Abstract
We evaluated the role of the tumor environment in the regulation of apoptosis of tumor-infiltrating neutrophils, the number of which correlates negatively with outcome, in patients with adenocarcinoma of the bronchioloalveolar (BAC) subtype. We examined three different parameters of apoptosis, namely morphological aspect, annexin-V expression, and DNA fragmentation. Bronchoalveolar lavage fluid (BALF) supernatants from patients with BAC significantly inhibited the 24-hour spontaneous apoptosis of normal peripheral blood neutrophils in vitro compared to BALF supernatants from control patients (64 ± 4% versus 90 ± 2% measured by annexin-V flow cytometry, P = 0.04). The alveolar neutrophil count correlated positively with the granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations in the patient’s BALF. Furthermore, neutralizing antibodies (Abs) against GM-CSF and G-CSF significantly inhibited BALF anti-apoptotic activity (15 to 40% and 34 to 63% inhibition, respectively), whereas neutralizing Abs against interleukin (IL)-8, IL-6, IL-1β and tumor necrosis factor-α had no significant effect. In an attempt to identify the cell origin of anti-apoptotic cytokines, we tested in vitro the effect of BAC cells (A549 cell line and primary culture derived from a patient’s BAC tumor) on the apoptosis of peripheral blood neutrophils. Cell-free supernatants from tumor cells did not inhibit neutrophil apoptosis. In contrast, cell-free supernatants from tumor cells previously exposed to conditioned media from peripheral blood mononuclear cells and alveolar macrophages significantly inhibited spontaneous neutrophil apoptosis. This inhibition was partially lifted when conditioned media from mononuclear cells were previously treated with Abs against IL-1β and tumor necrosis factor-α. As in vivo, neutralizing Abs against GM-CSF significantly inhibited the anti-apoptotic activity of cell culture supernatants, and combination with Abs against G-CSF had an additive effect. In vivo, GM-CSF and G-CSF were strongly expressed by tumor cells and moderately or not expressed by the normal epithelium, as assessed by immunohistochemical studies. These findings demonstrate that the tumor environment generates local conditions that prolong alveolar neutrophil survival through the production of soluble factors, thereby contributing to the persistence of the neutrophil alveolitis observed in BAC.
Lung cancer is the leading cause of cancer-related death in industrialized countries. 1 Its incidence has been increasing for several decades, mainly owing to an increase in peripheral lung adenocarcinoma. 2 Peripheral lung adenocarcinoma consists of a heterogeneous pattern of bronchioloalveolar (BAC), papillary, acinar, and solid adenocarcinomas rather than a pure histological subtype, although small tumors are mostly of the BAC subtype. 3,4 The long-term prognosis is uncertain, even in early TNM stages, possibly reflecting different patterns of tumor progression. 5 Indeed, these tumors can either extend outward, with luminal spread and preservation of the elastotic framework, or become invasive with formation of a desmoplastic reaction. 3,6,7 Other stroma-reaction features, such as high tumor microvessel density and tumor-infiltrating inflammatory cells, have also been evaluated as prognostic factors. 8-13
We have previously shown that the presence of increased numbers of tumor-infiltrating neutrophils is linked to poorer outcome in patients with adenocarcinoma of the BAC subtype 11 and that tumor cells drive local neutrophil recruitment and activation via C-X-C chemokine release. In the present study, we examined the role of the tumor microenvironment in promoting neutrophil survival and in the persistence of the deleterious neutrophil alveolitis observed in this subtype of adenocarcinoma. We focused our attention on the neutrophil anti-apoptotic activity of tumor-derived cytokines.
Materials and Methods
Clinical Samples
Patients with BAC
Between January, 1989, and January, 1999, BAC was diagnosed in 50 patients who were treated and followed-up in our chest department. The patients were 32 men and 18 women, with a mean age (±SD) of 64.8 ± 11 years (range, 34 to 81 years), and comprised 34 smokers and 16 nonsmokers. The diagnosis was based on previously published criteria; 13,14 bronchoalveolar lavage (BAL) was used as a diagnostic procedure in 41 cases. 15 Briefly, 200 ml of sterile saline in four 50-ml aliquots was infused into the radiologically abnormal segment or lobe. Fluid recovered by gentle suction was pooled, filtered through sterile gauze, and used for total cell counting. Differential cell counts and tumor cell counts were performed on cytospin preparations stained with modified Wright-Giemsa (Diff-Quick; Dade Behring, Paris, La Défense, France). Bronchoalveolar lavage fluid (BALF) containing >5% neutrophils was examined for the presence of apoptotic neutrophils (see below). For apoptosis assays and cytokine measurements, the remaining BALF was spun and the supernatant was aseptically removed and stored at −80°C.
Control Group
We used BALF supernatants obtained during a diagnostic procedure from six patients. The control patients were four men and two women aged 61 ± 7 years. Three were smokers. None had a history of neoplastic disease and all had normal results of BALF analysis (see below). Bronchoalveolar lavage was performed in the middle lobe and supernatants were stored at −80°C after centrifugation.
