Kunzo Orita

Characterization of anti-human interleukin-18 (IL-18)/interferon-γ-inducing factor (IGIF) monoclonal antibodies and their application in the measurement of human IL-18 by ELISA

Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel cytokine, which is a potent inducer of IFN-gamma production and plays an important role in Th1 responses. In order to develop a specific ELISA for the measurement of human IL-18, we established 13 anti-human IL-18 monoclonal antibodies and characterized them. 7 murine anti-human IL-18 mAbs and 6 rat anti-human IL-18 mAbs were obtained by fusion of splenocytes from mice or rats immunized with human IL-18, with SP2/0 myeloma cells. These antibodies were classified into 4 groups according to competitive binding ELISAs to the human IL-18 molecule. 1 murine mAb and all 6 rat mAbs neutralized IFN-gamma production induced by IL-18. A specific human IL-18 ELISA was developed using two neutralizing mAbs (#125-2H and #159-12B). This ELISA detects human IL-18 with a minimum detection limit of 10 pg/ml, but does not react with heat-denatured human IL-18. The ELISA does not show any cross-reactivity with other cytokines. Using this assay, human IL-18 was measurable in the plasma of leukemia patients. This ELISA would become a powerful tool for investigating the relationship between IL-18 and various diseases or analyzing the control mechanisms of IL-18 production from IL-18 producing cells.

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.

The p53 gene is a potent determinant of chemosensitivity and radiosensitivity in gastric and colorectal cancers

We previously reported that introduction of the wild-typep53 gene into human cancer cells with deletedp53 enhanced apoptosis induced by chemotherapy [Fujiwara et al. (1994) Cancer Res 54∶2287]. This suggests thatp53 status could be a potent determinant of the therapeutic efficacy of DNA-damaging cancer therapy. We analyzed 24 patients with gastric or colorectal cancer forp53 mutations and apoptotic changes in surgical specimens. Out of 11 patients with gastric cancer, 3 were treated with chemotherapeutic drugs before resection; 5 of 13 patients with colorectal cancer had 30 Gy radiation prior to surgery.p53 mutations were detected in 4 cases of gastric cancer (36.4%) and in 6 cases of colorectal cancer (46.2%) by immunohistochemical staining. The preoperative DNA-damaging therapies increased the number of apoptotic cells in wild-type-p53-expressing tumors; tumors with mutantp53, however, significantly showed fewer apoptotic cells compared with those expressing wild-typep53. Thep53-inducible WAF1/CIP1 protein was immunohistochemically observed in wild-type-p53-containing tumors, where-as mutant-p53-expressing tumors expressed no detectable WAF1/CIP1. Taken together, we conclude thatp53 mutations are associated with the poor response of chemotherapy and radiotherapy.

IFN-γ-Dependent and -Independent Mechanisms in Adverse Effects Caused by Concomitant Administration of IL-18 and IL-12

IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1β. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-γ level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-α was not induced. The involvement of IFN-γ in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-γ treatment of wild-type mice alleviated it. When IFN-γ-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-γ-dependent mechanisms but also IFN-γ-independent processes.

Adverse affect of blood transfusions on survival of patients with gastric cancer

The effect of perioperative blood transfusions on the survival rate of patients with gastric cancer was studied. The survival rate of the transfusion group was significantly lower than that of the nontransfusion group in each of the 5 postoperative years. When no adjuvant immunochemotherapy was performed postoperatively, the prognosis was definitely worse in the transfusion group than in the nontransfusion group. Furthermore, the survival rate of the transfusion group was lower than that of the nontransfusion group in both histopathologic classifications of gastric cancer, and it was lower to a statistically significant extent among the well‐differentiated types. These results indicate that transfusions might adversely affect postoperative survival of patients with gastric cancer.

ICAM-1 expression and the soluble ICAM-1 level for evaluating the metastatic potential of gastric cancer

ICAM‐1 plays an important role in cell–cell and cell–extracellular matrix interactions, especially tumor invasion and cytotoxicity of lymphocytes. In the present study, the relationship between metastasis of gastric cancer and ICAM‐1 expression by cancer cells or the serum level of s‐ICAM‐1 was (s‐ICAM‐1) was examined. ICAM‐1 was detected by immunohistochemic staining in 49.0% of 108 patients with gastric cancer. The ICAM‐1 expression rate was higher at a more advanced stage, based on lymph node metastasis, being 46.9% in node‐negative and 56.1% in node‐positive cases. In patients with liver metastasis, the rate was 90.9%, while it was 43.3% in patients without liver metastasis (p < 0.05). The serum s‐ICAM‐1 level was 262.1 ng/ml (median 205.5, range 176.0–271.0) in healthy subjects and 391.5 ng/ml (median 317.5, range 148.7–1,768.0) in gastric cancer patients (p < 0.001). The serum s‐ICAM‐1 level was significantly higher in patients with liver metastasis than in patients without liver metastasis (p < 0.0001). In addition, positive ICAM‐1 expression cases had significantly higher s‐ICAM‐1 levels than negative ones, 408.9 ± 188.4 and 308.1 ± 88.1 ng/ml, respectively. These results suggested that ICAM‐1 was overexpressed in cancer cells and released as s‐ICAM‐1, which would promote hematogenous metastasis by suppressing local anticancer immunity.

