Increased CD44 expression in human bronchial epithelial repair after damage or plating at low cell densities

Shih-Hsing Leir, Janice E. Baker, Stephen T. Holgate, and Peter M. Lackie

Southampton University Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have investigated the effect of mechanical damage, cell density, and cell-derived soluble mediators on CD44 expression in a model of bronchial epithelial repair. CD44 (all isoforms) and variant-containing isoforms (CD44v3, CD44v6, and CD44v9) were identified with flow cytometry and immunocytochemistry with image analysis. After mechanical damage, CD44 expression increased up to 500 µm from the wound edge and for up to 48 h in two human bronchial epithelium-derived cell lines, 16HBE14o- and NCI-H292. CD44 expression was unchanged by interferon-gamma and increased by <50% by tumor necrosis factor-alpha . To exclude other soluble factors, a Vaseline spacer was used to temporarily divide petri dishes, with cells at high density on one side and those at low density on the other. After the spacer was removed, the cells at low cell density growing in the shared medium expressed up to fourfold higher CD44, although cell proliferation was unchanged. Thus increased CD44 expression at low cell density was not mediated by soluble factors and may reflect functional involvement in cell motility, dedifferentiation, or altered cell-substrate adhesion in epithelial repair.

CD44 variants; bronchial epithelium; intercellular adhesion molecule-1; epithelial damage


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXPRESSION of CD44 is increased in areas of epithelial repair (24). In such situations, we would expect to see reduced cell-cell density, increased cell migration, and an increase in the levels of proinflammatory mediators such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma . Cell proliferation would also subsequently be increased. CD44 is increasingly considered to have a physiological role in cell migration (18, 23, 36, 37) as well as in cell adhesion and invasion in tumor cells (25). Changes in CD44 expression at low cell density could arise through a number of routes including alterations in the surrounding soluble mediators, altered cell proliferation, or changes in homotypic or heterotypic cell-cell contact. In this study, we aimed to distinguish between these possibilities.

Bronchial epithelial damage is characteristically increased in asthma and is increasingly recognized as an important feature of the disease (31). The epithelium of the human conducting airways is a key interface with the airborne environment. It functions as a barrier to inhaled pathogens and other noxious agents and through mucus secretion and cilial beating serves to clear deposited particulates from the airway. The epithelium in vivo comprises two cell layers and a number of differentiated cell types. After damage, rapid and effective repair of the epithelium should restore epithelial integrity, maintaining other functions in the interim insofar as possible. After epithelial damage in the airway, the events of epithelial repair include cell migration, spreading, and subsequent proliferation and redifferentiation (12, 13, 47, 48). In vitro, cells rarely achieve the density and degree of differentiation seen in vivo and probably reflect many of the characteristics of the repair process rather than those of the "normal" epithelium.

CD44 is a cell surface proteoglycan thought to be involved in cell-cell adhesion, cell-matrix adhesion interactions, lymphocyte activation and homing, and cell migration. CD44 is abundant in many tissues and can bind the extracellular glycosaminoglycan hyaluronate (HA) (2, 29, 34) as well as collagen I, collagen IV, and fibronectin (14, 21, 22). Variant forms of CD44 (CD44v) containing additional peptide sequences encoded by variant exons are more restricted in their distribution in nonmalignant tissues. These alternative isoforms are encoded by mRNA species derived from varying combinations of the exons in a single gene locus on chromosome 11p13. Of the 20 CD44 exons in this locus, 10 exons are always present in CD44 mRNA species, whereas 10 "variant" exons (9 in humans) can be alternatively spliced into nascent RNA.

Standard CD44 (CD44s) plays a role in the uptake and degradation of HA in macrophages and in the degradation of HA in lung and lymphoid tissue (10, 39). CD44s is important in organogensis (44) and the homing of lymphocytes to Peyer's patches (45). Epithelial expression of CD44 variants has been described in a wide range of normal epithelial tissues including the skin, digestive tract, and lung (27). It is likely that expression of CD44 variants contributes to the enhanced metastatic properties of some lymphomas and melanomas (16), possibly by modifying the migratory ability of cells. It is further becoming apparent that CD44 may be involved in the formation of structures such as microvilli on the cell surface (46). Increased expression of CD44 has been found in areas of repair in bronchial epithelium and is also increased in asthmatic subjects (24). In asthma, up to 60% of the epithelial area (30, 32) may have an altered structure associated with damage and repair processes, reflecting increased epithelial fragility in asthma. Intercellular adhesion molecule (ICAM)-1 is also expressed in human bronchial epithelium (38) and has been reported to be upregulated in asthma (40). Expression of ICAM-1 is highly responsive to IFN-gamma and TNF-alpha treatment (26), whereas CD44 is less so. Expression of other cell adhesion molecules has been seen to change with cell density, including upregulation of neural cell adhesion molecule in neuronal cells (6) and integrins in colon and bladder carcinoma cells (35).

