Alpha-melanocyte stimulating hormone prevents lipopolysaccharide-induced vasculitis by down-regulating endothelial cell adhesion molecule expression.

The neuroendocrine hormone alpha-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that alpha-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNFalpha in vitro and in vivo. In HDMEC, alpha-MSH (10(-8)/10(-12) M) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNFalpha as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, alpha-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, alpha-MSH effectively inhibited the transcription factor nuclear factor-kappaB activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of alpha-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that alpha-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.

Management of high-risk melanoma.

Improved treatment options for patients with high-risk melanoma are of great importance for clinicians who participate in the care of these patients. There remains an overall lack of response to existing treatment options, which continues to fuel the efforts of basic scientists and clinicians to pursue other approaches for the treatment of melanoma that is no longer limited to the skin. Continued investigation into the innovative and concurrent use of surgery, chemotherapy, immunotherapy, and radiation therapy holds significant promise for improved outcomes in the management of patients with this devastating disease.

The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells.

PURPOSE: Gram-negative bacterial infections of the eye can lead to corneal bacterial keratitis, visual impairment, and blindness. Many of these pathologic changes may be mediated by bacterially derived products such as lipopolysaccharide (LPS). In this investigation, it has been established for the first time that human corneal cells are capable of expressing the functional LPS receptor complex proteins, CD14 and Toll-like receptor 4 (TLR4). METHODS: CD14 and TLR4 mRNA expression in human corneal cells was determined by RT-PCR and Northern blot analysis, and cell surface expression of these proteins was measured by flow cytometry. LPS-mediated corneal cell activation was determined by measuring intracellular calcium mobilization. Cellular cytokine and chemokine secretion in response to LPS was measured by ELISA. The expression and localization of CD14 in whole human cornea was determined by immunohistochemistry. RESULTS: Human corneal epithelial, stromal, and endothelial cells expressed CD14 mRNA and cell surface CD14. LPS binding to cornea CD14 resulted in a rapid intracellular calcium response and the secretion of multiple proinflammatory cytokines and chemokines. CD14 mRNA expression in corneal epithelial cells was upregulated by LPS. In addition to CD14, corneal epithelial cells expressed the functional LPS receptor-signaling protein TLR4, which was also augmented by LPS. CONCLUSIONS: The cornea expresses functional CD14 and TLR4 LPS receptor proteins. Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections.

The neurosensory tachykinins substance P and neurokinin A directly induce keratinocyte nerve growth factor.

Nerve growth factor is an essential neurotrophic factor required for the growth and maintenance of cutaneous sensory nerves. In the skin, keratinocytes are a significant source of nerve growth factor; however, the regulation of cutaneous nerve growth factor production still remains to be fully understood. In this study we tested the hypothesis that neuropeptides released by cutaneous sensory nerves have the capacity to modulate directly the expression of keratinocyte nerve growth factor, which would have important implications for the maintenance and repair of nerves in the skin. In order to address this question experimentally we examined the effect of the neuropeptides, substance P and neurokinin A, on nerve growth factor expression in human keratinocytes and the murine keratinocyte PAM 212 cell line by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and the PC-12 nerve growth factor bioassay. The results of these studies indicated that substance P and neurokinin A can directly induce nerve growth factor mRNA expression and the secretion of bioactive nerve growth factor protein in both human and murine keratinocytes. The specificity of these responses was demonstrated using neuropeptide receptor antagonists and nerve growth factor blocking antibodies. Additional studies also demonstrated a significant in vivo upregulation of keratinocyte nerve growth factor expression in murine epidermis after the topical application of the neuropeptide releasing agent capsaicin. This is the first report demonstrating the induction of cutaneous nerve growth factor by sensory nerve-derived neuropeptides such as substance P and neurokinin A. This direct effect of the neurosensory system on keratinocyte nerve growth factor production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing.

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability.

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.

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.

Interleukin-18 expression and modulation in human corneal epithelial cells.

PURPOSE: The interferon-gamma-inducing factor Interleukin-18 (IL-18) is a recently described cytokine that appears to have multiple important pro-inflammatory effects including the induction of interferon-gamma (IFN-gamma) by activated T-cells. The expression of IL-18 by human cornea has not been previously reported. In the present study, we examine the possibility that human corneal epithelial cells are capable of producing this leukocyte-activating factor which may play an important role in IFN-gamma-dependent inflammation responses in the cornea. METHODS: Northern blot analysis and ELISA were used to investigate the in vitro expression of IL-18 mRNA and protein respectively in primary (HCEC) and transformed human corneal epithelial cells (HCET). To determine if IL-18 expression was modulated by pro-inflammatory mediators, cells were treated with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) or synthetic double stranded RNA (poly dI : dC). IL-18 bioactivity was determined in a leukocyte interferon-gamma induction assay and IL-18 was immunolocalized in whole human cornea by immunohistochemistry using a specific anti-IL-18 antibody. RESULTS: IL-18 mRNA and bioactive protein was constitutively expressed by human corneal epithelial cells and upregulated by PMA, LPS and poly dI : dC. The constitutive expression of IL-18 protein immunoreactivity was also demonstrated in the epithelial cells of whole human cornea tissue. CONCLUSIONS: This is the first study demonstrating that corneal epithelial cells are capable of producing the IFN-gamma inducing factor IL-18. Increased bioactive corneal IL-18 production can be induced by a number of pro-inflammatory agents and may play an important role in initiating gamma-interferon-mediated inflammatory responses in the cornea.

