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Hyaluronan (HA) is present at the apical surface of airway epithelium as a high-molecular-weight polymer. Since HA depolymerization initiates a cascade of events that results in kinin generation and growth factor processing, in the present work we used primary cultures of human bronchial epithelial (HBE) cells grown at the air–liquid interface (ALI) to assess hyaluronidase (Hyal) activity by HA zymography, gene expression by quantitative real-time PCR, and localization by confocal microscopy. Because TNF-α and IL-1β induce Hyals in other cells, we tested their effects on Hyals expression and activity. We found that Hyal-like activity is present in the apical and basolateral secretions from HBE cells where Hyals 1, 2, and 3 are expressed, and that IL-1β acts synergistically with TNF-α to increase gene expression and activity. Confocal microscopy showed that Hyals 1, 2, and 3 were localized intracellularly, while Hyal2 was also expressed at the apical pole associated with the plasma membrane, and in a soluble form on the apical secretions. Tissue sections from normal individuals and from individuals with asthma showed a Hyal distribution pattern similar to that observed on nontreated HBE cells or exposed to cytokines, respectively. In addition, increased expression and activity were observed in tracheal sections and in bronchoalveolar lavage (BAL) obtained from subjects with asthma when compared with normal lung donors and healthy volunteers. Our observations indicate that Hyal 1, 2, and 3 are expressed in airway epithelium and may operate in a coordinated fashion to depolymerize HA during inflammation associated with up-regulation of TNF-α and IL-1β, such as allergen-induced asthmatic responses.
This article addresses mechanisms of hyaluronan degradation in human airways associated with inflammatory responses.
Hyaluronan (HA) is a glycosaminoglycan widely distributed in tissues and is a normal constituent of airway secretions (1, 2). In the airways, HA is produced by submucosal glands and by superficial airway epithelial cells (3, 4), where it is synthesized by hyaluronan synthases (HAS) at the plasma membrane and released as a high-molecular-weight polymer into the extracellular space (5, 6). Although hyaluronan was discovered in 1934 (7) and its structure defined in 1951 (8), only recently it has become clear that, besides its structural role as a component of the extracellular matrix (ECM) (9), HA is involved in a broad range of biological processes associated with health and disease (for review see Refs. 10, 11).
In the lungs, HA is involved in injury and repair processes through its receptor CD44 (12), in interstitial fluid balance regulation due to its high water-binding capacity (13), and in the induction of inflammatory mediators in alveolar macrophages and lung fibroblasts (14, 15).
Most HA functions have been shown to be size-dependent: the high-molecular-weight molecules have been reported to exert anti-inflammatory and immunosuppressive effects (16), while smaller fragments stimulate gene expression and protein synthesis of pro-inflammatory mediators such as cytokines and chemokines (for review see Ref. 17). We have previously reported that high-molecular-weight HA found at the surface of airway epithelium is involved in airway homeostatic mechanisms by binding and regulating the activity of enzymes such as lactoperoxidase and tissue kallikrein (18, 19), while small HA fragments stimulate ciliary beating through a mechanism that involves the receptor for hyaluronic acid–mediated motility (RHAMM) (20, 21).
Hyaluronan fragments can be generated by reactive oxygen species (ROS)-induced depolymerization and/or by the activity of the hyaluronan degrading enzymes, the hyaluronidases (Hyals). ROS induce HA depolymerization in a variety of tissues, including skin (22), cartilage (23), and at the airway surface (21, 24, 25). In contrast, the role of Hyals in HA degradation at the airway surface and subepithelial tissues has not been previously explored.
Six hyaluronidase genes have been reported in humans: Hyal1, Hyal2, and Hyal3, clustered on chromosome 3p21.3, and Hyal4, PH20 and PHyal1, clustered on chromosome 7q31.3 (26). Among them, Hyals 1, 2, and 3 are widely expressed in somatic tissue and in cancer (26, 27). PH20 expression seems to be limited to testes, but its mRNA can be detected in other tissues and in malignancies (28, 29).
