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The fate of the contact sensitizer fluorescein isothiocyanate was traced by means of fluorescence spectrophotometry and flow cytometry. The hapten applied to one ear rapidly entered the circulation by way of local lymphatics and blood vessels. It was dispersed for several hours essentially as free hapten, released from a reservoir left behind at the site. Hapten molecules coupled to plasma proteins while circulating and reacted with white blood cells. Total cells of regional lymph nodes, spleen, and distant lymph nodes became fluorescent in successive order. Fluorescence of CD11c-positive dendritic cells exceeded significantly that of lymphoid cells. Total spleen cells and total nonregional lymph node cells were shown in vitro to drive committed lymph node cells to proliferation. The mechanism disclosed is proposed to counterbalance the action of epidermal Langerhans cells for regulation of contact hypersensitivity.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

We previously described the promotion of type 1 cytokine responses by the nucleoside analogue, ribavirin, in human T cells in vitro. In this study, we examined whether type 1 cytokine polarization by ribavirin in vivo could promote contact hypersensitivity (CHS) responses to dinitrofluorobenzene, a type 1 cytokine-mediated immune response. Unexpectedly, although type 1 cytokine responses were enhanced following ribavirin treatment in vitro and in vivo, the magnitude of CHS responses in BALB/c and C57BL/6 mice was influenced more by a second ribavirin-regulated pathway. The key regulatory molecule in this pathway was IL-10. Ribavirin-mediated suppression of IL-10 in BALB/c mice was associated with increased B7-2 expression and enhanced CHS responses, whereas enhanced IL-10 levels, following ribavirin administration, led to increased B7-1 expression and impaired CHS responses in C57BL/6 mice. The effect of ribavirin on the expression of B7 molecules and on CHS responses was neutralized by IL-10 administration in BALB/c and by anti-IL-10 Ab in C57BL/6. Thus, ribavirin controlled CHS responses directly through the modulation of IL-10 expression, and in vivo outcome was dictated by the preferential expression of either B7-1, an inappropriate costimulatory molecule in CHS, or B7-2, the predominant costimulatory molecule in CHS. Replacing dinitrofluorobenzene priming with IFN-alpha stimulation, we showed that the ribavirin-regulated pathway could function independent of Ag priming. Altogether, these data showed that, although ribavirin treatment induced a type 1 cytokine bias in contact allergen-primed BALB/c and C57BL/6 mice, in vivo CHS responses were dependent on ribavirin-mediated regulation of both IL-10 and preferential costimulatory signaling.

The immunomodulatory effects of allopurinol were investigated in a mouse contact hypersensitivity model. Allopurinol caused a time- and dose-dependent lethal effect in dinitrofluorobenzene (DNFB)-sensitized mice. Furthermore, allopurinol markedly increased ear swelling in the remaining mice. In contrast, TMX-67, a newly synthesized xanthine oxidase/xanthine dehydrogenase (XOD/XDH) inhibitor, had almost no effect on DNFB-sensitized mice. Allopurinol reduced both the spleen weight and white blood cell count in DNFB-sensitized mice without affecting the T cell subset of splenocytes. The production of interferon (IFN)-gamma, in the splenocytes of DNFB-sensitized mice was reduced by allopurinol administration. Death due to allopurinol was much lower in the non-sensitized mice than in the DNFB-sensitized mice. These findings indicate that allopurinol may interact with DNFB to enhance its toxicity and allopurinol might also modulate or enhance the inflammatory effect of DNFB. Also, DNFB may cause metabolic alterations via inflammation, leading to enhanced allopurinol toxicity.

Although regional lymph nodes (LN) have been extensively studied as rich sources of effector T cells in contact hypersensitivity (CH), it remains unknown whether T cell responses in the LN reflect those in effector skin sites. We previously showed that repeated elicitation of CH results in a shift in the time course of Ag-specific CH from a delayed-type hypersensitivity response to an early-type response, a reflection of a shift in cutaneous cytokine expression from a type 1 to a type 2 profile. To investigate whether repeated elicitation of CH could also drive T cell development to the type 2 phenotype in the regional draining LN, sequential cytokine gene expression after hapten application was assessed during both the acute and the chronic phase of CH. In the draining LN the shift to type 2 cytokine production was also observed, but more mixed patterns of responses were induced than in the corresponding skin sites. The chronic LN cells (LNC), when stimulated in vitro, produced markedly lower levels of type 1 cytokines and higher levels of type 2 cytokines than the acute LNC. A successful passive transfer of an early-type response by the LNC was only induced in the recipient mice when the skin sites chronically treated with hapten were elicited. These results indicate that an early-type response by regional LNC would take place only in a milieu with sufficient levels of type 2 cytokines.

Integrin alpha4beta7 has been associated with tissue-specific homing of malignant and inflammatory lymphocytes to gastrointestinal mucosa, whereas integrin alphaEbeta7 has been associated with intraepithelial lymphocytes in both the gut and the skin. This prompted us to examine the expression of alpha4beta7 on skin-infiltrating lymphocytes in 12 cases of patch/plaque stage cutaneous T cell lymphoma (CTCL) and in 4 cases of spongiotic dermatitis, which also display intraepidermal T cell accumulation. alpha4beta7 was found to be expressed on 64.8+/-7.4% of intraepidermal and 39.1+/-5.0% of intradermal T lymphocytes in CTCL. There was a significant positive correlation (r=0.58) between the degree of epidermotropism and the percentage of intraepidermal T cells expressing alpha4beta7. Similar findings were observed in spongiotic dermatitis, indicating that this result is not unique to malignant T cells. We evaluated staining of T cells in the same specimens for presence of alphaEbeta7 and observed a strong correlation between the expression of both beta7 integrins in each specimen. Staining with antibodies directed against the known ligands of alpha4beta7 was also performed on skin biopsies from CTCL patients. There was significantly increased dermal microvascular endothelial expression of vascular cell adhesion molecule-1 in lesional compared with nonlesional skin, and in nonlesional skin compared with skin of normal control subjects. Dermal and epidermal expression of the CS-1 domain of fibronectin was present but not increased in lesional biopsies compared with nonlesional or normal controls, whereas expression of mucosal addressin cell adhesion molecule-1 was not detectable in any skin biopsy specimens. In summary, alpha4beta7, like alphaEbeta7, is expressed at high levels on epidermotropic T cells and may interact with endothelial cell vascular cell adhesion molecule-1 as part of stepwise recruitment of lymphocytes from the blood to the epidermis.

