The Effect of Human Eosinophils on Cultured Human Nasal Epithelial Cell Activity and the Influence of Nedocromil Sodium In Vitro Jagdish L. Devalia, Raymond J. Sapsford, Csaba Rusznak, and Robert J. Davies Department of Respiratory Medicine, St. Bartholomew's Hospital, London, United Kingdom

Although there is increasing evidence of a pathogenic role for eosinophils in the airway epithelium, there is little direct evidence which demonstrates that eosinophils influence epithelial cell activity in humans. We have cultured human nasal epithelial cells in vitro and studied the effect of isolated human eosinophils on the ciliary beat frequency (CBF) and cell membrane integrity of these cells after incubation in the absence or presence of 0.1 I-tM phorbol 12-myristate 13-acetate (PMA) or 0.1 mg/ml opsonized latex beads and the absence or presence of to- 5 M nedocromil sodium. CBF was monitored by an analogue contrastenhancement technique, and cell damage was assessed by release of 51Cr from the cells. Cell cultures were also assessed for the percentage of eosinophil cationic protein (ECP) released into the medium at the end of incubation. Neither 0.1 I-tM PMA, 0.1 mglml opsonized latex beads, to- 5 M nedocromil sodium, nor eosinophils alone altered the CBF of the epithelial cells. PMA-stimulated eosinophils, however, attenuated the CBF significantly, from to.2 ± 0.3 to 8.8 ± 0.4 Hz (P < 0.05) after 15 h of incubation. Similarly, opsonized latex bead-stimulated eosinophils led to a significant attenuation of CBF from 9.2 ± 0.3 to 8.4 ± 0.3 Hz (P < 0.05), 6.9 ± 0.5 Hz (P < 0.001), and 7.5 ± 0.3 Hz (P < 0.001) after 2, 15, and 24 h of incubation, respectively. Of the two activating agents, only opsonized latex beads induced release of significantly greater amounts of ECP from eosinophils (30.4 ± 5.5% of total, compared with control values of 13.1 ± 2.6% of total; P < 0.05). Nedocromil sodium (10-5 M) abrogated both the PMA- and latex bead-stimulated eosinophil-induced attenuation of CBF and also blocked the release of ECP from the eosinophils. Studies of cell damage demonstrated that incubation of epithelial cells in the presence of eosinophils and PMA or opsonized latex beads led to significantly greater release of 51Cr (32.7 ± 5.7% and 31.5 ± 3.8%, respectively; P < 0.05) compared with incubation of the cells in the presence of eosinophils alone (14.4 ± 1.3%). These data suggest that "activated" eosinophils are capable of directly causing airway epithelial cell dysfunction and may be inhibited by agents such as nedocromil sodium.

The airway epithelium is thought to play an important physicochemical role in the etiology of airways disease. Pathologic examination of airway tissue of chronic asthmatic subjects (1) and studies of respiratory viral infections (2) have suggested that the frequently observed increase in airway hyperresponsiveness in these conditions may be associated with epithelial damage. Examination of asthmatic tissue has demonstrated that there is gross damage and infiltration of the epithelium by increased numbers of predominantly eosinophils and to a lesser extent neutrophils (3). Studies, par-

(Received in original form August 21, 1991 and in final form March 25, 1992) Address correspondence to: Dr. 1. L. Devalia, Department of Respiratory Medicine, St. Bartholomew's Hospital, London ECIA ?BE, United Kingdom. Abbreviations: ciliary beat frequency, CBF; eosinophil cationic protein, ECP; eosinophil peroxidase, EPO; fetal calf serum, FCS; major basic protein, MBP; medium 199, M199; platelet-activating factor, PAF; phorbol12myristate 13-acetate, PMA. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp, 270-277, 1992

ticularly by Gleich and colleagues, have suggested that the eosinophil is the chief effector cell of epithelial damage in bronchial asthma. These investigators have demonstrated that majorbasic protein (MBP), a principal constituent of the eosinophil granules, is present in elevated levels in the sputum of asthmatic patients but not nonasthmatic subjects (4) and is also found deposited in areas of damaged respiratory epithelium in patients who have died of asthma (5). In vitro studies have demonstrated that MBP and other eosinophil granule-associated proteins such as eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) , which are released on "activation" of eosinophils, are capable of damaging the respiratory epithelium of both animals and humans (6-8), and that the ciliated epithelial cells are mostly affected (9). Indeed, a recent study by Yukawa and colleagues has demonstrated that platelet-activating factor (PAF)activated, but not "nonactivated;' guinea pig eosinophils are capable of grossly disrupting the guinea pig ciliated tracheal epithelium in vitro (to). There is now increasing evidence to suggest that eosinophils may be equally important in the pathogenesis of al-

