American Journal of Respiratory Cell and Molecular Biology

Transforming growth factor (TGF)- β 1 is an important regulator of inflammation and fibrosis. TGF- β 1 is usually secreted as a biologically latent protein called latent TGF- β 1 (L-TGF- β 1). L-TGF- β 1 has no biologic effect unless L-TGF- β 1 is converted to its active form. Using a well-recognized model of lung injury induced by the antineoplastic antibiotic bleomycin (Blm), we demonstrated that 7 d after intratracheal Blm administration, total lung TGF- β was maximally increased. This induction was due to TGF- β 1 production by alveolar macrophages that, when explanted, generated increased quantities of L-TGF- β 1 complexed with the glycoprotein thrombospondin (TSP)-1. The TSP-1/L-TGF- β 1 complex was associated with CD36, a receptor for TSP-1. The association of TSP-1/L-TGF- β 1 to CD36 was critical for plasmin-mediated release of mature TGF- β 1. In this paper we show that, compared with administration of Blm by itself, when a synthetic peptide of CD36 between amino acids 93 and 110 is given concomitantly with Blm to rats, alveolar macrophages generate markedly less active TGF- β 1, the rats gain weight more rapidly, and there is less inflammation, collagen I and III, and fibronectin synthesis. These findings demonstrate a novel in vivo mechanism of activation of L-TGF- β 1 in lung injury and the importance of alveolar macrophage– derived active TGF- β 1 in the pathogenesis of pulmonary inflammation and fibrosis.

Before an increase in connective tissue synthesis after lung injury, there is an influx of inflammatory cells that are present not only in the alveoli but also in the interstitium (1). In inflammatory infiltrates the alveolar macrophages are activated to produce a number of proinflammatory and fibrogenic cytokines (2) such as transforming growth factor (TGF)-β (3-6), platelet-derived growth factor (7), insulin growth factor-1 (7), tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-5 (4, 5, 8). Of these cytokines, TGF-β1 has been demonstrated to regulate not only itself but also a variety of fibrogenic cytokines (9) that ultimately are important in the pathogenesis of inflammation and connective tissue synthesis (2-8).

Although TGF-β exists in three isoforms in mammals, it is the TGF-β1 isoform that has been most commonly associated with disorders characterized by inflammation and fibrosis (10). All the TGF-βs are initially synthesized as large precursors that are 390 to 414 amino acids in size (11). The intracellular and extracellular processing of the TGF-β1 isoform has been most extensively studied. Before the secretion of the TGF-β1, the intracellular protease furin cleaves the amino terminal end of the precursor between amino acids 278 and 279 (11), resulting in an amino terminal peptide called the latency-associated peptide (LAP) and a carboxy terminal protein called the mature TGF-β1. However, the cleaved 75-kD amino terminal portion of the protein noncovalently associates with the 25-kD carboxy terminal portion of the protein (11). When the carboxy terminal TGF-β1 peptide is secreted as a complex with the LAP it is called latent TGF-β1 (L-TGF-β1), and in this form L-TGF-β1 cannot interact with its receptor or have a biologic effect. In vitro, the LAP can be removed from its association with the TGF-β1 by pH < 2 or > 8, heat of 100°C (11), chaotropic agents, proteases such as plasmin (5, 6, 11), cathepsin A and D, endoglycosidase, sialic acid, reactive oxygen species, or high concentrations of glucose (11). Alternatively, L-TGF-β1 can be activated without removing the LAP by interacting with the glycoprotein thrombospondin (TSP)-1 (12) or the integrin αvβ6 (13). Although the expression of TGF-β1 has been associated with a number of inflammatory and fibrotic diseases, for TGF-β1 to be physiologically or pathologically significant in these disorders it must be present in a biologically active form. The in vivo activation of L-TGF-β1 in these instances is poorly understood.

