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The healing phase of contact hypersensitivity reactions is critically dependent on regulatory T cells (Tregs), but even the early inflammatory phase, that is, 6-24 hours after induction of a contact hypersensitivity reaction, is susceptible to Treg-mediated suppression. To investigate the underlying mechanisms, we injected Tregs before the challenge and analyzed the skin-infiltrating cells as early as 6 hours later. Early on, we found mainly neutrophils in the challenged skin, but only a few T cells. This influx of neutrophils was blocked by the injection of Tregs, indicating that they were able to prevent the first wave of leukocytes, which are responsible for starting an immune reaction. As an underlying mechanism, we identified that Tregs can tighten endothelial junctions by inducing intracellular cAMP, leading to protein kinase A-RhoA‒dependent signaling. This eventually reorganizes endothelial junction proteins, such as Notch3, Nectin 2, Filamin B, and VE-cadherin, all of which contribute to the tightening of the endothelial barrier. In summary, Tregs prevent the leakage of proinflammatory cells from and into the tissue, which establishes a mechanism to downregulate immune reactions.

Allergic contact dermatitis and its animal model, contact hypersensitivity, are T-cell-mediated inflammatory skin diseases that require activation of the innate immune system. Here we investigate the role of innate lymphoid cells (ILCs) during the elicitation phase of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity using EomesGfp/+ x Rorc(γt)-CreTg x Rosa26RYfp/+ reporter mice. Ear swelling responses, cutaneous ILC numbers, and cytokine production were determined at different time points. Functional analyses were performed in a CD90.1/.2 congenic adoptive transfer model that allowed selective antibody-mediated depletion of ILCs before hapten challenge, and in Rorasg/floxIl7rCre/+ mice, which lack ILC2. Hapten challenge induced early increases of natural killer cells in skin and ear draining lymph nodes corresponding to the peak ear swelling response. In contrast, ILC1, 2, and 3 showed a delayed increase in numbers corresponding to the contact hypersensitivity resolution phase. Hapten challenge induced increased marker cytokines in all ILC subtypes and an activated phenotype in ILC2. Depletion of all ILC resulted in a significantly enhanced ear swelling response. Similarly, ILC2-deficient mice (Rorasg/floxIl7rCre/+) displayed increased ear swelling responses on hapten challenge, suggesting that ILC2 act as negative regulators in the type 1-dominated immune response of contact hypersensitivity.

Allergic contact dermatitis, caused by nickel, is a delayed-type hypersensitivity reaction, and 14.5% of the general population may be affected in Europe. Among a wide range of cytokines, the IL-12 family has unique structural and immunological characteristics. Whereas IL-12p70 promotes T helper (Th) 1 cell polarization, IL-23 promotes Th17 cell development and both have been isolated from nickel-allergic patients. In this work, we were interested in understanding the mechanism behind nickel-induced Th17 cell development. We showed that nickel induced an early production of IL-23 in human monocyte-derived dendritic cells along with an increase in the expression of il-23p19 and il-12p40 mRNA. However, the production of a significant level of IL-12p70 required an additional signal such as IFN-γ. Moreover, nickel-treated monocyte-derived dendritic cells induced an increase in the percentage of IL-17A+ CD4+ T cells, an effect reduced by IL-23 neutralization. We then investigated the molecular mechanism of IL-23 production. Our results showed that toll-like receptor 4, p38 mitogen-activated protein kinase, and NF-κB were involved in IL-23 production induced by nickel. However, Jak-signal transducer and activator of transcription activation seems to maintain the IL-23/IL-12p70 balance by limiting IL-23 production and promoting Th1 polarization. These results indicate that nickel-induced Th17 cell development is dependent on the production of IL-23 by human monocyte-derived dendritic cells via toll-like receptor 4, p38 mitogen-activated protein kinase, NF-κB, and Jak-signal transducer and activator of transcription pathways.

Skin is the primary barrier between the body and the outside world, functioning not only as a physical barrier, but also as an immunologic first line of defense. A large number of T cells populate the skin. This review highlights the ability of these cutaneous T cells to regulate skin-specific environmental threats, including microbes, injuries, solar UV radiation, and allergens. Since much of this knowledge has been advanced from murine studies, we focus our review on how the mouse state has informed the human state, emphasizing the key parallels and differences.

Injection of regulatory T cells (Tregs) followed by sensitization with 2,4,6-trinitrochlorobenzene induced a transient increase in size and cellularity of skin-draining lymph nodes (LNs) in mice. This led us to hypothesize that Tregs may affect the trafficking of T cells from and to peripheral LNs. Two to three hours after sensitization, we found fewer CD8+ T cells expressing CD62L in LNs compared with untreated controls. Injection of wild-type Tregs prevented this down-regulation of CD62L. In contrast, Tregs devoid of the adenosine triphosphate (ATP)-degrading ecto-enzyme CD39 were unable to do so. As for the mechanism of CD62L regulation, we found that ATP, which is released in skin upon hapten-exposure, is inducing the protease ADAM17 in LN-residing T cells via engagement of P2X7 ATP receptors. ADAM17 cleaves CD62L from the surface of CD8+ T cells, which in turn provide a signal for T cells to leave the LNs. This regulation of CD62L is disturbed by the presence of Tregs, because Tregs remove extracellular ATP from the tissue by activity of CD39 and, therefore, abrogate the shedding of CD62L. Thus, these data indicate that the regulation of ATP turnover by Tregs in skin and LNs is an important modulator for immune responses.

T-cell-mediated immune responses play an important role in body protection. However, aberrantly activated immune responses are responsible for inflammatory and autoimmune diseases. The regulation of pathologic immune responses may be a potential therapeutic strategy for the treatment of these diseases. Despite that multiple pharmacologic properties of benzoxathiole derivatives have been defined, the molecular mechanisms underlying these properties remain to be clarified. Here, we demonstrated the benzoxathiole derivative 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one (BOT-4-one) regulated immune responses and ameliorated experimentally induced inflammatory skin diseases both in vitro and in vivo. BOT-4-one inhibited the differentiation of CD4(+) T-cell subsets by regulating the expression and production of T-cell lineage-specific master transcription factors and cytokines and activating the signal transducer and activator of transcription proteins. In addition, BOT-4-one inhibited TCR-mediated Akt and NF-κB signaling. Topical application of BOT-4-one ameliorated experimentally induced inflammatory skin diseases in mice models such as 2,4,6-trinitrochlorobenzene-induced contact and atopic dermatitis and IL-23-induced psoriasis-like skin inflammation. Our study demonstrated that BOT-4-one ameliorates inflammatory skin diseases by suppressing the pathogenic CD4(+) T cell differentiation and overall immune responses.

