Role of IFN-{gamma} and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury

Olivier Lesur,1,2 Marcel Brisebois,1,2,3 Alexandre Thibodeau,1,2,3 Frédéric Chagnon,1 Denis Lane,1 and Tamas Füllöp3

1Groupe de Recherche en Physiopathologie Respiratoire, 2Soins Intensifs Médicaux, and 3Groupe de Recherche en Gériatrie et Gérontologie, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

Submitted 30 October 2002 ; accepted in final form 3 August 2003


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In the present study, IFN-{gamma} exposure to primary cultures of rat type II epithelial cells (TIIP) upregulated membrane expression of the common {gamma}-chain of the IL-2 receptor (~2.5- to 4-fold increase) and redistributed receptor affinity in TIIP, as assessed by Western blot, cell, and tissue histochemistry and Scatchard analysis. As for restitution processes of the lung epithelium, functionality of IL-2R on TIIP was conditional to IFN-{gamma} exposure: 1) IFN-{gamma} priming promoted a fivefold increase of IL-2-driven TIIP locomotion (P < 0.05 vs. control at 100 U/ml) and 2) IFN-{gamma} coincubation with IL-2 reduced bleomycin-induced TIIP apoptosis in vitro by 25% (caspase-3 activity) and by ~70% (TdT-mediated dUTP nick end labeling/4',6'-diamidino-2-phenylindole assay) as well as in vivo by ~90% (caspase-3 activity; P < 0.05 vs. control). Sustained p42/44 extracellular signal-regulated kinase activity played a protective role in this process, whereas specific inhibition by PD-98059 (50 µM) significantly reversed bleomycin-induced TIIP apoptosis (P < 0.05 vs. control). From these in vitro and in vivo data, it is proposed that combinations of IFN-{gamma} and IL-2 can drive repair activity of TIIP by stimulating migration and preventing programmed cell death, both of which are speculated to be very fast restitution events after oxidant-induced acute lung injury.

interferon-{gamma}; interleukin-2 receptor


ACUTE LUNG INJURY (ALI) is observed in intensive care units on a daily basis and can evolve into a disastrous, life-threatening clinical pattern called acute respiratory distress syndrome (ARDS), with a mortality rate nearing 40% (5). Diffuse alveolar damage (DAD) is the related pathological entity observed in ARDS lung and is characterized by a major alteration of the air-blood interface (4, 58). Lung alveolar epithelial cells are usually the targeted injured cells of the distal air space in DAD, which become hyperactive during repair. Overlapping combinations of events, including spreading, migration, proliferation, differentiation, and apoptosis allow for either the onset or absence of restitution. Although an increasing number of cytokines are being recognized as effectors of these various processes leading to restitution (4, 39, 58), very little is known about restitution itself. On the other hand, residual epithelial cells bathe in the air space lining fluid, which contains increased and/or unbalanced amounts of pro- and anti-inflammatory cytokines (46). Accordingly, we were able to measure high concentrations of IL-2, a cytokine belonging to the proinflammatory family (55), in alveolar fluids of ARDS (40). Concomitant to this observation, IL-2 was also identified as an efficient antiapoptotic factor of polymorphonuclear neutrophil (PMN) death in vitro as well as in bronchoalveolar lavage fluids of ARDS patients (40, 47).

In fact, IL-2 exhibits various additional properties other than those identified in the immune system many years ago. Aside from modulating lymphocyte as well as PMN apoptosis (40, 47, 50), IL-2 can also target other cells in the nonimmune system and is already recognized as a direct modulator of the proliferation of several epithelial and nonepithelial cell cancers in vitro (14, 19, 43, 49, 61). Yet, its counterpart, the functional IL-2-receptor (IL-2R), consisting of various combinations of three known subunits, {alpha}, {beta}, and {gamma}, was first presumed to be exclusively present on lymphoid cell and macrophage membranes until it was later identified in several other cell types and tissues (19, 43, 55, 61). The {beta}- and {gamma}-subunits are shared by several IL receptors, including IL-4R, IL-7R, IL-9R, and IL-15R (55). Only combinations containing the {beta}/{gamma} complex (Kd 10-9 to 10-11 M) are capable of transducing the IL-2-mediated signal, whereas the {alpha}-chain is the binding subunit (55). It is recognized that intestinal and kidney epithelial cells as well as keratinocytes express functional IL-2R (12, 34, 41, 49, 53, 55). We have also previously characterized the presence of functional IL-2Rs in lung type II epithelial cells (or type II pneumocytes; TIIP). TIIP can respond to the combination of IFN-{gamma} and IL-2 by an increased proliferative activity (38). Although enhanced transcript expression of IL-2R{beta}{gamma} was observed, modulation of membrane expression was not screened nor were other functional activities assessed (38).

The signaling cascades of mitogen-activated protein (MAP) kinases are intimately committed in the control of cell growth/apoptosis and differentiation (15, 37, 56). It has become increasingly evident that alterations in these signaling pathways lead to very distinctive cell behaviors. MAP kinases include extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal kinases (JNKs), and p38 MAP kinases (60). Coordinated changes in these signaling pathways in an integrated network are targeted for the induction of apoptosis and can vary according to cell type (60).

Hence, the present study was designed to investigate the hypotheses that 1) IFN-{gamma} can modulate cellular expression of the signaling complex IL-2R{beta}{gamma}, 2) IFN-{gamma}-induced IL-2R modulation renders TIIP sensitive to IL-2 repair activity (i.e., migration and apoptosis), 3) p38, JNK, and ERK MAP kinases are differentially and coordinately involved in IL-2-driven prevention of bleomycin-induced TIIP apoptosis, and 4) ERK-sustained activation in TIIP is a rescue pathway of apoptosis and a marker of IL-2 preventive activity.


