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. Author manuscript; available in PMC: 2008 Mar 19.
Published in final edited form as: J Biol Chem. 2007 Dec 28;283(10):6058–6066. doi: 10.1074/jbc.M709273200

Extracellular Superoxide Dismutase Inhibits Inflammation by Preventing Oxidative Fragmentation of Hyaluronan*

Fei Gao ‡,1, Jeffrey R Koenitzer ‡, Jacob M Tobolewski ‡, Dianhua Jiang §, Jiurong Liang §, Paul W Noble §, Tim D Oury ‡
PMCID: PMC2268976  NIHMSID: NIHMS41589  PMID: 18165226

Abstract

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.

Hyaluronan/hyaluronic acid (HA)2 is a negatively charged, high molecular mass polysaccharide found predominantly in the extracellular matrix (ECM). Under physiologic conditions, hyaluronan exists as a high molecular mass polymer in excess of 106 Da. It does not induce inflammatory or proliferative genes as a native high molecular mass polymer. However, recent studies have found that low molecular mass hyaluronan fragments accumulate in tissues after injury (1, 2). These low molecular mass hyaluronan fragments have been shown to be capable of activating macrophages and inducing the expression of genes whose functions are relevant to chronic inflammation (3). Hyaluronan turnover and degradation increase during inflammation, and lower molecular mass species of hyaluronan accumulate. Importantly, this accumulation is detected prior to the influx of inflammatory cells and deposition of collagen, which suggests that low molecular mass hyaluronan accumulation is an early event in the development of inflammatory pulmonary disease (4). Although the mechanisms of hyaluronan turnover and degradation are still unknown, it is generally accepted that free radicals, especially the highly reactive hydroxyl radical, play an important role in the degradation process of hyaluronan (5, 6). Uchiyama et al. (7) showed that the oxidative reductive depolymerization reaction of hyaluronan proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents.

Asbestos is a group of naturally occurring mineral fibers that are associated with the development of both malignant (lung cancer, mesothelioma) and nonmalignant (asbestosis) diseases in the lung and pleura (8, 9). Both acute and chronic inflammatory responses are involved in asbestos-induced lung injury. Although the mechanisms of asbestos-induced lung injury are not fully understood, numerous studies suggest that reactive oxygen species may contribute to ECM degradation and enhanced inflammation and abnormal repair in asbestos-induced lung injury models (10, 11). An important factor in determining the surface and biological reactivity of asbestos fibers is their ability to participate in redox reactions that generate free radicals. Free radicals generated from asbestos fibers and/or damage by fibers are linked to cell signaling, inflammation, and a plethora of other responses associated with the pathogenesis of asbestos-associated diseases (10, 12–17). The oxidative stress-related nature of asbestos-associated lung injury makes it a good model to study the possible relationship between free radical-induced hyaluronan degradation and the possible protective effects of antioxidants in vivo. In fact, studies show that asbestos-induced lung injury is associated with excessive ECM turnover, and hyaluronan is one of the ECM components that accumulates in asbestos-induced lung injury (18, 19). Recent studies suggest that low molecular mass hyaluronan accumulates in sites of injury and induces chemokine gene expression in human alveolar macrophages from patients with idiopathic pulmonary fibrosis (3). Failure to remove ECM degradation products from the site of tissue injury results in the host succumbing to persistent inflammation that can progress to fibrosis.

Extracellular superoxide dismutase (EC-SOD) is the predominant extracellular SOD, and it is expressed at especially high levels in mammalian lungs compared with other tissues (20). This antioxidant scavenges the potentially harmful superoxide free radical, which suggests that it may play an important role in protecting against pulmonary diseases characterized by oxidative stress (13, 20). Previous studies have found that EC-SOD protects mice from bleomycin- and asbestos-induced lung injuries (10, 11, 21) and that it is significantly depleted from the lung parenchyma in response to these injuries (13, 22). Furthermore, EC-SOD knock-out mice show enhanced lung injury and inflammation compared with wild-type mice in both asbestos- and bleomycin-induced lung injuries (10, 13). Although the mechanisms in which EC-SOD prevents inflammation in response to lung injuries are not clear, its location in the extracellular matrix and ability to bind to negatively charged components in the matrix suggest that one mechanism may be to prevent oxidative modification/degradation of these matrix components. These properties of EC-SOD led to the hypothesis that one mechanism in which EC-SOD may prevent inflammation is by binding to hyaluronan and preventing oxidant-induced formation of proinflammatory low molecular mass hyaluronan fragments.

