Patients with Lung Cancer by Stimulation with Interferon-γ .... each well and counted in a gamma counter. ... (0.1 ml) was counted with a gamma counter.
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Heterogeneous Response Patterns of Alveolar Macrophages from Patients with Lung Cancer by Stimulation with Interferon-γ Ryozo Eifuku1, Takashi Yoshimatsu1, Ichiro Yoshino1, Mitsuhiro Takenoyama1, Satoru Imahayashi1, Tomoko So1, Takeshi Hanagiri1, Kikuo Nomoto2 and Kosei Yasumoto1 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 Received February 9, 2000; accepted May 8, 2000
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-γ (IFN-γ). Results: In seven patients (21.9%), AM showed no cytotoxicity even though AM were incubated with IFN-γ. In 20 (62.5%), AM showed substantial cytotoxicity in response to IFN-γ in a dose-dependent manner. In the other five (15.6%), relatively strong cytotoxicity was observed even without preincubation with IFN-γ. 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-γ dependent and -independent cytotoxicity were partially blocked either by anti-tumor necrosis factor-α (TNF-α) 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-α-mediated but not NO-mediated cytolysis, TNF-α 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-α and NO production.
Key words: alveolar macrophages – lung cancer – interferon-γ – tumor necrosis factor-α – nitric oxide
INTRODUCTION 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,
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-γ, interferon-γ; TNF-α; tumor necrosis factor α; LPS, lipopolysacharide; NMM-L-A, N-monomethyl-Larginine; NMM-D-A, N-monomethyl-D-arginine; ELISA, enzyme-linked immunosorbent assay; IL-1β, interleukin 1β; IL-12, interleukin 12; NO, nitric oxide; CM, culture medium
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-γ (IFN-γ) to become fully activated to produce H2O2, TNF-α, 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. © 2000 Foundation for Promotion of Cancer Research
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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-γ for activation and the mediators to exert cytotoxic activity.
MATERIALS AND METHODS 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-(2hydroxyethyl)-1-piperazineethanesulfonic acid,100 units/ml penicillin G, 100 µg/ml streptomycin sulfate and 10% heatinactivated (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 × 106/ml. These cells were cultured with various concentrations (10, 100 and 1000 units/ml) of recombinant interferon-γ (IFN-γ) for 48 h. To determine the purity of macrophages, these adherent cells were stained for nonspecific 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 × 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 × 104/ml. A constant number of 5 × 103 51Cr-labeled target cells were incubated with 2 × 105 effector cells treated with IFN-γ 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 experimental (c.p.m.) – spontaneous (c.p.m.) cytotoxicity (%) = ------------------------------------------------------------------------------------------------------------- × 100 maximum (c.p.m.) – spontaneous (c.p.m.)
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 × 103) were incubated with 1:2-diluted supernatants (0.2 ml) of the AM cultured with IFN-γ 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-monomethylL-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-α To assess the contribution of TNF-α to the cytotoxic activity of the AM, murine anti-human TNF-α 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-α, IL-1β AND IL-12 After 48 h of culture of AM with various concentrations of IFN-γ, 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 enzymelinked 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
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Figure 1. Cytotoxicity of AM against QG56. AM were examined for tumor cytotoxicity after pre-incubation with various concentrations of IFN-γ. (A) The cases whose AM cytotoxicity showed less than 10% and no response to IFN-γ. Three representative cases are shown among seven similar cases. (B) The cases whose AM cytotoxicity showed more than 10% response to IFN-γ. Four representative cases among 20 similar cases are shown. (C) The cases whose AM showed strong cytotoxicity even without any stimulation with IFN-γ. Three representative cases among five similar cases are shown. The E:T ratio was 40:1.
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 CYTOTOXIC ACTIVITY OF AM The cytotoxicity of AM against QG56 squamous lung cancer cell line fluctuated among patients before stimulation with IFN-γ. 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-γ (Fig. 1A). AM obtained from 20 cases showed substantial cytotoxicity in response to IFN-γ 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-γ (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-γ dependent cytotoxicity [20 cases (cases 8–27), 62.5%]; and (3) IFN-γ 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-1β AND IL-12 PRODUCTION BY ACTIVATED AM IN RESPONSE TO IFN-γ To analyze the relationship between the cytolytic potential and cytokine production of AM, the production of IL-1β and IL-12 by AM were examined in two cases with no cytotoxicity, in six cases with IFN-γ dependent cytotoxicity and in one case with
Table 1. Classification of cytotoxic activity of alveolar macrophages No cytotoxicity
IFN-γ-dependent cytotoxicity
IFN-γ-independent cytotoxicity
7* (21.9)†
20 (62.5)
5 (15.6)
*Number of patients. †% in the 32 cases.
