Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF β were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on <20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-γ-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-γ treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-β synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-β, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.

Idiopathic pulmonary fibrosis (IPF)3 is an enigmatic disease that is characterized by the exuberant deposition of collagenous material in the lower airspace (1). Although the etiopathogenesis of IPF is poorly understood, T lymphocytes (2), mast cells (3), and eosinophils (4) have all been implicated in the progression of this disease. A putative cytokine effector in IPF is IL-4, which can be released by T cells (5) and mast cells (6) and is postulated to contribute, in part, to the activation of fibroblasts to synthesize extracellular matrix (7). However, the lung-derived factors that facilitate the recruitment of immune cells to the lung and fibroblast activation during the inflammatory and fibrotic process remain poorly characterized (8). Detailed examinations of the soluble factors present in the bronchoalveolar lavage and in lung biopsies from IPF patients have revealed that levels of monocyte chemoattractant protein-1 (MCP-1) are consistently augmented (9, 10, 11), suggesting that MCP-1 may play a role in IPF.

The recent discovery that MCP-1 is involved in fibrotic events in the lung (reviewed in Ref. 12) and in the kidney (13) led us to hypothesize that alterations in MCP-1 generation by lung fibroblasts may predispose the lung to exaggerated infiltration by T cells and other mononuclear cells. The presence of these activated immune cells may then lead to the release of profibrotic mediators into the interstitial spaces of the lung. A more direct role for MCP-1 in fibrotic responses is derived from recent studies by Gharaee-Kermani et al. (14) who showed that MCP-1 contributes to de novo generation of type 1 procollagen through its effect on TGF-β synthesis by pulmonary fibroblasts. Alterations in MCP-1 generation by the lung fibroblast concomitant with its altered ability to respond to MCP-1 may then dictate the outcome of the pulmonary immune response, leading to a resolution of the inflammatory response without overt fibroblast activation or to persistent inflammation and a fibrotic response.

Thus, the aim of the present study was to examine the expression of MCP-1, TGF-β, and the MCP-1 receptor, C-C chemokine receptor 2 (CCR2), in lung fibroblasts derived from normal mice and from mice with phenotypically distinct pulmonary granulomas (15). We also examined whether these cultured fibroblasts had altered capacities to express type I and III procollagen mRNA. Previous studies have shown that the formation of persistent pulmonary granulomas in mice following the embolization of Sepharose beads coated with Schistosoma mansoni egg Ag (SEA) into mice sensitized with SEA (16) is dependent upon IL-4 (17). In the Th2-type model, the pulmonary granuloma is composed of mononuclear cells and eosinophils, and this granuloma does not resolve due, in part, to the progressive deposition of collagen. The Th1-type granuloma counterpart to this model is elicited by purified peptide derivative (PPD)-coated bead delivery to CFA-sensitized mice, and this granuloma is promoted by IFN-γ and IL-12 (16). The Th1-type granuloma is initially composed of mononuclear cells, and it resolves without collagen deposition by 8 days after PPD-bead administration. More recent studies have begun to elucidate the role of endogenous MCP-1 in the development of the Th2-type (18) and Th1-type (19) pulmonary granulomas. However, the potential contribution of lung fibroblasts to the generation of MCP-1 and TGF-β in either model has not been thoroughly examined, nor has any analysis of changes in CCR2, the only known C-C chemokine receptor that binds MCP-1 (20), on these cells been previously reported.

Recombinant murine IL-4 was purchased from R&D Systems (Minneapolis, MN), and recombinant murine IFN-γ was purchased from Genzyme (Cambridge, MA). Polyclonal goat anti-mouse CCR2 and anti-mouse β-actin Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and purified goat IgG was obtained from Cappel (West Chester, PA). Biotin-labeled secondary donkey anti-goat IgG was purchased from Jackson ImmunoResearch (West Grove, PA). The following immunoreagents were acquired from Caltag Laboratories (Burlingame, CA): FITC-labeled rat anti-mouse F4/80 mAb, FITC-labeled rat IgG2b and avidin-FITC. Streptavidin-peroxidase and chromagen substrate were both acquired from Bio-Rad (Richmond, CA). PCR primers for mouse CCR2, β-actin, and procollagen type I and III were purchased from Genosys Biotechnology (Woodland, TX) or from the University of Michigan Biomedical Research Core Facility (Ann Arbor, MI). All other reagents were obtained from the Sigma (St. Louis, MO).

Female CBA/J mice (5–7 wk of age) were purchased from The Jackson Laboratories (Bar Harbor, ME) and were housed under specific pathogen-free conditions with access to food and water at all times. Mouse usage was approved by the University Laboratory Animal Medicine facility at the University of Michigan Medical School. As previously described in detail (17), CBA/J mice were sensitized by receiving either a subcutaneous injection of CFA diluted 1:1 with normal saline or 3000 freshly isolated S. mansoni eggs suspended in 0.5 ml normal saline. S. mansoni eggs were isolated from separate CBA/J mice following their tail infection with 25–30 S. mansoni cercariae (Puerto Rican strain) as previously described (21). Sixteen days after sensitization, mice received 3000 Sepharose 4B beads covalently coupled to either PPD from Mycobacteria species or SEA i.v. Age-matched control CBA/J mice received saline at the time of sensitization and saline alone i.v. 2 wk later.