    MATERIALS AND METHODS
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Animals and Cells

Sprague-Dawley pathogen-free rats weighing 200–250 or 350 g were purchased from Charles River (St. Constant, PQ, Canada) and selected for cell isolation and experimental challenge, respectively. TIIP were purified as described previously (39) and maintained in plastic petri dishes for 48 h in RPMI 1640 with 10% FBS before being assayed. All animals received care in compliance with The Guide to the Care and Use of Experimental Animals from the Canadian Council of Animal Care (1993, CCAC, 2nd ed.), and the protocol was approved by our institution's internal animal ethics committee board.

Cytokines and Antibodies

Recombinant murine IFN-{gamma} and IL-2 were purchased from Pepro Tech. Rabbit anti-rat IL-2R{gamma}-chain M-20 and K-20 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). M-20 and K-20 are IgG polyclonal antibodies that recognize amino acid sequences mapping the COOH terminus and the NH2 terminus of mouse IL-2R{gamma}, as stated by the manufacturer. Rabbit anti-IL-2R{beta}-chain M-20 was also obtained from Santa Cruz Biotechnology and recognizes the IL-2R{beta}-chain from rat and mouse origin with sequence mapping at the COOH terminus of the precursor form of the mouse IL-2R{beta}-chain. Nonimmune normal rabbit antiserum was purchased from Santa Cruz Biotechnology. Neutralizing antibody NDS61 against rat IL-2R{alpha} (anti-CD25) was obtained from Serotec (Toronto, ON, Canada) and used as described previously (38). Anti-rat CD4 and CD45 (Biosource/Medicorp, Montreal, PQ, Canada) served as irrelevant antibodies in binding assays and immunohistochemistry. Anti-rat surfactant protein C (SP-C; M-10, Santa Cruz Biotechnology) was selected for further identification of TIIP in immunofluorescence (IF) assays.

Antibodies against phospho-p38 were obtained from Santa Cruz Labs, and antibodies against phospho-ERK, -JNK, and MEK/ERK inhibitor (PD-98059) were obtained from New England Biolabs (Mississauga, ON, Canada).


    IL-2R{beta}{gamma} Immunodetection
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Western blot and confocal microscopy analyses. TIIP were cultured in the presence or absence of IFN-{gamma} (400 U/ml) for 16–48 h. For Western blotting, cells were scraped and lysed for 30 min on ice (50 mM Tris, pH 7.4, 10% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 0.1 mg/ml aprotinin, 1 mM Na3V04, 10 mM NaF, 1 mM Na4P207, 1 mM PMSF, and 1x anti-protease cocktail; Boehringer Mannheim, Laval, PQ, Canada). Lysates were centrifuged at 13,000 g for 10 min at 4°C, and protein-normalized aliquots (25 µg) from supernatants were subjected to electrophoresis on 7.5% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked overnight in nonfat dry milk (NFDM) and incubated with rabbit anti-rat IL-2R{gamma} and -{beta} antibodies (K-20 and M-20), followed by exposure to donkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham, Oakville, ON, Canada). Specific bands were revealed using an ECL chemiluminescence kit (Amersham) according to the manufacturer's instructions and quantified as relative values using a densitometer analysis software (NIH Image, version 1.61).

Confocal microscopy analysis was carried out to confirm expression and distribution of IL-2R{gamma}{beta} on a cell to cell basis. TIIP were examined using a scanning confocal microscope (NORAN Instrument, Middleton, WI) equipped with a krypton/argon laser and coupled to an inverted microscope with a x40 oil-immersion objective (Nikon). TIIP were specifically plated on four-well Labtech slides (Nalge Nunc International, Naperville, IL) as described previously (38) and conditioned as detailed above for Western blotting assays. Cells were fixed with paraformaldehyde followed by quenching of free aldehydes for 30 min in 50 mM NH4Cl. Cells were permeabilized, and nonspecific sites were blocked in PTB (0.1% Triton X-100 and 5% BSA in PBS). Cells were then incubated for 2 h with rabbit anti-rat IL-2R{gamma} and -{beta} antibodies (K-20 and M-20) vs. isotypically matched nonspecific rabbit IgG at 2.0 µg/ml in PTB, followed by a 1-h incubation with FITC-conjugated goat anti-rabbit IgG (Sigma) 1:200 in PTB. After being washed and mounted, 15 randomly selected fields (at x40) of distinctive TIIP isolates were analyzed by microscopic examination for each experimental condition, using Image Analysis software (Intervision 2D analysis, NORAN Instruments). Relative expressions of IL-2R{beta}{gamma} for a given field were represented by the percentage of pixels ranging in intensity from 20 to 255 according to an eight-bit pseudocolor intensity scale. A baseline of 20 was arbitrarily chosen as the value of naturally occurring background cell fluorescence.

IL-2 binding assays. TIIP were harvested in lidocaine hydrochloride (2 mM, Sigma) after overnight preincubation in the presence or absence of IFN-{gamma} (400 U/ml). Human recombinant 3-[125I] iodotyrosyl IL-2 (specific activity 808 Ci/mmol, Amersham) alone or together with a 200-fold excess of nonlabeled IL-2 was added to normalized cell concentrations in microcentrifuge tubes and incubated at 4°C for similar sequential time intervals. Cells were then separated from unbound ligand by being layered onto 500 µl of fresh 20% sucrose and centrifuged for 3 min at 4°C. The tips of the microfuge tubes containing the cell pellets were cut, and cell-bound activity was measured in a gamma counter. Scatchard plot analysis was carried out after initial assessment of binding capacities with mixtures of 125I and unlabeled IL-2 in concentrations varying from 0.05 x 10-3 to 300 nM, with 30 min as the reference period of incubation. Control samples for binding specificity were run by coincubation with either blocking antibody (NDS61) to rat IL-2R or the isotype-matched anti-rat CD5 antibody (both 1:100, vol:vol).

