Characterization of Fibroblast-specific Protein 1 in Pulmonary Fibrosis
http://www.100md.com
美国呼吸和危急护理医学 2005年第4期
Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Division of Nephrology
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine
Department of Veterans Affairs Medical Center, Nashville, Tennessee
ABSTRACT
Because fibroblasts produce collagen and other extracellular matrix components that are deposited during tissue fibrosis, defining the behavior of these cells is critical to understanding the pathogenesis of fibrotic diseases. We investigated the utility of fibroblast-specific protein 1 (FSP1), a member of the calmodulin S100 troponin C superfamily, for identifying lung fibroblasts in a murine model of pulmonary fibrosis induced by intratracheal administration of bleomycin. Protein and mRNA expression of FSP1 was minimal in untreated lungs, but increased by 1 week after bleomycin administration and remained increased at 2 and 3 weeks after treatment. By immunohistochemistry, the number of FSP1+ cells increased in a dose-dependent manner in the lungs after bleomycin treatment. Colocalization of 1 procollagen and FSP1 in interstitial cells demonstrated that FSP1+ fibroblasts contribute to the deposition of collagen after bleomycin administration. In primary lung cell cultures, lung fibroblasts, but not macrophages or type II alveolar epithelial cells, expressed FSP1. FSP1 also identified fibroblasts in lung biopsy specimens from patients with documented usual interstitial pneumonitis. Therefore, FSP1 is an improved marker for lung fibroblasts that could be useful for investigating the pathogenesis of pulmonary fibrosis.
Key Words: -smooth muscle actin bleomycin collagen lung mouse
In pulmonary fibrosis, fibroblasts are largely responsible for the production and deposition of interstitial collagen and other extracellular matrix (1eC3). Therefore, defining the behavior of these cells is critical for understanding the pathobiology of fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF). Identification of fibroblasts in vivo is difficult because there are no widely accepted markers that are sensitive and specific for this cellular population.
Fibroblast-specific protein 1 (FSP1) is an 11-kD protein that belongs to the S100 superfamily of intracellular binding proteins (4, 5). FSP1, also identified as S100A4, mts1, 18A2, and pEL-98 (6eC10), identifies fibroblasts in the kidney during renal fibrosis induced by unilateral ureteral obstruction (5). Both FSP1 and FSP1 promoter-reporter constructs are expressed in primary fibroblasts and NIH 3T3 fibroblasts, but not in other cell types (5). The FSP1 promoter contains an element between eC187 and eC88 that is required for maximal expression, is not active in epithelial cells, and encompasses a novel domain from eC177 to eC173 that binds nuclear proteins in fibroblasts but not epithelial cells (11).
Members of the S100 superfamily are implicated in microtubule dynamics, cytoskeletal membrane interactions, calcium signal transduction, cell-cycle regulation, and cell growth and differentiation (12). Although the precise functions of FSP1 and its homologs are still being investigated, their interactions with nonmuscle myosin II (13), nonmuscle tropomyosin (10), actin (10, 14, 15), and tubulin (16eC18) suggest that FSP1 is important in mesenchymal cell shape and motility.
We hypothesized that FSP1 could serve as a sensitive and specific marker for lung fibroblasts. In these studies, we used a mouse model of pulmonary fibrosis induced by intratracheal injection of bleomycin (19eC22). Although the bleomycin model does not completely recapitulate the pathobiology of human IPF, it is a reproducible and instructive model for many aspects of pulmonary fibrosis and continues to be used by many investigators of lung fibrosis (23eC30). In addition to the mouse model, we evaluated FSP1 expression in lung biopsy specimens from patients diagnosed with IPF. Some of the results of these studies have been previously reported in the form of abstracts (31, 32).
METHODS
Detailed methods are provided in an online supplement.
Mouse Model
Wild-type C57BL/6 (Harlan-Sprague Dawley [Indianapolis, IN]) and transgenic C57BL/6 mice with green fluorescent protein (GFP) under control of the FSP1 promoter (FSP1.GFP) (33, 34) were used with experimental protocol approval by the Vanderbilt institutional animal care and utilization committee. After anesthesia with intramuscular ketamine (80 mg/kg) and xylazine (15 mg/kg), bleomycin (Blenoxane; Nippon Kayaku, Tokyo, Japan) was injected intratracheally (0.04, 0.06, or 0.08 U). At designated time points, lungs were harvested for histology or frozen tissue.
Histology and Immunohistochemistry
After harvest, lungs were formalin-fixed, paraffin-embedded, and cuts prepared for H&E and trichrome blue staining. Immunohistochemistry for FSP1 was performed using a biotinylated rabbit polyclonal antibody (5), -smooth muscle actin (-SMA) using a monoclonal antibody conjugated to alkaline phosphatase (Sigma-Aldrich, St. Louis, MO), and 1 type 1 procollagen using a goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunofluorescent detection of 1 type 1 procollagen was generated using a Texas Red conjugated donkey anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).
Cell Culture/Isolation and Immunocytochemistry
Primary lung fibroblasts, type II alveolar epithelial cells, and alveolar macrophages were isolated from untreated wild-type C57BL/6 mice (described in the online supplement), and immunocytochemistry for FSP1 and -SMA were performed.
