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Keywords:

  • Interstitial pneumonia;
  • CTL;
  • Apoptosis;
  • Pro-inflammatory cytokine

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Apoptosis is thought to be involved in lung epithelial cell damage in acute respiratory distress syndrome and interstitial pneumonia. Both the role of apoptosis and its underlying molecular mechanisms in human lung tissue remain unclear. To address these issues, we developed an in vitro assay in which a human lung epithelial cell line and a staphylococcal enterotoxin B (SEB)-reactive human CD8+ CTL line were co-cultured in the presence of SEB. SEB-stimulated CD8+ CTL induced apoptosis in the lung epithelial cell line primarily through the perforin/granzyme-mediated pathway. In these cells, apoptosis was initially independent of death receptor pathways. We also tested the effect of IFN-γ on modulation of apoptosis in lung epithelial cells. In IFN-γ-pretreated lung epithelial cells, CD95 (APO-1/Fas) activation as well as TNF-related apoptosis-inducing ligand (TRAIL) receptor and TNFR activation led to apoptosis. Furthermore, we found that the interaction of SEB-stimulated CD8+ CTL with lung epithelial cells induced an increase in TNF-α secretion. These results suggest an important role for bacterial superantigen-reactive CD8+ CTL in induction of lung epithelial cell apoptosis and in modulation of inflammatory processes in lung tissue.

Abbreviations:
BD-fmk:

t-Butoxy carbonyl-Asp-fluoromethylketone

CD95L:

CD95 ligand

CFSE:

Carboxyfluorescein diacetate succinimidyl ester

CHX:

Cycloheximide

CMA:

Concanamycin A

CVD-IP:

Interstitial pneumonia associated with collagen vascular disease

IPF:

Idiopathic pulmonary fibrosis

L-NMMA:

NG-Monomethyl-L-arginine

PI:

Propidium iodide

SEB:

Staphylococcal enterotoxin B

TRAIL:

TNF-related apoptosis-inducing ligand

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Injury of lung epithelial and endothelial cells promotes recruitment of circulating immune cells to lung tissue and enhances inflammatory reactions; furthermore, such injury also recruits fibroblasts to the lung and activates them to produce and deposit extracellular matrix 13. Severe damage of lung tissue and/or prolonged inflammation lead to progressive fibrosis of the lung 47. Apoptosis is thought to be involved in the mechanism of lung epithelial cell damage 812. Evidence for apoptosis has been seen in lung epithelial cells from patients with acute respiratory distress syndrome (ARDS) 8, 9, idiopathic pulmonary fibrosis (IPF) 10, 11, and interstitial pneumonia associated with collagen vascular disease (CVD-IP) 12. Furthermore, increased expression of CD95 (APO-1/Fas) 13, 14 on the surface of bronchiolar epithelial cells and alveolar epithelial cells from IPF 15 and CVD-IP 12 patients has been reported. In the acute phase (within 24 h of diagnosis) of septic ARDS patients, expression of perforin and granzyme B on lymphocytes and the presence of high levels of soluble CD95 ligand (CD95L) in the bronchoalveolar lavage fluid (BALF) have been detected. These findings are absent in late phase or septic non-ARDS patients 16. Such reports suggest the participation ofCD95/CD95L- and perforin/granzyme-mediated pathways in lung epithelial cell injury in ARDS and interstitial pneumonia. In animal models, apoptosis in lung epithelial cells has been observed but themechanism leading to apoptosis is controversial. It has been reported that soluble CD95-Fc fusion proteins and anti-CD95L Ab inhibit development of interstitial pneumonia. In addition, CD95- or CD95L-deficient mice show resistance to induction of interstitial pneumonia by bleomycin 17. In contrast, in staphylococcal enterotoxin B (SEB)-induced interstitial pneumonia in systemic lupus erythematosus-prone CD95-deficient MRL-lpr/lpr mice, significant resistance to induction of interstitial pneumonia is not observed. These mice show a somewhat milder mononuclear cell infiltration into the alveolar septal walls, a milder increase in pulmonary interstitial collagen fibers, and a more severe lymphocyte accumulation in the periarterial space than do MRL-+/+ mice (18 and our unpublished data). Enelow et al. have suggested a role for TNF-α-mediated apoptosis in injury of lung epithelial cells in an animal model in which activated CD8+ T cells were adoptively transferred into transgenic mice expressing a target antigen in lung epithelial cells 19, 20. Thus, T cell-mediated apoptosis of lung epithelial cells is under complex control and may depend on different agents stimulating T cells and on different immunological reactions of afflicted individuals. There are only a few studies that directly examine the mechanism of T cell-mediated apoptosis of human lung epithelial cells. Therefore, using a SEB-reactive human CD8+ CTL line, we developed an in vitro system to analyze CD8+ T cell-mediated apoptosis of human lung epithelial cells and focused on the investigation of cytokines participating in the development of interstitial pneumonia. Here, we show that human CD8+ CTL stimulated by SEB induce apoptosis in human lung epithelial cells and enhance the release of TNF-α upon interaction with lung epithelial cells. Stimulation of secretion of TNF-α may lead to enhanced inflammation and progression of fibrotic changes in lung tissue.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Phenotypic characteristics of T221 CTL and A549 cells and functional characteristics of T221 CTL

