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Biomedical subjects

K Takatsu

Publications and source records attributed to K Takatsu.

At least 163 records · Page 9Linked to original sources
PubMed1990

Interleukin 5 activity in sera from patients with eosinophilia.

Sera from 10 patients with eosinophilia contained eosinophil colony stimulating factor (Eo-CSF) activity. Using anti murine (m) interleukin-5 (IL-5) antibody, we demonstrated that this activity was mainly derived from IL-5. Administration of prednisolone to patients decreased both Eo-CSF activity in sera and the number of eosinophils in blood. These results extend our recent study demonstrating that T cells from eosinophilic patients produce IL-5 with IL-2 stimulation and may support the speculation that IL-5 is an important factor which induces eosinophilia.

Blood Cell Count↗
PubMed1990

Conversion of normal Ly-1-positive B-lineage cells into Ly-1-positive macrophages in long-term bone marrow cultures.

We obtained eight different cell lines in the long-term bone marrow culture system that showed a germ-line configuration of the joining (J) region segments of the Ig heavy-chain (IgH) genes. Their surface markers were CD45R+, Ly-1+, Lyb-2+, cIgM-, sIgM-, Ia-, Thy-1-, Mac-1-, and IL-2R (Tac)+. Use of very young mice and the presence of IL-5 were important for preferential promotion of the survival of B-lineage lymphocytes bearing the Ly-1 markers. When we treated two of them (J8 and J10) with 5-azacytidine for 24 h followed by co-culture with stromal cells and IL-5, they became Ly-1+, sIgM+ B cells, and Ly-1+, Mac-1+ macrophagelike cells, respectively. After other early lymphoid lines (J1, J8, and J13) were maintained by co-culture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene, although only J13 rearranged the kappa-light chain gene. Northern blot analysis revealed that these cell lines expressed C mu-mRNA, and lambda 5-mRNA, consistent with normal pre-B cells. Intriguingly, J1, J8, and J13 expressed c-fms mRNA constitutively. When J13 cells were co-cultured with ST2 and GM-CSF in place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspecific-esterase-positive, and showed phagocytosis of latex beads. These results support evidence for a close relationship between the myeloid and Ly-1+ B-cell pathways of differentiation, and indicate that our IL-5-dependent clones are multipotential intermediates in differentiation from pro-B cells to B cells and macrophages.

Animals↗
PubMed1990

Spontaneous T cell proliferation and release of soluble interleukin-2 receptors in patients with HTLV-I-associated myelopathy.

Patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) show increased serum levels of soluble interleukin-2 receptors (sIL-2R), a marker of T cell activation. We found that peripheral blood lymphocytes from HAM patients proliferated spontaneously and released sIL-2R when cultured in vitro. Spontaneous proliferation was observed in T cell populations (both CD4+ cells and CD8+ cells), but not in B cell-rich populations or monocyte-rich populations. There was a significant increase of IL-2 activity in the culture supernatants of peripheral blood mononuclear cells (PBMC) after 2-3 days cultivation. On the other hand, sIL-2R concentrations in the supernatants were much higher after 5 days of cultivation. Such spontaneous T lymphocytic proliferation and release of sIL-2R were also found in non-HAM HTLV-I carriers, but not as intensely as in HAM patients. HTLV-I infection causes T cell activation to release IL-2 and sIL-2R; such T cell responses may play a role in the pathogenesis of HTLV-I-associated myelopathy.

Adult↗
PubMed1990

In vivo changes of hemopoietic progenitors and the expression of the interleukin 5 gene in eosinophilic mice infected with Toxocara canis.