Analysis of FSP1+ Cells on Lung Sections
Ten sequential, nonoverlapping fields from FSP1 immunohistochemistry slides were scored for FSP1 expression on a 0eC4 scale, with the average representing the score. Evaluation for colocalization of GFP and 1 type 1 procollagen was performed using an LSM510 META Laser Scanning Microscope (Carl Zeiss Advanced Imaging Microscopy, Jena, Germany).
Morphometric Analysis
Morphometric analysis was performed on 10 nonoverlapping fields of H&E-stained lung tissue using Image-Pro Express software (Media Cybernetics, Silver Spring, MD) with measurement of tissue volume density (%) (35, 36).
Western Blot
Cytoplasmic protein extracts were prepared from frozen tissue and Western blots performed for FSP1 and -SMA using antibodies mentioned previously. Cell lysates from NIH 3T3 fibroblasts served as positive controls. Blots were analyzed by laser densitometry.
RNA Isolation and Northern Blots
RNA was extracted from frozen tissue. cDNA probes were generated by polymerase chain reaction using specific primer sets for FSP1, -SMA, and procollagen type 1. 18S ribosomal band cDNA was obtained (Ambion, Austin, TX). Probes were labeled with Strip-EZ DNA (Ambion). Twenty micrograms of RNA were separated on an agarose gel, transferred to membrane, and probed. Blots were analyzed by laser densitometry.
Whole-Lung GFP Expression
After harvest from FSP1.GFP mice, lungs were analyzed ex vivo for GFP expression using an IVIS Imaging System (Xenogen, Alameda, CA).
IPF Lung Samples for Immunohistochemistry
Surgical lung biopsies from two patients with usual interstitial pneumonitis (UIP) containing areas of active fibrosis and from two patients with lung cancer with areas of normal lung tissue were obtained after institutional review board approval and informed written consent. Diagnosis of IPF was made in accordance with the American Thoracic Society/European Respiratory Society Consensus Statement (37). FSP1 immunohistochemistry was performed as described previously.
Statistics
Statistical analyses were performed using analysis of variance (p < 0.05 considered significant). Results are presented as mean ± SEM.
RESULTS
To investigate induction of FSP1 during the development of lung fibrosis, we harvested mice at baseline and 1eC4 weeks after intratracheal instillation of bleomycin. In addition, we chose three different doses of bleomycin (0.04, 0.06, and 0.08 U) to produce a range of fibrotic responses in the lungs. Initially, we correlated the presence of FSP1-positive cells with the development of fibrosis on histologic sections. Figure 1 shows lung sections stained with trichrome blue to identify the time course for collagen deposition after treatment with 0.08 U of bleomycin. A slight increase in trichrome staining was identified by 1 week after bleomycin. By 2 weeks, patches of collagen deposition were present. At 3 weeks, maximal collagen deposition was identified with multiple foci of dense collagen accumulation. By 4 weeks, some remodeling was apparent with diminished trichrome staining compared with the 3-week time point. Lung sections from these mice were immunostained to identify FSP1+ fibroblasts (Figure 2). Lung tissue from untreated mice showed only rare interstitial cells that were FSP1+. After intratracheal bleomycin injection (0.08 U), FSP1+ cells increased in number by week 1 and peaked by 2eC3 weeks. Subsequently, FSP1+ cells decreased in number but were still greater than baseline at 4 weeks. Many individual FSP1+ cells were located in the interstitium, but in areas of prominent fibrosis multiple FSP1+ cells were present in groups. FSP1+ cells were oval- to elongated-shaped cells located in the interstitium in areas of collagen deposition, consistent with the appearance of fibroblasts. No peribronchial smooth muscle, arterial wall smooth muscle, endothelial, or airway epithelial cells were positive for FSP1.
To quantify the observations made by histologic evaluation, we determined tissue volume density as a measurement of architectural changes in the lungs after bleomycin. This assessment was performed on lung sections at baseline and weeks 1eC4 after bleomycin following each of the three bleomycin doses (Figure 3A). These findings were correlated with a semiquantitative assessment of FSP1+ cells in 10 nonoverlapping fields of lung parenchyma from each mouse (Figure 3B). Although the peak tissue volume density was similar in each treatment group, at 4 weeks, the amount of architectural change present (as assessed by this parameter) corresponded to bleomycin dose (p < 0.05 for differences between 0.04, 0.06, and 0.08 U). At time points before 4 weeks, substantial inflammation and edema induced by bleomycin treatment contribute to the increased tissue volume density measurement. By 4 weeks, however, inflammation and edema have largely subsided and fibrotic remodeling predominates. For comparison with lung morphometric analysis, FSP1 expression was scored on sections from the same lungs using a 0eC4 scale. The FSP1 score increased after bleomycin, peaking at 2 weeks after treatment with each bleomycin dose and subsequently returning toward baseline. FSP1 expression was dose-dependent with the greatest numbers of FSP1+ cells in the high-dose bleomycin group (p < 0.05 for differences between each dose at weeks 2 and 4, and for differences between the 0.04 unit dose and the two higher doses at weeks 1 and 3).
