Introduction

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Hyaluronan (HA) is a nonsulfated, linear glycosaminoglycan that is ubiquitously distributed in the extracellular matrix and has been implicated in development and wound healing as well as in tumor invasion and metastasis (1). HA is synthesized by cells of mesenchymal origin in response to growth factors and other stimuli (2). Recently, a family of related vertebrate genes encoding putative HA synthases (HAS1, HAS2, and HAS3) have been identified and cloned (3). The vertebrate HAS isoforms exhibit 55 to 71% sequence similarities, whereas homologous isoforms of human and mouse share about 96 to 99% complementary DNA (cDNA) sequence identities (8). The expression of each HAS gene is cell type-specific and is differentially regulated in response to external stimuli. The effects are dependent upon gene induction (9), but resident HAS proteins can also be activated in a pathway involving protein kinase (PK)C (10). Further, each HAS isoform possesses the ability to synthesize HA of a given size (11). Genes encoding HA-degrading enzymes, i.e., hyaluronidases (HYALs), have also been cloned and characterized (12, 13). Multiple HYAL genes exist but only three encoded proteins, termed PH-20 (14), HYAL1 (15), and HYAL2 (16), have been partially characterized. The different HYAL exhibit different catalytic mechanisms and substrate specificities (15, 16). HYAL has been shown to be elevated during prostate cancer progression (17) and in the early phase of lung injury (18). Until now, the molecular mechanisms controlling the activities of HAS isoforms and HYAL under normal and pathologic situations are not well understood.

Under normal conditions the biosynthesis and degradation of HA are regulated in a tissue-specific manner. However, the balance is altered in several disorders that lead to an accumulation of HA in tissues or in blood or other body fluids (19). HA is a common constituent of lung tissue, but in the early course of fibrotic lung injury a transient accumulation of HA is seen (22). Although HA has been suggested to be a lung injury marker, the mechanisms responsible for the perturbation of HA biosynthesis and catabolism during the process of the inflammation are poorly understood.

Our present study was aimed at investigating the mechanisms involved in the accumulation of HA in the lungs of irradiated rats and to assess the mode of action of growth factors on the expressions of HAS isoforms and HYALs.

	Materials and Methods

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Materials

Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline without Ca²⁺ and Mg²⁺ (PBS), and trypsin-ethylene-diaminetetraacetic acid (EDTA) (0.25 to 0.02%, wt/vol) were purchased from SVA (Statens Veterinärmedicinska Anstalt, Uppsala, Sweden). Recombinant platelet-derived growth factor (PDGF)- BB and transforming growth factor (TGF)-1, and immunoglobulin G fraction of rabbit antisera recognizing PDGF-BB, were generously provided by Drs C.-H. Heldin and P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden). [³H]HA (relative molecular mass [M_r] 0.97 × 10⁶, 68.2 µg/ml) was a generous gift from Dr. J. R. E. Fraser (Melbourne, Australia). Anti-TGF-, neutralizing all isoforms of TGF-, was purchased from R&D Systems (Abingdon, UK). HA of M_r 3.9 × 10⁶ was a kind gift from O. Wik (Q-Med, Uppsala, Sweden). HA oligosaccharides of defined sizes were prepared as described by Rahmanian and colleagues (25) and were designated by the number of monosaccharide residues per molecule, e.g., HA₆ stands for a hexasaccharide. DNase I was purchased from Boehringer Mannheim (Mannheim, Germany). Bovine testicular HYAL type IV-S, collagenase type IV, pepstatin, leupeptin, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). Aprotinin was purchased from Bayer (Leverkusen, Germany). Dormicum was purchased from F. Hoffmann-La Roche AG (Basel, Switzerland) and Hypnorm was purchased from Janssen (Burkinghamshire, UK). cDNAs for procollagen 1(I) and procollagen 1(III) were generously provided by Drs. A. McWhirter and K. Rubin (Uppsala University, Uppsala, Sweden). cDNAs for HAS2 and HAS3 were generously provided by Dr. A. P. Spicer (University of California).

Induction of Radiation-Induced Lung Injury

Adult male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) weighing about 200 g were used. The animals were divided into six groups (three rats in each group) and provided with standard chow and tap water ad libitum. The investigation was approved by the Ethical Committe for Animal Research in Uppsala, Sweden. Before radiation, each rat was anesthetized by intraperitoneal injection of a solution (100 µl/100 g body weight) composed of equal parts of Dormicum (2.5 mg/ml) and Hypnorm (10 mg/ml), and thereafter placed on a platform. The left lung was covered with a lead shield and the right lung was irradiated with ⁶⁰Co- ray in a field of 3 × 3 cm at an average exposure rate of 0.91 Gy/ min to a total dose of 30 Gy at a depth of 1.5 cm. Control animals were treated in the same way but were not irradiated. The animals were killed at 2, 4, 6, 8, and 10 wk after irradiation.

