Localization of Insulin-like Growth Factor-I in Lung Tissues of Patients with Fibroproliferative Acute Respiratory Distress Syndrome

Department of Medicine, Immunology Research Group, and Departments of Surgery, Pathology, Biochemistry and Molecular Biology, and Critical Care Medicine, University of Calgary, Health Sciences Center, Calgary, Alberta, Canada

	ABSTRACT

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Insulin-like growth factor-I (IGF-I) is elevated in human fibrotic lung diseases and in animal models of pulmonary fibrosis, implicating IGF-I in the pathogenesis of fibrotic lung disease. We questioned whether IGF-I protein levels were enhanced in fibroproliferative acute respiratory distress syndrome (FP-ARDS). Serial lung tissue sections from a biopsy database were immunohistochemically stained for IGF-I, IGF-I receptor, CD68, -smooth muscle actin, collagens I and III, and proliferating cell nuclear antigen. Our results show enhanced staining of IGF-I and IGF-I receptor, collagens I and III, smooth muscle actin, CD68, and proliferating cell nuclear antigen in FP-ARDS compared with control lung sections. In FP-ARDS specimens, prominent staining of IGF-I and IGF-I receptor was seen in alveolar and interstitial macrophages as well as in a variety of mesenchymal cells. There was a correlation between IGF-I staining and CD68-positive cells, suggesting macrophages as a potential source of the IGF-I protein present in lungs. IGF-I also correlated with enhanced collagen I, collagen III, and proliferating cell nuclear antigen immunoreactivity, suggesting that IGF-I may play a role in the extracellular matrix protein deposition and cellular proliferation seen in the lungs of individuals with FP-ARDS. Our results indicate that IGF-I is increased in FP-ARDS and may be an important mediator in the progression of acute lung injury to FP-ARDS.

　

Key Words: acute respiratory distress syndrome • insulin-like growth factor-I • macrophage • pulmonary fibrosis

	INTRODUCTION

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Ashbaugh and coworkers first described acute respiratory distresssyndrome (ARDS) in 1967 in ventilated patients with bilateral chest infiltrates and refractory hypoxemia (1). In the United States, ARDS has an incidence, at present, of 1.5 to 5.3 per 100,000 and a mortality rate from 30 to 50% (2). ARDS is characterized by epithelial and endothelial damage leading to pulmonary edema, hypoxemia, neutrophilic infiltration, denudation of type I epithelial cells, and formation of fibrinous exudates (hyaline membranes) (3). The disease often progresses to a fibroproliferative phase characterized by elevated mesenchymal cell numbers and profound extracellular matrix deposition in the interstitial and alveolar spaces. Persistent or fibroproliferative ARDS (FP-ARDS), also identified pathologically as organizing diffuse alveolar damage, is associated with increased cytokine, growth factor, and collagen protein levels, as well as reduced survival (4).

Macrophages have been thought to be important in the progression of acute lung injury to FP-ARDS as well as in other fibrotic lung diseases, as they are present in high numbers and secrete numerous proinflammatory mediators and growth factors. Several growth factors are secreted by macrophages, including transforming growth factor-ß, platelet-derived growth factor, fibroblast growth factor-2 (basic fibroblast growth factor), transforming growth factor-, and insulin-like growth factor-I (IGF-I) (5–10). These peptide growth factors influence mesenchymal cell migration, proliferation, and extracellular matrix deposition, thus implicating them in the progression of fibroproliferative lung disorders. In both clinical and experimental models of fibrotic lung disease, such mesenchymal growth factors have been shown to be elevated and are thought to potentiate disease (11). IGF-I is thought to be a progression-type growth factor stimulating cells to enter the G₁ phase, contributing to cell proliferation. Importantly, IGF-I is also a potent survival factor, acting by inhibiting the induction of apoptosis in a variety of cells (12). Studies have shown that IGF-I directly stimulates fibroblast proliferation and perhaps collagen synthesis (13–16). Increased cell proliferation, decreased apoptosis, and enhanced extracellular matrix deposition are important characteristics of fibrotic lung diseases, including FP-ARDS.

