Wanhong Ding

Catecholamines Inhibit the Antigen-Presenting Capability of Epidermal Langerhans Cells

The sympathetic nervous system modulates immune function at a number of levels. Within the epidermis, APCs (Langerhans cells (LC)) are frequently anatomically associated with peripheral nerves. Furthermore, some neuropeptides have been shown to regulate LC Ag-presenting function. We explored the expression of adrenergic receptors (AR) in murine LC and assessed their functional role on Ag presentation and modulation of cutaneous immune responses. Both purified LC and the LC-like cell lines XS52-4D and XS106 expressed mRNA for the ARs α1A and β2. XS106 cells and purified LC also expressed β1-AR mRNA. Treatment of murine epidermal cell preparations with epinephrine (EPI) or norepinephrine inhibited Ag presentation in vitro. Furthermore, pretreatment of epidermal cells with EPI or norepinephrine in vitro suppressed the ability of these cells to present Ag for elicitation of delayed-type hypersensitivity in previously immunized mice. This effect was blocked by use of the β2-adrenergic antagonist ICI 118,551 but not by the α-antagonist phentolamine. Local intradermal injection of EPI inhibited the induction of contact hypersensitivity to epicutaneously administered haptens. Surprisingly, injection of EPI at a distant site also suppressed induction of contact hypersensitivity. Thus, catecholamines may have both local and systemic effects. We conclude that specific ARs are expressed on LC and that signaling through these receptors can decrease epidermal immune reactions.

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administration of donor T cells to bone marrow chimeric mice with persistent host LC, but not mice whose LC have been replaced by donor cells, exhibit marked skin graft-vs-host disease, demonstrating that LC can trigger graft-vs-host disease.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-γ 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.

Augmentation of Cutaneous Immune Responses by ATPγS: Purinergic Agonists Define a Novel Class of Immunologic Adjuvants

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPγS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPγS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPγS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1β, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X1, P2X7, P2Y1, P2Y2, P2Y4, P2Y9, and P2Y11 receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.

Vaccinia Virus Infection Attenuates Innate Immune Responses and Antigen Presentation by Epidermal Dendritic Cells

ABSTRACT Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-α), and IFN-γ, it actively inhibited the production of proinflammatory cytokines TNF-α and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-α secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the ΔE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IκBα. These results suggest a novel role for the E3L protein as an antagonist of the NF-κB signaling pathway. ΔE3L-infected XS52 cells secreted higher levels of TNF-α and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. ΔK1L-infected cells secreted higher levels of TNF-α and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.

Vasoactive Intestinal Peptide Modulates Langerhans Cell Immune Function

Epidermal nerves lie in close proximity to Langerhans cells (LC) and are capable of releasing peptides that modulate LC function, including calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide. The neuropeptide vasoactive intestinal peptide (VIP) has also been found in cutaneous nerves and mRNA, for the VIP receptor vasoactive intestinal peptide receptor type 1, and vasoactive intestinal peptide receptor type 2 have been found in murine LC and the LC-like cell line XS106. We examined the effects of VIP on LC function and cutaneous immunity. VIP inhibited elicitation of a delayed-type hypersensitivity response in previously immunized mice by epidermal cells enriched for LC content pulsed with Ag in vitro. VIP also inhibited the ability of unseparated epidermal cells to present Ag to a T cell clone and hybridoma and the ability of highly enriched LCs to present to the T cell clone. Inhibition of presentation to the hybridoma was observed with an antigenic peptide that does not require processing, suggesting that VIP is active at a step independent of Ag processing. To elucidate the mechanism(s) by which VIP may mediate these effects, we determined the effects of VIP on LC cytokine production using the XS106 cell line as a surrogate for LC. VIP augmented the production of the IL-10 in LPS-stimulated XS106 cells while down-regulating IL-12 and IL-1β production. Thus, VIP, like pituitary adenylate cyclase-activating polypeptide and calcitonin gene-related peptide, down-regulates LC function and the associated immune response.

