Japanese Journal of Clinical Oncology 30:295-300 (2000)
© 2000 Foundation for Promotion of Cancer Research
Heterogeneous Response Patterns of Alveolar Macrophages from Patients with Lung Cancer by Stimulation with Interferon-
1Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and 2Department of Immunology, Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Background: Macrophages are considered to play an important role in the host defense against malignant tumors. In this study, cytotoxic activity of alveolar macrophages (AM) derived from 32 patients with lung cancer was investigated.
Methods: AM were aseptically obtained by lavage from resected lung and subsequently tested for cytolytic activity against QG56, a lung squamous cell line, following treatment with recombinant interferon-
(IFN-).
Results: In seven patients (21.9%), AM showed no cytotoxicity even though AM were incubated with IFN-. In 20 (62.5%), AM showed substantial cytotoxicity in response to IFN- in a dose-dependent manner. In the other five (15.6%), relatively strong cytotoxicity was observed even without preincubation with IFN-. Such a heterogeneous profile of the cytotoxicity of AM might be a reflection of various activated states of AM since the potential of cytotoxicity and that of IL-1 secretion were almost parallel. Both IFN- dependent and -independent cytotoxicity were partially blocked either by anti-tumor necrosis factor-
(TNF-) antibody or by the inhibitor of nitric oxide synthesis. However, those activities were completely abrogated by both treatments. Since the supernatant of AM culture exhibited TNF--mediated but not NO-mediated cytolysis, TNF- could mediate a bystander killing whereas NO acts in close contact with tumor cells.
Conclusion: The AM have anti-tumor cytotoxicity in lung cancer although the cytolytic potential is heterogeneous and that the tumor lysis by AM is mediated by both TNF- and NO production.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Macrophages are considered to play an important role in the host defense against malignant tumors (1). In human lung cancer, we had elucidated the functions and mechanisms of macrophages surrounding lung cancer such as tumor tissues, lung, pleural cavity or peripheral blood (2–5). In these studies, cytostatic activities of the macrophages against lung cancer cells decreased in patients with advanced disease and would give prognostic information. However, concerning alveolar macrophages (AM) obtained from resected lung specimens of lung cancer, the cytostatic activity was influenced by the smoking history of the patients, the site at which AM were obtained (tumor-bearing or non-tumor-bearing segments) and the degree of tumor burden (4). These data imply heterogeneity of the activation state of AM in patients with lung cancer.
Basically, macrophages require multiple stimuli such as lipopolysacharide (LPS) and interferon- (IFN-) to become fully activated to produce H2O2, TNF-, IL-1, proteases or lysozyme or to acquire cytotoxicity or mobility (6). On the other hand, macrophages are suppressed by tumor-derived factors (7,8). Therefore, macrophages may have heterogeneous functions at each activated state and may be influenced by the stage of cancer.
In the present study, we examined the cytotoxicity of AM derived from patients with lung cancer against cancer cells and further analyzed the requirement of IFN- for activation and the mediators to exert cytotoxic activity.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Patients
Thirty-two patients with resectable primary lung cancer comprised of 27 males and five females were included in this study. They had not received any anticancer therapy except one case who was given a bronchial arterial infusion chemotherapy prior to surgery. According to the histological classification, they included 20 adenocarcinomas, 11 squamous cell carcinomas and one adenosquamous cell carcinoma. Informed consent was obtained from all patients and their families about the experimental use of the surgical material.
Culture Medium
The culture medium (CM) consisted of RPMI 1640 (Nissui Seiyaku, Tokyo, Japan) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,100 units/ml penicillin G, 100 µg/ml streptomycin sulfate and 10% heat-inactivated (56°C for 30 min) fetal calf serum.
