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K Takatsu

Publications and source records attributed to K Takatsu.

At least 145 records · Page 8Linked to original sources
PubMed1991

Murine interleukin 5 receptor isolated by immunoaffinity chromatography: comparison of determined N-terminal sequence and deduced primary sequence from cDNA and implication of a role of the intracytoplasmic domain.

The murine interleukin 5 receptor (IL-5R) was identified by utilizing an immobilized IL-5 and an immobilized monoclonal antibody against the murein IL-5R (designated H7 mAb). The H7 mAb immunoaffinity-purified materials from the extract of cell-surface radioiodinated T88-M cells (an IL-5-dependent early B cell line) using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were reacted with an immobilized IL-5 matrix. SDS-PAGE of the adsorbed fraction revealed a single band at approximately 60 kDa. The binding of the 60 kDa protein to the immobilized IL-5 matrix was inhibited by the excess IL-5. The CHAPS-extract depleted of the 60 kDa protein by the absorption with H7 mAb did not contain any IL-5 binding proteins. Immunoaffinity procedure provided a final 7400-fold purification, based on an estimation of the content of the 60 kDa protein (approximate purity: 20%) from the silver-stained pattern of SDS-PAGE. Actin was copurified with the 60 kDa protein at an approximate ratio of 1:1, suggesting that the intracytoplasmic domain of the IL-5R may interact with actin. Furthermore, soluble IL-5R (molecular mass: 50 kDa) was purified by the H7 mAb-immunoaffinity chromatography. The purified soluble IL-5R was capable of inhibiting the binding of IL-5 to T88-M cells. Preparative SDS-PAGE followed by electroblotting onto a membrane permitted the determination of the N-terminal sequence of the IL-5R. The determined N-terminal sequence of the IL-5R and the deduced primary sequence from recently isolated cDNA were compared.

Actins↗
PubMed1991

T-cell-dependent accumulation of eosinophils in the lung and its inhibition by monoclonal anti-interleukin-5.

The transnasal administration of an extract of the parasite Ascaris suum to C57BL/6 mice for 3 weeks produced marked eosinophilia in the bronchoalveolar lavage (BAL) fluid. The oral administration of ciclosporin significantly suppressed the pulmonary eosinophilia. Athymic C57BL/6-nu/nu mice failed to develop pulmonary eosinophilia. These data indicate that the pulmonary eosinophilia caused by this parasite extract is T-cell-dependent. Genetically mast-cell-deficient (WB x C57BL/6) F1-W/Wv (W/Wv) mice developed marked eosinophilia in the BAL, which shows that mast cells are not necessary in the formation of lung eosinophilia in this model. Monoclonal antimurine interleukin-5 injected intraperitoneally clearly inhibited the infiltration of eosinophils in the lung, suggesting that T-cell-derived interleukin-5 is essential.

Animals↗
PubMed1991

Role of interleukin-5 in local accumulation of eosinophils in mouse allergic peritonitis.

The injection of ragweed pollen extracts into the peritoneal cavity of actively immunized mice caused an accumulation of eosinophils in the cavity. We investigated the role of interleukin-5 (IL-5), one of the T-cell-derived factors, in this in vivo local accumulation. Ciclosporin, an immunosuppressant that mainly suppresses T cell functions, dose-dependently inhibited the in vivo local accumulation. Injections of the neutralizing antibody to IL-5 into the cavity decreased the number of the accumulating eosinophils. When the peritoneal cells of the immunized mice were cultured in the presence of the antigen in vitro, IL-5 activity was detected in the culture supernatant. The results indicate that IL-5 plays an important role in the in vivo local accumulation of eosinophils after an antigen challenge.

Allergens↗
PubMed1991

[Genetic engineering of cytokine system on the tuberculosis research].

Murine IL-5 is an acidic glycoprotein with a molecular mass of 40 to 50 kda that consists of homodimer of which monomer comprises 113 amino acid residues. Murine IL-5 has pleiotropic activities on various target cells. IL-5 acts on committed B cells to induce differentiation into Ig-producing cells and on common progenitors for Ly-1+ pre-B cells and Ly-1+ macrophages to support their growth. It also supports growth and/or differentiation of eosinophil precursor and mature eosinophil. IL-5 specifically binds to its receptor (IL-5R) with high and low affinity. IL-5 signals appears to be transduced through the high affinity IL-5R that is composed of two different polypeptide chains; alpha chain and beta chain. The IL-5R alpha chain cDNA encodes p60 that binds IL-5 with low affinity. The IL-5R beta chain, p130/p140 does not bind IL-5 by itself, but binds to alpha chain/IL-5 complex to convert them to the high affinity IL-5R. The beta chain was identified as the IL-3R homologue, AIC2B. Both alpha and beta chain of IL-5R belong to a cytokine receptor superfamily.

