Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45+, CD13+, collagen 1+, CD34−). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2+/+ mice than from CCR2−/− mice. Transplant of CCR2+/+ bone marrow into CCR2−/− recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2+/+ lung fibrocytes also migrated to injured airspaces of CCR2−/− recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury. Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45+, CD13+, collagen 1+, CD34−). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2+/+ mice than from CCR2−/− mice. Transplant of CCR2+/+ bone marrow into CCR2−/− recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2+/+ lung fibrocytes also migrated to injured airspaces of CCR2−/− recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury. Pulmonary fibrosis is characterized by alveolar epithelial cell injury, hyperplasia, inflammatory cell accumulation, fibroblast proliferation, and deposition of extracellular matrix.1Thannickal VJ Toews GB White E Lynch JI Martinez F Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-471Crossref PubMed Scopus (583) Google Scholar, 2Lynch JI, Toews G: Idiopathic pulmonary fibrosis. Edited by Fishman A. 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International consensus statement.Am J Respir Crit Care Med. 2000; 161: 646-664Crossref PubMed Scopus (1003) Google Scholar In animals, the disease process can be modeled via the intratracheal administration of fluorescein isothiocyanate (FITC).7Christensen P Goodman R Pastoriza L Moore B Toews G Induction of lung fibrosis in the mouse by intratracheal instillation of fluorescein isothiocyanate is not T-cell dependent.Am J Pathol. 1999; 155: 1773-1779Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar Soluble monocyte chemoattractant protein-1 (CCL2) levels in the bronchoalveolar lavage (BAL) fluid peak by day 1 after FITC and remain elevated until day 7;8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar matrix-bound CCL2 is bioactive and evident through day 21 after FITC. CC chemokine receptor 2 (CCR2) is the high-affinity receptor for CCL2.9Kurihara T Bravo R Cloning and functional expression of mCCR2, a murine receptor for the CC-chemokines JE and FIC.J Biol Chem. 1996; 271: 11603-11607Crossref PubMed Scopus (105) Google Scholar, 10Yamagami S Tokuda Y Ishii K Tanaka H Endo N cDNA cloning and functional expression of a human monocyte chemoattractant protein 1 receptor.Biochem Biophys Res Commun. 1994; 202: 1156-1162Crossref PubMed Scopus (34) Google Scholar We previously demonstrated that mice that are deficient in CCR2 (CCR2−/− mice) are protected from the development of experimental pulmonary fibrosis induced either by FITC or bleomycin.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar However, recruitment of classical inflammatory cells (monocytes, macrophages, eosinophils, neutrophils, T, B, or NK cells) to the lung in response to FITC was not diminished when CCR2−/− mice were compared to CCR2+/+ mice.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar Because altered recruitment of classical inflammatory cells was not noted, we determined whether CCR2 played a role in mesenchymal cell recruitment to the lung in response to injury. A circulating population of cells (termed fibrocytes) that share leukocyte (CD45, CD34, CD13) and mesenchymal markers [collagen 1 (col 1), fibronectin] has been described.11Chesney J Metz C Stavitsky AB Bacher M Bucala R Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.J Immunol. 1998; 160: 419-425PubMed Google Scholar, 12Bucala R Spiegel LA Chesney J Hogan M Cerami A Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar, 13Abe R Donnelly SC Peng T Bucala R Metz CN Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562PubMed Google Scholar Fibrocytes cultured from peripheral blood migrate to skin wound chambers13Abe R Donnelly SC Peng T Bucala R Metz CN Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562PubMed Google Scholar and bronchial mucosa after antigen challenge.14Schmidt M Sun G Stacey M Mori L Mattoli S Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.J Immunol. 