Preparation of Bronchoalveolar Lavage Fluid Alveolar Macrophages and Lung Tissue Fibroblasts

Isolation of bronchoalveolar lavage fluid (BALF)-alveolar macrophages (AMs) was performed as described by Teder and Heldin (26). Briefly, the animals were tracheostomized under anesthesia at different time points after irradiation, and a catheter was inserted into the trachea. Vascular perfusion with 20 ml of PBS was performed via the right heart. Using an in situ technique, lungs were removed from the thorax and lavaged by intratracheal infusions of 7 ml of cold PBS (five times) under gravity at a hydrostatic pressure of 20 cm. The BALF (34 ml) was recovered into a polypropylene tube kept on ice and centrifuged at 820 × g at 4°C for 10 min, and the supernatants were kept frozen at 20°C until use. The cell pellet was gently dissolved in 3 ml PBS containing 0.3% bovine serum albumin (BSA), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml fungizone (PBS-BSA), and mixed with 7 ml Percoll solution (Pharmacia, Upjohn) (9 parts of Percoll diluted with 1 part of 10-fold concentrated PBS). The cell suspensions were then underlayered with a two-step Percoll gradient (15 ml of 65% Percoll solution, 20 ml 35% Percoll solution, and 5 ml PBS-BSA) in 50-ml tubes and centrifuged at 1,850 × g for 30 min. BALF-AMs were obtained from the interface between the 65 and 35% Percoll layers and were resuspended in 50 ml PBS-BSA, followed by centrifugation at 820 × g for 10 min to remove the Percoll solution. The total cell number was measured in a Bürker chamber and BALF-AMs were seeded into 35 × 10 cm cell culture dishes (1 × 10⁶ cells/dish) and cultured at 37°C in DMEM supplemented with 0.1% fetal calf serum (FCS) in a humidified atmosphere of 5% CO₂ for 24 h. At that time the conditioned media were collected and kept frozen at 20°C until use.

After bronchoalveolar lavage, the lung tissue was minced to about 3-mm³ pieces and subjected to enzymatic digestion (two cycles at 37°C, 30 min each cycle) in 5 ml trypsin-EDTA solution, each time supplemented with 1 mg/ml type IV collagenase, 1 mg/ ml BSA, and 100 µg/ml DNase I. Then 10 ml of DMEM medium containing 10% FCS was added to inhibit the proteolytic activity of trypsin. The dispersed lung cells were filtrated through a 75-µm sterile gauze and centrifuged at 200 × g for 10 min, and the cell pellets were gently resuspended in 4 ml of serum-free DMEM. Rat lung fibroblasts were separated from interstitial macrophages by plating the cells into 35 × 10 mm cell culture dishes for 15 min at 37°C in a humidified atmosphere of 5% CO₂. During this short culturing time, essentially only interstitial macrophages were attached to the culture dishes. The nonadherent cells, lung fibroblasts, were removed and reseeded in DMEM supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 4 mM L-glutamine (complete medium).

HA Binding Assay

The HA-binding capacity of HA cell surface receptors on rat BALF-AMs isolated at different times after irradiation was investigated essentially as described previously (24). The freshly isolated cells were washed once with DMEM containing 0.1% FCS and were cultured in 1 ml medium for 24 h. After four washes with PBS, 1 ml PBS containing 0.25 µg/ml [³H]HA in the absence or presence of 100 µg/ml of nonlabeled HA was added and the cells were incubated for 1 h at room temperature with gentle shaking, followed by four washings in PBS. The cell layers were then solubilized in 600 µl/well of 0.3 M NaOH and 1% sodium dodecyl sulfate (SDS) for 30 min at room temperature, followed by the addition of 100 µl 2 M HCl. The cell extracts were then mixed with scintillation cocktail and subjected to scintillation counting. The specific binding was determined by subtraction of radioactivity retained after addition of nonlabeled HA from the radioactivity detected in the absence of unlabeled HA.

