Biomedical subjects

K Takatsu

Publications and source records attributed to K Takatsu.

At least 37 records · Page 2Linked to original sources

PubMed2000

Control of B cell production by the adaptor protein lnk. Definition Of a conserved family of signal-modulating proteins.

Lnk is an SH2 domain-containing adaptor protein expressed preferentially in lymphocytes. To illuminate the importance of Lnk, we generated lnk(-/-) mice. Whereas T cell development was unaffected, pre-B and immature B cells accumulated in the spleens. In the bone marrow, B-lineage cells were proportionately increased, reflecting enhanced production of pro-B cells that resulted in part from hypersensitivity of precursors to SCF, the ligand for c-kit. Hence, Lnk ordinarily acts to regulate B cell production. Further characterization of lnk(-/-) mice also revealed that full-length Lnk is a 68 kDa protein containing a conserved proline-rich region and a PH domain. Lnk is a representative of a multigene adaptor protein family whose members act, by analogy with Lnk, to modulate intracellular signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Allergen-induced airway inflammation and bronchial responsiveness in interleukin-5 receptor alpha chain-deficient mice.

OBJECTIVE: The role of IL-5 receptor alpha chain (IL-5Ralpha) in the onset of bronchial hyperresponsiveness (BHR) to acetylcholine was investigated by testing IL-5Ralpha knockout (IL-5Ralpha KO) mice. METHODS: Mice were immunized with antigen at intervals of 12 days. Starting 10 days after the secondary immunization, mice were exposed to antigen three times every fourth day. Twenty-four hours after the last antigen challenge, bronchial responsiveness to acetylcholine was measured and bronchoalveolar lavage was carried out. RESULTS: Twenty-four hours after the last antigen inhalation, total and differential cells counts of bronchoalveolar lavage revealed a significant increase in eosinophils and lymphocytes in ovalbumin-exposed wild-type mice. In IL-5Ralpha KO mice, there was little increase of eosinophils in bronchoalveolar lavage fluid (BALF). The production of IL-5 in BALF increased in both mice after repeated antigen challenge, and there was no significant difference between wild-type and IL-5Ralpha KO mice. Similar to the BAL study, histological sections of lung tissue from ovalbumin-exposed wild-type mice exhibited airway eosinophilic inflammation, which was attenuated by the deficiency of IL-5Ralpha chain. There was no significant difference in serum antigen-specific IgE levels between wild-type and IL-5Ralpha KO mice after immunization nor antigen inhalation. Repeated antigen provocation caused BHR to acetylcholine in wild-type mice. In contrast, no BHR was observed in IL-5Ralpha KO mice after repeated inhalation of antigen. CONCLUSION: These findings indicate that IL-5Ralpha plays an important role in the development of antigen-induced airway eosinophilia and BHR in mice.

Acetylcholine↗

PubMed2000

Identification and characterization of a molecule, BAM11, that associates with the pleckstrin homology domain of mouse Btk.

Bruton's tyrosine kinase (Btk) is required for normal B cell development and signal transduction through cell surface molecules, and its defects lead to X-linked immune deficiency in mice and X-linked agammaglobulinemia in humans. In this report, we will describe the identification and characterization of a molecule, BAM11, which binds to the pleckstrin homology domain of Btk. A sequence homology search revealed that BAM11 has 89% homology, at the amino acid level, to human LTG19/ENL, that was originally identified as one of the fusion partners involved in chromosomal translocations of 11q23, MLL/ALL-1/HRX, in leukemia cells. Deletion mutants demonstrated that the region of BAM11 required for binding to Btk was localized between amino acid residues 240 and 256. Forced expression of a truncated form of BAM11 (amino acids 246-368) inhibited IL-5-induced proliferation by 50%, whereas forced expression of full-length BAM11 in Y16 cells did not affect the IL-5 responsiveness. We have also shown that BAM11 (amino acids 246-368) inhibited the kinase activity of Btk. These results suggest that the binding of BAM11 to Btk plays a regulatory role in the Btk signal transduction pathway. A cell fractionation study and analysis using EGFP-fused Btk protein demonstrated that a proportion of Btk is present within the nucleus.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2000

Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase.

To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (alpha, betaI, betaII, and delta) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.

Animals↗

PubMed1999

IL-5 induces IgG1 isotype switch recombination in mouse CD38-activated sIgD-positive B lymphocytes.

