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H Nakauchi

Publications and source records attributed to H Nakauchi.

At least 145 records · Page 8Linked to original sources
PubMed1991

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro.

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.

Animals↗
PubMed1991

Multipotent and committed CD34+ cells in bone marrow transplantation.

In order to study the role of CD34+ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34+ cells were also performed to evaluate their in vitro hematopoietic function. CD34+ cells were detectable in bone marrow cells on day 14. More than 80% of CD34+ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34+ cells. In normal bone marrow cells, CD34+, CD33+ cells amounted to about 40% of CD34+ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34+, CD33- cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34+, CD33+ cells as committed progenitors and CD34+, CD33- cells as multipotent stem cells have distinctive biological behaviors in BMT.

Antibodies, Monoclonal↗
PubMed1991

Effects of interleukin 3, interleukin 6, and granulocyte colony-stimulating factor on sorted murine splenic progenitor cells.

In order to examine the effect of recombinant growth factors on hemopoietic stem cells, these cells were enriched using wheat germ agglutinin (WGA) and monoclonal antibodies for lineage markers (Lin) such as B220, L3T4, Lyt-2, asialo GM1, Mac-1, and AL-21. Spleen colony-forming units (CFU-S) and in vitro colony-forming units were highly enriched in the fraction of WGA+Lin- spleen cells. To eliminate committed progenitor cells, spleen cells of 5-fluorouracil (5-FU)-treated mice were used. By this treatment, day-8 CFU-S disappeared but day-14 CFU-S were preserved. Day-14 CFU-S were also contained in the fraction of WGA+Lin- cells, which made up about 0.5% of total nucleated spleen cells. Moreover, this fraction contained primitive stem cells that could reconstitute the hemopoiesis of irradiated mice. Sorted WGA+Lin- spleen cells obtained from male 5-FU-treated mice were injected into lethally irradiated female mice. Southern hybridization using a mouse Y chromosome-specific probe showed that the bone marrow, spleen, and thymus of the recipients was reconstituted by male mouse-derived cells. When sorted WGA+Lin- spleen cells of the 5-FU-treated mice were cultured in vitro in the presence of recombinant interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), colony formation was observed only in wells with IL-3, whereas unfractionated spleen cells formed colonies in the presence of IL-3, IL-6, or G-CSF. However, IL-6 but not G-CSF acted synergistically on enriched hemopoietic stem cells in the presence of IL-3. These data suggest that G-CSF or IL-6 did not affect primitive stem cells independently but showed the effect on these cells indirectly or synergistically with IL-3.

Animals↗
PubMed1990

Target cells for granulocyte colony-stimulating factor, interleukin-3, and interleukin-5 in differentiation pathways of neutrophils and eosinophils.

To study the relationship between hematopoietic factors and their responsive hematopoietic progenitors in the differentiation process, both purified factors and enriched progenitors are required. We isolated total CD34+ cells, CD34+,CD33+ cells, and CD34+,CD33- cells individually from normal human bone marrow cells by fluorescence-activated cell sorter (FACS), and examined the effects of granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and IL-5 on in vitro colony formation of these cells. CD34+,CD33+ cells formed granulocyte colonies in the presence of G-CSF. Both CD34+,CD33+ cells and CD34+,CD33- cells formed granulocyte/macrophage colonies in the presence of IL-3. Eosinophil (Eo) colonies were only formed by CD34+,CD33- cells in response to IL-3, but scarcely formed by CD34+ cells in the presence of IL-5. We performed the two-step cultures consisting of the primary liquid culture for 6 days and the secondary methylcellulose culture, and serially examined changes in phenotypes of ,he cells cultured in the primary culture. CD34-,CD33+ cells derived from CD34+,CD33+ cells by preincubation with G-CSF or IL-3 formed Eo colonies in the presence of IL-5 but not IL-3. CD34-,CD33+ cells derived from CD34+,CD33- cells by preincubation with IL-3 also formed Eo colonies by support of IL-5 as well as IL-3. Both CD34+ cells gradually lost the CD34 antigen by day 6 of incubation with G-CSF or IL-3. Loss of this antigen was well-correlated with acquisition of susceptibility to IL-5. It was concluded that G-CSF supported the neutrophil differentiation of committed colony-forming cells, IL-3 supported that of both committed and multipotent colony-forming cells. G-CSF and IL-3 also supported the early stage of E. differentiation; IL-5 supported the late stage of that.

