Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0504288 on August 17, 2004

Published online before print August 17, 2004
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(Journal of Leukocyte Biology. 2004;76:1019-1027.)
© 2004 by Society for Leukocyte Biology

IL-4-induced macrophage-derived IGF-I protects myofibroblasts from apoptosis following growth factor withdrawal

Murry W. Wynes*,{dagger}, Stephen K. Frankel{ddagger},§ and David W. H. Riches*,{dagger},{ddagger},§,1

* Program in Cell Biology, Department of Pediatrics, and
{ddagger} Interstitial Lung Disease Program, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado; and
{dagger} Department of Immunology and
§ Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver

1 Correspondence: Program in Cell Biology, Department of Pediatrics, Neustadt Room D405, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: richesd{at}njc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of idiopathic pulmonary fibrosis (IPF) is associated with myofibroblast accumulation and collagen deposition in the lung parenchyma. Recent studies have suggested that the fibroproliferative response is associated with immune deviation toward a T helper cell type 2 (Th2) cytokine profile. In addition, myofibroblast accumulation may be the result of resistance to physiologic apoptosis. If and how these events are linked remain largely unknown. Insulin-like growth factor-I (IGF-I) is a fibroblast growth and survival factor that has long been implicated in the pathogenesis of IPF. We have previously shown that interstitial macrophage-derived IGF-I correlates with disease severity in IPF, and the Th2 cytokines interleukin (IL)-4 and IL-13 stimulate the expression and secretion of IGF-I by macrophages. In the present study, we tested the hypothesis that IL-4-induced, macrophage-derived IGF-I protects myofibroblasts from apoptosis. Using a growth factor withdrawal model of apoptosis in the myofibroblast cell line, CCL39, we demonstrate that conditioned media from IL-4-stimulated macrophages protect myofibroblasts from apoptosis. The survival effect is lost when IGF-I is immunodepleted from macrophage-conditioned media with IGF-I-specific antibodies. We also show that the protection of myofibroblasts by macrophage-derived IGF-I correlates with and is dependent on the activation of the prosurvival kinases Akt and extracellular signal-regulated kinase. These findings support the view that IL-4-stimulated, macrophage-derived IGF-I may contribute to the persistence of myofibroblasts in pulmonary fibrosis in the Th2-deviated environment of the fibrotic lung.

Key Words: monocytes/macrophages • cytokines • lung • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Idiopathic pulmonary fibrosis (IPF) is a progressive and invariably lethal interstitial lung disease with no known effective therapy. Pathologically, IPF is characterized by usual interstitial pneumonitis (UIP), which represents the "final common pathway" for a number of fibrosing lung diseases. IPF/UIP is characterized by extensive alveolar epithelial cell injury and an intense fibroproliferative response, which leads to excessive deposition of collagen within the lung parenchyma [1 , 2 ]. This collagen deposition is linked to the accumulation of {alpha}-smooth muscle actin-expressing myofibroblasts organized into so-called "fibroblastic foci," which are thought to represent the leading edge of the fibrotic response [3 , 4 ]. During normal wound repair, myofibroblasts contribute to the elaboration of matrix proteins and collagen and the contraction of newly formed granulation tissue before undergoing normal, physiologic apoptosis and clearance [5 ]. In contrast, in IPF, myofibroblasts persist and accumulate in the lung parenchyma, where they are thought to contribute to ongoing collagen synthesis [3 , 4 ]. However, the fundamental mechanisms that result in the accumulation of myofibroblasts in IPF remain poorly understood.

Several growth factors have been implicated in the fibroproliferative response that characterizes UIP/IPF, although their precise roles remain unclear [6 7 8 9 10 ]. Platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I), and fibroblast growth factor-2 have been implicated in fibroblast proliferation [6 , 8 , 11 , 12 ], and transforming growth factor-ß (TGF-ß) and IGF-I have been shown to stimulate collagen matrix synthesis [13 14 15 ] and prevent the induction of apoptosis [16 17 18 19 ]. Thus, survival factors may plausibly contribute to myofibroblast accumulation in IPF by preventing these cells from undergoing apoptosis.

It is now well established that tissue and bronchoalveolar lavage levels of IGF-I are increased in patients with IPF, as well as in bleomycin-induced pulmonary fibrosis in mice [6 7 8 , 20 ]. In the normal lung, IGF-I is expressed primarily by alveolar macrophages [21 ]. However, in IPF, IGF-I is also expressed by alveolar epithelial cells and by interstitial macrophages. In a study of lung biopsy specimens from patients with IPF, we found significant correlations between IGF-I expression by interstitial macrophages and parameters of disease severity, suggesting that IGF-I production by these cells may be important in the progression of IPF [21 ].