IGF-I, originally identified in the lung as alveolar macrophage-derived growth factor, is elevated in the bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis (IPF) and coal miners' pneumoconiosis (9). Studies have shown enhanced immunohistochemical staining of IGF-I in macrophages and epithelial cells of individuals with IPF (17). IGF-I mRNA levels are also enhanced in bleomycin-induced pulmonary fibrosis in mice as well as human IPF, implicating a role for the local production of IGF-I in the progression of fibrotic lung disease (18, 19).

We questioned whether IGF-I is present in the lungs of individuals with FP-ARDS for the following reasons: first, there is an active fibroproliferative process occurring in the lungs of individuals with FP-ARDS; second, the biologic roles of IGF-I include cell proliferation and collagen synthesis, which are both important in the fibroproliferative process seen in FP-ARDS; and third, IGF-I has been shown to be present in lungs in other fibroproliferative conditions such as IPF. We show, for the first time, enhanced IGF-I immunoreactivity in lung biopsy specimens of individuals with FP-ARDS. IGF-I immunoreactivity localized primarily to alveolar and interstitial macrophages, myofibroblasts, and epithelial cells. The elevated levels of IGF-I correlated positively with CD68, a macrophage marker, and with collagens I and III in the lung specimens. Results of the current study suggest that enhanced production of IGF-I in FP-ARDS, perhaps by macrophages and other cells in the lung, may play a role in the abnormal fibroproliferative process and pathogenesis of ARDS.

	METHODS

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Study Population
Eight patient biopsy specimens showing organizing diffuse alveolar damage were chosen by retrospective analysis of a large pathologic database (more than 125,000 specimens) and medical record screen in a tertiary care teaching hospital. Hamartoma biopsy specimens were similarly chosen as control specimens because adjacent lung tissue exhibited normal histology. Details of the study population can be found in RESULTS. The local institutional review board approved the use of all patient information and lung biopsy tissue for this study.

Immunohistochemical Staining
Serial sections, prepared from blocks of FP-ARDS and control lung tissue, were fixed, deparaffinized, and then stained for IGF-I and IGF-I receptor  subunit (IGF-IR) as described (17), with the exception that for IGF-I staining a 1:200 dilution of citric acid was used for antigen retrieval before the addition of mouse anti-human IGF-I monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Slides stained for IGF-IR were pretreated with a 1:1,000 diluted EDTA solution before the addition of rabbit anti–IGF-IR antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Serial sections were also stained with antibodies reactive to CD68 (Dako, Glostrup, Denmark), -smooth muscle actin (Biogenex, San Ramon, CA), proliferating cell nuclear antigen (PCNA; Novacastra, Newcastle on Tyne, UK), collagen I (Fuji Chemicals, Takaoka, Japan), and collagen III (Fuji Chemicals, Toyama, Japan), utilizing the automated staining services of Calgary Laboratory Services (Calgary, AB, Canada).

Morphometry and Quantification of Immunohistochemistry
The area fractions of selected features were determined on immunostained sections by a point-counting technique (20) using a Carl Zeiss (Thornwood, NY) Axioplan light microscope, drawing tube, and square lattice grid containing 240 intersects. Each intersection on the grid was scored for positive/negative intracellular staining and whether the feature was located in the interstitial or alveolar space. Four random fields were counted per slide (21). The area fraction of the feature of interest was calculated according to Equation 1:

　

where Z is the distance between two points on the grid and n is the number of points landing on the feature of interest.

The luminal surface length (LSL) of the alveolar wall basement membrane was determined by the mean linear intercept method (20), by counting intersections of the horizontal and vertical lines on the grid with the airspace basement membrane, according to Equation 2:

　

where BMI is the number of times the grid intersects with the basement membrane and X is the magnification factor.

