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Division of Pulmonary and Critical Care, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109 and * University of Texas, Austin, TX 78712
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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compared with FITC-treated CCR2+/+ mice.
Thus, CCR2 signaling promotes a profibrotic cytokine cascade following
FITC administration. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokine responses are mediated by the interaction of the soluble chemokine proteins with receptors belonging to the superfamily of 7-transmembrane G protein-coupled receptors. Eleven different CC family chemokine receptors have been characterized. CCR2 is the major MCP-1 receptor, and MCP-1 binds to CCR2 with high affinity (23, 24, 25).
We have recently characterized the use of intratracheal FITC challenge as a model of lung injury that results in lymphocyte-independent pulmonary fibrosis (26). FITC challenge results in acute lung injury, chronic inflammation, and the eventual deposition of extracellular matrix and scar tissue formation. This protocol results in areas of patchy inflammation and subpleural scarring characteristic of animal models of pulmonary fibrosis. In response to this insult, numerous cytokines, chemokines, and growth factors are produced that mediate the fibrotic/repair process. We demonstrate that the pro-fibrotic signaling cascade is diminished in CCR2-/- mice in two different models, suggesting that CCR2 signaling is part of a generalized pathway leading to pulmonary fibrosis in response to diverse insults.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CCR2+/+ (B6129F2/J; The Jackson Laboratory, Bar Harbor, ME), CCR2-/- mice (27) (B6129F2-Cmkbr2tm1Kuz), and CCR5-/- (28) (B6129F2-Cmkbr5tm1Kuz), bred at the University of Michigan, were housed under specific pathogen-free conditions in enclosed filter top cages. Clean food and water was given ad libitum. The mice were handled and maintained using microisolator techniques with daily veterinarian monitoring. The University of Michigan Committee on the Use and Care of Animals approved these experiments.
FITC and bleomycin injections
CCR2+/+ and CCR2-/- mice were anesthetized with sodium pentobarbital. The trachea was exposed and entered with a needle under direct visualization. For experiments using FITC injection, 21 mg of FITC (F-7250; Sigma, St. Louis, MO) was dissolved in 10 ml of sterile PBS, vortexed extensively, and sonicated for 30 s. This slurry was transferred to multiuse vials, and vortexed extensively before each 50-µl aliquot was removed for intratracheal injection using a 26-gauge needle. For bleomycin experiments, a single 30-µl injection containing 0.025 U of bleomycin (Sigma) diluted in normal saline was injected using a Tridak stepper (Brookfield, CT) and a 30-gauge needle.
Hydroxyproline assays
Mice were euthanized by CO2 asphyxiation and perfused via the heart with 5 ml of normal saline. Individual lung lobes were removed, taking care to avoid the large conducting airways. The isolated lobes were homogenized in 1 ml of PBS, and hydrolyzed by the addition of 1 ml of 12 N hydrochloric acid (HCl). Samples were then baked at 110° C for 12 h. Aliquots (5 µl) were then assayed by adding chloramine T solution for 20 min followed by development with Erlich’s reagent at 65°C for 15 min as previously described (29). Absorbance was measured at 550 nm, and the amount of hydroxyproline was determined against a standard curve generated using known concentrations of hydroxyproline standard (Sigma).
Histology
Animals were euthanized and perfused via the right ventricle with 3 ml of normal saline. Lungs were inflated with 1 ml of 10% neutral buffered formalin, removed, and fixed overnight in formalin before being dehydrated in 70% ethanol. Lungs were processed using standard procedures and embedded in paraffin. Sections (3–5 µm) were cut, mounted on slides, and stained with H&E or Masson’s trichrome blue for collagen deposition.