We have used a well-recognized model of lung injury induced by the antineoplastic antibiotic bleomycin (Blm) (3-6) to describe a novel mechanism of activation of L-TGF-β1 (4-6). After a single intratracheal dose of Blm, there is injury to the alveolar epithelium and endothelium, followed by recruitment and activation of inflammatory cells before epithelial cell regeneration, resolution of inflammation, and enhanced connective tissue synthesis (3-6). We have demonstrated that 7 d after Blm-induced lung injury and before collagen synthesis, activated alveolar macrophages were the primary source of a 30-fold increase in total lung TGF-β content (3-6). When these alveolar macrophages were explanted they generated active TGF-β1, whereas alveolar macrophages from normal saline-treated rats or those receiving no treatment secreted either no TGF-β1 or small amounts of L-TGF-β1 (4-6). In addition, 7 d after Blm injury alveolar macrophages released maximal quantities of the serine protease plasmin and the glycoprotein TSP-1 (5). The presence of α2-antiplasmin or aprotinin, both inhibitors of plasmin or anti–TSP-1 antibodies, inhibited the post-translational activation of alveolar macrophage–derived L-TGF-β1 (5, 6). It was also demonstrated that before the release of active TGF-β1 the L-TGF-β1 that was complexed with the TSP-1 interacted with the TSP-1 receptor CD36 on the alveolar macrophage (6). The CD36–TSP-1/L-TGF-β1 interaction appears critical to the activation process because in the presence of antibodies to CD36 that prevent the association of TSP-1 to CD36 the activation of L-TGF-β1 was totally abrogated. TSP-1 associates with CD36 by interaction of TSP-1 with the ectodomain of CD36 between amino acids 93 and 110. In the presence of a synthetic peptide of the ectodomain of CD36 that mimics the region between amino acids 93 to 110 the activation of L-TGF-β1 by explanted alveolar macrophages was also inhibited (6).

In this paper we demonstrate that compared with Blm administration alone the concomitant administration of Blm and the CD36 synthetic peptide 93-110 significantly reduces alveolar macrophage secretion of active TGF-β1, inflammatory lesions, and collagen I and III and fibronectin synthesis. These findings support the importance of biologically active TGF-β1 derived from alveolar macrophages in the pathogenesis of Blm-induced pulmonary inflammation and fibrosis. Further, these findings suggest that a TSP-1–CD36 plasmin–dependent mechanism is involved in the process of local activation of alveolar macrophage–derived L-TGF-β1.

Animals

Female Sprague–Dawley rats, free of respiratory disease and weighing between 250 and 300 g, were obtained from the University of Manitoba vivarium. In each experiment, all rats were matched for age and weight. In compliance with the Canadian Council on Animal Care (CCAC), the numbers of rats used were restricted to numbers that were as minimal as possible to adequately address the most relevant issues related to this work.

Reagents

Blm (Blenoxane) was purchased from Bristol-Myers Squibb (Evansville, IN). Neutralizing antibodies to TGF-β1-3 were obtained from Genzyme (Cambridge, MA). Antibody to procollagen I and III (Cedarlane Laboratories Inc., Hornby, ON, Canada) and fibronectin (Sigma, St. Louis, MO) were used for immunoblotting.

Preparation of Synthetic CD36 Peptides

The CD36 peptide YRVRFLAKENVTQDAEDNC (93-110), the scrambled peptide of 93-110 (RFAYLRKNVTENDEQAVCD), and the CD36 synthetic peptide (208-224) CADGVYKVFNGKDNISKV were synthesized (6), on the basis of the work of Leung and colleagues (14). The peptides were synthesized with an Applied Biosystems model 431A peptide synthesizer, using Fmoc (N-[9-Fluoreny-d-methoxycarbonyl]) chemistry, and were purified by reverse high-performance liquid chromatography using a C18 column.

Blm and Synthetic Peptide Administration

This procedure is described in detail in References 3–6. Briefly, rats were given normal saline, 1 μg of Blm, 1,600 μg of a CD36 synthetic peptide, or 1 μg of Blm concomitantly with 1,600 μg of a CD36 synthetic peptide in a total volume of 500 μl sterile normal saline. Rats used as controls received 1,600 μg of the CD36 synthetic peptide 93-110 or a CD36 peptide unrelated to the site of interaction of CD36 with TSP-1 mimicking the amino acid 208-224 sequence of the CD36 ectodomain (14). The quantity of 1,600 μg of peptide used was based on a pilot study demonstrating that quantities less than 1,600 μg of the peptide did not affect Blm toxicity in a significant manner. The CD36 synthetic peptide from amino acids 208 to 224 has previously been demonstrated not to inhibit activation of L-TGF-β1 (6). In addition, a scrambled peptide of the CD36 amino acid 93-110 (sequence given earlier) was also used. After administration of various agents, the rats were killed at different time intervals. For some experiments, 7 d after reagent administration was chosen as the time to harvest alveolar macrophages, on the basis of our findings that alveolar macrophages are maximally stimulated at this time to secrete active TGF-β1 (5). Weights and appearances of rats were recorded daily after receiving the various treatments.

