Naoki Kajiwara

IL-33 is a crucial amplifier of innate rather than acquired immunity

IL-33, a member of the IL-1-related cytokines, is considered to be a proallergic cytokine that is especially involved in Th2-type immune responses. Moreover, like IL-1α, IL-33 has been suggested to act as an “alarmin” that amplifies immune responses during tissue injury. In contrast to IL-1, however, the precise roles of IL-33 in those settings are poorly understood. Using IL-1- and IL-33-deficient mice, we found that IL-1, but not IL-33, played a substantial role in induction of T cell-mediated type IV hypersensitivity such as contact and delayed-type hypersensitivity and autoimmune diseases such as experimental autoimmune encephalomyelitis. Most notably, however, IL-33 was important for innate-type mucosal immunity in the lungs and gut. That is, IL-33 was essential for manifestation of T cell-independent protease allergen-induced airway inflammation as well as OVA-induced allergic topical airway inflammation, without affecting acquisition of antigen-specific memory T cells. IL-33 was significantly involved in the development of dextran-induced colitis accompanied by T cell-independent epithelial cell damage, but not in streptozocin-induced diabetes or Con A-induced hepatitis characterized by T cell-mediated apoptotic tissue destruction. In addition, IL-33-deficient mice showed a substantially diminished LPS-induced systemic inflammatory response. These observations indicate that IL-33 is a crucial amplifier of mucosal and systemic innate, rather than acquired, immune responses.

IL-33 can promote survival, adhesion and cytokine production in human mast cells

IL-33 is a recently identified member of the IL-1 family of molecules, which also includes IL-1 and IL-18. IL-33 binds to the receptor, T1/ST2/IL-1R4, and can promote cytokine secretion by Th2 cells and NF-κB phosphorylation in mouse mast cells. However, the effects of these molecules, especially IL-33, in human mast cells are poorly understood. Expression of the receptors for IL-1 family molecules, specifically, IL-1R1, IL-18R and T1/ST2, was detectable intracellularly in human umbilical cord blood-derived mast cells (HUCBMCs) by flow cytometry, but was scarcely detectable on the cells’ surface. However, IL-1β, IL-18 or IL-33 induced phosphorylation of Erk, p38 and JNK in naïve HUCBMCs, and IL-33 or IL-1β, but not IL-18, enhanced the survival of naive HUCBMCs and promoted their adhesion to fibronectin. IL-33 or IL-1β also induced IL-8 and IL-13 production in naïve HUCBMCs, and enhanced production of these cytokines in IgE/anti-IgE-stimulated HUCBMCs, without enhancing secretion of either PGD2 or histamine. Moreover, IL-33-mediated IL-8 production by HUCBMCs was markedly reduced by the p38 MAPK inhibitor, SB203580. In contrast to findings with mouse mast cells, IL-18 neither induced nor enhanced secretion of the mediators PGD2 or histamine by HUCBMCs. Our findings identify previously unknown functions of IL-33 in human mast cells. One of these is that IL-33, like IL-1β, can induce cytokine production in human mast cells even in the absence of stimuli of FcɛRI aggregation. Our findings thus support the hypothesis that IL-33 may enhance mast cell function in allergic disorders and other settings, either in the presence or absence of co-stimulation of mast cells via IgE/antigen–FcɛRI signals.

IL-33 induces IL-13 production by mouse mast cells independently of IgE-FcεRI signals

The IL‐1‐related molecules, IL‐1 and IL‐18, can promote Th2 cytokine production by IgE/antigen‐FcεRI‐stimulated mouse mast cells. Another IL‐1‐related molecule, IL‐33, was identified recently as a ligand for T1/ST2. Although mouse mast cells constitutively express ST2, the effects of IL‐33 on mast cell function are poorly understood. We found that IL‐33, but not IL‐1β or IL‐18, induced IL‐13 and IL‐6 production by mouse bone marrow‐derived, cultured mast cells (BMCMCs) independently of IgE. In BMCMCs incubated with the potently cytokinergic SPE‐7 IgE without specific antigen, IL‐33, IL‐1β, and IL‐18 each promoted IL‐13 and IL‐6 production, but the effects of IL‐33 were more potent than those of IL‐1β or IL‐18. IL‐33 promoted cytokine production via a MyD88‐dependent but Toll/IL‐1R domain‐containing adaptor‐inducing IFN‐β‐independent pathway. By contrast, IL‐33 neither induced nor enhanced mast cell degranulation. At 200 ng/ml, IL‐33 prolonged mast cell survival in the absence of IgE and impaired survival in the presence of SPE‐7 IgE, whereas at 100 ng/ml, IL‐33 had no effect on mast cell survival in the absence of IgE and reduced mast cell survival in the presence of IgE. These observations suggest potential roles for IL‐33 in mast cell‐ and Th2 cytokine‐associated immune responses and disorders.