To evaluate whether FSP1+ fibroblasts in the lungs are responsible, at least in part, for the deposition of collagen that occurs after intratracheal bleomycin, we performed studies using FSP1.GFP reporter mice. In these transgenic mice, we measured whole-lung fluorescence ex vivo (using an IVIS fluorescent imaging system) to show that FSP1 promoter-dependent GFP expression was increased at 2 weeks after bleomycin administration (Figure 4). These lungs were then sectioned and immunostained for 1 type 1 procollagen (Figure 5). By confocal microscopy, immunofluorescent detection of 1 type 1 procollagen (red) and GFP expression (green) were found to colocalize in cells located in areas of collagen deposition, indicating that FSP1+ cells participate in collagen production in the process of bleomycin-induced lung fibrosis. Although most GFP+ cells appeared to express 1 type 1 procollagen at 2 weeks after bleomycin, some GFP+ cells and some 1 type 1 procollagen+ cells did not colocalize these markers, indicating that not all FSP1+ cells are producing collagen and not all collagen producing cells express FSP1.
Together, these studies indicate that: (1) FSP1+ fibroblasts increase in a defined temporal pattern after bleomycin treatment; (2) maximum numbers of FSP1+ cells are present by 2 weeks after treatment, preceding maximal fibrotic changes; (3) numbers of FSP1+ cells correlate with the extent of lung fibrosis measured at 4 weeks after treatment; and (4) FSP1+ cells directly contribute to the collagen deposition that occurs in bleomycin-induced pulmonary fibrosis.
To further investigate the specificity of FSP1 for identifying fibroblasts in mouse lungs, we cultured primary lung fibroblasts and type II alveolar epithelial cells from control (untreated) mice. In addition, alveolar macrophages were obtained by lung lavage. Figure 6 shows immunocytochemistry for FSP1 in these different cell populations and in NIH 3T3 fibroblasts. NIH 3T3 fibroblasts and lung fibroblasts (Figures 6A and 6C) were positive for FSP1, whereas type II alveolar epithelial cells and alveolar macrophages were FSP1-negative (Figures 6E and 6F). No staining was identified in fibroblast cultures without the addition of primary (FSP1) antibodies (Figures 6B and 6D). In contrast to the FSP1 immunostaining pattern, -SMA staining was only seen in NIH 3T3 cells, but not in primary fibroblasts, type II epithelial cells, or macrophages (data not shown). In combination, these studies indicate that FSP1 is a specific marker for lung fibroblasts and that -SMA and FSP1 identify different cellular populations.
-SMA is commonly discussed as a marker for myofibroblasts in pulmonary fibrosis. In human forms of fibrotic lung disease such as IPF, -SMA has been used to identify interstitial myofibroblasts (38); however, much of the data using -SMA as a marker in animal models have been obtained in rat models of bleomycin-induced lung fibrosis (22, 39eC41). In mouse models, some investigators have identified -SMA as a myofibroblast marker (42, 43), but its correlation with important parameters of lung fibrosis is not well described. We compared FSP1 expression with -SMA expression on serial lung sections from mice in the 0.08-unit bleomycin group. Figure 7A shows -SMA immunostaining of a lung section obtained 2 weeks after bleomycin treatment. In this analysis, -SMA antibodies stained predominantly peribronchial and perivascular smooth muscle cells with little staining in or around the areas of fibrosis. In contrast, abundant FSP1+ cells were located in and around the areas of fibrosis (Figure 7b). Few cells appeared to stain positive for both FSP1 and -SMA. Therefore, -SMA does not appear to be a good marker for identifying effector cells in our model of mouse lung fibrosis.
We evaluated FSP1 mRNA expression in the lungs at baseline and 1eC4 weeks after bleomycin injection. Lungs were harvested at each time point and FSP1 mRNA was detected by Northern blot and normalized to the 18S RNA band (Figures 8A and 8B). In the 0.08-unit bleomycin group, FSP1 mRNA expression was increased at 1 week after treatment, remained elevated through Week 3 and declined by Week 4. This pattern was similar to FSP1 protein expression as assessed by Western blot (see below). We also compared FSP1 expression with mRNA expression of -SMA and procollagen type 1, a major form of collagen in lung fibrosis (Figure 8). Interestingly, -SMA mRNA expression was induced by 1 week after bleomycin in lung tissue and remained above baseline to 4 weeks. This increased mRNA expression of -SMA did not correlate with increased -SMA protein expression by Western blot (see below) or increased number of -SMA+ cells identified by immunohistochemistry (Figure 6). Procollagen type 1 mRNA expression increased by 1 week, preceding the collagen accumulation identified at 2 weeks after treatment. Expression of procollagen type 1 mRNA showed continued elevation above baseline at 2 and 3 weeks and returned to basal levels at 4 weeks.
