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There is increasing recognition of the role of Toll-like receptor 3 (TLR3) in noninfectious inflammatory diseases, but the function of TLR3 in inflammatory skin diseases is unclear. We investigated the functions of TLR3 in allergic and irritant contact dermatitis (ICD). The contact hypersensitivity (CHS) response was lower in Toll-like receptor 3 knockout (Tlr3 KO) mice, and was greater in TLR3 transgenic (Tg) mice than in wild-type (WT) mice after challenge with 2,4,6-trinitro-1-chlorobenzene. Adoptive transfer of immunized lymph node cells from Tlr3 KO mice induced CHS in WT recipients. In contrast, adoptive transfer of those from WT mice did not fully induce CHS in Tlr3 KO recipients. The ICD reaction following croton oil application was lower in Tlr3 KO mice, and was greater in TLR3 Tg mice than in WT mice. Maturation, migration, and antigen presentation of dendritic cells and proliferation of lymphocytes between WT mice and Tlr3 KO mice were comparable. These results show that TLR3 enhances antigen-independent skin inflammation in the elicitation phase of allergic contact dermatitis and in ICD.

Strong inhibition of NF-κB signaling in the epidermis results in spontaneous skin inflammation in mice and men. As there is evidence for linkage between polymorphisms within the NF-κB signaling pathway and human inflammatory skin phenotypes, we asked whether partial functional inhibition of NF-κB signaling in epidermal keratinocytes can modulate clinically relevant skin inflammation. We therefore mutated rela specifically in the epidermis of mice (RelA E−MUT mice). These mice show no inflammatory phenotype. Induction of contact allergy, but not croton oil–induced irritant dermatitis, resulted in stronger ear swelling and increased epidermal thickness in RelA E−MUT mice. Both contact allergen and croton oil treatment led to increased expression of calgranulins A and B (S100A8/ A9) in RelA E−MUT mice. Epidermal hyperproliferation in RelA E−MUT mice was non-cell autonomous as cultured primary epidermal keratinocytes from RelA E−MUT mice showed reduced proliferation compared with controls. These results demonstrate that epidermal RelA specifically regulates delayed-type hypersensitivity-induced skin inflammation. In addition, we describe here an essential but nonspecific function of RelA in the protection of epidermal keratinocytes from apoptosis. Our study identifies functions of NF-κB signaling in the epidermis and corroborates a specific role of epidermal keratinocytes in the regulation of skin inflammation.

Centuries ago, an obscure report on an unusual skin cancer launched the modern era of cancer research. Ever since, skin research has made key contributions to our understanding of the biology and biochemistry of cancer pathogenesis. In 1775, Percival Pott published his essay entitled Chirurgical observations Relative to the Cataract, the Polypus of the Nose, the Cancer of the Scrotum, [etc.] describing the origin of the ‘‘soot-wart’’, a cancer of the inferior surface of the scrotum occurring with high frequency in English chimney sweeps (Brown and Thornton 1957). Pott attributed the cause of this tumor to ‘‘the lodgement of soot in the rugae of the scrotum’’, thus providing in one description the first recognition of environmental and occupational cancer, the first identification of a human carcinogen, the first opportunity for cancer prevention, and a protocol for surgical treatment. Included in that 797 word essay was a remarkably detailed description of the course of the disease from a local confined lesion to invasive and then disseminated lethal cancer. These insights were little noticed for almost 150 years until (Yamagiwa and Ichikawa, 1918) reported that the chronic topical application of coal tar on rabbit ears produced cutaneous squamous cell carcinomas, the first chemical carcinogenesis experiment. By describing the evolution Figure 1 of individual lesions, Yamagiwa and Ichikawa concluded that cancer developed through multiple phenotypic stages and progressed even after the carcinogen was removed. Over the ensuing 20 years, many biologists and chemists sought to identify the active carcinogen in coal tar, culminating in 1932 with the report by a group led by Ernest Kennaway that 3,4 benzpyrene was the potent polycyclic aromatic hydrocarbon in coal tar that produced cancers when topically applied to mouse skin. Incorporated into this project was the isolation or synthesis of multiple carcinogenic hydrocarbons, all recognized by their action as skin carcinogens (summarized in Kennaway, 1955). At the same time, the availability of synthetic carcinogens and a reliable squamous cancer model emboldened a group of pathologists and biologists to methodically explore the pathogenesis of the neoplastic skin lesions. For the next two decades, these investigators discovered concepts regarding skin cancer development that dominate our current understanding of cancer development in almost all epithelial tissues. In particular, the work of Cecil Mottram, Friedewald and Rous (Rous later won the Nobel Prize for his work on the viral etiology of skin cancer), and Berenblum and Shubik led to the following conclusions on the biology of cancer: cancer occurs through multiple stages with distinct operational mechanisms and required sequence; cancer is more frequent when proliferating tissues are exposed to carcinogens; the extent of exposure or potency of the carcinogen determines the latent period for tumor development; tumor development does not require continuous exposure to carcinogenic agents and the effect of a subtumorigenic carcinogen exposure is permanent; tumors will develop after a subtumorigenic carcinogen exposure if the target tissue is subjected to regenerative hyperplasia as caused by repeated wounding or application of an irritant (Berenblum and Shubik, 1947, 1949; Mottram, 1944a, b; Berenblum and Haran, 1955; Berenblum, 1957; Friedewald and Rous, 1944). It was Friedewald and Rous who coined the terms ‘‘initiation’’ and ‘‘promotion’’ to distinguish the irreversible action of a carcinogen and the non-carcinogenic reversible activity of an irritant, still a major part of the cancer lexicon today. One of the important additions to the skin cancer protocols provided by Berenblum was the use of topical croton oil, a non-carcinogenic skin irritant that induced the outgrowth of tumors from skin that had been treated with a subtumorigenic dose of carcinogen (initiated). Croton oil (Crotonis Oleum) is prepared from the seeds of Croton tiglium, a tree belonging to the natural order Euphorbiales and family Euphorbiaceae. Croton oil had been used for centuries as folk medicine and was known to induce inflammation and peeling of skin when applied topically and diarrhea when taken internally. Its potency to promote tumors on skin catalyzed interest among cancer biologists to identify the active compounds contained within the complex mixture. In a series of brilliant chemical purifications and analyses, Van Duuren and Hecker almost simultaneously identified phorbol myristate acetate (12-O-tetradecanoyl-phorbol-13-acetate), commonly