Chromium exposure plays an important role in development of contact dermatitis. The prevalence of contact dermatitis among tannery workers and cement-exposed workers is high. This study was designed to determine the prevalence of contact dermatitis among some Egyptian workers exposed to chromium and to investigate the role of patch test and IgE immunoassay in diagnosis of contact dermatitis. Eighty-three male workers who were exposed to chromium were selected after application of certain exclusion criteria to be the target population of this study. Forty male workers away from exposure to chromium were taken to be the controls. All the exposed and non exposed workers were investigated through an interview questionnaire, clinical examination, patch test and determination of blood and urine chromium levels, absolute eosinophilic count and total IgE level. The results showed that there was no significant difference between exposed workers with clinically diagnosed contact dermatitis and the clinically free exposed workers regarding age and work duration. 7.7% of exposed workers with positive patch test suffered from contact dermatitis while 31.6% of exposed workers with negative patch test suffered from contact dermatitis. There was no statistically significant difference between clinically diagnosed contact dermatitis workers and clinically free workers regarding blood and urine chromium levels. IgE level and absolute eosinophilic count were statistically higher among exposed workers with contact dermatitis than among clinically free exposed workers. According to the results of this study, it is concluded that the diagnosis of skin hypersensitivity to chromium should depend upon the history of chromium exposure, clinical examination and a battery of investigations including IgE level, eosinophilic count and patch test.

In this study, we made a mouse model for contact hypersensitivity (CH) using oxazolone as a contact allergen and examined the expression of interleukin-18 (IL-18) in the diseased skin sites at both mRNA and protein levels. In the kinetic study by semiquantitative RT-PCR, IL-18 mRNA was constitutively produced in normal murine skin but increased significantly at 12 h and peaked at 24 h in the ear skin of CH mice. A positive correlation was confirmed between the IL-18 mRNA signal and CH, as measured by mouse ear swelling response. Histologically, in situ hybridization showed that the IL-18 mRNA signal was weakly observed in the dermis but not the epidermis of normal skin, whereas the IL-18 mRNA signal was found intensively in the dermis, particularly in inflammatory cell areas. Using IL-18-specific antibody immunostaining, it was further found that IL-18 protein production had a histologic location similar to that of IL-18 mRNA in both normal and CH mice. The present study suggests that IL-18 may be implicated in the pathogenesis of murine CH.

To gain a better understanding of the interaction of the model detergent sodium lauryl sulfate (SLS) with the stratum corneum, we investigated systematically the ultrastructural changes of the epidermal barrier and the nucleated parts of the epidermis after the occluded application of different concentrations of SLS in human. Different application models were investigated. Two of the three irritation procedures (long duration exposure and the repetitive exposure for 3 d) provoked damage of the nucleated parts of the epidermis and alterations of the lower parts of the stratum corneum. Here, the extrusion and transformation of lamellar body derived lipids into lamellar lipid bilayers were disturbed; however, the upper portions of stratum corneum displayed intact intercellular lipid layers that contradict the long-standing belief that surfactants damage the skin by delipidization. Furthermore, we investigated ultrastructurally and by measurement of transepidermal water loss the influence and protective capacity of a lipophilic barrier cream on acute irritant contact dermatitis. The irritant contact dermatitis was induced by the standardized cumulative short application model with two SLS concentrations (0.5% and 0.75%). The cumulative type of exposure simulates daily living more realistically. Because most of the previous tests have been performed on the human forearm or back, we analyzed whether the pattern of response was similar on both sites. The back showed a higher level of irritant reaction, but the pattern of irritant response proved to be similar to the forearm. Application of the barrier cream before and during irritation showed a decrease of transepidermal water loss enhancement with 0.5% SLS by 58% (back) and 49% (arm) and after irritation with 0.75% SLS by 56% (back) and 43% (arm). Because the experimental result correlated with the clinical experience, the development of the cumulative short exposure model might help to predict and to discriminate the efficacy of barrier creams.

Previous data suggested a role of endothelial selectins in skin homing of lymphocytes. In the current study, we have analyzed the expression and functional role of E-and P-selectin ligands on CD4+ T cells induced in vivo upon skin sensitization, using soluble selectin-Ig chimera and blocking Abs. Only low numbers of CD4+ cells expressing significant levels of E- or P-selectin ligands were present in s.c. lymph nodes of untreated mice (0.5-1.5% and 2-4%, respectively). Induction of a delayed-type hypersensitivity reaction increased the percentage of E-selectin-binding CD4+ cells in the draining lymph nodes up to 6 to 9% and that of P-selectin-binding cells up to 14%. The majority of E- and P-selectin-binding cells displayed an activated phenotype as judged by the increase in IL-2R, CD71, or cell size. The populations of E- and P-selectin-binding cells were largely overlapping; all E-selectin-binding cells also bound to P-selectin, whereas only a subfraction of P-selectin-binding cells reacted with E-selectin. Both E- and P-selectin-binding CD4+ cells, isolated by FACS, efficiently migrated into inflamed, but not normal skin, whereas P- or E-selectin ligand-negative CD4+ T cells did not. Abs against one of the two endothelial selectins partially inhibited the entry of isolated, ligand-positive cells, whereas a combination of Abs against both selectins almost completely abrogated skin homing. These data indicate that the expression of functional ligands for E- and for P-selectin is essential for homing of CD4+ T cells into the inflamed skin.

Human telomerase RNA (hTER) expression in skin was examined by in situ hybridization analysis. All newborn foreskins examined (n = 5) expressed hTER in epidermal basal cells at moderate levels. Telomerase RNA was not detectable in most adult specimens from sun protected areas (six of seven), whereas all samples obtained from sun exposed areas (n = 8) showed moderate hTER signals in epidermal basal cells. Telomerase RNA was also detected at moderate to strong levels in basal cells of psoriasis, contact dermatitis, and the proliferative cells of the anagen hair bulb. Basal cell carcinoma samples (14 of 15) had moderate to high hTER expression throughout the entire tumor, whereas squamous cell carcinomas (seven of eight) showed variable intensities of hTER expression but only in the cells at the periphery of tumor nests. All melanomas examined (n = 5) had moderate hTER expression in all tumor cells. The hTER signal intensities in skin tumors did not correlate with the age or sex of the donors, the clinical history of the lesions, or the histologic subtypes. To address whether hTER expression correlated with the proliferative state, sequential sections were stained with anti-Ki-67 antibody, a proliferation marker. In newborn foreskins, squamous cell carcinomas, and basal cell carcinomas, the distributions of hTER and Ki-67 were similar but not always identical. Telomerase RNA was more abundant than Ki-67 in the basal and suprabasal layer of newborn foreskins, suggesting that hTER expression is present both in actively cycling and in resting cells.

We investigated the effects of S-(4-dimethylaminobenzyl)-N-[(2S)-3-mercapto-2-methylpropionyl]-L- cysteine (SA6541), a potent leukotriene A4 hydrolase inhibitor. on early phase of carrageenan-induced dermatitis model. Carrageenan injection induced edema and neutrophil influx in the mouse ear. SA6541 inhibited edema formation and neutrophil influx. SA6541 also inhibited leukotriene B4 production but not prostaglandin E2 production in the mouse ear. On the other hand, indomethacin inhibited edema formation but not neutrophil influx. Indomethacin also inhibited prostaglandin E2 production but not leukotriene B4 production. Combination therapy with SA6541 and indomethacin strongly inhibited edema formation in comparison with treatment with either agent alone. These results suggest that leukotriene B4 may be important in the pathogenesis of dermatitis.

