INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The morphologic characteristics of idiopathic pulmonary fibrosis (IPF) comprise type I epithelial cell injury followed by alveolar inflammation, fibroblast proliferation, type II cell hyperplasia, alveolar collapse, and reepithelialization of the organized alveolar inflammation resulting in a thickened fibrotic interstitium. Alveolar and interstitial macrophages play an important role in the pathogenesis of lung fibrosis, in part by secreting fibrogenic cytokines and growth factors such as tumor necrosis factor- (TNF-), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor- (TGF-), and insulin-like growth factor I (IGF-I) (1). IGF-I, a progression-type growth factor for fibroblasts, has been widely implicated in disease pathogenesis in clinical and experimental models of pulmonary fibrosis. Previous work has shown that the levels of IGF-I in bronchoalveolar lavage fluid (BALF) are increased in patients with IPF, coal worker's pneumoconiosis (6), and systemic sclerosis (7) as compared with those in healthy control subjects. In addition, increased levels of IGF-I messenger RNA (mRNA) have been detected in lung tissues following the induction of bleomycin-induced pulmonary fibrosis in mice (8), suggesting a possible role for IGF-I in this animal model. Bitterman and colleagues (1) have shown that alveolar macrophages (AM) from patients with IPF secrete increased amounts of IGF-I as compared with macrophages from normal subjects. In addition, the level of IGF-binding protein 3, a major serum binding protein that enhances the action of IGF-I (9), is significantly increased in BALF from patients with IPF as compared with healthy controls (10). Thus, IGF-I is likely to play an important role in the development of pulmonary fibrosis by its ability to stimulate fibroblast proliferation (11) and augment collagen synthesis by these cells (12).

Although AM have been clearly shown to be an important source of IGF-I in the injured lung, considerably less is known about other sources of this growth factor, especially interstitial macrophages, fibroblasts, and alveolar epithelial cells. Interstitial macrophages are only occasionally present in the normal lung, although their numbers increase significantly during the fibrogenic response. Although interstitial macrophages probably play a role in scavenging matrix components and inhaled stimuli that become deposited within this tissue, these cells have also been shown to be an important source of bFGF following intratracheal instillation of asbestos fibers (13), and to play an important role in stimulating fibrogenesis in a silica-induced model of pulmonary fibrosis in the rat (14). Interstitial macrophages have also been proposed to be a source of macrophage inflammatory protein-1 (MIP-1), a neutrophil chemokine, in IPF (15). Thus, interstitial macrophages have the capacity to secrete several cytokines and growth factors that may regulate lung inflammation and fibrosis. Airway and alveolar epithelial cells have been shown to express a panoply of cytokines, chemokines, and growth factors during the fibrogenic response, including PDGF and TGF- (16, 17). However, it is not clear to what extent the various cell types that constitute the fibrotic lung, other than AM, express IGF-I. In addition, the relationship between the degree of IGF-I expression within the lung and the degree of clinical impairment in patients with IPF has not been previously defined.

The principal goal of the present study, therefore, was to determine the distribution of cell types expressing IGF-I in lung tissues obtained from patients with IPF, and to compare the findings with the distribution of IGF-I in normal lung tissue. An immunocytochemical approach, combined with morphometric analysis of IGF-I-expressing cells, was used to define the distribution of cell types expressing IGF-I, with a particular emphasis on alveolar and interstitial macrophages and alveolar epithelial cells. The data suggest that several cell types express IGF-I in IPF. However, only the level of expression of IGF-I by interstitial macrophages was related to the degree of disease severity as assessed by a clinical-radiologic-pathologic (CRP) score.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Population