The mechanism responsible for the increase in IGF-I expression by macrophages as well as the underlying fibroproliferative response is poorly understood, but recent studies have suggested that deviation toward a T helper cell type 2 (Th2) cytokine profile may be important [22 23 24 ]. Using interleukin (IL)-4–/– mice, Huaux et al. [25 ] showed that IL-4 plays an important role in collagen deposition during the fibroproliferative phase of bleomycin-induced pulmonary fibrosis and that this effect appears to be dependent on macrophages. In addition, Jakubzick et al. [26 ] showed that when IL-4- and IL-13-responsive cells were deleted in the lungs of bleomycin-treated mice, the number of pulmonary macrophages as well as the level of collagen deposition were significantly reduced, implying that IL-4 may act on macrophages to promote or sustain the fibroproliferative phase. Related to these findings, we recently reported that mouse macrophages synthesize increased amounts of IGF-I mRNA and protein in response to stimulation with IL-4 or IL-13 [27 ], and previous studies have shown that exposure to the antifibrotic cytokine, interferon-{gamma} (IFN-{gamma}), inhibits IGF-I expression by macrophages [28 , 29 ].

Given that IGF-I can protect cells from apoptosis and that macrophages are a source of IGF-I in lung tissues of patients with IPF, we hypothesized that macrophage-derived IGF-I may serve to protect myofibroblasts from apoptosis and hence contribute to myofibroblast accumulation in the IPF lung. Additionally, as IL-4 stimulates IGF-I expression by macrophages, we hypothesized that IL-4 would augment the protection of myofibroblasts from apoptosis by the elaboration of macrophage-derived IGF-I. To address these hypotheses, we used an established model of growth factor withdrawal-induced myofibroblast apoptosis using the lung myofibroblast cell line, CCL39 [30 , 31 ], and we show that IL-4-induced, macrophage-derived IGF-I protects myofibroblasts from apoptosis. We further demonstrate that this protection occurs via activation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
C3H/HeJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed at the accredited National Jewish Center Biological Resource Center (Denver, CO). This strain of mouse was used to avoid the possibility of stimulation by trace amounts of endotoxin contaminants. CCL39 cells were purchased from the American Type Culture Collection (Manassas, VA). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from BioWhittaker (Walkersville, MD) and Atlanta Biologicals (Norcross, GA), respectively. Mouse IFN-{gamma}, mouse IL-4, and anti-IGF-I antibodies (MAB791 and MAB291) were purchased from R&D Systems (Minneapolis, MN). IGF-I-specific, 96-well enzyme-linked immunosorbent assay (ELISA) plates were purchased from ELISA Tech (Denver, CO). Akt antibodies, phospho (Ser 473)-Akt (#9277) and total Akt (#9272), were purchased from Cell Signaling Technology (Beverly, MA). The phospho-specific ERK antibody was Anti-ACTIVE MAPK (#V8031) from Promega (Madison, WI), and the total ERK antibody was the Anti-MAP Kinase 1/2 (ERK1/2-CT, #06-182) from Upstate Biotechnology (Lake Placid, NY). LY294002 and PD98059 were purchased from Calbiochem (La Jolla, CA).

Cell culture
Monolayers of mouse bone marrow-derived macrophages were prepared as described previously [32 ]. Bone marrow cells from the tibias, femurs, and pelvises of mice were flushed with and grown in DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% (v/v) FBS, and 10% (v/v) L929 cell-conditioned medium as a source of macrophage-colony stimulating factor. The bone marrow cells were seeded in a six-well culture dish at 11 x 106 cells/plate with 4 ml medium/well and cultured at 37°C under a 10% (v/v) CO2 atmosphere for 5 days. Media (2 ml) were added on day 4, and on day 5, the media was exchanged with 3 ml fresh media. On the 6th day after initial seeding, the macrophages were washed twice with excess serum-free media, and then, 1 ml DMEM, supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1% (v/v) L929 cell-conditioned medium but no FBS, as serum has a profound effect on the survival of the CCL39 cells, was added to each well, followed by the addition of cytokine stimuli. CCL39 cells were seeded in 12-well culture dishes at 1.5 x 106 cells/plate with 2 ml/well DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) FBS and cultured under a 5% (v/v) CO2 atmosphere. The next day, the cells were washed twice with serum-free medium followed by the addition of 0.5 ml serum-free medium alone or macrophage-conditioned medium.

Western blotting
Detection of specific proteins within cell lysates was accomplished using Western blot analysis. Lysis buffer [200 µl; 50 mM Tris-HCl, pH 7.4, 136 mM NaCl, 10% (v/v) glycerol, 1% Nonidet P-40, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM Na3VO4] was added to each well of a 12-well plate containing CCL39 cells. The cells were scraped off the plate, and the method of Chan et al. [33 ] was followed using the antibodies stated in Materials.

