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Published ahead of print on October 9, 2003, doi:10.1165/rcmb.2003-0105OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 569-575, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0105OC

Macrolides Inhibit Epithelial Cell–Mediated Neutrophil Survival by Modulating Granulocyte Macrophage Colony–Stimulating Factor Release

Hideaki Yamasawa, Katsuhisa Oshikawa, Shoji Ohno and Yukihiko Sugiyama

Division of Pulmonary Medicine, Department of Medicine, Jichi Medical School, Tochigi, Japan

Address correspondence to: Hideaki Yamasawa, M.D., Division of Pulmonary Medicine, Department of Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan. E-mail: hyamasa{at}jichi.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrolides have been shown to be effective in treating diffuse panbronchiolitis (DPB), although the precise modes of action remain unclear. At sites of airway inflammation, respiratory epithelium is considered an active participant in regulating neutrophil survival. We therefore examined the effect of erythromycin, clarithromycin, azithromycin, and josamycin on both neutrophil survival and on epithelial-derived factors, which influence neutrophil longevity. Media conditioned with transiently tumor necrosis factor (TNF)-{alpha}–stimulated A549 human airway epithelial cells prolonged neutrophil survival compared with control media. The presence of dexamethasone during neutrophil culture led to further prolongation of neutrophil survival. In contrast, none of the tested macrolides modulated neutrophil survival, suggesting a lack of direct effect of these drugs. On the other hand, pretreatment of TNF-{alpha}–stimulated A549 cells by erythromycin, clarithromycin, azithromycin, or dexamethasone, but not josamycin, decreased the neutrophil survival–enhancing effects in a dose-dependent manner. Neutralizing antibodies to granulocyte macrophage colony–stimulating factor (GM-CSF) dampened the prolonged neutrophil survival observed in TNF-{alpha}–stimulated A549 conditioned media. Erythromycin, clarithromycin, azithromycin, and dexamethasone inhibited TNF-{alpha}–induced GM-CSF expression in A549 cells at both the protein and messenger RNA levels. These results suggest that macrolides inhibit epithelial cell–mediated neutrophil survival by modulating GM-CSF release, which may, at least in part, explain the effectiveness of this family of drugs on DPB.

Abbreviations: complementary DNA, cDNA • diffuse panbronchiolitis, DPB • enzyme-linked immunosorbent assay, ELISA • fetal calf serum, FCS • granulocyte colony-stimulating factor, G-CSF • granulocyte macrophage colony–stimulating factor, GM-CSF • interleukin, IL • lipopolysaccharide, LPS • messenger RNA, mRNA • phosphate-buffered saline, PBS • recombinant human, rh • reverse transcription-polymerase chain reaction, RT-PCR • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils are an essential component of the early inflammatory response, but under some circumstances, they also contribute to tissue injury. Upon stimulation, apoptotic neutrophils lose specific functions, including chemotaxis, degranulation, and respiratory burst (1). In addition, apoptotic neutrophils, unlike necrotic neutrophils, retain their membrane integrity and are swiftly recognized and ingested by phagocytes, preventing leakage of potentially injurious contents from dying cells (2). Thus, apoptosis of activated neutrophils and subsequent phagocytosis by macrophages and phagocytic parenchymal cells are considered to be important processes in the resolution of inflammation (2, 3).

At sites of inflammation, recruited neutrophils show delayed apoptosis and their life spans are markedly prolonged. In vitro studies of neutrophil survival have shown that a variety of endogenous and exogenous factors prolong the functional life span of neutrophils. These include granulocyte macrophage colony-stimulating factor (GM-CSF) (4, 5), granulocyte colony-stimulating factor (G-CSF) (6), interleukin (IL)-6 (7), IL-8 (8), and lipopolysaccharide (LPS) (5, 6). Airway epithelial cells indeed produce some of these factors and are shown to prolong neutrophil survival in vitro (9, 10). These findings suggest that airway epithelial cells are active participants in regulating neutrophil survival at sites of airway inflammation.

Several studies have demonstrated that long-term, low dosages of 14-membered ring macrolides, including erythromycin, clarithromycin, and roxithromycin, are effective for the treatment of chronic airway diseases, such as diffuse panbronchiolitis (DPB) and chronic sinusitis (11–13). Although the precise modes of action remain unclear, the effectiveness of this subclass of macrolides is attributed to mechanisms independent of antibacterial activity. One possible mechanism involves inhibiting neutrophil accumulation in airways by modulating cytokine production—especially IL-8, which is a major neutrophil chemoattractant released from airway epithelial cells or inflammatory cells (14, 15). Interestingly, recent reports have shown that 14-membered ring macrolides directly induce neutrophil apoptosis (16, 17), although conflicting reports also exist (18, 19). Thus, the modulation of the intrapulmonary kinetics of neutrophils by macrolides seems to be involved in their effectiveness in treating chronic airway diseases. However, the precise mechanisms of this favorable effect for the control of disease activity have yet to be fully elucidated.

