Skin Cell Types Research Articles

Antioxidant and anti‐inflammatory activities of melanocortin peptides

α‐Melanocyte‐stimulating hormone (α‐MSH) has previously been identified as a potent anti‐inflammatory agent in various tissues including the skin. It operates by binding to the melanocortin‐1 receptor (MC‐1R) which results in the elevation of cyclic AMP. α‐MSH opposes the action of several proinflammatory cytokines including tumour necrosis factor‐α (TNF‐α). We have shown that α‐MSH can inhibit TNF‐α‐stimulated activation of nuclear factor‐κB (NF‐κB) in human cultured melanocytes, melanoma cells, keratinocytes, fibroblasts, Schwann cells and olfactory ensheathing cells. It also inhibits TNF‐α‐stimulated upregulation of intercellular adhesion molecule‐1 (ICAM‐1) in many of these cells and can inhibit peroxide‐stimulated activation of glutathione peroxidase, suggesting an antioxidant role. α‐MSH is also able to stimulate intracellular calcium release in keratinocytes and fibroblasts (which do not readily show detectible cyclic AMP elevation) but only in the presence of PIA (an adenosine agonist). The carboxyl terminal tripeptides KPV/KP‐D‐V are reported to be the minimal sequences necessary to convey anti‐inflammatory potential, but evidence on how they act is not fully known. Stable transfection of Chinese hamster ovary cells with MC‐1R suggests that the KPV peptides operate by this receptor, at least by elevating intracellular calcium. Elevation of cyclic AMP by these tripeptides has not been detected in any cell type studied; however, calcium elevation can inhibit TNF‐α‐stimulated NF‐κB activity (as for cyclic AMP). In conclusion, the MSH peptides convey anti‐inflammatory and antioxidant activity in many cell types in skin and nerve, by counteracting proinflammatory cytokine signalling. The KPV peptides appear to act functionally via the MC‐1R and can also elevate intracellular calcium.

Clinical potential of Melanotan® (NDP‐α‐MSH) in skin protection – current status and future perspective

There is good epidemiological evidence that melanin in the skin protects against sunburn and skin cancer resulting from excessive ultraviolet radiation (UVR) exposure. Melanin is synthesized in a multistep biochemical pathway within specialized cell types in human skin called melanocytes situated in the epidermis. UV light causes an increase in melanocyte‐stimulating hormone (α‐MSH) receptor activity on cutaneous melanocytes (MC1‐R) that results in an increase of eumelanin within the epidermal melanocytes. Melanotan is a synthetic analogue of α‐MSH with potent melanogenic activity in animals and humans. We have recently completed a double‐blind, randomized controlled study in 80 healthy Caucasian adult subjects. A fixed dose of Melanotan (0.16 mg/kg/day) or placebo in a 3 : 1 ratio was given as three cycles of daily subcutaneous injections over 10 days each month for three consecutive months. Changes in skin pigmentation were quantified by serial skin reflectance measurements and UVR injury, as determined by ×3 MED exposure to broadband UVR, was assessed by measurement of apoptosis (sunburn cells). Results demonstrated that a highly significant increase in skin melanin was attained in all subjects on active treatment compared with placebo, and this was especially pronounced in subjects with lighter skin types (Fitzpatrick I and II; P < 0.0001). In these latter subjects, significant sunburn protection was also determined. The ability of Melanotan to stimulate melanin production without the associated UV‐induced skin damage may prove a useful adjunct to current photoprotective strategies to stem the marked rise in skin cancer incidence. Currently, we are studying the therapeutic efficacy of a slow‐release implant of Melanotan in skin disorders such as polymorphic light eruption and psoriasis.

Expression of C-type lectin receptors by subsets of dendritic cells in human skin.