Materials and Methods

Binding of Mouse EC-SOD to Hyaluronan EAH-Sepharose

Mouse EC-SOD was purified from mouse lungs (Pelfreeze) as described previously (23). A 5-ml HA-Sepharose column was prepared by coupling 50 mg of HA (Streptococcus sp., from Sigma) to 6 ml of EAH-Sepharopore™ 4B (Amersham Biosciences) and 0.12 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce) according to the method of Tengblad (24) and Barry et al. (25). Prior to coupling, HA was digested with 33 units of testicular hyaluronidase (Sigma) for 3 h in 50 ml of 0.15 m NaCl, 0.1 m sodium acetate at pH 5.8. For chromatography, purified mouse EC-SOD was dissolved in 1 ml of 0.05 m sodium acetate, pH 5.8, and applied to the HA-Sepharose column (5 ml). The column was washed 20 times with 1 ml of 0.05 m sodium acetate, pH 5.8, and the flow-through was collected. Bound proteins were eluted with 1 m NaCl in 100 mm Tris-HCl, pH 7.5 (for full-length EC-SOD) or by step elution with increasing concentrations of NaCl (0.1–1 m NaCl for proteolyzed EC-SOD). Fractions of 1 ml were collected and analyzed by Western blotting.

Proteolysis of Mouse EC-SOD

Purified mouse EC-SOD was incubated with trypsin (1:0.3 molar ratio of EC-SOD to active site-titrated trypsin) at 37 °C for 1 h as previously described (26). Trypsin was inhibited with 0.1 mm DCI at 25 °C for 30 min, and the samples were analyzed by nonreducing SDS-PAGE and transferred to Immobilon-P transfer membranes for EC-SOD detection.

Protection of ROS-mediated Hyaluronan Degradation by EC-SOD

ROS were generated by the Cu(II)/H2O2 system. This system was chosen because it generates both superoxide anions and hydroxyl radicals. The reaction mechanism has been described previously (27, 28). The reaction proceeds faster at alkaline pH, but was carried out at pH 7.4 to lower the reaction rate. 20 μg of pure high molecular mass hyaluronan from Streptococcus sp. in the range of 5.0 × 105 to 1.2 × 106 (Calbiochem) was added to 30 μl of 0.1 m NaH2PO4, pH 7.4, containing 50 μm CuSO4 and human EC-SOD purified from human aorta (26) or CuZn-SOD (Sigma) at the indicated concentrations. Formation of ROS was initiated by the addition of 100 μm H2O2. The reaction was allowed to proceed for 1 h at room temperature, and the reaction mixtures were analyzed by electrophoresis through a 0.7% agarose or 7% polyacrylamide gel. After electrophoresis, gels were stained in the dark at room temperature overnight with Stains-All dye (1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtha[1,2-d] thiazolium bromide; Fisher). DNA restriction fragments produced by BstEII digestion of λ DNA from New England Biolabs Inc. and dual color protein standard (Bio-Rad) were used as size markers. Bromphenol blue (Sigma) was used to track the sample movement.

Western Blot Analysis

Western analysis was performed as previously described (22). EC-SOD was detected with antibody (1:10,000 dilution) against mouse EC-SOD as previously described (11).

Neutrophil Chemotaxis Assay

A neutrophil chemotaxis assay using a modified Boyden chamber was used to evaluate the migration of purified human primary neutrophils (PMNs) across a transwell membrane in response to hyaluronan treated with ROS in the absence or presence of EC-SOD. Human PMNs were isolated as previously described from 50 ml of blood drawn from volunteers, as approved by the Institutional Review Board at the University of Pittsburgh (29).