Apoptosis assays
Phosphatidylserine exposure was determined by flow cytometry of fluorescein isothiocyanate (FITC)–Annexin-V-stained cells. Cells were harvested by trypsinization, washed in Ca2+-free phosphate-buffered saline (PBS), and stained with 5 µl FITC–Annexin-V solution (BD Bioscience PharMingen, San Diego, CA) for 30 min at 25°C according to the manufacturer’s instructions. The number of fluorescent cells and intensity of fluorescence were determined by flow cytometry using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The results were analyzed with Cell Quest software (Becton Dickson). Active caspase-3 was quantified by using FAM-Asp-Glu-Val-Asp (DEVD)-fluoromethyl ketone (FMK) fluorochrome inhibitor of caspase apoptosis detection kit (Immunochemistry Technologies, LLC, Bloomington, MN) according to the manufacturer’s instructions. The number of fluorescent cells and intensity of fluorescence were determined by flow cytometry as described above.

Immunodepletion of IGF-I
Conditioned supernatants from the stimulated macrophages were collected, and 2 µg/ml each IGF-I-specific antibody (see Materials) was incubated with the supernatants for 1 h, rotating at 25°C. In separate tubes, protein G agarose beads were washed once with excess PBS and once with excess serum-free medium. After removing the final wash from the beads, the macrophage supernatants containing the antibodies were added such that there was 1 ml supernatant per 25 µl packed beads. These were then rotated for another 1 h at 25°C. The beads were centrifuged down, and the supernatants were filtered through a 0.22-µM millipore filter. Supernatants (500 µl) were added to each well of CCL39 cells.

Determination of IGF-I protein levels in culture supernatants
An ELISA was used to determine the concentration of IGF-I protein in cell-culture supernatants and performed as previously reported [27 ]. The sensitivity of the assay was ~50 pg/ml. Culture supernatants were diluted 1:4 to place the samples in the middle of the standard curve. The ELISA was found to yield consistent results between and within assays.

Statistics
Results are expressed as means ± SEM. Statistical analyses using GraphPad InStat version 3.0b for Macintosh (GraphPad Software, San Diego CA) were performed by one-way ANOVA, and comparisons among groups were performed with Bonferroni’s multiple comparisons test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-4-stimulated macrophages express a soluble factor(s) that protects CCL39 cells from growth factor withdrawal-induced apoptosis
To address the role of macrophage-derived growth factors in protecting myofibroblasts from apoptosis, we used a previously characterized model of growth factor withdrawal-induced apoptosis in the lung myofibroblast cell line, CCL39 [30 , 31 ]. Macrophage monolayers were incubated in serum-free medium alone or were stimulated with IL-4 (2 ng/ml) for 26 h. The conditioned media were then removed and tested for their ability to protect CCL39 cells from growth factor withdrawal-induced apoptosis. Microscopic examination of the CCL39 cells showed that supernatants from unstimulated macrophages afforded some protection compared with serum-free medium, whereas supernatants from IL-4-stimulated macrophages provided greater protection (Fig. 1A ). CCL39 cells cultured in serum-free medium with IL-4 were not protected, demonstrating that IL-4 itself does not exhibit survival factor activity. We quantified the ability of macrophage-conditioned media to protect CCL39 cells from apoptosis by measuring Annexin-V staining. Figure 1B shows that when CCL39 cells were cultured in normal medium, less than 10% of the cells were phosphatidylserine (PS)-positive. However, when the cells were subjected to growth factor withdrawal, greater than 50% of the cells were PS-positive. Incubation with conditioned medium from unstimulated macrophages resulted in a modest protection from growth factor withdrawal-induced apoptosis. However, conditioned media from IL-4-stimulated macrophages afforded significant (P<0.001) protection from apoptosis compared with control cells incubated in serum-free medium. The level of protection against apoptosis was found to be related directly to the level of secreted macrophage survival factor(s) based on measurements of CCL39 cell apoptosis following incubation with serially diluted, IL-4-stimulated macrophage culture supernatants (data not shown).



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  		Figure 1. IL-4-stimulated macrophages express a soluble factor(s) that protects CCL39 myofibroblasts from growth factor withdrawal-induced apoptosis. (A) Light micrograph of CCL39 myofibroblasts cultured for 24 h in normal media (NM), serum-free media (SF), serum-free media with IL-4 (SF IL-4; 2 ng/ml), or conditioned media from unstimulated or IL-4 (2 ng/ml for 26 h)-treated macrophages (Mac Un and Mac IL-4, respectively). These images are representative of greater than three experiments. (B) Graphical representation of three independent flow cytometric experiments of CCL39 myofibroblasts stained with FITC-conjugated Annexin-V after culturing for 26 h in NM; SF with no cytokines, IL-4 (2 ng/ml), or IFN-{gamma} (20 U/ml); or conditioned media from macrophages (Mac), unstimulated (Unstim) or stimulated with IL-4 (2 ng/ml) or IFN-{gamma} (20 U/ml) for 26 h. The graph shows the mean ± SEM of three independent experiments for the percent of cells positive for Annexin-V.

