Hepatic Mononuclear Cells Research Articles

Decreased Tumor Necrosis Factor-alpha and Interleukin-1alpha Production from Intrahepatic Mononuclear Cells in Chronic Ethanol Consumption and Upregulation by Endotoxin

The relationship between the changes in liver pathology and the production of interleukin (IL)-1α, IL-6, and tumor necrosis factor-α (TNF-α) by intrahepatic mononuclear cells was studied in rats fed alcohol and subsequently exposed to lipopolysaccharide (LPS). Rats were fed 40% ethanol in drinking water, whereas control rats were provided with a chow diet with isocaloric or 2% sucrose drinking solutions for up to 20 weeks. Decreased IL-1α and TNF-α production in 24-hr culture supernatants of mononuclear cells isolated from liver perfusate was detected while IL-6 remained unchanged over 20 weeks. When animals were injected with LPS (1.0 μg/kg body weight), there was a 5-fold rise in ALT levels in the ethanol-fed group, but not in control groups. Increased IL-6 and TNF-α levels in the serum and supernatant of cultured intrahepatic mononuclear cells stimulated with or without LPS or concanavalin A was observed. There was a correlation between levels of ALT and TNF-α, but not IL-6. T cells and Kupffer cells were the major source of TNF-α in culture supernatants of hepatic perfusate mononuclear cells from ethanol-consuming rats injected LPS. In addition, pathological liver injury was evident, which suggests a pathogenic role for TNF-α in alcohol-induced liver disease.

Decreased Tumor Necrosis Factor‐α and Interleukin‐1α Production from Intrahepatic Mononuclear Cells in Chronic Ethanol Consumption and Upregulation by Endotoxin

The relationship between the changes in liver pathology and the production of interleukin (IL)-1alpha, IL-6, and tumor necrosis factor-alpha (TNF-alpha) by intrahepatic mononuclear cells was studied in rats fed alcohol and subsequently exposed to lipopolysaccharide (LPS). Rats were fed 40% ethanol in drinking water, whereas control rats were provided with a chow diet with isocaloric or 2% sucrose drinking solutions for up to 20 weeks. Decreased IL-1alpha and TNF-alpha production in 24-hr culture supernatants of mononuclear cells isolated from liver perfusate was detected while IL-6 remained unchanged over 20 weeks. When animals were injected with LPS (1.0 microg/kg body weight), there was a 5-fold rise in ALT levels in the ethanol-fed group, but not in control groups. Increased IL-6 and TNF-alpha levels in the serum and supernatant of cultured intrahepatic mononuclear cells stimulated with or without LPS or concanavalin A was observed. There was a correlation between levels of ALT and TNF-alpha, but not IL-6. T cells and Kupffer cells were the major source of TNF-alpha in culture supernatants of hepatic perfusate mononuclear cells from ethanol-consuming rats injected LPS. In addition, pathological liver injury was evident, which suggests a pathogenic role for TNF-alpha in alcohol-induced liver disease.

Cutting Edge: Critical Role of NK1+ T Cells in IL-12-Induced Immune Responses In Vivo

Abstract CD1-dependent NK1+ T cells rapidly produce IL-4 upon stimulation through the TCR. These cells may therefore play an important role in the initiation of Th2 responses. Here, we show that NK1+ T cells constitutively express receptors for IL-12 and IFN-γ, and that IL-12 induces production of perforin in these cells. Moreover, while IL-12 induces high levels of IFN-γ and cytotoxic activity of hepatic or splenic mononuclear cells against tumor cells, this effect of IL-12 is significantly reduced in CD1-deficient mice with impaired NK1+ T cells development. These results indicate that NK1+ T cells play a critical role in IL-12-induced production of IFN-γ to initiate Th1 immune responses and as IL-12-induced cytotoxic effector cells to initiate antitumor immunity.

The tumor-bearing state induces augmented responses of organ-associated lymphocytes to high-dose interleukin-2 therapy in mice.

