1 Novartis Horsham Research Centre, Horsham, West Sussex RH12 5AB, United Kingdom; and 2 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current (Isc), followed by a sustained inhibition of amiloride-sensitive Isc. These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in Isc was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.
trypsin; epithelial sodium channel; human airway
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INTRODUCTION |
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PROTEASE-ACTIVATED RECEPTORS (PARs) are a family of seven-transmembrane G protein-coupled receptors (7, 11). These receptors are activated by the proteolytic cleavage of a receptor-bound, NH2-terminal tethered ligand domain, which is then able to bind to the receptor and initiate signaling. To date, four PARs (PAR1-4) have been characterized (11). Thrombin activates PAR1, PAR3, and PAR4, whereas trypsin and mast cell tryptase activate PAR2 and possibly PAR4 (11). Synthetic peptides resembling the tethered ligand sequence of the PARs (with the exception of PAR3) can bind and activate the receptors and are useful tools for functional studies.
The functional relevance of PARs has been the subject of intense study
because of their widespread systemic distribution and potential roles
in numerous systems, including hemostasis (7), inflammation (5, 25), the regulation of smooth muscle tone (4, 20), and ion transport processes (1, 6,
24). PAR2 has been identified in a number of epithelial tissues
and has been observed to have both protective and proinflammatory activities. PAR2 has been described as a "sentry for inflammation" due to its linkage to a variety of cytoprotective pathways
(5). A protective role for PAR2 has been proposed in the
pancreatic duct epithelium in which PAR2 activation induced a
Cl secretory response that would potentially "flush"
bacterial toxins or microorganisms out of the system (24).
A similar PAR2-mediated activation of Cl
secretion
described in the M-1 kidney cortical collecting duct cell line might
also represent a flushing/diluting mechanism (1). PAR2
activation has further been demonstrated to trigger glandular mucin
secretion (15), another cytoprotective mechanism. However, with the exception of thrombin-mediated activation of PAR1 in the
vasculature, the relevant endogenous activators of PAR2 are still
largely the subject of speculation.
The regulation of ion transport processes in the airway epithelium is highly coordinated, and, dysregulation, as in cystic fibrosis, can be catastrophic to the normal functioning of the lung. The maintenance of a hydrated airway lining is essential for efficient mucociliary clearance (18), a primary defense mechanism for the removal of inhaled debris or microbes. The aim of the present study was to establish whether PAR2 could regulate ion transport processes in a differentiated culture of normal human bronchial epithelial cells (HBECs).
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METHODS |
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Cell Culture
HBECs (Biowhittaker, UK) were cultured using a modification of the method described by Gray and colleagues (9). Cells were seeded into plastic T-75 flasks and grown in bronchial epithelial cell growth medium (BEGM; Biowhittaker) supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant epidermal growth factor (0.5 µg/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 µg/ml), triiodothyronine (6.5 µg/ml), gentamycin (50 µg/ml), and amphotericin B (50 µg/ml). Medium was changed every 48 h until cells were 90% confluent. Cells were then passaged and seeded onto polycarbonate Snapwell inserts (Costar, UK) or 25-mm glass coverslips (Fisher Scientific) in differentiation media containing 50% DMEM in BGEM with the same supplements as above but without triiodothyronine and a final retinoic acid concentration of 50 nM (all-trans-retinoic acid). Cells were maintained submerged for the first 7 days in culture, after which time they were exposed to an apical air interface for the remainder of the culture period. Cells on coverslips were maintained submerged during the entire culture period. Cells on inserts were used between days 14 and 21 after establishment of the apical air interface, and cells on coverslips were used between days 7 and 14 after seeding. At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator. All studies were performed on cultures from three individual donors.Short-Circuit Current Measurements
