Respiratory Cell and Molecular Biology, Infection Immunity and Repair Division, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom
Submitted 2 August 2002 ; accepted in final form 7 August 2003
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ABSTRACT |
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hyaluronic acid; IFN-; wound healing; repair; bronchial epithelium
Proinflammatory cytokines released in the mucosa in allergic diseases upregulate the expression of cell adhesion molecules and induce leukocyte infiltration. Interferon- (IFN-
) and interleukin-1
(IL-1
) increase ICAM-1 expression on endothelial cells (7), which in animal models (43) promotes eosinophil infiltration into inflammatory sites. Tumor necrosis factor-
(TNF-
) plays a role as a pleiotropic cytokine found in increased concentration at sites of inflammation and has shown the ability to regulate neutrophil infiltration and eosinophil recruitment (28). The effect of cytokines on CD44 expression has been investigated in several cell types. Cytokine treatment of different cell types has been reported to have contrasting effects on CD44 expression. For instance, in several human colonic epithelial cell lines, IL-4 upregulated CD44 expression whereas TNF-
had no effect (41). However, other studies have shown that IL-4 inhibits CD44 expression, whereas TNF-
induces CD44 expression in monocytes (26). Standard CD44 (CD44s) is widely expressed in many cell types, but the expression of some variant isoforms is restricted to a few cell types and significantly correlated with metastasis (10). The differences between the effect of IL-4 and TNF-
on tumor epithelial cells and monocytes may be explained on the basis of a difference in CD44 variant expression.
In previous studies we have demonstrated that expression of CD44 is associated with epithelial damage and can be regulated by proinflammatory cytokines (24). Here, we have investigated the relationship between enhanced motility of airway epithelial cells and CD44. Using cytokine treatment and antibodies against CD44, we studied the role of CD44-mediated bronchial epithelial cell adhesion and migration. Evidence is presented indicating the involvement of CD44 in bronchial epithelial cell binding to HA and the importance of CD44 in cell migration. We have shown that cytokine-induced cell binding to HA and cell migration in an in vitro wounding model of epithelium involve changes in the functional activity of CD44 rather than altered levels of expression.
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MATERIALS AND METHODS |
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Cell migration after mechanical damage. 16HBE 14o- cells were cultured in 35-mm petri dishes embossed with a 2 x 2-mm grid (Nunc; Life Technologies, Paisley, UK). Two days after cell confluence, the epithelial monolayer was damaged with a pipette tip. After washing twice with warm HBSS, we added fresh culture media either with or without CD44s antibody. We assessed cell migration by measuring the decrease in surface area of the damaged area after 3, 6, and 9 h using images acquired with a cooled charge-coupled device camera (Digital Pixel, Brighton, UK) connected to a Leica DM IRB inverted microscope. The area of damage and the length of the wound margin in the image were measured with Scion Image software (Scion); the average distance moved by the wound margin was calculated from these values.
To evaluate the function of CD44 in epithelial repair processes, we mechanically damaged and cultured 16HBE 14o- cells grown on filter inserts (BD Falcon; BD Biosciences) with 1-µm pores in medium containing CD44 antibodies. Cultures were used when transepithelial resistance (TER) reached 400-600 · cm2. To avoid damaging the filter, we placed inserts in petri dishes to support the filter, and cells were damaged with a plastic Pasteur pipette. After being washed with medium warmed to 37°C, cells were cultured in medium containing 0, 5, or 25 µg/ml CD44s or 25 µg/ml CD44 variant (v) 9 antibodies, and cell repair was monitored by the measurement of TER with an EVOM epithelial voltohmmeter (World Precision Instruments, Sarasota, FL). After cell damage, TER were in the range of 60-85
· cm2.
Localization of CD44-blocking antibody in cell migration. To localize the site of binding of the antibody used to block cell migration, we washed the cultures three times with warm HBSS and then fixed them with cold methanol. Cells were then incubated with FITC-conjugated rabbit anti-mouse IgG for 45 min on ice. After being washed three times with PBS, cells were mounted in Mowiol (16% Mowiol in 33% glycerol-PBS solution; Harlow Chemical) containing Citifluor antifadent (Citifluor) agent and examined by fluorescence microscopy.
Adhesion assay. Ninety-six-well microplates (Costar) were coated with 50 µl of 1 mg/ml HA or 30 µl of 500 µg/ml bovine serum albumin (BSA) overnight at 4°C (all substrates from Sigma). After air-drying, microplates were blocked with 500 µg/ml heat-inactivated BSA for 30 min at room temperature, and the plates were washed with 100 µg/ml BSA/PBS three times for 30 min each time before use in adhesion assays.