Lung fibroblasts were cultured from CBA/J mice as previously described in detail (21). Briefly, whole lungs were removed from exsanguinated mice and transferred to RPMI 1640 containing 10% FBS (RPMI-10). The whole lungs were then mechanically separated into single-cell preparations, and contaminating RBC were lysed with hypotonic buffer (150 mM NH4Cl, 10 mM NaHCO3, 1 mM EDTA) for 2 min at 4°C. The remaining cells were added to 175 ml tissue culture flasks. These cells were grown at 37°C in a humidified CO2 incubator and fed DMEM containing 1% (v/v) antibiotic-antimycotic and 15% (v/v) FBS twice weekly. After a minimum of two passages, homogenous populations of fibroblasts were transferred to 6-well tissue culture plates for experiments. For convenience, lung fibroblasts isolated from the PPD-bead granuloma model are referred to as Th1-type fibroblasts, whereas fibroblasts isolated from the SEA-bead granuloma model are referred to as Th2-type fibroblasts. Lung fibroblasts grown from CBA/J mice that received saline during the sensitization and bead embolization periods are referred to as normal fibroblasts. Before use in any experiment, lung fibroblasts were transferred to two-well Labtek chamber culture slides, and these cells were stained for α-actin and desmin to confirm that the cells were of a fibroblast phenotype. All lung fibroblast cultures used in the following experiments were found to be factor VIII- and cytokeratin-negative and completely free of α-naphthyl acetate esterase-positive macrophages. As positive controls for esterase staining, splenic macrophages were cultured from mouse spleen as previously described (22). All cultured lung fibroblasts were used in these experiments up to the sixth passage, and the results presented herein were confirmed in fibroblasts from three separate primary cultures of normal and granulomatous lungs.

Five days before an experiment, each well in a 6-well tissue culture plate was initially seeded with ∼1.0 × 106 lung fibroblasts. When fibroblast confluence was reached, the growth medium was removed, and to these cells we then added IL-4 or IFN-γ suspended at 10 ng/ml in RPMI-10. IL-4 was added at 10 ng/ml because our previous studies showed that this concentration maximally promoted MCP-1 by cultured lung fibroblasts (22). Because Grandaliano et al. (23) showed that IFN-γ at 10 ng/ml maximally stimulated MCP-1 synthesis by mesangial fibroblasts, this concentration of cytokine was used in the present study. To address the role of MCP-1 in TGF-β synthesis and procollagen mRNA expression, MCP-1 synthesis was abolished in separate culture plates of normal, Th1-type, and Th2-type fibroblasts using purified phosphorothioated MCP-1 antisense oligonucleotide (5′-AAG CGT GAC AGA GAC CTG CAT AGT GGT GG-3′; 10 nM final concentration) during the treatment period. Our previous studies showed that this concentration of MCP-1 antisense oligonucleotide abolished MCP-1 production by cultured lung fibroblasts for 24 h (22). Purified phosphorothioated MCP-1 sense oligonucleotide (5′-CCA CCA CTA TGC AGG TCT CTG TCA CGC TT-3′; 10 nM final concentration) was added to other cultures of these same fibroblasts. Twenty-four hours later, cell-free supernatants were removed for ELISA measurements, and the adherent fibroblasts were washed with fresh RPMI-10 and prepared for RNA isolation, flow cytometry, or Western blot analysis.

Murine MCP-1 and TGF-β levels in cultures of normal, Th1-type, and Th2-type fibroblasts were determined in 50-μl supernatant samples using a standardized sandwich ELISA as previously described (24). Briefly, ELISA plates were coated with the appropriate cytokine capture Ab at a dilution of 1 μg/ml of coating buffer (0.6 M NaCl, 0.26 M H3BO4, 0.08 M NaOH, pH 9.6) for 16 h at 4°C. Excess capture Abs were washed away, and each plate was blocked for 90 min with 2% BSA-PBS at 37°C. After blocking, ELISA plates were washed with PBS-Tween 20 (0.05%; v/v), and samples (no dilution or 1:10; 50 μL volume) were added to wells in duplicate for 1 h at 37°C. Recombinant murine MCP-1 and TGF-β standard curves were used to calculate chemokine concentrations. The plates were then thoroughly washed and the appropriate biotinylated polyclonal rabbit anti-cytokine Ab (3.5 μg/ml) was added (21). After washing the plates 30 min later, streptavidin-peroxidase was added to each well for an additional 30 min. Chromagen substrate was subsequently added to each plate after it was thoroughly washed again, and plates were read on an ELISA plate scanner at 492 nm. The limit of detection for MCP-1 was consistently above 10 pg/ml, whereas the limit of ELISA detection of total TGF-β levels in each sample was consistently above 1 pg/ml.

Total RNA samples were prepared from cultured normal Th1- and Th2-type fibroblasts using guanidine isothiocyanate lysis as previously described (25). RNA from specific samples was reverse transcribed into cDNA using reverse transcription kit (Life Technologies, Rockville, MD) and oligo(dT) 12–18 primers. The amplification buffer contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, and 2.5 mM MgCl2. Specific oligonucleotide primers were added (200 ng/sample) to the buffer, along with 5 μl of reverse transcribed cDNA sample. The following oligonucleotide primers were used: CCR2 primer sequences: sense, 5′-CACGAAGTATCCAAGAGCTT-3′; antisense, 5′-CATGCTCTTCAGCTTTTTAC-3′; 422-bp product; β-actin primer sequences: sense, 5′-GCTCGGCCGTGGTGGTGAAGC-3′; antisense, 5′-GTGGGGCGCCCCAGGCACCA-3′; 450-bp product; procollagen type Iα primer sequences: sense, 5′-TCGTGACCGTGACCTTGCG-3′; antisense, 5′-GGATGAGTCGGCAGACACGGA-3′; 255-bp product; procollagen type III primer sequences: sense, 5′-GCTCAGAGTAGCACCATCAG-3′; antisense, 5′-GGCTGATGTACACATGCTCC-3′; 220-bp product.

The cDNA was amplified using the following cycling parameters. The mixture was first incubated for 5 min at 94°C and then cycled 35 times at 94°C for 30 s, 58°C for 45 s, and elongated at 72°C for 70 s. After amplification, the samples were separated on a 2% agarose gel containing 0.3 μg/ml of ethidium bromide, and bands were visualized and photographed using a translucent UV source.