Snapwell inserts were mounted in Costar vertical diffusion chambers and were bathed with continuously gassed Ringer solution (5% CO2 in O2; pH 7.4) maintained at 37°C containing (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose. The solution osmolarity was always between 280 and 300 mosmol/kgH2O for all physiological salt solutions used. Cells were then voltage clamped to 0 mV (EVC4000, World Precision Instruments). Transepithelial resistance (RT) was measured by either applying a 2-mV pulse at 60 s intervals or by measuring the potential difference under open-circuit conditions and calculating RT by Ohm's law. Data were recorded using a PowerLab workstation (ADInstruments, UK). For ClIntracellular Ca2+ Concentration Measurements
HBECs on 25-mm glass coverslips were incubated at room temperature with 5 µM fura 2-AM in a bath solution containing (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 using NaOH) for 45 min. Cells were washed with a fura 2-AM-free bathing solution and incubated at room temperature for 45 min to allow for the further hydrolysis of intracellular fura 2-AM to fura 2. Coverslips were then mounted in a 0.5-ml perfusion chamber, cells facing upward, on a heated stage fixed at 37°C. Fluorescence was measured with an inverted microscope (Nikon Diaphot 300) equipped with a ×40 oil-immersion objective. Fluorescence was monitored at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Images were acquired every 2 s using an intensified charge-coupled device camera (C4742-95; 640 × 512 pixels resolution; Hamamatsu Photonics, Bridgewater, NJ). The Ca2+ concentration was calculated by in vivo calibration using the following formula
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Compound Additions
The effects of PAR activators were studied on the spontaneous basal short-circuit current (Isc) established by the HBECs and on the development of a forskolin-dependent current following treatment with the epithelial Na+ channel blocker, amiloride. To investigate potential mechanisms of any PAR activity on these currents, experiments were also performed under ClRT-PCR and Cloning of PAR2 and Putative Endogenous PAR Activators
Unless otherwise stated, all reagents were used according to the manufacturer's instructions. Briefly, mRNA was extracted from HBEC cultures using a QIAGEN RNeasy Mini kit. The expression of mRNA transcripts for PAR2, human airway trypsin-like enzyme (HAT) and trypsinogen, was investigated using RT-PCR (Promega Access RT-PCR System). Samples were run in a Biometra T3 Thermocycler for 30 cycles using primers designed from GenBank sequences (Primer Express, PE Biosystems). Primers were synthesized by Sigma Genosys: 5'-cctgcagtggcaccatcc-3' (PAR2 forward), 5'-cagggagatgccaatggc-3' (PAR2 reverse), 5'-cctggcagtcaccatagctct-3' (HAT forward), 5'-agtcttcttgtactagtggg-3' (HAT reverse), 5'-acaagtcccgcatccaggt-3' (trypsinogen forward), and 5'-tggtgtccttaatccagtcc-3' (trypsinogen reverse). A further hot-start PCR was carried out on the RT-PCR products for HAT and trypsinogen to increase specificity and selectivity. Each reaction contained 5 µl of buffer (PCR reaction buffer containing MgCl2; Boehringer Mannheim), 1 µl of dNTP mix (Promega), 1 µl (300 µg) of forward primer, 1 µl (300 µg) of reverse primer, 1 µl of RT-PCR product (diluted 1/20), and 40 µl of diethyl pyrocarbonate-treated water. One microliter of Taq DNA polymerase (Boehringer Mannheim; 5 U/µl) was added to the tubes after the initial denaturation at 94°C while the reaction paused at 80°C. The hot-start PCR was run in a Biometra T3 Thermocycler (30 cycles of 60°C, 45 sHAT and trypsinogen transcripts were sequenced using the ABI Prism Big
Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied
Biosystems). Briefly, the bands from the appropriate reactions were
excised from the gels and the DNA was extracted using a QIA quick gel
extraction kit (Qiagen). Sequencing reactions were prepared and run in
a Biometra T3 Thermocycler (30 cycles of 96°C 30 s 50°C,
15 s
60°C 4 min). After ethanol precipitation, the final
product was resuspended in 25 µl of template suppression reagent (ABI
Prism DNA sequencing kit) and sequenced on an ABI Prism 310 sequencer,
and identity was confirmed by comparison with GenBank sequences. PAR2
was cloned using the TOPO cloning system and TOP10 One Shot Chemical
transformation kit (Invitrogen). Plasmids were analyzed by restriction
analysis, isolated using the QIAGEN Plasmid Mini kit (Qiagen), and
sequenced as described above.