 
In contrast to the effect of IL-4, conditioned medium from macrophages stimulated with IFN-{gamma} (20 U/ml) afforded no protection. Indeed, these conditioned media were consistently less protective than conditioned media from unstimulated macrophages (Fig. 1B) . It can also be seen in Figure 1B that neither IL-4 nor IFN-{gamma} afforded any direct protection to the CCL39 cells, indicating that neither cytokine alone is a survival factor. These findings suggest that macrophages secrete low levels of a survival factor(s) for the CCL39 myofibroblast cell line and that IL-4 greatly augments the production of the survival factor(s).

IGF-I is the survival factor produced by the macrophages that protect CCL39 myofibroblasts
As discussed earlier, we have previously shown that IL-4 stimulates the production of IGF-I by macrophages [27 ], and IFN-{gamma} inhibits IGF-I production [28 , 29 ]. To test the hypothesis that IGF-I is the survival factor present in macrophage-conditioned media, we depleted IGF-I from macrophage supernatants using IGF-I-specific antibodies and then tested the conditioned media for their ability to protect CCL39 cells from growth factor withdrawal-induced apoptosis. The efficiency of IGF-I depletion was confirmed by IGF-I ELISA on the macrophage-conditioned medium before and after immunodepletion. As can be seen in Figure 2A , the amount of IGF-I in IL-4-stimulated macrophage-conditioned medium was reduced from 12 ng/ml to 3 ng/ml following immunodepletion with anti-IGF-I antibodies. Depletion of IGF-I from conditioned media obtained from IL-4-stimulated macrophages resulted in a significant (P<0.001) reduction in the protection from apoptosis afforded by these conditioned media as demonstrated by Annexin-V staining (Fig. 2B) and increased the level of apoptosis to that seen in controls incubated in serum-free medium. This effect was not seen following "mock" immunodepletion with nonimmune IgG antibodies (Fig. 2B) . Immunodepletion of IGF-I from unstimulated, macrophage-conditioned medium also resulted in a modest but significant (P<0.05) reduction in the protection from apoptosis compared with undepleted, conditioned medium.



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  		Figure 2. IGF-I is the survival factor produced by the macrophages that protect CCL39 myofibroblasts. (A) Graph showing the concentration of IGF-I in the conditioned media of macrophages unstimulated (Unstim) or stimulated with IL-4 (2 ng/ml) for 26 h before (Mac) and after immunodepleting with IGF-I-specific antibodies (Mac ID IGF-I) or with nonimmune immunoglobulin G (IgG) control antibodies (Mac ID IgG). This graph represents the mean ± SEM of three independent experiments using an IGF-I-specific ELISA. (B) Graphical representation of three independent flow cytometric experiments of CCL39 myofibroblasts stained with FITC-conjugated Annexin-V after culturing for 26 h in serum-free media (SF) with no cytokines or IL-4 (2 ng/ml); conditioned media from macrophages (Mac), unstimulated or stimulated with IL-4 (2 ng/ml) for 26 h; conditioned media from unstimulated macrophages or those stimulated with IL-4 and then immunodepleted of IGF-I using IGF-I-specific antibodies (Mac ID IGF-I); or conditioned media from unstimulated or IL-4-stimulated macrophages that had been immunodepleted with nonimmune IgG control antibodies (Mac ID IgG). The graph shows the mean ± SEM for the percent of cells positive for Annexin-V.

 
Growth factor withdrawal also predictably resulted in caspase-3 activation in CCL39 cells (Fig. 3 ), and incubation with conditioned media from IL-4-stimulated macrophages significantly (P<0.001) reduced caspase-3 activation. Consistent with the Annexin-V staining results shown earlier, immunodepletion of IGF-I from IL-4-stimulated, macrophage-conditioned media likewise reversed the protective effect (Fig. 3) , and elevated levels of caspase-3 activation were restored. These findings confirm that macrophage-derived IGF-I is necessary for the protection of CCL39 cells from growth factor-induced apoptosis by macrophage-conditioned media. We also examined these supernatants for other survival factors (TGF-ß1 and PDGF-BB) by specific ELISAs, but we were unable to detect PDGF-BB or active TGF-ß1. In addition, neutralizing anti-TGF-ß1 antibodies had no effect on CCL39 cell apoptosis (data not shown). Thus, although TGF-ß and PDGF have been implicated in protection from apoptosis [19 , 34 ], the mechanism of protection by IGF-I from IL-4-stimulated macrophages likely occurs independently of TGF-ß and PDGF.



