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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 765-773, (2002)
© 2002 American Thoracic Society

Original Article

Basic Fibroblast Growth Factor and Its Receptors in Idiopathic Pulmonary Fibrosis and Lymphangioleiomyomatosis

Yoshikazu Inoue, Talmadge E. King, Jr., Elizabeth Barker, Elaine Daniloff and Lee S. Newman

Division of Environmental and Occupational Health Sciences and Pulmonary Division, Department of Medicine, National Jewish Medical and Research Center; Division of Pulmonary Science and Critical Care Medicine, Department of Medicine and Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, Colorado; Department of Diffuse Lung Diseases and Respiratory Failure, Clinical Research Center, National Kinki-Chuo Hospital for Chest Diseases, Osaka, Japan; and Department of Medicine, San Francisco General Hospital, San Francisco, California

Correspondence and requests for reprints should be addressed to Yoshikazu Inoue, M.D., Ph.D., Department of Diffuse Lung Diseases and Respiratory Failure, Clinical Research Center, National Kinki-Chuo Hospital for Chest Diseases, 1180 Nagasone-cho, Sakai, Osaka 591-8555, Japan. E-mail: giichi{at}kch.hosp.go.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Basic fibroblast growth factor (bFGF) is a potent mitogenic factor for smooth muscle cells, myofibroblasts, and fibroblasts, proliferation of which is a hallmark of idiopathic pulmonary fibrosis (IPF) and lymphangioleiomyomatosis (LAM). Mast cells produce bFGF and have been associated with pulmonary fibrosis. We hypothesize that smooth muscle cell/myofibroblast–like cells will be spatially associated with bFGF-containing mast cells and that bFGF receptors will be expressed on the effector cells in IPF and LAM. We performed quantitative immunohistochemistry for bFGF, mast cell tryptase, smooth muscle actin for smooth muscle cell/myofibroblast–like cells, and fibroblast growth factor receptors (Flg, Bek) and measured collagen and elastic fiber in lung sections from IPF (n = 14), LAM (n = 9), and control lung (n = 10). IPF and LAM lung contained more smooth muscle cell/myofibroblast–like cells than did control lung. bFGF-containing mast cells were abundant both in IPF and LAM and were associated with collagen, elastic fibers, and smooth muscle cell/myofibroblast–like cells in IPF. Flg was expressed on epithelial cells, endothelial cells, smooth muscle cell/myofibroblast–like cells, and macrophages in IPF. In LAM, Flg was expressed on epithelial cells adjacent to smooth muscle cell/myofibroblast–like cell aggregates. Bek was expressed dominantly on smooth muscle cell/myofibroblast–like cells in LAM and on smooth muscle cell/myofibroblast–like cells as well as neutrophils in IPF. These data suggest that mast cell–derived bFGF might exert fibrogenic, proliferative effects on smooth muscle cell/myofibroblast–like cells through its receptors.

Key Words: mast cells • extracellular matrix • myofibroblasts • fibroblast growth factor receptors • lung


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Basic fibroblast growth factor (bFGF) is a potent chemotactic and mitogenic factor for cells of mesodermal, ectodermal, and endodermal origin (1), including smooth muscle cells and myofibroblasts (2, 3). The bioactivity of bFGF is mediated through high-affinity fibroblast growth factor receptors (FGFR) such as FGFR1 (Flg) and FGFR2 (Bek) (4). High levels of bFGF have been found in bronchoalveolar lavage fluid and in lung tissue of patients with acute lung injury and pulmonary fibrosis (5, 6). Although alveolar macrophages, fibroblasts, human T-lymphocytes, and endothelial cells can all produce bFGF (7–9), mast cells are considered to be a major source of bFGF in the lungs of patients with chronic pulmonary fibrosis (6, 10). bFGF protein and/or mRNA have been detected in human mast cells (6, 10, 11) and in rat mast cells in pulmonary fibrosis (12). Recently, we demonstrated that mast cell bFGF is associated with both the degree and location of fibrosis in idiopathic pulmonary fibrosis (IPF), sarcoidosis, chronic beryllium disease (6), and silicosis (13).