In our in vitro assay system, the human type II lung epithelial cell line A549 was cultured with the human CD8+ T cell line T221 in the presence of SEB. T221 CTL expressed CD3, CD8, HLA-DR, CD95, and TNF-related apoptosis-inducing ligand (TRAIL) on their surface (Fig. 1A, E), and mRNA encoding TCR Vβ14 (Fig. 1B) which is reactive to SEB 21. Bacterial superantigens such as SEB can bind to specific TCR Vβ without strict antigen specificity and can stimulate many T cells 21, 22. SEB stimulates T221 CTL and activates transcription of perforin, granzyme A, granzyme B, CD95L, TNF-α, and IFN-γ, even in the absence of APC (Fig. 1C). IFN-γ contents in T221 CTL culture supernatants were also increased by stimulation with SEB (Fig. 1D). Expression levels of the TRAIL transcript in T221 CTL did not change upon SEB stimulation (Fig. 1C). As IFN-γ has been reported to sensitize A549 cells to agonistic anti-CD95 Ab- or TRAIL-induced apoptosis 2325, analysis of cell surface markers was performed on IFN-γ-treated A549 cells as well as on non-stimulated cells. Both non-stimulated and IFN-γ-treated A549 cells expressed CD95, TRAIL-R1, and TRAIL-R2. Expression of CD95 and TRAIL-R2 on the surface of A549 cells was slightly increased upon IFN-γ treatment (Fig. 1E). A549 cells did not express HLA-DR, even upon treatment with IFN-γ (Fig. 1E), in agreement with a previous report 26.

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Figure 1. Characterization of T221 CTL and A549 cells. (A) Two-color FACS analysis of CD3 and CD8 or CD4 and CD8 expression on T221 CTL. The proportion of cells in each quadrant is indicated. (B) Constitutive expression of TCR Vβ14 mRNA in T221 CTL. Total RNA from T221 CTL was examined for expression of mRNA for TCR Vβ14 by RT-PCR analysis. (C) mRNA expression of perforin, granzyme A, granzyme B, CD95L, TRAIL, TNF-α, and IFN-γ in T221 CTL. RT-PCR was performed on total RNA isolated from T221 CTL that were either not treated or treated with 0.1 μg/ml SEB for 2 or 4 h. (D) IFN-γ production of T221 CTL. IFN-γ levels in the supernatants of T221 CTL cultured with or without SEB were measured by ELISA. Error bars show SEM of duplicate wells. (E) Surface phenotype of T221 CTL and A549 cells not treated or treated with IFN-γ. Surface expression of HLA-DR, CD95, TRAIL, TRAIL-R1, -R2, -R3, and -R4 (open histograms) on T221 CTL and A549 cells treated or not treated with IFN-γ was analyzed by FACS as described in Sect. 4.2. Filled histograms represent the staining of isotype-matched control Ab. Graphs are representative of three (A–C, E) or two (D) independent experiments.

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2.2 Activated CD8+ CTL induce apoptosis in A549 cells

To examine whether CD8+ CTL induce apoptosis in A549 cells, we first assessed CD8+ CTL-mediated death of A549 cells using annexin V-FITC and propidium iodide (PI) staining, followed by FACS analysis. Annexin V-positive and PI-negative cells are early apoptotic cells; by contrast, annexin V-positive and PI-positive cells are late apoptotic or necrotic cells 27. Death of A549 cells determined by annexin V staining was detected as early as 4 h after start of the co-culture and progressed until 12 h (Fig. 2A). We observed a time lag of several hours between an increase in annexin V-positive and PI-negative cells and an increase in annexin V-positive and PI-positive cells (Fig. 2A).

Contamination of A549 cells by CTL (in the R1 gate; see Fig. 2A) may render the determination of the proportion of dead A549 cells inaccurate. Therefore, we determined by FACS analysis the proportion of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled and unlabeled A549 cells after culture with or without T221 CTL and SEB. We found that contamination of A549 cells by T221 CTL was less than 5.5% (Fig. 2B). We further examined the effect of the SEB dose and the E/T ratio on apoptosis of A549 cells. SEB was most effective at a concentration of 0.1 μg/ml to induce T221 CTL-mediated apoptosis in A549 cells (Fig. 2C), and A549 cells were killed in an E/T ratio-dependent manner (Fig. 2D). A JAM test done to analyze the breaking of cellular DNA revealed that T221 CTL-mediated death of A549 cells was accompanied by DNA fragmentation (Fig. 2E). The effect of the SEB dose on apoptosis in A549 cells determined by DNA fragmentation was similar to that assessed by the annexin V assay (Fig. 2C, E). These observations indicate that SEB-stimulated human CD8+ CTL induce apoptosis in human lung epithelial cells.

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Figure 2.  SEB-stimulated CD8+ CTL induce apoptosis in A549 cells. (A) Kinetic analysis of apoptosis in A549 cells. A549 cells were cultured with T221 CTL at an E/T ratio of 10:1 and with 0.1 μg/ml SEB. Cells were collected at the indicated time. Apoptosis of A549 cells in the R1 gate was analyzed as described in Sect. 4.3. The proportion of cells in each quadrant is indicated. (B) Determination of purity of gated (R1 in Fig. 2A) A549 cells in SSC versus FSC dot blots by FACS analysis. Unlabeled and CFSE-labeled A549 cells were cultured either in the presence of T221 CTL and SEB or in medium alone for 12 h. CFSE fluorescence intensity of cells in the R1 gate was analyzed by FACS as described in Sect. 4.3. The proportion of CFSE-positive cells in the R1 gate is shown in the upper right corner. (C) The effect of the dose of SEB on apoptosis in A549 cells. A549 cells were cultured for 12 h in the presence or absence of T221 CTL (E/T ratio = 10:1) and variable concentrations of SEB. Percentages of annexin V-positive cells in the R1 gate are shown. (D) The effect of the E/T ratio on apoptosis in A549 cells. A549 cells were cultured for 9 h in the presence or absence of T221 CTL at the indicated E/T ratios and 0.1 μg/ml SEB. The mean values of the percentages of annexin V-positive cells in the R1 gate from duplicate wells are shown. Error bars show SEM of duplicate wells. (E) DNA fragmentation in A549 cells assessed by the JAM test. [3H]Thymidine-labeled A549 cells were cultured with T221 CTL and variable concentrations of SEB. After 20 h of co-culture, cells were harvested. Percentages of fragmented DNA were calculated as described in Sect. 4.3. These data are representative of three (A–D) or two (E) independent experiments.

