Protective role of retinoic acid from antiproliferative action of TNF-alpha on lung epithelial cells

Valérie Besnard, Elodie Nabeyrat, Alexandra Henrion-Caude, Katarina Chadelat, Laurence Perin, Yves Le Bouc, and Annick Clement

Département de Pneumologie Pédiatrique, Institut National de la Santé et de la Recherche Médicale U515, Hôpital Trousseau Assistance Publique-Hôpitaux de Paris, Université Paris VI, 75012 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor (TNF)-alpha is a key molecule in lung inflammation. We have established the insulin-like growth factor binding protein 2 (IGFBP-2) as a marker associated with the growth arrest of lung alveolar epithelial cells (AEC). Here, we studied the effects of TNF-alpha on AEC proliferation and the putative protective role of retinoic acid (RA). We documented an antiproliferative action of TNF-alpha that was reversible only at 24 h and then became irreversible with induction of apoptosis. TNF-alpha treatment was associated with a dramatic induction of IGFBP-2. To discover the mechanism of action of IGFBP-2, we further tested the mitogenic potential of IGF-I to counteract TNF-alpha inhibition. Addition of IGF-I to the TNF-alpha containing medium did not stimulate proliferation, whereas des(1-3)IGF-I, an analog of IGF-I that bears low affinity for IGFBPs, was able to restore cell growth. Interestingly, we observed that RA abrogated TNF-alpha -induced growth arrest and that this effect was associated with a dramatic decrease in IGFBP-2 expression. These results suggest a protective role of RA from TNF-alpha antiproliferative action, through mechanisms involving modulation of IGFBP-2 production.

lung epithelial cells; inflammation; insulin-like growth factor; proliferation; retinoic acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LUNG INFLAMMATION is a critical response for the protection of the respiratory system from infectious as well as noninfectious insults. Although this response is usually beneficial, it can also be deleterious. In particular, chronic inflammation can be viewed as the result of uncontrolled proinflammatory events and inefficiency of repair mechanisms.

The multifunctional cytokine tumor necrosis factor (TNF)-alpha is one of the key mediators involved in the inflammatory response and its role in the course of inflammation has been extensively investigated (12, 22). Indeed, TNF-alpha displays a wide range of activities, including immunoregulatory and mitogenic functions, and consequently is a central component of the initiation and the progression of inflammatory processes.

Among the various structures that compose the lung, alveoli are especially sensitive to a number of insults. After injury, a rapid initiation of the repair processes is critical to maintain alveolar architecture integrity and gas exchange function. Repair of the alveolar structure is dependent on the proliferative response of the alveolar epithelial cells and the mesenchymal cells. Recent studies have provided some information on the influence of TNF-alpha on fibroblast proliferation during lung injury and repair (13, 23). In several experimental models, it has been reported that pulmonary inflammation and fibrosis could be prevented by injection of anti-TNF-alpha antibodies or TNF-alpha antagonists (35). Yet little is known on the influence of TNF-alpha on the alveolar reepithelialization process, whether beneficial or deleterious.

Repair of the alveolar epithelium is controlled by the ability of the stem cells, the type 2 cells, to proliferate and undergo transition into type 1 cells (1, 2). Among growth factors regulating lung cell proliferation, the insulin-like growth factors (IGF) are mitogenic peptides with an autocrine/paracrine action on lung epithelial cell proliferation (26, 37). The actions of IGF are regulated by a family of high-affinity IGF binding proteins (IGFBP) (19). IGFBPs display opposite effects on proliferation. They can exert antiproliferative action either through IGF-dependant mechanisms by sequestering the IGF from the IGF receptor or through IGF-independent mechanisms. Previously, we reported the involvement of several components of the IGF system in the control of proliferation of type 2 alveolar epithelial cells. Particularly, blocking of type 2 cell proliferation induced by various situations such as serum deprivation, oxidant exposure, or glucocorticoid treatment was found to be associated with accumulation of IGFBP-2 (5, 28, 29). Interestingly, we recently provided data indicating that retinoic acid (RA) could stimulate type 2 cell proliferation and that this effect was associated with a decrease in IGFBP-2 expression (30). Retinoids, including retinol and RA derivatives, have been shown to be involved in the processes of lung repair after injury (6, 7). Several reports have found vitamin A deficiency to be associated with extensive alterations of the epithelial structure that could be reversed by RA treatment. Massaro and Massaro (24) showed that postnatal treatment with RA increased the number of pulmonary alveoli in rats. Moreover, they provided data indicating that RA could reverse the effects of elastase-induced alveolar damage in rats (25). These results fit in well with the current understanding of the effect of retinoids, including retinol and RA, on lung repair.