To investigate the intracellular signaling mechanisms involved in the activation of APC by contact sensitizers, we studied the induction of tyrosine phosphorylation by these agents. Selective analysis of phosphotyrosine (p-tyr) in human Langerhans cells and different mononuclear cell types was achieved using a multicolor flow-cytometric technique. Stimulation with contact sensitizers revealed a distinct increase in p-tyr exclusively for MHC class II-positive cells. For different haptens, irritants, as well as activators of distinct signal transduction pathways, it was demonstrated that only strong sensitizers or the protein tyrosine phosphatase inhibitor sodium orthovanadate or cross-linking of MHC class II molecules were able to induce formation of p-tyr in human blood-derived dendritic cells serving as model for the dendritic cell family. This event required physiologic cell culture conditions and was blocked by specific inhibitors of protein tyrosine kinases. No evidence for the inhibition of protein tyrosine phosphatases by haptens was found. Western blot analysis of monocyte-enriched populations revealed an augmented phosphorylation of distinct proteins after hapten stimulation partly resembling the pattern noticed after cross-linking of HLA-DR molecules. In dendritic cells generated from mononuclear progenitors, the protein tyrosine kinase inhibitor genistein was able to block tyrosine phosphorylation as well as production of IL-1beta mRNA transcripts. Our data underline the unique capacity of haptens to activate APC and the important role of tyrosine phosphorylation for this process.

The NO synthases (NOS) generate NO from L-arginine. High concentrations of NO have been shown to be responsible for tissue injury and cell death, while low concentrations of NO induce vasodilatation and other signaling effects. We have investigated the involvement of NO in contact hypersensitivity (CHS) reactions. CHS induced by treatment of BALB/c mice with the contact allergen 2,4-dinitrofluorobenzene (DNFB) was significantly reduced by the NOS inhibitor N-methyl-L-arginine (L-NMA), but not by the stereoisomer D-NMA, as shown by reduced ear swelling responses and evaluation of ear tissue sections. The CHS response was also reduced by aminoguanidine, which is known to preferentially inhibit the inducible isoform of the enzyme (iNOS), suggesting that iNOS contributed to the inflammatory response. We therefore investigated whether iNOS was expressed by epidermal cells. Epidermal Langerhans cells produced low but significant amounts of iNOS mRNA at the single-cell level as indicated by RT-PCR. Likewise, keratinocytes expressed basic iNOS mRNA levels. Elicitation of a CHS response by DNFB in vivo resulted in enhanced iNOS mRNA expression in Langerhans cells and keratinocytes, with higher levels of expression in Langerhans cells. The enhanced mRNA expression in Langerhans cells correlated with iNOS protein production as shown by immunofluorescence staining of epidermal sheets performing double staining with anti-iNOS and anti-MHC class II antibodies. Our data suggest that epidermal cell-derived NO contributes to the ear swelling reaction in CHS.

Background: The chemokine eotaxin is produced at sites of allergic inflammation, binds selectively to the chemokine receptor CCR3 and attracts eosinophil and basophil leukocytes, which express high numbers of this receptor. Responses of T lymphocytes to eotaxin have not been reported so far. We have investigated the expression of CCR3 in T lymphocytes and analysed the properties and in vivo distribution of T lymphocytes expressing this receptor., Results: In search of chemokine receptors with selective expression in T lymphocytes, we have isolated multiple complementary DNAs (cDNAs) encoding CCR3 from a human CD4+ T-cell cDNA library. T-lymphocyte clones with selectivities for protein and non-protein antigens were analysed for expression of CCR3 and production of Th1- and Th2-type cytokines. Of 13 clones with surface CCR3, nine secreted enhanced levels of interleukin-4 and/or interleukin-5, indicating that CCR3 predominates in Th2-type lymphocytes. CCR3+ T lymphocytes readily migrated in response to eotaxin, and showed the characteristic changes in cytosolic free calcium. Immunostaining of contact dermatitis, nasal polyp and ulcerative colitis tissue showed that CCR3+ T lymphocytes are recruited together with eosinophils and, as assessed by flow cytometry, a large proportion of CD3+ cells extracted from the inflamed skin tissue were CCR3+. By contrast, CCR3+ T lymphocytes were absent from tissues that lack eosinophils, as demonstrated for normal skin and rheumatoid arthritis synovium., Conclusions: We show that T lymphocytes co-localizing with eosinophils at sites of allergic inflammation express CCR3, suggesting that eotaxin/CCR3 represents a novel mechanism of T-lymphocyte recruitment. These cells are essential in allergic inflammation, as mice lacking mature T lymphocytes were insensitive to allergen challenge. Surface CCR3 may mark a subset of T lymphocytes that induce eosinophil mobilization and activation through local production of Th2-type cytokines.

Objective: To study the immunoneurocrine network in inflammatory dermatoses, we investigated histochemically acute UV and acute contact dermatitis., Methods: Antibodies were applied to frozen and paraffin specimens of human skin after irradiation (n = 10), to positive patch tests (n = 10) and controls (n = 10) against: HSP 70, 72, 27, neuronal polypeptides (alpha-MSH, NSE, bombesin, PGP 9.5, NGF, NGF-R) and intermediate filaments (peripherin, NF 200, CK 19, 20)., Results: HSPs and alpha-MSH were upregulated in UV dermatitis in the epidermis compared to contact dermatitis and normal skin. Sunburn cells did not express HSPs or alpha-MSH in UV dermatitis. Neuronal markers and HSP 27 labeled more nerve fibers in UV than in contact dermatitis, except the increased staining for NGF, NGF-R and alpha-MSH in nerve fibers in contact dermatitis. In UV dermatitis, 50% of Merkel cells were suprabasal, but in contact dermatitis, basal, rounded and reduced in number., Conclusions: Merkel cells, HSPs and markers of neuroinflammation are of different importance in UV and contact dermatitis in vivo.

We examined the effects of dietary nucleoside-nucleotide mixture on synthesis of inflammatory cytokines, interleukin-8 and tumor necrosis factor-alpha, in sensitized and nonsensitized colitic rats. Sensitized and nonsensitized colitic rats that were fed a nucleoside-nucleotide mixture had greater colonic weight and macroscopic and microscopic damage scores than nucleoside-nucleotide-free sensitized and nonsensitized colitic rats. Increased colonic tumor necrosis factor-alpha and interleukin-8 concentrations were associated with increased colonic inflammation and ulceration in the nucleoside-nucleotide mixture-fed group. There was also increased ear thickness in the nucleoside-nucleotide mixture-fed sensitized and nonsensitized colitic rats, which correlated highly with increased tumor necrosis factor-alpha and interleukin-8 levels in the ear lobes. Nucleoside-nucleotide-free diets may suppress cytokine secretion, thereby reducing colonic damage and contact sensitivity responses in colitic rats.