Measurement of 24-Hour Spontaneous Polymorphonuclear Neutrophil (PMN) Apoptosis
PMNs were isolated from peripheral blood of healthy human donors by means of density gradient centrifugation (Polymorphonuclear cell separation media; Eurobio, Les Ulis, France). PMNs were then separated from erythrocytes by hypotonic shock and washed three times in sterile saline. This method yielded >97% pure PMNs as assessed by May Grünwald Giemsa staining. Normal peripheral venous-blood PMNs at a density of 1 × 106/ml were incubated for 24 hours in Dulbecco’s modified Eagle’s medium (Gibco, Cergy-Pontoise, France) with 5 mmol/L HEPES, 2 mmol/L l-glutamine, 10 5 units/L penicillin, 100 mg/L streptomycin (referred to as complete medium), and supplemented with 2% fetal bovine serum (FBS). At 24 hours the PMN preparation thus obtained was evaluated for apoptosis by using three different techniques.
Morphological Criteria for Apoptosis
One hundred μl of PMN preparation was cytospun. Cytospin slides stained with May Grünwald Giemsa were evaluated by two independent investigators by consensus. At least 200 PMNs were graded for apoptosis by using predetermined morphological criteria. PMNs were considered apoptotic if they showed dense condensation of chromatin in the form of either a single nucleus or nuclear fragments not connected by strands. 16 The results are expressed as the percentage of apoptotic PMNs.
Annexin V-Fluos Expression by Flow Cytometry
One μl of PMN preparation was washed once with ice-cold phosphate-buffered saline (PBS) and centrifuged at 200 × g for 5 minutes. The cell pellet was resuspended at a density of 1 × 10 6 cells/100 μl in labeling buffer (10 mmol/L HEPES/NaOH, 140 mmol/L NaCl, 5 mmol/L CaCl2, pH 7.4) containing 20 μl/ml of Annexin V-Fluos (Boehringer Mannheim, Meylan, France) and 20 μl/ml of propidium iodide (50 μg/ml) (Sigma-Aldrich, Saint Quentin Fallavier, France). Cells were incubated for 10 minutes at room temperature in the dark and analyzed within 1 hour by flow cytometry (Elite flow cytometer; Beckman Coulter, Villepinte, France) with gating set on forward scatter and side scatter to identify PMNs and to exclude cell debris. 17 The results are expressed as the percentage of PMNs that met criteria for apoptosis, ie, a strong annexin V-fluorescein isothiocyanate signal and a low propidium iodide signal (early apoptotic PMNs), or a strong annexin V-fluorescein isothiocyanate signal and a strong propidium iodide signal (late apoptotic PMNs).
DNA Fragmentation Analysis
Five μl of PMN preparation was washed once with PBS, gently resuspended in 0.5 ml of lysis buffer (50 mmol/L Tris, 10 mmol/L ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, and 250 μg/ml proteinase K), and incubated for 24 hours at 37°C with 5% CO2. The lysate was extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) and precipitated with two volumes of cold ethanol and 0.5 volume of 10 mol/L ammonium acetate at 4°C overnight. The DNA pellet was washed once with 75% ethanol, resuspended in 30 μl of TBE buffer (89 mmol/L Tris, 89 mmol/L boric acid, 2 mmol/L ethylenediaminetetraacetic acid, pH 8.0) containing 250 μg/ml of RNase (Sigma) and incubated at 65°C for 5 minutes. Then, 0.1 volume of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) was added to each sample, and electrophoresis was performed in 1.2% agarose gel at 50 V for 3 hours. After staining with ethidium bromide, DNA was visualized with UV light and photographed.
Modulation of 24-Hour Spontaneous PMN Apoptosis by Tumor Cells
Effect of Tumor Cell-Conditioned Media
To determine whether tumor cells can modulate PMN apoptosis, 1 × 10 6 PMNs were incubated for 24 hours in 1 ml of tumor cell-conditioned medium (CM) and evaluated for apoptosis. Tumor cell-CM was obtained with: 1) the A549 cell line (American Type Culture Collection, Rockville, MD) originally established from a person with BAC 18 and cultured in complete medium (see above) supplemented with 10% FBS; and 2) a primary culture (first to third passage) of tumor cells obtained from a pleural effusion specimen recovered from a patient with BAC, as previously described by Oie and colleagues. 19 Briefly, pleural fluid was centrifuged and pleural cells (tumor cells, 65%; inflammatory cells, 35%) were resuspended in Dulbecco’s modified Eagle’s medium and separated by density gradient centrifugation (Lymphocytes separation medium, MSL1077; Eurobio, Les Ulis, France). The interface layer containing tumor cells was harvested, washed twice in Dulbecco’s modified Eagle’s medium, and seeded into 25-cm 2 flasks in complete medium with 10% FBS. The purity of the tumor cells thus obtained was verified by May Grünwald Giemsa staining. Then, cultured cells were routinely passaged every 7 to 15 days. Tumor cells, ie, A549 or primary BAC cells, were resuspended at a density of 1 × 105/ml in complete medium with 2% FBS and cultured in 24-well plates (500 μl/well) for 72 hours at 37°C. The medium was then harvested and confluent monolayers of tumor cells were exposed to complete medium with 2% FBS or to CM from inflammatory cells, ie, peripheral blood mononuclear cells (PBMCs), PMNs, and alveolar macrophages (AMs), prepared as described below. Cell-free supernatants were collected after 24 hours of incubation and stored at −80°C.
