Production of tumor necrosis factor-alpha by alveolar macrophages of lung cancer patients

The abilities of human alveolar macrophages (AM) obtained from healthy donors and patients with lung cancer to produce tumor necrosis factor (TNF) were compared with those of their blood monocytes after activation with lipopolysaccharide (LPS). TNF activity was assayed by measuring cytotoxicity against actinomycin D-treated L929 cells and TNF was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA) with polyclonal and monoclonal antibodies against TNF-alpha. Unstimulated AM from healthy donors released variable amounts of TNF spontaneously, whereas blood monocytes did not. When treated with LPS for 24 h, AM and monocytes produced TNF dose-dependently, but TNF production by AM was significantly more than that by blood monocytes. This TNF activity was inhibited completely by monoclonal anti-TNF-alpha antibody. Macrophages generated by in vitro maturation of monocytes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) produced more TNF than freshly isolated monocytes. No difference was found in the abilities of AM from healthy donors and patients with lung cancer to produce TNF after activation stimuli. These observations suggest that human AM may be important in in vivo antitumor defense of the lung through TNF-alpha production.     

Human Alveolar Macrophages Augment Natural Killer Cell Stimulatory Factor (Interleukin-12)-inducible Killer Activity from Autologous Blood Lymphocytes

Interleukin-12 (IL-12), also known as natural killer cell stimulatory factor (NKSF), was found to induce cytotoxic activity from human blood T cells and NK cells. The present study was undertaken to examine the effect of human alveolar macrophages (AM) on induction by IL-12 cytotoxic cells from blood lymphocytes. AM were obtained by bronchoalveolar lavage from healthy donors. Highly purified lymphocytes (>99%) and monocytes (>90%) were also isolated by centrifugal elutriation from peripheral blood of the same donors. Cytotoxicity of lymphocytes was measured by 4-h ⁵¹Cr release assay. IL-12 stimulated blood lymphocytes to produce interferon γ (IFNγ) and tumor necrosis factor α (TNFα), and this effect was augmented by co-cultivation with monocytes or AM. AM-upregulated induction of cytotoxic lymphocytes was stimulated with IL-12, and this effect was significantly abrogated by addition of antibodies against IFNγ and TNFα. Induction by IL-12 of IFNγ production and cytotoxic activity of CD8⁺ cells was also augmented by co-cultivation with monocytes or AM. AM were more effective than monocytes in augmenting the cytotoxic activity of IL-12-stimulated lymphocytes and CD8⁺ cells. These observations suggest that in situ induction of IL-12-stimulated cytotoxic cells in the lung may be regulated by complex cytokine networks, depending on participation of monocytes and alveolar macrophages.     

Human alveolar macrophages: wheat germ agglutinin-dependent tumor cell killing

Human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors were examined for ability to cause lectin-dependent tumor cell killing. Of five plant and two animal lectins tested, only one lectin, wheat germ agglutinin (WGA), induced significant and reproducible lectin-dependent macrophage-mediated cytotoxicity (LDMC) against human bladder cancer (T-24) cells. There was no significant difference between the LDMCs of AM and blood monocytes. All 6 tumor cell lines tested were sensitive to various extents to LDMC induced by WGA. Quantitative analysis with WGA-FITC conjugate showed the presence of various levels of receptors for WGA on the surface of AM, monocytes and tumors. A relatively good correlation was found between the sensitivities of the tumor cells to LDMC mediated by AM and the numbers of receptors for WGA. Pretreatment of AM or monocytes with LPS did not affect their LDMC. These results indicate that a plant lectin, WGA which binds to both human AM and tumor cells, renders human AM cytotoxic to allogeneic tumor cells by a different mechanism(s) from that involved in the nonspecific tumor cytotoxicity of activated macrophages.     