Contact dermatitis is caused by epicutaneous exposure to environmentally and/or industrially derived allergens. As the exposure is unavoidable in many instances, therapeutic suppression of allergic inflammation appears to be of clinical relevance. It was recently reported that itraconazole (ITZ), an anti-fungal agent, may be of therapeutic importance in allergic skin diseases. Therefore, we were interested in the effect of ITZ on contact hypersensitivity (CHS). Mice (C3H/HeN or Balb/c) were administered with ITZ orally before sensitization or challenged with haptens (dinitrofluorobenzene or oxazolone). Consequently, the administration of ITZ before challenge, but not before sensitization, significantly suppressed the reaction. Intriguingly, ITZ failed to suppress the irritant dermatitis induced by croton oil or benzalkonium chloride, suggesting that it may affect molecule(s) rather selectively involved in the elicitation of CHS. To further analyze mechanisms involved, splenic T cells obtained from sensitized or naive mice were stimulated with plate-bound anti-CD3 in the presence or absence of ITZ and release of cytokines was tested by ELISA. T cells from hapten-immunized mice produced a significant amount of IFN-gamma, which was markedly suppressed by ITZ. Our study demonstrates that ITZ selectively suppresses the elicitation phase of CHS possibly via downmodulation of IFN-gamma.

Recent reports have suggested that chemical-induced allergic contact dermatitis may not be a traditional type IV hypersensitivity, in part due to the dual irritant and antigenic properties of sensitizing chemicals. In order to investigate the contribution of these properties to the molecular and cellular mechanism underlying allergic contact dermatitis, we evaluated oxazolone-induced changes in cell populations and cytokine production in the dermis of transgenic mice with impaired innate immunity (the FcgammaR subunit knockout mouse), and absent specific immunity (the athymic mouse), and the appropriate B6,129F2 and C57BL/6 control mice. Oxazolone and croton oil were applied in a single sensitizing dose, or in sensitizing and challenge doses, and the dermal response was evaluated by immunohistochemistry. In the wild type mice, with or without sensitization to oxazolone or croton oil, we observed mixed Th1/Th2 cytokine production and both CD4+ and CD8+ T lymphocytes; however, the neutrophil was the predominant cell in the dermis, even 72 h after final chemical application. Athymic mice displayed a similar neutrophil response with moderate Th1/Th2 cytokine production, and FcgammaR subunit knockout mice exhibited very mild dermatitis when treated with either oxazolone or croton oil. These results provide support for the hypothesis that allergic contact dermatitis is not a classic delayed type hypersensitivity, demonstrate the importance of the interaction between the irritant and antigenic properties of sensitizing chemicals in the development of allergic contact dermatitis, and suggest that the irritant effect of chemicals may be mediated through the cutaneous innate immune system.

In response to exogenous stimuli such as phorbol-12-myristate 13-acetate, ultraviolet B radiation, and lipopolysaccharide, human keratinocytes produce soluble mediators that are important in primary contact irritancy including cytokines that are associated with proinflammatory properties (interleukin-1 alpha [IL-1 alpha], tumor necrosis factor alpha), chemotaxis (IL-8), and growth activation (granulocyte/macrophage colony stimulating factor, IL-6, transforming growth factor alpha). We examined qualitative and quantitative changes in selected intracellular and secreted cytokines in human keratinocyte cultures in response to non-sensitizing contact irritants (croton oil, sodium lauryl sulfate, methyl salicylate, ethyl phenylpropiolate), sensitizing irritants (oxazolone, dinitrofluorobenzene), and ulcerative agents (phenol, benzalkonium chloride, chromium trioxide). The chemicals were also applied to mouse skin to assess whether the chemical-specific pattern of inflammation correlated with the in vitro production of keratinocyte-derived cytokines. Although all agents elicited neutrophils to the site of chemical application, time dependent and chemical-specific patterns of inflammation could be detected. Sodium lauryl sulfate, phenol, and croton oil induced increases in IL-8 production at non-cytotoxic concentrations in semi-confluent human keratinocyte cultures. Phenol and croton oil stimulated tumor necrosis factor alpha production, whereas croton oil was the only agent found to induce granulocyte/macrophage colony-stimulating factor production. Croton oil, phenol, benzalkonium chloride, and dinitrofluorobenzene induced the intracellular production of IL-1 alpha without a concomitant release into the medium. The release of cytokines occurred in parallel with a relative increase in cytokine-specific mRNA transcripts. Studies using neutralizing antibodies to tumor necrosis factor alpha and IL-1 alpha demonstrated that IL-8 induction by croton oil and phenol occurred directly rather than through autocrine circuits. These data suggest that a given pattern of cytokine production is chemical-specific and may predict the contribution of keratinocytes to skin inflammation.