For cytokine treatments, 16HBE 14o- cells or primary bronchial epithelial cells were cultured in 2% USG medium with cytokines (all from R & D Systems Europe, Abingdon, UK) for 18-24 h. The cells were then detached with nonenzyme cell dissociation solution (Sigma). The cell concentration was adjusted to 2.5 x 106 cells /ml and incubated with 50 µg/ml CD44 or isotype control antibodies for 45 min at 4°C. Cells were allowed to adhere to the wells of matrix-coated or BSA-coated plates (5 x 104 cells in 100 µl USG medium) for 1.5 h at 37°C in a CO2 incubator. Nonadherent cells were gently but consistently removed by triturating with an eight-channel electronic pipette three times with warm medium containing 5% FCS. We quantified adherent cells using Cell-Titer 96 AQueous MTS assay (Promega UK) by measuring the enzymatic conversion of a tetrazolium dye during a 2-h incubation at 37°C. Plate absorption at 490 nm was determined with a microplate reader, the result reflecting the remaining viable cell number per well.
Analysis of CD44 expression by flow cytometry. Cells were harvested by nonenzymatic cell dissociation solution and resuspended in HBSS containing 2% FCS and 0.1% sodium azide. Aliquots of 100 µl, containing 2 x 105 cells, were incubated for 1 h on ice with 30 µl (0.2 µg) of protein A-purified anti-CD44s, CD44v6, or CD44v9 antibodies from hybridoma culture supernatants (clones 25.32, FW11.9.2.2, and FW11.24.7.36, respectively; European Collection of Cell Cultures, Porton Down, UK) or CD44v3 (clone 3G5: R&D Systems Europe). Appropriate isotype controls were used in parallel. Thirty microliters of anti-mouse IgG (heavy and light chain) FITC-conjugated F(ab')2 fragment (0.3 µg total; Dako, Ely, UK) were added to each tube after washing and were incubated for 45 min on ice. Cells were washed and resuspended in 0.45 ml of HBSS with 2% FCS. Flow cytometry data analysis was carried out with a Becton Dickinson FACScan with Cell Quest software (Becton Dickinson); fluorescence was measured and displayed on a single-parameter histogram using a log scale.
Statistical analyses. All flow cytometry data were expressed as median fluorescence values. Statistical significance was assessed with a nonpaired two-group Student's t-test, paired two-group t-test (cell migration experiments), or the ANOVA test (cytokine treatments) using SPSS software.
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RESULTS |
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16HBE 14o- cells damaged with a plastic Pasteur pipette showed a decrease in TER from 600 to 100 · cm2. TER measurements give an indication of the overall quality and barrier function of the epithelium at a single time that will reflect all the repair activity up to that point. By this measurement during 16HBE 14o- cell repair, CD44 antibody concentrations as low as 5 µg/ml showed an inhibition in cell repair compared with controls without CD44s antibody (Fig. 3). Cells cultured with CD44v9 antibody, which is the same isotype as the CD44s antibody, showed no significant inhibition of cell repair (P > 0.05, Fig. 3). The TER was not changed when the same amount of CD44s antibody was added to undamaged cell layers either apically or basolaterally (results not shown).
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CD44s antibody inhibits cell binding. We assayed cell adhesion to different substrata by plating 16HBE 14o- cells onto plastic dishes coated with HA or BSA. Cells exhibited a higher binding to a HA- than to a BSA-coated surface. At 90 min after initial contact, 6-10% of total cells bound to HA, whereas <2% of total cells had bound to BSA (Fig. 4). The low proportion of adherent cells reflects the cells that are not detached by the washing protocol, indicating a relatively weak interaction.
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Serial dilutions of CD44 monoclonal antibodies (MAb; 6.25, 12.5, 25, 50, 100, and 200 µg/ml) were assayed for their ability to inhibit cell adhesion. High concentrations (>6.25 µg/ml) of CD44s antibody inhibited cell binding to HA but not BSA. The maximum inhibition of the CD44s MAb was found at concentrations >50 µg/ml, which gave a decrease in binding to HA to a level similar to the BSA control (Fig. 4). There was a dose-dependent inhibition in cells treated with CD44s antibody with a significant inhibitory effect (P < 0.01) on cell binding to HA at 12.5 µg/ml and above (Fig. 4).
Cytokines induce cell binding to HA in a CD44-dependent manner. We investigated the effect of TNF-, IFN-
, IL-1
, IL-4, and phorbol myristate acetate (PMA) on cell adhesion to an HA-coated substrate. These treatments increased 16HBE 14o- cell binding to an HA-coated surface by two- to threefold. Next we tested whether the blocking CD44 MAb reduced adhesion of bronchial epithelial cells to an HA-coated surface. As shown in Fig. 5, incubation with CD44s antibody decreased the cytokine-induced cell adhesion to HA in 16HBE 14o- cells by 45% (Fig. 5A), revealing a CD44s-dependent mechanism for this cytokine-induced cell binding to HA. Although primary bronchial epithelial cells (Fig. 5B) were generally less responsive to cytokine treatment, the effect of CD44 blocking was greater.
