Itsuo Iwamoto

Enhanced Th2 Cell-Mediated Allergic Inflammation in Tyk2-Deficient Mice

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-γ and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-αβ, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2−/−) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2−/− mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2−/− mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4+ T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2−/− mice. Adoptive transfer experiments revealed that CD4+ T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2−/− mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2−/− mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2−/− mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.

IL-4–Stat6 Signaling Induces Tristetraprolin Expression and Inhibits TNF-α Production in Mast Cells

Increasing evidence has revealed that mast cell–derived tumor necrosis factor α (TNF-α) plays a critical role in a number of inflammatory responses by recruiting inflammatory leukocytes. In this paper, we investigated the regulatory role of interleukin 4 (IL-4) in TNF-α production in mast cells. IL-4 inhibited immunoglobulin E–induced TNF-α production and neutrophil recruitment in the peritoneal cavity in wild-type mice but not in signal transducers and activators of transcription 6 (Stat6)–deficient mice. IL-4 also inhibited TNF-α production in cultured mast cells by a Stat6-dependent mechanism. IL-4–Stat6 signaling induced TNF-α mRNA destabilization in an AU-rich element (ARE)–dependent manner, but did not affect TNF-α promoter activity. Furthermore, IL-4 induced the expression of tristetraprolin (TTP), an RNA-binding protein that promotes decay of ARE-containing mRNA, in mast cells by a Stat6-dependent mechanism, and the depletion of TTP expression by RNA interference prevented IL-4–induced down-regulation of TNF-α production in mast cells. These results suggest that IL-4–Stat6 signaling induces TTP expression and, thus, destabilizes TNF-α mRNA in an ARE-dependent manner.

Proteolytic Processing of Stat6 Signaling in Mast Cells as a Negative Regulatory Mechanism

Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases. In this study, we show a novel regulatory mechanism of Stat6-mediated signaling in mast cells. When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells. The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription. The retrovirus-mediated expression of cleavage-resistant Stat6 mutants prolongs the nuclear accumulation of Stat6 upon IL-4 stimulation and enhances IL-4–induced gene expression and growth inhibition in mast cells. These results indicate that the proteolytic processing of Stat6 functions as a lineage-specific negative regulator of Stat6-dependent signaling in mast cells, and thus suggest that it may account for the limited role of Stat6 in IL-4 signaling in mast cells.

Indispensable Role of Stat5a in Stat6-Independent Th2 Cell Differentiation and Allergic Airway Inflammation

It is well-recognized that Stat6 plays a critical role in Th2 cell differentiation and the induction of allergic inflammation. We have previously shown that Stat5a is also required for Th2 cell differentiation and allergic airway inflammation. However, it is the relative importance and redundancy of Stat6 and Stat5a in Th2 cell differentiation and allergic airway inflammation are unknown. In this study we addressed these issues by comparing Stat5a-deficient (Stat5a−/−) mice, Stat6−/− mice, and Stat5a- and Stat6 double-deficient (Stat5a−/− Stat6−/−) mice on the same genetic background. Th2 cell differentiation was severely decreased in Stat6−/−CD4+ T cells, but Stat6-independent Th2 cell differentiation was still significantly observed in Stat6−/−CD4+ T cells. However, even in the Th2-polarizing condition (IL-4 plus anti-IFN-γ mAb), no Th2 cells developed in Stat5a−/−Stat6−/− CD4+ T cells. Moreover, Ag-induced eosinophil and lymphocyte recruitment in the airways was severely decreased in Stat5a−/−Stat6−/− mice compared with that in Stat6−/− mice. These results indicate that Stat5a plays an indispensable role in Stat6-independent Th2 cell differentiation and subsequent Th2 cell-mediated allergic airway inflammation.

IgE‐dependent enhancement of Th2 cell‐mediated allergic inflammation in the airways

T helper 2 (Th2) cell‐derived cytokines, including interleukin (IL)‐4, IL‐5 and IL‐13, play important roles in causing allergic airway inflammation. In contrast to Th2 cells, however, the role of IgE and mast cells in inducing allergic airway inflammation is not understood fully. In the present study, we addressed this point using transgenic mice expressing trinitrophenyl (TNP)‐specific IgE (TNP–IgE mice), which enable us to investigate the role of IgE without the influence of antigen‐specific T cell activation and other immunoglobulins. When the corresponding antigen, TNP–BSA, was administered intranasally to TNP–IgE mice, a large number of CD4+ T cells were recruited into the airways. In contrast, TNP–BSA administration did not induce eosinophil recruitment into the airways or airway hyperreactivity. Furthermore, when ovalbumin (OVA)‐specific Th2 cells were transferred to TNP–IgE mice and the mice were challenged with inhaled OVA, TNP–BSA administration increased OVA‐specific T cell recruitment and then enhanced Th2 cell‐mediated eosinophil recruitment into the airways. These results indicate that IgE‐induced mast cell activation principally induces CD4+ T cell recruitment into the airways and thus plays an important role in enhancing Th2 cell‐mediated eosinophilic airway inflammation by recruiting Th2 cells into the site of allergic inflammation.