Ten patients with a clinical diagnosis of IPF and a pathologic diagnosis of usual interstitial pneumonitis (UIP) were randomly selected to be enrolled in this study. Patients with a connective-tissue disease, left ventricular failure, an occupational and/or environmental exposure that may result in interstitial lung disease, or a history of ingestion of a drug or an agent known to cause pulmonary fibrosis were excluded from the study. All patients underwent open thoracotomy or thoracoscopic lung biopsy. In each subject, tissue was obtained from two different sites, the upper and lower lobes of the same lung (when technically feasible). None of the cases in this study demonstrated histologic features of eosinophilic granuloma, respiratory bronchiolitis, hypersensitivity pneumonitis, sarcoidosis, bronchiolitis obliterans and organizing pneumonia, diffuse alveolar damage, lymphocytic interstitial pneumonitis, or unclassifiable forms of chronic interstitial pneumonia. The study population consisted of seven males and three females with a median age of 64 yr. The subjects' lung biopsies showed varying degrees of interstitial fibrosis and honeycombing, the hallmarks of late-stage IPF (18). The clinical profiles of patients with IPF are listed in Table 1. Ten control subjects with no evidence of interstitial or other lung diseases were also enrolled. This group comprised six males and four females with a median age of 34 yr. Five of the control cases were donors for lung transplants who had died from traffic accidents, sudden cardiac arrest associated with Marfans' syndrome, suicide, and cerebrovascular accident (Table 2). The other five control cases had a history of beryllium exposure and were enrolled in a study in which transbronchial lung biopsies were obtained. All control lung specimens were normal by histology. Informed consent was obtained from each subject, and the study protocol was approved by the Institutional Human Subjects Review Board.

CRP Score

The CRP score was computed prior to open lung or thoracoscopic biopsy, and was determined for each IPF patient from the sum of points assigned to each of seven variables: dyspnea, chest radiography, spirometry, lung volumes, diffusing capacity for carbon monoxide (DL_CO), resting alveolar-arterial oxygen difference AaPO₂, and gas exchange during exercise, using the difference between AaPO₂ at the end of maximal exercise and that at rest. These values are corrected for maximum oxygen consumption achieved during exercise (expressed as the percent of predicted maximal oxygen consumption) (19). The highest (and thus worst) possible score is 100. In cases in which lung tissues were obtained at autopsy, the CRP scores were determined with the most current tests available, and the clinical score was assigned the worst possible score.

Immunohistochemical Staining

Monoclonal antihuman IGF-I antibody (IgG1) (Upstate Biotechnology, Lake Placid, NY), antihuman CD68 antibody (Dako, Carpinteria, CA), and antihuman cytokeratin antibody (Becton Dickinson, San Jose, CA) were used as primary antibodies. Mouse IgG1 (Sigma Chemical, St. Louis, MO) was used as a negative control. Biotinylated antimouse IgG (Vectastain kit; Vector Laboratories, Burlingame, CA) was used as the secondary antibody. Immunocytochemical assays for IGF-I, cytokeratin, and CD68 were performed as described previously (17, 20). Briefly, all lung tissues were fixed overnight in 10% vol/vol formalin and embedded in paraffin. Two-to-3-µm sectioned, deparaffinized lung tissues were then incubated with methanol containing 0.3% (vol/vol) hydrogen peroxide for 30 min to remove endogenous peroxidase activity. After incubation and washing with Tris-buffered saline (TBS), pH 7.4, lung tissues were digested with 0.1% (wt/vol) trypsin in 0.1% (wt/vol) CaCl₂ for 10 min at room temperature. The lung sections were then blocked with 1.5% (vol/vol) normal horse serum to reduce nonspecific binding, and were incubated with the primary antibodies or with IgG1 for 2 h at room temperature. After washing with TBS, the lung tissues were incubated with biotinylated antimouse IgG antibody for 30 min and then incubated to allow the avidin/biotinylated peroxidase complex to form for an additional 30 min. Subsequent color development was achieved by incubating the sections with 3,3'-diaminobenzidine (DAB) in the presence of hydrogen peroxide according to the manufacturer's instructions (DAB substrate kit for peroxidase; Vector Laboratories). The tissues were counterstained with hematoxylin and mounted with Permount. Hematoxylin and eosin (H&E) staining (21) was also done, for lung volume density, and Movat's pentachrome staining (22) was used for morphometric analysis of collagen/reticular fibers and elastin, and for confirmation of diseased or healthy lungs in all cases.