By Western blot from lung homogenates, FSP1 protein expression in lung tissue was minimal at baseline but was markedly upregulated by 1 week after intratracheal bleomycin (0.06 U) (Figure 9A), similar to findings by immunohistochemistry. In separate studies to identify the onset of FSP1 production, induction of FSP1 expression was not seen at Day 5 but was present at Day 7 (data not shown). Increased FSP1 protein levels persisted at Weeks 2 and 3. By 4 weeks after bleomycin, FSP1 expression returned near baseline. Quantitation by laser densitometry revealed that at Weeks 1, 2, and 3 after bleomycin, FSP1 protein expression was increased above baseline to a similar extent (Figure 9B). Groups treated with 0.04 or 0.08 U bleomycin demonstrated similar patterns of expression (not shown). In contrast to the marked induction of FSP1 in lungs after bleomycin, expression of immunoreactive -SMA did not change significantly from baseline through Week 4 (Figures 9c and 9d).
FSP1 mRNA and protein expression in lung tissue were inducible by bleomycin treatment and detectable 1eC3 weeks after intratracheal instillation. These characteristics are consistent with the proposed role of FSP1 in identifying the lung fibroblast population. In addition, the time course for FSP1 mRNA expression correlated with the duration of procollagen type 1 mRNA expression, consistent with our hypothesis that FSP1+ fibroblasts are important for the generation of lung fibrosis.
Because FSP1 identifies fibroblasts in the bleomycin model of lung fibrosis, we wanted to determine whether this marker could identify fibroblasts in human IPF. Therefore, we obtained lung tissue sections from two patients with documented UIP and two histologically normal lung sections and performed immunostaining for FSP1. Very few FSP1 positive cells were found on sections from normal human lung (not shown); however, positive staining for FSP1 was found on the lung biopsy sections from patients with UIP in areas with increased cellularity and active lung fibrosis (Figure 10). The shape and location of the identified cells appeared consistent with fibroblasts. Based on this finding, FSP1 has potential utility in identifying fibroblasts in human lung tissue in areas of ongoing fibrosis.
DISCUSSION
In these studies, we characterized FSP1 as a novel and useful marker for fibroblasts in pulmonary fibrosis. In normal murine lung parenchyma, FSP1 is expressed at very low levels, but is induced in the development of pulmonary fibrosis that follows intratracheal administration of bleomycin. We have demonstrated that the expression of FSP1 correlates well with collagen deposition and lung parenchymal architectural changes in this model. Furthermore, we have demonstrated that FSP1 can be used to identify fibroblasts in human lung tissue from patients with UIP/IPF, providing initial evidence that it can be used as a fibroblast marker in studies of human lung disease.
Prior studies in models of renal fibrosis have identified FSP1+ fibroblasts (5, 11, 33, 44, 45). At baseline, FSP1 expression is very low in the kidney, but after unilateral ureteral ligation, numerous FSP1+ cells appear in association with the development of renal fibrosis (5). In studies using a construct with the FSP1 promoter linked to thymidine kinase, FSP1+ cells were selectively eliminated by treatment with ganciclovir, resulting in decreased renal fibrosis (44). Although work in kidney models has revealed the specificity and sensitivity of FSP1 for fibroblasts in noncancerous tissues, FSP1 is often expressed in malignant cells (7, 34), likely related to the increased expression of cytoskeletal components favoring cell motility and tissue invasiveness.
Because fibroblasts produce collagen and other extracellular matrix components that are deposited during tissue fibrosis (1eC3, 22, 46), defining the behavior of these cells is critical to our understanding of fibrotic diseases. However, identification of fibroblasts in vivo is difficult with current tools because markers such as vimentin and -SMA lack specificity or sensitivity (47). Vimentin has been used as a marker for fibroblasts in studies of pulmonary fibrosis (47), but unfortunately does not distinguish fibroblasts from other cells of mesenchymal origin. In the lungs, several studies have investigated -SMA+ cells, identified as myofibroblasts (48, 49). Although myofibroblasts have been implicated as important collagen-producing cells in bleomycin models of lung fibrosis (22, 46) and in human forms of fibrotic lung disease (3), -SMA does not identify resting fibroblasts and is found in smooth muscle cells (50). Furthermore, in a recent study demonstrating that a large proportion of the collagen-producing lung fibroblasts in pulmonary fibrosis are derived from bone marrow progenitor cells, it was shown that these collagen-producing fibroblasts were -SMAeCnegative (51). In our experiments, immunohistochemistry for -SMA failed to identify a large population of fibroblasts in the murine lung during fibrogenesis, whereas immunohistochemistry for FSP1 readily identified a larger fibroblast population. In addition, primary lung fibroblasts uniformly expressed FSP1 but not -SMA.
FSP1 has substantial utility as a fibroblast marker in future studies in both animal models and in human forms of fibrotic lung disease. In animal studies, using FSP1 as a fibroblast marker will allow investigators to better identify fibroblasts in vivo, and thus design experiments to better elucidate fibroblast recruitment, proliferation, and responses to inflammatory and fibrotic stimuli. Specifically, using FSP1 to follow the recruitment and proliferation of fibroblasts may help elucidate the origins of effector fibroblasts in the lungs. For human studies, FSP1 identification of fibroblasts may help to determine the response of these cells to antifibrotic therapies, leading to the development of improved fibroblast-targeted therapeutic interventions.