Histology and Histochemistry

Fourteen days after Blm administration, a time previously reported to be associated with increased connective tissue synthesis and inflammation (1, 3), we obtained lungs for histology as previously described (3). At 24 h after fixation in 10% formalin the lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histology and Mason's Trichrome for distribution of collagen, as well as Alcian blue for location of proteoglycans. Because Blm-induced lung injury is patchy in nature, all lung segments were examined from each treatment group. All slides were blinded and two independent examinations (by authors N.K. and R.O.) were done and then collated. For the extent of lung involved, the lung sections were examined under low power and revealed the entire lung section that contained both normal lung and an inflammatory or fibrotic lesion. The proportion of lung section with inflammation was then reported as a percentage of the overall lung section. For grading the extent of staining with Mason's Trichrome or Alcian blue, 0 designated no staining, +1 designated detectable color (green for Mason's Trichrome and blue for Alcian blue), and +2 designated an extent of staining between +1 and +3. The grade +3 was reserved for those instances where there was extensive staining within an inflammatory and fibrotic lesion.

Differential Cell Count of Cells Obtained by Bronchoalveolar Lavage

Cells obtained by bronchoalveolar lavage (BAL), as previously described (4-6), were suspended at a concentration of 1 × 106 cells/ml and were used in a cytospin preparation (4). The cells were stained using Diff-Quik (Baxter Healthcare Corp., Miami, FL) fixative and nuclear/cytoplasmic stains (4). Five fields at high power were used to enumerate and identify cells as macrophages, polymorphonuclear leukocytes (PMNs), or lymphocytes (4).

Macrophage Cultures

The lungs were lavaged to obtain cells for culture of alveolar macrophages as previously described (4-6). Alveolar macrophages were maintained in serum-free media containing Gentamicin (4 mg/ 100 mls; Roussel, Montreal, PQ, Canada), Fungizone (100 μ1/ 100 mls; GIBCO BRL, London, ON, Canada), and 0.2% clotted bovine calf plasma (BCP) (National Biological Laboratory Limited, Dugald, MB, Canada). After 20 h of incubation at 37°C, 5% CO2, the media were collected in the presence of protease inhibitors—leupeptin, 0.5 μg/ml, from Amersham, Buckinghamshire, UK; and aprotinin and pepstatin, 1 μg/ml each, both from Sigma (Oakville, ON, Canada)—frozen at −80°C until ready for TGF-β quantitation (4-6).

CCL-64 Mink Lung Epithelial Growth Inhibition Assay for TGF- β

The CCL-64 growth inhibition assay to identify and quantitate TGF-β has been described elsewhere (3-6). Briefly, neutral conditioned media or conditioned media that were acidified and subsequently neutralized were added to subconfluent cells in α–Eagle's minimum essential medium, 0.2% BCP, 10 mM N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid at pH 7.4, and penicillin (25 μg/ml) and streptomycin (25 μg/ml), and cultured as 5 × 104 cells/0.5 ml in 24-well Costar dishes (Flow Laboratories, Inc.; Mississauga, ON, Canada). After 22 h the cells were pulsed with 0.25 μCi of 5-[125I]iodo 2′-deoxyuridine [125I]UdR (ICN Pharmaceutical, Costa Mesa, CA) for 2 to 3 h at 37°C, then lysed with 1 ml of 1 N NaOH for 30 min at room temperature. The [125I]UdR was then counted in a γ counter (LKB Instruments, Gaithersburg, MD). A standard curve using porcine TGF-β1 was included in each assay. For confirmation of TGF-β activity, neutralizing monoclonal antibody against TGF-β1-3 (Genzyme) was added before the addition of the conditioned media (3-6) and resulted in abrogation of all TGF-β activity.

Protein Extraction for Western Analysis

Untreated rats and rats treated with various agents were killed and the peripheral blood, heart, and blood vessels were removed as described earlier (3-6). The lungs were snap-frozen on dry ice with ethanol and stored at −80°C until protein extraction. Whole-lung protein extraction was performed as described previously (6). Briefly, the frozen lungs were pulverized in a chilled mortar and placed in tissue lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma). The samples were further homogenized in the presence of 0.5% Triton X-100, then centrifuged at 200 mg for 10 min at 4°C. The supernatant was collected and protein levels were determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).

