Stéphanie Sénéchal

Involvement of CCL18 in Allergic Asthma

Allergic asthma is associated with a pulmonary recruitment of Th type 2 cells, basophils, and eosinophils, mainly linked to chemokine production. CCL18 is a chemokine preferentially expressed in the lung, secreted by APCs, induced by Th2-type cytokines, and only present in humans. Therefore, CCL18 may be involved in allergic asthma. PBMC from asthmatics allergic to house dust mite cultured in the presence of Dermatophagoides pteronyssinus 1 (Der p 1) allergen secreted CCL18, 48 and 72 h after stimulation, whereas those from healthy donors did not. Part of CCL18 was directly derived from Der p 1-stimulated plasmacytoid dendritic cells, whereas the other part was linked to monocyte activation by IL-4 and IL-13 produced by Der p 1-stimulated T cells. In bronchoalveolar lavages from untreated asthmatic allergic patients, CCL18 was highly increased compared with controls. Functionally, CCL18 preferentially attracted in vitro-polarized Th2 cells and basophils, but not eosinophils and Th1 cells, and induced basophil histamine and intracellular calcium release. These data show a new function for CCL18, i.e., the recruitment of Th2 cells and basophils, and suggest that CCL18 may play a predominant role in allergic asthma.

Diesel Exposure Favors Th2 Cell Recruitment by Mononuclear Cells and Alveolar Macrophages from Allergic Patients by Differentially Regulating Macrophage-Derived Chemokine and IFN-γ-Induced Protein-10 Production

Diesel exhausts and their associated organic compounds may be involved in the recent increase in the prevalence of allergic disorders, through their ability to favor a type 2 immune response. Type 2 T cells have been shown to be preferentially recruited by the chemokines eotaxin (CCL11), macrophage-derived chemokine (MDC, CCL22), and thymus activation-regulated chemokine (CCL17) through their interaction with CCR3 and CCR4, respectively, whereas type 1 T cells are mainly recruited by IFN-γ-induced protein-10 (CXCL10) through CXCR3 binding. The aim of the study was to evaluate the effect of diesel exposure on the expression of chemokines involved in type 1 and 2 T cell recruitment. PBMC and alveolar macrophages from house dust mite allergic patients were incubated with combinations of diesel extracts and Der p 1 allergen, and chemokine production was analyzed. Diesel exposure alone decreased the constitutive IP-10 production, while it further augmented allergen-induced MDC production, resulting in a significantly increased capacity to chemoattract human Th2, but not Th1 clones. Inhibition experiments with anti-type 1 or type 2 cytokine Abs as well as cytokine mRNA kinetic evaluation showed that the chemokine variations were not dependent upon IL-4, IL-13, or IFN-γ expression. In contrast, inhibition of the B7:CD28 pathway using a CTLA-4-Ig fusion protein completely inhibited diesel-dependent increase of allergen-induced MDC production. This inhibition was mainly dependent upon the CD86 pathway and to a lesser extent upon the CD80 pathway. These results suggest that the exposure to diesel exhausts and allergen may likely amplify a deleterious type 2 immune response via a differential regulation of chemokine production through the CD28 pathway.

Chemokine-induced cutaneous inflammatory cell infiltration in a model of Hu-PBMC-SCID mice grafted with human skin.