To further understand the regulation of CD44 and its isoforms in bronchial epithelial damage and repair, we have used immunostaining techniques with fluorescence microscopy and flow cytometry to investigate the relationship between cell density, soluble factors, and the expression of CD44 in two bronchial epithelial cell lines.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. The CD44v9 monoclonal antibody (11.24) was a kind gift from Dr. U. Günthert (Basel Institute for Immunology, Basel, Switzerland). The following materials were purchased: RPMI 1640 medium, MEM, fetal calf serum (FCS), Ultroser G, Hank's balanced salt solution (HBSS), and Ca2+- and Mg2+-free HBSS from GIBCO BRL (Paisley, UK); nonenzyme cell dissociation solution, propidium iodide, and RNase A from Sigma (Dorset, UK); human IFN-gamma and TNF-alpha from Genzyme (Kent, UK); FITC-labeled anti-human CD54 antibody (ICAM-1; clone 84H10) from Serotec (Oxford, UK); anti-human CD44v3 antibody (clone BBA11) from R&D Systems Europe (Abingdon, UK); anti-CD44s (25.32) and CD44v6 (FW11.9.2.2) hybridoma from The European Collection of Cell Cultures (Porton Down, UK); and anti-mouse IgG heavy and light chain (H+L) FITC-conjugated F(ab')2 fragment from DAKO (Bucks, UK).

The bronchial carcinoma-derived cell line NCI-H292 (3) was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium containing 10% heat-inactivated FCS. The SV40-transformed bronchial epithelial cell line 16HBE14o- (8) was a gift from Dr. D. Gruenert (Cardiovascular Research Institute, University of California, San Francisco, CA) and was cultured in MEM supplemented with 10% heat-inactivated FCS. Cells were cultured at 37°C with 5% CO2, and plastics were obtained from Falcon (Becton Dickinson, Oxford, UK). The mouse monoclonal anti-human ICAM-1 antibody was used at a working dilution of 1:30; anti-human CD44v3 antibody (1:500), anti-mouse IgG (H+L) FITC-conjugated F(ab')2 (0.1 µg/tube), and anti-CD44s, anti-CD44v6, and anti-CD44v9 antibodies were purified from hybridoma supernatants and used at the optimal working dilution established by titration in the range of 20-30 µg/ml.

Cell damage. NCI-H292 or 16HBE14o- cells were cultured in 90-mm-diameter petri dishes at 2 × 104 or 4 × 104 cells/cm2. Approximately 48 h after confluence, the cell layer was damaged with a fine plastic pipette tip, which removed 40-50% of cells. The cells were harvested from 1 to 120 h after cell damage, labeled with antibodies, and analyzed by flow cytometry as in Flow cytometry analysis.

Cell density and culture of cells at different densities. To reduce the possible effects of nutrient depletion and soluble factors produced by cells at higher densities, low- and high-density cell cultures were grown in culture plates and shared the same medium. NCI-H292 or 16HBE14o- cells were cultured in 90-mm-diameter petri dishes that were initially divided into two parts by a plastic spacer. The gap between the plastic spacer and petri dish was sealed by sterile petroleum jelly (Vaseline), and cell suspensions were plated on either side of the divider. On one side, cells were plated at high cell density (1.0 × 105 to 0.3 × 104 cells /cm2) and on the other, at low cell density (0.125 × high cell density). The cells were allowed to settle and adhere for 12 h, after which the spacer was removed and the cells were cultured in the same medium until the highest cell density culture in the series had been confluent for 3 days, after which analysis of flow cytometry was performed (see Flow cytometry analysis). To examine the effect of the period of time in culture on CD44 expression after the cells were plated, 3 × 106 cells were plated in one side of a dish and 3.75 × 105 cells in the other. After 12 h, the spacers were removed, and the cells from each dish were collected as above but at different times.