Devalia, Sapsford, Rusznak et al.: Effect of Isolated Eosinophils on Cultured Human Nasal Epithelial Cells

lergic rhinitis, since they can readily be detected in nasal secretions after both natural exposure to birch pollen and acute exposure to allergen out of the pollen season (11, 12). Additionally, it has been demonstrated that the numbers of eosinophils correlate well with the occurrence of symptoms during the pollen season (11, 13) and that nasal symptoms and "activation" of the eosinophils are both attenuated by treatment with steroids in vivo (14, 15). Although there appears to be a close association between eosinophil infiltration and epithelial cell damage in bronchial asthma, this has not been found to be the case in seasonal allergic rhinitis. Because eosinophils are commonly found in nasal tissue in nonallergic eosinophilic rhinitis, or NARES, which is frequently associated with asthma and nasal polyposis (16, 17), it may be that epithelial damage only occurs when there is chronic or prolonged inflammation. In view of increasing evidence of the detrimental effects of "activated" eosinophils and their products, we have investigated the effect of incubating isolated human eosinophils, in the absence or presence of phorbo1 12-myristate 13acetate (PMA) or opsonized latex beads, on ciliary beat frequency (CBF) and cell membrane integrity of human nasal epithelial cells in vitro. In addition, we have also studied the influence of nedocromil sodium on eosinophil-induced changes in the CBF of the epithelial cells and the release of ECP from eosinophils in this system.

Materials and Methods All chemicals and reagents were of tissue culture grade and, unless stated otherwise, were obtained from the Sigma Chemical Co. (Poole, UK). Nasal Tissue Nasal turbinate tissue was obtained from patients presenting for turbinectomy, primarily for upper airway obstruction, at the Ear, Nose and Throat Department of St. Bartholomew's Hospital, London. After resection, the tissue was placed into chilled medium 199 (M199) (Northumbria Biologicals Ltd., Northumberland, UK) and brought back to the laboratory, where it was processed for tissue culture within 0.5 to 1 h of resection. Isolation and Culture of Nasal Epithelial Cells Nasal epithelial cells were cultured by an explant cell culture technique that we developed in our laboratory and have described fully elsewhere (18). Briefly, the epithelium was dissected away from the underlying tissue and, after further dissection into smaller sections of approximately 1 to 2 mm' in size, the tissue was "sterilized" by gently washing 3 times in prewarmed and pregassed M199 containing 1% (vol/vol) antibiotics/antimycotic solution. Two or three sections were transferred to untreated 6-cm diameter Falcon Primaria culture dishes (Becton Dickinson Ltd., Oxford, UK) and incubated at 37° C in a 5 % CO 2-air atmosphere in 2.5 ml of sterile and freshly prepared culture medium for a period of 2 to 3 days. After this period, all the explants were observed for cell outgrowth and the medium was changed at 48-h intervals until the cells had grown to confluency, normally by 3 to 4 wk. The identity of the epithelial cells was confirmed by sev-

