Hiromi Iwagaki

Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells.

Intercellular adhesion molecule‐1 (ICAM‐1, CD54) is a membrane glycoprotein and a member of the immunoglobulin superfamily. It plays a central role in cell to cell‐mediated immune responses and is a ligand for leukocyte function‐associated antigen‐1 (LFA‐1). We report here that a newly discovered cytokine, interferon‐γ‐inducing factor (IGIF) [H. Okamura et al. (1995) Nature 378, 88] recently proposed to be designated as IL‐18, selectively up‐regulates ICAM‐1 expression in KG‐1 cells, a human myelomonocytic cell line, in which IL‐18 also enhances interferon‐γ production. IL‐18 induced heterotypic aggregation between KG‐1 and Peer T cells, which was blocked by anti‐ICAM‐1 and/or LFA‐1 antibodies. Anti‐interferon‐γ antibody did not block the IL‐18‐induced up‐regulation of ICAM‐1 on KG‐1 cells. These results thus show that IGIF/IL‐18, enhances ICAM‐1 expression in KG‐1 cells in an interferon‐γ‐independent pathway, up‐regulates ICAM‐1 functions, and that IL‐18 might play a potential role in immunoregulation by mediating immune cell infiltration into the tissues. J. Leukoc. Biol. 64: 519–527; 1998.

Elevation of serum interleukin-18 levels and activation of kupffer cells in biliary atresia

BACKGROUND/PURPOSE Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel proinflammatory cytokine that can induce interferon gamma (IFN-gamma). In addition, IL-18 enhances intracellular adhesion molecule-1 (ICAM-1) expression as well as Fas ligand (FasL) expression, and induces apoptosis in hepatic injury. The aim of this study was to clarify the potential role of IL-18 in the pathogenesis of the progressive inflammation and fibrosis in biliary atresia (BA). METHODS Six children with BA before hepatic portoenterostomy (HPE), 13 with BA including 7 without jaundice and 6 with persistent jaundice after HPE, and 16 healthy controls were examined. Blood samples were obtained preoperatively from 6 patients, after HPE from 13, and after liver transplantation from 4. The IL-18 level was determined by an enzyme-linked immunosorbent assay (ELISA). Immunohistochemically, liver specimens from BA patients were studied using a monoclonal antibody to macrophage-associated antigen (CD68). RESULTS IL-18 levels were elevated in the patients before HPE compared with those of the controls (349+/-54 pg/mL v. 138+/-13 pg/mL, P<.0001). After HPE, extremely high concentrations of IL-18 were observed in patients with persistent jaundice (532+/-95 pg/mL, P<.0001), and the IL-18 levels were significantly high even in the patients without jaundice (249+/-29 pg/mL, P<0.005). The high IL-18 level lasted for a long time even in the patients without jaundice after HPE. In contrast, the IL-18 levels immediately decreased after liver transplantation. Immunohistochemically, the number of CD68-positive Kupffer cells was significantly higher, and the size was larger in the livers of the patients than in the controls. The proliferation of CD68-positive cells was much more conspicuous in the liver specimens obtained during liver transplantation than in those at the time of HPE. CONCLUSIONS Our findings showed elevation of serum IL-18 levels and activation of Kupffer cells in BA. IL-18 released from activated Kupffer cells might play an important role in the pathophysiology of the progressive inflammation and fibrosis in BA. Furthermore, IL-18 level may be related to the prognosis in patients with BA.