Determination of HA in Rat Lung Fibroblast Cultures and in BALF

Rat lung fibroblasts from nonirradiated rats (5 × 10⁴ cells/well in 12-well Falcon plates) grown in complete medium were cultured for 24 h in DMEM containing 0.1% FCS (starvation medium). At this time, medium was changed and the cultures were incubated in fresh starvation medium in the absence or presence of 50 ng/ml PDGF-BB, 5 ng/ml TGF-1, 10 nM phorbol 12-myristate 13-acetate (PMA), or BALF supernatants obtained at different time points after irradiation. After 24 h of incubation, the HA content in the conditioned media was measured using a commercial kit (HA Test 50; Kabi Pharmacia Diagnostics, Uppsala, Sweden). This kit is based on the high affinity of HA for a specific protein from nasal bovine cartilage (27). The content of HA in BALF at different times after irradiation was also measured by the same kit. To exclude the possibility that other HA binding proteins (such as tumor necrosis factor [TNF]-stimulated gene 6 and inter--trypsin inhibitor) compete for binding to HA some samples were pretreated with 100 to 200 µg proteinase K/ml at 37°C for 75 min in a total volume of 0.5 ml, followed by heating at 95°C for 10 min before the HA content detection assay. The measurements were performed in duplicate and the intervariability was less than 10%.

Northern Analysis

Subconfluent fibroblast cultures from nonirradiated rats (2 × 10⁶ cells/175 cm² Falcon flask, six bottles) were cultured for 24 h in complete medium and starved for another 24 h. At this time, fresh starvation medium was added and the cells were cultured for 6 h in the absence or presence of 50 ng/ml PDGF-BB, 5 ng/ml TGF-1, 10 nM PMA, 10% FCS, and BALF supernatants obtained at different times after irradiation (25% in relation to culture medium, vol/vol). Messenger RNAs (mRNAs) were prepared using a commercially available kit, essentially according to the instructions of the manufacturer (Maxi Message Maker; R&D Systems), except that the cells were lysed directly in the bottles with lysis buffer and harvested with a rubber policeman. Total RNAs from nonirradiated and irradiated lung tissues as well as from fibroblast cultures treated with HA of different molecular mass were prepared by using the acid guanidine thiocyanate-phenol-chloroform extraction method (28).

To quantify the expression of mRNAs for HAS1, HAS2, and HAS3, as well as for HYAL1 and HYAL2, in nonstimulated and stimulated cells, mRNAs (2 µg) were separated on 1% agarose gel containing formaldehyde and transferred overnight to Hybond N⁺ nylon membranes (Amersham, Sweden). After ultraviolet cross-linking fixation for 2 min, the membranes were prehybridized in Rapid-hyb buffer (Amersham) for 1 h at 65°C, according to the instructions of the manufacturer. Each membrane was sequentially hybridized for 3 h at 65°C (about 1.5 × 10⁶ counts per min/ml for each hybridization mixture) with [³²P]-labeled cDNA probes for each HAS and HYAL isoform (Mega Prime; Amersham). [³²P]-labeled cDNA probes for human HAS1 (hHAS1) (EcoRI/NotI digest of hHAS1 cDNA in pcDNA3 vector, 2,116- base pair [bp] nucleotide sequence [4]), mouse HAS2 (mHAS2) (EcoRI digest of mHAS2 cDNA in pCIneo, 926-bp nucleotide sequence [6]), mHAS3 (EcoRI digest of mHAS3 cDNA in pCIneo, 1,665-bp nucleotide sequence [7]), HYAL1 (EcoRI/HindIII digest of hHYAL1 cDNA in pCR3.1-Uni vector, 1,329-bp nucleotide sequence [15]), and HYAL2 (EcoRI digest of hHYAL2 cDNA in pZErO^rm-2 vector, 2,010-bp nucleotide sequence [16]) were used. The blots for total RNA quantification were hybridized with labeled cDNA probes representative for procollagen 1(I) (PstI/PvuII digest of human procollagen 1[I] cDNA in pUC8 vector, 327-bp nucleotide sequence [29]) and procollagen 1(III) (PstI digest of human procollagen 1[III] cDNA in pUC8 vector, 295-bp nucleotide sequence [30]), as well as with the HYAL1 and HYAL2 cDNA probes described earlier. After hybridization, the blots were washed two times for 20 min at 50°C in 2× saline sodium citrate (SSC)/0.1% SDS, two times for 30 min at 65°C in 1× SSC/0.1% SDS, and one time for 10 min at 65°C in 0.1× SSC/0.1% SDS. The blots were then exposed to X-ray film at 70°C with intensifying screens. After exposure, the membranes were stripped by boiling for 5 min in 0.1% SDS, exposed to X-ray film overnight to ensure the complete removal of the probe, and rehybridized. Variation in sample loading was evaluated by probing the membranes with [³²P]-labeled cDNA for human glyceraldehyde-3-phosphate dehydrogenase. The mRNA expression levels were quantified by a phosphoimager (Bio-Rad PhosphorImager and associated software).