Acknowledgments
The authors thank the Mouse Pathology and Immunostaining Core Facility at Vanderbilt University Medical Center for their assistance with the lung tissue slide preparations. They also thank Tamara Lasakow for editorial and Janet Shelton for electronic assistance with manuscript preparation.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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Department of Cell and Developmental Biology, Vanderbilt University School of Medicine
Department of Veterans Affairs Medical Center, Nashville, Tennessee
ABSTRACT
Because fibroblasts produce collagen and other extracellular matrix components that are deposited during tissue fibrosis, defining the behavior of these cells is critical to understanding the pathogenesis of fibrotic diseases. We investigated the utility of fibroblast-specific protein 1 (FSP1), a member of the calmodulin S100 troponin C superfamily, for identifying lung fibroblasts in a murine model of pulmonary fibrosis induced by intratracheal administration of bleomycin. Protein and mRNA expression of FSP1 was minimal in untreated lungs, but increased by 1 week after bleomycin administration and remained increased at 2 and 3 weeks after treatment. By immunohistochemistry, the number of FSP1+ cells increased in a dose-dependent manner in the lungs after bleomycin treatment. Colocalization of 1 procollagen and FSP1 in interstitial cells demonstrated that FSP1+ fibroblasts contribute to the deposition of collagen after bleomycin administration. In primary lung cell cultures, lung fibroblasts, but not macrophages or type II alveolar epithelial cells, expressed FSP1. FSP1 also identified fibroblasts in lung biopsy specimens from patients with documented usual interstitial pneumonitis. Therefore, FSP1 is an improved marker for lung fibroblasts that could be useful for investigating the pathogenesis of pulmonary fibrosis.
Key Words: -smooth muscle actin bleomycin collagen lung mouse
In pulmonary fibrosis, fibroblasts are largely responsible for the production and deposition of interstitial collagen and other extracellular matrix (1eC3). Therefore, defining the behavior of these cells is critical for understanding the pathobiology of fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF). Identification of fibroblasts in vivo is difficult because there are no widely accepted markers that are sensitive and specific for this cellular population.
Fibroblast-specific protein 1 (FSP1) is an 11-kD protein that belongs to the S100 superfamily of intracellular binding proteins (4, 5). FSP1, also identified as S100A4, mts1, 18A2, and pEL-98 (6eC10), identifies fibroblasts in the kidney during renal fibrosis induced by unilateral ureteral obstruction (5). Both FSP1 and FSP1 promoter-reporter constructs are expressed in primary fibroblasts and NIH 3T3 fibroblasts, but not in other cell types (5). The FSP1 promoter contains an element between eC187 and eC88 that is required for maximal expression, is not active in epithelial cells, and encompasses a novel domain from eC177 to eC173 that binds nuclear proteins in fibroblasts but not epithelial cells (11).
Members of the S100 superfamily are implicated in microtubule dynamics, cytoskeletal membrane interactions, calcium signal transduction, cell-cycle regulation, and cell growth and differentiation (12). Although the precise functions of FSP1 and its homologs are still being investigated, their interactions with nonmuscle myosin II (13), nonmuscle tropomyosin (10), actin (10, 14, 15), and tubulin (16eC18) suggest that FSP1 is important in mesenchymal cell shape and motility.
We hypothesized that FSP1 could serve as a sensitive and specific marker for lung fibroblasts. In these studies, we used a mouse model of pulmonary fibrosis induced by intratracheal injection of bleomycin (19eC22). Although the bleomycin model does not completely recapitulate the pathobiology of human IPF, it is a reproducible and instructive model for many aspects of pulmonary fibrosis and continues to be used by many investigators of lung fibrosis (23eC30). In addition to the mouse model, we evaluated FSP1 expression in lung biopsy specimens from patients diagnosed with IPF. Some of the results of these studies have been previously reported in the form of abstracts (31, 32).
METHODS
Detailed methods are provided in an online supplement.
Mouse Model
Wild-type C57BL/6 (Harlan-Sprague Dawley [Indianapolis, IN]) and transgenic C57BL/6 mice with green fluorescent protein (GFP) under control of the FSP1 promoter (FSP1.GFP) (33, 34) were used with experimental protocol approval by the Vanderbilt institutional animal care and utilization committee. After anesthesia with intramuscular ketamine (80 mg/kg) and xylazine (15 mg/kg), bleomycin (Blenoxane; Nippon Kayaku, Tokyo, Japan) was injected intratracheally (0.04, 0.06, or 0.08 U). At designated time points, lungs were harvested for histology or frozen tissue.
Histology and Immunohistochemistry
After harvest, lungs were formalin-fixed, paraffin-embedded, and cuts prepared for H&E and trichrome blue staining. Immunohistochemistry for FSP1 was performed using a biotinylated rabbit polyclonal antibody (5), -smooth muscle actin (-SMA) using a monoclonal antibody conjugated to alkaline phosphatase (Sigma-Aldrich, St. Louis, MO), and 1 type 1 procollagen using a goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunofluorescent detection of 1 type 1 procollagen was generated using a Texas Red conjugated donkey anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).
Cell Culture/Isolation and Immunocytochemistry
Primary lung fibroblasts, type II alveolar epithelial cells, and alveolar macrophages were isolated from untreated wild-type C57BL/6 mice (described in the online supplement), and immunocytochemistry for FSP1 and -SMA were performed.
