DOCK8 Expression in Regulatory T Cells Maintains their Stability and Limits Contact Hypersensitivity.

Chronic dermatitis is a hallmark of Dedicator of cytokinesis 8 (DOCK8) deficiency. The migration of DOCK8-deficient T cells to the skin and their survival there have been reported to be defective. Surprisingly, we found that Dock8-/- mice demonstrated an exaggerated contact hypersensitivity (CHS) response to oxazolone with increased ear swelling, T-cell infiltration, and expression of Ifng. To understand the mechanisms of persistent skin inflammation in DOCK8 deficiency, we examined mice with selective deficiency of DOCK8 in T cells or T regulatory cells (Tregs) and found that both have exaggerated CHS. Moreover, oral tolerance to oxazolone, mediated by Tregs, was impaired in Dock8-/- mice. Transfer of Tregs from oxazolone-sensitized wild-type mice, but not Dock8-/- mice, reduced the CHS response of Dock8-/- recipients. Lack of DOCK8 in Tregs resulted in their acquisition of a pathogenic FOXP3+T-bet+IFNγ+ phenotype at CHS sites and promoted their conversion into ex-Tregs. The transfer of Tregs from Dock8-/- mice increased the CHS response of wild-type recipients to oxazolone. Thus, DOCK8 expression in Tregs limits CHS by promoting Treg stability and fitness in inflamed skin. Interventions aimed at ameliorating Treg function may be useful in treating skin inflammation in DOCK8 deficiency.

Galectin-1 Expression in CD8+ T Lymphocytes Controls Inflammation in Contact Hypersensitivity.

Allergic contact dermatitis, also known as contact hypersensitivity, is a frequent T-cell‒mediated inflammatory skin disease characterized by red, itchy, swollen, and cracked skin. It is caused by the direct contact with an allergen and/or irritant hapten. Galectin-1 (Gal-1) is a ß-galactoside‒binding lectin, which is highly expressed in several types of immune cells. The role of endogenous Gal-1 in contact hypersensitivity is not known. We found that Gal-1‒deficient mice display more sustained and prolonged skin inflammation than wild-type mice after oxazolone treatment. Gal-1‒deficient mice have increased CD8+ T cells and neutrophilic infiltration in the skin. After the sensitization phase, Gal-1‒depleted mice showed an increased frequency of central memory CD8+ T cells and IFN-γ secretion by CD8+ T cells. The absence of Gal-1 does not affect the migration of transferred CD4+ and CD8+ T cells from the blood to the lymph nodes or to the skin. The depletion of CD4+ T lymphocytes as well as adoptive transfer experiments demonstrated that endogenous expression of Gal-1 on CD8+ T lymphocytes exerts a major role in the control of contact hypersensitivity model. These data underscore the protective role of endogenous Gal-1 in CD8+ but not CD4+ T cells in the development of allergic contact dermatitis.

Neutrophil-Derived Myeloperoxidase Facilitates Both the Induction and Elicitation Phases of Contact Hypersensitivity.

Background: Allergic contact dermatitis (ACD) is a common skin disorder affecting an estimated 15-20% of the general population. The mouse model of ACD is contact hypersensitivity (CHS), which consists of two phases: induction and elicitation. Although neutrophils are required for both CHS disease phases their mechanisms of action are poorly understood. Neutrophils release myeloperoxidase (MPO) that through oxidation of biomolecules leads to cellular damage. Objectives: This study investigated mechanisms whereby MPO contributes to CHS pathogenesis. Methods: CHS was induced in mice using oxazolone (OX) as the initiating hapten applied to the skin. After 7 days, CHS was elicited by application of OX to the ear and disease severity was measured by ear thickness and vascular permeability in the ear. The role of MPO in the two phases of CHS was determined utilizing MPO-deficient mice and a specific MPO inhibitor. Results: During the CHS induction phase MPO-deficiency lead to a reduction in IL-1ß production in the skin and a subsequent reduction in migratory dendritic cells (DC) and effector T cells in the draining lymph node. During the elicitation phase, inhibition of MPO significantly reduced both ear swelling and vascular permeability. Conclusion: MPO plays dual roles in CHS pathogenesis. In the initiation phase MPO promotes IL-1ß production in the skin and activation of migratory DC that promote effector T cell priming. In the elicitation phase MPO drives vascular permeability contributing to inflammation. These results indicate that MPO it could be a potential therapeutic target for the treatment of ACD in humans.