Detection and Quantification of HYAL Activity in Rat Lung Fibroblast Cultures

Fibroblast cultures obtained from nonirradiated rats were grown in complete medium containing 10% FCS for 24 h (1.5 × 10⁶ cells/ 175 cm² Falcon bottles), starved for another 24 h in medium containing 0.1% FCS, and then cultured for 24 h in starvation medium alone or in starvation medium supplemented with 5 ng/ml TGF-1, 50 ng/ml PDGF-BB, 10 nM PMA, or 10% FCS. The conditioned media were then removed and supplemented with protease inhibitors at a final concentration of 1 µg/ml each of aprotinin, pepstatin, and leupeptin, and 0.2 M of PMSF. After clearing by centrifugation at 1,000 × g for 10 min at 4°C, the media were dialyzed overnight at 4°C in 0.01 M sodium formate buffer, pH 3.7, containing 0.01 M NaCl, using a dialysis membrane of 12,000 to 14,000 molecular weight cutoff. The dialysates were concentrated 10-fold and centrifuged at 15,000 × g for 10 min at 4°C, and the supernatants were stored at 20°C until quantification of HYAL activity. After removal of the conditioned media, 1 ml lysis buffer (0.01 M formate, pH 3.7, containing 0.01 M NaCl, 0.1% Triton X-100, 50 U/ml DNase I, and protease inhibitors) was added and the cells were harvested from the bottles with a rubber policeman. The cell extract was left for 30 min on ice, followed by sonication utilizing a Soniprep 150 MSE (Manor Royal, Crawley, Sussex, UK). The extracts were then centrifuged at 10,000 × g for 15 min at 4°C to remove cell debris; supernatants were concentrated 10-fold and stored at 20°C until HYAL assay.

Detection of the HYAL activities in unstimulated and stimulated fibroblast cultures was performed using a microtiter-based assay essentially as described by Frost and Stern (31). Briefly, biotinylated HA was immobilized covalently in a 96-well Covalink-NH plate (10 µg/well) (Nunc Brand Products, Denmark). The plate was washed three times for 5 min each with PBS containing 2 M NaCl and 50 mM MgSO₄ and equilibrated for 10 min at room temperature with 0.1 M sodium formate buffer, pH 3.7, containing 0.1 M NaCl, 1% Triton X-100, and 5 mM saccharolactone (enzyme buffer). The buffer was then aspirated, and media samples as well as cell layer lysates, diluted 5- and 10-fold, respectively, with enzyme buffer, were added to the plate (100 µl/well), followed by incubation for 30 min at 37°C. Bovine testicular HYAL was used as a standard. The reaction was terminated by the addition of 6 M guanidine-HCl (200 µl/well). The plate was then washed three times for 5 min each in PBS containing 2 M NaCl, 50 mM MgSO₄, and 0.05% Tween 20. Streptavidin-biotinylated horseradish peroxidase complex (Amersham) diluted 1:1,600 in PBS containing 0.1% Tween 20 was added (100 µl/well) and samples were incubated for 30 min at room temperature. After three washes in PBS containing 2 M NaCl, 50 mM MgSO₄, 0.05% Tween 20, the samples were incubated for 14 min at room temperature in the dark with substrate solution composed of 0.4 mg/ml o-phenylenediamine (Sigma, Sweden) in 50 mM citrate phosphate, pH 5.0, and 0.012% H₂O₂ (100 µl/well). The reaction was stopped by adding 50 µl/well of 8 M H₂SO₄ and the product was measured by reading the absorbance at 492 nm using a Titertek Multiskan PLUS automated plate reader (EFLAB, Finland).

	Results

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BALF-AMs and Lung Tissue Fibroblast Cultures

Rats exposed to irradiation with ⁶⁰Co- ray, to a total of 30 Gy, tolerated the treatment well with no lethality. However, on the irradiated part of the right lung, blood patches appeared transiently between 2 and 4 wk after irradiation followed by shrinkage of the right lung after 10 wk. AM cultures were obtained from BALF at various times after irradiation, and were purified to about 95% purity (26). The obtained cultures exhibited typical macrophage morphology when examined with a light microscope and no differences were observed between AMs isolated from nonirradiated and irradiated rats (data not shown). The total number of AMs in BALF decreased about 43% compared with that of control rats at 2 wk after irradiation and then gradually increased, reaching control levels after 8 wk (Table 1). However, at 10 wk after irradiation, a decrease in the number of BALF-AMs to about half of that of control rats was observed, possibly due to the extensive shrinkage of right lungs not permitting complete lavage of the lungs.