Analysis of the expression of CCR2 on normal, Th1-, and Th2-type lung fibroblasts was accomplished using flow cytometry. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates using cold (i.e., 4°C) Ca2+- and Mg2+-free HBSS containing 5 mM EDTA. Single-cell suspensions were then obtained through rapid, repetitive pipetting of the fibroblasts. Primary goat anti-mouse IgG Ab directed against CCR2 at a dilution of 1 μg/ml HBSS containing 2% FBS and 0.5% sodium azide was then added to these suspensions for 30 min. Purified goat IgG at the same dilution was used as an appropriate control Ab. The fibroblasts were subsequently washed with HBSS, and a biotin-labeled secondary donkey anti-goat IgG (Jackson ImmunoResearch) diluted 1:200 was added for an additional 30 min. The secondary Ab was washed from the fibroblasts, and avidin-FITC was added for 15 min. In each flow cytometry experiment, all fibroblast cultures were screened for the presence of F4/80, a mouse macrophage marker, using an FITC-labeled anti-F4/80 mAb. An FITC-labeled rat IgG2b was used as a control. Following the last round of washes with HBSS, the fibroblasts were fixed in 2% paraformaldehyde. The fibroblasts were transferred to HBSS containing FBS and analyzed for chemokine receptor expression by the Biomedical Research Core Flow Cytometry Unit (University of Michigan, Ann Arbor, MI) using an EPICs XL cytometer (Coulter, Palo Alto, CA). A minimum of 5000 cells in each sample were analyzed, and all results were expressed as the percentage of CCR2-positive fibroblasts. Negative staining corresponded to the FITC levels measured in fibroblast preparations treated with the control Ab, whereas FITC staining greater than that observed in the control fibroblast preparations was considered positive staining for CCR2.

Analysis of the expression of total CCR2 protein in normal, Th1-, and Th2-type lung fibroblasts was also confirmed using Western blot analysis. Spleen cells from normal CBA/J mice were used as positive controls for CCR2 expression. Briefly, cultured fibroblasts and freshly dissected spleens were thoroughly washed with HBSS, and lysing buffer (100 mM Tris, 0.1% SDS, 0.1% Triton X-100, and 15% glycerol) chilled to 4°C was added to tissue culture plates containing the fibroblasts or whole spleens. The spleens were gently disrupted using a syringe plunger as previously described (22). All plates were maintained at 4°C and gently agitated on a plate rocker for 60 min. Samples were transferred to 1.5 ml Eppendorf tubes (Eppendorf Scientific, Westbury, NY) and were subsequently clarified through centrifugation at 10,000 × g for 30 min. Immunoprecipitation with anti-mouse CCR2 or anti-mouse β-actin polyclonal Abs was performed as previously described in detail (26). Immunoprecipitates were resolved in SDS/12.5% PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk in TBS and then incubated with goat anti-mouse IgG Ab directed against CCR2 or β-actin at a dilution of 1 μg/ml HBSS (containing 2% FBS and 1% Triton-X100) for 120 min at room temperature. Purified goat IgG at the same dilution was used as an appropriate control Ab. A peroxidase-labeled secondary donkey anti-goat IgG (Jackson ImmunoResearch) diluted 1:200 was added for 60 min. The reactions were developed using enhanced chemiluminescence (Pierce, Rockford, IL) according to the manufacturer’s instructions or using 3-amino-9-ethylcarbazole chromagen as previously described (27). β-actin levels were determined in each sample to ensure those equivalent quantities of protein from each was loaded onto the gels. CCR2 and β-actin bands were digitized and CCR2:β-actin ratios were calculated.

All experimental conditions were completed in triplicate for chemokine analysis and flow cytometry. Results are expressed as mean ± SEM of a minimum of three separate experiments. ANOVA and the Neuman-Keuls multiple comparison test was used to determine statistical significance between control and experimental groups; p < 0.05 was considered statistically significant.

Previous studies have shown that platelet-derived growth factor (PDGF)- stimulated murine fibroblasts are a source of MCP-1 (28) and that the de novo synthesis of MCP-1 is modulated by the pretreatment of these cells with IL-4 for 24 h (21). The aim of this experiment was to determine whether MCP-1 synthesis by murine lung fibroblasts was affected by the prior exposure of these cells to Th cytokine-mediated pulmonary granuloma responses. Six-well tissue culture plates were seeded with 1.0 × 106 fibroblasts/well, and no significant differences between the growth rates of these lung fibroblasts were apparent over a 24-h period (data not shown). All cultured lung fibroblasts used in the following experiments exhibited typical fibroblast morphology (Fig. 1,A), and no esterase-positive macrophages were detected in cultures after the second passage. Esterase-positive splenic macrophages present in separate cultures are shown in Fig. 1 B.

FIGURE 1.

α-Naphthyl acetate esterase staining in cultures of murine lung fibroblasts (A) and splenic macrophages (B). Esterase staining is indicated by the black pigmentation in the splenic macrophages (B), but similar staining was lacking from all cultures of normal Th1- and Th2-type lung fibroblasts.

FIGURE 1.

α-Naphthyl acetate esterase staining in cultures of murine lung fibroblasts (A) and splenic macrophages (B). Esterase staining is indicated by the black pigmentation in the splenic macrophages (B), but similar staining was lacking from all cultures of normal Th1- and Th2-type lung fibroblasts.

