Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Regulation of T helper cell responses during antigen presentation by norepinephrine-exposed endothelial cells.

Dermal blood vessels and regional lymph nodes are innervated by sympathetic nerves and, under stress, sympathetic nerves release norepinephrine (NE). Exposure of primary murine dermal microvascular endothelial cells (pDMECs) to NE followed by co-culture with Langerhans cells (LCs), responsive CD4+ T-cells and antigen resulted in modulation of CD4+ T-cell responses. NE-treatment of pDMECs induced increased production of interleukin (IL)-6 and IL-17A while down-regulating interferon (IFN)-γ and IL-22 release. This effect did not require contact between pDMECs and LCs or T-cells and depended upon pDMEC production of IL-6. The presence of NE-treated pDMECs increased the proportion of CD4+ T-cells expressing intracellular IL-17A and increased IL-17A mRNA while decreasing the proportion of IFN-γ- or IL-22-expressing CD4+ T-cells and mRNA levels for those cytokines. Retinoic acid receptor-related orphan receptor gamma (ROR-γt) mRNA was significantly increased in CD4+ T-cells while T-box transcription factor (T-bet) mRNA was decreased. Intradermal administration of NE prior to hapten immunization at the injection site produced a similar bias in draining lymph node CD4+ T-cells towards IL-17A and away from IFN-γ and IL-22 production. Under stress, release of NE may have significant regulatory effects on the outcome of antigen presentation through actions on ECs with enhancement of inflammatory skin disorders involving IL-17/T helper type 17 (Th17) cells.

Calcitonin Gene-Related Peptide-Exposed Endothelial Cells Bias Antigen Presentation to CD4+ T Cells toward a Th17 Response.

Calcitonin gene-related peptide (CGRP) is a neuropeptide with well-established immunomodulatory functions. CGRP-containing nerves innervate dermal blood vessels and lymph nodes. We examined whether CGRP regulates the outcome of Ag presentation by Langerhans cells (LCs) to T cells through actions on microvascular endothelial cells (ECs). Exposure of primary murine dermal microvascular ECs (pDMECs) to CGRP followed by coculture with LCs, responsive CD4(+) T cells and Ag resulted in increased production of IL-6 and IL-17A accompanied by inhibition of IFN-γ, IL-4, and IL-22 compared with wells containing pDMECs treated with medium alone. Physical contact between ECs and LCs or T cells was not required for this effect and, except for IL-4, we demonstrated that IL-6 production by CGRP-treated pDMECs was involved in these effects. CD4(+) cells expressing cytoplasmic IL-17A were increased, whereas cells expressing cytoplasmic IFN-γ or IL-4 were decreased by the presence of CGRP-treated pDMECs. In addition, the level of retinoic acid receptor-related orphan receptor γt mRNA was significantly increased, whereas 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 toward IL-17A and away from IFN-γ. 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.

Norepinephrine and adenosine-5'-triphosphate synergize in inducing IL-6 production by human dermal microvascular endothelial cells.

Endothelial cells (ECs) play important roles in cutaneous inflammation, in part, by release of inflammatory chemokines/cytokines. Because dermal blood vessels are innervated by sympathetic nerves, the sympathetic neurotransmitter norepinephrine (NE) and the co-transmitter adenosine-5'-triphosphate (ATP) may regulate expression of EC inflammatory factors. We focused on IL-6 regulation because it has many inflammatory and immune functions, including participation in Th17 cell differentiation. Strikingly, NE and ATP synergistically induced release of IL-6 by a human dermal microvascular endothelial cell line (HMEC-1). Adrenergic antagonist and agonist studies indicated that the effect of NE on induced IL-6 release is primarily mediated by ß2-adrenergic receptors (ARs). By real-time PCR IL-6 mRNA was also synergistically induced in HMEC-1 cells. This synergistic effect of NE and ATP was reproduced in primary human dermal endothelial cells (pHDMECs) and is also primarily mediated by ß2-ARs. Under conditions of stress, activation of the symphathetic nervous system may lead to release of ATP and NE by sympathetic nerves surrounding dermal blood vessels with induction of IL-6 production by ECs. IL-6 may then participate in immune and inflammatory processes including generation of Th17 cells. Production of IL-6 in this manner might explain stress-induced exacerbation of psoriasis, and perhaps, other skin disorders involving Th17-type immunity.