Cell confluence was defined as the point when the surface of the culture dish was covered in a layer of cells with no gaps visible by phase-contrast light microscopy. Subconfluent cultures are expressed as a percentage of the confluence culture, with the cell number counts in the subconfluent culture divided by the cell number at confluence. Cell number was established with a hemocytometer.

Flow cytometry analysis. For flow cytometry, cells were harvested by washing twice in Ca2+- and Mg2+-free HBSS and then incubated in nonenzymatic cell dissociation solution at 37°C until all the cells were detached. The cells were then resuspended in HBSS containing 2% FCS and 0.1% sodium azide (fluorescence-activated cell-sorting buffer) and adjusted to equal cell number per unit volume (2 × 106 cells/ml) for all cell densities. Aliquots of 2 × 105 cells were incubated for 1 h on ice with 30 µl of anti-ICAM-1 antibody or CD44 primary antibodies. Appropriate negative controls were set up for each antibody. For CD44 antibodies, 30 µl of anti-mouse IgG (H+L) FITC-conjugated F(ab')2 fragment (0.1 µg total) were added to each tube after the cells were washed, and the tubes were incubated for 45 min on ice. After the final antibody incubation, the 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 Lysis II software; fluorescence was measured and displayed on a single parameter histogram with a log scale.

For cell cycle analysis, 16HBE14o- cells grown at different cell densities, including 10%, 50%, and fully confluent, were collected by trypsinization, pelleted, and resuspended in HBSS to a final concentration of 1 × 106 cells/ml. Cells in 1 ml of cell suspension were ethanol fixed and resuspended in 1 ml of staining solution (50 µg/ml of propidium iodide and 100 U/ml of RNase A in PBS with 0.1% glucose). Samples were stained for 30 min at room temperature before analysis by flow cytometry.

Immunostaining. For immunofluorescent staining, 16HBE14o- cells were grown on glass coverslips (22 × 22 mm). When confluent, the cells were damaged with a plastic pipette tip pulled across the coverslip that removed the cells in a strip 150-200 µm wide. Cells on the coverslips were fixed after different periods in culture after damage and stained with anti-CD44 antibody (25.32) or an isotype control antibody followed by incubation with rabbit anti-mouse IgG (H+L) FITC-conjugated F(ab')2 fragment. Cells on the coverslips were then mounted in Mowiol (Harlow Chemical, Essex, UK) with Citifluor as an antifading agent (Citifluor, London, UK).

Image analysis. For each time point, duplicate samples were stained as in Immunostaining and measured at the same time. All the samples were kept in the dark and examined for the same short period of time to minimize fading. At three random points along the damaged edge in each sample, seven neighboring images at ×200 magnification (field size 75 × 135 µm, image size 765 × 512 pixels) were taken. To do this, a cooled charge-coupled device camera (Digital Pixel, Brighton, UK) connected to a Leica DMRB fluorescence microscope was used, images were then transferred to TIF format, and staining intensity was assessed with Scion Image software (Scion) with a Pentium II 233 MHz-based microcomputer. Fluorescence intensity was measured as the mean of pixel intensity in each rectangle.

Cytokine and cell density. Cells were cultured in 24-well plates at 1 × 105 cells/well for NCI-H292 cells and 2.5 × 105 cells/well for 16HBE14o- cells. When 60-70% confluent, the cells were serum deprived for 12 h and then changed into their normal medium containing 2% Ultroser G serum substitute instead of FCS. The cells were incubated with and without IFN-gamma (200 U/ml) and TNF-alpha (200 U/ml) for 24 h and then collected for flow cytometry analysis. These concentrations gave maximal ICAM-1 responses (data not shown) and have also been used in another study (27).

Statistical analyses. All flow cytometry data are expressed as median fluorescence values. Significance was assessed with a nonpaired two-group t-test or the ANOVA test (cytokine treatments).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CD44 expression is altered after cell damage. By flow cytometry, the median value of cell fluorescence intensity for total CD44 after 16HBE14o- or NCI-H292 cell layers were mechanically damaged was decreased 6 h after damage. After 12 h, the level was increased, and this was maintained until at least 48 h after damage. Other CD44 isoforms showed similar changes after cell damage (Fig. 1). ICAM-1 expression was not significantly changed after damage (data not shown).