To obtain inflammatory cell-CM, PBMCs and PMNs were recovered from peripheral blood of healthy donors by density gradient centrifugation (Polymorphonuclear cell separation medium, Eurobio). Briefly, PMNs were separated as above. The PBMC layer was washed three times in sterile saline solution. PBMCs comprised 65 ± 10% lymphocytes and 35 ± 13% monocytes as evaluated by flow cytometry. AMs were isolated from BALF of controls (see above). AMs accounted for >90% of BALF cells (May Grünwald Giemsa staining). Cells were separated from the remaining BALF by centrifugation. PBMCs, PMNs, and AMs were resuspended at a density of 5 × 105/ml in complete medium with 2% FBS, then plated in 24-well plates (500 μl/well) and cultured at 37°C in humidified 5% CO2/95% air. After 24 hours, inflammatory cell-CM was collected and stored at −80°C.
Effect of BALF from BAC Patients
One million PMNs were incubated for 24 hours in 1 ml of BALF supernatant from patients and controls at a final concentration of 50 to 90% in complete medium with 2% FBS, and were then examined for apoptosis.
Neutralization Studies
Conditioned media and BALF supernatants were preincubated for 45 minutes with several goat polyclonal neutralizing antibodies (Abs) directed against human 1) granulocyte-macrophage colony-stimulating factor (GM-CSF) (1:250 dilution; R&D Systems, Abington, UK), 2) granulocyte colony-stimulating factor (G-CSF) (1:125; R&D Systems), 3) interleukin (IL)-8 (1:20; R&D Systems), 4) IL-6 (1:200; R&D Systems), 5) tumor necrosis factor-α (TNF-α) (1:250; Genzyme Diagnostics, Cergy Saint-Christophe, France), 6) IL-1β (1:100; R&D Systems) and with irrelevant control goat polyclonal Abs (R&D systems). PMNs were then incubated for 24 hours at 37°C in Ab-treated CM or BALF supernatants and analyzed by means of flow cytometry. The results are expressed as the percentage inhibition of anti-apoptotic activity defined as follows:
 |
in which the anti-apoptotic activity of CM was:
 |
Enzyme-Linked Immunosorbent Assay (ELISA) Measurements
GM-CSF, G-CSF, IL-8, IL-6, IL-1β, and TNF-α concentrations were measured in CM and BALF supernatants by using commercially available ELISA assays (R&D Systems). The ELISA kits were used as indicated by the manufacturer and consistently detected GM-CSF, G-CSF, IL-8, IL-6, TNF-α, and IL-1β concentrations >3, 20, 10, 0.7, 4.4, and 1 pg/ml, respectively, in linear manner.
Immunohistochemical Studies of GM-CSF and G-CSF Expression in BAC and Normal Pulmonary Tissue
We examined tumoral and distant normal pulmonary tissues from three patients with BAC. Tissue fragments were immediately frozen in liquid nitrogen and stored at −80°C. Sections 4-μm thick were fixed in acetone and reacted with appropriate dilutions of monoclonal anti-human GM-CSF (Genzyme) and G-CSF Abs (R&D Systems). Isotype-matched Abs were used as controls (MOPC 21, IgG1; Sigma, Saint Louis, MO).
Positive cells were revealed by using the Vectastain ABC-alkaline phosphatase kit (Vector, Burlingame, CA) and Fast Red substrate. To test the specificity of immunostaining, Abs were omitted or replaced by an isotype-matched control. No positive cells were identified in these conditions. The intensity of immunostaining was graded from − (absent) to +++ (strongly positive). Complete agreement was obtained between the two independent observers.
Statistical Analysis
Spearman’s ρ coefficient was used for correlation studies between quantitative variables. Comparisons were made using the Mann-Whitney nonparametric test for unpaired data and the Wilcoxon nonparametric test for paired data. Statistical analysis was performed using one-way analysis of variance for parametric data sets. Data are presented as means ± SEM. P values of <0.05 were considered to denote statistical significance.
Results
Inhibition of Spontaneous PMN Apoptosis by A549 Cells
Conditioned media from unstimulated A549 tumor cells did not inhibit constitutive PMN apoptosis at 24 hours. Numerous PMNs showed morphological characteristics of apoptotic cells, such as condensed nuclei and apoptotic bodies, which were absent at T0 in cytospin preparations (Figure 1, A and C) ▶ . This phenomenon was accompanied by the emergence of annexing V labeling (Figure 1, B and D) ▶ and DNA fragmentation (Figure 2) ▶ . In contrast, cell-free supernatants from A549 cells exposed to CM from PBMCs or AMs inhibited 24-hour constitutive PMN apoptosis. In these conditions, PMNs were morphologically normal (Figure 1, E and G) ▶ . Flow cytometry showed the loss of annexin V labeling (Figure 1, F and H) ▶ , and DNA electrophoresis showed a decrease in DNA fragmentation (Figure 2) ▶ . Cell-free supernatants from A549 cell cultures exposed to PMN-CM did not inhibit 24-hour constitutive PMN apoptosis (Figure 1, I and J ▶ , and Figure 2 ▶ ).
Figure 1.
Effect of A549 cell line-CM on PMN apoptosis in vitro. PMNs were evaluated for apoptosis by morphological analysis (left; original magnification, ×400) and annexin-V labeling (right) at T0 and after a 24-hour incubation in cell-free supernatant from A549 cells previously exposed to CM from PBMCs, AMs, and PMNs, as described in Materials and Methods. At T0, PMNs had a normal morphology with a polysegmented nucleus (A) and appear in quadrant 3 (low annexin V-fluorescein isothiocyanate signal and low propidium iodide signal) (B). At 24 hours, PMNs were apoptotic, with dense condensation of chromatin in the form of either a single nucleus or nuclear fragments not connected by strands (C, arrows) and appeared in quadrant 1 (D) (high annexin V-fluorescein isothiocyanate signal but low propidium iodide signal). Cell-free supernatant from A549 cell line cultured with PBMC-CM (E and F) or AM-CM (G and H) inhibited PMN apoptosis, whereas cell-free supernatant from A549 cell line cultured with PMN-CM (I and J) did not. This figure is representative of 10 experiments.
