cDNA prepared from this RNA was used in a quantitative PCR (Taqman) assay

cDNA prepared from this RNA was used in a quantitative PCR (Taqman) assay. antigen-challenged lymphocytes. However, compared with CA-challenged wild-type mice, challenged CCR6-deficient mice had reduced airway resistance, fewer eosinophils round the airway, lower levels of interleukin 5 in the lung, and reduced serum levels of immunoglobulin E. Together, these data demonstrate that MIP-3 and CCR6 function in allergic pulmonary responses and suggest that these molecules might represent novel CCT241533 therapeutic targets for treatment of asthma. 0.05 was considered significant. Results and Conversation MIP-3 Is usually Induced by Allergen Challenge. We first investigated whether MIP-3 is usually expressed in the lungs of wild-type C57BL/6 mice after sensitization and challenge with CA. This allergen was used because of its clinical relevance and because it induces responses common of asthma, including production of Th2 cytokines, eosinophil infiltration, and airway hyperreactivity. Quantitative PCR analysis (Taqman) was performed on cDNAs derived from total lung RNA. In sensitized but unchallenged mice, only very low levels of MIP-3Cspecific mRNA were detected. However, shortly after CA challenge, these levels increased dramatically, peaking at 8 h after challenge (Fig. 1). These data show that MIP-3, the only known chemokine ligand for CCR6, is usually expressed in allergen-challenged lungs. Open in a separate window Physique 1 MIP-3 expression in allergen-challenged lungs. RNA was prepared from lungs harvested at the indicated occasions after challenge with CA. cDNA prepared from this RNA was used in a quantitative PCR (Taqman) assay. Values shown symbolize femtograms of MIP-3 cDNA per 25 ng total cDNA. CCR6?/? Mice Have Reduced Airway Hyperreactivity after CA Challenge. To investigate whether the induction of MIP-3 in lungs and lymph nodes was of functional importance in pulmonary allergic responses, we investigated the physiologic response of CCR6= 5 animals per treatment group. * 0.05. CCR6?/? Mice Have Reduced Peribronchial Eosinophil Accumulation. Previous studies have exhibited that airway hyperreactivity after multiple allergen difficulties is closely associated with the level of peribronchial eosinophil accumulation 19. To determine whether the conversation of MIP-3 and CCR6 is required for the eosinophilic response to CA, lung sections were prepared and eosinophils counted with high-power CCT241533 microscopy. This analysis revealed that CA-challenged CCR6 0.05. Analysis of Pulmonary Cytokines. One possible explanation for the decreased FGF3 eosinophil recruitment into the airway of CCR6 0.05. IgE Production. Another hallmark of the asthmatic response is an increase in circulating levels of IgE. To determine whether the reduced IL-5 and airway hyperreactivity seen in the CCR6 0.05. Immunization. To address the possibility that the reduced response in the lungs of CCR6 em ?/ /em ? mice was related to a reduced capacity to be immunized, we prepared lymphocyte cultures from your lymph nodes of CA-immunized mice and measured cytokine production after CA activation in vitro. Compared with lymphocytes prepared from nonimmunized mice, large increases in IL-4 production were seen in both challenged wild-type (590 11-fold) and challenged CCR6 em ?/ /em ? mice (481 7-fold). Similarly, IL-5 was increased in wild-type (6.2 0.4-fold) and CCR6 em ?/ /em ? mice (6.1 0.2-fold). Thus, the extent of immunization was comparable in the two groups of challenged mice. Taken together, the data from these studies support a role for CCR6 in allergic airway responses. In particular, CCR6 is required for normal allergen-induced eosinophil accumulation and for production of IL-5 and IgE. This blunted response in the lung mucosa is usually consistent with the diminished humoral response previously seen in the intestinal mucosa of CCR6 em ?/ /em ? mice and suggests that CCR6 might have a general function in mucosal immune responses. The expression of CCR6 in DCs, memory CCT241533 T cells, and B lymphocytes suggests that the diminished allergic response in CCR6 em ?/ /em ? mice might result from impaired function of one or more of these cell types. Thus, even though mechanism of CCR6 action in this model remains unclear, the data presented here identify CCR6 as a potential therapeutic target for individuals suffering from pathophysiologic responses to airway allergens. Acknowledgments This work was supported in part by National Institutes of Health grants HL31963 and AI36302..