Immunohistochemistry and immunogold labeling. Rats (350 g) were first anesthetized with xylazine/ketamine (20 and 50 mg/kg in, respectively) and then intratracheally instilled with 5,000 units of murine IFN-{gamma} (Pepro Tech) in PBS or with PBS alone (250 µl of total volume). Rats were allowed to recover overnight and then killed for lung sampling.

For IF studies, lung samples of ~1 cm3 were immediately fixed in 4% paraformaldhehyde at 4°C for 4 h, followed by an overnight wash in 20% cold sucrose, and then dehydrated and paraffin embedded. Five-micrometer slices were harvested and deposited on poly-L-lysine-coated slides. For histochemistry, rehydrated slides were incubated for 10 min at 37°C with pepsin (Digest-All3; Zymed, San Francisco, CA) for antigen unmasking. Nonspecific binding was prevented by incubation with 1% BSA-PBS. Slices were recovered with 0.1% BSA buffer containing anti-IL-2R K-20, M-20, and anti-rat SP-C M10 (2 µg/ml; Santa Cruz Biotechnology) or the irrelevant isotype-matched anti-rat CD5 antibody (both 1:100, vol:vol). Specific amplification was obtained by further incubation with monoclonal anti-rabbit Ig biotin conjugate (1:1,500, vol:vol) followed by QDot 605 streptavidin conjugate (Quantum Dot, Hayward, CA) for anti-IL-2R (red) and anti-goat FITC (Sigma, MO) for SP-C (green) stainings. Slides were mounted in Quantafluor medium (Kallestad) after being washed in buffer solution. Nuclear contrast was obtained by 4',6'-diamidino-2-phenylindole (DAPI) staining (2 µg/ml; Sigma).

For electron microscopy (EM) studies, semi-thick 100-µm cryostat sections, prepared from frozen lung specimens, were fixed for 1 h in 4% paraformaldehyde and exchanged overnight in 20% sucrose at 4°C. Sections were then exposed for 30 min to blocking buffer medium (0.002% Triton X-100, 0.02% NaN3, and 0.02 M glycine in PBS) and washed 4x 8 h in PBS at 4°C. Overnight incubation at 4°C was performed with K-20 or M-20 (2 µg/ml) or equivalent concentrations of nonimmune rabbit anti-serum, followed by serial washes and overnight exposure to gold-conjugated anti-rabbit IgG (10 nM in 1:10 dilution) at 4°C. Sections were postfixed in 3% glutaraldehyde followed by 1% osmium, washed in H2O2, and stained with 1% uranyl acetate in darkness before ethanol dehydration and Epon embedding. Ultrathin sections were harvested and stained with lead citrate before EM examination.

In addition to the procedures described above, specificity of immunostaining was further controlled by preincubating specific primary antibodies with terminal peptides of rat IL-2R{beta} and -{gamma} (10 µg/ml) used as immunogens (Santa Cruz Labs) and also by using anti-rat CD45 (Biosource) irrelevant antibodies in parallel experiments.

Functional Implication of IFN-{gamma}-Induced Alteration of IL-2R Expression on TIIP

TIIP migration in vitro. Migration of TIIP was performed as detailed previously (39). Cells were plated on plastic dishes for 48 h in RPMI 1640 with 10% FBS. Nonadherent cells were then removed, and the medium was replaced with either unsupplemented (control) or IFN-{gamma} (400 U/ml)-containing medium for an additional 24 h, before recovery of epithelial cells and locomotion challenge through 8-µm pore gelatin-coated polycarbonated filters (Nucleopore Canada, Toronto, ON) in 10-well Boyden chambers (Neuroprobe, Sin Can, Calgary, AL, Canada). Gradients for cell migration were created using an increasing scale of recombinant rat IL-2 concentrations (10–1,000 U/ml) in RPMI-0.1% BSA. Specificity of cell migration against IL-2 was assessed by preincubating epithelial cells for 30 min with blocking concentrations of NDS61 as described in binding assays.

TIIP apoptosis in vitro. TIIP were induced into programmed cell death by exposure to bleomycin (Bristol-Myers-Squibb Canada, 30 µg/ml) overnight (positive control) as established in preliminary assays. The influence of coexposing TIIP to IFN-{gamma} (400 U/ml), IL-2 (50 U/ml), or a combination of both, on bleomycin-induced apoptosis was observed using two different methodologies.

TdT-mediated dUTP nick end labeling assay. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed on paraformaldehyde-fixed cells. TUNEL reagents were purchased from Boehringer Mannheim. Briefly, TIIP were cultured on four-well Labtech (Nunc) slides and incubated with blocking solution (0.3% H2O2 in methanol) for 1 h at room temperature. Cells were then incubated with Triton X-100 for 2 min on ice to allow permeabilization. Subsequent end labeling with TdT (0.3 U/µl) in TdT buffer containing 2 µM fluorescein 16-uridine triphosphate was performed for 1 h at 37°C. TIIP were then incubated with anti-fluorescein antibody conjugated with horseradish peroxidase for 30 min at 37°C and stained with diaminobenzidine. DAPI nuclear counterstaining was processed in parallel, and TIIP were definitely considered as apoptotic and counted per se when observed to be both peroxidase positive and DAPI negative under an ultraviolet filter. Double-blind observations of five to ten x40 power fields were performed for a total of 500 cells in each condition. The % inhibition score was established by the ratio of TUNEL-positive and DAPI-negative cells to total nuclei.