Histamine Is a Potent Inducer of IL-18 and IFN-γ in Human Peripheral Blood Mononuclear Cells

Histamine (10−7 to 10−4 M) concentration-dependently stimulated the production of IL-18 and IFN-γ and inhibited the production of IL-2 and IL-10 in human PBMCs. Histamine in the same concentration range did not induce the production of IL-12 at all. The stimulatory or inhibitory effects of histamine on cytokine production were all antagonized by H2 receptor antagonists ranitidine and famotidine in a concentration-dependent manner, but not by H1 and H3 receptor antagonists. Selective H2 receptor agonists, 4-methylhistamine and dimaprit, mimicked the effects of histamine on five kinds of cytokine production. The EC50 values of histamine, 4-methylhistamine, and dimaprit for the production of IL-18 were 1.5, 1.0, and 3.8 μM, respectively. These findings indicated that histamine caused cytokine responses through the stimulation of H2 receptors. All effects of histamine on cytokine responses were also abolished by the presence of either anti-IL-18 Ab or IL-1β-converting enzyme/caspase-1 inhibitor, indicating that the histamine action is dependent on mature IL-18 secretion and that IL-18 production is located upstream of the cytokine cascade activated by histamine. The addition of recombinant human IL-18 to the culture concentration-dependently stimulated IL-12 and IFN-γ production and inhibited the IL-2 and IL-10 production. IFN-γ production induced by IL-18 was inhibited by anti-IL-12 Ab, showing the marked contrast of the effect of histamine. Thus histamine is a very important modulator of Th1 cytokine production in PBMCs and is quite unique in triggering IL-18-initiating cytokine cascade without inducing IL-12 production.

Prostaglandin E2 Inhibits IL-18-Induced ICAM-1 and B7.2 Expression Through EP2/EP4 Receptors in Human Peripheral Blood Mononuclear Cells

Costimulatory molecules play important roles in immune responses. In the present study we investigated the effects of PGE2 on the expression of ICAM-1, B7.1, and B7.2 on monocytes in IL-18-stimulated PBMC using FACS analysis. Addition of PGE2 to PBMC inhibited ICAM-1 and B7.2 expression elicited by IL-18 in a concentration-dependent manner. We examined the involvement of four subtypes of PGE2 receptors, EP1, EP2, EP3, and EP4, in the modulatory effect of PGE2 on ICAM-1 and B7.2 expression elicited by IL-18, using subtype-specific agonists. ONO-AE1–259-01 (EP2R agonist) inhibited IL-18-elicited ICAM-1 and B7.2 expression in a concentration-dependent manner with a potency slightly less than that of PGE2, while ONO-AE1-329 (EP4R agonist) was much less potent than PGE2. The EP2/EP4R agonist 11-deoxy-PGE1 mimicked the effect of PGE2 with the same potency. ONO-D1-004 (EP1R agonist) and ONO-AE-248 (EP3R agonist) showed no effect on IL-18-elicited ICAM-1 or B7.2 expression. These results indicated that EP2 and EP4Rs were involved in the action of PGE2. Dibutyryl cAMP and forskolin down-regulated ICAM-1 and B7.2 expression in IL-18-stimulated monocytes. As EP2 and EP4Rs are coupled to adenylate cyclase, we suggest that PGE2 down-regulates IL-18-induced ICAM-1 and B7.2 expression in monocytes via EP2 and EP4Rs by cAMP-dependent signaling pathways. The fact that anti-B7.2 as well as anti-ICAM-1 Ab inhibited IL-18-induced cytokine production implies that PGE2 may modulate the immune response through regulation of the expression of particular adhesion molecules on monocytes via EP2 and EP4Rs.

Stimulation of alpha7 nicotinic acetylcholine receptor inhibits CD14 and the toll-like receptor 4 expression in human monocytes.

