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Departments of
*
Pathology and
Internal Medicine, Division of Pulmonary and Critical Care, University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Pathology, Veteran Affairs Medical Center, Ann Arbor, MI 48105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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- treatment augmented CCR2 protein in normal and Th1-type
fibroblasts. All three fibroblast populations exhibited MCP-1-dependent
TGF-ß 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-ß, CCR2, and procollagen following their
participation in pulmonary inflammatory processes, and these changes
may be important during fibrosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-ß 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-ß, 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- 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-ß 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant murine IL-4 was purchased from R&D Systems
(Minneapolis, MN), and recombinant murine IFN- was purchased from
Genzyme (Cambridge, MA). Polyclonal goat anti-mouse CCR2 and
anti-mouse ß-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, ß-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).
Mice
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.
Normal and granulomatous lung fibroblast culture
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 
-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
-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.
Experimental protocol
Five days before an experiment, each well in a 6-well tissue
culture plate was initially seeded with 
1.0 x
106 lung fibroblasts. When fibroblast confluence
was reached, the growth medium was removed, and to these cells we then
added IL-4 or IFN- 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- 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-ß 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.
ELISAs
Murine MCP-1 and TGF-ß 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-ß 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-ß levels in each sample was consistently above 1 pg/ml.
Preparation of cDNA and RT-PCR amplification
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; ß-actin primer
sequences: sense, 5'-GCTCGGCCGTGGTGGTGAAGC-3'; antisense,
5'-GTGGGGCGCCCCAGGCACCA-3'; 450-bp product; procollagen type I
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.
Flow cytometry
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.
Immunoprecipitation, SDS-PAGE, and Western blot analysis
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 x g for 30 min. Immunoprecipitation with anti-mouse CCR2 or anti-mouse ß-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 ß-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). ß-actin levels were determined in each sample to ensure those equivalent quantities of protein from each was loaded onto the gels. CCR2 and ß-actin bands were digitized and CCR2:ß-actin ratios were calculated.
Statistical analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x
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. 1B.
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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- 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- for 24 h did not
alter their MCP-1 levels (Fig. 2).
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on CCR2 mRNA in normal, Th1-, and
Th2-type lung fibroblasts
CCR2 mRNA levels in normal, Th1, and Th2 fibroblast types
following 24 h of culture with or without IL-4 or IFN- 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. 3A) 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- for 24 h showed CCR2 mRNA. As
indicated by the CCR2:ß-actin ratios graphed below each RT-PCR gel,
there were no differences in the level of CCR2 mRNA between untreated
and IFN--treated normal fibroblasts. CCR2 mRNA was not present in
untreated or cytokine-treated Th1-type fibroblasts after 24 h in
culture (Fig. 3B). In contrast, Th2-type fibroblasts
contained CCR2 mRNA only following the addition of IL-4 for 24 h.
However, the CCR2:ß-actin ratio for Th2-type fibroblasts was
approximately one-half of that calculated in untreated and IL-4-treated
normal fibroblasts (Fig. 3C). Taken together, these findings
suggested that IL-4 and IFN- 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.
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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. 4A, 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- for 24 h did not change the percentage of
cells that expressed CCR2 (Fig. 4A). However, significantly
(p 0.05) fewer CCR2-positive fibroblasts
were present in IFN--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- for
24 h are shown in Fig. 4B.
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). 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- 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- for 24 h
before flow cytometry.
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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. 6A).
Following the immunoprecipitation of the lysates from each fibroblast
population, a single band of 86 kDa molecular mass was consistently
observed (Fig. 6B). 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 ß-actin
band did not differ between the untreated and cytokine-treated
fibroblast populations. However, based on the ratios of CCR2:ß-actin
protein signals, differences in the level of CCR2 protein expression in
the fibroblast populations were apparent (Fig. 6C). 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- 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.
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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 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:ß-actin ratio was two-fold higher after a 24-h
IL-4 treatment. IFN--treated fibroblasts lacked type I procollagen
gene expression (Fig. 7A). 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. 7B). The
ratios of procollagen:ß-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. 7C). The combined ratios of
procollagen:ß-actin indicated that procollagen gene expression was
increased the greatest in Th2-type fibroblasts compared with the other
fibroblast populations.
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Recent studies by Gharaee-Kermani et al. (14) showed
that MCP-1 induces the expression of TGF-ß by cultured rat
fibroblasts. This study suggested that MCP-1 promotes collagen
expression by fibroblasts in a TGF-ß-dependent manner. The aim of our
experiments was to determine whether TGF-ß 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. 8A, cell-free supernatants
from Th2-type fibroblasts under control conditions (i.e., exposed to
MCP-1 sense oligonucleotide) contained 4-fold more TGF-ß 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-ß
production by normal and Th2-type fibroblasts, but the antisense
treatment did not inhibit TGF-ß levels in cultures of Th1-type
fibroblasts. Following their exposure to IL-4 and MCP-1 sense
oligonucleotide for 24 h, TGF-ß release by normal fibroblasts
was increased 3-fold above levels measured in cultures that received
the sense oligonucleotide alone (Fig. 8B). IL-4 treatment
did not alter TGF-ß 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-ß levels in
these cultures. The presence of MCP-1 antisense oligonucleotide and
IL-4 for 24 h was associated with diminished or absent TGF-ß
levels in cultures of all three fibroblast populations. No TGF-ß was
detected in cultures of IFN--stimulated normal, Th1-, or Th2-type
fibroblasts, regardless of whether MCP-1 sense or antisense
oligonucleotide was present in these cultures (data not shown).
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to fibroblast cultures before mRNA isolations
and RT-PCR. Table I summarizes the
procollagen:ß-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- for 24 h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-ß, 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- 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- (C.M.H., unpublished observations).
Interestingly, differential MCP-1 generation in response to IFN- has
been shown previously in other cells. For example, IFN- 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- 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- 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- 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-ß. 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- 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-, 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-ß
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-ß
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-ß in these cultures. Consistent
with previous studies (48, 49), IFN- was a potent
inhibitor of TGF-ß 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- on CCR2. Considering that MCP-1 drives TGF-ß 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-ß (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-ß 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-1, has a prominent role in the
fibrotic process in a murine model of crescentic nephritis. TGF-ß
production by Th2-type fibroblasts was dependent on endogenous MCP-1
synthesis because the presence of MCP-1 antisense oligonucleotides
markedly reduced TGF-ß 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-ß 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-ß 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-ß 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cory M. Hogaboam, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor MI, 48109-0602. E-mail address:
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.
Received for publication March 26, 1998. Accepted for publication June 3, 1999.
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