Biomedical subjects

M Hatakeyama

Publications and source records attributed to M Hatakeyama.

At least 73 records · Page 4Linked to original sources

PubMed1991

A novel monoclonal antibody against murine IL-2 receptor beta-chain. Characterization of receptor expression in normal lymphoid cells and EL-4 cells.

A mAb specific for the murine IL-2R beta-chain (IL-2R beta) was produced by immunizing a rat with a rat transfectant cell line expressing a large number of cDNA-encoded murine IL-2R beta. The mAb, designated TM-beta 1, is specifically reactive with the murine IL-2R beta cDNA-transfectant but not with the recipient cell, and immunoprecipitates murine IL-2R beta of Mr 75 to 85 kDa. TM-beta 1 mAb completely abolished the high affinity IL-2 binding by inhibiting the ligand binding to IL-2R beta. Murine IL-2R beta was found to be constitutively expressed on a subpopulation of CD8+ T cells and almost all NK1.1+ NK cells in the spleen, whereas TM-beta 1 mAb inhibited the proliferation of spleen cells induced by 1 nM of IL-2. Interestingly, EL-4 cells that express murine IL-2R beta as detected by TM-beta 1 mAb can bind neither human nor murine IL-2 under the intermediate affinity conditions, although cDNA-directed human IL-2R beta expressed in the same EL-4 cells has been previously shown to manifest the intermediate affinity IL-2 binding. These results may imply that functional expression of IL-2R beta is differentially regulated between humans and mice. Finally, our neutralizing anti-IL-2R beta mAb TM-beta 1 will be useful not only for various in vitro studies but also for in vivo studies to directly investigate the possible involvement of the IL-2/IL-2R pathway in the generation and differentiation of T lymphocytes and NK cells.

Animals↗

PubMed1991

Monoclonal antibodies defining distinct epitopes of the human IL-2 receptor beta chain and their differential effects on IL-2 responses.

We have established and characterized five new monoclonal antibodies (mAbs) which specifically immunoprecipitate the human interleukin-2 receptor beta chain (IL-2R beta). One of them, TU30, recognizes the intracytoplasmic 'serine-rich region' of IL-2R beta that is critical for IL-2 signal transduction. The others, TU12, TU21, TU23 and TU25, completely inhibit IL-2 binding, as does the previously characterized TU27. However, reciprocal binding competition assays show that the epitopes recognized by the individual mAbs are different from each other. The mAbs inhibit the growth of IL-2-dependent cells. The magnitude of their inhibitory effects is dependent on not only the affinities of the mAbs for IL-2R beta but also upon the number of IL-2R alpha subunits expressed on IL-2-dependent cells. These mAbs should be useful in studying the structure and function of the IL-2R.

Animals↗

PubMed1991

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association.

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.

Adult↗

PubMed1991

The integrity of the conserved 'WS motif' common to IL-2 and other cytokine receptors is essential for ligand binding and signal transduction.

Recent studies have identified a new family of cytokine receptors, which is primarily characterized by the conservation of periodically interspersed four cysteine residues and the W-S-X-W-S sequence ('WS motif') within the extracellular domain. However, the role of such conserved structures still remains elusive, in particular that of the WS motif. Interleukin-2 (IL-2) is known to play a critical role in the clonal expansion of antigen-stimulated T lymphocytes, and the IL-2 signal is delivered by one of the receptor components, the IL-2 receptor beta (IL-2R beta) chain. The IL-2R beta chain, unlike the IL-2R alpha chain, belongs to this receptor family. In the present study, we analyzed the function of the WS motif of IL-2R beta (Trp194-Ser195-Pro196-Trp197-Ser198) with the use of site-directed mutagenesis. Our results indicate the critical role of the two Trp residues in the proper folding of the IL-2R beta extracellular domain and point to the general functional importance of the WS motif in the new cytokine receptor family.

Amino Acid Sequence↗

PubMed1991

Signal transduction through the human IL-2 receptor beta-chain expressed in IL-6-dependent mouse B cell hybridoma.