This study used MIB-1 monoclonal antibody to quantify the proliferating keratinocytes and endothelial cells and their proliferation fractions in cases of normal skin, acute and established plaque psoriasis, and acute and chronic spongiotic dermatitis. The number and the proliferation fraction of MIB-1 positive cells were higher in psoriatic and chronic spongiotic lesions than in normal skin (p < 0.05). Established plaque psoriasis had a higher number of proliferating keratinocytes and a higher keratinocytic proliferation fraction than did acute psoriasis (p < 0.05). The number of proliferating endothelial cells decreased as acute psoriatic lesion became chronic, but the number in acute spongiotic lesion increased as it became chronic. The endothelial proliferation fraction was higher in acute psoriasis than in established plaque psoriasis (p < 0.05). The ratio of keratinocytic proliferation fraction to endothelial cell proliferation fraction of the psoriatic and spongiotic lesions suggested the presence of different reaction patterns to inflammation in psoriasis and spongiotic dermatitis.

This paper describes the development of Ag-specific proliferation and the production of IFN-gamma and IL-2 during contact sensitivity (CS) to the hapten picryl chloride (PCl). Lymph node cells from mice immunized with PCl proliferate and produce IFN-gamma and IL-2 when re-exposed to the specific Ag in vitro. Time course experiments showed that the peak IFN-gamma production occurred at days 3 and 4 after immunization, with a sharp decline by day 6. In contrast, proliferation and IL-2 production peaked at day 3 but persisted up to day 10. Proliferation and IFN-gamma and IL-2 production displayed by immune lymph node cells were Ag-specific but required different cell populations. In fact, the production of IFN-gamma was due to a CD8+, gamma delta+ T cell, while proliferation and IL-2 production required the presence of a CD4+, alpha beta+ T cell. Furthermore, IFN-gamma production showed genetic (MHC) restriction, and finer analysis using congenic strains of mice indicated that the K molecule was the restricting element. This was confirmed by blocking the K molecule of the APC used to trigger IFN-gamma production with a specific mAb. In contrast, proliferation and IL-2 production were I-A-restricted, as demonstrated using congenic strains of mice and blocking the I-A molecule of the APCs. Further analysis using purified gamma delta+ cells revealed that these cells produced IFN-gamma in an Ag-specific and MHC (K)-restricted fashion. Injection of mice with a mAb to IL-2 blocked subsequent in vitro proliferation, as well as IL-2 and IFN-gamma production, while all three in vitro responses were unaffected by injection of a mAb to IFN-gamma given at the time of immunization. Furthermore, injection of mice with a mAb to IL-2 blocked the CS reaction when given at the time of immunization, while it had no effect when given at the time of challenge. Injection of mice with mAb to IFN-gamma at the time of challenge reduced but did not abolished CS, suggesting that IFN-gamma is important but not exclusively responsible for the CS reaction.

The study of the participation of T cells and the kinetic studies of cytokines by the RT-PCR method in the challenge phase of contact hypersensitivity reaction (CHR) were performed using skin tissues collected at regular intervals from mice sensitized with 2,4-dinitro-1-fluorobenzene (DNFB). The results obtained from these studies and the further analyses using anti-CD4 and CD8 antibodies showed that the revelation of phenotype of lymph node cells at the reaction site and T lymphocytes at the CHR site were found to play important roles. Furthermore, we examined cytokines formed at the CHR site on the mRNA level. Consequently, it was found that 24 h after the challenge IFN-gamma, IL-6 and IL-1 beta were expressed and 48 h after the challenge IL-2, IL-4 and TNF-alpha were expressed in addition to these cytokines. The results of the expression of IL-12 and those of the examination using IFN-gamma knock out mice suggested that the expression of IFN-gamma among several Th1 types of cytokine is especially important for the formation of CHR.

We examined the mechanism of the onset of contact hypersensitivity reaction (CHR) induced by several kinds of chemical substances in mice, focusing on cytokines. Before doing this experiment, we investigated the involvement of T cells in the formation of CHR using each primer of CD4 and CD8 (surface markers of lymphocytes) positive cells. It was suggested that both cells participate in the formation of CHR. On the basis of these results we analyzed cytokines produced in the reaction site on the mRNA level in with or without the presensitization of two kinds of strong contactants. Consequently, there was a tendency that the expression of mRNA of Th1 types of cytokines was commonly observed around 24 h after the reaction irrespective of the presence or the absence of presensitization. These date suggested that CHR can be presumed on the genetic level by the only one priming of strong contactants.

Many studies report that sunscreens effective against UVR-induced inflammation afford poor protection against immunosuppression. We have studied the relationship between photoprotection of inflammation and immunosuppression with monochromatic UVB (Philips TL01 tubes, lambda max = 311 nm) to remove possible confounding effects of differences in end point action spectra. Dose-response curves for edema and systemic suppression of contact hypersensitivity (CHS) in HRA.HRII-c/+/Skh mice showed that suppression of CHS was more sensitive to UVB irradiation by a factor of 2. The UVB dose-response curve for murine edema was similar to that for human erythema, with threshold doses of 773 mJ x cm(-2) and 632 mJ x cm(-2), respectively. The protection afforded by two UVB filters, octyl dimethyl para-aminobenzoic acid and 2-ethylhexyl-4'-methoxycinnamate, prepared in an identical vehicle, each with the same optical density at 311 nm, was tested in mice. We applied sunscreen to all exposed skin or to transpore tape above the irradiation cages, prior to exposure with 2.8 minimal edema doses. Topical or tape application of both sunscreens protected totally against edema but only partially against immunosuppression, with no significant difference in protection between the two application techniques (p > 0.4). A sunscreen protection factor of 4 in vivo was determined for 2-ethylhexyl-4'-methoxycinnamate for both edema and immunosuppression. Failure of the sunscreens to protect completely against immunosuppression was due to the ability of subedemal doses of UVB to induce substantial immunosuppression and not, as previously suggested, to any skin interaction.

Objective: p-Phenylenediamine (PPD) has been widely distributed as hair dye ingredient and may be responsible for contact dermatitis. Since not all the subjects exposed to PPD react to the substance, we tested a possible predisposing factor of cutaneous drug metabolism., Methods: Eighty-five patients were selected on the basis of their patch test result for PPD. The acetylator status of patients was estimated using HPLC analysis of urinary caffeine metabolites., Results: Among patients with a negative result for PPD, there were three groups, i.e. fast, intermediate and slow acetylators, just as in the healthy population. However, we could not find any rapid acetylator in the PPD-sensitive patient group, and slow acetylators were more often encountered in this group (p < 0.05)., Conclusion: The acetylator phenotype might be a good marker for the cutaneous sensitivity to PPD.