Mouse B cells express CD38, whose ligation by anti-CD38 Ab induces their proliferation and protection from apoptosis. We previously showed that stimulation of mouse splenic B cells with IL-5 together with CS/2, an anti-mouse CD38 mAb, induces production of IgG1 and IgM. Here we examined the role of IL-5 and CS/2 in the expression of germline gamma1 transcripts and the generation of reciprocal products forming DNA circles as byproducts of mu-gamma1 switch recombination. By itself, CS/2 induced significant expression of germline gamma1 transcripts in splenic naive B cells, whereas IL-5 neither induced nor enhanced germline gamma1 expression. Increased cellular content of reciprocal product, which is characteristic of mu-gamma1 recombination, was not observed after culturing B cells with CS/2, but increased reciprocal product, along with high levels of lgG1 secretion, was found when B cells were cultured with CS/2 plus IL-5. Although IL-4 did not, by itself, induce mu-gamma1 recombination in B cells stimulated with CS/2, in conjunction with CS/2 plus IL-5, IL-4 dramatically enhanced sterile gamma1 transcription and IgG1 production. These results demonstrate that CD38 ligation induces only germline gamma1 transcription and that IL-5 promotes both mu-gamma1 switch recombination and lgG1 secretion in an IL-4-independent manner.

ADP-ribosyl Cyclase↗

PubMed1999

Affinity modulation of very late antigen-5 through phosphatidylinositol 3-kinase in mast cells.

Adhesiveness of integrins is up-regulated rapidly by a number of molecules, including growth factors, cytokines, chemokines, and other cell surface receptors, through a mechanism termed inside-out signaling. The inside-out signaling pathways are thought to alter integrin affinity for ligand, or cell surface distribution of integrin by diffusion/clustering. However, it remains to be clarified whether any physiologically relevant agonists induce a rapid change in the affinity of beta1 integrins and how ligand-binding affinity is modulated upon stimulation. In this study, we reported that affinity of beta1 integrin very late Ag-5 (VLA-5) for fibronectin was rapidly increased in bone marrow-derived mast cells by Ag cross-linking of FcepsilonRI. Ligand-binding affinity of VLA-5 was also augmented by receptor tyrosine kinases when the phospholipase Cgamma-1/protein kinase C pathway was inhibited. Wortmannin suppressed induction of the high affinity state VLA-5 in either case. Conversely, introduction of a constitutively active p110 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) increased the binding affinity for fibronectin. Failure of a constitutively active Akt to stimulate adhesion suggested that the affinity modulation mechanisms mediated by PI 3-kinase are distinct from the mechanisms to control growth and apoptosis by PI 3-kinase. Taken together, our findings demonstrated that the increase of affinity of VLA-5 was induced by physiologically relevant stimuli and PI 3-kinase was a critical affinity modulator of VLA-5.

Androstadienes↗

PubMed1999

Measurement of serum levels of des-gamma-carboxy prothrombin in patients with hepatocellular carcinoma by a revised enzyme immunoassay kit with increased sensitivity.

BACKGROUND: Des-gamma-carboxy prothrombin (DCP) is a useful tumor marker for hepatocellular carcinoma (HCC). The conventional enzyme immunoassay (EIA) kit for DCP lacks adequate sensitivity to detect small HCC. Thus, a revised EIA kit for DCP has been developed. In this revised DCP kit, the blank value has been reduced, making it now possible to obtain a normal value. The authors used this revised EIA kit for DCP with increased sensitivity and evaluated its usefulness as a tumor marker for HCC. METHODS: Serum DCP and alpha-fetoprotein (AFP) levels were determined in 60 patients with HCC, 60 with cirrhosis, 57 with chronic hepatitis, and 273 normal subjects. The cutoff value for the revised DCP kit was determined to be 40 mAU/mL, and the values for the conventional DCP kit and AFP were 100 mAU/mL (0.1 AU/mL) and 20 ng/mL, respectively. RESULTS: The mean DCP value was 17.5 mAU/mL in the normal subjects, and the detection limit was 10 mAU/mL for this revised DCP kit. The positivity rate for DCP in patients with HCC was 60% by the revised DCP kit, in contrast to 40% by the conventional DCP kit. The sensitivity, specificity, and accuracy of the revised kit were 60%, 92.3%, and 81.4%, respectively, whereas those of the conventional kit were 40%, 98.3%, and 78.5%. Thirty-five percent of HCC tumors smaller than 2 cm and 78.1% of those larger than 3 cm were positive for DCP by the revised kit. The corresponding figures were 20% and 56.3% with the conventional kit. Twelve (33.3%) of the 36 HCC patients who were negative for DCP by the conventional kit were positive by the revised kit. When the revised DCP kit was used in combination with AFP, 86.7% of the HCC patients and 78.3% of the patients with solitary HCC were positive for at least 1 of these markers. CONCLUSIONS: The revised DCP kit is more useful than the conventional DCP kit as a tumor marker for HCC and should be used in combination with AFP.

