M1/M2 Macrophage Skewing is Related to Reduction in Types I, V, and VI Collagens with Aging in Sun-Exposed Human Skin.

Sun-exposed, aged human skin is fragile because of collagen fragmentation and loss. We recently reported that the balance of M1 and M2 macrophages is associated with chronic inflammation and related inflammaging in sun-exposed human skin. In this study, we analyzed its role in the maintenance of collagen matrix formation by performing histological analyses of human facial skin. In addition, RNA sequencing, protein assays, and functional assays revealed the details of the mechanism. The number of M2 macrophages was positively correlated with the abundance of type I collagen, whereas the M1/M2 ratio was negatively correlated with the abundance of type V and VI collagen, which are the essential minor collagens required for collagen assembly in the skin; however, there was no correlation with type III collagen. Furthermore, M2 macrophages induced the expression of the proteins required for the assembly of collagen fibrils, suggesting that the M1/M2 balance controls not only the quantity but also the quality of the collagen matrix. Indeed, M1 macrophages induced abnormal collagen fibrils consisting of types I, V, and VI collagens. Our results demonstrate the relationship between the M1/M2 balance and the dysregulation of collagen homeostasis in photoaged skin and suggest the possible involvement of macrophages in skin photoaging.

IL-34 Downregulation‒Associated M1/M2 Macrophage Imbalance Is Related to Inflammaging in Sun-Exposed Human Skin.

Macrophages can be polarized into two subsets: a proinflammatory (M1) or an anti-inflammatory (M2) phenotype. In this study, we show that an increased M1-to-M2 ratio associated with a decrease in IL-34 induces skin inflammaging. The total number of macrophages in the dermis did not change, but the number of M2 macrophages was significantly decreased. Thus, the M1-to-M2 ratio was significantly increased in sun-exposed aged skin and positively correlated with the percentage of p21+ and p16+ senescent cells in the dermis. The supernatant of M1 macrophages increased the percentages of senescence-associated ß-galactosidase‒positive cells, whereas the supernatant of M2 macrophages decreased the percentages of senescence-associated ß-galactosidase‒positive cells in vitro. Among the mechanisms that could explain the increase in the M1-to-M2 ratio, we found that the number of IL-34+ cells was decreased in aged skin and negatively correlated with the M1-to-M2 ratio. Furthermore, IL-34 induced the expression of CD206 and IL-10, which are M2 macrophage markers, in an in vitro assay. Our results suggest that a reduction in epidermal IL-34 in aged skin may skew the M1/M2 balance in the dermis and lead to low-grade chronic inflammation and inflammaging.

Regeneration of collagen fibrils at the papillary dermis by reconstructing basement membrane at the dermal-epidermal junction.

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

Basement Membrane Helps Maintain Epidermal Hyaluronan Content.

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix of most mammalian tissues, including the epidermis. It is synthesized in epidermis, and mainly metabolized after transfer to the liver via lymphatic vessels in the dermis following its passage through the basement membrane (BM) at the dermal-epidermal junction. The aim of the present study was to investigate the influence of BM integrity on the level of HA in the epidermis. Epidermal HA content was decreased in sun-exposed skin of older subjects, whose BM structure was impaired, compared with sun-exposed young skin and sun-protected skin, in which BM integrity was well maintained. In an organotypic culture model of sun-exposed facial skin, epidermal HA was increased in the presence of inhibitors of BM-degrading matrix metalloproteinases and heparanase. In a skin equivalent model treated with these inhibitors, HA content was increased in the epidermis, but decreased in conditioned medium. These findings suggest that the BM at the dermal-epidermal junction plays an important role in maintaining epidermal HA levels.

Decrease of laminin-511 in the basement membrane due to photoaging reduces epidermal stem/progenitor cells.

