John C. Ansel

Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems

Abstract: The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. It has been observed that certain skin diseases such as psoriasis and atopic dermatitis have a neurogenic component. Neuropeptides released by sensory nerves that innervate the skin and often contact epidermal and dermal cells can directly modulate functions of keratinocytes, Langerhans cells (LC), mast cells, dermal microvascular endothelial cells and infiltrating immune cells. Among these neuropeptides the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene related peptide (CGRP), vasoactive intestinal peptide (VIP) and somato statin (SOM) have been reported to effectively modulate skin and immune cell functions such as cell proliferation, cytokine production or antigen presentation under physiological or pathophysiological conditions. Expression and regulation of their corresponding receptors that are expressed on a variety of skin cells as well as the presence of neuropeptidespecific peptidases such as neutral endopeptidase (NEP) or angiotensinconverting enzyme (ACE) determine the final biological response mediated by these peptides on the target cell or tissue. Likewise, skin cells like keratinocytes or fibroblasts are a source for neurotrophins such as nerve growth factor that are required not only for survival and regeneration of sensory neurons but also to control responsiveness of these neurons to external stimuli. Therefore, neuropeptides, neuropeptide receptors, neuropeptidedegrading enzymes and neurotrophins participate in a complex, interdependent network of mediators that modulate skin inflammation, wound healing and the skin immune system. This review will focus on recent studies demonstrating the role of tachykinins, CGRP, SOM and VIP and their receptors and neuropeptide‐degrading enzymes in mediating neurogenic inflammation in the skin.

Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase.

Abstract: Proteinase‐activated receptor‐2 (PAR‐2) is a G‐protein coupled receptor. Tryptic proteases cleave PAR‐2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR‐2 is highy expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin in unknown, and the proteases that activate PAR‐2 in the skin have not been identified. We localized PAR‐2 in human skin by immunohistochemistry, examined PAR‐2 expression by RT‐PCR and RNA blotting, and investigated PAR‐2 activation by mast cell tryptase. PAR‐2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic‐like cells. PAR‐2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR‐2 mRNA was detected in normal human skin by RT‐PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNG2) increased [Ca2+]i in keratinocytes, measured using Fura‐2/AM. Although tryptase‐containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal‐epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR‐2 on keratinocytes and endothelial cells during inflammation.

Neutral Endopeptidase Terminates Substance P-Induced Inflammation in Allergic Contact Dermatitis

Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP−/− knockout and NEP+/+ wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP−/− animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP−/− mice, the acute inhibition of NEP in NEP+/+ animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP−/− compared with NEP+/+ animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.

A 15-year follow-up of AJCC stage III malignant melanoma patients treated postsurgically with Newcastle disease virus (NDV) oncolysate and determination of alterations in the CD8 T cell repertoire.

BackgroundThe development of effective adjuvant therapies for the treatment of high-risk melanoma patients is critical for the prevention of metastatic disease and improvement of patient survival. Active specific immunotherapy has been tested as an adjuvant treatment in numerous clinical trials with overall limited, but occasionally promising, success rates. Newcastle disease virus (NDV) oncolysate has been utilized as an adjunctive immunotherapeutic agent in the postsurgical management of these patients. A phase II study initiated in 1975 using adjuvant vaccine therapy composed of allogeneic and autologous human melanoma cells infected with live NDV (NDV oncolysate) in patients with AJCC stage III melanoma following therapeutic lymph node dissection has shown >60% survival rate at 10 years with no adverse effects. Continued long-term analysis of trials with promising early results as well as assessment of immunologic responses generated in these patients may result in improved therapeutic decisions for clinical trials in the future.Materials and MethodsWe analyzed the 15-year survival of patients treated postsurgically with NDV oncolysate in the phase II study described above. In an attempt to understand the immunological effects of this treatment, we have also carried out a comprehensive analysis of the peripheral blood T cell repertoire in these patients.ResultsThe overall 15-year survival of this group of patients is 55%. Previous studies have suggested that improved outcome in patients undergoing immunotherapy is correlated with increased numbers of CD8+CD57+ cells. In surviving patients, we observed a striking oligoclonality in the CD8+ T cell population in peripheral blood, which reflects clonal expansions in the CD8+CD57+ subset.ConclusionsThe data suggest that adjuvant vaccination with NDV oncolysates is associated with prolonged survival of patients with lymph node-positive malignant melanoma and that CD8+ T cells may be an important component of therapeutic efficacy.