A high-dose bolus regimen for interleukin(IL)-2 administration to cancer patients frequently causes serious side-effects in which various organs are involved. In order to reveal the mechanism of toxicities associated with this regimen, we compared the augmenting effect of high-dose IL-2 on murine organ-associated lymphocytes between neoplastic and non-neoplastic states. Intraperitoneal administration of IL-2 at a dose of 10(5) JRU (Japanese Reference Units) twice daily for 3 days led to the death of all the syngeneic MH134-hepatoma- or X5563-myeloma-bearing mice, whereas it had no lethal effect on non-tumor-bearing mice. Histological and morphometric analyses demonstrated that tumor-bearing mice displayed more extensive infiltration of large granular lymphocytes and agranular lymphocytes in the liver and lungs than did the non-tumor-bearing mice. Large granular lymphocytes had the ultrastructural characteristics of lymphokine-activated killer cells. Lymphocytes often underwent extravasation into the interstitial space and exhibited local proliferation without causing any direct injury to apposed parenchymal cells. Flow-cytometric analysis of hepatic mononuclear cells demonstrated that IL-2-receptor-beta (IL-2R beta)-bearing lymphocytes, i.e., natural killer cells and intermediate CD3 cells, were increased in number in the neoplastic state before the IL-2 injection. The present study indicates that the tumor-bearing state increases the number of organ-associated IL-2R beta + lymphocytes, which are then greatly amplified by the challenge of high-dose IL-2, leading to the functional disturbance of organs. We have further demonstrated here that an intermittent low-dose IL-2 regimen has a potential therapeutic effect on tumor regression without causing lethal side-effects.

Expansion of intermediate T cell receptor cells expressing interleukin-2 receptor alpha- beta+, CD8alpha+ beta+, and lymphocyte function-associated antigen-1+ in the liver in association with intrahepatic islet xenograft rejection from rat to mouse: prevention of rejection with anti-interleukin-2 receptor beta monoclonal antibody treatment.

The precise mechanisms involved in islet xenograft rejection remain unknown. The purpose of the present study was to determine cellular mechanisms responsible for islet xenograft rejection in the liver to facilitate finding a procedure for prevention of immune rejection. Hepatic mononuclear cells (MNC) as well as splenocytes, peripheral blood MNC, and thymocytes from streptozotocin-induced diabetic mice (BALB/c) rejecting the intrahepatic rat (Lewis) islet xenografts were isolated and examined by two-color FACS analysis. The characteristic finding of the hepatic MNC from the mice rejecting islet xenografts compared with mice receiving isografts was a significant increase in the yield as well as in the percentage of the cells expressing CD3+ interleukin-2 receptor (IL-2R) alpha- beta+, CD3+ CD8alpha+ beta+, and T cell receptor (TCR) alphabeta+ lymphocyte function-associated antigen-1+. The expression of CD3 and TCR alphabeta of these T cells was found to be of intermediate intensity (TCR(int) cells). The expansion of these TCR(int) cells occurred predominantly in the liver. There was no significant difference in the cells expressing CD3+ IL-2R alpha+, CD3+ CD4+, CD3+ TCRgammadelta+, CD3- IL-2Rbeta+ (natural killer cells), and B220+ (B cells). In vivo administration of anti-IL-2Rbeta monoclonal antibody directed to the expanded cells produced a prevention of rejection. These findings suggest that islet xenograft rejection in the liver from rat to mouse is an event for which the TCR(int) cells are responsible.

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice.

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.

Reducing environment protects sinusoidal lymphocytes isolated from normal human liver from apoptosis

Background/Aims: We previously reported that the populations of lymphocytes and the expression of activated antigens in human sinusoidal mononuclear cells were different from those in peripheral blood mononuclear cells. Attempts to culture these cells for further study failed because they died rapidly under standard culture conditions in vitro after isolation from the liver. In this study, we evaluated the characteristics of cell death and the effects of various culture conditions on the viability of these cells. Methods: Sinusoidal mononuclear cells were isolated from University of Wisconsin solution that had been perfused through the portal veins of normal healthy human livers harvested for transplantation into living related recipients. Results: 70% of sinusoidal mononuclear cells cultured in in vitro were nonviable within 48 h after isolation, while only 10% of peripheral blood mononuclear cells died under the same conditions. Sinusoidal mononuclear cells showed DNA ladder formation of DNa on electrophoresis and characteristic morphological pattern on electron microscopic examination that suggested they had died in an apoptotic manner. The addition of human liver extracts or 2-mercaptoethanol and reduced glutathione to the cultures rescued the sinusoidal mononuclear cells from apoptosis. Furthermore, diamide, a sulfhydryl group specific oxidant negated the effect of the liver extract. Conclusion: In comparison with peripheral blood mononuclear cells, human sinusoidal mononuclear cells were more subject to death by apoptosis ex vivo, which was reversed by exogenous agents producing reducing conditions. These results suggested that hepatic sinusoidal mononuclear cells might express a different sensitivity to redox environment than peripheral blood mononuclear cells.