Adult↗

PubMed1999

Deficiency of IL-5 receptor alpha-chain selectively influences the development of the common mucosal immune system independent IgA-producing B-1 cell in mucosa-associated tissues.

Deletion of IL-5R alpha-chain (IL-5R alpha-/-) selectively influenced the mucosal IgA responses in vivo. While levels of IgA in mucosal secretions were more reduced in IL-5R alpha-/- mice than in wild-type mice, the levels of IgA in serum were not changed. The frequency of IgA-producing cells was reduced in mucosal effector sites (e.g., intestinal lamina propria and nasal passage), but not in inductive sites such as Payer's patches and nasal-associated lymphoreticular tissues in IL-5R alpha-/- mice. IgA-committed (surface IgA+; sIgA+) B-1 cells mainly resided in mucosal effector tissues, while conventional sIgA+ B (B-2) cells formed in mucosal inductive sites of wild-type mice. In contrast, in the effector tissue of IL-5R alpha-/- mice, sIgA+ B-1 cells, but not sIgA+ B-2 cells in the inductive site, were significantly reduced. IL-5R alpha was more expressed on sIgA+ B-1 cells than was IL-6R, while both IL-5R alpha and IL-6R were expressed on sIgA+ B-2 cells in wild-type mice. sIgA+ B-1 cells produced high levels of IgA with rIL-5 rather than of rIL-6 in vitro. Taken together, the findings suggest that the IL-5/IL-5R signaling pathway is critically important for the development of common mucosal immune system independent sIgA+ B-1 cell in mucosal effector tissues in vivo.

Animals↗

PubMed1999

Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice.

BACKGROUND: TRAF6, a member of the tumour necrosis factor receptor-associated factor family, was first identified as a transducer of CD40 and interleukin-1 receptor (IL-1R) signals based on the interaction of TRAF6 with the cytoplasmic tail of CD40 and with the IL-1R associated kinase in vitro. However, the functions of TRAF6 in vivo remain unidentified. RESULTS: We show that TRAF6-/- mice exhibit severe osteopetrosis and are defective in osteoclast formation. In vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6-/- mice are unable to differentiate to functional osteoclasts in response to osteoclast differentiation factor (ODF). In bone marrow of TRAF6-/- mice, the number of sIgM+B220+ immature B cells is markedly reduced while the ratio of proB to preB cells is not affected. In contrast, development of thymocytes is not affected. Furthermore, TRAF6-/- mice are defective in lymph node organogenesis and IL-1 signalling in thymocytes. CONCLUSIONS: The results identify TRAF6 as an essential component of ODF signalling pathway, and also show that TRAF6 plays pivotal roles in immune and inflammatory systems in vivo.

Animals↗

PubMed1999

Sophoricoside analogs as the IL-5 inhibitors from Sophora japonica.

Fruits of Sophora japonica L. (Leguminosae) exhibited an inhibitory effect in the IL-5 bioassay of mIL-5-dependent Y16 proliferation. The isoflavonoids of sophoricoside, genistein, orobol, and genistin were isolated as the IL-5 inhibitors from fresh fruits of the plant by activity-guided fractionation. Among the IL-5 inhibitors, sophoricoside exhibited the highest inhibitory effect with 89% inhibition at 12.5 microM, 82% at 6.3 microM, 72% at 3.1 microM, 59% at 1.6 microM, and 24% at 0.8 microM where the 50% inhibition (IC50) was shown at the concentration of 1.5 microM. Oxyphenylbutazone as the positive control exhibited the IC50 value at the concentration of 31.7 microM. In the order of IC50 values the inhibitory potency in the IL-5 bioassay was sophoricoside > orobol (9.8 microM) approximately equal to genistin (10.6 microM) > genistein (51.9 microM). The position of O-glycosylation and number of hydroxy groups in the isoflavonoids seem to play an important role in the inhibitory effect in the IL-5 bioassay.