Figure 2.
Effect of A549 cell line-CM on PMN apoptosis in vitro. Electrophoresis of DNA extracted from normal PMNs showed fragmentation, ie, the ladder-like appearance of smaller fragments characteristic of apoptosis, when PMNs were incubated for 24 hours in cell-free supernatant from A549 cells (lane 3); absent at T0 (lane 2). At T24, cell-free supernatant from A549 cells cultured in presence of medium conditioned by PBMCs (lane 4) or AMs (lane 5), but not PMNs (lane 6), inhibited PMN apoptosis. Lane 1: Molecular weight marker. This figure is representative of five experiments.
To quantify this inhibition, apoptotic PMNs were counted using morphological criteria and flow cytometry (Table 1) ▶ . Based on morphological criteria, the proportion of apoptotic PMNs fell significantly from 40 ± 9% with A549-CM to 7 ± 2% (P < 0.01) and 2 ± 1% (P < 0.01) with cell-free supernatants from A549 cells exposed to PBMC-CM and AM-CM, respectively. Based on flow cytometry, the proportion of apoptotic PMNs fell significantly from 88 ± 2% to 27 ± 7% (P < 0.01) and 23 ± 3% (P < 0.01) in the same conditions (Table 1) ▶ . As shown in Figure 3 ▶ , the observed anti-apoptotic activity was predominantly related to tumor cell release, even if PBMC or AM release also contributed to the effect (Figure 3A) ▶ . Indeed, analysis of dose-response curves obtained with the different CM (Figure 3, B and C) ▶ showed that the anti-apoptotic activity derived from A549 cells stimulated by mononuclear cell-CM was respectively 10-fold and fourfold higher than that derived from PBMC-CM and AM-CM alone.
Table 1.
Measurement of PMN Apoptosis at 24 Hours
| Morphologic criteria | Annexin-V labeling | |
|---|---|---|
| A549-CM | 40 ± 9 | 88 ± 2 |
| A549 + PBMC-CM | 7 ± 2* | 27 ± 7* |
| A549+ AM-CM | 2 ± 1* | 23 ± 3* |
| A549+ PMN-CM | 35 ± 9 | 87 ± 2 |
PMN were isolated from whole blood and then incubated for 24 hours in cell-free supernatant from A549 cells cultured with or without CM from PBMCs, AMs, or PMNs as described in Materials and Methods. The data are the mean ± SEM percentage of cells with an apoptotic morphology or Annexin-V labeling. NS, not significant.
*P < 0.05 in the Mann-Whitney test, between PMNs cultured in A549-CM and A549 exposed to PBMC-CM or AM-CM. This figure is representative of 10 experiments.
Figure 3.
PMN anti-apoptotic activity from A549 cells exposed to mononuclear cell-CM. The effect of cell-free supernatants on PMN apoptosis in vitro was measured by flow cytometry (A). As described in Materials and Methods, PMNs were incubated for 24 hours with CM from A549 cells (open bars), PBMCs, or AMs (hatched bars), and also with cell-free supernatant from A549 cells previously exposed to CM from PBMCs or AMs (filled bars). Results expressed as percentage of apoptotic PMNs are means ± SEM of six experiments. Comparisons were made using the Wilcoxon nonparametric test. Dose-response curves (B and C) performed with PBMC-CM (hatched circles), AM-CM (hatched squares), and cell-free supernatant from A549 cells previously exposed to CM from PBMCs (filled circles) or AMs (filled squares) used at different concentrations (from pure to 1/10); each point represents the mean of two experiments.
Finally, we performed neutralization experiments against IL-1β and TNF-α to investigate their role in the ability of mononuclear cells-CM, ie, PBMCs and AMs, to induce neutrophil anti-apoptotic activity from A549-cells. As shown in Figure 4 ▶ , the proportion of apoptotic PMNs increased significantly from 34 ± 6% to 66 ± 2% (P < 0.01) when anti-IL-1β and anti-TNF-α Abs were previously added to PBMC-CM (Figure 4) ▶ . Similarly, this proportion increased from 28 ± 1% to 51 ± 3% (P < 0.01) when anti-IL-1β and anti-TNF-α Abs were added to AM-CM (Figure 4) ▶ . Interestingly, the inhibition obtained with anti-IL-1β and anti-TNF-α Abs was associated with a >90% reduction in G-CSF and GM-CSF production by A549 cells (data not shown). The fact that A549 cells did not release detectable amounts of IL-1β or TNF-α, either constitutively or after exposure to mononuclear cell-CM (Table 2) ▶ , eliminated the possibility of autocrine IL-1β and TNF-α production by A549 cells.
Figure 4.
Role of IL-1β and TNF-α in the induction of PMN anti-apoptotic activity from A549 cells exposed to mononuclear cell-CM. The effect of A549 cell-free supernatants on PMN apoptosis in vitro was measured by flow cytometry. As described in Materials and Methods, PMNs were incubated for 24 hours with CM from A549 cells (open bars), cell-free supernatant from A549 cells previously exposed to CM from PBMCs, or AMs (filled bars) and IL-1β- and TNF-α-neutralized PBMC-CM or AM-CM (hatched bars). Results (expressed as percentage of apoptotic PMNs) are means ± SEM of three experiments. Comparisons were made using the Wilcoxon test.