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MCP-1 accumulation in cultures of normal (i.e., lung fibroblasts grown from naive CBA/J mice), Th1-, and Th2-type granuloma fibroblast types over a 24-h period is shown in Fig. 2. MCP-1 levels in cultures of Th2-type fibroblasts exposed to culture media alone (i.e., control) were ∼2-fold greater (i.e., 42 ± 3 vs 20 ± 5 ng/ml; p ≤ 0.05) than levels present in cultures containing equivalent numbers of the other two fibroblast populations. Exposure of all three fibroblast cell types to 10 ng/ml of IL-4 significantly augmented the amount of MCP-1 detected in 24-h cultures by up to 2-fold above that detected under control conditions (Fig. 2). IFN-γ treatment (10 ng/ml) for 24 h also significantly enhanced the amount of immunoreactive MCP-1 in cultures of Th1- and Th2-type fibroblasts above levels detected under control conditions, but exposure of normal fibroblasts to IFN-γ for 24 h did not alter their MCP-1 levels (Fig. 2).

FIGURE 2.

MCP-1 generation by normal, Th1-, and Th2-type lung fibroblasts. All lung fibroblast types were added to six-well tissue culture plates at identical densities and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. MCP-1 was measured in cell-free supernatants using a specific ELISA. Data are mean ± SEM of five separate experiments.

FIGURE 2.

MCP-1 generation by normal, Th1-, and Th2-type lung fibroblasts. All lung fibroblast types were added to six-well tissue culture plates at identical densities and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. MCP-1 was measured in cell-free supernatants using a specific ELISA. Data are mean ± SEM of five separate experiments.

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CCR2 mRNA levels in normal, Th1, and Th2 fibroblast types following 24 h of culture with or without IL-4 or IFN-γ were examined using RT-PCR. Representative ethidium bromide-stained gels of RT-PCR products from all three fibroblast types are shown in Fig. 3, A–C. CCR2 mRNA was present in untreated normal fibroblasts (Fig. 3,A) at 24 h, but CCR2 mRNA was not detected in normal fibroblasts exposed to IL-4 for the same amount of time. Similar to untreated fibroblasts, normal fibroblasts exposed to IFN-γ for 24 h showed CCR2 mRNA. As indicated by the CCR2:β-actin ratios graphed below each RT-PCR gel, there were no differences in the level of CCR2 mRNA between untreated and IFN-γ-treated normal fibroblasts. CCR2 mRNA was not present in untreated or cytokine-treated Th1-type fibroblasts after 24 h in culture (Fig. 3,B). In contrast, Th2-type fibroblasts contained CCR2 mRNA only following the addition of IL-4 for 24 h. However, the CCR2:β-actin ratio for Th2-type fibroblasts was approximately one-half of that calculated in untreated and IL-4-treated normal fibroblasts (Fig. 3 C). Taken together, these findings suggested that IL-4 and IFN-γ regulated the CCR2 mRNA levels in a divergent fashion among the fibroblast populations. We subsequently determined the manner in which these cytokines affected CCR2 protein expression by the fibroblast populations.

FIGURE 3.

CCR2 and β-actin mRNA levels in normal (A), Th1-type (B), and Th2-type (C) fibroblasts. All three fibroblast types were seeded at identical densities in six-well tissue culture plates and treated as described in Materials and Methods. Depicted is a representative ethidium bromide-stained gel showing RT-PCR gel comparing β-actin and CCR2. The ratio of CCR2:β-actin expression is shown immediately below each panel and is representative of three similar experiments.

FIGURE 3.

CCR2 and β-actin mRNA levels in normal (A), Th1-type (B), and Th2-type (C) fibroblasts. All three fibroblast types were seeded at identical densities in six-well tissue culture plates and treated as described in Materials and Methods. Depicted is a representative ethidium bromide-stained gel showing RT-PCR gel comparing β-actin and CCR2. The ratio of CCR2:β-actin expression is shown immediately below each panel and is representative of three similar experiments.

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CCR2 is presently the only CCR that has been shown to bind MCP-1 and elicit a MCP-1-dependent effect (20). CCR2 levels on lung fibroblasts from normal CBA/J mice were examined first using flow cytometry. As shown in Fig. 4,A, 32 ± 5% of the fibroblasts exposed to culture media alone (i.e., control) for 24 h expressed CCR2, and this expression was modestly increased to 42 ± 3% when these cells were cultured in the presence of IL-4 for 24 h. Compared with the untreated fibroblasts, exposure of the fibroblasts to IFN-γ for 24 h did not change the percentage of cells that expressed CCR2 (Fig. 4,A). However, significantly (p ≤ 0.05) fewer CCR2-positive fibroblasts were present in IFN-γ-treated cultures compared with cultures exposed to IL-4. Representative histograms of CCR2 expression on normal fibroblasts left untreated or treated with either IL-4 or IFN-γ for 24 h are shown in Fig. 4 B.

FIGURE 4.

A, Representative flow cytometry results illustrating changes in the percentage of CCR2-positive lung fibroblasts cultured from normal CBA/J mice. After a minimum of three passages in culture, normal pulmonary fibroblasts were added to six-well tissue culture plates and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates and stained for CCR2 (see Materials and Methods). Cell-surface expression of CCR2 was determined by flow cytometry. τ, p ≤ 0.05 compared with IL-4-treated normal fibroblasts. B, Representative histograms of FITC staining corresponding to CCR2 expression on normal fibroblasts after 24 h in culture are shown. Negative staining corresponded to the FITC levels measured in fibroblast preparations treated with the control Ab, whereas FITC staining greater than that observed in the control fibroblast preparations was considered positive staining for CCR2. Similar results were obtained in five additional experiments.

FIGURE 4.

