TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.

Allergic contact dermatitis is a common skin disease associated with inflammation and persistent pruritus. Transient receptor potential (TRP) ion channels in skin-innervating sensory neurons mediate acute inflammatory and pruritic responses following exogenous stimulation and may contribute to allergic responses. Genetic ablation or pharmacological inhibition of TRPA1, but not TRPV1, inhibited skin edema, keratinocyte hyperplasia, nerve growth, leukocyte infiltration, and antihistamine-resistant scratching behavior in mice exposed to the haptens, oxazolone and urushiol, the contact allergen of poison ivy. Hapten-challenged skin of TRPA1-deficient mice contained diminished levels of inflammatory cytokines, nerve growth factor, and endogenous pruritogens, such as substance P (SP) and serotonin. TRPA1-deficient sensory neurons were defective in SP signaling, and SP-induced scratching behavior was abolished in Trpa1(-/-) mice. SP receptor antagonists, such as aprepitant inhibited both hapten-induced cutaneous inflammation and scratching behavior. These findings support a central role for TRPA1 and SP in the integration of immune and neuronal mechanisms leading to chronic inflammatory responses and pruritus associated with contact dermatitis.

Role of macrophage migration inhibitory factor in the Th2 immune response to epicutaneous sensitization.

We examined the role of macrophage migration inhibitory factor (MIF) in the generation of the Th2 response using MIF-deficient mice in a model of epicutaneous sensitization to ovalbumin. Lymph node cells from sensitized MIF-deficient mice produce lower levels of Th2 cytokines after antigen challenge when compared to their wild-type counterparts. Sensitized mice lacking MIF show less pulmonary inflammation after intranasal antigen exposure. Mice deficient in CD74, the MIF receptor, also are unable to generate an inflammatory response to epicutaneous sensitization. Examination of the elicitation phase of the atopic response using DO11.10 OVA TCR transgenic animals shows that T cell proliferation and IL-2 production are strongly impaired in MIF-deficient T cells. This defect is most profound when both T cells and antigen-presenting cells are lacking MIF. These data suggest that MIF is crucial both for the sensitization and the elicitation phases of a Th2-type immune response in allergic disease.

Immune sensitization to methylene diphenyl diisocyanate (MDI) resulting from skin exposure: albumin as a carrier protein connecting skin exposure to subsequent respiratory responses.

BACKGROUND: Methylene diphenyl diisocyanate (MDI), a reactive chemical used for commercial polyurethane production, is a well-recognized cause of occupational asthma. The major focus of disease prevention efforts to date has been respiratory tract exposure; however, skin exposure may also be an important route for inducing immune sensitization, which may promote subsequent airway inflammatory responses. We developed a murine model to investigate pathogenic mechanisms by which MDI skin exposure might promote subsequent immune responses, including respiratory tract inflammation. METHODS: Mice exposed via the skin to varying doses (0.1-10% w/v) of MDI diluted in acetone/olive oil were subsequently evaluated for MDI immune sensitization. Serum levels of MDI-specific IgG and IgE were measured by enzyme-linked immunosorbant assay (ELISA), while respiratory tract inflammation, induced by intranasal delivery of MDI-mouse albumin conjugates, was evaluated based on bronchoalveolar lavage (BAL). Autologous serum IgG from "skin only" exposed mice was used to detect and guide the purification/identification of skin proteins antigenically modified by MDI exposure in vivo. RESULTS: Skin exposure to MDI resulted in specific antibody production and promoted subsequent respiratory tract inflammation in animals challenged intranasally with MDI-mouse albumin conjugates. The degree of (secondary) respiratory tract inflammation and eosinophilia depended upon the (primary) skin exposure dose, and was maximal in mice exposed to 1% MDI, but paradoxically limited in mice receiving 10-fold higher doses (e.g. 10% MDI). The major antigenically-modified protein at the local MDI skin exposure site was identified as albumin, and demonstrated biophysical changes consistent with MDI conjugation. CONCLUSIONS: MDI skin exposure can induce MDI-specific immune sensitivity and promote subsequent respiratory tract inflammatory responses and thus, may play an important role in MDI asthma pathogenesis. MDI conjugation and antigenic modification of albumin at local (skin/respiratory tract) exposure sites may represent the common antigenic link connecting skin exposure to subsequent respiratory tract inflammation.