Abundant smooth muscle actin (SMA)–expressing cells, smooth muscle cells, or myofibroblasts in the interstitium are considered potentially significant contributors to the increased extracellular matrix and elastic recoil observed in advanced pulmonary fibrosis (14). During lung development, primordial fibroblasts appear to differentiate into myofibroblasts or smooth muscle cells in the sheep alveolus (15). bFGF, a potent growth factor for myofibroblasts, may control this differentiation as well these cells' extracellular matrix production (2, 3, 16, 17).

Analogously, lymphangioleiomyomatosis (LAM) can be thought of as hyperplastic disorders of smooth muscle cell/myofibroblast–like cells (18). LAM is characterized by a nodular proliferation of smooth muscle–like cells in the peribronchial, perivascular, and perilymphatic lung tissue, accompanied by cystic dilation of the alveolus, rupture of the alveolar wall, lymphangiectasis, and septal collagen fiber deposition (19). As in pulmonary fibrosis, smooth muscle–like cells in LAM express SMA (20). Thus, although the etiologies of IPF and LAM may be different, they share the common pathophysiologic features of smooth muscle cell/myofibroblast–like cell proliferation and extracellular matrix deposition.

On the basis of this line of reasoning, we hypothesize that bFGF-containing mast cells contribute to smooth muscle cell/myofibroblast–like cell hyperplasia in both IPF and LAM. To test this hypothesis, we performed quantitative morphometric immunohistochemical studies of smooth muscle cell/myofibroblast–like cells and bFGF-containing mast cells in relation to extracellular matrix. We localized the cells that possessed bFGF receptors in the lungs of patients with IPF and LAM and in control lungs.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Lung tissue was obtained from 14 patients with IPF (8 open lung biopsy, 2 lung transplantation, and 4 autopsy) and from 9 patients with LAM (7 open lung biopsy and 2 lung transplantation). The control lung specimens were obtained from the contralateral lung of healthy lung transplantation donors (n = 5) or from individuals who underwent transbronchial lung biopsy but were found to have no lung pathology (n = 5). The diagnosis of IPF was established according to previously described clinical and histologic criteria (21). Patients with collagen vascular disease, drug or chemical exposure, or other possible etiologies of interstitial lung disease such as bronchiolitis obliterans and organizing pneumonia, diffuse alveolar damage, lymphocytic interstitial pneumonia, nonspecific interstitial pneumonia, or nonclassified forms of chronic interstitial pneumonia were excluded from this study group. All patients with IPF had histology consistent with usual interstitial pneumonia. The diagnosis of LAM was based on a compatible history, physical examination, chest radiograph, computed tomographic scan of the lung, pulmonary physiologic evaluation, and open lung biopsy (22). Demographic description of the subjects is summarized in Table 1 .


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  		TABLE 1. Demographic data for study subjects

 
Informed consent was obtained from each subject, and the protocol was approved by our Institutional Human Subjects Review Board.

Sample Collection and Preparation
Lung tissue was fixed with 10% formalin or 4% paraformaldehyde and embedded in paraffin. One paraffin block was used for each subject. Each block contained two or three large specimens obtained by open lung biopsy or autopsy or a minimum of six specimens obtained by transbronchial lung biopsy. Each block was cut into 2-µm-thickness serial sections and laid on Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA) for immunohistochemistry, hematoxylin–eosin staining, and Movat's pentachrome staining (23).

Immunohistochemical Staining of bFGF, Tryptase, SMA, Bek, and Flg
We used monoclonal antibodies directed against bovine bFGF, type II (5 µg/ml, immunoglobulin G1-{kappa} [IgG1-{kappa}]) (Upstate Biotechnology Incorporation, Lake Placid, NY) for immunostaining of bFGF. This antibody is highly specific for bovine, human, rat, and mouse bFGF (6). We used anti–human mast cell–tryptase monoclonal antibody (1 µg/ml, subclass, IgG1-{kappa}; DAKO, Carpinteria, CA) to identify mast cells, anti-CD68 (3.6 µg/ml, PG-M1, IgG3-{kappa}, DAKO) to identify macrophages, and anti-SMA monoclonal antibody (1:500 dilution, HHF35, IgG1; Enzo Diagnostics, Farmingdale, NY) to identify smooth muscle cell/myofibroblast–like cells (14). Anti–human Bek rabbit polyclonal antibody (4 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) and anti–human Flg rabbit polyclonal antibody (2 µg/ml, Santa Cruz Biotechnology) were used to detect FGFR (24, 25).

