Biomedical subjects

K Inaba

Publications and source records attributed to K Inaba.

At least 37 records · Page 2Linked to original sources

PubMed1991

Mapping of ATP-dependent trypsin-sensitive sites on the beta chain of outer-arm dynein from sea urchin sperm flagella.

The beta chain of sea urchin outer-arm dynein showed a peculiar tryptic digestion pattern in the presence of ATP (or ADP) plus Vi. Examination of the molecular mass of the products formed by photocleavage of tryptic fragments indicated that the trypsin-sensitive sites on the 165-kDa ATP-binding polypeptide in the presence of ATP and Vi are located 15 kDa apart from its amino-terminus, 2 kDa apart from its carboxy-terminus, and near the middle portion between the adenine- and gamma-Pi-binding sites. On the other hand, the carboxy-terminal region of the beta chain, the 135-kDa polypeptide, was cleaved into a 96-kDa polypeptide by tryptic digestion in the presence of ATP and Vi. Peptide mapping of 135-kDa, 96-kDa, and carboxy-terminally truncated polypeptides of the 135-kDa polypeptide revealed that the 96-kDa region is located at the amino-terminal portion of the 135-kDa region. These results indicate that the changes of trypsin susceptibility of dynien beta chain caused by binding ADP and Vi occur not in local region but over an extensive region on the beta chain.

Adenosine Triphosphate↗

PubMed1991

Alteration of physiological activity of activated macrophages through L-arginine metabolism.

Our aim in this study was to define the effect of L-arginine on macrophages (M phi) in relation to the decay of tumoricidal activity of activated M phi. We found that the activated M phi retained their cytotoxicity when cultured in L-arginine-deficient medium but not in conventional medium. Such a decline of tumoricidal activity was associated with increase of glucose consumption and concomitant lactate production, resulting in M phi death. Addition of glucose to the culture medium of activated M phi appeared to cause only a slight delay of the decrease of tumoricidal activity and M phi death. These events were also coincident with a decrease of electron transport activity in mitochondria. Cytological observation by electron microscopy clearly showed the structural alteration or destruction of mitochondria, which preceded the changes of other physiological and functional activities. These results demonstrate that the L-arginine-dependent cytolytic activity against tumor target cells also impairs M phi functions and ultimately induces M phi death, which is primarily mediated by the inhibition of mitochondrial activity.

Animals↗

PubMed1991

Stimulation of erythrophagocytosis in mouse peritoneal macrophages by chondroitin sulfates: correlation with effect of phorbol esters.

Resident macrophages which were harvested from the mouse peritoneal cavity showed the attachment activity to opsonized erythrocytes (OE) without the treatment of chondroitin sulfates (CSA) or phorbol esters. Phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-diacetate) rapidly activated an opsonin-dependent erythrophagocytosis in resident macrophages, whereas CSA slowly activated it in vivo and in vitro. An additive effect of phorbol esters was observed in macrophages which were cultured with CSA in vitro or stimulated by the intraperitoneal injection of CSA for 1 or 2 day(s). In the case of macrophages stimulated by the intraperitoneal injection of CSA for 3 or 4 days the erythrophagocytic activity was at very high level and the additive effect of phorbol esters vanished. These results indicate that CSA plays a role in the induction of opsonin-dependent ingestion activity of resident macrophages.

Animals↗

PubMed1991

[Inhibition by Y-25130 of the von Bezold-Jarisch effect evoked by 5-HT or 2-methyl-5-HT in anesthetized rats].

Effect of Y-25130 on 5-hydroxytryptamine3 (5-HT3) receptors was investigated using the von Bezold-Jarisch effect (BJE) in anesthetized rats. Intravenous or intraduodenal administration of Y-25130 antagonized the BJE evoked by 5-HT and its effect was over 100 times more potent than that of metoclopramide. Y-25130 also completely blocked the BJE induced by 2-methyl-5-HT, a selective 5-HT3 receptor agonist. The BJE induced by 5-HT was not antagonized by spiperone, ketanserin, phenoxybenzamine, yohimbine and haloperidol, but antagonized by atropine. Atropine inhibited the bradycardia caused by electrical stimulation of the vagus nerve, but Y-25130 had no inhibitory effect. These results indicate that Y-25130 possesses a potent and selective 5-HT3 receptor antagonistic property.

Anesthesia↗

PubMed1990

The effect of immunosuppressive agents on the induction of nuclear factors that bind to sites on the interleukin 2 promoter.