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2.3 The perforin/granzyme pathway is involved in CD8+ CTL-mediated A549 cell death

To minimize the contamination of A549 cells by CTL, A549 cells were first labeled with CFSE and then subjected to the CTL assay. The proportion of annexin V-PE-positive cells in CFSE-positive cells was analyzed by FACS as shown in Fig. 3A. Contamination by T221 CTL was assumed to be minimal after preliminary experiments.

Two well-defined death pathways of CTL-mediated cytotoxicity are the perforin/granzyme- and the CD95/ CD95L-mediated pathways. Both pathways involve caspase activation 28. We first verified the participation of caspases in our CTL assay system. Expectedly, 10 μM t-butoxy carbonyl-Asp-fluoromethylketone (BD-fmk) significantly inhibited A549 cell death (Fig. 3B). Higher doses of BD-fmk up to 100 μM showed a similar effect. The nitric oxide (NO)-synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) was also utilized since 2–5 mM of the NO-releasing agent S-nitroso-N-acetylpenicillamine induced apoptosis of 11–22% of A549 cells in 20-h cultures as assessed by annexin V or Nicoletti analysis (data not shown). L-NMMA did not have any effect on CD8+ CTL-mediated A549 cell death (Fig. 3B). By contrast, 100 nM concanamycin A (CMA), which inhibits the perforin/granzyme pathway 29, showed over 60% inhibition of A549 cell death (Fig. 3C). These results indicate that perforin and granzymes participate to a large extent in CD8+ CTL-mediated A549 cell death. By contrast, 15 μg/ml of several inhibitors of the members of the TNF family, including NOK1 (anti-CD95L Ab), TRAIL-R2-Fc and TNF-R2-Fc, did not show any significant effect on A549 cell death (Fig. 3D). These results were similar when inhibitors were tested in longer culture periods (24 h). Thus, apoptosis induced in A549 cells by CD8+ CTL is primarily mediated by perforin/granzyme.

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Figure 3.  Inhibition assay of CD8+ CTL-mediated death of A549 cells. (A) FACS analysis of annexin V-PE fluorescence intensity on CFSE-labeled A549 cells. Death of CFSE-labeled A549 cells cultured in the presense or absence of T221 CTL (E/T ratio = 10:1) and 0.1 μg/ml SEB for 12 h was assessed by staining with annexin V-PE and by FACS analysis. The proportion of CFSE-positive and annexin V-PE-positive cells or CFSE-positive and annexin V-PE-negative cells in A549 cells (top panel) or A549 cells plus CTL in the presence of SEB (bottom panel) is indicated in each quadrant. (B–D) Analyses of inhibition of CD8+ CTL-mediated A549 cell death by several anti-apoptotic reagents or Ab. CFSE-labeled A549 cells were cultured with or without T221 CTL (E/T ratio = 10:1) and 0.1 μg/ml SEB in the presence or absence of 5 mM L-NMMA or 10 μM BD-fmk (B), variable concentrations of CMA (C), or either 15 μg/ml NOK1, 15 μg/ml TRAIL-R2-Fc, or 15 μg/ml TNF-R2-Fc (D) for 12 h, and then subjected to annexin V-PE staining and FACS analysis. Mouse IgG1 and human IgG1 (15 μg/ml) were used as controls for NOK1 or TRAIL-R2-Fc and TNF-R2-Fc, respectively. Values are means ± SEM of triplicate wells. Significant differences from responses obtained from co-cultures of A549 cells and CTL in the presence of SEB are shown by asterisks: *p<0.01, **p=0.001, and ***p<0.0001. All graphs are representative of more than three independent experiments.

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2.4 Induction of apoptosis in untreated or IFN-γ-treated A549 cells

Blocking CD95L, TRAIL, or TNF-α did not affect CD8+ CTL-mediated A549 cell death. Since increased IFN-γ production has previously been detected in the lungs of patients with asbestosis 30 and in the lungs of animal models of interstitial pneumonia 31, 32, we asked whether resistance to apoptosis mediated by ligands such as CD95L, TRAIL, or TNF-α could be modified by IFN-γ. Thus, we determined whether triggering of CD95, TRAIL-R, and TNFR in the absence or presence of IFN-γ induces apoptosis of A549 cells in order to evaluate the roles of death pathways from these receptors in CD8+ CTL-mediated A549 cell death in an interferon-containing environment. Activation of CD95 led to apoptosis of IFN-γ-pretreated A549 cells (Fig. 4A, D) in accordance with previous reports 23, 24. When protein A was utilized to cross-link anti-APO-1 Ab bound to CD95, apoptosis was induced not only in IFN-γ-pretreated but also in A549 cells that had not been pretreated with IFN-γ (Fig. 4D). TRAIL-R activation induced apoptosis in both IFN-γ-pretreated and unpretreated A549 cells in a dose-dependent manner (Fig. 4B). TNF-α also induced apoptosis in both IFN-γ-treated and nontreated A549 cells in the presence of cycloheximide (CHX) (Fig. 4C). Thus, activation of CD95, TRAIL-R, and TNFR led to apoptosis of A549 cells, and IFN-γ-treatment sensitized A549 cells to these death pathways.