Immunohistochemistry was performed by avidin–biotin complex peroxidase methods using VECTASTAIN Elite avidin–biotin complex kit (Vector Laboratories, Burlingame, CA) (6). After incubation in 0.3% hydrogen peroxide dissolved in methyl alcohol for 30 minutes, tissue sections for bFGF or Flg were treated with 1-mg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO) for 30 minutes at 37°C to reveal the antigens. Sections for Bek were treated with 1-mg/ml pepsin (Sigma) for 30 minutes at 37°C to reveal the antigen. After blocking with 1.5% normal horse serum for 30 minutes, we incubated sections with primary antibody at the appropriate dilution in 1.5% normal horse serum for 60 minutes, washed them with Tris-buffered saline, pH 7.3, and incubated them with biotinylated horse–anti–mouse IgG antibody for 30 minutes. For Bek and Flg, we incubated with biotinylated anti–rabbit IgG (Vector Laboratories) at room temperature. After washing with Tris-buffered saline, the sections were incubated with horseradish peroxidase–conjugated avidin–biotin complex for 30 minutes. We used 3'-diaminobenzidine (Vector Laboratories) as the substrate for peroxidase. Counterstaining was performed with hematoxylin.

The specificity of immunolabeling of bFGF was tested under various conditions. We omitted first antibody, second antibody, or avidin–biotin complex. We used normal mouse serum, normal nonimmune mouse IgG (Sigma), and anti–human transforming growth factor-ß monoclonal antibody (IgG1, Genzyme Corporation, Cambridge, MA) in lieu of the primary antibody. Sufficient recombinant human bFGF (Upstate Biotechnology Incorporation) and synthesized peptide of Flg or Bek (Santa Cruz Biotechnology) were each incubated for 60 minutes at room temperature in the presence of their respective antibodies, to absorb the antibody, and then were used in lieu of the primary antibody. To inhibit nonimmunologic binding of monoclonal antibody to heparin in mast cells, the buffers for dilution of antibody and washing solution were acidified to pH 6.0 using 2-[n-morpholino] ethanesulfonic acid (Sigma) buffer containing 150 mM NaCl, pH 6.0, and with a high-salt washing buffer (400 mM NaCl) (26).

In some experiments, we performed double immunohistochemistry by labeling sections with both anti-bFGF plus anti-tryptase, anti-bFGF plus anti-CD68, or anti-SMA plus anti-tryptase, using combined avidin–biotin complex–peroxidase and avidin–biotin complex–alkaline phosphatase methods (Vectastain avidin–biotin complex–alkaline phosphatase kit; Vector Laboratories). We used 3'-diaminobenzidine as the substrate for horseradish peroxidase and Vector Red (Vector Laboratories) as the substrate for alkaline phosphatase. Levamisole 1.25 mM (Vector Laboratories) was added to the alkaline phosphatase substrate (6).

Morphometric Analysis
We performed quantitative morphometric analysis of all lung histology specimens using computer-assisted video microscopy as described previously (6, 13, 27, 28). In brief, by this method we can determine the volume of each cell type or tissue component of interest relative to the total volume of lung tissue examined. To determine the volume density (VV) of interest (e.g., bFGF+ cells), microscopic images of each histology section were captured and stored in the computer. A grid (42 or 125 points) was superimposed on each image, points of interest intersecting the grid counted, and VV calculated. We counted 12 random fields from each section, from which we calculated a mean value for each section. The number of fields in each section was decided before quantitation by preliminary counting using transbronchial lung biopsy and open lung biopsy sections and by determining the minimum number of images with minimal variance (6, 13, 28, 29).

To normalize for potential artifacts created by compression or expansion of lung specimens due to fixation or biopsy method, VV of each component was corrected for the VV lung tissue (parenchyma) measured with hematoxylin–eosin–stained sections at x10 magnification with the 42-point grid by the following formula (6, 13, 29): Normalized VV = (VV Component of Interest x 100)/VV Lung Tissue. The VV bFGF+ cells and VV tryptase-immunopositive mast cells were calculated both by counting the grid points that hit immunopositive cells in the interstitium and in the alveolus at x40 magnification using a 125-point grid. The VV of total immunopositive cells was calculated by summing the VV interstitial and alveolar immunopositive cells.

To determine the degree of lung fibrosis, we quantified VV collagen/reticular fibers and VV elastic fibers on sections stained by Movat's Ppentachrome (23), measured at x40 magnification with a 42-point grid (6, 13).

