Ferret Tracheal Epithelial Cells Grown In Vitro Are Resistant to Lethal Injury by Activated Neutrophils Youngran Chung, Carolyn M. Kercsmar, and Pamela B. Davis Department of Pediatrics, Case Western Reserve University School of Medicine at Rainbow Babies and Children's Hospital, Cleveland, Ohio
Airway inflammation is often accompanied by accumulation of polymorphonuclear leukocytes (PMN) as well as epithelial sloughing. To determine whether PMN contribute to epithelial damage in inflammatory states, we examined the interaction of PMN and tracheal epithelial cells in culture. Ferret tracheal epithelial (FTE) cells were grown in primary culture on collagen-coated multiwell dishes. Confluent mono layers were loaded with (51Cr]04 and exposed to resting and activated neutrophils. There was no significant increase in cell death as assessed by (5ICr]04release over 8 h of exposure, at effector (PMN)-to-target cell (epithelial cell) ratios up to 90:1, whether PMN were activated by maximal activating concentrations of phorbol myristate acetate or formylmethionylleucylphenylalanine with or without cytochalasin B. This result was confirmed by using a pH]leucine release assay as well as by uptake of a supravital dye. However, exposure ofFTE cells to activated PMN for 4 h resulted in separation of adjacent cells and formation of gaps in the monolayer, without significant detachment of epithelial cells from the dish. Gap formation was prevented by aI-antitrypsin, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, or 10% serum, was mimicked by PMN elastase (24 JLg/ml), but not by hydrogen peroxide in concentrations up to 10 mM, or superoxide generated by xanthine/xanthine oxidase, and was reversible within 24 h of removal of elastase and exposure to fresh medium. We conclude that activated PMN do not kill FTE cells in culture. However, disruption of the epithelial cell monolayer probably by a proteolytic mechanism can result from exposure to activated PMN and may allow alteration of the epithelial barrier during airway inflammation.
In diseases such as cystic fibrosis, asthma, or bronchopulmonary dysplasia, there is persistent airway inflammation with accumulation of polymorphonuclear leukocytes (PMN). The airway inflammation in asthma and bronchopulmonary dysplasia is often accompanied by denudation of airway epithelium. Neutrophils can generate toxic O2 metabolites and contain many degradative enzymes that can injure tissue. For example, superoxide ion, hydrogen peroxide, and hypochlorous acid can kill cells and damage cellular membranes and DNA (1-5). Neutrophil enzymes, such as elastase, have been implicated in lung parenchymal damage in diseases (Received in original form June 13, 1990 and in revised form January 22, 1991) Address correspondence to: Carolyn M. Kercsmar, M.D., Pediatric Pulmonary Division, Rainbow Babies and Children's Hospital, 2101 Adelbert Road, #3001, Cleveland, OH 44106. Abbreviations: cytochalasin B, CB; N-methoxysuccinyl-Ala-Ala-Pro-Valchioromethylketone, CMK; Dulbecco's modified Eagle's medium, DMEM; Dulbecco's phosphate-buffered saline, D-PBS; fetal calf serum, FCS; formylmethionylleucylphenylalanine, FMLP; ferret tracheal epithelial cells, FTE cells; Hanks' balanced salt solution, HBSS; Madin Darby canine kidney cells, MDCK cells; phorbol myristate acetate, PMA; polymorphonuclear leukocytes, PMN. Am. J. Respir. CeU Mol. BioI. Vol. 5. pp, 125-132, 1991
such as emphysema (6) and bronchopulmonary dysplasia (7, 8). Cellular injuries mediated by PMN have been studied in isolated cell culture models of endothelial cells and, more recently, alveolar type II epithelial cells. In these studies, exposure to PMN causes target cell death (1, 2, 4, 9), as well as a variety of nonlethal changes such as cell detachment and reduction in protein and DNA synthesis (10). Because the airway epithelium forms a barrier against inhaled particles and plays an active role in mucus secretion, ion and water transport, and mucociliary clearance, injuries to these cells which alter such functions may be detrimental to maintenance of homeostasis. However, only a few studies examine the role of PMN in injury to the airway epithelium, and those data suggest that isolated airway epithelial cells are relatively resistant to the cytotoxic effect of PMN (11, 12). Therefore, we considered it important to define the nature of PMN-mediated airway epithelial injury. We designed this study to see whether PMN can kill or damage epithelial cells derived from the tracheas of ferrets. We selected the ferret because the ferret is already an established model for study of airway epithelial injury by influenza virus (13), and because the airway epithelium of the ferret changes during postnatal development (14), thus affording the opportunity
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for further studies of the developmental changes of the airway epithelial injury response. We used the epithelial cell culture model in order to isolate the epithelial cells from the influences of other cell types in the intact trachea.
Materials and Methods Reagents The following were purchased from Sigma Chemical Co. (St. Louis, MO): insulin, transferrin, hydrocortisone, epidermal growth factor, cholera toxin, endothelial cell growth factor, retinoic acid, Hepes, phorbol myristate acetate (PMA), formylmethionylleucylphenylalanine (FMLP), cytochalasin B (CB), at-antitrypsin, N-methoxysuccinyl-Ala-Ala-Pro-Valchloromethylketone (CMK), heparin, catalase, superoxide dismutase, xanthine, and xanthine oxidase. L-glutamine, Hanks' balanced salt solution (HBSS), and Dulbecco's phosphate-buffered saline (D-PBS) were obtained from GIBCO (Grand Island, NY); Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12, phosphatidyl ethanolamine, and trace elements were from Biofluids (Rockville, MD). Bovine collagen dispersion TD-211 was a generous gift from Ethicon (Somerville, NJ). Bovine anti-rabbit polyclonal antikeratin antiserum was purchased from Biomedical Tech. (Stoughton, MA),· and fluorescein-conjugated goat and rabbit antiserum from Cappell, Cooper Biomedical (Malvern, PA). Purified PMN elastase was obtained from Elastin Products (Pacific, MO). PMN (2 mg/ml), FMLP (10- 2 M), and CB (10 mg/ml) were dissolved in dimethyl sulfoxide and diluted to the appropriate concentrations immediately prior to use. Final dimethyl sulfoxide concentration in the medium used in the experiments was < 0.1 %. Ferret Tracheal Epithelial (FTE) Cell Culture Adult ferrets, Mustela putoriusfuro, 25 to 32 wk old, weighing 750 to 1,500 g, were obtained from Marshall Research Animals (North Rose, NY). Canine distemper virus vaccine was not administered within 2 wk prior to use. The animals were killed with intraperitoneal injection of pentobarbital solution (60 mg/kg). The trachea was excised from just below the larynx to the carina, clamped, and filled from the open end with 0.1% protease (Type XIV) in DMEM. After closing the open end, the trachea was placed in DMEM containing 100 U penicillin/streptomycin and 2.5 ",g/ml fungizone and incubated at 4° C for 18 h. The cells from the trachea were collected by flushing the protease out of the lumen. The trachea was then filled with 10 mM ethylenediaminetetraacetic acid and incubated at 37° C for 10 min before repeated flushing of the luminal surface with DMEM with 10% fetal calf serum (FCS). The trachea was also opened and the luminal surface lightly scraped before the final rinse. The cell suspension was centrifuged at 270 x g, washed, and the final cell pellet resuspended in culture medium (see below). Cell viability was> 95% as determined by acridine orange-ethidium bromide staining. FTE cells were plated in multiwell plastic culture plates (Costar, Cambridge, MA) coated with collagen and incubated in a 5 % CO 2-95 % air humidified incubator. Forty-eight hours after plating, the medium and any unattached cells were removed and fresh medium was added. Further medium changes occurred every 48 h. The growth medium was derived from formulation for epithelial cells by Wu (15) and