Western Analysis

The protein samples (25 μl) were electrophoresed on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis in a Mini-PROTEAN II Electrophoresis Cell (Bio-Rad). Protein molecular weight markers (Amersham) were run parallel to each blot as an indicator of the molecular weight. Equal loading of protein was evaluated using silver staining (not shown). The separated proteins were transferred at 50 V overnight onto nitrocellulose membrane (GIBCO BRL) in a Mini Trans-Blot chamber with transfer buffer (25 mM Tris-Cl, 192 mM glycine, and 20% methanol). The nitrocellulose membrane was blocked for 1 h using 5% instant skim-milk powder in Tris-buffered saline (TBS). For detection of procollagen I and III a 1:300 dilution of antibody was used, whereas a 1:1,000 dilution was used for fibronectin in 1% instant skim-milk powder. After washing, the nitrocellulose membrane was incubated with horseradish peroxidase linked with the secondary antibody (goat antimouse immunoglobulin G; Bio-Rad) as recommended by the manufacturer. Finally the washed blots were exposed to an enhanced chemiluminescence (ECL) detection system (Amersham) and recorded on an autoradiograph (Kodak X-Omat film). Before reprobing, the nitrocellulose membrane was incubated at 50°C for 30 min with a stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7). The blots were rinsed twice with TBS. To ensure the removal of antibodies, membranes were incubated with the ECL detection reagents and exposed to film (Kodak). No band was detected, confirming that all antibodies were stripped off the membrane. The same nitrocellulose membrane was blocked using 5% instant skim-milk powder in TBS for detection of procollagen III and fibronectin.

Cytotoxicity Assay

LY/5178Y mouse lymphoma cells were incubated with normal saline, Blm (1 μg/500 μl of normal saline), CD36 synthetic peptide 93-110 (1,600 μg/500 μl of normal saline), or Blm (1 μg /500 μl of normal saline) plus CD36 synthetic peptide 93-110 (1,600 μg) for 1 h at 37°C in media containing horse serum. The cytotoxic activity of the agents on the LY/5178Y cells was determined by a soft agar cloning assay and was expressed as the surviving cell fraction as described previously (15). Cloning efficiencies ranged from 35 to 65%. The cytotoxicities of the different treatments in the cell line were compared statistically by analysis of variance (ANOVA).

Statistical Analysis

Statistical analysis using ANOVA or Wilcoxon's rank signed nonparametric statistical test was done by Dr. Bob Tate, Biostatistical Unit, University of Manitoba.

Post-Translational Activation of Alveolar Macrophage–Derived L-TGF- β 1

Our previous work demonstrated that a single intratracheal dose of Blm resulted in generation of active TGF-β1 by explanted alveolar macrophages that was maximal 7 d after Blm administration (3-6). TGF-β2 and TGF-β3 from alveolar macrophages remained unchanged (4). Similar to our previous findings, alveolar macrophages obtained from rats 7 d after Blm administration secreted increased quantities of TGF-β1, which was 61.7 ± 8.6% in the active form (Figure 1A). Alveolar macrophages from rats that had received Blm concomitantly with the CD36 peptide 93-110 generated decreased quantities of active TGF-β1 while the quantity of total TGF-β1 released remained unchanged (Figure 1A). In this group of rats the percent of active TGF-β secreted was only 2.2 ± 2.1% of the total TGF-β1 released by these alveolar macrophages (Figure 1A). We reported previously that activated alveolar macrophages were the primary source of the 30-fold increase in total lung TGF-β 7 d after Blm administration (3, 4). In the present study total lung TGF-β was not quantitated because alveolar macrophages after CD36 synthetic peptide and Blm administration release increased quantities of total TGF-β compared with normal saline-treated rats. It was then unlikely that there would be a significant change in total lung TGF-β after Blm and CD36 peptide administration. Further, the method for quantitation of total lung TGF-β does not distinguish active from latent TGF-β (3, 4) and the quantitation of active TGF-β was more pertinent to the current study. The administration of a scrambled peptide of the amino acids between 93 and 110 (CD36 s93-110) of CD36 concomitantly with Blm did not affect the generation of active or L-TGF-β1 (Figure 1B). In addition, the administration of the control CD36 synthetic peptide 208-224 concomitantly with Blm had no significant effect on generation of active or L-TGF-β1 or the percent of active TGF-β secreted by alveolar macrophages compared with Blm-treated rats (Figure 1B). The CD36 peptide 93-110, CD36 s93-110, or 208-224 given alone (data not shown) or normal saline administration did not induce TGF-β1 production and had no effect on the generation of active or L-TGF-β1.