Morphometric Analysis

We performed quantitative morphometric analysis of lung biopsy specimens using computer-assisted video microscopy as described (23, 24). Briefly, to determine the volume density (Vv) of interest (e.g., IGF-I) a 125-point grid was superimposed on the captured ×400 magnification images. The points targeting the area of interest were counted, and the Vv of interest was then obtained. We counted 12 random consecutive images beginning at the center of each tissue section, and calculated a mean value of Vv for each section. The quantitation of collagen/reticular and elastin fibers was done with a 42-point grid on 12 randomly selected ×40-magnification images stained with pentachrome stain. To normalize for the effect of collapse or expansion of lung tissue during biopsy or tissue fixation, the Vv of interest was divided by lung volume density according to the following formula: Normalized Vv of interest = (Vv of interest/Vv of lung) × 100. The Vv of the lung was obtained as described earlier, except that a 42-point grid was overlaid on ×10-magnification images of H&E-stained lung sections. The data were thus expressed as normalized Vv of interest.

Statistical Analysis

The Mann-Whitney U test was used to detect any differences in normalized Vv of interest between IPF patients and control subjects. The correlation between the normalized Vv of interest and the composition of CRP score was assessed with Spearman's rank sum test. The morphometric data are expressed as the mean ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Immunocytochemical Localization of IGF-I

Tissue sections from lung biopsies and postmortem specimens obtained from 10 patients with IPF and 10 normal controls were stained with H&E or pentachrome stains to assess the overall histologic changes and degree of interstitial and alveolar fibrosis in the study population. As can be seen in Figure 1, H&E-stained sections of normal lung revealed well-preserved alveoli with no cellular infiltration or thickening of the interstitium (Figure 1A). In addition, as expected, there was little staining of collagen/reticular and elastin fibers in the interstitium of normal lung as revealed with the pentachrome stain (Figure 1B). In contrast, the histologic findings for lung specimens obtained from IPF patients were principally those of interstitial cellular infiltration with mononuclear cells (Figure 1C), extensive deposition of collagen/reticular fibers as revealed in the pentachrome-stained sections (Figure 1D), and significant type II cell hyperplasia and honeycombing (Figures 1C and 1D). These histologic findings in the IPF group are consistent with the pathologic diagnosis of UIP (24).

View larger version (104K): [in this window] [in a new window]   Figure 1.   Serial H&E and pentachrome stains of samples of normal (A and B) and IPF lung (C and D). The normal lung shows well-preserved alveoli with no cellular infiltration or thickening in interstitium in an H&E-stained section (A), and minimal staining of collagen/reticular and elastin fibers in a pentachrome-stained section (B). The histologic section of IPF lung shows interstitial cellular infiltration with mononuclear cells, interstitial fibrosis, and honeycombing with H&E stain (C ), and extensive deposition of collagen/reticular (black) and elastin (yellow) fiber with pentachrome stain (D). Original magnification: ×200.

To evaluate the distribution of cell types staining for IGF-I in normal lung tissue and in tissues obtained from IPF patients, four 3-µm serial sections were cut from each block and were subjected to immunocytochemical staining with antibodies directed against: (1) IGF-I; (2) CD68, an antigen corresponding to mouse macrosialin and expressed principally on macrophages (25); (3) cytokeratin, to define the distribution of epithelial cells (26); and (4) nonimmune mouse IgG1 as a negative isotype-matched control. As can be seen in the sections from two representative subjects (Patients 4 and 6) shown in Figures 2 and 3, IGF-I was widely expressed in specimens obtained from patients with IPF. In particular, heavy staining was detected in alveolar epithelial cells that showed colocalization of staining for cytokeratin. Abundant staining of IGF-I was also seen in AM that were CD68⁺. In addition, mononuclear cells within the thickened interstitium were found to costain for IGF-I and CD68, and could thus be defined as IGF-I⁺ interstitial macrophages. Endothelial cells and smooth-muscle cells were found to be only weakly stained for IGF-I in the tissue specimens from patients with IPF. In contrast to this extensive distribution of cells expressing IGF-I in IPF, only AM and a few columnar epithelial cells showed significant IGF-I staining in specimens from control subjects (Figure 4). As expected, the numbers of interstitial macrophages and type II alveolar epithelial cells were few in the tissue samples from normal lung, but when present were also stained for IGF-I.