Statistical Analysis
Dunn's nonparametric multicomparison procedure or Wilcoxon nonparametric rank sum procedure for ranked data was used to compare differences among all groups or among paired groups (30). For tests of association, we calculated Spearman's correlation coefficient ({rho}). All data are expressed as median (with range). We defined statistical significance at p values less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution and Morphometric Analysis of Collagen/Reticular Fibers, Elastic Fibers, and Smooth Muscle Cell/Myofibroblast–like Cells
In control lungs, small amounts of collagen/reticular fibers and elastic fibers were observed mainly around bronchovascular bundles, in a normal distribution (6, 27). In patients with IPF, collagen/reticular fibers and elastic fibers were observed in thickened interstitium. In patients with LAM, these fibers were observed among proliferated smooth muscle–like cells. Although the distribution of these extracellular matrix elements differed from each other, we analyzed the total amount of collagen/reticular fibers and elastic fibers. The VV of collagen/reticular fibers in IPF was significantly higher than in control lungs (p < 0.05) (Table 2) . We observed no significant difference in the VV elastic fibers among the groups.


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  		TABLE 2. Morphometric analysis of collagen/reticular fibers, elastic fibers, and smooth muscle actin–positive cells*

 
Smooth muscle cells adjacent to blood vessels and airways stained strongly with anti-SMA monoclonal antibody in all subjects. Proliferated smooth muscle–like cells in LAM and interstitial smooth muscle–like cells in IPF stained weakly, but specifically, in a granular pattern, suggesting immature smooth muscle cells or myofibroblasts (Figure 1A [control], Figure 2A [IPF], and Figure 3A [LAM]; brown color, arrows). VV SMA+ cells in IPF and LAM was significantly higher than in control lungs (p < 0.05) (Table 2).



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  		Figure 1 (top). Control lung: (A) double immunohistochemistry of tryptase (red color) and SMA (brown color); magnification, x20. Arrows indicate small smooth muscle cell/myofibroblast–like cells. (B) Immunohistochemistry of bFGF (brown color); magnification, x20. Arrows indicate bFGF-positive histiocyte-like cells (mast cells). (C) Immunohistochemistry of Flg (brown color); magnification, x20. (D) Immunohistochemistry of Bek (brown color); magnification, x20. All specimens are counterstained with hematoxylin.

 


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  		Figure 2 (bottom). IPF lung: (A) double immunohistochemistry of tryptase (red color) and SMA (brown color); magnification, x20. Arrows indicate smooth muscle cell/myofibroblast–like cells. (B) Immunohistochemistry of bFGF (brown color); magnification, x20. Arrows indicate bFGF-positive histiocyte-like cells (mast cells). (C) Immunohistochemistry of Flg (brown color, arrows); magnification, x20. (D) Immunohistochemistry of Bek (brown color, arrows); magnification, x20. All specimens are counterstained with hematoxylin (inset window shows high magnification [x40]).

 


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  		Figure 3. LAM lung: (A) double immunohistochemistry of tryptase (red color) and SMA (brown color); magnification, x20. Arrows indicate smooth muscle cell/myofibroblast–like cells. (B) Immunohistochemistry of bFGF (brown color); magnification, x20. Arrows indicate bFGF-positive histiocyte-like cells and epithelial cells. (C) Immunohistochemistry of Flg (brown color, arrows); magnification, x20. (D) Immunohistochemistry of Bek (brown color, arrows); magnification, x20. All specimens are counterstained with hematoxylin (inset window shows high magnification [x40]).

 
We observed a significant correlation between VV SMA+ cells and VV collagen or reticular fibers in IPF ({rho} = 0.55, p = 0.04, n = 14) and in LAM ({rho} = 0.73, p = 0.02, n = 9). There was a significant correlation between VV SMA+ cells and VV elastic fibers in IPF ({rho} = 0.65, p = 0.01, n = 15).

Immunohistochemistry and Morphometric Analysis of Tryptase and bFGF
As shown in Figures 1A (control), 2A (IPF), and 3A (LAM), tryptase+ cells (mast cells, red color) were located preferentially in the interstitium, not in the alveoli. We found abundant tryptase+ mast cells in the interstitium of lungs of patients with IPF and LAM. In control lungs, mast cells were observed mainly around blood vessels or bronchi. In IPF, mast cells were found mainly in thickened interstitium among smooth muscle cell/myofibroblast–like cells and among collagen or reticular fibers. In LAM, mast cells were found mainly in the interstitium around and within areas of smooth muscle cell/myofibroblast–like cell hyperplasia, together with collagen/reticular fiber deposition.