In addition to persistent neutrophil accumulation, prolongation of neutrophil survival by airway epithelial cells through the release of inflammatory mediators may also be one of the factors inducing dense neutrophil infiltration in airways, a characteristic of chronic airway diseases. Evidence of the clinical effectiveness of macrolide treatment for DPB implies that this family of drugs may also modulate neutrophil survival. In this study, we test whether macrolides directly or indirectly reduce neutrophil survival and, through this modulation of neutrophil survival, facilitate the resolution of chronic airway inflammation by examining the effects of several subclasses of macrolides, which influence neutrophil longevity, on both neutrophil survival and epithelial cell products.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Erythromycin, dexamethasone, ampicillin, and gentamycin were purchased from Sigma Chemical Co. (St. Louis, MO). Clarithromycin, azithromycin, and josamycin were generously donated by Abbott Japan Co. (Osaka, Japan), Pfizer Pharmaceutical Co. (Tokyo, Japan), and Yamanouchi Pharmaceutical Co. (Osaka, Japan), respectively. These drugs were dissolved in dimethyl sulfoxide and subsequently diluted to an appropriate concentration in medium. Recombinant human (rh) tumor necrosis factor (TNF)-{alpha} was purchased from PeproTech EC (London, UK). Neutralizing antibodies to rhGM-CSF, rhG-CSF, rhIL-6, and rhIL-8 were purchased from R&D Systems (Minneapolis, MN). All culture media and additives, unless specified otherwise, were purchased from BioWhittaker (Walkersville, MD).

Neutrophil Isolation
Peripheral blood was collected from healthy human donors. Neutrophils were isolated by dextran sedimentation and centrifugation on a Ficoll-Paque gradient (Amersham Biosciences, Uppsala, Sweden). The neutrophil layer was removed, washed in phosphate-buffered saline (PBS), and centrifuged. Contaminating erythrocytes were lysed with 0.9% buffered ammonium chloride. After washing in PBS, cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS). All solutions used for isolation and culturing of neutrophils were confirmed to be negative for endotoxin (< 5 pg/ml) by a Limulus lysate test (Seikagaku, Tokyo, Japan). Isolated neutrophils were consistently > 98% pure by modified Wright-Giemsa (Diff-Quick; International Reagents Co., Kobe, Japan), and > 98% viable as determined by trypan blue dye exclusion. Suspensions of freshly isolated neutrophils that showed signs of cellular activation (> 5% of neutrophils in clumps of two or more cells) were discarded.

Airway Epithelial Cell Culture and Generation of Conditioned Media
The human pulmonary type II-like alveolar epithelial cell line, A549, was purchased from the American Type Culture Collection (Rockville, MD). The cells were maintained in RPMI 1640 medium supplemented with 10% FCS at 37°C in a 5% CO2 incubator. To generate epithelial cell conditioned media, A549 cells were grown in 12-well culture plates (Corning Inc., Corning, NY) until confluent. Cells were then stimulated with TNF-{alpha} (10 ng/ml) or left untreated. After 3 h of incubation, the cells were washed twice with PBS and media were replaced with fresh culture media supplemented with 10% FCS to avoid direct effects of TNF-{alpha} on neutrophil survival. In some experiments, A549 cells were preincubated with various concentrations of dexamethasone, macrolides, or other antibiotics for 24 h before the 3-h TNF-{alpha} stimulation, then processed following the same procedure as above. Supernatants were harvested after an additional 24 h of incubation and stored at -80°C until being used for cell survival assays or analysis by means of enzyme-linked immunosorbent assay (ELISA).

Neutrophil Survival Studies
Purified neutrophils (2 x 106/ml) were incubated in 48-well plates (Corning Inc.) with conditioned media from resting or TNF-{alpha}–stimulated A549 cells (with or without pretreatment by dexamethasone, macrolides, or other antibiotics) that were obtained as described above. Control experiments with cells in culture media supplemented with 10% FCS were assayed in parallel. In some experiments, neutrophils were incubated with culture media or conditioned media from TNF-{alpha}–stimulated A549 cells in the presence or absence of macrolides or dexamethasone to examine the direct effect of the drugs on neutrophil survival. For studies involving neutralizing antibodies, antibodies to GM-CSF (1 µg/ml), G-CSF (1 µg/ml), IL-6 (1 µg/ml), or IL-8 (100 µg/ml) were added to conditioned media from TNF-{alpha}–stimulated A549 cells. The optimal antibody concentrations were determined by preliminary studies of neutralizing the survival activity of purified cytokine. In all experiments, neutrophil survival was assessed after 24 h of culture using an Annexin V apoptosis detection kit (BD PharMingen, San Diego, CA), according to the manufacturer's instructions. Briefly, cells were washed twice with PBS, incubated for 15 min in the dark at room temperature with annexin V–fluorescein isothiocyanate and propidium iodode, and analyzed with an FACScan flow cytometer (Becton Dickinson, San Jose, CA). Neutrophil survival is expressed as the percentage of live, non-apoptotic neutrophils (annexin V–negative, propidium iodode–negative). Additionally, in some experiments, neutrophil survival was assessed based on morphologic criteria for apoptosis. Aliquots of neutrophil suspensions were cytospun onto glass slides. Slides were stained with modified Wright–Giemsa and examined by light microscopy. At least 200 cells were graded for apoptosis using predetermined morphologic characteristics following Lee and colleagues (5).

ELISA Measurements
GM-CSF concentrations in conditioned media were measured using commercially available ELISA kits (R&D Systems). The ELISA kits were used as indicated by the manufacturer and consistently detected GM-CSF concentrations > 3 pg/ml in linear manner.