To provide information on the influence of TNF-alpha on the repair capacity of the alveolar epithelium, we chose to analyze the effect of TNF-alpha on the proliferative response of alveolar type 2 epithelial cells. Experiments were performed with a rat type 2 cell line that has been shown in previous studies to regulate some aspects of proliferation in a fashion similar to that of primary type 2 epithelial cells (8, 10). Our findings document an antiproliferative action of TNF-alpha , an effect that was associated with an increased expression of IGFBP-2. To document any protective role of RA to reverse the TNF-alpha effect, we also examined the consequence of RA treatment on TNF-alpha -induced growth inhibition of type 2 epithelial cells. We provide data indicating that RA was able to restore the proliferative capacity of the cells through mechanisms that involve a downregulation of IGFBP-2.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture

The type 2 cell line used in this study was derived from rat primary neonatal type 2 cells and has been extensively studied (10). Cells were grown in Earle's MEM (GIBCO-BRL, Grand Island, NY) supplemented with 4 mM glutamine, 50 units of penicillin/ml, 50 µg of streptomycin/ml, and 10% fetal bovine serum (Eurobio) in 5% CO2-95% air atmosphere at 37°C.

Cells treated with TNF-alpha were exponentially grown at a density of 4 × 104 cells/cm2, washed, and further cultured in serum-free medium containing various concentrations of recombinant human TNF-alpha (R&D System) for the indicated durations. Stock solutions of TNF-alpha were prepared at a concentration of 10 µg/ml in PBS containing 0.1% of BSA (Sigma, St. Louis, MO) and stored at -80°C. RA (Sigma) was prepared at a concentration of 10 mM in 100% ethanol and stored at -80°C. In all experimental conditions, cell viability was tested using trypan blue.

Cells treated with IGF-I or des(1-3)IGF-I were exponentially grown at a density of 4 × 104 cells/cm2, washed, and further cultured in medium containing either vehicle (10 mM HCl) or 10 nM recombinant human (rh) IGF-I protein (GroPep Pty) or 10 nM rh des(1-3)IGF-I protein (GroPep Pty), with or without TNF-alpha (10 ng/ml), in combination or not with RA (1 µM).

For experiments with conditioned medium, cells were washed three times with serum-free medium and incubated for an additional 8 h in serum-free medium.

For each protocol, three independent experiments were performed.

Proliferation Studies

Cell number assay. Cell proliferation was evaluated by measurement of cell number as previously described (10). Briefly, cells were harvested with trypsin-EDTA and counted in triplicate using a hemocytometer.

DNA synthesis assay. For autoradiography of labeled nuclei, cells were incubated for 24 h in medium containing 2 µCi/ml [methyl-3H]thymidine (60-70 Ci/mmol), as previously described (9). The plates were then washed three times with cold PBS, fixed with methanol, air dried, and coated with NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Twenty-four hours later, they were developed with Kodak Rapid Fix. After being stained with Giemsa, an average of 300 cells in random fields was examined at a magnification of ×400, and labeled nuclei were counted.