Analysis of FSP1+ Cells on Lung Sections
Ten sequential, nonoverlapping fields from FSP1 immunohistochemistry slides were scored for FSP1 expression on a 0eC4 scale, with the average representing the score. Evaluation for colocalization of GFP and 1 type 1 procollagen was performed using an LSM510 META Laser Scanning Microscope (Carl Zeiss Advanced Imaging Microscopy, Jena, Germany).
Morphometric Analysis
Morphometric analysis was performed on 10 nonoverlapping fields of H&E-stained lung tissue using Image-Pro Express software (Media Cybernetics, Silver Spring, MD) with measurement of tissue volume density (%) (35, 36).
Western Blot
Cytoplasmic protein extracts were prepared from frozen tissue and Western blots performed for FSP1 and -SMA using antibodies mentioned previously. Cell lysates from NIH 3T3 fibroblasts served as positive controls. Blots were analyzed by laser densitometry.
RNA Isolation and Northern Blots
RNA was extracted from frozen tissue. cDNA probes were generated by polymerase chain reaction using specific primer sets for FSP1, -SMA, and procollagen type 1. 18S ribosomal band cDNA was obtained (Ambion, Austin, TX). Probes were labeled with Strip-EZ DNA (Ambion). Twenty micrograms of RNA were separated on an agarose gel, transferred to membrane, and probed. Blots were analyzed by laser densitometry.
Whole-Lung GFP Expression
After harvest from FSP1.GFP mice, lungs were analyzed ex vivo for GFP expression using an IVIS Imaging System (Xenogen, Alameda, CA).
IPF Lung Samples for Immunohistochemistry
Surgical lung biopsies from two patients with usual interstitial pneumonitis (UIP) containing areas of active fibrosis and from two patients with lung cancer with areas of normal lung tissue were obtained after institutional review board approval and informed written consent. Diagnosis of IPF was made in accordance with the American Thoracic Society/European Respiratory Society Consensus Statement (37). FSP1 immunohistochemistry was performed as described previously.
Statistics
Statistical analyses were performed using analysis of variance (p < 0.05 considered significant). Results are presented as mean ± SEM.
RESULTS
To investigate induction of FSP1 during the development of lung fibrosis, we harvested mice at baseline and 1eC4 weeks after intratracheal instillation of bleomycin. In addition, we chose three different doses of bleomycin (0.04, 0.06, and 0.08 U) to produce a range of fibrotic responses in the lungs. Initially, we correlated the presence of FSP1-positive cells with the development of fibrosis on histologic sections. Figure 1 shows lung sections stained with trichrome blue to identify the time course for collagen deposition after treatment with 0.08 U of bleomycin. A slight increase in trichrome staining was identified by 1 week after bleomycin. By 2 weeks, patches of collagen deposition were present. At 3 weeks, maximal collagen deposition was identified with multiple foci of dense collagen accumulation. By 4 weeks, some remodeling was apparent with diminished trichrome staining compared with the 3-week time point. Lung sections from these mice were immunostained to identify FSP1+ fibroblasts (Figure 2). Lung tissue from untreated mice showed only rare interstitial cells that were FSP1+. After intratracheal bleomycin injection (0.08 U), FSP1+ cells increased in number by week 1 and peaked by 2eC3 weeks. Subsequently, FSP1+ cells decreased in number but were still greater than baseline at 4 weeks. Many individual FSP1+ cells were located in the interstitium, but in areas of prominent fibrosis multiple FSP1+ cells were present in groups. FSP1+ cells were oval- to elongated-shaped cells located in the interstitium in areas of collagen deposition, consistent with the appearance of fibroblasts. No peribronchial smooth muscle, arterial wall smooth muscle, endothelial, or airway epithelial cells were positive for FSP1.
To quantify the observations made by histologic evaluation, we determined tissue volume density as a measurement of architectural changes in the lungs after bleomycin. This assessment was performed on lung sections at baseline and weeks 1eC4 after bleomycin following each of the three bleomycin doses (Figure 3A). These findings were correlated with a semiquantitative assessment of FSP1+ cells in 10 nonoverlapping fields of lung parenchyma from each mouse (Figure 3B). Although the peak tissue volume density was similar in each treatment group, at 4 weeks, the amount of architectural change present (as assessed by this parameter) corresponded to bleomycin dose (p < 0.05 for differences between 0.04, 0.06, and 0.08 U). At time points before 4 weeks, substantial inflammation and edema induced by bleomycin treatment contribute to the increased tissue volume density measurement. By 4 weeks, however, inflammation and edema have largely subsided and fibrotic remodeling predominates. For comparison with lung morphometric analysis, FSP1 expression was scored on sections from the same lungs using a 0eC4 scale. The FSP1 score increased after bleomycin, peaking at 2 weeks after treatment with each bleomycin dose and subsequently returning toward baseline. FSP1 expression was dose-dependent with the greatest numbers of FSP1+ cells in the high-dose bleomycin group (p < 0.05 for differences between each dose at weeks 2 and 4, and for differences between the 0.04 unit dose and the two higher doses at weeks 1 and 3).