Groelke and associates (16) and consisted of equal volumes ofDMEM/Ham's F-12, supplemented with L-glutamine (2.5 ",M), insulin (5 ",g/ml), transferrin (5 ",g/ml), hydrocortisone (6 x 10- 8 M), cholera toxin (10 ng/ml), endothelial growth factor (30 ",g/ml), and conditioned medium. Conditioned medium was prepared by incubating confluent monolayers of fetal human lung fibroblasts (GM 5387; NIGMS Human Mutant Cell Repository, Camden, NJ) in a mixture ofDMEM/F-12 and 2 % FCS (Hyclone, Logan, UT) for 48 h. Three parts growth medium to one part conditioned medium resulted in a final serum concentration of 0.5 %. For cell counting, the monolayers were treated with 0.25% trypsin for 30 min. The cell suspension was centrifuged, and the pellet resuspended in crystal violet and stained for at least 24 h before counting on a hemacytometer. The epithelial nature of the cells was verified by their reaction with antibodies to cytokeratin and by their morphology using light and electron microscopy (17, 18). For keratin staining, cells grown on collagen-coated plastic multiwell plates were used. Confluent monolayers were washed 3 times in D-PBS. Cells were fixed at room temperature for 30 min with 10% buffered formalin, then permeabilized with cold methanol for 10 min. Cells were then washed with D-PBS and incubated for 1 h at 37° C with bovine anti-rabbit polyclonal antikeratin antiserum. Control cultures were incubated with normal rabbit serum. The antiserum was removed, and the monolayers were washed thoroughly with D-PBS and then incubated for 1 hat 37° C with fluoresceinconjugated goat and rabbit antiserum. Cultures were examined on a Nikon fluorescence microscope. PMN Isolation Human blood was anticoagulated with acid-citrate-dextrose and centrifuged at 200 x g for 20 min to remove platelet-rich plasma. PMN were separated by Ficoll Hypaques density centrifugation, dextran sedimentation, and hypotonic lysis of residual erythrocytes (19). The final pellet of PMN was resuspended in D-PBS containing 1 mg/ml glucose and 0.1 mg/ml bovine serum albumin. The final preparation contained at least 97 % PMN, and the cells were> 90 % viable by trypan blue exclusion.> Myeloperoxidase release and superoxide-specific chemiluminescence were measured according to methods described previously (20). Ster Cytotoxicity Assay Cytotoxicity was assessed by a "Cr release assay (9). When the FTE cells were at confluence (days 3 to 4), 4.8",Ci Na2(5'Cr]O. (New England Nuclear, Boston, MA) was added to each well (2-cm 2 surface area) containing 0.5 ml medium and incubated overnight. The monolayers were then washed 3 times with D-PBS. PMN were added to the wells and incubated for 15 min to allow sedimentation onto the monolayer, then DMEM or culture medium was added to a final total volume of 0.5 ml. A PMN-activating agent (PMA or FMLP) was added in a volume of 0.05 ml, and the plate was returned to the incubator. At the end of the incubation period, O.1-ml aliquots were removed and radioactivity was measured (LKB 1275 Minigamma Gamma counter; Pharmacia LKB Biotechnology, Piscataway, NJ). For the time course of "Cr release, aliquots were removed at various in-
Chung, Kercsmar, and Davis: Tracheal Epithelial Cells Resist Killing by Neutrophils
tervals, and an equal amount of fresh medium was added to keep the total volume constant. After the last sampling, the remaining medium was removed, 1% Triton X-lOO was added to the wells, and the plates incubated for 5 min. After mixing the contents of the wells, 0.1 ml of the Triton lysate was removed and the radioactivity measured. Calculations. 51Cr release for the well was determined by multiplying the counts in each O.1-ml aliquot by a factor determined by the total well volume at that time. The percentage of 51Cr released was calculated as A/A + T, where A = cpm in each O.1-ml aliquot corrected for sample volume and T = cpm released into the Triton lysate. For the time course experiments in which the same well was sampled repeatedly, total 51Cr released was corrected for 51Cr removed in previous aliquots. Cytotoxicity was confirmed by release of 3H from epithelial cells labeled with 0.6 JLCi pH]leucine for 18 h. The monolayer was washed 3 times with D-PBS and exposed to PMN. Aliquots taken at intervals were added to aqueous counting scintillant (Amersham Corp., Arlington Heights, IL), and the radioactivity was measured in a scintillation counter (TM Analytic, Elk Grove Village, IL). The percentage of 3H released was calculated as described for 51Cr release. Adherence Assay Freshly isolated PMN in HBSS without calcium or magnesium were exposed to Na2(51Cr]04 (1 JLCi/10 6 PMN). The PMN were vigorously agitated for 60 min at 37° C, then washed twice in cold HBSS. The 51Cr-labeled PMN (4 X 106/well) were added to the FTE monolayer. After 4 h incubation, the well was gently shaken and the medium removed. The well was then treated with Triton X-lOa. The amount of radioactivity in the Triton lysate was expressed as a percentage of total 51Cr-labeled PMN originally added and was taken as a measure of PMN adherent to the FTE monolayer. Detachment Assay Four hours after incubation of 51Cr-labeled FTE cells with activated PMN, the supernatant was removed and centrifuged at 16,000 x g for 30 s. The pellet contained intact epithelial cells. The amount of radioactivity in the pellet represented the amount of intact 51Cr-labeled epithelial cells that had been released from the plate. This was expressed as a percentage of total 51Cr uptake. To check if the PMN might have taken up the free released 51Cr during the incubation period, the following separate assay was done: 4 x 1Q6 PMN were incubated with free (51Cr]04 at the concentration that is usually released by FTE cells after 4 h. The PMN took up < 5% of free (51Cr]04' Electron Microscopy FTE cells grown in 12-well plastic dishes (growth area, 4 ern') were fixed at 4 0 C with 2.5% glutaraldehyde in phosphate buffer overnight and post-fixed with 2 % aqueous osmium tetroxide for 1 h. Routine dehydration with graded ethanols followed, 70 %, 95 %, and 100 %, three changes, 10 min each. Propylene oxide was used to lift cells from 12-well plastic plates (growth area, 4 cm-) to preserve intact monolayers. Monolayers were flat-embedded to maintain cross-
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15
O+---+----!c-------+----+--+----+------1 o 2 3 4 6 7 5 Days in Culture
Figure 1. Number of ferret trachealepithelial(FTE) cells in a well of area 2 ern'. Mean of three to seven different tracheas. Plating density was 12.5 x 104/cm2.
section orientation in Spurr embedding media. Ultrathin sections of monolayers were cut and stained with uranyl acetate and lead citrate. Sections were examined on JEM II 100 electron microscope. Statistics All results are expressed as the mean ± SEM. Statistical analyses (21) were performed using the Student's t test for unpaired data. Probability values of P < 0.05 were considered significant.
Results FTE Cell Culture Cells seeded at 1.25 X 105 cells/em- on collagen-coated plastic dishes grew to confluence by days 3 to 4, with an average doubling time of 29 h (Figure 1). All of the experiments were performed after cells reached confluence (days 4 to 5), when many of the cells in the monolayer had actively beating cilia. The epithelial nature of the cells was confirmed by their uniform fluorescence when treated with fluorescein-conjugated antikeratin antiserum (~ 95 % keratin-positive). In Control
0-.0
PMN + PMAo 25
'0
"
Ul
20
o
"
Q) ~
15
10
0+-----+-----+-----+-------1 o Time (hours)
Figure 2. Time course of % 51Cr release by FTE cell monolayers exposed to medium alone (control), or 4 x 106 polymorphonuclear leukocytes (PMN) + phorbol myristate acetate (PMA; 25 ng/ml) (effector-to-target ratio of 30:1). Mean of four separate experiments.
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20
"
15
III 0
e
~
U
10
:;:;
ill!
5
o
PN.
,",
c:oHTftOL n_&
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"_5
CONTROL
CON11lOL
n_5
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Figure 3. Release of 51Cr expressed as the percentage of total from monolayers of FTE cells after 4 h of incubation with: (1) PMN, (2) PMN + formylmethionylleucylphenylalanine (FMLP; 10-6 M), (3) PMN + PMA (25 ng/ml). Effector-to-target ratio =' 30:1. Controls represent the spontaneous release ofSlCr by epithelial cells exposed only to medium. There was no significant difference compared to the control of that day.
addition, electron microscopic studies showed cilia, microvilli, tonofilaments, and desmosomes, features consistent with the epithelial origin of the cells. PMN To ensure that the PMN were activated by the methods used in the experiments and that the PMN were not already preactivated during isolation, we measured superoxide-specific chemiluminescence and myeloperoxidase release from PMN prepared by the same methods. There was a 5-fold increase in chemiluminescence in PMN activated with PMA (25 ng/ml), compared to resting (unactivated) PMN. Myeloperoxidase release by PMA (25 ng/ml)-activated PMN was increased nearly 2-fold over that of resting PMN. Cytotoxicity Measurements Epithelial cell death was measured by release of 51Cr from monolayers previously labeled with pICr]04. FTE cell monolayers exposed to medium alone (control) for 4 h released 12 ± 1.5% (n = 15) of the total 51Cr. Addition of PMN (unactivated) to the monolayer did not alter 51Cr release. Activation of PMN by PMA did not increase the rate or extent of 51Cr release during an 8-h observation period (Figure 2). Activation of PMN by FMLP (1 x 10-6 M) also Figure 4. FTE monolayer disruption by activated PMN. Top panel: Controls: FTE cells exposed only to medium. Middlepanel:
TABLE I
Cells exposed to PMN (4 x 106/well)
PMN-induced cytotoxicity "Cr Released (% of total)
Control PMN + PMA + CB PMN + FMLP + CB
+ PMA (25 ng/ml)
Bottom panel: Cells exposed to PMA only. Bar
DMEM
Media
11.0 ± 0.5 10.5 ± 1.8 10.4 ± 1.7
10.0 ± 1.1 11.0 ± 2.0 8.5 ± 1.8
Definition ofabbreviations: PMN = polymorphonuclear leukocytes; DMEM = Dulbecco's modified Eagle's medium; PMA = phorbol myristate acetate; CB
= cytochalasin B; FMLP = formylmethionylleucylphenylalanine. Media = formulation described in text (contains 0.5% serum). Ferret tracheal epithelial cells were incubated for 4 h with PMN (4 x 106 cells/well) activated by PMA (25 ng/rnl) or FMLP (10 6 M) and CB (10 ",g/mI). Mean of four separate experiments. There was no significant difference (P> 0.10) between DMEM and media.