Flow cytometry analysis. The flow cytometric assay was performed after the DNA was stained by the propidium iodide method. Briefly, exponentially growing cells were treated with TNF-alpha and other reagents for the indicated durations. Floating and attached cells were harvested, washed, and centrifuged (1,000 g for 10 min). Pellets were resuspended in physiological serum (0.9% NaCl) to reach a cell concentration of 5 × 105-106 cells in 400 µl. The cell suspension was fixed in 4 ml 70% ethanol. The fixed cell suspension was allowed to stand for 16 h at 4°C. Cells were centrifuged and stained by the addition of propidium iodide-staining solution (50 µg/ml propidium iodide) in the presence of 100 µg/ml RNase A (Sigma) for 30 min at 37°C in the dark. DNA content was analyzed through a FACStar plus flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Apoptotic cells were defined as those exhibiting lower relative fluorescence than the G0/G1 peak.

Western Immunoblotting

The conditioned medium was then harvested, centrifuged (1,000 g for 10 min) to remove debris and unattached cells, desalted on Sephadex G-25 disposable columns (Amersham Pharmacia Biotech, Buckinghamshire, UK), and lyophilized (18, 30). The pellet was dissolved in a volume of 2× Laemmli buffer accordingly to the number of cells (40 µl for 8 × 105 cells). Equal volumes of samples were loaded for each experimental condition and were electrophoresed through an 11% SDS-polyacrylamide gel. Proteins were transferred onto 0.45-µm nitrocellulose (NC) membranes (Bio-Rad, Richmond, CA). Membranes were blocked 2 h at room temperature in PBS plus 0.2% Tween 20 (PBS-T) containing 10% skim milk. Incubation of the membrane was performed using the rabbit anti-bovine IGFBP-2 at 1:1,000 dilution (UBI, Lake Placid, NY) in 5% milk-PBS for 20 h at 4°C. Membranes were then washed three times in PBS-T and incubated for 1 h at 37°C with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Amersham Pharmacia Biotech) diluted at 1:6,000 in 5% milk-PBS. ECL was performed according to the manufacturer's instructions (ECL Western blotting, Amersham Pharmacia Biotech). Membranes were then exposed to autoradiography film (Hyperfilm-ECL, Amersham Pharmacia Biotech).

For IGF-II immunoblotting, 2 ml of conditioned medium (2 × 106 cells equivalent) were concentrated on Centricon C10 (Millipore, Bedford, MA), lyophilized, and loaded on a 15% gel under nonreducing conditions. rIGF-II (100 ng) and protein extracts (250 µg) from normal serum were used as controls and loaded on the same gel to measure IGF-II protein in conditioned media. After transfer, the blots were probed with an anti-rat IGF-II monoclonal antibody (UBI) at 1:500 dilution. This antibody was specific to rat and hIGF-II and showed less than 10% cross-reactivity with IGF-I.

Western Ligand Blotting

Conditioned media were collected after incubation of cells in basal medium and prepared as indicated above. The ligand blotting experiments were performed as previously described (29). Briefly, the lyophilized samples containing the secreted proteins were dissolved in a volume of 1× Laemmli buffer adjusted to cell number and analyzed on SDS-PAGE (11% polyacrylamide) under nonreducing conditions. The proteins were electrotransferred onto an NC filter, and the membranes were washed for 1 h at 4°C in 5 mM Tris · HCl, pH 7.4, and 150 mM NaCl (TBS) containing 0.2% Tween, then incubated for 48 h at 4°C with a mixture of 125I-IGF-I and 125I-IGF-II (200,000 counts/min each) in TBS and 1 mg/ml gelatin (Serva, Heidelberg, Germany). After being washed, the binding proteins were visualized by autoradiography. Relative molecular weight was estimated by running a prestained molecular-weight standard.

IGF-I Assay

Each conditioned medium were desalted on Sephadex G25 disposable columns (Amersham Pharmacia Biotech) and lyophilized. Then the lyophilysates were reconstituted with 2 ml of 0.01 N HCl and ultrafiltrated on Centricon C30 (Millipore). After lyophilization and reconstitution with RIA buffer (0.1 M phosphate buffer, pH 7.4, 1 mg/ml IGFBP-free BSA; Biomerieux, Paris, France), each sample were assayed by RIA using anti-IGF-1 antibodies provided by Drs. Closet, Frankenne, and Hennen (Liege, Belgium) as previously described (17, 18).

