend reverses at puberty, suggesting a regulation by sex hormones. In this review, we summarize the current knowledge on how sex hormones impact allergic inflammation and particularly describe the protective actions of androgens on the development and function of key immune cell subsets involved in allergic responses.Asthme et allergie : qu’en est-il des différences entre les hommes et les femmes ? L’asthme allergique est une maladie inflammatoire chronique des voies aériennes caractérisée par une hyperréactivité bronchique, une hyperproduction de mucus et un remodelage des voies aériennes. L’asthme, qui débute souvent avant l’âge de 5 ans, est la maladie chronique la plus fréquente chez l’enfant et touche environ 10 % de la population dans les pays industrialisés. Comme c’est le cas pour de nombreuses maladies allergiques, à l’âge adulte l’asthme touche préférentiellement les femmes comparées aux hommes. Paradoxalement, la pathologie est plus fréquente chez les garçons dans l’enfance, mais ce dimorphisme s’inverse à la puberté, suggérant une régulation par les hormones sexuelles. Dans cette mise au point, nous résumerons les données de la littérature concernant le biais de sexe dans l’asthme allergique et les données expérimentales en faveur d’un rôle protecteur des androgènes, sur le développement et la fonction de cellules immunitaires clés dans le développement des réponses allergiques.

Allergic asthma is a complex disease, often characterized by an inappropriate Th2 response to normally harmless allergens. Epithelial cells damaged or activated by the allergen produce IL-33, TSLP and IL-25, activating ILC2 and dendritic cells. The latter migrate into lymph nodes where they induce Th2-cell commitment. Th2 and other type 2 innate inflammatory cells trigger inflammation and airway hyper-reactivity. The toolbox consisting of the ion channels varies from one cellular type to another and depends on its activation state, offering the possibility to design novel drugs in the field of allergy. We will discuss about some channels as calcium, nonselective cation, potassium and chloride channels that appear as good candidates in allergy.

Introduction Les canaux calciques Cav1.2 et Cav1.3, sont selectivement presents sur les lymphocytes Th2 (LTh2) murins et humains (et non sur les Th1). Ils sont essentiels in vitro pour la reponse calcique et la production des cytokines Th2 (IL-4, -5 et -13) et in vivo pour l’induction d’un asthme allergique. Nous suspectons la proteine kinase C alpha (PKCα) d’etre un partenaire pour l’activation des canaux Cav1 dans les LTh2 et donc une nouvelle cible therapeutique dans l’asthme allergique. Methodes Les LTh2 sont stimules avec le PMA (activateur des PKCs) en presence ou pas de nicardipine, un inhibiteur des canaux calciques Cav1. Apres transfections des LTh2 avec des oligonucleotides (ODN) antisens PKCα (AS-PKCα), la secretion des cytokines Th2 a ete quantifiee par ELISA dans les surnageants et le signal calcique est analyse au champ large apres stimulation du TCR. L’effet de l’AS-PKCα a ete etudie dans deux modeles d’asthme : – Les souris BALB/c recoivent de l’ovalbumine (OVA) en alum par voie intraperitoneale (i.p.) puis inhalent l’OVA avec l’AS-PKCα ou un ODN controle ; – Les LTh2 prealablement transfectes avec l’AS-PKCα sont injectees en i.v. a des souris Balb/c qui inhalent l’OVA. Les lavages bronchoalveolaires (LBA), les IgE seriques, ainsi que les lesions histologiques pulmonaires sont analyses. Resultats Le PMA induit un signal calcique et la production des cytokines Th2 dans les LTh2, qui sont inhibes par la nicardipine. L’AS-PKCα diminue in vitro le signal calcique et la production de cytokines dans les LTh2 suite a l’engagement du TCR. In vivo, le nombre total de cellules dans le LBA, le score histologique et la concentration d’IgE seriques sont diminues dans le groupe AS-PKCα, dans le modele d’asthme actif comme dans le modele passif. Conclusions L’activation directe des PKCs induit la production des cytokines Th2 de facon dependante des canaux calciques Cav1. La PKCα joue un role preponderant dans la fonction des LTh2 et in vivo dans l’induction de l’asthme allergique. Du fait d’une large surface permettant les echanges alveolocapillaires, le poumon est l’organe ideal pour tester le potentiel therapeutique de ces ODN antisens PKCα inhales dans l’inflammation de l’asthme allergique.

At the onset of adolescence, asthma becomes less prevalent in males than in females, suggesting a protective role of male sex hormones. Here, Laffont et al. show that androgens negatively control ILC2 development and ILC2-driven lung inflammation in male mice., Prevalence of asthma is higher in women than in men, but the mechanisms underlying this sex bias are unknown. Group 2 innate lymphoid cells (ILC2s) are key regulators of type 2 inflammatory responses. Here, we show that ILC2 development is greatly influenced by male sex hormones. Male mice have reduced numbers of ILC2 progenitors (ILC2Ps) and mature ILC2s in peripheral tissues compared with females. In consequence, males exhibit reduced susceptibility to allergic airway inflammation in response to environmental allergens and less severe IL-33–driven lung inflammation, correlating with an impaired expansion of lung ILC2s. Importantly, orchiectomy, but not ovariectomy, abolishes the sex differences in ILC2 development and restores IL-33–mediated lung inflammation. ILC2Ps express the androgen receptor (AR), and AR signaling inhibits their differentiation into mature ILC2s. Finally, we show that hematopoietic AR expression limits IL-33–driven lung inflammation through a cell-intrinsic inhibition of ILC2 expansion. Thus, androgens play a crucial protective role in type 2 airway inflammation by negatively regulating ILC2 homeostasis, thereby limiting their capacity to expand locally in response to IL-33.