Stat5a is essential for the proliferation and survival of murine mast cells

The regulatory role of signal transducer and activator of transcription (Stat) 5a in the proliferation and survival of mast cells was determined using Stat5a-deficient (Stat5a–/–) mice. First, although the mast cells in Stat5a–/– mice were morphologically indistinguishable from those in wild-type (WT) mice, the number of peritoneal mast cells was significantly decreased in Stat5a–/– mice as compared with that in WT mice. Furthermore, the interleukin-3 (IL-3)-dependent development of bone marrow-derived mast cells (BMMCs) was markedly decreased in Stat5a–/– mice. Second, IL-3-induced but not stem cell factor (SCF)-induced proliferation of BMMCs was significantly diminished in Stat5a–/– mice as compared with that in WT mice. Moreover, survival rates of both peritoneal mast cells and BMMCs were significantly decreased with increased apoptotic cells in Stat5a–/– mice as compared with those in WT mice. Finally, mRNA of Bcl-x(L) was induced after IL-3 stimulation in WT BMMCs but not in Stat5a–/– BMMCs, which may account for the accelerated apoptosis in Stat5a–/– mast cells. These results indicate that Stat5a plays an important role in mast cell development, proliferation, and survival.

Lineage-specific negative regulation of STAT-mediated signaling by proteolytic processing.

Accumulating evidence suggests that STAT-mediated signaling plays critical roles in cell differentiation and/or cell expansion and that in turn, STAT-mediated signaling is regulated strictly by many mechanisms. In murine mast cells, when Stat6 is activated by IL-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease (namely Stat6-protease). Similarly, the activated Stat5 is cleaved by a protease (Stat5-protease) in the nucleus of myeloid progenitors. These STAT proteases cleave the corresponding STAT proteins at the carboxyl-terminus and the resultant STAT proteins function as dominant negative molecules. Functionally, Stat6-protease protects mast cells from Stat6-dependent growth inhibition while Stat5-protease maintains the immature state of myeloid progenitors. In addition, it has been shown that the activated Stat3 is cleaved in mature neutrophils. These findings indicate that the proteolytic processing of STAT proteins by the nucleus-associated protease functions as a lineage-specific negative-regulator of STAT-mediated signaling.

Tyk2 Is Essential for IFN-α-Induced Gene Expression in Mast Cells

Mast cells are recognized not only as the major effector cells of type I hypersensitivity reactions but also as an important player of innate immune response against bacterial infection. Type I IFNs are also involved in the response against bacterial infection. However, the role of type I IFNs and their associated Janus kinase Tyk2 in mast cell functions remains to be determined. In this study, we addressed this issue using Tyk2-deficient (Tyk2–/–) bone marrow-derived mast cells (BMMCs). When BMMCs from wild-type (WT) mice were stimulated with IFN-α, they expressed mRNA for IFN-γ-inducible protein 10 (IP-10) and monocyte chemoattractant protein-5 (MCP-5). Interestingly, IFN-α-induced expression of IP-10 and MCP-5 was severely decreased in Tyk2–/– BMMCs. In addition, IFN-α-induced Stat1 phosphorylation was decreased in Tyk2–/– BMMCs. On the other hand, IFN-α-induced Stat1 phosphorylation and IP-10 and MCP-5 expression were normal in Tyk2–/– fibroblasts. These results indicate that IFN-α induces the expression of TNF-α and the chemokines IP-10 and MCP-5 in mast cells and thatTyk2 plays a nonredundant role in IFN-α signaling in mast cells.

T Cell Vaccination Eliminates Antigen-Specific T Cells and Prevents Antigen-Induced Eosinophil Recruitment into the Tissue

Asthma is characterized by airway inflammation with prominent eosinophil infiltrates. In a murine model of asthma, antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4+ T cells and their cytokines, especially IL-5. In the present study, using ovalbumin-specific T cell receptor transgenic mice, we found that T cell vaccination, which was the administration of preactivated and attenuated antigen-specific T cells by the intraperitoneal route, prevented antigen-induced eosinophil recruitment into the airways. This effect was antigen-specific because ovalbumin-specific T cell vaccination did not affect BSA-induced eosinophil recruitment into the airways. We also found that antigen-specific IgE production as well as antigen-induced proliferation and cytokine production of splenocytes were diminished by T cell vaccination. Moreover, flow-cytometric analyses revealed that T cell vaccination eliminated antigen-specific T cells in the periphery. Together, these results indicate that T cell vaccination prevents antigen-induced eosinophil recruitment into the airways presumably by eliminating antigen-specific T cells.