=
for 4 h. 20 ~ro.
did not result in significant FTE cell killing (Figure 3). Priming the PMN by pretreatment with chemotactic concentrations of FMLP (1 x 10-8 M) followed by full activation with FMLP (1 x 10-6 M) or PMA (25 ng/ml) in the presence of CB (10 j.tg/ml) also did not result in significant cell killing. Cytotoxicity was not seen with addition of PMN at effector-target (PMN:FTE cells) ratio from 10:1 to 90:1. Substitution ofDMEM without additives for culture medium (which contains 0.5% serum) did not alter cytotoxicity for FTE cells (Table 1). The results of the 51Cr release assays
Chung, Kercsmar, and Davis: Tracheal Epithelial Cells Resist Killing by Neutrophils
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TABLE 2
Effect of activated PMN on detachment of FTE cells
Control PMN + PMN + PMN + PMA +
PMA + CB FMLP PMA CB
n
51Cr Released (% of total)
5 6 6 5 3
15.4 15.9 13.8 17.5 14.0
± 1.5 ± 4.4 ± 2.2 ± 4.0 ± 1.5
Detached Intact Cells (% of total)
6.8 8.0 5.0 8.0 3.0
± ± ± ± ±
1.4 2.6 1.0 2.0 0.4
Definition of abbreviations: FTE = ferret tracheal epithelial; for other abbreviations, see Table I. Supernatant from 51Cr-labeled FTE cells exposed to activated PMN for 4 h was removed and centrifuged. % detached represents the amount of radioactivity in the pellet (i.e., detached, intact cells) expressed as a percentage of total 51Cr initially incorporated in the monolayer. Control = DMEM, PMA (25 ng/ml), FMLP (10- 6 M), CB (10 ILg/ml). PMN-to-FTE cell ratio = 30:1.
were confirmed by other methods. The release of (3H]leucine from FTE monolayers previously labeled with (3H]leucine was no different from that seen with the 51Cr release assays (n = 2). Viability of the cells in the monolayer was > 98 % determined by acridine orange-ethidium bromide staining following 4 h incubation with PMN activated by PMA (n = 3). Monolayer Morphology We examined the morphology of FTE monolayers following exposure to activated PMN. FTE cell monolayers exposed to PMN activated by PMA or FMLP (and CB) at effector:target ratios of 30:1 and greater displayed separation of adjacent cells, leaving gaps in the monolayer (Figure 4). These changes were evident under phase-contrast microscopy after 4 h of incubation with the activated PMN. There was no such morphologic disruption of the monolayer with exposure to PMA, FMLP, CB, or with medium alone, nor to PMN without activators. These open spaces in the monolayer were not due to cell detachment. Activated PMN did not increase detachment of FTE cells at 4 h, the time at which the morphologic changes in the monolayer were apparent (Table 2). Ultrastructural examination of cultured FTE cells exposed to activated PMN revealed a grossly intact monolayer with only rare adherent PMN. However, compared to controls, FTE monolayers incubated with activated PMN had widened intercellular spaces and intercellular desmosomes and junctional complexes that appeared disrupted (Figure 5). In addition, the substratum to which the cells were attached (consisting of the applied collagen coating and probably macromolecules produced by the FTE cells) was much reduced in FTE cultures exposed to activated PMN (Figure 5). Mechanisms of Monolayer Disruption We tested whether gap formation in the FTE monolayer was due to some consequence of adherence of PMN to FTE cells, to proteolytic damage, or to oxidative mechanisms. 51Crlabeled PMN were used to assess PMN adherence to FTE cell monolayer. No more than 8 % of added PMN, either unactivated or activated by PMA (25 ng/ml) with or without CB (10 flg/rnl) , was adherent at the end of 4-h exposure (n = 3).
Figure 5. Ultrastructural findings in FTE monolayers following incubation with PMN. Top panel: Control cells, exposed to unactivated PMN, showing microvilli (M), tonofilaments (T), intercellular desmosomes (D), and multiple areas of tight intercellular membrane apposition (small arrows). Large arrows denote prominent culture substrate. Bar = 0.5 {lm. Middle panel: FTE monolayer exposed to PMN + PMA (25 {lg/ml) for 4 h, showing widened intercellular spacing and disrupted intercellular junctions (small arrows). Bar = 0.6{lm. Bottom panel: FTE monolayer exposed to PMN + PMA (25 {lg/ml) for 4 h. Arrow indicates thinned culture substrate. Bar = 0.7 {lm.
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effector:target ratio of 30:1 to 50:1). Gap formation was apparent after 4 h of incubation, and was similar in appearance to that seen with activated PMN (Figure 6, top panel). In five separate experiments, gap formation was completely inhibited by the addition of 10%, but not 1%, FCS or by the addition of aI-antitrypsin (1 mg/ml) (Figure 6, middle panel) simultaneously with elastase- or PMA-activated PMN. The specific elastase inhibitor, CMK (0.02 mM), also prevented elastase-induced gap formation (Figure 6, bottom panel) in two experiments. We examined the possibility that gaps formed in the monolayer secondary to degradation of the collagen coating on the culture plate. When FTE cells were plated onto collagen-coated culture wells that were previously incubated for 4 h with either activated or inactivated PMN or culture medium alone, there was no difference in the cell number from days 2 to 6 of culture, regardless of preincubation conditions. To test whether oxidative mechanisms contributed to gap formation, we tested two PMN-derived oxidants, exogenous hydrogen peroxide and superoxide generated enzymatically from xanthine and xanthine oxidase, and also tested to see if antioxidants prevented the gaps formed by activated PMN. Hydrogen peroxide (0.5 mM to 10 mM) or superoxide ion produced by xanthine (80 J,'g/ml) and xanthine oxi-
Figure 6. Effect of inhibitors on elastase-induced gap formation. Top panel: FTE cells exposed to PMN elastase (24 Itg/ml) for 4 h. Middle panel: Cells exposed to PMN elastase (24 Itg/ml) + ~I antitrypsin (l mg/ml) for 4 h. Bottom panel: Cells exposed to PMN elastase (24Itg/ml) + chloromethylketone (CMK; 0.02 mM). Bar = 20 Itm.
To examine whether the gap formation in the monolayer was due to a proteolytic mechanism, we tested whether these changes could be prevented by protease inhibitors or mimicked by PMN elastase. Monolayers were incubated with purified PMN elastase at various concentrations of 6, 10, and 24 J,'g/ml. No morphologic changes in the monolayer were evident at concentrations less than 24 J,'g/ml elastase (equivalent to the amount contained in 4 X 1()6 PMN [6], the number of PMN used in most of our experiments, or
Figure 7. Recovery of FTE monolayers from elastase. Top panel: FTE cells exposed to PMN elastase (24 Itg/ml) for 4 h. Bottom panel: Same field, 24 h after removal of elastase and replacement with fresh medium. Bar = 20 Itm.
Chung, Kercsmar, and Davis: Tracheal Epithelial Cells Resist Killing by Neutrophils
dase (I m Vlml) did not alter the integrity of the monolayer. Neither catalase (0.2 mg/ml) nor superoxide dismutase (300 Vlml) inhibited gap formation due to the PMA-activated PMN. When the medium containing elastase was removed after 4 h and replaced with fresh medium, the gaps in the monolayer disappeared in 24 h. Similarly, gaps formed in response to PMA-activated PMN also disappeared in 24 h (n = 4) (Figure 7).