RAG1, RAG2 and pre-T cell receptor alpha chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation.

Flow cytometric analysis of cell suspensions obtained from normal adult liver tissue at the time of transplantation revealed significant populations of T lymphocytes. These were examined for molecular evidence of local T cell maturation using reverse transcription-polymerase chain reaction to detect expression of recombination activation gene 1 (RAG1), RAG2 and pre-T cell receptor alpha chain (pTalpha), which occurs only in early thymocyte development. Four specimens of whole liver were positive for RAG1 and RAG2 expression, whereas peripheral blood mononuclear cells from the same individuals were negative. To localize RAG expression, immature (CD2+CD7+) and mature (CD45R0+) T cell subpopulations were isolated by magnetic separation from hepatic and peripheral blood mononuclear cell preparations. We detected the expression of RAG1, RAG2 and pre-TCRalpha in five specimens of hepatic CD2+CD7+ but not in CD45RO+ hepatic lymphocytes. Four out of six specimens of CD2+CD7+ cells from the peripheral blood were negative for RAG1 and RAG2 while all six specimens were positive for pTalpha expression. These results suggest that pre-T cells are trafficking from the bone marrow or the thymus to other tissues to continue differentiation and selection in the context of an appropriate cellular and molecular environment. The presence of immature populations of T cells in the adult liver and high levels of RAG expression suggests that the adult liver provides such an environment for extrathymic T cell maturation. These findings may have important implications for tolerance induction after liver transplantation and offer help in understanding the etiology of autoimmune liver disease.

C-kit+ stem cells and thymocyte precursors in the livers of adult mice.

Livers of the adult mice contain c-kit+ stem cells that can reconstitute thymocytes, multiple lineage cells, and bone marrow (BM) stem cells. Transfer of 1 x 10(7) hepatic mononuclear cells (MNC) and 5 x 10(4) hepatic c-kit+ cells of BALB/c mice induced DP thymocytes within a week in four Gy-irradiated CB17/-SCID mice, but 2 wk were required for BM cells or BM c-kit+ cells to produce DP thymocytes. Moreover, B cell-depleted BM cells or liver MNC of SCID mice that had been rescued by hepatic MNC of BALB/c mice again reconstituted thymus and B cells of other irradiated SCID mice. CD3- IL-2R beta- populations of both BM cells and hepatic MNC of C57BL/6 (B6) mice could generate T cells with intermediate TCR (mostly NK1.1-) in the liver of irradiated B6 SCID mice before thymic reconstitution (extrathymic T cells). Furthermore, transfer of liver c-kit+ cells of B6-Ly 5.1 mice into irradiated B6 SCID (Ly5.2) mice revealed that liver c-kit+ cells can reconstitute myeloid and erythroid lineage cells. These results strongly suggest that the liver contains pluripotent stem cells and serves an important hematopoietic organ even into adulthood.

Liver NK1.1+ CD4+ alpha beta T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis.