2003; 170: 380-389Google Scholar The findings that fibrocytes can differentiate into myofibroblasts,14Schmidt M Sun G Stacey M Mori L Mattoli S Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.J Immunol. 2003; 170: 380-389Google Scholar, 15Aiba S Tagami H Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids.J Cutan Pathol. 1997; 24: 65-69Crossref PubMed Scopus (81) Google Scholar and the fact that fibrocytes are present in fibrosing conditions such as asthma,14Schmidt M Sun G Stacey M Mori L Mattoli S Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.J Immunol. 2003; 170: 380-389Google Scholar nephrogenic fibrosing dermopathy,16Cowper SE Bucala R Nephrogenic fibrosing dermopathy: suspect identified, motive unclear.Am J Dermatopathol. 2003; 25: 358Crossref PubMed Scopus (151) Google Scholar and hypertrophic scarring15Aiba S Tagami H Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids.J Cutan Pathol. 1997; 24: 65-69Crossref PubMed Scopus (81) Google Scholar suggest a potentially pathogenic role for this cell type. We hypothesized that CCR2 is a critical regulator of fibrocyte recruitment and activation and that fibrocytes are mediators of lung fibrosis. C57BL/6 and B6/129F2 mice were from Jackson Laboratories (Bar Harbor, ME). CCR2−/− mice on the C57BL/6 or B6/129F2 background were bred in the University of Michigan Laboratory Animal Medicine facilities under SPF conditions and have been described previously.17Kuziel W Morgan S Dawson T Griffin S Smithies O Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2.Proc Natl Acad Sci USA. 1997; 94: 12053-12058Crossref PubMed Scopus (576) Google Scholar Mice were used at 6 to 8 weeks of age. The University Committee on the Use and Care of Animals approved these experiments. FITC inoculation was performed as previously described.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar Briefly, mice were anesthetized with sodium pentobarbital. The trachea was exposed and entered with a needle under direct visualization. FITC (21 mg, no. F-7250; Sigma, St. Louis, MO) was dissolved in 10 ml of sterile phosphate-buffered saline (PBS), vortexed extensively, and sonicated for 30 seconds. This slurry was transferred to multiuse vials and vortexed extensively before each 50-μl aliquot was removed for intratracheal injection using a 23-gauge needle. Mice were euthanized by CO2 asphyxiation and the trachea was exposed in a sterile manner. The trachea was cannulated with polyethylene tubing (PE50, Intramedic; Clay Adams, Parsippany, NJ) attached to a 25-guage needle on a tuberculin syringe, and the lungs were lavaged three times with 0.75 ml of sterile 1× PBS. The lavage fluid from a single mouse was combined, spun at 500 × g, and the supernatant removed. The cell pellet was resuspended in complete media and cultured for 10 to 14 days to allow mesenchymal cells to expand before analysis. Murine lungs were perfused with 5 ml of normal saline and removed using aseptic conditions. Lungs were minced with scissors in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Lungs from a single animal were placed in 10 ml of media in 100-cm2 tissue culture plates. Mesenchymal cells were allowed to grow out of the minced tissue, and when cells reached 70% confluence they were passaged using trypsin digestion. Fibroblasts were grown for 10 to 14 days (two to three passages) before being used and were always used at or before passage 3. Cells fixed for 10 minutes with 10% neutral buffered formalin were analyzed by IHC using CD45-PE or CD13-PE (BD Pharmingen, San Diego, CA), and col 1 (rabbit anti-mouse antisera; Accurate Chemical, Westbury, NY) with PE- (BD Pharmingen) or AMCA-coupled secondary reagents (Vector, Burlingame, CA). For CCR2 IHC, fixed cells were first treated with 1% trypsin before anti-CCR2 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were detected with a Vector alkaline phosphatase kit. Images were visualized on a Nikon Eclipse E600 microscope (Melville, NY) equipped for epifluorescence with appropriate filters. Images were captured on a digital SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI). Lung mesenchymal cells were grown from lung minces for a period of 10 to 14 days. The trypsinized cells were stained with anti-CD45 Abs coupled to magnetic beads (Miltenyi Biotech, Auburn, CA). Labeled cells were then sorted by binding the cell population to MS- or LS-positive selection columns using a SuperMacs apparatus (Miltenyi Biotech) according to the manufacturer's instructions. Cells are then washed extensively. CD45+ cells are retained on the column and can be removed by flushing the column with buffer once it is removed from the magnetic field. CD45− cells are collected in the original flow through. For extra purity, CD45+ cells were sometimes reapplied to a second LS-positive selection column. The absolute number of lung fibrocytes is determined by counting the cells that were retained on the column by a hemocytometer. IHC staining or flow cytometry staining on this population confirmed that these cells were CD45+, CD13+, and col 1+. For Figure 2b, cells were incubated for 15 minutes on ice with Fc block (BD Pharmingen, San Diego, CA) before surface staining with CD45-PE (BD Pharmingen). Cells were then washed and fixed/permeabilized using the Cytofix/Cytoperm kit from BD Pharmingen and stained for col 1 (rabbit anti-mouse col 1, Accurate) followed by a donkey anti-rabbit Cy5-coupled secondary (Research Diagnostics, Inc., Flanders, NJ). For Figure 2d, CD45+ cells were first purified from lung mince cultures on a magnet as described above. Then CD45+ cells were stained with CD45-PE or CD34-PE (clone RAM34, BD Pharmingen) followed by fixation/permeabilization and staining with col 1 (rabbit anti-mouse, Accurate) followed by a goat anti-rabbit FITC-secondary (Pharmingen). Cells were analyzed on the flow cytometer (FACScan; BD Biosciences, Mountain View, CA). Lung mince cultures were serum-starved for 24 hours before magnetic purification of CD45+ and CD45− cells. Chemotaxis on cells at 1 × 106/ml was performed in Boyden chambers to recombinant proteins (fibronectin at 100 μg/ml; Sigma) or CCL2 (50 ng/ml; R&D Systems, Minneapolis, MN) through gelatin-coated 5- to 8-μm filters. Checkerboard analysis proved the migration was directional. The protocol for syngeneic BMT has been previously described.18Cooke KR Krenger W Hill G Martin TR Kobzik L Brewer J Simmons R Crawford JM van den Brink MR Ferrara JL Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation.Blood. 1998; 92: 2571-2580PubMed Google Scholar, 19Down JD Mauch P Warhol M Neben S Ferrara JL The effect of donor T lymphocytes and total-body irradiation on hemopoietic engraftment and pulmonary toxicity following experimental allogeneic bone marrow transplantation.Transplantation. 1992; 54: 802-808Crossref PubMed Scopus (45) Google Scholar Recipient mice received 13 Gy of total body irradiation (137Cs source) delivered in two fractions separated by 3 hours.19Down JD Mauch P Warhol M Neben S Ferrara JL The effect of donor T lymphocytes and total-body irradiation on hemopoietic engraftment and pulmonary toxicity following experimental allogeneic bone marrow transplantation.Transplantation. 1992; 54: 802-808Crossref PubMed Scopus (45) Google Scholar Five million bone marrow cells and 1 × 106 nylon wool-purified T cells were resuspended in Leibovitz's L-15 medium (Life Technologies, Grand Island, NY) and transplanted intravenously (0.25 ml total volume). After transplantation, mice were housed in sterilized microisolator cages and were fed normal chow and autoclaved hyperchlorinated water for the first 2 weeks after BMT. Total lung collagen levels were determined by harvesting lungs from mice on day 21 after FITC or saline administration. Animals were euthanized, and perfused with 3 ml of normal saline before all five lung lobes were removed and snap-frozen in liquid nitrogen. Before analysis, lungs 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 hours. Aliquots (5 μl) were then assayed by adding chloramine T solution for 20 minutes followed by development with Erlich's reagent at 65°C for 15 minutes as previously described.