Papers by Hiromi Kubagawa
Blood, Jul 15, 1997
containing DJCm transcripts were detected in both fetal and lineage commitment occurs. These feat... more containing DJCm transcripts were detected in both fetal and lineage commitment occurs. These features were used to adult cells. Although D H Q52 DJCm transcripts were abundant better define the earliest stage of B-cell commitment in huin fetal CD34 " CD19 Ï cells, they were not detected in cells mans and to determine if these stages differ as a function of the same phenotype derived from adult bone marrow. In of human ontogeny. Fetal and adult bone marrow mononuboth fetus and adult, V H 3-and V H 6-containing VDJCm tranclear cells were sorted into B-lineage subpopulations on the scripts were detected only in the CD19 " subpopulations. basis of surface expression of the stem cell marker CD34, These data indicate that transcription of D H Q52-J H and DXPthe pan-B-cell marker CD19, and IgM and analyzed for tran-J H rearrangements differs during fetal and adult B lymphoscription and rearrangement of the IgH locus. The locus was poiesis. Moreover, in both fetus and adult, transcription of found to be transcriptionally active before surface expresunrearranged components of the IgH locus and DJ rearsion of CD19, as indicated by the presence of germline Im, rangements can proceed before the surface expression of Cm, and D H Q52 transcripts in the CD34 " CD19 Ï subpopula-CD19. tion. Transcripts from IgH alleles that had undergone DJCm ᭧ 1997 by The American Society of Hematology. rearrangements were also detected in the CD34 " CD19 Ï sub-From the Division of Developmental and Clinical Immunology Early pre-B cells, which express cytoplasmic m-chain and and the Comprehensive Cancer Center, Departments of Microbiolnuclear TdT, can be identified by surface expression of CD19 ogy, Pathology, and Medicine, University of Alabama at Birin the absence of surface IgM. Coexpression of surface CD19 mingham, Birmingham, AL. and the stem cell marker CD34 characterizes a population
The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rea... more The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple V(H) and V(L) gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.
The Journal of Immunology, Apr 1, 2007
PIR-A and PIR-B, paired immunoglobulin-like receptors encoded, respectively, by multiple Pira gen... more PIR-A and PIR-B, paired immunoglobulin-like receptors encoded, respectively, by multiple Pira genes and a single Pirb gene in mice, are relatives of the human natural killer (NK) and Fc receptors. Monoclonal and polyclonal antibodies produced against a recombinant PIR protein identified cell surface glycoproteins of ف 85 and ف 120 kD on B cells, granulocytes, and macrophages. A disulfide-linked homodimer associated with the cell surface PIR molecules was identified as the Fc receptor common ␥ (FcR ␥ c) chain. Whereas PIR-B fibroblast transfectants expressed cell surface molecules of ف 120 kD, PIR-A transfectants expressed the ف 85-kD molecules exclusively intracellularly; PIR-A and FcR ␥ c cotransfectants expressed the PIR-A/ FcR ␥ c complex on their cell surface. Correspondingly, PIR-B was normally expressed on the cell surface of splenocytes from FcR ␥ c Ϫ / Ϫ mice whereas PIR-A was not. Cell surface levels of PIR molecules on myeloid and B lineage cells increased with cellular differentiation and activation. Dendritic cells, monocytes/macrophages, and mast cells expressed the PIR molecules in varying levels, but T cells and NK cells did not. These experiments define the coordinate cellular expression of PIR-B, an inhibitory receptor, and PIR-A, an activating receptor; demonstrate the requirement of FcR ␥ c chain association for cell surface PIR-A expression; and suggest that the level of FcR ␥ c chain expression could differentially affect the PIR-A/PIR-B equilibrium in different cell lineages. . PIR expression in FcR␥c-deficient and wild-type mice. Bone marrow cells from FcR␥c-deficient (thick line) and wild type mice (thin line) were stained with 6C1 anti-PIR or control (dotted line) mAb, and the stained cells were analyzed as described in the legend for . Only the wild-type mice control staining is illustrated.
Journal of Clinical Immunology, Jun 30, 1983
Epstein-Barr virus (EBV) transformation was used to examine the differentiation potential of circ... more Epstein-Barr virus (EBV) transformation was used to examine the differentiation potential of circulating B cells from eight individuals with late-onset panhypogammaglobulinemia. Cytoplasmic and secreted immunoglobulins were evaluated by immunofluorescence and radioimmunoassay. EBV-infected cultures of B cells from patients and healthy controls generated similar numbers of IgM-secreting plasma cells, but relatively few IgG and IgA plasma cells were induced in cultures of patients' B cells. As further evidence of B-cell immaturity, approximately 90% of the IgA B cells in the eight patients coexpressed IgM. Clonal diversity of B cells from hypogammaglobulinemic patients was examined with a panel of mouse monoclonal antibodies directed against idiotypic and VH subgroup determinants. The frequencies of EBV-induced plasma cells exhibiting the different idiotypic and VH determinants were similar for patients and controls. The data suggest the continued generation of clonally diverse B cells that are capable of terminal plasma-cell differentiation when the normal triggering mechanisms are bypassed by EBV. The arrested differentiation at an immature B-cell stage in these hypogammaglobulinemic individuals would appear to reflect a defect in normal B-cell triggering.