Animals↗

PubMed1999

IgG1 production by sIgD+ splenic B cells and peritoneal B-1 cells in response to IL-5 and CD38 ligation.

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induces proliferation, IL-5 receptor alpha chain expression and tyrosine phosphorylation of Bruton's tyrosine kinase. Furthermore, stimulation of splenic B cells with IL-5 together with CS/2 induces Blimp-1 expression and differentiation into Ig-producing cells. Here we examined the role of IL-5 in IgG1 and IgA production by B cells isolated from the spleen and peritoneal cavity. CD38 recognized by CS/2 was expressed in the follicular mantle B cells surrounding the germinal center, sIgD+ splenic B cells and peritoneal B cells. IL-5 induced IgG1 production in splenic sIgD+ B cells stimulated with CS/2, while it was ineffective to induce IgA production. Among the various cytokines tested, only IL-5 had a synergistic effect on IgG1 production with CS/2. IL-5 could induce the generation of S micro-Sgamma1 reciprocal recombination DNA products in CS/2-stimulated B cells. IL-4 was ineffective to induce either micro-gamma1 switch recombination or IgG1 secretion with CS/2, demonstrating that IL-5 promotes both micro-gamma1 switch recombination and IgG1 secretion in an IL-4-independent manner. The peritoneal B-2 cells exhibited both IgG1 and IgA production in response to IL-5 plus CS/2, while B-1 cells produced IgG1. These results imply that the pattern of differentiation to Ig-producing cells seen with peritoneal B cells is not identical to the pattern seen with splenic B cells and that peritoneal B-2 cells contain precursors of IgA-producing cells responding to IL-5 plus CS/2.

ADP-ribosyl Cyclase↗

PubMed1999

Requirement of IL-5 for induction of autoimmune hemolytic anemia in anti-red blood cell autoantibody transgenic mice.

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activate B-1 cells in vivo in normal mice and anti-red blood cell autoantibody transgenic mice (HL mice). To assess the exact role of IL-5 in proliferation and activation of peritoneal B-1 cells, we analyzed IL-5 receptor alpha chain-deficient HL (IL-5Ralpha-/- x HL) mice generated by the cross between IL-5Ralpha-/- and HL mice. In IL-5Ralpha-/- x HL mice, Ig-producing B-1 cells in the peritoneal cavity were negligible, although the total number of B-1 cells in the peritoneal cavity were as many as 30% of that in HL mice. Moreover, LPS- or IL-10-induced differentiation of B-1 cells into antibody-producing cells was severely impaired in IL-5Ralpha-/- x HL mice. We also used in vivo 5-bromo-2'-deoxyuridine labeling to estimate the proliferation of B-1 cells in IL-5Ralpha-/- mice. The absence of IL-5Ralpha did not affect spontaneous proliferation of peritoneal B-1 cells. However, induced proliferation of peritoreal B-1 cells by oral administration of LPS was markedly impaired in IL-5Ralpha-/- mice. These results suggest that IL-5 is required for activation-associated proliferation of B-1 cells but not for their spontaneous proliferation and support the idea that IL-5 plays an important role on the induction of autoantibody production from B-1 cells.

Anemia, Hemolytic, Autoimmune↗

PubMed1999

Distinctive roles of Fyn and Lyn in IgD- and IgM-mediated signaling.

Src family kinases Fyn and Lyn associate with the B cell antigen receptor (BCR). Accumulating data show that Lyn plays important roles in BCR-mediated signaling, while the role of Fyn remains obscure. Here we dissected the role of Fyn and Lyn in BCR signaling using B cells from fyn(-/-), lyn(-/-) and fyn/lyn double-deficient (fyn(-/-)lyn(-/-)) mice. In contrast to previous reports, fyn(-/-) B cells were slightly hyporeactive to both anti-IgM and anti-IgD-dextran. Although lyn(-/-) B cells were hyper-reactive to anti-IgM, anti-IgD-induced proliferation was impaired in lyn(-/-) B cells. Most of the other phenotypes of fyn(-/-)lyn(-/-) mice were similar to that of lyn(-/-) mice, except that proliferative responses of B cells to various stimuli, such as BCR cross-linking and lipopolysaccharide, were significantly lower in fyn(-/-)lyn(-/-) mice than in lyn(-/-) mice. Finally, immune responses to thymus-independent type 2 antigen were affected in these mutant mice. These observations suggest that Fyn and Lyn are involved in B cell functions, and play similar, but partly distinct, roles in BCR signaling.