Resume L’asthme est une pathologie touchant plus de 4 millions de personnes en France. Le role des lymphocytes Th2 (LTh2) produisant de l’interleukine (IL)-4, de l’IL-5 et de l’IL-13 dans la physiopathologie de l’asthme allergique n’est plus a prouver. Les differentes populations de LT expriment des equipements distincts en composants regulant la [Ca2+]i. La comprehension de la signalisation calcique propre a chaque population de LT permettrait de concevoir des approches therapeutiques specifiques utiles pour le traitement des differentes maladies dans lesquelles elles sont impliquees, telles que l’asthme allergique pour les LTh2, sans engendrer d’immunosuppression globale. Notre equipe a demontre le role essentiel des canaux calciques Cav1.2 et Cav1.3 et de leurs sous-unites auxiliaires dans la fonction des LTh2 humains et murins et le developpement de l’asthme allergique chez la souris. Nous discuterons des potentialites therapeutiques qu’apporte l’identification d’une voie de signalisation calcique propre aux LTh2. Nous proposerons de cibler selectivement cette reponse calcique Th2 pour court-circuiter la reponse inflammatoire chronique caracteristique des maladies allergiques en evitant une immunosuppression globale puisque ces canaux sont exprimes selectivement dans les LTh2.

Calcium is a second messenger essential, in all cells, for most cell functions. The spatio-temporal control of changes in intracellular calcium concentration is partly due to the activation of calcium channels. Voltage-operated calcium channels are present in excitable and non-excitable cells. If the mechanism of voltage-operated calcium channels is well known in excitable cells the Ca2+ toolkit used in non-excitable cells to activate the calcium channels is less described. Herein we discuss about very similar pathways involving voltage activated Cav1 channels in two unrelated non-excitable cells; ectoderm cells undergoing neural development and effector Th2 lymphocytes responsible for parasite elimination and also allergic diseases. We will examine the way by which these channels operate and are regulated, as well as the consequences in terms of gene transcription. Finally, we will consider the questions that remain unsolved and how they might be a challenge for the future.

International audience; BACKGROUND: T-lymphocytes express not only the cell membrane calcium ORAI1 but also voltage-dependent Cav1 channels. In excitable cells, these channels are composed of the ion forming pore α1 and auxiliary subunits (β and α2δ) needed for proper trafficking and activation of the channel. We previously disclosed the role of Cav1.2 α1 in mouse and human Th2- but not Th1-cell functions and showed that knocking-down Cav1 α1 prevents experimental asthma OBJECTIVE: We investigated the role of β and α2δ auxiliary subunits on Cav1 α1 function in Th2 lymphocytes and on the development of acute allergic airway inflammation. METHODS: We used antisense oligonucleotides (CavβAS) to knockdown Cavβ and gabapentin, a drug that binds to and inhibits α2δ1 and α2δ2, to test their effects on Th2 functions and their capacity to reduce allergic airway inflammation. RESULTS: Mouse and human Th2-cells express mainly Cavβ1, β3 and α2δ2 subunits. CavβAS reduces TCR-driven calcium responses and cytokine production by mouse and human Th2, with no effect on Th1-cells. Cavβ is mainly involved in restraining Cav1.2 α1 degradation through the proteasome as a proteasome inhibitor partially restores the α1 protein level. Gabapentin impairs TCR-driven calcium response and cytokine production associated with the loss of α2δ2 protein in Th2-cells. CONCLUSIONS: These results stress the role of Cavβ and α2δ2 auxiliary subunits in the stability and activation of Cav1.2 channels in Th2 lymphocytes both in vitro and in vivo as demonstrated by the beneficial effect of CavβAS and gabapentin in allergic airway inflammation. CLINICAL IMPLICATIONS: The demonstration that auxiliary subunits are involved in calcium signaling through Cav1 channels and function of mouse and human Th2-lymphocytes supports their potential beneficial effect on allergic asthma.