Inactivation of the PD-1-Dependent Immunoregulation in Mice Exacerbates Contact Hypersensitivity Resembling Immune-Related Adverse Events.

Blockade of PD-1, an indispensable physiological immunoregulatory mechanism, enhances immune activities and is widely used in the immunotherapy of cancer. This treatment often accompanies inflammatory complication called immune-related adverse events (irAE), most frequently in the skin. To analyze how skin inflammation develops by the blockade of PD-1-dependent immunoregulation, we studied the exacerbation of oxazolone-induced contact hypersensitivity by PD-L1 blockade. The inactivation of PD-1 signaling enhanced swelling of the skin with massive CD8+ T cell infiltration. Among PD-1-expressing cells, T cells were the predominant targets of anti-PD-L1 mAb treatment since PD-L1 blockade did not affect skin inflammation in RAG2-/- mice. PD-L1 blockade during immunization with oxazolone significantly promoted the development of hapten-reactive T cells in the draining lymph nodes. The enhancement of local CD8+ T cell-dominant immune responses by PD-L1 blockade was correlated with the upregulation of CXCL9 and CXCL10. Challenges with a low dose of oxazolone did not demonstrate any significant dermatitis; however, the influence of PD-L1 blockade on T cell immunity was strong enough to cause the emergence of notable dermatitis in this suboptimal dosing, suggesting its relevance to dermal irAE development. In the low-dose setting, the blockade of CXCR3, receptor of CXCL9/10, prevented the induction of T cell-dominant inflammation by anti-PD-L1 mAb. This experimental approach reproduced CD8+ T cell-dominant form of cutaneous inflammation by the blockade of PD-L1 that has been observed in dermal irAE in human patients.

Increased IL-26 Expression Promotes T Helper Type 17- and T Helper Type 2-Associated Cytokine Production by Keratinocytes in Atopic Dermatitis.

Whereas atopic dermatitis (AD) is considered as a T helper 2 (Th2)-centered disease, IL-17-producing Th (Th17) cells are also activated in AD lesional skin. However, the relationship between Th17 responses and Th2 responses in AD is still to be elucidated. Although Th17 cells are increased in AD skin, the expression and function of IL-26, which is also produced by Th17 cells, in AD are still unknown. In this report, we demonstrated that IL-26 mRNA expression levels were elevated in AD lesional skin compared with healthy controls and that IL-26-producing cells were increased in AD lesional skin by immunohistochemistry. Furthermore, IL-26 promoted IL-8, IL-1ß, chemokine (C-C motif) ligand 20, IL-33, and ß-defensin 2 production in keratinocytes through phosphorylation of signal transducer and activator of transcription 1 and signal transducer and activator of transcription 3. Selective JAK inhibitors for JAK1, JAK2, and tyrosine kinase 2 blocked IL-26-induced cytokine production in keratinocytes. We also showed that injection of IL-26 exacerbated an oxazolone-induced AD mouse model and upregulated Th2 and Th17 cytokine expression in vivo. Because previous studies indicate that the above molecules induced by IL-26 can promote Th17 and/or Th2 immune responses, IL-26 may play an important role for bridging between Th17 and Th2 responses, resulting in the development of AD.

Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine.

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the "adaptive" characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system.

Transcriptome profiling reveals Th2 bias and identifies endogenous itch mediators in poison ivy contact dermatitis.

In the United States, poison ivy exposure is the most common naturally occurring allergen to cause allergic contact dermatitis (ACD). The immune and pruritic mechanisms associated with poison ivy ACD remain largely unexplored. Here, we compared skin whole transcriptomes and itch mediator levels in mouse ACD models induced by the poison ivy allergen, urushiol, and the synthetic allergen, oxazolone. The urushiol model produced a Th2-biased immune response and scratching behavior, resembling findings in poison ivy patients. Urushiol-challenged skin contained elevated levels of the cytokine thymic stromal lymphopoietin (TSLP), a T-cell regulator and itch mediator, and pruritogenic serotonin (5-HT) and endothelin (ET-1), but not substance P (SP) or histamine. The oxazolone model generated a mixed Th1/Th2 response associated with increased levels of substance P, 5-HT, ET-1, but not TSLP or histamine. Injections of a TSLP monoclonal neutralizing antibody, serotonergic or endothelin inhibitors, but not SP inhibitors or antihistamines, reduced scratching behaviors in urushiol-challenged mice. Our findings suggest that the mouse urushiol model may serve as a translational model of human poison ivy ACD study. Inhibiting signaling by TSLP and other cytokines may represent alternatives to the standard steroid/antihistamine regimen for steroid-resistant or -intolerant patients and in exaggerated systemic responses to poison ivy.