In previous studies, we showed that a number of aspects of the histopathology of irritant contact dermatitis arc profoundly influenced by the chemical nature of the irritant applied. We report here that this phenomenon also extends to the infiltration of leukocytes into the epidermis. Healthy volunteers were patch tested with the following irritants and their appropriate controls: benzalkonium chloride, sodium lauryl sulphate, crown oil, dithranol, nonanoic acid, and propylene glycol. After visually grading the intensity of the resulting inflammation, biopsies were removed and the major phenotypic classes of leukocytes identified immunocytochemically. Dermal and epidermal cell densities were determined, and the expression of several activation/proliferation antigens studied. We found a similar pattern of cellular infiltration in the dermis of all irritant groups; the densities of most of the cell types rising in line with the intensity of inflammation. Within the epidermis, however, there were marked differences in the patterns of cellular infiltration between the irritant groups, leading to poorer correlations between leukocyte density and visual grading. The greatest disparity occurred between croton oil and nonanoic acid biopsies, the former being characterized by the influx of large numbers of leukocytes, the latter showing remarkably little exocytosis. Infiltration of neutrophils occurred to varying degrees with all irritants, but a disproportionately large number were present in sodium lauryl sulphate biopsies. All control groups showed a rise in CD4 + cells, with distilled water also producing increases in CD11c+ cells and neutrophils. A selective influx of CD25 + cells occurred in the epidermis of both irritant and control groups. Our observations further highlight the heterogeneous nature of irritant contact dermatitis, and confirm previous findings that visually negative control patch tests show marked cellular reactivity.

In order to gain a greater insight into the complex mechanisms of action of different irritant chemicals on the skin, we have studied the behavior of epidermal CD1+ cells in experimentally induced irritant contact dermatitis. Healthy, human volunteers were patch tested for 48 h with the following six chemically unrelated irritants and their appropriate vehicle controls; benzalkonium chloride, sodium lauryl sulphate, dithranol, nonanoic acid, croton oil, and propylene glycol. After visually assessing and grading the resulting inflammatory reactions, punch biopsies were taken and the morphology and density of CD1+ cells in the epidermis studied using immunocytochemical techniques in combination with image analysis and electron microscopy. Statistically significant decreases in the epidermal density of CD1+ cells occurred in the responses to dithranol (p less than 0.05) and nonanoic acid (p less than 0.01). Importantly, these changes in density were not simply due to variations in the intensity of inflammatory response (r = 0.1157). Alterations in the length of the dendritic processes of CD1+ cells were also induced, and semi-quantitative analysis revealed significant decreases in dendrite length in the reactions to sodium lauryl sulphate (p less than 0.05), nonanoic acid (p less than 0.001), croton oil (p less than 0.05), and dithranol (p less than 0.005). Unlike epidermal density, however, this effect on cell morphology was directly related to the severity of inflammation (r = -0.74, p less than 0.01). Morphologic evidence of cellular injury to Langerhans cells was seen by electron microscopy in the majority of biopsies, although relatively few cells were affected in sodium lauryl sulphate and propylene glycol reactions. Benzalkonium chloride, unlike the other irritants, also induced a state of metabolic activation in a high proportion of epidermal Langerhans cells. Lymphocyte/Langerhans cell apposition was observed in most samples, but was particularly prevalent in the reactions to dithranol. The results of this study demonstrate that significant changes in the morphology and density of Langerhans cells occur in irritant contact dermatitis, some of which are directly influenced by the chemical nature of the irritant.

BALB/c mice were treated with the irritants croton oil (0.5%, 20%), sodium lauryl sulfate (15%), and benzalkonium chloride (25%), the contact sensitizers 2,4-dinitrofluorobenzene (DNFB, 0.3%) and picryl chloride (PCl, 1%), and the tolerogen 2,4-dinitrothiocyanatebenzene (DNTB, 2%). All irritants used produced degenerative alterations of Langerhans cells (LCs). After application of 0.5% croton oil, however, this degeneration was preceded by an activation of the cells with increased number of mitochondria and enlargement of nuclei. The DNFB and PCl application in sensitizing doses to nonsensitized animals resulted in a cellular activation similar to that observed for 0.5% croton oil. In addition, these LCs showed enhanced adsorptive endocytosis as demonstrated by increased numbers of Birbeck granules and coated vesicles. The endocytotic activity was more pronounced in DNFB-sensitized animals. The DNTB at a concentration that induced tolerance to DNFB did not cause either cellular or endocytotic activation of LCs. These results demonstrate that the contact sensitizers DNFB and PCl induce characteristic cellular reaction patterns of LCs, which may be related to their sensitizing property.