A, Representative flow cytometry results illustrating changes in the percentage of CCR2-positive lung fibroblasts cultured from normal CBA/J mice. After a minimum of three passages in culture, normal pulmonary fibroblasts were added to six-well tissue culture plates and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates and stained for CCR2 (see Materials and Methods). Cell-surface expression of CCR2 was determined by flow cytometry. τ, p ≤ 0.05 compared with IL-4-treated normal fibroblasts. B, Representative histograms of FITC staining corresponding to CCR2 expression on normal fibroblasts after 24 h in culture are shown. Negative staining corresponded to the FITC levels measured in fibroblast preparations treated with the control Ab, whereas FITC staining greater than that observed in the control fibroblast preparations was considered positive staining for CCR2. Similar results were obtained in five additional experiments.

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Flow cytometry of untreated and cytokine-treated Th1- and Th2-type lung fibroblasts for 24 h showed clear differences in the levels of CCR2 present on these cells. Approximately 40 ± 5% of the untreated Th2-type fibroblasts were positive for CCR2 after 24 h culture with RPMI-10 (Fig. 5). In contrast, ∼15% of the Th1-type fibroblasts were positive for CCR2 under similar culture conditions. Further differences in CCR2 on the cell surface of Th1- and Th2-type fibroblasts were apparent following exposure of these cells to IL-4. The percentage of CCR2-positive Th2-type fibroblasts following IL-4 treatment was significantly decreased by >50% from that observed in untreated cultures (Fig. 5). A 24-h treatment with IFN-γ also significantly reduced the percentage of CCR2-positive Th2-type fibroblasts from control levels of 40 ± 5% to 10 ± 2%. The low percentage of CCR2-positive Th1-type fibroblasts detected under control conditions was not changed following the exposure of these cells to either IL-4 or IFN-γ for 24 h before flow cytometry.

FIGURE 5.

Representative flow cytometry analysis demonstrating the percentage of CCR2-positive Th1- and Th2-type lung fibroblasts derived from CBA/J mice that had either Th1- or Th2-type pulmonary granulomatous inflammation for 8 days. After a minimum of three passages in culture, purified Th1- and Th2-type fibroblasts were added to six-well tissue culture plates and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates and stained for CCR2, and cell-surface expression of CCR2 was determined by flow cytometry (see Materials and Methods). Results shown are representative data from five similar experiments. ∗, p ≤ 0.05 compared with untreated Th2-type fibroblasts.

FIGURE 5.

Representative flow cytometry analysis demonstrating the percentage of CCR2-positive Th1- and Th2-type lung fibroblasts derived from CBA/J mice that had either Th1- or Th2-type pulmonary granulomatous inflammation for 8 days. After a minimum of three passages in culture, purified Th1- and Th2-type fibroblasts were added to six-well tissue culture plates and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates and stained for CCR2, and cell-surface expression of CCR2 was determined by flow cytometry (see Materials and Methods). Results shown are representative data from five similar experiments. ∗, p ≤ 0.05 compared with untreated Th2-type fibroblasts.

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To further examine changes in total CCR2 protein expression by fibroblasts from normal lungs and from Th1- and Th2-type granulomas, immunoprecipitates from all three fibroblast populations were subjected to Western blot analysis. Results from this analysis are shown in Fig. 6. Immunoprecipitation was not necessary to detect CCR2 protein in spleen cells (the CCR2-positive control), unlike the fibroblast populations in which this technique was required to reveal CCR2. In spleen cells, a major band of ∼43 kDa molecular mass and minor lighter bands were also detected (Fig. 6,A). Following the immunoprecipitation of the lysates from each fibroblast population, a single band of ∼86 kDa molecular mass was consistently observed (Fig. 6,B). The two-fold greater size of the CCR2 band present in the fibroblast populations may reflect the fact that CCR2 exists as a dimer in the fibroblast (29). The actin band was ∼80 kDa molecular mass. The size of the CCR2 and β-actin band did not differ between the untreated and cytokine-treated fibroblast populations. However, based on the ratios of CCR2:β-actin protein signals, differences in the level of CCR2 protein expression in the fibroblast populations were apparent (Fig. 6 C). When left untreated, Th2-type fibroblasts expressed two-fold more CCR2 protein than the other two populations. IL-4 augmented total CCR2 protein levels the greatest in Th1-type fibroblasts, but this treatment had no effect in normal fibroblasts. IFN-γ treatment increased total CCR2 protein in normal and Th1-type fibroblasts by ∼3-fold above those levels measured under control conditions, but this treatment did not alter CCR2 protein levels in Th2-type fibroblasts. Overall, CCR2 protein levels showed greater shifts in normal and Th1-type fibroblasts following cytokine treatment than in Th2-type fibroblasts in which total CCR2 protein levels appeared to be more tightly controlled.

FIGURE 6.

Representative Western blots of spleen cells (A) and normal, Th1-, and Th2-type fibroblasts (B) cultured from normal CBA/J mice or mice that had either Th1- or Th2-type pulmonary granulomatous inflammation for 8 days. Spleen cells were derived from normal mice and prepared for Western analysis as described in Materials and Methods. Immunoprecipitation was not required for the detection of CCR2 in spleen cells (A). After a minimum of three passages in culture, purified Th1- and Th2-type fibroblasts were added to six-well tissue culture plates and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates and processed for CCR2 and β-actin detection by immunoprecipitation and Western blot analysis (see Materials and Methods). Densitometry results depicting CCR2:β-actin protein ratios in the fibroblast populations are shown (C). Results are representative of three experiments with similar results.

FIGURE 6.