E-selectin (CD62E, formerly termed ELAM-1) is a cytokine-inducible adhesion molecule which mediates the binding of neutrophils, monocytes, and skin homing T-cells. The murine homologue of E-selectin has been cloned. A monoclonal antibody (21KC10) was used here to study immunohistochemically the expression and regulation of murine E-selectin in vitro and in vivo. As described for the human system, there was no staining of normal endothelium in skin and other tissues. LPS and tumour necrosis factor-alpha (TNF-alpha), but not interleukin-4 (IL-4) or interferon-gamma (IFN-gamma), induced a transient expression of E-selectin, both when injected in vivo and when added to endothelial cell lines in vitro. To analyse temporal expression of E-selectin under pathophysiological conditions in vivo, we chose two murine models of inflammation: allergic (ACD) and irritant contact dermatitis (ICD). Expression of E-selectin was found to be induced on vascular endothelium of post-capillary venules in both ACD and ICD. In ICD, maximal staining of endothelial cells occurred earlier than in ACD. Expression of E-selectin during ICD and ACD was then compared between strains of mice which differ with regard to the intensity of their inflammatory reaction. BALB/c mice, which in contrast to C57BI/6 mice show a denser infiltrate and prolonged influx of granulocyte and monocytes, revealed a more pronounced and more prolonged expression of E-selectin than C57BI/6 mice. This held true for both ACD and ICD, and in each case, peak expression of E-selectin was associated with the highest density of the leukocytic infiltrate. This study thus reveals regulatory mechanisms involved in the expression of murine E-selectin in vivo and in vitro. It also demonstrates a correlation between endothelial expression of E-selectin and the genetically determined intensity of the inflammatory response.

Tenascin is a large extracellular matrix glycoprotein which is found in limited regions of normal adult tissues including the skin. We investigated the induction of tenascin expression in mouse skin during hapten-induced dermatitis. In the dorsal skin, hapten application first induced a transient expression of tenascin in deeper regions of the skin. Its distribution then spread over the whole dermis corresponding to the infiltration of Mac-2-positive macrophages. In the ear, tenascin was consistently found in the subcutaneous tissue on the inner side, but very little was seen on the outer side. Tenascin did appear transiently, however, on both sides under hapten treatment. In the early phase of allergic contact dermatitis, no apparent induction of tenascin expression was observed in the swollen ear. However, there was an abundant tenascin expression on both sides during healing. Tenascin expressed under normal conditions was mostly the 180-kDa isoform, while the 230-kDa isoform was markedly induced during healing of the dermatitis. These results suggest that tenascin, particularly the larger 230-kDa isoform, may play important roles in the pathogenesis and healing of hapten-induced dermatitis.

Treatment of mice with multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol resulted in a preferential decrease in epidermal protein kinase C-beta 2 (PKC-beta 2) compared with PKC-alpha as determined by western analysis. When PKC-alpha was decreased by 40%, PKC-beta 2 could no longer be detected, suggesting that PKC-beta 2 is more sensitive to downregulation, and/or specific epidermal cell types that contain PKC-beta 2 are more sensitive to TPA/diacylglycerol. To address this issue, we isolated Langerhans cells (LCs) from epidermal cell suspensions with immunomagnetic beads and an antibody to the class II major histocompatibility complex. Northern blot analysis revealed a PKC-beta 2 signal in isolated LCs that was 40-fold greater than that observed in unfractionated epidermal cells, and no PKC-beta 2 signal was detected in epidermal cells depleted of LCs, indicating that PKC-beta 2 is expressed exclusively in LCs within the epidermis. Western blot analysis confirmed the presence of PKC-beta 2 in LCs. PKC-beta 2 was highly sensitive to downregulation, because a single application of TPA resulted in a 90% loss of PKC-beta 2 within 6 h without a decrease in the number of LCs. To determine whether the decreased level of PKC-beta 2 within LCs was associated with an alteration in contact hypersensitivity, we treated mice with only a single application of TPA, and 6 hours later mice were sensitized with 2,4-dinitrofluorobenzene on the same dorsal area. Subsequent challenge revealed a 60% decrease in contact hypersensitivity in TPA-treated mice. These data indicate that (i) within the epidermis, PKC-beta 2 is highly sensitive to downregulation and is exclusively expressed in LCs, and (ii) the downregulation of PKC-beta 2 is associated with impaired LC function with respect to contact hypersensitivity.

Retinoids have shown anti-inflammatory activity in some animal models and human diseases, although the mechanism by which retinoids elicit this activity is unknown. In this study, retinoids significantly attenuated, in a dose-dependent fashion, murine ear oedema induced by phorbol 12-myristate 13-acetate (PMA) or oxazolone. Dexamethasone inhibited both oedemas whereas ketoprofen reduced only that induced by PMA. PMA application or oxazolone-induced contact hypersensitivity markedly increased production of eicosanoids such as 6-keto-PGF1 alpha or LTB4. The anti-oedematous effects of retinoids were accompanied by inhibition of tissue eicosanoid levels. Besides, retinoids showed toxic effects on culture fibroblasts caused by an irritant effect on plasma membrane. However, when we used subtoxic doses, we demonstrated that retinoids in vitro could inhibited arachidonate mobilization and eicosanoid biosynthesis induced in fibroblast cultures by PMA, calcium ionophore A23187 or bradykinin. Thus, this paper reports the ability of retinoids to inhibit skin inflammatory processes induced by tumour promotors or immunological stimuli. Moreover, we have demonstrated that retinoids at non-cytotoxic doses may inhibit eicosanoid generation and arachidonic acid mobilization in 3T6 fibroblasts.

Previous work has indicated the importance of cytokine cascades in the induction of contact dermatitis, but there is little information on the cellular localization of cytokines in human skin, particularly during the early phases of the inflammatory response to contact allergens. Using in situ hybridization for mRNA and immunocytochemistry on biopsies from a series of 16 patients with known allergic contact dermatitis, we examined the kinetics of early cytokine production after challenge with relevant or irrelevant antigen. We show that epidermal keratinocytes from patients challenged in vivo with allergen, but not irrelevant antigen, rapidly synthesize (within 4 h) mRNA for interferon-gamma and produce immunoreactive interferon-gamma. Interleukin-1alpha and interleukin-8 mRNA were also detected but showed no correlation with relevant antigen challenge. This study demonstrates that keratinocytes can produce interferon-gamma and that this production is linked to challenge with relevant antigen in allergic contact dermatitis. These findings indicate that keratinocytes may amplify allergen-specific T-lymphocyte-triggered interferon-gamma dependent responses and might partially explain the speed of reaction in this common disease and other delayed hypersensitivity reactions involving the skin.

