Toshikazu Hada

Caspase-1-Independent, Fas/Fas Ligand–Mediated IL-18 Secretion from Macrophages Causes Acute Liver Injury in Mice

IL-18, produced as a biologically inactive precursor, is processed by caspase-1 in LPS-activated macrophages. Here, we investigated caspase-1-independent processing of IL-18 in Fas ligand (FasL)-stimulated macrophages and its involvement in liver injury. Administration of Propionibacterium acnes (P. acnes) upregulated functional Fas expression on macrophages in an IFNgamma-dependent manner, and these macrophages became competent to secrete mature IL-18 upon stimulation with FasL. This was also the case for caspase-1-deficient mice. Administration of recombinant soluble FasL (rFasL) after P. acnes priming induced comparable elevation of serum IL-18 in parallel with elevated serum liver enzyme levels. However, liver injury was not induced in IL-18-deficient mice after rFasL administration. These results indicate a caspase-1-independent pathway of IL-18 secretion from FasL-stimulated macrophages and its critical involvement in FasL-induced liver injury.

Excessive production of transforming growth-factor β1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth

Angiogenesis is an important part of tumor growth in vivo. We used the transfected Chinese hamster ovary (CHO) cells that overproduced recombinant transforming growth-factor beta 1 (TGF-beta 1) to examine the possible role of this factor in tumor growth and angiogenesis in a nude mouse model. The in-vitro proliferation of TGF-beta 1-transfected CHO cells was unaffected by the treatment of either recombinant TGF-beta 1 or an anti-TGF-beta 1 antibody. The TGF-beta 1-transfected cells grew more rapidly than the parental CHO cells when injected subcutaneously into nude mice. The tumors derived from the TGF-beta 1-transfected cells showed prominent tumor-associated angiogenesis, whereas the parental cells produced tumors without such angiogenesis. In addition, an anti-TGF-beta 1 neutralizing antibody was able to inhibit both growth and angiogenesis in the tumors derived from TGF-beta 1-transfected cells. These findings suggest that the overproduction of TGF-beta 1 by tumor cells can contribute to neovascularization and may help promote tumor development in vivo.

Local production and localization of transforming growth factor-beta in tuberculous pleurisy.

Transforming growth factor‐beta (TGF‐β) is one of the cytokines which play an immunosuppressive role in an inflammatory process. To investigate the local production of TGF‐β, we evaluated the levels of TGF‐β in tuberculous pleural effusions (TBPE) and non‐tuberculous benign pleural effusions (non‐TBPE) by the growth inhibition assay with MvILu mink lung epithelial cells. The mean level of TGF‐β in TBPE (46.1 ± 31.5 pM; mean ± s.d.) was higher than in non‐TBPE (21.7 ± 12.3 pM) (P<0.05). Although the level of interferon‐gamma (IFN‐γ) in TBPE measured by ELISA was significantly higher than in non‐TBPE. there was no significant difference in the levels of tumour necrosis factor‐alpha (TNF‐α) measured by ELISA between these two groups. Moreover, to elucidate localization of TGF‐β in tuberculous pleurisy, immunohistochemical studies of pleura, using the rabbit polyclonal antibody Ab39 against latent TGF‐β1 binding protein (LTBP) were performed. Results revealed that LTBP was localized in immature fibrotic areas where infiltrations of T lymphocytes and macrophages were absent. Importantly, the major sources of LTBP in these areas were thought to be mesothelial cells and fibroblasts. LTBP was not found in granulomas and mature fibrotic areas. Our data suggest that TGF‐β in tuberculous pleurisy may play important roles for regression of granulomatous inflammation and pleural fibrosis for tissue repair.

Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Emodin

Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time‐and dose‐dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (ΔΨm), followed by the activation of caspase–9 and caspase–3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide‐scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, ΔΨmdisruption and caspase activation may be involved in the apoptotic pathway induced by emodin.