The PMN pellet was resuspended in Dulbecco's modified Eagle's medium (Fisher) for cell counting. PMNs were used at a final concentration of 2.5 × 107 cells/ml. 24-well plates (BD Biosciences) were coated with 1 ml of pure high molecular mass hyaluronan (Calbiochem) at 5 mg/ml and washed with phosphate-buffered saline after overnight incubation. PMNs (5 × 106) were added to the upper chamber of transwell inserts with a 5-μm pore size (Corning Glass) to create the modified Boyden chamber (30). The lower chambers contained 0.6 ml of Dulbecco's modified Eagle's medium and various doses of ROS with or without the presence of EC-SOD (1000 units/well). ROS was generated using a CuSO4/H2O2 system as previously described (20). ROS are generated by adding 100 μm H2O2 to CuSO4 solution (0.1 m NaH2PO4, 50 μm CuSO4, pH 7.4), and superoxide production was determined by a colorimetric assay using WST-1 proliferation reagent (Roche Applied Science). If EC-SOD was present, it was added to wells prior to the addition of ROS. The wells were incubated for 2 h at 37 °C and 5% CO2. Lower chamber supernatants were collected on ice, and cell counts were performed on a Beckman Coulter Counter (Beckman Coulter, Fullerton, CA). Controls included the use of plates not coated with hyaluronan to ensure that chemotactic responses were not secondary to the generation of ROS alone and omission of hydrogen peroxide from the reaction to ensure that chemotaxis did not occur in response to hyaluronan-coated plates alone.

Animals

All animal experimental protocols were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Male C57BL/6 (Taconic, Germantown, NY) and EC-SOD knock-out mice (congenic with the C57BL/6 strain of mice), 8–10 weeks old and 5–6 mice per group, were treated with 0.1 mg of NIEHS crocidolite asbestos (>10 μm in length) or 0.1 mg of titanium dioxide (Sigma) by intratracheal instillation as previously described (11, 13). Mice were euthanized 14 days post-treatment. Lungs were removed, quick frozen by liquid nitrogen, and stored at −80 °C until used for biochemical analysis after bronchoalveolar lavage fluid (BALF) was obtained.

BALF Analysis

BALF was obtained by intratracheal administration and recovery of 0.8 ml of 0.9% saline. Lavage recovery from each animal was consistently 85–90% of that instilled. Total protein was determined using the Coomassie Plus protein assay (Pierce). Total white blood cell counts were measured using a Beckman Z1 Coulter Particle Counter (Beckman Coulter, Fullerton, CA). The remaining BALF were stored at −80 °C for further analysis.

Quantification of Hyaluronan Levels

BALF and serum hyaluronan were measured using the hyaluronan enzyme-linked immunosorbent assay kit (Echelon, Salt Lake City, UT). The concentration of hyaluronan in the sample was determined using a standard curve of known amounts of hyaluronan.

Sizing Hyaluronan in Lung Tissue

Lung tissues were digested with protease digestion buffer (0.15 m NaCl containing 1000 units/ml Pronase from Streptomyces griseus; Calbiochem) at 55 °C overnight, followed by inactivation of protease activity by boiling the samples at 100 °C for 10 min. Samples were concentrated with centrifugal filter (10,000 kDa cut-off; Millipore). Concentrated samples along with known molecular mass hyaluronan standards (1300, 500, 132, and 35 kDa) were electrophoresed on a 0.5% agarose gel or 7% polyacrylamide gel, stained with 0.005% Stain-All for overnight, and then destained with water for 2 days and exposing to amber light for 30 min. HA-agarose gels were photographed on a Geliance 600 Imaging System (PerkinElmer Life Sciences) and polyacrylamide gels were photographed on Kodak Gel Logic 2200 imaging system (New Haven, CT). The captured agarose gel images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD) to document hyaluronan bands on the gel. A standard curve was determined by the running distance of known molecular mass hyaluronan standards. Hyaluronan peaks in the samples were calculated against the standard curve. Band peaks were analyzed from triplicate sample for each group.