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Fig. 1.   CD44 isoform immunoreactivity measured by flow cytometry in 16HBE14o- (A) and NCI-H292 (B) cells after mechanical damage. , Standard CD44 (CD44s); , CD44 variant 3 (CD44v3); triangle  CD44 variant 6 (CD44v6); ×, CD44 variant 9 (CD44v9). Fluorescence intensity of CD44 isoform immunoreactivity was determined by flow cytometry as described in MATERIALS AND METHODS. Data are mean percentages of fluorescence intensity normalized to undamaged cells (100%).

Altered expression of CD44 after cell damage was also seen with indirect immunofluorescence (Fig. 2, A-C). The increase in the mean pixel intensity toward the wound edge in digitized images indicated an increase in CD44 immunoreactivity associated with cells in this area. After an initial small drop in intensity at 3 h, there was an overall increase in CD44 immunoreactivity up to 48 h, even up to 450 µm from the wound edge (Fig. 2, D and E). The CD44 immunostaining was stronger closer to the wound edge, particularly in the first 12 h (Fig. 2, A-C). At 36 (Fig. 2, D and E) and 48 (Fig. 2E) h, staining intensity was increased over a wider band around the track of the wound. Wound closure in these samples was seen in some areas of the wound by 12 h and was complete at 24 h.



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Fig. 2.   Quantitative evaluation of CD44s immunoreactivity in damaged areas. CD44s immunoreactivity on 16HBE14o- cells was measured by analysis of pixel intensity of 7 adjoining 75 × 135-µm rectangular areas along wound edge (B, 1-7). A-C: pattern of CD44s staining at 30 min, 3 h, and 12 h, respectively, after damage. D: change in mean pixel intensity of CD44 in wounded cultures at different times and distances from wound margin. E: change in mean pixel intensity against time. Areas were grouped and averaged. Each value is mean ± SE.

CD44 isoform expression is increased at lower cell densities. By flow cytometry, 16HBE14o- cells showed immunofluorescence significantly above control levels with antibodies to ICAM-1, total CD44, and CD44 isoforms (Fig. 3). Although ICAM-1 fluorescence was increased in high-density 16HBE14o- cultures (Fig. 3B), the level of CD44 immunoreactivity for all isoforms was greater in cells cultured at lower cell density (Fig. 3, C-F). Cells at lower density were also more immunoreactive for CD44v3, CD44v6, and CD44v9 (both cell lines; Fig. 4). With high and low cell density cocultures, cells were seeded at densities from 130 × 103 to 0.5 × 103 cells/cm2 (in the high cell density section). Before confluence was achieved, the cells at lower density in the cocultured preparations in all cases expressed more CD44 than those at high density (Figs. 3 and 4). A similar pattern was seen in NCI-H292 cells, although CD44 immunoreactivity was generally less in the NCI-H292 cells (Figs. 4B and 5B). The total CD44 expression decreased with cell density until the cells were confluent in both the 16HBE14o- and NCI-H292 cell lines (Fig. 4). There was good correlation of the CD44 expression between the lower densities of the "high-density" series and the higher densities of the "low-density" cultures (Fig. 5, overlapping lines). At confluence, the cells reached a density of ~1.5 × 105 cells/cm2 in 16HBE14o- and NCI-H292 cells. Maximum cell densities were 2.5 × 105 cells/cm2 in 16HBE14o- cells and 5.0 × 105 cells/cm2 in NCI-H292 cells. Little change in CD44 expression was seen after confluence (Fig. 5).


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Fig. 3.   Flow cytometric analysis of CD44 and its isoforms on 16HBE14o- cells. Cells from subconfluent (shaded area) and confluent (solid line) cultures were stained with CD44 antibodies, with a second antibody conjugated to FITC or intercellular adhesion molecule (ICAM)-1 antibodies directly labeled with FITC. Flow cytometry results show fluorescence intensity histogram on a log scale for 10,000 cells from each sample. A secondary antibody control for CD44 (A) shows no difference between confluent and subconfluent cells, whereas ICAM-1 (B), total CD44 (C), CD44v3 (D), CD44v6 (E), and CD44v9 (F) show different levels of staining under these conditions. For ICAM-1, another FITC-labeled antibody of the same isotype was used as a control (B, dotted line).



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Fig. 4.   CD44 expression in different cultures of 16HBE14o- (A) and NCI-H292 (B) cells at different levels of confluence. Flow cytometry for total CD44, CD44v3, CD44v6, and CD44v9 was performed on cultured bronchial cells grown to <60% confluence, 80-90% confluence, and full (100%) confluence (for >24 h). Results are medians ± SE from 4-5 separate cultures.