Membrane-associated interleukin 1 alpha as a mediator of tumor cell killing by human blood monocytes fixed with paraformaldehyde

Human blood monocytes isolated by centrifugal elutriation from healthy donors were tested for ability to produce membrane-associated antitumor monokine(s) in response to activation stimuli such as various types of interferon (IFN) and/or synthetic desmethyl muramyl dipeptide (norMDP). IFNs (alpha, beta, and gamma) and norMDP rendered blood monocytes cytotoxic to allogeneic A375 melanoma cells, as assayed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with any type of IFN for 16 h, and then fixed with paraformaldehyde, they did not show cytotoxicity to A375 cells, but when they were fixed after treatment with norMDP or lipopolysaccharide they showed significant cytotoxicity to A375 melanoma cells. This membrane-associated antitumor monokine induced by the synergistic actions of suboptimal concentrations of IFN-gamma and norMDP, was cytotoxic to HT-29 colon cancer cells as well as A375 melanoma cells, but not to actinomycin D-treated L-929 cells. The fixed monocyte-mediated cytotoxicity against A375 melanoma cells was completely inhibited by a specific anti-interleukin 1 alpha antiserum, but not by a specific anti-interleukin 1 beta antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated interleukin 1 alpha is involved through cell-to-cell contact in the host defense mechanism against cancer.     

Up- and down-regulation of human lymphokine (IL-2)-activated killer cell induction by monocytes, depending on their functional state

Studies were made to determine whether freshly isolated monocytes from healthy donors could influence the induction of lymphokine (IL-2)-activated killer (LAK) activity. Highly purified lymphocytes (greater than 99%) and monocytes (greater than 90%) were isolated by counter-flow centrifugal elutriation from peripheral blood. Lymphocytes incubated for 4 days with IL-2 showed significant LAK activity against natural killer (NK) cell-resistant target (Daudi) cells, whereas monocytes treated for 4 days with IL-2 and/or IFN-gamma were not cytotoxic. Under the experimental conditions used, addition of monocytes to the lymphocyte cultures resulted in significant augmentation of the LAK activity, depending on the density of monocytes added. In contrast, monocytes stimulated by lipopolysaccharide (LPS) markedly suppressed LAK activity induced by IL-2, depending on the dose of LPS added. Similar up- and down-regulations of LAK cell induction by monocytes were observed with 4 lines of human lung cancer cells as targets for LAK activity. Although supernatants from untreated monocytes did not increase LAK induction, supernatants from LPS-stimulated monocytes suppressed LAK induction. The regulatory role of monocytes could not be replaced by the addition of exogenous interleukin I (IL-I) or tumor necrosis factor (TNF). Prostaglandin E did not seem to play a regulatory role, since addition of indomethacin did not affect the regulation of LAK cell induction by monocytes. These results clearly indicate that human monocytes may cause up- or down-regulation of the expression of IL-2-induced LAK activity, depending on their functional state.     

Kinetics and function of tumor cytotoxic factor(s) produced by human blood monocytes activated to the tumoricidal state

Human monocytes obtained from healthy volunteers and isolated by centrifugal elutriation were not cytotoxic to allogeneic tumorigenic cells. These freshly isolated monocytes were rendered tumoricidal following interaction in vitro for 24 hours with greater than 0.01 micrograms lipopolysaccharide (LPS)/ml or over 1 microgram nor-muramyl dipeptide/ml. Monocytes activated by this procedure produced a soluble factor that lysed tumor cells. Full expression of tumor cell lysis required a minimum of 18 hours' exposure of tumor cells to the factor. The degree of tumor cytotoxic factor (TCF) production was closely related to the intensity of monocyte activation to become tumoricidal. Significant production of TCF by monocytes was detected in the supernatants after treatment for 3 hours with LPS. TCF was also released by activated monocytes when cocultivated with tumorigenic cells. Similarly, the level of TCF production correlated with the monocyte density. TCF destroyed human allogeneic tumor cell lines (melanoma, glioblastoma, colon carcinoma, prostatic carcinoma, and breast carcinoma), but it did not affect nontumorigenic cell lines (lung and skin fibroblasts). TCF activity was not blocked by superoxide dismutase, catalase, or protease inhibitors; it was destroyed by being heated at 100 degrees C for 2 minutes. The ability of activated monocytes to release TCF could enhance host defense against cancer.     

Improvement of Macrophage Dysfunction by Administration of Anti-transforming Growth Factor-β Antibody in EL4-bearing Hosts

An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor-β (TGF-β) was tried in EL4 tumor-bearing mice. TGF-β was detected in cell-free ascitic fluid from EL4-bearers, but not in tbat from normal mice, by western blot analysis. The ascites also showed growth-suppressive activity against MvlLn cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophagcs were suppressed in EL4-bearers, the abilities to produce nitric oxide and tumor necrosis factor-α (TNF-α) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4-bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non-tumor-bearing controls. TGF-β activity was abrogated by administration of anti-TGF-α antibody to EL4-bearing mice. While a large amount of TGF-β was detected in ascitic fluid from control EL4-bearers, little TGF-β was detectable in ascites from EL4-bearers given anti-TGF-β antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF-α on LPS stimulation in vitro , macrophages from EL4-bearers administered with anti-TGF-β antibody showed the same ability as normal macrophages. These results clearly indicate that TGF-β contributes to macrophage dysfunction and that the administration of specific antibody for TGF-β reverses macrophage dysfunction in EL4-bearing hosts.     

