Michio Fujiwara

Suppression of hepatic lesions in a murine graft-versus-host reaction by antibodies against adhesion molecules

BACKGROUND/AIMS The injection of parental CD4+ T cells into major histocompatibility complex (MHC) class II disparate F1 hybrid mice induced an autoimmune graft-versus-host reaction (GVHR) which is analogous to autoimmune liver diseases. The interaction of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) has been known to be profoundly involved in the trafficking of lymphocytes into the inflammatory tissues. The aim of this study was to clarify the role of VLA4 or VCAM-1 in the development of GVHR-induced hepatic lesions in our model. METHODS B6 T spleen cells were injected into (B6.C-H-2bm12xB6) F1 mice intravenously. Anti-VLA-4 mAbs and/or anti-VCAM-1 mAbs were injected intraperitoneally at a dose of 2.5 mg/kg of each mAbs per body weight of mouse. We examined the changes in GVHR-induced hepatic lesions, serum levels of antimitochondrial antibodies (AMA) and cytokine mRNA expressions of liver-infiltrating lymphocytes using H.E. and immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS Hepatic lesions of anti-VLA-4 mAbs-treated mice were inhibited compared with those of GVHR mice. However, the administration of mAbs did not interfere with the induction of splenomegaly, the invasion of CD4+, CD8+, B220+, or Mac-1+ cells around bile ducts, nor the production of AMA. Liver-infiltrating CD4+ T cells obtained from these treated mice did not alter the expression of T helper (Th)1 and Th2 cytokine mRNA. CONCLUSION The results suggest that treatment with antibodies against these adhesion molecules could inhibit the infiltration of lymphocytes without affecting the Th1/Th2 balance. The blockade of VLA-4-mediated cell infiltration into the liver in this model may have a possible novel therapeutic role of VLA-4 mAbs.

Induction of autoimmune disease by graft-versus-host reaction across MHC class II difference: modification of the lesions in IL-6 transgenic mice

We examined the effect of IL‐6 on the development of autoimmune diseases (primary biliary cirrhosis. Sjögrens syndrome) employing murine grari‐versus‐host reaction (GVHR) model with MHC class II disparity. For this purpose, we used IL‐6 transgenic(B6.6) mice in which a high level of IL‐6 was detected. C57B1/6 (B6) spleen T cells were injected into B6.6 mated with B6.C‐H‐2(bml2) mutant mice ((bmi2x B6.6)FI) and GVHR with MHC class II disparity was induced. The iransgenic hybrid mice with GVHR showed a larger spleen index and contained a higher serum level of IL‐6 than those without GVHR. Autoimmune‐like lesions in transgenic recipients became weakened compared with those in non‐transgenic (bml2 x B6)F1 recipients. In contrast, levels of antimitochondrial antibodies in (bm 12 x B6.6)FI GVHR group were signiiicantly higher than that of (bml2 X B6)FI GVHR group. These results indicate that lL‐6 excessively produced in vivo might regulate the progression of autotmmune diseases.

LPS-induced autoantibody response: I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes☆

Abstract Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro . The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.

Cytokine profile of liver-infiltrating CD4+ T cells separated from murine primary biliary cirrhosis-like hepatic lesions induced by graft-versus-host reaction

Background and Methods : We have previously reported that CD4+ T cells induced primary biliary cirrhosis (PBC)‐like hepatic lesions in mice with graft‐versus‐host reaction due to major histocompatibility complex class II disparity. To clarify the relationship between the cytokine profile produced by CD4+ T cells and the formation of hepatic lesions, we sorted CD4+ T cells from the liver by using flow cytometry and examined their cytokine mRNA expression at various times after GVHR induction. We also examined the associated changes in the serum levels of antimitochondrial antibodies (AMA).

Studies on the resistance to tolerance induction against human IgG in DDD mice: I. Organ differences of tolerogen susceptibility and cellular sites responsible for the resistance

Abstract Cellular sites of the tolerogen resistance in DDD mice against human IgG (HGG) were examined by reconstitution experiments in which cells of various lymphoid organs from tolerized mice were transferred into lethally irradiated syngeneic recipients with or without the supplement of an excess number of untreated T or B cells. It was shown that T cells but not B cells in the spleen and bone marrow-locating B cells were tolerogen resistant. Kinetic profiles of tolerance induction were compared among thymus, lymph node, and spleen T cells. Thymus cells fall into unresponsive state as early as 2 days after the tolerogen (tHGG) injection when only partial tolerance was observed in lymph node T cells. By 1 week of tolerogen treatment, the tolerant state was completed in both thymus cells and lymph node T cells, while spleen T cells showed marked resistance. Tolerance induced in thymus cells and spleen T cells was of relatively short duration and responsiveness was completely recovered by 5 weeks after the injection of tHGG. At this time lymph node T cells still showed hyporesponsiveness. The differences in tolerance inducibility were also shown among different lymphoid organs in tolerogen dose response. Lymph node T cells were very sensitive to tolerance induction, giving no response even by the injection of 0.01 mg of tHGG. Thymus cells were much less sensitive with the gradual loss of responsiveness by increasing the amount of tHGG. In contrast, spleen T cells showed gradual resistance with increasing amount of tHGG, indicating that some positive response was evoked in spleen T cells by a relatively high dose of tHGG. These results seem to suggest that the tolerogen resistance of spleen T cells may be due to their capability of showing positive response against the tolerogenic material. This was also suggested by the fact that the treatment with cyclophosphamide following the tolerogen injection diminished completely the responsiveness against the subsequent challenge immunization.