Animals↗

PubMed1999

Identification of amino acid residues of the T-cell epitope of Mycobacterium tuberculosis alpha antigen critical for Vbeta11(+) Th1 cells.

Stimulation of Mycobacterium tuberculosis-primed lymph node cells from C57BL/6 mice with alpha antigen (also known as antigen 85B and MPT59) induced cell proliferation, production of interleukin 2 and gamma interferon, and expansion of Vbeta11(+) CD4(+) T cells in conjunction with antigen-presenting cells in an I-A(b)-restricted manner. Using a series of 15-amino-acid peptides that overlapped each other by 5 amino acids and spanned the mature alpha antigen, we identified the antigenic epitope for alpha antigen-specific Vbeta11(+) Th1 cells. That peptide (peptide-25), which corresponds to amino acid residues 240 to 254 of alpha antigen, contains a motif that is conserved in I-A(b) and requires processing by antigen-presenting cells. Using peptide-25-reactive Vbeta11(+) T-cell clones and substituted peptide-25 mutants, we determined which amino acid residues within peptide-25 were critical for T-cell receptor (TCR) recognition. Our results showed that the amino acid residues at positions 245, 246, 248, 250, and 251 are important for recognition of TCRVbeta11 and that residues at positions 244, 247, 249, and 252 are I-A(b) contact residues. We also observed that active immunization of C57BL/6 mice with peptide-25 can lead to decreased bacterial load in the lungs of M. tuberculosis H37Rv-infected mice. These results should provide us with a useful tool for delineating the regulation of Vbeta11(+) Th1-cell development during M. tuberculosis infection and for developing a vaccine inducing a Th1-dominant immune response.

Acyltransferases↗

PubMed1999

Eosinophil and IgE responses of IL-5 transgenic mice experimentally infected with Nippostrongylus brasiliensis.

Eosinophil and IgE responses of interleukin (IL)-5 transgenic and normal C3H/HeN mice were studied after experimental infection with Nippostrongylus brasiliensis (Nb). Intestinal worms were recovered at day 5 post-infection (PI), and numbers of total white blood cells (WBC) and eosinophils, and total serum IgE and anti-hapten (dinitrophenyl) (DNP) specific IgE titers, were measured at days 0, 14 and 21 PI. IL-5 mice appeared resistant to Nb infection showing a significantly lower worm recovery rate than normal mice (P < 0.05). Total WBC and eosinophil counts (/mm3) were significantly increased in Nb infected normal mice (P < 0.05), but unchanged (total WBC) or decreased (eosinophils) in IL-5 mice at day 21 PI. The total serum IgE level remarkably increased in normal mice, but only a little in IL-5 mice at days 14 and 21 PI. Priming with DNP brought about more remarkable increases of the total and anti-DNP specific IgE in normal mice than in IL-5 mice. The results show that IL-5 mice are resistant to Nb infection, and that eosinophil and IgE responses in these mice are not augmented by Nb infection.

Animals↗

PubMed1999

Effect of overproduction of interleukin 5 on dinitrofluorobenzene-induced allergic cutaneous response in mice.

The effect of overproduction of interleukin (IL) 5 on the allergic cutaneous response was investigated in transgenic mice overexpressing IL-5. Five repeated topical applications of 2, 4-dinitrofluorobenzene (DNFB) to the ears of mice resulted in allergic dermatitis on the ears as well as significant elevation in dinitrophenol-specific IgE antibody and total IgE in the serum in both wild-type and transgenic mice. The development of dermatitis as measured by skin thickness and histopathological changes were potentiated in the transgenic mice. In IL-5 transgenic mice, significant accumulation of eosinophils in skin lesions was observed after five paintings of DNFB, and the magnitudes of eosinophilia and IL-5 messenger RNA expression were significantly higher than in wild-type mice. The dinitrophenol-specific and total IgE in the serum were higher in IL-5 transgenic mice. The late phase reaction of IgE antibody-mediated biphasic cutaneous response was potentiated in IL-5 transgenic mice. The magnitudes of vasopermeability increase by passive cutaneous anaphylaxis, serotonin, and platelet-activating factor were similar in both mice. These results indicate that overproduction of IL-5 resulted in the potentiation of DNFB-induced dermatitis by elevation of IgE production, IgE-mediated allergic late-phase cutaneous reaction, and eosinophilia in the skin lesion.