Caspase-3 activity assay. Adherent and nonadherent TIIP were scraped from six-well microtiter plates and centrifuged. Cell pellets were resuspended in 100 µl of lysis buffer (10 mM Tris, pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM NaPi, 10 mM NaPPi) and incubated for 30 min on ice. Cells were then centrifuged at 13,000 rpm for 10 min, and the supernatant was retrieved for caspase-3 assay. Forty micrograms of extracted proteins (48) were incubated with 20 µg of fluorescent substrate Ac-DEVD-AFC (Bio-Rad) in 1 ml of reaction buffer (10 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT) for 2 h at 37°C. AFC fluorescence was measured with a Hitachi F-2000 xenon lamp fluorescence spectrophotometer. Excitation wavelength was 395 nm, and the maximum emission wavelength was defined using an emission spectrum from 420 to 600 nm.

TIIP apoptosis in vivo. The time course of bleomycin-induced lung epithelial cell apoptosis in vivo was determined in preliminary experiments that demonstrated that cell death progressed continuously according to a sigmoidal slope reaching a plateau at day 5 postintratracheal instillation (data not shown). Hence, four groups of rats (350 g, n = 3) were first anesthetized with xylazine/ketamine (20 and 50 mg/kg im, respectively) and then intratracheally instilled with 250 µl of PBS, bleomycin (1.5 units), bleomycin (1.5 units) plus IFN-{gamma} (1,000 units) plus IL-2 (750 units), or IFN-{gamma} (1,000 units) plus IL-2 (750 units) without bleomycin, and left to recover until day 5. Rats were then killed, and lung samples were either extracted for caspase-3 assay or fixed and processed for TUNEL/DAPI assay as detailed above.

Signaling Patterns of MAPK Activation/Deactivation in Bleomycin-Induced TIIP Apoptosis: Effect of IFN-{gamma}/IL-2 Exposure

For this set of experiments, in vivo challenged lung tissues were first studied at day 5 postinstillation. Protein-normalized aliquots (25 µg) from supernatants of lysed specimens were subjected to electrophoresis on 10% polyacrylamide gels and transferred onto PVDF membranes for Western blotting. Membranes were blocked overnight in NFDM and incubated with rabbit anti-phosphorylated MAPK JNK, ERK, and p38, followed by secondary antibody incubation with either horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) for JNK and ERK or horseradish peroxidase-conjugated goat anti-mouse IgM for p38.

In parallel with these specimen assays, TIIP were isolated from healthy rats and cultured as described above. TIIP were further cultured for 24 h in RPMI-0.1% BSA in the presence or absence of bleomycin (50 µg/ml) with or without IL-2 (60 ng/ml) after conditioning or nonconditioning to IFN-{gamma} (400 U/ml). Membranes were first hybridized with anti-phosphorylated p44/42ERK antibodies and then washed before rehybridizing with anti-p44/42ERK protein antibodies for control loading (New England Biolabs). Specific bands were revealed using an ECL chemiluminescence kit (Amersham) according to the manufacturer's instructions and were quantified in relative values with densitometer analysis software (NIH Image, version 1.61). Concomitantly, TIIP cultured on four-well Labtech slides and exposed to the combination of bleomycin and IL-2/IFN-{gamma} were additionally submitted to incremental concentrations of the MEK/ERK specific inhibitor PD-98059 (5–50 µM) before assessing the potential inhibitory impact on cell apoptosis by TUNEL/DAPI assay as described above.


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 MATERIALS AND METHODS
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Primary cultures of TIIP are an incomparable tool for studying lung epithelial cell biology, which is not possible by the use of undifferentiated cell lines. The presence of the IL-2R{beta}{gamma} transduction complex is perceived as a pivotal element for receptor functionality in lymphoid cells, with IFN-{gamma} as an upregulator of its expression (38). Although we have previously reported the expression of IL-2R{alpha} and upregulation by IFN-{gamma} by TIIP, we were also able to observe transcript expression and upregulation of IL-2R{beta}{gamma} with IFN-{gamma} exposure by semiquantitative PCR (38).

IFN-{gamma} Upregulates Membrane Expression of the IL-2R Signaling Complex by Increasing the Presence of the {gamma}-Chain

IL-2R{gamma} was expressed by epithelial cells from rat lungs instilled with IFN-{gamma} in vivo as seen in Fig. 1, A–H. SP-C-containing TIIP also exhibited IL-R{gamma} membrane expression (Fig. 1, D, F, and G). IFN-{gamma} also enhanced IL-2R{gamma} protein expression by a magnitude of ~2.5- to 4-fold of that seen in isolated TIIP by Western blotting and confocal IF levels, respectively (Fig. 2, A and C). {gamma}-Chain overexpression with IFN-{gamma} exposure was not paralleled by similar enhanced expression of subunit {beta}, the other component of the IL-2R transduction complex (at the immunodetection level, Fig. 2B). However, binding assays further suggest the presence of two distinct populations of TIIP receptors respectively exhibiting intermediate and relatively low affinities with apparent approximate Kd of 0.37 ± 0.09 and 6.69 ± 1.2 nM, and an estimated receptor number of 1,593 ± 58 and 11,247 ± 159 per cell, respectively. IFN-{gamma} incubation altered the distribution of these two receptor populations with a Kd of 0.25 ± 0.02 and 8.87 ± 1 nM and an estimated receptor number of 2,213 ± 55 and 36,322 ± 38 per cell, respectively (Fig. 3, A and B). Specific binding was inhibited by competition with blocking anti-CD25 antibodies (P < 0.05 vs. control; Fig. 3C).