Proceedings of the National Academy of Sciences of the United States of America, May 13, 1997
An Fcalpha receptor probe of human origin was used to identify novel members of the Ig gene super... more An Fcalpha receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcalphaR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses.
Nature, Jan 27, 1983
Immunoglobulin gene expression is initiated in pre-B cells by rearrangements of heavy-chain varia... more Immunoglobulin gene expression is initiated in pre-B cells by rearrangements of heavy-chain variable genes V, D and J, for transcription together with the constant region gene Cμ (refs 1-7). The subsequent joining of light-chain V-J genes in the κ or λ families8,9 leads to formation of complete IgM molecules, which are then expressed on the surface of B cells. Heavy-chain isotype switching has been thought to occur later and to involve CH gene rearrangement via deletion of DNA between a switch site 5' to Cμ and a switch site 5' to the downstream Cγ, Cɛ or Cα gene expressed by a mature plasma cell10-15. On the other hand, isotype switching has been seen in human pre-B-cell leukaemias16,17 and in a murine pre-B-cell line before light-chain gene rearrangements and without Cμ gene deletion18,19. To explore further the isotype switching in pre-B cells, we used monoclonal antibodies in immunofluorescence assays to allow unambiguous assignment of the heavy-chain isotypes expressed by individual leukaemic cells in 11 pre-B-cell leukaemias. Switching in these leukaemic clones invariably led to expression of γ1 heavy-chain and κ light-chain determinants, occasionally together with γ4 and α. These results indicate a nonrandom order for heavy-chain isotype switching and for light-chain isotype expression, and also suggest that a mechanism exists for coordinating the two events.
The Journal of Immunology
Endoglin, a glycoprotein that is expressed by human endothelial cells, binds TGF-beta 1 and -beta... more 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.
The Journal of Immunology
In mice and chickens, J chain appears to be expressed only in activated B cells and plasma cells.... more 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.
Clinical & Experimental Immunology
IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In t... more IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age. Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures. Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency. When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal. The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced f...
Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by m... more Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection, PIR-B-deficient (PIR-B ؊/؊ ) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica Typhimurium (WB335). PIR-B ؊/؊ mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns of inflammatory lesions were found in their livers at 7-14 days postinfection: diffuse spreading along the sinusoids in PIR-B ؊/؊ mice vs nodular restricted localization in WT mice. PIR-B ؊/؊ mice have more inflammatory cells in the liver but fewer B cells and CD8 ؉ T cells in the spleen than WT mice at 14 days postinfection. PIR-B ؊/؊ bone marrow-derived macrophages (BMM) failed to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared with WT BMM. PIR-B ؊/؊ BMM also produced more nitrite and TNF-␣ upon exposure to Salmonella than WT BMM did. These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial infection.
How the Immune System Recognizes Self and Nonself, 2008
The authors have developed a ..gamma..2akappa mouse monoclonal antibody (FC6) against pre-B leuke... more The authors have developed a ..gamma..2akappa mouse monoclonal antibody (FC6) against pre-B leukemia cells with chromosomal translocation t(1;19). By immunofluorescence, FC6 recognized approx.20% of fresh marrow mononuclear cells (NMC), but <1% of fresh blood erythrocytes, granulocytes, MNC, and platelets in normal adults. However, approx.50% of mitogen-stimulated blood T cells expressed FC6 antigen on day 3 and all hemopoietic cultured cell
The Journal of Immunology
The innate immune system has developed to acquire a wide variety of pattern-recognition receptors... more The innate immune system has developed to acquire a wide variety of pattern-recognition receptors (PRRs) to identify potential pathogens, whereas pathogens have also developed to escape host innate immune responses. ITIM-bearing receptors are attractive targets for pathogens to attenuate immune responses against them; however, the in vivo role of the inhibitory PRRs in host-bacteria interactions remains unknown. We demonstrate in this article that Staphylococcus aureus, a major Gram-positive bacteria, exploits inhibitory PRR paired Ig-like receptor (PIR)-B on macrophages to suppress ERK1/2 and inflammasome activation, and subsequent IL-6 and IL-1β secretion. Consequently, Pirb(-/-) mice infected with S. aureus showed enhanced inflammation and more effective bacterial clearance, resulting in resistance to the sepsis. Screening of S. aureus mutants identified lipoteichoic acid (LTA) as an essential bacterial cell wall component required for binding to PIR-B and modulating inflammatory...