Concentric fibrosis and cellular infiltration around bile ducts induced by graft-versus-host reaction in mice : A role of CD8+ cells

We report in this paper on obvious fibrotic lesions in the liver of mice undergoing specified graft-versus-host reaction (GVHR). B6 CD8+ splenocytes were transferred into (bm 12 x bm 1)F1 mice to induce GVHR. Recipient mice had been thymectomized and administrated with anti-CD8 monoclonal antibody (mAb) to deplete CD8+ cells from the hosts. Two weeks after the mAb administration, recipient mice were injected with B6 CD8+ cells and sacrificed further two or four weeks later for analyzing hepatic lesions histopathologically. Light microscopic analyses revealed the presence of concentric fibrosis around both small and large duct levels and the infiltration of mononuclear cells into portal areas. Focal necrosis of hepatocytes was also detected electron-microscopically. These findings suggest that CD8+ T lymphocytes might play an important role in the induction of fibrotic lesions in the liver.

Studies on the resistance to tolerance induction against human IgG in DDD mice: IV. Transient tolerant state of T-cell precursors in bone marrow

Abstract Immunological reactivity of lethally irradiated mice reconstituted with syngeneic bone marrow cells from either tolerized or normal donors was investigated using human IgG (HGG) as antigen. Bone marrow cells from tolerized mice were prepared 2 or 3 weeks after the injection of the tolerogen. The antibody response of reconstituted mice against HGG or hapten-coupled HGG recovered gradually after the bone marrow cell transfer and within a few weeks reached a plateau or the level of control mice which had received bone marrow cells plus excess numbers of thymocytes. Immunological reactivity of bone marrow cells from tolerized mice was significantly lower than that of age-matched normal donors. Strain differences were observed in the rate of recovery from the hyporesponsive state: the lowered reactivity was transient in DDD mice, while more stable hyporesponsiveness was induced in C57BL/6 mice. Even in the latter strain of mice, the tolerant state did not last longer than 2 weeks after the cell transfer and completely disappeared by the third week. Since the recovery of responsiveness was dependent on the presence of thymus in the host, the reactivity was considered to be due to helper T cells recruited in the host from prethymic T-cell precursors (pre-T cells) in transferred bone marrow cells. The results suggest that pre-T cells carry an antigen recognition site and can be specifically rendered tolerant, though temporarily. In contrast to pre-T cells, hyporesponsiveness was not induced in antibody-forming cell precursors in bone marrow of DDD mice. Thus dichotomy in tolerogen susceptibility was observed between T- and B-lymphocyte precursors in bone marrow of DDD mice.

Studies on the resistance to tolerance induction against human IgG in DDD mice. III. Development of the resistance with age and cellular events.

Abstract Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.

Recipient-derived T cells participate in autoimmune-like hepatic lesions induced by graft-versus-host reaction.

Autoimmune-like hepatic lesions were induced by injection of CD4+ T cells from B10.Thy-1.1 mice into MHC class II-disparate (B10.Thy-1.1 x bm12)F1 mice. Hepatic lesions characterized by mononuclear cell accumulation in the portal area of the central vein and around interlobular bile ducts were observed in these recipients. The morphologic features of the lesions resembled primary biliary cirrhosis. The origin of T cells invading at the site of the hepatic lesions was immunohistochemically analyzed. It was shown that many recipient-derived T cells were present at the lesions and that some of them infiltrated the bile duct epithelia. Furthermore, the lesions were weakened when recipient-type T cells were depleted by thymectomy and the administration of anti-Thy-1.2 monoclonal antibody. Recipient-derived T cells were observed to take part in the formation of autoimmune hepatic lesions. These findings suggest the possibility that the tolerance of self-reactive T cells is abrogated by the graft-versus-host reaction.

Lipopolysaccharide-Induced Autoantibody Response

In spleen of normal mice, there are relatively large numbers of plaque-forming cells (PFC) against syngeneic or allogeneic red blood cells treated with bromelain (Br. MRBC) and the numbers of PFC remarkably increase by the injection of bacterial lipopolysacchaiide (LPS). In this report, age-related change in the anti-Br. MRBC PFC response was studied in vivo and in vitro. It was revealed that mean numbers of splenic anti-Br. MRBC PFC increased in both untreated and LPS-injected mice with age (3–14 months). The increase in anti-Br.MRBC PFC detected in LPS-stimulated spleen was dependent on the numbers of PFC in untreated spleen, but not related to the numbers of anti-TNP PFC nor the degree of 3H-thymidine incorporation assessed in in vitro culture. These results imply that enhanced anti-Br.MRBC PFC response was not merely due to polyclonal activation of B lymphocytes. Reasons for increased PFC numbers were analyzed in special references to suppressor activities. The experimental results were obtained suggesting that suppressors were not involved in anti-Br.MRBC PFC response. There were also no marked differences in the acceptability of suppressive signals between young and older spleen cells. It was concluded that hightened anti-Br.MRBC PFC response in older mice might be due to spontaneous increase in the size of Br.MRBC-reactive clone.