Tumor Necrosis Factor-α Gene Transfer Augments Anti-Fas Antibody-mediated Apoptosis in Human Glioma Cells

To effectively induce apoptosis in human glioma cells, we tried to transfer the tumor necrosis factor (TNF)-α gene into glioma cells to produce TNF-α locally in these cells. The stable transfectants of three gliorna cells (U251-SP, U251-MG, and T98G) were resistant to exogenous TNF-α, but their cell surface expression of the Fas antigen was dramatically enhanced by about 10 to 100-fold as compared with untransfected glioma cells exposed to exogenous TNF-α. The Fas antigen is a transmemhrane cytokine receptor protein of the nerve growth factor/TNF receptor superfamily. Although the untransfected glioma cells tested were resistant to anti-Fas antibody-mediated apoptosis, the TNF-α gene-transfected glioma cells exhibited high susceptibility to anti-Fas antibody-mediated apoptosis. Thus, TNF-α gene transfer combined with anti-Fas antibodies may be useful for the treatment of malignant glioma.     

Induction of in vitro tumoricidal activity in alveolar macrophages and monocytes from patients with lung cancer

Human alveolar macrophages (AMs) and blood monocytes were obtained from 65 smoking and nonsmoking normal volunteers and 29 patients with lung cancer. The oxidative metabolic response of these cells was measured by superoxide anion production after incubation with lipopolysaccharide. In addition, tumoricidal activity of AMs and monocytes was assessed against [3H]thymidine-labeled tumor target cells. Smoking was associated with depressed AM superoxide anion responses in normals but not in patients. In contrast, smoking appeared to slightly elevate monocyte superoxide anion activity. AMs and monocytes exposed to lipopolysaccharide or recombinant gamma-interferon showed tumoricidal activity in all groups. Mean cytotoxicity values of smoking patients versus smoking normals and exsmoking patients versus nonsmoking normals were not significantly different. Smoking, however, in both patients and normals was associated with significantly (P less than 0.005) depressed AM cytotoxicity levels (less than 40%) compared to nonsmoking volunteers and exsmoking patients. Activated AMs from cancer patients and normals were cytotoxic against three different tumorigenic cell lines but not against a nontumorigenic line. No correlation between monocyte and AM cytotoxic activity within single individuals was found. We conclude that AM and monocytes from smoking and exsmoking patients can be activated after exposure to immunomodulators; however, smoking may be slightly suppressive to cytotoxic responses. These studies provide a rationale for clinical trials of immunomodulators in patients with lung cancer.     

Enhancing Effect of an Inhibitor of Nitric Oxide Synthesis on Bacillus Calmette-Guérin-induced Macrophage Cytotoxicity against Murine Bladder Cancer Cell Line MBT-2 in vitro

We studied the effect of an inhibitor of nitric oxide (NO) synthesis, N^G-monomethyl-L-arginine (LNMMA), on the Bacillus Calmette-Guérin (BCG)-induced antitumor activity of murine peritoneal exudate cells (PEC) against murine bladder cancer cell line MBT-2 in vitro. L-NMMA enhanced BCG-induced cytotoxic activity of PEC, as well as interferon (IFN)-γ and tumor necrosis factor (TNF)-α production. The L-NMMA-induced enhancement was due to the prolonged survival of BCG in macrophages, because no enhancement of cytotoxicity was observed and neither IFN-γ nor TNF-α production was significantly enhanced by killed BCG. Anti-TNF-α antibody (Ab) and anti-IFN-γAb reduced the L-NMMA-induced enhancement of the cytotoxicity. The depletion of T cells from PEC reduced the production of both IFN-γ and TNF-α, as well as the enhancement of cytotoxicity induced by viable BCG plus L-NMMA. These results suggest that L-NMMA has an enhancing effect on BCG-induced macrophage cytotoxicity and the enhancement is partially mediated by T cells and their soluble products. Accordingly, NO inhibitor should be a valuable adjunct to BCG immunotherapy for bladder cancer.     