View larger version (127K): [in this window] [in a new window]   Figure 2.   Immunocytochemical stains of IGF-I (A), cytokeratin (B), CD68 (C ), and of IgG (D) as a negative control in serial lung sections from an IPF patient (Patient 4 [see Table 1]). Abundant staining of IGF-I was seen in alveolar and interstitial macrophages and alveolar epithelial cells. Original magnification: ×400.

View larger version (115K): [in this window] [in a new window]   Figure 3.   Immunocytochemical stains of IGF-I (A), cytokeratin (B), CD68 (C ), and of IgG (D) as a negative control in serial lung sections from an IPF patient (Patient 6 [see Table 1]). Abundant staining of IGF-I was seen in alveolar and interstitial macrophages and alveolar epithelial cells. Original magnification: ×400.

View larger version (123K): [in this window] [in a new window]   Figure 4.   Immunohistochemical stains for IGF-I in normal lung. AM (A) and columnar epithelial cells (B) contain IGF-I. Original magnification: ×400.

Quantitative Analyses of IGF-I and CD68

Sections from each IPF patient and each normal control were subjected to morphometric analyses to quantify the proportion of cells staining for IGF-I. As can be seen in Figure 5, within the interstitium, the normalized Vv of IGF-I⁺ mononuclear cells having the morphologic appearance of interstitial macrophages was significantly greater in samples obtained from IPF patients than from normal control subjects (3.44 ± 0.75% versus 1.27 ± 0.42%, respectively; p < 0.05). Similarly, the normalized Vv of interstitial CD68⁺ cells, defined herein as interstitial macrophages, was also greater in patients with IPF (4.64 ± 0.84% versus 1.57 ± 0.37%; p < 0.0001) (Figure 5). As would be expected, the percentages of both types of cell are similar for IPF patients and for normal controls, since CD68 staining was used to confirm that the cells having the morphologic appearance of macrophages were indeed macrophages. These findings are thus consistent with the interpretation that the increased expression of IGF-I in the interstitium of patients with IPF is due to increased numbers of infiltrating macrophages, a conclusion that was supported by the finding that the ratio of IGF-I⁺ cells to CD68⁺ interstitial macrophages was not significantly different between IPF patients and control subjects (81.57 ± 12.19% versus 62.20 ± 14.01%; p = 0.227). Because the normal control subjects and IPF patients were not age-matched, we also evaluated possible relationships between IGF-I staining in interstitial macrophages and age, using Spearman's rank-sum analysis. When IGF-I localization was compared with subject age for all subjects, no significant correlation was observed (r² = 0.06, p = 0.28). In addition, we quantified IGF-I staining in interstitial macrophages in age-matched subgroups of normal control subjects (n = 4) and IPF patients (n = 4), selected to have ages between 40 and 60 yr, with mean ages of 49.5 and 48 yr, respectively. Within these groups, the means ± SEM for IPF-I⁺ cells for the control and IPF subjects were 1.00 ± 0.53% and 5.77 ± 0.76% (p = 0.03), respectively, thus reflecting the trend in the two groups as a whole.

View larger version (13K): [in this window] [in a new window]   Figure 5.   The normalized Vv values of IGF-I+ interstitial macrophages and interstitial CD68+ cells are increased in the lungs of patients with IPF as compared with control subjects. *p < 0.05 and #p < 0.001 versus control subjects. Open circles: tissues obtained from lung transplant donors; closed circles: tissues obtained by transbronchial biopsy. All tissues from the IPF patient group were obtained by autopsy or by open lung or thoracoscopic biopsy. Open squares: never-smokers; closed squares: former-smokers. All control subjects were never-smokers.

In contrast to the normalized Vv observed for CD68⁺ interstitial macrophages, the normalized Vv of IGF-I⁺ AM was not different in the IPF patients and normal control subjects (5.79 ± 1.00% versus 3.89 ± 0.93%; p > 0.05). However, these values are by definition normalized to lung volume. Thus, although the absolute numbers of AM were increased in IPF patients as compared with normal control subjects (3.86 ± 2.01% versus 1.92 ± 1.01%; p = 0.019), a large variation was also detected in lung volume of the normal subjects and IPF patients (68.40 ± 6.17 versus 58.88 ± 18.37; p = 0.448). We did not quantify IGF-I staining of endothelial cells or smooth-muscle cells, since these cells were detected with a low frequency, thereby preventing the collection of sufficient data to allow adequate statistical evaluation.