Reverse Transcription-PCR for Hyaluronan Synthases

Lung total cellular RNA was isolated by an RNA isolation kit (Qiagen, Valencia, CA). After determining the concentration, 1 μg of RNA was reverse-transcribed into cDNA and amplified using Power SYBR Green kit (Applied Biosystems, Foster City, CA) according to the Applied Biosystems two-step reverse transcription-PCR procedure (n = 3/group). The reaction was performed by an Applied Biosystems 7300 real time PCR system. Mouse Has sequences were used for this analysis (31). The primers had the following sequences: Has1 forward, 5-AGTATACCTCGCGCTCCAGA-3, Has1 reverse, 5-AGCAGCAGTAGAGCCCAGAG-3; Has2 forward, 5-AACAGGGTGTTGAGTCTGGG-3; Has2 reverse, 5-TAAACCACACGGACACTGGA-3; Has3 forward, 5-CGGGTGAAGGAGAGACAGAG-3; Has3 reverse, 5-GCAATGAGGAAGAATGGGAA-3. PCR for glyceraldehyde-6-phosphate dehydrogenase was performed as a control, using the following primers: forward, 5-AATGCATCCTGCACCACCAA-3; reverse, 5-GTAGCCATATTCATTGTCATA-3.

Hyaluronan Zymography

Hyaluronidase activities were detected by hyaluronan zymography (32–34). Mouse serum was diluted with 5 volumes of 0.15 m NaCl, and the diluted samples were mixed with an equivalent volume of Laemmli's sample buffer containing 4% SDS and no reducing reagent. The mixtures were applied to 7% SDS-polyacrylamide gels containing 0.17 mg/ml HA. After electrophoresis at 25 mA, the gels were rinsed with 2.5% Triton X-100 for 2 h at room temperature. The gels were incubated with fresh incubation buffer (0.1 m sodium formate, 0.15 m NaCl, pH 3.5) for 24 h at 37 °C. Following incubation, the gels were rinsed with water and incubated with 0.1 mg/ml Pronase in 20 mm Tris-HCl (pH 8.0) for 1 h at 37 °C. The gels were rinsed again with 20% ethanol, 10% acetic acid solution for 10 min and stained with Alcian blue solution (0.5% Alcian blue in 20% ethanol and 10% acetic acid) overnight. After destaining with 20% ethanol and 10% acetic acid, the hyaluronan-hydrolyzing proteins were detected as transparent bands against a blue background, and the images were photographed with an Eastman Kodak Co. imaging system.

Statistical Analyses

Data are expressed as mean ± S.E. and analyzed using one-way analysis of variance, followed by group comparisons using Newman-Keuls multiple comparison tests using the computer program Prizm (GraphPad). Statistical significance is set at p < 0.05.

Results

Mouse EC-SOD Directly Binds to Hyaluronan

EC-SOD has a polycationic matrix-binding domain, which has high affinity for polyanionic components in the matrix, such as heparan sulfate. Because hyaluronan is also a polyanionic component in the matrix, we hypothesized that EC-SOD may directly bind to hyaluronan and that this interaction may protect hyaluronan from oxidative degradation. To determine if EC-SOD can bind to hyaluronan, purified mouse EC-SOD was applied to a hyaluronan EAH-Sepharose column. The bound protein was subsequently eluted by NaCl, and the collected fractions were analyzed by Western blotting. This analysis revealed that mouse EC-SOD directly binds to hyaluronan in vitro (Fig. 1A).

FIGURE 1. EC-SOD directly binds to hyaluronan.

FIGURE 1

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A, purified mouse EC-SOD was applied to a hyaluronan EAH-Sepharose column, and bound proteins were eluted with NaCl. B, mouse EC-SOD proteolyzed by trypsin. C, proteolyzed EC-SOD was applied to a hyaluronan EAH-Sepharose column and eluted by a series of concentrations of NaCl.

The Matrix-binding Domain of EC-SOD Is Necessary for Binding to Hyaluronan

To study the involvement of the EC-SOD matrix-binding domain in hyaluronan binding, this domain was removed by limited proteolysis with trypsin, as described previously (26). Proteolysis with trypsin produces a truncated EC-SOD subunit (Fig. 1B), which retains its full enzymatic activity but lacks the C-terminal matrix-binding domain. When this form of EC-SOD was added to the hyaluronan EAH-Sepharose column, it was unable to bind to the hyaluronan, as indicated by its presence only in the flow-through (Fig. 1C). This indicates that the matrix-binding domain of EC-SOD is responsible for the binding to hyaluronan.