Evans blue permeability assays
Lung permeability was determined by assessing tissue accrual of Evans blue as previously described (30). This assay measures the leak of Evans blue dye, injected into the tail vein 30 min before sacrifice, into pulmonary tissue. Thus, the ratio of dye found in the lung vs the plasma is a measure of vascular permeability and acute lung injury. Animals were administered 20 mg/kg Evans blue (Sigma) by tail vein injection 30 min before lung harvest. Lungs were perfused with 1 ml of PBS + 5 mM EDTA, followed by removal and snap-freezing in liquid nitrogen. Samples were homogenized in 2 ml of PBS. Evans blue was extracted from lung homogenates by incubating the sample in 2 volumes of formamide at 60°C for 18 h. The supernatant was then separated by centrifugation at 5000 x g for 30 min. Evans blue concentration in lung homogenate supernatants was quantitated by a dual wavelength spectrophotometric method at absorbencies of 620 and 740 nm, which allows for correction of contaminating heme pigments, as determined by the following formula: E620 (Evans blue) = E620 - (1.426 x E740 + 0.030). Data are presented as tissue value normalized to plasma value.
Bronchoalveolar lavage (BAL) and determination of protein concentration
Mice were euthanized via CO2 asphyxiation, the trachea cannulated with polyethylene tubing (PE50, Intramedic; Clay Adams, Parsippany, NJ) attached to a 25-gauge needle on a tuberculin syringe, and the lungs were lavaged twice with 0.75 ml of PBS/5 mM EDTA (Sigma) for a total lavage volume of 1.5 ml. In >95% of the mice, the recovery volume was 1.3–1.4 ml. The BAL fluid was spun at 1500 rpm, and the supernatant was removed. Total protein content in BAL fluid was measured using a modification of the Bradford protein assay (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.
Collagenase digestions of whole lung
Collagenase digestions can be used to analyze both resident and recruited populations of lung cells found both in the alveolar space and interstitium. This procedure has been optimized to purify lung leukocytes (31). Lungs were excised, minced, and enzymatically digested for 30 min using 15 ml/lung of digestion buffer (RPMI 1640, 5% FCS, antibiotics, 1 mg/ml collagenase (Boehringer Mannheim, Chicago, IL) and 30 µg/ml DNase (Sigma). The cell suspension and undigested fragments were further dispersed by repeated passage through the bore of a 10-ml syringe without a needle. The total cell suspension was pelleted, and any contaminating erythrocytes were eliminated by lysis in ice-cold NH4Cl buffer (0.829% NH4Cl, 0.1% KHCO3, and 0.0372% Na2 EDTA, pH 7.4). The pellet was resuspended in 5 ml of complete medium (RPMI 1640, 5% FCS, 1% penicillin/streptomycin) and dispersed by 20 passages through a 5-ml syringe. The dispersed cells were filtered through a Nytex filter (Tetko, Kansas City, MO) to remove clumps. The total volume was brought up to 10 ml with complete medium. An equal volume of 40% Percoll (Sigma) was added, and the cells were centrifuged at 3000 rpm for 30 min (room temperature) without a brake. The cell pellets were resuspended in complete medium, and leukocytes were counted on a hemacytometer in the presence of trypan blue. Cells were >90% viable by trypan blue exclusion. Cytospins of recovered cells were prepared for differential staining as described below. In addition, recovered leukocytes were analyzed by flow cytometry.
Differential staining
Cytospins of collagenase digestions were made by centrifuging 50,000 cells onto microscope slides using a Shandon Cytospin 3 (Astmoore, England). The slides were allowed to air dry and were stained using a modified Wright-Giemsa (WG) stain. For WG staining, the slides were fixed/prestained for 2 min with a one-step methanol-based WG stain (Harleco; EM Diagnostics, Gibbstown, NJ), followed by steps 2 and 3 of the Diff-Quick whole blood stain (Diff-Quick; Baxter Scientific, Miami, FL). This modification of the Diff-Quick stain procedure improves the resolution of eosinophils from neutrophils in the mouse. A total of 300 cells were counted from randomly chosen high power microscope fields for each sample. The differential percentage was multiplied by the total leukocyte number to derive the absolute number of monocyte/macrophages, neutrophils, and eosinophils per sample.