Quantitative Analysis of IGF-I and Cytokeratin

We next compared the expression of IGF-I by alveolar epithelial cells in tissue samples from IPF patients and normal control subjects. The normalized Vv of IGF-I⁺ alveolar epithelial cells was significantly greater in patients with IPF than in normal control subjects (5.99 ± 1.19% versus 4.05 ± 0.75%; p < 0.001). By contrast, the normalized Vv of IGF- I⁺ ciliated columnar epithelial cells was not different in the IPF patients and control subjects (0.63 ± 0.33% versus 1.73 ± 0.84%; p > 0.05).

Quantitative Analysis of IGF-I and Matrix

On the basis of previously reported studies discussed earlier, which have defined a role for IGF-I in fibroblast proliferation and collagen synthesis (7, 12), it seemed plausible that the increased expression of IGF-I, especially in the interstitium in IPF patients' lungs, would be related to the increased deposition of collagen and reticular fibers that is one of the hallmark features of IPF. As expected, the normalized Vv of collagen/ reticular and elastin fibers was significantly increased in patients with IPF as compared with normal control subjects (41.3 ± 7.3% versus 9.7 ± 5.2% [p < 0.001] and 17.3 ± 9.9% versus 8.9 ± 4.3% [p < 0.05], respectively). Next, we investigated the relationship between the extent of collagen/reticular and elastin fibers present in lung sections of IPF patients and normal control subjects to the staining pattern for IGF-I and CD68. As shown in Figure 6, the normalized Vv of IGF-I⁺ interstitial cells bearing the morphologic characteristics of interstitial macrophages and of CD68⁺ interstitial macrophages was correlated with the normalized Vv of collagen in both control subjects and IPF patients (r² = 0.3; p = 0.01 and r² = 0.25, p = 0.02, respectively), but was not correlated with the normalized Vv of elastin (Figure 6). The ratio of IGF-I⁺ interstitial cells exhibiting macrophage morphology to CD68⁺ interstitial macrophages did not correlate with either the normalized Vv of collagen or the normalized Vv of elastin.

View larger version (25K): [in this window] [in a new window]   Figure 6.   The correlation between the normalized Vv of IGF-I+ interstitial macrophages and CD68+ cells with both elastic and collagen fiber content. The amount of collagen fiber correlated well with both the normalized Vv of IGF-I+ interstitial macrophages and of the CD68+ cells. Open circles and closed circles denote idiopathic pulmonary fibrosis patients and control subjects, respectively. Open circles: tissues obtained from lung transplant donors; closed circles: tissues obtained by transbronchial biopsy. All tissues from the IPF patient group were obtained by autopsy or by open lung or thoracoscopic biopsy. Open squares: never-smokers; closed squares: former-smokers. All control subjects were never-smokers.

Relationship between the Level of Expression of IGF-I and Disease Severity

To determine whether a relationship existed between the degree of IGF-I staining and disease severity in IPF patients, we conducted analyses of the correlation between the degree of IGF-I staining in the different lung cells that were found to be positive for IGF-I and the CRP score, an objective cumulative evaluation of disease severity in these patients. The ratio of IGF-I⁺ interstitial cells exhibiting macrophage morphology to CD68⁺ interstitial macrophages was found to correlate with the CRP and with the less objective clinical evaluation of dyspnea (Figure 7). However, there was no correlation with any of the physiologic or radiologic parameters used in computing the CRP score, including perfusion or the presence of honeycomb lesions on chest radiographs (data not shown). The normalized Vv of IGF-I⁺ interstitial macrophages and CD68⁺ interstitial macrophages alone did not correlate with the degree of dyspnea. In addition, neither abnormalities in pulmonary function test results nor in arterial blood gas values correlated with the normalized Vv of any IGF-I⁺ cells (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 7.   Ratio of Vv of IGF-I+ interstitial macrophages to Vv of interstitial CD68+ cells was correlated with both clinical and CRP scores in patients with IPF. Open squares: never-smokers; closed squares: former-smokers.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The role of several mesenchymal cell growth factors, especially TGF- and PDGF, in the development of pulmonary fibrosis in humans and in experimental animal models is now well established (as reviewed by Chan and associates [27]). Although having received less experimental attention until quite recently, IGF-I is now also believed to play an important role in the development of pulmonary fibrosis. In particular, IGF-I has been shown to: (1) act as a progression-type growth factor for mesenchymal cells that in conjunction with PDGF can direct fibroblast proliferation (7); (2) stimulate collagen matrix synthesis by fibroblasts (12); (3) be present in cell-free BALF in patients with diagnosed pulmonary fibrosis of a variety of causes (6); and (4) is synthesized in increased amounts by AM in IPF. In the present study we applied quantitative morphometric analyses to investigate the localization of IGF-I in lung biopsy and postmortem specimens from patients with IPF and normal control subjects, and to determine possible linkages in IGF-I expression to objective measures of disease severity. The morphometric analyses used the Vv of the lung parenchyma for standardization, since Hyde and collaborators (24) have previously shown that this provides an accurate assessment of the pathologic features in IPF. Our major findings were that: (1) IGF-I is expressed principally by AM, interstitial macrophages, and alveolar type II epithelial cells in patients with IPF, whereas in normal controls staining for IGF-I was localized mainly to AM; and (2) the expression of IGF-I by interstitial macrophages was related to both the extent of collagen deposition and disease severity.