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Cytokine-induced cell binding to HA does not require increased CD44 protein. To determine whether the cytokine-induced changes in cell binding were due to changes in the expression of CD44s or altered expression of v3-, v6-, or v9-containing isoforms, CD44 protein expression was assessed on 16HBE 14o- cells. The levels of cell surface CD44 were compared in untreated cells or cells treated with different cytokines or PMA. Flow cytometry demonstrated similar or only slightly increased CD44 isoform expression in cytokine-stimulated cells. Cells treated with PMA showed >100% increase of CD44 isoform expression, whereas cells treated with IL-1 showed some increase in CD44s expression, and IL-1
and IL-4 treatments increased CD44v3 and CD44v9 expression (Fig. 6). No effect of these cytokines on CD44v6 expression was seen, and no significant effect was seen with IFN-
on cell surface CD44 expression.
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DISCUSSION |
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Cell migration and CD44. A high level of CD44 expression has been observed on the surface of the cells close to damage where epithelial migration is occurring (24). This is likely to reflect a role of CD44-mediated adhesion in migration, as has been shown in other systems, for example, in embryonic development (39, 47, 50) and tumor invasion (3, 51). Our results provide supporting evidence that CD44-mediated cell migration is involved in the response of bronchial epithelial cells to damage and mediated in the epithelial cell repair processes. Firstly, antibody against CD44s was able to inhibit the migration of cells to close a wound. Secondly, we have further demonstrated here that the functionally blocking antibody was bound to the highly migrating cells along the wound edge. Finally, as measured by TER, which reflects the reestablishment of cell-cell contact and tight junctions between cells, CD44s antibody inhibited the restoration of the barrier function of 16HBE 14o- cells cultured on filter inserts. During such migration processes, cells extend a leading edge that adheres to the substrate through cytoskeleton-associated cell surface receptor (22). CD44 has been shown to be highly expressed in the filipodia and lamellipodium, which are highly motile structures of fibroblasts (40), or on the leading and trailing edge of migrating murine mammary adenocarcinoma cells (21), indicating that CD44-mediated processes participate in cell motility. The ability of a CD44-blocking antibody, localized to the leading edge of the epithelial damage, to inhibit migration in our system indicates a similar role for CD44 in the migration of airway epithelium after damage. The recent observation that CD44 and matrix metalloproteinase-9 may be functionally associated on the surface of murine mammary carcinoma cells and keratinocytes also indicates that the subcellular organization of molecules on the cell surface is involved in adhesion/deadhesion processes through breaking cell-matrix bonds and regulating the overall migratory ability of the cells (48, 49). In a human breast cancer cell line, Herrera-Gayol and Jothy (14) have shown similar CD44 antibody inhibition of cell migration. Studies in dendritic cells have also shown that CD44 splice variant expression is obligatory for their migration and functioning (44). In this study by Weiss and colleagues (44), increased expression of CD44 isoforms encoded by exons v4, v5, v6, and v9 increased cell movement. Studies in glioma cells (19) have shown that HA can stimulate cell migration and promotes invasion via its interaction with CD44. In vitro studies have also shown that an increase of tumor cell invasion was seen with increasing concentrations of HA incorporated in a Matrigel matrix, when cell invasion could be inhibited by using CD44 MAb (19, 36). Together, these studies suggest that HA enhances migration and invasion of cells and that this mechanism does involve CD44-HA interaction.
CD44 and HA adhesion in epithelial cell repair. HA is frequently expressed at the sites of tissue damage and repair (4). In normal human lung, HA was localized in and around the smooth muscle bundles, whereas in the airways of severe asthma, a disease characterized by chronically compromised epithelial structure, it also appeared in the submucosa (38). Pirinen and coworkers (35) demonstrated that HA is not expressed on columnar cells in normal pseudostratified bronchial epithelium but weakly stains the basal epithelial cells. HA synthesis was increased with inflammation in the airways of animal models (31), and an increased level of HA in bronchoalveolar lavage fluid has been seen in persistent asthma (42). This increase of HA after damage and in asthma where CD44 levels are elevated (20), together with the current results, suggests that epithelial cell adhesion mediated by CD44-HA is enhanced during repair.