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Fig. 1. IL-2 receptor (IL-2R{gamma}) chain immunostaining in IFN-{gamma}-instilled rat lung tissue is shown. The {gamma}-chain was identified through the use of a polyclonal antibody raised against the NH2-terminal amino acid sequence of the precursor form of the mouse IL-2R{gamma}. Tissues were processed as described in MATERIALS AND METHODS. A: electron microscopy (EM) gold immunostaining demonstrating specific membrane labeling of a lung epithelial cell at the alveolar-capillary barrier (ACB) level (magnification, x65,000). BM, basement membrane; EN, capillary endothelial cell; EC, erythrocyte; C, capillary lumen; EP, epithelial cell. ACB overview (magnification, x12,000) is shown at bottom, left. B: high magnification (x90,000) of an EP labeling at the ACB level. C: negative control of EM gold immunostaining at the ACB level (magnification, x45,000) using an irrelevant (CD45) primary antibody as described in MATERIALS AND METHODS. D, F, G: fluorescence microscopy of rat lung distal air spaces: variable intensity of IL-2R{gamma} (red)- and surfactant protein C (green; with blue nuclear counterstaining)-combined expressions by TIIP (type II pneumocytes; focused cells are indicated by arrows) are presented (D, magnification, x400; F and G, magnification, x1,000). Respective negative controls, as described above for EM labeling, are shown in E (magnification, x400) and H (magnification, x1,000).

 


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Fig. 2. Modulation of IL-2R expression by TIIP: effect of IFN-{gamma} exposure. TIIP were either not exposed or exposed to IFN-{gamma} (400 U/ml) for 16–48 h. A: Western blot of cell extracts normalized for protein concentration before loading bar charts representing relative optical density (OD) values of IL-2R{gamma} expression as a function of time (a); corresponding scanned gel (1 out of 3 experiments, b). B: a and b, similar presentation as described above but with {beta}-chain. C: confocal image analysis as described in MATERIALS AND METHODS: a, control and assay panels (magnification, x40); b, IFN-{gamma}-exposed TIIP (magnification, x100). Shown are quantitative values of relative fluorescence intensity. *P < 0.05 vs. control. Abs, antibodies.

 


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Fig. 3. Scatchard plot analysis and specific binding of 125I-labeled IL-2 to TIIP at equilibrium. Cells were incubated for 30 min at 4°C in binding medium containing serial dilutions of 125I-IL-2 after no preexposure (A) or 16-h preexposure with 400 U/ml IFN-{gamma} (B). IL-2-binding assays were performed in the absence ({square}) or presence ({blacksquare}) of neutralizing anti-rat IL-2R (NDS61) antibodies 1:250 (vol:vol; solid lines vs. dashed lines). Binding data are expressed as bound/free IL-2 (pM) plotted against bound molecules per cell. C: IFN-{gamma}-preincubated TIIP were exposed to 125I-labeled IL-2 for 60 min at 4°C to detect nonspecific binding. Nonspecific binding was detected in the presence of 200-fold molar excess of cold IL-2. Competition assays were run in the presence of neutralizing anti-IL-2R (NDS61). Nonspecific binding was subtracted. Results from 3 repeated experiments in triplicate are shown. *P < 0.05 vs. condition without antibodies.

 

IFN-{gamma}-Mediated IL-2R{gamma} Overexpression Contributes in Revealing IL-2-Induced TIIP Promigratory Activity

These experiments were aimed at further demonstrating that INF-{gamma} not only enhances chain/subunit expression but is essential to IL-2R functionality in TIIP. Preincubation with IFN-{gamma} revealed IL-2-driven TIIP migration, which peaks at 100 U/ml (Fig. 4). The level of TIIP migration obtained by combining IL-2/IFN-{gamma} was significant and represented as much as two-thirds of total cell locomotion obtained with EGF, a well-recognized epithelial cell chemokinetic cytokine. TIIP locomotion was chemotactically driven without significant random migration on checkerboard analysis (data not shown). Specific neutralization of IL-2R in TIIP plated in the upper wells of Boyden chambers reversed this effect by >75% (Fig. 4).



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Fig. 4. Effect of IFN-{gamma} preexposure on IL-2-induced TIIP migration. Assays were performed in Boyden chambers, as described in MATERIALS AND METHODS. Cells were plated for 48 h in the presence or absence of murine IFN-{gamma} (400 U/ml) in culture medium. Migration of harvested cells was studied by exposure to a gradient of rat IL-2 (10–1,000 U/ml) compared with control conditions (C) and EGF-containing medium (25 ng/ml). Effect of coincubation of migrating TIIP with neutralizing antibodies (Abs, NDS61) is shown. Data are expressed as means ± SD of 6 independent assays performed in duplicate. *P < 0.05 vs. control.

 

IFN-{gamma}-Mediated IL-2R{gamma} Overexpression Is Essential to IL-2-Driven Inhibition of TIIP Apoptosis Triggered by the Oxidative Agent Bleomycin In Vitro and In Vivo

As hypothesized, the combination of IL-2/IFN-{gamma} was able to partially prevent bleomycin-induced TIIP apoptosis, whereas IL-2 or IFN-{gamma} alone did not influence TIIP apoptosis in unstimulated cultures (data not shown). Upstream TIIP caspase-3 activation was reduced by almost 25% when IL-2/IFN-{gamma} incubation was combined with bleomycin exposure (Fig. 5A). Downstream DNA alterations as assessed by TUNEL-positive and DAPI-negative cells were reduced by as much as 70% (Fig. 5B). In this respect, the level of TIIP apoptosis ranged from 47.8 ± 7% when induced by bleomycin comparative to a baseline value of 7.9 ± 4% in control conditions. Similarly, the combination of bleomycin with IL-2/INF-{gamma} intratracheal instillations reversed the activation of caspase-3 and TUNEL/DAPI index in vivo by almost 90% (Fig. 6, A and B).