As shown in Figures 1B (control), 2B (IPF), and 3B (LAM), bFGF was detected in interstitial histiocyte–like cells (arrows). bFGF was also detected on epithelial cells (especially adjacent to smooth muscle cell/myofibroblast–like cells in LAM), basement membrane (especially near linear deposition of bFGF in control tissue), endothelial cells, and weakly on smooth muscle cells. Consistent with our previous report (6), we confirmed that these histiocyte–like cells were mast cells by performing double immunohistochemistry using anti-bFGF antibody, anti-tryptase antibody, and anti-CD68 antibody (data not shown).

We observed significantly higher VV of interstitial tryptase+ mast cells in IPF (2.9 [1.3–5.0]) and in LAM (2.4 [1.4–4.3]) than in control tissue (0.4 [0.2–0.9]) (p < 0.05). The VV of alveolar tryptase+ cells in IPF, LAM, and control tissue showed no significant difference (0.2 [0–0.9], 0.3 [0–1.4], and 0.2 [0–0.4], respectively). The VV of total (interstitial + alveolar) tryptase+ mast cells in IPF (3.2 [1.4–5.1]) and LAM (2.9 [1.6–5.7]) were significantly higher than in control tissue (0.6 [0.3–0.9]) (p < 0.05).

The VV of interstitial bFGF+ cells in IPF (3.0 [1.8–5.4]) and LAM (2.7 [1.6–4.4]) was significantly higher than in control lung (0.7 [0.3–1.3]) (p < 0.05). The VV of alveolar bFGF+ cells in IPF (0.3 [0–0.9]) and LAM (0.4 [0.1–0.9]) were significantly higher than in control lung (0 [0–0.5]) (p < 0.05). The VV of total (interstitial + alveolar) bFGF+ cells in IPF and LAM was significantly higher than in the control lungs (p < 0.05) (see Figure 4) .



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  		Figure 4. ([VV total (alveolar + interstitial) bFGF+ cells]/VV lung tissue) x 100 is significantly increased in IPF (3.6 [2.2–5.9]) and in LAM (2.9 [1.9–5.2]) compared with Ctrl (0.8 [0.4–1.3]). Solid lines represent median values. Ctrl = control. (*p < 0.05).

 
We observed a strong correlation between VV total bFGF+ cells and VV total tryptase+ mast cells (n = 33, {rho} = 0.82, p < 0.001). We also found significant correlation between VV total bFGF+ cells and VV total tryptase+ mast cells in IPF (n = 14, {rho} = 0.82, p < 0.0005) and a trend of correlation between VV total bFGF+ cells and VV total tryptase+ mast cells in LAM (n = 9, {rho} = 0.60, p < 0.08). The ratios of VV total bFGF+ cells to VV total tryptase+ mast cells in IPF, LAM, and control tissue were 1.1 (0.8–1.6), 1.0 (0.8–1.6), and 1.2 (0.7–2.3), respectively (no significant difference), suggesting cell-specific colocalization of bFGF and tryptase (mast cells).

Immunohistochemistry and Morphometry of FGFR
As shown in Figures 1C (control), 2C (IPF), and 3C (LAM), Flg antibody bound to smooth muscle cell/myofibroblast–like cells, type-II epithelial cells, endothelial cells, and alveolar macrophages in all subject groups (arrows). Interestingly, we detected strong, significant staining for Flg in epithelial cells along the surface of smooth muscle cell/myofibroblast–like cell hyperplastic lesions in LAM (Figure 3C). As shown in Table 3 , in IPF, VV Flg+ epithelial cells, VV Flg+ endothelial cells, and VV Flg+ smooth muscle cell/myofibroblast–like cells and other interstitial cells were significantly higher than in control tissue (p < 0.05). In LAM, VV Flg+ epithelial cells and VV Flg+ smooth muscle cell/myofibroblast–like cells and other interstitial cells was significantly higher than in control tissue (p < 0.05). In summary, VV total Flg+ cells in IPF and LAM were significantly higher than in control tissue (p < 0.05, Figure 5) .