Preparation of AM
To obtain AM, non-tumor-bearing segments of resected lungs were irrigated with 200 ml of PBS passed through individual segmental bronchial trees. The recovered saline was centrifuged at 1500 r.p.m. for 5 min to obtain cell pellets. Contaminated RBC were lysed by treatment with 0.83% NH4Cl at 37°C for 10 min. The cells obtained were washed three times with HBSS. These cells were suspended in 10 ml of CM. This cell suspension was placed in a plastic culture dish and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After 1 h of incubation, non-adherent cells were removed by gentle washing three times with HBSS. Cells adhering to the plastic dish were detached from the culture dish by a jet stream of 10 ml of HBSS from a 26-gauge injection needle. These cells were resuspended in the CM to be adjusted to a final concentration of 2 x 106/ml. These cells were cultured with various concentrations (10, 100 and 1000 units/ml) of recombinant interferon- (IFN-) for 48 h. To determine the purity of macrophages, these adherent cells were stained for non-specific esterase (esterase stain kit; Muto Pure Chemical, Tokoyo, Japan) and counterstained with Giemsa. More than 90% of these adherent cells were identified as macrophgages.
Cytotoxic Assay
Cytolytic activity of AM against a lung cancer cell line (QG-56, squamous cell carcinoma; PC-10, small cell carcinoma) were assessed by an 18 h 51Cr release assay. Target cells (5 x 105) in 0.4 ml of the culture medium were labeled with 0.1 mCi of Na251CrO4 for 60 min at 37°C. After washing three times with the culture medium, target cells were prepared at a concentration of 5 x 104/ml. A constant number of 5 x 103 51Cr-labeled target cells were incubated with 2 x 105 effector cells treated with IFN- as indicated above in round-bottomed microtest plates in 0.2 ml of CM. Cultures in quadruplicate were incubated at 37°C for 18 h. Then 0.1 ml of supernatant was collected from each well and counted in a gamma counter. The percentage of cytotoxicity was calculated as
Target cells without effector cells were mixed with 0.1 ml of the culture medium to obtain spontaneous 51Cr release and with 0.1 ml of 1% Triton X-100 to obtain maximum 51Cr release. When the maximum release was less than 1000 c.p.m. or the spontaneous release from targets was more than 30% of the maximum release, the data were excluded from the study.
Blocking of Nitric Oxide Synthesis
To examine the contribution of the soluble factors secreted by the AM, targets (5 x 103) were incubated with 1:2-diluted supernatants (0.2 ml) of the AM cultured with IFN- for 48 h. Then following 48 h culture, the radioactivity of the supernatants (0.1 ml) was counted with a gamma counter. To assess the contribution of NO to the cytotoxicity of the AM, N-monomethyl-L-arginine (NMM-L-A) (Sigma, St. Louis, MO) (10–4 M) was added in the cytotoxicity assay to block the synthesis of NO. As a control, N-monomethyl-D-arginine (NMM-D-A) (Sigma) (10–4 M), an optical isomer, was added.
Neutralization of TNF-
To assess the contribution of TNF- to the cytotoxic activity of the AM, murine anti-human TNF- monoclonal Ab (Genzyme, Cambridge, MA) was added in the cytotoxicity assay at a concentration of 10 µg/ml. As a control, murine IgG1 was added at the same concentration.
Quantification of TNF-, IL-1ß and IL-12
After 48 h of culture of AM with various concentrations of IFN-, the supernatants were collected and stored frozen at –20°C. The concentration of each cytokine was measured with a commercially available enzyme-linked immunosorbent assay (ELISA) (Amersham International, Amersham, UK) according to the manufacturer’s recommended protocol. Briefly, the samples were pipetted into the wells that had been already coated with a murine monoclonal antibody specific for every cytokine and were then incubated at room temperature for 2 h. After washing away any unbound sample proteins, an enzyme-linked polyclonal antibody specific for every cytokine was added to the wells and the plates were incubated at room temperature for 2 h. Following washing to remove any unbound antibody–enzyme reagent, hydrogen peroxide and chromogen were added to the wells and the plates were incubated at room temperature for 20 min. The reaction was stopped by adding 1 M sulfuric acid to each well and the absorbance at 450 nm was measured using a dual-wavelength flying-spot scanning densitometer (CS-9300PC, Shimadzu, Kyoto, Japan).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Cytotoxic Activity of AM
The cytotoxicity of AM against QG56 squamous lung cancer cell line fluctuated among patients before stimulation with IFN-
. The same tendency was also observed in the cytotoxic activity of AM against PC10, small lung cancer cell line in each case (data not shown). In seven cases, AM showed no cytotoxicity even with preincubation with IFN- (Fig. 1A). AM obtained from 20 cases showed substantial cytotoxicity in response to IFN- in a dose-dependent manner (Fig. 1B). In the other five cases, relatively high cytotoxicity was observed, but the cytotoxicity was not affected by preincubation with IFN- (Fig. 1C).