Discussion Activated human neutrophils did not kill FTE cells at effector:target ratios as high as 90:1 over an 8-h exposure. This apparent lack of cytotoxicity was not a quirk of the 5lCr release assay, for similar results were obtained using release of previously incorporated (3H]leucine from the cells as an outcome measure and by supravital dye staining. The absence of cytotoxicity was not due to defective or preactivated neutrophils because we confirmed that the PMN, isolated by our methods, are activated by the effectors used. The activating agents PMA and FMLP were used at concentrations that maximally stimulate PMN oxidative burst and degranulation, and degranulation was further enhanced by addition of CB. Although our initial experiments were performed in culture medium that contains 0.5% serum, which might conceivably have quenched free radicals or inhibited proteases, comparison with experiments done in DMEM without additives revealed no difference in cytotoxicity of PMN for epithelial cells. Therefore, PMN specifically stimulated to produce O 2 radicals or release proteases are not cytotoxic to healthy FTE cells in culture. In contrast, endothelial cells in culture are readily killed by PMN, apparently by oxygen metabolites (1, 2,4). Our results also contrast with studies of type II alveolar epithelial cells that are killed by activated PMN by a mechanism that requires adherence of PMN to the epithelial monolayer (9). In our system, PMN adherence to FTE cells was minimal and did not increase with PMN activation. It may be that PMN do not readily adhere to intact healthy airway epithelial cells; similar results are obtained with Madin Darby canine kidney (MDCK) cells, an epithelial cell line that is resistant to PMN adherence unless the MDCK cells are first virusinfected (22). Our results do not exclude the possibility that damaged epithelial cells may be better targets for PMN adhesion, just as endothelial cells that are preinjured by hyperoxia are better targets for PMN (23, 24). In vivo, conditions in which PMN are recruited to the airway may be accompanied or initiated by epithelial damage, for example, viral infection or pollutant exposure (25). In these pathologic states, PMNinduced cytotoxicity may indeed be present. This hypothesis is supported by the finding that, in vitro, airway epithelial cells obtained from rhesus monkeys exhibited significant PMN-induced cytotoxicity only when first exposed to ozone (II). Although activated PMN did not kill the FTE cells, they caused obvious morphologic changes in the monolayer. Within 4 h of exposure to activated PMN, there was separation of cells leaving gaps in the monolayer. These gaps were formed as a result of activated PMN, since unactivated PMN, the activating agents themselves (PMA, FMLP), or CB alone did not induce such changes.
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The gaps did not result from cell death or detachment (Table 2). Therefore, the gaps in the monolayer probably resulted from separation of adjacent cells. This phenomenon may be similar to that observed by other investigators. Endothelial cells or epithelial cells (MDCK) pulled apart when exposed to oxygen metabolites (26, 27). Endothelial cell monolayer integrity is also disrupted by activated PMN by a nonoxidative mechanism (28). In both studies, the morphologic changes were also accompanied by increased endothelial or epithelial monolayer permeability. We tested three possible mechanisms of gap formation: one mediated by PMN adherence, oxidative damage by hydrogen peroxide or superoxide (known PMN products), and proteolytic damage. Gap formation was probably not a consequence of significant PMN adherence to FTE cells, because gap formation was observed under conditions that produced no change in PMN adherence. Oxidative products of activated PMN also appear to be unlikely to be responsible for the gap formation. Neither superoxide nor hydrogen peroxide, known PMN products, caused gap formation, even at concentrations much higher than would be generated by 4 X 106 activated PMN (a number sufficient for gap formation). Moreover, the morphologic changes caused by activated PMN were not inhibited by superoxide dismutase or catalase. Proteolytic damage seems a likely candidate mechanism. PMN elastase alone, in the amounts contained in 4 x 106 PMN (6), caused reversible gap formation in the monolayer with a time course similar to PMN-induced damage. Inhibitors of elastase, including ai-antitrypsin, CMK, and 10 % FCS prevented the damage to the monolayer induced by activated PMN in the presence of CB, or the damage from elastase itself. Therefore, the morphologic changes induced by activated PMN were likely in part due to proteolytic mechanisms. ai-antitrypsin and CMK not only inhibit proteolysis but can also block cationic sites (29), so it is not surprising that these agents completely prevented gap formation. Therefore, although the damage from elastase is largely proteolytic, the charge effects of elastase may also contribute to the morphologic changes seen in our experiments. Gap formation in our sy~tem may not precisely reflect a comparable process in vivo since our culture substrate is not identical to basement membrane. However, activated PMN did not completely remove or alter the collagen-coated substrate enough to alter cell adherence, suggesting that the mechanism of gap formation is not solely due to disruption of the artificial culture substratum. Although ultrastructural examination revealed that the culture substratum for the FTE cells was decreased in amount following exposure to activated PMN (Figure 5), it appeared that intercellular adherence rather than detachment from substrate accounts for our observations. Therefore, this culture system may be a useful model for studying this aspect of PMN-epithelial cell interactions. In conclusion, activated PMN do not kill airway epithelial cells but can cause reversible disruption of epithelial monolayer integrity by gap formation. The mechanism by which activated PMN cause this damage is largely proteolytic rather than oxidative and does not depend on PMN adherence to the monolayer. PMN elastase appears to be a major contributor to barrier disruption. Further studies are re-
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quired to see how such monolayer disruption might alter important epithelial functions and whether other nonlethal damage is done by activated PMN. Acknowledgments: We thank Ms. Cathy L. Silski for her excellent technical assistance and Ms. YoshieHervey for technical support. This study was supported in part by Training Grant HL-07415 and Grants HL-28386, DK-27651 (Cystic Fibrosis Core Center), and HL-28530 from the National Institutes of Health and by the Cystic Fibrosis Foundation.
15. 16. 17. 18.
References I. Martin, J. 1984. Neutrophils kill pulmonary endothelial cells by a hydrogen-peroxide-dependent pathway. In vitro model of neutrophilmediated lung injury. Am. Rev. Respir. Dis. 130:209-213. 2. Weiss, S. J., 1. Young, A. F. LoBuglio, A. Slivka, and N. F. Nimeh. 1981. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. CUn. Invest. 68:714-721. 3. Henson, P. M., and R. B. Johnson. 1987. Tissue injury in inflammation. Oxidants, proteinases, and cationic proteins. J. CUn. Invest. 79:669-674. 4. Vrani, 1., S. E. G. Frigiel, G. O. Till, R. G. Kunkel, U. S. Ryan, andP. A. Ward. 1985. Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical. Lab. Invest. 53:656-663. 5. Weiss, S. J. 1989. Tissue destruction by neutrophils. N. Engl. J. Med. 320:365-376. 6. Janoff, A. 1985. Elastases and emphysema. Am. Rev. Respir. Dis. 132:484-491. 7. Ogden, B. E., S. A. Murphy, G. C. Saunders, D. Pathak, andJ. D. Johnson. 1984. Neonatal lung neutrophils and elastase/proteinase inhibitor imbalance. Am. Rev. Respir. Dis. 130:817-821. 8. O'Brodovich, H. M., and R. B. Mellins. 1985. Bronchopulmonary dysplasia. Am. Rev. Respir. Dis. 132:694-709. 9. Simon, R. H., P. D. DeHart, and R. F. Todd. 1986. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J. Clin. Invest. 78: 1375-1386. 10. Ayars, G. H., L. C. Altman, H. Rosen, and T. Doyle. 1984. The injurious effect of neutrophils on pneumocytes in vitro. Am. Rev. Respir. Dis. 130:964-973. II. St. George, J. A., S. Lonning, R. J. McDonald, and D. M. Hyde. 1989. The role of the neutrophil in ozone induced injury to airway epithelium examined in vitro. Am. Rev. Respir. Dis. 139(Suppl.):A278. (Abstr.) 12. Bascom, R., J. Resau, D. Proud, and R. P. Schleimer. 1988. Modulation in vitro of hydrogen peroxide toxicity to human tracheal epithelial cells by neutrophils. Am. Rev. Respir. Dis. 139(Suppl.):A83. (Abstr.) 13. Francis, T., and C. H. Stuart-Harris. 1938. Studies on the nasal histology of epidemic influenza virus infection in the ferret. The development and repair of the nasal lesion. J. Exp. Med. 68:789-801. 14. Leigh, M. W., T. M. Gambling, J. L. Carson, A. M. Collier, R. E. Wood,
19. 20.
21. 22. 23. 24. 25. 26. 27.