Daily sunlight exposure damages the epidermal basement membrane (BM) and disrupts epidermal homeostasis. Inter-follicular epidermal stem cells (IFE-SCs) regulate epidermal proliferation and differentiation, which supports epidermal homeostasis. Here, we examine how photoaging affects the function of IFE-SCs and we identify key components in their cellular environment (niche). We found that sun-exposed skin showed a decrease of MCSP-positive and ß1-integrin-positive cells concomitantly with a decrease of laminin-511 at the dermal-epidermal junction (DEJ), as compared with sun-protected skin. Higher levels of laminin-511 were associated with not only increased efficiency of colony formation, but also higher expression levels of MCSP as well as other stem cell markers such as Lrig1, ITGB1, CD44, CD46, DLL1, and K15 in keratinocytes from skin of 12- to 62-year-old subjects. UVB exposure to cultured human skin impaired laminin-511 integrity at the dermal-epidermal junction and reduced MCSP-positive basal epidermal cells as well as K15-positive cells. Combined treatment with matrix metalloproteinase and heparanase inhibitors protected the integrity of laminin-511 and inhibited the reduction of MCSP-positive cells and K15-positive cells. These results suggest that photoaging may reduce the levels of MCSP-positive and K15-positive epidermal stem/progenitor cells in the epidermis via loss of laminin-511 at the dermal-epidermal junction.

Reduction in Human Epidermal Langerhans Cells with Age Is Associated with Decline in CXCL14-Mediated Recruitment of CD14+ Monocytes.

The skin provides the first line of physical and immunological defense against environmental insults. However, the age-related changes in the immune function of human skin are unclear. Here, we investigated the age-related changes in epidermal Langerhans cells (LCs), which play a sentinel role in the initiation of the immune responses in the skin. We found a significant reduction in the number of epidermal LCs in sun-protected skin with age. Among the possible explanations for this reduction, the number of CD14+ CD207+ CCR6+ dermal-resident monocytes that can differentiate into epidermal LCs was markedly reduced with age (P = 0.0057). Among the chemokines that can recruit these cells into the skin, the expression of CXCL14 was significantly down-regulated in epidermal keratinocytes with age. In addition, we discovered that young skin recruited a significantly higher number of monocytic THP-1 cells compared with old skin ex vivo. This recruitment was blocked by CXCL14 neutralizing antibody and conversely promoted by CXCL14 treatment. Collectively, our findings indicate that decreased CXCL14-mediated recruitment of CD14+ monocytes in human skin results in the reduction of epidermal LCs with age, and CXCL14 may provide a therapeutic target for the prevention of age-related reduction in LCs.

Label-free stimulated Raman scattering microscopy visualizes changes in intracellular morphology during human epidermal keratinocyte differentiation.

Epidermal keratinocyte (KC) differentiation, which involves the process from proliferation to cell death for shedding the outermost layer of skin, is crucial for the barrier function of skin. Therefore, in dermatology, it is important to elucidate the epidermal KC differentiation process to evaluate the symptom level of diseases and skin conditions. Previous dermatological studies used staining or labelling techniques for this purpose, but they have technological limitations for revealing the entire process of epidermal KC differentiation, especially when applied to humans. Here, we demonstrate label-free visualization of three-dimensional (3D) intracellular morphological changes of ex vivo human epidermis during epidermal KC differentiation using stimulated Raman scattering (SRS) microscopy. Specifically, we observed changes in nuclei during the initial enucleation process in which the nucleus is digested prior to flattening. Furthermore, we found holes left behind by improperly digested nuclei in the stratum corneum, suggesting abnormal differentiation. Our findings indicate the great potential of SRS microscopy for discrimination of the degree of epidermal KC differentiation.

Evaluation of psychological stress in confined environments using salivary, skin, and facial image parameters.

Detecting the influence of psychological stress is particularly important in prolonged space missions. In this study, we determined potential markers of psychological stress in a confined environment. We examined 23 Japanese subjects staying for 2 weeks in a confined facility at Tsukuba Space Center, measuring salivary, skin, and facial image parameters. Saliva was collected at four points in a single day to detect diurnal variation. Increases in salivary cortisol were detected after waking up on the 4th and 11th days, and at 15:30 on the 1st and in the second half of the stay. Transepidermal water loss (TEWL) and sebum content of the skin were higher compared with outside the facility on the 4th and 1st days respectively. Increased IL-1ß in the stripped stratum corneum was observed on the 14th day, and 7 days after leaving. Differences in facial expression symmetry at the time of facial expression changes were observed on 11th and 14th days. Thus, we detected a transition of psychological stress using salivary cortisol profiles and skin physiological parameters. The results also suggested that IL-1ß in the stripped stratum corneum and facial expression symmetry are possible novel markers for conveniently detecting psychological stress.

Direct control of regulatory T cells by keratinocytes.