Table 2.
Cytokine Levels in Culture Supernatants
| GM-CSF (pg/ml) | G-CSF (pg/ml) | IL-8 (ng/ml) | IL-6 (pg/ml) | TNF-α (pg/ml) | IL-1β (pg/ml) | |
|---|---|---|---|---|---|---|
| A549 | ND | 73 ± 8 | 0.5 ± 0.1 | 14 ± 4 | ND | ND |
| PBMC-CM | 70 ± 39 | 145 ± 31 | 27 ± 6 | 263 ± 89 | 342 ± 152 | 185 ± 113 |
| A549+ PBMC-CM | 227 ± 44 | 1777 ± 254 | 169 ± 22 | 2983 ± 1185 | 112 ± 42 | 152 ± 91 |
| AM-CM | 9 ± 3 | 106 ± 13 | 117 ± 41 | 451 ± 120 | 661 ± 384 | 1 ± 1 |
| A549+ AM-CM | 38 ± 18 | 194 ± 47 | 128 ± 41 | 1948 ± 506 | 227 ± 122 | 0.6 ± 0.6 |
| P value* | 0.04 | 0.06 | 0.04 | 0.06 | 0.06 | 0.28 |
| P value† | 0.01 | 0.10 | 0.86 | 0.01 | 0.10 | 0.10 |
Cytokine concentrations were measured by ELISA in CM from A549 cells, PBMCs, AMs, and A549 cells previously cultured with PBMC-CM or AM-CM. Results are means ± SEM. ND, not detectable. Wilcoxon’s test was used to compare cytokine concentrations in PBMC-CM versus A549 + PBMC-CM (*) and AM-CM versus A549 + AM-CM (†). This figure is representative of 10 experiments.
Inhibition of Spontaneous PMN Apoptosis by A549 Cell-Derived Cytokines
To determine whether cytokines were responsible for the anti-apoptotic activity of cell-free supernatants from A549 cells, concentrations of cytokines known to be involved in neutrophil survival were determined in cell culture supernatants by using specific ELISAs. As shown in Table 2 ▶ , unstimulated A549 tumor cells constitutively released moderate amounts of G-CSF, IL-8, and IL-6, but did not detectably release GM-CSF, TNF-α, or IL-1β. PBMC-CM and AM-CM induced GM-CSF release and also enhanced G-CSF and IL-6 release. Contrary to AM-CM, PBMC-CM enhanced IL-8 release by A549 cells.
We then used neutralizing Abs to examine the relative contributions of these cytokines to the anti-apoptotic activity of A549 cells. Neutralizing Abs to G-CSF, IL-6, IL-8, IL-1β, and TNF-α, used alone, had no significant effect on the anti-apoptotic activity of cell-free supernatants from A549 cells stimulated with PBMC-CM (data not shown). As shown in Figure 5 ▶ , top, anti-GM-CSF Ab inhibited this anti-apoptotic activity by 21% when used alone and by 51% when combined with anti-G-CSF Abs. The inhibition induced by anti-GM-CSF was not enhanced by simultaneous addition of each of the other anti-cytokine Abs (data not shown). Neutralizing Abs to G-CSF, IL-6, IL-1β, and TNF-α had no individual effect on the anti-apoptotic activity of cell-free supernatants from A549 cells stimulated with AM-CM (data not shown). Anti-GM-CSF and anti-IL-8 Abs inhibited anti-apoptotic activity by 35% and 21%, respectively, when used separately, and by 58% when combined (Figure 5 ▶ , bottom). However, the IL-8 effect was related to AM release rather than to tumor cell release (Table 2) ▶ .
Figure 5.
Role of A549 cell-derived GM-CSF and G-CSF on PMN apoptosis. Cell-free supernatants from A549 cells exposed to CM from PBMCs (top) or AMs (bottom) were preincubated with neutralizing polyclonal goat anti-human Abs against GM-CSF, G-CSF, or IL-8, used alone or in combination. PMNs were then incubated for 24 hours at 37°C in Ab-treated cell culture medium and analyzed for apoptosis by flow cytometry. Results (expressed as anti-apoptotic activity, see Materials and Methods) are means ± SEM of six experiments. Comparisons were made using the Wilcoxon test.
Inhibition of PMN Apoptosis in Patients with BAC
The mean percentage of neutrophils in BALF was 33 ± 5% in patients with BAC and only 1.2 ± 0.2% in controls (P = 0.03). In the 29 BALF samples showing neutrophil alveolitis exceeding 5%, the mean proportion of apoptotic PMNs was 11 ± 2% (median, 7%; range, 0.5 to 43%). In this group, the degree of PMN alveolitis correlated negatively with the percentage of apoptotic PMNs, as shown in Figure 6A ▶ (P = 0.024).
Figure 6.
Alteration of PMN apoptosis in patients with bronchioloalveolar carcinoma. A: Correlation between the percentage of apoptotic PMNs and the neutrophil count in BALF from patients with bronchioloalveolar carcinoma (BAC). Among the 41 patients with BAC, 29 had a neutrophil count >5% and could be evaluated for in vivo apoptosis by using morphological criteria, as described in Materials and Methods. Correlations were performed using Spearman’s ρ coefficient. B: Effect of BALF supernatant on PMN apoptosis in vitro. PMNs were incubated for 24 hours with BALF supernatant from controls (n = 6) or patients (n = 14) with BAC, and evaluated for apoptosis by flow cytometry. Comparisons were made using the Mann-Whitney test.