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  		TABLE 3. Morphometric analysis of flg expression

 


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  		Figure 5. (VV total Flg+ cells/VV lung tissue) x 100 in IPF (21.4 [17.1–31.2]) and in LAM (18.0 [9.8–42.8]) are significantly higher than in Ctrl (6.2 [3.5–22.4]). Solid lines represent median values. Ctrl = control. (*p < 0.05).

 
As shown in Figures 1D (control), 2D (IPF), and 3D (LAM), Bek was detected specifically and predominantly in the proliferative clusters of smooth muscle cell/myofibroblast–like cells of LAM. In IPF, it was detected on neutrophils (data not shown) and some smooth muscle cell/myofibroblast–like cells and weakly on epithelial cells, endothelial cells, and alveolar macrophages.

As shown in Table 4 , in LAM, the VV of Bek+ smooth muscle cell/myofibroblast–like cells and other interstitial cells were significantly higher than in either IPF or control tissue. As shown in Figure 6 , VV total Bek+ cells in LAM was significantly higher than in either IPF or control tissue (p < 0.05).


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  		TABLE 4. Morphometric analysis of bek expression

 


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  		Figure 6. (VV total Bek+ cells/VV lung tissue) x 100 in LAM (22.7 [3.8–33.5]) is significantly higher than in IPF (2.2 [0.9–9.8]) and in Ctrl (0.9 [0–3.2]). Solid lines represent median values. Ctrl = control. (* p < 0.05).

 
Relationship of Mast Cells, bFGF+ Cells, Flg+ Cells, and Bek+ Cells With Collagen/Reticular Fibers, Elastic Fibers, and SMA+ Cells
We found no significant correlation between VV total bFGF+ cells and VV total Flg+ cells in IPF ({rho} = -0.33) or in LAM ({rho} = -0.12). Notably, there was a strong correlation between VV total bFGF+ cells and VV total Bek+ cells in LAM ({rho} = 0.85, n = 9, p < 0.01) but not in IPF ({rho} = -0.54). We found no significant correlation between VV total Flg+ cells and VV total Bek+ cells in IPF ({rho} = 0.45) or in LAM ({rho} = -0.18).

As shown in Table 5 , in IPF, mast cells and bFGF+ cells correlated with the degree of fibrosis (collagen/reticular fibers, elastic fibers, smooth muscle cell/myofibroblast–like cells) (p = 0.05), although there was no significant correlation between VV total Flg+ cells or VV total Bek+ cells and VV collagen or reticular fibers, VV elastic fibers, or VV SMA+ cells. In LAM, we observed a significant negative correlation between VV total tryptase+ cells and VV elastic fibers ({rho} = -0.68, p = 0.05). Interestingly, there was a significant correlation between VV total Bek+ cells and VV SMA+ cells ({rho} = 0.77, p = 0.02), suggesting disease-specific immunostaining of Bek in the proliferative regions of smooth muscle cell/myofibroblast–like cells in LAM.


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  		TABLE 5. Relationship of tryptase-positive cells, bFGF-positive cells, flg-positive cells, and bek-positive cells with collagen or reticular fibers, elastic fibers, and smooth muscle actin–positive cells*

 
Specificity of Immunohistochemistry
We observed no specific immunostaining if we omitted primary antibody, secondary antibody, or avidin–biotin complex (data not shown). We saw no staining when normal mouse IgG was used instead of primary antibody (data not shown). Specific immunostaining of bFGF, Flg, and Bek disappeared or decreased after incubation of the antibodies with sufficient amount of recombinant human bFGF, synthesized peptide of Flg, and Bek, respectively (data not shown). There was no extinction or reduction of bFGF immunostaining on mast cells in the presence of buffer containing high salt (400 mM/L NaCl) or pH 6.0 MES buffer (data not shown) (6, 26).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we observed increased expression of mast cell–derived bFGF and its receptors in the lung tissue of patients with IPF and LAM. There were two different patterns of FGFR expression in these diseases. Specifically, in the patients with IPF, we found enhanced expression of Flg on epithelial cells, endothelial cells, and smooth muscle cell/myofibroblast–like cells, without an increase in Bek. In contrast, lung tissue from patients with LAM expressed Flg on the epithelium but expressed Bek on the smooth muscle cell/myofibroblast–like cells. These data support the hypothesis that mast cell–derived bFGF contributes to the pathogenesis of these disorders of smooth muscle cell/myofibroblast–like cell proliferation via FGFR.