EBI3 deficiency leads to diminished T helper type 1 and increased T helper type 2 mediated airway inflammation.

Despite extensive investigation of the signals required for development of T helper type 1 (Th1) and type 2 (Th2) immune responses, the mechanisms involved are still not well-defined. A critical role for Epstein-Barr virus-induced gene 3 (EBI3) in these responses has been proposed. EBI3, initially discovered as a transcriptionally activated gene in Epstein-Barr virus-infected B lymphocytes, codes for a subunit of the cytokine interleukin-27 (IL-27). While initial studies suggested that it had an important role in promoting Th1 responses, subsequent studies have revealed that EBI3 receptor signalling influences a variety of immune cell types and can inhibit both Th1 and Th2 responses. In the present study, we evaluated EBI3(-/-) mice for their ability to mount both Th1-mediated and Th2-mediated airway inflammatory responses. The EBI3(-/-) mice sensitized by exposure to inhaled ovalbumin plus a high dose of lipopolysaccharide, which normally results in Th1 responses in wild-type (WT) mice, instead developed Th2 type airway inflammation, with increased numbers of eosinophils. The EBI3(-/-) mice that were exposed to inhaled ovalbumin with a low dose of lipopolysaccharide, which induces Th2 responses in WT mice, showed a marked enhancement of these responses, with increased airway eosinophils, increased serum IgE levels and increased levels of Th2 cytokines (IL-4, IL-5 and IL-13) in culture supernatants of mediastinal lymph node cells. Increased production of Th2 cytokines was also seen when naive CD4(+) T cells from EBI3(-/-) mice were stimulated in vitro compared with cells from WT mice. These results provide the first evidence that EBI3 may play an inhibitory role in allergic asthma development.

TLR4 signaling in stromal cells is critical for the initiation of allergic Th2 responses to inhaled antigen.

Allergic asthma is an inflammatory lung disease driven by Th2. We have shown that both Th1 and Th2 sensitization to inhaled OVA depend on the presence and concentration of LPS, where high concentrations (LPS(hi)) induce Th1 and low concentrations (LPS(lo)), Th2. Stromal cells (SCs), such as airway SCs, exacerbate established airway disease; however, little is known about their role early during sensitization. In this study, using bone marrow chimeric mice to restrict TLR4 signaling to either the SC compartment (SC(+)HPC(-)) or the hematopoietic cell (HPC) compartment (SC(-)HPC(+)), we report that HPC TLR4 is necessary and sufficient for Th1 sensitization to OVA-LPS(hi), whereas TLR4 in both compartments is required for Th2 sensitization to OVA-LPS(lo). Surprisingly, although SC(+)HPC(-) mice were unable to generate a Th1 response to OVA-LPS(hi), they instead mounted a robust Th2 response, indicating that in the presence of higher concentrations of LPS, SC TLR4 is sufficient for Th2 sensitization. We show that the SC TLR4 response to LPS leads to induction of Th2-inducing dendritic cells that upregulate Notch ligand Jagged-1 but not Delta-4. Furthermore, airway SCs upregulate thymic stromal lymphopoietin in response to exposure to both OVA-LPS(lo) and OVA-LPS(hi). These studies demonstrate that SC TLR4 signaling is critically involved in Th2 but not Th1 sensitization to inhaled Ag.

The immune system and atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a complex pathogenesis. It is clinically well-defined and represents one manifestation of the atopic state, along with asthma, food allergy and/or allergic rhinitis. Within the last several decades, there has been much evidence to support the contribution of immune mechanisms in the pathogenesis of AD. It has also been documented that the prevalence of all atopic disease, including AD, has been increasing, although the environmental factors that may be contributing to this increase are not clearly defined. A better understanding of the underlying immunopathogenesis of AD should aid in better clinical management and development of new treatment options.

Lung/skin connections in occupational lung disease.

PURPOSE OF REVIEW: Exposure to occupational and environmental agents can cause a spectrum of lung diseases that are predominantly immune-mediated. Research and prevention have focused primarily on the respiratory tract. Recent studies, however, suggest that the skin may also be an important route of exposure and site of sensitization. This article highlights key findings, focusing on isocyanate asthma and chronic beryllium disease. RECENT FINDINGS: Occupational lung diseases such as isocyanate asthma and chronic beryllium disease continue to occur despite reduced airborne exposures. Although challenging to quantify, recent studies have documented isocyanate and beryllium skin exposure, even with the use of personal protective clothing. Factors that impair skin barrier function, such as trauma, may promote sensitization to such agents. Animal studies demonstrate that skin exposure to isocyanates and protein allergens is highly effective at inducing sensitization, with subsequent inhalation challenge eliciting asthmatic responses. Limited clinical studies suggest a similar role for human skin exposure to certain sensitizing agents. SUMMARY: Recent findings support a greater focus on the role of skin exposure in the development of certain occupational and environmental lung diseases. Although further research is needed, it is prudent to reduce both skin and inhalation exposures.