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and T. F. Boat. 1986. Postnatal development of tracheal surface epithelium and submucosal glands in the ferret. Exp. Lung Res. 10:153-169. Wu, R. 1986. In vitro differentiation of airway epithelial cells. In In Vitro Models of Respiratory Epithelium. L. J. Schiff, editor. CRC Press, Boca Raton, FL. 1-26. Groelke, J. W., J. J. Coalson, and J. B. Baseman. 1985. Growth requirements offerret tracheal epithelial cells in primary culture. Proc. Soc. Exp. Bioi. Med. 179:309-317. O'Guin, W. M., A. Schermer, and T. T. Sun. 1985. Immunofluorescence staining of keratin filaments in cultured epithelial cells. J. Tissue Culture Methods 9: 123-128. Sun, T.-T., R. Eichner, A. Schermer, D. Cooper, W. G. Nelson, and R. A. Weiss. 1984. Classification, expression and possible mechanisms of evolution of mammalian epithelial keratins: a unifying model. In The Cancer Cell, Vol. I: The Transformed Phenotype. A. Levine, W. Topp, G. Van de Woude et al., editors. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY. 169-176. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21:77-89. Tosi, M. F., and M. Berger. 1988. Functional differences between the 40 kDa and 50 to 70 kDa IgG Fc receptors on human neutrophils revealed by elastase treatment and antireceptor antibodies. J. Immunol. 141: 2097-2103. Stanton, A. G. 1987. Primer of Bio-Statistics. McGraw-Hill, New York. Ratcliffe, D. R., S. L. Nolin, and E. B. Cramer. 1988. Neutrophil interaction with influenza-infected epithelial cells. Blood 72: 142-149. Suttorp, N., and L. Simon. 1982. Lung cell oxidant injury: enhancement of polymorphonuclear leukocyte-mediated cytotoxicity in lung cells exposed to sustained in vitro hyperoxia. J. Clin. Invest. 70:342-350. MacGregor, R., E. Macarak, and N. Kefalides. 1978. Comparative adherence of granulocytes to endothelial monolayers and nylon fibers. J. Clin. Invest. 61:697-702. Holtzman, M. J., L. M. Fabbri, P. M. O'Byrne et al. 1983. Importance of airway inflammation for hyperresponsiveness induced by ozone. Am. Rev. Respir. Dis. 127:686-690. Welsh, M. J., D. M. Shasby, and R. M. Husted. 1985. Oxidants increase paracellular permeability in a cultured epithelial cell line. J. CUn. Invest. 76: 1155-1168. Shasby, D. M., S. E. Lind, S. S. Shasby, J. C. Goldsmith, and G. W. Hunninghake. 1985. Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis. Blood 65:605-614. Harlan, J. M., B. R. Schwartz, M. A. Reidy, S. M. Schwartz, H. D. Ochs, and L. A. Harker. 1985. Activated neutrophils disrupt endothelial monolayer integrity by an oxygen radical-independent mechanism. Lab. Invest. 52:141-150. Peterson, M. W., P. Stone, and D. M. Shasby. 1987. Cationic neutrophil proteins increase transendothelial albumin movement. J. Appl. Physiol. 62:1521-1530.
Airway inflammation is often accompanied by accumulation of polymorphonuclear leukocytes (PMN) as well as epithelial sloughing. To determine whether PMN contribute to epithelial damage in inflammatory states, we examined the interaction of PMN and tracheal epithelial cells in culture. Ferret tracheal epithelial (FTE) cells were grown in primary culture on collagen-coated multiwell dishes. Confluent mono layers were loaded with (51Cr]04 and exposed to resting and activated neutrophils. There was no significant increase in cell death as assessed by (5ICr]04release over 8 h of exposure, at effector (PMN)-to-target cell (epithelial cell) ratios up to 90:1, whether PMN were activated by maximal activating concentrations of phorbol myristate acetate or formylmethionylleucylphenylalanine with or without cytochalasin B. This result was confirmed by using a pH]leucine release assay as well as by uptake of a supravital dye. However, exposure ofFTE cells to activated PMN for 4 h resulted in separation of adjacent cells and formation of gaps in the monolayer, without significant detachment of epithelial cells from the dish. Gap formation was prevented by aI-antitrypsin, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, or 10% serum, was mimicked by PMN elastase (24 JLg/ml), but not by hydrogen peroxide in concentrations up to 10 mM, or superoxide generated by xanthine/xanthine oxidase, and was reversible within 24 h of removal of elastase and exposure to fresh medium. We conclude that activated PMN do not kill FTE cells in culture. However, disruption of the epithelial cell monolayer probably by a proteolytic mechanism can result from exposure to activated PMN and may allow alteration of the epithelial barrier during airway inflammation.
In diseases such as cystic fibrosis, asthma, or bronchopulmonary dysplasia, there is persistent airway inflammation with accumulation of polymorphonuclear leukocytes (PMN). The airway inflammation in asthma and bronchopulmonary dysplasia is often accompanied by denudation of airway epithelium. Neutrophils can generate toxic O2 metabolites and contain many degradative enzymes that can injure tissue. For example, superoxide ion, hydrogen peroxide, and hypochlorous acid can kill cells and damage cellular membranes and DNA (1-5). Neutrophil enzymes, such as elastase, have been implicated in lung parenchymal damage in diseases (Received in original form June 13, 1990 and in revised form January 22, 1991) Address correspondence to: Carolyn M. Kercsmar, M.D., Pediatric Pulmonary Division, Rainbow Babies and Children's Hospital, 2101 Adelbert Road, #3001, Cleveland, OH 44106. Abbreviations: cytochalasin B, CB; N-methoxysuccinyl-Ala-Ala-Pro-Valchioromethylketone, CMK; Dulbecco's modified Eagle's medium, DMEM; Dulbecco's phosphate-buffered saline, D-PBS; fetal calf serum, FCS; formylmethionylleucylphenylalanine, FMLP; ferret tracheal epithelial cells, FTE cells; Hanks' balanced salt solution, HBSS; Madin Darby canine kidney cells, MDCK cells; phorbol myristate acetate, PMA; polymorphonuclear leukocytes, PMN. Am. J. Respir. CeU Mol. BioI. Vol. 5. pp, 125-132, 1991
such as emphysema (6) and bronchopulmonary dysplasia (7, 8). Cellular injuries mediated by PMN have been studied in isolated cell culture models of endothelial cells and, more recently, alveolar type II epithelial cells. In these studies, exposure to PMN causes target cell death (1, 2, 4, 9), as well as a variety of nonlethal changes such as cell detachment and reduction in protein and DNA synthesis (10). Because the airway epithelium forms a barrier against inhaled particles and plays an active role in mucus secretion, ion and water transport, and mucociliary clearance, injuries to these cells which alter such functions may be detrimental to maintenance of homeostasis. However, only a few studies examine the role of PMN in injury to the airway epithelium, and those data suggest that isolated airway epithelial cells are relatively resistant to the cytotoxic effect of PMN (11, 12). Therefore, we considered it important to define the nature of PMN-mediated airway epithelial injury. We designed this study to see whether PMN can kill or damage epithelial cells derived from the tracheas of ferrets. We selected the ferret because the ferret is already an established model for study of airway epithelial injury by influenza virus (13), and because the airway epithelium of the ferret changes during postnatal development (14), thus affording the opportunity
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for further studies of the developmental changes of the airway epithelial injury response. We used the epithelial cell culture model in order to isolate the epithelial cells from the influences of other cell types in the intact trachea.
