Southampton University Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom
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ABSTRACT |
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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-
and increased by <50% by tumor necrosis factor-
.
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
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INTRODUCTION |
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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)- and interferon (IFN)-
. 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- and TNF-
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.
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MATERIALS AND METHODS |
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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- and TNF-
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.
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, 16HBE14oImmunostaining.
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-
(200 U/ml) and TNF-
(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).
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RESULTS |
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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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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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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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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- can enhance CD44 isoform expression.
IFN-
and TNF-
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-
and TNF-
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-
was
generally greater than that by IFN-
(Fig. 7). Only the increased
CD44 expression of CD44v9 due to TNF-
on 16HBE14o
cells
reached significance (P < 0.05; Fig. 7A). TNF-
increased total CD44 and all the isoforms examined on NCI-H292 cells
and, in conjunction with IFN-
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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DISCUSSION |
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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.
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- and TNF-
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-
and TNF-
. In
contrast, total CD44 and some of the CD44 isoforms were not affected by
IFN-
and TNF-
. 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-
and TNF-
treatment. In other
systems, TNF-
upregulates CD44 on endothelial cells (28). In
myelomonocytic cell lines, TNF-
predominantly upregulated
CD44v9- and IFN-
-induced CD44v6 expression (27). Furthermore,
IFN-
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-
and
TNF-
treatment (9).
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 |
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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.
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FOOTNOTES |
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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.
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