IL-33 and IL-33 Receptors in Host Defense and Diseases

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family, which includes IL-1 and IL-18. IL-33 is considered to be crucial for induction of Th2-type cytokine-associated immune responses such as host defense against nematodes and allergic diseases by inducing production of such Th2-type cytokines as IL-5 and IL-13 by Th2 cells, mast cells, basophils and eosinophils. In addition, IL-33 is involved in the induction of non-Th2-type acute and chronic inflammation as a proinflammatory cytokine, similar to IL-1 and IL-18. In this review, we summarize and discuss the current knowledge regarding the roles of IL-33 and IL-33 receptors in host defense and disease development.

Antimicrobial Peptides Human β-Defensins and Cathelicidin LL-37 Induce the Secretion of a Pruritogenic Cytokine IL-31 by Human Mast Cells

In addition to their microbiocidal properties, human β-defensins (hBDs) and cathelicidin LL-37 stimulate a number of mammalian cell activities, including migration, proliferation, and cytokine/chemokine production. Because hBDs and LL-37 cause mast cells to release pruritogens such as histamine and PGs, we hypothesized that these peptides would stimulate the secretion of a novel pruritogenic mediator IL-31, predominantly produced by T cells. hBDs and LL-37 enhanced IL-31 gene expression and IL-31 protein production and release in the human mast cell line LAD2, as well as in peripheral blood-derived cultured mast cells, suggesting that mast cells are another source of IL-31. Moreover, the expression of IL-31 was elevated in psoriatic skin mast cells, and hBD-2–4 and LL-37, but not hBD-1, enhanced its expression in vivo in rat skin mast cells. hBDs and LL-37 also induced the release of other pruritogenic mediators, including IL-2, IL-4, IL-6, GM-CSF, nerve growth factor, PGE2, and leukotriene C4, and increased mRNA expression of substance P. hBD– and LL-37–mediated IL-31 production/release was markedly reduced by pertussis toxin and wortmannin, inhibitors of G-protein and PI3K, respectively. As evidenced by the inhibitory effects of MAPK-specific inhibitors, hBD-2–4 and LL-37 activated the phosphorylation of MAPKs p38, ERK, and JNK that were required for IL-31 production and release. The ability of hBDs and LL-37 to stimulate the production and release of IL-31 by human mast cells provides a novel mechanism by which skin-derived antimicrobial peptides/proteins may contribute to inflammatory reactions and suggests a central role of these peptides in the pathogenesis of skin disorders.

Caspase-1, Caspase-8, and Calpain Are Dispensable for IL-33 Release by Macrophages

In addition to IL-1 and IL-18, IL-33 was recently identified as a member of the IL-1 cytokine family. rIL-33 can promote production of Th2-type cytokines by Th2 cells and mast cells in vitro. Administration of rIL-33 to mice results in increases in IgE secretion and eosinophilic inflammation. However, the precise immune cell source of IL-33 remains unclear. Moreover, although recombinant pro-IL-33 is cleaved by recombinant caspase-1 in vitro, as are pro-IL-1β and pro-IL-18, the involvement of caspase-1 in pro-IL-33 cleavage remains controversial. In this study, we show that mouse peritoneal macrophages, but not splenic dendritic cells, produced IL-33 upon stimulation with LPS. Likewise, mouse bone marrow cell-derived cultured mast cells also produced a small, but significant amount of IL-33 via FcεRI cross-linking, but not in response to stimulation with LPS. To our surprise, IL-33 release was found even in caspase-1-deficient, caspase-8 inhibitor-treated, and calpain inhibitor-treated macrophages. These observations suggest that caspase-1-, caspase-8-, and calpain-independent IL-33 production by macrophages and/or mast cells may contribute to the pathogenesis of Th2-type allergic inflammation.

Activation of human mast cells through the platelet-activating factor receptor

BACKGROUND In human subjects platelet-activating factor (PAF) concentrations are markedly increased in the plasma after anaphylactic reactions, and these correlate strongly with the severity of the response. The mechanism for the systemic spread of mast cell (MC) activation in anaphylaxis is often assumed to relate to the hematogenous spread of allergen, but this is implausible, and amplification mechanisms need to be considered. OBJECTIVE We have investigated the ability of PAF to induce human MC degranulation using skin, lung, and peripheral blood (PB)-derived cultured MCs and the signaling pathways activated in PB-derived MCs in response to PAF. METHODS The expression of PAF receptor was investigated by means of RT-PCR and Western blot analysis. Cell-signaling pathways in PB-derived MCs in response to PAF were investigated by analyzing the effect of various inhibitors and the silencing of phospholipase C (PLC) mRNA on PAF-mediated histamine release. RESULTS We show for the first time that PAF induces histamine release from human lung MCs and PB-derived MCs but not skin MCs. Activation of PAF receptor-coupled G(alphai) leads to degranulation through PLCgamma1 and PLCbeta2 activation in human MCs. PAF-induced degranulation was rapid, being maximal at 5 seconds, and was partially dependent on extracellular Ca(2+). CONCLUSION Our findings provide a mechanism whereby PAF mediates an amplification loop for MC activation in the generation of anaphylaxis.