T lymphocytes express not only cell membrane ORAI calcium release-activated calcium modulator 1 but also voltage-gated calcium channel (CaWe investigated the role of β and α2δ auxiliary subunits on CaWe used CaMouse and human TThese results stress the role of Ca

Resume Les lymphocytes T effecteurs incluent les lymphocytes Th1, Th2, Th17… qui produisent des cytokines et ont des fonctions differentes. Les Th2 producteurs d’(IL)-4, 5 et 13 permettent l’elimination des parasites, mais peuvent causer l’asthme allergique. Mise a part l’immunotherapie qui n’est pas generalisee, les traitements de l’asthme sont symptomatiques et de nouvelles pistes therapeutiques sont indispensables. La concentration de calcium intracellulaire ([Ca]i) est etroitement regulee dans les lymphocytes. Les variations de la [Ca]i controlent la majorite des fonctions lymphocytaires. Pourtant cette regulation differe selon le type de lymphocytes rendant imaginable de cibler une sous-population de lymphocytes impliquee dans une pathologie donnee. Les canaux calciques qui font entrer le Ca2+ dans la cellule sont des acteurs importants de la regulation de la [Ca]i comprenant classiquement les canaux ORAI1 mais aussi les canaux Cav1 (v pour dependant du voltage) codes par 4 genes de Cav1.1 a Cav1.4. Les canaux Cav1.2 et a un moindre degre Cav1.3 sont exprimes par les Th2 murins. L’inhibition de l’une ou l’autre de ces isoformes reduit considerablement le signal calcique et la production de cytokines par ces lymphocytes. Un melange d’oligonucleotides anti-sens ciblant chacune de ces isoformes previent l’asthme experimental. Nous avons aussi montre la selectivite d’expression de Cav1.2 dans les lymphocytes Th2 humains obtenus a partir de donneurs de sang. Notre but est maintenant de determiner le niveau d’expression des canaux Cav1 dans les lymphocytes de sujets asthmatiques et de determiner si ces canaux sont detectables dans les cellules inflammatoires chez les sujets asthmatiques, ce qui en ferait une cible therapeutique interessante dans l’asthme. Un autre objectif est d’identifier la voie de signalisation impliquant les canaux Cav1, ce qui pourrait nous permettre de pointer des molecules-cles les plus selectives possibles des Th2 dans une optique therapeutique.

Resume L’asthme allergique est une maladie inflammatoire pulmonaire chronique qui pourrait etre initiee par des lymphocytes T auxiliaires (Th) de type 2 (Th2) producteurs d’interleukine (IL)-4, 5 et 13. Toutefois, d’autres populations de Th comme les Th17 et les Th9 pourraient participer a la pathologie dans certaines formes d’asthme. L’augmentation de la concentration intracellulaire de Ca 2+ ([Ca]i) est necessaire a la plupart des fonctions des lymphocytes. Toutefois la signature definie par l’amplitude, la frequence et la localisation des variations de [Ca]i n’est pas la meme dans les sous-populations de lymphocytes ce qui peut interferer avec le type de genes induits et les fonctions lymphocytaires. Nous avons identifie des canaux calciques comme selectivement surexprimes dans les Th2 et dont l’inhibition abroge le developpement d’asthmes experimentaux sans immunosuppression globale. Nous discuterons des potentialites therapeutiques qu’apporte l’identification d’une voie de signalisation propre aux Th2 et peut-etre a d’autres cellules inflammatoires typiques de l’asthme.

Calcium signaling is essential for all the functions of T lymphocytes, including those of Th2 cells. Th2 lymphocytes producing interleukins 4, 5 and 13 orchestrate allergic diseases including asthma. T-cell activation induces an influx of Ca(2+) from the external medium through ORAI calcium channels although other calcium channels are likely to be involved. Among them, voltage-gated calcium (Ca(v)1) channels have been reported in some T-cell subsets including Th2 cells. The inhibition of Ca(v)1 channels abrogates T-cell receptor-driven calcium influx and interleukin production by Th2 cells. From a therapeutic point of view, the inhibition of Ca(v)1 channels prevents Th2-dependent experimental allergic asthma. In this review, we will discuss the singularities of calcium responses depending upon the T-cell subset and its state of activation.

Resume L’asthme allergique est une maladie inflammatoire pulmonaire ou les lymphocytes (L) Th2 jouent un role-cle. Par leur secretion d’interleukines (IL)-4, 5 et 13, ils participent a la reponse inflammatoire en permettant le recrutement et l’activation des eosinophiles et la production d’immunoglobulines IgE par les lymphocytes B (LB). Une hypothese rendant compte du developpement de la reponse Th2 dans l’asthme implique la production de cytokines lymphopoietine stromale thymique (TSLP), IL-25 et IL-33 par l’epithelium bronchique qui favoriseraient la differenciation des LTh2. Il est largement admis que l’engagement du recepteur T pour l’antigene (TCR) induit une liberation du calcium contenu dans le reticulum endoplasmique (RE), entrainant l’entree de calcium via les canaux Orai et probablement d’autres canaux calciques a la membrane plasmique. En consequence, le facteur de transcription NFAT ( Nuclear Factor of Activated T cells ) se localiserait dans le noyau, permettant la production de cytokines. Notre groupe a montre que les canaux calciques dependant du voltage (Ca v 1), normalement presents dans les cellules excitables, sont selectivement exprimes par les LTh2. La transfection de LTh2 avec des oligonucleotides antisens specifiques des canaux (Ca v 1AS) reduit l’expression des canaux, l’augmentation de la concentration calcique intracellulaire et la production de cytokines induites par engagement du TCR. De plus, l’inhalation de Ca v 1AS previent le developpement d’un asthme experimental chez la souris. Nous discutons des possibilites permettant d’expliquer le couplage entre le TCR et l’ouverture des canaux Ca v 1 dans les LTh2. Le decryptage de ces voies de signalisation pourrait aboutir a l’dentification de nouvelles cibles dans le traitement de l’asthme (canaux Ca v 1 et possiblement molecules les regulant).