To determine average thickness of the alveolar interstitium, Equation 3 was used:

　

The data for each feature were calculated per field view and were averaged for each patient. Sections stained for IGF-I, IGF-I receptor, -smooth muscle actin, CD68, collagen I, and collagen III were quantified by point count morphometry. The data were normalized for the variable interstitial thickness resulting from disease by expressing the positive staining as a percentage of total alveolar or interstitial area (21). PCNA immunoreactivity was subjectively graded by two independent investigators on a scale of 0 to 3, where 0 = no, 1 = weak, 2 = moderate, and 3 = strong reactivity, because intense staining of FP-ARDS sections made accurate point counting difficult.

Statistical Alanlysis
StatView statistical software (SAS Institute, Cary, NC) was used to determine statistical significance and to perform regression analysis. Morphometry and point count data were analyzed for significance by t test, and regression analysis of variance was used to determine relationships between the different immunohistochemically stained populations. Data were considered statistically significant when p  0.05.

	RESULTS

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Patient Population
The study population consisted of eight FP-ARDS and seven control lung biopsy specimens. Cases were selected by a text-based screening approach of a large pathologic database, collected between 1993 and 1998, of specimens identified as having organizing diffuse alveolar damage or ARDS. Specimens showing characteristic features of FP-ARDS (39 cases) were then screened for adequate sampling of lung tissue and sufficient material in the block for immunostaining of serial sections. Samples were discarded if confounding pulmonary pathology (e.g., overt infection, infiltration by tumor) existed. Those cases selected were further screened on the basis of medical records. Only those clinically identified as having ARDS (Pa_O2/FI_O2  200, diffuse bilateral infiltrates on chest X-ray, and no evidence of left ventricular failure clinically or a pulmonary arterial occlusion pressure  18 mm Hg) were further selected from the samples identified on the pathologic screen. Finally, samples were used for the current study only if there was no resolution of ARDS for 7 days after a clinical diagnosis. The resulting eight patient biopsy specimens were therefore identified as a persistent or FP-ARDS population. The clinical characteristics of the FP-ARDS population are included in  . Note that the FP-ARDS patient population had an average Pa_O2/FI_O2 value of 120.37 ± 11.53 and an average compliance of 26 ± 7.38 ml/cm H₂O. Three of the eight patients survived to discharge from the hospital. The control population was similarly obtained by searching the pathologic database for hamartoma lung resection specimens. Similar selection criteria were used for the control specimens. All patients selected in the control group were immediately extubated and did not require mechanical ventilation after surgery. All were discharged from hospital stay within 7 days of surgery. All but one control specimen had changes consistent with chronic obstructive pulmonary disease, whereas none displayed evidence of other lung conditions apart from the hamartoma. Where possible, we show intraoperative gas exchange data (blood gases were obtained from at least one patient during single-lung ventilation) as an indicator of lung function in this group. These outpatient elective individuals did not require ventilation or intensive care unit admission postoperatively. Serial sections of normal lung tissue adjacent to the hamartoma were used for immunohistochemical staining. The clinical characteristics of the control population are included in  . Note that the control patients had an average Pa_O2/FI_O2 value of 338.86 ± 49.02 (with a single lung-ventilated control patient-removed average Pa_O2/FI_O2 of 379.0 ± 36.32). Compliance measurements were not performed on these patients intraoperatively.

fig.ommitted TABLE 1. Clinical characteristics of fibroproliferative acute respiratory distress group

 