Reverse Transcription-Polymerase Chain Reaction
A549 cells, pretreated with or without macrolides (10 µg/ml each) or dexamethasone (1 µM) for 24 h, were stimulated with TNF-{alpha} (10 ng/ml). After 3 h of incubation, culture media were discarded and total RNA was isolated using TRIzol reagent (GibcoBRL, Gaithersburg, MD) according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using RNA PCR kits (Takara, Shiga, Japan) according to the manufacturer's instructions. Briefly, RT of total RNA into complementary DNA (cDNA) was performed at 55°C for 30 min. cDNA was synthesized in 20-µl reaction mixtures containing 1 µg of total RNA, 0.125 µM of oligo dT primer, 1 mM of each deoxynucleotide triphosphate, 20 U of RNase inhibitor, 5 U of reverse transcriptase, and the enzyme buffer supplied by the manufacturer. The cDNA mixture was subjected to PCR amplification in 100-µl reaction mixtures containing 2.5 U of Taq polymerase, 0.2 mM of each deoxynucleotide triphosphate, 0.2 µM of both antisense and sense primers, and the enzyme buffer. The following primers were designed based on the cDNA sequences: GM-CSF, 5'-GACACTGCTGCTGAGATGAA-3' (sense) and 5'-AGGGGATGACAAGCAGAAAG-3' (antisense); and ß-actin, 5'-GGGACCTGACTGACTACC-3' (sense) and 5'-CTCGTCATACTCCTGCTTGC-3' (antisense). The expected sizes of the PCR products were 267 bp for GM-CSF, and 547 bp for ß-actin. The cDNA was denatured for 2 min at 94°C followed by 27 cycles of amplification. Each cycle consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and primer extension at 72°C for 2 min. Amplification was stopped in the linear phase of the PCR to quantify the amount of messenger RNA (mRNA) in each sample. After amplification, PCR products were separated by electrophoresis on 3% agarose gels. Densitometric analysis was performed using Luminous Imager software (Aisin Cosmos R&D Co., Aichi, Japan).

Statistical Analysis
Data are expressed as mean ± SEM. Data were analyzed for statistical difference by paired t test (Statview, SAS Institute, Cary, NC). Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Macrolides Fail to Directly Modulate Neutrophil Survival
The following macrolides were tested in this study: erythromycin and clarithromycin, 14-membered ring macrolides; azithromycin, a 15-membered ring macrolide; and josamycin, a 16-membered ring macrolide. We first examined the direct effects of these macrolides or dexamethasone on neutrophil survival. Neutrophils incubated with culture media in the absence of drugs rapidly underwent spontaneous apoptosis (38.2 ± 4.5% of cells remained viable). The inhibitory effect of glucocorticoids on spontaneous neutrophil apoptosis has been described previously (20). Consistent with this finding, dexamethasone (1 µM) showed a potent effect on the prolongation of neutrophil survival (72.0 ± 4.2%). In contrast, all of the macrolides tested (10 µg/ml each) failed to modulate neutrophil survival. On the other hand, neutrophils incubated with conditioned media from resting A549 cells had only modest prolongation of neutrophil survival (52.6 ± 6.7%), whereas conditioned media from TNF-{alpha}–stimulated A549 cells induced markedly prolonged survival (69.4 ± 5.1%) compared with culture media alone. The presence of dexamethasone (1 µM) during neutrophil culture with conditioned media from TNF-{alpha}–stimulated A549 cells led to additional prolongation of neutrophil survival (83.8 ± 4.8%). None of the macrolides tested (10 µg/ml each), however, modulated neutrophil survival in media conditioned with TNF-{alpha}–stimulated A549 cells (Figure 1). Additionally, increasing the concentration of each macrolide, up to 100 µg/ml, failed to produce significant effects (data not shown). We also assessed neutrophil survival using morphologic criteria of apoptosis; the results showed the lack of direct effects of macrolides on neutrophil survival, in agreement with results obtained by annexin V binding (data not shown).



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  		Figure 1. Macrolides fail to directly modulate neutrophil survival. Neutrophils were incubated with culture media or media conditioned with TNF-{alpha}–stimulated A549 cells in the presence or absence of erythromycin (EM, 10 µg/ml), clarithromycin (CAM, 10 µg/ml), azithromycin (AZM, 10 µg/ml), josamycin (JM, 10 µg/ml), or dexameyhasone (DEX, 1 µM). Conditioned media from resting or TNF-{alpha}–stimulated A549 cells was generated as described in MATERIALS AND METHODS. Neutrophil survival was assessed using annexin V–binding with flow cytometry after 24 h of culture. Data are expressed as mean ± SEM of four experiments. *P < 0.05, **P < 0.01 compared with culture media alone; #P < 0.05 compared with TNF-{alpha}–stimulated A549 conditioned media alone. Conditioned media, CM.

 
Macrolides Decrease Epithelial Cell–Mediated Prolongation of Neutrophil Survival
We next examined the indirect effects of the drugs on neutrophil survival following the suppression of survival-enhancing factor production. The pretreatment of TNF-{alpha}–stimulated A549 cells by erythromycin, clarithromycin, and azithromycin inhibited prolongation of neutrophil survival in a dose-dependent manner. The inhibitory effects of each drug became significant at concentrations greater than 10 µg/ml compared with survival in conditioned media from cells without the pretreatment (Figures 2A, 2B, and 2C). Similar, but more potent, effects were observed for conditioned media from A549 cells pretreated with dexamethasone (Figure 2E). In contrast, no significant differences in neutrophil survival were observed in conditioned media from A549 cells pretreated with josamycin or in conditioned media from A549 cells without pretreatment (Figure 2D). Similarly, neither ampicillin nor gentamycin inhibited epithelial cell-mediated neutrophil survival (data not shown).