Recurrent and intractable chronic itch is a worldwide problem, but mechanisms, especially the neural mechanisms, underlying chronic itch still remain unclear. In this study, we investigated the peripheral and spinal mechanisms responsible for prolonged itch in a mouse model of allergic contact dermatitis induced by squaric acid dibutylester. We found that repeated exposure of mice to squaric acid dibutylester evoked persistent spontaneous scratching and significantly aberrant cutaneous and systemic immune responses lasting for weeks. Squaric acid dibutylester–induced itch requires both nonhistaminergic and histaminergic pathways, which are likely relayed by GRPR and NPRA in the spinal cord, respectively. Employing genetic, pharmacologic, RNAscope assay, and cell-specific ablation methods, we dissected a neural circuit for prolonged itch formed as Grpr+ neurons act downstream of Npr1+ neurons in the spinal cord. Taken together, our data suggested that targeting GRPR and NPRA may provide effective treatments for allergic contact dermatitis–associated chronic pruritus.

Obesity is a major risk factor for inflammatory dermatologic diseases, including atopic dermatitis and psoriasis. In addition, recent studies have shown that obesity impairs lymphatic function. As the lymphatic system is a critical regulator of inflammatory reactions, we tested the hypothesis that obesity-induced lymphatic dysfunction is a key regulator of cutaneous hypersensitivity reactions in obese mice. We found that obese mice have impaired lymphatic function, characterized by leaky capillary lymphatics and decreased collecting vessel pumping capacity. In addition, obese mice displayed heightened dermatitis responses to inflammatory skin stimuli, resulting in both higher peak inflammation and a delayed clearance of inflammatory responses. Injection of recombinant vascular endothelial growth factor-C remarkably increased lymphangiogenesis, lymphatic function, and lymphatic endothelial cell expression of chemokine (C-C motif) ligand 21, while decreasing inflammation and expression of inducible nitrous oxide synthase. These changes resulted in considerably decreased dermatitis responses in both lean and obese mice. Taken together, our findings suggest that obesity-induced changes in the lymphatic system result in an amplified and a prolonged inflammatory response.

The year 2014 marks the centennial of events that led to the First World War ("the war to end all wars") following the assassination of Archduke Ferdinand of the crumbling Austro-Hungarian Empire. It also marks the 120th anniversary of the birth of Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin. In this review, we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical backwater to a scientific discipline that can hold its own with any other specialty.

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.

Keratinocytes have a key role in all phases of allergic contact dermatitis. We have recently identified the possibility to use IL-18 production for the in vitro identification of contact allergens. The purpose of this study was to characterize the molecular mechanisms underlying allergen-induced IL-18 production, in order to identify the cellular source of reactive oxygen species (ROS) and the danger signals involved. The NCTC2544 cell line was exposed to three contact allergens, namely p-phenylenediamine (PPD), 2,4-dinitrochlorobenzene (DNCB), and citral, in the presence or absence of diphenylene iodonium (DPI), allopurinol, and rotenone to identify the source of ROS, and to anti-Toll-like receptor 4 antibody and glycirrizic acid to characterize the danger-associated molecular pattern molecules. In the case of PPD, the induction of IL-18 can be modulated by rotenone, allopurinol, and DPI. In the case of DNCB, rotenone completely prevents the induction of IL-18, whereas for citral, DPI completely prevents the induction of IL-18. We demonstrated the ability of all allergens tested to induce the release of high-mobility group protein B1 (HMGB1). Its sequester by glycirrizic acid significantly modulates PPD-induced IL-18 production and completely prevents DNCB- and citral-induced IL-18. We found that different intracellular sources of ROS are triggered by contact allergens, and an important role for HMGB1 in chemical allergen-induced IL-18 production was demonstrated.

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.

The cytokine IL-9, derived primarily from T-helper (Th)-9 lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also plays a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch test sites from non-atopic patients were assayed using qPCR and immunohistochemistry. Along with Th2 associated cytokines, IFN-γ, IL-4, and IL-17A, expression of IL-9, and PU.1, a Th9-associated transcription factor, were elevated when compared to paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+CD3+, and PU.1+CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. PBMC from nickel-allergic patients, but not non-allergic controls, show significant IL-9 production in response to nickel. Blocking studies with monoclonal antibodies to HLA-DR (but not HLA-A, B, C) or chloroquine significantly reduced this nickel-specific IL-9 production. Additionally, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9−/− mice, shows enhanced Th1 lymphocyte immune responses, when compared to WT mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.

Allergen-specific responses in atopic dermatitis (AD) are skewed toward a Th2 profile. However, individuals with AD have been shown to make effective virus-specific Th1 responses, raising the possibility that the skin itself contributes to driving the AD Th2 immunophenotype. Therefore, to explore the programming of immunological sensitization by the skin, we examined the outcome of sensitization through non-lesional skin of individuals with AD and healthy controls. Volunteers (controls, AD individuals with filaggrin gene (FLG) mutations (ADFM), and AD individuals without FLG mutations (ADWT)) were sensitized by cutaneous application of 2,4-dinitrochlorobenzene (DNCB), a small, highly lipophilic chemical sensitizer. At the doses tested, DNCB showed equal penetration into skin of all groups. Clinical reactions to DNCB were significantly reduced in AD. Although both controls and AD made systemic DNCB-specific Th1 responses, these were reduced in AD and associated with significantly Th2-skewed DNCB-specific T-cell responses. Th2 skewing was seen in both ADFM and ADWT, with no difference between these groups. After 3 months, DNCB-specific Th2 responses were persistent in individuals with AD, and Th1 responses persisted in controls. These data provide evidence that when antigen penetration is not limiting, AD skin has a specific propensity to Th2 programming, suggesting the existence of altered skin immune signaling that is AD-specific and independent of FLG status.