FACS analysis
Lung cells (1 x 106) from the collagenase digestions of individual animals were incubated for 15 min on ice in Fc block (BD PharMingen, San Diego, CA) before washing and centrifugation. Cells were stained in a 100-µl total volume with 1-µg combinations of the following Abs (obtained from BD PharMingen unless otherwise noted): CD45 (YW62.3; Caltag Laboratories, Burlingame, CA), CD4 (RM4-4), CD8 (53-6.7), CD19 (1D3), or DX5. Stained samples were stored in the dark at 4°C until analyzed on a flow cytometer (FACScan; BD Biosciences, Mountain View, CA). All samples were stained with CD45 to identify a leukocyte-specific gate. The absolute number of a type of leukocyte in the lungs was determined as the percentage of that cell type times the total number of cells (leukocytes) in the lung collagenase digestion. Mean data represent the averages of three independent experiments with a total of eight animals per group.
Lung RNA preparation
Whole lungs were perfused with saline, removed, snap-frozen in liquid nitrogen, and stored at -70°C until use. To prepare RNA, frozen lungs were homogenized in 3 ml of TRIzol reagent (Life Technologies, Gaithersburg, MD) and extracted according to manufacturer’s instructions.
RNase protection analysis of lung mRNA
RNase protection assays for lung GM-CSF and TNF- mRNA were
performed using pooled total RNA from mice treated with FITC. GM-CSF
was assayed with the Riboquant CK-1 probe set, and TNF- was assayed
with the CK-3b probe set (both obtained from BD PharMingen) according
to manufacturer’s recommendations for hybridization, digestion, and
electrophoresis. GAPDH quantification was performed on autoradiographs
developed from 1- to 3-h exposures, whereas GM-CSF and TNF-
quantifications were from 24-h exposures.
Densitometric analyses
A digital picture of each autoradiograph was taken, and band intensities were analyzed using NIH Image public domain software (developed at the Research Services Branch of the National Institute of Mental Health; available for download at http://rsb.info.nih.gov/nih-image). Specific cytokine band intensities were normalized to GAPDH controls to account for differences in total RNA loading in each sample.
Lung homogenates
Lung homogenates were prepared from experimental animals at indicated time points. Animals were euthanized and perfused with 3 ml of PBS via the heart. Isolated lung lobes were removed and snap-frozen in liquid nitrogen until analysis. Lung lobes were homogenized using a Tissue Tearor (Biospec Products, Bartlesville, OK) at setting 5 in 1 ml of PBS containing Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN). Samples were then sonicated for 10 s before cellular debris was removed by centrifugation for 10 min at 3000 rpm. Homogenates were then filtered through a 0.45 µm filter before protein analysis via ELISA. In addition, protein concentrations within lung homogenates were determined using the Bio-Rad protein assay (Hercules, CA) according to manufacturer’s instructions.
ELISA
The measurements for MCP-1, GM-CSF, and TNF- were performed
on lung homogenates using Opti-EIA kits obtained from BD PharMingen
according to manufacturer’s instructions. Sensitivity ranged from 10
to 50 pg/ml at the lower limit.
Statistics
Statistical significance was analyzed using the InStat 2.01 program (GraphPad Software) on a Power Macintosh G3. Student’s t tests were run to determine p values when comparing two groups. When comparing three or more groups, ANOVA analysis was performed with a post hoc Bonferroni test to determine which groups showed significant differences. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that mixtures of both acute and chronic
inflammatory cells persist in fibrotic areas following FITC
administration (26). Therefore, we reasoned that
mononuclear chemotactic factors might be elevated. To determine whether
a mononuclear chemotactic factor was being induced following FITC
injury, we examined lung homogenates at various time points post-FITC
inoculation for MCP-1 protein accumulation. Fig. 1
A documents that MCP-1 is
elevated in the CCR2+/+ wild-type animals at days
1 and 3. In addition, we analyzed CCR2-/-
animals for MCP-1 induction following FITC administration (Fig. 1B). Higher levels of MCP-1 ligand are noted in the absence
of the CCR2 receptor compared with CCR2+/+ mice.
