Contact Hypersensitivity Response In Mice Research Articles

Circadian rhythm affects the magnitude of contact hypersensitivity response in mice.

The circadian rhythm controls multiple biological processes, including immune responses; however, its impact on cutaneous adaptive immune response remains unclear. We used a well-established cutaneous type IV allergy model, contact hypersensitivity (CHS). We induced CHS using dinitrofluorobenzene (DNFB). Mice were sensitized and elicited with DNFB in the daytime or at night. In mice, a nocturnally active animal, we found that ear swelling increased when mice were sensitized at night compared with in the daytime. In addition, cell proliferation and cytokine production in the draining lymph nodes (LNs) were promoted when sensitized at night. We hypothesized that these differences were due to the oscillation of leukocyte distribution in the body through the circadian production of adrenergic hormones. Administration of a β2-adrenergic receptor (β2AR) agonist salbutamol in the daytime decreased the number of immune cells in blood and increased the number of immune cells in LNs. In contrast, a β2AR antagonist ICI18551 administration at night increased the number of immune cells in blood and decreased the number of immune cells in LNs. Accordingly, the severity of CHS response was exacerbated by salbutamol administration in the daytime and attenuated by ICI18551 administration at night. Our study demonstrated that the magnitude of adaptive CHS response depends on the circadian rhythm and this knowledge may improve the management of allergic contact dermatitis (ACD) in humans.

Diuretics combined with captopril modulate macrophage-induced humoral and contact hypersensitivity responses in mice

Abstract Introduction The over-activated inflammatory responses of macrophages (Mph) likely increases the risk of hypertension development, while clinically relevant diuretics administered alone or in combination with antihypertensive drugs may possibly exert the immunomodulatory effects. Thus, we investigated the impact of these drugs on macrophage-mediated immunity in mice. Methodology CBA mice were treated i.p. with captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) by 8 days. On the third day, mice were i.p. injected with mineral oil and 5 days later Mph were harvested to assess the generation of reactive oxygen species (ROS) and nitric oxide (NO). Mph were also pulsed with sheep red blood cells (SRBC) or hapten and transferred to naive mice for evaluation of their ability to induce humoral or contact hypersensitivity (CHS) reactions, respectively. Results Diuretics, when administered alone or with captopril, enhanced ROS, but decreased NO production by Mph. SRBC-pulsed Mph from mice treated with captopril combined with diuretics increased the secretion of antigen-specific antibodies by recipient B cells, while Mph of mice treated with hydrochlorotiazide or furosemide with captopril increased the number of antigen-specific B cells. Captopril reduced CHS ear swelling response and this effect was augmented by diuretics. Conclusions Our results showed that diuretics with or without captopril modulate the humoral and allergic cell-mediated immune responses by affecting the function of Mph. Further studies should investigate the clinical effect of these observations. The study was supported by budget funds for science in 2017-2021 under the “Diamond Grant” program (0168/DIA/2017/46).

CD5 on dendritic cells regulates CD4+ and CD8+ T cell activation and induction of immune responses.

The role of CD5 as a regulator of T cell signaling and tolerance is well recognized. Recent data show expression of CD5 on different subtypes of human dendritic cells, however its functional relevance in modulating DC mediated responses remains poorly understood. In this study, we show CD5 is expressed on CD11c+ DC from murine thymus, lymph node, spleen, skin and lung. Although the development of DC subpopulations in CD5-/- mice was normal, CD5-deficient DC produced significantly higher levels of IL-12 than wild type DC in response to LPS. CD5-/- DC, in comparison to CD5+/+ DC, enhanced the activation of CD4+ and CD8+ T cells in vitro and in vivo and induced significantly higher production of IL-2 and IFN-gamma by T cells. Consequently, CD5-/- DC were significantly more potent than wild type DC in the induction of anti-tumor immunity and contact hypersensitivity responses in mice. Restoration of CD5 expression in CD5-/- DC reduced IL-12 production and inhibited their capacity to stimulate T cells. Collectively, these data demonstrate that the specific expression of CD5 on DC inhibits the production of inflammatory cytokines and has a regulatory effect on their activity to stimulate T cells and induce immune responses. This study reveals a previously unrecognized regulatory role for CD5 on DC and provides novel insights into mechanisms for DC biology in immune responses.

