Teruaki Hamano

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.

Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma

Summary. Angiogenesis is a crucial process in the progression of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are multifunctional cytokines that potently stimulate angiogenesis including tumour neovascularization. Serum levels of VEGF and HGF were measured in 52 patients with MM by enzyme‐linked immunosorbent assay (ELISA). Serum levels of VEGF and HGF were elevated in MM patients compared with healthy controls (VEGF: mean 0·31 ng/ml and 0·08 ng/ml respectively, P < 0·01; HGF: mean 2·17 ng/ml and 0·45 ng/ml, respectively, P < 0·001). In serial samples taken after chemotherapy, serum VEGF and HGF levels were correlated with M‐protein levels. Serum levels of VEGF were higher in patients with extramedullary plasmacytomas than in patients without them (P < 0·05). They were also significantly higher in a group of patients who showed poor response to chemotherapy (P < 0·01). Serum levels of HGF were higher in patients with complications such as anaemia, hypercalcaemia and amyloidosis than in patients without these complications (P < 0·01, P < 0·05, P < 0·05 respectively). Both serum VEGF and HGF levels were significant predictors of mortality (P = 0·01, P = 0·02, respectively, log‐rank test). The present study demonstrated that serum levels of VEGF and HGF are significantly elevated and dependent on the severity of MM, suggesting that measurement of VEGF and HGF may be useful for assessing disease progression and for predicting the response to chemotherapy in MM patients.

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.

A case of pulmonary amyloidosis associated with multiple myeloma successfully treated with dimethyl sulfoxide.

A 57-year-old man with IgG multiple myeloma developed pulmonary infiltration caused by pulmonary amyloidosis, for which continuous transdermal dimethyl sulfoxide (DMSO) treatment was given. After 8 weeks from the start of DMSO treatment, a dramatic reduction of pulmonary infiltration as determined by chest roentgenogram was observed, and arterial blood gas levels were improved. No serious side effect of DMSO was encountered. We conclude that a therapeutic trial with transdermal DMSO administration brought about a marked regression of the pulmonary infiltrate.

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.

Biphasic anti-osteoclastic action of intravenous alendronate therapy in multiple myeloma bone disease

Multiple myeloma is a malignancy of plasma cells with osteolytic bone destruction. Bisphosphonates inhibit osteoclast activity and are widely used for the treatment of myeloma bone disease. We analyzed the changes in urinary cross-linked N-telopeptides of collagen (u-NTx) and urinary calcium (u-Ca) after bisphosphonate alendronate therapy in ten patients with myeloma bone disease. In all patients, the levels of u-Ca and u-NTx decreased within a week. After the maximum decrease of u-NTx, u-NTx started increasing in half of the patients. However, this further increase in u-NTx decreased again without any additional therapy. Disease severity and pretreatment u-NTx concentrations did not differ between patients with and without the rebound. Patients who did not have rebound had decreased bone marrow monocytes and decreased serum concentrations of interleukin 18, which is produced by monocytes. Our results suggest that impaired activity of monocytes, which are possible osteoclast precursors, is related to reduced bone destruction in multiple myeloma.

Gamma interferon-induced nitric oxide production in mouse CD5+ B1-like cell line and its association with apoptotic cell death

The in vitro effect of gamma interferon (IFN‐γ) on nitric oxide (NO) production in a mouse CD5+ B1‐like cell line, TH2.52, was studied. The TH2.52 cell line is the hybridoma line between mouse B lymphoma line and mouse splenic B cells and expresses a series of B1 markers. IFN‐γ induced a marked NO production in TH2.52 cells through the expression of an inducible type of NO synthase (iNOS). IFN‐γ‐induced NO production was triggered by the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway since it was inhibited by AG490, a JAK2 inhibitor. The growth of TH2.52 cells significantly was inhibited in the presence of IFN‐γ. A significant number of cells underwent apoptotic cell death, accompanied by the DNA fragmentation, annexin V binding, and caspase 3 activation. N(G)‐monomethyl‐L‐arginine, an iNOS inhibitor, prevented IFN‐γ‐induced cell death. Therefore, IFN‐γ‐induced NO production was possible in causing cell death in TH2.52 cells. Further, IFN‐γ‐induced NO production and cell death significantly were prevented by interleukin‐4, a representative Th2 cytokine. The immunological significance of IFN‐γ‐induced NO production in a mouse B1‐like cell line is discussed.

Change of Mouse CD5+ B1 Cells to a Macrophage-Like Morphology Induced by Gamma Interferon and Inhibited by Interleukin-4

ABSTRACT The in vitro effects of gamma interferon (IFN-γ) on the mouse CD5+ B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-γ, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-γ. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-γ-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-γ and IL-4 exhibited contradictory actions on morphological change of CD5+ B1 cells into macrophage-like cells. Differential regulation of CD5+ B1 cells by IFN-γ, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.

Suppression of mitogen- and alloantigen-induced proliferation by chronic lymphocytic leukemia cells of T-cell origin

Abstract Leukemic T cells from a patient with chronic lymphocytic leukemia (T-CLL cells), which could neither respond to mitogens and alloantigens nor stimulate responder cells in one-way mixed lymphocyte culture, markedly suppressed the mitogen- and alloantigen-induced proliferation of normal lymphocytes. This suppression was not attributed to the nonspecific effect of T cells or leukemic cells, and not due to the direct cytotoxic effect of the T-CLL cells. The suppressor activity of these cells was resistant to mitomycin C and a low or moderate dose of irradiation, but sensitive to a high dose of irradiation. They functioned in a genetically nonrestricted fashion. These functional T-CLL cells are suggested to be a neoplastic form of a T-cell subset which possessess suppressor activity.

Mouse B1 cell line responds to lipopolysaccharide via membrane-bound CD14

The role of membrane-bound CD14 in the response of mouse B1 cell lines to lipopolysaccharide (LPS) was studied. The surface profile of mouse TH2.52 B cells was positive for CD5, IgM, B220, CD11b and F4/80, suggesting that TH2.52 cells carried the typical phenotype of B1 cells. Furthermore, TH2.52 B1 cells were found to express membrane-bound CD14, which plays a critical role in LPS recognition. TH2.52 B1 cells responded to a very low concentration of LPS and exhibited: (i) augmentation of DNA synthesis; (ii) activation of nuclear factor (NF)-κB; and (iii) phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2). They were markedly inhibited by anti-CD14 antibody. Therefore, the expression of membrane-bound CD14 was suggested to provide high sensitivity to LPS for TH2.52 B1 cells.