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Antimicrobial Agents and Chemotherapy, June 1998, p. 1499-1502, Vol. 42, No. 6
Departments of
Medicine and Physical
Therapy,1
Laboratory
Medicine,2 and
Otopharyngology,
Received 30 October 1997/Returned for modification 25 February
1998/Accepted 8 April 1998
We evaluated the effect of roxithromycin on cytokine production and
neutrophil attachment to human airway epithelial cells. Roxithromycin
suppressed production of interleukin 8 (IL-8), IL-6, and
granulocyte-macrophage colony-stimulating factor. It inhibited neutrophil adhesion to epithelial cells. Roxithromycin modulates local
recruitment and activation of inflammatory cells, which may have
relevance to its efficacy in airway diseases.
Roxithromycin is a 14-member
macrolide antibiotic effective for the treatment of upper and lower
respiratory tract infections (10). Recent reports showed
that roxithromycin is also effective for the treatment of chronic
airway diseases such as diffuse panbronchiolitis, bronchial asthma, and
chronic sinusitis (6, 7, 13), although its precise actions
remain unclear. In the present study, we investigated if roxithromycin
had any effect on the production of cytokines, especially interleukin 8 (IL-8), by human bronchial epithelial cells (8) and if it
had any effect on the process of neutrophil adhesion onto these cells
in vitro.
Normal human bronchial epithelial cells were prepared by the
proteolytic digestion of bronchi as reported previously (9, 14,
15). The cells were plated onto collagen-coated 24-well flat-bottom tissue culture plates (Koken, Tokyo, Japan) and cultured in
hormonally defined Ham's F-12 medium (HD-F12) as reported previously (9, 14) until confluence. The cells were keratin positive but vimentin negative, showing that they were epithelial cells (9,
14). A human bronchial epithelial cell line, Bet-1A
(11), was cultured in HD-F12.
Roxithromycin was dissolved in methanol as stock solutions and further
diluted in sterile physiological saline. The methanol at the final
concentrations in each experiment showed no cytotoxicity or effect on
IL-8 release by bronchial epithelial cells (data not shown). Various
concentrations of roxithromycin were added to the cells simultaneously
with and without stimulation of IL-1 Roxithromycin showed an inhibitory action on IL-8 release by
unstimulated and IL-1 Next, neutrophil adhesion to human bronchial epithelial Bet-1A
cells was studied in vitro. Briefly, Bet-1A cells were cultured on
collagen-coated 96-chamber flat-bottom culture plates. Neutrophils were
isolated by density gradient Percoll centrifugation (>97% pure)
(5). When the epithelial cells reached confluency, the cells
were rinsed and purified neutrophils (2.0 × 105/ml)
were applied to culture plates. After 30 min of incubation, each well
was rinsed twice, and 0.01% Triton ×-100 was added for cell lysis.
Myeloperoxidase (MPO) activity was measured by spectrophotometric assay
as reported previously (5). There was a significant positive correlation between the actual number of the attached neutrophils per
three randomized high-power fields and MPO activity (r = 0.976; P < 0.001 [Pearson's correlation test]).
Human peripheral neutrophils adhered to epithelial cells, and the
pretreatment
of neutrophils with N-formyl-methionyl-leucyl-phenylalanine (FMLP)
(10
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Roxithromycin Inhibits Cytokine Production by and
Neutrophil Attachment to Human Bronchial Epithelial Cells In
Vitro
ABSTRACT
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. After 24 h the
supernatants were harvested for the measurement of cytokines. Specific
immunoreactivity for IL-8 in culture supernatants was measured with
enzyme-linked immunosorbent assay (ELISA) kits (R & D
Systems, Inc., Minneapolis, Minn.) (15).
-stimulated human primary bronchial epithelial cells in a concentration-dependent fashion (Fig.
1a). A lactic dehydrogenase release
assay, trypan blue dye exclusion test, and a colormetric 3-4, 5-dimethyl-thiazol-2-yl-2, 5-diphenyltetrazolium bromide assay
(17) showed that this effect was not due to cytotoxicity (data not shown). This antibiotic also showed an inhibitory action on
IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) release by IL-1 -stimulated human bronchial epithelial cells in a
concentration-dependent fashion as assessed by ELISA kits specific for
each cytokine (R & D Systems) (Fig. 1b). Northern blot analysis for
IL-8 mRNA was performed by the method described previously (1, 3,
14, 19). Roxithromycin decreased the steady-state levels of IL-8
mRNA in IL-1 (10 ng/ml)-stimulated Bet-1A cells in a
concentration-dependent fashion (Fig.
2).
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FIG. 1.
Effect of roxithromycin on IL-8, IL-6, and GM-CSF
release by human primary bronchial epithelial cells. (a) Roxithromycin
was added to primary bronchial epithelial cells simultaneously with
(left panel) and without (right panel) IL-1 (10 ng/ml). The
supernatants were harvested after 24 h for IL-8 measurement. (b)
Effect of roxithromycin on IL-6 and GM-CSF release by IL-1 (10 ng/ml)-stimulated primary human bronchial epithelial cells.
Simultaneous treatment with roxithromycin significantly inhibited IL-6
and GM-CSF release after 24 h. For all panels, an asterisk
represents a P of <0.01 compared to controls (ANOVA;
n = 5). Error bars, standard deviations.
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FIG. 2.