Invariant natural killer T (iNKT) cells expressing a CD1d-restricted invariant αβTCR have key regulatory roles in autoimmunity, pathogen immunity, and tumor surveillance, but their function in the control of allergic skin diseases remains poorly documented. Using a model of contact hypersensitivity (CHS) to the hapten DNFB, we show here that iNKT cell deficiency results in enhanced skin inflammation due to augmented hapten-specific IFN-γ-producing CD8(+) effectors in skin draining lymph nodes (dLNs) and their massive recruitment into the allergen-exposed skin. Adoptive transfer and antibody depletion experiments as well as in vitro studies revealed that iNKT cells (1) reduce the severity of CHS, even in presensitized mice, (2) require hapten presentation by CD1d(+) dendritic cells (DCs) to dampen skin inflammation, and (3) produce IL-4 and IL-13 after CD1d-dependent in vitro stimulation by hapten-loaded DCs only in the presence of IFN-γ released from activated CD8(+) effector T cells. In corollary, mice double deficient in IL-4 and IL-13 exhibit an exacerbated CHS. Finally, iNKT-suppressive function is independent of Foxp3(+) regulatory T cells (Tregs). These data highlight that, besides Foxp3(+) Tregs, iNKT cells are potent downregulators of CD8(+) T cell-mediated CHS, and underscore that both cell types are important for the regulation of allergic skin inflammation.

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a well-known mediator of skin pigmentation. More recently, it has been shown that α-MSH also exerts a strong anti-inflammatory and immunosuppressive activity. To elucidate the mechanisms underlying α-MSH-induced immunosuppression, we investigated whether α-MSH affects dendritic cell/T cell communication, as especially this interaction has an important role in the regulation of immune responses. Here, we show that α-MSH, by binding to MC-1R, induced tolerogenic dendritic cells, which were capable of expanding CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in vitro, as well as in vivo. Notably, those α-MSH-induced Tregs were functional as they efficiently inhibited cutaneous contact allergy and ongoing psoriasis-like skin inflammation in mice. Furthermore, α-MSH induced tolerogenic dendritic cells capable of generating functional Tregs in human blood. Interestingly, human Tregs expanded via α-MSH-stimulated dendritic cells suppressed the proliferation and cytokine secretion of pathogenic T-helper-17 (Th17) cells from individuals with psoriasis. Taken together, these data indicate that α-MSH induced immunosuppressive Tregs in vitro and in vivo, which inhibited disease progression in a mouse model of psoriasis-like skin inflammation and suppressed the activation and proliferation of effector T cells from subjects with psoriasis.

Vitamin D may be responsible for reducing the development and severity of autoimmune and allergic diseases. Topically applied 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) enhances the immunoregulatory ability of CD4+CD25+ T cells residing in the skin-draining lymph nodes (SDLNs) of mice. The mechanisms responsible were investigated by examining the expression of 84 cytokine and cytokine-related genes in a 96-well gene array. CD4+CD25+ cells isolated from the SDLNs of BALB/c mice, 24 and 96hours after topical treatment with 1,25(OH) 2 D 3 , consistently expressed increased IL-2 mRNA levels and also secreted enhanced quantities of IL-2 after ex vivo stimulation with phorbol 12-myristate 13-acetate and ionomycin. CD4+CD25+ cells from the lymph nodes of naive mice constitutively express the vitamin D receptor, allowing direct modulation by 1,25(OH) 2 D 3 . However, in vitro treatment with 1,25(OH) 2 D 3 did not modify the expression of 84 tested cytokine and cytokine-related mRNAs. It was only in the presence of IL-2 that 1,25(OH) 2 D 3 increased the expression of genes including IL-2 and TLR4. Further, 1,25(OH) 2 D 3 enhanced the ability of IL-2 to stimulate CD4+CD25+ cells to proliferate in vitro and also regulate contact hypersensitivity responses on adoptive transfer into naive mice. Therefore, 1,25(OH) 2 D 3 enabled by IL-2 can directly enhance the regulatory potential of CD4+CD25+ T cells to control immune disease.

Exposure to p -phenylenediamine (PPD) is associated with the development of T-cell-mediated allergic contact dermatitis. The purpose of this study was to define the nature of the interaction of PPD with the protein and the antigenic determinant that stimulates T cells. Mass spectrometry was employed to show that PPD oxidation products bind irreversibly to cysteine (Cys, position 34) in human serum albumin (HSA). A modified tryptic peptide was characterized with an increase in mass of 106Da, corresponding to the addition of PPD and not to the secondary products of self conjugation. Lymphocytes from 10 PPD-allergic patients, but not tolerant/naive individuals, were stimulated with PPD and PPD-modified HSA. A total of 70 PPD-specific and 10 PPD–HSA-specific CD4 + , CD8 + , and CD4 + CD8 + , Th2-secreting T-cell clones were generated from three allergic patients. In total, 40 clones were stimulated with both PPD and PPD-modified HSA. PPD-modified HSA triggered T-cell responses through a classical hapten mechanism involving processing. Presentation of PPD to several clones was dependent on protein complex formation (42 out of 48) and processing (32 out of 68); however, 12% of clones were triggered with PPD directly. These data identify Cys as the single target for PPD–HSA binding, and show that PPD protein adducts are antigenic determinants in patients with contact dermatitis.

Factors predisposing to individual susceptibility to contact allergic dermatitis are ill defined. This study was designed to characterize the response of allergic and tolerant individuals' T-lymphocytes after exposure to p-phenylenediamine (PPD). Peripheral blood mononuclear cells (PBMCs) from allergic patients proliferated when treated with PPD and Bandrowski's base (BB) and secreted IL-1alpha, -1beta, -4, -5, -6, -8, -10, and -13; IFN-gamma; tumor necrosis factor-alpha; MIP-1alpha/beta; MCP-1 (monocyte chemotactic protein-1); and RANTES. PBMCs from tolerant individuals were stimulated to proliferate only with BB, and they secreted significantly lower levels of Th2 cytokines. Principal component analysis showed that genes are differentially expressed between the patient groups. A network-based analysis of microarray data showed upregulation of T helper type 2 (Th2) gene pathways, including IL-9, in allergic patients, but a regulatory gene profile in tolerant individuals. Real-time PCR confirmed the observed increase in Th2 cytokine gene transcription in allergic patients. Purified CD4+ and CD8+ T cells from allergic patients were stimulated to proliferate and secrete Th2 cytokines following antigen exposure. Only CD4+ T cells from tolerant individuals were stimulated by BB, and levels of Th2 cytokines were 80% lower. The nature of the antigenic determinant stimulating PBMCs and levels of Th2 cytokines, including IL-9, was confirmed in a validation cohort. These studies show increased activity of Th2 cytokines in CD4+ and CD8+ T cells from individuals with allergic contact dermatitis.