Expression of Results and Statistical Analysis
Results are expressed as absolute changes in Isc (means ± SE). Measurements were taken either as peak changes or once responses had plateaued and were stable. Control inserts were run alongside all experiments for paired comparisons to be made owing to the potential day-to-day and interbatch variability of the Isc. A Student's t-test was used to compare between groups with statistical significance assumed when P < 0.05.Reagents
HBECs obtained from postmortem specimens were purchased from Biowhittaker, as were all media. All other cell culture reagents were purchased from Life Technologies (UK). PAR peptide agonists were purchased from Sigma Genosys (UK). Fura 2-AM was purchased from Molecular Probes. All other reagents were purchased from Sigma (UK). ![]() |
RESULTS |
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HBEC Characteristics
The culture methods employed produced a multilayered bronchial epithelial tissue that had differentiated to the extent that ciliated and goblet cells were identifiable. Goblet cells typically accounted for 20-25% of the total number of cells (data not shown). When voltage clamped to 0 mV, the cells displayed an Isc of 9.1 ± 0.3 µA/cm2 (n = 90) and an RT of 972 ± 34Trypsin and Thrombin on Basal Isc
Trypsin (1 µM), when added to the basolateral medium, caused a transient increase in Isc of 12.5 ± 1.5 µA/cm2 above baseline that was followed by a sustained reduction in Isc of 4.4 ± 0.5 µA/cm2 below the initial baseline and a 38.0 ± 3.3% increase in RT (n = 4; Fig. 1). The subsequent addition of amiloride (10 µM) to the apical medium reduced the Isc by a further 3.6 ± 0.3 µA/cm2, compared with a decrease of 5.8 ± 0.2 µA/cm2 in the paired control cells (P < 0.05; n = 6). The decrease in Isc below basal levels observed with the combination of trypsin and amiloride of 8.0 ± 0.4 µA/cm2 was significantly greater than the decrease of 5.8 ± 0.2 µA/cm2 seen with amiloride alone (P < 0.05), indicating that trypsin also inhibits a proportion of the amiloride-insensitive current.
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The addition of forskolin in the presence of amiloride caused an increase in Isc of 12.5 ± 0.4 µA/cm2 (n = 6) in control cells that was significantly attenuated in the trypsin-treated cells to 4.0 ± 0.6 µA/cm2 (n = 4; P < 0.01). Heat-inactivated trypsin added to the basolateral surface was without effect on any of the currents studied (data not shown). Likewise, there was no effect of trypsin when added apically or to thrombin (5 U/ml) added to either the apical or basolateral solution on the Isc (Fig. 1, C and D).
Dose Dependence of Trypsin and PAR Peptide Effects
The effects of trypsin on the ion transport characteristics described above were concentration dependent and observed over a concentration range of 100-1,000 nM (Fig. 2). These phenomena were also observed with the PAR2-activating peptide, SLIGRL-NH2 (10-100 µM; Figs. 2 and 3), and the mixed PAR1/2-activating peptide, SFLLRN-NH2 (15 and 100 µM), added to the basolateral membrane (Fig. 3). SLIGRL-NH2 (100 µM) caused a transient increase in Isc of 12.5 ± 0.8 µA/cm2, followed by a decrease below the initial basal current of 2.7 ± 0.5 µA/cm2 (n = 4). The residual amiloride-sensitive Isc of 1.7 ± 0.2 µA/cm2 was also significantly reduced when compared with the paired control amiloride-sensitive Isc of 2.9 ± 0.1 µA/cm2 (P < 0.05). The subsequent forskolin-stimulated current increase of 3.3 ± 0.3 µA/cm2 in the SLIGRL-NH2-stimulated group was also significantly reduced when compared with the paired control increase of 8.4 ± 0.3 µA/cm2 (P < 0.05). A scrambled control PAR2 peptide (LSIGRL-NH2, 100 µM) was without effect on the Isc. Basolateral SFLLRN-NH2 (100 µM), the mixed PAR1/PAR2 peptide, induced a transient increase in current of 3.5 ± 0.1 µA/cm2 followed by a decrease of 6.0 ± 0.3 µA/cm2 below the basal current (n = 4). The amiloride-sensitive current was reduced from 10.4 ± 0.7 µA/cm2 in the paired control group to 4.4 ± 0.3 µA/cm2 in the SFLLRN-NH2-treated cells (P < 0.05). SFLLRN-NH2 also reduced the forskolin-stimulated increase in Isc from 11.9 ± 0.8 µA/cm2 in paired control cells to 7.0 ± 0.4 µA/cm2 (P < 0.05). The selective PAR1 peptide, TFRIFD-NH2 (100 µM), was without effect (Fig. 3C; n = 4).
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Potential Mechanisms of PAR2-Mediated Effects on Ion Transport
Ca2+ dependence.
PAR2-mediated effects in other cell types have been shown to be
mediated by an increase in [Ca2+]i. This
signaling mechanism was investigated in HBECs using the fluorescent
Ca2+-sensitive dye fura 2 and by buffering intracellular
Ca2+ using BAPTA. Loading the cells with BAPTA (50 µM, 60 min) had no effect on the basal characteristics of the epithelium.