Brown Norway rats injected with aurothiopro-panolsulfonate sodium salt develop systemic autoimmunity. The aim of this study was to assess the influence of the sulfur-containing group in this experimental model of gold-induced autoimmunity. It was shown that the sulfur-containing group does not induce autoimmunity of itself, but potentiates the immunotoxic effects of gold.

Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions.Because dihydropyridines bind to and modulate voltage-dependent calcium (Ca(v)1) channel in excitable cells, we aimed at showing that Th2 cells selectively express functional Ca(v)1-related channels, the inhibition of which may prevent asthma.We looked for Ca(v)1 channel expression in Th2 and Th1 cells by real-time polymerase chain reaction and Western blotting. We sequenced the isoforms expressed by Th2 cells and tested whether Ca(v)1 antisense oligodeoxynucleotides (Ca(v)1AS) affected Ca(2+) signaling and cytokine production. Finally, we tested the effect of Ca(v)1AS in the passive asthma model by injection of ovalbumin-specific Th2 cells transfected with Ca(v)1AS into BALB/c mice challenged with intranasal ovalbumin and in the active model of asthma by intranasal delivery of Ca(v)1AS together with soluble ovalbumin in BALB/c mice previously immunized with ovalbumin in alum.We show that mouse Th2 but not Th1 cells expressed Ca(v)1.2 and Ca(v)1.3 channels. Th2 cells transfected with Ca(v)1AS had impaired Ca(2+) signaling and cytokine production, and lost their ability to induce airway inflammation on adoptive transfer. Furthermore, intranasal administration of Ca(v)1AS suppressed airway inflammation and hyperreactivity in an active model of asthma.These results indicate that Th2 cells selectively express Ca(v)1 channels that may be efficiently targeted in T lymphocytes to prevent experimental asthma.

Asthma is a chronic airway disease resulting from inappropriate Th2-cell biased activation. Interleukin (IL)-4, IL-5 and IL-13 produced by Th2 cells contribute to the inflammatory process. Attempts for inhibiting interleukin-4 or IL-5 gave disappointing results. The simultaneous inhibition of several Th2-cytokines could be a more promising issue. Several arguments support the concept that Th2-cells express selective markers that could be targeted in asthma. Our group showed that Th2-cells selectively up-regulated dihydropyridine-sensitive Ca(2+) (DHP-Ca) channels essential for Ca(2+) signaling and type-2 cytokine production. Indeed, DHP-Ca antagonist effectively prevented or even reverted airway inflammation, airway remodeling and airway hyperresponsiveness in experimental models of asthma. Although it remains to be formally demonstrated that human lung infiltrating T-lymphocytes in asthmatic patients express DHP- Ca(2+) channels, we hypothesize that targeting DHP-Ca channels in T-lymphocytes could represent an efficient strategy in the treatment of asthma.This review article also discussed patents relevant to the field.

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.

Les lymphocytes T CD4+ sont heterogenes en termes de fonctions et de production de cytokines. Les lymphocytes Th1 produisent de l’IL-2 et de l’interferon IFNy, et sont impliques dans l’elimination des organismes pathogenes intracellulaires. Au contraire, les lymphocytes Th2 produisent de l’IL-4 et de l’IL-5 et contribuent a l’elimination des helminthes. Ces sous-populations peuvent deriver d’un precurseur commun: la presence d’IL-12 et d’anticorps anti-IL-4 promeut la differenciation des lymphocytes Th1 tandis que la presence d’IL-4 et d’anti corps anti-IFNg favorise le developpement de lymphocytes Th2. La stimulation du TCR active une proteine kinase C qui controle une entree de calcium via des canaux de type L dans les lymphocytes Th2. La differenciation des lymphocytes Th2 mais pas celle des lymphocytes Th1 s’accompagne de l’expression des canaux L. Enfin, un inhibiteur des canaux L a ete utilise avec succes pour traiter une maladie autoimmune due a une activation exageree des lymphocytes Th2 chez le Rat.

Summary: The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune-mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases while BN rats are highly susceptible to Th2-mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC-dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC-independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL-4, IL-13, IL-10 and TGF-β in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1-mediated experimental autoimmune encephalomyelitis, and to Th2-mediated disorders triggered by gold salts as well as the difference in the CD45RChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

It is clear that functional heterogeneity of T cells may be explained by differential cytokine production. The aim of this paper was to review evidence for regulatory cells, generated after HgCl(2)-exposure. They differ from classical Th1 and Th2 cells, produce transforming growth factor-beta and interleukin-10 and exert their regulatory functions in a Th1/Th2-unrestricted fashion.