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eral techniques, including light microscopy, electron microscopy, and indirect immunoperoxidase staining techniques, as previously described (18). Isolation and Purification of Human Eosinophils Eosinophils were isolated from blood of atopic subjects, according to a modification of the method of Kloprogge and colleagues (19). Fifty milliliters of heparinized blood was mixed with 12.5 ml of 6% dextran (average mol. wt., 100,000 to 200,000) in 0.15 M saline and allowed to sediment at room temperature for 45 min. The leukocyte-rich, dextran-plasma fraction (huffy layer) was aspirated gently and washed twice by centrifugation at 500 x g for 10 min and resuspension in 15 ml buffered sterile Hanks' balanced salt solution, pH 7.4. The final washed cell pellet was resuspended in 1.5 ml isotonic Percoll of density 1.07 g/ml (Pharmacia, Milton Keynes, UK), supplemented with 10% fetal calf serum (FCS), and carefully "loaded" onto a discontinuous density Percoll gradient prepared with 2.0-ml aliquots of isotonic Percoll solutions of densities 1.085, 1.090, 1.095, and 1.105 g/ml. The Percoll gradient was centrifuged at 1,600 x g for 20 min at 20° C, and at the end of centrifugation 0.5-ml aliquots of the gradient were harvested from the bottom upward. A small volume of each aliquot was stained using Kimura's stain (20), and all the aliquots containing eosinophils with < 10% contaminating cells were pooled. This was made up to a final volume of 15 ml with sterile M199, as above, and then centrifuged at 500 x g for 10 min. The cell pellet was resuspended in 1.5 ml 0.87% ammonium chloride solution and incubated in an ice-bath for 10 min to lyse contaminating red blood cells. At the end of incubation, the cell suspension was made up to a final volume of 15 ml with sterile medium and washed twice by resuspension in fresh sterile culture medium and centrifugation at 500 X g for 10 min. The final washed eosinophil cell pellet was resuspended in 1.5 ml sterile complete culture medium, as above, and assessed for cell viability by trypan blue exclusion. Cell numbers and purity were determined after staining with Kimura's stain and counting in an improved Neubauer hemocytometer, and eosinophil suspensions of 95 % viability and 90% purity were used in further investigations. Estimation of CBF CBF of 2- to 3-wk-old cultures was measured by a modification of the analogue contrast-enhancement technique, using the Reece Scientific PCX on Screen Measurement System (Brian Reece Scientific Instruments, Newbury, UK), as previously described (18). Briefly, this involved transfer of the microscopic image of epithelial cells under different experimental conditions onto a television monitor via a video camera, followed by electronic processing of the video signal such that the contrast was dramatically increased to allow visualization of the specimen far beyond the resolution limits of the optical microscope. Television signals relating to differences in light intensity, resulting from ciliary motion at any specific point on the monitor screen, determined by positioning a cross-hair light-sensing probe directly over ciliated cells, were analyzed by use of a microcomputer incorporating a PCX Video Digitizer Card and specifically programmed for this application (Brian Reece Scientific In-

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struments). The differences in light intensities were computed in hertz (Hz). Before any treatment or monitoring, each culture was equilibrated for 2 min at room temperature and then monitored for the "baseline" CBF in six randomly chosen areas of ciliated cells, to give a mean CBF value. After specific treatment, the cultures were incubated at 37° C for up to 40 h, and at several times during incubation each culture was equilibrated and monitored for CBF as outlined above. All the experiments were commenced at about the same time of the day in order to account for effects of circadian rhythms in the cells. Estimation of ECP ECP was estimated in cultures studied for changes in CBF over a period of 24 h. At the end of incubation, medium was collected from each culture and the cells were washed gently with 1.0 ml fresh M199. The culture medium and the wash were pooled and centrifuged at 500 x g for 10 min at 4° C to separate the eosinophils and the medium, which were stored by freezing at -70° C, until assayed for ECP. Before assay, the eosinophil pellets were resuspended in 3.0 ml M199 and disrupted by vortexing. Cetyltrimethylammonium bromide (CTAB) was added to all samples, to a final concentration of 0.3 %, and 50-p.l aliquots of each sample were assayed in duplicate. ECP was estimated by a double-antibody radioimmunoassay, according to the manufacturer's instructions (Pharmacia), and ECP released into the medium was expressed as a percentage of the total in each culture. Estimation of Epithelial Cell Membrane Damage Epithelial cell membrane damage was investigated by assessing the release of 51Cr from cells radiolabeled with sodium chromate (Amersham International, Amersham, UK). Twoto three-wk-old cultures were used for these studies, and before experimentation explants were removed from each culture. The cells were incubated in the presence of 0.5 p.Ci/ml [51Cr]sodium chromate overnight, after which incubation the culture medium was decanted and the cells washed gently with M199. The cultures were exposed to eosinophils under different experimental conditions and then incubated for a further 24 h at 37° C in a 5 % COr-air atmosphere. At the end of incubation, the medium was collected and the cells were gently washed with 1.0 ml fresh ice-cold medium. The culture medium and the wash were pooled, and the adherent cells were scraped off into 1.0 ml M199. One hundredmicroliter aliquots of all samples were mixed with 3.0 ml of Ready-Solv'" CP scintillation cocktail (Beckman-RIIC Ltd., High Wycombe, UK) and assessed for 51Cr radioactivity, using a Beckman LS1800 scintillation counter (Beckman RIIC). All radioactivity released into the medium was expressed as a percentage of the total in the culture, according to the formula: Total cpm in medium Total cpm in medium

