Hiroaki Yanagawa

Serum levels of interleukin 6 in patients with lung cancer

Serum interleukin 6 (IL-6) levels were measured in 75 patients with lung cancer and in 20 patients with benign lung diseases. IL-6 was detectable in 29 patients with lung cancer (39%), but was not detectable in any of the patients with benign lung diseases. Serum C-reactive protein levels and plasma fibrinogen levels were significantly higher and serum albumin concentration was significantly lower in lung cancer patients with detectable serum IL-6 levels than in those without detectable serum IL-6 levels and in patients with benign lung diseases. On the other hand, no significant difference was observed in blood platelet counts in these three groups. Moreover, serum IL-6 levels were not significantly different in lung cancer patients with or without clinically demonstrated distant metastasis. These results suggest that IL-6 may be a mediator of various reactions including an inflammatory response in lung cancer patients.

Vascular endothelial growth factor in malignant pleural effusion associated with lung cancer.

Abstract The presence of vascular endothelial growth factor (VEGF) was examined by enzyme immunoassay in 60 cytology-documented malignant pleural effusions associated with primary lung cancer and 51 other benign and malignant pleural effusions. Exudative pleural effusions contained significantly higher amounts of VEGF than transudative pleural effusions. Among exudative pleural effusions, levels of VEGF in malignant pleural effusions associated with lung cancer were significantly higher than those of benign exudative pleural effusions. There was no significant difference in pleural VEGF in patients with different histological types or clinical stages of lung cancer. Serial measurement of pleural VEGF levels was performed in six lung cancer patients treated with intrapleural instillation of recombinant interferon γ, and reduction of pleural effusion was associated with decreasing pleural VEGF levels. These findings suggest that VEGF has a role in the accumulation of exudative pleural effusions, especially that of malignant pleural effusion associated with lung cancer.

Antiallergic and Anti-Inflammatory Effects of a Novel IκB Kinase β Inhibitor, IMD-0354, in a Mouse Model of Allergic Inflammation

Background: Nuclear factor (NF)-κB is a transcription factor known to regulate allergy-associated cytokine and chemokine production related to the induction of inflammation. IκB kinase β (IKKβ), which is responsible for activation of the NF-κB pathway, may be an ideal molecular target to inhibit this process. IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is an attractive novel IKKβ inhibitor that prevents the production of inflammatory cytokines in various diseases, although it is not known if IMD-0354 is effective against allergic inflammation. This study aimed to elucidate the antiallergic effects of a newly synthesized IKKβ inhibitor, IMD-0354, in a mouse model of allergic inflammation. Methods: We generated ovalbumin (OVA)-sensitized mice which were then challenged with OVA. IMD-0354 was administered intraperitoneally to therapeutic groups. Lung histopathology and the concentrations of cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and supernatants of lung homogenates were determined. Results: Administration of IMD-0354 ameliorated airway hyperresponsiveness and reduced the numbers of bronchial eosinophils and mucus-producing cells in OVA-sensitized mice. The total numbers of cells and eosinophils in BALF were also reduced by treatment with IMD-0354. Treatment with IMD-0354 inhibited the production of Th2 cytokines such as interleukin (IL)-5 and IL-13 and eotaxin in the airways and/or lungs of OVA-sensitized mice, but it did not affect the restoration of Th1 cytokines such as IL-12 and interferon-γ under the same experimental conditions. IgE production was also inhibited by IMD-0354. Conclusion: A specific IKKβ inhibitor, IMD-0354, improved allergic airway inflammation and hyperresponsiveness in mice. IMD-0354 may have therapeutic potential for bronchial asthma.

Therapeutic efficacy of mouse-human chimeric anti-ganglioside GM2 monoclonal antibody against multiple organ micrometastases of human lung cancer in NK cell-depleted SCID mice