Sunscreen preparations containing various chemical UV absorbers, para-aminobenzoic acid (PABA), 2 PABA derivatives, benzophenone or a combination of these were topically applied to the backs of C3H/HeN mice prior to their being irradiated with ultraviolet light in the UVB range. In all cases this treatment was effective in preventing the pathological skin changes associated with UVB irradiation. Histological evaluation of skin biopsies from mice treated with the sunscreen preparations and UVB irradiation showed little or no difference from normals in amount of hyperplasia, melanization or parakeratosis present. These histologic changes were observed in animals receiving UVB irradation in the absence of any sunscreen agent. Pretreatment with the various sunscreen agents did not, however, prevent the induction of tumor susceptibility as measured by the sustained growth of a UV-induced tumor which is immunologically rejected in normal syngeneic mice. These data show a clear distinction between the effects of UVB irradiation leading to histological changes in the epidermis and those leading to the state of tumor susceptibility in mice. The distinction was further corroborated by the finding that epidermal hyperplasia induced by repeated applications of croton oil had no significant enhancing or inducing effects on the induction of tumor susceptibility. In addition, the induction of tumor susceptibility. In addition, the induction of tumor susceptiblity is not due to wavelengths of light less than 320 nm since this effect was abrogated when the UVB radiation was filtered through glass. Possible mechanistic differences between the tumor susceptiblity generated in UVB and photoprotected UVB irradiated animals were observed, however, when we attempted to adoptively transfer the state of tumor susceptibility to normal animals. While it was readily transferable with splenic lymphoid cells from UVB irradiated animals, all attempts to transfer the tumor susceptibility from photoprotected animals have, to date, been unsuccessful.

A polar photoproduct of cholesterol oxidation, 7-ketocholesterol, was able to inhibit in a dose-dependent manner the mouse ear-swelling response to irritants such as croton oil or cantharidin. Its anti-inflammatory properties were much less than equivalent concentrations of hydrocortisone, but the oxidized sterol did not induce any systemic effects (as measured by thymolytic activity), as did topical hydrocortisone. It is concluded that 7-ketocholesterol has weak anti-inflammatory activity, and its mode of action may be different from that of glucocorticoids.

A mixture of the aliphatic alcohol, triacontanol, and other chemically associated naturally occurring alcohols was applied to the denuded dorsal cutaneous surface of guinea pigs to evaluate anti-inflammatory activity. In the setting ofa chemical irritation with 2% croton oil and in an allergic dermatitis created with dinitrochlorobeuzene sensitization and challenge, the triacontanol-containing preparation was significantly more effective than vehicle alone (DHL skin cream) but not as effective as 0.05% Diprolene® ointment. Lymphocyte stimulation was studied by tritiated thymidine uptake and morphologic examination for blast transformation. When triacontanol-containing compounds were solubilized in aqueous media, effects on lymphocytes were insignificant. When solubilized in ethanol, there was a marked effect on thymidine uptake but not on blast transformation when compared to parallel controls.

Formulated corticosteroids were assayed on human skin using as a measure of anti-inflammatory activity the suppression of blisters produced by kerosene and pustules provoked by croton oil. Vascoconstriction activity was evaluated at the same time. The rank order of anti-inflammatory efficacy corresponded reasonably well with clinical judgements of comparative effectiveness although the ranking was not always the same for the identical steroid in the two models. With some steroids ointments were superior to creams while the reverse was true for other. Vasoconstriction generally correlated with anti-inflammatory activity but strong exceptions occurred.

Phorbol ester acetate was produced by the esterification of the free hydroxyl of phorbol ester C (the tumor promoting agent from croton oil). The phorbol ester acetate was used to promote skin tumors in female mice that had previously received a subcarcinogenic dosage of 7,12 DMBA. The morphology of the tumor revealed an epidermal hyperplasia as well as a hyperkeratosis of the stratum corneum. There was a marked increase in the intercellular space in the strata basale and spinosum. These cells also demonstrated an increase in ribosomes and polysomes. Many of the mitochondria of these cells revealed intramitochondrial granules that resembled lipid droplets. Phorbol ester- 14 C acetate was prepared by acetyl-2- 14 C esterification of the free hydroxyl of the phorbol ester C. The phorbol ester- 14 C acetate was applied to papillomas that were initiated with 7,12 DMBA and promoted with phorbol ester acetate for 90 days. Grains due to the phorbol ester- 14 C acetate were observed overlying the loose chromatin of the nuclei and tonofibrils.

Either 3mg of 1,2- 3 H-cortisol (20 μc) or 5mg of 1,2- 3 H-testosterone (20 μc) was injected intramuscularly into young adult male rates, 1, 4 and 7 days after skin wounds were created by intradermal croton oil injection. Thirty minutes and 4 hours after injection of each labeled steroid, radioactivity was measured in wound tissue, in tissue adjacent to the wound, in a contralateral control site and in plasma. The radioactivity obtained from the tissues is expressed in 3 ways: (1) as radioactivity extracted from fresh tissue; (2) as radioactivity calculated for dry fat-free tissue; (3) as radioactivity calculated for "tissue water". The relative amount of radioactivity in wound, adjacent skin and control skin frequently varied with the manner of data expression. There was a difference between the distribution of radioactivity from cortisol and testosterone in control and adjacent skin. No distinct differences with wound age were observed in the distribution of either cortisol or testosterone between croton oil lesions, tissue adjacent to the wound and control skin. "Tissue water" radioactivities indicated that none of 3 tissues investigated concentrated cortisol and/or its metabolites. However, "tissue water" radioactivities of control and adjacent skin of rats given testosterone were similar to or greater than plasma "tissue water" radioactivities.