+ total cpm in cell pellet

Effect of Eosinophils on Ciliary Activity and Cell Membrane Damage of Epithelial Cells Before incubation, the eosinophils were equilibrated for 1 h at 37° C in a 5 % CO 2-air atmosphere and then co-cultured

with epithelial cells at a concentration of 0.3 x lQ6 eosinophils/cell culture, to give an eosinophil-to-epithelial cell ratio of approximately 1:5. Separate sets, containing a minimum of six cultures from different individuals, were incubated in the presence of either (1) eosinophils alone, (2) eosinophils plus an activator (PMA or opsonized latex beads), or (3) an activator alone, and each culture was monitored for effects on either CBF and ECP or release of 51Cr, as described above. At the end of incubation, the viability of eosinophils was also estimated by trypan blue exclusion in cultures investigated for effects on CBF. For studies investigating the effect of PMA, fresh solutions at final concentrations of 0.1 and 1.0 p.M PMA were prepared from a stock solution of 100 p.M PMA in M199. Before use, all solutions were sterilized by passing through 0.2-p.m Minisart micropore filters (Sartorius Ltd., Epsom, UK) and 2-ml aliquots used for the incubation of epithelial cell cultures in the absence or presence of eosinophils. Initially, the effect of PMA-activatedeosinophil-induced changes in CBF was studied over a period of 40 h. However, because of microbial contamination resulting occasionally during the later stages of incubation in these studies, all subsequent studies were carried out over an incubation period of 24 h and cultures were investigated for effects on CBF after 2, 15, and 24 h incubation and for release of ECP and 51Cr after 24 h of incubation. Similarly, for studies using opsonized latex beads as the eosinophil-stimulating agent, cultures were incubated in medium containing beads at a concentration of 0.1 mg/ml in the absence or presence of eosinophils and treated as above. Before use, however, an aqueous suspension of latex beads (mean diameter, 1.1 p.m) was sterilized by centrifugation at 1,500 x g for 3 min and resuspension in lO-ml aliquots of sterile M199 containing 1% (vol/vol) antibiotic/antimycotic solution several times. The washed beads were opsonized by incubating in 5 ml sterile FCS (Northumbria Biologicals) at 37° C overnight, and, after further centrifugation as above were collected and resuspended in sterile complete culture medium at a concentration of 10 mg/ml. This stock solution was diluted further with fresh complete culture medium to give a final suspension of 0.1 mg/ml. Effect of Nedocromil Sodium on Eosinophil-induced Changes in CBF of Epithelial Cells and Release of ECP from Eosinophils The protocol used for these studies was similar to the one described above except that epithelial cells were incubated with eosinophils in the absence or presence of either 0.1 p.M PMA or 0.1 mglml opsonized latex beads, and the effect of 1 X 10- 5 M nedocromil sodium (Fisons pIc., Loughborough, UK) was investigated on any eosinophil-induced changes in CBF. In studies investigating the effect of opsonized latex beadstimulated eosinophils, epithelial cell cultures were either exposed to nedocromil sodium at the same time of exposure to the eosinophils and the latex beads or were pretreated with nedocromil sodium for 2 h before exposure to the eosinophils and the latex beads. All cultures were incubated for 24 h, and the effect on CBF was studied after 2, 15, and 24 h of incubation, while release of ECP into the medium was studied after 24 h of incubation.

Devalia, Sapsford, Rusznak et al.: Effect of Isolated Eosinophils on Cultured Human Nasal Epithelial Cells

Statistical Analysis Data of CBF measurements were expressed as the mean over six areas within a culture and, at any specified time point during the incubation, were finally calculated as the mean ± SEM for a given treatment group. The mean difference between baseline and each time point, within any single treatment group, was compared using Student's paired t test. Changes between baseline and each time point were also compared across the treatment groups by one-way ANOVA in order to assess whether these changes were different for different treatments. Differences in means of 51Cr and ECP release were compared using a two-sample t test. All values of P < 0.05 were considered to be significant.

Results These studies demonstrate that functional ciliated human nasal epithelial cells could be cultured to confluence in vitro, and confirm our previous findings (18). Studies investigating the effect of PMA on the CBF of epithelial cells demonstrated that the higher concentration of 1.0 JLM itself attenuated the CBF of these cells significantly from a baseline value of 9.1 ± 0.3 Hz to 8.1 ± 0.2 Hz (P < 0.05) and 7.7 ± 0.2 Hz (P < 0.05), by 15 and 24 h of incubation, respectively. Consequently, this concentration was not used in any further investigations. The 0.1 JLM PMA concentration, however, did not have any significant effect on the CBF of these cells at any time during the culture period and was used in all subsequent investigations. Studies investigating the effect of PMA-stimulated eosinophils on epithelial cell CBF over a period of 24 h of incubation demonstrated that the effect on CBF was most prominent by 15 h of incubation and resulted in the attenuation of the CBF from a baseline value of 9.2 ± 0.3 to 7.5 ±. 0.4 Hz (P < 0.01) (Figure 1). Addition of 10- 5 M nedocromil so-

