中島 裕史

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.

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.

A short isoform of Stat6 (65-kDa Stat6), a product of proteolytic processing by an undefined protease (Stat6-protease) in the nucleus, downregulates Stat6-mediated signaling in mast cells. Similarly, Stat5-mediated signaling is downregulated by Stat5-protease in myeloid progenitors. These proteases share a number of characteristics, including their nuclear localization and susceptibility to protease inhibitors. Here, we further investigated these Stat proteases. Interestingly, the activity of Stat6-protease but not of Stat5-protease was inhibited by ONO-5046, an elastase inhibitor that inhibits the activity of neutrophil elastase (NE) and NE-related protease proteinase 3 (PR3). Although both NE and PR3 were able to cleave Stat6 in vitro, the cleavage sites of Stat6 by NE or PR3 differed from that by Stat6-protease in mast cells. In addition, both NE and PR3 could also cleave Stat5, but they differed from Stat5-protease in myeloid progenitors. These results suggest that Stat6-protease may belong to the elastase family but differs from NE or PR3. (C) 2003 Elsevier Inc. All rights reserved.

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. (C) 2003 Elsevier Ltd. All rights reserved.

Interleukin-25 (IL-25) is a recently described T helper 2 (TH2) cell–derived cytokine that belongs to the IL-17 family and induces the production of IL-4, IL-5, and IL-13 from an unidentified non–T-cell population. Here, we show that mast cells are also potent IL-25–producing cells. When bone marrow–derived mast cells were stimulated by immunoglobulin E cross-linking, IL-25 mRNA was induced within 30 minutes in a calcineurin-dependent manner, and the levels of IL-25 mRNA were comparable with those of activated TH2 cells. Production of IL-25 by mast cells was also detected at protein levels by immunoblotting. These results suggest that mast cells may enhance TH2-type immune response by producing IL-25.

Recent studies with gene knockout mice have demonstrated that T helper 2 (Th2) cell-derived cytokines, including IL-4, IL-5, and IL-13, play important roles in causing allergic airway inflammation. In addition to Th2 cytokines, IgE-dependent activation of mast cells has been suggested to play a role in allergic airway inflammation. In this review, we will discuss the role of IgE in Th2 cell-mediated allergic airway inflammation. We used IgE transgenic mice, which enabled us to investigate the role of IgE without the influence of activated T cells and other immunoglobulins. Whereas IgE cross-linking by antigens did not induce eosinophil recruitment into the airways or airway hyperreactivity, IgE cross-linking induced CD4+ T cell recruitment into the airways. In addition, when antigen-specific Th2 cells were transferred to IgE transgenic mice, IgE cross-linking significantly enhanced antigen-induced eosinophil recruitment into the airways. These findings suggest that IgE-dependent mast cell activation plays an important role in allergic airway inflammation by recruiting Th2 cells into the site of allergic inflammation. Copyright (C) 2003 S. Karger AG, Basel.

Recent studies with gene knockout mice have demonstrated that T helper 2 (Th2) cell-derived cytokines, including IL-4, IL-5, and IL-13, play important roles in causing allergic airway inflammation. In addition to Th2 cytokines, IgE-dependent activation of mast cells has been suggested to play a role in allergic airway inflammation. In this review, we will discuss the role of IgE in Th2 cell-mediated allergic airway inflammation. We used IgE transgenic mice, which enabled us to investigate the role of IgE without the influence of activated T cells and other immunoglobulins. Whereas IgE cross-linking by antigens did not induce eosinophil recruitment into the airways or airway hyperreactivity, IgE cross-linking induced CD4+ T cell recruitment into the airways. In addition, when antigen-specific Th2 cells were transferred to IgE transgenic mice, IgE cross-linking significantly enhanced antigen-induced eosinophil recruitment into the airways. These findings suggest that IgE-dependent mast cell activation plays an important role in allergic airway inflammation by recruiting Th2 cells into the site of allergic inflammation. Copyright (C) 2003 S. Karger AG, Basel.