There is increasing evidence that vascular remodeling and endothelial cell activation promote acute and chronic inflammation. Thrombospondin 1 (TSP-1) is a potent endogenous angiogenesis inhibitor thought to play an important role in maintaining cutaneous vascular quiescence. We first investigated TSP-1 expression in human and contact hypersensitivity (CHS) reactions and found that TSP-1 was upregulated in the inflamed skin of patients and in mice. To elucidate the function of TSP-1 in cutaneous inflammation, we induced CHS reactions in the skin of mice with targeted epidermal TSP-1 overexpression in TSP-1-deficient mice and in wild-type mice. We found decreased edema formation, angiogenesis, and inflammatory infiltrate in the inflamed skin of TSP-1 transgenic mice. Conversely, TSP-1-deficient mice exhibited an enhanced and prolonged inflammation, characterized by increased edema formation, enhanced vascular remodeling, and increased neutrophilic infiltrate, when compared with wild-type mice. Moreover, we found strong upregulation of the proinflammatory cytokines IL-1beta, macrophage inflammatory protein 2, and tumor necrosis factor-alpha in the inflamed skin of TSP-1-deficient mice. Our results indicate that TSP-1 downregulates cutaneous delayed-type hypersensitivity reactions by acting on several distinct pathways mediating skin inflammation.

Migration of Langerhans cells (LCs) from the skin to the lymph node is an essential step in the pathogenesis of allergic contact dermatitis (ACD). Therefore, inhibition of LC-migration could be a promising strategy to improve this skin disease. Effects of sphingosine-1-phosphate (S1P) and the immunomodulator FTY720 on LC trafficking is not well defined, yet. Thus, we investigated the action of topically administered S1P and FTY720 in a murine model of ACD. Most interestingly, FTY720 as well as S1P inhibited the inflammatory reaction in the elicitation phase of ACD. In the sensitization phase, FTY720, and S1P reduced the weight and cell count of the draining auricular lymph node, as well as immigrated dendritic cells provoked by repetitive topical administration of the hapten. Correspondingly, the density of LCs in the epidermis was higher in FTY720- and S1P-treated mice compared to vehicle treatment. A skin dendritic cell migration assay confirmed the significant inhibition of dendritic cell migration by FTY720 and S1P. These data supply conclusive evidence that the strategy of targeting the migratory response of LCs with locally acting S1P or FTY720 represents an emerging option in the treatment of allergic skin diseases like contact hypersensitivity and atopic dermatitis.

Neutralization of stratum corneum (SC) adversely impacts key epidermal functions, including permeability barrier homeostasis and SC integrity. Conversely, acidification of SC improves these functions in developmentally impaired (neonatal or aged) skin, and enhances function in normal skin. Hence, we hypothesized that acidification could alter the course of inflammatory dermatoses, which invariably exhibit an increased SC pH. Maintenance of a low pH by topical applications of the polyhydroxyl acid, lactobionic acid, during the repeated-challenge phase inhibited the development of oxazolone-induced atopic dermatitis (AD). Neither gross/histological dermatitis nor altered barrier function developed, and emergence of epidermal hyperplasia was prevented; however, cytokine generation decreased. Acidification also largely normalized the development of hapten-induced changes in eosinophil/mast cell densities, density of chemoattractant receptor-homologous molecule expressed on TH2-positive lymphocytes, and serum IgE levels. The pH-induced improvement in barrier function most likely accounts for the anti-inflammatory activity, which could be further attributed to normalization of both lamellar body secretion and lamellar bilayer formation. Acidification of SC alone substantially prevents development of barrier abnormalities and downstream immune abnormalities during the elicitation phase of murine AD. These results provide direct evidence for the “outside–inside” pathogenesis of AD and further suggest that maintenance of an acidic SC pH could prevent the emergence of AD in humans.

Udgivelsesdato: 2008-Nov-6 Mitogen-activated protein kinase (MAPK) AP kinase 2 (MK2) is a serine/threonine kinase that is phosphorylated and activated by p38 MAPK. MK2 regulates the expression of various proinflammatory cytokines including TNF-alpha, IL-1beta, IL-6, and IL-8. Recently, MK2 was demonstrated to be activated in lesional psoriatic epidermis. This study investigates for the first time the role of MK2 in skin inflammation using the model of oxazolone-induced acute allergic contact dermatitis in mice. We show that oxazolone treatment leads to increased expression and sustained activation of both p38 MAPK and MK2. The inflammatory response was determined by ear thickness, myeloperoxidase activity, and histology after oxazolone challenge. Pretreatment with the p38 MAPK inhibitor SB202190 and genetic ablation of MK2 inhibit this inflammatory response. In particular, IL-1beta and, to a smaller but significant extent, also TNF-alpha and IFN-gamma expression were decreased in MK2 knockout mice compared with wild-type mice. These results indicate that MK2 is a potential target for the treatment of inflammatory skin diseases.Journal of Investigative Dermatology advance online publication, 6 November 2008; doi:10.1038/jid.2008.322.