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar 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). CCR2+/+ or CCR2−/− mice were injected with saline or FITC on day 0. On day 7, lungs were removed, minced, and cultured for 14 days. At this time, CD45+ fibrocytes and CD45− fibroblasts were isolated via magnetic sorting and total numbers of each cell type were enumerated. Five thousand cells of each cell type were plated in 96-well flat-bottomed tissue culture dishes in complete media containing 10% fetal calf serum and antibiotics. Cells were cultured for 24, 48, or 72 hours before the addition of 10 μCi of 3H-thymidine for an additional 16 hours. Cells were harvested onto glass fiber filters using an automated cell harvester and filters were counted using a β-scintillation counter. Fibrocytes were purified from lung mince cultures by magnetic separation and labeled ex vivo with PKH-26 (Sigma) according to the manufacturer's instructions. Cells were washed extensively and reinfused (5 × 105) via tail vein into mice on day 4 after FITC. PKH-26-labeled cells were visualized using fluorescent light and appropriate filters in frozen sections 24 hours after infusion. Cellular mRNA pooled from cells from three mice was prepared using Trizol reagent (Invitrogen, Rockville, MD) according to the manufacturer's instructions. Total mRNA was converted to labeled cDNA using the SuperArray AmpoLabeling-LPR kit and hybridized to GEArray Q series mouse chemokines and receptors gene array according to the manufacturer's instructions (SuperArray Biosciences, Frederick, MD). Blots shown represent data from two to three experiments per cell type. Cellular mRNA was prepared using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Gene expression for col 1, col 3, and fibronectin was determined by reverse transcriptase (RT)-polymerase chain reaction (PCR) using the Promega Access RT-PCR kit (Promega, Madison, WI) according to manufacturer's instructions. The conditions for PCR were 95°C for 1 minute, 55°C for 1 minute, and 68°C for 1 minute. Extracellular matrix genes were amplified for 35 cycles. β-Actin was amplified for 25 cycles. Primer sequences were as follows: col 1 sense, TGGTGCCAAGGGTCTCACTGGC; col 1 anti-sense, GGACCTTGTACACCACGTTCACC; col 3 sense, GCAGTCCAACGTAGATGAATTGG; col 3 anti-sense, GAAGGCCTGGTGGACCAGCTGG; fibronectin sense, AAGTGTGATCCCCATGAAGCAACG; fibronectin anti-sense, CTCTCGAGAATCGTCTCTGTCAGC; β-actin sense, GTGGGGCTCCCCAGGCACCA; β-actin anti-sense, GCTCGGCCGTGGTGGTGAAGC. Cells were seeded (4 × 105/well) and serum-starved in 35-mm dishes. Cultures were exposed to treatments [interleukin (IL)-13 at 10 ng/ml or transforming growth factor (TGF)-β1 at 2 ng/ml or CCL2 at 10 ng/ml] all from R&D Systems for 24 hours and then were washed with ice-cold PBS and lysates were prepared, adjusted for protein concentration, and analyzed for col 1 expression by Western blot analysis as previously described.20Kolodsick JE Peters-Golden M Larios J Toews GB Thannickal VJ Moore BB Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation.Am J Respir Cell Mol Biol. 2003; 29: 537-544Crossref PubMed Scopus (258) Google Scholar Blots were stripped and reprobed for β-tubulin. Digital photographs of blots were taken and the density of the bands was calculated using the Image J 1.32 program available for free download from http://rsb.info.nih.gov/ij/. The density of the collagen signal for each lane was normalized to the density of β-tubulin in each lane. The values for the serum-free conditions were normalized to 1 in each experiment and the relative density for each condition was calculated relative to serum-free media for each experiment. Four experiments were averaged together to obtain statistical significance. In all graphs where error bars are shown, data represent mean ± SEM. Statistical significance was analyzed using the InStat v. 3 program (Graphpad Software) for Windows on a Dell GX260 computer. Student's t-tests were run to determine P values when comparing two groups. For three or more groups, analysis of variance was performed with a posthoc Bonferroni test. A value of P < 0.05 was considered significant. Cells obtained by BAL from wild-type C57BL/6 mice on days 0, 1, 3, 5, 7, 10, 14, 17, and 21 after FITC were cultured and analyzed for the expression of the fibrocyte markers, col 1, CD45, and CD13 (Figure 1a) by IHC. Virtually all cells that could be isolated from lavages of FITC-treated mice at all time points were positive for each of these markers. The dual expression of CD45 and col 1 was confirmed in BAL fibrocytes isolated after FITC (Figure 1b). The number of fibrocytes that can be cultured from BAL increases after FITC exposure (Figure 1c) and correlates with the kinetics of CCL2 expression within the BAL after FITC injury.