It has been demonstrated that purified recombinant interleukin 5 (rIL-5) supports the terminal differentiation and proliferation of eosinophilic precursors in vitro and plays an important role in increasing the functional activities of eosinophils. In this study, we examined the hemopoietic changes and analyzed murine (m) IL-5 mRNA expression in eosinophilic mice infected with the helminth Toxocara canis. In eosinophilic mice, eosinophils increased in number in both bone marrow and spleen. However, the number of eosinophilic precursors increased markedly in spleen cells of eosinophilic mice but remained relatively constant in the bone marrow. In the presence of granulocyte colony-stimulating factor (G-CSF), the number of granulocytic precursors increased in the spleen cells of eosinophilic mice. From these findings, the condition of eosinophilopoiesis in eosinophilic mice is accompanied by an increase in granulocyte-macrophage progenitors as well as eosinophil progenitors. Using Northern blot analysis, a weak but definite band corresponding to mIL-5 mRNA was detected in spleen cells of mice 4 and 5 days after helminthic infection. In addition, these data were confirmed by in vitro polymerase chain reaction (PCR) amplification of mRNA obtained from these spleen cells. Finally, injections of a monoclonal antibody against mIL-5 completely suppressed the blood eosinophilia in mice infected with T. canis. In conclusion, IL-5 is suggested to play a major role in eosinophilopoiesis in vivo.

Animals↗
PubMed1989

Rapid methods for purification of human recombinant interleukin-5 (IL-5) using the anti-murine IL-5 antibody-coupled immunoaffinity column.

A large scale production of human recombinant IL-5 (hIL-5) was performed by way of recombinant DNA technology. In this study, we transfected Chinese hamster ovary cells with pdKCR-hIL-5gene-dhfr plasmid and selected a cell line, with the use of methotrexate, producing large amounts of hIL-5. The recombinant hIL-5 thus obtained induced IgM production of murine B cell leukemia BCL1, and its activity was inhibited by TB13 anti-mouse IL-5 monoclonal antibody. hIL-5 could be purified from the cell-free supernatants of the transfectants with high recoveries by using anti-mouse IL-5 antibody-coupled immunoaffinity column in combination with a gel permeation column chromatography. N terminal amino acid sequence analysis of purified hIL-5 revealed that a single amino-terminal amino acid (isoleucine) is detected and hIL-5 consists of 115 amino acid residues.

Amino Acid Sequence↗
PubMed1989

Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production.

Effects of transforming growth factor beta (TGF-beta) on IgA production by LPS-stimulated B cells have been studied. TGF-beta itself could augment polyclonal IgA production in concomitant inhibition of polyclonal IgM and IgG1 production. Furthermore, TGF-beta and IL-5 additively augmented IgA production. TGF-beta exerted its activity early in the culture (by 2 d in a 5-d culture) and IL-5 was required late in the culture. Surface IgA- (sIgA-) B cells responded to TGF-beta for the development of IgA-secreting cells. By contrast, sIgA+ B cells, but not sIgA- B cells, responded to IL-5 for IgA production. These results suggest that TGF-beta has a differential role in the induction of IgA production from IL-5 on murine-activated B cells.

Animals↗
PubMed1989

Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5.

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.

Actins↗
PubMed1989

T cells from eosinophilic patients produce interleukin-5 with interleukin-2 stimulation.

Anti-murine (m) interleukin-5 (IL-5) antibody was found to inhibit eosinophil (Eo) colony formation stimulated by recombinant human (rh) IL-5, but did not inhibit the production of Eo stimulated by rh IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). Conditioned medium (CM) prepared from eosinophilic patients' T cells with interleukin-2 (IL-2) stimulation (T-IL-2-CM), was found to contain CFU-Eo growth-stimulating factor. Using anti-mIL-5 antibody, we demonstrated that T-IL-2-CM from patients with eosinophilia contained a significant amount of IL-5. We also detected IL-5 mRNA in T cells from eosinophilic patients with IL-2 stimulation. These results suggest that IL-5 plays an important role in the induction of selective eosinophilia in humans and that IL-5 is produced from T cells with IL-2 stimulation.

Adult↗
PubMed1989

Characterization of high-affinity receptors for interleukin 5 on interleukin 5-dependent cell lines.