We analyzed lung homogenates at days 7, 10, 14, and 21 as well, but did
not detect elevated levels of MCP-1 compared with baseline by ELISA at
these later time points in wild-type or CCR2-/-
animals. These data demonstrate that MCP-1 is induced following
FITC-mediated acute lung injury in both CCR2+/+
and CCR2-/- mice. The expression of the other
murine CCR2 ligands, MCP-3 and MCP-5, was analyzed by RT-PCR. MCP-3 is
not induced following FITC administration, but MCP-5 mRNA is induced
2-fold at day 7 post-FITC compared with baseline (data not
shown).
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Given the elevated expression of MCP-1 following FITC
administration, we hypothesized that MCP-1 signaling via its receptor,
CCR2, is involved in the fibrotic response to FITC. Therefore, we
examined the consequences of FITC administration in
CCR2-/- mice compared with wild-type,
CCR2+/+ mice. To quantitatively determine the
extent of fibrosis induced by FITC, we measured hydroxyproline as a
surrogate for lung collagen deposition. On day 21 post-FITC,
CCR2+/+ mice display higher levels of
hydroxyproline compared with CCR2-/- mice (Fig. 2A) on day 21
(p = 0.03). There were no significant
differences in the baseline levels of collagen found in
CCR2+/+ or CCR2-/- mice
injected with saline. Masson’s Trichrome staining of day 21 post-FITC
lungs in CCR2+/+ and
CCR2-/- mice was also performed (see Fig. 3, bottom panel). The
blue staining represents areas of mature collagen deposition. This
analysis confirms that CCR2-/- mice show
attenuated levels of collagen deposition. To determine whether the
protection was limited to FITC, the fibrotic response to bleomycin in
CCR2+/+ and CCR2-/- mice
was also studied. As seen in Fig. 2B,
CCR2-/- mice are similarly protected from
bleomycin-induced pulmonary fibrosis. The bleomycin-treated
CCR2-/- mice developed significantly less
pulmonary fibrosis (p = 0.0003) compared with
bleomycin-treated CCR2+/+ mice. Thus,
CCR2-/- mice are protected from pulmonary
fibrosis in two independent models.
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To determine whether the protection seen in
CCR2-/- mice was specific for a particular CCR,
we analyzed FITC-induced pulmonary fibrosis in
CCR5-/- mice. CCR5 is the receptor for the CC
chemokines macrophage-inflammatory protein (MIP)-1 and RANTES, both
of which are mononuclear cell chemotaxins. Fig. 4A demonstrates that
CCR5-/- mice show equivalent hydroxyproline
accumulation following FITC injection as wild-type mice, whereas
CCR2-/- mice are protected
(p = 0.001). Furthermore, analysis of
bleomycin-induced pulmonary fibrosis revealed that only
CCR2-/- mice are protected
(p = 0.0003), whereas the wild-type and
CCR5-/- mice are susceptible to
bleomycin-induced pulmonary fibrosis (Fig. 4B). Thus, the
protection from fibrotic insults is specific to the absence of the CCR2
receptor-signaling pathway.
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One explanation for the protection seen in
CCR2-/- mice following FITC injection would be
that the degree of initial lung injury in
CCR2-/- mice following FITC deposition is less
than in wild-type mice. We examined histology sections from lungs of
mice at days 0, 1, 3, 7, and 21 following FITC injection. The
histologic pattern of injury seen in CCR2+/+ and
CCR2-/- mice is indistinguishable throughout
day 7 (Fig. 3
). Both CCR2+/+ and
CCR2-/- mice show evidence of alveolar fluid
accumulation at day 1 and inflammation and hemorrhage at days 3 and 7.
