Takanori Kuroiwa

Hepatocyte growth factor ameliorates acute graft-versus-host disease and promotes hematopoietic function

Acute graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation (BMT) and is characterized by hematopoietic dysfunction, immunosuppression, and tissue injury in the skin, liver, and intestinal mucosa. Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, induces mitogenic and antiapoptotic activity in various epithelial cells and promotes hematopoiesis. Working in a murine model of acute GVHD, we performed repeated transfection of the human HGF cDNA into skeletal muscle and showed that this treatment inhibited apoptosis of intestinal epithelial cells and donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by acute GVHD. HGF also markedly suppressed IFN-gamma and TNF-alpha expression in the intestine and liver and decreased the serum IL-12. Furthermore, extramedullary hematopoiesis by donor cells was increased, and the survival rate was improved. These results suggest that HGF may be useful for controlling acute GVHD after allogeneic BMT.

The role of donor T cells for target organ injuries in acute and chronic graft‐versus‐host disease

Donor T cells are crucial for target organ injury in graft‐versus‐host disease (GVHD). We examined the effects of donor T cells on the target organs using a parent‐into‐F1 model of acute and chronic GVHD. Donor T cells showed engraftment in the spleen, small intestine and liver of mice with acute GVHD, causing typical GVHD pathology in these organs. Interferon‐γ and Fas ligand expression were up‐regulated, and host lymphocytes were depleted in the target organs of these mice. In contrast, donor T cells did not show engraftment in the small intestine of mice with chronic GVHD, and no GVHD pathology was observed in this organ. However, both donor T‐cell engraftment and GVHD pathology were observed in the spleen and liver of chronic GVHD mice, along with the up‐regulation of interleukin‐4 (IL‐4) and IL‐10 expression plus the expansion of host lymphocytes such as splenic B cells and hepatic natural killer (NK) 1.1+ T cells. Donor anti‐host cytotoxic T‐lymphocyte activity was observed in spleen cells from mice with acute GVHD, but not in spleen cells from mice with chronic GVHD. Transplantation of Fas ligand‐deficient (gld) spleen cells did not induce host lymphocyte depletion in target organs. These results indicate that donor T cells augment type 1 T helper immune responses and deplete the host lymphocytes from target organs mainly by Fas‐mediated pathways in acute GVHD, while donor T cells augment type 2 T helper immune responses and expand host splenic B cells and hepatic NK1.1+ T cells in chronic GVHD.

Hepatocyte growth factor prevents lupus nephritis in a murine lupus model of chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by the injection of DBA/2 mouse spleen cells represents histopathological changes associated with systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC) and Sjogrens syndrome (SS), as indicated by glomerulonephritis, lymphocyte infiltration into the periportal area of the liver and salivary glands. We determined the therapeutic effect of hepatocyte growth factor (HGF) gene transfection on lupus using this chronic GVHD model. Chronic GVHD mice were injected in the gluteal muscle with either HVJ liposomes containing 8 μg of the human HGF expression vector (HGF-HVJ liposomes) or mock vector (untreated control). Gene transfer was repeated at 2-week intervals during 12 weeks. HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis. While liver and salivary gland sections from untreated GVHD mice showed prominent PBC- and SS-like changes, HGF gene transfection reduced these histopathological changes. HGF gene transfection greatly reduced the number of splenic B cells, host B cell major histocompatibility complex class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 dendritic cells was significantly inhibited by the addition of recombinant HGF in vitro. These results suggest that HGF gene transfection inhibited T helper 2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of SLE, SS and PBC.

The protective role of host Toll-like receptor-4 in acute graft-versus-host disease.