It has been shown that the activity of both HA synthases and Hyals can be regulated by cytokines and growth factors (30, 31). Asthma and a variety of airways inflammatory responses are characterized by increased levels of the pro-inflammatory cytokines IL-1β and TNF-α (32, 33). Thus, it is likely that the expression and activity of Hyals can be induced by these cytokines in human airways, and therefore be responsible, at least in part, for the increased levels of soluble HA observed in the bronchoalveolar lavage (BAL) of individuals with asthma (34) with the associated decrease in HA average molecular size observed after allergen challenge (35).
In the present work we report that Hyal 1, 2, and 3 are expressed in the airway epithelium and that TNF-α induces up-regulation of both gene and protein expression as evidenced by QPCR and increased glycosidase activity in human airway epithelial cells in vitro. These effects were potentiated by simultaneous exposure to IL-1β. The biological relevance of these observations is supported by the evidence of augmented Hyal 1, 2, and 3 expressions in tissue sections obtained from lung donors with asthma, and the increased Hyal activity on the BAL of individuals with asthma after segmental allergen challenge when compared with normal lung donors and healthy volunteers, respectively.
All materials were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.
Human tracheas and main bronchi from donor lungs were obtained through the University of Miami Life Alliance Organ Recovery Agency with approval from the local Institutional Review Board. The trachea and main bronchi were opened at the membranous portion and the mucosa was dissected off the cartilage. Mucosal strips were digested with 0.05% protease (type 14) incubated in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Carlsbad, CA) and incubated at 4°C. Human bronchial epithelial (HBE) cells were released by vigorous shaking and harvested by centrifugation as previously described (25, 36).
Human tracheobronchial epithelial cells were counted and their viability was determined by trypan blue exclusion (viability was always > 80%). Cells (1 × 106; referred to as P0) were plated on collagen-coated plastic dishes (100 mm; Corning Costar Corporation, Cambridge, MA), and grown in bronchial epithelial growth medium (BEGM) (37), yielding undifferentiated airway epithelial cells. After reaching confluence, cells were dissociated with trypsin (referred to as P1 ), and 5 × 105 cells from P1 were plated onto 24-mm Transwell-clear culture inserts (Corning), coated with human placental collagen. The culture medium (ALI medium) contained 50% DMEM and 50% Lechner and LaVeck (LHC) basal medium supplemented with insulin (5 μg/ml), hydrocortisone (0.072 ng/ml), epidermal growth factor (0.5 ng/ml), triiodothyronine (T3, 6.5 ng/ml), transferrin (10 μg/ml), epinephrine (0.6 μg/ml), phospholethanolamine (0.5 μM), ethanolamine (0.5 μM), bovine pituitary extract (1% vol/vol), bovine serum albumin (0.5 mg/ml), CaCl2 (0.08 mM), trace elements, penicillin/streptomycin (100 μg/ml), and retinoic acid (1 μM) (36). Cells were grown in an incubator at 37°C in ambient air supplemented with 5% CO2. Their apical surface was exposed to air as soon as they reached confluence (~ 7 d). These conditions were maintained until the cells reached full re-differentiation, as evidenced by ciliary beating and the presence of mucus on the apical surface (~ 4 wk), and used for the experiments described below.
Each experimental condition described below was performed in individual cultures obtained from three different lung donors (n = 3). Fully differentiated HBE cultures grown at ALI were exposed basolaterally to ALI media containing TNF-α (20 ng/ml), IL-1β (2.5 ng/ml), or their combination (TNF-α 20 ng/ml + IL-1β 2.5 ng/ml) for 18 hours at 37°C. Cultures exposed to ALI media were used as controls. After treatments, basolateral media and apical secretions, obtained by washing with 500 μl of PBS (apical washes), were collected. Cell layers were treated with 1% Triton X-100 in 20 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA, 50 mM HEPES pH 7.8 (lysis buffer) containing 50 mM NaF, 1 mM sodium orthovanadate, 5 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, spun at 14,000 rpm for 5 minutes at 4°C, and stored at −20°C for later analysis.