Northern blot analysis showing effect of roxithromycin
(RXM) on IL-8 mRNA expression by IL-1 (10 ng/ml)-stimulated Bet-1A
cells in culture. Roxithromycin was added to the cells with IL-1 ,
and total cellular RNA was extracted after 12 h. (a) Roxithromycin
showed a concentration-dependent inhibition on the steady-state levels
of IL-8 mRNA. (b) Measurement of densitometric signals of IL-8
corrected by 
-actin transcripts showed a significant inhibition by
roxithromycin. *, P < 0.01 compared to controls
(ANOVA; n = 4). Error bars, standard deviations.
7 M) for 30 min, but not with lipopolysaccharide (10 ng/ml), IL-8 (5 ng/ml), or C5a (108 M), significantly
enhanced adhesion (by 245% ± 33.4%, 116% ± 14.7%, 92.8% ± 20.1%, and 71.2% ± 12.4%, respectively [for the first value,
P < 0.01]; baseline MPO activity = 100 [analysis of variance {ANOVA}; n = 4]. As for the
stimulation of epithelial cells, gamma interferon (IFN-
) (100 ng/ml)
and tumor necrosis factor alpha (10 ng/ml) significantly upregulated
neutrophil adhesion to the epithelial cells after 18 h (by 155% ± 10.5% and 191% ± 12.2%, respectively [P < 0.01]; baseline MPO activity = 100 [ANOVA; n = 4]). Confluent monolayers of Bet-1A cells were treated with IFN-
(100 ng/ml) with different concentrations of roxithromycin for 18 h. Then, the purified neutrophils with and without pretreatment of FMLP
were added to each well and incubated for 30 min. The adherence of
neutrophils onto epithelium was evaluated by MPO assay. As shown in
Fig. 3a, roxithromycin (1 to 25 µg/ml)
inhibited neutrophil adhesion to epithelial cells in a
concentration-dependent fashion when the neutrophils were pretreated
with 107 M FMLP for 30 min. Roxithromycin also showed an
inhibitory effect on adhesion of naive neutrophils, but it was
significant only at 25 µg/ml (Fig. 3b).
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FIG. 3.
Effect of roxithromycin on neutrophil adhesion onto
IFN- (100 ng/ml)-treated Bet-1A in vitro. Different concentrations
of roxithromycin were added to Bet-1A epithelial cells simultaneously
with IFN- (100 ng/ml) for 18 h. The purified neutrophils were
then added with and without 30 min of pretreatment with FMLP
(107 M). Roxithromycin showed a concentration-dependent
inhibitory effect on neutrophil-epithelium adhesion at a concentration
of no less than 1 µg/ml in FMLP-treated neutrophils (MPO assay) (a)
and at a concentration of 25 µg/ml in naive neutrophils (b). For both
panels, an asterisk represents a P of <0.05 compared to
controls (ANOVA; n = 4). Error bars, standard
deviations; OD, optical density.
To elucidate the key role of intercellular adhesion molecule-1 (ICAM-1)
on human bronchial epithelial cells (18), its expression on
Bet-1A was evaluated by a cell ELISA method. Briefly, Bet-1A cells were
cultured until confluency on 96-well plates, and the cells were treated
with IFN- (100 ng/ml) with and without roxithromycin for 18 h.
Then, the cells were rinsed and anti-human ICAM-1 monoclonal antibody
conjugated with horseradish peroxidase (British Biotechnology Products,
Ltd., Abingdon, United Kingdom) was added to each well and incubated
for 1 h at room temperature. After the wells were washed twice,
the substrate solution (tetramethylbenzidine) was added to quantify the
magnitude of ICAM-1 expression on the surfaces of the cells. The
epithelial cells expressed ICAM-1 molecules on their surfaces in a
manner that was upregulated by IFN- (100 ng/ml) (Fig.
4). Roxithromycin was shown to decrease
the magnitude of ICAM-1 expression on IFN- (100 ng/ml)-treated
epithelial cells (Fig. 4).
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Neutrophils play an important role in the pathogenesis of various airway inflammatory disorders (2). Their local recruitment is induced by chemokines such as IL-8 (20) and increased expression of adhesion molecules such as ICAM-1 on airway epithelial cells (18). Roxithromycin clearly decreased the number of neutrophils in bronchoalveolar lavage fluids from patients with airway inflammation (12). Its actions seem to be apart from its antibiotic activity, since it was effective even when the pathogens were absent or resistant to this antibiotic (12). Therefore, it is reasonable to assume that roxithromycin suppresses expression of chemokines relevant to cell recruitment into the airways. It has been reported that erythromcin and/or clarithromycin inhibited IL-6 and IL-8 production by normal bronchial epithelial cells (4, 16). Neutrophil adhesion to epithelium inhibits the access of the antiprotease system to neutrophil-derived enzymes and superoxides to epithelium. Therefore, this process is important for the prolongation of airway inflammation. In the present report, we have showed that roxithromycin, another 14-member macrolide antibiotic, has an inhibitory effect on IL-8 and ICAM-1 expression in human bronchial epithelial cells. These findings may explain, at least in part, the attenuating effect of this drug on local neutrophil recruitment and activation. Further study is necessary to elucidate the molecular mechanisms of this drug.
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ACKNOWLEDGMENTS |
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We are grateful to J. F. Lechner and C. C. Harris for supplying Bet-1A cells. We also thank Eisai Pharmaceutical Company for supplying roxithromycin.
This work was supported in part by a grant from the Diffuse Lung Disease Research Committee, Japan Ministry of Health and Welfare, and Manabe Medical Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Laboratory Medicine, University of Tokyo, School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 3-3815-5411. Fax: 3-3815-5954. E-mail: TAKIZAWA-PHY{at}h.u-tokyo.ac.jp.
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Antimicrobial Agents and Chemotherapy, June 1998, p. 1499-1502, Vol. 42, No. 6
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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