273

dium into the culture medium, however, completely blocked this PMA-stimulated eosinophil-induced attenuation of the CBF (Figure 1). Incubation of the epithelial cells in the presence of either eosinophils, PMA, or 10- 5 M nedocromil sodium alone did not affect the CBF of these cells over a 24-h incubation period. Analysis of variability in CBF within each treatment group demonstrated that this did not change during incubation. Analysis by ANOVA demonstrated that there were significant differences between the treatments at 15 and 24 h. These were significant between eosinophil- and PMAtreated cells compared with cells treated with either eosinophils, PMA, or 10-5 M nedocromil sodium alone. Additionally, there were significant differences between eosinophil- and PMA-treated cells and eosinophil-, PMA-, and 10- 5 M nedocromil sodium-treated cells (Table 1). Estimation of eosinophil viability at the end of incubation demonstrated that this was> 90% in all treatment groups. Analysis of the cultures for the amount of ECP released into the medium demonstrated that there was spontaneous release of 13.1 ± 2.6% ECP from untreated eosinophils by 24 h of incubation and that this was not significantly different from the release of 10.1 ± 1.5% ECP observed with eosinophils incubated in the presence of PMA. Like the PMA-stimulated eosinophils, the latex beadstimulated eosinophils also induced ciliary dyskinesia and a marked attenuation of the CBF of the epithelial cells. Incubation of the epithelial cells in the presence of both latex beads and eosinophils resulted in a progressively significant attenuation of the CBF from baseline values of 9.2 ± 0.3 Hz to 8.4 ± 0.3 Hz (P < 0.05),6.9 ± 0.5 Hz (P < 0.001), and 7.5 ± 0.3 Hz (P < 0.001), by 2, 15, and 24 h of incubation, respectively (Figure 2). Addition of 10- 5 M nedocromil sodium into the culture medium did not block the attenuation of CBF immediately, as was the case with PMA-stimulated

CBF (Hz) 12

m"Hi+.

10 Figure 1. Effect of (a) 0.3 x 106 eosinophils, (b) 0.1 /LM PMA, (c) 0.1 /LM PMA + 0.3 x 106 eosinophils, (d) 0.1 /LM PMA + 0.3 x 106 eosinophils + 10- 5 M nedocromil sodium, and (e) 10- 5 M nedocromil sodium on CBF of human nasal epithelial cells cultured over an incubation period of 24 h (n = 9 separate cultures for all treatment groups). ** P < 0.01 versus to.

8

6

4

2

o

o

2 1524

a

o

2 15 24

0 2 15 24 0 2 15 24 Time (hours) b e d

o

2 1524

e

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TABLE 1

Analysis of differences in mean CBF changes between baseline and 15 or 24 h in epithelial cell cultures by ANOVA* E

Mean of differences at To - TIs Mean of differences at To - T24

E+P

P

N

E+P+N

0.71

± 0.32

1.64

± 0.39

0.31

± 0.23

0.33

± 0.20

0.48

± 0.19

0.54

± 0.30

1.62

± 0.28

-0.24

± 0.26

-0.29

± 0.42

-0.41

± 0.21

Significance

F4 ,40 = 3.99 P < 0.01 F4•40 = 10.79 P < 0.005

* Cultures were exposed to 0.3 X 106 eosinophils (E), 0.3 x 106 eosinophi1s + 0.1 I'M PMA (E+ P), 0.1 I'M PMA (P), 0.3 x 106 eosinophils + 0.1 I'M PMA + 10-' M nedocromil sodium (E+P+N), or 10-' M nedocrornil sodium alone (N). CBF (Hz) 12

10

8

r+~K-~

,---l-+

-+-

X 106 eosinophils (n = 9), (b) 0.1 mg/ml opsonized latex beads (n = 6), (c) 0.1 mg/ml opsonized latex beads + 0.3 x 106 eosinophils (n = 8), (d) 0.1 mg/ml opsonized latex beads + 0.3 x 106 eosinophils + 10- 5 M nedocromil sodium (n = 8), and (e) 10- 5 M nedocromil sodium for 2 h before exposure to 0.1 mg/ml opsonized latex beads + 0.3 X 106 eosinophils (n = 6) on CBP of human nasal epithelial cells cultured over an incubation period of 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001 versus to.