Animals↗
PubMed1991

The role of interleukin-5 in protective immunity to Strongyloides venezuelensis infection in mice.

We depleted or neutralized interleukin-5 (IL-5) and IL-5 receptor of C57BL/6 mice, using rat anti-murine IL-5 monoclonal antibody (NC17) and anti-murine IL-5 receptor monoclonal antibody (H7). Mice treated with these monoclonal antibodies were infected with Strongyloides venezuelensis larvae. The time-course of faecal egg output and peripheral eosinophilia were monitored. In a primary infection, anti-IL-5 treatment did not affect faecal egg output, although the eosinophil count in peripheral blood was markedly reduced. There was no difference in intestinal worm burden or faecal egg output between anti-IL-5 treated and non-treated mice. In a secondary infection, worms were expelled from the small intestine of anti-IL-5-treated mice as well as from non-treated mice. Worm recovery from the lungs of mice treated with either anti-IL-5 or anti-IL-5 receptor monoclonal antibody was the same as that of normal controls. However, a marked reduction in worm recovery was observed in re-infected mice that had not been treated with monoclonal antibodies. Treatment with anti-IL-5 or anti-IL-5 receptor monoclonal antibody suppressed blood and tissue eosinophilia. Thus the results suggested that the host's protective immunity against tissue-migrating larvae was IL-5-dependent but intestinal immunity was not.

Animals↗
PubMed1990

Interleukin 5 and interleukin 3 induce serine and tyrosine phosphorylations of several cellular proteins in an interleukin 5-dependent cell line.

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and eosinophils. To elucidate the intracellular events via the IL-5 receptor, we have investigated IL-5-induced protein phosphorylation in an IL-5-dependent murine early B-lineage cell line (T88-M). The rapid phosphorylation of a 60-kDa protein at serine residues and of 140-, 92-, 53-, 48-, and 45-kDa proteins at tyrosine residues were induced by the stimulation of T88-M cells with IL-5. T88-M cells were found to proliferate in response to IL-3 as well as IL-5, and the phosphoproteins in T88-M cells could be induced by IL-3 stimulation in a similar manner of those induced by IL-5. Thus, these results suggest that the protein phosphorylation events in T88-M cells may be shared by both pathways via the IL-5 receptor and the IL-3 receptor.

Animals↗
PubMed1990

Role of IL-5 in IL-2-induced eosinophilia. In vivo and in vitro expression of IL-5 mRNA by IL-2.

We recently demonstrated in vivo that IL-5 was an important mediator of eosinophilia in mice with parasite infections. In this study, we examined whether or not IL-5 was actually responsible for the eosinophilia induced by injection of human rIL-2. Mice administered hIL-2 developed eosinophilia during the course of the series of injections. This eosinophilia could be suppressed by a single injection of mAb against murine IL-5. The number of eosinophilic precursors increased more in the spleen cells of the IL-2-treated mice in comparison to the control mice, although in bone marrow precursors showed little change. Similarly, the number of granulocytic precursors increased markedly in the spleen cells of IL-2-treated mice. In vitro polymerase chain reaction amplification of cDNA subfragments corresponding to IL-5 mRNA (reverse transcription-polymerase chain reaction), followed by Southern blot analysis, revealed enhancement of IL-5 mRNA expression in spleen cells 3 days after starting the human IL-2 injections. We also observed enhanced murine IL-5 mRNA expression in spleen cells stimulated with IL-2 in vitro. The level of murine IL-5 mRNA expression by IL-2-stimulated spleen cells from normal mice increased at 24 h, reached a plateau after 48 h, and decreased again within 72 h of starting culture. These results indicate that the eosinophilia induced by IL-2 in vivo is presumably mediated by IL-5 released from IL-2-stimulated lymphocytes.

Animals↗
PubMed1990

Analysis of Th1 and Th2 cells in murine gut-associated tissues. Frequencies of CD4+ and CD8+ T cells that secrete IFN-gamma and IL-5.