Inhibition of PMN Apoptosis by BALF from BAC Patients
To determine whether BALF supernatants from BAC patients inhibited neutrophil apoptosis, we incubated normal peripheral blood PMNs for 24 hours in BALF supernatants from patients (n = 14) and controls (n = 6) at a final concentration of 50% in complete media. These BAL samples were selected on the basis of availability, with no knowledge of neutrophil alveolitis or cytokine concentrations. As shown in Figure 6B ▶ , BALF from the patients inhibited PMN apoptosis relative to BALF from controls (apoptotic PMNs: 64 ± 4% versus 90 ± 2%, P = 0.04). This anti-apoptotic effect was concentration-dependent, as the proportion of apoptotic PMNs reached 49 ± 5% (n = 4), when BALF from the patients was used at a final concentration of 90%.
To evaluate whether the anti-apoptotic effects of BAC BALF depended on the cytokines previously shown to be involved in neutrophil survival in vitro, cytokine concentrations were determined using ELISA methods. As shown in Table 3 ▶ , only G-CSF was detectable in BALF from controls, whereas all of the cytokines were detected in BALF from the patients; in addition, G-CSF concentrations were significantly higher in BALF from patients than controls. Only GM-CSF concentrations correlated with BALF anti-apoptotic activity (P = 0.01, n = 14). However, a positive correlation was also noted between the BALF G-CSF concentration and the total neutrophil count (P = 0.01, n = 14) and the neutrophil percent (P = 0.01, n = 14), as well as between the BALF GM-CSF concentration and the neutrophil percent (P = 0.05, n = 14).
Table 3.
Cytokine Levels in BAL Fluid Supernatants from BAC Patients and Controls
| GM-CSF (pg/ml) | G-CSF (pg/ml) | IL-8 (pg/ml) | IL-6 (pg/ml) | TNF-α (pg/ml) | IL-1β (pg/ml) | |
|---|---|---|---|---|---|---|
| Patients (n = 14) | 2.5 [0–353] | 88 [0–566] | 219 [0–2612] | 55 [0–375] | 2 [0–21] | 1.4 [0–86] |
| Controls (n = 6) | ND | 9 [0–22] | ND | ND | ND | ND |
| P value | 0.01 | 0.01 | <0.01 | <0.01 | 0.02 | 0.02 |
Cytokine concentrations were measured by ELISA in bronchoalveolar lavage fluid supernatants from patients with bronchioloalveolar carcinoma and from healthy controls. Results are medians and ranges. ND, not detectable. P value calculated by the Mann-Whitney test.
To determine the contribution of the above cytokines to the anti-apoptotic activity of patient’s BALF, we added neutralizing Abs to the six BALFs with the highest anti-apoptotic activity (30 to 60% inhibition). Neutralizing Abs to IL-8, IL-6, IL-1β, and TNF-α had no significant effect. Combinations of anti-cytokine Abs did not further enhance PMN apoptosis (data not shown). By contrast, anti-GM-CSF and anti-G-CSF Abs inhibited 15 to 40% and 34 to 63% of the BALF anti-apoptotic activity, respectively, with an additive effect of the two Abs used in combination (Figure 7) ▶ . Moreover, the observed levels of G-CSF and GM-CSF in these BALF (65 to 566 pg/ml and 10 to 353 pg/ml, respectively, Figure 7 ▶ ) were in the range of concentrations of the corresponding recombinant cytokines (50 to 2000 pg/ml G-CSF and 5 to 200 pg/ml GM-CSF) preventing 24-hour neutrophil apoptosis in vitro (data not shown).
Figure 7.
Role of BALF GM-CSF and G-CSF on PMN apoptosis in vitro. BALF from six patients (patient 1 to patient 6) with bronchioloalveolar carcinoma (BAC) was preincubated with neutralizing polyclonal goat anti-human Abs against G-CSF (open bars), GM-CSF (hatched bars), or both (filled bars). PMNs were then incubated for 24 hours at 37°C with Ab-treated BALF and analyzed for apoptosis by flow cytometry. Results (expressed as percent inhibition of anti-apoptotic activity, see Materials and Methods) are means of two different experiments.
Inhibition of PMN Apoptosis by Primary Culture Supernatants of Tumor Cells from a BAC Patient
To confirm observations with the A549 cell line, we used tumor cells directly derived from the lung tumor of a patient with BAC (BAC cells). As shown in Figures 8 and 9 ▶ ▶ , results with BAC cells were similar to those obtained with A549 cells (Figures 3 and 4) ▶ ▶ . First, the proportion of apoptotic PMNs fell significantly from 82 ± 3% with BAC-CM to 20 ± 6% (P < 0.01) and 18 ± 6% (P < 0.01) with cell-free supernatants from BAC cells exposed to PBMC-CM and AM-CM, respectively. Second, neutralizing Abs against GM-CSF and G-CSF significantly inhibited the anti-apoptotic activity of cell-free supernatants from BAC cells exposed to PBMC-CM or AM-CM, with an additive effect of the two Abs used in combination, whereas neutralizing Abs against IL-8 had a significant effect only on the cell-free supernatant of BAC cells exposed to AM-CM (Figure 9) ▶ .