fig.ommitted TABLE 2. Clinical characteristics of control group

 
Lung Morphometry
Lung sections stained with hematoxylin and eosin were used to identify morphologic differences between patient groups. Each of the seven control lung specimens was moderately to well inflated and maintained normal lung architecture including normal alveolar structures with little cellular infiltration or thickening of the interstitium. Variable numbers of alveolar macrophages were seen in the alveolar spaces. Areas consistent with mild emphysema were seen in the control lung specimens, as all but one of the control patient population were cigarette smokers. Representative images of FP-ARDS lung morphometry are included in  and . Unlike the control population, the FP-ARDS lung biopsy specimens showed enhanced mononuclear interstitial infiltrates, interstitial edema, interstitial and intraalveolar fibrosis, and hyperplasia of type II cells. In addition, some cases showed focal neutrophilic infiltrates and hyaline membranes. Representative images of normal lungs are included in  and . Lung morphometry revealed that total interstitial thickness was increased in the FP-ARDS population (32.11 ± 5.69 µm) compared with control subjects (7.18 ± 0.88 µm, p = 0.018; ) with an increase in total interstitial area for FP-ARDS specimens (110 ± 6.8 mm²) versus control lungs (49.6 ± 5.9 mm², p = 0.001; ). At the same time, there was an overall decrease in lumenal surface length (4,139 ± 656 µm in FP-ARDS lung specimens versus 7,261 ± 876 µm in control subjects, p = 0.04; ) and alveolar area (71.77 ± 6.8 mm² in FP-ARDS lungs versus 132.05 ± 5.8 mm² in control lungs, p = 0.001; ) in FP-ARDS versus control populations.

fig.ommitted Figure 2. IGF-I immunohistochemistry. (A) Lung section (original magnification, x10) from control subject 6 shows normal alveolar architecture. Staining for IGF-I shows markedly positive immunostaining of a cluster of alveolar macrophages. Inset: Higher power (x40) shows positive immunoreactivity of the alveolar macrophages and also linear staining of the alveolar type I cells (arrow). (B) Section of open lung biopsy from FP-ARDS patient 4, immunostained for IGF-I. Low magnification (x10) shows thickening and fibrosis of the interstitium with chronic inflammatory cell infiltrates. Numerous strongly staining macrophages are seen in the alveolar lumen. Interstitial staining is also present but less strong. Inset: Higher magnification (x40) shows strong immunoreactivity of macrophage cell populations. (C) Lung section from FP-ARDS patient 8 shows IGF-I immunoreactivity of interstitial foamy macrophages (arrows) and interstitial myofibroblast-like cells (arrowhead). Inset: Higher magnification (x40) shows interstitial immunoreactive cells consistent with myofibroblasts and interstitial macrophages. (D) Lung section from FP-ARDS patient 4 shows type II cell hyperplasia (arrows). Inset: Higher magnification (x40) shows that the hyperplastic type II cells are positive for IGF-I. (E) Quantified IGF-I immunoreactivity as described in METHODS shows significantly elevated IGF-I immunoreactivity in FP-ARDS lung sections when compared with control lung sections in total field view (p = 0.003) and when alveolar (p = 0.009) and interstitial area (p = 0.007) differences are accounted for.

 

fig.ommitted Figure 3. IGF-I receptor immunohistochemistry. (A) Lung section (original magnification, x10) from control subject 6 shows normal alveolar architecture. Positive immunoreactivity for IGF-I receptor was seen in alveolar macrophages and alveolar type I cells (inset, x40). (B) Lung section from FP-ARDS patient 4 shows severe remodeling of the parenchyma. The lower magnification (x10) view shows IGF-I receptor immunostaining of type II cells and marginal interstitial cell staining. The higher magnification view (inset, x40) shows strong type II cell staining for IGF-I receptor. (C) Quantified IGF-I receptor immunoreactivity, as described in METHODS, shows significantly elevated IGF-I receptor immunoreactivity in FP-ARDS lung sections when compared with control lung sections, both in terms of the percentage positive per total field view (p = 0.02) and when alveolar (p = 0.009) and interstitial area (p = 0.02) differences are accounted for.

 

fig.ommitted Figure 4. CD68 immunohistochemistry. (A) Control lung section from patient 6 shows positive CD68 immunoreactivity of alveolar macrophages. Higher magnification (inset, x40) shows macrophage immunoreactivity. (B) FP-ARDS lung section from patient 1 shows that the majority of the immunoreactive cells within the alveolar lumen are macrophages. Higher magnification (inset, x40) shows that interstitial macrophages are also immunoreactive for CD68. (C) Quantified CD68 immunoreactivity as described in METHODS shows significantly elevated percent CD68 immunoreactivity in FP-ARDS lung sections when compared with control lung sections in both alveolar (p = 0.0004) and interstitial (p = 0.0007) areas.