What is the most promising strategy for the treatment of metastasizing melanoma?

The treatment of patients with metastasizing melanoma, still one of the most deadly diseases in modern medicine, ranks among the greatest challenges that a clinician has to face. Metastatic melanoma also is one of the most profound sources of clinical frustration, since it provides far more ultimately defeating experiences than clinical victories. At the same time, the fascinating biology of melanoma has invited the study of this neuroectodermal tumor as a model system for dissecting many of the key problems of modern oncology, ranging from molecular oncogenesis via the controls of tumor proliferation, apoptosis, invasion, metastasis, and angiogenesis to tumor immunosurveillance and tumor drug resistance. Together with the dire need to develop more effective treatment modalities for improving both life expectancy and quality of life of affected patients, this has made metastatic melanoma a favorite model for the exploration of innovative strategies for tumor management. Encouragingly, many of these have already generated very promising results in animal models. However, this impressive level of research progress in conquering melanoma in the animal room contrasts rather pitifully with the actual progress made on the ward. This CONTROVERSIES feature, therefore, critically and soberly reviews the state of the art of treating metastatic melanoma today (distinguishing between nodal and distant metastases), and sharply defines unresolved or comparatively neglected key problems. In addition, this feature highlights several novel, provocative, hitherto underappreciated, yet potentially promising treatment approaches that deserve systematic exploration. Hopefully, this will offer further inspiration for the design and pursuit of innovative anti-melanoma strategies off-the-beaten-track.

Expression of proopiomelanocortin peptides in human dermal microvascular endothelial cells: evidence for a regulation by ultraviolet light and interleukin-1.

Proopiomelanocortin peptides such as alpha-melanocyte-stimulating hormone and adrenocorticotropin are expressed in the epidermal and dermal compartment of the skin after noxious stimuli and are recognized as modulators of immune and inflammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte-endothelial interactions during cutaneous inflammation by the expression of cellular adhesion molecules and cytokines such as interleukin-1. This study addresses the hypothesis that human dermal microvascular endothelial cells express both proopiomelanocortin and prohormone convertases, which are required to generate proopiomelanocortin peptides. Semiquantitative reverse transcriptase polymerase chain reaction and northern blot studies revealed a constitutive expression of proopiomelanocortin mRNA by human dermal microvascular endothelial cells in vitro that was time- and concentration-dependently upregulated by interleukin-1 beta. Furthermore, irradiation of human dermal microvascular endothelial cells with ultraviolet A1 (30J per cm(2)) or ultraviolet B (12.5 mJ per cm(2)) enhanced proopiomelanocortin expression as well as the production and release of the proopiomelanocortin peptides adrenocorticotropin and alpha-melanocyte-stimulating hormone. In addition to proopiomelanocortin, prohormone convertase 1 mRNA expression was detected by reverse transcriptase polymerase chain reaction in unstimulated human dermal microvascular endothelial cells and was augmented after exposure to alpha-melanocyte- stimulating hormone, interleukin-1 beta, or irradiation with ultraviolet. These findings demonstrate that human dermal microvascular endothelial cells express proopiomelanocortin and prohormone convertase 1 required for the generation of adrenocorticotropin. Additionally, human dermal microvascular endothelial cells express mRNA for the prohormone convertase 2 binding protein 7B2. Taken together these findings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of alpha-melanocyte-stimulating hormone. As proopiomelanocortin peptides were found to regulate the production of human dermal microvascular endothelial cell cytokines and adhesion molecules and to have a variety of anti-inflammatory properties these peptides may significantly contribute to the modulation of skin inflammation. J Invest Dermatol 115:1021-1028 2000

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

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-1alpha mRNA in a dose and time dependent manner and this induction was inhibited by an NK-2R antagonist. Secretion of bioactive IL-1alpha 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.

Neural regulation of endothelial cell-mediated inflammation.