Representative Western blots of spleen cells (A) and normal, Th1-, and Th2-type fibroblasts (B) cultured from normal CBA/J mice or mice that had either Th1- or Th2-type pulmonary granulomatous inflammation for 8 days. Spleen cells were derived from normal mice and prepared for Western analysis as described in Materials and Methods. Immunoprecipitation was not required for the detection of CCR2 in spleen cells (A). After a minimum of three passages in culture, purified Th1- and Th2-type fibroblasts were added to six-well tissue culture plates and were left untreated or were exposed to IL-4 or IFN-γ for 24 h. Untreated and cytokine-pretreated fibroblasts were lifted from the tissue culture plates and processed for CCR2 and β-actin detection by immunoprecipitation and Western blot analysis (see Materials and Methods). Densitometry results depicting CCR2:β-actin protein ratios in the fibroblast populations are shown (C). Results are representative of three experiments with similar results.

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Sempowski et al. (30) recently demonstrated that murine lung fibroblasts respond to IL-4 by increasing type I procollagen gene expression. Differences in α type I and III procollagen gene expression were also evident among the fibroblast populations examined in the present study (Fig. 7, A–C). Type I procollagen mRNA was present in untreated and IL-4-treated normal fibroblasts, but the procollagen type I:β-actin ratio was two-fold higher after a 24-h IL-4 treatment. IFN-γ-treated fibroblasts lacked type I procollagen gene expression (Fig. 7,A). Type III procollagen gene expression was only present in untreated normal fibroblasts. Th1-type fibroblasts did not constitutively express either type of procollagen, but type I and type III procollagen mRNA were present in these fibroblasts following an IL-4 treatment (Fig. 7,B). The ratios of procollagen:β-actin showed that IL-4 augmented the mRNA levels for both procollagen types to a greater extent in these cells than in the normal fibroblasts exposed to IL-4. Th2-type fibroblasts also did not express type I or type III procollagen when left untreated, but both types of procollagen were present after only IL-4 treatment (Fig. 7 C). The combined ratios of procollagen:β-actin indicated that procollagen gene expression was increased the greatest in Th2-type fibroblasts compared with the other fibroblast populations.

FIGURE 7.

Procollagen type I and III gene expression in normal (A), Th1-type (B), and Th2-type (C) fibroblasts. All three fibroblast types were seeded at identical densities in six-well tissue culture plates and treated as described in Materials and Methods. Depicted is a representative ethidium bromide-stained gel showing RT-PCR gel with procollagen I and III products. The ratio of procollagen I or III:β-actin expression is shown immediately below each panel and is representative data from three separate experiments.

FIGURE 7.

Procollagen type I and III gene expression in normal (A), Th1-type (B), and Th2-type (C) fibroblasts. All three fibroblast types were seeded at identical densities in six-well tissue culture plates and treated as described in Materials and Methods. Depicted is a representative ethidium bromide-stained gel showing RT-PCR gel with procollagen I and III products. The ratio of procollagen I or III:β-actin expression is shown immediately below each panel and is representative data from three separate experiments.

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Recent studies by Gharaee-Kermani et al. (14) showed that MCP-1 induces the expression of TGF-β by cultured rat fibroblasts. This study suggested that MCP-1 promotes collagen expression by fibroblasts in a TGF-β-dependent manner. The aim of our experiments was to determine whether TGF-β accumulation after 24 h in culture varied among the types of fibroblasts examined in this study and whether this accumulation was dependent on endogenous MCP-1 generation. MCP-1 levels in all cultures of fibroblasts were not affected by MCP-1 sense oligonucleotide, but the presence of MCP-1 antisense oligonucleotide abolished MCP-1 production by all three fibroblast populations for 24 h. As shown in Fig. 8,A, cell-free supernatants from Th2-type fibroblasts under control conditions (i.e., exposed to MCP-1 sense oligonucleotide) contained 4-fold more TGF-β than similar supernatants removed from similarly treated normal or Th1-type fibroblasts. Furthermore, the addition of a MCP-1 antisense oligonucleotide to additional cultures significantly inhibited TGF-β production by normal and Th2-type fibroblasts, but the antisense treatment did not inhibit TGF-β levels in cultures of Th1-type fibroblasts. Following their exposure to IL-4 and MCP-1 sense oligonucleotide for 24 h, TGF-β release by normal fibroblasts was increased 3-fold above levels measured in cultures that received the sense oligonucleotide alone (Fig. 8 B). IL-4 treatment did not alter TGF-β production in cultures of Th1-type fibroblasts containing MCP-1 sense oligonucleotide, whereas the same treatments in cultures of Th2-type fibroblasts markedly decreased TGF-β levels in these cultures. The presence of MCP-1 antisense oligonucleotide and IL-4 for 24 h was associated with diminished or absent TGF-β levels in cultures of all three fibroblast populations. No TGF-β was detected in cultures of IFN-γ-stimulated normal, Th1-, or Th2-type fibroblasts, regardless of whether MCP-1 sense or antisense oligonucleotide was present in these cultures (data not shown).

FIGURE 8.

TGF-β generation by normal, Th1-, and Th2-type lung fibroblasts. All lung fibroblast types were added to six-well tissue culture plates at identical densities and were left untreated (A) or were exposed to IL-4 (B) for 24 h. In addition, MCP-1 sense or antisense oligonucleotides were added to these wells during this time (see Materials and Methods). TGF-β was measured in cell-free supernatants using a specific ELISA. No TGF-β was detected in any of the fibroblast cultures exposed to IFN-γ for 24 h (data not shown). Data are mean ± SEM from three separate experiments.

FIGURE 8.

TGF-β generation by normal, Th1-, and Th2-type lung fibroblasts. All lung fibroblast types were added to six-well tissue culture plates at identical densities and were left untreated (A) or were exposed to IL-4 (B) for 24 h. In addition, MCP-1 sense or antisense oligonucleotides were added to these wells during this time (see Materials and Methods). TGF-β was measured in cell-free supernatants using a specific ELISA. No TGF-β was detected in any of the fibroblast cultures exposed to IFN-γ for 24 h (data not shown). Data are mean ± SEM from three separate experiments.