Impaired production of tumor necrosis factor in breast cancer

Spontaneous and lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF) by peripheral blood macrophages was investigated in breast cancer. Whereas spontaneous TNF production by macrophages derived from patients with breast cancer was comparable with the one found in healthy controls (P greater than 0.1), LPS-stimulated macrophages derived from patients in the disease-free interval as well as with metastatic breast cancer were found to produce significantly lower amounts of TNF, as compared with macrophages derived from healthy control individuals (P less than 0.0005). However, the production of TNF did not significantly differ between the two patient populations (P greater than 0.05). The impairment of LPS-induced TNF production did not depend upon such characteristics of the primary tumor as size, axillary lymph node and estrogen receptor status, or upon the fact of administration of adjuvant chemotherapy and, in patients with metastatic disease, hormone treatment. To further investigate cytokine production by macrophages, spontaneous and LPS-induced interleukin-1 (IL-1) production was investigated also. However, no difference was found between patients and controls concerning IL-1 generation. The authors thus conclude that LPS-induced TNF production was impaired in breast cancer independent of the presence of detectable metastatic disease, whereas IL-1 production remained unimpaired.     

In Vitro Effects of Lithium Chloride on TNFα and IL-6 Production by Monocytes from Breast Cancer Patients

Abstract

It is well known that lithium chloride (LiCI) is able to trigger human monocytes to release tumor necrosis factor alpha (TNFα). In this study we have evaluated the In Vitro effect of LiCI on TNFα and interleukin-6 (IL-6) release by monocytes from patients affected by non-metastatic (BCa/M0) and metastatic breast cancer (BCa/M1), preincubated with autologous serum (sPt). Our data demonstrate that monocytes from cancer patients (BCa) treated with LiCI released lower amounts of TNFα compared to those from healthy donors (HD). Preincubation in autologous serum (sPt) impaired TNFα production by monocytes treated with LiCI. On the contrary, our data indicate that IL-6 production by monocytes from BCa was not impaired. Moreover, the results obtained from the same cells, preincubated in sPt and treated with LiCl, indicate that serum factors may synergize with LiCI treatment in releasing IL-6.     

Autonomous Expressions of Cytokine Genes by Human Lung Cancer Cells and Their Paracrine Regulation

Cell-to-cell interaction between tumors and host inflammatory cells is important for the subsequent cancer progression or regression. We examined the expressions of mRNAs for various proinflammatory cytokines by nine human lung cancer cell lines and the influences of cytokines on their gene expressions. The cytokines used were interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF) and monocyte chemotactic and activating factor. Gene expressions of cytokines were measured by Northern blot analysis. Substantial expressions of cytokine genes were detected in several lung cancer cell lines such as RERF-LC-MS, RERF-LC-OK and VMRC-LCD, although the levels of expression of each cytokine varied in different cell lines. Four lung cancer cell lines (RERF-LC-MS, RERF-LC-OK, A549 and YO-88) were used to examine the effects of exogenous cytokines (IL-1β, TNF-α and GM-CSF) on cytokine gene expressions by the cells. TNF-α and IL-1β caused significant changes in the levels of mRNA expressions of certain cytokines. Moreover, on stimulation with TNF-α, RERF-LC-OK cells produced IL-6 extracellularly. These extensive differences in the levels of gene expressions and productions of cytokines could have profound effects on the interactions between human lung cancer cells and the corresponding host cells.     

Suppression by Interleukin 4 of Activation of Human Blood Monocytes to the Tumoricidal State

The effect of interleukin 4 (IL-4) on expression of antitumor activity of blood monocytes purified by counter-flow centrifugal elutriation from healthy donors was examined. The blood monocytes were incubated for 24 h in medium with lipopolysaccharide, interferon γ (IFN-γ) or desmethyl muramyl dipeptide (norMDP) or with IFN-γ and norMDP in the presence of IL-4, and then their tumoricidal activity was assayed by measuring ¹²⁵IUdR release from human melanoma (A375) cells. Irrespective of activation stimulus, addition of IL-4 to cultures of monocytes and activators resulted in dose-dependent suppression of the tumoricidal activity of monocytes against parent A375 melanoma cells and the variant cells, A375-R resistant to IL-1 and tumor necrosis factor α. IL-4 suppressed the early induction phase of monocyte activation. Rabbit anti-IL-4 antisera completely blocked the IL-4-mediated suppression of monocyte activation to the tumoricidal state. These findings suggest that IL-4 is important in vivo in down-regulation of anti-tumor expression of monocytes.     

