Induction of Macrophage Insulin-Like Growth Factor-I Expression by the Th2 Cytokines IL-4 and IL-13

Murry W. Wynes and David W. H. Riches
J Immunol October 1, 2003, 171 (7) 3550-3559; DOI: https://doi.org/10.4049/jimmunol.171.7.3550

Abstract

Macrophage-derived insulin-like growth factor-I (IGF-I) has long been implicated in the pathogenesis of the interstitial lung disease, idiopathic pulmonary fibrosis, in part, by its ability to 1) stimulate the proliferation and survival of fibroblasts and myofibroblasts and 2) promote collagen matrix synthesis by these cells. However, little is known about the mechanisms that stimulate the expression of IGF-I by macrophages. Previous studies have shown that the development of pulmonary fibrosis is accompanied by enhanced expression of Th2-profile cytokines, especially IL-4, and diminished expression of Th1 cytokines, including IFN-γ. In addition, in vitro studies have shown that IFN-γ down-regulates the expression of IGF-I. Thus, the paucity of IFN-γ in the fibrotic lung may favor increased growth factor production by allowing Th2 cytokines to predominate. In view of these findings, we investigated the hypothesis that Th2 cytokines stimulate the expression of IGF-I by macrophages. Incubation with IL-4 or IL-13 led to concentration- and time-dependent increases in the expression of IGF-I mRNA and the secretion of IGF-I protein by mouse macrophages as a consequence of increased transcription of IGF-I pre-mRNA. Exposure of macrophages to IL-4 in the presence of IFN-γ inhibited the increase in the expression of IGF-I. Studies using STAT6-deficient macrophages indicated that the increase in IGF-I expression was dependent on STAT6. In addition, the down-regulation of IGF-I expression by IFN-γ was absent in STAT1-deficient macrophages. Collectively, these findings define a homeostatic mechanism in which Th2 cytokines promote, and Th1 cytokines inhibit, the expression of IGF-I by macrophages.

Idiopathic pulmonary fibrosis (IPF),3 an interstitial lung disease of the lower respiratory tract of unknown origin, is associated with high morbidity and mortality; currently, there is no effective therapy (1). The disease is characterized by the accumulation of macrophages, neutrophils, and lymphocytes within the alveolar and interstitial compartments (2). The inflammatory response leads to damage and elimination of alveolar type I epithelial cells and to hyperplasia and hypertrophy of type II epithelial cells. These events result in the denudation of, and subsequent injury to, the alveolar basement membrane resulting in the collapse and fusion of the alveolar units. During the repair process, the lung is remodeled and reorganized by an exuberant fibroproliferative response that ultimately leads to an impairment of gas exchange between the alveoli and pulmonary capillaries. Central to the fibroproliferative response is the proliferation, differentiation, and accumulation of fibroblasts and myofibroblasts which engage in the synthesis of type I and type III collagens, especially in areas of actively evolving fibrosis (3, 4, 5). These latter events are controlled by a plethora of cytokines and growth factors, though principal among these are platelet-derived growth factor, TGF-β, and insulin-like growth factor-I (IGF-I) (6, 7, 8, 9, 10, 11).

IGF-I, especially macrophage-derived IGF-I, has long been implicated in the pathogenesis of IPF. Originally described as alveolar macrophage-derived growth factor (AMDGF), IGF-I levels in bronchoalveolar lavage fluid are significantly increased in patients with IPF and other fibroproliferative lung diseases (9, 11). Postmortem and open lung biopsy specimens from patients with IPF have been shown to express elevated levels of IGF-I mRNA (9) and alveolar macrophages from IPF patients secrete increased amounts of IGF-I compared with normal subjects (8, 9, 10). A morphometric evaluation of IGF-I expression in IPF patients showed that IGF-I is also expressed by alveolar epithelial cells and interstitial macrophages (12). In addition, parameters of disease severity were found to be associated with the expression of IGF-I by interstitial macrophages emphasizing the potential importance of IGF-I production by these cells in the progression of IPF (12). However, while there are abundant data to suggest that IGF-I plays a role in the regulation of pulmonary fibrosis, little is known about the stimuli or mechanisms that increase IGF-I expression by macrophages.

The role of the adaptive immune system in the pathogenesis of pulmonary fibrosis has not been completely resolved. However, several studies have suggested that skewing of Th1 and Th2 cytokine profiles is associated with the development of granulomatous and fibroproliferative disorders, respectively, in the pulmonary interstitium and conducting airways. In studies of lung biopsy specimens from IPF patients, Wallace et al. (13, 14) showed that IL-4 was expressed in the pulmonary parenchyma by alveolar type II epithelial cells. In addition, Ando et al. (15) reported that the expression of IL-4 was prominently associated with advancing areas of fibrosis compared with end-stage honeycombing. Animal model studies of interstitial fibrosis using bleomycin, silica, and thoracic irradiation support these conclusions (16, 17, 18). In contrast, expression of the Th1 cytokine, IFN-γ, has been found to be reduced or absent in IPF yet is prominent in granulomatous lung disorders such as sarcoid (13, 19, 20). These findings are consistent with previous observations that IFN-γ 1) inhibits IGF-I production by macrophages (21), 2) inhibits collagen synthesis by fibroblasts (22), 3) is efficacious in ameliorating pulmonary fibrosis in the bleomycin model in mice (23), and 4) modestly improves pulmonary function and survival in IPF patients (24). Thus, the available data are supportive of the hypothesis that the development of pulmonary fibrosis is dependent upon skewing toward a Th2-type cytokine profile (25). In view of this hypothesis, the aim of the present study was to investigate the mechanisms governing the regulation of IGF-I by the prototypic Th2 cytokine, IL-4. In contrast to previous studies showing that the Th1 cytokines, IFN-γ and IFN-β, inhibit IGF-I expression (21, 26), we now show that IL-4 stimulates the expression of IGF-I through a mechanism that is dependent on the transcription factor, STAT6. Furthermore, we show that in contrast to IL-4, IFN-γ inhibits IGF-I expression in a STAT1-dependent fashion.