Materials and Methods Reagents The following were purchased from Sigma Chemical Co. (St. Louis, MO): insulin, transferrin, hydrocortisone, epidermal growth factor, cholera toxin, endothelial cell growth factor, retinoic acid, Hepes, phorbol myristate acetate (PMA), formylmethionylleucylphenylalanine (FMLP), cytochalasin B (CB), at-antitrypsin, N-methoxysuccinyl-Ala-Ala-Pro-Valchloromethylketone (CMK), heparin, catalase, superoxide dismutase, xanthine, and xanthine oxidase. L-glutamine, Hanks' balanced salt solution (HBSS), and Dulbecco's phosphate-buffered saline (D-PBS) were obtained from GIBCO (Grand Island, NY); Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12, phosphatidyl ethanolamine, and trace elements were from Biofluids (Rockville, MD). Bovine collagen dispersion TD-211 was a generous gift from Ethicon (Somerville, NJ). Bovine anti-rabbit polyclonal antikeratin antiserum was purchased from Biomedical Tech. (Stoughton, MA),· and fluorescein-conjugated goat and rabbit antiserum from Cappell, Cooper Biomedical (Malvern, PA). Purified PMN elastase was obtained from Elastin Products (Pacific, MO). PMN (2 mg/ml), FMLP (10- 2 M), and CB (10 mg/ml) were dissolved in dimethyl sulfoxide and diluted to the appropriate concentrations immediately prior to use. Final dimethyl sulfoxide concentration in the medium used in the experiments was < 0.1 %. Ferret Tracheal Epithelial (FTE) Cell Culture Adult ferrets, Mustela putoriusfuro, 25 to 32 wk old, weighing 750 to 1,500 g, were obtained from Marshall Research Animals (North Rose, NY). Canine distemper virus vaccine was not administered within 2 wk prior to use. The animals were killed with intraperitoneal injection of pentobarbital solution (60 mg/kg). The trachea was excised from just below the larynx to the carina, clamped, and filled from the open end with 0.1% protease (Type XIV) in DMEM. After closing the open end, the trachea was placed in DMEM containing 100 U penicillin/streptomycin and 2.5 ",g/ml fungizone and incubated at 4° C for 18 h. The cells from the trachea were collected by flushing the protease out of the lumen. The trachea was then filled with 10 mM ethylenediaminetetraacetic acid and incubated at 37° C for 10 min before repeated flushing of the luminal surface with DMEM with 10% fetal calf serum (FCS). The trachea was also opened and the luminal surface lightly scraped before the final rinse. The cell suspension was centrifuged at 270 x g, washed, and the final cell pellet resuspended in culture medium (see below). Cell viability was> 95% as determined by acridine orange-ethidium bromide staining. FTE cells were plated in multiwell plastic culture plates (Costar, Cambridge, MA) coated with collagen and incubated in a 5 % CO 2-95 % air humidified incubator. Forty-eight hours after plating, the medium and any unattached cells were removed and fresh medium was added. Further medium changes occurred every 48 h. The growth medium was derived from formulation for epithelial cells by Wu (15) and
Groelke and associates (16) and consisted of equal volumes ofDMEM/Ham's F-12, supplemented with L-glutamine (2.5 ",M), insulin (5 ",g/ml), transferrin (5 ",g/ml), hydrocortisone (6 x 10- 8 M), cholera toxin (10 ng/ml), endothelial growth factor (30 ",g/ml), and conditioned medium. Conditioned medium was prepared by incubating confluent monolayers of fetal human lung fibroblasts (GM 5387; NIGMS Human Mutant Cell Repository, Camden, NJ) in a mixture ofDMEM/F-12 and 2 % FCS (Hyclone, Logan, UT) for 48 h. Three parts growth medium to one part conditioned medium resulted in a final serum concentration of 0.5 %. For cell counting, the monolayers were treated with 0.25% trypsin for 30 min. The cell suspension was centrifuged, and the pellet resuspended in crystal violet and stained for at least 24 h before counting on a hemacytometer. The epithelial nature of the cells was verified by their reaction with antibodies to cytokeratin and by their morphology using light and electron microscopy (17, 18). For keratin staining, cells grown on collagen-coated plastic multiwell plates were used. Confluent monolayers were washed 3 times in D-PBS. Cells were fixed at room temperature for 30 min with 10% buffered formalin, then permeabilized with cold methanol for 10 min. Cells were then washed with D-PBS and incubated for 1 h at 37° C with bovine anti-rabbit polyclonal antikeratin antiserum. Control cultures were incubated with normal rabbit serum. The antiserum was removed, and the monolayers were washed thoroughly with D-PBS and then incubated for 1 hat 37° C with fluoresceinconjugated goat and rabbit antiserum. Cultures were examined on a Nikon fluorescence microscope. PMN Isolation Human blood was anticoagulated with acid-citrate-dextrose and centrifuged at 200 x g for 20 min to remove platelet-rich plasma. PMN were separated by Ficoll Hypaques density centrifugation, dextran sedimentation, and hypotonic lysis of residual erythrocytes (19). The final pellet of PMN was resuspended in D-PBS containing 1 mg/ml glucose and 0.1 mg/ml bovine serum albumin. The final preparation contained at least 97 % PMN, and the cells were> 90 % viable by trypan blue exclusion.> Myeloperoxidase release and superoxide-specific chemiluminescence were measured according to methods described previously (20). Ster Cytotoxicity Assay Cytotoxicity was assessed by a "Cr release assay (9). When the FTE cells were at confluence (days 3 to 4), 4.8",Ci Na2(5'Cr]O. (New England Nuclear, Boston, MA) was added to each well (2-cm 2 surface area) containing 0.5 ml medium and incubated overnight. The monolayers were then washed 3 times with D-PBS. PMN were added to the wells and incubated for 15 min to allow sedimentation onto the monolayer, then DMEM or culture medium was added to a final total volume of 0.5 ml. A PMN-activating agent (PMA or FMLP) was added in a volume of 0.05 ml, and the plate was returned to the incubator. At the end of the incubation period, O.1-ml aliquots were removed and radioactivity was measured (LKB 1275 Minigamma Gamma counter; Pharmacia LKB Biotechnology, Piscataway, NJ). For the time course of "Cr release, aliquots were removed at various in-
Chung, Kercsmar, and Davis: Tracheal Epithelial Cells Resist Killing by Neutrophils
tervals, and an equal amount of fresh medium was added to keep the total volume constant. After the last sampling, the remaining medium was removed, 1% Triton X-lOO was added to the wells, and the plates incubated for 5 min. After mixing the contents of the wells, 0.1 ml of the Triton lysate was removed and the radioactivity measured. Calculations. 51Cr release for the well was determined by multiplying the counts in each O.1-ml aliquot by a factor determined by the total well volume at that time. The percentage of 51Cr released was calculated as A/A + T, where A = cpm in each O.1-ml aliquot corrected for sample volume and T = cpm released into the Triton lysate. For the time course experiments in which the same well was sampled repeatedly, total 51Cr released was corrected for 51Cr removed in previous aliquots. Cytotoxicity was confirmed by release of 3H from epithelial cells labeled with 0.6 JLCi pH]leucine for 18 h. The monolayer was washed 3 times with D-PBS and exposed to PMN. Aliquots taken at intervals were added to aqueous counting scintillant (Amersham Corp., Arlington Heights, IL), and the radioactivity was measured in a scintillation counter (TM Analytic, Elk Grove Village, IL). The percentage of 3H released was calculated as described for 51Cr release. Adherence Assay Freshly isolated PMN in HBSS without calcium or magnesium were exposed to Na2(51Cr]04 (1 JLCi/10 6 PMN). The PMN were vigorously agitated for 60 min at 37° C, then washed twice in cold HBSS. The 51Cr-labeled PMN (4 X 106/well) were added to the FTE monolayer. After 4 h incubation, the well was gently shaken and the medium removed. The well was then treated with Triton X-lOa. The amount of radioactivity in the Triton lysate was expressed as a percentage of total 51Cr-labeled PMN originally added and was taken as a measure of PMN adherent to the FTE monolayer. Detachment Assay Four hours after incubation of 51Cr-labeled FTE cells with activated PMN, the supernatant was removed and centrifuged at 16,000 x g for 30 s. The pellet contained intact epithelial cells. The amount of radioactivity in the pellet represented the amount of intact 51Cr-labeled epithelial cells that had been released from the plate. This was expressed as a percentage of total 51Cr uptake. To check if the PMN might have taken up the free released 51Cr during the incubation period, the following separate assay was done: 4 x 1Q6 PMN were incubated with free (51Cr]04 at the concentration that is usually released by FTE cells after 4 h. The PMN took up < 5% of free (51Cr]04' Electron Microscopy FTE cells grown in 12-well plastic dishes (growth area, 4 ern') were fixed at 4 0 C with 2.5% glutaraldehyde in phosphate buffer overnight and post-fixed with 2 % aqueous osmium tetroxide for 1 h. Routine dehydration with graded ethanols followed, 70 %, 95 %, and 100 %, three changes, 10 min each. Propylene oxide was used to lift cells from 12-well plastic plates (growth area, 4 cm-) to preserve intact monolayers. Monolayers were flat-embedded to maintain cross-
127
15
O+---+----!c-------+----+--+----+------1 o 2 3 4 6 7 5 Days in Culture
Figure 1. Number of ferret trachealepithelial(FTE) cells in a well of area 2 ern'. Mean of three to seven different tracheas. Plating density was 12.5 x 104/cm2.
section orientation in Spurr embedding media. Ultrathin sections of monolayers were cut and stained with uranyl acetate and lead citrate. Sections were examined on JEM II 100 electron microscope. Statistics All results are expressed as the mean ± SEM. Statistical analyses (21) were performed using the Student's t test for unpaired data. Probability values of P < 0.05 were considered significant.
