Sensitization Phase Of Contact Hypersensitivity Research Articles

Tolerogenic phenotype of dendritic cells is induced after hapten sensitization followed by attenuated contact hypersensitivity response in atopic dermatitis model NC/Nga mice

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are common inflammatory diseases. We previously reported attenuated contact hypersensitivity (CHS) responses in AD model mice using 2,4-dinitrofluorobenzene, reflecting clinical experiments. However, previous studies have not addressed the commonality of findings across haptens and mechanisms focused on dendritic cells (DCs). Thus, this study evaluated CHS responses to fluorescein isothiocyanate (FITC) and DC migration and maturation in the sensitization phase of CHS in AD. CHS responses to FITC were compared between NC/Nga mice without and with AD induction (non-AD and AD mice, respectively). T-cell responses and DC migration and maturation after FITC-induced sensitization were examined in the draining lymph nodes of non-AD and AD mice. AD mice demonstrated reduced CHS responses to FITC under decreased T-cell proliferation following sensitization and interferon-γ production by hapten-specific T cells compared with non-AD mice. In addition, the number of FITC+CD11c+MHC class IIhigh migratory DCs 24 h after FITC sensitization was comparable between non-AD and AD mice. However, FITC+CD11c+MHC class IIhigh migratory DCs in AD mice exhibited lower expression levels of CD80 and CD86 and higher expression levels of PD-L1 and mRNA of transforming growth factor beta than non-AD mice. These findings suggest that attenuated CHS responses may be hapten-independent and the induction of the tolerogenic phenotype of hapten-bearing DCs can contribute to reduced T-cell proliferation after sensitization and CHS responses in AD.

An inhibitory immunoreceptor, Allergin-1, suppresses FITC-induced type 2 contact hypersensitivity

Although allergic contact dermatitis (ACD) is the most common T cell-mediated inflammatory responses against an allergen in the skin, the pathogenesis of ACD remains incompletely understood. In the sensitization phase in ACD, hapten-bearing dermal dendritic cells (DCs) play a pivotal role in the transport of an antigen to the lymph nodes (LNs), where they present the antigen to naïve T cells. Here we report that Allergin-1, an inhibitory immunoreceptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region, is highly expressed on dermal DCs. Mice deficient in Allergin-1 exhibited exacerbated fluorescein isothiocyanate (FITC)-induced type 2 contact hypersensitivity (CHS) such as ear swelling and skin eosinophilia. Allergin-1-deficient mice also showed larger numbers of CD4+ T cells and FITC-bearing DCs and greater expressions of type 2 cytokines, including IL-5, IL-10 and IL-13, in the draining LNs than did wild type mice. In sharp contrast, Allergin-1-deficient mice showed comparable level of type 1 CHS induced by 2,4-dinitrofluorobenzene (DNFB). These results suggest that Allergin-1 on dermal DC inhibits type 2, but not type 1, immune responses in the sensitization phase of CHS.

Pituitary adenylate cyclase-activating polypeptide promotes cutaneous dendritic cell functions in contact hypersensitivity

Sensory nerves regulate cutaneous local inflammation indirectly through induction of pruritus and directly by acting on local immune cells. The underlying mechanisms for how sensory nerves influence cutaneous acquired immune responses remain to be clarified. This study aimed to explore the effect of peripheral nerves on cutaneous immune cells in cutaneous acquired immune responses. We analyzed contact hypersensitivity (CHS) responses as a murine model of delayed-type hypersensitivity in absence or presence of resiniferatoxin-induced sensory nerve denervation. We conducted ear thickness measurements, flow cytometric analyses, and mRNA expression analyses in CHS. CHS responses were attenuated in mice that were denervated during the sensitization phase of CHS. By screening neuropeptides, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA expression was decreased in the dorsal root ganglia after denervation. Administration of PACAP restored attenuated CHS response in resiniferatoxin-treated mice, and pharmacological inhibition of PACAP suppressed CHS. Flow cytometric analysis of skin-draining lymph nodes showed that cutaneous dendritic cell migration and maturation were reduced in both denervated mice and PACAP antagonist-treated mice. The expression of chemokine receptors CCR7 and CXCR4 of dendritic cell s was enhanced by addition of PACAP invitro. These findings indicate that a neuropeptide PACAP promotes the development of CHS responses by inducing cutaneous dendritic cell functions during the sensitization phase.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.