Multi-walled carbon nanotube oxidation dependent keratinocyte cytotoxicity and skin inflammation

BackgroundThe effects of carbon nanotubes on skin toxicity have not been extensively studied; however, our lab has previously shown that a carboxylated multi-walled carbon nanotube (MWCNT) exacerbates the 2, 4-dinitrofluorobenzene induced contact hypersensitivity response in mice. Here we examine the role of carboxylation in MWCNT skin toxicity.ResultsMWCNTs were analyzed by transmission electron microscopy, zetasizer, and x-ray photoelectron spectroscopy to fully characterize the physical properties. Two MWCNTs with different levels of surface carboxylation were chosen for further testing. The MWCNTs with a high level of carboxylation displayed increased cytotoxicity in a HaCaT keratinocyte cell line, compared to the MWCNTs with intermediate levels of carboxylation. However, neither functionalized MWCNT increased the level of in vitro reactive oxygen species suggesting an alternative mechanism of cytotoxicity. Each MWCNT was tested in the contact hypersensitivity model, and only the MWCNTs with greater than 20% surface carboxylation exacerbated the ear swelling responses. Analysis of the skin after MWCNT exposure reveals that the same MWCNTs with a high level of carboxylation increase epidermal thickness, mast cell and basophil degranulation, and lead to increases in polymorphonuclear cell recruitment when co-administered with 2, 4-dinitrofluorobenzene.ConclusionsThe data presented here suggest that acute, topical application of low doses of MWCNTs can induce keratinocyte cytotoxicity and exacerbation of allergic skin conditions in a carboxylation dependent manner.

065 Circadian rhythm affects the severity of contact hypersensitivity response in mice

065 Circadian rhythm affects the severity of contact hypersensitivity response in mice

Regulatory B cells induced by ultraviolet B through toll-like receptor 4 signalling contribute to the suppression of contact hypersensitivity responses in mice.

Ultraviolet (UV) B irradiation is known to suppress contact hypersensitivity (CHS) responses in mouse models by suppressing immune responses. However, the cellular mechanisms responsible for UVB-induced systemic suppression remain unclear. Regulatory B cells have been reported to play an inhibitory role during CHS. It is presently unknown whether regulatory B cells contribute to the effect of UVB phototherapy. To investigate the inductive effect of UVB on regulatory B cells and the underlying mechanisms by using a CHS mouse model. CHS was induced with oxazolone, and evaluated by histopathology, flow cytometry, and quantitative real-time polymerase chain reaction. We found that UVB irradiation induced regulatory B cell expansion and ameliorated CHS. UVB-induced regulatory B cells contribute to systemic immunosuppression by inhibiting the proliferation of T cells. Moreover, we determined that toll-like receptor (TLR) 4, the expression of which was upregulated in B cells after UVB exposure, played an essential role in the induction of regulatory B cells. Our data identified regulatory B cells as regulators of UVB-induced immunosuppression in CHS, and suggest the importance of the UVB-TLR4 axis in the generation of regulatory B cells.

Abstract 2221: Honokiol stimulates immune reactivity in UV-irradiated skin through DNA demethylation-dependent functional activation of dendritic cells in mice