Materials and Methods

Materials

C3H/HeJ, BALB/c, and STAT6 knockout mice were obtained from The Jackson Laboratory (Bar Harbor, ME). STAT1 knockout and svj129 control mice were a generous gift from Dr. R. Schreiber (Washington University, St. Louis, MO). The mice were housed at the National Jewish Center Biological Resource Center (Denver, CO). DMEM and FBS were purchased from BioWhittaker (Walkersville, MD) and Atlanta Biologicals (Norcross, GA), respectively. IFN-γ was generously provided by Genentech (San Francisco, CA). IL-4 was purchased from Genzyme (Cambridge, MA) initially and subsequently from R&D Systems (Minneapolis, MN). IGF-I exon 4 cDNA was kindly provided by Dr. P. Rotwein (University of Oregon Health Sciences Center, Portland, OR). The GAPDH and 18S probes were restriction-digested from plasmids purchased from the American Type Culture Collection (Manassas, VA). Cycloheximide and actinomycin D were purchased from Sigma-Aldrich (St. Louis, MO). IGF-I intron 5 primers were synthesized by Genelink (Thornwood, NY). Restriction enzymes were purchased from Promega (Madison, WI).

Primary macrophage isolation and culture

Monolayers of mouse bone marrow-derived macrophages were prepared as previously described (27). Briefly, 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 M-CSF. The bone marrow cells were seeded in six-well culture dishes at 2 × 106 cells/well with 4 ml of medium/well and cultured at 37°C under a 10% CO2 atmosphere for 5–7 days. Fresh media were added on days 5 and 6. Unless indicated, most experiments were conducted with macrophages from C3H/HeJ mice. This strain of mouse was selected to circumvent the possibility of exposure to trace amounts of endotoxin that occasionally contaminate commercially available cytokines and other stimuli.

RNA isolation and Northern analysis

Total RNA was isolated from bone marrow-derived macrophages using TRIzol from Life Technologies (Grand Island, NY) and the level of mRNA expression was then determined by Northern blot analysis as described (28). Fifteen micrograms of total RNA were electrophoresed through a denaturing 1.2% (w/v) agarose gel, transferred to a nylon membrane, UV cross-linked, and hybridized at 42°C for 12–18 h with 5 × 106 cpm of [32P]IGF-I exon 4, GAPDH, or 18S probes. The membrane was washed and exposed to Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ) at −70°C. Band intensities were quantified using the Storm 860 PhosphorImager in phosphor mode and Image Quant software (Molecular Dynamics, Sunnyvale, CA).

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 of bone marrow-derived macrophages. IGF-I-specific 96-well ELISA plates were purchased from ELISA Tech (Denver, CO). The plates had been coated with Ab MAB792 (R&D Systems), a hamster mAb directed against mouse IGF-I. After washing the blocking solution from the wells, 125 μl of diluted culture supernatant or culture medium spiked with increasing concentrations of recombinant mouse IGF-I (R&D Systems) were added to each well and incubated at 4°C overnight. After thorough washing, anti-human IGF-I polyclonal goat Ab conjugated with biotin (BAF291 from R&D Systems) was added and incubated for 2 h at 25°C followed by washing. Subsequently, streptavidin-HRP was added for 1 h and after washing, substrate was added for 10–15 min followed by stop solution. The absorbance at 450 nm was ascertained on a plate reader and the concentration of IGF-I was determined by interpolation of the standard curve generated with recombinant mouse IGF-I. 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 both between and within assays. Replica supernatants were also sent to the National Hormone and Peptide Program (Torrance, CA) and were assayed for IGF-I protein by radio-immunoassay using an Ab directed against human IGF-I to independently confirm the results of the ELISA. Both assays yielded qualitatively and quantitatively similar results.