Background. Mutations in Toll-like receptor (TLR)-4 have been associated with the hyporesponsiveness of macrophages to lipopolysaccharide, possibly reducing the risk of acute graft-versus-host disease (GVHD). However, TLR-4 mutations may also increase the risk of intestinal damage and microbial infection, thereby accelerating acute GVHD. Methods. In this study, we investigated the role of TLR-4 in triggering acute GVHD using C3H/HeJ mice with disrupted TLR-4 and C3H/HeN mice with intact TLR-4 as recipients in an acute GVHD model. Results. TLR-4 expression was significantly increased in the intestines and livers from acute GVHD mice. TLR-4-mutant C3H/HeJ hosts that received C57BL/6 (B6) donor cells developed significantly more severe GVHD than TLR-4-intact C3H/HeN hosts receiving B6 donor cells. Antibiotic treatment prolonged the survival of C3H/HeN-host GVHD mice but reduced the survival of C3H/HeJ-host GVHD mice. C3H/HeJ-host GVHD mice showed increased lipopolysaccharide levels in the blood, donor cell and CD68+ cell infiltration, tumor necrosis factor-&agr; mRNA expression, and more apoptotic cells in the intestine compared with C3H/HeN host GVHD mice. In contrast, intestinal cyclooxygenase-2, prostaglandin E2, and hepatocyte growth factor expression in C3H/HeJ-host GVHD mice were significantly decreased compared with C3H/HeN-host GVHD mice. Conclusions. Our results indicated that host TLR-4 is crucial for the induction of tissue protective factors and for protection against intestinal cell apoptosis during acute GVHD.

Graft-versus-host-disease-associated donor cell engraftment in an F1 hybrid model is dependent upon the Fas pathway.

The graft‐versus‐host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell‐mediated attack on host lymphohaematopoietic populations, resulting in the reconstitution of the host with donor cells. We examined Fas–Fas ligand (FasL) interactions in donor and host haematopoietic cells over a prolonged period of parental‐induced GVHD. Fas expression on bone marrow cells of both donor and host origin increased at 2 weeks. Host cell incubation with anti‐Fas antibody induced apoptosis, and the number of haematopoietic progenitor cells decreased. Fas‐induced apoptosis by the repopulating donor cells, however, did not increase until 12 weeks, when more than 90% of the cells were donor cells. The expression of various cytokines, such as interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), and FasL gene expression in the bone marrow increased concomitantly. To examine directly whether FasL has a major role in the development of donor cell engraftment, FasL‐deficient (gld) mice were used as donors. Injection of B6/gld spleen cells induced significantly less host lymphohaematopoietic depletion, resulting in a failure of donor cell engraftment. Furthermore, injection of IFN‐γ gene knockout (gko) B6 spleen cells failed to augment Fas and FasL expression in recipient mice, resulting in a failure of donor cell engraftment. This suggests that the induction of apoptosis by Fas–FasL interactions in host cells may contribute to a reconstitution of the host with donor cells and that donor‐derived IFN‐γ plays a significant role for Fas–FasL interactions in host cells during parental‐induced GVHD.

Effect of FK506 on donor T-cell functions that are responsible for graft-versus-host disease and graft-versus-leukemia effect.

Background. FK506 is a potent immunosuppressive agent that is used in human graft-versus-host disease (GvHD) prevention. However, the precise mechanisms for GvHD prevention and the effect on graft-versus-leukemia (GvL) activity are unknown. This study was undertaken to determine the effect of FK506, given at clinically relevant doses, on donor T-cell functions responsible for GvHD and GvL activity. Methods. The effect of FK506 on GvHD prevention and GvL activity was investigated using a murine model of allogeneic bone-marrow transplantation in which mice were injected with a P815 leukemic cell line. The regulatory role of FK506 on donor T cells was tested by analysis of donor T-cell expansions in the spleen and donor anti-host T-cell proliferative and cytotoxic responses. mRNA expression of type 1 T helper (Th1), Fas ligand (L), and granzyme B were also evaluated in target organs of GvHD. Results. FK506 significantly prolonged the survival of GvHD mice when given at the trough level of 17.6 ng/mL, whereas it also blocked GvL effect in P815-injected GvHD mice. FK506 reduced the expansion of donor CD8+ and, to a lesser extent, CD4+ T cells in the spleen and inhibited donor anti-host T-cell proliferative and cytotoxic responses. It also inhibited the induction of Th1, FasL, and granzyme B mRNA expression in target organs of GvHD. Conclusions. FK506 inhibits both GvHD and GvL activity when given at clinical doses by inhibiting donor T-cell expansion, donor anti-host T-cell reactivity, and Th1 immune responses.