International immunology, 2014
The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune se... more The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcμR(-) B6/lpr than FcμR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcμR(+) B6/lpr mice, were reduced to normal B6 levels in FcμR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcμR(-) B6/lpr mice compared with either FcμR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1999
To explore the phylogenetic history of the murine paired Ig-like receptors of activating (PIR-A) ... more To explore the phylogenetic history of the murine paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) types, we isolated PIR homologues from a rat splenocyte cDNA library. The rat (ra) PIR-A and raPIR-B cDNA sequences predict transmembrane proteins with six highly conserved extracellular Ig-like domains and distinctive membrane proximal, transmembrane, and cytoplasmic regions. The raPIR-B cytoplasmic region contains prototypic inhibitory motifs, whereas raPIR-A features a charged transmembrane region and a short cytoplasmic tail. Southern blot analysis predicts the presence of multiple Pira genes and a single Pirb gene in the rat genome. Although raPIR-A and raPIR-B are coordinately expressed by myeloid cells, analysis of mRNA detected unpaired expression of raPIR-A by B cells and raPIR-B by NK cells. Collectively, these findings indicate that the structural hallmarks of the Pir gene family are conserved in rats and mice, yet suggest divergence of PIR regulatory el...
Current topics in microbiology and immunology, 1999
A distinguishing feature of the immune system is the ability to respond rapidly and efficiently t... more A distinguishing feature of the immune system is the ability to respond rapidly and efficiently to pathogenic insult. Cellular immune responses, which may include proliferation, elaboration of cytokines, and cytotoxicity, are initiated by receptor-ligand interactions. To maintain immune system homeostasis, the responses initiated by activating receptors are normally counterbalanced by signals propagated through ligation of corresponding inhibitory receptors. Protein motifs that nucleate the activating or inhibitory cascade following receptor ligation have been identified in the cytoplasmic tails of many immune system molecules (Weiss and Schles-singer 1998). The immunoreceptor tyrosine-based activation motif (ITAM), with the consensus sequence of D/E-x7-D/E-x-x-Y-x-x-L/I-x7-Y-x-x-L/I, was first identified in the signal transducing components of the antigen receptor complexes on B and T lymphocytes (Reth 1989; Cambier 1995). The immunoreceptor tyrosine-based inhibitory motif (ITIM), with the consensus sequence of I/V/L/S-x-Y-x-x-V/L, which is the focus of this volume, was originally described in the cytoplasmic tails of the low affinity Fc receptor for IgG antibodies (FcγRUB) on B cells and the killer inhibitory receptors (KIR) on NK cells (Ravetch 1994; Daéron 1997; Vély and Vivier 1997).
Acta pathologica japonica, 1980
Systemic pathological alterations were studied in thirty-seven autopsied patients with Kawasaki d... more Systemic pathological alterations were studied in thirty-seven autopsied patients with Kawasaki disease. Systemic vasculitis was the most characteristic pathological finding and was present in all the patients. In addition to the vasculitis, there was a high incidence of inflammatory lesions in various organs and tissues: in the heart, endocarditis, myocarditis, and pericarditis; in the digestive system, stomatitis, sialoduct-adenitis, catarrhal enteritis, hepatitis, cholangitis, pancreatitis, and pancreas ductitis; in the respiratory system, bronchitis and segmental interstitial pneumonia; in the urinary system, focal interstitial nephritis, cystitis, and prostatitis; in the nervous system, aseptic leptomeningitis, choriomeningitis, gangliontis, and neuritis; in the hematopoietic system, lymphadenitis, splenitis, and thymitis. Dermatitis, panniculitis or myositis were also observed in some patients. Therefore, Kawasaki disease is a systemic inflammatory disease which mainly affects...
Journal of immunology (Baltimore, Md. : 1950), 1982
The clonal origin of an IgA1 kappa B cell leukemia in a 71-year-old man (WF) was examined using a... more The clonal origin of an IgA1 kappa B cell leukemia in a 71-year-old man (WF) was examined using a monoclonal anti-Id antibody and a panel of monoclonal anti-VH antibodies. Immunofluorescent studies revealed that all surface IgA1 kappa + leukemic cells in WF's blood and 10% of the IgM+ B cells in his bone marrow expressed the WF Id. Three percent of the IgA1 kappa + leukemic cells in blood also expressed gamma-chains in their cytoplasm. Approximately 0.1%, 1%, and 10% of bone marrow mononuclear cells, respectively, expressed mu-chains, gamma-chains, and alpha-chains in their cytoplasm, but no detectable light chains or surface immunoglobulins. These mu, gamma, and alpha-positive cells had the convoluted nucleus and narrow cytoplasm characteristic of normal mu+ pre-B cells. Sequential isotype switching among this unusual pre-B population was indicated by co-expression of mu-chains and alpha-chains by 11% and 63%, respectively, of the gamma pre-B cells. These pre-B cells and the su...
Uploads
Papers by Hiromi Kubagawa