ABSTRACT The lipopolysaccharide (LPS)-receptor complex, CD14/toll-like receptor 4, is known to play a role in the immune responses during sepsis. Excessive inflammation and tumor necrosis factor (TNF)-&agr; synthesis have been reported to cause morbidity and mortality in endotoxemia and sepsis. Cell-to-cell interaction through the engagement between intercellular adhesion molecule 1, B7.1, and CD40 on monocytes and their ligands on T cells has been suggested to play a role in the inflammatory response such as TNF-&agr; and interleukin 10 production. Nicotine, with the stimulation of the nicotinic acetylcholine receptor &agr;7 subunit (&agr;7-nAChR), has now become the focus of attention because of its anti-inflammatory effects. However, little is known about the mechanism of the inhibitory effects induced by nicotine on the LPS-induced immune responses. In the present study, we found that nicotine suppressed the expression of CD14, toll-like receptor 4, intercellular adhesion molecule 1, B7.1, and CD40 on monocytes and the production of TNF-&agr;, but not interleukin 10, in human peripheral blood mononuclear cells in the presence of LPS. The actions of nicotine were reversed by a nonselective and a selective &agr;7-nAChR antagonist, mecamylamine and &agr;-bungarotoxin, respectively. Therefore, nicotine might inhibit the LPS receptor complex expression via &agr;7-nAChR, thus leading to a decrease in the adhesion molecule expression and TNF-&agr; production. Moreover, we demonstrated that a nuclear factor-&kgr;B and a p38 mitogen-activated protein kinase inhibitor mimicked the actions of nicotine in the presence of LPS. These results suggested that the nuclear factor-&kgr;B and p38 mitogen-activated protein kinase might be involved in the actions of nicotine.

Serum interferon-gamma-inducing factor/IL-18 levels in primary biliary cirrhosis

Primary biliary cirrhosis is an autoimmune disease of the liver in which T helper 1 cytokines predominate over those of T helper 2 in the pathogenesis. Interleukin‐18 (IL‐18), for which the gene was recently cloned, is a novel T helper 1 cytokine, which augments interferon‐gamma production. We designed this study to clarify the role of IL‐18 in primary biliary cirrhosis and to examine whether serum IL‐18 level can be a prognostic indicator for the disease. Serum IL‐18 levels were measured using an enzyme linked immuno sorbent assay with mouse monoclonal antibodies. Twenty‐two healthy volunteers, 31 patients with primary biliary cirrhosis (Scheuers stage I, 13; II, 10; and IV, 8), 20 patients with autoimmune hepatitis, 11 patients with virus‐related liver cirrhosis and six patients with obstructive jaundice were enrolled. Significant differences of serum IL‐18 levels were observed between patients with Scheuers stage IV and those with stage I, or II, virus‐related liver cirrhosis and obstructive jaundice (P < 0·05). The IL‐18 levels in primary biliary cirrhosis increased according to the disease progression, and fell promptly after living‐related liver transplantation. Moreover, serum IL‐18 levels in primary biliary cirrhosis were correlated with serum bilirubin concentrations and the Risk scores of the Mayo Clinic prognostic model for the disease. The IL‐18 levels observed in patients with autoimmune hepatitis were also elevated, and correlated with the activity of the disease. These results indicate that serum interleukin‐18 levels reflect the severity of primary biliary cirrhosis, the activity of autoimmune hepatitis, and may be an additive prognostic indicator in primary biliary cirrhosis.

Expression of Phosphorylated Ser70 of Bcl-2 Correlates with Malignancy in Human Colorectal Neoplasms