Psychological state is related to the remission of the Boolean-based definition of patient global assessment in patients with rheumatoid arthritis

Abstract Objectives. To evaluate whether the psychological state is related to the Boolean-based definition of patient global assessment (PGA) remission in patients with rheumatoid arthritis (RA). Methods. Patients with RA who met the criteria of swollen joint count (SJC) ≤ 1, tender joint count (TJC) ≤ 1 and C-reactive protein (CRP) ≤ 1 were divided into two groups, PGA remission group (PGA ≤ 1 cm) and non-remission group (PGA > 1 cm). Anxiety was evaluated utilizing the Hospital Anxiety and Depression Scale-Anxiety (HADS-A), while depression was evaluated with HADS-Depression (HADS-D) and the Center for Epidemiologic Studies Depression Scale (CES-D). Comparison analyses were done between the PGA remission and non-remission groups in HADS-A, HADS-D and CES-D. Results. Seventy-eight patients met the criteria for SJC ≤ 1, TJC ≤ 1 and CRP ≤ 1. There were no significant differences between the PGA remission group (n = 45) and the non-remission group (n = 33) in age, sex, disease duration and Steinbrockers class and stage. HADS-A, HADS-D and CES-D scores were significantly lower in the PGA remission group. Conclusions. Patients with RA who did not meet the PGA remission criteria despite good disease condition were in a poorer psychological state than those who satisfied the Boolean-based definition of clinical remission. Psychological support might be effective for improvement of PGA, resulting in the attainment of true remission.

Effect of ursodeoxycholic acid on expression of Fas ligand and tumor necrosis factor-alpha in the liver of mice with acute graft-versus-host disease

Abstract Background: The Fas-Fas-ligand (FasL) system and tumor necrosis factor-alpha (TNF-α) are involved in immune-mediated liver injury. They have also been shown to play a critical role in the pathogenesis of acute graft-versus-host disease (GVHD). Objective: The aim of the present study was to determine whether continuous oral administration of ursodeoxycholic acid (UDCA) could inhibit Fas-FasL and TNF-α expression in the liver of mice with acute GVHD. Methods: BDF1 mice were assigned to 1 of 2 groups. In the UDCA group, mice were administered UDCA 50 mg/kg once daily for 3 weeks via gastric tube in 200 μL of sodium hydroxide (1 mol/L at pH 8.3). In the control (placebo) group, mice were administered 200 μL of sodium hydroxide (1 mol/L at pH 8.3) for 3 weeks. Acute GVHD was induced 1 week after administration of UDCA or placebo. To determine the serum concentration of TNF-α, serum was assayed using an enzyme-linked immunosorbent assay. A reverse transcribed polymerase chain reaction procedure was performed to investigate the expression of FasL and TNF-α mRNA in the liver. Individual bile acids were separated and quantified by high-performance liquid chromatography to determine if UDCA composition was enriched in the bile after administration of UDCA. Results for group means were compared using the Student t test. Significance was set at P Results: There were 5 mice in each group. Oral administration of UDCA resulted in a 34.0% enrichment of UDCA in the serum of mice given UDCA and in 1.9% of mice given placebo. The mean UDCA composition in the bile was 32.1% in mice given UDCA and 9.2% in mice given placebo. Although treatment with UDCA did not affect phenotypic change of hepatic lymphocytes caused by acute GVHD, it significantly ( P P Conclusions: These findings suggest that UDCA suppresses both FasL- and TNF-α-mediated hepatotoxic pathways in acute GVHD-induced liver injury. Further studies are needed to determine the role of UDCA plus immunosuppressive drugs in liver injury caused by acute GVHD.