Taku Kouro

PI3K and Btk differentially regulate B cell antigen receptor-mediated signal transduction

Phosphoinositide-3 kinase (PI3K) is thought to activate the tyrosine kinase Btk. However, through analysis of PI3K−/− and Btk−/− mice, B cell antigen receptor (BCR)-induced activation of Btk in mouse B cells was found to be unaffected by PI3K inhibitors or by a lack of PI3K. Consistent with this observation, PI3K−/− Btk−/− double-deficient mice had more severe defects than either single-mutant mouse. NF-κB activation along with Bcl-xL and cyclin D2 induction were severely blocked in both PI3K−/− and Btk−/− single-deficient B cells. Transgenic expression of Bcl-xL restored the development and BCR-induced proliferation of B cells in PI3K−/− mice. Our results indicate that PI3K and Btk have unique roles in proximal BCR signaling and that they have a common target further downstream in the activation of NF-κB.

A Protein Associated with Toll-Like Receptor 4 (PRAT4A) Regulates Cell Surface Expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

The Activation of the JAK2/STAT5 Pathway Is Commonly Involved in Signaling through the Human IL-5 Receptor

The JAK (Janus kinase) family of protein tyrosine kinases and the STATs (signal transducers and activators of transcription) have been shown to be activated in response to a number of cytokines and growth factors. In this study, we evaluated the activation of JAK/STAT pathway upon human interleukin-5 (hIL-5) stimulation of two different hIL-5-responsive cell lines, hIL-5 receptor alpha-subunit (hIL-5R alpha) cDNA-transfected TF-1 (TF-h5R alpha) and butyric-acid-treated YY-1 (YY-Bu), and peripheral eosinophils. Immunoprecipitation and electrophoretic mobility shift analysis revealed that tyrosine phosphorylation of JAK2 and activation of STAT5 were induced upon stimulation with hIL-5 in all three cell types, while STAT1 activation was only observed in eosinophils. These results indicate that JAK2/STAT5 activation is a common JAK/STAT pathway for hIL-5-mediated signal in these cells.

Interleukin 5 Plays an Essential Role in Elicitation of Contact Sensitivity through Dual Effects on Eosinophils and B-1 Cells

Background: Elicitation of contact sensitivity (CS) depends on B-1-cell-derived antigen-specific immunoglobulin M (IgM) antibodies that recruit CS effector T cells into the local tissue, which is followed by infiltration of antigen-nonspecific mononuclear cells and polymorphonuclear cells, such as neutrophils and eosinophils. In this study, we investigated the role of interleukin (IL)-5, which has broad effects on both eosinophils and B-1 cells, in elicitation of CS. Methods: IL-5 receptor α-chain-deficient (IL-5Rα–/–) mice and IL-5Rα+/+ mice were contact sensitized with oxazolone hapten. Four days later, mice were challenged with the same hapten, and ear swelling responses were measured at 24 h after challenge. Eosinophil infiltration into the local tissue was determined by examination of skin histology and eosinophil peroxidase activity. To investigate the role of IL-5 in B-1 cell activation, the number of oxazolone-specific IgM-producing cells in the spleen was determined by enzyme-linked immunospot assay. Results: Ear swelling responses in IL-5Rα–/– mice were about half of those in IL-5Rα+/+ mice, and nearly no eosinophil infiltration was observed in IL-5Rα–/– mouse skin. Eosinophil peroxidase activity in the sensitized and challenged IL-5Rα–/– mice was about 11 times less than that in immunized IL-5Rα+/+ mice. Contact sensitization significantly increased in numbers of oxazolne-specific IgM-producing cells in IL-5Rα+/+ mouse spleen, but not in IL-5Rα–/– mouse spleen. Conclusion: We conclude that IL-5-dependent activation of eosinophils and B-1 cells is necessary for induction and elicitation of CS. These findings provide a new insight into complicated mechanisms of CS elicitation and suggest a novel role of IL-5 in the regulation of immune responses.