Statistical Analysis

Results were reported as the means ± SE. Data were analyzed using ANOVA, followed, if possible, by Mann-Whitney U-test for multiple comparisons against control conditions. Significance was assigned for P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of TNF-alpha on Type 2 Alveolar Epithelial Cell Proliferation

We first assessed the effects of TNF-alpha on type 2 alveolar cell proliferation. Exponentially growing cells were washed and cultured for 24, 48, and 72 h in serum-containing medium with or without TNF-alpha (10 ng/ml). We evaluated the rate of proliferation by counting the number of viable cells. Treatment with TNF-alpha was associated with a rapid block of cell proliferation (Fig. 1A). This growth arrest was associated with a decreased number of cells that could initiate DNA synthesis: the percentage of labeled nuclei was 53 ± 6% at 72 h vs. 98 ± 2% in conditions without TNF-alpha . We also tested the effects of TNF-alpha on cells cultured in the absence of serum (basal medium). Proliferative cells were washed and cultured for the indicated durations in basal medium with or without various concentrations of TNF-alpha . As shown in Fig. 1B, TNF-alpha induced a block of proliferation at all concentrations with a more prominent effect at 10 ng/ml. Indeed, a significant decrease in cell number was observed after 24 h of culture in the presence of TNF-alpha compared with culture conditions without TNF-alpha . This was associated with a lower number of cells that could initiate DNA synthesis, as assessed by the amount of [3H]thymidine incorporation. After 48 h of culture with TNF-alpha , the percentage of labeled nuclei was 49 ± 8% (vs. 78 ± 6% in conditions without TNF-alpha , P < 0.05).


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Fig. 1.   Effects of tumor necrosis factor (TNF)-alpha on lung alveolar epithelial cell proliferation. A: proliferative cells were cultured in serum-containing medium with diluent solution (control) or with TNF-alpha (10 ng/ml). At the end of culture, cells were harvested, and cell number was measured using a hemocytometer. B: proliferative cells were cultured in basal medium with diluent solution (control) or with various concentrations of TNF-alpha . At the end of culture, cells were harvested, and cell number was measured using a hemocytometer. Results are expressed as the means ± SE of 3 experiments performed in triplicate. * P < 0.05 vs. control conditions.

We then examined restimulation of epithelial cell proliferation after TNF-alpha treatment. Cells were first cultured in basal medium with or without TNF-alpha (10 ng/ml) for 24 or 48 h and then replaced in serum-containing medium for an additional 24 to 72 h (Fig. 2). In the experimental conditions of a 24-h TNF-alpha treatment, cells rapidly resumed proliferation (Fig. 2A). By contrast, after a 48-h TNF-alpha treatment, no increase in cell number could be observed when cells were returned to serum-containing medium (Fig. 2B).


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Fig. 2.   Reversibility of TNF-alpha effects on lung alveolar epithelial cell proliferation. Proliferative cells were cultured for 24 (A) or 48 h (B) in basal medium with diluent solution (control) or with TNF-alpha (10 ng/ml) and were then replaced in serum-containing medium for an additional 24-72 h. At the end of culture, cell number was determined. Results are expressed as means ± SE of 3 experiments performed in triplicate. * P < 0.05 vs. no restimulation conditions at 24 or 48 h.

Based on these data and to assess the putative protective action of RA, we chose to use culture conditions without serum (30, 31).

Modulation by RA of TNF-alpha Effects on Cell Proliferation

Based on previous results indicating that RA treatment could stimulate proliferation of alveolar epithelial cells under basal conditions in a dose-dependent manner, we asked whether the effects of TNF-alpha on cell growth could be modulated by RA (30). Proliferative cells were washed and cultured for the indicated durations in serum-free medium with or without TNF-alpha (10 ng/ml), in combination or not with RA (1 µM) (Fig. 3). As previously reported, when serum-deprived cells were treated with RA, an induction of proliferation was observed with a significant increase in cell number (30). Interestingly, the stimulatory effect of RA on cell proliferation could still be observed in the presence of TNF-alpha for 24 and 48 h. This was associated with a high number of cells that could initiate DNA synthesis, as assessed by [3H]thymidine incorporation experiments. After 48 h of culture with TNF-alpha in the presence of RA, the percentage of labeled nuclei was 68 ± 5% (vs. 49 ± 2% in condition without TNF-alpha , P < 0.05).