Keratinocyte Proline-Rich Protein Deficiency in Atopic Dermatitis Leads to Barrier Disruption.

Atopic dermatitis is a common inflammatory skin disease caused by the interaction of genetic and environmental factors. By allelic copy number analysis at missense single-nucleotide polymorphisms on 26 genes with copy number variation, we identified a significant association between atopic dermatitis and human KPRP. Human KPRP expression, which was localized to the upper granular layer of epidermis, was significantly decreased in atopic dermatitis compared with normal skin. KPRP was histologically colocalized with loricrin and was mainly detected in cytoskeleton fractions of human keratinocytes. To further investigate the role of KPRP in skin, Kprp-knockout mice were generated. Heterozygous knockout (Kprp+/-) mice exhibited reduced KPRP expression to level a similar to that of human AD lesional skin. Kprp+/- mice showed abnormal desmosome structure and detachment of lower layers of the stratum corneum. Percutaneous inflammation by topical application of croton oil or oxazolone was enhanced, and epicutaneous immunization with ovalbumin induced a high level of IgE in Kprp+/- mice. Our study, started from allelic copy number analysis in human AD, identified the importance of KPRP, the decrease of which leads to barrier dysfunction.

Mature IgDlow/- B cells maintain tolerance by promoting regulatory T cell homeostasis.

A number of different B cell subsets have been shown to exhibit regulatory activity using a variety of mechanisms to attenuate inflammatory diseases. Here we show, using anti-CD20-mediated partial B cell depletion in mice, that a population of mature B cells distinguishable by IgDlow/- expression maintains tolerance by, at least in part, promoting CD4+Foxp3+ regulatory T cell homeostatic expansion via glucocorticoid-induced tumor necrosis factor receptor ligand, or GITRL. Cell surface phenotyping, transcriptome analysis and developmental study data show that B cells expressing IgD at a low level (BDL) are a novel population of mature B cells that emerge in the spleen from the transitional-2 stage paralleling the differentiation of follicular B cells. The cell surface phenotype and regulatory function of BDL are highly suggestive that they are a new B cell subset. Human splenic and peripheral blood IgDlow/- B cells also exhibit BDL regulatory activity, rendering them of therapeutic interest.

Visualization of T Cell-Regulated Monocyte Clusters Mediating Keratinocyte Death in Acquired Cutaneous Immunity.

It remains unclear how monocytes are mobilized to amplify inflammatory reactions in T cell-mediated adaptive immunity. Here, we investigate dynamic cellular events in the cascade of inflammatory responses through intravital imaging of a multicolor-labeled murine contact hypersensitivity model. We found that monocytes formed clusters around hair follicles in the contact hypersensitivity model. In this process, effector T cells encountered dendritic cells under regions of monocyte clusters and secreted IFN-γ, which mobilizes CCR2-dependent monocyte interstitial migration and CXCR2-dependent monocyte cluster formation. We showed that hair follicles shaped the inflammatory microenvironment for communication among the monocytes, keratinocytes, and effector T cells. After disrupting the T cell-mobilized monocyte clusters through CXCR2 antagonization, monocyte activation and keratinocyte apoptosis were significantly inhibited. Our study provides a new perspective on effector T cell-regulated monocyte behavior, which amplifies the inflammatory reaction in acquired cutaneous immunity.

The role of toll-like receptor 3 in chronic contact hypersensitivity induced by repeated elicitation.