Gold salts are beneficial in the treatment of rheumatoid arthritis but may induce immune-mediated disorders in predisposed patients. Gold salts induce Th2-dependent autoimmunity in Brown-Norway (BN) rats but not in Lewis (LEW) rats. The aim of this study was to define molecular targets of gold salts and to approach why LEW rats are resistant. Gold salts act on early steps of transduction in T cells from BN and LEW rats since they trigger tyrosine phosphorylation of numerous proteins including p56(lck) and a calcium signal which results in IL-4 and IFN-gamma expression by BN and LEW T cells. However, the IL-4 response was favored in BN spleen cells in vitro and in vivo. IFN-gamma, produced in part by CD8(+) cells, contributes to the resistance of LEW rats since gold salt-injected LEW rats receiving anti-CD8 or anti-IFN-gamma mAb displayed the parameters characteristics of gold salt-induced Th2 autoimmunity although to a lesser extent than in BN rats. Gold salts transduce a signal in BN and LEW spleen cells resulting in IL-4 and IFN-gamma gene transcription with a preferential IL-4 response in BN rats, a Th2-prone strain, while IFN-gamma contributes to the resistance of LEW rats.

Th1 and Th2 cells produce different cytokines and have distinct functions. Th1/Th2 cell differentiation is influenced, among other factors, by the nature of TCR-MHC interactions. However, how the TCR transduces a signal resulting in IFN-gamma or IL-4 production is a matter of debate. For example, some authors reported a loss of calcium signaling pathway in Th2 cells. We used a T cell hybridoma producing IL-4 upon weak TCR stimulation and both IL-4 and IFN-gamma for strong TCR engagement as a model to study how TCR signaling pathways are differentially activated in both conditions of stimulation and how this influences the production of cytokines. We show that: (1) the calcium response is identical following weak and strong TCR stimulation; (2) mitogen-activated protein kinase(MAPK) activation is a gradual phenomenon depending upon the strength of TCR activation; (3) a calcium response, even weak, triggers IL-4 expression; (4) IFN-gamma synthesis requires not only a calcium response but also MAPK activation. The MAPK pathway is dispensable for IL-4 production, although it amplifies IL-4 synthesis upon strong TCR stimulation; (5) TCR-induced IL-4 production also depends on calcium signaling in Th2 cells, while IFN-gamma synthesis is dependent, in addition, on MAPK activation in Th1 cells.

Les lymphocytes T CD4+ sont heterogenes en termes de fonctions et de production de cytokines. Les lymphocytes Thl produisent de l’IL-2 et de l’IFNγ et sont impliques dans l’elimination des agents pathogenes intracellulaires. Au contraire, les lymphocytes Th2 produisent de l’IL-4 et de l’IL-5 et contribuent a l’eradication des helminthes. Cet article decrit les voies de signalisation activees apres stimulation via le recepteur T pour l’antigene (TCR) et tente de comprendre lesquelles interviennent dans la synthese de telle ou telle cytokine. Une nouvelle voie impliquee dans la production d’IL-4 est decrite. Elle couple le TCR a une PKC qui controle une entree de calcium via des canaux calciques sensibles a la dihydropyridine, vraisemblablement apparentes aux canaux de type L des cellules excitables. Ce signal calcique est suffisant pour initier la transcription d’IL-4. Au contraire, la production d’IFNγ requiert absolument l’activation des MAP-kinases.

> La reconnaissance du peptide antigenique par son recepteur (TCR) entraine une augmentation de la concentration de Ca2+ dans le cytosol ([Ca2+]i), necessaire a la differenciation et aux fonctions effectrices des lymphocytes (L)T. La stimulation via le TCR permet le recrutement de molecules adaptatrices et le couplage a des enzymes, dont la phospholipase Cγ, qui produit de l’inositol 1,4,5 tri-phosphate (IP3) et du diacylglycerol. L’IP3 en se fixant a ses recepteurs du reticulum endoplasmique libere les stocks de Ca2+ dans le cytosol (Figure 1). L’entree de Ca2+ a partir du milieu extracellulaire reconstitue ces stocks et maintient une signalisation soutenue. Les canaux calciques responsables sont definis comme des SOC (store-operated Ca2+ channels) mais leur identite moleculaire n’est pas completement elucidee. Un des principaux facteurs de transcription active par la voie calcique est NFAT (nuclear factor of activated T cells). La calcineurine, une phosphatase regulee par le Ca2+ dephosphoryle NFAT, ce qui permet sa localisation nucleaire et l’expression des genes cibles.

Mercuric chloride (HgCl2) induces T helper 2 (Th2) autoreactive anti-class II T cells in Brown Norway rats. These cells produce interleukin (IL)-4 and induce a B cell polyclonal activation that is responsible for autoimmune disease. In Brown Norway rats, HgCl2 triggers early IL-4 mRNA expression both in vivo and in vitro by T cells, which may explain why autoreactive anti-class II T cells acquire a Th2 phenotype. The aim of this study was to explore the transduction pathways by which this chemical operates. By using two murine T cell hybridomas that express IL-4 mRNA upon stimulation with HgCl2, we demonstrate that: 1) HgCl2 acts at the transcriptional level without requiring de novo protein synthesis; 2) HgCl2 induces a protein kinase C-dependent Ca2+ influx through L-type calcium channels; 3) calcium/calcineurin-dependent pathway and protein kinase C activation are both implicated in HgCl2-induced IL-4 gene expression; and 4) HgCl2 can activate directly protein kinase C, which might be one of the main intracellular target for HgCl2. These data are in agreement with an effect of HgCl2 which is independent of antigen-specific recognition. It may explain the T cell polyclonal activation in the mercury model and the expansion of pathogenic autoreactive anti-class II Th2 cells in this context.