C-type lectins are cell surface receptors that recognize carbohydrate structures which are often part of microbial pathogens. Several of these molecules are expressed on dendritic cells and are involved in antigen uptake. Expression of C-type lectins on dendritic cells of the human skin, i.e. Langerhans cells of the epidermis and dermal dendritic cells, has been incompletely studied to date. We therefore investigated C-type lectins in situ and on dendritic cells obtained by migration from skin explants by immunofluorescence and flow cytometry. Emphasis was laid on expression patterns of DEC-205/CD205 and BDCA-2, a marker for plasmacytoid dendritic cells. Langerhans cells in situ expressed low levels of DEC-205. Expression was upregulated upon maturation in skin explant organ culture. Most dermal dendritic cells were found to be positive for DEC-205 and DC-SIGN/CD209. Few BDCA-2-expressing cells were found in most skin samples. They were located in small groups in the dermis close beneath the basement membrane. The vast majority of all types of dendritic cells in normal human skin was of immature phenotype, i.e. did not express DC-LAMP/CD208. It is concluded that normal appearing human skin harbors different subsets of dendritic cells including few scattered BDCA-2-expressing cells, presumably plasmacytoid dendritic cells, expressing variable sets of C-type lectin receptors. This may critically contribute to the capacity of the skin immune system to flexibly respond to the world of microbial pathogens.

Role of extracellular adenosine triphosphate in human skin.

The nucleotide adenosine triphosphate (ATP) has long been known to drive and participate in countless intracellular processes. Extracellular ATP and its metabolite adenosine have also been shown to exert a variety of effects on nearly every cell type in human skin. Knowledge of the sources and effects of extracellular ATP in human skin may help shape new therapies for skin injury, inflammation, and numerous other cutaneous disorders. The objective of this review is to introduce the reader to current knowledge regarding the sources and effects of extracellular ATP in human skin and to outline areas in which further research is necessary to clarify the nature and mechanism of these effects. Extracellular ATP seems to play a direct role in triggering skin inflammatory, regenerative, and fibrotic responses to mechanical injury, an indirect role in melanocyte proliferation and apoptosis, and a complex role in Langerhans cell-directed adaptive immunity.

Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders.

Recently melanogenic paracrine or autocrine cytokine networks have been discovered in vitro between melanocytes and other types of skin cells. These include endothelin (ET)-1, granulocyte macrophage colony stimulating factor, membrane-type stem cell factor (SCF) and growth-related oncogene-alpha for interactions between keratinocytes and melanocytes, and hepatocyte growth factor and soluble type SCF for interactions between fibroblasts and melanocytes. These networks are also associated with corresponding receptors expressed on melanocytes, including ET B receptor and the SCF receptor, c-KIT. Consistent with in vitro findings on the melanogenic paracrine or autocrine cytokine networks, we have found that the up- or down-regulation of such networks is intrinsically involved in vivo in the stimulation of melanocyte functions in several epidermal hyper- or hypo-pigmentary disorders. These are ET-1/ET B receptor as well as membrane type SCF/c-KIT for ultraviolet B-melanosis, granulocyte macrophage colony stimulating factor for ultraviolet A-melanosis, ET-1/ET B receptor as well as membrane type SCF for lentigo senilis, growth related oncogene-alpha for Riehl's melanosis, sphingosylphosphorylcholine for hyperpigmentation in atopic dermatitis, ET-1 for seborrhoeic keratosis, soluble type SCF as well as hepatocyte growth factor for dermatofibroma and café-au-lait macules, and c-KIT for vitiligo vulgaris. These unveiled regulatory mechanisms involved in the abnormal up- or down-regulated levels of lesional melanocyte function provide new insights into therapeutic tools utilizing blockage of responsible cytokine networks.

Role of Extracellular Adenosine Triphosphate in Human Skin

Background:The nucleotide adenosine triphosphate (ATP) has long been known to drive and participate in countless intracellular processes. Extracellular ATP and its metabolite adenosine have also been shown to exert a variety of effects on nearly every cell type in human skin. Knowledge of the sources and effects of extracellular ATP in human skin may help shape new therapies for skin injury, inflammation, and numerous other cutaneous disorders.Objective:The objective of this review is to introduce the reader to current knowledge regarding the sources and effects of extracellular ATP in human skin and to outline areas in which further research is necessary to clarify the nature and mechanism of these effects.Conclusion:Extracellular ATP seems to play a direct role in triggering skin inflammatory, regenerative, and fibrotic responses to mechanical injury, an indirect role in melanocyte proliferation and apoptosis, and a complex role in Langerhans cell-directed adaptive immunity.