N-acetyl-S-farnesyl-l-cysteine suppresses chemokine production by human dermal microvascular endothelial cells.

Isoprenylcysteine (IPC) molecules modulate G-protein-coupled receptor signalling. The archetype of this class is N-acetyl-S-farnesyl-l-cysteine (AFC). Topical application of AFC locally inhibits skin inflammation and elicitation of contact hypersensitivity in vivo. However, the mechanism of these anti-inflammatory effects is not well understood. Dermal microvascular endothelial cells (ECs) are involved in inflammation, in part, by secreting cytokines that recruit inflammatory cells. We have previously shown that the sympathetic nerve cotransmitter adenosine-5'-triphosphate (ATP) and adenosine-5'-O-(3-thio) triphosphate (ATPγS), an ATP analogue that is resistant to hydrolysis, increase secretion of the chemokines CXCL8 (interleukin-8), CCL2 (monocyte chemotactic protein-1) and CXCL1 (growth-regulated oncogene α) by dermal microvascular ECs. Production of these chemokines can also be induced by the exposure to the proinflammatory cytokine TNFα. We have now demonstrated that AFC dose-dependently inhibits ATP-, ATPγS- and TNFα-induced production of CXCL1, CXCL8 and CCL2 by a human dermal microvascular EC line (HMEC-1) in vitro under conditions that do not affect cell viability. Inhibition of ATPγS- or TNFα-stimulated release of these chemokines was associated with reduced mRNA levels. N-acetyl-S-geranyl-l-cysteine, an IPC analogue that is inactive in inhibiting G-protein-coupled signalling, had greatly reduced ability to suppress stimulated chemokine production. AFC may exert its anti-inflammatory effects through the inhibition of chemokine production by stimulated ECs.

Pituitary adenylate cyclase-activating peptide and vasoactive intestinal polypeptide bias Langerhans cell Ag presentation toward Th17 cells.

Epidermal Langerhans cells (LCs) are dendritic APCs that play an important role in cutaneous immune responses. LCs are associated with epidermal nerves and the neuropeptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) inhibit LC Ag presentation for Th1-type immune responses. Here, we examined whether PACAP or VIP modulates LC Ag presentation for induction of IL-17A-producing CD4(+) T cells. Treatment with VIP or PACAP prior to in vitro LC Ag presentation to CD4(+) T cells enhanced IL-17A, IL-6, and IL-4 production, decreased interferon (IFN)-γ and interleukin (IL)-22 release, and increased RORγt and Gata3 mRNA expression while decreasing T-bet expression. The CD4(+) T-cell population was increased in IL-17A- and IL-4-expressing cells and decreased in IFN-γ-expressing cells. Addition of anti-IL-6 mAb blocked the enhanced IL-17A production seen with LC preexposure to VIP or PACAP. Intradermal administration of VIP or PACAP prior to application of a contact sensitizer at the injection site, followed by harvesting of draining lymph node CD4(+) T cells and stimulation with anti-CD3/anti-CD28 mAbs, enhanced IL-17A and IL-4 production but reduced production of IL-22 and IFN-γ. PACAP and VIP are endogenous mediators that likely regulate immunity and immune-mediated diseases within the skin.

Calcitonin gene-related peptide inhibits chemokine production by human dermal microvascular endothelial cells.

This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP8-37 and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced IκBα degradation and NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-κB activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-κB pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation.

Muramyl dipeptide induces Th17 polarization through activation of endothelial cells.