Antigens, CD34↗
PubMed1990

Colony formation of clone-sorted human hematopoietic progenitors.

To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34+ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34+ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.01% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34+ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34+ cells. Approximately 15% of PB CD34+ cells formed colonies in which erythroid bursts were predominant. CD34+ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34+, CD33+ cells formed GM and Mac colonies 7 to 10 times as often as CD34+, CD33- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34+, CD13- cells or CD34+, CD33- cells. The expression of HLA-DR on CD34+ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34+ cells between BM and PB. About 10% of CD34+ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34+, CD33+ fraction than in CD34+, CD33- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.

Antigens, CD34↗
PubMed1990

Comparative structure of two duplicated T1a class I genes (T10c and 37) of the murine H-2d MHC. Implications on the evolution of the T1a region.

The class I Ag encoded in the Qa/T1a regions of the murine MHC are much less polymorphic, and usually have a more restricted tissue distribution than the classical histocompatibility class I Ag, encoded by genes located in the H-2K, D, and L loci. The isolation of a quasi-ubiquitously expressed, poorly polymorphic class I gene of the T1a region of the H-2d mouse MHC, namely gene 37 (or T18d), has been recently reported. We describe the nucleotide sequence of a closely related gene, T10c gene, the counterpart of the gene 37 in the large duplicated parts of T1a region of the BALB/c (H-2d) MHC. The T10c gene structure and sequence are very similar to those of gene 37, but T10c gene is most likely a pseudogene. In A/J mouse strain, there appears to be a single gene related to 37, which is also found expressed in a variety of tissues. We show that this gene is likely to be a chimeric one derived from T10c for its 3' part, and from a gene closely related to gene 37 for its 5' part, which potentially encodes for an unusual class I molecule composed of the first two domains. Finally, Southern blot analysis of a number of wild mice and related animals suggests that a gene closely related to the present T10c gene may be the ancestor of this subfamily of class I genes characterized by the presence of an unusual second domain.

Animals↗
PubMed1990

The dose dependence of glucocorticoid-inducible gene expression results from changes in the number of transcriptionally active templates.

Glucocorticoid hormones induce the transcription of genes having glucocorticoid response elements in a dose dependent manner. To determine whether this dose dependence represents a response of individual templates or of the mass of templates, we introduced a bacterial beta-galactosidase gene linked to the glucocorticoid-inducible enhancer/promoter of the mouse mammary tumor virus (MTV) into Ltk- cells and obtained stable transformants containing a single or a few templates per cell. Visual inspection and flow cytometry analysis by enzyme histochemistry assay for beta-galactosidase revealed that individual cells showed very heterogeneous beta-galactosidase activity after 48 h induction with dexamethasone. When the glucocorticoid concentration was increased, an increasing cell population producing beta-galactosidase was observed. These phenomena were probably not due to heterogeneity of template copy number or to a predetermined cellular state among individual cells, since cells forming a single small colony gave similar results. This was also supported by data showing that recloned cells retained both their responsiveness to the glucocorticoid hormone and their digestion pattern in Southern blotting analyses. These results indicate that the dose dependent increase of glucocorticoid-inducible gene expression is caused by an increase in the number of transcriptionally active templates.

Animals↗
PubMed1990

The cytoplasmic domain of the CD8 alpha-chain is required for its interaction with p56lck.

The CD8 glycoprotein expressed on the surface of CTLs is a heterodimer composed of alpha (Lyt-2) and beta (Lyt-3) chains. Recent studies have shown that CD4 and CD8 are physically associated with a T cell-specific protein-tyrosine kinase p56lck. Our previous experiments have suggested strongly that p56lck interacts directly with CD4 and CD8 molecules. The present report using cytoplasmic deletion mutants of the CD8 alpha-chain gene has extended our observations to demonstrate unequivocally that the cytoplasmic domain of the CD8 alpha chain is responsible for interaction with p56lck. The data has also confirmed the importance of the conserved twelve amino acid sequence motif of the CD8 alpha cytoplasmic domain in complex formation with p56lck.