Non-covalent association between at least two polypeptides, alpha (p55) and beta (p70), yields a high-affinity interleukin 2 receptor (IL-2R). Recent findings suggest that the beta-chain can mediate IL-2 signals on its own, while the alpha-chain is not involved in IL-2 signal transduction. To study the role of the beta-chain, directly, we transfected with the human IL-2R beta-chain cDNA a murine IL-6-dependent B cell hybridoma, F12-28, which originally did not express IL-2R. We established a stable transformant, beta E12, expressing the beta-chain (Kd = 1300 pM, 3000 sites/cell) in the absence of any detectable alpha-chain. We showed that (i) beta E12 manifested the intermediate affinity IL-2 binding, which was completely blocked with anti-human beta-chain antibody (Mik-beta 1); (ii) beta E12 acquired an ability to proliferate in response to IL-2 (greater than 0.1 nM) in a dose-dependent manner. These results clearly demonstrate that the beta-chain itself is directly involved in IL-2 signal transduction in the absence of the alpha-chain. Our results also suggest that a certain IL-6-dependent B cell line possesses cellular components) capable of transducing IL-2 signals.

Animals↗

PubMed1991

Signal transduction by interleukin 2 receptor beta chain: importance of the structural integrity as revealed by site-directed mutagenesis and generation of chimeric receptors.

The functional interleukin 2 receptor (IL-2R) consists of at least two IL-2 binding cell surface molecules, IL-2R alpha and IL-2R beta, the latter component being responsible for the intracellular growth signal transduction. In this study we attempted to identify the critical amino acid residues in the cytoplasmic domain of human IL-2R beta for such signal transduction by expressing mutated IL-2R beta cDNAs in a pro-B cell line, BAF-B03. We demonstrate that a single amino acid substitution within the 'serine-rich' cytoplasmic region of IL-2R beta (i.e. Leu299 changed to Pro) completely abrogates the receptor function in growth stimulation, but not in ligand binding. We also show that the murine erythropoietin receptor (EPO-R) is functional in BAF-B03, but that chimeric receptors, essentially possessing the IL-2R beta extracellular and a homologous region derived from EPO-R cytoplasmic domain, are not capable of transducing the IL-2-induced signal.

Amino Acid Sequence↗

PubMed1990

Characterization of the heterodimeric complex of human IL-2 receptor alpha.beta chains reconstituted in a mouse fibroblast cell line, L929.

The receptor for IL-2 has been known to exist in three forms on the basis of their affinities to IL-2: high, intermediate, and low affinity forms. Two IL-2R components have been identified as IL-2R alpha (p55, Tac Ag) and IL-2R beta (p70-75) chains, both bind IL-2 with low and intermediate affinities, respectively. Recently, we cloned human IL-2R beta chain cDNA and demonstrated that the cDNA product binds IL-2 with intermediate affinity and forms high affinity IL-2R with coexpressed IL-2R alpha chain in a human T cell line, Jurkat. In this study, we report the establishment of the mouse fibroblast transformants expressing either the IL-2R beta chain alone or both the IL-2R alpha and IL-2R beta chains. In contrast to lymphoid cells, significant IL-2 binding was not detected in the transformants expressing the IL-2R beta chain alone at IL-2 concentrations (50 pM to 10 nM) generally utilized. Nonetheless, the transformants expressing both IL-2R alpha and IL-2R beta chains displayed two forms of the IL-2R with high and low affinities to IL-2. However, neither IL-2 internalization nor signal transduction via the high affinity IL-2R complex were observed in the L929 transformants. Those findings suggest that the interaction of the IL-2R beta chain with the IL-2R alpha chain occurs in the absence of additional lymphoid specific component(s) to form high affinity IL-2R, but that this interaction is insufficient for IL-2 internalization and signal transduction just as observed in lymphoid cells. The experimental approach described here may allow further dissection of the molecular architecture of the IL-2R complex in the ligand binding, internalization, and signal transduction.

Animals↗

PubMed1990

The IL-2 receptor beta-chain (p70). Ligand binding ability of the cDNA-encoding membrane and secreted forms.

