Introduction

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Lung disorders such as bronchopulmonary dysplasia (1, 2), idiopathic pulmonary fibrosis (3), and occupational lung diseases (4) commonly involve abnormal remodeling of the extracellular matrix (ECM). Fibrotic changes, which are a consequence of repair, impair normal gas exchange in the lung. The processes by which the ECM is altered after pulmonary injury have been advanced using a model of lung injury induced by intratracheal bleomycin (5, 6). In this model, lung injury results both in an inflammatory infiltrate consisting largely of macrophages (7, 8) and in ECM remodeling with an increased production of fibronectin (9, 10), collagen (11), and hyaluronan (HA) (12). Fibronectin expression is maximal at 7 d and gradually declines to normal levels by 28 d (13), whereas collagen content increases at 7 d and remains elevated even 28 d after injury (14). The expression of HA appears to be more tightly regulated and coincides with early macrophage accumulation in the lung after injury (12). Maximal accumulation of HA occurs at 4 d, both in the alveolar interstitium (15) and in bronchoalveolar lavage (BAL) (16). Levels decrease rapidly thereafter.

HA, a nonsulfated glycosaminoglycan, consists of a polymer of repeating disaccharide units of N-acetyl glucosamine and glucuronic acid (17). An increased recovery of HA in BAL has been found in various disease states such as sarcoidosis (18), occupational lung disorders (19), and adult respiratory distress syndrome (20). HA has also been shown to increase dramatically in the alveolar interstitium (15, 21) as well as in BAL (16, 22) after intratracheal instillation of bleomycin in rats. Further, the increased recovery of HA temporally correlates with an influx of inflammatory cells (16).

The accumulation of inflammatory cells at sites of injury requires their activation, adhesion to endothelium, and subsequent transmigration into the injured tissue. Although the precise role that HA plays in these processes is not clear, HA has been implicated in tissue responses to injury. Thus, HA is involved in monocyte activation (23), in leukocyte adhesion to endothelium (26, 27), and in smooth-muscle cell migration after wounding (28). In the present study, we document the content and localization of HA after injury with intratracheal bleomycin in rats. We show that maximum motility of BAL macrophages coincides with elevated HA concentrations, and we document the efficacy of peptides that bind HA in inhibiting macrophage motility and chemotaxis in vitro. Importantly, the administration of HA-binding peptide before bleomycin-induced lung injury inhibits the motility of macrophages isolated by BAL and their accumulation in the lung after injury, as well as steady-state collagen  (I) messenger RNA (mRNA) levels and hydroxyproline content in the injured lung. Together, these data implicate HA as a critical component in the inflammatory and fibrotic response to pulmonary injury.

	Materials and Methods

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Animals

Ethical standards of animal care were followed and approval of the research protocol was obtained from The University of Manitoba Central Animal Care Committee (Winnipeg, MB, Canada), The Institutional Animal Care and Use Committees of The Joseph Stokes Jr. Research Institute (Children's Hospital of Philadelphia, Philadelphia, PA), and the University of Pennsylvania School of Medicine (Philadelphia, PA). After anesthesia using a cocktail of 80 mg/kg ketamine, 40 mg/kg xylazine, and 0.05 mg/kg atropine administered intraperitoneally, 250- to 300-gram male Sprague-Dawley rats were treated intratracheally with 8 U/kg bleomycin sulfate (Bristol-Myers Squibb, Princeton, NJ) in 0.25 ml of normal saline or with 0.25 ml normal saline alone as previously described (14). The animals were killed at 2, 4, 7, 10, and 14 d after treatment. The main pulmonary artery of each animal was flushed with Hanks' balanced salt solution (HBSS) (GIBCO BRL, Burlington, ON, Canada) and the lungs were harvested for further analysis.

Isolation of Macrophages

For certain experiments, BAL was performed as previously described (8) using a total of 30 to 40 ml of sterile, lipopolysaccharide (LPS)-negative HBSS. Fluid recovery was consistent in all animals with 80 to 90% of lavage fluid recovered after instillation. The cells obtained by BAL were resuspended in -modified Eagle's medium (GIBCO BRL) with 0.5 U/ml insulin (beef and pork zinc suspension; Novo Laboratories Ltd., Willowdale, ON, Canada) and 4 µg/ml transferrin (Sigma Chemical Co., St. Louis, MO). The cells were incubated at 37°C in 5% CO₂ for 15 min to promote macrophage plating, and cell locomotion was measured in the first 2 h after harvest using time-lapse cinemicrography as described later.