Cannabinoid Receptor 2 Modulates Maturation of Dendritic Cells and Their Capacity to Induce Hapten-Induced Contact Hypersensitivity.

Contact hypersensitivity (CHS) is an established animal model for allergic contact dermatitis. Dendritic cells (DCs) play an important role in the sensitization phase of CHS by initiating T cell responses to topically applied haptens. The cannabinoid receptors 1 (CB1) and 2 (CB2) modulate DC functions and inflammatory skin responses, but their influence on the capacity of haptenized DCs to induce CHS is still unknown. We found lower CHS responses to 2,4-dinitro-1-fluorobenzene (DNFB) in wild type (WT) mice after adoptive transfer of haptenized Cnr2−/− and Cnr1−/−/Cnr2−/− bone marrow (BM) DCs as compared to transfer of WT DCs. In contrast, induction of CHS was not affected in WT recipients after transfer of Cnr1−/− DCs. In vitro stimulated Cnr2−/− DCs showed lower CCR7 and CXCR4 expression when compared to WT cells, while in vitro migration towards the chemokine ligands was not affected by CB2. Upregulation of MHC class II and co-stimulatory molecules was also reduced in Cnr2−/− DCs. This study demonstrates that CB2 modulates the maturation phenotype of DCs but not their chemotactic capacities in vitro. These findings and the fact that CHS responses mediated by Cnr2−/− DCs are reduced suggest that CB2 is a promising target for the treatment of inflammatory skin conditions.

IL-25 enhances TH17 cell–mediated contact dermatitis by promoting IL-1β production by dermal dendritic cells

In addition to thymic stromal lymphopoietin and IL-33, IL-25 is known to induce TH2 cytokine production by various cell types, including TH2 cells, TH9 cells, invariant natural killer T cells, and group 2 innate lymphoid cells, involved in TH2-type immune responses. Because both TH2-type and TH17-type cells/cytokines are crucial for contact hypersensitivity (CHS), IL-25 can contribute to this by enhancing TH2-type immune responses. However, the precise role of IL-25 in the pathogenesis of fluorescein isothiocyanate-induced CHS is poorly understood. We investigated the contribution of IL-25 to CHS using Il25-/- mice. CHS was evaluated by means of measurement of ear skin thickness in mice after fluorescein isothiocyanate painting. Skin dendritic cell (DC) migration, hapten-specific TH cell differentiation, and detection of IL-1β-producing cells were determined by using flow cytometry, ELISA, and immunohistochemistry, respectively. In contrast to thymic stromal lymphopoietin, we found that IL-25 was not essential for skin DC migration or hapten-specific TH cell differentiation in the sensitization phase of CHS. Unexpectedly, mast cell- and non-immune cell-derived IL-25 was important for hapten-specific TH17 cell-mediated rather than TH2 cell-mediated inflammation in the elicitation phase of CHS by enhancing TH17-related, but not TH2-related, cytokines in the skin. In particular, IL-1β produced by dermal DCs in response to IL-25 was crucial for hapten-specific TH17cell activation, contributing to induction of local inflammation in the elicitation phase of CHS. Our results identify a novel IL-25 inflammatory pathway involved in induction of TH17 cell-mediated, but not TH2 cell-mediated, CHS. IL-25 neutralization can be a potential approach for treatment of CHS.

Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity

Aquaporins (AQPs, AQP0-12) are membrane water/glycerol channel proteins that participate in various cellular functions in many cell types. AQP9 is expressed in several immune cells including neutrophils; however, its role in cutaneous immune response remains unknown. Here we show the involvement of AQP9 in skin allergic inflammation using AQP9 knockout (AQP9−/−) mice and the contact hypersensitivity (CHS) model. First, AQP9−/− mice showed impairment of CHS responses to 2,4-dinitrofluorobenzene (DNFB) compared with wild-type (WT) mice. The sensitization phase of CHS was examined by evaluating the immune cells accumulated in skin draining lymph nodes (LNs) after DNFB application. Adoptive transfer of AQP9−/− LNs cells into the ears of WT recipients resulted in impaired CHS response, indicating the defect of sensitization in AQP9−/−. IL-17 production was decreased in AQP9−/− compared with WT LNs cells, though the T cell proliferation and IFN-gamma production levels were comparable. WT mice showed high neutrophil accumulation in LNs at 18 h after DNFB application, but it was diminished in AQP9−/− mice. In accordance, we found the attenuation of AQP9−/− neutrophil migration to LNs after DNFB application. Furthermore, the neutrophil migration in response to CCR7 ligands was suppressed in AQP9−/− using chemotaxis chambers. Finally, we found that the administration of Gr-1 (neutrophil) antibody during the sensitization phase suppressed IL-17 production from LNs cells as well as CHS response in WT mice, suggesting the implication of neutrophils for IL-17 production during the development of CHS. These data suggest that AQP9-expressing neutrophils play an important role in the sensitization phase of CHS through their migration function.

Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity.

Allergic contact dermatitis and its animal model, contact hypersensitivity (CHS), are T cell-mediated inflammatory skin diseases induced by contact allergens. Though numerous cellular and molecular players are known, the mechanism of chemical-induced sensitization remains poorly understood. Here, we identify neutrophils as crucial players in the sensitization phase of CHS. Genetic deficiency of neutrophils caused by myeloid-specific deletion of Mcl-1 or antibody-mediated depletion of neutrophils before sensitization abrogated the CHS response. Neutrophil deficiency reduced contact allergen-induced cytokine production, gelatinase release, and reactive oxygen species production in naive mice. Mast cell deficiency inhibited neutrophil accumulation at the site of sensitization. In turn, neutrophils were required for contact allergen-induced release of further neutrophil-attracting chemokines, migration of DCs to the draining lymph nodes, and priming of allergen-specific T cells. Lymph node cells from mice sensitized in the absence of neutrophils failed to transfer sensitization to naive recipients. Furthermore, no CHS response could be induced when neutrophils were depleted before elicitation or when normally sensitized lymph node cells were transferred to neutrophil-deficient recipients, indicating an additional role for neutrophils in the elicitation phase. Collectively, our data identify neutrophils to be critically involved in both the sensitization and elicitation phase of CHS.

Natural Killer T Cells Are Essential for the Development of Contact Hypersensitivity in BALB/c Mice

Contact hypersensitivity (CHS) has been widely used to study cutaneous immune responses, as a prototype of delayed-type hypersensitivity. Although natural killer T (NKT) cells have been assumed to have an important role in CHS, their role is controversial. Here, we report the role of NKT cells in the sensitization phase of CHS, by promoting the survival and maturation of dendritic cells (DCs) in the draining lymph nodes (LNs). The CHS response was attenuated with Cd1d1(-/-) and Traj18(-/-) BALB/c mice in which NKT cells were absent. In the draining LNs, the number of effector T cells and cytokine production were significantly reduced with NKT cell-deficient mice. NKT cells activated and colocalized with DCs in the draining LNs after sensitization. The number of migrated and mature DCs was reduced in NKT cell-deficient mice 72 hours after FITC application. In in vitro experiments, activated NKT cells enhanced bone marrow-derived DC (BMDC) survivability via tumor necrosis factor (TNF) production from BMDCs. In addition, TNF production from BMDCs was partially suppressed by the neutralizing anti-CD54 or CD154 antibodies. Our data demonstrate that DC-NKT interaction has a pivotal role in the sensitization phase of CHS.

Nonmetal Haptens Induce ATP Release from Keratinocytes through Opening of Pannexin Hemichannels by Reactive Oxygen Species

Although extracellular adenosine 5'-triphosphate (eATP) has a crucial role in the sensitization phase of contact hypersensitivity (CHS), the mechanism by which hapten causes keratinocyte cell death and ATP release is unknown. We examined the time course of cell death, reactive oxygen species (ROS) production, and ATP release in HaCaT cells and in normal human keratinocytes after exposure to nonmetal haptens, NiCl2, or irritants. Both haptens and irritants caused cell death of keratinocytes but with different time courses. N-acetylcysteine (NAC) significantly reduced only nonmetal hapten-induced cell death as assessed by propidium iodide exclusion. We examined the effects of antioxidants and pannexin (Panx) inhibitors on cell death, ROS production, and ATP release by chemical-treated HaCaT cells. Nonmetal hapten-induced cell death, but not NiCl2- or irritant-related cell death, was dependent on reactivity to thiol residues in the cells. NAC reduced cell death and ATP release, whereas antioxidants and Panx inhibitors did not inhibit cell death but significantly attenuated ATP release. Panx1 small interfering RNA (siRNA) also suppressed ATP release from hapten-exposed HaCaT cells. Intraperitoneal injection of a Panx1 inhibitor attenuated murine CHS. These findings suggest that nonmetal hapten reactivity to thiol residues causes membrane disruption of keratinocytes and ROS production that leads to ATP release through opening of Panx hemichannels.