General Appearance and Weight of the Rats

Rats that had been given normal saline, or one of the synthetic peptides alone, or no treatment appeared healthy and gained weight with normal aging as observed during the course of the experiments (Figure 2) and no rats from these groups died. Rats that had received Blm looked generally unwell, characterized by poor ambulation and activity in the cage. In addition, these rats lost considerable amounts of weight (Figure 2). The death rate in Blm-treated rats was approximately 11 to 13%. However, rats that had received both Blm and the CD36 synthetic peptide 93-110 concomitantly looked generally better, were more active in their cages, did not lose as much weight, and had a more prompt weight gain (Figure 2). There were no deaths in this group. Rats that had received the CD36 peptide 208-224 concomitantly with Blm had the characteristics of rats treated with Blm alone, as described earlier (data not shown). However, rats that had received Blm concomitantly with the scrambled peptide of CD36 between amino acids 93 and 110 lost weight in excess of rats treated with Blm alone (data not shown). It is possible that rats treated with Blm alone or those treated with Blm and the scrambled peptide would have had a greater weight loss than demonstrated in Figure 2. However, to comply with the CCAC these rats were force-fed pureed, high-calorie food and hydrated with intraperitoneal injections of normal saline when weight loss of > 10% from the previous day was observed.

Differential Cell Count in BAL Fluid

Normally the cells retrieved by BAL contain greater than 95% macrophages while PMNs, lymphocytes, and other cells make up the rest of the cell population (1, 4). After Blm-induced lung injury there is an increase in not only PMNs but also lymphocytes, basophils, mast cells, and other cells (1, 4). The differential cell count in BAL fluid is in keeping with previous findings in rats treated with normal saline or the CD36 synthetic peptides where macrophages were predominantly present while PMNs and lymphocytes were less in number (Figure 3). After Blm administration the percent and absolute numbers of PMNs were increased. However, when the CD36 synthetic peptide 93-110 was administered with Blm the percent and absolute numbers of PMNs decreased while the percent and absolute numbers of macrophages increased (Figure 3). The total number of inflammatory cells after the use of Blm concomitantly with CD36 synthetic peptide 93–110 was 6.3 × 106 ± 1.2. However, when Blm alone was used the number of cells retrieved was 8.4 × 106 ± 0.1 (P value for these comparisons is ⩽ 0.01).

Histologic Changes

No inflammatory or fibrotic lesions were observed at any time interval in lungs of rats that had received normal saline or the CD36 synthetic peptides 93-110 (not shown). In agreement with numerous previous reports (3-6), rats that had received Blm 14 d earlier had patchy areas of inflammation and fibrosis in several lobes but the most pronounced lesions were in the right lower lobe (Figure 4A). The patchy lesions were characterized by an increase in interstitial and alveolar inflammatory cells as well as the presence of interstitial fibroblasts and early granulation tissue with extensive staining, using Mason's Trichrome for collagen and Alcian blue for proteogylcan (Figures 4B and 4C). The extent of staining with Alcian blue and Mason's Trichrome in Figures 4B and 4C is representative of grade +3 (Table 1). However, 14 d after Blm and CD36 synthetic peptide 93-110 administration there was evidence of less inflammation and fibrosis, inasmuch as the size of lesions was reduced and occupied a smaller volume of the overall section (Table 1; Figures 4D–4F). In addition, the number of cells retrieved by BAL was lower 7 d after administration of Blm and the CD36 synthetic peptide, suggesting that there is less inflammation when the peptide is administered. After Blm and concomitant CD36 synthetic peptide 93-110 administration, the presence of Alcian blue and Mason's Trichrome within the lesion was minimal and is representative of a grade +1 (Figures 4D and 4E; Table 1).