Figure 2. Effect of (a) 0.3

r+

6

4

2

o

o

2 1524

a

o

2 15 24

0 2 15 24 0 2 15 24 Time (hours) b e d

o

2.1524

e

TABLE 2

Analysis of differences in mean CBF changes between baseline and 2, 15, or 24 h in epithelial cell cultures by ANOVA* E

Mean of differences at To - T2 Mean of differences at To - TIs Mean of differences at To - T24

E+LB

E+LB+N

E+LB+pN

LB

-0.21

± 0.20

0.77

±

0.23

0.90

± 0.22

-0.52

± 0.30

-0.09

± 0.13

0.57

± 0.29

2.32

± 0.35

0.64

± 0.22

0.16

± 0.12

0.35

± 0.14

0.52

± 0.29

1.74

± 0.16

-0.19

± 0.29

-0.20

± 0.15

0.33

± 0.18

Significance

F4•32 = 7.82 P < 0.005 F4 •32 = 10.87 P < 0.005 F4 •32 = 12.85 P < 0.005

* Cultures were exposed to 0.3 x 106 eosinophils(E), 0.3 x 106 eosinophils + 0.1 mg/ml opsonized latex beads (E+LB), 0.3 x 106 eosinophils + 0.1 mg/ml opsonized latex beads + 10-' M nedocromil sodium (E+LB+N), 10-' M nedocrornil sodium for 2 h before exposure to 0.3 x 106 eosinophils + 0.1 mg/ml opsonized latex beads (E+LB+pN), and 0.1 mg/ml opsonized latex beads alone (LB).

eosinophils, but appeared to reverse this effect over the course of 24 h of incubation (Figure 2). Preincubation of the epithelial cells with 10- 5 M nedocromil sodium for 2 h before exposure to latex beads and eosinophils, however, blocked the latex bead-stimulated eosinophil-induced attenuation of CBF (Figure 2). Incubation of the epithelial

cells in the presence of either eosinophils, latex beads, or 10- 5 M nedocromil sodium alone did not affect the CBF of these cells over a 24-h incubation period. As demonstrated for investigations with PMA, there was little difference in variability of CBF within each treatment group during incubation. ANOVA demonstrated that there

Devalia, Sapsford, Rusznak et al.: Effect of Isolated Eosinophils on Cultured Human Nasal Epithelial Cells

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% ECP in medium

40 40

30 30

20 20

10 10

o o

a

b

c

Figure 3. Release ofECP into culture medium after 24 h of incubation from cultures exposed to (a) 0.3 X 1()6 eosinophils (n = 7), (b) 0.3 X 1()6 eosinophils + 0.1 mg/ml opsonized latex beads (n = 9), and (c) 0.3 X 1()6 eosinophils + 0.1 mg/ml opsonized latex beads + 10- 5 M nedocromil sodium (n = 6). * P < 0.05 versus to.

a

b

c

d

Figure 4. Release of IICr into medium after 24 h of incubation from (a) untreated cell cultures, (b) cultures exposed to 0.3 X 106 eosinophils, (c) cultures exposed to 0.3 X 106 eosinophils + 0.1 J.tM PMA, and (d) cultures exposed to 0.3 x 1()6 eosinophils + 0.1 mg/ml opsonized latex beads (n = 6 separate cultures for all treatment groups). * P < 0.05 versus to.

Discussion were significant differences between cells incubated in the presence of eosinophils and latex beads compared with cells incubated in the presence of eosinophils, latex beads, or 10- 5 M nedocromil sodium alone, or in the presence of 00sinophils, latex beads, and 10- 5 M nedrocromil sodium together, at all time points investigated (Table2). As for studies with PMA, these studies also demonstrated that> 90% of eosinophils excluded trypan blue at the end of the incubation. Analysis of ECP released into the culture medium revealed that incubation of the epithelial cells and eosinophils in the presence of opsonized latex beads induced significantly greater release of ECP (30.4 ± 5.5 %; P < 0.05) compared with incubation in presence of eosinophils alone (12.9 ± 2.4% ECP) (Figure 3). Treatment with 10-5 M nedocromil sodium blocked the latex bead-induced release of ECP observed after 24 h of incubation. Studies investigating the release of IICr from epithelial cells after 24 h of incubation demonstrated that there was spontaneous release of 16.5 ± 1.2 % 51Cr from cells incubated under normal conditions, and that this was not significantly different from a release of 14.4 ± 1.3% IICr observed from cells incubated in the presence of eosinophils alone. Incubation of cultures with eosinophils in the presence of either 0.1 Jl.M PMA or 0.1 mg/ml opsonized latex beads, however, led to significantly greater release of 51Cr (32.7 ± 5.7 % and 31.5 ± 3.8 %, respectively; P < 0.05) (Figure 4).