Results FTE Cell Culture Cells seeded at 1.25 X 105 cells/em- on collagen-coated plastic dishes grew to confluence by days 3 to 4, with an average doubling time of 29 h (Figure 1). All of the experiments were performed after cells reached confluence (days 4 to 5), when many of the cells in the monolayer had actively beating cilia. The epithelial nature of the cells was confirmed by their uniform fluorescence when treated with fluorescein-conjugated antikeratin antiserum (~ 95 % keratin-positive). In Control
0-.0
PMN + PMAo 25
'0
"
Ul
20
o
"
Q) ~
15
10
0+-----+-----+-----+-------1 o Time (hours)
Figure 2. Time course of % 51Cr release by FTE cell monolayers exposed to medium alone (control), or 4 x 106 polymorphonuclear leukocytes (PMN) + phorbol myristate acetate (PMA; 25 ng/ml) (effector-to-target ratio of 30:1). Mean of four separate experiments.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
20
"
15
III 0
e
~
U
10
:;:;
ill!
5
o
PN.
,",
c:oHTftOL n_&
Pll!N + FMLP
"_5
CONTROL
CON11lOL
n_5
,-,
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Figure 3. Release of 51Cr expressed as the percentage of total from monolayers of FTE cells after 4 h of incubation with: (1) PMN, (2) PMN + formylmethionylleucylphenylalanine (FMLP; 10-6 M), (3) PMN + PMA (25 ng/ml). Effector-to-target ratio =' 30:1. Controls represent the spontaneous release ofSlCr by epithelial cells exposed only to medium. There was no significant difference compared to the control of that day.
addition, electron microscopic studies showed cilia, microvilli, tonofilaments, and desmosomes, features consistent with the epithelial origin of the cells. PMN To ensure that the PMN were activated by the methods used in the experiments and that the PMN were not already preactivated during isolation, we measured superoxide-specific chemiluminescence and myeloperoxidase release from PMN prepared by the same methods. There was a 5-fold increase in chemiluminescence in PMN activated with PMA (25 ng/ml), compared to resting (unactivated) PMN. Myeloperoxidase release by PMA (25 ng/ml)-activated PMN was increased nearly 2-fold over that of resting PMN. Cytotoxicity Measurements Epithelial cell death was measured by release of 51Cr from monolayers previously labeled with pICr]04. FTE cell monolayers exposed to medium alone (control) for 4 h released 12 ± 1.5% (n = 15) of the total 51Cr. Addition of PMN (unactivated) to the monolayer did not alter 51Cr release. Activation of PMN by PMA did not increase the rate or extent of 51Cr release during an 8-h observation period (Figure 2). Activation of PMN by FMLP (1 x 10-6 M) also Figure 4. FTE monolayer disruption by activated PMN. Top panel: Controls: FTE cells exposed only to medium. Middlepanel:
TABLE I
Cells exposed to PMN (4 x 106/well)
PMN-induced cytotoxicity "Cr Released (% of total)
Control PMN + PMA + CB PMN + FMLP + CB
+ PMA (25 ng/ml)
Bottom panel: Cells exposed to PMA only. Bar
DMEM
Media
11.0 ± 0.5 10.5 ± 1.8 10.4 ± 1.7
10.0 ± 1.1 11.0 ± 2.0 8.5 ± 1.8
Definition ofabbreviations: PMN = polymorphonuclear leukocytes; DMEM = Dulbecco's modified Eagle's medium; PMA = phorbol myristate acetate; CB
= cytochalasin B; FMLP = formylmethionylleucylphenylalanine. Media = formulation described in text (contains 0.5% serum). Ferret tracheal epithelial cells were incubated for 4 h with PMN (4 x 106 cells/well) activated by PMA (25 ng/rnl) or FMLP (10 6 M) and CB (10 ",g/mI). Mean of four separate experiments. There was no significant difference (P> 0.10) between DMEM and media.
=
for 4 h. 20 ~ro.
did not result in significant FTE cell killing (Figure 3). Priming the PMN by pretreatment with chemotactic concentrations of FMLP (1 x 10-8 M) followed by full activation with FMLP (1 x 10-6 M) or PMA (25 ng/ml) in the presence of CB (10 j.tg/ml) also did not result in significant cell killing. Cytotoxicity was not seen with addition of PMN at effector-target (PMN:FTE cells) ratio from 10:1 to 90:1. Substitution ofDMEM without additives for culture medium (which contains 0.5% serum) did not alter cytotoxicity for FTE cells (Table 1). The results of the 51Cr release assays
Chung, Kercsmar, and Davis: Tracheal Epithelial Cells Resist Killing by Neutrophils
129
TABLE 2
Effect of activated PMN on detachment of FTE cells
Control PMN + PMN + PMN + PMA +
PMA + CB FMLP PMA CB
n
51Cr Released (% of total)
5 6 6 5 3
15.4 15.9 13.8 17.5 14.0
± 1.5 ± 4.4 ± 2.2 ± 4.0 ± 1.5
Detached Intact Cells (% of total)
6.8 8.0 5.0 8.0 3.0
± ± ± ± ±
1.4 2.6 1.0 2.0 0.4
Definition of abbreviations: FTE = ferret tracheal epithelial; for other abbreviations, see Table I. Supernatant from 51Cr-labeled FTE cells exposed to activated PMN for 4 h was removed and centrifuged. % detached represents the amount of radioactivity in the pellet (i.e., detached, intact cells) expressed as a percentage of total 51Cr initially incorporated in the monolayer. Control = DMEM, PMA (25 ng/ml), FMLP (10- 6 M), CB (10 ILg/ml). PMN-to-FTE cell ratio = 30:1.
were confirmed by other methods. The release of (3H]leucine from FTE monolayers previously labeled with (3H]leucine was no different from that seen with the 51Cr release assays (n = 2). Viability of the cells in the monolayer was > 98 % determined by acridine orange-ethidium bromide staining following 4 h incubation with PMN activated by PMA (n = 3). Monolayer Morphology We examined the morphology of FTE monolayers following exposure to activated PMN. FTE cell monolayers exposed to PMN activated by PMA or FMLP (and CB) at effector:target ratios of 30:1 and greater displayed separation of adjacent cells, leaving gaps in the monolayer (Figure 4). These changes were evident under phase-contrast microscopy after 4 h of incubation with the activated PMN. There was no such morphologic disruption of the monolayer with exposure to PMA, FMLP, CB, or with medium alone, nor to PMN without activators. These open spaces in the monolayer were not due to cell detachment. Activated PMN did not increase detachment of FTE cells at 4 h, the time at which the morphologic changes in the monolayer were apparent (Table 2). Ultrastructural examination of cultured FTE cells exposed to activated PMN revealed a grossly intact monolayer with only rare adherent PMN. However, compared to controls, FTE monolayers incubated with activated PMN had widened intercellular spaces and intercellular desmosomes and junctional complexes that appeared disrupted (Figure 5). In addition, the substratum to which the cells were attached (consisting of the applied collagen coating and probably macromolecules produced by the FTE cells) was much reduced in FTE cultures exposed to activated PMN (Figure 5). Mechanisms of Monolayer Disruption We tested whether gap formation in the FTE monolayer was due to some consequence of adherence of PMN to FTE cells, to proteolytic damage, or to oxidative mechanisms. 51Crlabeled PMN were used to assess PMN adherence to FTE cell monolayer. No more than 8 % of added PMN, either unactivated or activated by PMA (25 ng/ml) with or without CB (10 flg/rnl) , was adherent at the end of 4-h exposure (n = 3).
Figure 5. Ultrastructural findings in FTE monolayers following incubation with PMN. Top panel: Control cells, exposed to unactivated PMN, showing microvilli (M), tonofilaments (T), intercellular desmosomes (D), and multiple areas of tight intercellular membrane apposition (small arrows). Large arrows denote prominent culture substrate. Bar = 0.5 {lm. Middle panel: FTE monolayer exposed to PMN + PMA (25 {lg/ml) for 4 h, showing widened intercellular spacing and disrupted intercellular junctions (small arrows). Bar = 0.6{lm. Bottom panel: FTE monolayer exposed to PMN + PMA (25 {lg/ml) for 4 h. Arrow indicates thinned culture substrate. Bar = 0.7 {lm.