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Fig. 5.   Cell density and CD44 expression. Cells were seeded at 0.5 × serially reducing cell densities in pairs at high and low (<FR><NU>1</NU><DE>8</DE></FR> of high cell density) cell densities in the 2 halves of a petri dish. Points labeled with the same roman numerals are high and low cell density cultures from the same petri dish. Because the maximum low density was higher than the minimum high density, there is some overlap. Cells were harvested for flow cytometry analysis, and final cell density was calculated as described in MATERIALS AND METHODS. Data are medians of fluorescence intensity of CD44 isoforms on 16HBE14o- (A) and NCI-H292 (B) cells.

CD44 expression on cell lines is not affected by length of culture. Cells collected at the same final cell density but cultured for different lengths of time showed the same relationship of final cell density to total CD44 expression (Fig. 6) as seen for cells seeded at different densities but cultured for a consistent period of time (Fig. 5). There was a relative reduction in CD44 expression on cells collected after 1 day, which probably reflected the time taken to recover from passaging the cells on day 0. Other CD44 isoforms showed the same pattern (data not shown). Thus CD44 expression was dependent on final cell density and not on the time of culture before collection (Fig. 6).


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Fig. 6.   Plating time and CD44 expression on 16HBE14o- (A) and NCI-H292 (B) cells. Cells were plated at constant high (solid line) and low (<FR><NU>1</NU><DE>8</DE></FR> of high cell density; dashed line) cell densities in the 2 halves of a petri dish. Nos. above symbols, period of culture for the sample in days. Median fluorescence is plotted against final cell density at the time when the cells were collected for flow cytometry.

Cell proliferation is unaffected at different densities. To determine the effect of cell density on proliferation, cell cycle analyses were carried out for different densities. There was no significant difference in the percentage of cells in the G2/M phase cell population at 10% subconfluence (23.7 ± 2.6), 50% subconfluence (17.1 ± 3.2), or full confluence (18.3 ± 2.4).

TNF-alpha can enhance CD44 isoform expression. IFN-gamma and TNF-alpha at 200 U/ml increased the expression of ICAM-1 relative to control values on confluent cells by up to 1.8-fold (16HBE14o- cells, Fig. 7A) or 17-fold (NCI-H292 cells, Fig. 7B). The large increase in ICAM-1 on NCI-H292 cells reflects a relatively low baseline ICAM-1 expression. The effect of IFN-gamma and TNF-alpha at the same concentrations on CD44 and its isoforms was less marked (Fig. 7), never exceeding 1.2-fold (16HBE14o- cells) or 1.35-fold (NCI-H292 cells). In contrast to the ICAM-1 response, the change in CD44 induced by TNF-alpha was generally greater than that by IFN-gamma (Fig. 7). Only the increased CD44 expression of CD44v9 due to TNF-alpha on 16HBE14o- cells reached significance (P < 0.05; Fig. 7A). TNF-alpha increased total CD44 and all the isoforms examined on NCI-H292 cells and, in conjunction with IFN-gamma treatment, increased total CD44. On NCI-H292 cells, the most marked effect was an increase in CD44v6 and CD44v9 (P < 0.01; Fig. 7B).


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Fig. 7.   Effect of treatment on 16HBE14o- (A) and NCI-H292 (B) cells with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha for 24 h on ICAM-1 (right axis) and CD44 (left axis) levels measured by flow cytometry. Values are means ± SE for each antibody from 5 separate experiments normalized to 100% for untreated cells. Significant difference between samples: * P < 0.05; ** P < 0.01. NS, not significant.

Because the effect of IFN-gamma and TNF-alpha on the expression of CD44 might be mediated by a change in cell density induced by these cytokines, the cell density after IFN-gamma and TNF-alpha treatment was compared with untreated cells at 24 h. No changes in cell density were seen (data not shown) that might account for the changes in CD44 with these treatments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study with a mechanical damage model, CD44 expression was increased after cell damage and showed a predictable time course. A local increase in CD44 was seen at the wound margin, particularly in the 150 µm proximal to the wound track, corresponding to those cells previously shown to be involved in repair (48). Quantification by image analysis also revealed a general increase in CD44 up to 500 µm from the track of the wound and up to 48 h after wounding. An increase in the population mean fluorescence intensity for CD44 was also seen by flow cytometry. In the mechanically wounded epithelium, the small decrease in CD44 expression shortly after damage seen by flow cytometry may reflect reduced expression on damaged cells. Increased CD44 expression on cells at low density, even when they are grown in the same medium as cells at high density, suggests that neither soluble mediators nor nutrient depletion are responsible, and this is consistent with the hypothesis that upregulation of CD44 expression in a damaged area of epithelium reflects low cell density rather than the influence of soluble mediators. The later increase away from the damaged edge indicates that the response is propagated away from the wound margin to undamaged cells. In a similar way, CD44v6 has been reported to be upregulated on smooth muscle cells in injured arteries in vivo (20).