Lymphatic Dysfunction Impairs Antigen-Specific Immunization, but Augments Tissue Swelling Following Contact with Allergens

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

Inducible Nitric Oxide Synthase Downmodulates Contact Hypersensitivity by Suppressing Dendritic Cell Migration and Survival

Nitric oxide (NO) has several important roles in various physiological settings; one of the NO synthases, inducible NO synthase (iNOS), is induced by external stimulation of the skin. A prototypic example of external stimulation is hapten exposure, which induces the T-cell-mediated immune response known as contact hypersensitivity (CHS). We herein report on cutaneous dendritic cell (DC) function in the presence of an iNOS-specific inhibitor during the sensitization phase of CHS. First, we examined epidermal cell (EC) suspensions using flow cytometry with an iNOS antibody and confirmed that iNOS was expressed in the cytoplasm of Langerhans cells (LCs). We then studied the role of iNOS in CHS, and found that responses to DNFB were enhanced by the addition of an iNOS inhibitor during sensitization. Similarly, the iNOS inhibitor augmented FITC-induced migration of cutaneous DCs, including Langerin(+) LCs and Langerin(-) dermal DCs, to draining lymph nodes. Finally, we showed that iNOS inhibitor enhanced LC survival in vitro. We concluded that NO suppresses migration and survival of cutaneous DCs, resulting in a downmodulation of CHS.

17.BETA.-Estradiol Enhances Interleukin-18 mRNA Expression after Sensitization of Mice with Contact Hypersensitivity

To clarify the mechanism underlying the enhancing effect of 17β-estradiol (E2) on contact hypersensitivity (CHS) and the expression of interferon (IFN)-γ in mice, the mRNA expression levels of interleukin (IL)-18 were evaluated. Female BALB/c mice aged 3 weeks were ovariectomized, administered 3.2 μg of E2, and sensitized by 50 μl of 3% 4-ethoxymethylene-2-phenyl-2-oxazolin-one (OXA). Seven days later, CHS was elicited by the application of 7.5 μl of 1% OXA on the ear auricles. The auricles, cervical lymph nodes and spleens were excised, and gene expression was evaluated by reverse transcription-polymerase chain reaction. E2 enhanced the expression of IL-18 mRNA in the spleen on the following day and in the ear auricles on days 4 and 7 after sensitization with OXA. The preadministration of an antibody against IL-18 receptor suppressed the CHS and reduced IFN-γ mRNA expression in E2-administered mice. IL-18 was present in the dermis of the ear skin and absent in the epidermis. E2 also enhanced the expression of IFN-γ and IL-18 mRNAs in splenocytes cultured with lipopolysaccharide (LPS). IL-18 protein was detected by flow cytometry in CD4+, CD8+ and NKG2+ lymphocytes among splenocytes cultured with LPS. These results suggest that E2 enhances lymphocyte activation in the sensitization phase of CHS, and that IFN-γ mRNA expression is enhanced in the elicitation phase of CHS.

Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate

Many different types of phthalate ester are used as plasticizers and are thus found in the air. There have been several studies that suggest an association between allergies and phthalate esters. We previously found that di-butyl phthalate (DBP) has an adjuvant effect in a mouse contact hypersensitivity model, in which fluorescein isothiocyanate (FITC) is involved as an immunogenic hapten. We examined whether other phthalate esters enhance the process of sensitization to FITC by facilitating the trafficking of FITC-presenting dendritic cells or macrophages from skin sites to draining lymph nodes. Mice were epicutaneously sensitized with FITC dissolved in acetone containing a phthalate ester. Sensitization was evaluated as ear swelling after a challenge with FITC. Draining lymph node cells obtained 24 h after skin sensitization were examined for FITC fluorescence by means of flow cytometry. FITC-positive cells were characterized with anti-CD11c and anti-CD11b by three-colour flow cytometry. When mice were sensitized with FITC in acetone containing DBP or di-n-propyl phthalate (DPP), strong enhancement of the ear-swelling response was observed. Di-methyl phthalate (DMP) and di-ethyl phthalate (DEP) were less effective but produced some enhancement. Consistent enhancement was not observed with di-(2-ethylhexyl) phthalate or di-isononyl phthalate. Upon sensitization in the presence of DBP or DPP, the number of FITC-positive dendritic cells (total CD11c+ as well as CD11c+/CD11b+) was increased in draining lymph nodes. As to the other four phthalate esters, there was no significant increase in the FITC-positive cell number in the draining lymph nodes. During the process of sensitization to FITC, DBP, and DPP exert strong adjuvant effects that are associated with enhancement of trafficking of antigen-presenting dendritic cells from the skin to draining lymph nodes.