|
These results indicate that the cytotoxic potential of AM derived from patients with lung cancer is heterogeneous among patients, which was possibly due to activated states of AM in vivo. We therefore classified the cytotoxic potential of AM as follows: (1) no cytotoxicity [seven cases (cases 1–7), 21.9% of the total cases]; (2) IFN-
dependent cytotoxicity [20 cases (cases 8–27), 62.5%]; and (3) IFN- independent cytotoxicity [five cases (cases 28-32), 15.6%] (Tables 1 and 2). However, the cytotoxic potential did not correlate with any prognostic pattern as shown in Table 2.
|
|
IL-1ß and IL-12 Production by Activated AM in Response to IFN-
To analyze the relationship between the cytolytic potential and cytokine production of AM, the production of IL-1ß and IL-12 by AM were examined in two cases with no cytotoxicity, in six cases with IFN-
dependent cytotoxicity and in one case with IFN- independent cytotoxicity. In the no cytotoxicity group, the production of IL-1ß was 
100 pg/ml and did not show an IFN- dependent increase. No production of IL-12 was observed. In the IFN--dependent cytotoxicity group, a large amount of IL-1ß was produced depending on the concentration of IFN- . The production of IL-12 was minimally observed only after stimulation with a high concentration of IFN-. In the IFN- -independent cytotoxicity group, a substantial level of IL-1ß production was observed without any stimulation with IFN-. IL-12 was not produced even with maximum stimulation with IFN- (Fig. 2). These results suggested that the level of cytotoxic activity of AM was regulated by the degree of secretion of IL-1ß by AM.
|
Blocking of Cytotoxicity of AM by Anti-TNF-
Ab and NO Synthesis InhibitorTNF-
and NO are well known as cytolytic molecules of macrophages (9,10). Therefore, the role of these molecules in the cytotoxicity of AM was examined (Fig. 3). The IFN--dependent cytotoxicity was partially blocked by the addition of anti-TNF- antibody or NMM-L-A, an inhibitor of nitric oxide syntheses, in the assay. However, the activity was completely abrogated when both treatments were combined. The addition of NMM-D-A, an optical isomer of NMM-L-A, did not show any effect on the cytotoxicity.
|
Cytotoxic Activity of the Culture Supernatant of AM
To confirm the contribution of the soluble cytotoxic mediators secreted by the AM such as TNF-
or NO, the supernatant of the AM cultured with IFN- was examined for cytotoxicity. As shown in Fig. 4, cytotoxic activity against QG56 was exerted by the supernatant in the IFN--dependent cytotoxicity group. Moreover, the cytotoxic activity was abrogated completely by addition of anti-TNF- Ab.
|
TNF-
Production of AM in Response to IFN-The production of TNF-
by AM was examined in the IFN--dependent cytotoxicity group. As shown in Fig. 5, TNF- was produced in response to the IFN- treatments. These results suggested that the IFN--dependent cytotoxicity of AM was mediated by TNF-.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
We analyzed the cytotoxicity against tumor cells of AM derived from patients with lung cancer and classified the cytotoxicity into three categories as follows: (a) no cytotoxicity; (b) IFN-
-dependent cytotoxicity; and (c) IFN--independent cytotoxicity. Such a variety of cytotoxicity of AM may be attributed to several factors, such as degree of tumor burden, infection or smoking, which may influence the activation state of the AM. Previously, we reported that cytostatic activity against lung cancer cells of AM is negatively influenced by cigarette smoking or tumor progression (4). On the other hand, several investigators reported that AM derived from the host with smoking history, pulmonary infection or cancer were more cytotoxic than AM from normal non-smokers (11,12). In this study, at least three patterns of cytotoxic activity of AM were identified by the response pattern to IFN- stimulation. Interestingly, such a profile of cytotoxic potential correlated well with the degree of secretion of IL-1 but not IL-12. It has been reported that NO, TNF- and IL-1 produced by such activated AM in response to IFN- may cause suppression of T cell function (13) and cachexia in patients (14,15). Moreover, secretion of IL-1 by AM may drive the humoral immune response through Th2 activation but not Th1, then suppress cellular immune response such as induction of cytotoxic T cells against cancer (16–18).