Figure 1 shows the morphologic changes of fibroblasts obtained from nonirradiated and irradiated rat lung tissues at 2 wk after irradiation, after 1 or 2 wk in culture. During these culturing periods, fibroblasts from nonirradiated lung tissue exhibited a characteristic fibroblastic morphology, whereas fibroblasts from irradiated lung tissue exhibited enlarged cytoplasm and nonproliferating ability.

View larger version (134K): [in this window] [in a new window]   Figure 1.   Morphology of lung tissue fibroblasts isolated from nonirradiated and irradiated rats. Cells were isolated from nonirradiated lung tissue and from lung tissue 2 wk after irradiation as described in MATERIALS AND METHODS. Light microscopy photographs were taken after 1 and 2 wk of culture (original magnification: ×250).

HA Content in BALF from Irradiated and Nonirradiated Rats

As shown in Table 1, a marked increase of the HA content in BALF was observed at 6 wk after irradiation compared with that of BALF from nonirradiated rats, followed by a decrease to almost normal levels at 10 wk after irradiation. Pretreatment of BALF at 6 wk after irradiation with proteinase K had no effect on the quantification of HA levels (data not shown), showing that HA binding proteins, such as TNF-stimulated gene 6 and inter--trypsin inhibitor did not interefere with the assay. These results clearly demonstrate that radiation-induced lung injury was accompanied by an accumulation of HA during the early phase.

Expression of HA Cell Surface Receptors on BALF-AMs

To explain the mechanism behind the HA accumulation seen in BALF by 6 wk after irradiation, we investigated the HA binding capacity of BALF-AMs from irradiated and nonirradiated rats. We observed a decrease in the HA binding capacity of BALF-AMs at 4 and 6 wk after irradiation compared with that of nonirradiated rats; the ability of AMs to specifically bind [³H]HA at 4 and 6 wk after irradiation was about 3-fold lower than that of AM isolated from nonirradiated rats (Figure 2). The binding of [³H]HA was inhibited by about 98% in the presence of a 400-fold excess of unlabeled HA (data not shown). However, the decrease in the specific HA binding capacity of BALF-AMs after irradiation was transient and returned almost to control levels at 10 wk after irradiation. Thus, a decrease in the expression of cell-surface HA receptors on the BALF-AMs after irradiation leading to a decreased clearing of HA may partly account for the accumulation of HA during the early phase of lung injury.

View larger version (19K): [in this window] [in a new window]   Figure 2.   HA binding capacity of BALF-AMs isolated at different times after irradiation. [3H]HA (0.25 µg/ml) was added in the absence or presence of nonlabeled HA (100 µg/ml; Mr 3.9 × 106) to BALF-AM cultures (1 × 106 cells). The specific binding was determined by subtraction of the radioactivity retained after the addition of nonlabeled HA. The data shown are means ± standard error of the mean (SEM) from three different experiments.

Effects of BALF from Irradiated and Nonirradiated Rats, PDGF-BB, TGF-, and PMA on HAS Gene Expression and HA Synthesis in Fibroblast Cultures

The transient accumulation of HA in BALF (Table 1) prompted us to investigate whether BALF from irradiated rats contains factors that stimulate HA production and induce HAS gene expression in normal rat lung fibroblasts compared with that of BALF from nonirradiated rats. As shown in Figure 3a, addition of 25% BALF (vol/vol) from nonirradiated rats to the culture medium increased HA production about 3-fold compared with the HA production by fibroblast cells grown in starvation medium, suggesting a constitutive release of HA-stimulating factors by the resident cells in BALF. BALF obtained at different times after irradiation exhibited higher stimulatory activity than BALF from nonirradiated rats. The HA-stimulatory activity in BALF peaked at 6 wk after irradiation; at this time the stimulatory effect of 25% BALF was as high as that of 10% FCS (data not shown). These results indicate that additional HA-stimulatory mediators are present in BALF after irradiation compared with that of control animals. High concentrations of BALF from both control and irradiated rats suppressed HA synthesis, suggesting the presence of HAS activity inhibitory factors (Figure 3a).

View larger version (44K): [in this window] [in a new window]   Figure 3.   Effects of BALF at different times after irradiation on HA production and HAS gene expression in normal rat lung fibroblasts. (a) Growth-arrested fibroblast cultures (5 × 104 cells/ well in 12-well plates) were stimulated with different concentrations of BALF obtained 2, 4, 6, 8, and 10 wk after irradiation and from nonirradiated rats. At 24 h later, the amount of HA in culture media was determined using a commercial HA kit. Values are the average of two separate experiments ± variation. BALF obtained from nonirradiated rats (open squares) and rats 2 wk (filled squares), 4 wk (open circles), 6 wk (filled circles), 8 wk (open triangles), and 10 wk (filled triangles) after irradiation. (b) Quiescent rat lung fibroblasts (2 × 106 cells/175 cm2 bottle) were stimulated for 6 h with 25% BALF obtained at different time periods after irradiation or from control rats. mRNAs were prepared and subjected to Northern blotting analysis as described in MATERIALS AND METHODS. The data shown are representative of one from two separate experiments.