Cyclosporin A (CSA), FK506, and glucocorticosteroids all inhibit the production of lymphokines by decreasing lymphokine gene expression. Previous experiments have defined six different sites that may contribute to the transcriptional control of the interleukin 2 (IL-2) promoter, and for each, active nuclear binding factors are induced upon mitogenic stimulation. While dexamethasone markedly blocks the increase in IL-2 mRNA in stimulated human blood T cells, we found that the drug does not block the appearance of factors that bind to the transcriptional control sites termed AP-1, AP-3, NF-kB, OCT-1, B site, and NF-AT. In contrast, both CSA and FK506 have similar effects: the drugs cause modest decreases in AP-3 and NF-kB, and markedly decreases in the activity of AP-1 and NF-AT. Therefore, CSA and FK506, while chemically different, seem to act upon a similar pathway that leads to IL-2 gene expression, whereas glucocorticoids do not affect this pathway.

Anti-Bacterial Agents↗

PubMed1990

Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of major histocompatibility complex class II molecules and expression of invariant chain.

Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a T-T hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence of chloroquine or cycloheximide. The latter, in contrast, improves the efficacy of antigen pulsing in anti-Ig-stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cells types in the level of biosynthesis of MHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation.

Animals↗

PubMed1990

Granulocyte-macrophage colony-stimulating factor enhances macrophage accessory function in con A-stimulated T-cell proliferation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to augment various macrophage (M phi) functions, including antigen presentation in the antibody-producing response. We investigated the augmentative effect of GM-CSF on M phi A-cell activity in concanavalin A-stimulated T-cell proliferation. Pretreatment with GM-CSF of peritoneal M phi enhanced the T-cell proliferative response. This effect of GM-CSF was dose dependent and GM-CSF supplementation was needed at the beginning of M phi culture. We observed that GM-CSF induced M phi spreading and firm attachment accompanied with enlargement of the cytoplasm, but could not induce de novo expression of Ia antigen. GM-CSF treatment enabled M phi to produce more interleukin (IL)-1 and IL-6 upon stimulation with lipopolysaccharides or polyinosinic-polycytidylic acid, but was unable to stimulate M phi directly. This was confirmed by Northern blot analysis. These results indicate that GM-CSF augments M phi A-cell activity through the enhancement of the capacity of M phi to produce IL-1 and IL-6.

Animals↗

PubMed1990

Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen.

Dendritic cells are a specialized but trace population of antigen presenting cells that always have been enriched by multi-step procedures over a period of 1 or more days in tissue culture. Here we describe the isolation of dendritic cells from fresh mouse spleen suspensions using the FACS and a monoclonal antibody, N418, to the p150/90 member of the leukocyte integrin family (Metlay et al., 1990). By two color fluorescence activated cell sorter (FACS) analyses, the trace N418+ subset expressed most of the surface markers, including the 33D1 antigen, that are characteristic of dendritic cells isolated by other methods. An exception was that small amounts of Fc receptors, CD4 and F4/80 antigen were detected initially, but these diminished upon culture. In functional assays, sorted N418+ cells from fresh spleen were at least 30 times more active than N418- cells in presenting antigen to T cells. The assays were stimulation of the primary mixed leukocyte reaction and presentation of exogenous protein antigens to sensitized populations of lymph node T cells. The viability and MLR stimulating function of the sorted populations both were increased upon exposure to the cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). These results indicate that dendritic cells can be enriched from fresh isolates of mouse spleen using the FACS, and that when this is done, many of the distinctive features of dendritic cells - phenotype, APC function, and sensitivity to appropriate cytokines - are apparent.

Animals↗

PubMed1990

Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ.

T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen-reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.

Animals↗

PubMed1990

Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins.

We monitored the APC function of cells taken from the spleen and peritoneal cavity of mice that had been given protein antigens via the intravenous or intraperitoneal routes. Using the mAb 33D1 and N418 to negatively and positively select dendritic cells, we obtained evidence that dendritic cells are the main cell type in spleen that carries the protein in a form that is immunogenic for antigen-specific T cells. In vivo pulsed macrophages were not immunogenic and did not appear capable of transferring peptide fragments to dendritic cells.

Animals↗

PubMed1990

Inhibition of tumor cell proliferation by natural suppressor cells present in murine bone marrow.