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar Approximately 15 to 25% of the mesenchymal cells that are present in 10- to 14-day cultures of lung mince cells express the fibrocyte markers CD45 and CD13 (Figure 2a). Flow cytometry analysis of lung mince cultures demonstrates the dual expression of surface CD45 and intracellular col 1 (Figure 2b) on the fibrocyte population. The col I-positive, CD45-negative population of cells in the lung mince cultures represents effector fibroblasts. The level of extracellular matrix proteins being produced by fibrocytes relative to fibroblasts is appreciably lower when analyzed either by flow cytometry for intracellular col 1 expression (Figure 2b) or by RT-PCR for the expression of col I, col III, or fibronectin genes (Figure 2c). Fibrocytes isolated from alveolar spaces or lung mince cultures are 2% positive for CD34 (Figure 2d). Fibrocytes were purified from lung mince cultures via magnetic separation on CD45 beads and stained by flow cytometry for the expression of CD45 (Figure 2d, B), col 1 (Figure 2d, C), or dual expression of col 1 and CD34 (Figure 2d, D). It should be noted that under the culture conditions used in this study, both fibrocytes and fibroblasts express α-smooth muscle actin. Cultures of minced lungs from naïve mice contain 2.76 ± 0.8 × 106 lung fibrocytes whereas cultures from day 7 FITC-treated mice contain 4.64 ± 1.0 × 106 lung fibrocytes after the same length of culture (n = 3, P < 0.05). Thus, lung fibrocytes increase in number in the lung interstitium in response to FITC challenge. Interstitial lung mesenchymal cells are a mixture of lung fibrocytes and fibroblasts. Fibroblasts are col 1+, CD45−, CD13−, and CD34−. These two cell populations were purified from B6/129F2 mice by magnetic sorting for CD45 and were analyzed for chemokine receptor expression via hybridization to SuperArray gene arrays for mouse chemokines and receptors. Lung fibrocytes from this genetic background express modest levels of CXCR4 and CCR1, and high levels of CCR2, CCR5, and CCR7 (Figure 3a). CXCR4, CCR5, and CCR7 have previously been reported on human peripheral blood fibrocytes,13Abe R Donnelly SC Peng T Bucala R Metz CN Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562PubMed Google Scholar however CCR2 expression has not previously been evaluated. The fibroblast population expressed only CXCR4 (not shown). Lung fibrocytes from B6/129F2 mice were separated from fibroblasts in lung mince cultures via two rounds of positive selection on anti-CD45-coupled magnetic beads. The postsort lung fibrocyte population was 99.5% positive for CD45, CD13, and col 1. In contrast, the fibroblast population was 99.5% positive for col 1 but negative for both CD45 and CD13 (not shown). IHC staining confirmed the presence of CCR2 protein on the lung fibrocytes, but not fibroblasts (Figure 3b). Purified lung fibrocytes migrated in response to CCL2 in vitro whereas the fibroblast population did not (Figure 3c). Both lung fibrocytes and fibroblasts were able to migrate in response to the potent mesenchymal cell chemoattractant fibronectin. CCR2−/− mice are protected from FITC- and bleomycin-induced pulmonary fibrosis on the B6/129F2 background.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar CCR2−/− mice are also protected on the C57BL/6 background. C57BL/6 mice accumulate 7.3 ± 0.5 μg/ml hydroxyproline (collagen surrogate) per g of body weight in their lungs after FITC challenge compared to 4.2 ± 0.77 μg/ml hydroxyproline per g of body weight in the B6/CCR2−/− mice (n = 8, P < 0.05). Interestingly, the overall magnitude of the fibrotic response to FITC in the C57BL/6 background is higher than previously noted in the B6/129F2 background.8Moore B Paine R Christensen P Moore T Sitterding S Ngan R Wilke C Kuziel W Toews G Protection from pulmonary fibrosis in the absence of CCR2 signaling.J Immunol. 