To evaluate whether FSP1+ fibroblasts in the lungs are responsible, at least in part, for the deposition of collagen that occurs after intratracheal bleomycin, we performed studies using FSP1.GFP reporter mice. In these transgenic mice, we measured whole-lung fluorescence ex vivo (using an IVIS fluorescent imaging system) to show that FSP1 promoter-dependent GFP expression was increased at 2 weeks after bleomycin administration (Figure 4). These lungs were then sectioned and immunostained for 1 type 1 procollagen (Figure 5). By confocal microscopy, immunofluorescent detection of 1 type 1 procollagen (red) and GFP expression (green) were found to colocalize in cells located in areas of collagen deposition, indicating that FSP1+ cells participate in collagen production in the process of bleomycin-induced lung fibrosis. Although most GFP+ cells appeared to express 1 type 1 procollagen at 2 weeks after bleomycin, some GFP+ cells and some 1 type 1 procollagen+ cells did not colocalize these markers, indicating that not all FSP1+ cells are producing collagen and not all collagen producing cells express FSP1.
Together, these studies indicate that: (1) FSP1+ fibroblasts increase in a defined temporal pattern after bleomycin treatment; (2) maximum numbers of FSP1+ cells are present by 2 weeks after treatment, preceding maximal fibrotic changes; (3) numbers of FSP1+ cells correlate with the extent of lung fibrosis measured at 4 weeks after treatment; and (4) FSP1+ cells directly contribute to the collagen deposition that occurs in bleomycin-induced pulmonary fibrosis.
To further investigate the specificity of FSP1 for identifying fibroblasts in mouse lungs, we cultured primary lung fibroblasts and type II alveolar epithelial cells from control (untreated) mice. In addition, alveolar macrophages were obtained by lung lavage. Figure 6 shows immunocytochemistry for FSP1 in these different cell populations and in NIH 3T3 fibroblasts. NIH 3T3 fibroblasts and lung fibroblasts (Figures 6A and 6C) were positive for FSP1, whereas type II alveolar epithelial cells and alveolar macrophages were FSP1-negative (Figures 6E and 6F). No staining was identified in fibroblast cultures without the addition of primary (FSP1) antibodies (Figures 6B and 6D). In contrast to the FSP1 immunostaining pattern, -SMA staining was only seen in NIH 3T3 cells, but not in primary fibroblasts, type II epithelial cells, or macrophages (data not shown). In combination, these studies indicate that FSP1 is a specific marker for lung fibroblasts and that -SMA and FSP1 identify different cellular populations.
-SMA is commonly discussed as a marker for myofibroblasts in pulmonary fibrosis. In human forms of fibrotic lung disease such as IPF, -SMA has been used to identify interstitial myofibroblasts (38); however, much of the data using -SMA as a marker in animal models have been obtained in rat models of bleomycin-induced lung fibrosis (22, 39eC41). In mouse models, some investigators have identified -SMA as a myofibroblast marker (42, 43), but its correlation with important parameters of lung fibrosis is not well described. We compared FSP1 expression with -SMA expression on serial lung sections from mice in the 0.08-unit bleomycin group. Figure 7A shows -SMA immunostaining of a lung section obtained 2 weeks after bleomycin treatment. In this analysis, -SMA antibodies stained predominantly peribronchial and perivascular smooth muscle cells with little staining in or around the areas of fibrosis. In contrast, abundant FSP1+ cells were located in and around the areas of fibrosis (Figure 7b). Few cells appeared to stain positive for both FSP1 and -SMA. Therefore, -SMA does not appear to be a good marker for identifying effector cells in our model of mouse lung fibrosis.
We evaluated FSP1 mRNA expression in the lungs at baseline and 1eC4 weeks after bleomycin injection. Lungs were harvested at each time point and FSP1 mRNA was detected by Northern blot and normalized to the 18S RNA band (Figures 8A and 8B). In the 0.08-unit bleomycin group, FSP1 mRNA expression was increased at 1 week after treatment, remained elevated through Week 3 and declined by Week 4. This pattern was similar to FSP1 protein expression as assessed by Western blot (see below). We also compared FSP1 expression with mRNA expression of -SMA and procollagen type 1, a major form of collagen in lung fibrosis (Figure 8). Interestingly, -SMA mRNA expression was induced by 1 week after bleomycin in lung tissue and remained above baseline to 4 weeks. This increased mRNA expression of -SMA did not correlate with increased -SMA protein expression by Western blot (see below) or increased number of -SMA+ cells identified by immunohistochemistry (Figure 6). Procollagen type 1 mRNA expression increased by 1 week, preceding the collagen accumulation identified at 2 weeks after treatment. Expression of procollagen type 1 mRNA showed continued elevation above baseline at 2 and 3 weeks and returned to basal levels at 4 weeks.
By Western blot from lung homogenates, FSP1 protein expression in lung tissue was minimal at baseline but was markedly upregulated by 1 week after intratracheal bleomycin (0.06 U) (Figure 9A), similar to findings by immunohistochemistry. In separate studies to identify the onset of FSP1 production, induction of FSP1 expression was not seen at Day 5 but was present at Day 7 (data not shown). Increased FSP1 protein levels persisted at Weeks 2 and 3. By 4 weeks after bleomycin, FSP1 expression returned near baseline. Quantitation by laser densitometry revealed that at Weeks 1, 2, and 3 after bleomycin, FSP1 protein expression was increased above baseline to a similar extent (Figure 9B). Groups treated with 0.04 or 0.08 U bleomycin demonstrated similar patterns of expression (not shown). In contrast to the marked induction of FSP1 in lungs after bleomycin, expression of immunoreactive -SMA did not change significantly from baseline through Week 4 (Figures 9c and 9d).
