Seiji Kamijo

IL-33–Mediated Innate Response and Adaptive Immune Cells Contribute to Maximum Responses of Protease Allergen–Induced Allergic Airway Inflammation

How the innate and adaptive immune systems cooperate in the natural history of allergic diseases has been largely unknown. Plant-derived allergen, papain, and mite allergens, Der f 1 and Der p 1, belong to the same family of cysteine proteases. We examined the role of protease allergens in the induction of Ab production and airway inflammation after repeated intranasal administration without adjuvants and that in basophil/mast cell stimulation in vitro. Papain induced papain-specific IgE/IgG1 and lung eosinophilia. Der f 1 induced Der f 1–specific IgG1 and eosinophilia. Although papain-, Der f 1–, and Der p 1–stimulated basophils expressed allergy-inducing cytokines, including IL-4 in vitro, basophil-depleting Ab and mast cell deficiency did not suppress the papain-induced in vivo responses. Protease inhibitor–treated allergens and a catalytic site mutant did not induce the responses. These results indicate that protease activity is essential to Ab production and eosinophilia in vivo and basophil activation in vitro. IL-33–deficient mice lacked eosinophilia and had reduced papain-specific IgE/IgG1. Coadministration of OVA with papain induced OVA-specific IgE/IgG1, which was reduced in IL-33–deficient mice. We demonstrated IL-33 release, subsequent IL-33–dependent IL-5/IL-13 release, and activation of T1/ST2-expressing lineage−CD25+CD44+ innate lymphoid cells in the lung after papain inhalation, suggesting the contribution of the IL-33–type 2 innate lymphoid cell–IL-5/IL-13 axis to the papain-induced airway eosinophilia. Rag2-deficient mice, which lack adaptive immune cells, showed significant, but less severe, eosinophilia. Collectively, these results suggest cooperation of adaptive immune cells and IL-33–responsive innate cells in protease-dependent allergic airway inflammation.

Cytokine milieu modulates release of thymic stromal lymphopoietin from human keratinocytes stimulated with double-stranded RNA

BACKGROUND Thymic stromal lymphopoietin (TSLP) plays a key role in allergic diseases, such as atopic dermatitis (AD) and asthma. TSLP is highly expressed by keratinocytes in skin lesions of patients with AD, but environmental triggers for its release from keratinocytes with endogenous factors are not well understood. Patients with AD, in whom allergic sensitization is already established, are susceptible to viral dissemination. OBJECTIVES We investigated TSLPs release from primary human keratinocytes stimulated with a Toll-like receptor (TLR) 3 ligand, polyinosinic-polycytidylic acid, which mimics viral double-stranded RNA (dsRNA), and its modulation by cytokines. METHODS Primary human keratinocytes were stimulated with TLR ligands, cytokines, or both. TSLP released into culture supernatants was measured by means of ELISA. RESULTS Stimulation of keratinocytes with dsRNA induced release of TSLP and upregulated gene expression of TSLP and other cytokines and chemokines. The release of TSLP was enhanced by the addition of IL-4, IL-13, and/or TNF-alpha. With or without the T(H)2/TNF cytokines, the dsRNA-induced release of TSLP was upregulated by IFN-alpha and IFN-beta and suppressed by IFN-gamma, TGF-beta, or IL-17. CONCLUSIONS The effect of the TLR3 ligand on keratinocytes suggests contribution of viral dsRNA to skin inflammations under the influence of a cytokine milieu. The results imply that viral dsRNA and a T(H)2 cytokine milieu might promote T(H)2-type inflammation through an induction of TSLP expression, suggesting that a vicious cycle exists between AD with T(H)2-type inflammation and viral infections and a possible blockade of this cycle by other cytokine milieus provided by cells, such as T(H)1, regulatory T, and T(H)17 cells.

Staphylococcus aureus membrane and diacylated lipopeptide induce thymic stromal lymphopoietin in keratinocytes through the Toll-like receptor 2–Toll-like receptor 6 pathway