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  		Figure 3. Caspase-3 activity is reduced in CCL39 myofibroblasts that are protected by macrophage-derived IGF-I. Graphical representation of three independent flow cytometric experiments of CCL39 myofibroblasts stained with FAM-DEVD-FMK [caspase-3 fluorochrome-labeled inhibitor of caspases (FLICA)] after culturing for 24 h in normal media (NM); serum-free media (SF) with no cytokines or IL-4 (2 ng/ml); conditioned media from macrophages (Mac), unstimulated or stimulated with IL-4 (2 ng/ml) for 26 h; conditioned media from unstimulated macrophages or those stimulated with IL-4 and then immunodepleted of IGF-I using IGF-I-specific antibodies (Mac ID IGF); or conditioned media from unstimulated or IL-4-stimulated macrophages that had been immunodepleted with nonimmune IgG control antibodies (Mac ID IgG). The graph shows the mean ± SEM for the percent of cells with active caspase-3.

 
CCL39 myofibroblasts consume macrophage-derived IGF-I
A characteristic feature of survival factors is that they are consumed by the cells they protect, leading to a decline in the amount of survival factor in the culture supernatant [35 , 36 ]. In view of the importance of macrophage-derived IGF-I in protecting the CCL39 lung myofibroblast cell line from growth factor withdrawal-induced apoptosis, we next determined if IGF-I was consumed by the CCL39 cells. IGF-I levels were measured in macrophage-conditioned media before and after culture with CCL39 cells for 26 h. As can be seen in Figure 4 , the concentration of IGF-I in the supernatants from IL-4-stimulated macrophages was reduced from 12 ng/ml to 3 ng/ml (P<0.001) after incubation with CCL39 cells. In contrast, the concentration of IGF-I was unchanged when the conditioned media were incubated at 37°C for 26 h in the absence of CCL39 cells, indicating the IGF-I had not simply bound to the tissue-culture plates or decayed. Thus, IGF-I is consumed by CCL39 cells, as would be expected for a survival factor.



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  		Figure 4. Protected CCL39 myofibroblasts consume the macrophage-derived IGF-I. Graph showing the concentration of IGF-I in the conditioned media of macrophages before (Mac) and after culturing on the myofibroblasts (Mac-CCL39) or cell-free culture dishes (Mac-Plastic) for 26 h. The macrophages were unstimulated (Unstim) or stimulated IL-4 (2 ng/ml) for 26 h. The graph represents the mean ± SEM of three independent experiments using an IGF-I-specific ELISA.

 
The prosurvival kinases Akt and ERK are activated in CCL39 cells by macrophage-derived IGF-I
Several distinct pro-survival signaling pathways may be activated by IGF-I; therefore, we sought to determine whether macrophage-conditioned media activated the pro-survival signaling pathways leading to the activation of Akt, ERK, and nuclear factor (NF)-{kappa}B. CCL39 cells were incubated with macrophage-conditioned media, and lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed for active and total Akt, active and total ERK, and inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}). Incubation under conditions of growth factor withdrawal alone or in the presence of IL-4 (2 ng/ml) or IFN-{gamma} (20 U/ml) alone did not activate Akt (Fig. 5A ). However, incubation with conditioned medium from unstimulated macrophages induced Akt phosphorylation. Further, incubation with conditioned medium from IL-4-stimulated macrophages augmented this response (Fig. 5A) , whereas incubation with conditioned medium from IFN-{gamma}-stimulated macrophages reduced the level of Akt phosphorylation compared with that seen with IL-4-stimulated, macrophage-conditioned medium. To determine if IGF-I in the macrophage supernatants was responsible for activating Akt, IGF-I was immunodepleted from the conditioned media before addition to the CCL39 cells. Depletion of IGF-I reduced the ability of the macrophage-conditioned media to phosphorylate Akt to ~25% of the level seen after mock depletion with nonimmune IgG. The same blots were stripped and reprobed for total Akt to verify equal loading. In all the experiments, we saw a decrease in total Akt, where there was abundant active Akt. These results indicate that IGF-I, present in the macrophage-conditioned media, is responsible for the activation of Akt in CCL39 cells.



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  		Figure 5. The pro-survival kinases Akt and ERK are activated in CCL39 myofibroblasts by macrophage-derived IGF-I. (A) Western blot analysis of ~3 µg postnuclear lysate from CCL39 myofibroblasts blotted with antibodies specific for phospho-Akt (p-Akt) and total Akt. The myofibroblasts were cultured for 1 h in normal media (NM); serum-free media (SF) with no cytokines, IL-4 (2 ng/ml), or IFN-{gamma} (20 U/ml); conditioned media from macrophages (Mac), unstimulated or stimulated for 26 h with IL-4 (2 ng/ml) or IFN-{gamma} (20 U/ml); conditioned media from macrophages immunodepleted of IGF-I with IGF-I-specific antibodies (ID IGF-I Mac); or conditioned media from macrophages immunodepleted with nonimmune IgG control antibodies (ID IgG Mac). These blots are representative of three separate experiments. (B) Same as in A, except the CCL39 myofibroblasts were cultured for 15 min, and the lysates were blotted for phospho-ERK (p-Erk) and total ERK. These blots are representative of three separate experiments.