Figure 8.
PMN anti-apoptotic activity of primary cultured tumor cells from a patient with BAC (BAC cells), exposed to mononuclear cell-CM. The effect of cell-free supernatants on PMN apoptosis in vitro was measured by flow cytometry. As described in Materials and Methods, PMNs were incubated for 24 hours with CM from BAC cells (open bars), PBMCs, or AMs (hatched bars), and also with cell-free supernatant from BAC cells previously exposed to CM from PBMCs or AMs (filled bars). Results (expressed as percentage of apoptotic PMNs) are means ± SEM of three experiments. Comparisons were made using the Wilcoxon n test.
Figure 9.
Role of G-CSF and GM-CSF produced by primary cultured tumor cells from a patient with BAC (BAC cells) in PMN apoptosis. Effect of neutralizing antibodies on apoptosis in vitro. Cell-free supernatants from BAC cells exposed to CM from PBMCs (top) or AMs (bottom) were preincubated with neutralizing polyclonal goat anti-human Abs against GM-CSF, G-CSF, or IL-8, used alone or in combination. PMNs were then incubated for 24 hours at 37°C in Ab-treated cell culture and analyzed for apoptosis by flow cytometry. Results (expressed as anti-apoptotic activity, see Materials and Methods) are means ± SEM of three experiments. Comparisons were made using the Wilcoxon test.
Cellular Origin of GM-CSF and G-CSF in the Tumor Environment
To determine the cellular origin of GM-CSF and G-CSF in the tumor environment in vivo, we performed an immunohistochemical study of both tumoral and adjacent normal lung tissue (Figure 10) ▶ . The results obtained with three different specimens were similar. Tumor cells strongly expressed GM-CSF (+++), whereas interstitial mononuclear cells (++), AMs (++), and resident cells (++) expressed it less intensely. G-CSF was also strongly expressed by tumor cells and less intensely by interstitial mononuclear cells (++) and AMs (++) but not by resident cells. The expression of the two cytokines was homogenous and cytoplasmic. In normal lung tissue distant from the tumor, GM-CSF was also expressed by normal epithelial cells (++) and interstitial mononuclear cells (+), whereas G-CSF was only expressed by bronchiolar cells (+).
Figure 10.
GM-CSF and G-CSF expression in bronchioloalveolar carcinoma in vivo. Histological analysis and immunohistochemical evaluation of cytokine expression in tumor and adjacent normal tissue obtained by surgical excision of the right upper lobe of a patient with bronchioloalveolar carcinoma (BAC). A: H&S-stained paraffin section showing the typical aspect of a BAC tumor; note the presence of neutrophils at the luminal pole. B: Negative control using nonimmune mouse IgG on tumor tissue. C–H: Immunostaining using monoclonal Abs and the Vectastain ABC-alkaline phosphatase technique. C: Immunodetection of GM-CSF in tumor tissue showing that tumor cells are strongly positive (+++). D: Immunodetection of G-CSF in tumor tissue showing that tumor cells are also strongly positive for this cytokine (+++). E and F: Immunodetection of GM-CSF in normal tissue distant from the tumor, showing that this factor is moderately expressed in normal lung and mostly produced by bronchiolar epithelium (++). G and H: Immunodetection of G-CSF in normal tissue distant from the tumor; only normal bronchiolar cells express the cytokine (+). Original magnifications: ×200 (A, C, G, and H); ×125 (B and D to F). al, Alveolar lumen; bl, bronchiolar lumen.
Discussion
BALF supernatants from patients with BAC protected peripheral blood neutrophils from spontaneous apoptosis in a concentration-dependent manner. No such effect was observed with BALF supernatants from controls. Significant anti-apoptotic activity was observed with all but one of the patients’ BALF supernatants. This anti-apoptotic effect might have been underestimated, as the epithelial lining fluid recovered by BAL is usually 100-fold diluted. 20 Several soluble factors with neutrophil anti-apoptotic activity, such as cytokines, complement factor 5a, or eicosanoids 16,21,22 were probably contained in BALF supernatants from the patients but not the controls. We focused on epithelium-derived cytokines, and especially those known to have neutrophil anti-apoptotic activity in vitro, namely GM-CSF, G-CSF, IL-1β, IL-8, IL-6, and TNF-α. 21,23-26 All these cytokines were present in patients’ BALF, whereas only G-CSF (at a significantly lower concentration) was found in controls. Importantly, G-CSF and GM-CSF concentrations in patients correlated with the intensity of neutrophil alveolitis, and GM-CSF concentrations also correlated with neutrophil anti-apoptotic activity. Both GM-CSF and G-CSF seem to be involved in the anti-apoptotic effect, because Abs against the two cytokines significantly and independently inhibited BALF supernatant anti-apoptotic activity. Our use of peripheral blood neutrophils instead of alveolar neutrophils could limit the in vivo relevance of these findings. Indeed, endothelial transmigration by neutrophils could modulate their sensitivity to apoptosis, through adhesion molecule engagement. 27 We thus counted apoptotic alveolar neutrophils in BALF cytospin preparations from patients with BAC. Interestingly, we found a negative correlation between the degree of alveolar neutrophil apoptosis and that of neutrophil alveolitis, suggesting that an alteration of programmed neutrophil death might contribute to the persistence of neutrophil alveolitis in vivo.