BACKGROUND Staphylococcus aureus heavily colonizes the lesions of patients with atopic dermatitis (AD) and is known to trigger a worsening of AD. However, the exact mechanism by which S. aureus promotes AD is unknown. Thymic stromal lymphopoietin (TSLP), which is highly expressed by keratinocytes in skin lesions of patients with AD and bronchial epithelial cells in asthmatic patients, represents a critical factor linking responses at interfaces between the body and the environment to allergic type 2 immune responses. OBJECTIVES We sought to examine the ability of synthetic lipopeptides and S. aureus to induce TSLP expression in human keratinocytes and identify the pathway of induction. METHODS We stimulated primary human keratinocytes with lipopeptides and S. aureus-derived materials. The release and gene expression of TSLP were measured by means of ELISA and quantitative PCR, respectively. RESULTS Diacylated lipopeptide upregulated the expression of TSLP and other proinflammatory molecules. Heat-killed S. aureus and the subcellular fractions of S. aureus induced TSLPs release, with the membranous fraction having the greatest activity. Small interfering RNA-mediated knockdown of either Toll-like receptor (TLR) 2 or TLR6 inhibited the diacylated lipopeptide- and S. aureus membrane-induced TSLP gene expression. S. aureus membrane- and diacylated lipopeptide-induced release of TSLP was enhanced by T(H)2/TNF-α cytokines and partially suppressed by IFN-γ and TGF-β. CONCLUSIONS The results suggest that ligands for the TLR2-TLR6 heterodimer in S. aureus membranes, including diacylated lipoproteins, could promote T(H)2-type inflammation through TSLP production in keratinocytes, providing an overall picture of the vicious cycles between colonization by S. aureus and AD in the T(H)2-skewed sensitization process, exacerbation of the disease, or both.

Involvement of TNF-Like Weak Inducer of Apoptosis in the Pathogenesis of Collagen-Induced Arthritis

TNF-like weak inducer of apoptosis (TWEAK) is a type II membrane protein belonging to the TNF family that regulates apoptotic cell death, cellular proliferation, angiogenesis, and inflammation. However, the role of TWEAK in the pathogenesis of rheumatoid arthritis (RA) remains unclear. In this study, we have investigated the effect of neutralizing anti-TWEAK mAb on the development of collagen-induced arthritis (CIA), a well-established murine model of RA. Administration of anti-TWEAK mAb significantly ameliorated paw swelling, synovial hyperplasia, and infiltration of inflammatory cells. The levels of proinflammatory chemokines such as MCP-1 and MIP-2 in serum and knee joints were reduced by this treatment. Consistently, recombinant TWEAK enhanced the proliferation of MCP-1 and MIP-2 production by synovial cells from CIA mice in vitro. Histological examination also revealed that the treatment with anti-TWEAK mAb suppressed the development of small vessels in synovial tissues. These results indicated anti-inflammatory and antiangiogenic effects of the TWEAK blockade in CIA, which may be also beneficial for the treatment of RA.

Characterization of Proteases, Proteins, and Eicosanoid-Like Substances in Soluble Extracts from Allergenic Pollen Grains

Background: Pollen is an important trigger of seasonal rhinitis, conjunctivitis, and/or allergic asthma, and an exacerbating factor in atopic dermatitis. Pollen grains contain allergen proteins, enzymes, and bioactive lipid mediators, the latter two possibly involved in the pathogenesis of allergic diseases through IgE-independent mechanisms. Methods: We analyzed the patterns of release of endopeptidases from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and birch, ragweed, and two grasses, Kentucky blue and cultivated rye, using synthetic substrates, class-specific inhibitors, and zymography. The proteins released were analyzed by gel electrophoresis. Eicosanoid-like substances were measured by enzyme-linked immunosorbent assays for prostaglandin E2 and leukotriene B4. Results: Major fractions of proteins, eicosanoid-like substances, and at least one molecular species of serine endopeptidase were released into phosphate-buffered saline from the pollen grains at 37°C within 25 min or 60 min without sonication. In the Cupressaceae/Taxodiaceae family, sonication was necessary for the release of other proteins and another serine endopeptidase. In birch, ragweed, and the grasses, most of the serine and cysteine endopeptidases were released without sonication. Proteases released within 25 min digested gelatin and/or casein differently among plant species. Conclusions: Grains of allergenic pollen release proteases, which can digest not only short synthetic substrates but also protein substrates, along with eicosanoid-like substances and proteins. The release of these components could contribute to the formation of a microenvironment optimum for initiation of the sensitization or the exacerbation of pollen allergy in tissues exposed to pollen grains.

Extracellular Double-Stranded RNA Induces TSLP via an Endosomal Acidification- and NF-κB-Dependent Pathway in Human Keratinocytes

Double-stranded RNA (dsRNA) causes keratinocytes to release thymic stromal lymphopoietin (TSLP), which plays a key role in allergic diseases. Endosomal Toll-like receptor 3 (TLR3) and cytosolic RIG-like receptors (RLRs) and PKR have been reported to recognize dsRNA. Here, we demonstrate that dsRNA induces TSLP in keratinocytes via an endosomal acidification-dependent and NF-κB-mediated pathway. After treatment with pharmacologic inhibitors or transfection with small interfering RNAs (siRNAs), primary human keratinocytes were stimulated. Bafilomycin A1, which inhibits endosomal acidification to block the TLR3 pathway, blocked the dsRNA-induced expression of TSLP, IL-8, IFN-β, and other molecules including the dsRNA sensors, whereas it did not inhibit diacyllipopeptide-induced expression of TSLP and IL-8. The dsRNA-induced gene expression of TSLP depended on RelA, a component of NF-κB, but not IRF3, similar to IL-8 but different from IFN-β, which depended on both IRF3 and RelA. The results indicate that endosomal acidification and the subsequent activation of NF-κB are necessary to sense extracellular dsRNA, suggesting the importance of the TLR3-NF-κB axis to trigger production of TSLP against the self dsRNA released from damaged cells or viral dsRNA, in the epidermis, relating to skin inflammation including atopic dermatitis (AD).