 

fig.ommitted Figure 5. -Smooth muscle actin immunohistochemistry. (A) Lung section from control patient 6 shows staining of smooth muscle within a small pulmonary vessel (also in higher magnification inset, x40). Occasional myofibroblasts were seen within the pulmonary interstitium (not illustrated). (B) Lung section from FP-ARDS patient 6 stained for -smooth muscle actin. The thickened interstitium shows marked smooth muscle actin positivity that, on higher magnification (inset, x40), shows intracytoplasmic staining of spindle-shaped cells consistent with myofibroblasts and activated fibroblasts. Extracellular smooth muscle actin is not observed. (C) Quantified -smooth muscle actin immunoreactivity as described in METHODS shows significantly elevated immunoreactivity in the interstitial space of FP-ARDS lung sections when compared with control lung sections (p = 0.0004).

 

fig.ommitted   Figure 6. Collagen I and III immunohistochemistry. Collagen I immunostaining of a lung section from control patient 5 (A) shows staining in the adventitial tissues of a small blood vessel and patchy immunoreactivity within the alveolar interstitium. (B) Collagen I immunoreactivity in a lung section from FP-ARDS patient 5 shows diffuse staining with collagen fibrils throughout the interstitium. Higher magnification (inset, x40) shows that collagen I is located extracellularly. Collagen I staining was also seen within some alveolar lumena (not illustrated). A similar pattern of collagen III immunoreactivity was observed in lung sections from control patients, for example, control patient 5 (C), and from FP-ARDS patients, for example, FP-ARDS patient 5 (D). (E) Virtually no PCNA immunoreactivity was seen in the lungs of control patients, as shown in control subject 5. (F) Lung section from FP-ARDS patient 8 stained for PCNA shows the majority of nuclei are stained positively within the interstitium. Higher magnification (inset, x40) shows that cells with morphologies consistent with macrophages, fibroblasts, and myofibroblasts, as well as type II cells, are all reactive for PCNA staining.

 

fig.ommitted Figure 1. Mathematical description of lung morphometry. Displayed are averages of control (open bars) and FP-ARDS (solid bars) groups + SEM. Overall, there was decreased lumenal surface length (p = 0.04) (A), increased interstitial thickness (p = 0.02) (B), decreased alveolar area (p = 0.001) (C), and increased interstitial area (p = 0.001) (D) in the FP-ARDS lung specimens compared with control lung specimens.

 
IGF-I and IGF-IR
To evaluate the presence of IGF-I protein in normal lungs and in FP-ARDS lungs, serial lung biopsy sections were immunohistochemically stained with anti–IGF-I and anti–IGF-I receptor  subunit (IGF-IR) antibodies. IGF-IR is the integral receptor for the biologic effects of IGF-I. As can be seen in the representative images, IGF-I is widely expressed in alveolar macrophages as well as in epithelial cells (inset arrow) in control lungs  . IGF-I is represented in a variety of cell types in FP-ARDS lungs , including alveolar and interstitial macrophages, epithelial cells, myofibroblast-like cells, and endothelial cells. A lung biopsy from a patient with IPF was used for IGF staining control, as previous reports have indicated enhanced IGF-I in lung biopsy specimens from patients with IPF (results not shown) (17). The total IGF-I staining per field of view was significantly increased in FP-ARDS lungs versus control lungs (4.01 ± 0.33% versus 28.92 ± 2.09% [FP-ARDS], p = 0.003) . When positive staining was normalized to alveolar area and interstitial area to account for morphometric differences between patient populations, significant increases in IGF-I staining in both alveolar (1.66 ± 0.20% versus 24.39 ± 3.99% [FP-ARDS], p = 0.009) and interstitial compartments (0.52 ± 0.25% versus 23.15 ± 3.87% [FP-ARDS], p = 0.007) were observed .