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effector:target ratio of 30:1 to 50:1). Gap formation was apparent after 4 h of incubation, and was similar in appearance to that seen with activated PMN (Figure 6, top panel). In five separate experiments, gap formation was completely inhibited by the addition of 10%, but not 1%, FCS or by the addition of aI-antitrypsin (1 mg/ml) (Figure 6, middle panel) simultaneously with elastase- or PMA-activated PMN. The specific elastase inhibitor, CMK (0.02 mM), also prevented elastase-induced gap formation (Figure 6, bottom panel) in two experiments. We examined the possibility that gaps formed in the monolayer secondary to degradation of the collagen coating on the culture plate. When FTE cells were plated onto collagen-coated culture wells that were previously incubated for 4 h with either activated or inactivated PMN or culture medium alone, there was no difference in the cell number from days 2 to 6 of culture, regardless of preincubation conditions. To test whether oxidative mechanisms contributed to gap formation, we tested two PMN-derived oxidants, exogenous hydrogen peroxide and superoxide generated enzymatically from xanthine and xanthine oxidase, and also tested to see if antioxidants prevented the gaps formed by activated PMN. Hydrogen peroxide (0.5 mM to 10 mM) or superoxide ion produced by xanthine (80 J,'g/ml) and xanthine oxi-
Figure 6. Effect of inhibitors on elastase-induced gap formation. Top panel: FTE cells exposed to PMN elastase (24 Itg/ml) for 4 h. Middle panel: Cells exposed to PMN elastase (24 Itg/ml) + ~I antitrypsin (l mg/ml) for 4 h. Bottom panel: Cells exposed to PMN elastase (24Itg/ml) + chloromethylketone (CMK; 0.02 mM). Bar = 20 Itm.
To examine whether the gap formation in the monolayer was due to a proteolytic mechanism, we tested whether these changes could be prevented by protease inhibitors or mimicked by PMN elastase. Monolayers were incubated with purified PMN elastase at various concentrations of 6, 10, and 24 J,'g/ml. No morphologic changes in the monolayer were evident at concentrations less than 24 J,'g/ml elastase (equivalent to the amount contained in 4 X 1()6 PMN [6], the number of PMN used in most of our experiments, or
Figure 7. Recovery of FTE monolayers from elastase. Top panel: FTE cells exposed to PMN elastase (24 Itg/ml) for 4 h. Bottom panel: Same field, 24 h after removal of elastase and replacement with fresh medium. Bar = 20 Itm.
Chung, Kercsmar, and Davis: Tracheal Epithelial Cells Resist Killing by Neutrophils
dase (I m Vlml) did not alter the integrity of the monolayer. Neither catalase (0.2 mg/ml) nor superoxide dismutase (300 Vlml) inhibited gap formation due to the PMA-activated PMN. When the medium containing elastase was removed after 4 h and replaced with fresh medium, the gaps in the monolayer disappeared in 24 h. Similarly, gaps formed in response to PMA-activated PMN also disappeared in 24 h (n = 4) (Figure 7).
Discussion Activated human neutrophils did not kill FTE cells at effector:target ratios as high as 90:1 over an 8-h exposure. This apparent lack of cytotoxicity was not a quirk of the 5lCr release assay, for similar results were obtained using release of previously incorporated (3H]leucine from the cells as an outcome measure and by supravital dye staining. The absence of cytotoxicity was not due to defective or preactivated neutrophils because we confirmed that the PMN, isolated by our methods, are activated by the effectors used. The activating agents PMA and FMLP were used at concentrations that maximally stimulate PMN oxidative burst and degranulation, and degranulation was further enhanced by addition of CB. Although our initial experiments were performed in culture medium that contains 0.5% serum, which might conceivably have quenched free radicals or inhibited proteases, comparison with experiments done in DMEM without additives revealed no difference in cytotoxicity of PMN for epithelial cells. Therefore, PMN specifically stimulated to produce O 2 radicals or release proteases are not cytotoxic to healthy FTE cells in culture. In contrast, endothelial cells in culture are readily killed by PMN, apparently by oxygen metabolites (1, 2,4). Our results also contrast with studies of type II alveolar epithelial cells that are killed by activated PMN by a mechanism that requires adherence of PMN to the epithelial monolayer (9). In our system, PMN adherence to FTE cells was minimal and did not increase with PMN activation. It may be that PMN do not readily adhere to intact healthy airway epithelial cells; similar results are obtained with Madin Darby canine kidney (MDCK) cells, an epithelial cell line that is resistant to PMN adherence unless the MDCK cells are first virusinfected (22). Our results do not exclude the possibility that damaged epithelial cells may be better targets for PMN adhesion, just as endothelial cells that are preinjured by hyperoxia are better targets for PMN (23, 24). In vivo, conditions in which PMN are recruited to the airway may be accompanied or initiated by epithelial damage, for example, viral infection or pollutant exposure (25). In these pathologic states, PMNinduced cytotoxicity may indeed be present. This hypothesis is supported by the finding that, in vitro, airway epithelial cells obtained from rhesus monkeys exhibited significant PMN-induced cytotoxicity only when first exposed to ozone (II). Although activated PMN did not kill the FTE cells, they caused obvious morphologic changes in the monolayer. Within 4 h of exposure to activated PMN, there was separation of cells leaving gaps in the monolayer. These gaps were formed as a result of activated PMN, since unactivated PMN, the activating agents themselves (PMA, FMLP), or CB alone did not induce such changes.
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The gaps did not result from cell death or detachment (Table 2). Therefore, the gaps in the monolayer probably resulted from separation of adjacent cells. This phenomenon may be similar to that observed by other investigators. Endothelial cells or epithelial cells (MDCK) pulled apart when exposed to oxygen metabolites (26, 27). Endothelial cell monolayer integrity is also disrupted by activated PMN by a nonoxidative mechanism (28). In both studies, the morphologic changes were also accompanied by increased endothelial or epithelial monolayer permeability. We tested three possible mechanisms of gap formation: one mediated by PMN adherence, oxidative damage by hydrogen peroxide or superoxide (known PMN products), and proteolytic damage. Gap formation was probably not a consequence of significant PMN adherence to FTE cells, because gap formation was observed under conditions that produced no change in PMN adherence. Oxidative products of activated PMN also appear to be unlikely to be responsible for the gap formation. Neither superoxide nor hydrogen peroxide, known PMN products, caused gap formation, even at concentrations much higher than would be generated by 4 X 106 activated PMN (a number sufficient for gap formation). Moreover, the morphologic changes caused by activated PMN were not inhibited by superoxide dismutase or catalase. Proteolytic damage seems a likely candidate mechanism. PMN elastase alone, in the amounts contained in 4 x 106 PMN (6), caused reversible gap formation in the monolayer with a time course similar to PMN-induced damage. Inhibitors of elastase, including ai-antitrypsin, CMK, and 10 % FCS prevented the damage to the monolayer induced by activated PMN in the presence of CB, or the damage from elastase itself. Therefore, the morphologic changes induced by activated PMN were likely in part due to proteolytic mechanisms. ai-antitrypsin and CMK not only inhibit proteolysis but can also block cationic sites (29), so it is not surprising that these agents completely prevented gap formation. Therefore, although the damage from elastase is largely proteolytic, the charge effects of elastase may also contribute to the morphologic changes seen in our experiments. Gap formation in our sy~tem may not precisely reflect a comparable process in vivo since our culture substrate is not identical to basement membrane. However, activated PMN did not completely remove or alter the collagen-coated substrate enough to alter cell adherence, suggesting that the mechanism of gap formation is not solely due to disruption of the artificial culture substratum. Although ultrastructural examination revealed that the culture substratum for the FTE cells was decreased in amount following exposure to activated PMN (Figure 5), it appeared that intercellular adherence rather than detachment from substrate accounts for our observations. Therefore, this culture system may be a useful model for studying this aspect of PMN-epithelial cell interactions. In conclusion, activated PMN do not kill airway epithelial cells but can cause reversible disruption of epithelial monolayer integrity by gap formation. The mechanism by which activated PMN cause this damage is largely proteolytic rather than oxidative and does not depend on PMN adherence to the monolayer. PMN elastase appears to be a major contributor to barrier disruption. Further studies are re-
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
quired to see how such monolayer disruption might alter important epithelial functions and whether other nonlethal damage is done by activated PMN. Acknowledgments: We thank Ms. Cathy L. Silski for her excellent technical assistance and Ms. YoshieHervey for technical support. This study was supported in part by Training Grant HL-07415 and Grants HL-28386, DK-27651 (Cystic Fibrosis Core Center), and HL-28530 from the National Institutes of Health and by the Cystic Fibrosis Foundation.
15. 16. 17. 18.
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