The Role of α‐MSH as a Modulator of Cutaneous Inflammation

Abstract: Among various neuropeptides such as substance P, calcitonin gene‐related peptide and others, α‐melanocyte‐stimulating hormone (α‐MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 (MC‐1R), which is specific for α‐MSH and ACTH, is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells α‐MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL‐2, IFN‐γ, TNF‐α, and IL‐1. It downregulates the expression of costimulatory molecules such as CD86 and CD40 and induces the production of suppressor factors such as the cytokine synthesis inhibitory factor IL‐10. On endothelial cells α‐MSH is capable of downregulating the LPS‐induced expression of adhesion molecules such as vascular cell adhesion molecule (VCAM) and E‐selectin. Moreover, the LPS‐induced activation of transcription factors such as NFκB is downregulated by α‐MSH. In a mouse model i.v. or topical application of α‐MSH was found to inhibit the induction phase as well as the effector phase of contact hypersensitivity (CHS) reactions and to induce hapten‐specific tolerance. These findings indicate that the production of immunosuppressing neuropeptides such as α‐MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin.

Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-1 expression and function

There is increasing evidence that sensory nerves may participate in cutaneous inflammatory responses by the release of neuropeptides such as substance P (SP). We examined the direct effect of SP on human dermal microvascular endothelial cell (HDMEC) intercellular adhesion molecule 1 (ICAM-1) expression and function. Our results indicated that, although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMEC intracellular Ca2+ levels, primarily by the activation of NK-1R. Immunohistochemistry studies likewise demonstrated that HDMEC predominantly expressed NK-1R. The addition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1 mRNA levels, followed by a fivefold increase in ICAM-1 cell surface expression. This functionally resulted in a threefold increase in51Cr-labeled binding of J-Y lymphoblastoid cells to HDMEC. In vivo studies demonstrated a marked increase in microvascular ICAM-1 immunostaining 24 and 48 h after application of capsaicin to the skin. These results indicate that neuropeptides such as SP are capable of directly activating HDMEC to express increased levels of functional ICAM-1 and further support the role of the cutaneous neurological system in modulating inflammatory processes in the skin.There is increasing evidence that sensory nerves may participate in cutaneous inflammatory responses by the release of neuropeptides such as substance P (SP). We examined the direct effect of SP on human dermal microvascular endothelial cell (HDMEC) intercellular adhesion molecule 1 (ICAM-1) expression and function. Our results indicated that, although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMEC intracellular Ca2+ levels, primarily by the activation of NK-1R. Immunohistochemistry studies likewise demonstrated that HDMEC predominantly expressed NK-1R. The addition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1 mRNA levels, followed by a fivefold increase in ICAM-1 cell surface expression. This functionally resulted in a threefold increase in 51Cr-labeled binding of J-Y lymphoblastoid cells to HDMEC. In vivo studies demonstrated a marked increase in microvascular ICAM-1 immunostaining 24 and 48 h after application of capsaicin to the skin. These results indicate that neuropeptides such as SP are capable of directly activating HDMEC to express increased levels of functional ICAM-1 and further support the role of the cutaneous neurological system in modulating inflammatory processes in the skin.

Trauma and alkali burns induce distinct patterns of cytokine gene expression in the rat cornea

Cytokines such as the interleukins (IL) and tumor necrosis factor alpha (TNF alpha) have traditionally been associated with paracrine regulation of immune reactions. These proteins also have properties suggestive of functional roles in the inflammatory and reparative responses to tissue injury. In this study, mRNA levels for IL-1 alpha, IL-1 beta, IL-6, TNF alpha, interferon gamma, transforming growth factor beta 1, and CD4 were monitored in rat corneas at times from 1 hour through 2 weeks after incisional trauma or alkali burns. Transcripts for IL-1 alpha, TNF alpha, and TGF beta 1 were present in most corneal samples; whereas those for IFN gamma and CD4 were not detected. As early as 1 hour following either of these non-immunologic forms of injury, expression of IL-6 mRNA levels was induced. Only in corneas with alkali burns did IL-6 induction persist from days 1 through 7. The alkali-injured corneas also had markedly increased IL-1 beta mRNA levels from days 1 through 7. These observations indicate that cytokine mRNA is induced in the cornea by trauma without an apparent immunologic stimulus. Our data are consistent with the hypothesis that corneal tissues respond to different types of injury with different patterns of cytokine gene expression.