Similarly, IGF-IR staining was enhanced in FP-ARDS lung sections versus control lung sections as shown in representative images  . Cells consistent with alveolar and interstitial macrophages, epithelial cells, and interstitial cells stained positive for IGF-IR. Total IGF-IR staining was significantly enhanced in FP-ARDS lung sections when compared with control lung sections (2.37 ± 0.54% versus 8.63 ± 1.00% [FP-ARDS], p = 0.02;. Again, to compensate for tissue volume differences, the data was normalized to the alveolar and interstitial areas. The data show significantly elevated IGF-IR staining in alveolar (1.01 ± 0.20% versus 6.09 ± 1.03% [FP-ARDS], p = 0.009) and interstitial areas (0.34 ± 0.24% versus 5.90 ± 2.22% [FP-ARDS], p = 0.02 .

Actin and CD68
CD68 is a member of a family of lysosomal membrane proteins whose expression is limited nearly completely to monocyte/macrophage cell types (22, 23), and is useful in identifying macrophages in tissue specimens. -Smooth muscle actin is expressed in fibrotic tissue myofibroblasts and activated fibroblasts, both of which are known to be major producers of extracellular matrix proteins (24, 25). We used the point-counting method described above to quantify the degree of immunostaining for CD68 and -smooth muscle actin in an effort to examine the numbers of macrophages and myofibroblasts/fibroblasts, respectively, in the lung sections. Representative images of CD68 immunostaining in control lungs  and FP-ARDS lungs  are included. As shown in , there was a significant increase in CD68 immunoreactivity in both alveolar (1.84 ± 0.66% versus 35.19 ± 4.14% [FP-ARDS], p = 0.0004) and interstitial areas (1.53 ± 0.43% versus 12.815 ± 1.35% [FP-ARDS], p = 0.0007), indicating macrophage infiltration in FP-ARDS lung biopsy specimens. There was no actin immunoreactivity in control or FP-ARDS biopsy specimens in the alveolar areas; however, there was a significant increase in actin immunoreactivity in the interstitial areas of FP-ARDS biopsy specimens when compared with control subjects (2.27 ± 0.58% versus 17.05 ± 3.67% [FP-ARDS], p = 0.0004)  . Representative images of -smooth muscle actin immunostaining in control  and FP-ARDS  lung sections are included.

Collagen I, Collagen III, and PCNA
Collagen deposition is the hallmark feature of fibrotic lung disorders and contributes to the thickening of the interstitium as well as decreased lung compliance and impaired gas exchange. Serial lung sections were stained immunohistochemically for collagen I and III peptides; representative images of collagen I are included in  , and representative images of collagen III are included in . Collagen I and III immunoreactivity was quantified by the point-counting method described in METHODS. When comparing normal lung biopsy specimens with FP-ARDS lung specimens, immunoreactivity for interstitial collagens I and III was enhanced in FP-ARDS versus the control population (53 ± 6.7% versus 84.5 ± 8.92% [FP-ARDS], p = 0.019 for collagen I and 67.7 ± 5.44% versus 90.14 ± 4.62% [FP-ARDS], p = 0.005 for collagen III; graphic data not shown). These results are consistent with lung remodeling or fibrosis on pathologic examination.

To determine whether the increase in mesenchymal tissues was associated with cell proliferation, we stained lung serial biopsy specimens for PCNA expression. PCNA reactivity was blindly scored on a scale of 0 to 3 by two independent investigators. Control lung sections showed little PCNA staining (0.64 ± 0.092); however, FP-ARDS lung specimens showed significantly more immunoreactivity (2.44 ± 0.199, p < 0.0001) in a variety of cell types, including macrophages, epithelial cells, fibroblasts, myofibroblasts, and endothelial cells .