Natural suppressor (NS) cells, which are Thy-1-, immunoglobulin-, and nonadherent cells with relatively low density (1.063 to 1.075 g/ml), inhibit not only the proliferation of spleen cells which have been stimulated by allogeneic cells or mitogens but also the proliferation of tumor cell lines. Cell-to-cell contact is not necessary for NS cells to exert NS activity. Being radioresistant, DNA synthesis is not necessary for NS cells to suppress proliferation. However, protein synthesis is necessary, since puromycin blocks NS cell activity. In addition, NS cells were found to secrete a factor which inhibits DNA synthesis. Of the various cytokines tested, interleukin 3 and granulocyte-macrophage colony-stimulating factor enhance NS activity. These results suggest that NS cells play an important role in the suppression of not only immune responses but also tumor growth.

Animals↗

PubMed1990

Growth factors involved in lymphocyte differentiation.

We report here that an interleukin-3-dependent precursor B-cell line, LyD9, differentiated in vitro into mature B cells, producing immunoglobulin (Ig)M and IgG by co-culture with bone marrow stroma cells. Induced LyD9 cells underwent heterogenous immunoglobulin gene rearrangement and synthesized mRNAs encoding immunoglobulin mu (mu), gamma (gamma) and kappa (kappa) chains. LyD9 was also shown to differentiate into myeloid cells. We have established an interleukin-4-dependent derivative clone K-4 that is an intermediate between myeloid-lymphoid cells and the LyD9 clone. This differentiation required direct contact between LyD9 and stromal cells.

Animals↗

PubMed1990

Functional analyses of thymic CD5+ B cells. Responsiveness to major histocompatibility complex class II-restricted T blasts but not to lipopolysaccharide or anti-IgM plus interleukin 4.

The function of thymic B cells in several standard in vitro assays was investigated. Thymic B cells, 75% of which were CD5+, showed a poor responsiveness to the mitogens LPS or anti-mu plus IL-4. Both proliferation and antibody formation were much lower in thymic than splenic B cell cultures. However, CD5- B cells purified using a cell sorter responded well to B cell stimulants, whereas purified CD5+ thymic B cells did not, indicating that CD5+ thymic B cells were unresponsive to B cell growth factor or LPS. Thymic B cells could be activated polyclonally by direct interaction with alloreactive T blasts, as manifested by DNA synthesis and antibody formation. These findings indicate that CD5+ thymic B cells may not be stimulated via sIg and IL-4, but require instead direct interaction with T blasts.

Animals↗

PubMed1990

Effect of human recombinant interleukin-6 on the proliferation of mouse hepatocytes in the primary culture.

Effects of various cytokines on the proliferation of mouse hepatocytes were investigated. Human recombinant IL-6 not only enhanced the proliferation of mouse hepatocytes in the presence of epidermal growth factor, but also without epidermal growth factor. However, other human or mouse cytokines such as recombinant IL-1, IL-2, IL-3, IL-4, IFN-beta and IFN-gamma, which are known to regulate immune responses and/or hematopoiesis, had no effect on the proliferation of hepatocytes. These results suggest that IL-6 plays a crucial role in regulating the regeneration of hepatocytes after hepatitis or partial hepatectomy.

Animals↗

PubMed1990

Interferon-alpha and interferon-beta induce Ia expression in limited protein synthesis condition.

We investigated the regulatory effect of interferons (IFNs) on macrophage (m phi) Ia antigen expression. Unlike type II IFN (IFN-gamma), type I IFNs (IFN-alpha and IFN-beta) were incapable of inducing the accumulation of Ia mRNA. However, when cultured with type I IFN plus cycloheximide (CHX), M phi accumulated significant amounts of Ia-specific mRNA, which was translated into Ia molecules expressed on the cell surface after the removal of CHX. Significant enhancement of Ia mRNA accumulation and the Ia expression were observed also in M phi stimulated with IFN-gamma plus CHX. Type I IFNs suppressed IFN-gamma-dependent Ia expression through the inhibition of Ia mRNA accumulation, but such suppression was not directed to the post-transcriptional step in M phi that had accumulated Ia mRNA in response to stimulation by type I or type II IFN plus CHX. These results seem to indicate that type I IFNs can induce Ia mRNA accumulation but that they also induce some inhibitory protein, the synthesis of which precedes it and is dominant, so that the Ia expression is masked.

Animals↗

PubMed1990

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella coupled with ATP hydrolysis.