We demonstrate herein evidence that IL-12-activated alpha beta T cells with intermediate TCR (NK1+ TCRint cells) in the liver inhibit metastases in the lung as well as in the liver metastases of i-v. injected tumors. IL-12 administration enhanced NK1 expression of NK1+ TCRint cells (NK1high) and increased CD4 weakly positive (CD4low) TCRint cells, while both CD4+ TCRint cells and double-negative TCRint cells were proportionally diminished. Accordingly, the major parts of NK1high TCRint cells are CD4low cells, and most of these cells are V beta 8+ cells. The cytotoxic assays of IL-12-stimulated hepatic mononuclear cells after treatment with respective Abs and complement in vitro and after sorting revealed that CD4low NK1high TCRint cells are cytotoxic effectors. When IL-12-stimulated hepatic mononuclear cells (but not splenocytes) were transferred into tumor-preinjected mice, EL-4 cell metastases in the liver as well as 3LL cell metastases in the lung were inhibited. The antimetastasis of hepatic mononuclear cells transfer was abrogated by the depletion of NK1+ cells, CD3+ cells, or CD4+ cells but not CD8+ cells before transfer. Moreover, transfer of these cells of nude mice into tumor-preinjected mice also inhibited metastases in both organs. Although NK1+ TCRint cells are nearly absent in the hepatic vein blood, a significant proportion of NK1high TCRint cells appeared by IL-12 administration. These results demonstrate that IL-12-stimulated liver NK1high TCRint cells, including extrathymic ones, are major effectors against tumor metastasis and suggest that the cells migrate and inhibit lung metastases.

Interleukin-12 induces cytotoxic NK1+ alpha beta T cells in the lungs of euthymic and athymic mice.

We recently reported that interleukin-12 (IL-12) stimulated hepatic NK1.1 Ag+ alpha beta T cells with intermediate T-cell receptor (TCR; NK1+ TCRint cells) and enhanced their NK1 expression (NK1high TCRint), and that these cells acquire strong major histocompatibility complex (MHC) unrestricted cytotoxicity in C57BL/6 mice, both +/+ and nu/nu. In the present study, we find that although murine lung normally has few NK1+ TCRint cells, NK1high TCRint cells are induced in+/+ and nu/nu mice after systemic administration of IL-12; these cells exhibit strong MHC unrestricted cytotoxicity against NK-sensitive and -resistant targets. A small number of NK1high TCRint cells was also found in peripheral blood after increased amounts of IL-12 were administered. Cytotoxicity tests in vitro revealed that the cytotoxic activity of the lung mononuclear cells (MNC) of C57BL/6 mice induced by IL-12 was abrogated by the depletion of either NK1+ or CD3+ cells, but not of CD8+ cells, as reportedly was the case of hepatic MNC, suggesting that NK1high TCRint cells are an antimetastatic population not only in the liver but also in the lung of mice. IL-12 injection into mice markedly elevates serum interferon-gamma (IFN-gamma) levels. However, although IL-12-induced cytotoxicity of NK1high TCRint cells was significantly reduced by anti-IFN-gamma antibody injection (which decreased serum IFN-gamma to an undetectable level), the appearance of NK1high TCRint cells in the lung and liver was not so affected. These results suggest that IFN-gamma is an important mediator of the cytotoxicity of NK1high TCRint cells but is not an essential factor for induction of these cells. We also added data showing that IL-12 has a broad antimetastatic effect against various liver and lung metastatic tumours intravenously injected into several strains of mice, including NK-deficient bg/bg mice. It can be considered that, in addition to NK cells, CD8+ cytotoxic T cells and gamma delta T cells, NK1+ TCRint cells can be categorized as one of the cytotoxic effector populations. These novel type cells distinct from regular T cells may play an important role in monitoring intra- and perivascular areas.

LPS induces NK1.1+ alpha beta T cells with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells.