 
We also determined the effects of macrophage-conditioned media on ERK phosphorylation. As can be seen in Figure 5B , incubation under conditions of growth factor withdrawal alone or in the presence of IL-4 (2 ng/ml) or IFN-{gamma} (20 U/ml) alone did not directly activate ERK, However, ERK was phosphorylated in the presence of conditioned media from macrophages with the conditioned medium from IL-4-stimulated macrophages producing the greatest ERK activation. Depletion of IGF-I markedly reduced the activation of ERK by conditioned media from unstimulated and IL-4-stimulated macrophages. Equal loading was verified by reprobing for total ERK. In multiple experiments, we were unable to detect any activation of I{kappa}B kinase as reflected by degradation of I{kappa}B{alpha}, suggesting that NF-{kappa}B was not involved in the protection against apoptosis by macrophage-conditioned media (data not shown). Collectively, these data suggest that the protection of CCL39 cells conferred by macrophage-derived IGF-I is accompanied by activation of the pro-survival kinases Akt and ERK but not by the activation of NF-{kappa}B.

Akt and ERK are necessary for the survival effect of macrophage-conditioned medium
We next determined if Akt and/or ERK activation were necessary for the survival of the CCL39 cells by macrophage-conditioned media by inhibiting the activation of these kinases with LY294002 and PD98059, respectively [37 , 38 ]. The efficacy and specificity of these inhibitors were verified by showing that the activation of each kinase was blocked by the appropriate inhibitor (Fig. 6A ). We then determined the ability of LY294002 and PD98059 to reverse the protection against apoptosis conferred by conditioned media from unstimulated and IL-4-stimulated macrophages. In the presence of LY294002 or PD98059 alone, conditioned media from unstimulated and IL-4-stimulated macrophages were less protective than was seen in the absence of the inhibitors (Fig. 6B) . We also examined the combined effect of both inhibitors. Incubation with both inhibitors reversed the protective effects of unstimulated conditioned medium to the level seen with serum-free medium (Fig. 6B) . However, the combination of both inhibitors did not increase the caspase-3 activity above that which was seen with LY alone when using IL-4-stimulated, macrophage-conditioned medium. These data suggest that the activation of Akt and ERK contributes to the ability of macrophage-derived IGF-I to protect CCL39 cells from apoptosis following growth factor withdrawal.



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  		Figure 6. Akt and ERK are necessary for the survival effect of macrophage-conditioned medium. (A) Western blot analysis of ~3 µg postnuclear lysate from CCL39 myofibroblasts blotted with antibodies specific for phospho-Akt, total Akt, phospho-ERK, and total ERK. All the myofibroblasts were first cultured for 1 h in serum-free media (SF) with nothing, vehicle (Veh), LY294002 (LY; 20 µM), PD98059 (PD; 20 µM), or LY294002 (20 µM) plus PD98059 (LY/PD; 20 µM). Following this pretreatment, serum-free media with nothing or conditioned media from macrophages stimulated for 26 h with IL-4 (2 ng/ml) containing vehicle, LY294002 (20 µM), PD98059 (20 µM), or LY294002 (20 µM) plus PD98059 (20 µM) were added for 1 h (Akt blots) or 15 min (ERK blots). (B) Graphical representation of three independent flow cytometric experiments of CCL39 myofibroblasts stained with FAM-DEVD-FMK (caspase-3 FLICA). All the cells were initially pretreated for 1.5 h with serum-free media containing vehicle, LY294002 (20 µM), PD98059 (20 µM), or LY294002 (20 µM) plus PD98059 (20 µM). The cells were then cultured with normal media (NM), SF, or conditioned media from macrophages (Mac), unstimulated or stimulated with IL-4 (2 ng/ml for 26 h), containing the stated inhibitor or vehicle. Four hours later, the cells were redosed with inhibitor in serum-free media for a final concentration of 30 µM. Approximately 19 h later, the caspase-3 FLICA assay was performed. The graph shows the mean ± SEM for the percent of cells with active caspase-3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although recent studies have shed new light on the basic mechanisms underlying the pathogenesis of IPF, many issues remain poorly understood (as reviewed in ref. [1 ]). In particular, although it has been recognized that the accumulation of fibroblasts and myofibroblasts within fibroblastic foci is associated with a worse prognosis [39 ], little is known about the mechanisms that allow these cells to persist and accumulate. At the conclusion of normal wound healing or during postnatal alveolarization in the newborn lung, myofibroblasts are rapidly eliminated as a consequence of apoptosis and clearance [5 , 40 ]. Why this process fails to occur in IPF is not clear, although the continued presence of growth factors and survival factors such as PDGF, TGF-ß, and IGF-I [6 7 8 9 10 ] may plausibly account for the increased accumulation of myofibroblasts and increased collagen deposition that characterizes this disease. In addition, immune deviation toward a Th2 cytokine profile has been shown to predominate in the lung parenchyma of patients with IPF [23 , 24 ], and this may favor a fibroproliferative response. Based on our previous work showing that increased IGF-I expression by pulmonary interstitial macrophages in IPF patients is associated with increased collagen matrix deposition and deterioration in clinical course [21 ], and IGF-I expression by macrophages is increased by the Th2 cytokines IL-4 and IL-13 [27 ], the major goal of this study was to address the hypothesis that macrophage-derived IGF-I protects myofibroblasts from apoptosis.