The eosinophil is regarded as an important effector cell in the pathogenesis of asthma and possibly also allergic rhinitis, since it is thought to interact with and damage the airway epithelium. It has not, however, proved easy to demonstrate a direct association between the eosinophil-epithelial interaction and epithelial damage in vivo because of the presence of other cell types. Human airway epithelial cell cultures offer an ideal in vitro model system for study of the epithelium and its interaction with other cell types in the etiology of airway diseases and the underlying mechanism(s) of inflammation. We have recently developed a technique for the culture of human bronchial/nasal epithelial cells and demonstrated that these cells are similar to one another and in many respects also closely resemble the cells found in vivo (18). Using the model of cultured human nasal epithelial cells, we have studied the effect of isolated human eosinophils on ciliary activity and cell membrane integrity of these cells in vitro. For these studies, although eosinophils were separated according to their densities as described by others (19), the final eosinophil preparations were used as mixed populations of "total" purified eosinophils. This was important because although some researchers have suggested that hypodense eosinophils represent the activated form of eosinophils and therefore may be more appropriate in the etiology of airways disease (21), a recent study by Kauffman and colleagues (22) has suggested that hypodense eosinophils isolated from blood of asthmatic subjects are an artifact of the isolation procedure. These researchers haveinvestigated both eosinophils and neutrophils isolated from blood of asthmatic subjects and have demonstrated that the

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numbers of both hypodense eosinophils and neutrophils were greatly increased if the erythrocytes were removed by sedimentation before separation on discontinuous Percoll gradients. In contrast, hypodense eosinophils or neutrophils could not be isolated from blood that had not been subjected to erythrocyte sedimentation before centrifugation on the Percoll gradients. Our studies have, however, demonstrated that human eosinophils are indeed capable of directly influencing both the ciliary activity and cell membrane integrity of human nasal epithelial cells. Our investigations of the effects on CBF have demonstrated that ciliary activity of the epithelial cells is affected in a time-dependent fashion and is significantly attenuated by 2 to 15 h of incubation. Studies of effects on release of 51Cr have demonstrated that this is significantly increased by the end of incubation, suggesting that epithelial cell membrane is damaged. More importantly, these studies have demonstrated that activation of the eosinophils is a prerequisite for effects on cilia and cell membrane to be observed. Overall, our findings are in agreement with the findings of others (6-10) and suggest that substantial eosinophil-induced injury to airway epithelial cells, particularly ciliated epithelial cells, may play an important role in the pathogenesis of airway disease characterized by eosinophilia. Ayars and colleagues (7) have investigated the direct toxicity of eosinophil granule-associated proteins to human nasal epithelium in vitro and have demonstrated that an EPO/H 20 2-generating system and MBP are capable of causing marked nasal epithelial cell lysis in a concentrationand time-dependent manner. Furthermore, these investigators have demonstrated that the EPO/H20 2-generating system is much more potent than MBP in mediating cell injury. More recently, Yukawa and colleagues (10) have studied the effects of purified intraperitoneal guinea pig eosinophils on guinea pig tracheal epithelium in vitro and have reported findings similar to our own with this animal model. These researchers have demonstrated that although PAF or eosinophils alone do not have any significant effect on epithelial cells, PAF-activated eosinophils disrupt the epithelium and significantly attenuate the CBF of the epithelial cells. Furthermore, these investigators have also demonstrated that the effect on CBF can be blocked by the PAF antagonist WEB 2086, suggesting that eosinophil activation is necessary for any eosinophil-mediated effects at the epithelium. The effect of PAF-activated eosinophils on CBF noted by these researchers over a 24-h incubation period was comparatively greater than that observed for PMA- or opsonized latex beadactivated eosinophils in our model. Although this disparity may be due to the different activators used in these studies, it is more likely to have been a consequence of differences in culture conditions and the numbers of eosinophils used. Yukawa and colleagues have investigated the effect of eosinophils in numbers that were 16- to 160-fold greater than those used in our studies and have demonstrated that the eosinophils do indeed elicit dose-dependent effects in their guinea pig tracheal ring model (10). Also contrary to our findings, these researchers have demonstrated that the viability of the eosinophils decreased significantly in their system. It is possible that this may be an additive effect of the culture conditions used, since the viability of the untreated eo-