RT-PCR

Total RNA was isolated as described above and 30 μg were treated with 2.5 U of RNase-free DNase (Promega) for 15 min at 37°C to remove any genomic DNA contamination. The RNA was extracted with 1 volume of phenol/chloroform and the aqueous phase was extracted again with 1 volume of chloroform. The aqueous phase was then ethanol-precipitated and the resulting pellet was washed and resuspended in diethylpyrocarbonate-treated water. Reverse transcription was preformed using the RetroScript kit purchased from Ambion (Austin, TX). The concentrations listed below refer to the stock concentrations supplied by the manufacturer. Briefly, 1 μg of purified total RNA was mixed with 4 μl of dNTP mix (2.5 mM each dNTP) and 2 μl of random decamers (50 μM), in a volume of 16 μl. The sample was heated to 75°C for 4 min and then placed on ice for 1 min. Then, 2 μl of basic buffer (100 mM Tris-HCl, pH 8.3, containing 500 mM KCl and 15 mM MgCl2), 1 μl of placental RNase inhibitor (10 U/μl), and 1 μl of M-MLV reverse transcriptase (100 U/μl) were added and mixed. The sample was incubated at 44°C for 1 h, denatured at 92°C for 10 min, and then stored at −20°C. Two microliters of the reverse transcription reaction were used as template for the PCR. The following were added to the template: 41 μl of water, 5 μl of 10× Vent buffer (200 mM Tris-HCl, pH 8.5, containing 100 mM KCl, 100 mM (NH4)SO4, 20 mM MgSO4, and 1% (v/v) Triton X-100), 0.5 μl of MgSO4 (100 mM), 1 μl of dNTPs (2.5 mM each), 0.2 μl of the IGF-I intron 5 sense primer, 5′-GCAGAGGCAACTCAGAAAATCAGAGGTG-3′ (0.5 μg/ml), 0.2 μl of the IGF-I intron 5 antisense primer, 5′-TTAAGAAGGGGAGGGCATGTCTTGTTGC-3′ (0.5 μg/ml), and 0.25 μl of Vent polymerase (2 U/μl; New England Biolabs, Beverly, MA). To amplify 18S ribosomal RNA as a control, the reaction was modified to contain 37 μl of water, 0.8 μl of a mixture of 5′ and 3′ 18S primers (0.5 μM), and 3.2 μl of a mixture of 5′ and 3′ 18S competimers (0.5 μM). The reaction mixtures were amplified using the following parameters: 94°C for 5 min; 28 and 32 cycles (94°C for 30 s, 59°C for 30 s, 72°C for 1 min); and 4°C hold. The PCR products were then electrophoresed through a 1.5% (w/v) agarose gel, stained with Vistra Green (Amersham Pharmacia Biotech) at 1/10,000 (v/v) for 1 h, and scanned in the Strom 860 Phosphor/Fluorimager (Molecular Dynamics) using the blue fluorescence/chemifluorescence mode. The intensity of the bands in the resulting image was quantified with Image Quant (Molecular Dynamics).

NO2− assay

Nitrite anion (NO2−) accumulation in cultured macrophage supernatants, which is directly proportional to the amount of NO· produced, was determined by using the Greiss reagent (1% (w/v) sulfanilamide, 0.1% (w/v) naphthylethylene diamine, 2.5% (v/v) phosphoric acid) method (29). One-hundred microliters of the culture supernatants were mixed with an equal volume of Greiss reagent and incubated at room temperature for 10 min. The absorbance at 550 nm was then determined in a spectrophotometer.

Results

IL-4 stimulates IGF-I expression by mouse macrophages

We investigated the effect of IL-4 on IGF-I expression by incubating monolayers of mouse macrophages with increasing concentrations of IL-4 (0.006–6 ng/ml) for 10 h and performing Northern blot analysis on total RNA using a [32P]IGF-I exon 4-specific probe. This probe detects all IGF-I transcripts including the IA (exon 1 or 2 and exons 3, 4 and 6) and IB variants (exon 1 or 2 and exons 3, 4, 5 and 6). Exon 1 contains promoter 1, whereas exon 2 contains promoter 2, and their use is mutually exclusive. The observed multiple transcript sizes (7.5, 1.8, and 1.0 kb) are due to differences in the length of the 3′-untranslated region (i.e., the length of exon 6) and the length of the poly-A tail (30, 31) and are consistent with previous studies investigating IGF-I mRNA expression by mouse macrophages (28, 32). As can be seen in Fig. 1⇓A, IL-4 increased IGF-I transcript levels in a dose-dependent manner with an initial increase being seen at 0.03 ng/ml IL-4 and maximal stimulation being detected with 0.3 ng/ml IL-4. IL-4 increased the expression of each of the basal transcript species. Two additional transcript species also became apparent at concentrations of IL-4 >0.1 ng/ml (Fig. 1⇓A). PhosphorImager analysis revealed that when used at a concentration of 0.03 ng/ml, IL-4 increased the 7.5-kb transcript by 50% and the 1 kb by 2-fold, whereas when used at 0.3 ng/ml, IL-4 increased both transcript levels by >4-fold. IL-4 had no effect on the expression of the housekeeping gene GAPDH (Fig. 1⇓A). The time course of induction of IGF-I transcript expression by IL-4 was determined by stimulating macrophages with a fixed concentration of 1 ng/ml IL-4 for time intervals ranging from 1–30 h before determination of IGF-I mRNA levels by Northern blot analysis. As can be seen in Fig. 1⇓B, an increase in IGF-I expression was detected 2 h after the addition of IL-4, while maximal transcript expression was observed between 12 and 16 h poststimulation. PhosphorImager analysis indicated that the 7.5-kb transcript was increased by 3-fold after 2 h of stimulation and 5-fold after 12 h of stimulation with IL-4. The changes in IGF-I mRNA expression induced by IL-4 were not limited to macrophages derived from C3H/HeJ mice and were also observed in macrophages obtained from C3H/HeN, svj129, and BALB/c mice (data not shown). In addition, IL-4 also increased the expression of IGF-I mRNA in the mouse alveolar macrophage cell line, MH-S (data not shown). We investigated the physiological significance of the IL-4-stimulated increase in IGF-I mRNA levels by determining whether IGF-I protein was also expressed and secreted. Macrophages were incubated with 2 ng/ml IL-4 or medium alone for 26 h and IGF-I protein levels in the culture supernatants were determined by ELISA. As can be seen in Fig. 1⇓C, incubation in medium alone led to the basal secretion of ∼0.8 ng/ml IGF-I, a finding consistent with the observed basal expression of IGF-I mRNA. In contrast, incubation with IL-4 increased the secretion of IGF-I to ∼2.2 ng/ml. Thus, IL-4 stimulated the expression of both IGF-I mRNA and protein.