Environmental challenges to epithelial cells trigger gene expression changes that elicit context-appropriate immune responses. We found that the chromatin remodeler Mi-2ß controls epidermal homeostasis by regulating the genes involved in keratinocyte and immune-cell activation to maintain an inactive state. Mi-2ß depletion resulted in rapid deployment of both a pro-inflammatory and an immunosuppressive response in the skin. A key target of Mi-2ß in keratinocytes is the pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP). Loss of TSLP receptor (TSLPR) signaling specifically in regulatory T (Treg) cells prevented their activation and permitted rapid progression from a skin pro-inflammatory response to a lethal systemic condition. Thus, in addition to their well-characterized role in pro-inflammatory responses, keratinocytes also directly support immune-suppressive responses that are critical for re-establishing organismal homeostasis.

The effect of calprotectin on TSLP and IL-25 production from airway epithelial cells.

BACKGROUND: Calprotectin is a heterodimer complex of the S100A8 and S100A9 proteins, and has various functions as an innate mediator at the sites of inflammation. The aim of this study was to elucidate the roles of calprotectin in the eosinophilic chronic rhinosinusitis (ECRS). METHODS: Allergen-induced production of calprotectin was evaluated in cultured normal human bronchial epithelial (NHBE) cells by ELISA and RT-PCR. We then examined the roles of calprotectin on Alternaria alternata (Alternaria)-induced production of thymic stromal lymphopoietin (TSLP) and IL-25 in NHBE cells. The extracellular concentration and allergen-induced secretion of calprotectin in cultured primary nasal epithelial (PNE) cells were examined and compared between patients with ECRS and non-eosinophilic chronic rhinosinusitis (NECRS). RESULTS: Alternaria, house dust mites, protease from Staphylococcus aureus, papain, trypsin, polyinosinic:polycytidylic acid and lipopolysaccharide stimulated calprotectin production in the cultured NHBE cells. The combination of calprotectin and ATP stimulated the production of TSLP and IL-25 in NHBE cells, and calprotectin stimulated Alternaria-induced production of TSLP and IL-25, which was suppressed by blocking P2 purinergic receptors and by treatment with siRNA for S100A8, S100A9 or calprotectin receptors (Toll-like receptor 4 or receptor for advanced glycation end products). Allergen-induced calprotectin production was significantly stimulated in PNE cells from patients with ECRS. CONCLUSIONS: These results indicate that calprotectin enhances the allergen-induced Th2-type inflammatory responses in airway epithelial cells via the secretion of TSLP and IL-25, and that calprotectin secreted by the epithelial cells may be involved in the pathogenesis of ECRS.

In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy.

Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

High-throughput, high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system.

Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. To develop a comprehensive screening method for novel pigmentation regulators, we used immortalized melanocytes and keratinocytes in co-culture to screen large numbers of compounds. High-throughput screening plates were subjected to digital automated microscopy to quantify the pigmentation via brightfield microscopy. Compounds with pigment suppression were secondarily tested for their effects on expression of microphthalmia transcription factor (MITF) and several pigment regulatory genes, and further validated in terms of non-toxicity to keratinocytes/melanocytes and dose-dependent activity. The results demonstrate a high-throughput, high-content screening approach, which is applicable to the analysis of large chemical libraries using a co-culture system. We identified candidate pigmentation inhibitors from 4000 screened compounds including zoxazolamine, 3-methoxycatechol and alpha-mangostin, which were also shown to modulate expression of MITF and several key pigmentation factors and are worthy of further evaluation for potential translation to clinical use.

S100A9 is a novel ligand of EMMPRIN that promotes melanoma metastasis.

The calcium-binding proteins S100A8 and S100A9 can dimerize to form calprotectin, the release of which during tissue damage has been implicated in inflammation and metastasis. However, receptor(s) mediating the physiologic and pathophysiologic effects of this damage-associated "danger signal" are uncertain. In this study, searching for candidate calprotectin receptors by affinity isolation-mass spectrometry, we identified the cell surface glycoprotein EMMPRIN/BASIGIN (CD147/BSG). EMMPRIN specifically bound to S100A9 but not S100A8. Induction of cytokines and matrix metalloproteases (MMP) by S100A9 was markedly downregulated in melanoma cells by attenuation of EMMPRIN. We found that EMMPRIN signaling used the TNF receptor-associated factor TRAF2 distinct from the known S100-binding signaling pathway mediated by RAGE (AGER). S100A9 strongly promoted migration when EMMPRIN was highly expressed, independent of RAGE, whereas EMMPRIN blockade suppressed migration by S100A9. Immunohistologic analysis of melanomas revealed that EMMPRIN was expressed at both the invasive edge of lesions and the adjacent epidermis, where S100A9 was also strongly expressed. In epidermal-specific transgenic mice, tail vein-injected melanoma accumulated in skin expressing S100A9 but not S100A8. Together, our results establish EMMPRIN as a receptor for S100A9 and suggest the therapeutic use in targeting S100A9-EMMPRIN interactions.