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Fig. 3.   Modulation by retinoic acid (RA) of TNF-alpha effects on lung alveolar epithelial cell proliferation. Proliferative cells were washed and cultured for the indicated durations in basal medium with diluent solution (control), TNF-alpha (10 ng/ml), RA (1 µM), or with TNF-alpha (10 ng/ml) in combination with RA (1 µM). A: at the end of culture, cells were harvested, and cell number was measured using a hemocytometer. Results are expressed as the means ± SE of 3 experiments performed in triplicate. * P < 0.05 vs. control conditions. B: cells were labeled with [3H]thymidine as described in MATERIALS AND METHODS. Results are expressed as means ± SE of 3 experiments performed in triplicate. * P < 0.05 vs. control conditions.

To document the effect of TNF-alpha on apoptosis, we stained cells with propidium iodide and analyzed them by flow cytometry (Table 1). The percentage of apoptotic cells gradually increased with the duration of TNF-alpha treatment: after 48 h the mean percentage was 53.6 ± 5.7%. When cells were treated with RA, the stimulatory effect on type 2 cell proliferation was associated with a significant decrease in the percentage of cells undergoing apoptosis (P < 0.05 vs. control conditions). Finally, when type 2 cells were treated with TNF-alpha in combination with RA, the increase in apop totic cell number was completely abolished within 16 h of treatment (P < 0.05 vs. TNF-alpha conditions).

                              
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Table 1.   Effects of TNF-alpha and RA on lung alveolar epithelial cell apoptosis

Taken together, these results indicated that RA could abrogate the effects of TNF-alpha on cell growth and on apoptosis. To determine whether the stimulatory action of RA on cell proliferation could still be observed after an initial exposure to TNF-alpha , proliferative cells were washed and cultured for 48 h in basal medium with or without TNF-alpha (10 ng/ml), RA (1 µM) being added to the cultures for the first 24 h or the last 24 h (Fig. 4). The increase in cell number was significantly higher in the experimental conditions where RA was added during the first 24 h of TNF-alpha treatment.


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Fig. 4.   Proliferative cells were washed and cultured for the indicated durations in basal medium with diluent solution (control) or TNF-alpha (10 ng/ml) in presence of RA (1 µM) for 24 h or 48 h. For the 48-h conditions, RA was either not added (-) or added during the 48 h of culture (+RA 0-48 h), during the first 24 h of culture (+RA 0-24 h), or during the last 24 h of culture (+RA 24-48 h). At the end of culture, cells were harvested, and cell number was measured using a hemocytometer. Results are expressed as means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. control conditions; P < 0.05 vs. TNF-alpha without RA at 48 h; diamond P < 0.05 vs. TNF-alpha with RA for 48 h.

Modulation by RA of TNF-alpha Effects on IGFBP-2 Secretion

To gain some insights into the mechanisms involved in the effects of TNF-alpha on cell proliferation and the protective role of RA, we explored the effects of TNF-alpha and RA on IGFBP-2 expression. Indeed, type 2 cell growth arrest induced under our experimental conditions was constantly associated with an increased expression of IGFBP-2 (5, 28, 29). Conversely, low level of IGFBP-2 was secreted from cells allowed to proliferate in serum containing medium. Thus in cells cultured in basal medium with or without TNF-alpha , in combination or not with RA, secreted IGFBP was assessed by Western blotting. Results are shown in Fig. 5. IGFBP-2 antiserum recognized a 32-kDa band. Using ligand blotting analysis, we confirmed that this 32-kDa band was the only IGFBP accumulated in conditioned media of type 2 cells (data not shown). Compared with low levels in proliferative cells, the secretion of IGFBP-2 was increased at the various time points in the presence of TNF-alpha at a significantly higher level than in the conditioned media of serum-deprived cells. Interestingly, when cells were treated with TNF-alpha in combination with RA, results showed that RA was able to inhibit TNF-alpha induction of IGFBP-2 secretion. As previously reported, the induction of IGFBP-2 protein was reduced when serum-deprived cells were cultured in the presence of RA (30).