Interleukin 21 (IL-21) has recently been identified as a multifunctional cytokine that induces the proliferation of T cells and B cells and differentiation of natural killer cells. To determine whether IL-21 regulates IL-4–mediated immune responses, we examined the effect of IL-21 on antigen-specific IgE production in mice. We also examined the effect of IL-21 on IL-4–induced IgE production from B cells and antigen-induced T-helper 2 (Th2) cell differentiation. The in vivo injection of IL-21 prevented antigen-specific IgE but not IgG2a production on immunization. IL-21 did not affect Th2 cell differentiation or IL-4 production from CD4+ T cells but directly inhibited IL-4–induced IgE production from B cells at single-cell levels. Moreover, IL-21 inhibited IL-4–induced germ line Cε transcription in B cells without the inhibition of signal transducer and activator of transcription 6 (Stat6) activation. Taken together, these results indicate that IL-21 down-regulates IgE production from IL-4–stimulated B cells through the inhibition of germ line Cε transcription and thus suggest that IL-21 may be useful for the treatment of IgE-dependent allergic diseases.

1 Medium-chain triglyceride (MCT) is often administered to patients with Crohn's disease (CD) or short-bowel syndrome. However, little is known about the effects of medium-chain fatty acids (MCFAs) and MCT on intestinal inflammation. In this study we examined whether caprylic acid, one of the MCFAs, and MCT suppress IL-8 secretion by differentiated Caco-2 cells. 2 We found for the first time that caprylic acid and MCT suppress IL-8 secretion by Caco-2 cells at the transcriptional level when precultured together for 24 It. We also tried to clarify the mechanism of IL-8 gene inhibition by examining the activation of NF-kappaB and other transcription factors by electrophoretic mobility shift assay (EMSA), and found that caprylic acid did riot modulate their activation. 3 The result of dual-luciferase assay using Caco-2 cells transfected with IL-8 promoter/luciferase reporter plasmid revealed that caprylic acid inhibited the activation of IL-8 promoter. 4 Similar results were observed when cells were precultured with the well-known potent histone deacetylase inhibitor trichostatin A (TSA). 5 We examined the state of H4 acetylation in IL-8 promoter using the technique known as chromatin immunoprecipitation (Chr-IP). TSA rapidly induced H4 acetylation in IL-8 promoter chromatin, whereas caprylic acid did not. These results suggest that the inhibition of IL-8 gene transcription induced by caprylic acid and TSA does not necessarily require the marked suppression of transcription factors, and the mechanism of inhibition of IL-8 gene transcription may be different between caprylic acid and TSA.

Serine/threonine kinase Akt/protein kinase B, the cellular homologue of the transforming viral oncogene v-Akt, plays a central role in the regulation of cell survival and proliferation. We have previously demonstrated that the proto-oncogene TCL1 is an Akt kinase coactivator. TCL1 binds to Akt and mediates the formation of oligomeric TCL1-Akt high-molecular-weight protein complexes in vivo. Within these protein complexes, Akt is preferentially phosphorylated and activated. The MTCP1/TCL1/TCL1b oncogene activation is the hallmark of human T-cell prolymphocytic leukemia (T-PLL), a form of adult leukemia. In the present study, using a PCR-generated random TCL1 library combined with a yeast two-hybrid screening detecting loss of interaction, we identified D16 and 174 as amino acid residues mediating the association of TCL1 with Akt. Based on molecular modeling, we determined that the betaC-sheet of TCL1 is essential for TCL1 homodimerization. Studies with mammalian overexpression systems demonstrated that both Akt association and oligomerization domains of TCL1 are distinct functional domains. In vitro kinase assays and overexpression experiments in mammalian cells demonstrated that both TCL1-Akt interaction and oligomerization of TCL1 were required for TCL1-induced Akt activation and substrate phosphorylation. Assays for mitochondrial permeability transition, nuclear translocation, and cell recovery demonstrated that both Akt association and homodimerization of TCL1 are similarly needed for the full function of TCL1 as an Akt kinase coactivator in vivo. The results demonstrate the structural basis of TCL1-induced activation of Akt, which causes human T-PLL.