Minoxidil: mechanisms of action on hair growth.

We have known for over 30 years that minoxidil stimulates hair growth, yet our understanding of its mechanism of action on the hair follicle is very limited. In animal studies, topical minoxidil shortens telogen, causing premature entry of resting hair follicles into anagen, and it probably has a similar action in humans. Minoxidil may also cause prolongation of anagen and increases hair follicle size. Orally administered minoxidil lowers blood pressure by relaxing vascular smooth muscle through the action of its sulphated metabolite, minoxidil sulphate, as an opener of sarcolemmal KATP channels. There is some evidence that the stimulatory effect of minoxidil on hair growth is also due to the opening of potassium channels by minoxidil sulphate, but this idea has been difficult to prove and to date there has been no clear demonstration that KATP channels are expressed in the hair follicle. A number of in vitro effects of minoxidil have been described in monocultures of various skin and hair follicle cell types including stimulation of cell proliferation, inhibition of collagen synthesis, and stimulation of vascular endothelial growth factor and prostaglandin synthesis. Some or all of these effects may be relevant to hair growth, but the application of results obtained in cell culture studies to the complex biology of the hair follicle is uncertain. In this article we review the current state of knowledge on the mode of action of minoxidil on hair growth and indicate lines of future research.

Skin biopsy in Lafora disease: genotype-phenotype correlations and diagnostic pitfalls.

Lafora disease is characterized by pathognomonic inclusions, Lafora bodies (LB), in neurons and other cell types. In skin, LB have been reported in either eccrine sweat glands or in apocrine sweat glands. The disease is caused by mutations in either the EPM2A gene or in a second yet-unknown gene. Here the authors determine whether a genotype-phenotype correlation exists between the genetic form of the disease and the skin cell type affected by LB formation. Also is described an important source of false positivity in the use of axillary biopsies for disease diagnosis.

Biological effects of glycolic acid on dermal matrix metabolism mediated by dermal fibroblasts and epidermal keratinocytes.

Glycolic acid (GA), one of the alpha-hydroxy acids, is widely used as an agent for chemical peeling. Although there are several reports about the clinical effects of GA in the literature, its biological mechanism remains mostly unclear, and there are only a few reports about its effects on skin rejuvenation mediated by keratinocytes. The aim of this study was to investigate the effect of GA on the dermal matrix metabolism of keratinocytes and fibroblasts using in vitro and ex vivo systems. Our study shows that GA not only directly accelerates collagen synthesis by fibroblasts, but it also modulates matrix degradation and collagen synthesis through keratinocyte-released cytokines. We confirm that IL-1alpha is one of the primary mediators for matrix degradation released from keratinocytes after GA treatment. These results suggest that GA contributes to the recovery of photodamaged skin through various actions, depending on the skin cell type.

The Importance of Cationic Amino Acid Transporter Expression in Human Skin

Inducible nitric oxide synthase and arginase activities are acknowledged as important players in human skin epidermal function. For proper enzyme function the substrate availability of L-arginine for both enzymes and thus its transport across the cell membrane via the y+-system (also named cationic amino acid transporters) is critical. Here, we examine the expression of cationic amino acid transporters and their functional role in modulating inducible nitric oxide synthase and arginase activities in human skin and primary keratinocytes, fibroblasts and endothelial cells as well as their impact on keratinocyte proliferation. Skin biopsies were found to express constitutively both cationic amino acid transporter-1 and cationic amino acid transporter-2 mRNA, an expression pattern known to occur in hepatocytes and muscle cells only. To determine the cellular components expressing cationic amino acid transporter, we analyzed the expression patterns in the different human skin cell types in vitro, i.e., in fibroblasts, dermal endothelial cells, and keratinocytes as well as in the HaCaT cell line. An ubiquitous cationic amino acid transporter-1 mRNA expression was found in all cells, whereas constitutive cationic amino acid transporter-2 mRNA expression occurs in resident keratinocytes and dermal endothelial cells only. De novo induction of cationic amino acid transporter-2 and inducible nitric oxide synthase by proinflammatory cytokines was seen in fibroblasts and HaCaT. Competitive inhibition of the cationic amino acid transporter-mediated L-arginine transport by culturing primary human keratinocytes in the presence of increased L-lysine concentration led to decreased inducible nitric oxide synthase and arginase activities with a concomitant significant decrease in keratinocyte proliferation. In summary, our results demonstrate that human keratinocytes constitutively express cationic amino acid transporters 1 and 2 and that cationic amino acid transporter mediated L-arginine influx, is essential for both inducible nitric oxide synthase and arginase enzyme activities, which in turn modulate proliferation and differentiation of human epidermal skin cells.