Cytokines, CD44, and epithelial adhesion. We found that wound healing of airway epithelial cells was inhibited by CD44 antibody at 3 h but not at 6 h, indicating that CD44 is involved in the early period of cell adhesion but that other mechanisms are in place by 6 h. The low proportion of cells binding in our adhesion assay suggests that this is not a strong interaction, but this does not necessarily reflect the number of cells that adhere in this way in tissue nor the importance of this binding. It was particularly interesting to observe that epithelial adhesion sensitive to a CD44-blocking antibody was altered by treatment with proinflammatory cytokines without necessarily altering the level of CD44 expressed. This might reflect changes in the predominant CD44 isoforms expressed and corresponding alterations in the overall function of CD44 on the cell, by the insertion of peptide sequences encoded by alternatively spliced variant exons. For example, the CD44v9-containing variant expression seen in this study was increased by treatment of cells with IL-1, although the total CD44 expression was not affected. PMA treatment increased expression of all three cell surface CD44 variants and also cell binding to HA. Cytokine treatments did not always result in a proportionate increase in all isoforms. Increases in CD44v3 and CD44v6 were less in cytokine-treated cells. CD44v6, for example, was not increased in cells treated with any of the cytokines used, whereas CD44v9 showed significant increase with IL-1
and IL-4. Such a differential expression presumably reflects alternative splicing of CD44 mRNA to regulate CD44 variant expression.
Cytokines might also increase the ability of CD44 to bind to HA through other independent or complementary mechanisms. TNF--increased CD44 binding to HA in monocytes for example has been correlated with modified glycosylation of CD44 (27). Studies using a CD44- cell line transfected with a wild-type CD44 or a mutation of the serine 325 phosphorylation residue demonstrated that this mutation blocks CD44-mediated cell migration on HA but not cell binding to HA (32). Using a "Penetratin" sequence linked to the sequence from CD44 cytoplasmic domain (amino acid 318-340) containing phosphorylated or nonphosphorylated spanning at serine 325, this phosphopeptide showed the inhibition of cell migration on HA without altering the level of cell surface CD44 expression (32). CD44 interactions with ezrin/radixin/moesin proteins mediating linkage to the actin cytoskeleton are also modulated by phosphorylation, and this is required for CD44-dependent directional cell motility (23). PMA upregulation of CD44-mediated migration is thought to act though a PKC pathway (13), and our PMA results show enhanced CD44-mediated adhesion. In parallel with this, PMA induces increased CD44 expression. Of particular interest, the cytokines examined are almost equipotent to PMA in inducing adhesion but do not stimulate CD44 expression. Thus increased CD44-mediated adhesion does not require more CD44 expression, and this is consistent with the short time scale of the early effects of CD44 in wound healing. Cytokine stimulation in these epithelial cells, by IFN-
for example, therefore appears to act either through an entirely independent parallel pathway, or the stimulation of CD44 expression diverges upstream of the IFN-
pathway. IFN-
has been previously shown to have the potential to regulate CD44, although this is seen only in certain cell lines and can lead to downregulation as well as upregulation (29).
Interactions of HA and CD44 in airway inflammation. Although our studies have focused on the interactions of CD44 and HA, other ligand partners are known for both HA and CD44. CD44 functions as a coreceptor in inflammatory cell activation, whereas HA has a key role in providing an ephemeral extracellular structural scaffold in repair. Studies of CD44-HA interactions have shown that a hyaloadherin link module in the NH2-terminal region of CD44 plays a role as an HA-binding site (reviewed in Ref. 1). It is unclear if blocking using our CD44 antibody affects other HA-binding sites and if HA-binding peptides from aggrecan could abolish the binding between HA and CD44, since there are HA-binding domains in the region other than the Link module region (2). Other HA-receptor interactions are likely to be equally complex, and the high inherent charge of HA can only confound this. A further level of complexity is shown by the physical separation of HA-binding molecules. The receptor for hyaluronan-mediated motility (RHAMM) is another important receptor for HA and is expressed on the luminal surface of ciliated airway epithelial cells (9). HA bound to RHAMM is involved in innate airway defense by helping to retain enzymes inhibiting bacterial colonization and also by promoting ciliary clearance through stimulating ciliary beating (9). Once epithelial damage occurs, ciliated cells are lost (20), and repair predominantly involves CD44+ basal epithelial cells, often accompanied by an influx of inflammatory cells. A wide range of previous studies have demonstrated that increased levels of cytokines are found at sites of inflammation and also that these can be involved in regulating the immune response in airway inflammation (5, 22, 37). Our results show that this could modulate the epithelial repair processes by altering the interaction between CD44 and its ligands. The cytokine-mediated cross-talk between the immune system and the resident epithelial cells could play an important role in a number of human diseases. CD44-HA interactions in repairing epithelium would be particularly affected, and the increase of HA levels in bronchoalveolar lavage fluids in asthmatic patients might reflect increased epithelial turnover and repair in this inflammatory situation. In conclusion, we suggest that CD44-HA-mediated adhesion is a general modulator of epithelial repair that can be affected by the local cytokine environment without requiring altered expression of CD44.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address for Shih-Hsing Leir: Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford 0X3 9DS, UK.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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