RNA from HBE cultures were extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to manufacturer's instructions. Quantitative real-time PCR (QPCR) was performed using pre-made TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) with TaqMan MGB probes, FAM labeled Hyal1 (ID:Hs00738390_m1), Hyal2 (ID:Hs00186841_m1), Hyal3 (ID:Hs00185910_m1), and Ph20 (Hs00162139_m1). The QPCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) following the manufacturer's instructions. Thermal cycling was performed using ICycler IQ apparatus (Bio-Rad). The comparative CT method (ΔΔCT) was used for relative quantification. All results were normalized using the housekeeping gene GAPDH (Hs99999905_m1).
Samples were assessed for Hyal activity by zymography as described (39). Briefly, samples were electrophoresed in 10% SDS-polyacrylamide gels containing 0.17 mg/ml of HA (vitreous humor; US Biological, Swampscott, MA). After electrophoresis, gels were washed in 50 mM HEPES buffer pH 7.4 containing 3% Triton X-100 and subsequently incubated in 0.15 M NaCl–0.1 M sodium formate, pH 4.2 at 37°C for 18 hours. Gels were then stained with 0.5% Alcian blue in 3% acetic acid where hyaluronidase activity was visualized as clear bands on the blue background. For the assessment of relative activity, intensities of the bands were recorded using the GelDoc XRS system (Bio-Rad) and analyzed using Quantity One software (Bio-Rad).
After removing culture media, filter inserts were fixed in 4% paraformaldehyde in PBS, pH 7.4 for 20 minutes at room temperature (RT) and permeabilized with 0.05% Triton X-100 in PBS for 10 minutes at RT. After washing with PBS, each filter was blocked with 5% BSA in PBS for 1 hour at room temperature. Primary antibodies diluted in blocking solution were used as 1:500 dilutions for the following antibodies: rabbit anti-Hyal1 antiserum (a generous gift from Dr. Lokeshwar, University of Miami Miller School Of Medicine ), and mouse anti-Hyal2 and Hyal3 antiserum (both from Novus Biologicals, Littleton, CO). Visualization was achieved using Alexa 555– or Alexa 488–labeled anti-rabbit IgG (4 μg/ml) and Alexa 488– or Alexa 555–labeled anti-mouse IgG (4 μg/ml; Molecular Probes, Carlsbad, CA). Nuclei were visualized with 4′6-diamidine-2-phenylindole (DAPI; Molecular Probes). Samples were mounted on slides with gel/mount (Biomeda, Foster City, CA) and images were obtained using a Zeiss LSM-510 confocal laser scanning microscope (Zeiss LSM, Thornwood, NY) at the University of Miami Analytical Imaging Core Facility.
Tissue sections of human trachea were obtained from normal organ donors and from organ donors with asthma through the University of Miami Life Alliance Organ Recovery Agency with approval from the local Institutional Review Board. Paraffin-embedded sections were hydrated and incubated in EDTA 2 mM pH 8.0 for 15 minutes at 100°C (antigen retrieval) followed by acetone at −20°C for 10 minutes and blocked with Image-iTTM FX (Signal Enhancer; Invitrogen) following manufacturer's instructions. Immunolocalization was preformed as described above in the cell culture section.
BAL, kindly provided by Dr. Ndukwu (University of Chicago), were obtained from healthy volunteers (n = 3) and from volunteers with asthma (n = 3) 24 hours after segmental allergen challenge (or saline instillation on the contra lateral lung) as described (41). Hyal activity was assessed by HA-zymography as described above, where equal amounts of protein were loaded per well on the HA-acrylamide gels.
All n refer to the number of different HBE cultures used for each study where cells from each culture were obtained from only one lung donor. Data were expressed as means ± SEM. Differences between multiple groups were compared using a one-way ANOVA followed by the Tuckey Kramer honestly significant difference test. Levene test was used to analyze the homogeneity of variances. Significance was accepted at P < 0.05.
To test if primary cultures of HBE cells grown at the ALI had the ability to degrade HA, we determined glycosidase activity by HA-zymography. Cell lysates, apical washes, and basolateral media were collected (n = 3) and activity was determined as described in Materials and Methods. As shown in Figure 1A, clear bands were detected in apical washes, cell lysates, and basolateral media, indicating that Hyal-like activity is present in all three compartments. The apparent migration pattern (50–65 kD) shows that the band corresponding to apical washes appear to migrate slower than the one observed in cell lysates, indicating that more than one molecular species are expressed by these cells.