Langerhans cells (LCs) have to leave the epidermis to migrate into the regional lymph node (LN) after receiving immunogenic signals in contact sensitivity. There should be some molecules responsible for migration of LC in this process. In order to determine whether LN cells can generate such regulatory molecules, we made a search of such molecules in the culture supernatant of LN cells obtained from hapten-painted mice by using a modified Boyden chamber method. The molecule of LC chemotaxis appeared in the culture supernatant at 24 h after hapten painting and its production declined with time thereafter. Antibodies against both ICAM-1 and LFA-1 partially inhibited the chemotactic activity, but this was not the case with anti-TNF-alpha antibody, anti-GM-CSF antibody, fibronectin RGDS. RGES peptide or laminin. The molecule was heat labile and appeared as a molecule of 45-68 kDa on high performance liquid chromatography. Our results suggest that LN cells generate one of the chemotactic molecules of LCs, by which LC can migrate to the regional LN in contact sensitivity.

Epidermal cells, both keratinocytes and Langerhans cells, are able to synthesize and secrete a variety of cytokines, many of which influence or are essential for the induction of skin sensitization and the elicitation of local inflammatory reactions. It has been proposed that it may prove possible to distinguish between contact allergens and skin irritants as a function of differential induction or upregulation of epidermal cytokine expression. In the present study we have addressed this by examination of the local cutaneous production of interleukin 6 (IL-6) following topical exposure of mice to oxazolone, a potent contact allergen, or to benzalkonium chloride (BZC), a skin irritant that is considered not to have a significant potential to cause skin sensitization. Both oxazolone and BZC could induce the production of IL-6 as measured by enzyme-linked immunosorbent assay in homogenates prepared from treated skin. However, when these chemicals were applied at concentrations that resulted in equivalent cutaneous inflammatory responses, based on induced oedema, only oxazolone provoked the production in skin of IL-6. Moreover, under these conditions exposure only to oxazolone resulted in the secretion by draining lymph node cells of measurable concentrations of this cytokine. These data suggest that the ability of oxazolone to stimulate local IL-6 production is not secondary simply to the induction of local inflammatory responses. As such, the results support the possibility that skin allergens and skin irritants may stimulate variable patterns of epidermal cytokine production.

Vascular leakage induced by intradermal injection of endotoxin, zymosan-activated plasma (ZAP) and platelet-activating factor (PAF) was measured in nine Thoroughbreds using 125-iodine human serum albumin (125I-HSA) as a marker in the blood. ZAP and PAF produced dose-dependent increases in vascular permeability with the maximum occurring within the first 15 min after injection. The vascular leakage induced by endotoxin was also dose-dependent, but the maximum occurred 2 h after intradermal injection. Intradermal sites previously injected with endotoxin were refractory to a second injection of endotoxin for up to 5 days. However, sites injected with endotoxin and re-injected with either ZAP or PAF remained responsive with increased vascular leakage compared to saline injected control sites re-injected with either ZAP or PAF. Diminished response to endotoxin challenge may contribute to the poor prognosis of endotoxaemia in the horse.

Fluoroquinolones are widely used for the treatment of bacterial infection. However, some members of the group cause UV-dependent dermatitis. Therefore, animal models are required to screen and predict if a derivative is likely to cause photodermatitis. Under anaesthesia, the hair on the dorsal side of rats was shaved, then a fluoroquinolone derivative, Y-26611 or ofloxacin was administered subcutaneously (SC) followed by irradiation of a 3 cm diameter circular area of the skin with UV-B(ultraviolet-B) for 45 min. Peak photodermatitis was observed 24 h after irradiation in Y-2611-treated skin. Leucocyte infiltration into the skin, mostly polymorphonuclear leucocytes, was verified by histological techniques. O2.- radical generated by activated leucocytes was detected directly from the skin by SC application of a sensitive O2.- -dependent luminescence reagent, MCLA. O2.- -dependent photon emission (chemiluminescence) from the skin was amplified by phorbol myristate acetate and was markedly suppressed by superoxide dismutase. This is the first report of direct detection of chemiluminescence from the skin of a living animal.

The non-invasive electrical impedance technique used in this study reflects structural changes in a tissue, and provides an estimate of the level of oedema by a simple impedance index. Sodium lauryl sulphate (SLS), dissolved in water at concentrations of 0.1, 0.5 and 2.0%, was applied for 24 h in 12 mm Finn chambers on both volar forearms of 12 healthy volunteers. An unoccluded area was used as a reference site. Readings from all sites were taken before the application of the irritant, and 24 h after its removal. After the last reading, a 3-mm punch biopsy was taken from each test site for histological examination. The results obtained from electrical impedance measurements at five different skin depths were correlated with those obtained from histological examination, visual scoring and transepidermal water loss (TEWL). For all of the methods used the responses were proportional to the concentration of the irritant. Statistically significant changes of electrical impedance were found for all depths and concentrations, except for 0.1% SLS at the most superficial depth. The histological changes were focused in the epidermis, and mainly consisted of oedema. Alterations in the thickness of the epidermis due to oedema were used as a quantitative parameter for correlation with the assessment of irritation using the electrical impedance technique. For the detection of irritant reactions, TEWL and electrical impedance are more sensitive than visual scoring, and selection of the optimum depth penetration further increases the sensitivity of the electrical impedance measurement.

The skin lesions of patients with atopic dermatitis provide a model to study immunoregulation in human allergy. To determine the local cytokine pattern of cells present (both endogenous and recruited) at the site of disease, we extracted RNA from skin biopsy specimens from patients with atopic dermatitis, allergic contract dermatitis, and positive tuberculin reactions and used PCR to assay for cytokine mRNA. cDNAs were normalized to the intensity of the CD3 delta PCR product as a marker of T cell mRNA. We found overexpression of IL-10 mRNA in atopic dermatitis lesions, in comparison with allergic contact dermatitis lesions and tuberculin reactions. In contrast, IL-4 mRNA was most strongly expressed in allergic contact dermatitis lesions and IFN-gamma mRNA was the predominant cytokine in tuberculin reactions. Using an anti-IL-10 mAb with immunoperoxidase, we localized IL-10 protein to large mononuclear cells in the dermal infiltrate of atopic lesions. After immunomagnetic sorting of mononuclear cell populations from PBMC of atopic dermatitis subjects, IL-10 mRNA as measured by PCR was found to be strongly expressed in CD14+ cells. Spontaneous release of IL-10 from PBMC-derived adherent cells was greater in atopic dermatitis donors than normal controls. We therefore renormalized skin biopsy cDNA according to the level of beta-actin PCR product, as a marker of total cellular mRNA, and found by PCR that IL-10 was nevertheless greatest in atopic dermatitis subjects. We conclude that the relative overexpression of IL-10 in atopic dermatitis greatest in atopic dermatitis subjects. We conclude that the relative overexpression of IL-10 in atopic dermatitis may contribute to the up-regulation of humoral responses and the down-regulation of Th1 responses.