Endothelial cells (ECs) express the nucleotide-binding oligomerization domain (Nod) receptor 2, which recognizes the bacterial derivate muramyl dipeptide (MDP). MDP stimulation of these cells enhances their IL-6 production and may thus contribute to the immune and inflammatory activities in the skin. However, whether ECs are capable of influencing the development of T cell priming and its polarization remains unknown. We report that in vitro the murine bEnd.3 EC line induces, following MDP stimulation, a Th17 polarization at the expense of Th1 and Th2 polarization in the setting of Langerhans cell (LC) Ag presentation to responsive T cells as assessed by IL-17, IL-6, IFN-γ, and IL-4 production. Interestingly, IL-22 production, which has been associated with Th17 priming, was not influenced by MDP-treated bEnd.3 cells, illustrating differential regulation of this cytokine from IL-17. Additional analysis confirmed a significantly increased percentage of IL-17(+)CD4(+) T cells by flow cytometry and an increased mRNA level of the specific Th17 transcription factor retinoic acid-related orphan receptor γt in cocultures of LCs and responsive T cells in the presence of activated bEnd.3 cells. Experiments using the RNA interference technique to knockdown IL-6 in bEnd.3 cells confirmed that IL-6 produced by bEnd.3 cells stimulated by MDP is at least partially involved in Th17 polarization. Our data suggest that activated ECs are capable of influencing LC Ag processing and presentation to T cells and induce a Th17 polarization. These results are important for the understanding of Th17-related disorders of the skin such as psoriasis.

Calcitonin gene-related peptide biases Langerhans cells toward Th2-type immunity.

Langerhans cells (LC) are epidermal dendritic cells capable, in several experimental systems, of Ag-presentation for stimulation of cell-mediated immunity. LC have been considered to play a key role in initiation of cutaneous immune responses. Additionally, administration of donor T cells to bone marrow chimeric mice with persistent host LC, but not mice whose LC have been replaced by donor cells, exhibit marked skin graft-vs-host disease, demonstrating that LC can trigger graft-vs-host disease. However, experiments with transgenic mice in which regulatory elements from human langerin were used to drive expression of diphtheria toxin, resulting in absence of LC, suggest that LC may serve to down-regulate cutaneous immunity. LC are associated with nerves containing the neuropeptide calcitonin gene-related peptide (CGRP), and CGRP inhibits LC Ag-presentation in several models including presentation to a Th1 clone. We now report that CGRP enhances LC function for stimulation of Th2 responses. CGRP exposure enhanced LC Ag presentation to a Th2 clone. Upon presentation of chicken OVA by LC to T cells from DO11.10 chicken OVA TCR transgenic mice, pretreatment with CGRP resulted in increased IL-4 production and decreased IFN-gamma production. CGRP also inhibited stimulated production of the Th1 chemokines CXCL9 and CXCL10 but induced production of the Th2 chemokines CCL17 and CCL22 by a dendritic cell line and by freshly obtained LC. Changes in production of these chemokines correlated with the effect of CGRP on mRNA levels for these factors. Exposure of LC to nerve-derived CGRP in situ may polarize them toward favoring Th2-type immunity.

"Soluble" adenylyl cyclase-generated cyclic adenosine monophosphate promotes fast migration in PC12 cells.

In a model for neuronal movement, PC12 cells undergo fast migration in response to nerve growth factor (NGF) and phorbol ester (PMA). We previously showed that NGF increases intracellular cAMP via activation of soluble adenylyl cyclase (sAC). In this report, we demonstrate that sAC activation is an essential component of NGF- + PMA-induced fast migration in PC12 cells. Interestingly, PMA also raises intracellular cAMP but does so by stimulating transmembrane adenylyl cyclases (tmAC); however, this tmAC-generated cAMP does not contribute to fast migration. Therefore, cells must possess independent pools of cAMP capable of modulating distinct functions.