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Figure 4.  Apoptosis of untreated and IFN-γ-treated A549 cells mediated by the members of the TNFR family. (A–C) A549 cells not treated or treated with IFN-γ were washed and cultured for a further 7 h with variable concentrations of anti-APO-1 Ab (A), LZ-TRAIL (B), or with TNF-α either alone or in the presence of 10 μg/ml CHX (C). Apoptosis was assessed by annexin V-FITC and PI staining as described in Sect. 4.3. (D) A549 cells nontreated or pretreated with IFN-γ were washed and further cultured in the presence or absence of 1 μg/ml anti-APO-1 Ab, 10 ng/ml protein A, or both for 20 h. Apoptosis was assessed by measuring the subdiploid DNA content using FACS analysis. Values are the means ± SEM of triplicate wells. Significant differences between nontreated and IFN-γ-pretreated A549 cells in anti-APO-1 Ab-induced apoptosis, and between anti-APO-1 Ab stimulation and anti-APO-1 Ab + protein A stimulation in the induction of apoptosis in IFN-γ-pretreated A549 cells are shown by asterisks: ***p<0.0001. All graphs are representative of three independent experiments.

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2.5 Cytokine levels in supernatants of A549 and CD8+ CTL co-cultures

Several cytokines and/or chemokines released during the process of lung epithelial cell injury may contribute to the development of interstitial pneumonia. We were particularly interested in whether apoptosis of lung epithelial cells is accompanied by production and/or release of soluble factors causing interstitial pneumonia. Accordingly, we examined TNF-α and IL-1β in the supernatants of co-cultures of A549 cells and T221 CTL. Large amounts of TNF-α were detected only in the supernatants of A549 cells cultured with T221 CTL and SEB (Fig. 5). We did not detect IL-1β in any supernatant examined (data not shown). These results suggest that the interaction of CD8+ CTL and lung epithelial cells provokes a modification of inflammatory processes in lung tissue by changing the levels of TNF-α.

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Figure 5. Measurement of TNF-α levels in supernatants of A549 and CTL co-cultures. A549 cells not treated (nontreated) or treated with human IFN-γ (IFN-γ pretreated) were washed and then further cultured with or without T221 CTL and/or SEB for 24 h. Supernatants were collected and subjected to sandwich ELISA for TNF-α. Error bars show SEM of duplicate wells. Significant differences from responses obtained from culture of T221 CTL + SEB are shown by asterisks: ***p<0.0001. Data are representative of two independent experiments.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The pathogenesis of interstitial pneumonia is complex, and the mechanism of the development of fibrotic tissue is largely unclear. To gain insight into inflammatory and fibrogenic processes in the lung, we devised a model system in which antigen (here SEB)-stimulated CTL (T221) were co-cultured with alveolar type II epithelial cells (A549). This in vitro model mimics the interaction of autoreactive T cells with epithelial cells in the lung in vivo. We then asked whether epithelial cells could be destroyed by the CTL and undergo apoptosis. Since increased IFN-γ production had previously been found in the lungs of patients with asbestosis 30 and in the lungs of animal models of bacterial superantigen-induced pneumonitis 31 and bleomycin-induced pneumonitis 32, epithelial cells were employed that were either treated or untreated with IFN-γ. We showed that SEB-stimulated human CD8+ CTL induced apoptosis in the human lung epithelial cell line A549 primarily through the perforin/granzyme pathway. Although epithelial cells were primarily insensitive todeath receptor-induced apoptosis, sensitization could be obtained upon IFN-γ treatment. Thus, IFN-γ secreted by lung-infiltrating autoreactive T cells might be important in regulating sensitivity and resistance of epithelial cells to apoptosis. The biological action of IFN-γ during the development of interstitial pneumonia is complicated. IFN-γ treatment has been reported to be effective in patients with IPF 33 and CVD-IP 34. However, there are several reports describing the deteriorating effect of IFN-γ in patients with end-stage IPF 35 or pneumonitis induced by radiation therapy for lung cancer 36, 37. Further investigations to clarify the biological role of IFN-γ in the development of interstitial pneumonia are needed.

In addition, we investigated whether apoptotic epithelial cells were capable of releasing pro-inflammatory cytokines. We determined the levels of TNF-α and IL-1β in the supernatants oflung epithelial cells cultured with CD8+ CTL and SEB. We assume that SEB-stimulated CTL may be further activated to release TNF-α by interaction with lung epithelial cells or that theymay stimulate lung epithelial cells to produce TNF-α. TNF-α produced in the alveolar space affects a variety of cells in an autocrine and/or paracrine manner 38. For example, TNF-α stimulates macrophages to produce IL-1, IL-6, IL-8, and TNF-α 3942 and accelerates extravasation of leukocytes by activating endothelial cells39, 43, leading to recruitment of inflammatory cells into the lung. Thus, TNF-α plays a critical role in the development of interstitial pneumonia by enhancing inflammatory reactions and stimulating growth of fibroblasts 1, 3.

In conclusion, using an in vitro model system of lung fibrosis, we showed that human SEB-stimulated CTL induce apoptosis in human lung epithelial cells primarily through a perforin/granzyme-mediated pathway. This interaction leads to an increase in TNF-α secretion. Thus, apoptosis induced by CTL and other mechanisms appears to play an important role in modulating inflammatory responses and cytokine-mediated progression towards fibrotic changes. Understanding the molecular mechanisms regulating the injury of lung tissue as well as the consecutive pro-inflammatory and fibrogenic responses following such injury is essential to develop more efficient therapies for interstitial pneumonia.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Cells and culture conditions