Amino Acid Sequence↗
PubMed1990

Expression and function of the transfected CD8 alpha chain in murine T cell hybridomas.

Expression and function of mouse and human CD8 (mCD8 and hCD8) alpha chain molecules in mouse T cell hybridomas were analyzed. The expression of cytolytic T lymphocyte-derived CD8 molecules was suppressed in hybridomas established by fusing the BW5147 thymoma to a CD8+ cytolytic T lymphocyte clone, while expression of CD4 remained intact in BW x CD4+ helper T cell hybridomas. However, hybridomas established by fusing a cytolytic T cell clone with BW5147 cell lines, transfected with either the mCD8 alpha or hCD8 alpha chain, expressed the T cell-derived mCD8 beta chain as a CD8 heterodimer (mCD8 alpha/mCD8 beta or hCD8 alpha/mCD8 beta). These data suggest that negative regulatory mechanisms for the mCD8 alpha gene in BW thymoma failed to suppress mCD8 beta gene expression, indicating different regulatory mechanisms for the tightly linked mCD8 alpha and mCD8 beta genes. Analysis of the antigen reactivity of the hybridomas revealed that the human CD8 alpha chain failed to increase the mouse T cell receptor - class I MHC interaction, even as a heterodimeric form with mCD8 beta molecules. However, both the human CD8 alpha homodimer and the heterodimeric form with mCD8 beta were found to be capable of suppressing the class II-restricted T cell response.

Animals↗
PubMed1989

A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B-cell colony formation and an inhibitory effect of IL-1 alpha.

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7-induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.

Animals↗
PubMed1989

Isolation and characterization of the mouse CD8 beta-chain (Ly-3) genes. Absence of an intervening sequence between V- and J-like gene segments.

We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no "TATA" box nor "CAAT" box; however, three "GC" boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes.

Amino Acid Sequence↗
PubMed1989

Expression of Leu M1 antigen on a monoclonal B cell line established from a patient with rheumatoid arthritis.

The purpose of this study is to show that anti-Leu M1 antibody (anti-CD15), which has different staining characteristics in lymphoid and non-lymphoid cells, reacted against the surface antigen of a defined monoclonal B cell line. This antibody recognizes the sugar moiety, lacto-N-fucopentaose (LNF-III), which is linked to the cell membrane protein in several kinds of cells, but not in B cells. However, a human monoclonal B-cell line (TKS-1) which was established from the peripheral blood of a patient with rheumatoid arthritis, expressed the Leu M1 antigen spontaneously. The analysis of surface markers using a fluorescence-activated cell sorter (FACS) has revealed that the surface markers of TKS-1 were anti-mu, delta, kappa, HLA-DR, DQ, Leu 12 (CD19) and Leu M1 (CD15). TKS-1 cells were not reactive with any of the following antibodies: anti-OK M1 (CD11b), Leu M2, Leu M3 (CD14), Leu M4, Leu 1 (CD5), Leu 2 (CD8), Leu 3 (CD4), Leu 4 (CD3), Leu 7 and Leu 11 (CD16). In addition, TKS-1 was positive to Epstein-Barr nuclear antigen, weakly positive to non-specific esterase without staining inhibition by NaF, and negative to peroxidase. TKS-1 cells produced IgM in the culture supernatant and have kappa-light chain rearrangement in its DNA. As shown in other studies, distribution of Leu M1 is very wide. This antigen is not a specific immunodiagnostic marker to distinguish the cell type. We conclude that it is possible to express Leu M1 antigen on the membrane of a B-cell lineage cell.

Antibodies, Monoclonal↗
PubMed1989

Altered immune responsiveness in patients with chronic glomerulonephritis.