The development of distant metastases to multiple organs is a critical problem in the treatment of human lung cancer. In this study, we evaluated the therapeutic efficacy of a mouse‐human chimeric anti‐ganglioside GM2 (GM2) monoclonal antibody (MAb), KM966 against metastasis formation of GM2‐positive human lung cancer cells inoculated intravenously (i.v.) into natural killer (NK) cell‐depleted severe combined immunodeficient (SCID) mice. GM2‐positive human small cell lung cancer (SCLC), SBC‐3 cells (1 × 106), injected through a tail vein into NK cell‐depleted SCID mice, formed large number of metastatic colonies in the liver, kidneys and lymph nodes by 42 days after inoculation (day 42). KM966, but not control MAb, given on days 2 and 7, almost completely inhibited metastasis formation of SBC‐3 cells in the liver, kidneys and lymph nodes in a dose‐dependent fashion. Moreover, treatment with KM966 at advanced stages of metastasis (even from day 28) significantly suppressed multiple organ metastases of SBC‐3 cells. The anti‐metastatic effect of KM966 in vivo was mainly due to an antibody‐dependent cell‐mediated cytotoxicity (ADCC) reaction mediated by macrophages of the SCID mice. Our findings suggest that the mouse‐human chimeric anti‐GM2 MAb, KM966 may be useful for eradicating multiple organ micrometastases of lung cancer in humans.Int. J. Cancer 78:480–485, 1998.

Cytolysis of human dendritic cells by autologous lymphokine-activated killer cells: participation of both T cells and NK cells in the killing

Dendritic cells (DC) play a key role in the initiation of immune response by stimulating the naive T cells. The fate of DC after the initiation of immune response is not clearly understood. Although there are few reports implicating natural killer (NK) cells in the elimination of DC, killing of DC by LAK cells, and specifically by T cells, has not been studied. In this study, we observed that DC, generated from monocytes, in vitro in the presence of granulocyte‐macrophage colony‐stimulating factor, interleukin‐4 (IL‐4), and tumor necrosis factor α were susceptible to cytolysis by lymphokine‐activated killer (LAK) cells induced in the presence of IL‐2 and IL‐15 but not IL‐12 alone. However, LAK cells induced by a combination of IL‐12 and suboptimal dose of IL‐2 were cytotoxic to DC. When purified lymphocytes were activated with IL‐2, the CD8+/CD57− fraction (T‐LAK), but not the CD8−/CD57+ fraction (NK‐LAK) was cytotoxic to autologous DC. However, when unseparated peripheral blood mononuclear cells were used to generate LAK cells, both T‐LAK and NK‐LAK fractions showed equal cytotoxicity against autologous DC. Monoclonal antibodies against CD54, CD11a, and CD18 significantly inhibited the cytolysis, indicating that the killing involves the engagement of CD54 with its ligands. J. Leukoc. Biol. 65: 764–770; 1999.

Synthesis and Secretion of Interleukin-15 by Freshly Isolated Human Bronchial Epithelial Cells

Background: Interleukin-15 (IL-15), which shares many functional activities of IL-2, is proposed as a potential modulator of T and natural killer (NK) cell-mediated inflammatory diseases. Since IL-15 gene is expressed in various cell types including epithelial cells, we examined how proinflammatory modulators affect IL-15 gene expression in both freshly isolated human bronchial epithelial cells (HBECs) and the human bronchial epithelial cell line BEAS-2B. Methods: HBECs were obtained from 25 patients with primary lung cancer by bronchial brushing under bronchofiberscopy. The expressions of IL-15 and its receptor were examined using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis and enzyme-linked immunosorbent assay. Results: IL-15 mRNA was constitutively expressed in the cells and was upregulated by several proinflammatory cytokines such as IL-1β, tumor necrosis factor-α, interferon-γ (IFN-γ) and lipopolysaccharide. In addition, IFN-γ but not other cytokines induced the synthesis and secretion of IL-15 protein. Investigation of IL-15 receptor expression using RT-PCR showed that IL-15Rα and IL-2Rβ chains but not IL-2Rα or γ chain were constitutively expressed in these cells. Conclusions: Bronchial epithelial cells may contribute to T and NK cell-mediated airway inflammation through IL-15 production.

Interleukin‐4 Downregulates Interleukin‐6 Production by Human Alveolar Macrophages at Protein and mRNA Levels

Effects of interleukin‐4 (IL‐4) on IL‐6 production by human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined at the protein and gene levels. IL‐6 production was quantitated by enzyme immunoassay (EIA) and bioassay using the IL‐6 dependent murine hybridoma cell line MH60.BSF2. Results showed that when activated with LPS, AM released significantly more biologically active IL‐6 than blood monocytes. Human rIL‐4 significantly suppressed IL‐6 production by AM and monocytes stimulated with LPS. Northern blot analysis revealed that IL‐4 reduced the expression of IL‐6 mRNA in LPS‐stimulated AM and monocytes. The inhibitory effect was most pronounced when IL‐4 was added with LPS or within the first 4 hr after LPS to AM or monocytes. The suppressive effect of IL‐4 was completely neutralized by pretreatment with anti‐IL‐4 antibody. IL‐4 also showed a suppressive effect on IL‐6 production by macrophages generated in vitro by maturation of blood monocytes with granulocyte‐macrophage colony stimulating factor (GM‐CSF). These observations suggest that IL‐4 may play a critical role in in situ regulation of immune responses through suppression of IL‐6 production.