Antibodies

Fluorescein isothiocyanate (FITC)-conjugated ED-1 antibody was obtained commercially (Serotec, Raleigh, NC). This antibody has previously been shown to be specific for rat macrophages (29), and was used in immunofluorescence studies to quantify macrophage content in lung sections.

HA-Binding Peptides

To observe its effect on macrophage motility, a peptide (YKQKIKHVVKLK, 2 µg/ml) mimicking one of the HA-binding domains of the HA receptor RHAMM (receptor for HA-mediated motility, aa 401-411 [30]) was synthesized on an Applied Biosystems peptide synthesizer (Model 431A; Foster City, CA) using Fmoc chemistry, and was used to inhibit the locomotion of isolated alveolar macrophages as described later. A scrambled peptide (YLKQKKVKKHIV, 2 µg/ml) containing the same amino acids but in a random, non-HA binding order was used as a control for these experiments. The purity of the peptides was determined using mass spectroscopy, and the optimal concentration was determined in preliminary experiments using time-lapse cinemicrography to measure the effect of the peptides on the random locomotion of several macrophage cell lines (data not shown).

In experiments involving chemotaxis of rat alveolar macrophages to 5% fetal calf serum (FCS) and the in vivo treatment of animals with peptides, a synthetic peptide mimicking the optimal HA-binding motif (RGGGRGRRR, 80 mg/kg [31]) was administered subcutaneously to rats 1 h before instillation of intratracheal bleomycin. Control animals were treated with a scrambled peptide that consisted of the same amino acids but did not conform to the HA-binding motif (RGRRGRGRG, 80 mg/kg). We have previously shown this dose of peptide to be effective in limiting inflammation in a rat model of skin wound healing (32).

Immunocytochemistry

After inflation to 25 cm H₂O, lungs from at least five animals at each time point and for each treatment group were harvested for immunocytochemistry and were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and processed to obtain 5-µm sections. HA was localized using an avidin-biotin-peroxidase histochemical technique as described previously (33). Briefly, the sections were incubated overnight with the biotinylated HA binding region of aggrecan (bHABP) (1:300 dilution; Seikagaku Corp., Tokyo, Japan) isolated from bovine nasal cartilage. This bHABP recognizes all molecular-weight forms of HA greater than six-mer disaccharide units (Seikagaku Corp.). After washes, bHABP was detected using horseradish peroxidase-conjugated streptavidin. The color of the reaction product obtained using diaminobenzadine (Sigma) (10 mg/ml in 0.05 M Tris-buffered saline) was enhanced with 0.5% copper sulfate in 0.9% NaCl for 10 min. Sections were counterstained with 0.25% methyl green for 15 min, cleared in n-butanol and xylene, and then mounted in Permount (Fisher Scientific, Pittsburgh, PA). Specificity of staining for HA was confirmed both by incubation of the probe with excess HA before staining (Figure 1a), and by pretreatment of the sections with Streptomyces hyaluronidase to degrade HA before staining (data not shown).

View larger version (15K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 1.   Macrophage and HA changes after bleomycin injury. (a) The number of macrophages per hpf was determined at various times after intratracheal instillation of either bleomycin or saline using ED-1 staining to identify rat macrophages. The number of ED-1-positive cells per hpf in each treated condition is presented as the fold-change from untreated animals used as controls. A trend toward increased macrophage number was noted in saline-treated animals (dotted line) at 4 d, and bleomycin-treated animals (solid line) showed a 4- to 5-fold increase in macrophages per hpf at 4 and 7 d (*P < 0.05). Data represent macrophage numbers determined independently by three blinded observers counting five fields per section with three sections analyzed per condition from three separate experiments each. (b) The velocities of macrophages isolated by BAL at various times after intratracheal instillation of either bleomycin or saline were measured by time-lapse cinemicrography. A significant 3-fold increase in velocity was observed in macrophages isolated 4 d after saline instillation (*P < 0.01 versus untreated controls). However, after bleomycin injury a more dramatic 6-fold increase in macrophage velocity was noted from 2 to 7 d, with maximum velocity occurring at 4 d (#P < 0.001 compared with untreated and saline controls at the same time point). Values represent mean and standard error of five animals studied for each condition at each time point with at least 20 cells tracked for each animal studied. (c) The HA content of BAL from saline- or bleomycin-treated animals was determined using a volume of lavage equal to the total lung capacity for each animal. Compared with uninjured controls, total lung HA content was increased 2- to 3-fold in saline-treated animals on Day 4 (#P < 0.05, n = 5/time point). However, in bleomycin-injured animals, lung HA content was increased 250-fold on Day 4 and 200-fold on Day 7 as compared with controls (*P < 0.01, n = 5/time point). Lung HA content decreased thereafter to baseline by 14 d.