Differentiation of bone marrow cells in culture and in vivo

Recently, the bone marrow has been demonstrated to include progenitor cells that give rise to a large variety of cell types. We found that 3.5–4% of adult rat bone marrow cells express hepatocyte growth factor (HGF) receptor (c-Met) and α-fetoprotein (AFP). Using a hepatocyte growth medium supplemented with hepatocyte growth factor and epidermal growth factor, we succeeded in inducing hepatocyte-like cells from the bone marrow cells in culture. The cells expressed albumin as shown by immunocytochemistry and mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, the late markers of mature hepatocytes. Furthermore, in a skin reconstitution system in vivo, we showed that mouse bone marrow cells differentiated to several different cell types of skin within 3 weeks when combined with mouse embryonic skin cells.

Decreased expression of keratinocyte beta1 integrins in chronically sun-exposed skin in vivo.

Chronic exposure to ultraviolet (UV) radiation induces changes in the skin structure which are mostly found in the superficial dermis and at the dermal-epidermal junction. Keratinocytes and fibroblasts contribute both to the synthesis and to the degradation of the molecules important for the integrity of this skin site. While several studies have reported on alterations of dermal components and of the functions of fibroblasts in vivo and in vitro after UV exposure, recent data suggested that keratinocytes could be the main skin cell type involved in the photoageing process. In this study, we analysed the expression of two keratinocyte molecules namely, beta1 integrin (a proliferation marker) and involucrin (a differentiation marker) in sun-exposed and sun-protected facial skin of 16 healthy patients undergoing facial lifting. Methods included histology, immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analysis. Sun-exposed skin displayed the characteristic morphological and molecular features of dermal photoageing, compared with sun-protected skin, including dermal elastosis, diminished fibrillin and type VII collagen expression. Analysis of the epidermis in sun-exposed vs. sun-protected skin showed no histological differences, but dramatic changes in the expression of beta1 integrin and involucrin. In sun-exposed skin, expression of beta1 integrin protein by epidermal basal cells was reduced, paralleling a downregulation of beta1 integrin mRNA, whereas involucrin protein expression was greatly enhanced in the superficial epidermal cell layers. Interestingly, the ratio between involucrin and beta1 integrin protein expression was consistently increased in sun-exposed skin sites. Collectively these results demonstrate that epidermal homeostasis is impaired by chronic UV exposure, and define beta1 integrin expression as a molecular marker of the epidermal photoageing process.

An efficient gene transduction system for studying gene function in primary human dermal fibroblasts and epidermal keratinocytes.

One of the critical challenges for cellular genetic studies in primary human skin cells is lack of a gene delivery system that provides efficient transduction and sustained expression of the transgenes. Due to the limited time of survival in culture, the processes of drug selection and clonal expansion for establishing gene stably expressing cell lines are not a realistic option for primary skin cells. We have examined various gene transduction techniques in primary dermal fibroblasts and epidermal keratinocytes of human skin. We report here that vectors based on the human immunodeficiency virus (HIV, lentivirus) offer more than 90% gene transduction efficiency and sustained expression of transgenes in both human skin cell types. In contrast, most of the commonly used techniques have at best 30% transduction efficiency in these cells. Using two previously reported migration control genes, protein kinase Cdelta and p38alpha-MAPK, as examples, we provide evidence that the unprecedented efficiency of the lentiviral system enables a clear detection of the genes' dominant negative effects, which are otherwise greatly compromised by ordinary transfection techniques. We believe that a wide application of this gene transduction system will greatly benefit studies of gene function in human skin cells.