We used a growth factor withdrawal model of apoptosis in the CCL39 lung myofibroblast cell line to demonstrate that IL-4 enhances macrophage production of a factor that protects myofibroblasts from apoptosis. This survival factor was shown to be IGF-I. To clarify the mechanisms involved in the protection of CCL39 cells from apoptosis by macrophage-derived IGF-I, we investigated the activation and role of the pro-survival kinases Akt and ERK and transcription factor NF-{kappa}B. Akt and ERK were rapidly phosphorylated by conditioned media from IL-4-stimulated macrophages, and I{kappa}B{alpha} degradation was unaffected, suggesting that Akt and ERK but not NF-{kappa}B are involved in the protection against apoptosis. Pharmacologic inhibitors that block the activation of both kinases indicated that Akt and ERK were necessary to fully protect CCL39 cells from apoptosis.

The antiapoptotic properties of Akt and ERK have been studied in some detail. Whereas survival factor withdrawal leads to Forkhead transcription factor, FKHRL1, dephosphorylation, nuclear localization, target gene activation, and apoptosis, Akt-dependent phosphorylation of FKHRL1 results in its inactivation via association with 14-3-3 proteins and sequestration in the cytoplasm [16 ]. As a result, IGF-I promotes the survival of cerebellar granule neurons by down-regulating Bim expression via FKHRL1 phosphorylation [18 ]. In addition, IGF-I-induced, Akt-dependent phosphorylation of Bad inhibits its proapoptotic activity by blocking the interaction with Bcl-2 or Bcl-XL [41 , 42 ]. Akt can also directly inhibit apoptosis by phosphorylating caspase-9 at residue Ser198 [43 ]. ERK activation in CCL39 cells is necessary for the protection against apoptosis following loss of anchorage and serum removal [31 ]. Additionally, ERK activates Rsk2, a member of the pp90 ribosomal S6 kinase family, which in turn phosphorylates Bad at Ser 112 to block apoptosis in cerebellar granule neurons [44 ]. ERK has also been shown to directly phosphorylate caspase-9 at Thr 125, thereby blocking caspase-9 processing and subsequent caspase-3 activation [45 ]. Thus, activation of Akt and ERK signaling pathways results in multiple mechanisms that collectively protect cells from apoptosis.

