Kozo Yokomuro

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.

Detection of nephritis strain-associated streptokinase by monoclonal antibodies

Monoclonal antibodies (MAbs) N-59 and RU-1 were produced by immunisation of mice with streptokinase secreted by Streptococcus group A, type 12, strain A374 isolated from a patient with post-streptococcal glomerulonephritis (PSGN) and were characterised by Western blot analysis. MAb N-59 recognised antigenic determinants shared by both nephritis strain-associated streptokinase (NSA-SKase) and streptokinase of Streptococcus group C (C-SKase); MAb RU-1 reacted only with NSA-SKase. All nephritis-associated group A streptococcal strains tested reacted with MAb N-59; 87.5% of these strains reacted with MAb RU-1. MAb N-59 reacted with SKase produced by group G streptococcal strains isolated from patients with PSGN, and MAb RU-1 recognised SKase in two out of three of these strains.

Antigen-Driven Clonal Accumulation of Peritoneal γδ T Cells in vivo

How the clonality of γδ T cells changes in response to exogenous antigens is uncertain. Here we analyzed kinetics of Vγ1.1 and Vγ2 T cell clonality after intraperitoneal injection of purified protein derivatives (PPD) by the heterogeneity of the third complementarity determining region (CDR3) length in Vγ1.1-Jγ4-Cγ4 and Vγ2-Jγ1-Cγ1 junctions. The V-J junctions were analyzed in intrahepatic lymphocytes (IHL), spleen cells, and peritoneal exudate cells (PEC) by polyacrylamide gel electrophoresis. γδ T cells expressing Vγ1.1 and Vγ2 genes were heterogeneous in normal mice. Accumulation of specific Vγ1.1 T cell clones was transiently detected 7 days after the injection in PEC, but no accumulation was observed in IHL and spleen cells. The accumulated clones disappeared by 4 weeks. Transient accumulation of Vγ2 T cell clones was also observed in PEC at the early phase after the injection. These results suggest that γδ T cells with specific TCR respond to PPD and temporary accumulate in the peritoneal cavity, bu...

Mouse parenchymal liver cells in culture secrete a growth inhibitor for myeloma cells

METHODS Growth inhibitory activity in the conditioned medium of mouse parenchymal liver cells was examined in three strains of myeloma cells. RESULTS Two strains of myeloma cells were highly sensitive to a low concentration of mouse parenchymal liver cell derived growth inhibitor, whereas one strain was resistant to the same concentration. Interferon-alphabeta and transforming growth factor-beta activity were detected in mouse parenchymal liver cells, while interferon-gamma and tumor necrosis factor-alpha were not. The growth suppression exerted by mouse parenchymal liver cell derived growth inhibitor in the three myeloma strains was distinct from that exerted by transforming growth factor-beta, tumor necrosis factor-alpha, interferon-alphabeta and interferon-gamma. The mouse parenchymal liver cell derived growth inhibitor was eluted with a peak activity in the 18 kDa range and focused into pI values of 3.8-4.0, and it was lost when mouse parenchymal liver cells were treated with heat or trypsin. CONCLUSION These results indicate that mouse parenchymal liver cell derived growth inhibitor differs from the well-characterized growth inhibitors, transforming growth factor-beta, tumor necrosis factor-alpha, interferon-alphabeta and interferon-gamma.

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.

Suppression of liver regeneration resulting from intravenous injection of splenic glass adherent cells activated by poly I:C.

Syngeneic spleen cells (SPCs) were treated with poly I:C in the presence of DEAE-dextran for 2 h in vitro for their activation (IC-SPCs). When these IC-SPCs were inoculated into 70% partially hepatectomized mice, liver regeneration of the mice was strongly suppressed. The extent of suppression was dependent upon the number of injected cells and upon the concentration of poly I:C for in vitro treatment. Among IC-SPCs, the suppressive activity was predominantly caused by splenic glass adherent cells pretreated with poly I:C (IC-SACs) than by pretreated splenic T cells (IC-T). IC-SACs additionally cultured without poly I:C for 24 h lost their suppressive activity; however, when they were cultured in the presence of indomethacin, they could retain their activity, and interleukin 1 (IL 1) activity in culture supernatant was greater in the group cultured with indomethacin than without it. These results suggest that the suppressive activity of IC-SACs was not solely caused by the poly I:C carried into the mice with IC-SACs but by the activated IC-SACs themselves. The peak of liver cell mitosis in mice injected with IC-SACs was delayed 24 h as compared with non-injected groups, but IC-SACs-injected mice did not lose their regenerative potential. These findings demonstrate that the activated immune system may have some regulatory effects on liver regeneration.

Quantitative analysis of cytokine gene expression in the liver.

The relative levels of cytokine gene expression in the liver were analysed, focusing on IL‐2, IL‐4, IL‐5, IL‐6 and IFN‐γ compared to those in the spleen and Peyers patch by using the reverse transcriptase polymerase chain reaction (RT‐PCR). The levels of expression of cytokines in the liver mononuclear cells (MNC), especially that of IL‐6, were significantly higher than in other organs when mice were reared under specific pathogen‐free (SPF) or conventional conditions. Both the spleen and Peyers patch MNC expressed little of any of the cytokines, except for IL‐4 in Peyers patch MNC. The liver MNC produced significant amounts of IL‐6 in the culture supernatant upon concanavalin A stimulation. These findings suggest that the liver is a potent IL‐6‐ producing organ, which may relate to B cell differentiation, liver regeneration and the induction of acute phase proteins.

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.

Role of F4/80+Mac-1high adherent non-parenchymal liver cells in concanavalin A-induced hepatic injury in mice

Aim:  Non‐parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80+Mac‐1high+ cells were involved in the regulative feedback‐modulated regulation of inflammatory responses during concanavalin A (Con A)‐induced hepatitis.