The high affinity IL-2R is composed of at least two distinct subunits; alpha (p55 or Tac)- and beta (p70)-chains. Recent cDNA expression studies with lymphoid cells unequivocally demonstrated that the IL-2R beta is an indispensable component for the ligand internalization and the signal transduction via the high affinity IL-2R. Furthermore, these studies confirmed that the IL-2R beta singly expressed on T lymphoid cells represents the intermediate affinity IL-2R. In the present study, however, we show 1) the IL-2R beta expressed on a mouse fibroblast line, NIH-3T3, did not bind IL-2 under the intermediate affinity conditions, 2) nevertheless, the IL-2R beta coexpressed with the IL-2R alpha not only formed the high affinity receptor but also internalized IL-2. By the use of competitive sandwich ELISA to study the IL-2 binding ability of the IL-2R beta molecule itself, we also show that the IL-2R beta present in the detergent-solubilized cell extracts and the secreted IL-2R beta (p37) produced by the truncated cDNA are actually capable of binding IL-2, but with a much reduced affinity compared with the intact IL-2R beta expressed on the lymphoid cells. These findings lead us to postulate a more complex feature of the IL-2R than ever speculated; the IL-2R beta molecule having on its own minimal IL-2 binding ability requires a putative gamma-chain specifically present on lymphoid cells to generate the functional intermediate affinity IL-2R. The gamma-chain, however, seems not to be required as far as the formation of the high affinity IL-2R by the alpha- and beta-chains and the ligand internalization through it are concerned.

Amino Acid Sequence↗

PubMed1990

The human interleukin-2 receptor beta-chain gene: genomic organization, promoter analysis and chromosomal assignment.

The chromosomal gene for the human interleukin-2 receptor beta-chain (IL-2R beta) was isolated and characterized. The entire IL-2R beta gene is composed of ten exons spanning about 24.3 kilobases, in which the protein is encoded by the exons 2-10. The cysteine rich extracellular region which displays a significant evolutionary resemblance to other cytokine receptors, as well as growth hormone and prolactin receptors, is encoded primarily by exons 3 and 4, whereas the membrane proximal, cysteine poor domain showing a homology with type III modules of fibronectin is encoded by exon 7. Sequence analysis of the 5'-flanking region revealed the presence of potential binding sites for transcription factors such as Octamer binding factors, AP-1, AP-2 as well as the 'GC-clusters'. At least five potential cap sites were identified by S1 mapping analysis. The 850 bp DNA sequence of the 5'-flanking region exhibited constitutive promoter activity when it was linked upstream of the HSV-tk reporter gene and then transfected into YT cells, a human leukemic cell line. By applying the RFLP linkage analysis, the IL-2R beta gene has been assigned to chromosome 22q12-13.

Base Sequence↗

PubMed1990

Role of interleukin 2 receptor beta chain in initiating anti-CD3 and interleukin 2-induced proliferation of human resting T cells.

We have examined the role of isolated interleukin 2 receptor (IL2R) beta chains expressed by human resting T cells in the early period of primary T cell activation induced by soluble OKT3 monoclonal antibody (mAb) and exogenous IL2. In the initial 3-day-stimulation phase, high IL2 concentrations were required, in association with soluble OKT3 mAb, to induce the formation of IL2R alpha/beta heterodimers, while later, low IL2 concentrations were sufficient to promote cell growth. When added during the initial phase, TU27 mAb directed at the IL2 binding site of IL2R beta chain substantially inhibited the appearance of functional high-affinity IL2R. Lo-Tact-1 mAb directed at the IL2 binding site of the IL2R alpha chain had only a marginal effect. Strong induction of IL2R alpha mRNA occurred within 3 days upon OKT3 and IL2 stimulation even in the presence of Lo-Tact-1 mAb, but not in the presence of TU27 mAb. OKT3 alone failed to induce significant IL2R alpha gene transcription and that induced by IL2 alone was very weak. The constitutive expression of IL2R beta mRNA was visualized in resting T cells. It remained at a rather stable level, at least during the initial stimulation period which was examined herein. Given the fact that OKT3 alone was ineffective in up-regulating IL2R beta mRNA expression and that pre-incubation of the cells with OKT3 alone did not allow them to respond to high concentrations of IL2, it is highly probable that isolated IL2R beta chains constitutively expressed by CD8+ T cells (the main reactive cells in this system) are primarily responsible for the initial interaction of IL2 with these cells. Such an interaction will result in the formation of high-affinity IL2R and in the initiation of cell proliferation provided that a CD3-derived co-signal is simultaneously delivered to the cells.