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella in relation to ATP hydrolysis was examined by tryptic digestion. Tryptic digestion of the beta chain in the presence of 2 mM ATP (ADP) and 100 microM vanadate (Vi) or in the presence of 4 mM ATP gamma S produced different polypeptides from in the case of no addition. The difference was similar to the result previously reported for 21S outer-arm dynein heavy chains [Inaba, K. & Mohri, H. (1989) J. Biol. Chem. 264, 8384-8388]. Unlike the tryptic digestion pattern of 21S dynein heavy chains, however, the 135-kDa polypeptide was consistently produced from the beta chain, even in the presence of ATP (ADP) and Vi. The tryptic digestion pattern of the 21S particle reconstituted from the separated a chain, the beta/IC1 complex and the IC2/IC3 complex [Tang, W.-J.Y., Bell, C.W., Sale, W.S., & Gibbons, I.R. (1982) J. Biol. Chem. 257, 508-515] was similar to that of intact 21S dynein; the 135-kDa polypeptide was only slightly produced in the presence of ATP and Vi. The digestion rate constant of the 135-kDa polypeptide from the beta chain in the presence of ATP and Vi was significantly decreased as compared with in the case of 21S dynein or that of the reconstituted 21S particle. These results suggest that the trypsin sensitivity of the 135-kDa region of the beta chain changes with the association of the beta/ICI complex with the alpha chain and the IC2/IC3 complex in the presence of ATP and Vi.

Adenosine Triphosphate↗

PubMed1990

Thymic dendritic cells and B cells: isolation and function.

The thymus is the primary organ in which T cells undergo rearrangement of T cell receptor alpha and beta genes, positive selection for affinity to self MHC products, and elimination (negative selection) of reactivity to self antigens. These events require an interaction of the developing T cell with other cell types in the thymus. The latter include epithelial cells, macrophages, dendritic cells, and the recently described thymic B cells the majority of which are CD5+. Here we review the identification and isolation of thymic dendritic cells and CD5+ B cells. We consider phenotype, ontogeny, and function, including possible contributions to the induction of self tolerance. Thymic dendritic cells are similar to spleen dendritic cells, but are larger and exhibit a few differences in phenotype. Dendritic cells from both organs are equally potent accessory cells for the MLR and lectin-induced, T cell proliferation. Thymic dendritic cells have higher levels of Fc receptors and support anti-CD3 dependent mitogenesis. Thymic CD5+ B cells share phenotypic features with peritoneal CD5+ B cells. However thymic B cells neither proliferate nor form antibody producing cells in response to the stimulation with LPS or anti-IgM plus IL-4, but do respond to stimulation with MHC class II-restricted helper T cells. Thymic dendritic cells and CD5+ B cells both appear at a similar time in ontogeny, about 14 d of gestation, which is the time T cell differentiation begins to take place. Dendritic cells from spleen, which are potent activators for peripheral T cells, are also potent inactivators for thymic-derived cytotoxic T cells. A correlation between reactivity to MIs products and the expression of TCR-V beta genes is well documented, and B cells are the primary APC for this antigen. Therefore, thymic CD5+ B cells may be a good tool for the investigation of tolerance to M1s products.

Animals↗

PubMed1990

Dendritic cells as antigen presenting cells in vivo.

The biology of antigen presenting cells (APC) traditionally is studied in tissue culture systems using T cells that have been expanded beforehand by stimulation with antigen. Here we consider the distinctive roles of dendritic cells for sensitizing or priming T cells both in vitro and in vivo. Several functions of dendritic cells have been identified in tissue culture that are pertinent to T cell sensitization. These include the ability to a) capture and retain foreign antigens in an immunogenic form, b) bind antigen-specific resting lymphocytes, and c) activate T cells to produce lymphokines and undergo long term clonal growth. Dendritic cells have several properties in vivo that also would contribute to APC function. These are a) their widespread tissue distribution permitting access to antigens in most organs, b) the capacity to home via the blood stream and afferent lymph to the T-dependent areas of spleen and lymph node, and c) the ability to capture antigen in antigen-pulsed animals. Dendritic cells bearing antigen have been administered in situ to initiate responses like contact sensitivity, graft rejection, and antibody formation. A most striking recent example is that, when dendritic cells are pulsed with protein antigens in vitro and administered to immunologically naive mice, there is direct priming of antigen-specific T cells that are restricted to the MHC of the injected APC.

Animals↗