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows for selective pharmacological suppression of effector memory T (T(EM)) cells without affecting the function of naïve and central memory T cells. We here investigated whether PAP-1, a small molecule Kv1.3 blocker (EC50=2 nM), could suppress allergic contact dermatitis (ACD). In a rat model of ACD, we first confirmed that the infiltrating cells in the elicitation phase are indeed CD8+ CD45RC- memory T cells with high Kv1.3 expression. In accordance with its selective effect on T(EM) cells, PAP-1 did not impair sensitization, but potently suppressed oxazolone-induced inflammation by inhibiting the infiltration of CD8+ T cells and reducing the production of the inflammatory cytokines IFN-gamma, IL-2, and IL-17 when administered intraperitoneally or orally during the elicitation phase. PAP-1 was equally effective when applied topically, demonstrating that it effectively penetrates skin. We further show that PAP-1 is not a sensitizer or an irritant and exhibits no toxicity in a 28-day toxicity study. Based on these results we propose that PAP-1 could potentially be developed into a drug for the topical treatment of inflammatory skin diseases such as psoriasis.

Leukocyte extravasation is a finely tuned process, in which transmigration is the final step. Transmigration depends on molecules located at borders of endothelial cells; e.g., junctional adhesion molecules (JAM-A, -B and -C). In vivo blockade of JAM-A lead to decreased migration of monocytes into the skin. In contrast, the role of JAM-B and -C in development of cutaneous inflammation is unknown. We therefore elicited an allergic contact dermatitis in mice using 2,4-dinitro-1-fluorobenzene. RT-PCR and immunofluorescent staining of healthy skin revealed a constitutive JAM-B (66.4%+/-6.7% of all vessels) and -C expression (88.6+/-13.2%), which remained constant after induction of contact dermatitis. Functional studies, in which either JAM-B or -C neutralizing antibodies were injected into sensitized mice prior to allergen challenge showed a concentration-dependent reduction of the contact dermatitis. Decreased ear swelling was accompanied by reduction of leukocyte infiltration as analyzed by hematoxylin and eosin (H&E) histology and enzyme activity. Combined antibody treatment at doses of 1.25 mg per kg bodyweight lead to additive inhibition of allergic contact dermatitis, indicating that JAM-B and -C may have distinct functions. In conclusion, interactions with JAM-B and -C are essential for development of cutaneous inflammation.

Reflectance confocal microscopy (RCM) allows non-invasive visualization of human skin in vivo. It has been used to describe the histopathological features of acute contact dermatitis (CD). This work was designed to investigate the kinetics of both allergic and irritant CD (ACD and ICD) in vivo. Eighteen subjects with a prior diagnosis of ACD were patch tested with the specific allergen sodium lauryl sulfate as an irritant, and appropriate controls. RCM, transepidermal water loss (TEWL), and fluorescence excitation spectroscopy (FES) were performed at several time points within 2 wk after patch removal. After removal of the Finn chambers at 48 h, superficial epidermal changes, primarily involving the stratum corneum, and increased epidermal thickness were mainly present in ICD. ACD, on the other hand, showed microvesicle formation peaking at 96 h following patch removal. Both ACD and ICD showed exocytosis and similar degrees of spongiosis on RCM. TEWL and FES demonstrated a significant difference between ACD and ICD. RCM, TEWL, and FES are valuable non-invasive tools to quantitatively study the kinetics of the pathophysiology of acute CD reactions in vivo and monitor the changes at a cellular level.

Environmental exposures are important for development of allergic contact dermatitis, but genetic factors have been proposed to be of additional importance for contact sensitization. Recently genetic factors were shown to be of significance for hand eczema. In this study, a sample of twins recruited on the basis of hand eczema has been evaluated with respect to influence of genetic factors on development of nickel sensitization. A total of 1076 individual twins were patch tested and underwent clinical examination, and in the final genetic statistical analysis 630 females were available, of which 146 had a positive patch test to nickel. The aggregation of nickel allergy among twin pairs was measured by the casewise concordance and the twin odds ratio. The twin odds ratio were adjusted for effects of risk factors known to be associated with nickel allergy, namely, wet work, atopic dermatitis, and self-reported hand eczema. There was a small tendency for larger odds ratio among monozygotic twins than among dizygotic twins, which was not statistically significant. As a result of the statistical analysis, it is concluded that allergic nickel contact dermatitis is mainly caused by environmental and only to a lesser degree genetic factors. The selection of twins on the basis of hand eczema may theoretically influence the prevalence of nickel allergy and concordance estimates, which should be considered before extrapolating the data to a random population-based twin sample.

UVB radiation has been shown to induce T cell tolerance most likely via modulation of the function of antigen-presenting cells like dendritic cells (DC), which are therefore of interest for vaccination therapy. Since little is known about the effects of UVB-irradiated dendritic cells (UVB-DC) on CD8(+) T cells, which are the dominant effectors in various allergic and autoimmune diseases, we have investigated the potential of low dose UVB (100-200 J per m(2)) irradiated bone marrow-derived dendritic cells to induce tolerance in murine CD8(+) T cells specific for the contact allergen trinitrophenyl (TNP) or for a viral peptide. In contrast to the previously reported successful tolerization of primed CD4(+) Th1 cells, neither naïve CD8(+) T cells nor CD8(+) Tc1 effector cells or established CD8(+) T cell clones could be tolerized by TNP-modified or peptide-pulsed UVB-DC in vitro or in vivo. We observed, however, a reduced capacity of UVB-DC to prime naïve CD8(+) T cells. Our data demonstrate an important difference in the susceptibility of CD4(+) and CD8(+) T cells for tolerance induction using low-dose UVB-irradiated DC and have implications for DC therapy of CD8(+) T cell-mediated diseases.

In this issue, Simonsson and colleagues shed light on the chemical mechanisms determining hapten formation in the skin, which precede the elicitation of an antigen-specific immune response in allergic contact dermatitis. Combining fluorescence microscopy, proteomics, and mass spectrometry, the investigators identified keratins K5 and K14, particularly cysteine 54 of K5, in the human basal epidermal layer as the major molecular targets of caged thiol-reactive fluorescent haptens (i.e., bromobimanes). Anti-keratin antibody responses in mice exposed to bromobimanes suggest the generation of immunogenic epitopes by cysteine-reactive haptens. Although many issues await further investigation, Simonsson and co-workers' observations advance our understanding of the molecular basis of hapten-protein complex formation in skin.