From the present results, the cytotoxic mediation of IFN--activated AM was ascribed to TNF- and NO. TNF- causes fragmentation of DNA of TNF-sensitive (TNF receptor-positive) cells (19). Since the culture supernatant of the activated AM included an adequate concentration of TNF-, cytotoxicity mediated by TNF- might be able to work in a bystander fashion. NO was identified as a macrophage metabolite of L-arginine and causes inhibition of mitochondrial respiration of target cells (20). Such a reactive nitrogen intermediate was induced in macrophages by stimulation with IFN- and bacterial products (21), but the release of NO is more dependent on a triggering signal (IFN-) than a priming signal (i.e. bacteria) (22). Individual differences in in vivo suffering of priming/triggering of the AM, therefore, may be affected the variety of cytotoxic activity. NO-mediated killing was not detected in the pooled culture supernatants of AM. This was possibly due to the short half-life of NO (10). Therefore, it might be suggested that NO-mediated cytotoxicity requires sustained exposure of target cells to NO and continuous release of NO by activated macrophages.
Alleva et al. (23) reported that tumor cell-derived factors such as interleukin-10, transforming growth factor ß and prostaglandin E2 suppress the production of NO and TNF- by macrophages. In the present study, the AM derived from seven cases did not exhibit any cytotoxicity even when treated with a high concentration with IFN-. In these cases, tumor-derived factors may also negatively affect the cytotoxicity of the AM.
Andreesen et al. reported adoptive transfer of tumor cytotoxic macrophages generated from circulating blood monocytes by stimulation with IFN- in vitro for advanced metastasized cancer, although the therapeutic benefits were unsatisfactory (24). The AM may be a better alternative for the therapeutic effector source since the AM are potentially in a more highly activated state than the peripheral monocytes and recovery of the AM is easy and adequate in number (up to 109).
|
Acknowledgments |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
We thank Ms Kinue Nishida, Maki Mori and Miki Kiyofuji for their help.
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Ryozo Eifuku, Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, Iseigaoka 1–1, Yahatanishi-ku, Kitakyushu 807-8555, Japan Abbreviations: AM, alveolar macrophage; IFN-
, interferon-; TNF-; tumor necrosis factor ; LPS, lipopolysacharide; NMM-L-A, N-monomethyl-L-arginine; NMM-D-A, N-monomethyl-D-arginine; ELISA, enzyme-linked immunosorbent assay; IL-1ß, interleukin 1ß; IL-12, interleukin 12; NO, nitric oxide; CM, culture medium 
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
1 Levy MH, Wheelock EF. The role of macrophages in defense against neoplastic disease. Adv Cancer Res 1974;20:131–63.[Medline]
2 Takeo S, Yasumoto K, Nagashima A, Nakahashi H, Sugimachi K, Nomoto K. Role of tumor-associated macrophages in lung cancer. Cancer Res 1986;46:3179–82.
3 Nakahashi H, Yasumoto K, Nagashima A, Yaita H, Takeo S, Motohiro A, et al. Antitumor activity of macrophages in lung cancer patients with special reference to location of macrophages. Cancer Res 1984;44:5906–9.
4 Kuda T, Yasumoto K, Yano T, Nakahashi H, Sugimachi K, Nomoto K. Role of antitumor activity of alveoler macrophages in lung cancer patients. Cancer Res 1987;47:2199–202.
5 Nagashima A, Yasumoto K, Nakahashi H, Takeo S, Yano T, Nomoto K. Antitumor activity of pleural cavity macrophages and its regulation by pleural cavity lymphocytes in patients with lung cancer. Cancer Res 1987;47:5497–500.