Interleukin 5 (IL-5) is a glycosylated polypeptide that acts as a key factor for B-cell growth and differentiation. We demonstrated previously that there are two classes (high and low affinity) of IL-5 receptors on murine chronic B-cell leukemic cells (BCL1-B20). Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a polypeptide of Mr 92,500. In this study, we analyzed characteristics of high-affinity IL-5 receptors on IL-5-dependent early B-cell lines (T-88 and T88-M), mouse myeloma cells (MOPC-104E), and BCL1-B20 cells. All cell lines had two classes of IL-5 binding sites, but T88-M cells bore the highest number of high-affinity receptors. The number of high-affinity IL-5 receptors on BCL1-B20 cells could be up-regulated 3-fold by lipopolysaccharide and down-regulated by IL-5. Disuccinimidyl tartarate crosslinking of 35S-labeled IL-5 to the receptors on the T88-M and lipopolysaccharide-stimulated BCL1-B20 cells revealed two major 35S-labeled components of Mr 92,500 and Mr 160,000, even when the binding of 35S-labeled IL-5 was carried out under high-affinity conditions (100 pM 35S-labeled IL-5). The Mr 92,500 component, but not the Mr 160,000 component, was detected in the lysates of MOPC-104E and T-88 cells, both of which bore a large number of low-affinity receptors and a limited number of high-affinity receptors. The results suggest that the Mr 92,500 component represents the complex of IL-5 with the low-affinity Mr 46,500 receptor, whereas the high-affinity receptor consists of the Mr 46,500 peptide and an additional peptide.

Adjuvants, Immunologic↗
PubMed1989

Interferon-gamma inhibits the proliferation but not the differentiation of murine B cells in response to IL-5.

Interferon-gamma (IFN-gamma) is supposed to be produced by type 1 helper T cells (TH1) and inhibits IL-4-dependent B cell growth and differentiation. IL-5 (T cell-replacing factor, TRF), is a T cell-derived lymphokine which is predominantly produced by type 2 helper T cells (TH2) and regulates proliferation and differentiation of activated B cells. In this study, the effect of IFN-gamma on IL-5-dependent B cell growth and differentiation has been studied using murine chronic B cell leukemic cells (BCL1), normal splenic B cells, and cloned early B cell line. IFN-gamma selectively inhibits the IL-5-mediated proliferation of activated B cells as well as cloned early B cell lines at a low concentration (2 U/ml) in which polyclonal IgM production was not affected. This inhibitory effect of IFN-gamma occurs within 24 h after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to reverse totally the IFN-gamma-mediated suppressive effects if it was added later than 24 h after the onset of the culture. On the contrary, IL-5-mediated IgM secretion of BCL1 and IgA formation of LPS-stimulated normal B cells were relatively resistant to the suppressive effect of IFN-gamma. IFN-gamma does not affect the receptor expression for IL-5. Interestingly, IL-4-mediated IgG1 formation of LPS-stimulated B cells was markedly suppressed by IFN-gamma at 10 U/ml. These results strongly suggest that IFN-gamma may have differential effects on IL-5-mediated B cell triggering.

Animals↗
PubMed1989

Purification and characterization of recombinant human interleukin 5 expressed in Chinese hamster ovary cells.

Recombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40,000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22,000 and 20,000. After treatment with endoglycosidase F, a band with an apparent Mr of 18,000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond(s) might be important for the biological activity of rhIL-5.

Amino Acid Sequence↗
PubMed1989

Mechanisms of the interleukin 5-induced differentiation of B cells.

Interleukin 5 (IL5), a lymphokine produced by T cells, induces differentiation of B cell chronic leukemia BCL1-B20 cells into IgM-producing cells accompanied with growth arrest. To elucidate the intracellular mechanisms, the roles of Ca2+ mobilization and protein phosphorylation in the activation of the cells were examined. F(ab')2 fragment of anti-immunoglobulin (anti-Ig), which cross-links membrane-bound Ig, and calcium ionophore A23187 caused a rapid increase in the intracellular free calcium concentration [( Ca2+]i), whereas these stimulants did not give rise to differentiation of the cells. In contrast, treatment with IL5 did not affect either [Ca2+]i or the rates of Ca2+ uptake from the outside and release from the inside of the cells. Analysis by two-dimensional gel electrophoresis revealed that the in vitro phosphorylation of acidic 80-, 60-, and 45-kDa proteins was induced upon stimulation with IL5. Treatment with IL5 also caused a marked decrease in the in vitro phosphorylation of an acidic 100-kDa protein which was highly phosphorylated in the unstimulated state. Addition of phorbol 12-myristate 13-acetate (PMA) to the culture inhibited IL5-mediated differentiative responses. Therefore, these results suggest that Ca2+ mobilization is not involved but activities of stimulatory and inhibitory kinases may be involved in the IL5-mediated differentiation process.