Interleukin-1 receptor antagonist in pleural effusion due to inflammatory and malignant lung disease

Interleukin (IL)-1 is a key cytokine in inflammatory reactions. To clarify the mechanism of inflammation in the pleural cavity, we investigated the contribution of IL-1 and its antagonism to inflammatory processes in the pleural cavity. Interleukin-1 receptor antagonist (IL-1Ra) levels as well as IL-1 beta and interferon-gamma (IFN-gamma) levels were measured by enzyme immunoassay in pleural effusions from 70 patients. Pleural macrophages were also examined as possible sources of these cytokines in 10 patients. IL-1Ra was detectable in 28 patients (40%) out of 70 patients with pleural effusions. Patients with tuberculosis had significantly higher IL-1Ra as well as IFN-gamma levels in pleural effusion than patients with lung cancer. Transudative pleural effusions had low or undetectable IL-IRa levels. On the other hand, IL-1 beta levels were low, except in cases of parapneumonic pleural effusion. Spontaneous production of IL-1Ra pleural macrophages was observed in six patients, and IL-4 significantly augmented its production. Although spontaneous production of IL-1 beta was observed in only two patients, pleural macrophages produced significant amounts of IL-1 beta in response to lipopolysaccharide in all 10 patients examined. These results suggest that interleukin-1 receptor antagonist regulates various reactions by interleukin-1 in pleural effusion, and that pleural macrophages may act in situ as a source of interleukin-1 receptor antagonist.

Augmentation by interleukin-18 of MHC-nonrestricted killer activity of human peripheral blood mononuclear cells in response to interleukin-12

Interleukin (IL)-18 is a novel cytokine with pleiotropic functions. In the present study, we examined the induction of the killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines upon treatment with IL-18 in combination with IL-12. Cytotoxic activity was measured by standard (51)Cr release assay. IL-18 (100 ng/ml) was found to significantly augment IL-12-induced killer activity in a MHC-nonrestricted manner against allogeneic NK-resistant Daudi cells and lung cancer cell lines: SBC-3, RERF-LC-AI and A549. IL-18 could augment IL-12-induced killer activity both at the optimal as well as suboptimal doses of the latter. However, IL-18 was found to have little effect on the killer activity of MNC induced by optimal or suboptimal dose of IL-2 or IL-15. Treatment of MNC with IL-18 in combination with IL-12 for a period of more than 4 days was observed to optimally induce the killer activity. As for induction of IFN-gamma production by MNC, IL-18 augmented that induced by IL-2 and IL-15, as well as that induced by IL-12. These results show the potential of IL-18 in combination with IL-12 for clinical application in treatment of cancer.

Soluble Fas in malignant pleural effusion and its expression in lung cancer cells

Soluble Fas (sFas) has the ability to block Fas‐mediated apoptosis, suggesting that sFas at tumor sites might inhibit tumor cell‐killing by immune effector cells. We examined the sFas level in pleural effusion associated with lung cancer. The level of sFas in malignant pleural effusion was significantly higher than those in transudate and tuberculous pleural effusion. There was no significant difference in the sFas concentration among various histological types of lung cancer. The cytotoxicity mediated by anti‐Fas agonistic antibody against Jurkat cells was inhibited by the addition of malignant pleural effusion, being inversely correlated with the sFas concentration. When Fas expression was examined using flow cytometry, eight of ten (80%) lung cancer cell lines expressed cell surface Fas. On the other hand, sFas protein and mRNA were detected in six of ten (60%) lung cancer cell lines, but there was no correlation between Fas and sFas expression. Furthermore, although the expressions of Fas and sFas were clearly detected in tumor cells derived from malignant effusion, the sFas expression was down‐regulated in an in vitro culture. These results suggest that sFas in malignant pleural effusion is at least in part produced by lung cancer cells, and might play a role in local immunosuppression by tumor cells. (Cancer Sci 2003; 94: 302–307)