Autoreactive anti–MHC class II T cells are found in Brown Norway (BN) and Lewis (LEW) rats that receive either HgCl2 or gold salts. These T cells have a T helper cell 2 (Th2) phenotype in the former strain and are responsible for Th2-mediated autoimmunity. In contrast, T cells that expand in LEW rats produce IL-2 and prevent experimental autoimmune encephalomyelitis, a cell-mediated autoimmune disease. The aim of this work was to investigate, using T cell lines derived from HgCl2-injected LEW rats (LEWHg), the effect of these autoreactive T cells on the development of Th2-mediated autoimmunity. The five LEWHg T cell lines obtained protect against Th2-mediated autoimmunity induced by HgCl2 in (LEW × BN)F1 hybrids. The lines produce, in addition to IL-2, IFN-γ and TGF-β, and the protective effect is TGF-β dependent since protection is abrogated by anti-TGF-β treatment. These results identify regulatory, TGF-β–producing, autoreactive T cells that are distinct from classical Th1 or Th2 and inhibit both Th1- and Th2-mediated autoimmune diseases.

The role of voltage-dependent calcium (Cav1) channels is prominent in excitable cells while store-operated calcium channels (SOCC) were considered as characteristic of non-excitable cells. Cav1 channels are implicated in excitation transcription. Store-operated calcium channels (SOCC) activity is increased during cardiac stress and would contribute to Ca2+ influx and expression of genes responsible for cardiac hypertrophy and heart failure (Luo et al., 2012). Several lines of evidence now show the importance of Cav1 channels in non-excitable cells including lymphocytes (reviewed in Robert et al., 2011, 2013). Cav1 channels are defined by their voltage sensitivity and their sensitivity to drugs as dihydropyridines, phenylalkylamines, benzothiazepines, known to alter T-cell functions. However the drug concentrations needed were higher compared to excitable cells. The absence of cell membrane depolarization upon activation and possible non-specific effects of the drugs questioned the putative role of Cav1 channels in T-cells. Cav1 channels are formed by the ion forming pore α1 subunit encoded by four genes conferring some tissue-specific expression pattern in excitable cells. Cav1.1 is characteristic of skeletal muscle cells. Cav1.2 is found in neurons, heart, and smooth muscle cells while Cav1.3 is detected in neuroendocrine cells. Cav1.2 and Cav1.3 can be found in the same tissues even if their role is not redundant as shown by the differential phenotypes of Cav1.2 and Cav1.3 null mice. Cav1.4 is the retinal form. Cav1 channel isoforms differ by their sensitivity to depolarization and to antagonizing drugs such as dihydropyridines (DHP) as well as by their inactivation properties (Lipscombe et al., 2004). For example, Cav1.4 channels activate at more negative potentials than Cav1.3 and Cav1.2, which highlights the potential involvement of Cav1.4 in non-excitable cells as mast cells (McRory et al., 2004) and more recently in mouse T-lymphocytes (Omilusik et al., 2011).

CD4+ helper T (Th) lymphocytes orchestrate the immune response and include several types of effectors such as Th1, Th17 and Th2 cells. They fight against intracellular, extracellular pathogens and parasites respectively. They may also cause distinct immunopathological disorders. Th1 and Th17 are implicated in the development of autoimmune diseases while Th2 cells can initiate allergic diseases. These subsets differ by their TCR-associated signaling. In addition, the regulation of intracellular calcium concentration is not the same in Th1, Th2 and 17 cells. Our group showed that Th2 cells selectively overexpressed voltage-activated calcium (Cav1)-related channels. An increasing number of groups report the presence of Cav1-related products in T-lymphocyte subsets. This is a matter of debate since these calcium channels are classically defined as activated by high cell membrane depolarization in excitable cells. However, the use of mice with ablation of some Cav1 subunits shows undoubtedly an immune phenotype raising the question of how Cav1 channels are regulated in lymphocytes. We showed that knocking down Cav1.2 and/or Cav1.3 subunits impairs the functions of Th2 lymphocytes and is beneficial in experimental models of asthma, while it has no effect on Th1 cell functions. Beyond the role of Cav1 channels in T-lymphocytes, the identification of key components selectively implicated in one or the other T cell subset paves the way for the design of new selective therapeutic targets in the treatment of immune disorders while preserving the other T-cell subsets. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Calcium signaling is essential for all the functions of T lymphocytes, including those of Th2 cells. Th2 lymphocytes producing interleukins 4, 5 and 13 orchestrate allergic diseases including asthma. T-cell activation induces an influx of Ca(2+) from the external medium through ORAI calcium channels although other calcium channels are likely to be involved. Among them, voltage-gated calcium (Ca(v)1) channels have been reported in some T-cell subsets including Th2 cells. The inhibition of Ca(v)1 channels abrogates T-cell receptor-driven calcium influx and interleukin production by Th2 cells. From a therapeutic point of view, the inhibition of Ca(v)1 channels prevents Th2-dependent experimental allergic asthma. In this review, we will discuss the singularities of calcium responses depending upon the T-cell subset and its state of activation.