To investigate which of the three HAS isoforms was responsible for the increase in HA in BALF (Table 1), which has been proposed to be closely connected to HA deposition in the lung interstitium (32), the expression of the three HAS genes in the irradiated right lung and nonirradiated left lung was studied. However, we were unable to detect any expression, possibly due to the low number of copies of HAS mRNAs in rat lung tissue (data not shown). Therefore, we investigated the expression of HAS mRNAs in fibroblast cultures. Cultures of cells isolated from nonirradiated lung tissue were incubated for 6 h in media containing 25% BALF that was obtained from nonirradiated and irradiated rats 4, 6, and 10 wk after irradiation. The addition of BALF from irradiated rats resulted in about a 2-fold increase of the 4.8-kb transcript of rHAS2 over that from nonirradiated rats; the 3.5-kb transcript of rHAS2 was only slightly induced (Figure 3b). The mRNA expression of rat HAS1 (rHAS1) and rHAS3 was hardly detected. These results indicate that growth factors and/or cytokines present in BALF from irradiated animals stimulate HA synthesis by lung fibroblasts primarily through upregulation of the rHAS2 gene.

Previous studies revealed that production of growth factors such as TGF-1 and PDGF-BB by resident cells in injured lung tissue are responsible for the overproduction of extracellular matrix compounds characteristic of pulmonary fibrosis (33, 34). Therefore, we investigated whether growth factors such as TGF-1 and PDGF-BB, as well as the tumor promotor PMA, stimulate HA synthesis in lung fibroblasts isolated from nonirradiated rats. We found that TGF-1, PDGF-BB, and PMA are potent stimulators of HA synthesis; the stimulatory activity for each of the three stimuli was about 67% of that of 10% FCS (Figure 4a). To investigate whether the stimulatory effects are at the transcriptional level, we studied the expression of the three rHAS genes using Northern blotting. Treatment of the cells with PDGF-BB led to a 3-fold increase in the expression of rHAS2 mRNA, whereas TGF-1 and PMA induced about a 2-fold increase over the control levels. A slight induction of rHAS3 expression in response to PDGF-BB was also seen. rHAS1 gene could not be detected. These results indicate that TGF-1, PDGF-BB, and PMA mediate their stimulatory effects on HA synthesis in rat lung fibroblast cultures primarily through upregulation of the rHAS2 gene.

View larger version (44K): [in this window] [in a new window]   Figure 4.   Effects of TGF-1, PDGF-BB, and PMA on HA production and HAS gene expression. (a) Quiescent lung fibroblasts from nonirradiated rats were cultured in DMEM containing 0.1% FCS or in medium supplemented with TGF-1 (5 ng/ml), PDGF-BB (50 ng/ml), PMA (10 nM), or 10% FCS for 24 h. The amount of HA in the conditioned media was determined using a commercial HA kit. The data shown are means ± variation from two separate experiments. (b) mRNAs were prepared from quiescent cells cultured as in a for 6 h and then subjected to Northern blotting analysis as described in MATERIALS AND METHODS. The data shown are representative of one of two separate experiments.

Expression of HYAL Isoforms in Nonirradiated and Irradiated Rat Lung Tissues

A possible mechanism through which the HA content in BALF (Table 1) could be lowered, after a transient increase, would be by increased HYAL activities. We investigated this possibility by using Northern blotting on lung tissues obtained from nonirradiated rats as well as from rats 4, 6, and 10 wk after irradiation (Figure 5). We found induced expression of both HYAL1 and HYAL2 mRNAs which peaked at 4 wk after irradiation, followed by marked decreases to levels below the constitutive expression seen in lung tissues from nonirradiated rats (Figure 5). These results suggest that the decrease of HA content in BALF to almost normal level during pulmonary fibrosis may be due to an increase in HYAL1 and HYAL2 during the irradiation alveolitis before the subsequent progressive fibrosis.

View larger version (33K): [in this window] [in a new window]   Figure 5.   Expression of HYAL1 and HYAL2 in irradiated and nonirradiated rat lung tissues. (a) Total RNAs from nonirradiated and irradiated rat lung tissues obtained at different times after irradiation were extracted and subjected to Northern blotting as described in MATERIALS AND METHODS. The data shown are representative of one from three separate experiments. (b) Quantification of the expression levels by PhosphorImaging of the Northern blot.