Close modal

To further explore the role of endogenous MCP-1 synthesis in the expression of procollagen mRNA gene by normal, Th1-, and Th2-type fibroblasts, MCP-1 sense or antisense oligonucleotide was added with or without IL-4 or IFN-γ to fibroblast cultures before mRNA isolations and RT-PCR. Table I summarizes the procollagen:β-actin ratios from all three fibroblast types following the addition of MCP-1 antisense oligonucleotide for 24 h. Untreated and cytokine-challenged normal and Th2-type fibroblasts showed a marked dependence upon endogenous MCP-1 for the expression of procollagen type I and procollagen type III as evidenced by diminished or absent procollagen mRNA levels for both fibroblast populations. Conversely, the expression of procollagen by Th1-type fibroblasts under IL-4 stimulation was only marginally affected by the inhibition of MCP-1 by the MCP-1 antisense oligonucleotide treatment. Furthermore, the presence of MCP-1 antisense oligonucleotide appeared to promote the expression of both procollagen mRNAs in Th1-type fibroblasts exposed to IFN-γ for 24 h.

Table I.

Effect of MCP-1 antisense oligonucleotide on procollagen mRNA expression in normal, Th1, and Th2-type fibroblastsa

Fibroblast TypeFibroblast Treatment
NoneIL-4IFN-γ
Procollagen type I    
Normal 
Th1-type 1.9 1.6 
Th2-type 0.5 
Procollagen type III    
Normal 
Th1-type 1.4 1.8 
Th2-type 
Fibroblast TypeFibroblast Treatment
NoneIL-4IFN-γ
Procollagen type I    
Normal 
Th1-type 1.9 1.6 
Th2-type 0.5 
Procollagen type III    
Normal 
Th1-type 1.4 1.8 
Th2-type 
a

All fibroblasts were treated with MCP-1 antisense oligonucleotide with or without cytokines for 24 h before the isolation of mRNA and RT-PCR analysis. Data shown are procollagen: β-actin ratios.

The present study demonstrates that murine lung fibroblasts derived from normal lung and from Th1- and Th2-type pulmonary granuloma models display unique patterns of MCP-1, CCR2, type I and III procollagen, and TGF-β expression. All three fibroblast populations were examined after a 24-h control, IL-4, or IFN-γ treatment. The data presented in the present report suggest that the lung fibroblast is a very dynamic cell that is capable of altering its production of MCP-1 and its ability to respond to MCP-1 through alterations to CCR2 mRNA and protein. Such changes may be very important in determining the nature of the participation of this cell in chronic pulmonary inflammatory diseases such as IPF that are characterized by increased MCP-1, TGF-β, and the sustained deposition of extracellular matrix (9, 11).

The BALB/c-3T3 fibroblast cell line was first identified as a source of MCP-1/JE following activation with PDGF (28). Early studies also suggested that MCP-1 production by fibroblasts and other cells such as epithelial (31), and endothelial (32) cells could be augmented by cytokine treatment. Previous characterization of lung fibroblasts have shown that these cells express IL-4 receptors (30, 33), and our present findings showed that lung fibroblasts from normal and granulomatous lungs responded to IL-4 by increasing MCP-1 synthesis. IFN-γ did not increase MCP-1 synthesis by normal fibroblasts, although concomitant studies have shown that these cultured fibroblasts increase ICAM-1 expression in response to IFN-γ (C.M.H., unpublished observations). Interestingly, differential MCP-1 generation in response to IFN-γ has been shown previously in other cells. For example, IFN-γ augmented MCP-1 synthesis in normal mesangial cells (23), but it did not enhance MCP-1 production by resting human monocytes (34) or human periodontal fibroblasts (35). Both granuloma fibroblast populations derived showed increased MCP-1 generation in response to IFN-γ treatment for 24 h. Thus, the differential MCP-1 generation by the fibroblast populations in response to Th cytokine signals raises the possibility that the pulmonary fibroblast can exert divergent effects during pulmonary inflammatory events.

Before eliciting inflammatory effects, MCP-1 must interact with a seven-transmembrane domain G protein-coupled receptor. Presently, CCR2 is the only chemokine receptor that has been recognized to specifically bind MCP-1 (20), but other ligands for this receptor exist and include MCP-2, -3, -4, and -5 (36, 37, 38). CCR2 is unique from other chemokine receptors because the amino-terminal extracellular domain of this receptor confers its selectivity (39, 40). Previous studies have also documented that cytokines modulate the expression of CCR2 by monocytes (41, 42) and T cells (43). The present study also revealed key differences in the ability of IL-4 and IFN-γ to modulate CCR2 mRNA and protein levels in normal and granuloma fibroblasts. The discrepancies between CCR2 mRNA and protein levels among the fibroblast populations examined may be explained, in part, by the unique roles these cells acquire during their respective inflammatory reactions. The Th1-type fibroblast appears to be very limited in its ability to respond directly to exogenous MCP-1 because of the paucity of cell-surface expression of CCR2 by these cells. However, it should be noted that the Th1-type fibroblasts exhibited more total CCR2 protein levels than similar numbers of Th2-type fibroblast populations, particularly following an IL-4 or IFN-γ treatment. Presumably, MCP-1 generation by Th1-type pulmonary fibroblasts assists in the recruitment of the mononuclear cells that characterize the Th1-type (or PPD) pulmonary lesion (44), and these fibroblasts do not appear to require MCP-1 for their own synthetic purposes. Conversely, CCR2 expression in Th2-type fibroblasts facilitates the synthetic involvement by these cells in the fibrotic granulomatous response dominated by Th2 cytokines. In the absence of cytokine stimulation, Th2-type fibroblasts produced more MCP-1 and responded in kind to MCP-1 through the generation of profibrotic TGF-β. In addition, these fibroblasts also expressed the greatest amount of cell-surface CCR2 compared with the other fibroblast populations. Because fibroblasts are mobile during pulmonary inflammatory reactions (4, 45) and demonstrate chemotactic activity in the presence of MCP-1 (46, 47), it is possible that decreased cell-surface expression of CCR2 by Th2-type pulmonary fibroblasts exposed to IL-4 or IFN-γ limits their migration within the lung. Decreased cell-surface levels of CCR2 on Th2-type fibroblasts may also indicate that this cell is internalizing this receptor. The mechanisms by which IL-4, IFN-γ, and other factors such as LPS (42) and IL-2 (43) regulate CCR2 in many cell types are not immediately apparent, but studies are underway to investigate intercellular mechanisms through which CCR2 levels in pulmonary fibroblasts are regulated by cytokines.