In our study, the numbers of interstitial macrophages, as defined morphologically and by their staining pattern with antibodies directed against CD68, were significantly increased in IPF patients as compared with normal control subjects, and these cells were found to be positively stained for IGF-I protein. Furthermore, the increased number of IGF-I⁺ cells in the interstitium was completely accounted for by increased macrophage numbers. Despite their increased numbers, the relative importance of interstitial macrophages in the pathogenesis of pulmonary fibrotic responses has received only modest consideration. Adamson and Bowden (13, 28) provided the first suggestion that interstitial macrophages may play a more significant role in the fibrotic response than do AM. This conclusion was based on observations in two experimental systems that the accumulation of silica or asbestos fibers by interstitial macrophages was associated with the development of fibrosis, whereas the accumulation of these particulates by AM and neutrophils resulted in little or no fibrosis even in focal regions where the particles were lodged. More recently, interstitial macrophages have also been shown to express increased amounts of PDGF-B and TGF- mRNA and protein in both lung biopsy specimens obtained from IPF patients (16, 17) and in animal models of pulmonary fibrosis (29. 30). These findings thus support the importance of interstitial macrophages as a source of growth factors, including IGF-I, within this tissue. Since interstitial macrophages are in relatively close proximity to fibroblasts, the release of fibrogenic cytokines from macrophages may have a more profound effect on fibroblast proliferation and collagen synthesis in this tissue than do cytokines released from alveolar macrophages. This issue may be of particular significance to areas of the injured lung in which type II epithelial cell hypertrophy and hyperplasia have led to the reepithelialization of denuded basement membranes. An intact alveolar monolayer has been shown to prevent the translocation of molecules such as trypan blue, albumin, PDGF, and ₂-macroglobulin from alveolar surfaces to the interstitium (31). Thus, it is plausible that the hypertrophic alveolar epithelium may function as a barrier to growth factors produced by cells present in the air spaces.

Although the normal control subjects and IGF-I patients in our study were not age-matched, several lines of evidence suggest that the higher level of expression of IGF-I in the IPF patients was unrelated to the increased mean age of this group. First, we observed no relationship between the degree of IGF-I expression by interstitial macrophages and subject age. Second, in a comparison involving age-matched subgroups of a small number of controls and IPF patients, the increased numbers of IGF-I⁺ interstitial macrophages in the IPF group remained representative of the whole group. Third, previously reported studies of the levels of circulating IGF-I have documented an age-related linear decline in serum IGF-I levels in both males and females (32, 33). We also investigated the potential role of cigarette-smoking background within the IPF group, which comprised never-smokers and ex-smokers. There was no significant difference in IGF-I⁺ interstitial macrophages in the two subgroups according to smoking history, suggesting that ex-smoking status did not influence the results. In addition, although all of the IPF patients underwent open lung or thoracoscopic lung biopsies (or yielded autopsy specimens), some of the control subjects received transbronchial lung biopsies. However, no significant differences were noted in the level of IGF-I⁺ interstitial macrophages with respect to the type of lung biopsy.