More than 20 fibroblast growth factors have been identified. Fibroblast growth factors elicit their biologic activities by interacting with cell surface tyrosine kinase receptors. Four closely related receptors, designated FGFR1, FGFR2, FGFR3, and FGFR4, have been isolated. These multiple receptors are the product of alternative splicing. Each fibroblast growth factor can bind more than one type of FGFR. bFGF binds to FGFR1 and FGFR2 IIIc isoform with high affinity and to FGFR4 with low affinity (4, 31). FGFR2 IIIb isoform, also known as keratinocyte growth factor receptor, is a spliced variant of FGFR2. FGFR2 binds acidic fibroblast growth factor and bFGF with high affinity and does not interact with keratinocyte growth factor. FGFR2 IIIb isoform binds acidic fibroblast growth factor and keratinocyte growth factor with high affinity and bFGF with 20-fold lower affinity. The antibody directed against Bek that we used labels both FGFR2 IIIb and IIIc isoforms. Although the receptors' isoforms exhibit different affinities for the various fibroblast growth factors, most forms are functional in binding. In our data, there was no significant morphologic correlation between bFGF+ cells and Flg+ or Bek+ cells in IPF. However, bFGF+ cells were associated with Bek+ cells in LAM. We speculate that other fibroblast growth factors—such as acidic fibroblast growth factor or keratinocyte growth factor, which are also important for fibroproliferative response—may have caused the correlations between bFGF and FGFR to be low.

The developmental regulation of Flg and Bek has been well studied, although their expression in disease states has not. Flg and Bek are expressed diffusely throughout the primitive ectoderm before gastrulation, but with the onset of gastrulation, expression of the two receptors becomes distinct. Flg expression is most prominent in mesenchyme, whereas Bek expression is strongest in surface ectoderm and in the epithelium of several developing organs. Flg and Bek exhibit a complementary pattern of expression during organ formation (32). Our findings suggest the need for future research to clarify the regulatory mechanism of these receptors in disease.

bFGF is a heparin-binding growth factor and requires heparin-like molecules to bind its surface receptors. Released bFGF is bound to heparin-like molecules on basement membrane and extracellular matrix (33). Stored bFGF is released by enzymatic activities in inflammation (34). In disease states, we and others have observed the disproportionate induction of one or more bFGF isoforms. In fibrotic lung disorders, we reported an increase in the 17.8-kD isoform of bFGF (6). The mechanism of bFGF release is still unclear because bFGF lacks the signal sequence for secretion (35). One proposed manner of bFGF release from mast cells is through piecemeal degranulation (6, 36). In chronic inflammatory states, such as pulmonary fibrosis, sarcoidosis, collagen vascular diseases–related lung fibrosis, and the pneumoconioses, mast cells appear to release their contents slowly and partially.

bFGF and Its Receptors in IPF
Collagen/reticular fibers and elastic fibers, smooth muscle cell/myofibroblast–like cells, and epithelial cells accumulate in the lungs of patients with IPF (6, 13, 37). Above all, myofibroblasts and fibroblasts play important roles in the fibrotic process (38, 39). Gailit and coworkers (40) reported that mast cell mediators induce SMA expression and contraction of myofibroblasts. We have shown that bFGF+ mast cells accumulate in the areas of extracellular matrix deposition and of smooth muscle cell/myofibroblast–like cell proliferation.

As further support for the role of mast cell bFGF in fibrosis, Liebler and colleagues (12) reported that bFGF helps direct cell proliferation following bleomycin-induced lung injury in rats and that mast cells may represent a source of bFGF during the fibroproliferative late stage after lung injury. Studies using mast cell–deficient mice (WBB6F1-W/Wv) show less silica-induced, ozone-induced, and fungal-antigen–induced fibrosis and inflammation (41–43). However, bleomycin still can induce pulmonary injury in these WBB6F1-W/Wv mice (44). Mast cells are strongly associated with pulmonary fibrosis (45–48). In acute lung injury or in the acute phase of pulmonary fibrosis, bFGF may be produced by mast cells, macrophages, endothelial cells, and smooth muscle cells (7, 12) and thus may contribute to aberrant cell proliferation and extracellular matrix production.