Pimecrolimus and tacrolimus differ in their inhibition of lymphocyte activation during the sensitization phase of contact hypersensitivity

As reported previously, oral administration of the calcineurin inhibitors (CNI) pimecrolimus and tacrolimus resulted in equipotent inhibition of the elicitation phase of contact hypersensitivity (CHS) in mice. The sensitization phase was inhibited by tacrolimus but was unaffected by pimecrolimus, even at higher doses. The kinetics of lymph node hyperplasia and up-regulation of T and B cell activation antigens were analyzed to obtain a better understanding of the divergent CNI profile in CHS. Lymph node (LN) cells of CNI-untreated and treated mice were examined with flow cytometry at various time points after sensitization with oxazolone. LN hyperplasia and drug levels were also determined. Sensitization induced a higher portion of LN cells expressing the activation antigens CD25, CD69 and CD134 and an increase in activated B cells (B220(+)/CD40(+)) compared to naïve mice. Up-regulation of these markers was completely or profoundly blocked with tacrolimus, whereas pimecrolimus at the three-fold higher dose caused significantly less inhibition. Tacrolimus also completely blocked the sensitization-associated increase of CD11c(+) antigen presenting cells (APC) in LN, whereas pimecrolimus showed significantly less inhibition. In contrast to tacrolimus, LN weight and cellularity were not affected by pimecrolimus at any time point after sensitization. Concentration of tacrolimus in blood and in the draining LN substantially exceeded that of pimecrolimus by factors 6.7-14 and 5.6-5.8, respectively, at the same dose levels. In contrast to tacrolimus, systemic treatment of mice with pimecrolimus only weakly interferes with lymphocyte activation and does not affect hyperplasia of the draining lymph nodes during sensitization.

Distribution of MGL1 Binding Sites and MGL1/2-positive Cells in Lymph Nodes during the Sensitization Phase of Contact Hypersensitivity

In the sensitization phase of delayed-type hypersensitivity, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs), and this migration was previously suggested to be an essential process in the establishment of the immune response to peripheral antigens. We hypothesize that the interactions between MGL1 and its ligands determine the localization of MGL1/2-positive cells within the LNs. In the present report, distribution of MGL1 binding sites was immunohistochemically examined by means of biotinylated recombinant MGL1 and compared with the distribution of MGL1/2-positive cells after epicutaneous sensitization of the mice with fluorescein isothiocyanate. The binding sites of MGL1 were mainly detected on the subcapsular sinus, interfollicular regions, and the T/B boundary. The distribution overlapped with the area where MGL1/2-positive cells that had migrated from dermis localized after sensitization with the antigen.

Identification of Sialoadhesin as a Dominant Lymph Node Counter-receptor for Mouse Macrophage Galactose-type C-type Lectin 1

In the sensitization phase of contact hypersensitivity in mice, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs) where they accumulate in the subcapsular sinus, interfollicular regions, and areas surrounding high endothelial venules. We hypothesize that the interactions between MGL1 and its ligands determine the localizations of MGL1-positive cells within the LNs. In the present study, our major aim was to isolate MGL1 counter-receptor(s) from lysates of LNs using affinity chromatography with immobilized recombinant MGL1. Fractions bound and eluted with EDTA were analyzed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. One of the predominant components was sialoadhesin (Sn, Siglec-1). Sn from lysates of LNs was immobilized on microtiter plates precoated with anti-Sn monoclonal antibody, and binding of recombinant MGL1 and adhesion of cells expressing MGL1 were tested. The binding of recombinant MGL1 to Sn was shown to be dependent on Ca2+ and N-glycans on Sn. MGL1-transfected Chinese hamster ovary cells adhered to the Sn-coated plates, whereas mock transfectants did not. Immunohistochemical localization of anti-Sn monoclonal antibody in LN coincided with the subcapsular sinus area to which recombinant MGL1 was bound. Furthermore, the distribution of MGL1+ cells after sensitization with FITC was demonstrated to overlap with that of Sn within the subcapsular sinus of draining LNs. These results suggest that Sn acts as an endogenous counter-receptor for MGL1.