Hyaluronan Is Highly Sensitive to ROS-mediated Degradation and EC-SOD Prevents Its Degradation

A fixed amount of hyaluronan was exposed to ROS generated by the Cu(II)/H2O2 system. Analysis of hylauronan molecular mass distribution in these samples revealed that the copper solution or H2O2 alone does not induce any hyaluronan degradation, whereas the ROS generated by the combination decreased the average molecular size of the hyaluronan. These studies indicate that ROS led to degradation of hyaluronan in a dose-related manner (Fig. 2, A and B). The agarose gel used in Fig. 2A shows the shift of the major hyaluronan molecular mass from high to low, whereas the polyacrylamide gel used in Fig. 2B shows the degraded hyaluronan that is unable to be captured in Fig. 2A. To examine the functional relevance of the EC-SOD/hyaluronan interaction and the importance of the EC-SOD matrix-binding domain, we exposed a fixed amount of pure high molecular mass hyaluronan to ROS in the presence or absence of purified human EC-SOD and CuZn-SOD. Notably, co-treatment with EC-SOD was found to inhibit ROS-mediated hyaluronan degradation (Fig. 2C) in a dose-response manner. 300 units of EC-SOD can totally prevent ROS-induced hyaluronan degradation in our system. The EC-SOD gene is ∼60% homologous to Cu-Zn SOD, especially in the region of the active site (35), whereas compared with EC-SOD, CuZn-SOD lacks the matrix-binding domain, and it is not as efficient as EC-SOD at preventing ROS-induced hyaluronan degradation, since higher concentrations are needed to get the same protection effects (Fig. 2C). This suggests that the EC-SOD matrix-binding domain enhances the protective effects provided by EC-SOD.

FIGURE 2. Hyaluronan is extremely sensitive to ROS-mediated degradation but can be protected by EC-SOD and CuZn-SOD.

FIGURE 2

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Pure high molecular mass hyaluronan was incubated with a Cu(II)/H2O2 system, and samples were subsequently analyzed by agarose gel (A) or PAGE (B). C, pure high molecular mass hyaluronan was incubated with the Cu(II)/H2O2 system in the presence or absence of different amounts of purified human EC-SOD or CuZn-SOD.

EC-SOD Inhibits Neutrophil Chemotaxis Induced by Preventing Oxidative Hyaluronan Fragmentation

Isolated human neutrophils were added to a modified Boyden chamber and incubated for 2 h. Neutrophil immigration across a 0.5-μm transwell membrane was measured using the Beckman Coulter Counter. ROS-induced hyaluronan fragmentation led to an increase in PMN chemotaxis across the transwell membrane compared with samples without ROS (Fig. 3) (p < 0.05), and EC-SOD inhibited this response (p < 0.05 versus hyaluronan with ROS). Optimal hyaluronan fragment-induced chemotaxis effect was inhibited by 1000 units/ml purified human EC-SOD preadministered before ROS generation. There was no significant difference between the control samples and hyaluronan with both ROS and EC-SOD (Fig. 3). No chemotaxis was observed with high molecular mass hyaluronan alone or ROS in the absence of hyaluronan (not illustrated).

FIGURE 3. EC-SOD inhibits neutrophil chemotaxis by inhibiting oxidative fragmentation of hyaluronan.

FIGURE 3

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The number of neutrophils migrated versus the amount of ROS generated by the Cu(II)/H2O2 system added to the hyaluronan-coated plates was analyzed. *, statistically significant difference between hyaluronan and ROS group and other groups using a one-way analysis of variance with Newman-Keuls multiple comparison test (p < 0.05). There are no differences between the treatment group of hyaluronan and ROS with EC-SOD and other control groups.

Asbestos-induced Lung Injury and Inflammation Are Exacerbated in EC-SOD-null Mice

Asbestos exposure of EC-SOD gene knock-out mice and wild-type mice provides an excellent in vivo model to elucidate the role of EC-SOD in hyaluronan degradation in response to free radical-induced lung injury. Previous studies from our laboratory found that EC-SOD gene knock-out mice show more severe lung injury compared with wild-type mice after asbestos treatment (11, 13). In our current study, cytologic analysis of BAL fluid revealed a greater number of inflammatory cells, primarily neutrophils in BAL fluid from asbestos-treated wild-type mice compared with TiO2-treated controls that was further augmented in EC-SOD knock-out mice (Fig. 4A). These results indicate that EC-SOD knock-out mice have enhanced asbestos-induced lung inflammation, which is consistent with previous studies (11).