A simple and sensitive bioassay for the detection of human interleukin-18/interferon-γ-inducing factor using human myelomonocytic KG-1 cells

Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel cytokine which plays an important role in Th1 responses. Here we describe a simple, sensitive bioassay for human IL-18 using the human myelomonocytic cell line, KG-1, which produces IFN-gamma in response to human IL-18. IFN-gamma production induced by human IL-18 was completely blocked by an antibody against human IL-18. Human IL-18 could be measured in a concentration range from approximately 100 to 10,000 pg/ml, and intra- and inter-assay coefficient variations were both below 15%. It was possible to measure human IL-18 in human serum, cell lysate or culture supernatant by this bioassay. Thus, the human IL-18 bioassay can be expected to be useful in the investigation of the relationship between human IL-18 and various diseases or in analyzing the mechanisms of human IL-18 secretion from IL-18 producing cells.

Immunotherapy of solid cancer using dendritic cells pulsed with the HLA-A24-restricted peptide of carcinoembryonic antigen.

Abstract. Carcinoembryonic antigen (CEA), an oncofetal glycoprotein overexpressed in most gastrointestinal and lung cancers, is a candidate molecule for cancer immunotherapy. Recently, a CEA-derived 9-mer peptide, CEA652 (TYACFVSNL), has been identified as the epitope of cytotoxic T lymphocytes restricted with human leukocyte antigen (HLA)-A24, which is present in 60% of the Japanese population and in some Caucasians. The authors performed a clinical study of a vaccine using autologous dendritic cells (DCs) pulsed with CEA652 and adjuvant cytokines, natural human interferon alpha (nhuIFN-α), and natural human tumor necrosis factor alpha (nhuTNF-α), for the treatment of patients with CEA-expressing advanced metastatic malignancies. Ten HLA-A24 patients with advanced digestive tract or lung cancer were enrolled in the study to assess toxicity, tolerability and immune responses to the vaccine. DCs were generated from plastic adherent monocytes of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMCs) in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Generated DCs showing an immature phenotype were loaded with CEA652 and injected into patients intradermally and subcutaneously with 50% of the dose administered by each route every 2 weeks for a total of ten vaccinations. The total dose of administered DCs ranged from 2.7×107 cells to 1.6×108 cells. Adjuvant cytokines, i.e., 1×106 U/body of nhuIFN-α and nhuTNF-α, were administered to patients twice a week during the vaccination period. No severe toxicity directly attributable to the treatment was observed, and the vaccine was well tolerated. In the delayed-type hypersensitivity (DTH) skin test, two patients showed a positive skin response to peptide-pulsed DCs after vaccination, although none of the patients tested positive prior to vaccination. In the two patients who showed a positive skin response disease remained stable for 6 and 9 months respectively. These results suggest that active immunization using DCs pulsed with CEA652 peptide in combination with the administration of adjuvant cytokines is a safe and feasible treatment procedure.

Preoperative cell-mediated immune status of gastric cancer patients

The status of cell‐mediated immunity was studied in 360 patients with gastric cancer before surgery. For the skin test, tuberculin and DNCB were employed. For the in vitro test, the blastogenesis of peripheral blood lymphocytes to phytohemagglutinin (PHA), the ratio of rosette‐forming T cells to sheep blood red cells, and the macrophage migration inhibitory factor against autochthonous tumor antigens were measured. As a result, it was found that in progressive gastric cancers the cell‐mediated immunity decreased specifically or nonspecifically, especially the DNCB reaction; and the blastogenesis against PHA showed an inverse correlation to the advance of gastric cancer.

NF-ATc2 induces apoptosis in Burkitt's lymphoma cells through signaling via the B cell antigen receptor

Cross‐linking of the B cell antigen receptor (BCR) with an anti‐IgM antibody has been shown to induce dramatic apoptosis in type I Burkitts lymphoma (BL) cells. However, the apoptotic mechanism triggered via BCR remains unknown. Here we reports a mechanism of BCR ligation‐induced apoptosis involving protein phosphatase calcineurin and its specific substrate, transcriptional factor NF‐AT. In response to BCR cross‐linking, endogenous calcineurin was rapidly activated, and this facilitated nuclear translocation of NF‐ATc2, a subtype of NF‐AT members. Interestingly, nuclear‐imported NF‐ATc2 functioned pro‐apoptotically in BL cells. The effect of NF‐ATc2 was efficiently blocked with FK506, which prevented its nuclear translocation through inactivation of calcineurin. In addtion, TR3 induction during BCR cross‐linking was reduced by FK506 and the VIVIT peptide, which is a highly selective inhibitor for NF‐AT. This strongly suggests that activation of NF‐ATc2 by calcineurin is essential for TR3 recruitment, and that TR3 can be considered as a candidate for death effector in BCR‐mediated apoptosis. Therefore, NF‐ATc2 plays a crucial role in BCR‐mediated apoptosis in type IBL, providing greater insight into unique BL characteristics through BCR signaling.