The depigmenting C57BL/6J-mivit/mivit) (mivit/mivit) mouse, a congenic mutant of the C57BL/6 strain, exhibits an isolated, single immune deficiency. It is unable to mount a normal immune/inflammatory response upon epicutaneous application of DNFB or TNCB, although it does respond normally to oxazalone. The present investigations have been carried out to further study this deficiency. In vivo, C57BL/6 mice could be sensitized by the epicutaneous application of the hapten TNCB, the subcutaneous injection of hapten(TNBS)-conjugated C57BL/6, and hapten conjugated mivit/mivit epidermal cells. In the mivit/mivit mice, however, only subcutaneous injection of haptenized C57BL/6 epidermal cells caused an immune response. The response of these mivit/mivit mice could be documented only by adoptive transfer of splenic lymphocytes into naive C57BL/6 animals which then reacted to challenge doses of TNCB. These observations suggest that mivit/mivit epidermal cells can process and present and mivit/mivit T lymphocytes can react to the antigen. We postulated the presence of a deficient in vivo interaction between epidermal cells and T lymphocytes in the mivit/mivit mice. ICAM-1 is an important adhesion signal regulating epidermal cell/T-lymphocyte interaction. Its expression in mivit/mivit mice was studied using YN1/1 antibody against MALA-2, the murine counterpart of human ICAM-1. In contrast to C57BL/6 animals, the mivit/mivit epidermis essentially did not stain with the antibody after hapten challenge. In vitro after stimulation with TPA or IFN-gamma, the mivit/mivit epidermal cells expressed significantly lesser amounts of ICAM-1 than the C57BL/6 epidermal cells. Lower expression of ICAM-1 by mivit/mivit epidermal cells has also been demonstrated both by direct staining and by flow cytometry. The binding of lymphocytes to mivit/mivit epidermal monolayers, which were stimulated to express ICAM-1 by IFN-gamma, was decreased compared to that of C57BL/6 epidermal cells. We conclude that the muted contact sensitization response detected in vivo in the mivit/mivit mice at least partly results from lower expression of ICAM-1 and thus defective epidermal cell/T-lymphocyte interaction.

In vivo histamine release in cutaneous allergic reactions was chronologically examined using the skin microdialysis technique. Antigen challenge was made in ovalbumin-sensitized guinea pigs 1 h after the implantation of the microdialysis probe. A marked histamine release after intradermal injection of ovalbumin solution was observed in the early phase (up to 40 min). No isolated delayed histamine release was observed thereafter over 8 h. Also, in an atopic volunteer, a marked release of histamine after intradermal injection of mite extract was observed only in the early phase, although erythema and induration at the site of injection were continuously observed in the late phase (up to 4 h). These findings suggest that the skin microdialysis technique is a useful, simple technique for chronological determination of chemical mediators released in the allergic skin in vivo.

Many skin diseases such as eczema and psoriasis are characterised by a chronic inflammatory skin condition. In this respect they resemble other chronic diseases such as rheumatoid arthritis, bronchial asthma, ulcerous colitis and Crohn's disease. A persistent accumulation, predominantly of T-lymphocytes constitutes the central pathophysiological feature of such diseases. The past 15-20 years have witnessed the characterisation of an extensive series of peptides known as cytokines. These are soluble, relatively low molecular weight peptides which at low concentrations mediate regulation of cellular receptors, new phenotype expression, secretion and migration. Many cytokines have been found to be present in conjunction with skin diseases, and it is suggested that they are involved in the development of inflammation.

A Helium-Neon (He-Ne) laser with a wavelength of 632.8 nm is known to have photobiological effects and is widely used for reducing the pain of herpes zoster and accelerating wound healing, however the cellular mechanism and effect of the He-Ne laser are poorly understood. The present study was designed to examine the influence of He-Ne laser irradiation on irritant and allergic contact dermatitis of the mouse ear and on histamine release from rat peritoneal mast cells. Irradiation was applied with a He-Ne laser (12.2 J/cm2) at 1 h, 10 min, 5 min and 0 min before, and 5 min, 6 hs and 24 hs after a challenge of an irritated contact dermatitis (ICD) or allergic contact dermatitis (ACD) was made on the right ears of ICR-mice. Twenty-four hours after the challenge, the swelling of the ear was measured with a dial thickness gauge, and the anti-inflammatory effect of He-Ne laser irradiation was expressed as an ear thickness ratio (ETR). Although the laser did not decelerate the ETR from ICD, the allergic response was decelerated. Irradiation at 5 min after the challenge of contact dermatitis increased the thickness ratio. Next, the influence of the He-Ne laser on histamine release from Wistar-rat peritoneal mast cells was observed. The spontaneous histamine release was inhibited by laser irradiation, while substance P and compound 48/80-induced histamine release were not inhibited. From these results, it can be suggested that He-Ne laser irradiation has an anti-inflammatory effect on cutaneous inflammation.

To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensitizer (2,4-dinitrofluorobenzene, 3-n-pentadecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Interleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, interleukin-8 production was not detectable in unstimulated normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immunogenic or inflammatory signals in man.

The production of H2O2 by cells in cold paraformaldehyde-fixed frozen sections of inflammatory lesions was histochemically demonstrated by incubating them with diaminobenzidine (DAB) for 2 to 6 h. Catalase (150 micrograms/ml, about 1400 U/ml) inhibited the reaction, indicating that H2O2 was required to produce the chromogenic DAB product. Granulocytes (PMNs and eosinophils) were the main types of cells stained by the DAB reaction. Positive staining of macrophages was less frequent. The H2O2 was produced by metabolic enzymes that were still active after cell death and mild fixation. An atmosphere of 95 to 100% oxygen enhanced the specific DAB reaction, and an atmosphere of 100% nitrogen eliminated it. The DAB histochemical reaction to detect H2O2 requires the presence of peroxidases to produce the colored reaction product. Within our tissue sections, such peroxidases were evidently present in excess, because addition of low concentrations of H2O2 significantly increased the reaction product. Although some of the H2O2 produced by the granulocytes may have been derived from the dismutation of superoxide (O2-), the NADPH oxidase pathway for O2- formation did not seem to be involved: NADPH oxidase, a rather labile enzyme, should not be active after mild fixation, and diphenyleneiodonium (100 microM), an inhibitor of flavine-requiring NADPH oxidase, did not inhibit the reaction. Reactive nitrogen intermediates were also not involved, because NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthetase, did not appreciably inhibit the reaction. We conclude that stable, non-flavine-requiring oxidases, possibly cyclooxygenases or lipoxygenases, produced the H2O2 measured histochemically by our DAB reaction. These studies were made on tissue sections of acute dermal inflammatory lesions produced in rabbits by the topical application of 1% sulfur mustard [bis(2-chloroethyl) sulfide] in methylene chloride. Both intact PMNs and disintegrating PMNs in the base of the crust produced H2O2. Despite the production of H2O2 and the presence of peroxidase activity, no tissue damage was seen microscopically near the H2O2-producing cells, which indicates that the tissues are well protected by the antioxidants present in this self-limiting inflammatory reaction.