CD44 and cell density. After damage, a rapid response to restore airway epithelial structure is often critical to prevent infection and ingress of harmful environmental agents. Local loss of epithelial cells such as is frequently seen in asthma results in locally reduced cell density that is only restored relatively late in the repair response. CD44 immunoreactivity was approximately three- to fourfold higher in low-density compared with high-density (confluent) cultures. All cells continued to express some CD44 at high densities, but the percentage expressing CD44 isoforms containing CD44v6 or CD44v3 fell to 65%, indicating that different isoforms are differentially regulated in relation to cell density. Although the pattern of CD44 expression with cell density is similar between the two cell lines, the absolute cell density at which the reduction in CD44 was seen was higher in NCI-H292 cells. The level of expression for all CD44 isoforms was lower in NCI-H292 than in 16HBE14o- cells, and the maximum cell density achieved in NCI-H292 cells in culture was also higher. This may represent differences in cell size or the way in which the cells of this carcinoma-derived cell line "pack" together.

The observation of the same density-related CD44 expression trends irrespective of the previous period of growth in culture again suggests that cell-cell contact at higher cell densities is likely to regulate CD44 expression. The effect of cell density was greatest before the cells became confluent, and little further reduction was seen after confluence, although the cell density continued to increase. Taken together, these results indicate that CD44 expression is inversely correlated with cell density and that the effect is likely to be mediated through cell-cell contact. This parallels results from tissue samples where Lackie et al. (24) have previously observed that cells that are more differentiated, particularly columnar epithelial cells, do not express CD44.

Cell adhesion and cell density. Although the regulation of cell adhesion molecules has been extensively studied, most of the work has been devoted to the effects of cytokines (7, 17, 38, 42), growth factors (41), and extracellular matrix (33). Little is known about the direct effects of cell density on adhesion molecule expression. Upregulation of neural cell adhesion molecule in neuronal cells at high density has been observed (6) and is suggested to enhance aggregate formation. Studies in bladder and colonic carcinoma cell lines (35) have also shown that integrin expression changed with cell density and that these changes varied between cell types and different integrin molecules.

The two- to fourfold change in CD44 expression with cell density supports a link with cell-cell contact, whereas the contrasting increase in ICAM-1 expression at higher cell densities suggests that cell adhesion molecule expression is actively regulated. This also indicates the importance of controlling for cell density in experiments looking at the expression and regulation of CD44 and other adhesion molecules on cultured cells. If cell density is not controlled in experimental procedures, then the current studies would indicate that variation in cell density may increase variability in the results or might even introduce a systematic variation in CD44 expression linked to cell density rather than to other experimental conditions.

Effects of IFN-gamma and TNF-alpha on CD44 and ICAM-1. Although upregulation of ICAM-1 by inflammatory cytokines is well documented, less is known about the role of cytokines in regulating CD44 expression. We have confirmed that ICAM-1 was strongly upregulated in 16HBE14o- and NCI-H292 cell lines by IFN-gamma and TNF-alpha . In contrast, total CD44 and some of the CD44 isoforms were not affected by IFN-gamma and TNF-alpha . The contrasting increase in ICAM-1 expression at higher cell densities indicates that nutrient depletion is not responsible and confirmed that these cytokines were active when added to cell cultures. This confirms the findings of a previously published study (5) reporting CD44 expression in bronchial cell lines that have also shown no effect with IFN-gamma and TNF-alpha treatment. In other systems, TNF-alpha upregulates CD44 on endothelial cells (28). In myelomonocytic cell lines, TNF-alpha predominantly upregulated CD44v9- and IFN-gamma -induced CD44v6 expression (27). Furthermore, IFN-gamma downregulated total CD44 and CD44v9 and upregulated CD44v6 in some carcinoma and keratinocyte epithelial cell lines (27). In melanoma cells, little change in CD44 expression was seen with IFN-gamma and TNF-alpha treatment (9).