Thus, it appears that the acute lung injury phase of the fibrotic
process is similar between CCR2+/+ and
CCR2-/- mice. However, when histological
sections are examined at day 21 post-FITC,
CCR2+/+ mice demonstrate areas of mononuclear
cell inflammation and consolidation reported earlier for wild-type mice
(26). These areas are much less pronounced in the
CCR2-/- mice. This histological evidence
suggests that despite similar early injury, the fibrotic process is
minimized in the CCR2-/- mice.
To determine quantitatively the level of acute lung injury,
CCR2+/+ and CCR2-/- mice
were injected with FITC, and BAL fluid was collected at days 1, 3, and
7 following FITC inoculation. Lung permeability increases when alveolar
epithelial and endothelial cells are damaged, allowing fluid to
accumulate in the alveolar space. Therefore, total protein
concentrations in the BAL fluid were determined as a measure of plasma
leak into the alveolar space. BAL fluid protein increased dramatically
1 day post-FITC, confirming equivalent early leak in both
CCR2+/+ and CCR2-/- mice
(Fig. 5A). At day 3, the
protein concentration in the CCR2-/- mice is
less than that seen in the CCR2+/+ mice
(p = 0.03), but by day 7, the protein
concentrations in the BAL were again equivalent.
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B demonstrates
that the vascular permeability seen in CCR2+/+
and CCR2-/- mice was similar at all time points
tested following FITC inoculation. Both groups of mice show evidence of
similar acute lung injury early following FITC administration that then
returns to baseline in both groups of mice. Thus the protection seen in
CCR2-/- mice cannot be explained by differences
in early lung injury as measured by capillary leak or edema. The inflammatory response to FITC is similar in CCR2+/+ and CCR2-/- mice
Having determined that the protection seen in CCR2-/- mice could not be attributed to differences in acute lung injury induced by FITC, we examined whether the inflammatory response was different in the two groups of FITC-treated mice. The magnitude and composition of the inflammatory response was determined by performing collagenase digestion and leukocyte purification on whole lungs from treated animals at days 0, 7, 14, and 21. At baseline, both CCR2+/+ and CCR2-/- mice show equivalent numbers of resident leukocytes in their lungs (14.08 and 14.02 x 106, respectively). At day 7, there is a significant increase in the number of leukocytes compared with baseline in both groups of mice (24.22 x 106, p = 0.0001 in the CCR2+/+ and 25.13 x 106, p = 0.0001 in the CCR2-/- animals). By day 14, the number of leukocytes in the CCR2+/+ mice returned to baseline (14.84 x 106); however, leukocyte cell number was still modestly elevated compared with baseline in the CCR2-/- mice (19.7 x 106, p = 0.009). By day 21, the cell numbers in both CCR2+/+ and CCR2-/- mice are not statistically different from baseline levels (16.01 x 106 in the CCR2+/+ and 17.7 x 106 in the CCR2-/- mice). In all cases, SE values were <10% and represent data pooled from eight mice in three separate experiments.
To determine whether the composition of the inflammatory response to
FITC differed between the CCR2+/+ and
CCR2-/- mice, flow cytometry and differential
analyses were performed to identify leukocyte subpopulations. Fig. 6 demonstrates that there were no
statistical differences between any of the leukocyte subpopulations
analyzed between CCR2+/+ and
CCR2-/- mice at the peak of inflammation (day
7). Similarly, no differences were noted in the composition of the
inflammatory cells at days 14 or 21 (data not shown). Fig. 7 shows representative staining profiles
of the lymphocyte-gated population analyzed in
CCR2+/+ and CCR2-/- mice
at day 7 post-FITC. The staining profiles (percentage of positive
cells, and the relative fluorescent intensity of positive cells) are
similar in both groups. The same was true of the analyses at days 14
and 21 (data not shown). Thus, the protection in
CCR2-/- mice cannot be explained by
differential recruitment of inflammatory cells.