Epithelial cell-derived IL-25, but not Th17 cell-derived IL-17 or IL-17F, is crucial for murine asthma.

IL-17A, IL-17F, and IL-25 are ligands for IL-17RA. In the current study, we demonstrated that IL-25–deficient mice—but not IL-17A–, IL-17F–, IL-17A/F–, IL-23p19–, or retinoic acid-related orphan receptor (ROR)-γt–deficient mice—showed significant suppression of 1) the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, 2) airway hyperresponsiveness to methacholine, and 3) OVA-specific IgG1 and IgE levels in the serum during OVA-induced Th2-type/eosinophilic airway inflammation. The IL-25 deficiency did not affect lung dendritic cell migration or Ag-specific memory–Th2 cell expansion during Ag sensitization. Adoptive transfer of T cells, mast cells, or bone marrow cells from IL-25–deficient mice revealed that induction of Th2-type/eosinophilic airway inflammation was dependent on activation of lung epithelial cells and eosinophils by IL-25 produced by airway structural cells such as epithelial cells but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells. Therefore, airway structural cell-derived IL-25—rather than Th17 cell-derived IL-17A and IL-17F—is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase of Th2-type/eosinophilic airway inflammation. It is not required for Ag-specific Th2 cell differentiation in the sensitization phase.

Interleukin-17 Accelerates Allograft Rejection by Suppressing Regulatory T Cell Expansion

Background— Interleukin-17 (IL-17), which is predominantly produced by T helper 17 cells distinct from T helper 1 or T helper 2 cells, participates in the pathogenesis of infectious, autoimmune, and allergic disorders. However, the precise role in allograft rejection remains uncertain. In the present study, we investigated the role of IL-17 in acute allograft rejection using IL-17-deficient mice. Methods and Results— Donor hearts from FVB mice were heterotopically transplanted into either C57BL/6J-IL-17-deficient (IL-17−/−) or -wild-type mice. Allograft survival was significantly prolonged in IL-17−/− recipient mice due to reduced local inflammation accompanied by decreased inflammatory cell recruitment and cytokine/chemokine expression. IL-17−/− recipient mice exhibited decreased IL-6 production and reciprocally enhanced regulatory T cell expansion, suggesting a contribution of regulatory T cells to prolonged allograft survival. Indeed, allografts transplanted into anti-CD25 mAb-treated IL-17−/− recipient mice (regulatory T cell-depleted) developed acute rejection similar to wild-type recipient mice. Surprisingly, we found that gamma delta T cells rather than CD4+ and CD8+ T cells were key IL-17 producers in the allografts. In support, equivalent allograft rejection was observed in Rag-2−/− recipient mice engrafted with either wild-type or IL-17−/− CD4+ and CD8+ T cells. Finally, hearts transplanted into gamma delta T cell-deficient mice resulted in decreased allograft rejection compared with wild-type controls. Conclusions— During heart transplantation, (1) IL-17 is crucial for acceleration of acute rejection; (2) IL-17-deficiency enhances regulatory T cell expansion; and (3) gamma delta T cells rather than CD4+ and CD8+ T cells are a potential source of IL-17. IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.

Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines

Catestatin, a neuroendocrine peptide with effects on human autonomic function, has recently been found to be a cutaneous antimicrobial peptide. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu and Arg374Gln. Given reports indicating that antimicrobial peptides and neuropeptides induce mast cell activation, we postulated that catestatin might stimulate numerous functions of human mast cells, thereby participating in the regulation of skin inflammatory responses. Catestatin and its naturally occurring variants caused the human mast cell line LAD2 and peripheral blood‐derived mast cells to migrate, degranulate and release leukotriene C4 and prostaglandins D2 and E2. Moreover, catestatins increased intracellular Ca2+ mobilization in mast cells, and induced the production of pro‐inflammatory cytokines/chemokines such as granulocyte–macrophage colony‐stimulating factor, monocyte chemotactic protein‐1/CCL2, macrophage inflammatory protein‐1α/CCL3 and macrophage inflammatory protein‐1β/CCL4. Our evaluation of possible cellular mechanisms suggested that G‐proteins, phospholipase C and the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (ERK) are involved in catestatin‐induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G‐protein inhibitor), U‐73122 (phospholipase C inhibitor) and U0126 (ERK inhibitor), respectively. We also found that human mast cells express the α7 subunit of the nicotinic acetylcholine receptor at both the mRNA and protein levels. Given that silencing the α7 receptor mRNA and an α7‐specific inhibitor did not affect catestatin‐mediated activation of mast cells, however, we concluded that this receptor is not likely to be functional in human mast cell stimulation by catestatins. Our finding that the neuroendocrine antimicrobial peptide catestatin activates human mast cells suggests that this peptide might have immunomodulatory functions, and provides a new link between neuroendocrine and cutaneous immune systems.