Abstract It has recently been shown that CD4+CD25+ T cells are immunoregulatory T cells that prevent CD4+ T-cell–mediated organ-specific autoimmune diseases. In this study, the regulatory mechanism of CD4+CD25+ T-cell development were investigated using T-cell receptor (TCR) transgenic mice. It was found that CD4+CD25+ T cells preferentially expressed the endogenous TCRα chain in DO10+ TCR transgenic mice compared with CD4+CD25− T cells. Moreover, it was found that CD4+CD25+ thymocytes were severely decreased in DO10+ TCR-α−/− mice in positively selecting and negatively selecting backgrounds, whereas CD4+CD25− thymocytes efficiently developed by transgenic TCR in DO10+ TCR-α−/− mice in positively selecting backgrounds, indicating that the appropriate affinity of TCR to major histocompatibility complex (MHC) for the development of CD4+CD25+ thymocytes is different from that of CD4+CD25− thymocytes and that a certain TCR–MHC affinity is required for the development of CD4+CD25+ thymocytes. Finally, it was found that, in contrast to thymus, CD4+CD25+ T cells were readily detected in spleen of DO10+TCR-α−/− mice in positively selecting backgrounds and that splenic CD4+CD25+ T cells, but not CD4+CD25+ thymocytes, were significantly decreased in B-cell–deficient mice, suggesting that B cells may control the peripheral pool of CD4+CD25+ T cells. Together, these results indicate that the development of CD4+CD25+ T cells in thymus and the homeostasis of CD4+CD25+ T cells in periphery are regulated by distinct mechanisms.

SUMMARY Atopic disorders are caused by disregulated activation of T helper 2 (Th2) cells that produce IL-4 and IL-5. Because the presence of IL-4 potently augments the differentiation of naive T cells into Th2 cells, it is important to seek the cell population which provides IL-4 for naive T cells. Recently, a unique subpopulation of T cells, natural killer (NK) T cells, has been shown to produce a large amount of IL-4 upon activation, suggesting their regulatory role in initiation of Th2 cell differentiation. To determine whether NK T cells play a regulatory role in human Th2 cell-mediated atopic diseases, we analysed the frequency of invariant Vα24JαQ CD4−CD8− double-negative (DN) T cells, human NK T cells, in patients with atopic asthma and atopic dermatitis. We also studied cytokine production from Vα24+ Vβ11+ DN T cells, which comprise most of Vα24JαQ DN T cells. We found that the invariant Vα24JαQ DN T cells were greatly diminished in patients with asthma and atopic dermatitis. On the other hand, there was no significant difference in Vα24+ CD4+ T cells possessing invariant Vα24JαQ TCR between healthy subjects and atopic patients. We also found that Vα24+ Vβ11+ DN T cells from healthy subjects predominantly produced interferon-gamma (IFN-γ) but not IL-4 upon activation. These results suggest that NK T cells may not be essential for human atopic disease and that the disappearance of NK T cells, most of which produce IFN-γ, may be involved in the pathogenesis of atopic diseases.