As has been generally realized, "systemic stress" acts as a stimulant on the adrenal cortex, and the cortico-adrenal hormones play a part in systemic defense. Such a hormonal effect can be demonstrated inter alia by sensitizing animals to proteins and subsequently exposing them to cold or to other injurious factors. The anaphylactic shock is far less severe in previously treated animals (1—4). To study the cutaneous response in animals during and after" sytemic stress," a number of guinea-pigs were exposed to cold, or to subcutaneous formalin injections and 2, 4-dinitrochlorobenzene (DNB) was applied in test doses to the skin of the sensitized animals, while those not sensitized were treated with a primary irritant, viz. croton oil.

It has been conjectured that the melanocytes of the hair bulb can be made to migrate by different methods such as vibrapuncture (1), production of blisters and also dermabrasion. Pegun (2) suggested that the depigmented areas of vitiligo could be colonized by the melanocytes of the pigmented bulb in addition to repig-mentation from epidermal melanocyte sources. Increased consideration of the relationship between the melanocytes and the hair follicle comes also from the statement by Szabo (3) that the regional frequency distribution of hair follicles is similar to that of nevi, and that distribution of skin appendages, in general, is similar to that of melanomas. He further found, in his experiments with mouse skin, that after application of dibenzanthracene followed by Croton oil, the melanocytes of the hair follicle “may invade the surrounding dermis and develop into a blue nevus-like structure” (3).

Recent work by Doberauer (1) has suggested that histochemically the character of the healing of experimental wounds was different in the aged as opposed to the young rat. We have reported that wounds in an old animal contained more cells, more blood vessels and the eschars were firmer and better organized than wounds and eschars from the young rat (2).

In vivo effects of retinoids on epidermal differentiation were investigated by analyzing cytoskeletal proteins in rhino mice treated topically with all-trans-retinoic acid (RA) and other retinoids (13-cis-retinoic acid, etretinate, TTNPB). Nondisulfide-linked cytoskeletal proteins, including keratins from the epidermal "living layers", were first selectively extracted using 9.5 M urea; subsequently, keratins of the stratum corneum were isolated using 9.5 M urea plus a reducing agent. Gel electrophoresis and immunoblot analysis showed that urea extracts of epidermis from vehicle-treated skin were composed predominantly of four major keratins (analogous to human epidermal keratins K1, K5, K10, and K14), and the keratin filament-associated protein filaggrin. In contrast, extracts of epidermis from retinoid-treated skin contained additional keratins (K6, K16, and K17) and almost no detectable filaggrin. Furthermore, similar analysis of stratum corneum keratins demonstrated that extracts from RA-treated skin did not contain the partially proteolyzed keratins typically observed in stratum corneum extracts of control animals. Hyperplasia-inducing agents (salicylic acid, croton oil) caused an increase in keratins K6, K16, and K17, but they did not effect filaggrin or alter proteolysis of stratum corneum keratins. The result that RA induced expression of keratins K6, K16, and K17, as commonly expressed in hyperproliferative epidermis, is consistent with the notion that retinoids increase epidermal cell proliferation in the basal and/or lower spinous layers. The findings that topical RA decreased filaggrin expression and reduced proteolysis of stratum corneum keratins, despite increased size and number of granular cells and the presence of an anucleate stratum corneum, suggest that topical RA may also modulate a later stage of epidermal differentiation involved in stratum corneum formation.

The mechanism of irritant dermatitis and the immunologic consequences of such reactions are unclear. We evaluated the number and function of epidermal antigen-presenting cells contained in epidermal cell suspensions obtained from normal and irritant patch test reaction sites. Application of sodium lauryl sulfate or croton oil to human skin in vivo resulted in a progressive depletion in the number of epidermal OKT6+HLA-DR+ (T6+DR+) Langerhans cells (LC) from 3.1 +/- 0.2% of total epidermal cells (EC) to 1.2 +/- 0.1% after 8 d (mean values +/- SEM, N = 9). Between 1-4 d irritant patch test sites demonstrated an influx of non-Langerhans cell T6-DR+ cells. These cells were not DR+ keratinocytes but appeared to be of bone marrow derivation because they expressed the marker, HLe1. Among bone marrow derived cells, the T6-DR+EC appeared to be of monocyte, macrophage lineage, because they expressed the determinant recognized by the OKM5 (M5) antibody. Despite the induction of M5+DR+EC the total number of DR+EC showed progressively decreasing percentages over an 8-d period. Partial recovery to 73 +/- 12% of control value was observed at 2 weeks, with full recovery by 4 weeks after challenge. Concomitantly with the depletion of DR+EC, the capacity of EC to present alloantigens to T cells decreased. This reduction in antigen-presenting cell activity was strongly correlated to the reduction in total DR+ EC (r = 0.94, p less than 0.05). Thus, the capacity of irritants such as croton oil to abrogate the function of epidermal antigen-presenting cells may be related to the tumor promoting potential of these agents.

The histological (1), biological (2) and clinical (3) nature of the inflammatory response has been a subject of intensive interest during the past two decades. Despite this concern, little is known of the chemistry of this vital process. For this reason, we have studied some alterations in the chemical composition of the connective tissue (skin) in response to chemically induced local inflammation (4, 5). This paper describes further the results of our studies of the chemistry of local inflammation resulting from the intradermal injection of a chemical irritant (croton oil) in the rat.