Skin exposure to isocyanates: reasons for concern.

OBJECTIVE: Isocyanates (di- and poly-), important chemicals used worldwide to produce polyurethane products, are a leading cause of occupational asthma. Respiratory exposures have been reduced through improved hygiene controls and the use of less-volatile isocyanates. Yet isocyanate asthma continues to occur, not uncommonly in settings with minimal inhalation exposure but opportunity for skin exposure. In this review we evaluate the potential role of skin exposure in the development of isocyanate asthma. DATA SOURCES: We reviewed the published animal and human literature on isocyanate skin-exposure methods, workplace skin exposure, skin absorption, and the role of skin exposure in isocyanate sensitization and asthma. DATA EXTRACTION: We selected relevant articles from computerized searches on Medline, U.S. Environmental Protection Agency, Occupational Safety and Health Administration, National Institute for Occupational Safety and Health, and Google databases using the keywords "isocyanate," "asthma," "skin," "sensitization," and other synonymous terms, and our own extensive collection of isocyanate publications. DATA SYNTHESIS: Isocyanate production and use continues to increase as the polyurethane industry expands. There is substantial opportunity for isocyanate skin exposure in many work settings, but such exposure is challenging to quantify and continues to be underappreciated. Isocyanate skin exposure can occur at work, even with the use of personal protective equipment, and may also occur with consumer use of certain isocyanate products. In animals, isocyanate skin exposure is an efficient route to induce sensitization, with subsequent inhalation challenge resulting in asthma-like responses. Several lines of evidence support a similar role for human isocyanate skin exposure, namely, that such exposure occurs and can contribute to the development of isocyanate asthma in certain settings, presumably by inducing systemic sensitization. CONCLUSIONS: Integrated animal and human research is needed to better understand the role of skin exposure in human isocyanate asthma and to improve diagnosis and prevention. In spite of substantial research needs, sufficient evidence already exists to justify greater emphasis on the potential risks of isocyanate skin exposure and the importance of preventing such exposures at work and during consumer use of certain isocyanate products.

MyD88-dependent induction of allergic Th2 responses to intranasal antigen.

MyD88 is a common Toll-like receptor (TLR) adaptor molecule found to be essential for induction of adaptive Th1 immunity. Conversely, innate control of adaptive Th2 immunity has been shown to occur in a MyD88-independent manner. In this study, we show that MyD88 is an essential innate component in the induction of TLR4-dependent Th2 responses to intranasal antigen; thus we demonstrate what we believe to be a novel role for MyD88 in pulmonary Th2 immunity. Induction of the MyD88-independent type I IFN response to LPS is defective in the pulmonary environment. Moreover, in the absence of MyD88, LPS-induced upregulation of costimulatory molecule expression on pulmonary DCs is defective, in contrast to what has been observed with bone marrow-derived DCs (BMDCs). Reconstitution of Th2 responses occurs upon adoptive pulmonary transfer of activated BMDCs to MyD88-deficient recipients. Furthermore, the dependence of Th2 responses on MyD88 is governed by the initial route of antigen exposure; this demonstrates what we believe are novel site-specific innate mechanisms for control of adaptive Th2 immunity.

IL-4-dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88.

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed "collateral priming," in which naive CD4(+) T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.

Human gamma/delta T-cell proliferation and IFN-gamma production induced by hexamethylene diisocyanate.