HA Content

BAL using LPS-negative saline to a volume equal to total lung capacity (36 cc/kg) was obtained from each animal studied. Lavage recovery from each animal was consistently 85 to 90% of that instilled, and was centrifuged to separate the cellular compartment. The HA content of the supernatant was determined using the HA50 kit (Kabi-Pharmacia, Uppsala, Sweden) as per manufacturer's instructions. Five animals were examined for each condition at each time point and compared with five untreated animals used as controls.

Immunofluorescence and Quantitation of Macrophage Accumulation

To quantify macrophage accumulation in the lung after treatments, sections were examined by immunofluorescence using FITC-labeled ED-1 antibody as previously described (28), except that no secondary antibody was required. The number of ED-1- positive cells per high-power field (hpf) (original magnification, ×400) were counted by three independent and blinded observers in five randomly selected fields per section in three sections for each condition from at least three separate experiments. For immunofluorescence localization of HA, bHABP was detected by Texas Red-conjugated streptavidin. Confocal microscopic images were obtained using a computer-interfaced, laser-scanning microscope (Leica TCS 4D) in the Confocal Core Facility at the Children's Hospital of Philadelphia. Simultaneous wavelength scanning allowed superimposition of fluorescent labeling with FITC and Texas Red fluorophores at wavelengths of 488 and 568 nm, respectively. Laser power was fixed at 75% for all image acquisition. Image output was at 1,024 × 1,024 pixels.

Time-Lapse Cinemicrography

Alveolar macrophages, freshly isolated by BAL from five animals for each condition at each time point, were monitored for their motility using Metamorph/Image I (Universal Imaging Corp., West Chester, PA) as previously described (28). Initially, cells from each sample of BAL were allowed to plate for 15 min at 37°C, after which the medium was changed. The motility of at least 20 cells per animal studied was followed in each experiment for the first 2 h after plating with mean velocities calculated every 10 min. The effect of peptides was assessed in independent experiments (n = 5 animals each per treatment group and condition) by the addition of HA-binding peptides (2 µg/ml) to the medium 15 min before measurement of cell motility as described earlier. Scrambled peptides (2 µg/ml) were used as controls for these inhibition studies.

Chemotaxis Assay

The chemotactic assay used was a colorometric Boyden chamber assay described in detail elsewhere (34), with slight modifications. Briefly, a 96-well chemotaxis chamber with a lower recess large enough to hold a microtiter plate (Neuro Probe, stock #MBA96, Cabin John, MD) and an 8-µm framed filter (Neuro Probe, stock #PFD5/A) were used. The microtiter plate was filled with chemoattractants and controls and placed in the recess of the chemotaxis chamber. The framed filter was then placed on top of the filled microtiter plate. The chamber was then closed and 200 µl of a cell suspension (1.0 × 10⁶ cells/ml) in RPMI 1640 medium without FCS was then added to the wells of the upper plate. The chamber was incubated at 37°C for 16 h in 5% CO₂ in air. After incubation, the medium in the wells of the upper plate was replaced with 200 µl of phosphate-buffered saline (PBS) containing 20 µM ethylenediaminetetraacetic acid and incubated at 4°C for 30 min. Cells remaining on the top of the polycarbonate membrane were removed with cotton Q-tips. Cells that had migrated into or through the filter were collected by centrifugation using a rotor for 96-well plates at 500 × g for 10 min. Defined medium was removed and replaced with complete medium (RPMI 1640 with 10% FCS) for 1 to 3 h at 37°C. The number of displaced cells was quantified by using the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). The assay uses (3-(4,5-dimethylthiazol-2-y1)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) and the electron coupling reagent phenazine methosulfate to form a soluble formazan proportional to proliferative cell number (30 µl/well). Cells were incubated with MTS for 1.5 to 2 h and absorbance was measured at 490 nm using an Elx 800 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). In each experiment, five replicates were analyzed for each condition and each experiment was repeated at least three times.