Under these conditions, the initial transient increase in
Isc observed following trypsin (1 µM) was
reduced from 23.5 ± 2.9 µA/cm2 to 1.1 ± 0.4 µA/cm2 (P < 0.05, n = 4;
Fig. 4). There was, however, no effect of
BAPTA loading on the decrease in Isc, with
trypsin causing reductions in Isc of 5.6 ± 0.4 µA/cm2 and 5.2 ± 0.1 µA/cm2 in
the absence and presence of loaded BAPTA, respectively
(P > 0.05, n = 4). A similar
phenomenology was observed with SLIGRL-NH2 after BAPTA
loading with the transient response to basolateral SLIGRL-NH2 (30 µM) being reduced from 5.5 ± 1.4 µA/cm2 to 2.7 ± 0.3 µA/cm2
(P < 0.05, n = 4), while the
inhibitory phase remained unaffected (control 3.9 ± 0.8 µA/cm2 vs. BAPTA loaded 2.8 ± 0.2 µA/cm2, P > 0.05). The trypsin-mediated
effects on [Ca2+]i were determined using fura
2. HBECs, grown on coverslips, were loaded with fura 2, and
fluorescence was measured while cells were bathed in
Ca2+-containing solution. The
[Ca2+]i was calculated as described in
METHODS. The basal Ca2+ concentration was
43 ± 13 nM. Addition of trypsin (1 µM) caused a rapid increase
in [Ca2+]i to 321 ± 51 nM. After
1.5-4 min, [Ca2+]i returned to a level
that was not significantly different from the basal level (41 ± 24 nM compared with 43 ± 13 nM, P > 0.05, n = 28 cells, 5 experiments; Fig.
5).
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Cl-free conditions.
Cl
-free conditions were used to establish whether the
PAR2-mediated effects on Isc were dependent on
Cl
transport. In the absence of Cl
, trypsin
(1 µM) caused an increase in Isc of 9.2 ± 0.4 µA/cm2 followed by a sustained decrease of
3.5 ± 0.1 µA/cm2 (n = 6) below the
initial basal current (Fig. 6). Under
Cl
-free conditions, SLIGRL-NH2 (100 µM)
caused an increase in Isc of 8.2 ± 0.4 µA/cm2 and a sustained decrease in current of 2.0 ± 0.2 µA/cm2 (n = 4).
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Basolateral membrane K+ currents.
These experiments were used to establish whether K+
channels on the basolateral membrane played a role in the PAR2-mediated effects on Isc. Permeabilization of the apical
membrane with nystatin caused an immediate increase in K+
currents (IK) under the established
apical-to-basolateral K+ gradient (Fig.
7). The addition of either trypsin (1 µM) or SLIGRL-NH2 (30 µM) induced a transient increase
in Isc of 246.1 ± 8.0 µA/cm2
(n = 6) and 39.5 ± 5.6 µA/cm2
(n = 4) above the nystatin-induced baseline,
respectively. The trypsin- and SLIGRL-NH2-induced transient
increases then decreased to new baselines of 21.4 ± 0.8 µA/cm2 and 14.8 ± 3.6 µA/cm2 below
the nystatin-permeabilized baseline, respectively.
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Expression of PAR2 and Putative Endogenous PAR2 Activators in the HBEC Model
RT-PCR of isolated RNA from the HBECs used in these studies revealed the expression of PAR2 (Fig. 8). The identities of endogenous activators of PAR2 in the airway are unknown and the subject of speculation. Consequently, we examined the expression of two putative endogenous PAR2 activators in the cell culture system employed for the Isc studies. RT-PCR and subsequent sequencing revealed the expression of trypsinogen and HAT in the cultured cells (Fig. 8).