We propose that deviation toward a Th2 cytokine profile (i.e., elevated IL-4 and/or IL-13) in IPF is important in the pathological persistence of myofibroblasts via up-regulation of macrophage-derived IGF-I. In 1992, Stein et al. [46 ] proposed the concept of "alternatively activated" macrophages while investigating the role of IL-4 in the regulation of the macrophage mannose receptor. Alternatively activated macrophages are induced by IL-4 and IL-13 but not by IL-10 and TGF-ß and express a gene repertoire characterized by increased expression of arginase, IL-1 receptor antagonist (IL-1R{alpha}), and the phagocytic receptor dectin [47 , 48 ]. In contrast, classically activated macrophages are induced by exposure to Th1-deviating stimuli including IFN-{gamma}, IL-12, lipopolysaccharide, and tumor necrosis factor-{alpha} and exhibit increased expression of IFN-ß, nitric oxide synthase 2, and CC chemokine receptor 7 [47 ]. This pattern of gene expression enables classically activated macrophages to participate in pathogen elimination and inflammation. Although the functions of alternatively activated macrophages are not as clearly defined, recent studies suggest that alternatively activated macrophages play an important role in wound repair and fibrosis [49 50 51 ] and the elimination of parasites [52 ]. It is striking that the pattern of expression of IGF-I appears to coincide with the phenotype and responses of alternatively activated macrophages by being stimulated by IL-4 and IL-13 but not IL-10 and TGF-ß [27 ] and by being inhibited by IFN-{gamma} [28 , 29 ]. In our model, elevated levels of IL-4 and/or IL-13 stimulate alveolar and interstitial macrophages to produce increased amounts of IGF-I, which prevent myofibroblasts from undergoing apoptosis, and enable continued production of collagen. This is supported by recent reports that provide insights into the role and mechanism of action of IL-4 and IL-13 in bleomycin-induced pulmonary fibrosis. In studies with IL-4–/– mice, Huaux et al. [25 ] suggested that IL-4 plays a dual role in the development of pulmonary fibrosis. They noted that during the early stages of bleomycin-induced injury, IL-4–/– mice exhibited an increase in T cell accumulation in the lung and increased mortality compared with wild-type mice, suggesting that at early time-points, the anti-inflammatory activity of IL-4 may be important in limiting injury. However, during the later fibroproliferative stage, IL-4–/– mice had significantly lower levels of fibrosis compared with wild-type mice, suggesting that IL-4 is important in the development of this later fibrotic response. They also found that stimulation of fibroblasts with IL-4 did not promote fibroblast proliferation, myofibroblast differentiation, or type I collagen production, suggesting that the effect of IL-4 was indirect. As the number of macrophages and monocytes was also reduced in the IL-4–/– mice, the authors suggested that the profibrotic activity of IL-4 was mediated indirectly by macrophage growth factor production. Another study addressed the role of IL-4- and IL-13-responsive cells in the development of bleomycin-induced pulmonary fibrosis [26 ]. IL-4 binds two receptor complexes, the high-affinity IL-4R{alpha}/IL-2R common {gamma}-chain heterodimer and the IL-4R{alpha}/IL-13R{alpha}1 heterodimer, whereas IL-13 binds the high-affinity IL-4R{alpha}/IL-13R{alpha}1 complex or the monomeric receptors IL-13R{alpha}1 and IL-13R{alpha}2. The authors created an IL-13–Psuedomonas exotoxin (IL-13-PE) fusion protein to selectively delete pulmonary IL-4- and IL-13-responsive cells. Intranasal instillation of the IL-13-PE fusion protein significantly reduced the number of macrophages, and those that were present appeared to be less "activated," as revealed by a non-"foamy" appearance and a decreased production of CCL6 and macrophage-inflammatory protein-2. In addition, there was significantly reduced pulmonary fibrosis following bleomycin instillation. Thus, these data suggest that IL-4- and IL-13-responsive macrophages play a role in mediating the pro-fibrotic effect of these cytokines. Elevated levels of IL-4 have been shown to be associated with macrophages and mononuclear cells and localized to areas of remodeling and fibrosis [22 , 23 ].

IGF-I is not the only pro-survival growth factor in the lungs of patients with IPF or in mice with bleomycin-induced fibrosis. IGF-I and TGF-ß can independently protect myofibroblasts from apoptosis in vitro (this study and ref. [19 ]), which raises the question of their functional significance and/or redundancy in vivo. Although cytokines are expressed by alveolar macrophages and alveolar epithelial cells, activation of latent TGF-ß requires the epithelial integrin {alpha}vß6 [53 ], suggesting that active TGF-ß is present in the alveolus or at the epithelial surfaces. In contrast, IGF-I is also expressed by interstitial macrophages [21 ] at areas of intense fibroproliferation. Similarly, asbestosis studies in sheep have shown that TGF-ß expression is primarily restricted to alveolar macrophages along with alveolar and airway epithelial cells and matrix, whereas IGF-I expression is also detected in the interstitium [54 ]. Thus, although both cytokines may be present and promote myofibroblast survival at some locations, they may also act independently at other locations.

Collectively, these findings raise the question of how myofibroblast accumulation in IPF may be controlled or reversed. One possible approach would be to control IL-4-mediated up-regulation of IGF-I by macrophages, thus limiting the availability of IGF-I for myofibroblasts, which may lead to physiologic myofibroblast apoptosis. Alternatively, further investigations are necessary to determine how to actively induce myofibroblast apoptosis, even in the presence of IGF-I, to reverse the now inevitable, fatal course of IPF.


   ACKNOWLEDGEMENTS
 
This work was supported by Public Health Service Grants HL68628, HL55549, and HL65326 from the National Heart, Lung and Blood Institute of the National Institutes of Health (NIH). M. W. W. was supported by Institutional T-32 Training Grant AI00048 from NIH. S. K. F. was supported by K08 Grant HL67848 from the National Heart, Lung and Blood Institute of NIH. The authors acknowledge Linda Remigio and Benjamin Edelman for outstanding technical assistance and thank Dr. Jay Westcott from ELISA Tech (Denver, CO) for developing the mouse IGF-I ELISA.

Received May 13, 2004; revised July 20, 2004; accepted July 23, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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