FIGURE 1.
FIGURE 1.

IGF-I expression is up-regulated by IL-4. A, Concentration dependence. Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and GAPDH after stimulation with IL-4 (0.006–6 ng/ml) for 10 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, Time dependence. Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and GAPDH after stimulation with 1 ng/ml IL-4 for 1–30 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. C, IGF-I protein levels. IGF-I protein levels in culture supernatants from bone marrow-derived macrophages stimulated with IL-4 (2 ng/ml) or incubated in medium alone for 26 h. The values represent the mean from three independent experiments ± the SEM.

IL-4 is the prototypic Th2 cytokine. However, several other cytokines including IL-13, TGF-β, and IL-10 are also involved in either the regulation of Th2 responses, or in the effector functions that characterize Th2 responses. Therefore, we investigated whether other Th2-profile cytokines were also capable of up-regulating the expression of IGF-I by stimulating mouse macrophages with IL-4, IL-10, IL-13, or TGF-β. We also exposed macrophages to combinations of these cytokines (all at 5 ng/ml) for 18 h before quantifying the level of expression for IGF-I mRNA. As can be seen in Fig. 2⇓A, while TGF-β and IL-10 failed to up-regulate the level of IGF-I expression, IL-13 behaved similarly to IL-4 by increasing the expression of IGF-I. Costimulation with IL-4 and either IL-13, IL-10, or TGF-β resulted in transcript levels that were similar to the levels observed with IL-4 or IL-13 alone. Fig. 2⇓B shows that the levels of IGF-I protein secreted into culture supernatants recapitulate the pattern of expression of IGF-I transcripts. Collectively, these findings indicate that the stimulation of IGF-I by Th2-profile cytokines is facilitated by IL-4 and IL-13 and that their effects oppose the previously described inhibitory effects of the Th1 cytokines IFN-γ and IFN-αβ (21, 26). Thus, the levels of IL-4/IL-13 and IFNs play a key role in the differential regulation of IGF-I expression.

FIGURE 2.
FIGURE 2.

IGF-I transcripts are up-regulated by IL-4 and IL-13 but not by IL-10 or TGF-β. A, Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and GAPDH after stimulation with of IL-4, IL-10, IL-13, or TGF-β (all 5 ng/ml) for 18 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, IGF-I protein levels in culture supernatants from bone marrow-derived macrophages stimulated with IL-4 (2 ng/ml), IL-10 (5 ng/ml), IL-13 (5 ng/ml), TGF-β (2 ng/ml), or IL-4 with either IL-10, IL-13, or TGF-β for 26 h. The values represent the mean from three independent experiments ± the SEM.

Mutual antagonism of IGF-I expression by IL-4 and IFN-γ

IL-4 and IFN-γ exhibit mutual antagonism in many systems and this effect most commonly arises as a consequence of the sequence of exposure to each cytokine. Therefore, we determined the consequences of sequential or coincubation with IL-4 and IFN-γ on the expression of IGF-I by macrophages. Macrophage monolayers were pretreated with either 20 U/ml IFN-γ or 1 ng/ml IL-4 for 2 h and then stimulated with 1 ng/ml IL-4 or 20 U/ml IFN-γ, respectively, for 16 h. IGF-I expression was determined by Northern blot analysis with the exon 4 probe. As can be seen in Fig. 3⇓A, coincubation with IFN-γ and IL-4 did not change the level of IGF-I mRNA expression compared with basal levels, although when used alone, these concentrations of cytokines decreased and increased IGF-I levels, respectively. Similar effects were also seen when the cells were simultaneously exposed to both cytokines (Fig. 3⇓A). Fig. 3⇓B shows that the levels of IGF-I protein after stimulating macrophages with either 20 U/ml IFN-γ, 2 ng/ml IL-4, 5 ng/ml IL-13, or IFN-γ in the presence of either IL-4 or IL-13 for 26 h. As was seen with IGF-I mRNA expression, IFN-γ alone decreased IGF-I protein levels, while both IL-4 and IL-13 increased IGF-I protein expression. Also, as seen with IGF-I mRNA, coincubation with IFN-γ and either IL-4 or IL-13 did not change the level of IGF-I protein expression from that produced by unstimulated cells. Thus, each cytokine mutually represses the response to the other cytokine emphasizing that the presence or absence of each cytokine is important in determining the level of IGF-I expression.

FIGURE 3.
FIGURE 3.

Concurrent stimulation with IFN-γ and IL-4 does not alter the IGF-I transcript expression levels from basal levels. A, Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and 18S after stimulation with 20 U/ml IFN-γ or 1 ng/ml IL-4 for 16 h either with or without pretreatment with 20 U/ml IFN-γ or 1 ng/ml IL-4 for 2 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, IGF-I protein levels in culture supernatants from bone marrow-derived macrophages stimulated with 20 U/ml IFN-γ, 2 ng/ml IL-4, 5 ng/ml IL-13, or IFN-γ with either IL-4 or IL-13 for 26 h. The values represent the mean from three independent experiments ± the SEM.