Neutral endopeptidase inhibition in diabetic wound repair

In response to cutaneous injury, sensory nerves release substance P, a proinflammatory neuropeptide. Substance P stimulates mitogenesis and migration of keratinocytes, fibroblasts, and endothelial cells. Neutral endopeptidase (NEP), a cell surface metallopeptidase, degrades substance P. Chronic nonhealing wounds and skin from patients with diabetes mellitus show increased NEP localization and activity. We hypothesized that increased NEP may retard wound healing and that NEP inhibition would improve closure kinetics in an excisional murine wound model. NEP enzyme activity was measured in skin samples from mutant diabetic mice (db/db) and nondiabetic (db/–) littermates by degradation of glutaryl‐ala‐ala‐phe‐4‐methoxy‐2‐naphthylamine. Full‐thickness 6‐mm dorsal excisional wounds treated with normal saline or the NEP inhibitor thiorphan (10 µM or 25 µM) for 7 days were followed until closure. Histological examination and NEP activity were evaluated in a subset of wounds. NEP activity in unwounded db/db skin (20.6 pmol MNA/hr/µg) significantly exceeded activity in db/–skin (7.9 pmol MNA/hr/µg; p = 0.02). In db/db mice, 25 µM thiorphan shortened time to closure (18.0 days; p < 0.05) compared to normal saline (23.5 days). NEP inhibition did not alter closure kinetics in db/–mice. While the inflammatory response appeared enhanced in early wounds treated with thiorphan, blinded histological scoring of healed wounds using a semiquantitative scale showed no difference in inflammation. Unwounded skin from diabetic mice shows increased NEP activity and NEP inhibition improved wound closure kinetics without affecting contraction, suggesting that its principal effect was to augment epithelialization. (WOUND REP REG 2002;10:295–301)

Cutaneous allergic contact dermatitis responses are diminished in mice deficient in neurokinin 1 receptors and augmented by neurokinin 2 receptor blockage

Sensory neuropeptides such as neurokinin A (NKA) or particularly substance P (SP) by neurokinin receptor (NK‐R) activation modulate skin and immune cells functions during neurogenic inflammation. In this study, we examined the relative importance of SP/NK‐1Rs or NKA/NK‐2Rs in a murine model for allergic contact dermatitis (ACD) and tested if the functional absence of NK‐Rs will impair inflammatory response in vivo. Mice lacking NK‐1Rs (C57BL/6J‐NK‐1R•/•) displayed a significantly reduced ACD inflammatory ear swelling response to dinitrofluorobenzene (DNFB) with histological less edema and 50% fewer infiltrating leukocytes compared with the ACD response in wild‐type (+/+) animals. In NK‐1R+/+ mice, transient NK‐1R inhibition impaired ACD sensitization. In vitro haptenized bone marrow‐derived dendritic cells from NK‐1R+/+ mice matured in the presence of an NK‐1R antagonist displayed a reduced capability to induce T cell proliferation in vitro and ACD after adoptive transfer into naïve wild‐type mice in vivo. By contrast, NK‐2R inhibition significantly enhanced the ACD response in NK‐1R null or in wild‐type mice, whereas epicutaneous application of NK‐2R agonists diminished the ACD inflammation. Thus, NK‐1R and SP are required for antigen sensitization and a full inflammatory response to cutaneous allergens and NKA and the NK‐2R mediate a contrasting anti‐inflammatory role in ACD. Thus, SP, NKA, NK‐1R, and NK‐2R have important but differential roles in the regulation of cutaneous inflammatory responses.

Substance P induction of murine keratinocyte PAM 212 interleukin 1 production is mediated by the neurokinin 2 receptor (NK‐2R)

Abstract: The neurological system plays an important role in modulating some inflammatory skin diseases. Neuro‐cutaneous interactions may be mediated by the release of neuropeptides such as substance P (SP) which activate immunocompetent cells in the skin by binding to high affinity neurokinin receptors (NKR). Since epidermal keratinocytes produce a variety of cytokines and are intimately associated with cutaneous sensory fibers, we tested the ability of these cells to participate in the cutaneous neuroimmune system by the secretion of potent cytokines such as interleukin 1 (IL‐1) in response to released SP. RT‐PCR studies demonstrated that cultured PAM 212 murine keratinocytes expressed mRNA for NK‐2R but not NK‐1R. Correspondingly, the addition of SP to these cells resulted in a rapid increase in intracellular Ca2+ levels that could be specifically blocked by an NK‐2R antagonist. NK‐2R was also shown in normal mouse epidermis by immunohistochemistry. SP augmented the expression of PAM 212 keratinocyte IL‐1α mRNA in a dose and time dependent manner and this induction was inhibited by an NK‐2R antagonist. Secretion of bioactive IL‐1α by the PAM 212 keratinocytes was likewise stimulated by SP in a dose dependent manner. These data support the hypothesis that SP released from cutaneous sensory nerves contributes to neuroimmune inflammatory responses in the skin by modulating the expression and release of cytokines from epidermal keratinocytes.