Several investigators have demonstrated that IL-4 interacts with specific cell-surface receptors on fibroblasts causing these cells to deposit extracellular matrix such as collagen (7, 30), although the exact cellular mechanism through which this effect is mediated has not been elucidated. Type III collagen is the predominate isoform during the intermediate proliferative stage of IPF, whereas type I collagen is the predominate collagen found in the later stages of interstitial fibrosis (1). From the present study, it was apparent that IL-4 was a potent inducer of MCP-1 and TGF-β production and altered procollagen gene expression in all three fibroblast types. Th2-type fibroblasts derived from a fibrotic pulmonary granuloma model exhibited the greatest combined gene expression of procollagen type I and III after an IL-4 stimulus. Th1-type fibroblasts also expressed procollagen type I and III after IL-4 treatment, suggesting that these cells retain their ability to respond to a profibrotic stimulus, perhaps through a mechanism that is not completely dependent on IL-4-induced changes in MCP-1 and TGF-β synthesis. Data obtained from the MCP-1 antisense oligonucleotide experiments support this hypothesis because abolition of MCP-1 synthesis in cultures of Th1-type fibroblasts failed to affect the ability of these cells to generate procollagen type I and III after IL-4 stimulation. Interestingly, normal fibroblasts were the only fibroblasts that showed procollagen mRNA in the absence of cytokine stimulation, suggesting that the granulomatous response may alter the ability of the Th1- and Th2-type fibroblasts to generate extracellular matrix in the absence of an activating signal such as IL-4. This was further illustrated by the absence of procollagen I and III mRNA in untreated Th2-type fibroblasts despite the presence of greater than 40 ng/ml of MCP-1 and 30 pg/ml of TGF-β in these cultures. Consistent with previous studies (48, 49), IFN-γ was a potent inhibitor of TGF-β and procollagen gene expression in all three fibroblast populations studied. The present study suggests that this inhibitory effect may have been partially mediated through the effects of IFN-γ on CCR2. Considering that MCP-1 drives TGF-β synthesis by pulmonary fibroblasts (Ref. 14 and the present study), the regulation of MCP-1 activity through CCR2 during pulmonary fibrotic responses driven by Th2-type cytokines may be very important in modulating collagen deposition within the interstitial spaces of the lung. Although MCP-1 is an important chemoattractant in chronic models of lung injury (50), this C-C chemokine may also be involved in the deposition of extracellular matrix during interstitial fibrotic lung disease.

Interstitial fibrotic disease is an example of a chronic inflammatory disease in which the broad modulatory actions of MCP-1 may be relevant. Although increased MCP-1 in clinical IPF (9, 11, 51), experimental bleomycin-induced pulmonary fibrosis (52, 53, 54), and experimental particulate yeast cell wall-induced fibrosis (55) has been documented, an association between MCP-1 and profibrotic TGF-β (56, 57, 58, 59) was only recently shown. Interestingly, the activated fibroblast has been previously recognized as being an important source of MCP-1 and TGF-β during the pulmonary fibrotic process (60). Direct evidence for the involvement of MCP-1 in the development of interstitial fibrosis came from immunoneutralization studies by Lloyd et al. (13) who showed that MCP-1, but not RANTES nor macrophage inflammatory protein-1α, has a prominent role in the fibrotic process in a murine model of crescentic nephritis. TGF-β production by Th2-type fibroblasts was dependent on endogenous MCP-1 synthesis because the presence of MCP-1 antisense oligonucleotides markedly reduced TGF-β levels in these cultures. MCP-1 dependence was also shown with respect to procollagen mRNA expression in normal and Th2-type fibroblasts. Therefore, the production of TGF-β and procollagen mRNA by cultured pulmonary fibroblasts varies among the lung fibroblast types, and these discrepancies may relate to the type of pulmonary environment these cells were isolated from. Therefore, it is plausible that the regulation of pulmonary fibrosis in these models occurs at the level of the pulmonary fibroblast through MCP-1-dependent TGF-β and procollagen synthesis.

Thus, the present study supplies strong evidence that lung fibroblasts are altered in their ability to express MCP-1, CCR2, type I and III procollagen, and TGF-β after their exposure to distinct pulmonary inflammatory events. Considering recent evidence that MCP-1 (13, 14) and CCR2 (61) are involved in the deposition of extracellular matrix by fibroblasts, alterations in the synthesis of and the response to MCP-1 may be of great importance in the progression of clinical interstitial fibrotic diseases such as IPF.

We thank Robin G. Kunkel for her artistic contributions to this manuscript.

1

This work was supported by National Institutes of Health Grants 1P50HL60289, HL35276, and P01-HL31963.

3

Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; CCR2, C-C chemokine receptor 2; MCP-1, monocyte chemoattractant protein-1; PDGF, platelet-derived growth factor; PPD, purified peptide derivative; RPMI-10, RPMI 1640 plus 10% FBS; SEA, Schistosoma mansoni egg Ag.

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