Functional characteristics of the skin surface of children approaching puberty: age and seasonal influences.

The aim of this study was to determine differences in the functional properties of the stratum corneum of children and adults, focusing on the influence of approaching puberty. Biophysical measurements were made of the stratum corneum of 32 healthy Japanese children aged 10-14 years and their mothers in summer and the following winter. The children showed significantly lower skin surface hydration. Stratum corneum barrier function, evaluated in terms of trans-epidermal water loss, was poorer on the forearm in the children than in the adults regardless of season. By contrast, the stratum corneum barrier of the cheek, which was better in the children, tended to become poorer when the children reached puberty. Although the immaturity of the cornified envelopes of the superficial corneocytes, which ratio increased significantly in winter, was not different from that of adults, the corneocytes were significantly smaller in the children, suggesting a more rapid turnover of the stratum corneum. The amount of skin surface lipid, which was measured only on the cheek, remained low until 13 years of age, but at 14 years of age it increased remarkably, approaching adult levels. We conclude that, until puberty, most functional characteristics of the skin of children remain distinct from those of adults.

Glucocorticoids enhance Toll-like receptor 2 expression in human keratinocytes stimulated with Propionibacterium acnes or proinflammatory cytokines.

Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-alpha, and IL-1alpha. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis.

Extracellular ATP has stimulatory effects on the expression and release of IL-6 via purinergic receptors in normal human epidermal keratinocytes.

Extracellular ATP regulates proliferation and differentiation, functioning as an important messenger via purinergic (P2) receptors in keratinocytes. In this study, we investigated the effects of ATP on cytokine production in cultured normal human epidermal keratinocytes (NHEKs). Adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), adenosine 5'-O-2-(thio)diphosphate (ADPbetaS), ADP, ATP, and 2', 3'-O-(4-benzoyl-benzoyl) ATP (BzATP) significantly increased the release of IL-6. The P2 antagonists, suramin-, reactive blue 2-, and periodate-oxidized ATP, inhibited ATP-induced IL-6 release, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, adenosine 3'-phosphate 5'-phosphate, 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, and pertussis toxin did not. SQ22563, an adenylate cyclase inhibitor, inhibited ATP-induced IL-6 release. ATPgammaS, ADPbetaS, ATP, and BzATP significantly increased the intracellular cAMP content. Reverse transcription-PCR showed expression of P2Y1, P2Y2, P2Y4, P2Y11, P2Y12, P2Y13, P2X1, P2X4, P2X5, P2X6, and P2X7 receptor subtypes. Additionally, UVB radiation evoked the release of ATP from NHEKs. The release of IL-6 and the expression of IL-6 mRNA were increased after UVB radiation, and these increases were also inhibited by P2 receptor antagonists. These results suggest that cAMP-generating P2Y receptors are likely functional in ATP-induced IL-6 production in NHEKs. Furthermore, in UVB-radiated cells, we note the possibility that P2 receptor antagonists may reduce skin inflammation.

Neuropeptide control mechanisms in cutaneous biology: physiological and clinical significance.

The skin as a barrier and immune organ is exposed to omnipresent environmental challenges such as irradiation or chemical and biologic hazards. Neuropeptides released from cutaneous nerves or skin and immune cells in response to noxious stimuli are mandatory for a fine-tuned regulation of cutaneous immune responses and tissue maintenance and repair. They initialize host immune responses, but are equally important for counter regulation of proinflammatory events. Interaction of the nervous and immune systems occurs both locally - at the level of neurogenic inflammation and immunocyte activation - and centrally - by controlling inflammatory pathways such as mononuclear activation or lymphocyte cytokine secretion. Consequently, a deregulated neurogenic immune control results in disease manifestation and frequently accompanies chronic development of cutaneous disorders. The current understanding, therapeutic options, and open questions of the role that neuropeptides such as substance P, calcitonin gene-related peptide, vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide, neuropeptide Y, or others play in these events are discussed. Progress in this field will likely result in novel therapies for the management of diseases characterized by deregulated inflammation, tissue remodeling, angiogenesis, and neoplasm.