Abstract We have previously shown that CD4+ T cell–mediated allergic inflammation is diminished in signal transducer and activator of transcription (Stat)5a-deficient (Stat5a−/−) mice. To determine whether Stat5a regulates T helper cell differentiation, we studied T helper (Th)1 and Th2 cell differentiation of Stat5a−/−CD4+ T cells at single-cell levels. First, Th2 cell differentiation from antigen-stimulated splenocytes was significantly decreased in Stat5a−/− mice as compared with that in wild-type mice. Further, Th2 cell differentiation was also impaired in Stat5a−/− mice even when purified CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 antibodies in the presence of interleukin-4. Moreover, the retrovirus-mediated gene expression of Stat5a in Stat5a−/−CD4+ T cells restored the Th2 cell differentiation at the similar levels to that in wild-type CD4+ T cells. In addition, interleukin-4 normally phosphorylated Stat6 in CD4+ T cells from Stat5a−/− mice. Second, the development of CD4+CD25+ immunoregulatory T cells was impaired in Stat5a−/− mice, as indicated by a significant decrease in the number of CD4+CD25+ T cells in Stat5a−/− mice. Furthermore, the depletion of CD4+CD25+ T cells from wild-type splenocytes significantly decreased Th2 cell differentiation but increased Th1 cell differentiation, whereas the depletion of CD4+CD25+ T cells from Stat5a−/−splenocytes had no significant effect on the Th1 and Th2 cell differentiation. Together, these results indicate that the intrinsic expression of Stat5a in CD4+ T cells is required for Th2 cell differentiation and that Stat5a is involved in the development of CD4+CD25+ immunoregulatory T cells that modulate T helper cell differentiation toward Th2 cells.

Regulation of subcellular localization of Smad proteins is supposed to be critical for the effective initiation and maintenance of TGF-β signaling. Recently, Smad anchor for receptor activation (SARA) has been identified as a Smad2 binding protein. SARA regulates the subcellular localization of Smad2 and is required for TGF-β/Smad2-mediated signaling. In this study, we determined whether the interaction between SARA and Smad3 is essential for TGF-β/Smad3-mediated signaling. We found that a mutant Smad3 (Smad3NS) that lacked the binding to SARA was phosphorylated by TGF-β type I receptor at the similar level to that in wild-type Smad3 (Smad3WT). Smad3NS also formed complexes with Smad4 and translocalized into the nucleus. Moreover, Smad3NS and Smad3WT equally enhanced TGF-β-induced transcription. Therefore, these findings indicate that, in contrast to SARA/Smad2 interaction, SARA/Smad3 interaction is not essential for TGF-β/Smad3-mediated signaling. © 2001 Academic Press.

The regulatory roles of the common cytokine receptor γ chain (γ(c))- and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γ(c)-) and Jak3-deficient (Jak3-) mice. Although the mast cells in γc- and Jak3- mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc- and Jak3- mice as compared with that in wild-type mice. Among γ(c)-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow-derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γ(c)- and Jak3-mice. In addition, IL-4Rα, γ(c), and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γ(c)- and Jak3-mice. These results indicate that γ(c)- and Jak3-dependent signaling is essential for IL-4- and IL-9-induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γ(c)- and Jak3-dependent manner. (C) 2000 by The American Society of Hematology.

Antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4+ T cells and their cytokines, especially IL-5. In this study, we found that the antigen-induced airway eosinophilia was diminished in Stat5a-deficient (Stat5a(-/-)) mice and Stat5b-deficient (Stat5b(-/-)) mice. We also found that antigen-induced CD4+ T-cell infiltration and IL-5 production in the airways were diminished in Stat5a(-/- ) mice and Stat5b(-/-) mice. Moreover, antigen-induced proliferation of splenocytes was diminished in Stat5a(-/-) mice and Stat5b(-/-) mice, suggesting that the generation of antigen-primed T cells may be compromised in Stat5a(-/-) mice and Stat5b(-/-) mice and this defect may account for the diminished antigen-induced T-cell infiltration into the airways. Interestingly, IL-4 and IL-5 production from anti-CD3-stimulated splenocytes was diminished in Stat5a(-/-) mice and Stat5b(-/-) mice. However, antigen- specific IgE and IgG1 production was diminished in Stat5a(-/-) mice but not in Stat5b(-/-) mice, whereas antigen-specific IgG2a production was increased in Stat5a(-/-) mice, suggesting the enhanced Th1 responses in Stat5a(-/-) mice. Finally, we found that eosinophilopoiesis induced by the administration of recombinant IL-5 was also diminished in Stat5a(-/-) mice and Stat5b(-/-) mice. Together, these results indicate that both Stat5a and Stat5b are essential for induction of antigen-induced eosinophil recruitment into the airways and that the defects in antigen-induced eosinophil recruitment in Stat5a(-/-) mice and Stat5b (-/-) mice result from both impaired IL-5 production in the airways and diminished IL-5 responsiveness of eosinophils. (C) 2000 by The American Society of Hematology.