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Fig. 5.   Modulation by RA of TNF-alpha effects on IGFBP-2 secretion in lung alveolar epithelial cells. Samples of conditioned media of proliferative cells (P) cultured for the indicated durations in basal medium with diluent solution (control), TNF-alpha (10 ng/ml), RA (1 µM), or with TNF-alpha (10 ng/ml) in combination with RA (1 µM) were analyzed by Western immunoblotting using a polyclonal antibody specific to bovine insulin-like growth factor binding protein (IGFBP)-2, as described in MATERIALS AND METHODS. A: autoradiogram of signals for IGFBP-2 from a representative experiment. An additional band was detected which will need to be further characterized. B: the histogram shows a quantitative representation of IGFBP-2 protein levels obtained from laser densitometric analysis of 3 independent experiments. Results were expressed in arbitrary units (a.u.). * P < 0.05 vs. control conditions.

Effects of IGF-I and des(1-3)IGF-I on Cell Proliferation

To further document the role of IGFBP-2 in the antiproliferative action of TNF-alpha , we investigated whether IGFBP-2 could modulate interactions between IGF and IGF type 1 receptor that mediate mitogenic response, through influences on both the bioavailability and distribution of IGFs in the extracellular environment. Therefore, we performed experiments using IGF-I and the amino terminally truncated IGF-I analog des(1-3)IGF-I, which can bind to IGF-IR with the same affinity as native IGF-I but bears very low affinity for IGFBPs. We examined the effects of IGF-I and des(1-3)IGF-I on cell proliferation in the absence or presence of TNF-alpha and/or RA. Based on previous data, concentrations of 10 nM IGF-I and des(1-3)IGF-I were used (29). Proliferative cells were washed and cultured for the indicated durations in basal medium containing vehicle, IGF-I, or des(1-3)IGF-I, with or without TNF-alpha (10 ng/ml), in combination or not with RA (1 µM). We evaluated the rate of proliferation by counting the number of viable cells. As shown in Fig. 6, A and B, in control conditions as well as in conditions with TNF-alpha alone, no effect of IGF-I on cell number could be observed. By contrast, a significant increase in cell number was documented when IGF-I was replaced by des(1-3)IGF-I. When serum-deprived cells were treated with RA in the presence or absence of TNF-alpha , both IGF-I and des(1-3)IGF-I could stimulate cell proliferation (Fig. 6, C and D).


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Fig. 6.   Effects of insulin-like growth factor (IGF)-I and des(1-3)IGF-I on lung alveolar epithelial cell proliferation. Proliferative cells were washed and cultured for the indicated durations in basal medium containing either the vehicle (hatched bars), 10 nM IGF-I (open bars) or 10 nM des(1-3)IGF-I (solid bars) with (A) diluent solution (control), (B) TNF-alpha (10 ng/ml), (C) RA (1 µM), or (D) TNF-alpha (10 ng/ml) in combination with RA (1 µM). At the end of culture, cells were harvested, and cell number was measured using a hemocytometer. Results are expressed as the means ± SE of 3 experiments performed in triplicate. * P < 0.05 vs. vehicle conditions.

Effects of TNF-alpha on IGF Production

To determine whether the antiproliferative action of TNF-alpha was associated with changes in the expression of IGFs, we examined the effects of TNF-alpha on IGF-I and IGF-II production. Cells were cultured in basal medium with or without TNF-alpha in combination or not with RA for 24 h or 48 h. The conditioned media were recovered and assayed for IGF-I by RIA and for IGF-II by Western blotting. Results obtained for IGF-I are shown in Fig. 7. Serum-deprived cells progressively accumulated IGF-I in the conditioned media. The presence of TNF-alpha significantly lowered the release of IGF-I. Treatment with RA did not significantly affect the level of IGF-I either in the absence or presence of TNF-alpha compared with proliferative cells. For IGF-II, the level of expression was barely detectable and was not modified by TNF-alpha and/or RA treatments (data not shown). These data indicate that IGF-I is not responsible for the proliferative response induced by RA after TNF-alpha treatment.