Abstract Solar ultraviolet radiation (UVR), and in particular UVB spectrum (290-320 nm), are considered as a complete carcinogen for cutaneous malignancies. The over-exposure of UVR suppresses the development of allergic contact hypersensitivity (CHS) response in both laboratory animals as well as in humans. CHS response is considered to be a prototypic T-cell mediated immune response. UVR-induced suppression of immune sensitivity has been implicated in skin cancer risk. Therefore, the treatment options which can inhibit UVB-induced suppression of immune sensitivity may be useful in the management of skin cancers, including both melanoma and non-melanoma. Previously, we have shown that topical treatment of honokiol inhibits UVB induced immune suppression, which was associated with the reduction in UVB-induced inflammatory mediators in the mouse skin. Here, we have further determined the underlying mechanism of action of honokiol on UVR-induced immunosuppression including its effect on epigenetic regulators and their relationship with immune sensitivity in mouse skin. Topical treatment of C3H/HeN mice with honokiol (0.5 and 1.0 mg/cm2 skin area) in hydrophilic-cream based topical formulation significantly inhibits UVB-induced suppression of CHS response (P<0.01-0.005), which was associated with reduced levels of DNA methylation as well as Dnmt activity in the mouse skin compared with non-honokiol-treated and UVB-exposed control mice. To characterize the cell population responsible for the honokiol mediated inhibition of UVB-induced immunosuppression, we used an adoptive transfer approach. DNA-methylated (most of cells) dendritic cells (DCs), isolated from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) with and without honokiol (0.5 and 1.0 mg/cm2 skin area) treatment, were transferred into naïve recipient mice. The CHS response of the recipient mice to DNFB was then measured. Honokiol treatment of UVB-exposed donor mice relieved this suppression of the CHS response in naïve mice, while the recipient mice, obtained DNA-methylated DCs from UVB-exposed donor mice that were not treated with honokiol, showed significant suppression in the CHS response. The restoration of CHS response in mice receiving DCs from honokiol treated, UV-exposed donor mice was also associated with enhanced secretion of Th1-type cytokines compared with the Th1-type cytokines from the DCs obtained from non-honokiol-treated and UVB-exposed donor mice. These data suggest that honokiol mediated inhibition of UVB induced suppression of CHS response is associated with functional activation of dendritic cells and that is dependent on DNA demethylation in DCs. Note: This abstract was not presented at the meeting. Citation Format: Santosh K. Katiyar, Tripti Singh, Harish C. Pal, Ram Prasad. Honokiol stimulates immune reactivity in UV-irradiated skin through DNA demethylation-dependent functional activation of dendritic cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2017-2221

Honokiol inhibits ultraviolet radiation-induced immunosuppression through inhibition of ultraviolet-induced inflammation and DNA hypermethylation in mouse skin

Ultraviolet (UV) radiation exposure induces immunosuppression, which contributes to the development of cutaneous malignancies. We investigated the effects of honokiol, a phytochemical found in plants of the genus Magnolia, on UVB-induced immunosuppression using contact hypersensitivity (CHS) as a model in C3H/HeN mice. Topical application of honokiol (0.5 and 1.0 mg/cm2 skin area) had a significant preventive effect on UVB-induced suppression of the CHS response. The inflammatory mediators, COX-2 and PGE2, played a key role in this effect, as indicated by honokiol inhibition of cyclooxygenase-2 (COX-2) expression and PGE2 production in the UVB-exposed skin. Honokiol application also inhibited UVB-induced DNA hypermethylation and its elevation of the levels of TET enzyme, which is responsible for DNA demethylation in UVB-exposed skin. This was consistent with the restoration of the CHS response in mice treated with the DNA demethylating agent, 5-aza-2′-deoxycytidine, after UVB exposure. There was no significant difference in the levels of inhibition of UVB-induced immunosuppression amongst mice that were treated topically with available anti-cancer drugs (imiquimod and 5-fluorouracil). This study is the first to show that honokiol has the ability to inhibit UVB-induced immunosuppression in preclinical model and, thus, has potential for use as a chemopreventive strategy for UVB radiation-induced malignancies.

Estrogen inhibits acute and chronic contact hypersensitivity response in mice

Estrogen inhibits acute and chronic contact hypersensitivity response in mice

Epidermal NLRP10 contributes to contact hypersensitivity responses in mice.

The nucleotide binding and oligomerization domain-like receptor (NLR) protein NLRP10 is highly expressed in the epidermis and contributes to cell-autonomous responses against invasive bacteria. To investigate the role of NLRP10 in inflammatory responses of the skin we analyzed the effect of full-body and keratinocyte-specific depletion of NLRP10 in croton oil-induced irritant contact dermatitis (ICD) and 1-fluoro-2,4-dinitrobenzene (DNFB)-induced contact hypersensitivity (CHS) in mice. Nlrp10(-/-) mice were phenotypically normal and skin repair after wounding was not affected by lack of NLRP10. Similarly, we did not detect a contribution of NLRP10 to the ICD response induced by croton oil. In contrast, Nlrp10(-/-) mice showed significantly reduced inflammation in the DNFB-induced CHS response as compared to control animals. Microscopic analysis revealed significantly reduced numbers of CD4(+) and CD8(+) T cells in the infiltrates of animals lacking NLRP10 expression after CHS challenge. Epidermis-specific deletion of Nlrp10 by keratin-14 promotor driven Cre-recombinase was sufficient to account for this phenotype, although lymphocyte recruitment seemed to be unaltered in animals lacking NLRP10 expression in keratinocytes. Taken together, we provide evidence that NLRP10 contributes to T-cell-mediated inflammatory responses in the skin and highlight a physiological role of NLRP10 in epidermal keratinocytes.