Pharmacologic suppression of the effector phase of contact hypersensitivity appears to have major relevance with regard to treatment of type IV reactions like contact dermatitis. Recently, tumor necrosis factor alpha has been shown to be a critical mediator in hapten-induced irritant and contact hypersensitivity reactions, thus offering new possibilities, for therapeutic intervention. Pentoxifylline, a methylxanthine derivative used in the treatment of vascular disorders, currently has been found to suppress the production of tumor necrosis factor alpha by human and murine leukocytes. Therefore, the effect of pentoxifylline on the elicitation phase of contact hypersensitivity was studied. Intraperitoneal injection of pentoxifylline into sensitized Balb/c and C3H/HeN mice before application of the challenging hapten dose resulted in a significant reduction of the outcome of the contact hypersensitivity reaction. The suppressive effect of pentoxifylline was dose dependent and maximally pronounced upon injection 3 h before hapten application. In contrast to the effector phase of contact hypersensitivity, induction of contact hypersensitivity was not affected by pentoxifylline when injected into naive mice before performance of sensitization. In addition, irritant dermatitis induced by 1% croton oil or 5% benzalkonium chloride was suppressed by pentoxifylline as well. These data suggest a potential pharmacologic intervention, with pentoxifylline as a means to treat contact dermatitis.

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

The dermal chemical response to croton oil induced experimental wounds has been shown to involve both the accumulation of glycoproteins within the lesion and the disappearance of insoluble collagen from the site of injury (1). Associated with this loss of collagen from the wound was a smaller decrease in the concentration of insoluble collagen from the apparently uninjured skin distant from the site of local injury (2). The chemical changes appropriate to this “distant dermal collagen response” (3) to local croton oil injury were duplicated in the skin of uninjured animals which had received exogenous Cortisol (4). This “distant dermal collagen response” to local injury was also found in the skin of animals which had been subjected to sham adrenalectomy, daily intraperitoneal injections of large amounts of isotonic saline and other forms of chemical injury and trauma (5). From these findings it was argued that the trauma of croton oil induced injuries produced sufficient stress to result in losses of insoluble collagen from the uninjured skin. Since croton oil produced wounds are stressful, at least to this extent, the degree of stress produced by two croton oil induced injuries to one animal should be considerably greater than that produced by a single injury alone. The effect of duplicate injuries upon the kinetics of the chemical response to experimental wounds was studied and the results compared with those obtained from either a single injury alone or from singly injured animals which had also received physiological doses of stress hormone (Cortisol). The comparisons of these data are presented below.

Interleukin (IL) 10 is a recently discovered cytokine, originally isolated from T-helper 2 (Th2) cells, which inhibits cytokine production of T-helper 1 (Th1) cells. Because Th1 cells appear to be of importance during the contact hypersensitivity reaction (CHS) we hypothesized that IL-10 might modulate the outcome of CHS in vivo . Intraperitoneal injection of murine recombinant IL-10 (1000 ng) into naive mice 24, 72, or 120 h before sensitization by epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) did not affect ear swelling when ears were challenged 5 d later. However, intraperitoneal injection of IL-10 into already sensitized mice 24 h before challenge resulted in a significant suppression of the ear swelling response, suggesting that under the conditions employed IL-10 is able to block the effector phase, but not the induction phase of CHS in vivo . The suppression could be reversed by the concurrent injection of an IL-10 antibody. Moreover, heat inactivation of native IL-10 resulted in loss of the inhibitory capacity. When mice were sensitized by subcutaneous injection of trinitrophenyl-coupled spleen cells (DTH) instead of epicutaneous application of the hapten (CHS), intraperitoneally-injected IL-10 suppressed the effector phase, but also the induction phase of DTH. IL-10 did not inhibit the toxic ear-swelling response induced by topical application of two irritants tested (croton oil or benzalkonium chloride). The capacity of IL-10 to suppress the effector phase of CHS and DTH supports an important role for this cytokine in the downregulation of type IV immune reactions in vivo . The finding that IL-10 suppresses the induction of DTH, but not of CHS, further suggests that CHS and DTH are related but distinct immune reactions.

Interleukin 18 induces both T helper 1 and T helper 2 cytokines, proinflammatory cytokines, chemokines, and IgE and IgG1 production. A role of interleukin 18 in inflammatory cutaneous reactions is still unclear, however. Here we generated keratin 5/interleukin 18 transgenic mice overexpressing mature murine interleukin 18 in the skin using a human keratin 5 promoter. In the contact hypersensitivity model, trinitrochlorobenzene elicited a stronger ear swelling in keratin 5/interleukin 18 transgenic mice compared with control littermate wild-type or immunoglobulin/interleukin 18 transgenic mice in which mature interleukin 18 was expressed by B and T cells under the control of the immunoglobulin promoter. Application of an irritant, croton oil, induced stronger and more sustained ear swelling in keratin 5/interleukin 18 transgenic mice than in immunoglobulin/interleukin 18 transgenic or wild-type mice. Repetitive topical application (weekly for six consecutive weeks) of trinitrochlorobenzene to their ears also elicited a stronger cutaneous inflammation in keratin 5/interleukin 18 transgenic mice than seen in immunoglobulin/interleukin 18 transgenic or wild-type mice. After these six trinitrochlorobenzene applications, the expression of interferon-gamma, interleukin-4, and CCL20 mRNA in the ear tissue was increased and dermal changes, such as acanthosis and eosinophilic, neutrophilic, and mast cell infiltration, were greater in keratin 5/interleukin 18 transgenic mice than in wild-type mice. Furthermore, the repetitive application elicited a significant increase in serum IgE levels and the number of B cells in the draining lymph node in keratin 5/interleukin 18 transgenic mice. These results suggest that overexpression of interleukin 18 in the skin aggravates allergic and nonallergic cutaneous inflammation, which is accompanied by high expression of T helper 1 and T helper 2 cytokines and chemokines in the skin.