BACKGROUND: The human immune response to isocyanate, a leading cause of occupational asthma, remains incompletely characterized, including the cell types involved and the form of the chemical that acts as an antigen. OBJECTIVE: The purpose of this investigation was to characterize human T cells that respond to hexamethylene diisocyanate (HDI), an aliphatic isocyanate routinely used in the automobile body industry. METHODS: Human T-cell lines were generated and characterized from peripheral blood of HDI-exposed and HDI-unexposed subjects, using two different HDI antigens, HDI-conjugated albumin and HDI-exposed human airway epithelial cells (NCI-H292). Flow cytometry was used to characterize the phenotype of HDI-responsive T cells. ELISA and intracellular staining techniques were used to evaluate HDI-induced cytokine production. DNA sequence analysis of T-cell receptors was used to further define clonal populations of HDI-responsive T cells. RESULTS: HDI antigen preparations but not "mock exposed" control antigens lead to increased proliferation of specific cell types, CD3+CD4-CD8(dim) and/or CD3+CD4-CD8- cells, from HDI-exposed but not from HDI-unexposed subjects. These HDI-responsive T cells expressed unique oligoclonal gamma/delta rather than alpha/beta T-cell receptors, with characteristics suggestive of antigen-mediated selection and specificity. The HDI-stimulated gamma/delta T cells were associated with T(H)1-like cytokines and produce IFN-gamma but not IL-5 or IL-13. CONCLUSIONS: These data are the first to demonstrate that HDI can selectively stimulate gamma/delta T cells with the potential to modulate the human immune response to exposure.

Differential roles for CD4 and CD8 T cells after diisocyanate sensitization: genetic control of TH2-induced lung inflammation.

BACKGROUND: Exposure to diisocyanates is a major cause of occupational asthma. We previously developed a novel mouse model of diisocyanate-induced asthma involving epicutaneous sensitization to hexamethylene diisocyanate (HDI) that demonstrates many features of the human disease, including airway eosinophilia and mucus hypersecretion. OBJECTIVE: To determine what factors are critical for the development of HDI-induced airway inflammation, we investigated the strain distribution of this response and the roles of CD4(+) and CD8(+) T cells. METHODS: Mice were epicutaneously exposed to HDI and then challenged with HDI, either by means of inhalation to induce airway inflammation or on the ear to induce contact hypersensitivity (CHS). Lymph node cytokine production and serum antibodies were also measured. RESULTS: Induction of airway eosinophilia was highly dependent on the mouse strain used, with C57BL/6, A/J, CBA, C3H, and C57BL/10 mice all having significantly fewer eosinophils than BALB/c mice. HDI-specific antibodies and lymph node IL-5 and IL-13 production were also diminished in non-BALB/c strains. In contrast, CHS to HDI developed in all strains tested. Studies in mice deficient in either CD4(+) or CD8(+) T cells revealed that CD4(+) T cells were critical for HDI-induced airway eosinophilia, whereas CD8(+) T cells were the major effector cells in CHS. CONCLUSION: The data suggest that, in contrast to CHS, induction of T(H)2 responses after epicutaneous exposure to diisocyanates is strongly genetically influenced. Furthermore, the lung inflammatory response to inhaled HDI appears to depend primarily on effective generation of these CD4(+) T(H)2 responses, as is the case in atopic asthma.

To respond or not to respond: T cells in allergic asthma.

The incidence of allergic asthma has almost doubled in the past two decades. Numerous epidemiological studies have linked the recent surge in atopic disease with decreased exposure to infections in early childhood as a result of a more westernized lifestyle. However, a clear mechanistic explanation for how this might occur is still lacking. An answer might lie in the presently unfolding story of various regulatory T-cell populations that can limit adaptive immune responses, including T helper 2 (T(H)2)-cell-mediated allergic airway disease.

IL-13 is necessary, not simply sufficient, for epicutaneously induced Th2 responses to soluble protein antigen.

Th2 responses are clearly involved in the pathogenesis of atopic disease. Thus, understanding the factors responsible for Th2 sensitization at sites of allergen exposure, such as airway and skin, is crucial for directing therapeutic or preventive strategies. Contrary to other models of Th2 sensitization to proteins, we have reported that Th2 responses induced by epicutaneous exposure to OVA are IL-4 independent. Combined deficiency of both IL-4 and IL-13 signaling did prevent Th2 generation, suggesting that IL-13 was mediating these IL-4-independent responses. It was not clear, however, whether IL-13 was simply replacing the need for IL-4 in genetically deficient mice or if IL-13 played a unique role. In the present study, we show that Th2 responses induced by epicutaneous OVA exposure (including lung inflammatory responses after inhaled Ag challenge, OVA-specific IgG1, and draining lymph node IL-5 production) are impaired in IL-13-deficient (IL-13(-/-)) mice compared with wild type. In contrast, i.p. sensitization of IL-13(-/-) mice resulted in responses equivalent to wild type. Generation of contact hypersensitivity to dinitrofluorobenzene, which involves Th1 and CD8(+) effector cells, was also intact in IL-13(-/-) mice. Taken together, the data indicate that IL-13 is the major inducer of Th2 generation in the cutaneous microenvironment, being required independently of IL-4. This fact, in combination with the known abundance of IL-13 in atopic dermatitis skin lesions, emphasizes the potentially important role of the skin as a site for Th2 sensitization to environmental allergens, particularly in atopic individuals.

Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen.

Allergic asthma is an inflammatory lung disease initiated and directed by T helper cells type 2 (Th2). The mechanism involved in generation of Th2 responses to inert inhaled antigens, however, is unknown. Epidemiological evidence suggests that exposure to lipopolysaccharide (LPS) or other microbial products can influence the development and severity of asthma. However, the mechanism by which LPS influences asthma pathogenesis remains undefined. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 (Th1) responses, it is unclear if TLRs are needed for Th2 priming. Here, we report that low level inhaled LPS signaling through TLR4 is necessary to induce Th2 responses to inhaled antigens in a mouse model of allergic sensitization. The mechanism by which LPS signaling results in Th2 sensitization involves the activation of antigen-containing dendritic cells. In contrast to low levels, inhalation of high levels of LPS with antigen results in Th1 responses. These studies suggest that the level of LPS exposure can determine the type of inflammatory response generated and provide a potential mechanistic explanation of epidemiological data on endotoxin exposure and asthma prevalence.

Resident lung antigen-presenting cells have the capacity to promote Th2 T cell differentiation in situ.

Antigen exposure via airway epithelia is often associated with a failure to prime or with the preferential priming of Th2 cells. We previously reported that the intranasal delivery of a Th1-inducing antigen promoted Th2-dominated responses, rather than the expected Th1 responses. Thus, we proposed that when pulmonary T cell priming is induced, the lung microenvironment might intrinsically favor the generation of Th2 types of responses. To establish a potential mechanism for such preferential priming, we examined the initial interactions between antigens and resident antigen-presenting cells (APCs) within the lung. We show that intranasally delivered antigens are preferentially taken up and can be presented to antigen-specific T cells by a resident population of CD11c(bright) APCs. Most of these antigen-loaded APCs remained within lung tissues, and migration into secondary lymphoid organs was not crucial for T cell priming to occur within the pulmonary tract. Furthermore, these pulmonary APCs demonstrated a marked expression of IL-6 and IL-10 within hours of antigen uptake, suggesting that resident tissue APCs have the capacity to promote Th2 T cell differentiation in situ.

A novel mouse model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge.

BACKGROUND: Exposure to diisocyanates, a group of highly reactive, low-molecular-weight compounds, is a major cause of occupational asthma. In contrast to mouse models of atopic asthma, previous mouse models of diisocyanate-induced asthma have failed to show lung inflammation with characteristics of human disease. OBJECTIVE: Our goal was to establish a novel mouse model of diisocyanate-induced asthma in which lung inflammation reminiscent of that seen in human asthma is generated after inhaled antigen challenge. METHODS: BALB/c mice were epicutaneously sensitized to hexamethylene diisocyanate (HDI) and then challenged with an HDI-protein conjugate administered by means of an intranasal droplet. RESULTS: HDI sensitization resulted in development of contact hypersensitivity and HDI-specific antibody production. Most importantly, however, vigorous inflammatory responses with characteristics of human asthma were generated in the lung after inhaled HDI challenge. Challenge of sensitized, but not unsensitized, mice resulted in airway eosinophilia, mucus hypersecretion, and production of T(H)1-type (IFN-gamma) and T(H)2-type (IL-4, IL-5, and IL-13) cytokines by lung inflammatory cells. Despite the mixed T(H)1/T(H)2 response induced by HDI sensitization, use of cytokine-deficient mice revealed that airway eosinophilia was mediated by T(H)2 cytokines and not by IFN-gamma. CONCLUSION: We report a novel mouse model of diisocyanate-induced asthma that, in contrast to previous models, demonstrates antigen-induced lung inflammation with characteristics of human disease. This model will allow investigation of the immunopathogenesis of diisocyanate-induced asthma and should provide insight into this common form of occupational disease.