Stat5 gene dosage in T cells modulates CD8+ T-cell homeostasis and attenuates contact hypersensitivity response in mice.

Contact hypersensitivity assay (CHS) faithfully models human allergies. The Stat5 transcription factors are essential for both lymphocyte development and acute immune responses. Although consequences of Stat5 ablation and transgenic overexpression for the lymphocyte development and functions have been extensively studied, the role of Stat5 gene dosage in contact allergies has not been addressed. We investigated the effect of Stat5 gene dosage modulation in contact allergies using CHS in mice. Transgenic animals heterozygous for the germline Stat5 null allele were subjected to CHS. To dissect cell type sensitive to Stat5 gene dosage, animals with Stat5 haplo-insufficiency in T cells, where one Stat5 allele was removed by Lck-Cre-mediated deletion (Stat5(ΔT/+)), were tested by CHS. Frequency of T cells, B cells, and monocytes were analyzed in Stat5(ΔT/+) and wild-type animals by flow cytometry. Proliferation of Stat5(ΔT/+) CD8(+) T cells was studied in vitro by stimulation with IL-4 and IL-2 cytokines, and changes in the expression of Stat5 target genes were assayed by quantitative real-time PCR assay. Haplo-insufficiency of Stat5 in T cells leads to the reduction in CD8(+) T cells in all lymphoid organs and attenuates CHS response. Stat5(ΔT/+) CD8(+) T cells failed to fully activate Stat5-dependent expression of cell cycle/survival target genes, such as Bcl2 and Pim1, and to proliferate efficiently in response to IL-2 and IL-4 cytokine. Our data identify Stat5 as a dose-dependent regulator of CD8(+) T-cell functions in contact allergies and suggest that modulation of Stat5 dosage could be used to target contact allergies in humans.

Abstract 1082: Silymarin, a phytochemical from milk thistle, inhibits UVB-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T cells .

Abstract Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the risk of skin cancers, including melanoma and non-melanoma. Topical treatment of mouse skin with silymarin, a bioactive phytochemical from milk thistle (Silybum marianum L. Gaertn.), inhibits UVB radiation-induced skin tumor development and immunosuppression in mice. To identify the molecular mechanisms underlying its effect on immune system, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. CHS response is considered as a prototypic T-cell mediated immune response. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in xeroderma pigmentosum complementation Group A (XPA)-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8+ or CD4+ cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8+ T cells, and reduced secretion of Th2 cytokines by CD4+ cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4+ regulatory T-cell activity, and stimulation of CD8+ effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression in mice. This property of silymarin can be used as an alternative strategy to augment immune system and that may help to prevent or delay the risk of skin cancers in high-risk human population. Citation Format: Mudit Vaid, Ram Prasad, Tripti Singh, Craig A. Elmets, Hui Xu, Santosh K. Katiyar. Silymarin, a phytochemical from milk thistle, inhibits UVB-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1082. doi:10.1158/1538-7445.AM2013-1082

Silymarin inhibits ultraviolet radiation-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T cells

Silymarin inhibits UVB-induced immunosuppression in mouse skin. To identify the molecular mechanisms underlying this effect, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in XPA-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8+ or CD4+ cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8+ T cells, and reduced secretion of Th2 cytokines by CD4+ T cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4+ regulatory T-cell activity, and stimulation of CD8+ effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression.

Anti-inflammatory and immunomodulatory mechanisms of artemisinin on contact hypersensitivity

Artemisinin (Art) is a sesquiterpene trioxane lactone from Artemisia annua L., which has been shown to affect immune responses. However, the underlying mechanism remains elusive. In this study, we examined the anti-inflammatory and immunomodulatory effects of Art in a mouse model of contact hypersensitivity (CHS), a T-cell-mediated cutaneous inflammatory reaction. The data showed that topical administration of Art could suppress CHS response and Con A-induced T cell proliferation effectively. Further experiments indicated that Art induced the generation of regulatory T cells (Tregs) and impaired the phosphorylation of AKT, associated with the up-regulation of p38 MAPK activation. Moreover, Art also exerted a strikingly inhibitory effect on IL-17 production, and diminished the level of IL-6 paralleled with an enhancement of TGF-β, which effects were coupled with a significant reduction of STAT3 activation. These data reveal that Art could effectively block CHS response in mice by inducing the generation of Tregs and suppressing the development of Th17, indicating the potential of Art to be applied as an effective therapeutic agent for treating immune-related diseases.