The time course, dose response, components of inflammation, and involvement of putative mediators of inflammation in irritation induced by different chemicals was compared using a mouse ear swelling technique. Differences in time courses of inflammation produced by the irritants were not solely due to differences in rates of penetration. Changes in blood flow and permeability of vessels were phasic with different numbers of phases induced by different irritants. Effects of antagonists, synthesis, inhibitors, and depleting agents of putative inflammatory mediators on intensity of inflammation varied for different irritants. These studies demonstrate that all chemicals do not produce skin irritation by a common inflammatory pathway. J Invest Dermatol 88:24s—31s, 1987

The lymph transports tissue-resident dendritic cells (DCs) to regional lymph nodes (LNs), having important roles in immune function. The biological effects on tissue inflammation following lymphatic flow obstruction in vivo , however, are not fully known. In this study, we investigated the role of the lymphatic system in contact hypersensitivity (CHS) responses using k-cyclin transgenic (kCYC +/- ) mice, which demonstrate severe lymphatic dysfunction. kCYC +/- mice showed enhanced ear swelling to both DNFB and FITC, as well as stronger irritant responses to croton oil compared with wild-type littermates. Consistently, challenged ears of kCYC +/- mice exhibited massive infiltrates of inflammatory cells. In contrast, DC migration to regional LNs, drainage of cell-free antigen to LNs, antigen-specific IFN-γ production, and lymphocyte proliferation were impaired during the sensitization phase of CHS in kCYC +/- mice. Transfer experiments using lymphocytes from sensitized mice and real-time PCR analysis of cytokine expression using challenged ear revealed that ear swelling was enhanced because of impaired lymphatic flow. Collectively, we conclude that insufficient lymphatic drainage augments apparent inflammation to topically applied allergens and irritants. The findings add insight into the clinical problem of allergic and irritant contact dermatitis that commonly occurs in humans with peripheral edema of the lower legs.

Adenosine 3',5'-monophosphate (cyclic AMP) has been localized in mouse epidermal cells using an immunofluorescent technique. Within 10 min following the intraperitoneal injection of isoproterenol or 30 hr following the topical application of croton oil in acetone, staining was clearly visible in the cytoplasm of the basal cells.

Our previous studies demonstrated that psoralen plus longwave ultraviolet radiation (PUVA) treatment inhibited certain T-lymphocyte functions, such as locomotive ability. To further analyze the effects of PUVA on T-lymphocyte function, we investigated the ability of mouse spleen cells to produce interleukin 2 (IL-2) after treatment of the mice in vivo with PUVA. Interleukin 2 production was impaired in cells from PUVA-treated mice compared with those from UVA-irradiated, 8-methoxypsoralen-treated, or normal mice. This impairment was not dose dependent, over the dose range of UVA (2–20J/cm 2 ) examined. Interleukin 2 production was markedly suppressed on day 3 after PUVA and returned to normal by day 7 after the treatment. Topical treatment of the mice with croton oil did not affect IL-2 production of their spleen cells. This result indicates that cutaneous inflammation per se may not be responsible for the suppressive effect of PUVA on IL-2 production. Addition of exogenous IL-1 did not reconstitute the decreased ability of spleen cells to produce IL-2 in vitro, indicating that PUVA affected primarily IL-2 producing cells. These suggest that impaired IL-2 production may account for some of the immune dysfunction observed in PUVA-treated animals.

Previous studies demonstrated that BPO can promote chemically initiated tumor formation in SENCAR mice. In addition, a number of chemicals have been shown to promote and/or enhance UVR induced carcinogenesis. This study examined the effect of BPO on UVR initiated tumor formation. One hundred and forty-eight Uscd mice received 270 mJ/cm 2 of UVB radiation to the posterior halves of their backs 3 times a week for 8 weeks. Four weeks later the mice were divided into 4 groups. Group I received croton oil in acetone applications to the back 5 times a week for the duration of the study. Group II received acetone, Group III received the BPO diluent, and Group IV received the BPO in an aqueous diluent applications as in Group I. One mouse in Group II (acetone) and one in Group IV (BPO) developed tumors in unirradiated skin. In the UVR initiated skin 38% of the survivors developed tumors in Group I (croton oil), whereas 5% did in Group II (acetone), 8% in Group III (BPO base), and 8% group IV (BPO). Thus under the circumstances of this study croton oil did promote UV initiated tumor formation but BPO did not. These results are consistent with those recently reported by Iversen.