Localization of Calcineurin/NFAT in Human Skin and Psoriasis and Inhibition of Calcineurin/NFAT Activation in Human Keratinocytes by Cyclosporin A

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

Effects of micronutrient supplements on u.v.-induced skin damage.

Development of an orally-administered systemic agent that could reduce the effects of u.v. exposure on skin could potentially have a major effect on the incidence of skin cancers and photo-ageing. A number of micronutrients have been suggested to have metabolic properties that could induce this protection, and our data indicate that n-3 polyunsaturated fatty acids are particularly effective in this role. The mechanisms of action of n-3 polyunsaturated fatty acids appear to depend on their anti-inflammatory properties, acting to reduce the u.v.-induced release of cytokines and other mediators from a variety of skin cell types.

MRNA Expression of Multiple Cytochrome P450 Isozymes in Four Types of Cultured Skin Cells

Background: Many drugs are known to induce allergic reactions in the skin. The metabolic activation of drugs resulting in the formation of protein adducts is thought to be a first step in the induction of these allergic reactions. We postulated that dermal tissue might be a site of drug activation by cytochrome P450 (CYP) isozymes. Methods: Messenger RNA was extracted from cultured Langerhans cells, keratinocytes, fibroblasts and melanocytes from 6 individuals, and CYP mRNA expression was analyzed by RT-PCR. Results: CYP1A1, 1B1 and 2E1 were found in all four cell types. CYP2A6, 2C, 2D6, 3A5, 3A7 and 4B1 mRNA was expressed in a cell-type- and/or individual-specific manner. CYP1A2, 2A7, 2B6 and 3A4 mRNA was not detectable. Conclusions: The mRNA for a variety of CYP isozymes was expressed in all four types of skin cells examined. These CYP enzymes may be involved in the pathogenesis of drug-induced allergic reactions in the skin.

Biomarkers of Human Skin Cells Identified Using DermArray DNA Arrays and New Bioinformatics Methods

Biomarker genes of human skin-derived cells were identified by new simple bioinformatic methods and DNA microarray analysis utilizing in vitro cultures of normal neonatal human epidermal keratinocytes, melanocytes, and dermal fibroblasts. A survey of 4405 human cDNAs was performed using DermArray DNA microarrays. Biomarkers were rank ordered by “likelihood ratio” algorithms and stringent selection criteria that have general applicability for analyzing a minimum of three RNA samples. Signature biomarker genes (up-regulated in one cell type) and anti-signature biomarker genes (down-regulated in one cell type) were determined for the three major skin cell types. Many of the signature genes are known biomarkers for these cell types. In addition, 17 signature genes were identified as ESTs, and 22 anti-signature biomarkers were discovered. Quantitative RT–PCR was used to verify nine signature biomarker genes. A total of 158 biomarkers of normal human skin cells were identified, many of which may be valuable in diagnostic applications and as molecular targets for drug discovery and therapeutic intervention.

Biochemical Characterization of Endothelin-converting Enzyme-1α in Cultured Skin-derived Cells and Its Postulated Role in the Stimulation of Melanogenesis in Human Epidermis

