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Biomedical subjects

H Kubagawa

Publications and source records attributed to H Kubagawa.

At least 19 recordsLinked to original sources
PubMed1996

The J chain gene is transcribed during B and T lymphopoiesis in humans.

In mice and chickens, J chain appears to be expressed only in activated B cells and plasma cells. In humans, studies based mainly on transformed cells suggest that J chain expression may initiate during earlier stages in B lineage differentiation. In the present study, we isolated a series of hematopoietic subpopulations from human fetal and adult tissues by immunofluorescence cell sorting and examined each subpopulation for J chain expression by reverse transcriptase-PCR. In fetal and adult bone marrow, J chain transcripts were detected at all stages of B lineage differentiation, including the progenitor (CD34+/CD19-) and pro-B (CD34+/CD19+) cell subpopulations. J chain mRNA was also detected during fetal thymocyte development: double negative (CD4-/CD8-) through single positive (CD4+ or CD8+) cell subpopulations. The J chain message was not detected in peripheral CD3+ T cells, CD14+ monocytes, and CD56+ NK cells from either fetal or adult samples. The nucleotide sequence of J chain PCR products from CD34+/CD19- bone marrow progenitors and CD4+/CD8- thymocytes proved identical to the previously reported sequence of functionally spliced J chain mRNA. These results suggest that the J chain gene is transcriptionally active during early stages of both B cell and T cell differentiation, before the expression of their respective Ag receptors.

Adult↗
PubMed1996

B cells are generated throughout life in humans.

This analysis of B cell development as a function of age reveals a relatively widespread distribution of progenitor B (pro-B), pre-B, and B cells in fetal tissues, and thus supports the idea of a multifocal origin of B lineage cells during embryonic development. From mid-gestation onward, the bone marrow is the major site of B cell generation in humans. A relatively constant ratio of bone marrow precursors to B cells of immature phenotype (CD24highCD10+CD20lowIgD-) is maintained from mid-gestation through the eighth decade of life. The persistence of recombinase gene activity in pro-B cells further attests the sustained production of B cells in bone marrow. Interestingly, a subpopulation of B cells with mature phenotype (CD24lowCD10-CD20highIgD+) accumulates in the bone marrow during childhood, and this becomes the predominant B cell subpopulation in adult bone marrow. This mature population of bone marrow B cells may represent a subpopulation of recirculating B cells that have undergone selection in the periphery.

Adult↗
PubMed1995

Differential expression of endoglin on fetal and adult hematopoietic cells in human bone marrow.

Endoglin, a glycoprotein that is expressed by human endothelial cells, binds TGF-beta 1 and -beta 3 with high affinity. It was originally identified with the 44G4 mAb that was produced against a human pre-B cell line. We now report that another anti-pre-B cell mAb, 29-G8, reacts with pro-B and pre-B leukemic cells, but not with mature B and T cells, and recognizes a different epitope of endoglin. The 29-G8 mAb bound specifically to recombinant endoglin and immunoprecipitated a phosphorylated homodimeric glycoprotein with subunits of M(r) 95,000 from the 697 pre-B cell line. This new Ab removed all of the molecules identified by the prototypic 44G4 anti-endoglin Ab, but the reverse was not true. A subpopulation of 29-G8+ endoglin molecules on this pre-B cell line was unreactive with the 44G4 mAb, thus suggesting that these anti-endoglin Abs see different epitopes that may discriminate different species of endoglin molecules. Flow cytometric analysis with the 29-G8 mAb revealed two endoglin-positive subpopulations in fetal bone marrow: early B-lineage precursor cells (CD19+ and CD34+), and proerythroblasts (CD71+ and glycophorin A+). In adult bone marrow, only the proerythroblast subpopulation was observed. Stromal cells derived from fetal bone marrow also reacted strongly with the 29-G8 and 44G4 Abs, and these cells responded with enhanced proliferation after stimulation with either TGF-beta 1 or the anti-endoglin Abs. Thus, endoglin, a specialized component of the TGF-beta receptor system, may play a physiologic role in the stromal-hemopoietic cell interactions occurring during development.

Adult↗
PubMed1993

Characterization of an IgM Fc-binding receptor on human T cells.