FSP1 mRNA and protein expression in lung tissue were inducible by bleomycin treatment and detectable 1eC3 weeks after intratracheal instillation. These characteristics are consistent with the proposed role of FSP1 in identifying the lung fibroblast population. In addition, the time course for FSP1 mRNA expression correlated with the duration of procollagen type 1 mRNA expression, consistent with our hypothesis that FSP1+ fibroblasts are important for the generation of lung fibrosis.
Because FSP1 identifies fibroblasts in the bleomycin model of lung fibrosis, we wanted to determine whether this marker could identify fibroblasts in human IPF. Therefore, we obtained lung tissue sections from two patients with documented UIP and two histologically normal lung sections and performed immunostaining for FSP1. Very few FSP1 positive cells were found on sections from normal human lung (not shown); however, positive staining for FSP1 was found on the lung biopsy sections from patients with UIP in areas with increased cellularity and active lung fibrosis (Figure 10). The shape and location of the identified cells appeared consistent with fibroblasts. Based on this finding, FSP1 has potential utility in identifying fibroblasts in human lung tissue in areas of ongoing fibrosis.
DISCUSSION
In these studies, we characterized FSP1 as a novel and useful marker for fibroblasts in pulmonary fibrosis. In normal murine lung parenchyma, FSP1 is expressed at very low levels, but is induced in the development of pulmonary fibrosis that follows intratracheal administration of bleomycin. We have demonstrated that the expression of FSP1 correlates well with collagen deposition and lung parenchymal architectural changes in this model. Furthermore, we have demonstrated that FSP1 can be used to identify fibroblasts in human lung tissue from patients with UIP/IPF, providing initial evidence that it can be used as a fibroblast marker in studies of human lung disease.
Prior studies in models of renal fibrosis have identified FSP1+ fibroblasts (5, 11, 33, 44, 45). At baseline, FSP1 expression is very low in the kidney, but after unilateral ureteral ligation, numerous FSP1+ cells appear in association with the development of renal fibrosis (5). In studies using a construct with the FSP1 promoter linked to thymidine kinase, FSP1+ cells were selectively eliminated by treatment with ganciclovir, resulting in decreased renal fibrosis (44). Although work in kidney models has revealed the specificity and sensitivity of FSP1 for fibroblasts in noncancerous tissues, FSP1 is often expressed in malignant cells (7, 34), likely related to the increased expression of cytoskeletal components favoring cell motility and tissue invasiveness.
Because fibroblasts produce collagen and other extracellular matrix components that are deposited during tissue fibrosis (1eC3, 22, 46), defining the behavior of these cells is critical to our understanding of fibrotic diseases. However, identification of fibroblasts in vivo is difficult with current tools because markers such as vimentin and -SMA lack specificity or sensitivity (47). Vimentin has been used as a marker for fibroblasts in studies of pulmonary fibrosis (47), but unfortunately does not distinguish fibroblasts from other cells of mesenchymal origin. In the lungs, several studies have investigated -SMA+ cells, identified as myofibroblasts (48, 49). Although myofibroblasts have been implicated as important collagen-producing cells in bleomycin models of lung fibrosis (22, 46) and in human forms of fibrotic lung disease (3), -SMA does not identify resting fibroblasts and is found in smooth muscle cells (50). Furthermore, in a recent study demonstrating that a large proportion of the collagen-producing lung fibroblasts in pulmonary fibrosis are derived from bone marrow progenitor cells, it was shown that these collagen-producing fibroblasts were -SMAeCnegative (51). In our experiments, immunohistochemistry for -SMA failed to identify a large population of fibroblasts in the murine lung during fibrogenesis, whereas immunohistochemistry for FSP1 readily identified a larger fibroblast population. In addition, primary lung fibroblasts uniformly expressed FSP1 but not -SMA.
FSP1 has substantial utility as a fibroblast marker in future studies in both animal models and in human forms of fibrotic lung disease. In animal studies, using FSP1 as a fibroblast marker will allow investigators to better identify fibroblasts in vivo, and thus design experiments to better elucidate fibroblast recruitment, proliferation, and responses to inflammatory and fibrotic stimuli. Specifically, using FSP1 to follow the recruitment and proliferation of fibroblasts may help elucidate the origins of effector fibroblasts in the lungs. For human studies, FSP1 identification of fibroblasts may help to determine the response of these cells to antifibrotic therapies, leading to the development of improved fibroblast-targeted therapeutic interventions.
Acknowledgments
The authors thank the Mouse Pathology and Immunostaining Core Facility at Vanderbilt University Medical Center for their assistance with the lung tissue slide preparations. They also thank Tamara Lasakow for editorial and Janet Shelton for electronic assistance with manuscript preparation.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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