6 Adams DO, Hamilton TA. The cell biology of macrophage activation. Annu Rev Immunol 1984;2:283–318.[Web of Science][Medline]
7 Tsunawaki S, Sporn M, Nathan CF. Comparison of transforming growth factor-beta and a macrophage-deactivating polypeptide from tumor cells. Differences in antigenicity and mechanism of action. J Immunol 1989;142:3462–7.[Abstract]
8 Tsunawaki S, Sporn M, Ding A, Nathan C. Deactivation of macrophages by transforming growth factor-beta. Nature 1988;334:260–2.[Medline]
9 Urban JL, Shepard HM, Rothstein JL, Sugarman BJ, Schreiber H. Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages. Proc Natl Acad Sci USA 1986;83:5233–7.
10 Stuehr DJ, Nathan CF. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor cells. J Exp Med 1989;169:1543–55.
11 Kan-Mitchell J, Hengst JCD, Kemph RA, Rothbart RK, Simons SM, Brooker AS, et al. Cytotoxic activity of human pulmonary alveolar macrophages. Cancer Res 1985;45:453–8.
12 Swinburne S, Moore M, Cole P. Human bronchoalveolar macrophage cytotoxicity for cultured human lung-tumor cells. Br J Cancer 1982;46:625–34.[Web of Science][Medline]
13 Bingisser RM, Tilbrook PA, Holt PG, Kees UR. Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. J Immunol 1998;160:5279–334.
14 Oliff A. The role of tumor necrosis factor in cachexia. Cell 1988;54:141–2.[Web of Science][Medline]
15 Moldawere LL, Georgieff M, Lundholm KG. Interleukin 1, tumor necrosis factor and the pathogenesis of cancer cachexia. Clin. Physiol 1987;7:263–74.[Web of Science][Medline]
16 Mosmann TR, Coffman RC. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989;7:145–73.[Web of Science][Medline]
17 Weaver CT, Hawrylowicz CM, Unanue ER. T helper subsets require the expression of distinct costimulatory signals by antigen-presenting cells. Proc Natl Acad Sci USA 1988;85:8181–5.
18 Desmedt M, Rottiers P, Dooms H, Fiers W, Grooten J. Macrophage induce cellular immunitiy by activating Th1 cell responses and suppressing Th2 cell responses. J Immunol 1998;160:5300–8.
19 Larrick JW, Wright SC. Cytotoxic mechanism of tumor necrosis factor-. FASEB J 1990;4:3215–23.[Abstract]
20 Stuehr DJ, Gross SS, Sakuma R, Nathan CF. Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide. J Exp Med 1989;169:1011–20.
21 Ding A, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J Immunol 1988;141:2407–12.[Abstract]
22 Nathan CF, Root RK. Hydrogen peroxide release from mouse peritoneal macrophages. Dependence on sequential activation and triggering. J Exp Med 1977;146:1648–62.
23 Alleva DG, Burger CJ, Elgert KD. Tumor-induced regulation of suppressor macrophage nitric oxide and TNF- production. J Immunol 1994;153:1674–86.[Abstract]
24 Andreesen R, Scheibenbogen C, Brugger W, Krause S, Meerpohl HG, Leser HG, et al. Adoptive transfer of tumor cytotoxic macrophages generated in vitro from circulating blood monocytes: a new approach to cancer immunotherapy. Cancer Res 1990;50:7450–6.
Received February 9, 2000; accepted May 8, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J.-P. Herbeuval, C. Lambert, O. Sabido, M. Cottier, P. Fournel, M. Dy, and C. Genin Macrophages From Cancer Patients: Analysis of TRAIL, TRAIL Receptors, and Colon Tumor Cell Apoptosis J Natl Cancer Inst, April 16, 2003; 95(8): 611 - 621. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Crowther, N. J. Brown, E. T. Bishop, and C. E. Lewis Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors J. Leukoc. Biol., October 1, 2001; 70(4): 478 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Hickman-Davis, F. C. Fang, C. Nathan, V. L. Shepherd, D. R. Voelker, and J. R. Wright Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L517 - L523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Hickman-Davis, P. O'Reilly, I. C. Davis, J. Peti-Peterdi, G. Davis, K. R. Young, R. B. Devlin, and S. Matalon Killing of Klebsiella pneumoniae by human alveolar macrophages Am J Physiol Lung Cell Mol Physiol, May 1, 2002; 282(5): L944 - L956. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||