We recently reported that systemic administration of IL-12 into mice activates NK1.1+ alpha beta T cells with intermediate TCR (NK1+TCRint) and induces strong MHC-unrestricted cytotoxicity in C57BL/6 mice. In the present report, we examined the effect of LPS on Kupffer cells and NK1+TCRint, cells in C57BL/6 mice. Administration of LPS, as well as synthetic lipid A analogue (ONO-4007), but not detoxified LPS, induces the increase of NK1 expression of NK1+TCRint cells (NKlhighTCRint) and the acquisition of strong MHC-unrestricted cytotoxicity of these cells against NK-sensitive and NK-resistant targets as does IL-12 administration. LPS as well as ONO-4007 induced IL-12 mRNA in hepatic mononuclear cells, mainly in plastic-adherent Kupffer cells. LPS-induced cytotoxicity of hepatic mononuclear cells was greatly reduced by in vivo injections of anti-IL-12 Ab, to a lesser extent by anti-IFN-gamma Ab, but not by anti-IL-1 nor anti-TNF-alpha Ab. Pretreatment of mice with LPS induced inhibition of hepatic metastases of i.v. injected EL4 cells in C57BL/6 euthymic and athymic mice and this antimetastasis was inhibited by injection of anti-IL-12 Ab. This antimetastatic effect of LPS in the liver was also observed in different strains of mice and tumors, In contrast to IL-12, however, LPS was not so effective when administered after tumor inoculation. These results revealed that LPS (lipid A) stimulates NK1+TCRint cells through IL-12 production from Kupffer cells and suggest that bacterial components, probably including those from intestine, are activators of Kupffer cells and NK1+TCRint, cells in the liver. It is also suggested that the host condition as well as LPS-induced cytokines other than IL-12 may affect antitumor effect induced by LPS in the liver.

No evidence for emergence of autoreactive V β6 + T cells in Mls-1 a mice following exposure to a thymotoxic dose of 2,3,7,8-tetrachlorodibenzo- p-dioxin

Tolerance to minor lymphocyte stimulatory-1 a (Mls-1 a) antigens is associated with clonal deletion of cells carrying the T cell receptor variable region Vβ6. Thymic epithelial cells may contribute to the intrathymic negative selection of potentially autoreactive Vβ6 + cells. Because 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) acts on thymic epithelial cells, we hypothesized that it may interfere with intrathymic selection processes. In the present study, this was addressed by exposing Mls-1 a DBA/2 mice to a single thymotoxic dose of TCDD. The emergence of Vβ6 + cells in thymus, spleen, and mesenteric lymph nodes was investigated during the subsequent recovery of TCDD-induced thymic atrophy. In addition, the extrathymic differentiation of T lymphocytes in the liver was studied. TCDD exposure resulted in a severe thymic atrophy, and an increase in hepatic mononuclear cells. However, we were not able to demonstrate any emergence of potentially autoreactive mature Vβ6 + T cells, that differentiated either intrathymically or extrathymically, in TCDD-exposed DBA/2 mice.

Effect of splenectomy on hepatic metastasis of colon carcinoma and natural killer activity in the liver

We have previously demonstrated that administration of killed streptococcal preparation (OK432), a biological modifier, increased the number of asialo GM1-positive cells in the liver, enhanced NK activity of hepatic mononuclear cells, and reduced the number of hepatic metastases of colon 38 adenocarcinoma that were inoculated into the superior mesenteric vein of C57BL/6 strain mice. In the present study, to clarify the role of the spleen in immune surveillance of the liver, the effect of splenectomy on hepatic metastasis of colon carcinoma and on hepatic NK activity has been examined. The number of hepatic metastasis increased in the splenectomized mice, compared with that in sham-operated mice. Administration of OK432 increased the number of asialo GM1-positive cells in the liver and enhanced NK activity of hepatic mononuclear cells in both groups, but NK activity of hepatic mononuclear cells in the splenectomized mice was less than that of the sham-operated mice. An enhanced NK activity of these cells was abolished by treatment with anti-asialo-GM1 antibody plus complement in vitro. Interleukin-2 mRNA expression was increased in the spleen 2 hr after OK432 administration and persisted until 8 hr, but was scarcely noted in the liver. On the other hand, NK activity of hepatic mononuclear cells in the asialo GM1-positive cell-depleted (previous administration of antiserum against asialo GM1) mice was enhanced after OK432 administration in the sham operated and splenectomized mice, but an enhanced NK activity in these mice was only partially or not at all abolished by treatment with anti-asialo GM1 antibody plus complement in vitro, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic NK1.1 Ag+ alpha beta T cells with intermediate TCR induced in the liver of mice by IL-12.