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Fig. 5. Preventing the effect of IL-2 on bleomycin-induced TIIP apoptosis in vitro. All cells were exposed to bleomycin (30 µg/ml, positive control) with or without IFN-{gamma} (400 U/ml), IL-2 (50 U/ml), or a combination of both as described in MATERIALS AND METHODS. TIIP were then either extracted for interaction with fluorescent caspase-3 substrate (A) or processed for peroxidase TdT-mediated dUTP nick end labeling (TUNEL) assay with 4',6'-diamidino-2-phenylindole (DAPI) nuclear counterstaining (B). A: inhibition of caspase-3 activity in bleomycin-exposed TIIP (positive control) with cytokine exposure. Bleomycin exposure usually induced a 4.5 ± 0.5-fold increase of cellular extract OD. B: inhibition of genomic fragmentation in bleomycin-exposed TIIP (positive control) with cytokine exposure. Effect of neutralizing antibodies (Abs, NDS61) is shown (top); TIIP were considered apoptotic when exhibiting both positive peroxidase nuclear staining and negative DAPI staining (bottom; magnification, x400). Open arrows indicate living cells, and closed arrows indicate apoptotic cells. Bleomycin exposure usually induced a 10 ± 1-fold increase of peroxidase-positive/DAPI-negative cultured cells (27 ± 0.8%, n = 8). Data are expressed as means ± SD and are representative of at least 4 independent assays performed in duplicate. For each condition, 20 randomly selected fields corresponding to almost 1,000 cells were screened by 2 different readers (blinded to experimental conditions) and provided a consistent reproducibility (<5% interobserver variability). *P < 0.05 vs. control; §P < 0.05 vs. absence of antibody exposure.

 


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Fig. 6. Preventing the effect of IL-2/IFN-{gamma} combination on bleomycin-induced lung epithelial cell apoptosis in vivo. Data presented are from 3 separate experiments performed in duplicate. Agents were instilled intratracheally at the following concentrations: bleomycin (1.5 U/ml), IFN-{gamma} (1,000 units), and IL-2 (750 units). Experimental rats were killed at day 5 as described in MATERIALS AND METHODS. Lung tissues were either extracted for caspase-3 assay as described in Fig. 5A or processed for TUNEL/DAPI assay (B). A exhibits modulation of caspase-3 activity with PBS instillation (solid bars) or with various combinations of the above agents (gray bars). *P < 0.05 vs. bleomycin intratracheal instillation. B illustrates respective lung areas from PBS-instilled (left; negative control), bleomycin-instilled (middle), and bleomycin + IL-2/IFN-{gamma}-instilled lungs (right; magnifi-cation, x400). Similar criteria to that described in Fig. 5 were used to record and differentiate apoptotic from nonapoptotic cells.

 

IL-2-Driven Inhibition of Bleomycin-Induced TIIP Apoptosis Involves Sustained Activation/Phosphorylation of ERK MAPK

Constitutive phosphorylation of MAPK was generally low in lung tissue with the exception of JNK (Fig. 7). Bleomycin instillation induced unequivocal and sustained activation of all three MAP kinases at day 5. Instillation of the IL-2/IFN-{gamma} cytokine combination did not significantly influence JNK but did increase p38 and pERK phosphorylation. On the other hand, IL-2/IFN-{gamma} in the presence of bleomycin clearly reduced JNK and p38 activation (by 31 and 43%, respectively), whereas ERK was increased by a factor of several hundredfold (for ERK1) to more than a hundredfold (for ERK2) in total lung extracts (Fig. 7). Of note, addition of IL-2/IFN-{gamma} to bleomycin in instillates triggered more ERK2 (~7-fold) than ERK1 (~2-fold) activation. Average OD obtained in vivo experiments for control, bleomycin-instillated lungs, bleomycin and IL-2/IFN-{gamma}-instillated lungs, and IL-2/IFN-{gamma} lungs were: 767 ± 27, 970 ± 53, 610 ± 150, 550 ± 84 (for pJNK); 11 ± 8, 479 ± 242, 380 ± 22, 40 ± 1 (for p-p38); 41 ± 4, 162 ± 31, 251 ± 157, 67 ± 10 (for p-ERK1); and 19 ± 3, 35 ± 7, 63 ± 12, 11 ± 3 (for p-ERK2), respectively. IFN-{gamma} instilled alone or together with bleomycin did not significantly alter MAPK activation patterns compared with conditions in combination with IL-2 (data not shown). In vitro assays using isolated TIIP confirmed a sustained overactivation/phosphorylation of ERK (ERK2 over ERK1, similar to that in vivo) after a 24-h exposure to the combination of bleomycin and IL-2/IFN-{gamma} (~1.6-fold increase vs. bleomycin alone, P < 0.05; Fig. 8). Concomitant blockage of ERK activation by addition of the specific inhibitor PD-98059 was able to restore bleomycin-induced cell apoptosis by almost 50% (at 50 µM; Fig. 9).



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Fig. 7. MAPK signaling in IL-2/IFN-{gamma}-driven prevention of bleomycin-induced lung apoptosis in vivo. Experimental rats were killed at day 5 as described in MATERIALS AND METHODS and in Fig. 6. Total lung extracts were run on SDS gels for Western blot. Data shown are from 3 separate experiments performed in duplicate and represent lung protein extracts of rats intratracheally instilled with the following solutions: lane 1, PBS (control); lane 2, bleomycin; lane 3, bleomycin with IFN-{gamma} and IL-2; and lane 4, IFN-{gamma} and IL-2. Bands of phosphorylated MAPK p-JNK, p-p38, and p-ERK1/ERK2 are shown with corresponding relative OD values. JNK, c-jun NH2-terminal kinases.

