IL-36a and Pulmonary Inflammation Figure 8. Incubation with IL-36a enhances the ability of splenic CD11c+ cell mediated CD4+ T cell proliferation. A) Flow cytometric evaluation of CD4+ T cell proliferation responses induced by IL-36a stimulated splenic CD11c+ cells. Splenic CD11c+ cells were incubated with increasing doses of IL-36a for 2 h. Following incubation, media containing IL-36a was removed and CFSE-labeled CD4+ T cells were co-cultured with IL-36a stimulated CD11c+ cells. CFSE dilution was used to evaluate T cell proliferation responses 96 h following co-culture. CD4+ T cell proliferation was proportional to the concentration of IL-36a used for stimulating CD11c+ cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments. B) Flow cytometric evaluation of antigen-specific CD4+ T cell proliferation responses induced by IL-36a stimulated splenic CD11c+ cells. Splenic CD11c+ cells were incubated with increasing doses of IL-36a and 100 ng/mL OVA323-339 for 2 h. Following incubation, media containing IL-36a and the OVA peptide was removed and CFSE-labeled CD4+ T cells from OTII TCR transgenic mice were co-cultured with IL-36a stimulated, OVA peptide pulsed CD11c+ cells. CFSE dilution in the culture was used to evaluate T cell proliferation responses 96 h following co-culture. CD4+ T cell proliferation was proportional to the concentration of IL-36a used for stimulating CD11c+ cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments. doi:10.1371/journal.pone.0045784.g008 chemokines in airway epithelial cells in vitro [56–58] and in vivo [52]. Furthermore, overexpression of IL-1b in the airway epithelium increased the mRNA expression of CXCL1, CXCL2, CCL2 and CCL7 chemokines and the accumulation of neutro- phils in the lungs of mice [59]. Increased mRNA expression of CXCL1 and CXCL2 in the lungs of IL-36a instilled wildtype as well as IL-1ab2/2 mice in the current study suggests that IL-36a can induce the expression of neutrophil-specific chemokines in the lungs independent of IL-1a and IL-1b. We have previously demonstrated that a single intratracheal instillation of IL-36c induces airway hyperresponsiveness in response to aerosolized methacholine [38]. However, in the current study, a single i.t. instillation of a similar amount of IL-36a did not increase airway hyperresponsiveness. This discrepancy could be due to inherent differences in the ability of IL-36a and IL-36c in inducing AHR. For example, we have shown that unlike IL-36c, IL-36a is not constitutively expressed in lungs [38]. Furthermore, the magnitude of induction of IL-36a mRNA in response to cytokines and other inflammatory stimuli appears to be consistently lower than that of IL-36c mRNA expression in airway epithelial cells [39], bone marrow derived dendritic cells (BMDCs) and T cells [51]. Therefore, it is possible that a higher concentration of IL-36a is required to produce similar magnitude of responses driven by IL-36c at a lower concentration. In addition, the discrepancy could also be due to differences in AHR- specific genetic susceptibilities between A/J mice used in our previous report [38] and the C57BL/6 strain of mice used in this study. PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e45784