IGF-I Immunohistochemistry Relationships
As macrophages have been shown to be a source of IGF-I protein in several other fibrotic lung diseases, we attempted to determine whether increased IGF-I immunostaining correlated with macrophage infiltration as measured by CD68 immunostaining. As shown in  , there was a significant positive correlation between IGF-I and CD68 staining by simple linear regression in both alveolar (p = 0.0036) and interstitial compartments (p = 0.0014). This was confirmed by aligning serial sections immunostained for IGF-I and CD68 (data not shown). These data imply that infiltrating macrophages may be a source of IGF-I in the lungs of individuals with ARDS.

fig.ommitted Figure 7. Regression analysis show significant positive correlations between (A) IGF-I and CD68 in alveolar spaces (p = 0.0036); (B) IGF-I and CD68 interstitial spaces (p = 0.0014); (C) total IGF-I and collagen I (p = 0.019); (D) total IGF-I and collagen III (p = 0.005); and (F) IGF-I and PCNA (p < 0.001). No positive correlation was seen between (E) IGF-I and -smooth muscle actin (p = 0.55).

 
As mentioned, IGF-I has been shown to induce collagen synthesis in a variety of systems, and enhanced collagen deposition has been shown to be a predictor of poor outcome in patients with ARDS (26); therefore, we sought to determine whether there was a relationship between the degree of IGF-I and collagen immunostaining in the lung biopsy specimens. As shown in , IGF-I immunoreactivity positively correlated with both collagen I (p = 0.019) and collagen III (p = 0.005) immunostaining in the lung biopsy specimens, indicating a potential role of IGF-I in the induction of collagen synthesis in the lungs of individuals with ARDS.

As myofibroblast accumulation has been noted in fibrotic lung diseases, and has been associated with the enhanced deposition of extracellular matrix (24, 25), we attempted to correlate the degree of IGF-I reactivity to -smooth muscle actin (a marker of myofibroblasts) in the biopsy specimens. As shown in , a correlation between IGF-I and actin was not observed in the biopsy specimens (p = 0.55).

Cellular proliferation is another hallmark of fibroproliferation, and IGF-I is known to promote cell cycle progression. Therefore, we sought to determine whether there was a relationship between the degree of IGF-I immunoreactivity and PCNA immunoreactivity in the lung biopsy specimens. As shown in , a significant positive correlation exists between IGF-I and PCNA immunoreactivities (p < 0.001), indicating that IGF-I may play a global role in inducing cell proliferation rather than strictly in the proliferation or differentiation of myofibroblasts.

	DISCUSSION

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Growth factors including transforming growth factor-ß, basic fibroblast growth factor, platelet-derived growth factor, connective tissue growth factor, keratinocyte growth factor, and transforming growth factor- have been shown to be present in fibroproliferative lung disorders such as IPF, fibrosis resulting from inorganic dust inhalation, and pulmonary complications of systemic sclerosis (11). These growth factors have been shown in animal models and in in vitro systems to contribute to the fibroproliferation and/or extracellular matrix deposition seen in these fibrotic lung diseases (27). Similar to these fibrotic lung diseases, the fibroproliferative phase of acute respiratory distress syndrome is also characterized by excessive mesenchymal cell proliferation and extracellular matrix protein deposition, but to date little is known of the factors that contribute to the progression of FP-ARDS. The results presented in this study show enhanced immunostaining for IGF-I, IGF-IR, CD68, -smooth muscle actin, collagen I, collagen III, and PCNA in FP-ARDS lung biopsy specimens when compared with control patient biopsy specimens. These results indicate a potential role for IGF-I in the pathogenesis of FP-ARDS.

We show that IGF-I and CD68 immunoreactivities correlate positively in the lung specimens analyzed. Previous reports have shown that macrophages secrete enhanced levels of IGF-I protein in fibrotic lung diseases (9). In addition, macrophages have been shown to possess mRNA for IGF-I (18, 19, 28). Taken together, these data support the notion that macrophages may be an important source of IGF-I in the lungs of individuals with fibrotic lung diseases, including FP-ARDS.