Table 1. Histologic changes after intratracheal treatment

Intratracheal TreatmentPercent of Lung Involved in Inflammation and FibrosisExtent of Staining with:*
Mason's TrichromeAlcian Blue
Blm (1 μg)40.5 ± 2.2+2.9 ± 0.01+3 ± 0
Blm (1 μg) plus CD36  synthetic peptide  93-110 (1,600 μg) 8.0 ± 2.4+0.9 ± 0.4+0.8 ± 0.4

* 0: no staining, +1: detectable color of the Mason's Trichrome or Alcian blue, +2: staining between +1 and +2, and +3: extensive staining with Mason's Trichrome and Alcian blue within the lesion. The percent of lung involved after Blm compared with Blm plus CD36 synthetic peptide 93-110 has a P value of ⩽ 0.002. The extent of staining with Mason's Trichrome or Alcian blue after treatment compared with Blm plus CD36 synthetic peptide 93-110 has a P value ⩽ 0.001. The statistical analysis for this data was done using Wilcoxon's rank signed nonparametric statistical test.

Expression of Connective Tissue Proteins

After a single intratracheal dose of Blm there is increased expression of collagen I and III, decorin, fibronectin, and a variety of other connective tissue proteins (16). Total protein extracted from lungs of rats treated with Blm was 2-fold higher than protein obtained from untreated rats, or from those treated with normal saline or one of the CD36 synthetic peptides alone. This increase in protein extracted is expected to represent not only the aforementioned connective tissue proteins but also the protein content of inflammatory cells and influx of proteins from the circulation. However, the total protein content of lungs from rats that had received Blm concomitantly with the CD36 synthetic peptide 93-110 was approximately 50% less than that obtained when Blm alone was administered. It should be noted that to comply with the CCAC's requirement to use as few rats as possible, the entire time course was not done for experiments used to determine the effects of the CD36 synthetic peptide on connective tissue synthesis after Blm-induced lung injury. Instead, the changes in expression of procollagen I and III and fibronectin were done using a time course where rats were killed at regular time intervals consisting of 4, 7, 14, 21, and 28 d after Blm or normal saline administration (data not shown). Procollagen III and fibronectin were maximally expressed 7 d after Blm treatment, whereas procollagen I was increased in expression 7 and 14 d after Blm administration (data not shown). To determine the effects of CD36 synethetic peptide 93-110 on collagen III and fibronectin expression, rats were killed at 4 and 7 d. To detect changes in collagen I synthesis, rats were killed 7 and 14 d after Blm and CD36 synthetic peptide 93-110 administration (Figure 5A). Further, rats that had received Blm concomitantly with scrambled peptide CD36 amino acids 93-110 demonstrated extreme morbidity, and experiments on these rats were abbreviated. The expression of procollagen I was reduced (Figure 5A) in rats treated with Blm and the CD36 synthetic peptide 93-110 compared with rats that had received Blm alone or those that had received Blm and the scrambled peptide of CD36 93-110 or CD36 peptide 208-224 (Figure 5A). The expression of procollagen III and fibronectin was reduced in rats that received Blm and the CD36 peptide 93-110 (Figures 5B and 5C).

Cytotoxicity of Blm when Combined with the CD36 Synthetic Peptide 93-110

The intratracheal administration of Blm results in a random distribution of the drug (1, 3). The alveolar epithelial and endothelial cell injury that follows is in the areas of Blm deposition (17). When the Blm was combined in the same syringe with the CD36 synthetic peptide, the deposition of both substances was likely to be in the same distribution. The combination of Blm and the CD36 synthetic peptide 93-110 did not affect the potency of Blm toxicity to Blm-sensitive LY/5178Y lymphoma cells (Figure 6). For this reason the initial in vivo Blm injury to the alveolar epithelial and endothelial cells is not likely to be affected when Blm and the CD36 synthetic peptide 93-110 are administered together. It is of note that the administration of Blm concomitantly with the CD36 synthetic peptide 93-110 did not totally abrogate the generation of active TGF-β1, inflammation, or connective tissue synthesis. There was, however, a reduction in all these parameters, which supports the probability that Blm administration leads to pulmonary toxicity that can be ameliorated by the effects of the CD36 synthetic peptide 93-110.