Hydroxyproline Assay

To estimate total lung collagen content, hydroxyproline was measured in at least three animals per condition using previously described procedures with modifications (35). The lung vasculature was perfused free of blood by injecting 10 ml of PBS into the right ventricle. The whole lung was then excised, weighed, and minced, and then hydrolyzed in 2 ml of 6 N HCl at 110°C overnight. The resulting hydrolyate was neutralized with 2 ml of 6 N NaOH and filtered through a 0.45-mm nylon membrane, and 100 ml were then added to 1 ml of 1.4% chloramine T (Sigma), 10% n-propanol, and 0.5 M sodium acetate, pH 6.0. After 20 min incubation at room temperature, 1 ml of Erlich's solution (1 M p-dimethylaminobenzaldehyde in 70% n-propanol and 20% perchloric acid) was added and the resulting solution incubated at 65°C for 15 min. Absorbance was then measured at 550 nm and the amount of hydroxyproline was determined against a standard curve produced using known concentrations of hydroxyproline. To further analyze collagen deposition, sections of the lungs of at least three animals per condition from three separate experiments were examined by standard Masson's trichrome staining. Localization of lung collagen was determined in control and in saline- and bleomycin-injured animals, as well as in peptide and scrambled peptide-treated animals 14 d after treatments.

Reverse Transcriptase/Polymerase Chain Reaction

mRNA was isolated from lungs obtained from at least three animals at each time point for each treatment group using Micro-Fast Track (Invitrogen, San Diego, CA). The quality of extracted RNA was determined by denaturing gel electrophoresis. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (Clontech, Palo Alto, CA) per manufacturer's instructions. Briefly, using an oligo(dT) 18 primer, complementary DNA (cDNA) was generated from 0.2 µg of mRNA. This amount of mRNA per sample was heat-denatured in diethylpyrocarbonate-treated water for 2 min at 70°C and incubated at 42°C for 1 h in a total volume of 20 µl of 20-pmol primers, 0.5 mM of each deoxynucleotide triphosphate, 1 U/µl of ribonuclease inhibitor, and 200 units/µg murine Maloney leukemia virus reverse transcriptase (RT). The reaction was stopped by heating to 94°C for 5 min to destroy any DNA synthetic activity. The reaction mixture was diluted to a final volume of 100 µl and aliquots were stored at 80°C until further use.

To determine the relative quantity of the  chain of collagen type I and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples, polymerase chain reaction (PCR) was used to coamplify each cDNA using primers specific for each protein. The primers used to detect collagen 2(I) were as described by Power and colleagues (36): sense primer for collagen 2(I), 5' CCC ACG TAG GTG TCC TAA AGT 3'; antisense primer for collagen 2(I), 5' CCG TGG TGC TAA AAT 3'. The primers used to detect GAPDH were derived from the rat cDNA sequence as published by Tso and associates (37): sense primer for GAPDH, 5' AAG GTG AAG GTC GGA GTC AAC G 3'; antisense primer for GAPDH, 5' GGC AGA GAT GAT GAC CCT TTT GGC 3'. PCR amplification of specific cDNAs was achieved using 2.5 units Taq DNA polymerase and 0.2 µM primers in a 100-µl reaction mixture. PCR conditions were 94°C for 1 min for denaturation, 60°C for 1 min for annealing, and 2 min at 72°C for extension, run for 30 cycles. These conditions were determined previously to provide for linear exposure and amplification of these sequences (data not shown). PCR products were electrophoresed in a 1% agarose gel and transferred to nylon membranes (Amersham, Oakville, ON, Canada). The identity of all products was confirmed by Southern blotting using internest probes.

Statistical Analysis

Statistical comparisons were carried out using analysis of variance with Fisher's exact test. A P value less than 0.05 was considered significant.

	Results

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Macrophage Accumulation and Motility in Relation to Lavage HA after Bleomycin

Using ED-1 staining to label macrophages, we first quantified the accumulation of macrophages in the lung after intratracheal treatments. Uninjured control animals had low macrophage counts in the lung (6.6 ± 1.6 macrophages per hpf). Whereas saline-treated animals tended to have an increase in macrophage accumulation at 4 d, bleomycin-treated animals showed a 4- to 5-fold increase in macrophages at 4 and 7 d after injury (P < 0.05, Figure 1a). These data are consistent with previous literature describing the composition of the cellular infiltrate after injury in this model (7, 8, 38).

To determine changes in macrophage motility after bleomycin injury, we used time-lapse cinemicrography to measure the locomotion of macrophages in the first 2 h after isolation by BAL from control and bleomycin-treated animals. Macrophages obtained 2 to 7 d after bleomycin treatment showed significantly higher velocities than did saline-treated controls, with highest velocities noted at 4 d (P < 0.01, Figure 1b). Macrophages isolated from saline-treated animals 4 d after treatment also showed a small but significant increase in cell locomotion as compared with untreated controls (P < 0.05, Figure 1b). This may represent injury as a result of instillation of saline into the lungs.