To further investigate Hyals gene expression in these cells, QPCR (Figure 1B) studies were performed using mRNA obtained from cell cultures from three different lung donors. We found that Hyal1, Hyal2, and Hyal3 are normally expressed by these cells, while we did not detect PH20 mRNA expression (data not shown). The fact that Hyal activity was present at the apical surface (evidenced by zymography on apical washes) and in basolateral media, suggests that Hyals likely participate in HA depolymerization at the luminal pole and subepithelial areas of human airways.
To determine protein expression and cellular localization of the individual Hyals in airway epithelial cells, fully differentiated ALI cultures (n = 3) were labeled using primary antibodies generated against Hyal1, Hyal2, and Hyal3. Consistent with QPCR data, positive staining for the three Hyals were observed in HBE cells (Figure 2). Z-stack reconstruction images provided a more detailed view of the cellular localization of these enzymes (Figure 2, upper panels). Hyal1 (Figures 2A and 2B, red), Hyal2 (Figure 2A, green), and Hyal3 (Figure 2B, green) immunoreactivities were visualized intracellularly, inside vesicle-like structures as reported (42, 43). In addition, frequent co-localizations were observed between Hyal1 and 2 (Figure 2A) and Hyal1 and Hyal3 (Figure 2B).
Hyal2 was also observed at the apical surface, consistent with the glycosyl-phosphatidylinositol (GPI) anchored form reported for this enzyme in airway epithelial cell lines (44). Overall, these findings suggest the participation and a possible cooperation between different Hyals in HA degradation in the airway epithelium.
To determine if the observed Hyals distributions in cell cultures were consistent with localization in human airway epithelium, we used human trachea sections for immunohistochemical analysis. As observed in Figure 3, the three Hyals tested were expressed in the airways. Hyal1 (Figures 3C and 3D) and Hyal3 (Figures 3G and 3H) appeared to be distributed intracellularly as observed in HBE cultures.
Hyal2 labeling (Figures 3E and 3F) was visible inside the cells and at the epithelial surface, where it appears to be associated with the cell surface and also in a “free form” at the mucus layer (Figure 3, arrows). These data are consistent with the distribution pattern found in cell cultures and confirm that HBE cells are an appropriate model to evaluate Hyals expression and regulation.
Since HA depolymerization is associated with airway inflammatory responses, to test if pro-inflammatory cytokines that have been reported to regulate Hyals in other tissues (31, 45) have similar effect in airway epithelium, HBE cells were incubated with media alone (controls) or media containing TNF-α and/or IL-1β. As depicted in Figure 4, exposure to TNF-α alone resulted in increased Hyals1, 2, and 3 mRNA expressions, but only increases in Hyal1 expression achieved statistical significance (1.6 ± 0.21) from control cells (P < 0.05) on the three cultures tested. Exposure to IL-1β did not result in changes in gene expression in any of the three Hyals tested. In contrast, when cells were treated with TNF-α + IL-1β, all three (Hyal1 [2.3 ± 0.22], Hyal2 [2.2 ± 0.21], and Hyal3 [1.9 ± 0 0.25]) expressions were significantly increased when compared with controls (P < 0.05). These results suggest that IL-1β and TNF-α act in a synergistic fashion to induce Hyals gene expression in airway epithelial cells.
To further investigate if the stimulatory effect on Hyals gene expression observed after cytokine exposure resulted in increased in glycosidase activity, we evaluated Hyal activity by HA-zymography in cell lysates obtained from cells exposed to TNF-α and/or IL-1β. While TNF-α induced an increase of 1.5 ± 0.2 (P < 0.05) in Hyal activity, no effect was observed with IL-1β in accordance with gene expression results. Likewise, the exposure to both TNF-α + IL-1β induced a higher increase (2.2 ± 0.3, P < 0.05) in enzymatic activity with respect to controls (Figure 5). These data further confirmed the synergistic/potentiating effect between IL-1β and TNF-α on Hyal activity in airway epithelial cells.