An animal model (the rabbit) was used to define which of 8 chemicals caused pustule formation on topical application. Large occlusive chambers (diameter 12 mm), petrolatum as the vehicle and wrapping contributed to efficient occlusion and pustulation. Sodium lauryl sulfate and mecuric chloride gave reproducible results and clear dose-responses indicating that this pustulation is an expression of primary irritancy. Ammonium fluoride pustulation was not reproducible; croton oil pustules were more difficult to evaluate due to simultaneous erythema and edema. Sodium arsentate, nickel sulfate and potassium iodide pustules developed at sites where the skin barriers had been damaged by a stab injury. Benzalkonium chloride caused yellow staining and edema but not pustules. Because of lack of epidemiologic data, we do not know how frequently similar findings occur in man.

Understanding the relationships between chemical and ultraviolet light (UVL) cancer formation has been clouded by the influence of light energy on the carcinogen and/or the substrate (1—5). Recent studies indicate that under appropriate conditions, carcinogenic and sub-carcinogenic amounts of UVL will stimulate cancer formation initiated by a single appheation of the chemical carcinogen 7, 12-dimethyl benz (a) anthracene (6—8). The resultant responses simulate the pattern described for carcinogenic summation (9, 10). In the present study we further examined the influence of chemical stimulation on UVL carcinogenesis

Sunlight is now deemed to be the principal cause of hyperplasia of the elastic fibers in the exposed skin of adult Caucasians (1, 3). Although this elastosis increases with age, and is commonly termed senile elastosis, solar elastosis is a better name since it emphasizes etiology.In a histologic survey of cutaneous elastic tissue throughout the human life span, we became aware that the quantity increases with age even in unexposed areas, in Negroes as well as Caucasians. Although modest in amount when compared to advanced solar elastosis, it is truly an age dependent change.Sams and his co-workers (4) succeeded in producing elastosis in the skin of albino mice after less than twelve months of erythemo-genic radiation with ultraviolet light. Treatment with croton oil alone did not produce this effect, indicating that chronic inflammation by itself is not responsible. By irradiating the unexposed skin of elderly subjects we sought to learn whether aging, was accompanied by any particular predisposition to the formation of elastic tissue.METHODFour Caucasian males, aged 79, 80, 80 and 82 participated. Each exhibited some elastotic changes on the exposed areas. Four 2” X 2” squares were marked off on the lower back area and were treated as follows :1. One square was irradiated every other day with a bank of four Westinghouse FS-20 sunlamps, the principal emission of which is in the erythemo-genic band of sunlight, 290 to 320 nm.2. One square was irradiated every other daywith a bank of four Westinghouse Black Lamps, one half hour after 0.1 per cent 8-methoxypsoralen in hydrophilic ointment was applied to the skin. This combination produces an intense phototoxic reaction, mediated by the deeply penetrating long, ultraviolet rays.3. One square was irradiated every other day with a Hanovia hot quartz ultraviolet lamp whose line spectrum includes short, middle, and long ultraviolet radiation.4. One square was sprayed for 30 seconds every other day with ethyl chloride in order to produce non specific inflammation. (The aged skin was found to be quite refractory to croton oil irritation.)The exposure times were individually adjusted and gradually increased to a maximum which would have initially produced severe burns. Two subjects were treated for six months and two for 12 months. At the end of this time punch biopsies (8 mm) were removed from each of the four sites as well as from an unexposed control area.Frozen sections were cut transversely and horizontally and were stained for elastic with acid orcein using the thick section technique described previously (5) ; this technique allows three dimensional visualization so that both the amount and the architecture of the elastic fibers can be assessed. In addition, formalin fixed paraffin sections were stained with hematoxylin and eosin, Hale's acid ferrocyanide, periodic acid-Schifi reagent, and Mallory's trichrome. Frozen sections were stained for ATPase, DNP dehydrogenese, acid phosphatase, alkaline phosphatase, and succinic dehydrogenase.RESULTSThe irradiated areas became deeply tanned and thickened, most prominently in the photo-toxic sites. The area sprayed with ethyl chloride also showed some hyperpigmentation and slight scaling. Orcein stained sections failed to show any increase or anatomic alteration in elastic tissue. In the H&E stained specimens there was acanthosis and an obvious increase in both clear cells and in melanin. In addition, the areas sprayed with ethyl chloride showed a mild perivascular lymphocytic infiltration. None of the histochemically stained sections showed changes in the experimental sites.225226 THE JOURNAL OF INVESTIGATIVE DERMATOLOGYDISCUSSIONIntense ultraviolet irradiation of the skin of elderly males not only failed to induce elastosis, but was without effect on either the structure or the enzymic systems of the epidermis and dermis. There are several ways of interpreting this negative result. Unlike albino mice, the skin of our subjects became deeply pigmented within a few weeks. Melanin acts as an effective solar shield. Clinically, it is skin which fails to tan that exhibits the marked damage associated with aging. Because of the protection afforded by melanin, the production of elastosis might well require years of irradiation, unless “non-tanners” are used. Another possibility is that the decreased density and cytologic activity of fibroblasts, the presumed source of elastic fibers, may diminish synthetic activity. Decreased elastic synthesis might be an age dependent change. We do know from our histologic survey that elastosis is already well advanced in the second and third decades. Unfortunately, we did not irradiate young subjects.SUMMARYFour elderly individuals were exposed to various wavelengths of ultraviolet irradiation ranging from the long to the short, in an attempt to produce elastosis artificially. Periods of exposure up to one year were insufficient to produce any changes in the elastic tissue framework.