Systemic administration of IL-12 greatly reduced the hepatic metastases of i.v.-injected liver metastatic EL4 tumor cells in C57BL/6 +/+ and nu/nu mice. Cytotoxic assay in vitro revealed that administration of IL-12 greatly enhanced cytotoxicity of hepatic mononuclear cells (MNC) against various NK- sensitive and -resistant tumor targets, including EL4 cells, whereas only slight or moderate augmentation of the cytotoxicity was observed in splenocytes in normal and nude mice. After IL-12 administration, hepatic MNC increased in number and showed vigorous proliferation in vitro. Hepatic MNC of control C57BL/6 +/+ mice contain alpha beta T cells with intermediate TCR (TCRint) as well as alpha beta T cells with bright TCR, whereas hepatic MNC of nu/nu mice have only TCRint cells. These TCRint cells are found to be NK1.1 Ag+ (NK1+ TCRint). Systemic administration of IL-12 into normal and nude mice markedly augments the NK1 expression of NK1+ TCRint cells (NK1high TCRint), which is comparable to or brighter than that of NK cells in the liver, whereas alpha beta T cells with bright TCR or gamma delta T cells in the liver are NK1-. Depletion of either NK1.1+ or CD3+ cells, but not CD8+ cells, of hepatic MNC from IL-12-treated normal mice by respective Abs and C in vitro abrogate their cytotoxicity. These results revealed that TCRint cells are potent cytotoxic effector cells and suggest that NK1high TCRint cells are the main antimetastatic population in the liver, and that TCRint cells are functionally different from regular T cells with bright TCR.

Synchronous Expansion of Intermediate TCR Cells in the Liver and Uterus during Pregnancy

The immunophysiology of pregnancy was investigated in relation to how extra- and intrathymic pathways of T cell differentiation are modulated during pregnancy. Pregnancy was produced in syngeneic and allogeneic combinations in mice. In both cases, a prominent decrease in the number of thymocytes and a mild increase in the number of hepatic mononuclear cells (MNC) were induced during the periparturient period. The most striking change was the increase in the number of intermediate TCR cells with extrathymic T cell properties in the liver. When MNC, yielded by the uterus were examined, an expansion of intermediate TCR cells was also observed in this organ during pregnancy. The major populations in the pregnant uterus were NK cells and intermediate TCR cells, the latter of which contained double-negative CD4 -8 - cells and consisted of both αβ and γδ T cells. All of these properties coincided with those of extrathymic T cells in the liver. Reflecting extrathymic T cell differentiation, MNC in the liver and pregnant uterus expressed mRNA of RAG-1 and RAG-2. To determine the role of hormonal regulation in this phenomenon, the effects of various pregnancy-associated hormones on thymocytes and hepatic MNC were examined in nonpregnant mice. Of the hormones tested, estrogen was found to induce a response similar to that seen in pregnant mice. These results suggest that a local immune response in the uterus during pregnancy is a phenomenon occurring synchronously with systemic immune systems.

Induction of unresponsiveness of antigen-specific T lymphocytes by oral administration of cedar pollen extract in mice.

T lymphocyte unresponsiveness, induced in mice by a single gastric intubation of 0.2 ml cedar pollen extract (CPE, containing 4 micrograms protein/ml)/mouse daily for 3 to 28 consecutive days, was evaluated by the absence of a proliferative response of popliteal lymph node (PLN) T lymphocytes to CPE in vitro. T lymphocyte unresponsiveness increased with the period of gastric intubation of CPE and reached more than 80% of the control on day 28. The unresponsiveness to CPE was antigen-specific and T lymphocyte-mediated. In vitro CPE-specific T lymphocyte proliferation was significantly suppressed by intestinal intraepithelial lymphocytes (IELs) and hepatic mononuclear cells (MNCs), but not by spleen cells or PLN T lymphocytes from mice fed CPE for 28 days. The effector activity of IELs and MNCs was obviously antigen-specific. These results suggest that lymphocytes in the intestine and liver of mice fed CPE would be involved in the induction and maintenance of CPE-specific T lymphocyte unresponsiveness.

