Ayako Mabuchi

Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes)

The activity of macrophage colony‐stimulating factor (M‐CSF) was found in the culture supernatant of mouse parenchymal liver cell fractions in a bone marrow colony‐forming assay. The activity of an M‐CSF‐like substance purified by a four‐step procedure was neutralized by goat anti‐mouse M‐CSF antiserum. M‐CSF mRNA was detected in cellular UNA prepared from cultured parenchymal liver cell fractions by Northern blot analysis and also in cultured parenchymal liver cells by in situ hybridization. These results indicate that parenchymal liver cells have the capacity to produce M‐CSF. We discuss the role of M‐CSF in hematopoiesis, the immune response, and other biological phenomena.

The liver and the hematolymphoid system: I. The regulation of nylon-passed spleen cell proliferation by active factors released from syngeneic nonparenchymal liver cells.

Nylon‐passed spleen cells were found to proliferate when cultured with syngeneic nonparenchymal adherent liver cells and their culture supernatants. The supernatants contained IL‐1, IL‐6, GM‐CSF, and IFN (α + β) activities but not IL‐2 and IL‐3 activities. The IFN level was higher in early culture sup (2–24 hr) than in later culture sup (48–72 hr). Proliferation was greatly increased by anti‐IFN (α + β) serum in the spleen cells cultured in the earlier sup. This antiserum increased the spleen cell proliferation only slightly in the later culture sup. This suggests that nonparenchymal liver cells produce two factors, one having a suppressor, and the other an enhancer action, with IFN being one of the suppressor factors. With culture time, DNA synthesis of spleen cells increased and IL‐2 and IL‐3 activities were generated in the culture sup. Cells proliferated during culture were found to be morphologically lymphocytes, granulocytes, and macrophages. The mechanisms by which nonparenchymal liver cells regulate the hematolymphoid system are discussed based on our observations.

Unresponsiveness of Intrahepatic Lymphocytes to Bacterial Superantigen: Rapid Development of Suppressive Mac-1high Cells in the Mouse Liver

We previously found that a small dose (2 μg per mouse) of staphylococcal enterotoxin B (SEB) induced early emerging unresponsiveness in intrahepatic‐lymphocyte populations (IHLs). The purpose of this study was to reveal the inducing role of accessory cells involved in IHLs in this phenomenon. IHLs prepared at 3 to 24 hours after SEB injection failed to proliferate in response not only to SEB but also to SEA, representing ligand‐nonspecific unresponsiveness, whereas spleen cells (SPCs) and mesenteric lymph‐node cells showed transient proliferation. Unresponsiveness in IHLs was related to a deficit of their accessory cell function as measured by coculture of irradiated IHLs and antigen‐specific, type 1 T‐helper (Th1) clone cells. High levels of nitrite were detected in the culture supernatant. Supplement of NG‐monomethyl‐l ‐arginine lowered nitrite levels and concurrently restored the proliferative response of Th1 cells, indicating the involvement of nitric oxide in suppression. Adherent cells prepared from IHLs well reproduced these results. As shown by flow cytometry, Mac‐1high Ia+ cells, which mainly included F4/80+ cells (macrophages) and a minor population of CD11c+ cells (dendritic cells), increased in proportion in IHLs but not in SPCs at 6 to 24 hours. Depletion of Mac‐1high cells from IHLs with antibody‐coated magnetic beads recovered the proliferative response. Depleted Mac‐1high cells had a monocytoid appearance. In immunostained sections, Kupffer cells came to highly express both Mac‐1 and Ia at 12 hours. These results indicate that Mac‐1highIa+ adherent cells, largely Kupffer cells activated by SEB, nonspecifically suppress the proliferation of Th1 cells via nitric oxide production before manifestation of ligand‐specific unresponsiveness.

Role of the liver in T cell differentiation--generation of CD3-CD4+/CD8+TCRbeta- cells and CD3-4-8-TCRbeta+ cells from CD4-8-TCRbeta- athymic nude bone marrow cells by culture with parenchymal liver cells.

To investigate the influence of the liver on differentiation of hematopoietic stem cells/ pro‐T cells, TN‐NWP‐BMC (athymic nude bone marrow cells that were treated with anti‐TCRβ, anti‐CD4, and anti‐CD8 Abs plus complement and then passed through a nylon wool column) were cultured on parenchymal liver cells. After culture for 2.5 days, CD3–4–8–TCRβ+ cells and CD3–CD4+/CD8+TCRβ– cells were developed from TN‐NWP‐BMC. TCRVβ8+ cells comprised 19.9% of CD3–4–8–TCRβ+ cells, and Vβ8 mRNA was detected in the CD3–4–8–TCRβ+ cells by reverse transcriptase‐polymerase chain reaction. The CD3–CD4+/CD8+ TCRβ– cells contained not only single‐positive cells but also CD4+8+ double‐positive cells. The CD8 protein consisted of 88.9% CD8α+β–, 10.1% CD8α+β+, and 1% CD8α–β+ molecules. From these results and the finding of co‐expressed antigens, CD3–4–8–TCRβ+ cells and CD3–CD4+/CD8+TCRβ– cells appear to be immature cells not committed to a certain cell lineage. J. Leukoc. Biol. 63: 575–583; 1998.