In searching for pharmacologic agents able to reduce xenobiotic-induced skin irritation, we found that cyclosporine A exacerbates the skin irritation induced by tributyltin. We previously demonstrated the involvement of interleukin-1 alpha and tumor necrosis factor alpha in tributyltin-induced skin irritation. Here, we show that cyclosporine A (28 mg per kg), at a dose that results in systemic immunosuppression, potentiates tributyltin-induced skin irritation through increased tumor necrosis factor alpha production, associated with increased tributyltin-induced activation of transcription factor nuclear factor kappa B in cyclosporine-A-treated mice. On the other hand, under the same experimental conditions, cyclosporine A prevented the elicitation phase of oxazolone-induced contact allergy, but was ineffective in preventing benzalkonium-chloride-induced skin irritation. Using a murine keratinocyte cell line (HEL30) we demonstrated, also in vitro, that the cyclosporine A potentiates tributyltin-induced nuclear factor kappa B activation and cytokine production, this being preceded by an increase in cellular oxidative activity, essential for nuclear factor kappa B activation, that is time and dose (0.1-10 microM) dependent. This effect was not exclusive to tributyltin but could be extended to other mitochondrial poisons such as sodium arsenate. It has been reported that cyclosporine A binds to cyclophilins. An 18-mer antisense phosphorothioate oligodeoxynucleotide was used to target mitochondrial cyclophilin D mRNA. After 24 h exposure to the oligonucleotide, the amount of cyclophilin D in the cells was decreased by 54% as judged by Western blot analysis. Cyclophilin D suppression prevented cyclosporine A potentiation of tributyltin-induced cellular oxidative activity, indicating the key role of the binding of cyclosporine A to mitochondrial cyclophilin D in mediating this effect.

Interleukin-17 is a T-cell-derived cytokine, detected in skin affected by allergic contact dermatitis and psoriasis, which regulates keratinocyte expression of adhesion molecules and chemokines. In this study, we have analyzed whether interleukin-17 production segregates with a particular T helper (Th) cell subset, and have examined the capacity of interleukin-17 to modulate the activation of keratinocytes induced by Th1 and Th2 cytokines. A panel of 80 nickel-specific CD4+ T cell clones (36 Th0, 30 Th1, and 14 Th2) was isolated from peripheral blood or lesional skin of allergic contact dermatitis patients. Significant amounts (50 pg per ml) of interleukin-17 were released by about 50% of activated Th0, Th1, and Th2 cells. Interleukin-17 alone and in cooperation with interleukin-4, or to a lesser extent with interferon-gamma, decreased the interleukin-1 receptor antagonist to interleukin-1alpha ratio in the supernatants as well as in cell lysates from keratinocytes. In addition, interleukin-17 stimulated the release of growth-regulated oncogene-alpha, granulocyte-macrophage colony stimulating factor, and interleukin-6, with synergistic or additive effects when used together with interferon-gamma or interleukin-4. Interleukin-17 and interleukin-4 also increased stem cell factor release, a function that was inhibited by interferon-gamma. Moreover, interleukin-17 and interleukin-4 enhanced interferon-gamma-induced expression of intercellular adhesion molecule 1, but not CD40, on keratinocytes. The constitutive expression of interleukin-17 and interferon-gamma receptors on keratinocytes was not modulated by interleukin-17, interferon-gamma, or interleukin-4, whereas the interleukin-4 receptor was significantly downregulated by interferon-gamma. As a whole, the results indicate that interleukin-17 can participate relevantly in T-cell-mediated skin immune responses by amplifying both interferon-gamma- and interleukin-4-induced activation of keratinocytes.

Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Differentiation between allergic and irritant contact dermatitis reactions is difficult, as both inflammatory diseases are clinically, histologically, and immunohistologically very similar. Previous studies in mice revealed that the chemokine IP-10 is exclusively expressed in allergic contact dermatitis reactions. In the present study, we investigated whether the mRNA expression of IP-10 and the related CXCR3 activating chemokines, Mig and IP-9 are also differentially expressed in human allergic contact dermatitis and irritant contact dermatitis reactions. Skin biopsies from allergic (13 cases) and sodium lauryl sulfate-induced irritant patch test reactions (13 cases), obtained 1–72 h after patch testing, were studied by means of an in situ hybridization technique. Results of chemokine mRNA expression were correlated with clinical scoring, histology, and immunohistochemical data including the proportion of inflammatory cells expressing CXCR3, the receptor for IP-10, Mig, and IP-9, and ICAM-1 and HLA-DR expression on keratinocytes. IP-10, Mig, and IP-9 mRNA were detected in seven of nine allergic contact dermatitis reactions after 24–72 h, but not in sodium lauryl sulfate-induced irritant contact dermatitis reactions. ICAM-1 expression by keratinocytes was only found in allergic contact dermatitis reactions and correlated with chemokine expression. Moreover, up to 50% of the infiltrating cells in allergic contact dermatitis expressed CXCR3, in contrast to only 20% in irritant contact dermatitis reactions. In conclusion, we have demonstrated differences in chemokine expression between allergic contact dermatitis and irritant contact dermatitis reactions, which might reflect different regulatory mechanisms operating in these diseases and may be an important clue for differentiation between allergic contact dermatitis and irritant contact dermatitis reactions.

This study investigates the relative effects of low-dose solar-simulated ultraviolet, ultraviolet A, and ultraviolet B radiation on the elicitation of contact hypersensitivity to nickel in nickel-allergic volunteers. A xenon arc lamp with changeable filters was used to irradiate groups of volunteers daily, on separate areas of their lower backs, with both solar-simulated ultraviolet (ultraviolet B, ultraviolet AII + ultraviolet AI) and ultraviolet A (same ultraviolet AII content but twice the ultraviolet AI as the solar-simulated ultraviolet spectrum) for 1 and 2 d; 3, 4, and 5 d; and from 1 to 4 wk. A fourth group was irradiated for 1-5 d with the ultraviolet B component of solar-simulated ultraviolet. Following the final irradiation in each group, nickel-containing patches were applied to both ultraviolet-treated sites and adjacent, unirradiated control sites. Erythema caused by nickel contact hypersensitivity at each site was quantitated 72 h later with a reflectance erythema meter. By comparing the nickel reactions of irradiated and unirradiated skin, ultraviolet immunosuppression was assessed with the different spectra and durations of ultraviolet exposure. We found significant immunosuppression with daily doses of ultraviolet B and ultraviolet A equivalent to approximately 6 min of summer sun exposure, and that ultraviolet A and ultraviolet B exerted their maximal immunosuppressive effects at different times. Solar-simulated ultraviolet-induced immunosuppression was significant after one exposure, near-maximal after two exposures and remained elevated thereafter. Ultraviolet B-induced immunosuppression was lower than that induced by solar-simulated ultraviolet, but followed a similar time-course. In contrast, ultraviolet A-induced immunosuppression was transient, peaking after three exposures. Immune responses returned towards normal with subsequent ultraviolet A exposure, suggesting that an adaptive mechanism may prevent immunosuppression by continued ultraviolet A irradiation.