Effects of TGF-1, PDGF-BB, and PMA on HYAL1 and HYAL2 Activities and Gene Expression

In an attempt to elucidate whether cytokines released during the early phase of lung injury are also responsible for the induction of HYAL, we studied the effects of known growth factors on HYAL1 and HYAL2 gene expression as well as enzymatic activities in normal lung fibroblast cultures (Figure 6). We found that both media and cell fractions possessed HYAL activity at pH 3.7 (Figure 6a). The HYAL activities present in the media were about 20-fold higher than those detected in cell layers. The HYAL activity was markedly higher in quiescent cells compared with that of cells stimulated with 10% FCS (Figure 6a). PDGF-BB and PMA showed no stimulatory effect on HYAL activities present both in media and cell fractions, as compared with unstimulated cells. Interestingly, TGF-1 inhibited the enzymatic activities considerably. As shown in Figure 6b, TGF-1, PDGF-BB, PMA, and 10% FCS induced the expression of mRNA for HYAL2 but had no effect on the HYAL1 gene. Thus, it is most likely that HYAL2 is involved in the regulation of HA content in lung tissue.

View larger version (38K): [in this window] [in a new window]   Figure 6.   Effects of TGF-1, PDGF-BB, and PMA on HYAL activity and HYAL1 and HYAL2 gene expression. (a) Fibroblasts were isolated and grown in medium supplemented with 10% FCS. Growth medium was replaced by starvation medium containing 0.1% FCS 24 h before stimulation. The cells were then stimulated with TGF-1 (5 ng/ml), PDGF-BB (50 ng/ml), PMA (10 nM), and 10% FCS for 24 h. Control cells were cultured in starvation medium. HYAL activities in media and cell layers were detected as described in MATERIALS AND METHODS. The data are from one representative experiment of two experiments performed; values are the average of duplicates ± variation. (b) mRNAs were prepared from quiescent and cells stimulated for 6 h as in a and subjected to Northern blotting analysis as described in MATERIALS AND METHODS. The data shown are representative of one of two separate experiments.

Effects of HA and HA Fragments on Collagen I and Collagen III Gene Expression

There is an interesting possibility that increased concentration of HA fragments, resulting from the activity of HYAL, is important in the fibrotic process. Therefore, we investigated the effects of HA fragments on procollagen 1(I) and procollagen 1(III) gene expression (Figure 7a). We found that at a concentration of 100 µg/ml, HA fragments of a size of six to 18 saccharide units caused an upregulation of both collagen I and collagen III genes after 6 h of stimulation. In contrast, HA of a molecular mass of 3.9 × 10⁶ slightly suppressed the expression of collagen I and III genes. The effects of HA on the induction of collagen I gene expression were biphasic. High molecular-mass HA induced the procollagen 1(I) gene at low concentrations, up to 50 µg/ml, but inhibited the gene expression at 100 µg/ ml. It did not affect the expression of procollagen 1(III) gene appreciably (Figure 7b). It should be noted that procollagen 1(I) is much more abundantly expressed in lung fibroblasts because the blots hybridized with procollagen 1(I) were exposed to X-ray film for only 20 min, whereas that hybridized with procollagen 1(III) was exposed for 8 h. Thus, it is likely that the HA-dependent and HA fragment-dependent induction of procollagen 1(I) gene may be involved in the progress of pulmonary fibrosis.

View larger version (37K): [in this window] [in a new window]   Figure 7.   Effects of HA and HA fragments on procollagen type I and III gene expression. (a) Quiescent rat lung fibroblasts were incubated in DMEM containing 0.1% FCS or medium supplemented with 100 µg/ml of HA6, HA16, HA8-16, or larger than HA18 oligosaccharides, as well as high molecular-mass HA of 3.9 × 106 kD, for 6 h. (b) Quiescent rat lung fibroblasts were incubated in DMEM containing 0.1% FCS or medium containing 1, 10, 50, and 100 µg/ml of 3.9 × 106 kD HA for 6 h. Total RNAs were extracted and subjected to Northern blotting analysis as described in MATERIALS AND METHODS. The data shown are from one representative experiment of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies revealed that pulmonary lesions induced by irradiation are characterized by an early transient accumulation of HA in BALF, which has been suggested to be a good marker for the irradiation-induced inflammation (32, 35). In the present study, we investigated the cellular mechanisms behind the early accumulation of HA followed by its clearence and the deposition of collagen in this experimental animal model. The finding that the number of BALF-AMs decreased at 2 wk after irradiation, followed by an increase 6 wk thereafter (Table 1), is in good agreement with previous observations (38). It has been proposed that irradiation diminishes the pool of macrophage precursor cells situated in lung interstitial space. Similarily, lung fibroblasts isolated from the irradiated lung 2 wk after irradiation were not able to proliferate in culture (Figure 1). Fibroblasts isolated at 4, 6, and 8 wk after irradiation still grow slower than normal lung fibroblasts and more than 50% of the cells did not attach after subculturing, thereby preventing continuous culturing (data not shown). It appears very likely that radiation causes damage of alveolar interstitial tissue fibroblasts not just in a direct manner but also indirectly by infiltration of activated AMs and other inflammatory cells that release mediators that affect the resident connective tissue cells.

