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Y Hitoshi

Publications and source records attributed to Y Hitoshi.

52 records · Page 3Linked to original sources
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

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

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

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

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↗
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

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

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

IL-4, but not IL-5, can act synergistically with B cell activating factor (BCAF) to induce proliferation of resting B cells.

B cell activating factor (BCAF) was initially identified in the supernatant of the murine T helper cell clone 52-3 (52-3 SN) because of its ability to promote activation and proliferation of resting B cells in the absence of any other costimulus. In this paper, we show that 52-3 T helper cells also secrete IL-4 and IL-5 and we have analyzed the influence of these two lymphokines on B cell proliferation induced by BCAF-containing 52-3 SN. Using the neutralizing anti-IL-4 monoclonal antibody 11B11, we observed partial inhibition of B cell proliferation. 52-3 SN free of IL-4 prepared using an immunoabsorbent column was still able to induce significant B cell proliferation. Although recombinant IL-4 alone does not induce B cell proliferation, it increased the proliferation induced by IL-4-free 52-3 SN. Kinetic studies showed that IL-4 is required at the start of B cell cultures in order to exert optimal synergistic effects. In contrast, anti-IL-5 monoclonal antibody NC17 did not affect the B cell proliferative activity of 52-3 SN whether or not IL-4 was present. When 52-3 SN was tested on dextran-sulfate-activated B cells, IL-5 and BCAF activities were detected but only the IL-5 activity was neutralized by monoclonal antibody NC17. These results demonstrate that (i) BCAF-containing SN can induce proliferation of resting B cells independently of IL-4 and IL-5, and (ii) IL-4, but not IL-5, can act synergistically with BCAF to induce B cell proliferation.

Animals↗
PubMedDate not supplied

Maintenance of CD5+ B cells at an early developmental stage by interleukin-5: evidence from immunoglobulin gene usage in interleukin-5 transgenic mice.

We have characterized the development and expansion of CD5+ B cells in interleukin-5 (IL-5) transgenic mice in terms of autoantibody production and immunoglobulin gene usage. CD5+IL-5R alpha+ B cells maintained in the presence of IL-5 secreted fewer autoantibodies and had fewer N nucleotides at the 3' end of the D elements compared with CD5- B cells. The reduction in nucleotides, along with the finding that CD5+IL-5R alpha+ B cells in IL-5 transgenic mice use Q52 families more frequently than age-matched control B cells, also suggests that these cells have the characteristics of fetus-type B cells and represent an early stage of B-cell development. All of the VH11 families were expressed with JH1 and the Q52 families were frequently expressed with JH1. Furthermore, JH proximal DQ52 was frequently used in IL-5 transgenic mice. All of these characteristics in terms of immunoglobulin gene usage have been described for CD5+ B cells. These results suggest that IL-5 maintains CD5+ B cells that have a fetus-type of immunoglobulin gene usage. This cytokine could be responsible for prolonging the life span of immature CD5+ B cells, which subsequently mature to CD5- B cells that secrete polyreactive natural antibodies.

Animals↗