In order to detect abnormalities in humoral immunity and to determine immunogenetic traits underlying chronic glomerulonephritis, sera from 260 patients who had chronic glomerulonephritis and who were undergoing hemodialysis were tested for naturally occurring antibodies against mycoplasma and 22 different viruses. Among the 23 microorganisms tested, antibody titers were significantly lower against 12, higher against 3, and no different against 8 when compared with titers of 43 normal subjects. The data were analyzed further by plotting each in a 23-dimensional space according to their standardized antibody titers. Multivariate cluster analysis by the Ward's method revealed 3 large clusters differing from each other in natural antibody titers, and one of the clusters included 74% of the normal controls, while two other distinct clusters comprised the majority of the patients. The level of BUN, creatinine, and duration of hemodialysis treatment did not differ significantly among patients in these three different clusters. Our study suggests that patients with chronic glomerulonephritis being treated by hemodialysis have altered levels of naturally occurring antibodies to microorganisms. This alteration is not caused by just the uremic state or hemodialysis but immunogenetic regulation may also play a part.

Adolescent↗
PubMed1989

The distinct subgroup of patients with rheumatoid arthritis shown by Ig G3-reactive rheumatoid factor.

The reactivity of rheumatoid factor (RF) with immunoglobulins of the IgG3 subclass was examined in 49 patients with rheumatoid arthritis (RA) using two types of IgG3 myeloma (routine and IgG3m-15 allotype). Among 49 patients, serum from eight cases showed positive reactivity with both types of IgG3 myeloma by radio-immunoassay (RIA). The isotype of IgG3-reactive RF was not specific; it belonged to the IgM class as well as the IgG subclasses IgG1, IgG2 and IgG4. The patients with IgG3-reactive RF belonged to the clinically-severe classification of RA, having a high erythrocyte sedimentation rate (ESR), high titre in the RA hemagglutination (RAHA) test, and above all they had low levels of complement. Generally, it is concluded that patients with IgG3-reactive RF have serious arthritis and that IgG3-reactive RF might play an important role in the inflammatory process. Furthermore, it was also shown that the RF-reactive site was not associated with the protein-A binding site of IgG3, since RF reacting with IgG3m-15 reacted similarly with routine IgG3, regardless of the difference of the protein-A binding activity. This was confirmed by adding protein-A to the reaction of RF and IgG3m-15 which binds with protein-A. This suggests that the actual reactive site of RF is different to the site that binds protein-A.

Adult↗
PubMed1989

Generation of osteoclasts from isolated hematopoietic progenitor cells.

A variety of studies have shown that osteoclasts originate from bone marrow, but their exact progenitors and differentiation pathway remain unclear. The treatment of mice with a high dose of 5-fluorouracil (5-FU) results in an enrichment for primitive hematopoietic progenitors; using this procedure, we prepared a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro. When spleen cells from mice pretreated in vivo with 5-FU were cultured in the presence of methylcellulose medium containing recombinant interleukin-3 (rIL-3), small colonies consisting of blast cells with little sign of differentiation developed on day 7 of culture. We lifted these blast colonies, pooled them, and replated them as secondary methylcellulose cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of the cells formed colonies comprising various combinations of neutrophils, macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We replated such blast cells into microtiter wells and cultured them in the presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) (100 U/mL) plus 1.25(OH)2D3 (10(-7) mol/L). Multinucleated cells appeared from day 14 of culture and approximately 100 giant cells per well were scored on day 21 of culture. Parathyroid hormone (1 U/mL) also induced the multinucleated cell formation. May-Grunwald-Giemsa staining revealed the large cells containing many nuclei in their cytoplasm, which is characteristic of bone-resorbing cells or osteoclasts. These cells showed a tartrate-resistant acid phosphatase (TRAP) activity. Calcitonin caused a striking shape change in these cells and suppressed the formation of multinucleated cells. Moreover, electron microscopy shows that these cells were able to resorb fetal calvariae. In the presence of r granulocyte-colony stimulating factor, r macrophage-colony stimulating factor, or r interleukin-6 plus 1.25(OH)2D3, formation of TRAP-positive multinucleated cells was lower compared with the support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did not form the multinucleated cells as described above, even in the presence of rIL-3 and 1.25(OH)2D3. Moreover, to exclude the possibility that osteoclasts generated from non-blast cells, we performed a cloning experiment from one isolated blast cell and demonstrated that single cells differentiate into osteoclasts or macrophages in the presence of rIL-3 with or without 1.25(OH)2D3. This system will provide a useful model for further analysis of osteoclast formation in vitro.

Animals↗