The existence of an IgM receptor on human T cells has been suggested by T cell rosetting with IgM-coated erythrocytes. In this study we used immunofluorocytometry to demonstrate that 1) after short-term culture, a majority of the T cells can bind IgM at easily detectable levels, 2) human and mouse IgM preparations bind to human T cells but other Ig isotypes do not, 3) the IgM binding is saturable and inhibitable only by Ig of IgM isotype and 4) the Fc portion of IgM is involved in the binding to a protease-sensitive cell surface protein. Biochemical analysis of the T cell receptor for IgM reveals a cell surface protein of 60 kDa in comparison with the 58 kDa Fc mu receptors (Fc mu R) on B-lineage cells. Although Fc mu R expression is up-regulated after B cell activation, the reverse is true after T cell activation. In addition, the T cell Fc mu R is relatively resistant to phospholipase C treatment. These results indicate that T- and B-lineage cells express Fc mu R with different biologic characteristics.

Animals↗
PubMed1993

Expression of surrogate light chain receptors is restricted to a late stage in pre-B cell differentiation.

Surrogate light chain (psi LC) genes are transcriptionally active in progenitor B (pro-B) cells before immunoglobulin genes are rearranged. Current hypothetical models suggest that the psi LC proteins may couple with surrogate or conventional heavy chain proteins to form cell surface receptors that signal the progressive differentiation of pro-B, precursor B (pre-B), and immature B cells. Monoclonal antibodies were produced and used to examine the synthesis, expression, intermolecular interaction, and function of psi LC during B cell differentiation. The results indicate that, while psi LC production spans several developmental stages, cell surface expression is confined to a relatively late stage in normal pre-B cell differentiation, during which receptor cross-linkage does not impede cell growth or B cell differentiation.

Animals↗
PubMed1993

Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.

We describe a novel cytoplasmic tyrosine kinase, termed BPK (B cell progenitor kinase), which is expressed in all stages of the B lineage and in myeloid cells. BPK has classic SH1, SH2, and SH3 domains, but lacks myristylation signals and a regulatory phosphorylation site corresponding to tyrosine 527 of c-src. BPK has a long, basic amino-terminal region upstream of the SH3 domain. BPK was evaluated as a candidate for human X-linked agammaglobulinemia (XLA), an inherited immunodeficiency characterized by a severe deficit of B and plasma cells and profound hypogammaglobulinemia. BPK mapped to within 100 kb of a probe defining the polymorphism most closely linked to XLA at DXS178. Reduction in or the absence of BPK mRNA, protein expression, and kinase activity was observed in XLA pre-B and B cell lines. BPK is likely the XLA gene and functions in pathways critical to B cell expansion.

Agammaglobulinemia↗
PubMed1993

Recombinant soluble IgA Fc receptor: generation, biochemical characterization, and functional analysis of the recombinant protein.

We previously described the cloning of a human myeloid cell surface receptor for the Fc region of immunoglobulin A (Fc alpha R). In the present study, a soluble version of the Fc alpha R (solFc alpha R) was generated by removing the transmembrane and cytoplasmic coding regions from full-length Fc alpha R cDNA and ligating into a mammalian expression vector. COS-7 cells transfected with the solFc alpha R plasmid secreted a protein that inhibited both immunoglobulin A (IgA) and anti-Fc alpha R monoclonal antibody (mAb) binding to Fc alpha R+ U937 cells. Furthermore, the solFc alpha R bound specifically to and could be eluted from an anti-Fc alpha R mAb-immunoaffinity column, retaining biological activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the recombinant full-length Fc alpha R migrates over a molecular mass range of approximately 40-60 kd, consistent with the reported size and heterogeneity of the naturally occurring myeloid cell surface Fc alpha R. The solFc alpha R ran on SDS-PAGE as a smaller band (37-55 kd) that reduced to two bands of 23 and 25 kd following N-glycanase treatment, indicating that the Fc alpha R is a heavily glycosylated protein. The biochemical data, coupled with flow cytometry studies showing that the recombinant Fc alpha Rs bind to five different anti-Fc alpha R mAbs, clearly demonstrate that the cloned Fc alpha R corresponds directly to the major Fc alpha R species expressed on human monocytes, neutrophils, and myeloid cell lines. The generation of soluble receptor protein will permit investigations of the role of Fc alpha R in IgA-mediated immunoregulation, effector functions, and disease.

Antigens, CD↗
PubMed1993

Shared idiotypes in mesangial deposits in IgA nephropathy are not disease-specific.