Induction of Specific Tolerance by Hepatic Double-Negative CD4-8-αβ T Cells of Mice Immunized with Allogeneic Cells via the Portal Vein in Vitro

We immunized AKR/n (H-2k) spleen cells in BALB/c (H-2d) mice via the portal vein (pv) and investigated the role of hepatic mononuclear cells (MNC) in the induction of alloantigen-specific immune tolerance. MNC in the liver and spleen of pv-administered mice were demonstrated to abrogate the responses to AKR/n alloantigens in allogeneic MLR. On the contrary, MNC in the liver and spleen of mice administered subcutaneously with the same antigens showed greater responses than those of control mice. The tolerance induced by pv administration was alloantigen-specific and appeared earlier in hepatic MNC than in splenic MNC. Furthermore, hepatic MNC of pv-administered mice had a suppressive effect when these cells were added to allogeneic MLR, in which mitomycin C (MMC)-treated AKR/n splenic MNC were used as stimulator and control BALB/c splenic MNC were used as responder. Splenic MNC of pv-administered mice and hepatic MNC of control mice did not show such suppressive effects. Such suppression was alloantigen-specific, since no suppression was induced when hepatic MNC of pv-administered mice were added to a system using MMC-treated C57BL/6 (H-2b) splenic MNC. The alloantigen-specific suppression induced by hepatic MNC was abrogated by a depletion of TcR-αβ+ cells but not of CD4+, CD8+, nor B220+ cells from hepatic MNC. These results suggested that alloantigen-specific suppressor cells appeared predominantly in the hepatic MNC of pv-administered mice and displayed the phenotype of TcR-αβ+CD4-8- double-negative T cells, although alloantigen-specific tolerance was induced in both hepatic and splenic MNC.

Elevated production of interleukin 6 by hepatic MNC correlates with ICAM-1 expression on the hepatic sinusoidal endothelial cells in autoimmune MRL/lpr mice

MRL/lpr mice, which are a model of SLE and rheumatoid arthritis in humans, develop profound lymphadenopathy resulting from the accumulation of CD3 + 4 −8 − double-negative (DN) αβT cells in peripheral lymphoid tissues. We previously indicated that these DN αβT cells preferentially proliferate in the liver and migrate to the periphery. In this study, we analyzed whether any kind of cytokine was produced by hepatic mononuclear cells (MNC) in MRL/lpr mice. The evidence obtained indicates that interleukin 6 (IL-6) was vigorously produced by hepatic MNC in diseased MRL/lpr mice under unstimulated conditions. MNC in the spleen of these mice produced small amounts of IL-6, while those in the lymph nodes did not produce any appreciable amounts of IL-6. These activities of hepatic MNC in diseased MRL/lpr mice were almost completely neutralized by anti-mouse IL-6 monoclonal antibody (mAb). On the other hand, immunohistochemical staining of light- and electron-microscopic analyses revealed that the intracellular cell adhesion molecule 1 (ICAM-1) was expressed on the hepatic sinusoidal endothelial cells of diseased MRL/lpr mice. Moreover, ICAM-1 was newly induced in the hepatic sinusoids of control C3H/He mice by an intravenous injection of 50 units of recombinant mouse IL-6. These data suggest that ICAM-1 expressed on the hepatic sinusoidal endothelial cells in MRL/lpr mice is induced by IL-6, which is produced by hepatic MNC, and that such ICAM-1 may be responsible for the saturation of inflammatory cells and the proliferation of lymphocytes in the liver of MRL/lpr mice.

Age-associated increase of CD5+ B cells in the liver of autoimmune (NZB x NZW) F1 mice.

The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5+ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB x NZW) F1 mice. An age-associated increase of CD5+ B cells was demonstrated in the liver of these mice. Although CD5+ B cells (i.e., CD5+IgM+ and CD5+B220+) constituted a minor population of hepatic mononuclear cells (MNC) (< 5%) when mice were young (8 weeks), a large population of CD5+ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age-dependent increase of CD5+ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5+ B cells was the same as that of CD5+ B cells in the peritoneal cavity and spleen, showing dull-CD5, bright-IgM and dull-B220. High levels of CD5+ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5-day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM- and IgG-autoantibodies against double-stranded DNA and single-stranded DNA, despite the increased proportion of CD5+ B cells. On the other hand, peritoneal exudate cells produced only IgM-, but not IgG-, autoantibodies, whereas splenic cells were able to produce both IgM- and IgG-autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)