Transcriptional regulation of IGF-I expression by IL-4

Because the expression of many growth factors and cytokines are subject to regulation at multiple levels, we next determined whether the increase in IGF-I expression by IL-4 was regulated 1) at the transcriptional level, 2) by changes in mRNA stability, or 3) by both mechanisms. This question was first addressed by investigating the effects of the transcriptional inhibitor, actinomycin D, on changes in IGF-I transcript expression induced by IL-4. Macrophage monolayers were stimulated with 1 ng/ml IL-4 in the presence or absence of 5 μg/ml actinomycin D for up to 10 h and IGF-I transcript levels were analyzed by Northern blotting. The viability of the cells was assessed by trypan blue dye exclusion and was found to be >98% in cells exposed to actinomycin D for 10 h in either the presence or absence of IL-4. As can be seen in Fig. 4⇓A, stimulation with IL-4 in the presence of actinomycin D inhibited the increase in IGF-I expression at both 5 and 10 h compared with IL-4 stimulation alone. In the absence of IL-4, the basal level of IGF-I was modestly depressed in the presence of actinomycin D consistent with a requirement for basal transcription from this gene (Fig. 4⇓A). PhosphorImager analysis confirmed that in the presence of actinomycin D, IL-4 was unable to up-regulate IGF-I transcripts (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

Transcription is required for IL-4 to up-regulate IGF-I transcripts. A, Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and 18S after treatment with 1 ng/ml IL-4 in the presence or absence of 5 μg/ml actinomycin D for 5 or 10 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, PhosphorImager analysis of the blot shown in A, plotted relative to the unstimulated condition.

These findings were independently confirmed by measuring IGF-I pre-mRNA levels in response to stimulation with IL-4. Pre-mRNA transcripts are rapidly processed to mRNA (33) making the level of pre-mRNA a bona fide indicator of the degree of ongoing IGF-I transcription. Therefore, we used PCR amplification of an IGF-I intronic sequence to quantify the levels of IGF-I pre-mRNA. Mouse macrophages were stimulated with IL-4 (1 ng/ml) for 12 h and total RNA was isolated and treated with DNase to digest contaminating genomic DNA. The samples were then reverse-transcribed and the resulting cDNA was used as a template for PCR using primers specific for intron 5 of the IGF-I gene (illustrated schematically in Fig. 5⇓A). In addition, a control PCR was performed using primers and competimers specific for 18S ribosomal RNA. Fig. 5⇓B shows that the PCR products obtained with the IGF-I intron 5 primers are of the expected size (350 bp) while the 18S ribosomal RNA primers led to the amplification of a product with an expected size of 495 bp. Fig. 5⇓, B and C, show that stimulation with IL-4 increased the level of expression of IGF-I pre-mRNA by an average of 2.4-fold compared with unstimulated cells confirming that the increased expression of IGF-I by IL-4 occurs at the level of transcription. Because the down-regulation of IGF-I expression by IFN-γ has also been shown to occur at the level of transcription (26), we also quantified IGF-I pre-mRNA levels in IFN-γ-stimulated (20 U/ml, 18 h) macrophages as a control. Fig. 5⇓, B and C, show that IFN-γ down-regulated IGF-I pre-mRNA levels by an of average of 65% compared with unstimulated cells. No PCR products were detected in the absence of reverse transcriptase, ruling out the possibility that genomic DNA was being amplified.

FIGURE 5.
FIGURE 5.

IGF-I pre-mRNA is down-regulated by IFN-γ and up-regulated by IL-4. A, Schematic representation of the IGF-I gene showing the location of the intron 5-specific primers used for the PCR. B, Storm-generated image of PCR products using cDNA template made from bone marrow-derived macrophages after stimulation with 20 U/ml IFN-γ for 18 h or 1 ng/ml IL-4 for 12 h. Either 28 or 32 cycles of PCR were performed. The expected size of the IGF-I or 18S products are shown on the right. The last three lanes received no reverse transcriptase during the making of the cDNA. C, Storm analysis of the gel shown in B, plotted relative to the unstimulated condition. This is a representative experiment of greater than three independent experiments.

We also investigated the effect of IL-4 on the stability of IGF-I transcripts by determining the rate of decay of IGF-I transcripts in IL-4-stimulated macrophages. The cells were stimulated with IL-4 (1 ng/ml) for 10 h before the addition of the transcriptional inhibitor actinomycin D (5 μg/ml) and then lysed 0, 4, 8, and 12 h later. Fig. 6⇓A shows that IL-4 increased IGF-I transcript expression as shown earlier. Following the addition of actinomycin D, the levels of both the 7.5-kb and the 1-kb IGF-I transcripts began to decline by 8 h and were reduced to ∼45% of the initial levels by 12 h (Fig. 6⇓B). A similar rate of decline of IGF-I transcript levels was also seen in unstimulated cells in the presence of actinomycin D (Fig. 6⇓). Collectively, these findings indicate that IL-4 does not affect the stability of IGF-I mRNA and thus stimulates IGF-I expression by increasing the rate of transcription of the IGF-I gene.