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  		Figure 2. Macrolides decrease epithelial cell–mediated prolongation of neutrophil survival. Conditioned media were generated from A549 cells pretreated with or without various concentrations of: (A) EM, (B) CAM, (C) AZM, (D) JM, or (E) DEX for 24 h prior to 3 h of TNF-{alpha} stimulation as described in MATERIALS AND METHODS. Neutrophils were incubated with conditioned media from each condition and the survival was assessed using annexin V–binding with flow cytometry after 24 h of culture. Data are expressed as mean ± SEM of four experiments. *P < 0.05, **P < 0.01 compared with TNF-{alpha}–stimulated A549–conditioned media without pretreatment.

 
Anti–GM-CSF Antibodies Inhibit Epithelial Cell–Mediated Prolongation of Neutrophil Survival
We confirmed that pretreatment with dexamethasone and some subclasses of macrolides abated the epithelial cell–mediated prolongation of neutrophil survival observed in TNF-{alpha}–stimulated A549 cell–conditioned media, and hypothesized that this was accomplished through the modulation of soluble survival-enhancing factor production in TNF-{alpha}–stimulated A549 cells. In vitro studies of neutrophil survival have identified numerous factors, including GM-CSF, G-CSF, IL-6, and IL-8, that prolong neutrophil survival (4–8). Therefore, we determined the relative contribution of these cytokines to neutrophil survival in our experimental system using neutralizing antibodies. Treatment with anti–GM-CSF antibodies significantly decreased neutrophil survival prolongation by TNF-{alpha}–stimulated A549 cell–conditioned media (from 73.2 ± 4.0% viable cells for conditioned media from TNF-{alpha}–stimulated A549 cells to 54.5 ± 4.4% for the same conditioned media with the addition of anti–GM-CSF antibody). This finding suggests a pivotal role for this cytokine in enhancing neutrophil survival in this system. In contrast, none of the other antibodies tested, including anti–G-CSF, anti–IL-6, and anti–IL-8, had inhibitory effects on epithelial cell-mediated prolongation of neutrophil survival (Figure 3).



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  		Figure 3. Inhibition of prolonged neutrophil survival by neutralizing anti–GM-CSF antibodies (Ab). Conditioned media from TNF-{alpha}–stimulated A549 cells were supplemented with neutralizing antibodies to GM-CSF (1 µg/ml), G-CSF (1 µg/ml), IL-6 (1 µg/ml), or IL-8 (100 µg/ml) and the survival was assessed using annexin V–binding with flow cytometry after 24 h of culture. Data are expressed as mean ± SEM of four experiments. *P < 0.05 compared with TNF-{alpha}–stimulated A549–conditioned media without anti–GM-CSF Ab.

 
Macrolides Inhibit GM-CSF Release from TNF-{alpha}–Stimulated A549 Cells
We next measured the concentration of GM-CSF by ELISA in A549 cell–conditioned media generated by various culture conditions. Conditioned media from resting A549 cells contained very little if any GM-CSF (below the detection limit of the ELISA used), whereas TNF-{alpha} stimulation induced significant production of GM-CSF by A549 cells. The pretreatment of TNF-{alpha}–stimulated A549 cells by drugs inhibitory to epithelial cell–mediated neutrophil survival—erythromycin, clarithromycin, or azithromycin—showed dose-dependent decreases in the concentration of GM-CSF in each conditioned media (Figures 4A, 4B, and 4C). A significant inhibitory effect was detected at concentrations greater than 10 µg/ml for each drug. Pretreatment by dexamethasone, even at the lowest concentration tested (0.01 µM), significantly inhibited GM-CSF production, and at concentrations greater than 1 µM, the level of GM-CSF production dropped below the detection limit (Figure 4E). In contrast, only the highest concentration of josamycin (100 µg/ml) showed a slightly inhibitory effect (Figure 4D). Neither ampicillin nor gentamycin showed significant effects on GM-CSF production (data not shown).



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  		Figure 4. Release of GM-CSF by TNF-{alpha}–stimulated A549 cells and its inhibition by macrolides. A549 cells were pretreated with or without various concentrations of: (A) EM, (B) CAM, (C) AZM, (D) JM, or (E) DEX for 24 h prior to 3-h TNF-{alpha} stimulation and then media were replaced with fresh media. Supernatants were harvested after an additional 24 h of incubation. GM-CSF concentration in supernatants was measured by ELISA. Data are expressed as mean ± SEM of four experiments. *P < 0.05, **P < 0.01 compared with TNF-{alpha}–stimulated A549–conditioned media without pretreatment. Not detected, ND.

 
Macrolides Inhibit TNF-{alpha}–Induced GM-CSF mRNA Expression in A549 Cells
Lastly, we examined the effects of the macrolides or dexamethasone on the expression of GM-CSF mRNA in A549 cells by RT-PCR. GM-CSF mRNA expression was not detected in resting A549 cells, but was detected in TNF-{alpha}–stimulated A549 cells. This induction of GM-CSF mRNA was markedly inhibited by pretreatment with dexamethasone (1 µM), as was the production of GM-CSF protein. The pretreatment by erythromycin, clarithromycin, and azithromycin (10 µg/ml each) also showed modest inhibitory effects on the expression of GM-CSF mRNA. However, josamycin (10 µg/ml) failed to show an inhibitory effect, similar to the lack of effect on GM-CSF protein production (Figures 5A and 5B).