There is increasing evidence that the cutaneous neurosensory system can directly modulate inflammatory responses in the skin by the release of neuropeptides such as substance P (SP). Dermal microvascular endothelial cell (DMEC) cellular adhesion molecule (CAM) expression plays a key role in directing leukocyte trafficking during cutaneous inflammatory responses. In recent studies, our laboratory examined the direct effect of SP on DMEC CAM expression and function in vitro and in vivo. Our studies indicate that DMEC express high affinity functional receptors for SP. After exposure to SP, DMEC expressed significant levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which was accompanied by increased binding to leukocytes expressing the appropriate integrin counter receptors for these CAM. We then determined the in vivo effect of released neuropeptides on DMEC CAM expression. Our results indicate that the topical cutaneous application of the neuropeptide-releasing agent capsaicin resulted in increased ICAM-1 and VCAM-1 immunostaining of microvascular cells in the skin of human volunteers. Little is known regarding the cellular regulatory events by which SP modulates DMEC CAM expression. Our studies indicate that SP-induced cellular Ca+2 signals led to the activation of the NF-kappaB pathway, resulting in nuclear translocation of p65/p50 heterodimers that bind to high-affinity tandem kappaB sites on the VCAM-1 promoter, whereas SP activation induced NF-AT activation and ICAM-1 DNA binding. Thus, these studies further support the role of the cutaneous neurologic system in modulating inflammatory processes in the skin.

The role of alpha-MSH as a modulator of cutaneous inflammation.

Among various neuropeptides such as substance P, calcitonin gene-related peptide and others, alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 (MC-1R), which is specific for alpha-MSH and ACTH, is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells alpha-MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL-2, IFN-gamma, TNF-alpha 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 alpha-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 NF kappa B is downregulated by alpha-MSH. In a mouse model i.v. or topical application of alpha-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 alpha-MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin.

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.

Substance P activates coincident NF-AT- and NF-kappa B-dependent adhesion molecule gene expression in microvascular endothelial cells through intracellular calcium mobilization.

Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the -191/-170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-alpha), and NF-kappa B (p65/p50) to tandem NF-kappa B binding sites at -76/-52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-kappa B. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-kappa B activation and binding. These data demonstrate coincident activation of NF-AT and NF-kappa B via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses.

Effect of ultraviolet light on the release of neuropeptides and neuroendocrine hormones in the skin: mediators of photodermatitis and cutaneous inflammation.

Ultraviolet (UV) irradiation of the skin causes both inflammation and alterations in the skin immune system. There is increasing experimental evidence that UV-induced skin inflammation is influenced by the sensory nervous system and the neuroendocrine system in the skin. The resulting complex network of cytokines, chemokines, neuropeptides, neuropeptide-degrading enzymes, neurohormones, and other inflammatory mediators mediate photodermatitis and cutaneous inflammation. Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) are released from sensory nerves innervating the skin upon UV exposure. In addition, a variety of cells in the skin produce increased neuroendocrine hormones such as proopiomelanocortin (POMC) peptides and their receptors as well as neurotrophins after UV exposure. Neuropeptides and neurohormones are capable of directly or indirectly mediating UV-induced cutaneous neurogenic inflammation by the induction of vasodilatation, plasma extravasation, and augmentation of UV-induced cytokine, chemokine, or cellular adhesion molecule expression required for activation and trafficking of inflammatory cells into the inflamed tissue. Neuropeptides and neurotrophins may also play a role in the repair of cutaneous UV injury. In addition to proinflammatory effects, UV-induced neuropeptides and neurohormones such as CGRP and alpha-melanocyte-stimulating hormone may have immunosuppressive effects in the skin. This review will focus on the role that SP, CGRP, POMC peptides, and their receptors may play in modulating UV-induced inflammation in the skin.

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

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 highly expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin is 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 (SLIGKVNH2) 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 expression and distribution in human skin and wounds.

Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.

VCAM-1 expression on human dermal microvascular endothelial cells is directly and specifically up-regulated by substance P.

Sensory nerves in skin are capable of releasing multiple neuropeptides, which modulate inflammatory responses by activating specific cutaneous target cells. Extravasation of particular subsets of leukocytes depends upon the regulated expression of cellular adhesion molecules such as VCAM-1 on microvascular endothelial cells. We examined the direct effect of cutaneous neuropeptides on the expression and function of human dermal microvascular endothelial cell (HDMEC) VCAM-1. A significant increase in VCAM-1 immunostaining of microvascular endothelium was observed in vivo following capsaicin application to human skin. Multiple cutaneous sensory C-fiber-released neuropeptides were evaluated for their ability to induce VCAM-1 cell surface expression on HDMEC. Only substance P (SP) was found to be capable of inducing HDMEC VCAM-1 expression. This SP-mediated VCAM-1 induction appeared to be a direct effect that did not require the release of other HDMEC-derived soluble factors. Increased HDMEC VCAM-1 mRNA expression was detected 1 h after the addition of SP, with peak mRNA increase at 6-9 h postinduction. FACS studies demonstrated a 6.5-fold increase in endothelial cell surface VCAM-1 expression detectable 16 h after addition of SP, which was specifically blocked by a neurokinin-1 receptor antagonist. Increased VCAM-1 cell surface expression on SP-treated HDMEC resulted in a 4-fold increase in the functional binding of 51Cr-labeled MOLT-4 T cells. These data indicate that SP is capable of directly and specifically up-regulating functional endothelial VCAM-1 expression and thus may play a key role in modulating certain inflammatory responses in the skin.