In addition to heavy IGF-I-staining of interstitial macrophages, the results of the present study indicated heavy IGF-I staining of alveolar epithelial cells. A previously reported study also detected increased IGF-I staining of alveolar type II epithelial cells in biopsy samples obtained from patients with early-stage IPF (as characterized by diffuse alveolar damage), whereas biopsy specimens from patients with late-stage disease (defined by the presence of interstitial fibrosis and honeycombing) failed to show any IGF-I staining of the alveolar epithelium (34). The basis of the discrepancy between this and the finding in the present study, however, is unclear, since the patient population evaluated in our study uniformly had the diagnosis of UIP, associated with extensive collagen deposition and honeycombing.

IGF-I has also been reported to be expressed by alveolar epithelial cells in other situations. For example, in response to hyperoxic lung injury in the rat, total lung IGF-I mRNA levels increased significantly over those of controls. Moreover, the increased expression of IGF-I was localized by in situ hybridization and immunocytochemistry to areas of the alveolar and airway epithelium and peribronchial cells (35, 36). The potential role of epithelial cell-derived IGF-I in the pathogenesis or progression of IPF remains unknown. Previous work with neonatal mice and rats has shown that IGF-I is produced in the lung around the time of birth, and stimulates the proliferation and growth of the alveolar epithelium (37). The importance of IGF-I to lung development and function has been elegantly demonstrated by targeted deletion of the IGF-I receptor gene in mice, which die from respiratory failure associated with atelectasis shortly after birth. Given the suggestion that reexpression of IGF-I by resident lung cells during lung injury may recapitulate patterns of fetal lung development (35), it is possible that epithelial-derived IGF-I acts in an autocrine or paracrine fashion to stimulate (probably in conjunction with other growth factors) the proliferation of alveolar type II epithelial cells, and may contribute to the type II cell hypertrophy and hyperplasia seen in IPF. Alternately, transgene- and adenovirus-mediated overexpression of cytokines and growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF- and TGF- has been shown to result in the development of focal and/or more generalized fibrotic responses in mice (38). Thus, it is possible that the increased expression of IGF-I by alveolar epithelial cells may also enhance the alveolar and/or interstitial fibrotic responses seen in IPF.

Although the ability of IGF-I to stimulate the proliferation of and collagen synthesis by fibroblasts suggests that this growth factor may contribute to disease pathogenesis or progression, we felt it important to determine any relationships between the types and numbers of cells that stained positively for IGF-I and the degree of disease severity as assessed by: (1) an objective CRP score; (2) a less objective assessment of dyspnea; and (3) the degree of collagen fiber deposition. A significant positive correlation was observed between the number of interstitial cells positive for both IGF-I⁺ and CD68⁺ and disease severity as determined by both the CRP score and the assessment of dyspnea. These findings are consistent with the previously reported findings of Watters and colleagues (41), who detected a significant correlation between histologic findings and CRP score in patients with IPF. However, we found no correlation between the expression of IGF-I by any cell type and the individual components of the CRP score, including radiologic parameters as assessed by chest radiography. This finding is also consistent with earlier observations by Watters and colleagues (41), who found no correlation between the pathologic findings in patients with IPF and radiologic findings on chest radiography. Furthermore, high-resolution computed tomograms of the chest, although having greatly improved resolution over chest radiographs for the interpretation of parenchymal lung lesions, have been studied by Hirose and colleagues (23), who found that the central and peripheral lines on the tomographic scan could not distinguish thickening caused by increased cell content from that caused by other phenomena. The results of the present study did, however, reveal a significant correlation between the expression of IGF-I by interstitial macrophages and the degree of collagen deposition, in accord with earlier reports by Goldstein and colleagues that IGF-I stimulates collagen synthesis by embyonic lung fibroblasts (12).

In summary, the findings in the present study indicate that IGF-I is present principally in AM, interstitial macrophages, and alveolar epithelial cells in biopsy specimens from patients with IPF. However, the presence of IGF-I in interstitial macrophages, where this growth factor may contribute to fibroblast proliferation and collagen synthesis within the interstitium, appears to be of greatest significance to disease severity.