After Ag and/or mitogen stimulation, cloned mouse Th1 and Th2 cells produce different cytokines that contribute to induction of particular B cell isotype responses. In this regard, IL-5 produced by Th2 cells has been shown to enhance IgA synthesis in LPS-triggered splenic (SP) B cell or in unstimulated Peyer's patch (PP) B cell cultures. This raises the possibility that Th2 cells may occur in higher frequency in gut-associated tissues, because B cells in these areas are committed to IgA synthesis. We have used an ELISPOT assay to detect individual T cells producing IFN-gamma or IL-5. For the IL-5 assay, the mAb TRFK-5 and biotinylated TRFK-4 were used in coating and detection, respectively, whereas the mAb R4-6A2 and biotinylated XMG 1.2 were similarly used for enumeration of IFN-gamma-specific spot forming cells (SFC). Specificity of each assay was tested by using Con A-activated, cloned Th1 (H66-61) or Th2 (CDC-25) cells, where the Th1 cells only produced IFN-gamma SFC and the Th2 cells only gave IL-5-specific spots. Further, preincubation of biotinylated TRFK-4 or XMG 1.2 with rIL-5 or IFN-gamma, respectively, abrogated the formation of specific spots when tested with Con A-activated SP CD4+ T cells. Both IFN-gamma and IL-5 were produced de novo, because treatment of T cells with cycloheximide inhibited both IFN-gamma and IL-5 SFC. We have assessed the numbers of T cells spontaneously secreting these cytokines in PP and in lamina propria and intraepithelial lymphocyte (LPL and IEL) populations. Moderate levels of IL-5 SFC occurred in the IEL subset, whereas higher levels existed in the LPL population. Although significant numbers of IFN-gamma SFC (Th1-type) were also seen in LPLs, the frequency of IL-5 SFC was always higher (Th1:Th2 in LPL = 1:3). In IELs, equal numbers of IFN-gamma and IL-5 SFC were seen. Interestingly, CD8+ IEL T cells produced these two cytokines. In contrast, T cells freshly isolated from PP, an IgA inductive site, contained smaller numbers of IL-5- or IFN-gamma-secreting cells and SP T cells had essentially no SFC. When PP or SP T cells were stimulated with Con A, significant and approximately equal numbers of IFN-gamma- and IL-5-producing cells appeared.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Distribution of IL-5 receptor-positive B cells. Expression of IL-5 receptor on Ly-1(CD5)+ B cells.

mAb to murine IL-5R were prepared by means of fusion between mouse myeloma cells and spleen cells from a rat immunized with membrane-enriched fractions of IL-5-dependent early B cell line (T88-M). Two mAb (H7 and T21) were selected for their competitive inhibition of receptor binding by 35S-labeled IL-5 and of IL-5 biologic activities. The number of binding sites recognized by the mAb on different cell lines correlated with IL-5 responsiveness. Most surface IgM+ peritoneal B cells were H7+ and more than 70% were also Ly-1(CD5)dull+, and responded to IL-5 for polyclonal IgM production in a high frequency. A significant proportion of splenic B cells reacted with these mAb, although lower number (one-log less) than peritoneal B cells and a small proportion of H7dull+ splenic B cells seems to be Ly-1(CD5)dull+, 1 of 200 splenic B cells responded to IL-5 for IgM production. These results suggest that IL-5R+ B cells may consist of a subpopulation of B cells. Intriguingly, lymphoid populations of bone marrow cells were stained with H7 and T21, whereas myeloid populations were brightly stained with only T21. Finally, both H7 and T21 mAb specifically precipitated a protein of a Mr 60,000 from 125I-labeled cell lysates of IL-5R+ T88-M cells. The IL-5R with similar size (Mr 55,000 to 60,000) was precipitated from the cell lysates of peritoneal B cells. T21 mAb but not H7 mAb precipitated a protein of a Mr 110,000 from the cell lysates of bone marrow cells.

Animals↗
PubMed1990

Detection of individual mouse splenic T cells producing IFN-gamma and IL-5 using the enzyme-linked immunospot (ELISPOT) assay.

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and IL-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or IL-5, and should be useful for detection of cytokine secretion at the single cell level.

Animals↗
PubMed1990

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5.

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.

Animals↗
PubMed1990

Retroviral interleukin 5 gene transfer into interleukin 5-dependent growing cell lines results in autocrine growth and tumorigenicity.

Two interleukin 5 (IL5)-specific retroviral expression vectors have been constructed containing the neomycin gene as selectable marker and either the mouse IL5 cDNA region or the rat genomic IL5 gene under the control of the thymidine kinase promoter. High viral titer supernatants derived from the transfected or infected packaging cell line psi 2 were used to infect the two cell lines B13 and T88M whose growth is dependent on exogenous IL 5. Infection resulted in G418 resistance and IL 5-independent growth with a high frequency. Clones were established which secrete between 2 and greater than 1000 U IL5. The proliferation of the IL5 autocrine growing cells could be inhibited by an antibody directed against the IL5 receptor indicating that they grow as a result of the endogenously produced IL5. Regardless of the amount of IL5 they produced, all of the clones were highly tumorigenic in nucle mice. The phenotype of the tumors was indistinguishable from that of the injected cells. T88M or B13 cells infected with a control virus neither produced IL5, nor became factor independent, nor produced tumors. Together, the IL5 gene transfer and expression into IL5-dependent growing cells are in accordance with the "autocrine growth" hypothesis and contrast analogous experiments with IL4.

Animals↗
PubMed1990

Molecular cloning and expression of the murine interleukin-5 receptor.