 


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Fig. 8. MAPK ERK signaling in IL-2/IFN-{gamma} prevention of bleomycin-induced lung epithelial cell apoptosis in vitro. TIIP were isolated, cultured, and exposed to experimental conditions as described in MATERIALS AND METHODS. Cell extracts were obtained after an exposure time of 24 h to bleomycin, bleomycin with IFN-{gamma} and IL-2, or control conditions, and run on SDS gels for Western blot. A: bars represent OD values of phospho-ERK obtained in 4 independent experiments and expressed as means ± SD. *P < 0.05 vs. control and §P < 0.05 vs. bleomycin alone. B: the gel shown is representative of the above experiments. Blot shown was first probed with anti-pERK1/2 and then stripped and reprobed with anti-ERK1/2. Values of OD ratio were quantified by NIH Image analyzer.

 


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Fig. 9. Effect of MEK/ERK pathway inhibition on bleomycin-induced lung epithelial cell apoptosis in vitro. TIIP were isolated, cultured, and exposed to experimental conditions as described in MATERIALS AND METHODS and in Fig. 5B. In these experiments, cellular apoptosis recorded after combinations of bleomycin and IL-2/IFN-{gamma} exposures (as described in MATERIALS AND METHODS and in Fig. 8) was considered as the (positive) control baseline condition. Apoptosis was assessed using the TUNEL/DAPI assay. The reversal effect of coincubating TIIP with incremental concentrations of PD-98059 (5–50 µM), a MEK/ERK pathway-specific inhibitor, is shown and compared with the apoptosis cell index obtained at baseline (in %). Data are expressed as means ± SD of 4 independent assays performed in duplicate. *P < 0.05 vs. positive control.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 IL-2R{beta}{gamma}...
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-2Rs consist of various combinations of three subunits, namely {alpha}, {beta}, and {gamma}, in which the {beta}{gamma}-chains/subunits are the functional signaling pairing of the receptor (55). In a previous study, we described membrane expression of the {alpha}-connection unit in the alveolar epithelial cells L2 and TIIP (38) and established the upregulatory activity of IFN-{gamma} on this particular subunit as well as on the three recognized subunit transcripts. We also established that this TIIP-bearing IL-2R was fully functional, as demonstrated by proliferation assays (38). In the present work, IFN-{gamma} is now described as an upregulator of the {gamma}-subunit but not the {beta}-subunit. In addition, we provide further evidence of IL-2R activity on TIIP whereby IFN-{gamma} preexposure leads to IL-2-induced TIIP migration and partially prevents bleomycin-induced TIIP apoptosis in vitro and lung cell apoptosis in vivo.

Experimental exposure of animals to oxidant stress, such as hyperoxia or bleomycin, causes a form of ALI very similar to human ARDS, characterized by edema and inflammatory cell recruitment and activation along with the release of a myriad of cytokines, proteases, and reactive oxygen species. Recent studies suggest that the major histopathological features of oxidant-mediated lung injury both in vivo and in vitro are not only cell apoptosis but also failure to resolve inflammation and tissue repair (27). The balance between epithelial cell migration/proliferation and programmed cell death is an essential homeostatic mechanism in most living tissues (3, 4, 58). It is also critically essential in branching morphogenesis of epithelia during organogenesis (17, 32) and in tissue repair processes after injury (3, 4, 16, 17, 23, 24, 32, 35, 57).

Many cytokines have already been identified as growth inducers for TIIP (4, 39), some of which also exhibit promigratory properties (38, 39). Interestingly, several conditions, such as the combination with extracellular matrix (ECM) proteins or primering exposure to other molecules or cytokines, can reveal the chemokinetic activity of certain cytokines, which is totally inefficient outside of that given context (presumably by changing cell activation states or receptor membrane patterns). For instance, IL-1{beta} and TNF-{alpha} can prime EGF-induced TIIP migration and repair (18, 39), whereas ECM laminin (39) and fibronectin (29, 39) further promote EGF-, hepatocyte growth factor (HGF)-, and keratinocyte growth factor-induced TIIP migration (28, 29, 39). IFN-{gamma} alleviates EGF-induced (39) but optimizes HGF-induced TIIP migration by upregulating c-Met/HGF receptor expression (28, 29, 39, 44). Herein, IFN-{gamma} was shown to generate TIIP sensitivity to IL-2 at a level of locomotion close to that observed with EGF, the best known cytokine promoter of lung epithelial cell migration. This functional alteration is likely linked to a modified pattern of IL-2R expression with special upregulation of membrane {gamma}c expression of the signaling complex. In an in vitro model, IL-2 has already been described as enhancing intestinal epithelial cell restitution by a combination of migration and spreading, independent of cell proliferation or IFN-{gamma} preexposure (14). Unfortunately, epithelial cell migration initiated by remnant cell clusters presumably occurs within the very first hours from the time of insult and is hence very difficult to assess in vivo at the present time.

Apoptosis or programmed cell death is also perceived as an important pathway of lung tissue restitution. Although epithelial cell shedding is considered an initial marker of lung injury, cell apoptosis may be largely responsible for the disappearance of TIIP hyperplasia during the latter resolution phase of ALI (3, 4, 6, 16, 20, 21, 57). Apoptosis can be induced by many different signals, some from interaction with hormones, cytokines, or other growth factors, others in response to oxidative stress and other forms of cell injury (2, 53). Apoptosis is easier to monitor in vivo and in vitro compared with cell migration and, more importantly, can be assessed by a number of tools. In the present study, the combination of IFN-{gamma} and IL-2 was able to prevent lung epithelial cell apoptosis both in vitro and in vivo.