Animals↗

PubMed1998

T-cell-receptor signalling in inositol 1,4,5-trisphosphate receptor (IP3R) type-1-deficient mice: is IP3R type 1 essential for T-cell-receptor signalling?

Stimulation of T-cells via the T-cell receptor (TCR) complex is accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i). Recently, it was reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of inositol 1,4,5-trisphosphate receptor (IP3R) type 1 (IP3R1), failed to demonstrate increased [Ca2+]i or interleukin-2 production after TCR stimulation and was also resistant to apoptotic stimuli. This cell line lacked IP3R1 expression, but expressed the type-2 and -3 receptors, IP3R2 and IP3R3 respectively [Jayaraman, Ondriasova, Ondrias, Harnick and Marks (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6007-6011, and Jayaraman and Marks (1997) Mol. Cell. Biol. 17, 3005-3012]. The authors concluded that IP3R1 is essential for TCR signalling and suggested that Ca2+ release via IP3R1 is a critical mediator of apoptosis. To establish whether a loss of IP3R1 function in T-cells occurred in vivo and in vitro, we investigated Ca2+ signalling after TCR stimulation and the properties of T-cells using IP3R1-deficient (IP3R1-/-) mice. As IP3R1-/- mice die at weaning, we transplanted bone marrow cells of IP3R1-/- mice into irradiated wild-type mice. Western blot analysis showed that the recipient IP3R1-containing (IP3R1+/+) lymphocytes were replaced by the donor IP3R1-/- lymphocytes after transplantation and that expression of IP3R2 and IP3R3 was unaltered. In contrast with the previous reports, T-cells lacking IP3R1 were able to mobilize Ca2+ from intracellular Ca2+ stores after stimulation via the TCR. We observed no significant differences between IP3R1+/+ and IP3R1-/- T-cells in terms of the number of thymocytes and splenocytes, the proportion of the T-cell phenotype, proliferative response to anti-CD3 monoclonal antibody (mAb) stimulation and cell viability. Therefore IP3R1 is not essential for T-cell development and function.

Animals↗

PubMed1998

JAK2 and JAK1 constitutively associate with an interleukin-5 (IL-5) receptor alpha and betac subunit, respectively, and are activated upon IL-5 stimulation.

The human interleukin-5 receptor (hIL-5R) consists of a unique alpha subunit (hIL-5Ralpha) and a common beta subunit (betac) that activate two Janus kinases (JAK1 and JAK2) and a signal transducer and activator of transcription (STAT5). The precise stoichiometry of the hIL-5R subunits and the role of JAK kinases used in IL-5 signaling were investigated. We analyzed the interaction between hIL-5Ralpha and betac by immunoprecipitation using anti-hIL-5Ralpha and anti-betac monoclonal antibodies. The binding of JAK1 and JAK2 to each hIL-5R subunit was also evaluated in the hIL-5-responsive cell line, TF-h5Ralpha. It was observed that IL-5 stimulation induced the recruitment of betac to hIL-5Ralpha, although in the absence of IL-5 the subunits remain independent. In the absence of IL-5, JAK2 and JAK1 were associated with hIL-5Ralpha and betac, respectively. IL-5 stimulation resulted in tyrosine phosphorylation of JAK2, JAK1, betac, and STAT5. Moreover, IL-5-induced dimerization of IL-5R subunits caused JAK2 activation and betac phosphorylation even in the absence of JAK1 activation. Furthermore, tyrosine phosphorylation of JAK1 was dependent on the activation of JAK2. Detailed study of the C-terminal truncated cytoplasmic domain of hIL-5Ralpha revealed that the cytoplasmic stretch at position 346-387, containing the proline-rich region, is necessary for JAK2 binding. These observations suggest that activation of hIL-5Ralpha-associated JAK2 is indispensable for the IL-5 signaling event.

Humans↗