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  		Figure 5. Macrolides inhibit TNF-{alpha} induced GM-CSF mRNA expression in A549 cells. A549 cells were pretreated with or without EM (10 µg/ml), CAM (10 µg/ml), AZM (10 µg/ml), JM (10 µg/ml), or DEX (1 µM) for 24 h prior to 3 h of TNF-{alpha} stimulation, and then total RNA was isolated. GM-CSF mRNA expression was determined by using RT-PCR. (A) Analysis of RT-PCR products by 3% agarose gel electrophoresis. Data are representative of four separate experiments. (B) Relative levels of GM-CSF mRNA expression. The level of GM-CSF mRNA is normalized to each ß-actin internal control, and then presented as a percentage value of the TNF-{alpha}–stimulated A549 cells without pretreatment. Data are expressed as mean ± SEM of four separate experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has demonstrated that TNF-{alpha}–stimulated airway epithelial cells produce the factors that prolong neutrophil survival and that GM-CSF is the main contributing factor for this effect on survival. We also have demonstrated that 14- and 15-membered, but not 16-membered ring macrolides indirectly decrease neutrophil survival through inhibition of GM-CSF release from activated airway epithelial cells without exerting direct modulating effects.

The direct effect of macrolides on the modulation of neutrophil survival is still inconsistent. Previous studies reported that 14-membered ring macrolides induced neutrophil apoptosis (16, 17). On the other hand, one recent study by Tsuchihashi and colleagues demonstrated the lack of the modulating effects of this subclass of macrolides on both spontaneous neutrophil apoptosis and apoptosis of LPS-stimulated neutrophils (19). Consistent with the latter report, the present study found that erythromycin and clarithromycin failed to alter the rate of spontaneous apoptosis and directly modulate prolongation of neutrophil survival through activated epithelial cell–derived conditioned media. Percentages of apoptotic neutrophils were assessed only morphologically by electron microscope (16) or light microscope (17) examination in the previous studies. In contrast, our study and the study by Tsuchihashi and colleagues (19) utilized annexin-V labeling, which reacts with phosphatidylserine on the outer leaflet of the cell membrane to assess neutrophil apoptosis. Thus, the differences in the methods used for the assessment of neutrophil apoptosis may explain the discrepancy between the observed effects. We also showed the lack of modulating effects of a 15-membered ring macrolide, azithromycin, which is inconsistent with the recent study by Koch and colleagues (18). Different incubation times of neutrophils may account for the inconsistency in observed proapoptotic properties of azithromycin; neutrophil apoptosis was assessed after only 1 h of incubation in the Koch and colleagues study (18) compared with after 24 h in our study. Although further detailed time-course studies may be needed to resolve this issue, our study suggests that macrolides do not affect the rate of phosphatidylserine externalization of neutrophils, at least after the longer (24 h) incubations used in our study.

GM-CSF is shown to potently enhance neutrophil survival in vitro. Airway epithelial cells produce multiple proinflammatory mediators including GM-CSF and are involved in the prolongation of neutrophil survival in the airways (9, 10). In the present study, in contrast to the lack of the direct effects on neutrophil survival, erythromycin, clarithromycin, and azithromycin, but not josamycin–inhibited epithelial cell–mediated prolongation of neutrophil survival. Neutralizing anti–GM-CSF antibody decreased this neutrophil survival prolongation, suggesting a pivotal role for this cytokine in enhancing neutrophil survival in our experimental models. Furthermore, we demonstrated that all the tested macrolides, except josamycin, dose-dependently inhibited GM-CSF production by TNF-{alpha}–stimulated A549 cells. The inhibitory effects of the drugs were also observed at mRNA levels of GM-CSF. These lines of evidence clearly prove that 14- and 15-membered ring macrolides inhibit airway epithelial cell–mediated neutrophil survival through the modulation of GM-CSF release. The concentrations of the drugs that exerted these effects were slightly higher than the clinically achievable level in plasma (21, 22). However, macrolides, including erythromycin, clarithromycin, and azithromycin, were shown to achieve much higher concentrations in the intrapulmonary regions (21, 22). Thus, it is possible to say that these effects of macrolides occur in vitro at clinically relevant concentrations.

Josamycin showed an inhibitory effect on GM-CSF release at the highest concentration (100 µg/ml) tested, but the decrease in epithelial cell–mediated neutrophil survival was not statistically significant. In view of our conclusion that neutrophil survival is reduced by macrolide inhibition of GM-CSF release, this negative result is contradictory. However, the modest nature of the inhibition on GM-CSF release by josamycin may be responsible for this statically negative result. Therefore, further studies are necessary to elucidate whether the finding in this study was anomalous. Although not tested in the present study, much higher concentrations of josamycin (> 100 µg/ml) may have an inhibitory effect similar to that of other macrolides on epithelial cell–mediated neutrophil survival through a more pronounced inhibition of GM-CSF release. Even if this speculation is correct, however, the clinical significance of this phenomenon should be interpreted with caution as our preliminary data show that josamycin affects the proliferation and viability of A549 cells at high concentrations, indicating a possible cytotoxic effect of this drug.