Purification and immunohistochemical tissue localization of human xanthine oxidase

Xanthine oxidase was purified 1600-fold from human liver cytosol. The purified enzyme was shown as a single band of 300 kDa on polyacrylamide gel electrophoresis and 150 kDa on SDS-PAGE. Using this purified enzyme, polyclonal antibody against xanthine oxidase was raised in a rabbit. On Ouchterlonys double immunodiffusion method, the raised antibody and the human liver cytosol made a precipitation line stained by activity stain and protein stain, respectively. With the raised anti-xanthine oxidase sera, the immunohistochemical localization of xanthine oxidase in human tissues was examined. Immunostaining of frozen hepatic tissue section showed that the cytoplasm of hepatocytes and endothelial lining cells were stained. In a number of other tissues, the xanthine oxidase antigen was detected only in the endothelial lining cells from heart, kidney, brain, aorta, lung and mesentery, except for the duodenal mucosa cells. A possible role for xanthine oxidase in the endothelial cells from various human tissues in the pathogenesis of reperfusion injury was suggested.

Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor

Hepatoma‐derived growth factor (HDGF) is highly expressed in tumor cells, and stimulates their proliferation. In the present study, we investigated the role of HDGF in tumorigenesis and elucidated the mechanism of action. Stable transfectants of NIH3T3 cells overexpressing HDGF did not show significant anchorage‐independent growth in soft agar assay. However, these stable transfectants overexpressing HDGF generated sarcomatous tumors in nude mice. These tumors were red‐colored macroscopically, and histologically showed a rich vascularity. Immunohistochemical analysis using CD31 antibody showed new vessel formation. Recombinant HDGF stimulated proliferation of human umbilical vein endothelial cells in a dose‐dependent manner, and stimulated tubule formation. Furthermore, vascular endothelial growth factor (VEGF) was detected immunohistochemically in the tumor tissues. Transient expression of HDGF induced both VEGF gene and protein expression as demonstrated by a reporter assay using VEGF gene promoter. The administration of anti‐VEGF neutralizing antibody significantly suppressed, but did not block, the tumor growth of HDGF‐overexpressing cells in nude mice. Thus, these findings suggested that HDGF‐induced tumor formation in vivo involves induction of VEGF as well as direct angiogenic activity.

NS-398, a selective cyclooxygenase 2 inhibitor, inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines

Cyclooxygenase 2 (COX‐2) has been suggested to be associated with liver carcinogenesis. Several reports have shown that NSAIDs inhibit the growth of hepatocellular carcinoma cell lines. There is little evidence of how COX‐2 inhibitors regulate the proliferation of hepatocellular carcinoma cells or the mechanism involved. In our study, we investigated the growth‐inhibitory mechanism of a selective COX‐2 inhibitor, NS‐398, in 4 hepatocellular carcinoma cell lines by studying cell growth, COX‐2 and proliferating cell nuclear antigen (PCNA) expression, cell cycle distribution and the evidence of apoptosis. NS‐398 inhibited the growth of all 4 cell lines in a time‐ and dose‐dependent manner and the inhibitory effects were independent of the level of COX‐2 protein expression. PCNA expression was downregulated by NS‐398 in a dose‐independent manner. NS‐398 caused cell cycle arrest in the S phase with a reduction in cell numbers and cell accumulation in the G0/G1 phase, for all 4 cell lines. No evidence of apoptosis was observed in our present study. Our findings suggest that a selective COX‐2 inhibitor might serve as an effective tool for the chemoprevention and treatment of hepatocellular carcinomas. A reduction in cell number in the S phase may be an important event in cell cycle arrest caused by NS‐398 in hepatocellular carcinoma cell lines.

Tumor markers in pleural effusion diagnosis.