The role of interleukin-4 as a regulator of immune responses in the skin is investigated with regard to the outcome of contact hypersensitivity reaction in interleukin-4-deficient BALB/C mice. In previous studies conflicting results were obtained concerning the role of interleukin-4 in contact hypersensitivity reactions supporting either a proinflammatory or rather an inhibitory function of this cytokine. Interleukin-4 deficient BALB/C mice sensitized to 2,4-dinitrochlorobenzene showed after challenge a significant reduction in magnitude and duration of the contact hypersensitivity response in comparison with wild-type mice. This attenuation was accompanied by a significant reduction of edema and cellular infiltrates in the dermis and a lacking induction of IL-10 mRNA expression in skin. Also, adoptive transfer experiments revealed that BALB/C mice failed to exhibit contact hypersensitivity after injection of lymph node cells obtained from sensitized interleukin-4 deficient mice. To examine further the role of the contact allergen used to induce the contact hypersensitivity response, mice were also sensitized and challenged with Oxazolone. Here a similar magnitude and duration of contact hypersensitivity in both the interleukin-4 deficient mice and BALB/C control mice was observed. This indicates that the contact hypersensitivity response to 2,4-dinitrochlorobenzene and Oxazolone may partly evolve on different pathways being dependent and independent of interleukin-4. Our results clearly show that the complete loss of endogenous interleukin-4 expression in BALB/C mice is associated with an impaired manifestation of contact hypersensitivity response to 2,4-dinitrochlorobenzene, implying an important proinflammatory function of this cytokine.

To investigate the mechanisms underlying the expression of allergic contact dermatitis, we compared the characteristics of nickel (Ni)-specific T cell responses in 10 patients with allergic contact dermatitis to Ni and in 10 healthy, nonallergic individuals. CD4+ T cells purified from peripheral blood of both allergic and nonallergic subjects proliferated similarly to NiSO4 in vitro, with the responses mostly restricted to CD4+ CD45RO+ memory T cells. In contrast, Ni-specific CD8+ T cell responses were detected only in allergic patients. Limiting dilution assay confirmed a high frequency of Ni-specific CD4+ T cells in both individual categories, and of Ni-specific CD8+ T cells in allergic patients, but not in nonallergic persons. Ni-specific CD4+ T cell clones prepared from nonallergic subjects displayed lower interferon-gamma and higher interleukin-10 production compared with T cell clones from allergic patients. The T cell skin-homing receptor, cutaneous lymphocyte-associated antigen, was expressed on the large majority of specific CD4+ clones from both the groups. Finally, Ni-specific CD8+ clones prepared from patients also expressed the cutaneous lymphocyte-associated antigen receptor, and released high interferon-gamma and no interleukin-4. In aggregate, the results suggest that the presence of specific CD8+ T cells and a distinct pattern of cytokine release (e.g., an augmented production of interleukin-10) by CD4+ T cells can be important elements in determining whether a hapten induces allergy or a silent immune response.

Two patients with histologically proven mycosis fungoides, a malignancy of phenotypically mature T cells, received a topical challenge with mechlorethamine to areas of clinically uninvolved skin to exclude possible hypersensitivity reactions to this chemotherapeutic agent. In both patients, allergic contact dermatitis (ACD) developed at the sites of the application and resolved completely after withdrawal of mechlorethamine. The lesions were biopsied and analyzed for the presence of clonal T-cell receptor (TCR)-gamma gene rearrangements using two polymerase chain reaction (PCR)-based assays involving denaturing gradient gel electrophoresis (PCR/DGGE) and ribonuclease protection analysis (PCR/RPA). The former method has a clonal detection threshold of 10(-3)-10(-2), while the latter has a sensitivity of 10(-5). In both cases, the ACD lesions were polyclonal by PCR/DGGE. In contrast, PCR/RPA detected tumor-specific TCR-gamma gene rearrangements in these same lesions. This indicates that the ACD lesions contained tumor cells at a density within the 10(-5)-10(-2) range. Analysis of peripheral blood mononuclear cells from both patients failed to detect the malignant clone and showed the same result as blood from four normal individuals. The normal skin from one skin patient also lacked detectable TCR-gamma gene rearrangements. These results indicate that mycosis fungoides tumor are present within ACD lesions induced in mycosis fungoides patients and that this phenomenon does not appear to be due to the ubiquitous presence of detectable levels of these tumor cells in the blood or skin. These findings might be explained by nonspecific recruitment of malignant T cells to sites of local inflammation mediated by non-neoplastic antigen-specific T cells. Alternatively, they might be due to the local proliferation of very rare tumor cells in apparently normal skin in response to cytokines generated during the ACD reaction. In either case, the present study offers evidence that the malignant cells in myosis fungoides retain at least some capability of responding in vivo to physiologic stimuli.