IL-5-Induced Hypereosinophilia Suppresses the Antigen-Induced Immune Response via a TGF-β-Dependent Mechanism

Although eosinophils play an essential role in allergic inflammation, their role has recently been under controversy. Epidemic studies suggest that hypereosinophilia induced by parasite infection could suppress subsequent Ag sensitization, although the mechanism has not been fully clarified. In this study, we investigated whether eosinophils could suppress the Ag-specific immune response in the airway. BALB/c mice were sensitized and airway challenged with OVA. Systemic hypereosinophilia was induced by delivery of an IL-5-producing plasmid. IL-5 gene delivery suppressed the Ag-specific proliferation and cytokine production of CD4+ T cells in the spleen. IL-5 gene delivery before OVA sensitization significantly suppressed airway eosinophilia and hyperresponsiveness provoked by subsequent OVA airway challenge, while delivery during the OVA challenge did not suppress them. This IL-5-induced immune suppression was abolished in eosinophil-ablated mice, suggesting an essential role of eosinophils. IL-5 treatment increased the production of TGF-β1 in the spleen, and we demonstrated that the main cellular source of TGF-β1 production was eosinophils, using eosinophil-ablated mice and depletion study. TGF-β1, but not IL-5 itself, suppressed the Ag-specific immune response of CD4+ T cells in vitro. Furthermore, IL-5 treatment enhanced phosphorylation of Smad2 in CD4+ T cells. Finally, a TGF-β type I receptor kinase inhibitor restored this IL-5-induced immune suppression both in vitro and in vivo. These results suggest that IL-5-induced hypereosinophilia could suppress sensitization to Ag via a TGF-β-dependent mechanism, thus suppressed allergic airway inflammation. Therefore, hypereosinophilia could reveal an immunosuppressive effect in the early stage of Ag-induced immune response.

Expression of IL-5Rα on B-1 cell progenitors in mouse fetal liver and involvement of Bruton's tyrosine kinase in their development

B-1 cells are a subset of B cells responsible for the production of natural antibodies. Although the amount of natural antibody is tightly regulated, how this regulation occurs remains unknown. We examined the expression of IL-5 receptor, a cytokine receptor critical for homeostatic proliferation of B-1 cells, on B-1 cell progenitors in the fetal liver. We identified B-1 progenitors expressing low levels of IL-5 receptor alpha chain (IL-5Ralpha) and eosinophil progenitors expressing higher levels of IL-5Ralpha in the fetal liver. Moreover, the number of these B-1 progenitors were significantly reduced in the fetuses of mice deficient in Brutons tyrosine kinase (Btk), even though IL-5 and thymic stroma lymphopoietin signaling are intact in early B lineage cells in Btk-deficient mice. These data suggest that IL-5 is possibly involved in B-1 cell development and an uncharacterized, Btk-dependent regulatory signaling pathway is involved in unexpectedly early stages of B-1 cell differentiation.

Participation of intercellular adhesion molecule-2 (CD102) in B lymphopoiesis

The survival and fate of blood cell precursors is dependent on their communication with stromal cells of various types within bone marrow. Monoclonal antibodies have proven to be powerful tools for identifying molecules responsible for such interactions and we now describe one that selectively blocks B lymphopoiesis. The BF/32 antibody inhibited the establishment, but not the maintenance of long-term bone marrow cultures capable of lymphocyte production. However, there was no obvious effect on lymphocyte-stromal cell adhesion or responsiveness of pre-B cells to intereleukin-7. Furthermore, the reagent had no influence on myeloid precursors or myeloid bone marrow cultures. Injection of adult mice with BF/32 reduced B lineage precursors within bone marrow, but spared mature B cells. Moreover, the reagent did not alter responsiveness of mature B cells to activating stimuli. The 60 kDa protein recognized by this antibody was widely expressed on lymphocytes. Amino terminal protein sequencing and transfection experiments identified it as the murine homologue of ICAM-2 (CD102).