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The differences in the fibrotic response between
CCR2+/+ and CCR2-/- mice
could not be explained by differences in the magnitude or the
composition of the cellular recruitment. To determine whether
differences in cellular activation were responsible for the protection
of the CCR2-/- mice, we analyzed the expression
of two molecules known to be involved in early inflammatory responses
and fibrotic processes: TNF- and GM-CSF. We chose to evaluate the
levels of TNF- and GM-CSF at day 14 post-FITC inoculation. At this
time point, fibroproliferative changes are evident as measured by
collagen deposition. We analyzed total lung RNA from
CCR2+/+ and CCR2-/- mice
at day 14 post-FITC to quantitate mRNA for TNF- and GM-CSF using
ribonuclease protection assays. Fig. 8
shows the results of these assays. Densitometry analysis was performed,
and cytokine-specific mRNA levels were normalized to GAPDH control mRNA
levels. These analyses demonstrate that, collectively, GM-CSF mRNA
levels are 3 times higher in CCR2-/- mice
compared with CCR2+/+ mice treated with FITC.
Conversely, levels of TNF- mRNA are 11 times higher in
CCR2+/+ mice compared with
CCR2-/- mice at day 14 post-FITC.
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and GM-CSF at day 21 post-FITC. Fig. 9 shows the results of these experiments,
and verifies that TNF- protein levels are still elevated in
CCR2+/+ mice at day 21 (Fig. 9A),
whereas GM-CSF levels are elevated in CCR2-/-
mice at day 21 (Fig. 9B). Thus, the imbalance in pro- and
anti-fibrotic mediators is maintained at later time points.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several aspects of the animal model used in this study are worthy of
mention. First, the use of animals that are genetically deficient in
CCR2 offers a definitive system for studying the role of CCR2 receptor
signaling in the evolution of pulmonary fibrosis. This approach avoids
the use of neutralizing Abs, thereby eliminating the concerns about
neutralization efficiency and complement-mediated tissue injury.
Second, there is no evidence of altered collagen deposition at baseline
in the CCR2-/- and
CCR2+/+ mice (Figs. 2 and 3). Third, the
protection is related to CCR2 absence regardless of the fibrotic
insult. Finally, the FITC model offers several advantages for the study
of pulmonary fibrosis. The model is T cell independent. Histologic
abnormalities are peripheral in location, patchy in nature, and
nonresolving; these histologic features characterize human
IPF.
CCR2 is expressed on monocytes, activated T cells, B cells, NK cells,
fibroblasts, and mast cells (32, 33, 34). Expression of CCR2
is inhibited in monocytes and T cells by LPS and IFN-
(35, 36) and up-regulated by IL-2, IL-4, IL-10, and IL-12 in T cells
(35). Although MCP-1 is the principal ligand for CCR2,
other ligands include MCP-2, MCP-3, MCP-4, MCP-5, and HIV Tat
(37, 38, 39, 40, 41). However, in mice, only MCP-1, MCP-3, and MCP-5
bind CCR2. We have demonstrated that both MCP-1 and MCP-5 are elevated
following a fibrotic insult with FITC. Our ELISA data in lung
homogenates show that the peak expression of MCP-1 is at day 1 in
wild-type mice. By RT-PCR analysis, we know that MCP-5 mRNA levels are
increased at day 7 following FITC. It has been previously reported that
MCP-1 levels are elevated following bleomycin treatment in rats from
days 3 to 21 (42). We have extended these results to the
murine model of bleomycin-induced fibrosis. Analyzing MCP-1 expression
by ELISA in lung homogenates, we can demonstrate elevated levels of
MCP-1 from days 1 to 14 postbleomycin, with the peak being day 3 (data
not shown). Interestingly, the kinetics of MCP-1 expression differs
during FITC- and bleomycin-induced fibrosis. Although our studies do
not directly address the ligand involved in CCR2-mediated effects, our
data demonstrate that regulation of CCR2 expression and/or signaling
have profound effects on fibrotic processes.