The inhibitory effects of dexamethasone on airway epithelial cell–mediated neutrophil survival were more potent than those of macrolides, likely due to its potency for suppressing GM-CSF protein release and mRNA expression by TNF-{alpha}–stimulated A549 cells. However, in contrast to macrolides, which failed to show significant direct effects on neutrophil survival, dexamethasone directly prolonged neutrophil survival. It is speculated that neutrophil survival at sites of inflammation is altered by balance between such direct and indirect effects of the drugs. In this study, we demonstrated that neutrophil survival incubated with culture media in the presence of dexamethasone was almost similar to that prolonged by conditioned media from TNF-{alpha}–stimulated A549 cells (Figure 1). This finding implies that the direct survival-enhancing effect of dexamethasone is potent, and under some conditions, the net effect of this drug may even favor neutrophil survival. Our findings in vitro therefore suggest that dexamethasone treatment could provoke the prolongation of neutrophil survival, leading to an increase in neutrophil numbers at sites of inflammation in the airways. In fact, some studies have documented unchanged or even increased numbers of neutrophils in the airways of patients with asthma, who are undergoing anti-asthma therapy with long-term inhaled glucocorticoid (23–25).

Although the precise modes of action remain unclear, clinical findings suggest that the effectiveness of macrolides for chronic airway diseases depends on the mechanisms independent of their antibacterial activities. Previous studies have demonstrated anti-inflammatory action of macrolides: inhibition of neutrophil adhesion to epithelial cells (26), inhibition of intraepithelial mucus production (27, 28), inhibition of the production of proinflammatory cytokines by a variety of inflammatory cells (29–32), and inhibition of IL-8 release by bronchial epithelial cells and other inflammatory cells (14, 15). These effects have been considered to contribute to the clinical effectiveness of this family of drugs in treating chronic airway diseases. Knowledge concerning the molecular mechanism of the anti-inflammatory activities of macrolides has also been accumulating. Recent studies have demonstrated that erythromycin or clarithromycin suppresses the IL-8 promoter through transcription factors, including activator protein–1 and nuclear factor–{kappa}B in bronchial epithelial cells or monocytes (33–35). On the other hand, it is well established that glucocorticoids are powerful anti-inflammatory agents that suppress many phlogistic cellular responses. In fact, glucocorticoids are shown to have some of the above-mentioned activities to a similar or even greater degree than macrolides (27, 28, 31). Nevertheless, in contrast to macrolides, no report has described the clinical effectiveness of glucocorticoids for the treatment of chronic airway diseases. What is the reason for the ineffectiveness of this drug despite its powerful anti-inflammatory action? One possible explanation is the impairment of pulmonary defense against bacterial infections by long-term glucocorticoid treatment. Furthermore, in addition to this immunologically deleterious effect, neutrophil survival prolongation and subsequent failure to reduce neutrophil numbers may be one of mechanisms responsible for the ineffectiveness of glucocorticoids.

Previous studies demonstrated a significant reduction in bronchoalveolar lavage fluid neutrophil percentages in patients with DPB after treatment with erythromycin (14, 36). As the number of neutrophils in tissue reflects a balance between rates of influx and removal, the modulation of this balance by macrolides may contribute to their effectiveness. Down-regulation of IL-8 mRNA expression and protein release by 14-membered ring macrolides are assumed to be the possible mechanisms of the inhibition of neutrophil influx into airways (15, 19). In addition, one recent study reported the upregulation of alveolar macrophage phagocytic ability of apoptotic neutrophils by 14- or 15-membered ring macrolides (37). Thus, macrolides may help resolve chronic airway inflammation by inhibiting the influx of and promoting the removal of neutrophils. Furthermore, a process of apoptosis is essential for the safe clearance of activated neutrophils by macrophages. This implies that the life span of neutrophils is also one of the important factors regulating neutrophil number at sites of inflammation. There have been no published studies in regard to the life span of neutrophils in airways in patients with DPB so far. However, a recent study has demonstrated the strong expression of GM-CSF protein by bronchiolar epithelial cells (38), suggesting the possibility of neutrophil survival prolongation through this cytokine in patients with DPB.

In conclusion, we demonstrated evidence of the interaction between neutrophils and airway epithelial cells in neutrophil survival prolongation. This suggests the usefulness of the modulating neutrophil survival-enhancing factor release for controlling neutrophil longevity and, subsequently, airway inflammation. More importantly, 14- and 15-membered ring macrolides inhibited the release of GM-CSF by activated airway epithelial cells and provided indirect reduction in neutrophil survival time. The inhibition of epithelial cell-mediated neutrophil survival through this mechanism may, at least in part, explain the effectiveness of these macrolides on chronic airway diseases. A limitation of the present study is that A549 cells may not actually represent normal respiratory epithelium. Thus, future studies utilizing primary cultures of human airway epithelial cells or in vivo models will be important for determining the in vivo relevance of our findings.


   Acknowledgments
 
The authors thank Ms. Tomoko Ikahata for her excellent technical assistance.