To document changes in lavage HA concentrations after bleomycin injury, we quantified the HA content in BAL of bleomycin-treated animals and controls. Compared with uninjured controls, animals receiving intratracheal saline showed a significant 2- to 3-fold increase in lavage HA content 4 d after treatment (P < 0.05). Bleomycin-injured animals, however, had a maximal 200- to 250-fold increase in lavage HA as compared with controls at 4 and 7 d (P < 0.001, Figure 1c). HA content of lavage from injured animals decreased to baseline by 14 d (Figure 1c).

HA Content Increases in Macrophages after Bleomycin Lung Injury

We next examined the localization of tissue HA in lungs after bleomycin-induced injury (Figure 2). In normal rat lungs, HA was localized to the subepithelial matrix and surrounding bronchiolar and arterial smooth-muscle cells, and on arterial endothelium (Figure 2b). At 4 d after injury, macrophages accumulating within air spaces of injured areas of the lung were strongly positive for HA (Figure 2c). Sections either pretreated with hyaluronidase (data not shown) or probed with bHABP that had been preincubated with excess HA (Figure 2a) did not exhibit staining, thereby confirming the specificity of the probe for HA. To confirm that the HA was indeed in macrophages, we double-labeled sections with FITC-conjugated ED-1, a rat macrophage-specific antibody (Figure 2d), and bHABP identified by Texas Red-conjugated streptavidin (Figure 2e). Although HA was found in other areas as described earlier, HA was colocalized to accumulating macrophages after bleomycin injury (Figure 2f).

View larger version (61K): [in this window] [in a new window]   Figure 2.   Histochemical localization of HA after bleomycin injury. (a) Control section of normal, uninjured rat lung stained using bHABP that had previously been preincubated with excess unlabeled HA. The lack of staining confirmed the specificity of the probe for HA. (b) Constitutive expression of HA (brown color) occurred in the subepithelial matrix of bronchioles and around bronchiolar smooth muscle. (c) At 4 d after bleomycin injury, maximal staining for HA was seen in macrophages accumulating in injured areas of the lung (arrows). These changes were not observed in saline-treated animals (data not shown). (d-f  ) Colocalization (f, yellow) of FITC-ED-1 to identify rat macrophages (d, green) and Texas Red-bHABP to bind HA (e, red) indicates that HA is localized to macrophages. Staining for HA in other areas is also noted. The inset in f   shows a higher-power image of a macrophage close to the HA staining noted in the subepithelial matrix. Arrows indicate macrophages. The photomicrographs shown are representative of five independent experiments. Original magnification: ×400 for all photomicrographs except the insert in f, which was ×1,000.

HA Is Necessary for Increased Macrophage Motility after Bleomycin Injury

To investigate the role of HA in macrophage motility, the mean velocity of alveolar macrophages isolated from bleomycin- or saline-treated animals 7 d after treatment was measured in the presence of HA-binding peptide mimicking the first HA-binding domain of RHAMM (aa 401-411 [30]). HA-binding peptide significantly (P < 0.01) inhibited the increased locomotion of macrophages obtained from animals after bleomycin treatment (Figure 3). A non- HA-binding scrambled peptide, used as a control, had no effect on the motility of these cells (Figure 3).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Effect of HA-binding peptide on macrophage motility after injury. As noted earlier, cells isolated 7 d after bleomycin had a significantly greater mean cell velocity compared with those isolated from saline-treated controls (*P < 0.01 versus control and saline). Treatment with a scrambled peptide had no effect on cell velocity. HA-binding peptide mimicking the first HA-binding domain of RHAMM blocked the increase in velocity observed in macrophages isolated from bleomycin-treated animals (#P < 0.01 versus bleomycin and bleomycin plus scrambled peptide). Values represent mean and standard errors of five animals studied for each condition with at least 20 cells tracked per animal studied.

We have previously described a basic HA-binding motif (BX₇B), where two basic amino acids (usually lysine or arginine) flank seven nonacidic residues (31). By using arginine and glycine, we created a synthetic peptide mimicking the optimal HA-binding motif (RGGGRGRRR [31], Peptide A). Preliminary experiments studying the motility of BAL-isolated macrophages showed that these cells had decreasing cell velocities with time after isolation (data not shown). We therefore employed a chemotaxis assay using 5% FCS as a chemoattractant to test the effect of this synthetic peptide on rat alveolar macrophages isolated by BAL and allowed to become quiescent overnight. Peptide A significantly inhibited the chemotaxis of macrophages to 5% FCS by 43 ± 1% compared with the scrambled peptide (RGRRGRGRG) control (n = 5, P < 0.001). We therefore decided to use these peptides in vivo to determine their effects on macrophage accumulation and collagen deposition after bleomycin-induced lung injury.