Cupressaceae Pollen Grains Modulate Dendritic Cell Response and Exhibit IgE-Inducing Adjuvant Activity In Vivo

Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.

TSLP expression induced via Toll-like receptor pathways in human keratinocytes.

The skin epidermis and mucosal epithelia (airway, ocular tissues, gut, and so on) are located at the interface between the body and environment and have critical roles in the response to various stimuli. Thymic stromal lymphopoietin (TSLP), a cytokine expressed mainly by epidermal keratinocytes (KCs) and mucosal epithelial cells, is a critical factor linking the innate response at barrier surfaces to Th2-skewed acquired immune response. TSLP is highly expressed in skin lesions of atopic dermatitis patients. Here, we describe on Toll-like receptor (TLR)-mediated induction of TSLP expression in primary cultured human KCs, placing emphasis on experimental methods used in our studies. Double-stranded RNA (TLR3 ligand), flagellin (TLR5 ligand), and diacylated lipopeptide (TLR2-TLR6 ligand) stimulated human KCs to express TSLP and Staphylococcus aureus membranes did so via the TLR2-TLR6 pathway. Atopic cytokine milieu upregulated the TLR-mediated induction of TSLP. Culturing in the absence of glucocorticoid before stimulation enhanced the TSLP expression. Extracellular double-stranded RNA induced TSLP via endosomal acidification- and NF-κB-dependent pathway. Specific measurement of the long TSLP transcript, which contributes to the production of the TSLP protein, rather than total or the short transcript is useful for accurate detection of functional human TSLP gene expression. The results suggest that environment-, infection-, and/or self-derived TLR ligands contribute to the initiation and/or amplification of Th2-type skin inflammation including atopic dermatitis and atopic march through the induction of TSLP expression in KCs and include information helpful for understanding the role of the gene-environment interaction relevant in allergic diseases.

NADPH oxidase activity in allergenic pollen grains of different plant species

Pollen is an important trigger of allergic diseases. Recent studies have shown that ragweed pollen NAD(P)H oxidase generates reactive oxygen species (ROS) and plays a prominent role in the pathogenesis of allergies in mouse models. Here, we demonstrated that allergenic pollen grains showed NAD(P)H oxidase activity that differed in intensity and localization according to the plant families. The activity occurred at the surface or in the cytoplasm in pollen of grasses, birch, and ragweed; in subpollen particles released from ragweed pollen; and at the inner surface or in the cytoplasm but not on the outer wall, which was sloughed off after the rupture, of pollen of Japanese cedar and Japanese cypress. The activity was mostly concentrated within insoluble fractions, suggesting that it facilitates the exposure of tissues to ROS generated by this enzyme. The extent of exposure to pollen-generated ROS could differ among the plant families.

Epicutaneous Allergic Sensitization by Cooperation between Allergen Protease Activity and Mechanical Skin Barrier Damage in Mice

Allergen sources such as mites, insects, fungi, and pollen contain proteases. Airway exposure to proteases induces allergic airway inflammation and IgE/IgG1 responses via IL-33-dependent mechanisms in mice. We examined the epicutaneous sensitization of mice to a model protease allergen, papain; the effects of tape stripping, which induces epidermal barrier dysfunction; and the atopic march upon a subsequent airway challenge. Papain painting on ear skin and tape stripping cooperatively promoted dermatitis, the skin gene expression of proinflammatory cytokines and growth factors, up-regulation of serum total IgE, and papain-specific IgE/IgG1 induction. Epicutaneous sensitization induced T helper (Th) 2 cells and Th17 differentiation in draining lymph nodes. Ovalbumin and protease inhibitor-treated papain induced no or weak responses, whereas the co-administration of ovalbumin and papain promoted ovalbumin-specific IgE/IgG1 induction. Wild-type and IL-33-deficient mice showed similar responses in the epicutaneous sensitization phase. The subsequent airway papain challenge induced airway eosinophilia and maintained high papain-specific IgE levels in an IL-33-dependent manner. These results suggest that allergen source-derived protease activity and mechanical barrier damage such as that caused by scratching cooperatively promote epicutaneous sensitization and skin inflammation and that IL-33 is dispensable for epicutaneous sensitization but is crucial in the atopic march upon a subsequent airway low-dose encounter with protease allergens.