2001; 167: 4368-4377PubMed Google Scholar To compare the chemokine receptor profile on fibrocytes purified from both genetic backgrounds in which CCR2−/− mice are protected, we analyzed the chemokine receptor profile of fibrocytes purified via magnetic selection from lung mince cultures of saline- or FITC-treated C57BL/6 mice. Figure 4 demonstrates that fibrocytes from C57BL/6 mice express CCR1, CCR2, CCR5, and CCR7 as did fibrocytes from B6/129F2 mice (Figure 3a). There were some notable differences however. Fibrocytes from C57BL/6 mice also expressed low levels of CCR3 and had reduced expression of both CXCR4 and CCR7 compared to the fibrocytes from the B6/129F2 mice. Of note, the chemokine receptor profile of fibrocytes purified from FITC-treated mice was not different from the profile seen on fibrocytes from saline-treated mice on either genetic background. Thus, fibrocytes from two different genetic backgrounds express CCR2 and the chemokine receptor profile does not change in response to FITC. Wild-type and CCR2−/− mice on both the C57BL/6 or B6/129F2 backgrounds were injected with FITC on day 0. On day 4 after FITC, BAL cell pellets were cultured and analyzed for the dual expression of col 1 and CD45. The absolute number of lung fibrocytes present in cultures from injured airspaces of wild-type mice were significantly greater than noted in cultures from CCR2−/− mice (Figure 5a). The absolute number of lung fibrocytes in C57BL/6 mice was higher than in B6/129F2 mice correlating with differences observed in the magnitude of the fibrotic response to FITC between these strains. Accumulation of lung fibrocytes in injured airspaces is dependent on expression of the CCL2 receptor, CCR2. To be certain that differences in fibrocyte numbers noted in BAL cell cultures were not because of proliferative differences between CCR2+/+ and CCR2−/− cells, we purified fibrocytes via CD45 magnetic selection and fibroblasts from lung mince cultures of CCR2+/+ or CCR2−/− mice treated with saline or FITC. Purified fibrocytes and fibroblasts were cultured for 24 to 72 hours and proliferation was assessed. Figure 5b shows that the proliferation of CCR2+/+ and CCR2−/− fibrocytes and fibroblasts were similar after 48 hours of culture. No differences were noted in the proliferation of the CCR2+/+ or CCR2−/− cells at any time point tested from either FITC- or saline-treated animals. Furthermore, the addition of CCL2 to cells in culture did not alter proliferation (not shown). Thus the differences in fibrocyte accumulation in the BAL of CCR2+/+ and CCR2−/− mice treated with FITC likely reflect differences in recruitment, and not expansion in culture. Bone marrow-derived fibroblasts can be incorporated into fibrotic lung lesions.21Epperly M Guo H Gretton J Greenberger J Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis.Am J Resp Crit Care Med. 2003; 29: 213-224Google Scholar, 22Hashimoto N Jin H Liu T Chensue S Phan S Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (659) Google Scholar CCR2−/− mice were lethally irradiated and reconstituted with a BMT from CCR2+/+ mice. Mice were allowed to recover from the BMT for at least 7 weeks. At 7 weeks, macrophages purified from the peripheral blood, spleen, and BAL were donor-derived (CCR2-positive, not shown). CCR2+/+ mice, CCR2−/− mice, or CCR2−/− mice that had received a CCR2+/+ BMT (CCR2+/+ BMT) were injected with FITC and BAL was performed on day 4 after FITC. BAL cell pellets were cultured and cells that dually expressed CD45 and col 1 were enumerated (Figure 6a). Recruitment of lung fibrocytes in response to FITC was restored in the CCR2+/+ BMT mice. Lung fibrocytes cultured from the CCR2+/+ BMT mice were positive for CCR2 (not shown) indicating donor origin. CCR2−/− mice were given BMT from either CCR2−/− or CCR2+/+ donors. Mice were rested at least 7 weeks after BMT and each group was injected intratracheally with either saline or FITC. Lungs were harvested 21 days later and hydroxyproline assays were performed (Figure 6b). CCR2−/− mice that received either a CCR2+/+ BMT or a CCR2−/− BMT showed an equivalent level of hydroxyproline in response to saline injection (compare open bar to stippled bar). CCR2−/− mice that received a CCR2−/− BMT were prote