sinophils was also attenuated by approximately 40% of the starting viability by the end of incubation in their model. Overall, our studies have also demonstrated that opsonized latex beads, but not PMA, induced the release of ECP from the eosinophils into the culture medium. While this difference in the PMA- or latex bead-induced release of ECP could be a result of the "degree of activation" induced by these agents, the possibility that these agents may be operating through different mechanism(s) cannot be ruled out. However, similar studies investigating the release of other cytotoxic proteins or compounds from the eosinophils would have to be conducted before this could be said with any degree of certainty. Studies of eosinophils incubated in the presence of serumcoated latex beads demonstrated that these released significantly greater amounts of ECP compared with eosinophils incubated in the absence of opsonized latex beads. This is in agreement with the findings of others who have suggested that phagocytosis of opsonized particles by eosinophils leads to degranulation of the eosinophils via complement receptor activation. Winqvist and colleagues have studied the release of ECP from eosinophils after stimulation by serum-coated Sephadex beads and suggested that interaction through the C3 receptors is a prerequisite for ECP release from human eosinophils (23). Indeed, studies with C3b-coated Sepharose beads have demonstrated that the Sepharose-C3b-stimulated eosinophils release greater amounts of EPO than do the control nonstimulated eosinophils (24). Studies investigating the mechanisms of PMA-induced effects in effector cells such as polymorphonuclear neutrophils have suggested that PMA inhibits neutrophil granule exocytosis and degranulation but otherwise induces a respiratory burst, resulting in the generation of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals, which leads to tissue damage and propagation of inflammation (25, 26). Although some studies have suggested that PMA triggers the burst of oxidative metabolism through activation ofNADPH oxidase (27), others have suggested that activation of protein kinase C may be more important (25, 28, 29). We have also investigated the effect of nedocromil sodium, a nonsteroidal antiasthmatic drug, on eosinophil-induced changes in the CBF of the cells in our model but have limited these studies to investigating the effect of a single concentration of 1 X 10-5 M nedocromil sodium because of the requirement for very large numbers of the epithelial cell cultures for these studies. This particular concentration of nedocromil sodium, however, was chosen for study since it has been shown by others to inhibit both the complementinduced eosinophil granule protein release (24) and eosinophil "activation" (30) in vitro. Our findings of the effect of nedocromil sodium suggest that this agent blocks the activation of eosinophils and are in agreement with the findings of others (25, 30, 31). Although studies investigating the mechanisms of nedocromil sodium activity have demonstrated that this compound has multiple pharmacologic actions, in addition to anti-inflammatoryeffects on eosinophils and neutrophils (32-33), the precise mechanism through which it accomplishes a clinically relevant therapeutic role in asthma or allergic rhinitis is not clear.

Devalia, Sapsford, Rusznak et al.: Effect of Isolated Eosinophils on Cultured Human Nasal Epithelial Cells

Our studies would suggest that because nedocromil sodium blocks eosinophil activation by both PMA and opsonized latex beads, both of which are thought to act at the eosinophil cell membrane-bound enzyme level, then it is likely that this compound is acting at the eosinophil cell membrane level. Indeed, the finding that nedocromil sodium did not block the opsonized latex bead-stimulated eosinophil-induced changes in CBF immediately but did block these effects when the epithelial cells were pretreated with nedocromil sodium before incubation with eosinophils and opsonized latex beads suggests that nedocromil sodium may be exerting an effect not only on the eosinophil but also on the epithelial cell. This is in agreement with the findings of other recent studies that have demonstrated that nedocromil sodium is capable of blocking toluene diisocyanate-induced release of 15-hydroxyeicosatetraenoic acid from human bronchial epithelial cells (34) and attenuating endotoxin- and cigarette smoke-induced release of neutrophil chemotactic activity from bovine bronchial epithelial cells (35) in vitro, and suggests that this compound may play an active anti-inflammatory role at the epithelial level in the lower respiratory tract in vivo. Acknowledgments: The writers thank the National Asthma Campaign (UK), the Joint Research Board of St. Bartholomew's Hospital, London (UK), and Fisons pIc. for financial assistance. We also thank Pharmacia Biosystems Ltd. (UK) for the gift of reagents needed for analysis of ECP.

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