Received in original form March 24, 2003

Received in final form September 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Whyte, M. K., L. C. Meagher, J. MacDermot, and C. Haslett. 1993. Impairment of function in aging neutrophils is associated with apoptosis. J. Immunol. 150:5124–5134.[Abstract]
  2. Savill, J. S., A. H. Wyllie, J. E. Henson, M. J. Walport, P. M. Henson, and C. Haslett. 1989. Macrophage phagocytosis of aging neutrophils in inflammation: programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83:865–875.
  3. Hall, S. E., J. S. Savill, P. M. Henson, and C. Haslett. 1994. Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose–specific lectin. J. Immunol. 153:3218–3227.[Abstract]
  4. Lopez, A. F., D. J. Williamson, J. R. Gamble, C. G. Begley, J. M. Harlan, S. J. Klebanoff, A. Waltersdorph, G. Wong, S. C. Clark, and M.A. Vadas. 1986. Recombinant human granulocyte-macrophage colony–stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J. Clin. Invest. 78:1220–1228.
  5. Lee, A., M. K. Whyte, and C. Haslett. 1993. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J. Leukoc. Biol. 54:283–288.[Abstract]
  6. Colotta, F., F. Re, N. Polentarutti, S. Sozzani, and A. Mantovani. 1992. Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80:2012–2020.[Abstract/Free Full Text]
  7. Biffl, W. L., E. E. Moore, F. A. Moore, and C. C. Barnett, Jr. 1995. Interleukin-6 suppression of neutrophil apoptosis is neutrophil concentration dependent. J. Leukoc. Biol. 58:582–584.[Abstract]
  8. Kettritz, R., M. L. Gaido, H. Haller, F. C. Luft, C. J. Jennette, and R. J. Falk. 1998. Interleukin-8 delays spontaneous and tumor necrosis factor-alpha-mediated apoptosis of human neutrophils. Kidney Int. 53:84–91.[CrossRef][Medline]
  9. Daffern, P. J., M. A. Jagels, and T. E. Hugli. 1999. Multiple epithelial cell-derived factors enhance neutrophil survival: regulation by glucocorticoids and tumor necrosis factor-alpha. Am. J. Respir. Cell Mol. Biol. 21:259–267.[Abstract/Free Full Text]
  10. Wislez, M., J. Fleury-Feith, N. Rabbe, J. Moreau, D. Cesari, B. Milleron, C. Mayaud, M. Antoine, P. Soler, and J. Cadranel. 2001. Tumor-derived granulocyte-macrophage colony–stimulating factor and granulocyte colony–stimulating factor prolong the survival of neutrophils infiltrating bronchoalveolar subtype pulmonary adenocarcinoma. Am. J. Pathol. 159:1423–1433.[Abstract/Free Full Text]
  11. Nagai, H., H. Shishido, R. Yoneda, E. Yamaguchi, A. Tamura, and A. Kurashima. 1991. Long-term low-dose administration of erythromycin to patients with diffuse panbronchiolitis. Respiration (Herrlisheim) 58:145–149.
  12. Kudoh, S., A. Azuma, M. Yamamoto, T. Izumi, and M. Ando. 1998. Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. Am. J. Respir. Crit. Care Med. 157:1829–1832.
  13. Shirai, T., A. Sato, and K. Chida. 1995. Effect of 14-membered ring macrolide therapy on chronic respiratory tract infections and polymorphonuclear leukocyte activity. Intern. Med. 34:469–474.[Medline]
  14. Kadota, J., O. Sakito, S. Kohno, H. Sawa, H. Mukae, H. Oda, K. Kawakami, K. Fukushima, K. Hiratani, and K. Hara. 1993. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am. Rev. Respir. Dis. 147:153–159.[Medline]
  15. Takizawa, H., M. Desaki, T. Ohtoshi, S. Kawasaki, T. Kohyama, M. Sato, M. Tanaka, T. Kasama, K. Kobayashi, J. Nakajima, and K. Ito. 1997. Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells. Am. J. Respir. Crit. Care Med. 156:266–271.[Abstract/Free Full Text]
  16. Aoshiba, K., A. Nagai, and K. Konno. 1995. Erythromycin shortens neutrophil survival by accelerating apoptosis. Antimicrob. Agents Chemother. 39:872–877.[Abstract]
  17. Inamura, K., N. Ohta, S. Fukase, N. Kasajima, and M. Aoyagi. 2000. The effects of erythromycin on human peripheral neutrophil apoptosis. Rhinology 38:124–129.[Medline]
  18. Koch, C. C., D. J. Esteban, A. C. Chin, M. E. Olson, R. R. Read, H. Ceri, D. W. Morck, and A. G. Buret. 2000. Apoptosis, oxidative metabolism and interleukin-8 production in human neutrophils exposed to azithromycin: effects of Streptococcus pneumoniae. J. Antimicrob. Chemother. 46:19–26.[Abstract/Free Full Text]
  19. Tsuchihashi, Y., K. Oishi, H. Yoshimine, S. Suzuki, A. Kumatori, T. Sunazuka, S. Omura, K. Matsushima, and T. Nagatake. 2002. Fourteen-member macrolides suppress interleukin-8 production but do not promote apoptosis of activated neutrophils. Antimicrob. Agents Chemother. 46:1101–1104.[Abstract/Free Full Text]
  20. Liles, W. C., D. C. Dale, and S. J. Klebanoff. 1995. Glucocorticoids inhibit apoptosis of human neutrophils. Blood 86:3181–3188.[Abstract/Free Full Text]