In the present investigation, the effects of selective inhibitors of 5-lipoxygenase (5-LO), zileuton and TZI-41127, E-6080, AA-861 and antagonists of leukotriene B4 (LTB4) receptors, SC-41930, and SC-51146 and a selective cyclooxygenase inhibitor, indomethacin, were examined in TPA-induced acute mouse dermal inflammation. Topical application of all these agents, except indomethacin, resulted in marked attenuation of TPA-induced edema and influx of neutrophils reflected in myeloperoxidase measurements. Topically applied SC-41930 attenuated TPA-induced edema and neutrophil influx in a dose-related manner. Oral administration of LTB4 receptor antagonists either as a pre-treatment or post-treatment attenuated TPA-induced edema and influx of neutrophils. The O-demethyl analog of SC-41930, SC-37920, which was nearly 1000-fold less active than SC-41930 in LTB4 receptor binding assays, was inactive in inflammation assays, suggesting a role for LTB4 in this response. Zileuton and TZI-41127 were more effective as anti-inflammatory agents following oral administration than after i.p. administration. Intraperitoneally administered indomethacin attenuated edema response but not influx of neutrophils. Taken together, these results suggest a role for leukotrienes in acute inflammation induced by TPA and possible utility of this model to test in vivo 5-LO inhibitors and LTB4 receptor antagonists.

Azelastine hydrochloride (AZE) is an anti-allergic drug that inhibits the release of various chemical mediators from mast cells. We compared the immunosuppressive effects of AZE and FK-506 in vivo and in vitro. Topical application of AZE strongly inhibited the efferent phase of contact hypersensitivity, as did application of FK-506. In in vitro experiments, we found that 1) the suppression by AZE on interleukin (IL)-2 production from splenic T cells was partial and considerably large amounts of IL-2 were still produced, even in the presence of 10(-5) M of AZE, which was in sharp contrast to the observed marked inhibition of [3H]-TdR incorporation; 2) AZE significantly inhibited the phorbol myristate acetate-induced IL-2 responsiveness; 3) AZE did not inhibit the IL-2 receptor alpha expression of activated T cells; and 4) the significant inhibitory action was still observed even when AZE was added at 48 h after the initiation of culture. In regard to FK-506, we found that 1) FK-506 completely blocked the production of IL-2; 2) exogeneous IL-2 consistently restored the FK-506-induced inhibition; 3) FK-506 affected the phorbol myristate acetate-induced IL-2 responsiveness very little, if any; and 4) the significant suppression was observed only when FK-506 was added within 24 h after the initiation of culture. Thus, AZE exerts its in vitro immunosuppressive activity preferentially by interfering with the IL-2 responsiveness, with partial inhibition of IL-2 production. Conversely, FK-506 acts as a strong inhibitor of IL-2 production without a prominent effect on IL-2 responsiveness. The immunosuppressive activity of AZE shown in vitro may also be operative in vivo and may be applicable for topical use.

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.

The expression of the murine IP-10 and MCP-1 genes has been examined in the skin of mice during contact hypersensitivity reactions to the hapten trinitrochlorobenezene (TNCB). In both naive and passively sensitized animals, challenge with TNCB resulted in elevated expression of both genes as early as 4 h as detected by Northern hybridization analysis. Twenty-four hours after challenge, expression was markedly reduced in naive animals but remained elevated in sensitized animals. This prolonged expression of chemokine gene products correlates with the tissue swelling response generally used as a measure of delayed-type hypersensitivity (DTH) in this model and suggests that the continued expression of these genes results from the stimulation of hapten-specific T helper cells. Examination of cell type expression patterns by in situ hybridization using 3H-radiolabeled riboprobes confirmed the results of Northern hybridization experiments. Both genes were expressed predominantly in cells exhibiting the morphology of connective tissue fibroblasts, although the distribution of cells positive for IP-10 mRNA expression differed from that of cells expressing MCP-1 mRNA. IP-10 expression was localized almost exclusively to a population of connective tissue cells surrounding the fur follicle. MCP-1 expression was rarely found associated with fur follicles but instead was distributed throughout the dermis in cells embedded in the collagenous extracellular matrix. Surprisingly, neither endothelial cells lining the small vessels located deep within the dermis nor epidermal keratinocytes were positive under any of the conditions utilized in the present study. Expression of both IP-10 and MCP-1 has been previously reported in a variety of distinct cell types in vitro. The present results indicate that only a subset of the cell types with such potential are stimulated to express these chemokine genes in vivo during hapten-mediated DTH responses, implying the presence of subtle cell type- and tissue-specific control mechanisms.

T-cell activation probably plays the most important role in hyperproliferation of keratinocytes in psoriasis. We present here our results concerning the interacting immunocompetent cells and their phenotypic and functional characteristics in relation to psoriasis pathology. Immunohistochemical analysis of skin biopsies from psoriasis patients, did indeed show that hyperproliferation of keratinocytes is associated with increased vasculature and increased influx of MHC class II molecules expressing immunocompetent cells. Furthermore, in psoriasis, several adhesion molecules and other relevant activation markers were found to be upregulated even in the non-lesional psoriatic skin, indicating that psoriatic skin in general is in an activated state. This interpretation is further supported by the observation that the expression of several AR and other relevant activation markers when compared with those in non-lesional skin from contact dermatitis are increased in a significant manner in the non-lesional skin of psoriasis patients. We have then followed up our investigations by generating T-cell lines from lesional psoriatic skin and studied their adhesion patterns on cultured endothelial cells in order to get better insight into the migration pattern of different T cell subsets in psoriasis pathology. Our results indicate that different T-cell subsets CD4+, CD8+ (both TCR-alpha beta+) CD4-/CD8+ TCR-gamma delta+ and CD4-CD8-TCR-gamma delta (V delta 1-) T-cells can easily be generated from psoriatic patients. In a comparative kinetic study using unstimulated and stimulated cultured human umbilical vein endothelial cells, we observed that TCR-gamma delta T cells showed different adhesion properties from that of TCR-alpha beta+ T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Perforin (Pf), a pore-forming protein, is a cytolytic protein of killer cells. Its deposition in lesioned skin has not been studied., Objective: The purpose of this study is to show Pf deposition in the lesioned skin and Pf expression in the dermal infiltrates of various inflammatory skin diseases., Methods: Frozen specimens obtained from 29 patients with 5 different diseases were immunohistochemically stained., Results: Granular deposition of Pf was found in the lesioned skin in 2 out of 5 cases of viral vesicles and in 3 out of 7 cases of erythema multiforme. In the cases with Pf deposition, the percentages of Pf+ cells in the dermis were higher than in those without deposition., Conclusion: Pf released from natural killer cells or cytotoxic T lymphocytes may play a role in tissue damage.