Effector CD4(+) T lymphocytes generated in response to antigens produce endogenous opioids. Thus, in addition to their critical role in host defenses against pathogens, effector CD4(+) T lymphocytes contribute to relieving inflammatory pain. In this study, we investigated mechanisms of opioid release by antigen-experienced effector CD4(+) T cells that leave draining lymph nodes and come back into the inflammatory site. Effector antigen-primed CD4(+) T lymphocytes generated in vitro were intravenously injected into nude mice previously immunized with either cognate or irrelevant antigens in complete Freund adjuvant (CFA). CFA-induced mechanical hyperalgesia was only reduced in mice immunized with cognate antigen. Thus, antinociceptive activity of effector CD4(+) T cells requires the presence of the antigen for which they are specific within the inflammatory site. Accordingly, analgesia was inhibited by neutralizing cognate T cell receptor-mediated interaction between effector CD4(+) T lymphocytes and antigen-presenting cells at the site of inflammation. Analgesia was observed by transferring effector CD4(+) T lymphocytes with Th1 or Th2 phenotype, suggesting that antinociceptive activity is a fundamental property of effector CD4(+) T lymphocytes irrespective of their effector functions. Based on the use of agonists and antagonists selective for each of the opioid receptor subclasses, we showed that analgesia induced by T cell-derived opioids is elicited via activation of δ-type opioid receptors in the periphery. Thus, the antinociceptive activity is a fundamental property associated with the effector phase of adaptive immunity, which is driven by recognition of the cognate antigen by effector CD4(+) T lymphocytes at the inflammatory site.

Calcium signalling is essential for most of the biological T-cell activities, including in Th2 lymphocytes, a T-cell subset that produce interleukin 4, 5 and 13 and which is involved in allergic diseases. T-cell receptor engagement induces the production of inositol trisphosphate that binds to its receptor, releasing intracellular Ca(2+) stores. STIM in the endo (sarco) plasmic reticulum (ER/SR) is a Ca(2+) sensor that perceives the depletion of intracellular Ca(2+) stores, localizes near the cell membrane and allows the activation of ORAI, the main calcium channels at the cell membrane. However, other calcium channels at the membrane of intracellular compartments and at the cell membrane can also contribute to the TCR-driven intracellular Ca(2+) rise. Among them, voltage-dependent calcium (Ca(v)1) channels have been reported in several types of T-lymphocytes, although how they are gated in these non-excitable cells remains unsolved. We have shown that Cav1 channel expression was selectively up regulated in Th2 lymphocytes. In this review, we will discuss about the diversity of the Ca(2+) channels responsible for Ca(2+) homeostasis in the different cell subsets and the interactions between these molecules, which can account for the variety of the calcium responses depending upon the functions of effector T-cells.

Subcutaneous injections of mercuric chloride induce an autoimmune glomerulonephritis with both granular and linear IgG deposits along the glomerular capillary wall and proteinuria. This disease is due to a T cell dependent polyclonal B cell activation responsible for production of antibodies against self (glomerular basement membrane, immunoglobulins, DNA, myeloperoxydase) and non self (sheep red blood cells, trinitrophenol (TNP)) components. Increase in serum IgE concentration is the hallmark of this disease. To determine if mercury vapours have pathogenic effects is an important problem of public health. The aim of this study was, first to compare the effects of mercury vapour exposure to those of mercury injections and, second, to compare the effects of high doses to those of low doses of mercury. Two exposure levels were studied corresponding to a mercury absorption of 13.1 mumol/week per kg body wt. and 1.7 mumol/week per kg body wt. during a 5-week period. It will be shown that, whereas the mercury concentration in the kidneys was similar in injected--and vapour exposed--rats, the mercury concentration in blood at the end of the exposure was about twice as high in the injected animals. Blood concentration of mercury was related to dose level but kidney content of mercury was similar in all groups, in spite of a dose difference by a factor of seven between low and high exposure. Mercury vapour and HgCl2 injections both trigger autoimmunity to the same extent and, in both cases the extent of autoimmune manifestations was dose-dependent.