The human alveolar type II epithelial cell line A549 was cultured in DMEM supplemented with 10 mM Hepes, 5 mM L-glutamine, 100 μg/ml gentamycin and 10% FCS (all from GIBCO BRL, Eggenstein, Germany) in 5% CO2 at 37°C. If not otherwise indicated, A549 cells were removed from the wells by trypsinization (trypsin-EDTA; GIBCO BRL) and collected. The human alloreactive CD8+ T cell line T221 was kindly provided by Dr. Elisabeth Märker-Hermann (Johannes Gutenberg University of Mainz, Mainz, Germany) and was maintained in RPMI 1640 medium (GIBCO BRL) containing 10 mM Hepes, 2 mM L-glutamine, 100 μg/ml gentamycin, 20 U/ml human IL-2 (kindly provided by Dr. Hansjörg Hauser, German Research Center for Biotechnology, Braunschweig, Germany) and 10% human AB serum (ICN, Meckenheim, Germany) in 5% CO2 at 37°C. T221 CTL were stimulated with PHA (1 μg/ml; Sigma-Aldrich, Deisenhofen, Germany) every 3 weeks in the presence of the irradiated EBV-immortalized B cell lines PLH and HMY-2 [kindly provided by Dr. Reinhardt Schwartz-Albiez and Dr. Gerd Moldenhauer (German Cancer Research Center, Heidelberg, Germany), respectively] and irradiated PBMC. T221 CTL were separated from feeder cells by density gradient centrifugation before use in each experiment.

4.2 Immunostaining and flow cytometry

A549 cells that were either left untreated or treated with 1,300 U/ml human IFN-γ (Upstate Biotechnology, Lake Placid, NY) for 24 h were collected by rubber policemen rather than by trypsinization. Both T221 CTL and A549 cells were stained with mouse anti-human HLA-DR-PE (BD PharMingen, Hamburg, Germany) or isotype-matched mouse IgG2a-PE (Dianova, Hamburg, Germany). For detection of CD95, TRAIL, and TRAIL-R, cells were stained with mouse IgG1 Ab against human CD95 44, TRAIL, TRAIL-R1, -R2, -R3, -R4 (Alexis Biochemicals, San Diego, CA), or isotype-matched mouse IgG1 as a control, followed by biotinylated goat anti-mouse IgG1 and then streptavidin-PE (all from BD PharMingen). Two-color staining of T221 CTL was done using mouse anti-human CD3-FITC and CD8-PE mAb or mouse anti-human CD4-FITC and CD8-PE mAb (all from BD PharMingen). Surface staining was determined on a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany), and data were analyzed with CellQuest software (Becton Dickinson).

4.3 Cytotoxicity assay

A549 cells were cultured at a concentration of 2×105 cells/ml in 100 μl DMEM containing 10% FCS in flat-bottom 96-well plates in the presence or absence of 1,300 U/ml human IFN-γ for 24 h. Cells were then washed twice and cultured for a further 7 h with the agonistic anti-CD95 Ab anti-APO-1 (IgG3) (which recognizes an epitope on the extracellular domain of human CD95 13), leucine zipper (LZ)-TRAIL (which was produced essentially as described 45 and which is a stable trimer of TRAIL that induces apoptosis upon binding to TRAIL-sensitive cells 45), or with TNF-α (Biomol, Hamburg, Germany), either alone or in the presence of CHX (Boehringer Mannheim, Mannheim, Germany). Cells were collected, washed twice with cold PBS and double-stained with FITC-conjugated annexin V (Boehringer Mannheim) and 50 μg/ml PI (Sigma-Aldrich), according to the manufacturer's instructions. Apoptosis of gated (R1 gate in Fig. 2A) cells in side scatter (SSC) versus forward scatter (FSC) dot blots was then analyzed by FACS. In one experiment, A549 cells treated or untreated with human IFN-γ as described above were cultured for a further 20 h in medium alone or medium supplemented with anti-APO-1, protein A (Sigma-Aldrich), or a combination of the two. Apoptosis was verified by FACS analysis using the method of Nicoletti et al. 46. Briefly, cells were collected, washed and resuspended in a buffer containing 0.1% (v/v) Triton X-100, 0.1% (w/v) sodium citrate (both from Sigma-Aldrich), and 50 μg/ml PI. After incubation at 4°C in the dark for at least 14 h, apoptotic nuclei were quantified by FACS.

In co-culture assays, A549 cells were seeded into 100 μl DMEM containing 10% FCS in U-bottom 96-well plates at a density of 0.4×105 cells/ml. After 24 h, the cell number in arepresentative well was counted, and T221 CTL were added to each well at the indicated E/T ratios in 100 μl DMEM containing 10% FCS and 20 U/ml human IL-2 after two washes of A549 cells. Plateswere centrifuged at 800 rpm for 2 min, and cells were further incubated in the presence or absence of SEB (Sigma-Aldrich). Cells were collected at the indicated times of incubation, washed twice with cold PBS, double-stained with FITC-labeled annexin V and PI and analyzed by FACS as described above. To assay for contamination of T221 CTL in the R1 gate (see Fig. 2A), A549 cells were labeled with 2.5 μM CFSE (Molecular Probes, PoortGebouw, The Netherlands) at room temperature for 10 min, washed four times and cultured at a density of 0.4×105 cells/ml in 100 μl DMEM with 10% FCS in U-bottom 96-well plates for 24 h. T221 CTL equivalent to ten times the number of A549 cells were added to some wells, and cells were further cultured for 12 h in the presence of 0.1 μg/ml SEB as described above. The percentage of CFSE dye-positive cells in the R1 gate was analyzed by FACS.

To analyze the roles of several death systems in the killing of A549 cells by activated CD8+ T cells, NO synthase inhibitor L-NMMA (Sigma-Aldrich), vacuolar H+-ATPase inhibitor CMA (Calbiochem, Bad Soden, Germany), neutralizing Ab against CD95L NOK1 (BD PharMingen; 47), TRAIL-R2-Fc protein 45, TNFR2-Fc protein (EnbrelTM; obtained by the Heidelberg University, Pharmacy, Heidelberg, Germany), or isotype-matched control mouse IgG1 (BD PharMingen) or human IgG1 (Sigma-Aldrich) were preincubated with T221 CTL for 30 min. A549 cells were also preincubated with the broad-spectrum caspase inhibitor BD-fmk (Enzyme System, Livermore, CA) or L-NMMA for 30 min. In the apoptosis inhibition assay, A549 cellswere first labeled with 2.5 μM CFSE, then washed and subsequently cultured with T221 CTL and SEB in the presence or absence of several apoptosis-inhibiting reagents to minimize contamination ofA549 cells by T221 CTL. Collected cells were washed, stained with annexin V-PE (BD PharMingen) according to the manufacturer's instructions and then analyzed by FACS without gating in SSC versus FSCdot blots.