Of note, IL-15, a monocyte-derived cytokine functionally linked to IL-2 as well as sharing two of its three membrane receptors, acts as a protective agent in kidney tubular and skin epithelium (8, 9, 41, 53). Indeed, IL-15-/- kidneys exhibit enhanced epithelial apoptosis in a model of T cell-dependent nephritis in which tubular epithelial cells constitutively express {alpha}-, {beta}-, {gamma}-chain receptor (53). On the other hand, IL-2 withdrawal clearly triggers T cell apoptosis with Ras and CD18 dependencies (50), whereas its withdrawal is otherwise contributive (with IL-15) in preventing PMN programmed cell death (10, 40, 47).

IFN-{gamma}, in addition to upregulating IL-2R expression, is already known to upregulate potential antigen presenting cell and immune functions of lung and intestinal epithelial cells by increasing ICAM-1, polymeric Ig receptor expression, and class II major histocompatibility molecules (26, 50). Although IFN-{gamma} exhibits proapoptotic properties in transformed epithelial cells (33, 59), it is not an obvious programmed cell death inducer in normal cells and tissues. Indeed, neither IFN-{gamma} nor IL-2 was proapoptotic for TIIP by themselves in this study (data not shown). IFN-{gamma} also exhibits some antifibrotic potential in a bleomycin-induced lung fibrosis model, often attributed to transforming growth factor-{beta} downregulation (22). Extreme levels of alveolar epithelial cell apoptosis can disorganize lung tissue homeostasis. Excessive apoptosis is a limiting event in the repair process and is an inciting condition to fibrosis (3, 16, 35, 36), whereas failing apoptosis, on the other hand, favors sustained altered phenotype with dysmetaplasia and propensity to cancer development, both conditions documented in idiopathic pulmonary fibrosis.

There is nothing clearly established as to the control of apoptosis in alveolar epithelial cells other than the fact that the Bcl-2 family does not appear to be involved and that Fas is expressed by these cells (2024, 35, 36). Fas ligation can induce massive epithelial cell apoptosis in liver and lung, with subsequent organ failure and fibrosis into the lung (23, 24, 32, 35, 36). In bleomycin-induced fibrosis (a model of T cell-dependent fibrosis), prevention of excessive scarring can be achieved by either administration of a soluble form of Fas (fusion protein), by anti-Fas ligand antibody (23, 24, 32, 35, 36), or by an angiogenesis converting enzyme inhibitor (57). Expression of functional Fas is obvious in TIIP in vitro as well as in vivo (23, 24, 57). However, infiltration of lung tissues by lymphocytes expressing Fas ligand or soluble forms of Fas ligand may also contribute to the induction of alveolar epithelial cell apoptosis after bleomycin intratracheal instillation in animals (23, 24) or in ARDS patients (42). Interestingly, Fas ligation-induced massive hepatocyte apoptosis is prevented by IL-15 as well as by HGF (8, 9, 32). In this study, IL-2 was able to reduce bleomycin-induced alveolar epithelial cell apoptosis by almost two-thirds, as assessed by TUNEL assay.

Mechanisms behind this prevention are not well understood. MAPK are upstream kinases involved in cytokine-induced cell signaling, whereas JNK, p38 kinase, and ERK are differentially activated during cell signaling, depending on the functional pathway that is targeted (56, 60). Oxidants have been shown to activate JNK, which in turn leads to phosphorylation of its targets c-jun and activates transcription factor ATF-2 (56, 60). Herein, lung tissues exposed to oxidant bleomycin all exhibited activation of MAPK at day 5 postchallenge, whereas coinstillation with IL-2/IFN-{gamma} further induced ERK-sustained phosphorylation. This latter event was correlated with a decreased apoptotic index. In these lungs, TIIP apoptosis prevention was associated with sustained ERK activation/phosphorylation but with normalized levels of p38 and JNK activities (data not shown). Moreover, selective inhibition of MEK/ERK phosphorylation was not related to caspase-3 activity (data not shown) but instead with downstream DNA alteration. In this respect, one of the more quoted functions of MAPK pathway is its role in apoptosis. p38 And JNK are usually inversely coordinated in ERK activation, and this relative balance may determine susceptibility to apoptosis induction, although there are no preset rules: resulting signaling routes can be cell/tissue specific, trigger specific, and/or time dependent (11, 25, 45, 52). Sustained ERK activation/phosphorylation is mostly related to apoptosis prevention in epithelial repair and branching/morphogenesis (17, 25, 31). For instance, hyperoxia, ultraviolet, or cadmium carcinogen exposures have enabled us to determine ERK-sustained activation as a rescue pathway for epithelial cell apoptosis (7, 11, 13, 30).

In summary, the combination of IL-2/IFN-{gamma} is operative for epithelial cell repair activity, based on enhanced functional IL-2R expression by TIIP. Its antiapoptotic effect on TIIP appears to be mediated by sustained ERK activation. This observation is relevant in that it may have a potential therapeutic impact after oxidant injury, such as bleomycin exposure, and in the prevention of fibrosis consecutive to repair failure of the epithelium.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by the Centre de Recherche Clinique Sherbrooke and the Association Pulmonaire du Québec. O. Lesur is a Research Scholar of the Fonds de Recherche en Santé du Québec.


    FOOTNOTES
 

Address for reprint requests and other correspondence: O. Lesur, Groupe de Recherche en Physiopathologie Respiratoire, Centre de Recherche Clinique, CUSE Sherbrooke, Québec, Canada, J1H 5N4 (E-mail: olivier.lesur{at}USherbrooke.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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