The Sensitizers Nickel Sulfate and 2,4-dinitrofluorobenzene Increase CD40 and IL-12 Receptor Expression in a Fetal Skin Dendritic Cell Line

Dendritic cells (DCs) are antigen-presenting cells (APCs) capable of capturing haptens and to process and present them to T lymphocytes. In order to sensitize T cells for contact hypersensitivity (CHS), skin DCs suffer a maturation process with modifications on their surface molecules. The aim of this work was to evaluate changes induced by two contact sensitizers, 2,4-dinitrofluorobenzene (DNFB) and nickel sulfate (NiSO4), and a non-sensitizer 2,4-dichloronitrobenzene (DCNB), on the protein levels of two activation markers, CD40 and IL-12 receptor (IL-12R), in a mouse skin dendritic cell line (FSDC). The expression of CD40 and IL-12R proteins was evaluated by western blot assay and direct immunofluorescence microscopy. The results showed that CD40 and IL-12R expression increased significantly after cell exposure to NiSO4 and DNFB, although DNFB exhibited a stronger activity. There was no effect with DCNB. The epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), also used in the experiments, slightly increased the expression of both CD40 and IL-12R and when tested together with the sensitizers the effect was partially additive. The results suggest that the sensitizers DNFB and NiSO4 are directly involved on the changes of the surface markers CD40 and IL-12R in skin DCs, during the sensitization phase of CHS, and this effect may be enhanced by GM-CSF. In contrast, no effect was observed with DCNB.

JAK/STAT pathways are not involved in the direct activation of antigen-presenting cells by contact sensitizers.

JAK/STAT pathways are described as the major mechanisms by which cytokine receptors transduce intracellular signals. The signalling mechanisms in antigen-presenting cells (APC) in the sensitization phase of contact hypersensitivity are poorly understood. The aim of this study was to clarify whether well-established JAK/STAT signalling pathways might be activated directly by contact sensitizers as described previously for tyrosine kinases and some MAP kinases. As a model of epidermal APC, human monocytes and human monocyte-derived dendritic cells were stimulated with the structurally unrelated contact sensitizers MCI/MI, thimerosal, TNCB and formaldehyde. The phosphorylation states of the transcription factors STAT1, STAT3, STAT4, STAT5 and STAT6 were determined by Western blot analysis using phosphospecific antibodies. In contrast to the positive controls performed with the cytokines IFN-gamma, IL-10, IFN-alpha, GM-CSF and IL-4, no significant increase in the phosphorylation of STAT molecules was recognized in hapten-treated cells. These results suggest that contact allergens do not directly activate common JAK/STAT pathways. Therefore the activation of APC in the early sensitization phase of contact hypersensitivity by haptens does not involve signals normally delivered by JAK-associated cytokine receptors.

Peripheral blood granulocyte activity following contact sensitization of rats with dinitrochlorobenzene

Contact hypersensitivity (CHS) reaction is a classic example of a cell-mediated reaction. As the afferent phase of the reaction includes inflammation, CHS is a suitable model for investigating non-specific immunity. Some aspects of granulocyte activity in the afferent phase of experimentally induced CHS to dinitrochlorobenzene (DNCB) in two genetically different rat strains, AO and DA were examined in this study. A shift in the ratio of granulocytes to lymphocytes in favour of granulocytes and an increase in granulocyte survival were noted in DA rats. Granulocytes from both strains demonstrated increased levels of NBT reduction and an increase in their adhesion to plastic. Decreased granulocyte adhesion in the presence of monoclonal antibodies to β 2 integrins ( anti-CD11b/c and anti-CD18) points to the contribution of these molecules to granulocyte adhesiveness during the sensitization phase of CHS. Stimulation of adhesion in the presence of anti-CD11a antibody, points to a differential modulation of adhesion molecule activity during the afferent phase of CHS. Changes in functional activity of granulocytes demonstrated in this study might contribute to the development of CHS in rats.