FIGURE 4. EC-SOD knock-out mice show increased lung inflammation and increased hyaluronan levels in the BALF and serum after asbestos exposure compared with wild-type mice.

FIGURE 4

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A, total white blood cells in BALF isolated from control and asbestos-treated wild-type (WT) and EC-SOD null mice (KO). B, serum hyaluronan levels measured by ELISA. C, total hyaluronan levels in BAL fluid measured by ELISA. D and E illustrate sizing of hyaluronan in mouse lung tissues treated with asbestos or TiO2. Error bars, S.E. *, significant results (p < 0.05, Student's t test).

Asbestos-induced Hyaluronan Accumulation Is Significantly Greater in EC-SOD Knock-out Mice than in Wild-type Mice in both BAL Fluid and Serum

Hyaluronan levels were significantly elevated in the serum of asbestos-treated EC-SOD knock-out mice compared with controls and with asbestos-treated wild-type mice (Fig. 4B). BAL fluid hyaluronan levels were also significantly elevated in both EC-SOD knock-out mice and wild-type mice after asbestos injury (Fig. 4C). Notably, total BAL fluid hyaluronan levels were elevated to an even greater extent in asbestos-treated EC-SOD knock-out mice (Fig. 4C) compared with the asbestos-treated wild-type mice. Furthermore, sizing of hyaluronan in the lung tissue shows that the hyaluronan accumulating in EC-SOD knock-out mice has a lower molecular mass compared with wild-type mice with the same treatment (Fig. 4, D and E). The total hyaluronan amounts in BAL fluid are lower than in the lung tissues, and due to the low concentration of BAL fluid hyaluronan, sizing is difficult to achieve. Fig. 4D shows the sizing result of hyaluronan in lung tissues using agarose gel electrophoresis; the actual hyaluronan size was obtained by comparison with the known hyaluronan standards, and the results are from triplicate samples. Fig. 4E shows the results using polyacrylamide gel in order to get a clearer image that asbestos injury leads to fragmentation of hyaluronan in vivo. Fig. 4E shows that hyaluronan in the lungs of asbestos-treated mice has a widespread smear compared with hyaluronan in the lungs of TiO2-treated mice. Although actual sizes of hyaluronan are difficult to determine from the polyacrylamide gel, it shows that the hyaluronan in lungs treated with titanium dioxide is greater than 250 kDa, whereas the hyaluronan in asbestos-treated lungs ranges from above 250 to less than 37 kDa. This sizing assay is not quantitative, preventing direct comparisons of the amounts of fragmentation between wild type and knock-out mice. However, these results do indicate that hyaluronan is being fragmented after asbestos injury and, taken together with the quantitative assays showing increased hyaluronan accumulation in the BALF and sera in EC-SOD knock-out mice compared with wild type mice, do support our hypothesis that EC-SOD plays an important role in preventing asbestos-induced lung injury by inhibiting ROS-induced fragmentation of hyaluronan.

Hyaluronan Synthases and Hyaluronidase Activities Are Not Increased in the Process of Hyaluronan Accumulation

Hyaluronan synthase levels were studied by using reverse transcription-PCR. Although hyaluronan accumulated in both BAL fluid and serum, especially in EC-SOD knock-out mice treated with asbestos, reverse transcription-PCR results from lung samples show no increase in major pulmonary hyaluronan synthases (Fig. 5, A–C). This suggests that increased hyaluronan levels are not due to increases in newly synthesized hyaluronan. In addition, hyaluronidase activity also shows no difference between wild-type mice and EC-SOD knock-out mice in the different treatment groups. Notably, there is no difference between asbestos-treated mice and TiO2-treated mice (Fig. 5D). This result suggests that lower molecular mass hyaluronan accumulating after asbestos injury is not due to increased hyaluronidase activity.

FIGURE 5. Hyaluronan synthases and hyaluronidase activities are not increased in the process of hyaluronan accumulation.

FIGURE 5

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A–C, hyaluronan synthase 1–3 mRNA levels were measured by real time PCR. No changes were found between EC-SOD knock-out (KO) mice and wild-type (WT) mice with either TiO2 or asbestos treatment. D, hyaluronidase activity in the serum was evaluated by zymography, and there was no difference between EC-SOD knock-out mice and wild-type mice with either TiO2 or asbestos treatment. Results were from triplicate samples.

