All animal studies were approved by the Institutional Animal Care and Use Committee of the Weill Cornell Medical College. Reagents CGRP was purchased from Bachem; a fragment of cOVA (cOVA323C339) was obtained from Peptides International; anti-mouse CD3 mAb along with isotype controls was obtained from R&D Systems and anti-mouse CD28 mAb from BD Biosciences. effects. CD4+ cells expressing cytoplasmic IL-17A were increased while cells expressing cytoplasmic IFN- or IL-4 were decreased by the presence of CGRP-treated pDMECs and the level of retinoic acid receptor-related orphan receptor t mRNA was significantly increased while T-bet and GATA3 expression was inhibited. Immunization at the site of intradermally administered CGRP led to a similar bias in CD4+ T cells from draining lymph node cells towards IL-17A and away from IFN-. Actions of nerve-derived CGRP on ECs may have important regulatory effects on the outcome of Ag presentation with consequences for the expression of inflammatory skin disorders involving Th17 cells. Introduction Neurologic status, including emotional state, influences immune function. The GSK1278863 (Daprodustat) primary and secondary lymphoid organs including the spleen, thymus and lymph nodes are innervated and dendritic cells and lymphocytes express receptors for peptide and non-peptide products of nerves (1C5). Additionally, many studies have demonstrated that stress can have immunoregulatory effects both in humans and animals and these effects are mediated, at least in part, by neuroendocrine pathways (3C14). Also, there are reports that stress may exacerbate psoriasis and GSK1278863 (Daprodustat) atopic dermatitis (6C8) and an atopic dermatitis-like rash in an animal model (11). The importance of the nervous system to inflammatory skin disease is highlighted by the findings that psoriasis clears in denervated skin (15, 16) and that Vax2 some animal models of psoriasiform dermatitis depend on innervation for their expression (17, 18). Endothelial cells (ECs)3, which line blood vessels within the dermis, contribute to cutaneous immunity and inflammation through many mechanisms. Amongst these is the ability to release cytokines and chemokines as well as expression of adhesion molecules involved in recruitment of inflammatory cells out of the vasculature and into the interstitium (19C23). In this regard, we have recently reported that the vasodilator and peptide neurotransmitter calcitonin gene-related peptide (CGRP) inhibits the stimulated expression of the chemokines CXCL8, CCL2 and CXCL1 by human dermal microvascular ECs (24). CGRP is a 37 amino acid neuropeptide generated by tissue-specific alternative processing of the calcitonin gene and is widely distributed in organs of the immune system as well as the central and peripheral nervous system (25). Of particular interest, in a murine model of psoriasiform dermatitis, in which the Tie2 receptor tyrosine kinase is over-expressed in keratinocytes, denervation of skin results in loss of the psoriasiform phenotype but administration of CGRP to the animal inhibits this loss (17), suggesting a key role for CGRP in the phenotype observed. In this regard, it has been reported that in lesions of psoriasis ECs have CGRP on their surface (26). Furthermore, both sympathetic and sensory nerves are associated with dermal vessels (27, 28) and also innervate lymph nodes (29). Moreover, recent evidence indicates that sympathetic neurotransmitters, including norepinephrine, regulate immune and inflammatory responses (30, 31). Lymphocytes and APCs trafficking through the skin and exiting the vasculature GSK1278863 (Daprodustat) to enter the interstitium of the dermis are closely associated with ECs during these processes. Furthermore, release of EC-derived factors on the abluminal side of vessels would be able to interact with immune cells in the interstitium, particularly those in a perivascular arrangement. Thus, we asked whether CGRP modulates the ability of ECs acting as bystanders, to regulate the outcome of Ag presentation by Langerhans cells (LCs) to CD4+ T cells. LCs are dendritic APCs that reside in the epidermis that, depending on circumstances, can present Ag for induction or regulation of arms of the immune response (32, 33). They were chosen as APCs for this study because their function has previously been shown to be directly regulated by neuropeptides (34C40) and, when stimulated by Ag, they traffic through EC-lined lymphatics to regional lymph nodes (41). Additionally, there is evidence that they can present Ag for generation of Th17 helper T cells (42, 43), believed to be important in the pathogenesis of certain inflammatory skin disorders including psoriasis (44, 45). In this regard, LCs are believed to play a role in some other inflammatory dermatoses (46). We have now examined the effects of adding CGRP-treated or non-treated ECs to Ag presenting cultures of LCs and responding T cells, an environment perhaps similar to that in the dermis or regional lymph nodes during a local immune reaction, on the generation of T GSK1278863 (Daprodustat) helper cell subtypes. Materials and Methods Mice Six- to 12-wk-old female BALB/c (H-2d) and DO11.10 chicken OVA (cOVA) TCR transgenic (Tg) mice on a BALB/c background [C.Cg-Tg(DO11.10)10Dlo/J] mice were purchased from the Jackson Laboratory. GSK1278863 (Daprodustat) The DO11.10 mice carry MHC class II-restricted, rearranged TCR and chain genes that encode a TCR that recognizes a fragment of cOVA (cOVA 323C339) presented by I-Ad (47, 48). All animal studies were.