During thymocyte development,, T-cell receptor (TCR) alpha beta-mediated intracellular signals can elicit two entirely different cellular responses: positive selection (resulting in rescue from death and maturation or differentiation) and negative selection (induction of apoptosis). Here, Hiroshi Nakajima and colleagues discuss how survival signals that ave dependent on the common cytokine receptor gamma drain (gamma(c)) might affect the TCR-driven selection process in thymocytes, underscoring the potential role of cytokines in this process.

CD4+ T cells are thought to play an important role in airway inflammation in both atopic and non-atopic asthma. However, the mechanism by which T cells are activated in non-atopic asthma, where there is no causative antigen identified, is unknown. To elucidate this issue, we analysed T cell receptor (TCR) Vβ gene clonotypes of T cells in the bronchoalveolar lavage fluids (BALF) of non-atopic asthmatics using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and a sequencing method. We found that the numbers of TCR Vβ gene clonotypes of T cells in the BALF of non-atopic asthmatics were significantly increased compared with those of peripheral blood lymphocytes (PBL). We also found that there were several shared amino acid motifs in complementarity-determining region 3 (CDR3) of TCR Vβ genes from those T cell clones in BALF of non-atopic asthmatics, whereas these shared motifs were not found in the same Vβ family genes from PBL in the patients. Moreover, a conserved amino acid sequence was detected in two patients who shared a common HLA-DR allele. These results indicate that the infiltrating T cells in the airways of nonatopic asthmatics recognize relatively limited epitopes of antigens and are clonally expanded by antigen-driven stimulation.

VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)α−/− murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-γ locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Rα to rearrangement of the TCR-γ locus requires the γc receptor chain and the γc-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-γ locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Rα−/− thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Rα−/− thymocytes. Instead, the TCR-γ locus was shown to be methylated in IL-7Rα−/− thymocytes. Treatment of IL-7Rα−/− precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-γ gene rearrangement. This data supports the model that IL-7R promotes TCR-γ gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.

We have analyzed the immune system in Stat5-deficient mice. Although Stat5a−/− splenocytes have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b−/− splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained by diminished expression of the IL-2 receptor β chain (IL-2Rβ), which is a component of the receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b−/− mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the diminished proliferation, basal as well as IL-2– and IL-15–mediated boosting of NK cytolytic activity was also greatly diminished. These data indicate an essential nonredundant role for Stat5b for potent NK cell–mediated proliferation and cytolytic activity.

Mice lacking the common cytokine receptor gamma-chain (gamma(c)) gene exhibit defective development of NK cells and CD8(+) T cells and greatly diminished production of IFN-gamma, Because resistance of SCID mice to Toxoplasma gondii requires IL-12-dependent IFN-gamma production by NK cells, we expected that gamma(c)-deficient mice would succumb rapidly to the parasite, Surprisingly, however, most gamma(c)-deficient mice survived the acute phase of T. gondii infection. As in wild-type mice, this resistance required IL-12 and IFN-gamma; nevertheless, whereas wild-type mice depleted of CD4(+) T cells survived, anti-CD4(+) treated gamma(c)-deficient mice displayed diminished production of IFN-gamma and all succumbed to acute infection. These data not only reveal a role for CD4(+) T lymphocytes in IFN-gamma-dependent host defense but also establish SCID and gamma(c)-deficient mice as powerful complementary tools for assessing the function of NK vs CD4(+) T cells in immunopathophysiologic responses.

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γ _c ) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro -translated wild-type γ _c , but not γ _c containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γ _c in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γ _c . Moreover, in single positive CD4 ⁺ thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γ _c blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γ _c represents a mechanism by which γ _c -dependent signaling can be controlled.