The vasoconstrictive peptide endothelin-1 (ET-1) is expressed in human epidermis at the gene and protein levels and plays an important role in stimulating pigmentation via its increased secretion by keratinocytes following ultraviolet B (UVB) irradiation. However, one or more biological mechanisms underlying the secretion of ET-1 by keratinocytes in human skin have never been evaluated. In mammalian endothelial cells, a membrane-bound neutral metalloproteinase, termed endothelin-converting enzyme (ECE), catalyzes the specific cleavage of the inactive precursor Big ET to produce mature active ET, which leads in turn to the secretion of ET by those cells. To clarify the potential involvement of ECE in the processing and secretion of ET-1 by human keratinocytes, we synthesized the N-terminal peptide of human ECE-1alpha and generated a rabbit polyclonal antibody (alphaPEPT6) that specifically recognizes ECE-1alpha. Reverse transcription PCR and Western blotting analysis revealed that significant expression of ECE-1 transcripts and ECE-1alpha protein occurs in human keratinocytes. When ECE activity was assayed in extracts of human keratinocytes at pHs ranging from 5.0 to 8.0, the enzymatic profile had an optimal neutral pH of 7.0 and was sharply pH-dependent. Furthermore, when extracts of human keratinocytes were treated with alphaPEPT6, ECE activity was significantly reduced compared with extracts treated with the prebleed serum of alphaPEPT6, which supports the notion that ECE-1alpha is a major metalloproteinase with ECE activity in human keratinocytes. The exogenous addition of the pro-inflammatory cytokine interleukin-1alpha significantly increased expression of ECE-1 transcripts in cultured human keratinocytes, which suggests an association with post-inflammatory hyperpigmentation. Collectively, our results demonstrate for the first time that ECE-1alpha is expressed at significant levels in various types of human skin cells (including keratinocytes) and that it plays a constitutive role in the processing and UVB-inducible secretion of ET-1 by human keratinocytes, which leads to the stimulation of pigmentation in the epidermis.

The attachment of Schistosoma mansoni cercariae to human skin cells.

Most of our knowledge about the process of penetration of skin, by cercariae of Schistosoma mansoni, has been gained from studies carried out in vivo with laboratory animals. Human skin is significantly different from that of other animals but there are obvious practical difficulties in directly studying attachment and penetration with human skin. Techniques have been developed which enable a 3-dimensional skin equivalent to be grown in tissue culture, made from different types of human skin cells. The aim of the present study was to investigate cercarial interactions with confluent cultures of the individual skin cell types that make up normal human skin and which will be used to construct a multi-component model. Cercariae behaved differently towards the various cell types tested. They responded least to monolayers of endothelial cells and most to primary keratinocytes, derived from human foreskin and differentiated at an air/liquid interface. This study demonstrates, therefore, that cercariae are capable of distinguishing between different types of skin cells and they preferentially attach to differentiated cells which form the epidermis.

PARP Determines the Mode of Cell Death in Skin Fibroblasts, but not Keratinocytes, Exposed to Sulfur Mustard

Sulfur mustard is cytotoxic to dermal fibroblasts as well as epidermal keratinocytes. We demonstrated that poly(ADP-ribose) polymerase (PARP) modulates Fas-mediated apoptosis, and other groups and we have shown that PARP plays a role in the modulation of other types of apoptotic and necrotic cell death. We have now utilized primary dermal fibroblasts, immortalized fibroblasts, and keratinocytes derived from PARP(-/-) mice and their wildtype littermates (PARP(+/+)) to determine the contribution of PARP to sulfur mustard toxicity. Following sulfur mustard exposure, primary skin fibroblasts from PARP-deficient mice demonstrated increased internucleosomal DNA cleavage, caspase-3 processing and activity, and annexin V positivity, compared to those derived from PARP(+/+) animals. Conversely, propidium iodide staining, PARP cleavage patterns, and random DNA fragmentation revealed a dose-dependent increase in necrosis in PARP(+/+) but not PARP(-/-) cells. Using immortalized PARP(-/-) fibroblasts stably transfected with the human PARP cDNA or with empty vector alone, we show that PARP inhibits markers of apoptosis in these cells as well. Finally, primary keratinocytes were derived from newborn PARP(+/+) and PARP(-/-) mice and immortalized with the E6 and E7 genes of human papilloma virus. In contrast to fibroblasts, keratinocytes from both PARP(-/-) and PARP(+/+) mice express markers of apoptosis in response to sulfur mustard exposure. The effects of PARP on the mode of cell death in different skin cell types may determine the severity of vesication in vivo, and thus have implications for the design of PARP inhibitors to reduce sulfur mustard pathology.