We also show enhanced staining for IGF-IR in lung sections from individuals with FP-ARDS, suggesting that IGF-I potentially acts in an auto- or paracrine manner in the lungs of individuals with FP-ARDS. The enhanced staining for IGF-IR was present in epithelial cells, fibroblasts, endothelial cells, and macrophages and indicates that IGF-I may have multiple cellular targets in the lung on which to exert its effects. Interestingly, basic fibroblast growth factor has been shown to enhance IGF-I receptor expression (29), indicating that in a complex profibrotic environment, such as that in the lungs of individuals with FP-ARDS, where multiple growth factors and inflammatory substances are present, the induction of IGF-IR supports a role for IGF-I in the pathogenesis of lung fibrosis. Further, because we observed that a variety of cells including macrophages, fibroblasts, epithelial cells, and endothelial cells were immunoreactive for IGF-I protein and IGF-IR, it is possible that IGF-I may in fact be bound to its receptor on these target cells. This supports the multicellular target role of IGF-I throughout the lung in pulmonary fibroproliferation. This hypothesis is also supported by the positive immunoreactivity of IGF-I receptor on multiple cell types in the lung biopsy specimens. Alternatively, IGF-I may be produced by this variety of cell types in addition to macrophages, and be acting in an autocrine manner in the lungs of individuals with FP-ARDS. Support for the latter conclusion has come from the fact that fibroblasts and epithelial cells have been shown to possess IGF-I mRNA in various systems (8, 19).

Extracellular matrix synthesis is a hallmark feature of fibrotic lung disease. It is believed that during the fibroproliferative phase, enhanced collagen III expression in the lung is associated with developing fibrosis, whereas collagen I production is thought to be associated with remodeling of fibrotic tissue (30). In our patients with ARDS, we saw enhanced collagen I immunoreactivity and even greater levels of collagen III immunoreactivity in the FP-ARDS biopsy specimens. IGF-I has been reported to be able to stimulate fibroblast proliferation and collagen synthesis (14–16, 31). Indeed, we see a significant relationship between IGF-I reactivity and both collagen I and collagen III in the lung biopsy specimens analyzed. This suggests that IGF-I may contribute to the deposition of extracellular matrix proteins, specifically the collagens, in ARDS.

After lung injury, epithelial damage may be a persistent insult that leads to inflammation and the development of fibrosis. Migration and division of intact epithelial cells and mesenchymal cells, including fibroblasts and myofibroblasts, is critical to restore denuded epithelial barrier and damaged interstitial structures, respectively. IGF-I is well known to have mitogenic and antiapoptotic functions. Evidence that IGF-I may contribute to cellular division in the lungs of individuals with FP-ARDS is supported by a significant positive correlation between IGF-I and PCNA immunoreactivities in the lung biopsy specimens. IGF-I has also been shown to play a role in altering focal adhesions and actin structures in mammary epithelial cells, which may contribute to cell migration (32), suggesting that IGF-I may potentiate epithelial cell migration over denuded epithelium and fibroblast migration to areas of fibrosis.

In this study, we see enhanced IGF-I and IGF-I receptor staining in lung biopsy samples from individuals with FP-ARDS (organizing diffuse alveolar damage). The enhanced staining for IGF-I correlated with CD68 immunostaining, implicating the infiltrating macrophages as a potential major source of IGF-I in the lungs of individuals with FP-ARDS. Further, IGF-I immunoreactivity correlated with enhanced collagen I, collagen III, and PCNA reactivities, suggesting that IGF-I may contribute to the deposition of extracellular matrix proteins and to the general fibroproliferation seen in the lungs of individuals with FP-ARDS. Although increased IGF-I and IGF-I receptor levels indicate a role for IGF-I in FP-ARDS, the bioavailability of IGF-I in these lung sections cannot be determined with certainty in this study as the availability of IGF-I to cells is dependent on the presence of a number of IGF-I–binding proteins and IGF-I–binding protein proteases (33).

Taken together, our results suggest that IGF-I may play a significant role in the proliferation of a variety of cell types in the lung, and may contribute to the fibrotic process in the lungs of individuals with FP-ARDS. We are continuing our investigation of the roles of IGF-I in the lungs of patients with FP-ARDS.

	Acknowledgments

 
The authors acknowledge the technical assistance of Susan Hui (Immunopathology Department, Foothills Medical Center and Calgary Laboratory Services) and the fruitful discussion with and assistance of Dr. Christopher Doig in the statistical analysis.

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