Our findings documenting participation of CCR2 signaling in the
generation of pulmonary fibrosis are at variance with a previously
reported study using anti-CCR2 neutralizing Abs (43).
In those experiments, rabbit anti-CCR2 Ab was injected at days 0,
3, and 6 following bleomycin instillation (3 mg/kg). Animals were
assessed for survival and hydroxyproline accumulation over a 15-day
period. No effect of the CCR2 neutralization was noted on survival or
fibrosis measurements. Several aspects of the model used by these
investigators differ from our own. First, a much higher dose of
bleomycin was used in this study compared with our experiments. The
bleomycin dose resulted in 50% mortality between days 10 and 15,
whereas in our experiments the doses of bleomycin and FITC used
resulted in <10% mortality at day 21. Therefore, it is possible that
CCR2 plays a different role in response to high doses of injurious
agents. Alternatively, the fact that CCR2 was only neutralized for the
first week of injury in these experiments more likely explains the
discrepancy with our work in the knockout animals that are devoid of
CCR2 signaling throughout the course of the disease. Our data document
that CCR2-mediated effects are prominent during the fibrotic processes
rather than in the initial phases of injury. The fact that we have
documented protection from pulmonary fibrosis in
CCR2-/- mice using two different model systems
(bleomycin and FITC) to induce fibrosis strengthens our findings.
Our data demonstrate that CCR5-/- mice are not
protected from FITC or bleomycin-induced pulmonary fibrosis despite the
fact that the CCR5 ligands MIP-1 and RANTES are induced in animal
models of pulmonary fibrosis (Ref. 43 , and our unpublished
observations). These data demonstrate that the signaling cascade via
CCR2 is not shared with the homologous CCR5 chemokine receptor. These
observations highlight the importance of a specific chemokine receptor,
CCR2, in the generation of a pro-fibrotic signaling cascade.
One possible explanation for the protection in CCR2-/- mice could have been that the absence of a chemotactic receptor diminished the cellular recruitment. However, our data demonstrate that this is not the case. Despite the absence of the CCR2 receptor, there is no difference in the magnitude or composition of the inflammatory response generated to FITC compared with CCR2+/+ mice. This data can best be explained by the fact that chemotactic signals for leukocytes are redundant. Many different chemokines can recruit the same populations of leukocytes, and many chemokines can bind to shared chemokine receptors. Given this, it is not surprising that other chemotactic signals might substitute in the CCR2-/- mice.
Although similar numbers and types of leukocytes are recruited to the
lung in both CCR2+/+ and
CCR2-/- mice following FITC administration, the
function of the cells is altered in CCR2-/-
mice compared with wild-type mice. These data support a role for CCR2
beyond that of a chemotactic receptor, and suggest that MCP-1 signal
transduction via CCR2 plays an important role in cellular
activation. The differences in cellular function are best exemplified
by the alterations in the expression of TNF- and GM-CSF following
FITC inoculation in wild-type and CCR2-/-
mice.
The fact that TNF- expression is enhanced in
CCR2+/+ mice was expected given that TNF- has
been documented to be a pivotal cytokine involved in the fibrotic
process. It is well established that inhibition of TNF- signaling
can diminish the fibrotic response to bleomycin (44, 45, 46, 47);
therefore, it is not surprising that this mediator is also involved in
the fibrotic response to FITC. However, the fact that the
CCR2-/- mice recruited identical numbers and
subsets of leukocytes to their lung in response to FITC, yet failed to
activate TNF- production, is striking.