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Fig. 7.   IGF-I production by lung alveolar epithelial cells. Samples of conditioned media from proliferative cells (P) cultured for the indicated durations in basal medium with diluent solution (control), with TNF-alpha (10 ng/ml), RA (1 µM), or with TNF-alpha (10 ng/ml) in combination with RA (1 µM) were assayed by RIA as described in MATERIALS AND METHODS. Results are expressed as the means ± SE of 3 experiments performed in triplicate. * P < 0.05 vs. control conditions.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The alveolar microenvironment after acute lung injury plays a critical role in the repair process and, therefore, in the proper healing of the gas exchange structure. Repair is initiated by an extensive proliferative response that leads to reepithelialization of the alveolar surface. The mechanisms involved in this response imply the contribution of a number of mediators, including the major proinflammatory cytokine TNF-alpha . The role of TNF-alpha on the repair potential of the alveolar epithelium has been so far poorly investigated. In the present study we provide data indicating that TNF-alpha exerts an antiproliferative action on lung alveolar epithelial type 2 cells. We also show that TNF-alpha -induced growth arrest is associated with an increased expression of IGFBP-2. Moreover, we demonstrate that the antiproliferative effects of TNF-alpha are reversed by RA, which is associated with a downregulation of IGFBP-2.

The effects of TNF-alpha on cell proliferation have been reported to vary depending on the cell types (11, 13, 14, 34, 39). In the present work, we showed that TNF-alpha treatment of lung epithelial cells accelerated the decrease in the labeling index and growth arrest. This growth arrest was reversible only for a maximal duration of a 24-h TNF-alpha exposure. When cells were exposed to TNF-alpha for 48 h, they could not resume proliferation when replaced in serum-containing medium. This observation was associated with a dramatic increase in number of apoptotic cells. These results suggest that TNF-alpha is able to trigger two steps in the responses to inflammatory injury: first a reversible growth arrest of lung epithelial type 2 cells followed by an apoptotic process when lesions are more important. Such biphasic response has already been observed in rat fetal brown adipocytes maintained in primary culture where TNF-alpha treatment resulted in an inhibition of proliferation and induction of apoptosis (36). Also, histological findings of extensive damage of the alveolar epithelium reported in transgenic mice that overexpressed TNF-alpha in the alveolar epithelial type 2 cells are consistent with our present observation of antiproliferative effects of TNF-alpha on type 2 epithelial cells (27, 41).

There is now evidence for an important role of retinoids in the development, maturation, and homeostasis of the lung. Based on the current understanding of RA action, it seems that RA displays anti-inflammatory properties that take place at the various steps of the inflammatory response (16, 33). In a vitamin A deficiency rat model, Baybutt et al. (3) showed that scattered inflammation was observed in the vitamin A-deficient animals. RA influence also occurs at later stages of the inflammatory response by promoting re-epithelialization. In previous studies, we provided data indicating that RA could stimulate alveolar epithelial cell proliferation in serum-free conditions, i.e., in the absence of growth factors (30). In the present work, we demonstrate that RA could also exert mitogenic effects on epithelial cells in inflammatory conditions. Indeed, we found that the antiproliferative action of TNF-alpha was no longer observed in the presence of RA. Addition of RA at different time points revealed that RA action occurred mainly within the first 24 h of TNF-alpha treatment, suggesting an early function in the modulation of cell proliferation.