Discussion

In this study, we identify a possible mechanism by which EC-SOD inhibits inflammation after asbestos-induced lung injury. EC-SOD was first discovered in 1982 (36). It was shown to be the predominant superoxide dismutase in extracellular fluids, and it is highly expressed in the lung and other systemic vessels (over 70% of the total SOD activity in some vessels) (37). One important characteristic of EC-SOD discovered during the initial purification is that EC-SOD has a strong affinity for heparin (38, 39). Further studies revealed that the C-terminal domain of EC-SOD is essential for its interaction with heparin and heparan sulfate (40–42). These negatively charged proteoglycans interact in an electrostatic fashion with the positively charged matrix-binding domain in the C-terminal region of EC-SOD (43). Furthermore, other extracellular components, such as type I collagen, have been found to also bind EC-SOD. The original immunochemical studies of EC-SOD in the lung found that EC-SOD associated with type I collagen in the alveolar septa (44). Subsequent studies demonstrated that EC-SOD specifically binds to type I collagen through the heparin/matrix-binding domain, and the bound EC-SOD significantly protects type I collagen from oxidative fragmentation (45). However, whether EC-SOD can bind and protect other extracellular matrix components from oxidative stress is still unclear. Our current study demonstrates that EC-SOD also binds to negatively charged hyaluronan and significantly protects this matrix component from ROS-induced degradation. Importantly, the EC-SOD matrix binding domain is necessary for EC-SOD to bind to hyaluronan. We found that CuZn-SOD, lacking the matrix-binding domain, shows less protection against ROS-induced hyaluronan degradation compared with EC-SOD, which has the matrix binding domain (Fig. 2C). These results suggest that the ability of EC-SOD to tightly bind to hyaluronan via the matrix-binding domain enhances its protective effects against ROS-induced fragmentation. Notably, the EC-SOD matrix binding domain is sensitive to proteolytic removal, and proteolytic removal of the EC-SOD matrix-binding domain is thought to regulate the removal of this antioxidant enzyme from cell surfaces and the extracellular matrix of tissues. Thus, increased proteolytic activity may contribute to enhanced ROS-mediated tissue injury.

The role of hyaluronan in inflammatory conditions of the lung has been studied previously (46, 47). Inflammatory injuries in the lung have also been shown to lead to an increase in hyaluronan in the BAL fluid (48, 49). Savani et al. (50) suggested that lung injury results in an increase in hyaluronan that, at least in part, mediates the inflammatory response to the injury. The molecular mass of hyaluronan is an important factor that determines hyaluronan function in lung injury. High molecular mass hyaluronan inhibits chemotaxis and phagocytosis, whereas lower molecular mass hyaluronan fragments stimulate chemokine expression and play an important role in enhancing lung inflammation (51). Studies show that after lung injury, hyaluronan fragments accumulate and stimulate alveolar macrophages to produce chemokines that recruit subsets of inflammatory cells (3). In addition, in the bleomycin model of interstitial lung disease, which also involves ROS, the accumulation of hyaluronan has been shown to occur in both the alveolar and interstitial spaces and has been proposed as an important mechanism for edema formation (4). In our current study, ROS-induced hyaluronan fragments showed chemotactic effects to neutrophils, and this chemotactic effect was blocked by EC-SOD administration prior to ROS generation. This suggests that EC-SOD may inhibit the neutrophil chemotactic effect by preventing ROS-induced hyaluronan fragmentation.

Although hyaluronan fragments are currently believed to be associated with inflammatory responses, their mechanisms of action are still under investigation. Hyaluronan has been shown to bind several different molecules, including CD44 (52, 53), RHAMM (receptor for HA-mediated motility) (54), link protein (55), versican (56), aggrecan (57), neurocan (58), and many others. Although CD44 is generally considered to be a primary hyaluronan receptor, HA-CD44 interaction represents one of the multiple mechanisms by which hyaluronan and CD44 may regulate cellular activities. Many observations appear to support the notion that HA-CD44 interaction participates in the rolling of leukocytes over endothelial cells, thus promoting leukocyte homing (59, 60). However, although inhibitors to CD44 showed an important role for CD44 in leukocyte homing, these inhibitors did not block hyaluronan responses, suggesting that other non-CD44 mechanisms are involved in hyaluronan fragment-mediated inflammatory responses.