In Vivo Effects of HA-Binding Peptide on Bleomycin-Induced Lung Injury

Rats were treated subcutaneously 1 h before bleomycin injury with 80 mg/kg of either Peptide A or its scrambled control, and compared with non-peptide-treated controls. In separate experiments, we assessed the effects of these peptide treatments on the motility of macrophages isolated by BAL at 4 d after treatment (Figure 4a), and macrophage accumulation as determined by the number of ED-1-positive cells 7 d after treatment (Figure 4b). As described earlier, macrophages from animals 4 d after bleomycin had an approximately 4-fold increase in mean cell velocity as compared with control animals (Figure 4a). Peptide A treatment of animals before bleomycin injury resulted in inhibition of alveolar macrophage motility to baseline also at 4 d (Figure 4a). Treatment of animals with scrambled peptide had no effect on bleomycin-induced increases in macrophage motility, thereby confirming the specificity of Peptide A (Figure 4a). As described earlier, macrophage accumulation in the lung was significantly increased in animals given bleomycin alone (Figure 4b). Treatment with Peptide A resulted in a significantly decreased macrophage accumulation as compared with those animals given bleomycin alone or those treated with the scrambled peptide control (Figure 4b).

View larger version (11K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 4.   In vivo effects of Peptide A on macrophages after bleomycin. (a) The motility of BAL cells 4 d after injury. Macrophages from bleomycin-treated animals showed increased velocities as compared with those from control and saline-treated animals (*P < 0.001). Animals pretreated with scrambled Peptide A showed the same velocity as macrophages obtained from animals injured with bleomycin. However, macrophages from animals treated with Peptide A before bleomycin-induced injury had velocities similar to those of control or saline-treated animals and showed significantly lower velocities than either bleomycin-injured or scrambled peptide-treated controls (#P < 0.001). Values represent mean and standard error with three animals studied for each condition and at least 20 cells tracked per animal studied. (b) ED-1-positive cells per hpf in lung sections. Bleomycin treatment resulted in increased ED-1-positive macrophages per hpf in the lung. Peptide A treatment of animals resulted in significantly decreased numbers of ED-1-positive cells per hpf (*P < 0.05 versus bleomycin and bleomycin plus scrambled peptide), whereas animals treated with scrambled peptide had similar numbers of macrophages as those treated with bleomycin alone.

We used three approaches to investigate the effect of Peptide A treatment on collagen deposition. We determined both the steady-state mRNA content of the 2 chain of collagen type I by RT-PCR of mRNA obtained from lungs at 4 d (Figure 5a) and the hydroxyproline content of whole lungs at 14 d (Figure 5b) after treatment. Further, using trichrome staining of lung sections, we examined the effects of treatments on collagen deposition and lung architecture also at 14 d (Figure 6). An increase in the steady-state mRNA content of collagen type 2(I) was noted by 4 d after bleomycin injury (Figure 5a). This increase in collagen mRNA was inhibited by treatment of rats with Peptide A before intratracheal bleomycin (Figure 5a). Scrambled peptide treatment of rats before injury had no effect on collagen type 2(I) mRNA content (Figure 5a). As expected, hydroxyproline content of rat lungs 14 d after bleomycin was significantly increased as compared with controls (Figure 5b). Peptide A treatment before injury resulted in significantly lower hydroxyproline content at 14 d as compared both with bleomycin injury alone and with bleomycin and scrambled peptide treatment (Figure 5b). Trichrome staining of lung sections (Figure 6) confirmed the increase in collagen deposition and loss of normal lung architecture 14 d after injury. However, Peptide A treatment of animals before bleomycin resulted in considerably decreased trichrome staining for collagen and a preservation of more normal lung architecture as compared with injured animals given scrambled peptide (Figure 6). Collectively, these data indicate that HA-binding peptide treatment resulted in decreased macrophage accumulation and collagen deposition after bleomycin-induced lung injury.

View larger version (33K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Figure 5.   In vivo effects of Peptide A on collagen accumulation after bleomycin. (a) Lung collagen type 2(I) mRNA at 4 d. An increase in collagen type 2(I) mRNA content was observed as early as 4 d after injury. This increase was completely inhibited by Peptide A, whereas the scrambled peptide control had no effect on the mRNA content of this collagen. Data shown are representative of two separate experiments giving similar results. (b) Hydroxyproline content of lungs at 14 d. Both the bleomycin-alone and bleomycin-plus-scrambled peptide groups had increased hydroxyproline content as compared with saline-treated animals (*P < 0.05, n = 4/condition). HA-binding peptide treatment resulted in significantly decreased hydroxyproline as compared with animals given either bleomycin alone or bleomycin with scrambled peptide treatment (#P < 0.05, n = 4/condition).