The antigenic specificity of the mesangial IgA in IgA nephropathy (IgAN) remains unknown. Because shared antigenic specificities may be reflected in the usage of shared idiotypes, we prepared five monoclonal anti-idiotypic antibodies (MoAbs) specific for the mesangial IgA eluted from the kidney of an IgAN patient. All five MoAbs reacted with the same idiotype, which proved to be of a public nature. Although the idiotype could be identified in the mesangial deposits of the majority of IgAN patients studied, it was not specific for the disease because it was also found in the glomerular deposits of other types of glomerulonephritis. The idiotype was also expressed in polyethylene glycol precipitates of sera and in pokeweed mitogen-induced plasma cells from both IgAN patients and healthy controls. The conclusion that no disease-specific idiotypes are present in the renal eluate was further supported by the failure to produce polyclonal anti-idiotypic antibodies by immunizing a rabbit with the eluted mesangial IgA. Our results support the concept that mesangial IgA deposits in IgAN are of a polyclonal nature.

Antibodies, Monoclonal↗
PubMed1993

Signal transduction in human B cells initiated via Ig beta ligation.

Ig alpha and Ig beta heterodimers are non-covalently associated with Ig to compose the antigen receptor complexes on B cells. The demonstration that different sets of tyrosine kinases bind to the cytoplasmic tails of Ig alpha and Ig beta suggests that Ig alpha and Ig beta may activate distinct second messenger pathways. In this study, we examined the effects of mAbs against an exposed epitope of human Ig beta on pre-B and B cell triggering. Cross-linkage of Ig beta on B cells leads to activation of tyrosine kinases, hydrolysis of phosphatidylinositides, and elevation of intracellular Ca2+, effects qualitatively identical to those of anti-mu mAbs. Our observations thus indicate that cross-linking of Ig beta does not segregate signal transduction pathways connected with the cytoplasmic tails of Ig alpha and Ig beta. Ig alpha ligation has been reported to be more effective in triggering pre-B than B cells, whereas our results indicated that Ig beta ligation is more efficient in triggering B than pre-B cells. In addition to their activation properties, the anti-Ig beta mAbs effectively modulated B cell receptor complexes and blocked terminal differentiation of all plasma cell isotypes. The findings support the idea that anti-Ig beta could serve as a universal B cell immunosuppressant.

Animals↗
PubMed1993

Definition of immunoglobulin A receptors on eosinophils and their enhanced expression in allergic individuals.

Fc alpha receptors (Fc alpha R), detected by the binding of IgA and by anti-Fc alpha R antibodies, were found to be differentially expressed on eosinophils and neutrophils. Neutrophils were the major granulocyte population expressing Fc alpha R, and they expressed much higher levels of Fc alpha R than eosinophils. The expression of Fc alpha R by eosinophils could be upregulated approximately threefold by Ca2+ ionophore treatment in a dose- and time-dependent manner. This effect, which was blocked by a chelating agent, was not duplicated by other cellular stimuli. Eosinophils in allergic individuals displayed enhanced Fc alpha R expression, whereas neutrophils did not. The Fc alpha R on eosinophils had a higher molecular mass (70-100 kD) than those identified on neutrophils (55-75 kD). However, removal of N-linked carbohydrates from Fc alpha R of eosinophils and neutrophils revealed a major protein core of 32 kD for both cell types. The data indicate that expression of Fc alpha R molecules with a characteristic glycosylation pattern is upregulated on eosinophils in allergic individuals.

Adult↗
PubMed1992

Heterogeneity of immunoglobulin-associated molecules on human B cells identified by monoclonal antibodies.

Two covalently linked transmembrane molecules, encoded in mice by the mb-1 and B29 genes, have been defined as integral components of the antibody receptor units expressed on B cells. We have produced monoclonal antibodies against an exposed extracellular epitope on the putative human equivalent of the mouse B29 product. These antibodies, CB3-1 and -2, were used to show that cytoplasmic expression of this molecule begins in human pro-B cells (terminal deoxynucleotidyltransferase-positive, mu chain-negative), whereas surface expression coincides strictly with surface immunoglobulin expression of all isotypes. Immunochemical analysis of the human immunoglobulin-associated molecules revealed greater molecular heterogeneity than has been noted for the murine analogues. This molecular heterogeneity of immunoglobulin-associated molecules varied as a function of differentiation stage and the immunoglobulin isotypes expressed by B-lineage cells. Our data support the hypothesis that biochemical heterogeneity of the surface immunoglobulin-associated molecules may contribute to the variability in biological effects of antigen receptor crosslinkage on B cells of different maturational stages. Because the CB3 antibodies are capable of down-modulating the antigen receptors on all B cells, they may prove therapeutically useful as universal B-cell suppressants.