Importantly, promoting leukocyte homing is not the only function for CD44; it also is involved in hyaluronan fragment clearance from the inflamed lung by alveolar macrophages (61). Failure to clear hyaluronan fragments leads to unremitting inflammation, and hematopoietic CD44 has been shown to be necessary to clear hyaluronan fragments that are produced after lung injury (53, 62, 63). Additional studies show that TLR2 and TLR4 (Toll-like receptors 2 and 4) are responsible for macrophage inflammatory gene expression in response to hyaluronan fragments (61). Another hyaluronan receptor, RHAMM, which is also important in inflammatory responses mediated by hyaluronan, has been shown to modulate macrophage inflammatory responses in the lung after injury (64) and can compensate for CD44 in inflamed CD44 knock-out mice (65). Thus, the inflammatory responses toward hyaluronan fragments appear to be regulated by multiple mechanisms. Therefore, a single blocking agent may not be sufficient to block the many factors involved in hyaluronan fragment-induced inflammation.

Although the mechanisms in which EC-SOD inhibits pulmonary fibrosis are still unclear, EC-SOD is known to inhibit inflammation. Our current study shows that oxidative fragmentation of hyaluronan induces inflammatory cell chemotaxis and that EC-SOD can inhibit this response (Fig. 3). This suggests that EC-SOD may inhibit inflammation in part by preventing oxidative fragmentation of hyaluronan and other extracellular matrix components. This hypothesis is further supported by in vivo studies using an asbestos-induced lung injury mouse model. Asbestos-induced lung injury is characterized by inflammation and a slow insidious progression. The molecular mechanisms underlying asbestosis are largely unknown. However, inflammation, excessive extracellular matrix turnover, cytokine release (61, 64, 66–68), and ROS (69) are known to contribute to the pathogenesis of this disease. The inflammatory response was found to correlate with the accumulation of hyaluronan in the sera (Fig. 4B) and BAL fluid (Fig. 4C). Notably, both the asbestos-induced lung inflammation and hyaluronan levels were found to be significantly greater in EC-SOD knock-out mice than wild-type mice, consistent with previous studies that showed that EC-SOD protects against asbestos-induced lung injury (11, 70). Notably, increases in inflammatory cells could also contribute to further increases in the levels of ROS, which can further damage the ECM in the lung. Sizing the hyaluronan from lung tissue (Fig. 4, D and E) and BAL fluid further suggested that low molecular mass hyaluronan accumulated in the lung after asbestos treatment. There appear to be two mechanisms for depolymerizing hyaluronan: enzymatic and nonenzymatic (71). Hyaluronidases are the main enzymes responsible for enzymatic degradation of hyaluronan, and zymography is one of the most sensitive methods to detect hyaluronidase activity. Hyaluronan can also be degraded into smaller fragments in a nonenzymatic way by exposure to reactive oxygen intermediates (72), which is believed to be an important mechanism for generating HA fragments at sites of inflammation (73). Our study shows that hyaluronidase activity did not change due to asbestos and TiO2 treatment, which further supports the previous reports that ROS is an important mechanism for generating hyaluronan fragments at sites of inflammation.

Overall, our current studies suggest that EC-SOD can prevent the mobilization of hyaluronan from the matrix to the airspace and plasma after asbestos-induced lung injury in vivo. In conjunction with the in vitro studies, these studies suggest that at least one mechanism in which EC-SOD inhibits inflammation in response to lung injury is by inhibiting ROS-induced hyaluronan fragmentation.

Footnotes

*

This work was supported by NIEHS, National Institutes of Health Grant F32 ES015383-01 (to F. G.) and National Institutes of Health Grant R01 HL63700 (to T. D. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

The abbreviations used are: HA, hyaluronan; EC-SOD, extracellular superoxide dismutase; PMN, human polymorphic neutrophil; ECM, extracellular matrix; ROS, reactive oxygen species; BALF, bronchoalveolar lavage fluid.

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