To explore if Hyals are up-regulated in the airways in conditions that are associated with increases of TNF-α and IL-1β in vivo such as allergen-induced airway hyperreactivity (46), we assessed Hyal activity on BAL obtained from healthy volunteers and from volunteers with asthma 24 hours after segmental allergen challenge (41, 47). Equal amounts of protein were loaded in each well where Hyal activity was analyzed by HA-zymography as described on Materials and Methods. As seen in Figure 6, basal Hyal activity (saline) is increased in individuals with asthma when compared with normal subjects. In addition, Hyal activity in response to allergen challenge was visibly higher in the asthma group (allergen). These data provide in vivo evidence that Hyal activity is increased in airway inflammatory responses associated with TNF-α and IL-1β.
To test if the increases in Hyal activity observed on the BAL of individuals with asthma were due to infiltrated leukocytes or/and activated macrophages alone, or were associated with increases in epithelial Hyals expression, double immunolabeling for Hyal1 (green) and Hyals 2 or 3 (red) was performed in human tracheal sections obtained from subjects without asthma, as depicted in Figures 7A, 7B, 7F, and 7G, and from subjects with asthma (Figures 7C–7E and 7H–7J) donors, as described in Materials and Methods. We intentionally used three lung donors, each showing different characteristics of pathological findings in asthmatic airways: (1) denuded epithelium with abundant leukocyte infiltration, (2) intact epithelium, and (3) with increased numbers of goblet cells (associated with hypersecretion in these patients). In all of the asthmatic tissues, Hyal1 and 2 (Figures 7C–7E) and Hyal1 and 3 (Figures 7H–7J) expression were clearly increased when compared with those from individuals without asthma (Hyal1 and 2, Figures 7A and 7B) and Hyal1 and 3 (Figures 7F and 7G). Hyal1 staining resulted particularly strong in infiltrated leukocytes (Figures 7C and 7H, green arrows) and in the interstitial space in subepithelial tissues. Hyal2 and Hyal3 immunoreactivity were prominently visible in ciliated cell in both shedded and intact epithelium (Figures 7C and 7H, yellow arrows) of subjects with asthma. In addition, Hyals 1 and 2 were found increased at the apical pole of cells and at the ciliary border of epithelium from subjects with asthma (Figures 7D, 7E, and 7J). No staining for Hyals 1, 2 or 3 was observed in goblet cell (Figures 7D, 7E, 7I, and 7J, white arrows), suggesting that Hyals expression is limited to the ciliated and basal cells of airway epithelium. These data suggests that the airway epithelial cells can contribute to the increased Hyal activity observed on the BAL of individuals with asthma.
We have previously shown that HA is present at the apical pole of airway epithelial cells (3, 18), where it plays important roles in both health and disease (18, 21, 24, 25). In the present study we show that Hyal1, Hyal2, and Hyal3 are expressed in primary cultures of HBE cells grown at the ALI and are up-regulated by TNF-α and IL-1β. These in vitro findings correlated in vivo with observations on tissue sections and BAL obtained from individuals with asthma, suggesting that Hyals participate in HA fragmentation at the airway lumen during inflammatory responses.
HA zymography from samples obtained from HBE cells confirmed that Hyal activity is present in these cultures not only in the cell layers but also in the basolateral media and in apical washes. The presence of Hyal activity in the basolateral media suggests that Hyals produced by airway epithelial cells can be involved in the HA degradation in the subepithelial tissues as well. These results, together with the data indicating that Hyal1, 2, and 3 are expressed by HBE cells, suggest that more than one Hyal isoenzyme participates in HA degradation in airway epithelium and at the mucus layer.
Differences in the Hyal isoenzymes degradation properties have been described (see Ref. 26). For instance, Hyal1 is active only at acidic pH (optimum pH 3.8–4.3 [48, 49]) and degrades HA of any molecular size generating mainly tetrasaccharides/hexasaccharides (42, 50), while Hyal2 cleaves high-molecular-weight HA into fragments of approximately 20 kD (42). The optimum pH for Hyal2 activity is still controversial and appears to depend on the Hyal2 form expressed: a free form of Hyal2 has been shown to be active under acidic conditions (50, 51), while membrane-associated Hyal2 is active under acid-neutral conditions (pH 6.0–7.0 ).