Neuropeptides concentrations in the skin of a murine (NC/Nga mice) model of atopic dermatitis.

BACKGROUND: It has been reported that the expression of neuropeptides (NPs), and the density and structure of peripheral nerves in atopic dermatitis (AD) are different from those in normal skin. OBJECTIVE: We investigated the role of NPs, in the development of AD with quantitative study of substance P (SP) and calcitonin gene-related peptide (CGRP) in the skin of AD-model mice. METHODS: We measured the NPs in the skin of mice (NC/Nga as AD-model mice, BALB/c and C57BL/6 as control) by enzyme-linked immunosorbentassay (ELISA). Peripheral nerve fibers and SP in the skin were stained by immunohistochemical staining, using anti-PGP9.5 antibody and anti-SP antibody. RESULTS: Under conventional condition, SP concentration in AD-like skin lesions of NC/Nga mice was higher than that in non-affected skin of the same mice. Under specific pathogen-free condition, SP concentration in the skin of NC/Nga mice was higher than that in the skin of BALB/c and C57BL/6 mice. In contrast, CGRP concentration in the skin lesions was lower than that in non-affected skin of NC/Nga mice. SP was detected not only in the nerve fibers in the dermis but also in mast cells in the inflammatory areas. CONCLUSIONS: The skin of NC/Nga mice contains more SP congenitally, and environmental factors may aggravate this abnormal condition. We hypothesize that increase of SP accompanied with a decrease of CGRP in the skin may play important roles in the pathogenesis and development of AD.

Regulation of plasma substance P and skin mast cells by odorants.

BACKGROUND: Mast cells stimulate inflammation and itch sensation in the skin by releasing various mediators when they are activated. Stress exacerbates some skin diseases. We have reported that inhalation of certain odorants modulates immune reactions in the skin. OBJECTIVE: The possible usage of odorants in the regulation of skin inflammation and itch sensation was to be examined. METHODS: Female volunteers were subjected to interview stress with or without odorant inhalation. Mice were immobilized while inhaling odorants. Toluidene blue-stained sections were analyzed for activated mast cells. Plasma substance P level was determined by enzyme-linked immunoassay. RESULTS: Interview stress induced plasma substance P only in volunteers who did not inhale odorants containing 2% 1,3-dimethoxy-5-methyl benzene (DMMB). Immobilization stress induced mast cell activation in mice and the activation was blocked by exposure to DMMB. CONCLUSIONS: Stress causes mast cell activation via an increase in substance P. The effect of stress is suppressed by inhalation of DMMB.

Stress augmented ultraviolet-irradiation-induced pigmentation.

It was reported that adrenocorticotropic hormone stimulates melanogenesis in cultured melanocytes. Stress (high population density and restraint stress) induced a significant increase in adrenocorticotropic hormone levels in plasma and skin compared to control. The serum obtained from HR-1 x HR/De F1 female mice subjected to stress showed significantly increased tyrosinase activity in human melanocytes compared to that from nonstressed mice. The increase in tyrosinase activity was inhibited in the presence of 10 nM corticostatin, an adrenocorticotropic hormone inhibitor. The aim of this study was to examine whether adrenocorticotropic hormone released into the circulation under stressful conditions is associated with the regulation of ultraviolet-induced pigmentation. Mice divided into three groups were housed for 22 d under the following conditions: five mice per cage (control); 10 mice per cage (high population density); restraint stress 4 h per d. The animals were exposed to ultraviolet-B irradiation (72 mJ per cm2, thrice per wk). After ultraviolet-B irradiation, delayed tanning was marked in stressed mice. The number of dihydroxyphenylalanine-positive melanocytes also significantly increased in stressed animals. Pretreatment with 100 microg of corticostatin inhibited the augmentation of the stress-induced pigmentary response and the increase in dihydroxyphenylalanine-positive melanocytes after ultraviolet irradiation. Adrenocorticotropic hormone released by stress may activate tyrosinase in melanocytes, resulting in the augmentation of ultraviolet-induced pigmentation. These results suggest that adrenocorticotropic hormone is at least partly responsible for the sensitivity of the pigmentary response after ultraviolet irradiation under stressful conditions.