Exposure of skin to ultraviolet (UV) radiation can lead to diverse biologic effects, including inflammation, sunburn cell formation, alterations of cutaneous immune cells, and impaired induction of contact hypersensitivity responses. The molecular mechanisms of these UV-induced effects are not completely understood. We investigated the ability of sunscreens and liposomes containing the DNA excision repair enzyme T4 endonuclease V to prevent these effects of UV radiation. The use of T4N5 liposomes, which increase the repair of cyclobutyl pyrimidine dimers, provides an approach for assessing the role of DNA damage in the effects of UV radiation on the skin. Exposing C3H mice to 500 mJ/cm2 UVB radiation from FS40 sunlamps resulted in skin edema, sunburn cell formation, and morphologic alterations and decreased numbers of Langerhans cells and Thy-1+ dendritic epidermal T cells. In addition, the induction of contact hypersensitivity after application of 2,4-dinitrofluorobenzene on UV-irradiated skin was diminished by 80%. Applying sunscreens containing octyl-N-dimethyl-p-aminobenzoate, 2-ethylhexyl-p-methoxycinnamate, or benzophenone-3 before this dose of UV irradiation gave nearly complete protection against all of these effects of UV irradiation. In contrast, topical application of T4N5 liposomes after UV irradiation had no effect on UV-induced skin edema and only partially protected against sunburn cell formation and local suppression of contact hypersensitivity, although its ability to protect against alterations in dendritic immune cells was comparable to that of the sunscreens. These results suggest that DNA damage is involved in only some of the local effects of UV radiation on the skin. In addition, T4N5 liposomes may be a useful adjunct to sunscreens because they can reduce some of the deleterious effects of UV radiation on skin even after a sunburn has been initiated.

Interleukin-10 (IL-10) is known to play a major role in suppressing immune and inflammatory responses by inhibiting the production of proinflammatory cytokines. Therefore, we hypothesized that IL-10 might be capable of suppressing allergic contact hypersensitivity. BALB/c mice were sensitized on the dorsal skin with dinitrofluorobenzene and challenged on the ears 6 d later. The effect of IL-10 on the elicitation phase of contact sensitization was determined by its intradermal injection into the pinnae of the mice at doses of 0.1-100 ng. At 24 and 48 h after challenge, ear swelling was suppressed in a dose-dependent manner by injection of IL-10 at doses of 10-100 ng. Maximal inhibition of ear swelling (46.9%) was observed after injection of 100 ng of IL-10. IL-10-injected ear skins showed less inflammatory cell infiltration and decreased edema at the dermis compared with controls. Because IL-10 is known to inhibit Th1 cytokines such as interferon-gamma, we examined whether the suppressive effect of IL-10 on ear swelling was accompanied by IL-10-induced inhibition of interferon-gamma. We found that IL-10 application suppressed interferon-gamma mRNA upregulation in challenged skin. Our results suggest that IL-10 significantly modifies the elicitation of allergic contact sensitivity reactions.

Interactions of CD28 (on T cells) with its recently identified ligand B7/BB1 (on antigen-presenting cells) have been shown to activate T cells via a major histocompatibility complex/Ag-independent “alternative” pathway, leading to an amplification of T- cell – mediated immune responses. The in vivo relevance of these molecules for cutaneous immunity is presently unknown. These findings prompted us to study the expression of B7/BB1 and CD28 in normal human skin and in selected T-cell – mediated inflammatory skin diseases. Biopsies were obtained from lesional skin of patients with allergic contact dermatitis, lichen planus, and, as control, from basal cell carcinoma and from healthy controls. Serial cryostat sections were stained with a panel of MoAbs directed against CD28, B7/BB1, CD3, CD1a, and KiM8 using immunohistochemistry (ABC technique). CD28 expression was observed in the majority of dermal and epidermal CD3+ T cells in contact dermatitis and lichen planus. In normal skin and basal cell carcinoma, CD28 was expressed only occasionally by perivascular T cells. In allergic contact dermatitis and lichen planus, B7/BB1-expression was found on dermal dendritic cells, on dermal macrophages, on Langerhans cells, focally on keratinocytes, and occasionally on dermal T cells. No B7/BB1 immunoreactivity was detected in normal skin and basal cell carcinoma. These findings indicate that T-cell – mediated skin diseases are accompanied by an influx of CD28+ T cells and an upregulation of B7/BB1 on cutaneous antigen- presenting cells, keratinocytes, and on some T cells. We speculate that “alternative” T cell-activation via the B7/CD28 pathway may contribute to the pathogenesis of these skin diseases.

T lymphocytes play critical roles in a series of important immune responses, including delayed-type hypersensitivity and contact sensitivity. Metal haptens such as nickel are among the most common sensitizers. In general, T cells recognize fragments of proteins complexed to MHC molecules. Haptens, however, are too small to be antigenic by themselves. Recent experiments have suggested that hapten-specific T cells recognize hapten-modified MHC-peptide complexes. Here I review the data supporting this hypothesis and speculate on the relevance of these findings for the understanding of chemical-induced hypersensitivities.

Oral administration of nickel or chromium to naive guinea pigs results in immune unresponsiveness to subsequent induction of allergic contact hypersensitivity. Such "oral tolerance" depends on the oral dose, is antigen specific, T-suppressor-cell mediated, and very persistent. In contrast, oral antigen administration to sensitized animals results at best in transient desensitization. Here we report that even non-sensitizing epicutaneous skin contacts prevented the subsequent induction of oral tolerance. These data support the view that primed T cells are less sensitive to suppressor T-cell function than naive T cells.

To avoid unsuspected and unwanted consequences of excess hapten during epicutaneous sensitization, optimal sensitizing doses of dinitrofluorobenzene (DNFB) were determined for several ultraviolet B radiation (UVB)–resistant and UVB- susceptible strains of mice. Using these doses of hapten applied epicutaneously or injected intracutaneously into normal or UVB-exposed body wall skin, it was determined that four consecutive daily exposures to UVB prevented contact hypersensitivity induction in all mice when optimal sensitizing doses of DNFB were applied epicutaneously. By contrast, UVB-resistant, but not UVB-susceptible, mice developed contact hypersensitivity when an optimal sensitizing dose of DNFB was injected intracutaneously into UVB-irradiated skin. Moreover, whereas UVB-susceptible mice failed to develop contact hypersensitivity when an optimal sensitizing dose of DNFB was painted on skin exposed to a single dose of UVB, UVB-resistant mice did develop contact hypersensitivity under similar circumstances. Based on these results, it is concluded that 1) conventional doses of epicutaneously applied haptens induce contact hypersensitivity with the aid of antigen-presenting cells derived from both the epidermis and the dermis, 2) the phenomenon of UVB susceptibility is mediated by cells and molecules within the dermis when conventional doses of hapten and UVB radiation are employed, and 3) UVB susceptibility is mediated by cells and molecules within the epidermis when optimal sensitizing doses of hapten and a single exposure to UVB are employed.