In a murine model of airway late phase reaction, we have previously shown that Ag-induced eosinophil recruitment into the tissue of sensitized mice is mediated by CD4(+) T cells and IL-5. To determine whether IFN-alpha regulates Ag-induced eosinophil recruitment into tissue, we studied the effects of rIFN-alpha, endogenous IFN-alpha production induced by poly I.C, and anti-IFN-alpha mAb on the eosinophil infiltration of the trachea induced by Ag inhalation in mice. The i.p. administration of rIFN-alpha prevented Ag-induced eosinophil infiltration in the trachea of sensitized mice. In addition, the inhibitory effect of rIFN-alpha on Ag-induced eosinophil infiltration was not recovered by pretreatment with anti-IFN-gamma mAb. The administration of rIFN-alpha also inhibited Ag-induced CD4(+) T cell infiltration in the trachea. However, the administration of rIFN-alpha did not significantly affect blood eosinophil counts nor the number of blood and splenic CD4(+) T cells. On the other hand, pretreatment with anti-murine IFN-alpha mAb enhanced Ag-induced eosinophil and CD4(+) T cell infiltration in the trachea. Finally, endogenous IFN-alpha/beta production induced by poly I.C also inhibited Ag-induced eosinophil infiltration in the trachea and this inhibition of the eosinophil infiltration was abrogated by pretreatment with anti-IFN-alpha/beta mAb. These results indicate that IFN-alpha suppresses Ag-induced eosinophil and CD4(+) T cell recruitment into tissue.

To determine the role of vascular cell adhesion molecule 1 (VCAM-1)/very late activation antigen 4 (VLA-4) and intercellular adhesion molecule 1 (ICAM-1)/lymphocyte function-associated antigen 1 (LFA-1) interactions in causing antigen-induced eosinophil and T cell recruitment into the tissue, we studied the effect of the in vivo blocking of VCAM-1, ICAM-1, VLA-4, and LFA-1 by pretreatment with monoclonal antibodies (mAb) to these four adhesion molecules on the eosinophil and T cell infiltration of the trachea induced by antigen inhalation in mice. The in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, prevented antigen-induced eosinophil infiltration into the mouse trachea. On the contrary, the in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, increased blood eosinophil counts after antigen challenge, but did not affect blood eosinophil counts without antigen challenge in sensitized mice. Furthermore, the expression of VCAM-1 but not ICAM-1 was strongly induced on the endothelium of the trachea after antigen challenge. In addition, pretreatment with anti-IL-4 mAb decreased the antigen-induced VCAM-1 expression only by 27% and had no significant effect on antigen-induced eosinophil infiltration into the trachea. The in vivo blocking of VCAM-1 and VLA-4 inhibited antigen-induced CD4+ and CD8+ T cell infiltration into the trachea more potently than that of ICAM-1 and LFA-1. In contrast, regardless of antigen challenge, the in vivo blocking of LFA-1, but not of ICAM-1, increased blood lymphocyte counts more than that of VCAM-1 and VLA-4. These results indicate that VCAM-1/VLA-4 interaction plays a predominant role in controlling antigen-induced eosinophil and T cell recruitment into the tissue and that the induction of VCAM-1 expression on the endothelium at the site of allergic inflammation regulates this eosinophil and T cell recruitment.

A 44-year-old woman with scleroderma and Sjogren's syndrome developed altered consciousness, acute renal failure, and rhabdomyolysis. She had no history of trauma, seizures, alcohol abuse, hyperthermia, or other possible causative factors for rhabdomyolysis. A high serum salicylate level indicated a diagnosis of salicylate intoxication. Medical history after recovery revealed chronic salicylate ingestion for severe headaches. This is possibly the first reported case of rhabdomyolysis caused by chronic salicylate intoxication. Continuous hemodiafiltration early in hospitalization was an effective treatment.