BACKGROUND: Accumulating evidence suggests that Toll-like receptor (TLR)-3 signaling is involved in non-infectious immune and inflammatory reactions as well as in viral infections. The skin of patients with atopic dermatitis (AD) is often infected with virus and bacteria, leading to the aggravation of atopic symptoms. These findings suggest TLR3 signaling may be involved in the pathogenesis of AD, but the exact role of TLR3 in AD remains to be defined. OBJECTIVE: The purpose of this study was to investigate the role of TLR3 in chronic contact hypersensitivity reactions induced by repeated elicitation, resembling the features of AD. METHODS: Wild-type (WT) and Toll-like receptor 3 knockout (Tlr3 KO) mice were sensitized, and chronic contact hypersensitivity reactions were elicited in their ear skin via repeated application of a hapten, 2,4,6-trinitro-1-chlorobenzene (TNCB) or oxazolone. RESULTS: The Tlr3 KO mice exhibited less ear swelling, less leukocyte infiltration into the skin, and lower serum total IgE levels than WT mice after hapten challenge. The Tlr3 KO mice also displayed lower expression levels of inflammatory cytokines (interleukin (IL)-33, IL-4, IL-10, and interferon-ɤ in their TNCB-treated ear skin than WT mice. CONCLUSION: These results showed that TLR3 deficiency suppressed the development of chronic contact hypersensitivity reactions, suggesting that TLR3 signaling may participate in the pathogenesis of AD.

Topical application of Dermatophagoides farinae or oxazolone induces symptoms of atopic dermatitis in the rabbit ear.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of the epidermis, and dermal infiltration of inflammatory cells. Appropriate animal models that recapitulate human AD and allow the analysis of disease processes in a reliable manner are essential to the study of AD. In this study, we established two AD models in rabbits by applying an allergen, Dermatophagoides farinae (Der f), or a hapten, oxazolone (OXZ). Application of the allergen or hapten induced a rapid onset and a chronically sustained AD-like skin lesion. The clinical symptoms, which include skin erythema, scaling, papula and edema, of AD-like rabbit skin were similar to those in human AD. Histological analysis showed that allergen- or hapten-treated rabbit skin showed increased epidermal thickening and inflammatory cell infiltration. Furthermore, PCNA and keratin 10 (K10) staining revealed excessive proliferation and insufficient differentiation of the epidermis in the rabbit AD-like skin. Western blot analysis showed decreased expression of thymic stromal lymphopoietin (TSLP), an AD cytokine, in the rabbit AD-like skin. Our results suggest that the allergen- or hapten-induced rabbit AD models have pathological features of human AD-like symptoms and will be useful for evaluating both pathogenic mechanisms and potential therapeutic agents for human AD.

Effect of simultaneous exposure to mixture of two skin sensitizers on skin sensitization response in guinea pigs and mice.

Skin sensitization resulting in allergic contact dermatitis is a common occupational health issue. In this study, the effect of mixing two skin sensitizers on the skin sensitization response was investigated. Skin sensitizers are generally classified into T helper type 1 (Th1) or T helper type 2 (Th2), depending on the induced cytokine profile. Dinitrochlorobenzene (DNCB) and oxazolone (Oxa) are Th1 skin sensitizers and phthalic anhydride (PA) and toluene diisocyanate (TDI) are Th2 skin sensitizers. We investigated the effect on skin sensitization response to mixtures of three pairs of these sensitizers: DNCB and Oxa, DNCB and PA, and PA and TDI, using guinea pig maximization test and mouse ear swelling test. In guinea pigs sensitized with the mixture of DNCB and Oxa or PA and TDI, there were changes of skin sensitization response to DNCB and Oxa, and that to PA. On the other hand, there was no mixture effect in guinea pigs sensitized with the mixture of DNCB and PA. The skin sensitization responses were decreased in mice sensitized with the mixtures of DNCB and Oxa or PA and TDI, whereas the mixture effect was not observed in mice sensitized with the mixture of DNCB and PA. The present findings revealed that mixture effect on the skin sensitization response was observed after simultaneous exposure to two skin sensitizers, and the effect was determined by combinations of mixed skin sensitizers.

Continuous high-dose antigen exposure preferentially induces IL-10, but intermittent antigen exposure induces IL-4.