Thus IFN-gamma and TNF-alpha do not appear to be important regulators of bronchial epithelial CD44 expression, and the changes seen in response to treatment were not significant compared with those associated with changes in cell density.

Putative mechanisms of CD44 variation with cell density and its functional significance. The time course of increased CD44 expression is consistent with transcriptional control and clearly does not require the presence of other (nonepithelial) cell types. This supports the suggestion that the increased expression of CD44 seen in airway epithelium in asthma (24, 30) is associated with repair processes rather than with the associated inflammation.

The kinetics of CD44 expression seen in the present study provide some indication of the functional significance of the observed changes. In vitro, CD44 was upregulated from 12 to 72 h after damage, suggesting that it is not important in the immediate response to damage but is rather involved in repair, corresponding to the start of cell migration (25). Expression continues to be elevated during the repair process after migration is complete. During epithelial repair, as cell density increased, we would expect to initially see increased cell motility, cell contact, and a trend toward cell differentiation. These changes are correlated with reducing CD44 expression. CD44 might thus enhance cell migration in repair as seen in dendritic (43) and melanoma (15) cells and implicated in epithelial cells (18). The fact that CD44 expression is sustained longer than the initial repair response presumably reflects the time taken for these processes to be completed after repair and the time lag in returning to a normal phenotype.

Changes in cell-matrix adhesion might be expected to result from increased CD44 expression and could be further affected by the differing extracellular matrix affinities of the variants of CD44 expressed and the relative importance of HA as a ligand. However, because in vivo CD44 does not appear to be in areas of close cell-matrix contact (24), it is possible that altered CD44 expression is more involved in cell-cell interactions in repair. The CD44v3 variant, which contains the predominant CD44 glycosaminoglycan modification site, was expressed on less cells than total CD44 or CD44v9 at high cell densities. This is of particular interest because CD44v3 may enhance the response to heparin binding growth factors on cells (4, 19).

Increased CD44 has been previously associated with cell proliferation in normal and neoplastic human colorectal epithelial cells (1), proliferation being increased during repair and reduced at cell confluence. However, the in vitro systems used in this study differ significantly from the intestinal system, which is more stratified and has morphologically defined areas of cell proliferation. A previous study of airway epithelial repair after damage (48) showed that proliferation is maximal in the region 160-400 µm from the wound edge at 48 h. In the present study, CD44 was increased in this area at this time; however, the marked increase closer to the wound edge and at earlier times suggests that the increased CD44 expression is related to other processes. In the current model at different cell densities, cultures showed no significant difference in the percentage of cells in the G2/M phase cell population (range 6%), whereas CD44 expression varied two- to threefold, suggesting that the expression of CD44 is cell proliferation independent. In common with CD44v3, the number of cells expressing CD44v6 was significantly reduced at confluence. CD44v6 expression in tumors has been correlated with malignancy (11, 16) and could reflect its involvement in cell migration.

In conclusion, CD44 expression on cell lines derived from bronchial epithelium was increased on subconfluent cells, appeared to be correlated with epithelial repair processes, and was cell density dependent. CD44 expression is likely to be controlled by cell-cell-mediated signaling rather than by soluble factors. CD44 isoforms are thus likely to be involved in cell migration and other processes occurring in the mid to late phases of epithelial repair.


    ACKNOWLEDGEMENTS

We thank Dr. D. Gruenert (Cardiovascular Research Institute, University of California, San Francisco, CA) for the 16HBE14o- cell line and Dr. U. Günthert (Basel Institute for Immunology, Basel, Switzerland) for the CD44v9 antibody.


    FOOTNOTES

This work was supported by Medical Research Council Programme Grant G8604034 (to S. T. Holgate) and financial support from Rhone-Polenc Rohrer (London, UK).

S.-H. Leir was the recipient of an Overseas Research Studentship.

Part of this work was previously presented in abstract form at the American Society for Cell Biology meeting in 1997.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Lackie, Southampton Univ. Medicine, Level D Centre Block, Southampton General Hospital, Tremona Rd., Southampton SO16 6YD, UK (E-mail: p.m.lackie{at}soton.ac.uk).

Received 6 July 1999; accepted in final form 19 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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