Norio Nakamura

Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis.

The Fas ligand is predominantly expressed in activated T lymphocytes and is one of the major effector molecules of cytotoxic T lymphocytes and natural killer cells. Previously, we found excessive apoptosis of epithelial cells and infiltrating lymphocytes expressing Fas ligand mRNA in the lung tissue of bleomycin-induced pulmonary fibrosis in mice. Here we demonstrated that the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of this model and that lpr and gld mice were resistant against the induction of pneumopathy. These results suggest that the Fas-Fas ligand pathway plays an essential role in the development of pulmonary fibrosis and that preventing this pathway could have therapeutic value in lung injury and fibrosis.

Acute Toxicity of an Anti-Fas Antibody in Mice

By histopathologic examination of various organs in 3 normal strains, C3H/HeN, ICR, and DBA/1J, of mice treated intravenously once with anti-Fas antibody (Jo2), we failed to determine any target organ, except the liver, responsible for the acute lethality induced by the Fas/anti-Fas antibody interaction. However, we could show the presence of Fas-mediated apoptosis in other organs aside from the liver and normal mouse strain differences in susceptibility to anti-Fas antibody. Among these strains, C3H/HeN was the most susceptible to the antibody, followed by ICR and DBA/1J. We observed Fas-mediated apoptosis in the liver, spleen, thymus, lymph nodes, Peyers patch, intestine, skin, coagulation glands, ovary, uterus, and vagina in all 3 strains and additionally in the epididymides and seminal vesicles in the DBA/1J strain. We also demonstrated that Fas-mediated apoptosis of small lymphocytes in the mantle zone of splenic lymphatic follicles preceded that of the hepatocytes or thymic cells. Since cellular damage was most severe in the liver among all the apoptotic organs in the 3 mouse strains, liver injury induced by anti-Fas antibody is speculated to play a significant role in the death.

Inhibitory effect of M50054, a novel inhibitor of apoptosis, on anti-Fas-antibody-induced hepatitis and chemotherapy-induced alopecia

M50054, 2,2-methylenebis (1,3-cyclohexanedione), was identified as a novel inhibitor of apoptosis (programmed cell death) using an in vitro cell death assay system induced in human Fas-expressing WC8 cells by soluble human Fas ligand. Furthermore, M50054 inhibited the apoptotic cell death of U937, a human monocytic leukemic cell line, induced by anticancer agents such as etoposide; it was also confirmed that M50054 inhibited apoptotic features such as DNA fragmentation and phosphatidylserine exposure in these cells. These anti-apoptotic effects were attributable to inhibition of caspase-3 activation. Additionally, M50054 significantly inhibited anti-Fas-antibody-induced elevation of plasma alanine aminotransferase and aspartate aminotransferase. Alopecia (hair loss) symptoms were also significantly improved with topical treatment with M50054. In conclusion, M50054 inhibits apoptosis induced by a variety of stimuli via inhibition of caspase-3 activation, and may thus be effective for hepatitis and chemotherapy-induced alopecia.

Overproduction of the bleomycin‐binding proteins from bleomycin‐producing Streptomyces verticillus and a methicillin‐resistant Staphylococcus aureus in Escherichia coli and their immunological characterisation

The bleomycin‐binding proteins designated BLMA and BLMS, which confer resistance to bleomycin (Bm), from Bm‐producing Streptomyces verticillus ATCC15003 and a methicillin‐resistant Staphylococcus aureus B‐26, respectively, were overexpressed in Escherichia coli. The present study showed that both BLMA and BLMS quench the antibacterial activity of Bm by the binding to the drug. To immuno‐chracterize the Bm‐binding proteins, we constructed a monoclonal antibody against BLMA. The antibody, designated 893‐12, did not cross react to BLMS and another Bm‐binding protein from tallysomycin‐producing Streptoalloteichus hindustanus. Although the ability of Bm to cleavage DNA was eliminated by a binding of BLMA to Bm, as shown by Sugiyama et al. [Gene 151 (1994) 11–15], the Bm‐induced DNA degradation was restored by pre‐incubation of BLMA with the anti‐BLMA monoclonal antibody.

Expression of FasL and Fas Protein and Their Soluble Form in Patients with Hypersensitivity Pneumonitis