To examine whether activated CD8+ T cells induce DNA fragmentation in A549 cells, a JAM test was performed as described previously 48. Briefly, A549 cells were labeled by incubation with 10 μCi/ml [3H]thymidine (NEN, Neu-Isenburg, Germany) for 12 h at a density of 3×105 cells/ml. Cells were collected, washed, cultured at a density of1×105 cells/ml in 100 μl DMEM with 10% FCS in flat-bottom 96-well plates for 7 h, and then T221 CTL were added to each well at an E/T ratio of 5:1. The final volume per well was 200 μl DMEM with 10% FCS. Cells were centrifuged at 800 rpm for 2 min and co-cultured for 20 h. Cells were then harvested using a LKB/Wallace 1295–001 cell harvester (LKB Wallace, Turku, Finland).To collect all adherent cells, cells were trypsinized and re-harvested. Radioactivity bound to the filter was measured by scintillation counting in a 1205 betaplate counter (LKB Wallace). Percentages of DNA fragmentation were calculated using the following formula: DNA fragmentation (%) = (cpmspontaneous–cpmexperimental)/cpmspontaneous × 100. The value for cpmspontaneous was determined by incubating A549 cells with medium only.

4.4 Reverse transcription-PCR analysis

Total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany). cDNA was synthesized using Superscript II reverse transcriptase (GIBCO BRL) according to the manufacturer's instruction. PCR amplification was performed for 23–33 cycles with the following primer pairs: β-actin (sense: 5′-GAGGCCCAGAGCAAGAGAGG-3′; antisense: 5′-TCACCGGAGTCCATCACGAT-3′),TCR Vβ14 (sense: 5′-GTCTCTCGAAAAGAGAAGAGGAAT-3′; antisense: 5′-TTCTGATGGCTCAAACAC-3′), perforin (sense: 5′-CAGCCAACTTTGCAGCCCAGAAG-3′; antisense: 5′-GCAGGTCGTTAATGGAGGTGTGA-3′), granzyme A (sense: 5′-AGAGCTGCTCACTGTTGGG-3′; antisense: 5′-AGGAGACAATGCCCTGGG-3′), granzyme B (sense: 5′-TGCAGGAAGATCGAAAGTGCG-3′; antisense: 5′-GAGGCATGCCATTGTTTCGTC-3′), CD95L (sense: 5′-ATGTTTCAGCTCTTCCACCTACAGAAGGA-3′; antisense: 5′-ACCAGAGAGAGCTCAGATACGTTGACATA-3′), TRAIL (sense: 5′-CCCAATGACGAAGAGAGTATGAA-3′; antisense 5′-ACCATTTGTTTGTCGTTCTTTGTG-3′), TNF-α (sense: 5′-ACAAGCCTGTAGCCCATGTT-3′; antisense: 5′-AAAGTAGACCTGCCCAGACT-3′), IFN-γ (sense: 5′-GCAGAGCCAAATTGTCTCCT-3′; antisense: 5′-ATGCTCTTCGACCTCGAAAC-3′). To ensure that PCR analyses were in the linear range, reactions were performedusing various numbers of cycles for each primer pair, and products were analyzed by electrophoresis on 2% agarose gels. β-Actin expression levels were used to normalize the amounts of cDNA template. In all reverse transcription (RT)-PCR analyses, negative controls in which cDNA synthesis was done without reverse transcriptase showed no specific band after PCR.

4.5 Preparation of supernatants

A549 cells were plated (2×105 cells/well) in 24-well plates in DMEM with 10% FCS in the presence or absence of human IFN-γ (1,300 U/ml) and cultured for 24 h. After three washes, cells were cultured for a further 24 h in DMEM with 0.5% FCS and 40 U/ml human IL-2 in the presence or absence of SEB (0.3 μg/ml) and T221 CTL (E/T ratio = 8:1). Supernatants were collected, centrifuged for 10 min at 5,000 rpm, filtered through 0.22-μm filters and either used immediately or frozen at –80°C until further use. The supernatant from T221 CTL cultured for 24 h in the presence or absence of SEB (0.3 μg/ml) at the same cell density and in the same medium as described above was also collected.

4.6 ELISA assay

Cytokine analyses were carried out using sandwich ELISA for IFN-γ (Endogen, Woburn, MA) and TNF-α (BD PharMingen), according to the manufacturer's instructions.

4.7 Statistics

Statistical analysis was performed using the unpaired two-tailed Student's t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The authors thank Dr. Elisabeth Märker-Hermann for her generous gift of the CTL line, Drs. Reinhardt Schwartz-Albiez and Gerd Moldenhauer for providing EBV-immortalized B cell lines, and Dr. Hansjörg Hauser for providing human IL-2. We express gratitude to Drs. Takafumi Ohmura and Hisako Miyakawa for critical reading of the manuscript.