FIGURE 6.
FIGURE 6.

IL-4 does not increase the stability of IGF-I transcripts. A, Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and 18S after treatment with 1 ng/ml IL-4 for 10 h followed by 5 μg/ml actinomycin D for 0, 4, 8, or 12 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, Graphic representation of the PhosphorImager data from the blot shown in A showing the percentage of IGF-I transcripts remaining after treatment with actinomycin D.

Requirement of STAT6 and STAT1 in the regulation of IGF-I expression by IL-4 and IFN-γ

STAT6 is the major transcription factor that regulates the expression of IL-4-responsive genes. To address the role of STAT6 in the increased expression of IGF-I induced by IL-4, we compared IGF-I expression levels in macrophages from wild-type (BALB/c) and STAT6-deficient mice following stimulation with IL-4 (1, 2.5, or 5 ng/ml) for 18 h. Macrophages were also stimulated with IFN-γ (100 U/ml) as a control. As shown in Fig. 7⇓, A and B, IL-4 increased IGF-I transcript levels in the wild-type (BALB/c) macrophages, but not in the STAT6-deficient macrophages. The lack of STAT6 had no effect on the basal level of IGF-I expression as demonstrated by the comparable levels of IGF-I in the absence of stimulus in both the wild-type and STAT6-deficient cells. Additionally, STAT6 deficiency did not alter the ability of IFN-γ to down-regulate IGF-I. Fig. 7⇓C shows the results of Western blots confirming the absence of native-STAT6 and the lack of IL-4-dependent tyrosine phosphorylation of STAT6 in the STAT6-deficient cells.

FIGURE 7.
FIGURE 7.

STAT6 is required for IL-4 to up-regulate IGF-I transcripts. A, Northern blot analysis of total RNA (15 μg/lane) from wild-type (BALB/c) or STAT6-deficient bone marrow-derived macrophages hybridized with probes specific for IGF-I and GAPDH after treatment with IL-4 (0–5 ng/ml) or IFN-γ (100 U/ml) for 18 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, PhosphorImager analysis of the blot shown in A, plotted relative to the unstimulated condition. C, Western blot analysis of whole cell lysates from wild-type (BALB/c) or STAT6-deficient bone marrow-derived macrophages. The wild-type macrophages were stimulated with 1 ng/ml IL-4 for 15 min and the STAT6-deficient macrophages were stimulated with IL-4 (1 or 5 ng/ml) for 15 min. The blots were probed with Abs specific for total STAT6 or phosphorylated STAT6. Representative blots of three independent experiments.

Because STAT1 is the major transcription factor in IFN-γ gene expression, we also addressed the role of STAT1 in the IFN-γ-induced down-regulation of IGF-I by investigating IGF-I expression levels in macrophages from STAT1-deficient mice. Macrophage monolayers from wild-type (svj129) and STAT1-deficient mice were stimulated with IFN-γ (30, 60, or 100 U/ml), or with IL-4 (10 ng/ml) as a control, for 18 h before measuring IGF-I expression. Fig. 8⇓, A and B, show that IFN-γ down-regulated IGF-I transcript levels in wild-type cells in a dose-dependent manner but was without effect in the STAT1-deficient macrophages. In contrast, the ability of IL-4 to up-regulate IGF-I expression was unimpaired in the STAT1-deficient cells. As a control to verify that STAT1 functional activity was absent in these cells, we quantified nitrite anion (NO2−) production by wild-type (svj129) and STAT1-deficient macrophages. Macrophages from STAT1-deficient and wild-type mice were costimulated with 20 ng/ml TNF-α and 20 U/ml IFN-γ for 18 h and NO2− accumulation in the supernatant was measured as previously described (34). Fig. 8⇓C shows that costimulation with TNF-α and IFN-γ promoted NO2− production in the wild-type macrophages but not in the STAT1-deficient cells.

FIGURE 8.
FIGURE 8.

STAT1 is required for IFN-γ to down-regulate IGF-I transcripts. A, Northern blot analysis of total RNA (15 μg/lane) from wild-type (svj129) or STAT1-deficient bone marrow-derived macrophages hybridized with probes specific for IGF-I and GAPDH after treatment with IFN-γ (0–100 U/ml) or IL-4 (10 ng/ml) for 18 h. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, PhosphorImager analysis of the blot shown in A, plotted relative to the unstimulated condition. C, STAT1 is required for NO2− production. NO2− assay on supernatant from wild-type (svj129) or STAT1-deficient bone marrow-derived macrophages stimulated with 20 U/ml IFN-γ and/or 20 ng/ml TNF-α for 18 h.

The finding that the reciprocal changes in the expression of IGF-I by IL-4 and IFN-γ is dependent on STAT6 and STAT1, respectively, raised the question of whether the effects of these cytokines were regulated by the expression of additional gene products. To address this question, we determined whether de novo protein synthesis was required for IL-4 and IFN-γ to mediate their regulatory effects on IGF-I transcript expression. Macrophage monolayers were pretreated with cycloheximide for 1 h and then stimulated with 1 ng/ml IL-4 or 20 U/ml IFN-γ for 10 h. The viability of the cells was assessed by trypan blue dye exclusion and was found to be >98% in cells exposed to cycloheximide for 10 h in either the presence or absence of IL-4 or IFN-γ. As can be seen in Fig. 9⇓A, pretreatment with cycloheximide had no effect on the ability of either IL-4 or IFN-γ to increase or decrease IGF-I transcript levels, respectively. PhosphorImager analysis of the blot showed that IFN-γ decreased IGF-I transcript levels by >60% and IL-4 was able to increase IGF-I levels by almost 4-fold in the presence of cycloheximide (Fig. 8⇑B). Thus, STAT6 and STAT1 regulate the expression of IGF-I by IL-4 and IFN-γ, respectively. In addition, the regulation of IGF-I expression by these transcription factors occurs independently of de novo protein synthesis ruling out the possibility that they serve to promote the expression of additional genes that indirectly regulate the expression of IGF-I.