Cancer disease predictive diagnosis: BAT/CD3-positive lymphocytes in cancer patients

BAT is an immune-activating monoclonal antibody produced against Daudi cell membranes and selected for stimulating lymphocyte proliferation. The anti-tumor activity of BAT is related to its immunostimulatory properties. Both T and NK cells mediate the anti-tumor activity of BAT. CD4-positive T cells respond to BAT activation by proliferation and INF-gamma production. The aim of the study was to assess the probability that the BAT monoclonal antibody binding capacity to T cells is a marker for different cancers. Human peripheral blood T cells from colon, breast and prostate cancer patients, as well as healthy volunteer donors, were tested for the percentage of binding to BAT mAb (BAT/CD3 cells) by FACS analysis. All patients were tested before undergoing surgery or treatment, and their diagnosis was confirmed by histology. The results showed that the percentage of BAT monoclonal antibody binding to CD3-positive T cells in the peripheral blood was different in cancer patients with diverse tumor types. We found that lymphocytes from the blood of healthy donors contained 25% BAT/CD3 cells. In colon and breast cancer patients, a significant decrease to 13 and 11% of BAT/CD3 cells was found. In contrast, these cells increased ><50% in patients with prostate cancer. These findings may have a potential diagnostic significance and also assist in the evaluation of strategies for the therapeutic use of BAT for different cancer patients.     

The MHC class-II and CD44 molecules are involved in the induction of tumour necrosis factor (TNF) gene expression by human monocytes stimulated with tumour cells

Tumour necrosis factor alpha (TNF) mRNA is detected in the macrophage infiltrate surrounding the tumour, but the cellular/ molecular interactions leading to TNF gene expression in macrophages are unknown. The in vitro system in which human blood monocytes are stimulated with human cancer cells for TNF release was used to study such interactions. Monoclonal antibodies (MAbs) against various adhesion molecules (LFA-I, LFA-3, (CAM-I, VNR, VLAβI chain) were unable to block TNF production in co-culture of monocytes with a human pancreatic carcinoma (HPC) cell line. However, anti-CD44 and anti-HLA-DR MAbs effectively blocked TNF release and TNF-mRNA induction in monocytes. Pre-incubation of monocytes with anti-HLA-DR and tumour cells with anti-CD44 MAbs had a similar effect. It was concluded that CD44 molecules are involved in tumour-monocyte interactions and that HLA-DR determinants of monocytes are engaged in signal transduction for TNF gene activation. These findings may suggest that certain surface determinants of tumour cells act as ligands for MHC class-II molecules and induce TNF production in monocytes.     

Effect of Gene Transfer of Tumor Necrosis Factor Receptors into Human Lung Carcinoma Cell Line

The human lung adenocarcinoma cell line A549 is known to be resistant to tumor necrosis factor alpha (TNF-α)-mediated tumor cell lysis in spite of the expression of 55 kDa TNF receptor (TNF-R55) mRNA and its cell surface protein. In this study, we investigated the mechanism of TNF-α resistance and the role of two types of TNF receptors (TNF-R55 and TNF-R75 (75 kDa TNF receptor)). TNF-R55 or TNF-R75 cDNA was transfected into A549 cells. In addition, a C-terminal deletion mutant of TNF-R75 which lacks the intracellular domain of TNF-R75 was also transfected into A549 cells. We assessed the TNF-α-mediated tumor cell lysis of these transfected clones, and found that the cytotoxic effect increased in transfected clones highly expressing TNF-R55, but not in low-expression clones. As for TNF-R75, the cytotoxic effect of TNF-α was observed in TNF-R75-transfected clones even when expression was low. Furthermore, the cytotoxic effect was also observed in clones transfected with the deletion mutant of TNF-R75, as well as the complete TNF-R75. These results indicate that a certain level of expression of TNF-R55 is necessary for obtaining TNF-α-mediated tumor cell lysis in the absence of TNF-R75. On the other hand, the expression of TNF-R75 strongly induces TNF-α-mediated cytotoxicity through TNF-R55 in the absence of an intracellular signal via TNF-R75.     

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.