Tetracycline suppresses ATPγS-induced CXCL8 and CXCL1 production by the human dermal microvascular endothelial cell-1 (HMEC-1) cell line and primary human dermal microvascular endothelial cells

Abstract:  Tetracyclines (TCN) have powerful anti‐inflammatory properties in addition to their anti‐microbial effects. These anti‐inflammatory effects are thought to play a role in inhibiting cutaneous inflammation in patients with rosacea and acne; however, the mechanism(s) of this action remains poorly understood. We have previously shown that adenosine‐5′‐triphosphate (ATP)γS, a hydrolysis‐resistant ATP analogue, augments secretion of pro‐inflammatory messengers by a human dermal microvascular endothelial cell line (HMEC‐1). ATP released by the sympathetic nerves during stress may stimulate release of pro‐inflammatory chemokines by dermal vessel endothelial cells, resulting in recruitment of inflammatory cells and exacerbation of inflammatory skin disease. Here we demonstrate that TCN inhibits ATPγS‐induced release of pro‐inflammatory mediators by HMEC‐1 cells and primary human dermal microvascular endothelial cells. TCN dose‐dependently inhibited ATPγS‐induced augmentation of CXCL8 (interleukin‐8) and CXCL1 (growth‐regulated oncogene‐α) production by HMEC‐1 cells and primary human dermal endothelial cells in vitro. TCN and ATPγS did not affect HMEC‐1 cell viability as determined by trypan‐blue exclusion and cell counts. Inhibition of production of inflammatory mediators by endothelial cells may be one mechanism by which TCN improves inflammatory skin diseases. The ability to inhibit release of inflammatory mediators induced in HMEC‐1 cells by purinergic agonists may be a useful way to screen for potential therapeutic agents for cutaneous inflammation.

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.

Altered cutaneous immune parameters in transgenic mice overexpressing viral IL-10 in the epidermis

IL-10 is a pleiotropic cytokine that inhibits several immune parameters, including Th1 cell-mediated immune responses, antigen presentation, and antigen-specific T cell proliferation. Recent data implicate IL-10 as a mediator of suppression of cell-mediated immunity induced by exposure to UVB radiation (280-320 nm). To investigate the effects of IL-10 on the cutaneous immune system, we engineered transgenic mice that overexpress viral IL-10 (vIL-10) in the epidermis. vIL-10 transgenic mice demonstrated a reduced number of I-A(+) epidermal and dermal cells and fewer I-A(+) hapten-bearing cells in regional lymph nodes after hapten painting of the skin. Reduced CD80 and CD86 expression by I-A(+) epidermal cells was also observed. vIL-10 transgenic mice demonstrated a smaller delayed-type hypersensitivity response to allogeneic cells upon challenge but had normal contact hypersensitivity to an epicutaneously applied hapten. Fresh epidermal cells from vIL-10 transgenic mice showed a decreased ability to stimulate allogeneic T cell proliferation, as did splenocytes. Additionally, chronic exposure of mice to UVB radiation led to the development of fewer skin tumors in vIL-10 mice than in WT controls, and vIL-10 transgenic mice had increased splenic NK cell activity against YAC-1targets. These findings support the concept that IL-10 is an important regulator of cutaneous immune function.

Langerhans cell expression of neuropeptide Y and peptide YY

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides with a variety of known functions. The role of these peptides in the skin is largely unknown, although NPY-like immunoreactivity has been reported in the epidermis. The recent report that these peptides have antimicrobial properties suggests that NPY and PYY may contribute to the skins defense mechanisms against invading microorganisms. We have demonstrated that Langerhans cells (LC) and a certain BALB/c epidermis-derived dendritic cell line contain mRNA for NPY and PYY using RT-PCR. Furthermore, this dendritic cell line as well as an epidermis-derived dendritic cell line from A/J mice were found to produce NPY and PYY and LC produced PYY, as assessed by radioimmunoassay. These data suggest that the protective function of LC include not only antigen presentation, but also production of antimicrobial peptides.

Pituitary adenylate cyclase-activating polypeptide inhibits cutaneous immune function

Epidermal nerves are closely associated with Langerhans cells (LC) and may be able to release factors, such as calcitonin gene‐related peptide and epinephrine, that affect LC function. LC and the LC‐like cell line XS106 express mRNA for the pituitary adenylate cyclase‐activating polypeptide (PACAP) receptors VPAC1 and VPAC2. We examined whether PACAP regulates cutaneous immunity. Intradermal administration of PACAP prior to application of a contact sensitizer at the injected site inhibited the induction of contact hypersensitivity. Pretreatment of murine epidermal cells enriched for LC content (∼12% LC) with PACAP inhibited their ability to elicit delayed‐type hypersensitivity in previously immunized mice. In vitro, PACAP suppressed the ability of both murine epidermal cells and highly purified LC (∼95%) to present antigen to a T cell clone and hybridoma. Furthermore, in LC and the XS106 cell line, PACAP inhibited the LPS/GM‐CSF‐induced stimulation of IL‐1β secretion and augmented IL‐10 production. PACAP also down‐regulated CD86 expression in LPS/GM‐CSF‐stimulated XS106 cells. The immunosuppressive effects of PACAP may be due to modulation ofcytokine production and CD86 expression.