In an attempt to mimic the tumor environment and identify the respective roles of tumor cells and inflammatory cells in the prolongation of neutrophil survival, we tested the effect of A549 cell-derived cytokines in vitro on the 24-hour spontaneous apoptosis of peripheral blood neutrophils after culturing A549 cells under basal conditions and with media conditioned by several types of inflammatory cells. Although A549 cells constitutively released several of the anti-apoptotic cytokines tested (G-CSF, IL-8, and IL-6), A549 cell CM obtained under basal conditions did not prevent neutrophil apoptosis. By contrast, CM of A549 cells exposed to inflammatory cells (PBMCs and AMs) inhibited 24-hour spontaneous neutrophil apoptosis by 68% and 78%, respectively. This phenomenon was also observed when primary culture of BAC cells directly derived from lung tumors was used instead of the long-term established A549 cell line. Media directly conditioned by PBMCs and AMs also had anti-apoptotic activity, but less than tumor cells.
As in in vivo experiments, only tumor-derived GM-CSF and G-CSF were clearly involved in the in vitro prolongation of neutrophil survival. IL-8 had an additive effect on GM-CSF in the AM model but was released by AMs and not by tumor cells. Moreover, the similar pattern of results obtained with A549 cells and primary cultured BAC cells directly derived from lung tumor, as well as with patients’ BALF supernatants, strongly supports the in vivo relevance of our in vitro findings. Unlike Daffern and colleagues, 28 we found that IL-6 did not contribute to the anti-apoptotic activity of tumor epithelial cells. However, these latter authors used nasal-derived primary epithelial cells previously stimulated by high concentrations of recombinant TNF-α. 28 In the same way, IL-1β and TNF-α did not play a role in neutrophil survival in our study, but these cytokines were not produced by the A549 cell line whatever the stimulus used. By contrast, we showed that PBMCs and AMs derived IL-1β and TNF-α contribute to the release of neutrophil anti-apoptotic factors (GM-CSF and G-CSF) by tumor epithelial cells, as previously suggested by other studies. 28-30 Furthermore, TNF-α up-regulation of GM-CSF receptor expression on PMNs might also indirectly contribute to this phenomenon. 31 Finally, the fact that anti-GM-CSF and anti-G-CSF Abs did not completely abrogate the anti-apoptotic effect of tumor cell CM suggests that cytokines or soluble factors not studied here could partly explain our findings. 21,28
To examine the respective roles of tumor cells and tumor-infiltrating inflammatory cells in the production of anti-apoptotic factors in vivo, we then evaluated G-CSF and GM-CSF protein expression in specimens of tumorous and distant normal pulmonary tissue from patients with BAC. We observed strong expression of both cytokines by tumor cells. GM-CSF, but not G-CSF, was strongly expressed by the tumor-associated stroma reaction thickening the tumor alveolar wall. In distant normal lung tissue, GM-CSF was detected in epithelial cells as well as in monocytes/macrophages, fibroblasts, and endothelial cells, whereas G-CSF was only detected in bronchial cells. However, immunostaining of these two cytokines was less intense in distant normal lung than in tumoral areas. The accumulation of GM-CSF and G-CSF in tumoral areas, demonstrated by immunochemical studies and BALF cytokine measurements, suggest a possible up-regulation of GM-CSF and G-CSF production by the tumor microenvironment. Our in vitro results support the hypothesis that mononuclear cells such as AMs and PBMCs, which have close contacts with tumor cells in vivo, 11,13 might contribute to this cell’s previously reported paracrine feedback loop between tumoral and inflammatory. 32-35 It is also reasonable to suppose that mononuclear cell-derived TNF-α and IL1-β influence GM-CSF and G-CSF production by tumoral epithelia in vivo.
Although the mechanisms by which neutrophils influence the prognosis of BAC is unclear, it has been postulated that the persistence of neutrophil alveolitis would result in persistent release of proinflammatory mediators such as cytokines, proteases, and reactive oxygen and nitrogen species that can damage DNA and activate oncogenes. 36,37 We have also previously postulated that neutrophils might be involved in luminal tumor spread by promoting tumor cell shedding. 11 In addition, GM-CSF itself could be involved in tumor progression through its proliferative effect on type II alveolar epithelial cells, possible precursors of BAC. 38,39 Lastly, in vivo alterations of phlogistic phagocytic clearance of apoptotic neutrophils also results in a lack of release of anti-inflammatory mediators such as transforming growth factor-β, 40,41 which functions as a classic tumor suppressor. 42
In conclusion, we demonstrate that the BAC tumor microenvironment generates local conditions that prolong alveolar neutrophil survival through the production of soluble factors, and thereby contributes to the persistence of the deleterious neutrophil alveolitis observed in this setting. We also provide additional evidence of possible cooperation between tumor and inflammatory cells in tumor progression, possibly warranting clinical trials of local anti-inflammatory adjuvant therapy.
Footnotes
Address reprint requests to Pr. J. Cadranel, Service de Pneumologie et de Réanimation Respiratoire, Hôpital Tenon, 4 rue de la chine, 75020 Paris, France. E-mail: jacques.cadranel@tnn.ap-hop-paris.fr.
Supported by grants from “Pneumologie developpement” and “La Ligue Nationale contre le cancer–Comite de Paris.”
M. Wislez is a doctoral fellow of “L’Académie de Médecine” and “Les Fonds d’Etude et de Recherche du Corps Médical des Hôpitaux de Paris”
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