Antibodies, Monoclonal↗

PubMed1990

Purification of human C-reactive protein by immunoaffinity chromatography using mouse monoclonal antibody.

Human C-reactive protein (CRP) was purified from sera with a high concentration of CRP by immunoaffinity chromatography using mouse monoclonal antibody (mAb) to CRP (No. 18) and ion exchange on DEAE-Sephacel. CRP bound to the immunoaffinity column was eluted by 0.5 M acetate buffer, pH 5.0 containing 1.5 M NaCl. Final recovery of CRP was 90%. No significant immunochemical differences were observed between CRP obtained by the immunoaffinity chromatography and CRP purified by the method of Hokama and Riley.

Animals↗

PubMed1990

Murine interleukin 2 receptor beta chain: dysregulated gene expression in lymphoma line EL-4 caused by a promoter insertion.

The functional, high-affinity interleukin 2 receptor (IL-2R) consists of at least two receptor components, IL-2R alpha (p55) and IL-2R beta (p70-75). The cDNA encoding the murine IL-2R beta has been isolated by using the previously cloned cDNA for human IL-2R beta as a probe. Analysis of the cDNA revealed that the murine IL-2R beta shows a marked homology with the human IL-2R beta and that it is also structurally related to other cytokine receptors such as erythropoietin receptor. The cDNA-directed murine IL-2R beta formed high-affinity IL-2R in conjunction with the endogenous IL-2R alpha in a murine pro-B-cell line and could transduce IL-2-induced growth signal. In mouse lymphoma line EL-4, the IL-2R beta gene was found to be rearranged by the insertion of the long terminal repeat sequence of an intracisternal A particle, giving rise to constitutive expression of the IL-2R beta mRNA.

Amino Acid Sequence↗

PubMed1990

Microlamellar structures in lobular glomerulonephritis associated with monoclonal IgG lambda paraproteinemia. A case report and review of the literature.

We report a 49-year-old woman who developed lobular glomerulonephritis with prevalent deposition of material positive for IgG, C1q and lambda light chain, but which was not stained by Congo red. Glomeruli revealed massive electron-dense deposits with a microlamellar structure in the mesangial matrix and peripheral capillary loops. Clinically, the patient had nephrotic syndrome, microhematuria and hypertension. No Bence-Jones protein or cryoglobulin was found in the urine or serum. Anti-DNA antibody was positive, but systemic lupus erythematosus (SLE) was ruled out by repeated serological examinations. Immunoelectrophoresis of blood and urine revealed increased IgG-lambda paraprotein, but no free light chains were found. We reviewed 54 cases reported in the literature, which showed organized crystalline structures on ultrastructural examination, but were unassociated with amyloidosis, SLE, cryoglobulinemia or multiple myeloma. The present patient is the first reported to have exhibited a combination of glomerulonephritis with organized deposits, monoclonal IgG lambda paraproteinemia, and the presence of anti-DNA antibody.

Female↗

PubMed1990

[A case of mixed connective tissue disease exacerbated by griseofulvin complicating aseptic meningo-encephalitis and disseminated intravascular coagulation].

A variety of drugs have been implicated in causing or exacerbating SLE (drug-related lupus). Griseofulvin, the most commonly used agent in the treatment of fungal infections in the skin, has been shown to be a possible SLE-activating drug in several case reports. In the literature, however, any similar drug reactions have not been reported in MCTD, despite sharing features of SLE. The first case of drug (griseofulvin)-exacerbated MCTD was described and discussed. The patient was a 36-year-old woman with a six-year history of MCTD and subclinical Sjögren's syndrome. She developed aseptic meningo-encephalitis and disseminated intravascular coagulation after two weeks of continuous oral administration of griseofulvin 375 mg per day for trichophytosis of the feet. Following cessation of griseofulvin with anticoagulants, antibiotics, and high dose steroids at the same time, she greatly improved within one week.

Adult↗

PubMed1990

Binding of low density lipoprotein (LDL) to C-reactive protein (CRP): a possible binding through apolipoprotein B in LDL at phosphorylcholine-binding site of CRP.

