Takashi Haku

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.

Interleukin-10 is a potent inhibitor of tumor cytotoxicity by human monocytes and alveolar macrophages

The effects of purified human interleukin‐10 (IL‐10) on the expression of antitumor activity of human monocytes and alveolar macrophages (AMs) obtained by centrifugal elutriation and bronchoalveolar lavage, respectively, from the same healthy donors were examined. Monocytes and AMs were incubated for 16 h in medium with lipopolysaccharide (LPS) in the presence or absence of IL‐10 or IL‐4, and then their tumoricidal activity was assayed by measuring 125I‐IUdR release from human melanoma (A375) cells. Addition of IL‐10 to cultures of monocytes or AMs with LPS resulted in dose‐dependent suppression of their cytotoxicity against A375 cells, the suppression of the activity of monocytes being the higher. IL‐10 also suppressed the synergistic effects of interferon‐γ and desmethyl muramyldipeptide in activation of monocytes. IL‐10 inhibited the early induction phase of monocyte activation but not the effector phase (monocyte‐mediated cytotoxicity). IL‐10 plus IL‐4 inhibited the antitumor activities of AMs and monocytes much more than either IL‐10 or IL‐4 alone. IL‐10 and IL‐4 at sub‐optimal concentrations also showed synergistic inhibitory effects. These findings suggest that IL‐10 may be important in vivo in down‐regulating the antitumor activities of monocytes and AMs in the lung by inhibiting their productions of antitumor effector molecules. J. Leukoc. Biol. 55: 437–442; 1994.

Production of IL-1 and its receptor antagonist is regulated differently by IFN-gamma and IL-4 in human monocytes and alveolar macrophages

Interleukin-4 (IL-4) has previously been found to downregulate interleukin-1 (IL-1) production, but to upregulate the production of IL-1 receptor antagonist (IL-1ra) in human monocytes stimulated with lipopolysaccharide (LPS). In the present study we wanted to determine whether the production of IL-1ra in human monocytes and alveolar macrophages (AMs) is regulated differently at the protein and messenger ribonucleic acid (mRNA) levels by IL-4 and interferon-gamma (IFN-gamma). AMs and monocytes obtained from healthy donors by bronchoalveolar lavage and centrifugal elutriation were stimulated with LPS in the presence or absence of IL-4 or IFN-gamma, and the expression of mRNA for IL-1 and IL-1ra was measured by Northern blot analysis. The production of IL-1 and IL-1ra was quantitated by enzyme immunoassays (EIAs). Spontaneous IL-1ra production was seen in AMs after incubation for 4 h in medium alone, but not in blood monocytes, at both the protein and mRNA levels. The spontaneous expression of the IL-1ra gene in AMs was augmented by incubation with IL-4. Interleukin-1 beta (IL-1 beta) production by LPS-stimulated AMs and monocytes was upregulated by IFN-gamma, but downregulated by IL-4. Interestingly, when stimulated with LPS, IFN-gamma inhibited IL-1ra production by monocytes, but up-regulated its production in human AMs at the protein and mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Induction Mechanism of Human Blood CD8+ T Cell Proliferation and Cytotoxicity by Natural Killer Cell Stimulatory Factor (Interleukin‐12)

Natural killer cell stimulatory factor (NKSFJ IL‐12) has been found to induce cytotoxic activity of human blood T cells. In the present study, the effect of NKSF on induction of cytotoxic CD8+ T cells in the presence or absence of monocytes was examined. Highly purified lymphocytes (>99%) and monocytes (>90%) were isolated hy centrifugal elutriation from peripheral blood of normal donors. Then, CD8+ cells were isolated with antibody‐bound magnetic beads from purified lymphocytes. The cytotoxicity of CD8+ cells was measured by 51Cr release assay for 4 h. NKSF enhanced the proliferative response of CD8+ cells stimulated with suboptimal concentrations of interleukin‐2 (IL‐2), but rather inhibited their proliferative and cytotoxic responses on stimulation with an optimal concentration of IL‐2. NKSF stimulated CD8+ cells to produce interferon 7 (IFNγ) irrespective of the presence of added IL‐2, and this effect was augmented by co‐cultivation with monocytes. Blood monocytes upregulated induction of cytotoxic CD8+ cells stimulated with NKSF alone, and this effect was abolished by addition of antibody against IFNγ, but not of antibody against tumor necrosis factor a. Induction of NKSF‐inducible cytotoxic CD8+ cells was inhibited by addition of transforming growth factor β, but not of IL‐4. These observations suggest that in situ induction of NKSF‐stimulated cytotoxic CD8+ cells may be regulated by complex cytokine networks, depending on the participation of monocytes.

Induction by Interleukin-15 of Human Killer Cell Activity against Lung Cancer Cell Lines and Its Regulatory Mechanisms

Interleukin (IL)‐15 is a novel cytokine with IL‐2‐like activity. In the present study, we examined IL‐15‐mediated induction of killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines, and the regulatory mechanisms of this induction by IL‐15. Cytotoxic activity was measured by 51Cr release assay. IL‐15 at concentrations of more than 10 ng/ml induced significant killer activity of blood MNC against a small cell lung cancer cell line (SBC‐3), as well as Daudi cells, and 50 ng/ml was considered its optimal concentration. A time course study revealed that an incubation period of 4–6 days was optimal for induction of killer activity. MNC cultured with IL‐15 also exhibited killer activity against other lung cancer cell lines (H‐69, N‐291 and PC‐9 cells). IL‐15 and IL‐12 had additive effects on induction of killer activity against SBC‐3 cells. On the other hand, IL‐15 had no synergistic or additive effect on induction of killer activity by IL‐2. Fresh human monocytes isolated by centrifugal elutriation augmented the development of killer activity of lymphocytes stimulated by IL‐15. As a humoral regulatory factor, IL‐4 had a suppressive effect on induction of killer activity by IL‐15. IFN‐γ, IL‐1β, TNF‐α, IL‐6 or IL‐10 had no effect on induction of killer activity by IL‐15 at the optimal concentration. These results suggest that IL‐15 has potential for the immunotherapy of ling cancer.