The finding of enhanced expression of Flg on epithelial, endothelial, and smooth muscle cell/myofibroblast–like cells in IPF is consistent with the evidence that these cells contribute to the fibrogenic response in IPF (14, 49, 50). Interestingly, whereas Bek receptor was expressed on some interstitial cells in IPF lung, total Bek expression was not significantly increased. At this time, we have no explanation for this differential expression. Future in vitro studies of cells that differentially express these receptors may help clarify this divergence in receptor expression in IPF.

It is intriguing that neutrophils in IPF lung express Bek. Recent data suggest that addition of bFGF to long-term bone marrow cultures results in an increase in the number of neutrophils (51). Increased number of neutrophils in bronchoalveolar lavage fluid is associated with poor prognosis in IPF (52). The inhibition of neutrophil elastase inhibits pulmonary fibrosis (53). Proteolysis of bFGF by human neutrophil elastase has been reported (54) and leads us to speculate that bFGF may affect neutrophils via the Bek receptor in IPF.

bFGF and Its Receptors in LAM
Consistent with prior literature, in the lungs of patients with LAM, we found abundant SMA+ cells in regions of collagen deposition, although to a lesser extent than we saw in IPF (19, 20). A novel finding of our study is that bFGF was strongly expressed on mast cells in addition to epithelial cells at these sites of smooth muscle cell/myofibroblast–like cell proliferation. The expression of both Bek and Flg is increased in LAM but in different patterns. Bek+ cells significantly correlated with SMA+ cells, although there was no significant correlation between Flg+ cells and SMA+ cells. We found strongest staining of Flg on epithelial cells in the areas of smooth muscle cell/myofibroblast–like cell proliferation, suggesting autocrine expression of bFGF and specific Bek staining on the proliferated smooth muscle cell/myofibroblast–like cells. Our colocalization data in LAM suggest that the bFGF system may play an important role in LAM as in IPF. How bFGF influences mast cells and smooth muscle cell/myofibroblast–like cells via the different FGFR requires future study.

It is important to note that LAM is a disease of women. As such, we cannot fully exclude the possibility of a sex-specific difference in FGFR expression. We observed no significant differences in men and women in our control group. However, the small number of control subjects limits our conclusions on sex effects. We are aware of no published data on sex-related differences in FGFR expression.

Regarding the observed differences in FGFR expression in IPF and LAM, it is important to acknowledge that IPF occurs in a much older patient population. Our limited data cannot fully exclude the possibility of age/sex–related effect on FGFR expression in the lung because our control group was best matched for age with the LAM subjects rather than with the IPF subjects. We are not aware, however, of any published data suggesting an age-effect for FGFR expression.

In the present study, we analyzed combined data, using samples obtained by open lung biopsy, lung transplant, and autopsy together because of the limited number of samples. We compared results for each variable between the samples obtained by open lung biopsy, lung transplant, and autopsy in IPF and between the samples obtained by open lung biopsy and lung transplant in LAM. There was no significant difference between the groups in IPF. However, there was trend toward relatively higher VV of total tryptase-positive cells and total bFGF-positive cells in transplant specimens compared with open lung biopsy LAM specimens (data not shown). Further study is necessary to confirm the fibroproliferative difference between the disease stages in LAM.

In this study, we demonstrated spatial association between extracellular matrix, smooth muscle cell/myofibroblast–like cells, and bFGF/FGFR–expressing cells in IPF and LAM. Although our data are descriptive, they suggest one of the possible mechanisms by which mast cell–derived bFGF may elicit a fibroproliferative response in these idiopathic diseases. Future study is necessary to examine the functional significance of these findings.


   Acknowledgments
 
The authors thank Rubin Tuder, M.D., for technical discussion, Janet E. Henson, Martin Wallace, and Lynn Cunningham for preparing the samples, Dolly Kervitsky for assistance in data collection, Nina Rice for preparing the manuscript, Satoru Yamamoto, M.D., Masaji Okada, M.D., and Mitsunori Sakatani, M.D., for useful discussions.


   FOOTNOTES
 
Supported in part by U.S. Public Health Service grant R29ES-04834 (L.S.N.); Specialized Center of Research (SCOR) grant HL-27353 (T.E.K. and L.S.N.); and grants-in-aid for Cancer Research (11–13) and Diffuse Lung Diseases from the Ministry of Health, Labor, and Welfare, Japan (Y.I.).

Received in original form October 5, 2000; accepted in final form May 17, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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