Changes were noted in the expression of GM-CSF in FITC-treated CCR2+/+ and CCR2-/- animals. GM-CSF is a known mitogen for alveolar epithelial cells (48), and can influence the number and activity of alveolar macrophages (Refs. 49, 50 , and R. Paine, unpublished observations). Furthermore, neutralization of GM-CSF has been shown to worsen bleomycin-induced pulmonary fibrosis in both mice and rats (51, 52). Similarly, GM-CSF-/- animals have increased bleomycin-induced pulmonary fibrosis (53). In these experiments, the increased pulmonary fibrosis in GM-CSF-/- mice was shown to be due, in part, to diminished expression of the anti-fibrotic lipid mediator, PGE2 (53). Thus, GM-CSF is a molecule known to be important in many aspects of the repair and re-epithelialization response following an acute lung injury leading to fibrosis. The elevated expression of GM-CSF in CCR2-/- mice is evident both at days 14 and 21 post-FITC, supporting a role for GM-CSF in the re-epithelialization process.
In sum, these data suggest that CCR2 activation leads to the generation
of a variety of mediators involved in the fibrotic process. We do not
believe that the protection seen in the CCR2-/-
mice can be explained solely by the absence of TNF- expression. In
fact, TNF- is a gene that is known to be induced by GM-CSF
(54). Therefore, the finding of reduced TNF- in the
face of increased GM-CSF in the CCR2-/- mice
would not have been predicted. In the absence of CCR2 activation
following a fibrotic insult, the signaling cascade is altered to favor
the protective phenotype characterized by diminished TNF- and
increased GM-CSF. Thus, absence of CCR2 leads to a complex circumstance
whereby a pro-fibrotic mediator is diminished and an anti-fibrotic
mediator is enhanced.
Mediators other than those evaluated in our studies might be affected
by CCR2 signaling. MCP-1 can stimulate fibroblast deposition of
collagen via the up-regulation of TGF-
(55). TGF- is
known to be up-regulated in the bleomycin model system (56, 57), and we anticipate that the same might be true for the FITC
model. Thus alterations in TGF- may contribute to the protection
seen in the CCR2-/- mice. The imbalance in
TNF- production would favor the expression of pro-angiogenic CXC
chemokines in the wild-type mice. Neutralization of the CXC chemokine
MIP-2 has been shown to ameliorate bleomycin-induced fibrosis
(58). Lastly, the fact that MCP-1 has been shown to
induce/contribute to the expression of IL-4 (32, 59, 60)
could suggest that in the absence of CCR2 signaling, the balance in
T1/T2 cytokines is skewed to favor the production of the
anti-fibrotic T1 cytokines such as IFN-.
The study of IPF in humans is difficult for at least three reasons. First, the disease is idiopathic in nature in most instances, therefore, the insults that lead to the development of fibrosis are largely unknown. Second, the inability to perform serial sampling in humans makes it very difficult to study the natural history of the disease in a particular patient. Third, patients rarely present for treatment until late in the course of the disease process. Even in clinical situations such as sepsis and ARDS, where one can predict that patients will be at risk for the development of pulmonary fibrosis, it is difficult to determine which patients will actually progress to pulmonary fibrosis. CCR2-/- mice are protected from the development of pulmonary fibrosis, suggesting that therapies directed at blockade of the CCR2 receptor might provide a useful therapeutic option for clinical intervention. Additionally, CCR2 signaling does not appear to be involved in the acute inflammatory phase or the early lung injury phases of the disease, but rather, in the orchestration of the expression of pro-fibrotic mediators. This suggests that anti-CCR2 therapies could prove useful for patients presenting at later stages of the disease progression. Furthermore, the observation that CCR2-/- mice are protected from pulmonary fibrosis induced by two different agents, bleomycin and FITC, gives hope that this strategy may be widely applicable for this idiopathic human disease.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bethany B. Moore, University of Michigan, 6301 MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0642. E-mail address: Bmoore{at}umich.edu
3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; MCP-1, monocyte-chemoattractant protein-1; BAL, bronchoalveolar lavage; ARDS, acute respiratory distress syndrome; WG, Wright-Giemsa; MIP, macrophage-inflammatory protein.
Received for publication October 17, 2000. Accepted for publication August 8, 2001.
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