Animals↗
PubMed1989

Establishment of IL-5-dependent early B cell lines by long-term bone marrow cultures.

We established two different IL-5-dependent Ly1+ early B cell lines in long-term bone marrow culture system. One of them (J-87) is stromal cell (ST2) dependent and the other (T-88) is ST2 independent. Both J-87 and T-88 are B220+, Ly1+, sIgM-, Ia-, Thy1-, and IL-2R+, and respond to IL-3 and IL-5 in the presence of ST2. The T-88 can proliferate only in response to IL-5 in the absence of ST2. Southern blot analysis using JH probe revealed that configuration of IgH gene of both cell lines shows rearranged pattern. Binding assay for radiolabeled IL-5 to T-88 demonstrated that T-88 has two classes of IL-5 binding sites (low and high affinity) on the membrane. These data strongly suggest that there are IL-5-sensitive stages at both stromal cell-dependent and stromal cell-independent phases in early B cell development.

Animals↗
PubMed1989

Interleukin-5 induces maturation but not class switching of surface IgA-positive B cells into IgA-secreting cells.

There is a body of evidence suggesting that IgA production is regulated by helper T cells or their products. To elucidate molecular mechanisms of IgA production, the role of lymphokines in the in vitro antigen-specific and polyclonal IgA responses was examined. Supernatants from antigen-stimulated T cells or mitogen-stimulated T-cell clones can enhance 2,4-dinitro phenyl (DNP)-specific IgM, IgG1 and IgA production in cultures of DNP-ovalbumin (OVA)-stimulated T-cell-depleted spleen cells from DNP-keyhole limpet haemocyanin-primed mice. The IgA enhancement was inhibited by anti-IL-5 monoclonal antibody. Purified recombinant IL-5 could also enhance anti-DNP IgA production in a dose-dependent manner. This enhancing effect was not substituted by IL-1, IL-2, IL-3 or IL-4. Polyclonal IgA secretion of lipopolysaccharide-stimulated normal B cells was augmented preferentially by IL-5, but not by IL-4. Surface IgA-positive (sIgA+) B cells, but not surface IgA-negative B cells, responded to IL-5 for the development of IgA-secreting cells. Limiting-dilution analysis revealed that IL-5 increases the frequency of IgA-secreting cells in sIgA+ B-cell populations. These results indicate that IL-5 plays an essential role in the antigen-specific and polyclonal IgA formation as a maturation-inducing factor rather than class-switching factor.

Animals↗
PubMed1988

Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication.

T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1-B20 (in vitro line) (a murine chronic B cell leukemic cell line previously shown to differentiate into IgM-secreting cells in response to IL-5) within 10 min at 37 degrees C. There are two classes of binding sites with high affinity (Kd = 66 pM) and low affinity (Kd = 12 nM) for IL-5 and an average number of binding sites for high affinity and for low affinity were 400 and 7,500 per cell, respectively. The specificity of binding of radiolabeled IL-5 has been confirmed by demonstrating that only unlabeled IL-5 and anti-IL-5 mAb but not by IL-1, IL-2, IL-3, IFN-gamma, and GM-CSF inhibit radiolabeled IL-5 binding to BCL1-B20 cells. Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a membrane polypeptide of Mr 46,500 to which IL-5 is crosslinked. A variety of cell types have been surveyed for the capacity to bind specifically radiolabeled IL-5 with high affinity. BCL1 cells MOPC104E (murine myeloma cell line) expressed IL-5-R, whereas BAL. 17 and L10 A (B cell lymphoma) did not. T cell line, mastocytoma cell line, or macrophage tumor cell line did not display detectable levels of IL-5-R. were hardly detectable on normal resting B cells but were expressed on LPS-activated B cells, fitting the function of IL-5 that acts on activated B cells for their differentiation into Ig-secreting cells. Intriguingly, early B cell lines (J-87 and T-88) that grow in the presence of IL-5 expressed significant but low numbers of high-affinity binding sites for IL-5. The biological effects of IL-5 were mediated by high-affinity binding sites. The identification and characterization of IL-5-R should provide new insight into the apparent diverse biological activities of IL-5.