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DISCUSSION |
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PAR2 Activation From the Basolateral Surface
The bronchial epithelial cells used in this study displayed a spontaneous Isc that was 60-80% amiloride sensitive, as previously described for human airway epithelial cultures (8, 9, 29). Robust effects on ion transport were only observed with PAR2 activators (trypsin and PAR2-activating peptides) and not with PARs 1, 3, and 4 (thrombin or TFRIFD-NH2). The responses to trypsin and the PAR2-activating peptides were the same, suggesting that they share a common target, and it is unlikely that the trypsin-induced effect is via cleavage of a protein other than PAR2. PAR2 activation caused a transient increase in Isc followed by an inhibition of amiloride-sensitive Isc. The observation that the combination of PAR2 activator and amiloride caused a greater reduction in basal current than amiloride alone suggests that PAR2 activation also attenuates some of the amiloride-insensitive current. The subsequent forskolin-stimulated increase in Isc was also attenuated. Furthermore, PAR2-induced effects were only observed upon basolateral addition of the activators. It is interesting to note that in tissue sections of human airways, PAR2 was not exclusively localized to the basolateral membrane by immunohistochemistry (4). However, PAR2 was resolved exclusively to the basolateral membrane both functionally and histologically in pancreatic duct epithelium (24). The basolateral dependency for the PAR2 responses in this study is likely to represent receptor localization to the basolateral surface and not a differential membrane coupling to second messenger systems. Studies of P2Y2 agonist effects on ion transport in a variety of airway epithelia have described a similar phenomenology to that observed here, irrespective of apical or basolateral administration (8, 14, 22, 23). Like PAR2, P2Y2 receptors mediate their effects via an increase in intracellular Ca2+ as part of the signal transduction cascade in human airway epithelium (26). Therefore, the appropriate signaling apparatus, with respect to Ca2+ mobilization as well as Ca2+-activated ion channels, is located in both the apical and basolateral membranes. However, PAR2 agonists failed to have any detectable effects when added to the apical side, suggesting a lack of apically located receptors in the cultured cells.PAR2-Induced Transient Increase in Isc
Trypsin and the PAR2-activating peptides (SLIGRL-NH2 and SFLLRN-NH2) caused a transient increase in basal Isc. In the majority of tissues, PAR2 couples to phospholipase CPAR2-Induced Sustained Decrease in Isc
The sustained decrease in Isc observed after the resolution of the transient increase in current was apparent with both trypsin and PAR2 peptide activation. The sustained decrease in Isc was observed in ClPAR2-Induced Attenuation of Forskolin-Stimulated Isc
The inhibition of the forskolin-stimulated increase in Isc by PAR2 activation could also be explained in terms of an effect on basolateral K+ conductance. Forskolin is expected to activate both cystic fibrosis transmembrane conductance regulator and a basolateral KThe location of the PAR2 receptor to the basolateral surface implies that the endogenous ligand (protease) is either released in an autocrine manner by the epithelial cells or by an infiltrating inflammatory cell, or alternatively, the protease is perhaps a product of pathogens colonizing the airway mucosa. The most obvious source of a tryptic protease would be the mast cell, which is able to secrete mast cell tryptase, a recognized activator of PAR2 (7). Mast cell tryptase was not used in these studies because of the quantities that would have been required to achieve the working concentration in the Ussing chamber. It has also been noted that the cleavage site for human PAR2 (KGR/SLI) bears similarity to the activation site of complement factor C1s (KQR/IIG), raising the potential for complement activation to produce an activator of PAR2 (12). Cocks et al. (4) identified trypsinogen in human bronchial biopsies by immunohistochemistry, and we have confirmed the presence of mRNA for this trypsin precursor in our HBEC model. However, it remains to be determined whether an endogenous kinase (e.g., enterokinase) exists within the lung that would convert inactive trypsinogen to active trypsin. We have also identified the expression of HAT, a tryptic enzyme (28) that could potentially cleave and activate PAR2 in an autocrine manner. This enzyme has previously been identified in submucosal glands and in the sputum/bronchial lavage samples of patients suffering from chronic airway diseases (30). To our knowledge, the studies shown in Fig. 8 are the first to demonstrate the expression of HAT in HBECs. The presence of HAT in sputum and bronchial lavage samples would, however, localize the enzyme to the apical surface of the epithelium, and it remains to be shown whether it is also secreted to the basolateral side where it could activate PAR2.
This is the first description of a role for PAR2 in the regulation of
ion transport processes in the airway epithelium and represents a
putative mechanism for the endogenous control of basal Na+
reabsorption and both basal and stimulated anion secretion. Under basal
conditions, Na+ absorption represents the major ion
transport process (2) and plays a central role in the
regulation of airway surface liquid (ASL) volume (17). It
is important to note that Cl secretion has not been
detected under basal, unstimulated conditions in the human airway
(2). The PAR2-mediated inhibition of basal amiloride-sensitive Isc, if physiologically
relevant, would therefore be predicted to increase the ASL volume and
potentially improve mucociliary clearance (17, 18). The
physiological relevance of the PAR2-mediated attenuation of the basal
amiloride-insensitive Isc and cAMP-stimulated
Cl
secretion is unknown and may simply reflect a
consequence of K+-channel inhibition to achieve the
blockade of Na+ absorption. In summary, a PAR2-mediated
decrease in Na+ absorption in the airway epithelium adds
further evidence to a role for PAR2 as a first line of defense in
epithelia. Further studies will be required to determine whether this
mechanism is of functional relevance in vivo.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Danahay, Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, United Kingdom (E-mail: henry.danahay{at}pharma.novartis.com).
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.
Received 17 July 2000; accepted in final form 20 December 2000.
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