FIGURE 9.
FIGURE 9.

IFN-γ and IL-4 do not require de novo protein synthesis to exert their regulatory effects on IGF-I transcripts. A, Northern blot analysis of total RNA (15 μg/lane) from bone marrow-derived macrophages hybridized with probes specific for IGF-I and GAPDH after treatment with 20 U/ml IFN-γ or 1 ng/ml IL-4 for 10 h in the presence or absence of 10 or 20 μg/ml cycloheximide. Cycloheximide was added 1 h before cytokine stimulation. The multiple IGF-I mRNA species are shown. Representative blot of three independent experiments. B, PhosphorImager analysis of the blot shown in A, plotted relative to the unstimulated condition.

Discussion

In 1983, Bitterman et al. (8) found that alveolar macrophages from patients with IPF spontaneously released a peptide growth factor capable of promoting fibroblast proliferation. The growth factor was found to function as a progression factor for fibroblast proliferation and was termed AMDGF. Later work revealed AMDGF to be IGF-I (10). Since that time, other studies have shown that alveolar epithelial cells and interstitial macrophages are also capable of producing IGF-I in IPF (12), and that the level of expression of IGF-I by interstitial macrophages is associated with increased disease severity (12). These studies collectively emphasize the need to further our understanding of the pathophysiology of IGF-I in pulmonary fibrosis and underscore the importance of macrophages, especially interstitial macrophages, in the development and progression of this debilitating group of diseases. However, little is known about the mechanisms that govern the expression of IGF-I by macrophages. Initial studies by Noble et al. (28) showed that exposure of mouse macrophages to hyaluronnan, a glycosaminoglycan component of the extracellular matrix, stimulates the expression of IGF-I following its recognition by CD44. Arkins et al. (35) showed that M-CSF and IL-3 are capable of inducing IGF-I expression during monocyte-macrophage development. In addition, Lake et al. (21) and Arkins et al. (26) have established that the expression of IGF-I is down-regulated by IFN-γ and IFN-αβ. In view of the latter findings, combined with indications that the cytokine profile of the lung is skewed toward a Th2 profile in pulmonary fibrosis (13, 25), the goal of the present study was to investigate the role of Th2 cytokines in the regulation of IGF-I expression by macrophages and to determine their mechanism of action. A major finding of this study is that the Th2-profile cytokines IL-4 and IL-13 stimulate the expression of IGF-I by mouse macrophages, while other Th2-related cytokines, namely IL-10 and TGF-β, do not. This pattern of cytokine responsiveness has also been observed in other systems. For example, Relic et al. (36) have shown that IL-4 and IL-13, but not IL-10, protect human rheumatoid synovial cells from NO-induced apoptosis. Similarities in the responses to IL-4 and IL-13 have also been observed in the expression of eotaxin by human airway smooth muscle cells (37) and TGF-β2 production by human bronchial epithelial cells (38). These similarities are most likely the result of signaling by the IL-4R α-chain, and the IL-13R α1-chain, IL-4R α-chain heterodimer, respectively (39, 40). Thus, combined with our earlier observations that the Th1 cytokines IFN-γ and IFN-αβ down-regulate the expression of IGF-I, the finding that IL-4 and IL-13 stimulate IGF-I expression establishes a homeostatic mechanism for the regulation of IGF-I expression by immune regulatory cytokines.

In seeking to understand the fundamental mechanisms by which IL-4 stimulates the expression of IGF-I, we have established this to occur at the level of IGF-I transcription. This conclusion is based on findings that the increase in IGF-I expression induced by IL-4 was: 1) blocked by the transcriptional inhibitor, actinomycin D, 2) accompanied by an increase in IGF-I transcription as reflected by an increase in the level of IGF-I intron 5 containing pre-mRNA, and 3) not associated with a change in the stability of spliced IGF-I transcripts. Furthermore, based on results obtained with STAT6-deficient macrophages, we have also determined that STAT6 is necessary for the increase in IGF-I expression following stimulation with IL-4. In addition, the requirement for STAT6 is most likely due by a direct effect of STAT6 on IGF-I transcription because IL-4 was found to increase IGF-I transcript expression in the presence of the protein synthesis inhibitor, cycloheximide, thus ruling out the possibility of de novo translation of newly expressed mRNAs. This conclusion is also consistent with the widespread role of STAT6 in IL-4-stimulated gene expression including such genes as the IL-1R antagonist (41) and Bcl-xL (42). Interestingly, the 5′-flanking region of the IGF-I gene contains three sites conforming to the basic STAT6 consensus cis-element located ∼0.4–0.5 kb upstream of the major IGF-I transcriptional start site in exon 1. However, preliminary studies in which ∼1.5 kb of the 5′-flanking region of the mouse IGF-I gene were fused to a luciferase reporter gene failed to show IL-4 responsiveness when transfected into RAW264.7 cells (M. W. Wynes and D. W. H. Riches, unpublished observations). Thus, while it remains possible that this site is necessary for IL-4-stimulated IGF-I expression, additional IL-4-responsive sites located outside the boundaries of the proximal promoter are also speculated to be necessary to promote IGF-I transcription in response to IL-4.