Amphiregulin is Not Essential for Induction of Contact Hypersensitivity

Amphiregulin (AR) is expressed in Th2 cells, rather than Th1 cells, and plays an important role in Th2 cell/cytokine-mediated host defense against nematodes. We also found earlier that AR mRNA expression was strongly upregulated in inflamed tissue during Th2 cell/cytokine-mediated fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS), suggesting a contribution of AR to the induction of those responses. To elucidate the role of AR in the induction of FITC- or dinitrofluorobenzene (DNFB)-induced CHS, AR-deficient mice were sensitized and/or challenged with FITC or DNFB epicutaneously. The levels of FITC-mediated skin dendritic cell (DC) migration and FITC-specific lymph node cell proliferation and cytokine production were assessed by flow cytometry, [3H]-thymidine incorporation and ELISA, respectively, after FITC sensitization. The degree of ear swelling, the activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in inflammatory sites and the levels of FITC-specific immunoglobulin (Ig) in sera were determined by histological analysis, colorimetric assay and ELISA, respectively, after FITC challenge. DC migration and FITC-specific lymph node cell proliferation and cytokine production were normal in the AR-deficient mice. Ear swelling, tissue MPO and EPO activities and FITC-specific serum Ig levels were also similar in AR-deficient and -sufficient mice. Amphiregulin is not essential for the induction of FITC- or DNFB-induced CHS responses in mice.

Glucocorticoids Enhance Toll-Like Receptor 2 Expression in Human Keratinocytes Stimulated with Propionibacterium acnes or Proinflammatory Cytokines

Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-alpha, and IL-1alpha. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis.

The Endogenous Danger Signal Uric Acid Augments Contact Hypersensitivity Responses in Mice

Objective: The danger hypothesis proposes that the immune system responds not only to foreign antigens but also to damaged cells or tissues. Recently, uric acid crystals (monosodium urate, MSU) from necrotic cell lysates were identified as a danger signal for dendritic cells (DCs). Our aim was to determine whether MSU modulates immune responses in the skin. Method: We analyzed the effect of MSU on trinitrochlorobenzene-induced contact hypersensitivity responses using BALB/c mice administered potassium oxonate, an uricase inhibitor, to prevent MSU degradation. Ear swelling response after elicitation and activation profiles of DCs and T cells in draining lymph nodes after sensitization were assessed. Results: Intradermal administration of MSU augmented the ear swelling response in potassium oxonate-administered mice and enhanced expression of CD86 and CD40 molecules on DCs in the lymph nodes. Activation of DCs was followed by an increase in CD69+ and CD44+ T cells in CD4+ and/or CD8+ subsets in the lymph nodes 4 days after trinitrochlorobenzene sensitization. Conclusion: These observations demonstrate that MSU is an endogenous danger signal, which augments the contact hypersensitivity response in mice. MSU released from damaged skin may act as an endogenous adjuvant to augment immune response.

Induction of Regulatory T Cells by Leflunomide in a Murine Model of Contact Allergen Sensitivity

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8+ and CD4+ T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide (N-[4-trifluoro-methylphenyl]-5-methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. The loss of CHS responses was shown to be antigen-specific. Adoptive transfer of leukocytes from LF-treated mice into naïve mice resulted in a loss of CHS responsiveness. Transfer of both CD4+ and CD8+ cells was required for maximal loss of CHS responses, with CD8+ cells playing a major role. Significantly enhanced levels of IL-10 mRNA were detected in CD8+ T cells, but not in CD4+ T cells, following LF treatment of mice. LF also suppressed CHS responses in mice previously sensitized and challenged with hapten, when administered together with the hapten. Our data suggest that LF induces a long-lived tolerance in mice by inducing CD8+ and CD4+ regulatory T cells.

Augmentation of Cutaneous Immune Responses by ATPγS: Purinergic Agonists Define a Novel Class of Immunologic Adjuvants

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPgammaS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPgammaS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPgammaS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1beta, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X(1), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(9), and P2Y(11) receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.

Primary Defect in UVB-Induced Systemic Immunomodulation Does Not Relate to Immature or Functionally Impaired APCs in Regional Lymph Nodes

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.