To provide information on the mechanisms potentially involved in the TNF-alpha -induced growth arrest of lung epithelial cells, we focused on the IGF system, and mainly on IGFBP-2. The interest for IGFBP-2 is explained by previous reports indicating that IGFBP-2 is the main IGFBP produced by these cells. IGFBP-2 production is dramatically increased in conditions of serum deprivation or glucocorticoid and oxidant exposure. Data reported herein show for the first time an increase in IGFBP-2 expression upon TNF-alpha treatment. From these results, it could be suggested that the growth inhibitory action of TNF-alpha may involve a reduction of IGF-I bioavailability and bioactivity resulting from the competition for IGF-I between IGFBP-2 and the IGF type I receptor. Effects of TNF-alpha on the IGF system have been reported in situations where TNF-alpha was associated with growth arrest (4, 32, 38). In particular, Katz et al. (20) showed that TNF-alpha together with interferon-gamma exerted an antiproliferative action on human salivary gland tumor cells through an increase of the bound form of IGF-1 with IGFBP-3, which led to reduce the availability of IGF-1 and, consequently, its mitogenic effect. Our results using des(1-3)IGF-I showed that, in situations of growth arrest associated with a dramatic accumulation of IGFBP-2, IGF-I could not stimulate proliferation, whereas des(1-3)IGF-I was able to promote cell growth. These data provide evidence for a role of IGFBP-2 in the process of type 2 cell growth arrest through mechanisms involving a competition for IGF-I between IGFBP-2 and IGF-1R.

In addition, our results demonstrate that TNF-alpha effects on IGFBP-2 expression are modulated by RA. Indeed, the decreased production of IGFBP-2 by RA was associated with a mitogenic effect of both IGF-I and des(1-3)IGF-I. The observations that RA could modulate IGFBP-2 expression and abrogate TNF-alpha -induced growth arrest of lung epithelial cells suggest that RA may have a dominant effect. The mechanisms involved in this protective action of RA need to be further studied. Several reports have documented the influence of RA on the IGF system and have shown that RA may regulate IGFBPs at the transcriptional and/or posttranscriptional level in a cell-specific manner (15, 21, 40). Kim et al. (21) investigated the effects of RA on IGFBPs in human hepatoma cells. They found that RA treatment decreased IGFBP-1 and IGFBP-3 mRNA in PLC/PRF/5 cells and caused a downregulation of phosphorylated IGFBP-1 in PLC/PRF/5 and Hep G2 cells. Our present observation of a protective action of RA could also involve changes in TNF-alpha receptor expression. Indeed, Totpal et al. (42) showed that RA treatment led to a downregulation of p60 and p80 forms of TNF-alpha receptors, which subsequently desensitized the cells to TNF-alpha . Studies are currently being pursued to characterize the mechanisms by which RA could reverse TNF-alpha induced growth arrest in alveolar epithelial cells.

To conclude, our results demonstrate that TNF-alpha exerts an antiproliferative action on lung epithelial cells through mechanisms that involve IGFBP-2. We also show that RA can restore the proliferative capacity of the cells through pathways that include downregulation of IGFBP-2. This protective action of RA towards TNF-alpha may have important consequences in vivo. Indeed, the present data raise the possibility that patients with inflammatory diseases might benefit from vitamin A supplementation during inflammatory injury. At this stage, further studies are required to characterize the signaling pathways and the factors involved, particularly the mechanisms by which RA plays a protective role against TNF-alpha -induced lung injury.


    ACKNOWLEDGEMENTS

We thank Marie-Claude Miesch for technical assistance.


    FOOTNOTES

V. Besnard was supported by a grant from the Fondation pour la Recherche Médicale. This work was supported by Association Claude Bernard, Chancellerie des Universités de Paris (Legs Poix), Ligue Nationale contre le Cancer (Comite de Paris), Association pour la Recherche contre le Cancer, University Paris VI.

Address for reprint requests and other correspondence: A. Clement, Département de Pneumologie Pédiatrique, Hôpital Trousseau, 26, Ave Dr. Netter, 75012 Paris, France (E-mail: annick.clement{at}trs.ap-hop-paris.fr).

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.

10.1152/ajplung.00368.2001

Received 18 September 2001; accepted in final form 23 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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