IL-10 plays a critical role in the induction of specific T-cell tolerance. To date, whether IL-10 induction by antigen application is dose- or time-dependent remains unclear. In this study, IL-10 induction by allergen exposure was investigated in the several schedules. Oxazolone was repeatedly applied to mouse ear, and mRNA of inflammatory cytokines in lesional skins was measured. The results indicated that continuous high-dose antigen exposure induces IL-4 as well as abundant IL-10 production. Monocytes/dendritic cells and T cells are major source of IL-10. Allergen-specific immunotherapy is resumed before antigen scattering: preseason. We evaluated safe-loading dose of allergens in preseasonal therapy focusing Tr1 induction. Restarting immunotherapy with high dose effectively augmented IL-10 expression accompanied with further induction of IL-4 and inflammatory cytokines. Therefore, the protocol restarting with low-dose antigen is preferential to obviate the risk of exacerbation or anaphylaxis.

Topical cholesterol treatment ameliorates hapten-evoked cutaneous hypersensitivity by sustaining expression of 11ß-HSD1 in epidermis.

Changes in the stratum corneum extracellular matrix impair epidermal barrier function and may cause dermatoses. The aim of this study was to examine the effect of exogenous cholesterol application on skin barrier function and cutaneous inflammation. Skin barrier-disrupted or hapten-stimulated mice were treated with topical cholesterol. The effect of topical cholesterol application on an oxazolone (OXA)-induced hypersensitivity reaction was evaluated. Topical application of cholesterol efficiently decreased transepidermal water loss in areas of barrier-disrupted skin and ameliorated OXA-induced cutaneous hypersensitivity. These favourable effects may have resulted from sustained expression of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in the cholesterol-treated skin. As 11ß-HSD1 is known to produce active cortisol, topical cholesterol may attenuate contact hypersensitivity by normalizing secretion of hormonally active cortisol from the skin.

Contact hypersensitivity to oxazolone provokes vulvar mechanical hyperalgesia in mice.

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition.

Anti-inflammatory effect of a retrovirus-derived immunosuppressive peptide in mouse models.

BACKGROUND: Short dimeric or mulitmeric peptides derived from a highly conserved stretch of amino acids from gammaretroviral envelope proteins has been found to have immunosuppressive properties in vitro. Here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders. RESULTS: The anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of 17 amino acids was tested in two murine skin inflammation models, a TPA-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. Overall, mice (n = 24) treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of 28.8% in ear thickness (range 20.1-42.5), whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect (p = 0.028). Furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mRNA of the pro-inflammatory cytokines TNF-α, IL-17C, and IL-6 as well as the chemokine CXCL2 compared to mice treated with control peptides. CONCLUSION: Using two murine skin inflammation models, we show that an immunosuppressive retroviral peptide is capable of reducing inflammatory disorders. The results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation.

Lymphatic vessel function in head and neck inflammation.

BACKGROUND: Serious infections of the head and neck cause lymphedema that can lead to airway compromise and oropharyngeal obstruction. Lymphangiogenesis occurs in the head and neck during infection and after immunization. The goal of this project was to develop tools to image lymphatic vessels in living animals and to be able to isolate individual lymphatic endothelial cells in order to quantify changes in single cells caused by inflammation. METHODS: The ProxTom transgenic red-fluorescent reporter mouse was developed specifically for the purpose of imaging lymphatic vessels in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in development and for the maintenance of lymphatics in adulthood. Mice were immunized and their lymphatic vessels in lymph nodes were imaged in vivo. Individual lymphatic endothelial cells were isolated by means of their fluorescence. RESULTS: The ProxTom transgene has the red-fluorescent reporter td-Tomato under the control of Prox1 regulatory elements. tdTomato was faithfully expressed in lymphatic vessels coincident with endogenous Prox1 expression. We show lymphangiogenesis in vivo after immunization and demonstrate a method for the isolation of lymphatic endothelial cells by their tdTomato red-fluorescence. CONCLUSIONS: The faithful expression of the red-fluorescent reporter in the lymphatic vessels of ProxTom means that these mice have proven utility for in vivo study of lymphatic vessels in the immune response. ProxTom has been made available for distribution from the Jackson Laboratory: http://jaxmice.jax.org/strain/018128.html .

Intranasally administered antigen 85B gene vaccine in non-replicating human Parainfluenza type 2 virus vector ameliorates mouse atopic dermatitis.

Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.