In order to discriminate between malignant and benign effusions, the values of carcinoembryonic antigen (CEA), ferritin, beta2‐microglobulin (BMG), acid‐soluble glycoprotein (ASP), tissue polypeptide antigen (TPA), adenosine deaminase (ADA), and immunosuppressive acidic protein (IAP) were measured in the pleural fluid of 54 patients with lung cancer, 20 with malignancies other than lung cancer, 18 with tuberculous pleurisy, and 22 with benign diseases other than tuberculosis. CEA levels in malignant effusions were significantly higher than those in benign effusions. At a cutoff level of 5 ng/ml, 68% of the patients with lung cancer and 44% of the patients with other malignancies showed elevated pleural fluid CEA levels. In 13 lung cancer cases with negative pleural fluid cytology, nine cases had elevated pleural fluid CEA levels. The mean pleural fluid BMG level of patients with benign diseases was significantly higher than that of patients with malignant diseases, but there was a marked overlap between those with malignant and benign diseases. No significant differences were found in the pleural fluid ferritin, ASP, TPA, and IAP levels between malignant and benign conditions. ASP and IAP pleural fluid levels showed significant correlations with the pleural fluid C‐reactive protein (CRP) concentrations suggesting that they also reflect inflammatory activity. The mean ADA activity in tuberculous effusion was significantly higher than that resulting from other causes of pleural effusion.

Relation between interleukin-18 and PGE2 in synovial fluid of osteoarthritis: a potential therapeutic target of cartilage degradation.

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic joint changes. Interleukin (IL)-18 is a potent inducer of prostaglandin (PG) E2 in vitro. We determined the relation between IL-18 and PGE2 in synovial fluid (SF) of human OA, and discussed the role of IL-18 in the pathogenesis of OA and also its therapeutic consequences. SF was collected from 30 patients with knee OA. The concentrations of IL-18 and other cytokines including IL-1&bgr;, tumor necrosis factor (TNF)-&agr;, IL-6, and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of PGE2 was also assessed by inhibitory ELISA. The average value of IL-18 was 248 ± 310 pg/mL. The average value of PGE2 was 93 ± 103 pg/mL. There was a relatively strong correlation between IL-18 and PGE2 (r = 0.78, p = 0.0001). In contrast, IL-1&bgr; was undetectable (cutoff point of 20 pg/mL), except for one case. TNF-&agr; was also undetectable (cutoff point of 20 pg/mL), except for two cases. The average value of IL-6 was 1,310 ± 2,623 pg/mL (n = 17), whereas IL-8 was 5,208 ± 6,031 pg/mL (n = 5). Furthermore, IL-6 and IL-8 correlated with IL-18 (r = 0.69, p = 0.0024 and r = 0.87, p = 0.0527, respectively). Our results suggest that IL-18 could play a major role in vivo in inducing the production of PGE2, which in turn can cause cartilage degradation in OA pathogenesis. Thus, targeting this cytokine appears to be an important therapeutic approach in OA.

Stimulation of hyaluronan synthesis by tumor necrosis factor-α is mediated by the p50/p65 NF–κB complex in MRC-5 myofibroblasts

Abstract The lesions of fibrocontractive diseases result from an excessive myofibroproliferative response to numerous forms of inflammatory stimuli, which elicit the net deposition of extracellular matrix (ECM) in the interstitium of the affected tissue. Hyaluronan (HA), reported to be a key player supporting cellular migration and adherence, is a major component of ECM that undergoes dynamic regulation during inflammation. The molecular regulation of HA biosynthesis by inflammatory cytokines on myofibroblasts is not yet completely understood. Here we report the biochemical characteristics of the lung myofibroblast cell line MRC-5, and we demonstrate that the production of HA by this cell line is inducible by the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), at the message level of HA synthase (HAS). In TNF-α-stimulated MRC-5 cells, DNA-binding and competition experiments indicated that the predominant NF–κB binding activity detected with nuclear extract-stimulated cells is mediated by the p50/p65 complex. Using antisense oligonucleotides, we confirmed that the TNF-α-stimulation of HA synthesis by MRC-5 cells is dependent on the activation of the p50/p65 NF–κB complex. These findings indicate that TNF-α production within inflamed tissues may enhance the HA synthesis via the transcriptional induction of HAS on myofibroblasts, thereby providing a provisional matrix for supporting cellular migration and adhesion, and that the p50/p65 NF–κB complex that plays an important role in the regulation of HA production by TNF-α might be an appropriate target for therapeutic compounds to treat tissue fibrosis accompanied by inflammation.