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  • 1
    Kovacs, E. J., Fibrogenic cytokines: the role of immune mediators in the development of scar tissue. Immunol. Today 1991. 12: 1723.
  • 2
    Ryu, J. H., Colby, T. V. and Hartman, T. E., Idiopathic pulmonary fibrosis: current concept. Mayo Clin. Proc. 1998. 73: 10851101.
  • 3
    Coker, R. K. and Laurent, G. J., Pulmonary fibrosis: cytokines in the balance. Eur. Respir. J. 1998. 11: 12181221.
  • 4
    Adamson, I. Y. R., Young, L. and Bowden, D. H., Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am. J. Pathol. 1988. 130: 377383.
  • 5
    Mason, R. J. and Williams, M. C., Type II alveolar epithelial cells. In Crystal, R. G. and West J. B. (Eds.) The lung. Raven Press, New York 1991, pp 235246.
  • 6
    Bitterman, P. B., Pathogenesis of fibrosis in acute lung injury. Am. J. Med. 1992. 92: S3943.
  • 7
    Ward, P. A. and Hunninghake, G. W., Lung inflammation and fibrosis. Am. J. Respir. Crit. Care Med. 1998. 157: S123129.
  • 8
    Bardales, R. H., Xie, S., Schaefer, R. F. and Hsu, S., Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am. J. Pathol. 1996. 149: 845852.
  • 9
    Guinee, D. G. Jr., Fleming, M., Hayashi, T., Woodward, M., Zhang, J., Walls, J., Koss, M., Ferrans, V. and Travis, W., Association of p53 and WAF1 expression with apoptosis in diffuse alveolar damage. Am. J. Pathol. 1996. 149: 531538.
  • 10
    Kuwano, K., Kunitake, R., Kawasaki, M., Nomoto, Y., Hagimoto, N., Nakanishi, Y. and Hara, N., p21Waf1/Cip1/Sdi1 and p53 expression in association with DNA strand breaks in idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 1996. 154: 477483.
  • 11
    Kuwano, K., Hagimoto, N., Maeyama, T., Fujita, M., Yoshimi, M., Inoshima, I., Nakashima, N., Hamada, N., Watanabe, K. and Hara, N., Mitochondria-mediated apoptosis of lung epithelial cells in idiopathic interstitial pneumonias. Lab. Invest. 2002. 82: 16951706.
  • 12
    Kunitake, R., Kuwano, K., Miyazaki, H., Kawasaki, M., Hagimoto, N., Fujita, M., Kaneko, Y. and Hara, N., Expression of p53, p21 (Waf1/Cip1/Sdi1) and Fas antigen in collagen vascular and granulomatous lung disease. Eur. Respir. J. 1998. 12: 920925.
  • 13
    Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Moller, P., Falk, W., Debatin, K. M. and Krammer, P. H., Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 1989. 245: 301305.
  • 14
    Yonehara, S., Ishii, A. and Yonehara, M., A cell-killing monoclonal antibody (anti-fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J. Exp. Med. 1989. 169: 17471756.
  • 15
    Kuwano, K., Miyazaki, H., Hagimoto, N., Kawasaki, M., Fujita, M., Kunitake, R., Kaneko, Y. and Hara, N., The involvement of Fas-Fas ligand pathway in fibrosing lung diseases. Am. J. Respir. Cell Mol. Biol. 1999. 20: 5360.
  • 16
    Hashimoto, S., Kobayashi, A., Kooguchi, K., Kitamura, Y., Onodera, H. and Nakajima, H., Upregulation of two death pathways of perforin/granzyme and FasL/Fas in septic acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 2000. 161: 237243.
  • 17
    Kuwano, K., Hagimoto, N., Kawasaki, M., Yatomi, T., Nakamura, N., Nagata, S., Suda, T., Kunitake, R., Maeyama, T., Miyazaki, H. and Hara, N., Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis. J. Clin. Invest. 1999. 104: 1319.
  • 18
    Shinbori, T., Matsuki, M., Suga, M., Kakimoto, K. and Ando, M., Induction of interstitial pneumonia in autoimmune prone mice by intratracheal administration of superantigen staphylococcal enterotoxin B. Cell. Immunol. 1996. 174: 129137.
  • 19
    Liu, A. N., Mohammed, A. Z., Rice, W. R., Fiedeldey, D. T., Liebermann, J. S., Whitsett, J. A., Braciale, T. J. and Enelow, R. I., Perforin-independent CD8+ T cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-α: relative insensitivity to Fas ligand. Am. J. Respir. Cell Mol. Biol. 1999. 20: 849858.
  • 20
    Zhao, M. Q., Amir, M. K., Rice, W. R. and Enelow, R. I., Type II pneumocyte-CD8+ T cell interactions: relationship between target cell cytotoxicity and activation. Am. J. Respir. Cell Mol. Biol. 2001. 25: 362369.
  • 21
    Marrack, P. and Kappler, J., The staphylococcal enterotoxins and their relatives. Science 1990. 248: 705711.
  • 22
    White, J., Herman, A., Pullen, A. M., Kubo, R., Kappler, J. W. and Marrack, P., The Vβ-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 1989. 56: 2735.
  • 23
    Wen, L.-P., Madani, K., Fahrni, J. A., Duncan, S. R. and Rosen, G. D., Dexamethasone inhibits lung epithelial cell apoptosis induced by IFN-γ and Fas. Am. J. Physiol. 1997. 273: L921929.
  • 24
    Coulter, K. R., Doseff, A., Sweeney, P., Wang, Y., Marsh, C. B., Wewers, M. D. and Knoell, D. L., Opposing effect by cytokines on Fas-mediated apoptosis in A549 lung epithelial cells. Am. J. Respir. Cell Mol. Biol. 2002. 26: 5866.
  • 25