The presence of CD36 synthetic peptide 93-110 in cultures of alveolar macrophages obtained after Blm-induced lung injury prevents the conversion of L-TGF-β1 to active TGF-β1 (6). The present report is the first to describe that when the same CD36 synthetic peptide is administered to rats with Blm compared with administration of Blm alone, there is a reduction of inflammation and connective tissue synthesis induced by Blm. The administration of the CD36 synthetic peptide 93-110 with Blm results in decreased secretion of active TGF-β1 by explanted alveolar macrophages. It is of note that after Blm-induced lung injury the overexpression of TGF-β1 is seen almost exclusively in alveolar macrophages (3). No TGF-β1 was observed in alveolar epithelial cells or interstitial inflammatory cells (3). It then follows that in vivo the effect of the CD36 synthetic peptide 93-110 most likely inhibits the activation of alveolar macrophage–derived L-TGF-β1. The reduction of active TGF-β1 from alveolar macrophages may then lead to amelioration of Blm-induced inflammation and connective tissue synthesis.

The mechanisms by which the in vitro presence of (6) or in vivo administration of the CD36 synthetic peptide 93-110 with Blm diminishes the release of active TGF-β by explanted alveolar macrophages may be similar. It had previously been demonstrated that the amino acid sequence CSVTCG of TSP-1 interacts with the CD36 in the region of the amino acids 93 to 110 of CD36 (18). The CSVTCG region in the type 1 repeats of TSP-1 is also important for the release of active TGF-β1 by alveolar macrophages because the addition of the synthetic TSP-1 peptide CSVTCG to cultures of activated alveolar macrophages inhibits the activation of L-TGF-β1 (N. Khalil and colleagues, unpublished data). It has been demonstrated that the interaction of the various domains of TSP-1, with the ligands may depend on the conformational state of the TSP-1 molecule (19). When TSP-1 is soluble the sequence CSTVCG, present in two sites within the type 1 repeats of TSP-1, is more exposed than when TSP-1 is associated with a cell surface or matrix proteins (19). We demonstrated previously that alveolar macrophages generate TSP-1/L-TGF-β1 complexes after in vivo Blm injury (6). It is conceivable that when the CD36 synthetic peptide 93-110 is present either in vitro or in vivo, the peptide associates with the CSVTCG region of TSP-1 in the TSP-1/L-TGF-β1 complex. The association of the CD36 synthetic peptide 93-110 with the CSVTCG region in TSP-1 of the TSP-1/L-TGF-β1 complex may interfere with the TSP-1/L-TGF-β1 interaction with the CD36 receptor on the surface of the alveolar macrophage (6). Because the association of the TSP-1/L-TGF-β1 with the alveolar macrophage CD36 receptor is critical to the activation of the L-TGF-β1 by plasmin (5, 6), prevention of this association may diminish the activation of L-TGF-β1 in vivo.

The alveoli normally contain greater than 95% macrophages. After Blm administration the number of macrophages remains the same but decreases in percentage due to an increase in the number of PMNs (1, 4). However, when the CD36 peptide 93-110 was administered with Blm, there was a reduction in numbers of PMNs so that an increase in the percentage of alveolar macrophages was observed. The reduction in numbers of PMNs could be due to a number of reasons. TGF-β1 is a potent chemoattractant of PMNs (20), and when the CD36 synthetic peptide 93-110 was administered the decrease in active TGF-β1 in the alveolar space could lead to a reduction in PMN recruitment to the alveoli. The interaction of TSP-1 with CD36 could also be important in areas of injury in recruitment and activation of PMNs (21, 22). Blm administration injures endothelial cells (17), and TSP-1 that is released by injured endothelial cells (21, 22) binds to the same cells, where it functions to recruit PMNs and localize the PMNs to the endothelial cells and activates PMNs on endothelial cells to release reactive oxygen intermediates (21, 22). Because Blm injures the endothelial cells (17), this role of TSP-1–mediated recruitment of PMNs could contribute to lung injury induced by Blm administration. The binding of TSP-1 to endothelial cells is mediated by the association of TSP-1 to CD36, which is located on the endothelial cells (23). It is then conceivable that the presence of the CD36 synthetic peptide 93-110 may prevent the association of TSP-1 to endothelial cells, resulting in reduction in the recruitment of PMNs.