Purpose: Bcl-2 is a model apoptosis suppressor postulated to promote tumorigenesis. Recently, it has been reported that Bcl-2 undergoes phosphoregulation of its Ser70 to substantially alter its molecular function. Previous studies further suggest that such phospho-Bcl-2 regulation may influence tumor progression in colorectal and other cancers; however, phosphorylation status of the Ser70 of Bcl-2 (pSer70) in vivo in tumors remains obscure. To elucidate this question that may suggest the biological role, we molecularly screened a panel of human colorectal adenomas and adenocarcinomas for endogenous expression of pSer70 Bcl-2. Experimental Design: An antibody specific against pSer70 Bcl-2 was generated for thorough immunohistochemical examination of paraffin-embedded tumor specimens, allowing detection of the endogenously expressed antigen among a range of Bcl-2-positive colorectal neoplasms, including 75 tubular adenomas, 114 adenocarcinomas, and 15 cases of cancer in adenomas. Results: Loss of pSer70 Bcl-2 expression was observed in adenocarcinomas in a differentiation-dependent manner (positivities: well differentiated 63%, moderately differentiated 52%, and poorly differentiated 12%), whereas tubular adenomas maintained their expression (positivity 88%). Interestingly, an inverse correlation was found between expression of pSer70 Bcl-2 and Ki-67 antigen in those cases of cancer in adenoma (P < 0.01). It was further observed that loss of pSer70 Bcl-2 expression was associated with significantly shorter survival (P < 0.05) and correlated with clinical stages and lymph node metastasis (P < 0.05 and P < 0.05, respectively). Conclusions: Loss of pSer70 Bcl-2 expression is closely linked to biological aggressiveness in colorectal tumors and represents a statistically significant molecular index for prognosis of patients with these tumors.

Effect of nicotine on IL-18-initiated immune response in human monocytes

Nicotine is thought to inhibit the production of proinflammatory cytokines from macrophages through an anti‐inflammatory pathway that is dependent on nicotinic acetylcholine receptor α7 subunit (α7‐nAChR). IL‐18, an important proinflammatory cytokine, is reported to induce the expression of adhesion molecules on monocytes, thus enhancing cell‐to‐cell interactions with T‐cells and contributing to IL‐18‐initiated cytokine production. Accordingly, inhibition of IL‐18 suppresses systemic inflammatory responses. In the present study, we found that nicotine inhibited the IL‐18‐enhanced expression of ICAM‐1, B7.2, and CD40 on monocytes, and the production of IL‐12, IFN‐γ, and TNF‐α by PBMC. A nonselective and a selective α7‐nAChR antagonist, mecamylamine, and α‐bungarotoxin abolished the effects of nicotine, suggesting that this depends on α7‐nAChR stimulation. It is reported that nicotine induces prostaglandinE2 (PGE2) production in PBMC through the up‐regulation of cyclooxygenase (COX)‐2 expression. PGE2 is known to activate the EP2/EP4‐receptor, leading to an increase in cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity. Consistent with this, we found that COX‐2 and PKA inhibitors prevented the effects of nicotine on adhesion molecule expression and cytokine production, indicating that the mechanism of action of nicotine may be via endogenous PGE2 production.

MAP kinase-mediated proliferation of DLD-1 carcinoma by the stimulation of protease-activated receptor 2.

Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.

Preoperative proximal splenic artery embolization: a safe and efficacious portal decompression technique that improves the outcome of live donor liver transplantation.

Terminal liver cirrhosis is associated with marked severe portal hypertension, which increases the risk of intraoperative hemorrhage and graft hyper‐perfusion, especially, in small‐for‐size graft. In cases with developed collateral vessels, we often face difficulties in perihepatic dissection with blood stanching against bleeding during recipient hepatectomy. For aseptic preoperative portal decompression, we established the proximal splenic artery embolization (PSAE) technique. Sixty adult living donor liver transplantation recipients with viral/alcoholic hepatic failure were divided into two groups; PSAE group (n = 30) and non‐PSAE (n = 30). In the PSAE group, the splenic artery was embolized proximal to the splenic hilum 12–18 h before surgery. PSAE enabled shortening of operating time, reduced blood loss, led to less need for transfusion, and significantly reduced the post‐transplant portal venous velocity and ascites. PSAE was not associated with complications, e.g., splenic infarction, abscess, or portal thrombosis. Six of the non‐PSAE patients required additional surgical intervention to resolve postoperative hemorrhage and three patients required secondary PSAE for arterial‐steal‐syndrome. The hospital mortality rate of PSAE patients (3.3%) was significantly better than that of the PSAE group (13.3%, P < 0.05). Preoperative noninvasive PSAE makes more efficient use of portal decompression; thus, it can potentially contribute to improvement of outcome.