    Kim, K. B., Choi, Y. H., Kim, I. K., Chung, C. W., Kim, B. J., Park, Y. M. and Yung, Y. K., Potentiation of Fas- and TRAIL-mediated apoptosis by IFN-γ in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element. Cytokine 2002. 20: 283288.
  • 26
    Redondo, M., Ruiz-Cabello, F., Concha, A., Hortas, M. L., Serrano, A., Morell, M. and Garrido, F., Differential expression of MHC class II genes in lung tumour cell lines. Eur. J. Immunogenet. 1998. 25: 385391.
  • 27
    Vermes, I., Haanen, C., Steffens-Nakken, H. and Reutelingsperger, C., A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods. 1995. 184: 3951.
  • 28
    Krammer, P. H., CD95 (APO-1/Fas)-mediated apoptosis: live and let die. Adv. Immunol. 1999. 71: 163210.
  • 29
    Kataoka, T., Shinohara, N., Takayama, H., Takaku, K., Kondo,S., Yonehara, S. and Nagai, K., Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 1996. 156: 36783686.
  • 30
    Robinson, B. W., Rose, A. H., Hayes, A. and Musk, A. W., Increased pulmonary gamma interferon production in asbestosis. Am. Rev. Respir. Dis. 1988. 138: 278283.
  • 31
    Fujiki, M., Shinbori, T., Suga, M., Miyakawa, H. and Ando, M., Role of T cells in bronchoalveolar space in the development of interstitial pneumonia induced by superantigen in autoimmune-prone mice. Am. J. Respir. Cell Mol. Biol. 1999. 21: 675683.
  • 32
    Segel, M. J., Izbicki, G., Cohen, P. Y., Or, R., Christensen, T. G., Wallach-Dayan, S. B. and Breuer, R., Role of interferon-γ in the evolution of murine bleomycinlung fibrosis. Am. J. Physiol. Lung Cell Mol. Physiol. 2003. 285: L12551262.
  • 33
    Ziesche, R., Hofbauer, E., Wittmann, K., Petkov, V. and Block, L. H., A preliminary study of long-term treatment with interferon gamma-1b and low-dose prednisolone in patients with idiopathic pulmonary fibrosis. N. Engl. J. Med. 1999. 341: 12641269.
  • 34
    Hein, R., Behr, J., Hundgen, M., Hunzelmann, N., Meurer, M., Braun-Falco, O., Urbanski, A. and Krieg, T., Treatment of systemic sclerosis with gamma-interferon. Br. J. Dermatol. 1992. 126: 496501.
  • 35
    Honore, I., Nunes, H., Groussard, O., Kambouchner, M., Chambellan, A., Aubier, M., Valeyre, D. and Crestani, B., Acute respiratory failure after interferon-gamma therapy of end-stage pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 2003. 167: 953957.
  • 36
    Shaw, E. G., Deming, R. L., Creagan, E. T., Nair, S., Su, J. Q., Levitt, R., Steen, P. D., Wiesenfeld, M. and Mailliard, J. A., Pilot study of human recombinant interferon gamma and accelerated hyperfractionated thoracic radiation therapy in patients with unresectable stage IIIA/B nonsmall cell lung cancer. Int. J. Radiat. Oncol. Biol. Phys. 1995. 31: 827831.
  • 37
    van Zandwijk, N., Groen, H. J., Postmus, P. E., Burghouts, J. T., ten Velde, G. P., Ardizzoni, A., Smith, I. E., Bass, P., Sahmoud, T., Kirkpatric, A., Dalesio, O. and Giaccone, G., Role of recombinant interferon-gamma maintenance in responding patients with small cell lung cancer. A randomised phase III study of EORTC Lung Cancer Cooperative Group. Eur. J. Cancer 1997. 33: 17591766.
  • 38
    Gern, J. E. and Busse, W. W., Relationship of viral infections to wheezing illness and asthma. Nat. Rev. Immunol. 2002. 2: 132138.
  • 39
    Eigler, A., Sinha, B., Hartman, G. and Endres, S., Taming TNF: stratagies to restrain this proinflammatory cytokine. Immunol. Today 1997. 18: 487492.
  • 40
    Ware, L. B. and Matthay, M. A., The acute respiratory distress syndrome. N. Engl. J. Med. 2000. 342: 13341349.
  • 41
    Pope, R. M., Apoptosis as a therapeutic tool in rheumatoid arthritis. Nat. Rev. Immunol. 2002. 2: 19.
  • 42
    O'Shea, J. J., Ma, A. and Lipsky, P., Cytokines and autoimmunity. Nat. Rev. Immunol. 2002. 2: 3745.
  • 43
    Ebnet, K., Kaldjian, E. P., Anderson, A. O. and Shaw, S., Orchestrated information transfer underlying leukocyte endothelial interactions. Annu. Rev. Immunol. 1996. 14: 155177.
  • 44
    Dhein, J., Daniel, P. T., Trauth, B. C., Oehm, A., Moller, P. and Krammer, P. H., Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface antigens. J. Immunol. 1992. 149: 31663173.
  • 45
    Walczak, H., Degli-Esposti, M. A., Johnson, R. S., Smolak, P. J., Waugh, J. Y., Boiani, N., Timour, M. S., Gerhart, M. J., Schooley, K. A., Smith, C. A., Goodwin, R. G. and Rauch, C. T., TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 1997. 16: 53865397.
  • 46
    Nicoletti, I., Migliorati, G., Paggliacci, M. C., Grignani, F. and Riccardi, C., A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 1991. 139: 271279.
  • 47
    Kayagaki, N., Kawasaki, A., Ebata, T., Ohmoto, H., Ikeda, S., Inoue, S., Yoshino, K., Okumura, K. and Yagita, H., Metalloproteinase-mediated release of human Fas ligand. J. Exp. Med. 1995. 182: 17771783.
  • 48
    Böhm, C., Hanski, M.-L., Gratchev, A., Mann, B., Moyer, M. P., Riecken, E.-O. and Hanski, C., A modification of the JAM test is necessary for a correct determination of apoptosis induced by FasL+ adherent tumor cells. J. Immunol. Methods 1998. 217: 7178.