Intrapleural instillation of interferon γ in patients with malignant pleurisy due to lung cancer

Abstract The effect of intrapleural instillation of recombinant human interferon γ (IFNγ) at increasing doses of (1–12) × 106 U was examined in six patients with cytologically positive pleural effusion due to lung cancer. Intrapleural instillation was repeated up to three times. Clinically, no reaccumulation of pleural effusion was observed in one patient and disappearance of lung cancer cells from the pleural effusion was seen in two other patients. No severe side-effects were observed. Considerable levels of IFNγ remained in the pleural effusion as well as in patients’ serum up to 7 days after instillation of 2 × 106 U and higher doses. The total cell number showed a transient decrease on day 1 of therapy. Levels of pro-inflammatory cytokines, such as tumor necrosis factor α, interleukin(IL)-1β and IL-6, in the pleural effusion remained almost stable after IFNγ instillation. On the other hand, intrapleural IL-1 receptor antagonist levels were remarkably elevated by the instillation of IFNγ. IL-2- and IL-12-inducible killer activity of pleural mononuclear cells tended to increase slightly. Despite the inability of IFNγ to control pleural effusion in this treatment schedule, IFNγ instilled by an intrapleural route had a potential local antitumor activity. Moreover, since IFNγ persists in pleural effusions for a long time after a single instillation, such a therapy in combination with other fibrogenic biological response modifiers can be promising.

Intrapleural therapy with MDP-Lys (L18), a synthetic derivative of muramyl dipeptide, against malignant pleurisy associated with lung cancer

N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys (L18), romurtide) is a synthetic muramyl dipeptide derivative, and has immunomodulating activities including activation of cells of monocyte-macrophage lineage. We examined the effect of intrapleural instillation of MDP-Lys (L18) against malignant pleurisy associated with lung cancer. Six patients with cytologically-positive malignant pleural effusion (four with adenocarcinoma, one with small cell carcinoma and one with large cell carcinoma) were treated with single intrapleural instillation of MDP-Lys (L18) of 200 microg. Clinically, no reaccumulation of pleural effusion for at least 4 weeks was observed in four patients. No major side effects were observed. Total cell number elevated remarkably 4 h after instillation, and main increased population was that of neutrophils. Levels of chemotactic cytokines, such as interleukin (IL)-8, and monocyte chemotactic protein (MCP)-1 and levels of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, elevated in pleural effusion, and peak IL-1beta and IL-6 levels tended to be higher in clinical responders than non-responders. These results suggest MDP-Lys (L18) instilled by intrapleural route had a potential local immunomodulatory activity. Further study is warranted to further determine the critical factors which correlate with the clinical response.

Production of interleukin-10 by alveolar macrophages from lung cancer patients.

Interleukin (IL)-10 is known to be an autoregulatory factor of functions of monocyte macrophages. The purpose of this study was to determine whether IL-10 production by alveolar macrophages (AMs) is altered in patients with lung cancer. AMs were obtained by bronchoalveolar lavage from 25 patients with lung cancer and 14 control patients. The production of IL-10 by AMs was quantitated by enzyme immunoassay with or without stimulation with lipopolysaccharide (LPS). No significant difference in spontaneous and LPS-stimulated IL-10 production by AMs was observed between lung cancer patients and control patients (mean +/- SEM; 288.0 +/- 56.7 vs. 249.6 +/- 58.4 pg ml-1). IL-10 production of LPS-stimulated AMs was not impaired even in lung cancer patients with systemic metastasis. IL-4 failed to suppress LPS-induced production of IL-10 by AMs both in control patients and in lung cancer patients. In eight patients with lung cancer, IL-10 production by AMs was estimated before and after systemic chemotherapy and IL-10 production by LPS-stimulated AMs tended to increase after systemic chemotherapy from 152.3 +/- 51.9 to 278.0 +/- 112.8 pg ml-1. As IL-10 is a potent inhibitor of tumour angiogenesis, an important process of tumour progression, these results suggest that, even in advanced cancer patients, macrophages can produce potent angiogenesis inhibitor and systemic chemotherapy may augment this inhibitory activity in the lung.

Generation of killer activity by interleukin-12 of mononuclear cells in malignant pleural effusions due to lung cancer

Abstract In the present study, we examined the ability of interleukin (IL)-12 to generate an antitumor effect in the tumor-growing site. Mononuclear cells (MNC) were obtained from 12 malignant pleural effusions due to lung cancer in the tumor-growing site. Non-major-histocompatibility-complex-restricted killer activity, examined by 4-h 51Cr release assay against Daudi lymphoma cells as well as various lung cancer cell lines (H69 and PC-9), and in vitro production of interferon γ (IFNγ), measured by enzyme immunoassay, were investigated as mediators of antitumor effects of host cells activated by IL-12. IL-12 induced killer activity of MNC in pleural effusions (pleural MNC) dose-dependently. Moreover, pleural MNC produced a signficant amount of IFNγ in response to IL-12. The killer activities of IL-12-activated blood MNC were higher than those of pleural MNC. The supernatants of pleural effusions of these untreated patients suppressed killer induction by IL-12 of blood MNC of healthy volunteers. These observations suggest that MNC present at the site of growing tumors may act as effector cells against lung cancer in the presence of IL-12.