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.

Amino Acid Sequence↗
PubMed1990

Structural comparison of murine T-cell (B151K12)-derived T-cell-replacing factor (IL-5) with rIL-5: dimer formation is essential for the expression of biological activity.

T-cell-replacing factor (TRF)/IL-5 is a T-cell-derived glycoprotein which has pleiotropic activity on lymphoid and myeloid cells. IL-5 polypeptide translated into Xenopus oocytes are heterogeneous in molecular size (40,000 to 60,000 under nonreducing conditions) and yields a monomeric form (Mr of 25,000 to 30,000) under reducing conditions (J. Immun., 140, 1175-1181, 1988). We purified T-cell-derived TRF and rIL-5 using anti-TRF/IL-5 antibody-coupled affinity column from supernatants of a T-cell hybridoma B151K12 and supernatants of HeLa cells, respectively, which had been transfected with murine IL-5 cDNA, and determined their partial N-terminal amino acid sequence (27 residues for B151-TRF and 13 residues for rIL-5). A single amino acid sequence of each sample obtained beginning from methionine that was identical to that predicted from IL-5 cDNA. This finding supports the notion that secreted B151-TRF polypeptide consists of 113 amino acids. Purified B151-TRF supported eosinophilopoiesis of human bone marrow cells as effective as mouse rIL-5 and human rIL-5. B151-TRF competitively inhibited 35S-labeled rIL-5 binding to target cells to the same extent at rIL-5. Treatment of purified rIL-5 and B151-TRF with reducing reagents such as 2-ME, sodium borohydride or dithiothreitol produced a monomeric form of IL-5 which did not exert a biological activity. Reduction and alkylation of rIL-5 caused the loss of binding to its target cells. These results strongly suggest that B151-TRF exists as a homodimer and its primary structure and secondary structures are identical to those of rIL-5. Moreover, the formation of inter-molecular disulfide bond(s) linked by two pairs of cystein residues is essential for the expression of the biological activity of mouse IL-5.

Amino Acid Sequence↗
PubMed1990

Characterization of the murine interleukin 5 receptor by using a monoclonal antibody.

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. The receptor for IL-5 has been identified as two cross-linked complexes on T88-M cells (a murine IL-5-dependent early B cell line). In this study the IL-5 receptor was directly characterized by utilizing an immobilized IL-5 column and a rat monoclonal antibody, designated H7, directed against the IL-5 receptor. H7 completely inhibited specific binding of 35S-labeled IL-5 to T88-M cells, and bound to IL-5-responsive cells, e.g. T88-M, BCL1-B20 (a chronic B-cell leukemia), and MOPC104E (a myeloma), whereas H7 did not bind to IL-5-non-responsive cells, e.g. X5563 (a myeloma), FDC-P1 (an IL-3-dependent line), and MTH (an IL-2-dependent CTLL). H7 could barely bind to T88-M cells in the presence of IL-5, and immunoprecipitated a major band with an Mr of approximately 60 kd from the extract of surface-radioiodinated T88-M cells. The precipitation of this 60 kd molecule was inhibited by the addition of IL-5. Analysis with immobilized IL-5 also revealed that a 60 kd molecule bound specifically to IL-5-coupled beads compared with control beads. Furthermore, no additional molecule with a higher Mr that was recognized by H7 appeared under non-reducing, compared with reducing, conditions. The 60 kd molecule recognized by H7 could be digested with N-glycanase to yield a protein band of approximately 55 kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Elevated expression of proto-oncogenes during interleukin-5-induced growth and differentiation of murine B lineage cells.

Interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. To elucidate IL-5-mediated intracellular mechanisms, we have established IL-5-dependent and -independent murine early B cell lines, J6 and MJ88-1, respectively, and examined the effect of IL-5 on the expression of proto-oncogenes during proliferation. Two- to 3.5-fold increases in the levels of c-myb, c-myc, c-fos, and c-fms mRNA were observed in J6 cells, compared with those in MJ88-1 cells. Further, a role of IL-5 in the proto-oncogene expression during differentiation was examined by using thymidine-treated murine B-cell chronic leukemia BCL1-B20 cells with growth arrest. After 4-day culture, the amount of IgM secreted from BCL1-B20 cells was augmented 4-6 fold in the presence of IL-5. Although expression of c-myb, c-fos, and c-fms mRNA did not change, only c-myc mRNA expression was elevated within 30 min of stimulation with IL-5 and reached a maximal level by 1 hr. Addition of phorbol 12-myristate 13-acetate (PMA) or IL-4 to the culture of BCL1-B20 cells inhibited both the IL-5-mediated augmentation of IgM secretion and the elevated expression of c-myc mRNA. These findings suggest that the IL-5 signal may be associated with the up-regulation of c-myc expression.

Animals↗