Background: Hypersensitivity pneumonitis (HP) is characterized by a lymphocytic alveolitis and loosely formed granulomas in lung biopsy specimens. HP improves or disappears altogether after cessation of antigen exposure. The Fas-Fas ligand (FasL) system is one of the representative systems of apoptosis-signaling receptor molecules, and is involved in various inflammatory diseases. We hypothesized that the Fas-FasL system may be associated with this disorder. Methods: We examined the expression of FasL and Fas proteins in lung tissues from patients with HP using immunohistochemistry. We also measured the soluble form of FasL (sFasL) and sFas levels in serum and bronchoalveolar lavage fluid (BALF) from patients with HP using enzyme-linked immunosorbent assay (ELISA). Furthermore, we also measured the cytotoxic activity of BALF sFasL in vitro. Results: FasL was detected in infiltrating mononuclear cells, and Fas was detected in infiltrating mononuclear cells, alveolar macrophages, and epithelioid cells in HP, whereas FasL was not detected and Fas was detected in few alveolar macrophages in controls. The levels of sFasL and sFas in BALF, but not in serum, were significantly increased in HP compared with controls. BALF of HP that included high levels of sFasL had no cytotoxic activity for bronchiolar epithelial cells in vitro. Conclusions: In HP, there is an upregulation of FasL and Fas in lung tissues. Since there is no incidence of apoptosis and no cytotoxic activity for lung epithelial cells in BALF from patients with HP, the increased levels of BALF sFasL and sFas may reflect the activation and sequestration of inflammatory cells rather than apoptosis.

The expression of Fas and Fas ligand, and the effects of interferon in chronic liver diseases with hepatitis C virus

In viral hepatitis, binding of Fas ligand (FasL) with Fas expressed on the surfaces of infected hepatocytes induces apoptosis, removing hepatitis virus along with infected hepatocytes. We measured serum concentrations of soluble Fas (sFas) and FasL (sFasL), expression of membrane-bound FasL, and expression of FasL-mRNA in patients with chronic hepatitis C without cirrhosis (CH-C) and chronic hepatitis C with liver cirrhosis (LC-C). In CH-C, sFasL concentrations were lower and FasL-mRNA expression was significantly less than in volunteers. In LC-C, sFas concentrations were significantly greater than in healthy volunteers, while sFasL, membrane-bound FasL expression, and FasL-mRNA expression did not show significant differences. We also examined these variables over 24 h following the first interferon (IFN) treatment in patients with CH-C. Serum concentrations of sFas and sFasL, and FasL-mRNA expression increased markedly beyond amounts present before IFN injection until 12 h after IFN injection. However, membrane-bound FasL expression decreased until 6 h, followed by an increase until 24 h. Our findings suggest that the ratio of membrane-bound FasL to sFasL may be regulated to remove virally infected cells in CH-C. In addition, apoptosis mediated by the Fas/FasL system may be influenced by IFN injection for treatment of CH-C.

Increased levels of soluble Fas ligand in CSF of rapidly progressive HTLV-1-associated myelopathy/tropical spastic paraparesis patients

The interaction of Fas ligand (FasL) with Fas-bearing cells induces apoptosis and contributes to the negative regulation of peripheral T-cell responses. Membrane-bound FasL is cleaved by a matrix metalloproteinase-like enzyme and converted to a soluble form (sFasL). Recent studies suggest that such sFasL can cause systemic tissue damage. Here we report that serum and CSF levels of soluble FasL (sFasL) are markedly higher in three active phase patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). All of these patients showed higher sFasL levels in CSF than in serum. Although the HTLV-1 proviral load of patients showed no correlation with serum or with CSF sFasL, CSF sFasL levels of 14 HAM/TSP patients correlated with the anti-HTLV-1 antibody titer and neopterin concentration in CSF. These results indicate that sFasL mediated mechanisms may contribute to the inflammatory process and subsequent spinal tissue damage seen in HAM/TSP patients.

Serum levels of soluble Fas ligand in patients with acute and fulminant hepatitis : relationship with soluble Fas, tumor necrosis factor α and TNF receptor-1

We measured the serum levels of soluble Fas ligand (sFasL) using a newly developed ELISA system in patients with acute hepatitis (AH), acute severe hepatitis (ASH) and fulminant hepatitis (FH). In addition, we also evaluated the relationship between the serum levels of sFasL, soluble Fas (sFas), soluble tumor necrosis factor α (sTNFα), and soluble TNF receptor-1 (sTNFR1). The median serum sFasL level was significantly increased in patients with acute liver disease compared to that in normal subjects; however, there were no significant differences among patients with AH, ASH, and FH. The serum sFasL levels varied according to the cause of hepatitis and were particularly high in patients with hepatitis A viral infection. On the other hand, the median serum sFas level was also significantly increased in patients with acute liver disease compared to that in normal subjects; however, there were no significant differences among patients with the three clinical forms or various causes of hepatitis. The serum sFasL levels decreased rapidly following improvement in liver functions, whereas the serum sFas levels decreased more gradually and remained abnormally high even at follow-up. The serum sFasL levels were not correlated with the levels of serum transaminases or sFas, but were significantly correlated with those of sTNFα (r=0.483, P<0.001) and sTNFR1 (r=0.324, P<0.05). On the other hand, serum sFas levels were significantly correlated with those of sTNFR1 (r=0.319, P<0.05) alone. Our results suggest that the serum sFasL level increases in the acute phase of hepatitis and the level varies according to the cause of hepatitis. Our findings also suggest that the Fas-FasL system and cytokine networks may be closely associated with early process of hepatic necrosis.