Antibodies, Monoclonal↗
PubMed1992

Molecular heterogeneity of Fc alpha receptors detected by receptor-specific monoclonal antibodies.

Fc alpha receptors (Fc alpha R) were isolated from a human monocytic cell line and used to raise four mAb with receptor specificity. The antibodies were used to identify the types of white blood cells that express Fc alpha R and the molecular heterogeneity of the receptor molecules. Nonpolymorphic epitopes, outside of the Fc alpha-binding site, were recognized only on blood cells of granulocyte and monocyte/macrophage lineages. The molecules identified, both by the antibodies and by the IgA ligand, were glycoproteins ranging in relative molecular mass from 55 to 75 kDa. However, one antibody detected a subpopulation of Fc alpha R molecules characterized by relatively restricted size heterogeneity. A complex glycosylation pattern was revealed by the resolution of discrete 32- and 36-kDa molecular species after removal of N-linked oligosaccharides and by evidence for O-linked carbohydrate moieties on at least a portion of the Fc alpha R molecules. In biosynthetic studies, all four anti-Fc alpha R antibodies and the IgA ligand bound a single 32-kDa core protein present in tunicamycin-treated cells, and the exceptional antibody again recognized molecules with relatively restricted glycosylation in the nontreated cells. These antibodies and native IgA ligands thus provide complementary reagents for definition of the complex structure and function of Fc alpha R in systemic IgA antibody responses.

Antibodies, Monoclonal↗
PubMed1992

Molecular cloning and expression pattern of a human gene homologous to the murine mb-1 gene.

The mouse mb-1 gene was originally identified based on its restricted expression in B lineage cells. Predicted structural homology with the gamma chain of the CD3 complex on T cells led to the suggestion that the MB-1 protein might associate with surface Ig(sIg) on B cells and be involved in signal transduction. Other studies identified at least two proteins that are noncovalently associated with sIgM, one of which has recently been shown to be the product of the mb-1 gene. To identify genes specifically expressed in normal human B cells we constructed a B minus T lymphocyte subtraction library and isolated a cDNA clone highly homologous to murine mb-1 (m-mb-1). A full-length cDNA was subsequently isolated and found to encode a membrane glycoprotein of 226 amino acids. It included a leader sequence (32 amino acids), an extracytoplasmic domain (111 amino acids) containing six potential N-glycosylation sites and three cysteine residues for potential inter- or intrachain disulfide linkages, a transmembrane domain (22 amino acids), and an intracytoplasmic domain (61 amino acids). The amino acid sequence homology between human and mouse mb-1 was especially striking (approximately 92%) in the intracytoplasmic, transmembrane, and membrane-proximal extracellular domains but was less marked (approximately 42%) in the remaining extracytoplasmic portion. Interestingly, part of the 3'-untranslated region was also highly conserved between species, suggesting an important role for this region in the regulation of mb-1 expression. The human mb-1 (h-mb-1) cDNA hybridized with a mRNA species of approximately 1.2 kb on Northern blots. Similar to m-mb-1, the h-mb-1 transcripts could be detected in pre-B cell lines and fetal bone marrow, in normal, mitogen activated- and transformed B cells but not in myeloma plasma cells. h-mb-1 was not expressed in peripheral T cells nor by cells of other hemopoietic lineages or in brain, heart, muscle, lung, and kidney. Surprisingly, however, low levels of h-mb-1 transcripts were detectable in two early T lineage cell lines and in the fetal thymus. This suggests that mb-1 may have other functions in addition to its role in signal transduction in B lineage cells.

Amino Acid Sequence↗
PubMed1991

VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

Blotting, Northern↗
PubMed1991

Normal pre-B cells express a receptor complex of mu heavy chains and surrogate light-chain proteins.

Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions.

Antibody Formation↗
PubMed1990

Biochemical nature of an Fc mu receptor on human B-lineage cells.

An IgM-binding protein of approximately 60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (Fc microR). The Fc microR can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation. Receptor expression is not directly linked with IgM production, as both mu- pre-B cells and isotype-switched B cells may express the Fc microR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with O-linked, but no N-linked, oligosaccharide. The Fc microR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fc microR is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens.

B-Lymphocytes↗