Previous studies on bleomycin-induced lung injury in rats demonstrated that transient accumulation of HA in BALF during the early phase of lung fibrosis is caused by overproduction of HA by the lung mesenchymal cells in response to released mediators and to an impairment of the function of HA receptors on AMs (24, 26). Notably, the marked transient increase in HA level (about 30-fold; Table 1) is in accordance with previous observations where HA content in BALF has been proposed as a marker for estimation of interstitial inflammation (32). In the present study, we showed that AMs isolated at various time points after irradiation exhibit a different capacity to bind labeled HA, suggesting effects on CD44 HA receptors of AMs by released mediators (Figure 2). We further investigated which of the three HAS isoforms is responsible for the increased deposition of HA during the early phase of lung injury. In accordance with our previous observations on foreskin fibroblasts and mesothelial cells (9), HAS2 gene expression and HA production were markedly activated in response to TGF-1 and PDGF-BB (Figure 4b). These factors have been shown to play important roles in connective tissue remodeling (39, 40). In an attempt to investigate which factor could suppress the inductive effects of BALF, we studied the effects of neutralizing antibodies against PDGF-BB and TGF-. Although both PDGF-BB and TGF-1 stimulated HA synthesis of lung fibroblast cultures (Figure 4a), antibodies against PDGF-BB failed to inhibit HA stimulating activity whereas TGF- antibodies inhibited about 40% of the activity (data not shown). Thus, during the inflammatory state rat lung fibroblasts in vivo are mainly activated by mediators other than TGF- and PDGF-BB to synthesize HA. Interestingly, external stimuli such as PMA, which only slightly induced HAS2 gene expression, stimulated HA production dramatically, suggesting modulation of HAS2 protein activity at the post-translational level (Figure 4). This finding is in agreement with our previous observations that PMA acting via PKC activates HAS in a manner independent of gene induction (10). In addition, other studies have revealed that mast cell infiltration parallels HA accumulation in BALF, and further, that degranulation of mast cells enhances the proliferative rate of interstitial lung fibroblasts (35, 41). Further characterization of factors released in the early events of lung repair should help us to understand how signal transduction pathways lead to the fibroproliferative response.

During inflammatory conditions, HA not only is increased but has also been shown to be more polydisperse, with a preponderance of low molecular-weight forms. The accumulation of lower molecular-weight forms of HA has been postulated to occur by several mechanisms, including enzymatic cleavage (22, 42). Studies using bleomycin-induced lung injury in hamsters and rats revealed that the specific activity of HYAL did not change significantly after the bleomycin treatment (18, 26), yet the total enzymatic activity increased about 2.5-fold (18). In the present study, we carefully monitored the expressions of HYAL1 and HYAL2 in radiation-induced injured lung tissue. Our finding that radiation-induced HYAL1 and HYAL2 gene expression increased at 4 wk after irradiation further indicated that upregulation of HA degradation involves increased synthesis of HYAL molecules. Previous observations revealed that HYAL can stimulate HAS activity of intact cells (43). The peak of HYAL isoforms precedes the increase of HA in BALF and thus may contribute to an initial increase in HA content in BALF and lung tissue in addition to providing HA fragments.

The HA content decreases at 8 and 10 wk after irradiation to the level before irradiation, and is gradually replaced by collagen, elastin, and proteoglycans (44). In experimental animals, collagen synthesis is enhanced and gene expression of types I and III collagen is markedly activated (47). It is possible that HA and/or HA fragments resulting from the action of HYAL could play an important role in lung tissue remodeling. Our findings that HA oligosaccharides induce expression of types I and III collagen, whereas HA, with high molecular weight, suppressed the expression of type I and III collagen, strongly support this hypothesis. Other mediators shown to be responsible for the increase in collagen synthesis are TGF- and PDGF-BB (48, 49). Thus, the regulation of HAS2 and HYAL2 in response to mediators released during the healing process after lung injury is likely to be part of the mechanism leading to the fibroproliferative response. Increased knowledge about the precise mechanism involved in the development of lung fibrosis may make it possible to intervene therapeutically in patients.