IFN-γ independent cytotoxicity. In the no cytotoxicity group, the production of IL-1β was ∼100 pg/ml and did not show an No production of IL-12 was IFN-γ dependent increase. observed. In the IFN-γ-dependent cytotoxicity group, a large amount of IL-1β was produced depending on the concentration of IFN-γ . The production of IL-12 was minimally observed only after stimulation with a high concentration of IFN-γ. In the IFN-γ -independent cytotoxicity group, a substantial level of IL-1β production was observed without any stimulation with IFN-γ. IL-12 was not produced even with maximum stimulation with IFN-γ (Fig. 2). These results suggested that the level of cytotoxic activity of AM was regulated by the degree of secretion of IL-1β by AM. BLOCKING OF CYTOTOXICITY OF AM BY ANTI-TNF-α AB AND NO SYNTHESIS INHIBITOR TNF-α 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-γdependent cytotoxicity was partially blocked by the addition of anti-TNF-α 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-α or NO, the supernatant of
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Table 2. Patients’ profiles and cytotoxic pattern of AM Case
Age (years)
Gender*
1
78
F
2
62
3
63
4
56
5
75
6 7
Brinkmann index
Histology†
T
N
M
Stage
250
Ad
1
0
0
IA
M
760
Sq
1
0
0
IA
M
840
Ad
2
0
0
IB
M
740
Ad
2
0
0
IB
M
1000
Sq
2
0
0
IB
70
M
1500
Sq
2
1
0
IIB
63
M
920
Ad
2
2
0
IIIA
8
76
F
108
Ad
1
0
0
IA
9
62
M
1680
Sq
1
0
0
IA
10
54
M
0
Ad
1
0
0
IA
11
61
F
0
Ad
1
0
0
IA
12
47
M
1740
Ad
1
0
0
IA
13
76
M
1200
Ad
1
0
0
IA
14
46
M
900
Ad
1
0
0
IA
15
73
M
450
Sq
2
0
0
IB
16
72
F
200
Ad-Sq
2
0
0
IB
17
76
M
1120
Ad
2
0
0
IB
18
74
M
530
Sq
2
0
0
IB
19
69
M
0
Ad
2
0
0
IB
20
64
M
1320
Ad
1
1
0
IIA
21
82
M
860
Sq
3
0
0
IIB
22
52
M
1225
Ad
2
2
0
IIIA
23
71
M
2000
Sq
3
1
0
IIIA
24
53
F
0
Ad
1
2
0
IIIA
25
76
F
108
Ad
2
2
0
IIIA
26
73
M
530
Sq
3
2
0
IIIA
27
70
M
5000
Ad
4
2
1
IV
28
85
M
1000
Ad
1
0
0
IA
29
67
M
60
Ad
2
0
0
IB
30
60
M
860
Ad
2
2
0
IIIA
31
69
M
850
Sq
3
2
0
IIIA
32
53
M
1400
Sq
3
3
0
IIIB
Cytotoxic pattern of AM
No cytotoxicity
IFN-γ-dependent cytotoxicity
IFN-γ-independent cytotoxicity
*M, male; F, female. †Ad, adenocarcinoma; Sq, squamous cell carcinoma; Ad-Sq, adenosquamous cell carcinoma.
the AM cultured with IFN-γ was examined for cytotoxicity. As shown in Fig. 4, cytotoxic activity against QG56 was exerted by the supernatant in the IFN-γ-dependent cytotoxicity group. Moreover, the cytotoxic activity was abrogated completely by addition of anti-TNF-α Ab. TNF-α PRODUCTION OF AM IN RESPONSE TO IFN-γ The production of TNF-α by AM was examined in the IFN-γdependent cytotoxicity group. As shown in Fig. 5, TNF-α was
produced in response to the IFN-γ treatments. These results suggested that the IFN-γ-dependent cytotoxicity of AM was mediated by TNF-α.
DISCUSSION 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;
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Figure 2. IL-1β and IL-12 production of AM in response to IFN-γ. The supernatants of the AM pre-stimulated with various concentrations of IFN-γ for 48 h were examined for the concentrations of IL-1β and IL-12 by ELISA. (open square) Cytotoxicity of AM against QG56. The E:T ratio was 40:1. (open diamond) IL-1β production of AM (pg/ml). (open circle) IL-12 production of AM (pg/ml) (A) The representative case with no cytotoxicity of AM. (B) The representative case with IFN-γ-dependent cytotoxicity of AM. (C) The one case with IFN-γ-independent cytotoxicity of AM.
Figure 3. Blocking of tumor cytotoxicity by the AM by anti-TNF-α Ab and NO inhibitor. Anti-TNF-α Ab and/or NMM-L-A was added in the cytotoxicity assay of the IFN-γ-activated AM against QG56. NMM-D-A was used as an isomeric control of NMM-L-A. Representative data from three similar cases are shown.
(b) IFN-γ-dependent cytotoxicity; and (c) IFN-γ-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-γ stimulation. Interestingly, such a profile of cytotoxic potential correlated well with the degree of secretion of IL-1 but not IL-12. It has
Figure 4. Cytotoxic activity of the culture supernatant of AM. The AM-derived cytotoxic factors were examined by the cytotoxicity assay using a 1:2-diluted supernatant of the AM cultured with IFN-γ. The data from three cases (A, B and C) are shown.
been reported that NO, TNF-α and IL-1 produced by such activated AM in response to IFN-γ 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-γactivated AM was ascribed to TNF-α and NO. TNF-α causes fragmentation of DNA of TNF-sensitive (TNF receptorpositive) cells (19). Since the culture supernatant of the activated AM included an adequate concentration of TNF-α, cytotoxicity mediated by TNF-α 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-γ
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References
Figure 5. TNF-α production of AM in response to IFN-γ. The supernatants of the AM cultured with various concentrations of IFN-γ for 48 h were subjected to ELISA for detection of TNF-α by ELISA. The data from three cases (D, E and F) are shown. n.d.: Not done.
and bacterial products (21), but the release of NO is more dependent on a triggering signal (IFN-γ) 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 β and prostaglandin E2 suppress the production of NO and TNF-α 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-γ. 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-γ 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 We thank Ms Kinue Nishida, Maki Mori and Miki Kiyofuji for their help.
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