  21. Conte, J. E., Jr., J. A. Golden, S. Duncan, E. McKenna, and E. Zurlinden. 1995. Intrapulmonary pharmacokinetics of clarithromycin and of erythromycin. Antimicrob. Agents Chemother. 39:334–338.[Abstract/Free Full Text]
  22. Rodvold, K. A., M. H. Gotfried, L. H. Danziger, and R. J. Servi. 1997. Intrapulmonary steady-state concentrations of clarithromycin and azithromycin in healthy adult volunteers. Antimicrob. Agents Chemother. 41:1399–1402.[Abstract]
  23. Hoshino, M., and Y. Nakamura. 1996. Anti-inflammatory effects of inhaled beclomethasone dipropionate in nonatopic asthmatics. Eur. Respir. J. 9:696–702.[Abstract]
  24. Pedersen, B., R. Dahl, R. Karlstrom, C. G. Peterson, and P. Venge. 1996. Eosinophil and neutrophil activity in asthma in a one-year trial with inhaled budesonide: the impact of smoking. Am. J. Respir. Crit. Care Med. 153:1519–1529.[Abstract]
  25. Tanizaki, Y., H. Kitani, M. Okazaki, T. Mifune, F. Mitsunobu, and I. Kimura. 1993. Effects of long-term glucocorticoid therapy on bronchoalveolar cells in adult patients with bronchial asthma. J. Asthma 30:309–318.[Medline]
  26. Kawasaki, S., H. Takizawa, T. Ohtoshi, N. Takeuchi, T. Kohyama, H. Nakamura, T. Kasama, K. Kobayashi, K. Nakahara, Y. Morita, and K. Yamamoto. 1998. Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro. Antimicrob. Agents Chemother. 42:1499–1502.[Abstract/Free Full Text]
  27. Takahashi, Y., T. Shimizu, and Y. Sakakura. 1997. Effects of indomethacin, dexamethasone, and erythromycin on endotoxin-induced intraepithelial mucus production of rat nasal epithelium. Ann. Otol. Rhinol. Laryngol. 106:683–687.[Medline]
  28. Goswami, S. K., S. Kivity, and Z. Marom. 1990. Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro. Am. Rev. Respir. Dis. 141:72–78.[Medline]
  29. Khan, A. A., T. R. Slifer, F. G. Araujo, and J. S. Remington. 1999. Effect of clarithromycin and azithromycin on production of cytokines by human monocytes. Int. J. Antimicrob. Agents 11:121–132.[CrossRef][Medline]
  30. Schultz, M. J., P. Speelman, S. Zaat, S. J. van Deventer, and T. van der Poll. 1998. Erythromycin inhibits tumor necrosis factor alpha and interleukin 6 production induced by heat-killed Streptococcus pneumoniae in whole blood. Antimicrob. Agents Chemother. 42:1605–1609.[Abstract/Free Full Text]
  31. Morikawa, K., H. Watabe, M. Araake, and S. Morikawa. 1996. Modulatory effect of antibiotics on cytokine production by human monocytes in vitro. Antimicrob. Agents Chemother. 40:1366–1370.[Abstract]
  32. Sugiyama, Y., K. Yanagisawa, S. I. Tominaga, and S. Kitamura. 1999. Effects of long-term administration of erythromycin on cytokine production in rat alveolar macrophages. Eur. Respir. J. 14:1113–1116.[Abstract]
  33. Abe, S., H. Nakamura, S. Inoue, H. Takeda, H. Saito, S. Kato, N. Mukaida, K. Matsushima, and H. Tomoike. 2000. Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding site in human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 22:51–60.[Abstract/Free Full Text]
  34. Desaki, M., H. Takizawa, T. Ohtoshi, T. Kasama, K. Kobayashi, T. Sunazuka, S. Omura, K. Yamamoto, and K. Ito. 2000. Erythromycin suppresses nuclear factor-kappaB and activator protein-1 activation in human bronchial epithelial cells. Biochem. Biophys. Res. Commun. 267:124–128.[CrossRef][Medline]
  35. Kikuchi, T., K. Hagiwara, Y. Honda, K. Gomi, T. Kobayashi, H. Takahashi, Y. Tokue, A. Watanabe, and T. Nukiwa. 2002. Clarithromycin suppresses lipopolysaccharide-induced interleukin-8 production by human monocytes through AP-1 and NF-kappa B transcription factors. J. Antimicrob. Chemother. 49:745–755.[Abstract/Free Full Text]
  36. Ichikawa, Y., H. Ninomiya, H. Koga, M. Tanaka, M. Kinoshita, N. Tokunaga, T. Yano, and K. Oizumi. 1992. Erythromycin reduces neutrophils and neutrophil-derived elastolytic-like activity in the lower respiratory tract of bronchiolitis patients. Am. Rev. Respir. Dis. 146:196–203.[Medline]
  37. Yamaryo, T., K. Oishi, H. Yoshimine, Y. Tsuchihashi, K. Matsushima, and T. Nagatake. 2003. Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages. Antimicrob. Agents Chemother. 47:48–53.[Abstract/Free Full Text]
  38. Todate, A., K. Chida, T. Suda, S. Imokawa, J. Sato, K. Ide, T. Tsuchiya, N. Inui, Y. Nakamura, K. Asada, H. Hayakawa, and H. Nakamura. 2000. Increased numbers of dendritic cells in the bronchiolar tissues of diffuse panbronchiolitis. Am. J. Respir. Crit. Care Med. 162:148–153.[Abstract/Free Full Text]



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