Recently, certain chemokines and chemokine receptors have been preferentially associated with the selective recruitment in vitro of type 1 T cells, such as IP-10 and its receptor CXCR3, or type 2 T cells such as monocyte-derived chemokine (MDC) and eotaxin and their receptors CCR4 and CCR3. Very few models have provided confirmation of these findings in vivo. Taking advantage of the humanized SCID mouse model grafted with autologous human skin, the ability of the chemokines IP-10, MDC, eotaxin, and RANTES to stimulate cell recruitment was investigated. Intradermal IP-10 injection resulted in an influx of CD4+ T lymphocytes but also surprisingly in the recruitment of dendritic cells. MDC recruited mainly CD8+ T lymphocytes, and had little effect on eosinophils. As predicted, eotaxin was a potent inducer of eosinophil and basophil migration, also recruiting CD4+ T cells. RANTES, a ubiquitous chemokine associated with both type 1 and type 2 profiles, was able to recruit all cell types. CXCR3-positive cells were preferentially recruited by IP-10, whereas CCR3- and CCR4-positive cells were predominantly found after injection of eotaxin and MDC. Thus, in a human environment in vivo, some chemokines have the ability to recruit cells expressing chemokine receptors preferentially expressed on type 1 or type 2 cells. Further investigations revealed that MDC and eotaxin induced the recruitment of type 2, but not type 1, cytokine-producing cells. RANTES, on the other hand, induced the migration of both type 1 and type 2 cytokine-secreting cells, whereas IP-10 did not induce the recruitment of either subtype. These studies provide detailed information on the properties of MDC, eotaxin, IP-10, and RANTES as chemotactic molecules in skin in vivo. The use of the humanized SCID mouse model grafted with human skin is validated as a useful model for the evaluation of chemokine function in the inflammatory reaction, and suggests that therapeutic targeting of certain chemokines might be of interest in diseases associated preferentially with a type 1 or type 2 profile.

CCR3-Blocking Antibody Inhibits Allergen-Induced Eosinophil Recruitment in Human Skin Xenografts from Allergic Patients

Eosinophil, basophil, and T helper 2 (TH2) cell recruitment into tissues is a characteristic feature of allergic diseases. These cells have in common the expression of the chemokine receptor CCR3, which may represent a specific pathway for their accumulation in vivo. Although animal models of allergic reactions are available, findings cannot always be extrapolated to man. To overcome these limitations, we have developed a humanized mouse model of allergic cutaneous reaction using severe combined immunodeficiency mice engrafted with skin and autologous peripheral blood mononuclear cells from allergic donors. Intradermal injection of the relevant allergen into human skin xenografts from allergic individuals induced a significant recruitment of human CD4+ T cells, basophils, and TH2-type cytokine mRNA-expressing cells, as well as murine eosinophils. Human skin xenografts, atopic status, and autologous peripheral blood mononuclear cell reconstitution were all mandatory to induce the allergic reaction. Next, we addressed the role of CCR3 in the endogenous mechanisms involved in the inflammatory cell recruitment in this experimental model of allergic cutaneous reaction. In vivo administration of an anti-human CCR3-blocking antibody selectively reduced accumulation of eosinophils but not that of CD4+ cells, basophils, or cells expressing mRNA for TH2-type cytokines. These findings establish a new in vivo model of humanized allergic reaction and suggest that eosinophil migration is mediated mainly through CCR3. Finally, these results suggest that this model might be useful to test human-specific antiallergic modulators.

Role of CCL17 in the Generation of Cutaneous Inflammatory Reactions in Hu-PBMC-SCID Mice Grafted with Human Skin

CCL17 may be of interest in skin inflammation, because it mainly attracts T cells expressing the cutaneous homing receptor and binds the chemokine receptor CCR4, preferentially expressed on Th-2 cells. We evaluated the in vivo effect of CCL17 injection in a humanized mouse model. (125)I-CCL17 injection into human skin grafted on severe combined immunodeficient (SCID) mice reconstituted with peripheral blood mononuclear cells resulted in a rapid transportation of CCL17 from the skin to the homolateral lymph nodes, followed 3 hours later by a lymph node infiltration of human memory CD4+ cells and dendritic cells. Intradermal injection of CCL17 resulted 24 hours later in a cutaneous recruitment of human memory CD4+ cells, monocytes, and basophils, but also murine eosinophils. In SCID mice reconstituted with polarized Th-1 or Th-2 cells, intradermal injection of CCL17 resulted in the recruitment of IL-4+ Th-2 cells but not of IFN-gamma+ Th-1 cells, whereas CCL17 was able to recruit both subsets in vitro. These results suggest that, in a humanized in vivo model, CCL17 is sufficient per se to induce a lymph node recruitment of memory CD4+ and dendritic cells and a cutaneous recruitment of Th-2-type cells, stressing it as an important actor in the initiation and development of Th-2-associated skin inflammation.

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.

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.