Hyaluronidase 2 localization seems to be tissue specific: while it is exclusively intralysosomal in chondrocytes (43), there is also evidence that can be expressed bound to the plasma membrane through a GPI anchor in airway epithelial cell lines (53).
We found that in our primary cultures as well as in human tracheal tissue sections, Hyal2-like immunoreactivity was present intracellularly, associated with the apical epithelial surface and as a free form at the mucus layer, suggesting that in human airways, both forms are likely expressed.
The distribution pattern of Hyals 1 and 2 that we found in primary cultures and in tracheal tissue sections agrees with the differential contribution of these enzymes in intracellular and extracellular HA degradation, respectively, as has been proposed in keratinocytes (54).
A seminal work from Harada and Takahashi (52) on HEK 293 cells showed that intracellular and extracellular Hyals 1 and 2 activities are dependent on the association with CD44 (52). If the mechanism mentioned above operates in airway epithelial cells, other considerations need to be in place: CD44 is found at the basolateral compartment in normal airway epithelium (55) and therefore could not participate in hyaluronidase activity at the apical surface. The crystal structure of Hyal1, recently determined (56), evidenced EGF-like domains, close to the catalytic site. These domains, characteristic of protein–protein interaction, could be involved in Hyals interaction with other surface receptors, as has been shown for the tyrosine receptor RON (44). We speculate that RON (21), other apically expressed HA receptors such as RHAMM (19, 21), or other undetermined protein may regulate Hyal activity on the epithelial surface.
In the present work we report that TNF-α and IL-1β (known to regulate HA metabolism [30, 31] and to be associated with inflammation [57, 58], chronic obstructive pulmonary disease , and asthma ) stimulate Hyals gene expression and activity. The induction of Hyals 1, 2, and 3 by TNF-α and the potentiation by IL-1β is a phenomenon that has been previously reported. For instance, TNF-α and IL-1β have synergistic effects to stimulate matrix metalloprotease-3 (60) in the trabecular meshwork; prostaglandin E (PGE) and collagenase production in fibroblasts (61), and PGE2 and cyclooxygenase activity in chondrocytes (62). Although we did not determine the nature of the synergistic effect induced by TNF-α + IL-1β in our cells, it has been suggested that these cytokines can be involved in transcriptional versus post-transcriptional events (62) in lymphocytes, where IL-1β induces TNF receptor expression and up-regulates mRNA transcription and membrane expression (63). Asthma, and particularly allergen exposure in these patients, is characterized by synthesis and release of IL-1β and TNF-α (46). Consistent with in vitro data, we found that Hyal activity was augmented in the BAL of individuals with asthma and further increased after segmental allergen challenge.
We have previously shown that exogenous sources of ROS are involved in HA depolymerization in the airway lumen (19, 21, 25). In the present study we provide additional evidence that airway epithelial cells themselves can participate in HA degradation at the lumen and in subepithelial tissues, particularly during inflammatory responses in which TNF-α and IL-1β cytokines are up-regulated.
The specific roles of individual Hyals on HA metabolism in the airway lumen as well as in epithelial and subepithelial tissues under normal and pathological conditions remains to be elucidated.
The authors thank Dr. Vinata Lokeshwar from the Departments of Urology, Cell Biology and Anatomy at the University of Miami Miller School Of Medicine for providing the Hyal1 antibodies and for critical reading of the manuscript. The authors are particularly thankful to Dr. Ndukwu from the University of Chicago for providing the BAL samples. They also thank Drs. Gregory Conner, Matthias Salathe, and Nevis Fregien for helpful comments and support. This work was possible with the support of University of Miami Life Alliance Organ Recovery Agency, and the Analytical Imaging Core at the University of Miami and to the Histology Laboratory at the University of Miami Hospital and Clinics, Sylvester Comprehensive Cancer Center.
This work was supported by National Institutes of Health grant HL073156, James and Esther King Team Science Program and Biomedical Research grants 07KB-02-12292 (to R.M.F.) and 07KN-02-12324, and AHA Scientist Development grant 635093N (to S.M.C.-M.).
Originally Published in Press as DOI: 10.1165/rcmb.2007-0361OC on April 3, 2008
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.