In contrast to the stimulatory effects of IL-4 and IL-13 on IGF-I expression, IFN-γ was found to repress both basal and IL-4-stimulated IGF-I expression through a mechanism that requires the transcription factor, STAT1. In addition, exposure to IL-4 prevented the down-regulation of IGF-I expression seen in response to IFN-γ. The ability of IL-4 and IFN-γ to mutually antagonize their respective responses has also been seen in other settings. For example, IFN-γ inhibits IL-4-stimulated IgE class-switching in B cells (43, 44, 45), while IL-4 represses the transcription of the IFN-γ-induced genes IRF-I (46, 47) and CD40 (48) in macrophages. Two separate mechanisms have been proposed to contribute to these effects. First, both IFN-γ and IL-4 have been shown to induce the expression of suppressor of cytokine signaling-1, which inhibits signaling by both cytokines (43, 49, 50). However, this mechanism cannot account for the observed effects of IFN-γ and IL-4 on IGF-I expression because these responses occur in the presence of the protein synthesis inhibitor, cycloheximide. Second, both the IL-4-dependent activation of STAT6 and the IFN-γ-dependent activation of STAT1 use transcriptional coactivators such as CREB binding protein/p300, which are usually of limited abundance (51, 52). Horvai et al. (53) have shown that IFN-γ down-regulates transcription of the macrophage scavenger receptor gene by a mechanism in which tyrosine phosphorylated STAT1 preferentially binds CREB binding protein/p300 and prevents the coactivator from interacting with AP-1 thereby inhibiting the AP-1-dependent expression of the scavenger receptor. We speculate that the STAT1-dependent down-regulation of the basal and IL-4-stimulated expression of IGF-I by macrophages may involve a fundamentally similar mechanism.

The finding that Th1 and Th2 cytokines regulate IGF-I expression in a reciprocal and mutually restrictive fashion emphasize the importance of the concentrations of Th1 and Th2 cytokines in the regulation of IGF-I expression in the lung environment. Previous studies in IPF and sarcoid have shown that while IL-4 and IFN-γ are coexpressed in sarcoid, IFN-γ expression is reduced or absent in IPF allowing IL-4 expression to dominate (14, 19). The results of the present study suggest that these conditions favor the increased expression of IGF-I by macrophages, a condition that is seen in lung biopsy specimens obtained from IPF patients (12).

The possible consequences of increased macrophage expression of IGF-I are several-fold. As discussed earlier, IGF-I stimulates many responses that are consistent with its proposed role in the pathogenesis and progression of IPF including acting as a progression factor for fibroblast proliferation (8) and stimulating collagen matrix synthesis (22). However, recent studies have also revealed the importance of IGF-I as a survival factor that acts to inhibit apoptosis in a wide variety of cell types including myofibroblasts, and skeletal and smooth muscle cells (54, 55). In vivo studies have confirmed these findings. For example, Barton et al. (56) used mdx mice to show that transgenic overexpression of IGF-I protects mice from the loss in skeletal and diaphragmatic muscle in a model of Duchenne muscular dystrophy. Similarly, George et al. (57) showed that β cell-specific transgenic expression of IGF-I under the influence of the insulin promoter protected mice from insulitis and diabetes induced by streptozotocin. These findings suggest that while growth factor expression plays an important role in fibroblast proliferation and differentiation into myofibroblasts, increased expression of IGF-I in alveolar and interstitial tissues in IPF and other fibroproliferative diseases may also serve to inhibit fibroblast and myofibroblast apoptosis. Thus, understanding the mechanisms that regulate the expression of IGF-I in fibroproliferative lung diseases can be expected to shed new light on the pathogenesis of these lesions.

Acknowledgments

We acknowledge Linda Remigio and Fukun Hoffmann for outstanding technical assistance, and Dr. Robert Schreiber (Washington UniversitySchool of Medicine, St. Louis, MO) for graciously providing the STAT1-deficient mice. Also, we thank Dr. Jay Wescott (ELISA Tech, Denver, CO) for developing the mouse IGF-I ELISA. Lastly, we thank Dr. Peter Rotwein (University of Oregon Health Sciences Center, Portland, OR) for providing the IGF-I exon 4 cDNA probe.

Footnotes

  • ↵1 This work was supported by Public Health Service Grants HL68628, HL65326, HL55549, and Specialized Center of Research Grant HL56556 from the National Heart, Lung, and Blood Institute, National Institutes of Health. M.W.W. was supported, in part, by Institutional T-32 Training Grant AI00048 from the National Institutes of Health.

  • ↵2 Address correspondence and reprint requests to Dr. David W. H. Riches, Program in Cell Biology, Department of Pediatrics, Neustadt Room D405, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: richesd{at}njc.org

  • ↵3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; IGF, insulin-like growth factor; AMDGF, alveolar macrophage-derived growth factor.

  • Received March 21, 2003.
  • Accepted July 29, 2003.

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