View larger version (190K): [in this window] [in a new window]   Figure 6.   Lung collagen staining and architecture after peptide treatment. Trichrome staining of lung sections was used to examine collagen deposition and lung architecture. Compared with saline-treated animals (a), increased collagen staining was noted in animals given bleomycin (b), or bleomycin plus scrambled peptide (c) 14 d after treatment. At the same time point, HA-binding peptide treatment resulted in decreased collagen staining and a more normal lung architecture (d). Data shown are representative of at least three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The histologic and pathophysiologic changes observed after intratracheal instillation of bleomycin in rodents include the death of epithelial cells and an influx initially of neutrophils followed by macrophages and then fibroblasts in the injured lung (5). Injury is accompanied by profound changes in the synthesis and secretion of a number of cytokines and growth factors (39), and in components of the extracellular matrix (13, 21). The macrophage appears to be a key component in the process of fibrosis after lung injury (7, 38). The mechanisms by which macrophages accumulate at sites of injury have received considerable attention. Circulating monocytes undergo activation (40, 41) and become adherent at sites of endothelial activation (42, 43), a process that localizes the activated cells close to the site of injury. Migration of macrophages through the endothelium and subsequently in the affected tissue results in their accumulation in injured areas (44). This process is highly ordered and requires the involvement of several distinct families of molecules including selectins, integrins, and monocyte-specific chemoattractants (45).

In the present study, we demonstrated increased HA in macrophages accumulating in areas of the lung injured by intratracheal instillation of bleomycin. Coincident with the elevated HA, the motility of macrophages isolated from injured lungs was also increased. Using HA-binding peptide, we showed that HA is required for the increased macrophage motility observed after injury. The ability of HA-binding peptide to decrease macrophage motility and accumulation, and to limit the increases in lung collagen deposition that normally occur after bleomycin injury, suggests that HA plays a critical role in the inflammatory process and fibrotic consequences of acute lung injury.

The effect of HA-binding peptide on cell motility is most likely occurring through sequestration of HA, thereby competitively inhibiting surface receptors. There are several potential sites of action of HA-binding peptide to exert its anti-inflammatory effects in vivo. For instance, HA, acting via the cell-associated HA receptor CD44, is known to activate monocytes to produce insulin-like growth factor-I (23). CD44, presumably acting thorough its ability to bind HA, has also been implicated in novel rolling and adhesion pathways leading to leukocyte adhesion to activated endothelium (26, 27). We have previously shown that inflammatory cell chemotaxis and random migration is dependent on RHAMM (32). RHAMM-HA interactions may therefore be responsible for leukocyte transmigration through the endothelium and for chemotaxis within injured areas of the lung. Thus, administration of HA-binding peptide would be predicted to sequester HA and interrupt its interaction with HA receptors, resulting in decreased monocyte activation, adhesion, and migration. Further, induction of proinflammatory molecules such as chemokines and inducible nitric oxide synthase by HA-CD44 interactions (24, 25, 46) would also be inhibited. The decreased accumulation of macrophages and decreased collagen deposition observed in the lungs of animals treated with HA-binding peptide support these contentions.

We note that maximum lung HA content on Day 4 coincided with maximum motility of macrophages isolated by lavage. HA concentrations decreased thereafter. The dynamics of HA-receptor interactions in this response to injury are unclear at present, but several authors have suggested that internalization of HA by receptors may be required for the biologic actions of HA (47). It is possible that the decreasing content of HA after intratracheal bleomycin occurs due to receptor-mediated internalization and is required for the inflammatory and/or fibrotic response to injury. Changes in the expression of certain CD44 isoforms after bleomycin-induced lung injury in rats have recently been reported (51). Although the authors found no correlation between the presence of CD44 isoforms and the extent of pulmonary injury, reactive changes in epithelial and nonepithelial cells were found (51). The expression of the standard form of CD44 was increased in alveolar macrophages and in the interstitium of the lung in areas of thickened alveolar wall, whereas the expression of CD44v6, thought to be the epithelial form, was decreased in type II pneumocytes (51).