Healthy human serum reacted with C-reactive protein of human (hCRP) or rat (rCRP) immobilized on Sepharose 4B in the presence of Ca2+. The bound serum proteins were eluted with 0.1 M ethylenediaminetetraacetic acid, trisodium salt (EDTA) dissolved in 10 mM Tris-HCl buffer, pH 8.0, containing 140 mM NaCl. The eluted proteins from the hCRP column was found to be low density lipoprotein (LDL), 90% of its protein being apo B. The one from the rCRP column contained also apo B by 36.7%. The effect of phosphorylcholine (PC), phosphorylethanolamine, phosphorylserine, galactose and high density lipoprotein (HDL) on the binding of LDL and apo B to CRP was investigated by enzyme-linked immunosorbent assay. Among them, only PC strongly inhibited the binding. One of the two available mouse monoclonal antibodies to hCRP (#19), which interferes with the binding of PC to CRP molecule, prevented CRP from binding LDL or apo B. In contrast, the other antibody (#17), which does not affect the binding of PC to CRP, did not inhibit the binding of LDL or apo B to CRP. It was therefore concluded that LDL binds to CRP by apo B moiety at the PC-binding site of CRP molecule.

Animals↗

PubMed1989

A restricted cytoplasmic region of IL-2 receptor beta chain is essential for growth signal transduction but not for ligand binding and internalization.

The functional, high affinity form of interleukin-2 receptor (IL-2R) is composed of two receptor components, the IL-2R alpha (p55) and IL-2R beta (p70-75) chains. Unlike the IL-2R alpha chain, the IL-2R beta chain contains a large cytoplasmic domain that shows no obvious tyrosine kinase motif. In the present study, we report the establishment of a system in which the cDNA-directed human IL-2R beta allows growth signal transduction in a mouse pro-B cell line. This system enabled us to identify a unique region within the cytoplasmic domain of the human IL-2R beta chain essential for ligand-mediated signal transduction. We also demonstrate that certain cytoplasmic deletion mutants in the IL-2R beta chain, although deficient in signal transduction, can still form high affinity IL-2R in conjunction with endogenous mouse IL-2R alpha chain; the mutants are still able to internalize the ligand as well.

Amino Acid Sequence↗

PubMed1989

Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's.

Interleukin-2 (IL-2) binds to two distinct receptor molecules, the IL-2 receptor alpha (IL-2R alpha, p55) chain and the newly identified IL-2 receptor beta (IL-2R beta, p70-75) chain. The cDNA encoding the human IL-2R beta chain has now been isolated. The overall primary structure of the IL-2R beta chain shows no apparent homology to other known receptors. Unlike the IL-2R alpha chain, the IL-2R beta chain has a large cytoplasmic region in which a functional domain (or domains) mediating an intracellular signal transduction pathway (or pathways) may be embodied. The cDNA-encoded beta chain binds and internalizes IL-2 when expressed on T lymphoid cells but not fibroblast cells. Furthermore, the cDNA gives rise to the generation of high-affinity IL-2 receptor when co-expressed with the IL-2R alpha chain cDNA.

Amino Acid Sequence↗

PubMed1989

Ligand-dependent selection of the receptor gene: segregation of IL-2 binding activity and anti-Tac reactivity by a single amino acid alteration in the Tac antigen (p55).

The Tac antigen (p55, CD25) is a 55 kDa glycoprotein that binds interleukin 2 at low affinity (Kd congruent to 10-50 nM). Expression of the Tac antigen is induced in the activated human T cells to constitute the functional, high-affinity IL-2 receptors (IL-2Rs) (Kd congruent to 10 pM) in conjunction with p70-75. A monoclonal antibody, anti-Tac, recognizes this molecule and inhibits the binding of IL-2 to both high- and low-affinity IL-2Rs. This observation indicates that IL-2 and anti-Tac binding sites are located close to each other within the Tac molecule. In this report, by utilizing a novel approach, we selected cDNAs encoding the Tac antigen variants whose reactivity with anti-Tac is greatly reduced, while retaining their IL-2 binding activity. Each of the mutant cDNAs contained a point (G----A) mutation resulting in an amino acid substitution at the particular amino-terminal portion of the Tac molecule (Asp-4). These results demonstrate that N-terminal amino acid Asp-4 is involved in the epitope recognized by anti-Tac, and that IL-2 binding site and anti-Tac binding site are structurally separable from each other in the Tac molecule.

Amino Acid Sequence↗