Production of B cell differentiation factors by mouse parenchymal liver cells

Previously we reported that most antibody secreting cells secreted IgA in the liver. Here we assessed the possibility that parenchymal liver cells (PLC) produced factors, transforming growth factor (TGF)‐p and IL‐5, which participate in the differentiation of B cells to IgA‐secreting cells. We showed that TGF‐p activity was present in the culture supernatant of PLC. and IL‐5 activity was in the lysate of PLC. Moreover, it was confirmed that IL‐5 protein produced by PLC was mainly localized in the cell membrane by histochemical staining. The findings that both TGF‐P and IL‐5 were produced by PLC should provide useful information concerning the fact that IgA‐secreting cells were dominant in the liver.

Separation of RRBC-RFC and Non-Rosette Forming Cells (non-RFC) of Guinea Pig T-Cell Populations and Their Functional Difference

Guinea pig lymph node cells suspension (LNC‐O) was filtered through a glass wool column and the effluent (LNC‐G) was further filtered through a nylon column. In this effluent (LNC‐NE) about 30 per cent of the lymphocytes was identified as non‐rosette forming cells (non‐RFC). The non‐RFC fraction was separated from LNC‐NE fraction by Ficol‐Conray specific gravity centrifugation of effluent cells reacted previously to rabbit red blood cells (RRBC). The upper layer after centrifugation, designated non‐RFC fraction, was separated. In this fraction 96% of the cells were lymphocytes and about 95% of them were non‐RFC, which lacked receptors for rabbit red blood cells (RRBC) or EAC and detectable surface immunoglobulin by conventional techniques.

Antigen-specific T cell cluster formation on antigen-pulsed macrophage monolayers in mice.

We describe the quantitative measurement of antigen‐specific clusters formed by antigen‐pulsed macrophages and immunized T cells in mice. We have found the peripheral blood T cells show very little non‐specific adhesion to macrophages in mice. By using this population of lymphocytes in the peripheral blood as the source of immunized T cells, we could quantitate antigen‐specific cluster formation. On OVA‐pulsed monolayers of peritoneal exudate macrophages from normal BALB/c mice, syngeneic peripheral blood T cells from donors immunized with the same antigen develop 20–40 clusters per 1,000 macrophages, whereas the same T cells on non‐pulsed monolayers develop only 0–5 cluster‐like accumulations of cells. On antigen‐pulsed monolayers of macrophages from allogeneic (C57BL/6 or A/J) mice, clusters are developed only in the negative range (0–5/1,000 macrophages). Considering the observation by Braendstrup et al, these data seem to suggest that histocompatibility between macrophages and T cells is required to develop antigen‐specific T cell clusters on antigen‐pulsed macrophage monolayers, and that the genetic restriction of immune responsiveness may be directly expressed in this initial form of cellular interaction between antigen‐bearing macrophages and specific T cells.

211 Concanavalin A-Induced Hepatitis in Mice Is Prevented by Depletion of Gut Bacteria Using Antibiotics

L or hyposmotic conditions, as measured by LDH and HMGB1 release. Upon hyposmotic stress of hepatocytes, cell death occurred in the absence of caspase activation. Similar to primary hepatocytes, K18-DE mice treated with Fas-L had elevated serum necrosis markers (LDH and HMGB1) as compared to their WT counterparts. K18-DE mutation renders the K18 obligate heteropolymer, K8, less susceptible to phosphorylation at its p38 stress kinase site K8 S79, without altering the binding of K8/K18 to p38 kinase. Notably, K8 S79 phosphorylation plays an important role in mediating keratin filament reorganization upon stress. Conclusions: Caspase digestion of K18 plays an essential role in promoting apoptosisassociated keratin filament collapse in the liver. Keratin phosphorylation-mediated filament reorganization appears to be critical in protecting hepatocytes from necroapoptosis, and provides a mechanism for susceptibility to tissue injury in human IF mutations that involve caspase cleavage sites.

Abstracts of selected papers presented at the 77th General Meeting of the Japanese Society of Gastroenterology

M E E T I N G OF T H E J A P A N E S E S O C I E T Y OF G A S T R O E N T E R O L O G Y March 28-30, 1991-Tokyo, Japan Chairman: Yutaka MATSUO, M.D.

Suppression of IgE antibody response by preadministration of antigen-pulsed spleen cells. II: Characteristics of immediate tolerance induction

Intravenous administration of syngeneic spleen cells (SPC), briefly pulsed with antigen in vitro, resulted in a profound state of IgE antibody unresponsiveness. One of the mechanisms of this unresponsiveness is responsible for an immediate tolerance which is induced without any suppressor cells. Characteristics of this immediate tolerance were investigated. Administration of antigen-pulsed spleen cells 4 h before the immunization, suppressed the production of IgE antibody triggered by the subsequent immunization. Pretreatment with cyclophosphamide had no effect on this rapid suppression, and this suppressive state could not be transferred to normal syngeneic recipients by the injection of spleen cells from the tolerant mice used in our experiment. These observations suggest that suppressor cells do not play an important role in immediate tolerance. The extent of this immediate tolerance induced by the injection of antigen-pulsed SPC depends on the number of antigen-pulsed SPC and the dose of antigen to which SPC had been exposed. Injection route of antigen-pulsed SPC has a great influence on the induction of immediate tolerance. The order of suppressive extent is intravenous, greater than intraperitoneal greater than subcutaneous. This suppression is specific to the antigen pulsed to SPC. Carrier-specific T cells are the major target of suppression in immediate tolerance. Antigen-pulsed T cells induce immediate tolerance most effectively in the subpopulations of SPC.