Teder and coworkers (52, 53) proposed mechanisms for the increased production of HA after bleomycin injury in rats. They showed that fibroblasts exposed to BAL from injured animals increased their production of HA, a response that was largely abrogated by blocking antibodies to transforming growth factor (TGF)-1 (52). Further, alveolar macrophages obtained 5 d after bleomycin injury bound less [³H]HA than did those from saline-treated controls, suggesting lower HA receptor expression in macrophages after injury. This lower expression of HA receptors was also thought to contribute to elevated HA because less HA was internalized by these cells (52, 53). However, several investigators have shown that growth factors such as TGF-1 and platelet-derived growth factor-BB increase both HA production and HA receptor expression (54). Additionally, increased expression of CD44 after bleomycin injury has been reported (51, 53). We are currently completing studies on the expression of RHAMM in bleomycin-induced lung injury and have also noted an increased synthesis and expression of this HA receptor in accumulating macrophages (Savani and colleagues, manuscript in preparation). Alternatively, three mammalian HA synthases have recently been cloned and partially characterized (57), and both lung fibroblast (58) and alveolar macrophage (59) hyaluronidases have been described. There is currently little to no information as to the coordinate changes in content or activity of these enzymes after bleomycin-induced lung injury. Therefore, the exact mechanisms of HA turnover in the lung after injury remain unclear.

The increase in HA accumulation observed during tissue responses to injury are paradoxical to the large body of information citing the anti-inflammatory effects of exogenously administered HA in vitro and the use of HA as an anti-inflammatory agent in vivo. Indeed, HA at doses of 1 mg/ml or greater has been reported to inhibit inflammatory cell chemotaxis (60), phagocytosis (60, 61), elastase release (62), and respiratory burst activity (61). HA also acts as an anti-inflammatory and antifibrotic agent in rheumatoid and osteoarthritis (63), and in repair of tympanic membrane perforations (64). In addition, HA accelerates cutaneous wound healing (65) and reduces adhesion formation after intra-abdominal surgery (66). On the other hand, HA has been linked to the process of macrophage maturation (23), and its expression is greatly increased during inflammatory conditions such as myocardial infarction (67) and arthritis (68), and during transplant rejection (69). Further, removal of HA with early treatment of myocardial infarction with hyaluronidase results in reduced myocardial fibrosis and infarct size (70). These discrepancies could be explained by dose-dependent differences in the action of HA and/or by differing effects of low and high molecular-weight (mw) HA. For instance, it is known that the effect of HA on fibroblast cell motility is dose-dependent, with maximal stimulation of locomotion at 0.01 µg/ml (71). Further, 6- to 25-saccharide-long HA units stimulate angiogenesis, whereas large-mw HA does not (72), and macrophage activation occurs with low- rather than high-mw HA (24). In bleomycin-induced lung injury, the mw of HA obtained by BAL is 0.2 to 0.7 × 10⁶ (52, 73), whereas exogenously administered HA is considerably larger at 10⁹ mw (Sigma).

In lungs injured by bleomycin, increased HA in macrophages coincides with the temporal increase in TGF-1 expression observed in the same cells after similar injury (14). This similarity in expression suggests in vivo regulation of HA synthesis and accumulation in macrophages by TGF-1. Additionally, the ability of HA-binding peptide to inhibit macrophage motility predicts the likely regulation of macrophage motility by HA in vivo. In fibroblasts, HA-receptor interaction is required for TGF-1-stimulated increases in cell locomotion (55). TGF-1 may thus be responsible for an autocrine system in which production of this growth factor results in upregulation of HA and its receptors in macrophages, thereby mediating their migration to sites of injury. Our data, however, showing a decrease in macrophage accumulation with HA-binding peptide treatment, suggest that HA precedes and is at least partly responsible for the inflammatory response to acute lung injury. Whether low mw HA regulate TGF-1 gene expression in macrophages is currently unknown.

In conclusion, we demonstrate here that HA is a critical player in the macrophage response to lung injury and plays a pivotal role in the development of pulmonary inflammation and fibrosis seen after administration of intratracheal bleomycin in rats. Using current knowledge together with our data, a model for the role of HA in the response to lung injury can be developed (Figure 7). Injury results in an increase in HA and a subsequent inflammatory response. Sequestration of HA using the described peptide blocks HA-dependent macrophage activation, adhesion, migration, and chemokine production. Modulation of macrophage responses using HA-binding peptides in vivo may provide potentially powerful tools to reduce both pulmonary inflammation and fibrotic changes after human lung injury.

View larger version (16K): [in this window] [in a new window]   Figure 7.   A model for the involvement of HA in pulmonary inflammation and fibrosis after lung injury. Lung injury results in an increase in HA that, at least in part, mediates the inflammatory response to the injury. Macrophages accumulating in injured areas of the lung, also partly through HA-mediated regulation, produce cytokines and growth factors, leading to fibrosis. Use of HA-binding peptide to sequester HA would then decrease the accumulation and activity of inflammatory cells in the lung and dampen subsequent fibrosis.