Brown-Norway (BN) rats injected with HgCl2 develop a systemic autoimmune disease associated with a polyclonal B cell activation, due to autoreactive T cells specific for self-class II molecules, while Lewis (LEW) rats injected with HgCl2 do not exhibit autoimmunity and develop a non-antigen-specific, CD8-mediated immunosuppression assessed by a depression of T cell functions, and a protection against experimental autoimmune encephalomyelitis (EAE). Resistance to HgCl2-induced autoimmunity is not due to these suppressor cells since treatment with an anti-CD8 monoclonal antibody (mAb) did not allow autoimmunity to appear. The absence of autoimmunity in this strain could result from the absence of autoreactive T cells, or from quantitative or qualitative differences of these cells between susceptible and resistant strains. In the present study, we show that CD4+ anti-class II T cells are present in HgCl2-injected LEW rats and are as frequent as in BN rats when assessed by limiting dilution analysis. LEW CD4+ autoreactive T cell lines were derived. They proliferated in the presence of normal class II-bearing cells, secreted interleukin 2, and did not induce B cells to produce immunoglobulins. Transfer of one of these lines, LEW Hg A, into normal LEW rats led to the appearance of CD8+ cells responsible for a non-antigen-specific immunosuppression that induced complete protection from EAE. Immunosuppression was abrogated after treatment with an anti-CD8 mAb. In vitro, CD8+ cells from rats injected with the LEW Hg A T cell line proliferated in the presence of activated T cells whatever their origin. We conclude that HgCl2 induces CD4+ autoreactive T cells that proliferate in the presence of class II+ cells in susceptible BN as well as in resistant LEW rats. But while these cells collaborate with B cells to produce autoantibodies in BN rats, they initiate in LEW rats a suppressor circuit involving antiergotypic CD8+ suppressor cells.

Spontaneously hypertensive (SH) rats immunized with mouse renin produce anti-renin antibodies, responsible for down-modulation of blood pressure, associated with an infiltration of kidneys by mononuclear cells. In this work, anti-renin T cells from SH rats immunized with renin have been stimulated in vitro, and we have studied in vitro and in vivo their effect on anti-renin antibody production by normal syngeneic B cells. We show that, in vitro, renin-activated T cells induce a renin-specific antibody response without addition of exogenous renin. Anti-renin T cells injected into naive SH rats trigger normal B cells to secrete high amounts of monospecific anti-renin IgG antibodies as early as day 5. These antibodies interfere with the homeostasis of the renin-angiotensin system leading to the normalization of blood pressure without any nephritis. These results show that anti-renin B cells are either not tolerant per se or in a reversible state of anergy. Our results also suggest that anti-renin B cells constitutively express renin-derived peptides in such a way that they may be stimulated by activated anti-renin T cells; these cells express IL-4 mRNA indicating that IL-4 could play a role in the differentiation of B cells.

International audience; Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor α expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor α, exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS.

In the companion paper (J. Rossert et al., Cell. Immunol., 137, 1991), we showed by using limiting dilution analysis that Lewis (LEW) rats injected with HgCl2 and immunized with myelin ( LEWHg MYE ) exhibit anti-basic protein CD4+ T helper cells (Th), at least 10-fold more frequent CD8+ T suppressor cells (Ts), and T contrasuppressor cells (Tcs). These Tcs cells were shown to be CD4+ T cells adhering to Vicia villosa (VV) lectin and allowed Th cells to proliferate despite the presence of Ts cells. The CD8+ Ts cells might be responsible for the protection from experimental allergic encephalomyelitis (EAE) observed in about 70% of LEW rats injected with HgCl2. The concomitant presence of CD4+ Tcs cells might explain that 30% of the rats escaped this protection. The aim of this work is to demonstrate in vivo the roles of CD8+ Ts cells and Tcs cells in mercury-induced protection from EAE. It will be shown that LEWHg/MYE rats depleted of CD8+ cells as well as LEWHg/MYE rats transferred with VV lectin-adherent Tcs cells develop EAE. These data demonstrate that CD8+ Ts cells are responsible for HgCl2-induced protection and that Tcs cells are involved in the control of Ts cells in vivo.

Mercuric chloride (HgCl 2 ) induces in Lewis (LEW) rats a non-antigen-specific immuno-suppression and is able to down-modulate experimental allergic encephalomyelitis in about 70% of the rats. The aim of the present study was to determine the frequencies of lymph node cells involved in the proliferative response to myelin basic protein in rats injected with HgCl 2 and immunized with myelin by using limiting dilution analysis (LDA). Highly frequent CD8+ T suppressor cells and at least 10-fold less frequent protein basic-specific T helper cells were detected in these rats. A third cell type allowing the proliferative response of Th cells in spite of Ts cells was also demonstrated. These cells, which could act as contrasuppressor cells, were CD4+ and adhered to Vicia villosa lectin; their frequency was in the same range as that of T helper cells. These data illustrate the potential role of different levels of T cell immunoregulatory activity in autoimmunity and the major interest of LDA in their analysis.

This unit describes methods for inducing autoimmune disease in Brown Norway rats through HgCl(2) injections as well for assessing parameters that characterize the disease by serum IgE concentration assays, anti-laminin antibody measurement, and renal immunofluorescence studies to detect autoantibodies. Also covered are disease induction using autoreactive CD4(+) T(H)2 anti-self MHC class II molecules and preparation of T cell lines. IL-4 is produced very early after the first HgCl(2) injection (beginning at day 3, peaking at day 14, and continuing up to day 30). Thus, IL-4 mRNA expression may be detected in spleen and lymph nodes from HgCl(2)-injected BN rats. The fact that HgCl(2) induces in vitro mRNA IL-4 gene expression in normal BN T cells but not in LEW T cells is probably crucial to susceptibility to the development of autoimmunity in the sense that it may condition the development of autoreactive T cells into pathogenic T(H)2 cells; a test for this condition is therefore also included.