Affinity Labels↗
PubMed1988

Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor.

The recent molecular cloning of the complementary DNA encoding T cell--replacing factor (TRF) has demonstrated that a single molecule is responsible for B cell growth factor II (BCGF-II) activity and eosinophil differentiation activity. It has been proposed that this molecule be called interleukin 5 (IL-5). We previously reported that purified rIL-5 supports the terminal differentiation and proliferation of eosinophilic precursors. In this study, we examined the effects of IL-5 on functional activities of mature eosinophils. IL-5 maintained the viability of mature eosinophils obtained from peritoneal exudate cells of mice infected with parasites. It also induced superoxide anion production in a dose-dependent manner. The Boyden's chamber Millipore assay revealed that IL-5 had a marked chemokinetic effect on eosinophils in a dose-dependent manner. Moreover, IL-5 was found to be an eosinophil chemotactic factor by the checkerboard assay. In conclusion, IL-5 is suggested to play an important role in increasing the functional activities of eosinophils as well as their production in allergic and parasitic diseases.

Animals↗
PubMed1988

Molecular properties and regulation of mRNA expression for murine T cell-replacing factor/IL-5.

We previously cloned cDNA for a T cell-replacing factor (TRF) that has been defined as a T cell-derived lymphokine that acts on activated B cells as a B cell growth and differentiation factor. Based on the diverse activities of rTRF on different target cells, we proposed that TRF be called IL-5. In this study, the molecular characteristics of TRF/IL-5 prepared by rDNA technology and TRF/IL-5 mRNA expression in various T cell lines and normal T cells have been studied. Specific immunoassay showed that rTRF/IL-5, which is transiently translated in vitro by rabbit reticulocyte lysate, has an apparent m.w. of 14,000. By contrast, active forms of rTRF/IL-5 translated in Xenopus oocytes has an apparent m.w. of 45,000 to 50,000 in the nonreducing condition and migrates to the m.w. of 25,000 to 30,000 under the reducing condition, indicating that active form of rTRF/IL-5 consists of dimer forms. The rTRF/IL-5 does not show detectable levels of IL-2, IL-3, and B-cell stimulatory factor 1 (IL-4) activities. Northern blot hybridization of poly (A)+ RNA from constitutively TRF-producing B151K12 T cell hybridoma revealed a single 1.7-kb band hybridizing to the cloned murine TRF/IL-5 cDNA. The expression of TRF/IL-5 mRNA in B151K12 was augmented by the stimulation with PMA plus calcium ionophore. In contrast, neither thymoma BW5147 nor IL-2-producing T cell hybridoma A55, both of which produced an undetectable level of TRF, expressed detectable levels of TRF/IL-5 mRNA. Stimulation of EL 4 and D9 cells with PMA and Con A, respectively, induced an increase in the levels of TRF/IL-5 mRNA expression accompanied by TRF/IL-5 production, whereas both cell lines did not show significant gene expression in the absence of the stimulation. In spleen cells from Mycobacterium tuberculosis-primed mice, significant expression of TRF/IL-5 mRNA was detected only when the cells were stimulated with relevant Ag, PPD. Normal spleen cells stimulated with Con A showed a significant, but approximately four-fold less expression of TRF/IL-5 mRNA. Molecular and functional properties of TRF/IL-5 will be discussed.

Animals↗