CD36 is an 88-kD membrane glycoprotein that functions as a receptor for TSP-1 collagen and erythrocytes infected with Plasmodium falciparum (24). The interaction of CD36 with TSP-1 has been described to be important for a number of functions such as platelet aggregation, platelet-monocyte adhesion, platelet tumor cell adhesion, and macrophage uptake of apoptotic cells (24). Of these functions relevant to Blm lung injury are the observations that platelet aggregation has been reported to occur in early wounding (25) and is important in Blm-induced injury and inflammation (26). Further, platelet-aggregation (27) or platelet monocyte adhesion may result in activation and release of cytokines (27). The presence of the CD36 synthetic peptide 93-110 in vitro prevents platelet aggregation (14). In vivo, the presence of the CD36 synthetic peptide 93-110 could result in inhibition of platelet aggregation, platelet-monocyte adhesion, and release of cytokines, and thus less inflammation and fibrosis. These effects of the CD36 synthetic peptide 93-110 could also contribute to the amelioration of Blm-induced inflammation and fibrosis.

Over the past few years a number of treatments, such as anti–TNF-α antibodies (28), antioxidants (29), interferon-γ (30), and pirfenidone (31), have been demonstrated to ameliorate the Blm-induced pulmonary inflammation and fibrosis. Nonetheless, Giri and associates confirmed the importance of TGF-β1 in the pathogenesis of Blm-induced inflammation and fibrosis in mice when they demonstrated that administration of TGF-β1 antibodies and Blm resulted in less lung injury and fibrosis (32). However, the location of the neutralizing effects of the anti–TGF-β1 antibodies was not apparent from Giri and coworkers' study (32). On the basis of the current work and that of others, the administration of TGF-β1 antibodies could have neutralized the TGF-β1 generated by endothelial cells (33) and/or alveolar epithelial cells (13), as well as alveolar macrophages (3-6).

Our work has shown that the generation of plasmin in addition to the cellular localization of L-TGF-β1 (5, 6), is important for the activation of alveolar macrophage–derived L-TGF-β1. However, there are other mechanisms of activation of L-TGF-β1 that are independent of proteases. For example, reactive oxygen intermediates (11), hyperglycemia (11), and, in some but not all circumstances (34), TSP-1 can activate L-TGF-β1 in the absence of proteases (12). In addition, Munger and associates (13) described another protease-independent activation of L-TGF-β1 that may be important in Blm-induced lung injury (13). It was observed that the RGD (arginine–glycine–aspartic acid) sequence in the LAP interacts with the integrin αvβ6 on alveolar epithelial cells (13), leading to conformational changes of the L-TGF-β1 and exposing the site on TGF-β1 that interacts with the TGF-β receptor type II (13). It is not known whether Blm administration to rats, a different species than mice, induces αvβ6 on alveolar epithelial cells and is therefore important in the activation of L-TGF-β1. It is also not known whether Blm injury induces CD36 expression on rat alveolar epithelial cells that subsequently binds the TSP-1/L-TGF-β1 complex and results in plasmin-mediated activation of L-TGF-β1. Regardless of the expression of αvβ6 or CD36 on rat epithelial structures, we have demonstrated that the reduction of alveolar macrophage–derived active TGF-β1 or the potential for interruption of the CD36/ TSP-1 interactions is associated with a decrease in inflammation and fibrosis after Blm-induced lung injury. Collectively, our findings not only suggest a novel in vivo mechanism of activation of L-TGF-β1 but also support the importance of alveolar macrophage–derived active TGF-β1 in pulmonary fibrosis.

The work in this manuscript was supported by a grant from the Medical Research Council of Canada to one author (N.K.). The authors thank Dr. Arnold Greenberg for reviewing the manuscript, Dr. Bob Tate for the statistical analysis, Mrs. Stephanie Moorehouse for her technical assistance, and Ms. Carolin Hoette for typing the manuscript.

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Address correspondence to: Dr. Nasreen Khalil, M.D., Div. of Respiratory Medicine, University of British Columbia, 655 W. 12th Ave., Vancouver, BC, V5Z 4R4 Canada. E-mail:
Abbreviations: analysis of variance, ANOVA; bronchoalveolar lavage, BAL; bleomycin, Blm; Canadian Council on Animal Care, CCAC; hematoxylin and eosin, H&E; latency-associated peptide, LAP; latent TGF-β1, L-TGF-β1; polymorphonuclear leukocyte, PMN; Tris-buffered saline, TBS; transforming growth factor, TGF; thrombospondin, TSP.

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