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Am. J. Respir. Crit. Care Med., Volume 165, Number 8, April 2002, 1113-1118

Erythromycin Inhibits Rhinovirus Infection in Cultured Human Tracheal Epithelial Cells

Tomoko Suzuki, Mutsuo Yamaya, Kiyohisa Sekizawa, Masayoshi Hosoda, Norihiro Yamada, Satoshi Ishizuka, Akiko Yoshino, Hiroyasu Yasuda, Hidenori Takahashi, Hidekazu Nishimura, and Hidetada Sasaki

Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai; Virus Center, Clinical Research Division, Sendai National Hospital, Sendai; and Department of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To examine the effects of erythromycin on rhinovirus (RV) infection in airway epithelium, primary cultures of human tracheal epithelial cells were infected with the RV major subgroup, RV14, and the minor subgroup, RV2. Infection was confirmed by increases in viral RNA of the infected cells and viral titers of the supernatants. RV14 upregulated the expression of the mRNA and protein of intercellular adhesion molecule-1 (ICAM-1), the major RV receptor, and it increased the cytokine production. Erythromycin reduced the supernatant RV14 titers, RV14 RNA, the susceptibility to RV14 infection, and the production of ICAM-1 and cytokines. Erythromycin also reduced the supernatant RV2 titers, RV2 RNA, the susceptibility to RV2 infection, and cytokine production, although the inhibitory effects of erythromycin on the expression of the low-density lipoprotein receptor, the minor RV receptor, were small. Erythromycin reduced the nuclear factor-kappa B activation by RV14 and decreased the number of acidic endosomes in the epithelial cells. These results suggest that erythromycin inhibits infection by the major RV subgroup by reducing ICAM-1 and infection by both RV subgroups by blocking the RV RNA entry into the endosomes. Erythromycin may also modulate airway inflammation by reducing the production of proinflammatory cytokines and ICAM-1 induced by RV infection.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Keywords: asthma; common cold; ICAM-1; low-density lipoprotein receptor; erythromycin

Prospective studies have shown an association between asthma attacks and infection by various viruses including rhinoviruses (RVs), influenza viruses, and respiratory syncytial viruses (1, 2). Studies using polymerase chain reaction (PCR)- based diagnostics have demonstrated that RVs are responsible for 80% to 85% and 45% of the asthma flares in 9 to 11 year old children and adults, respectively (1, 2).

Macrolide antibiotics inhibit the production of intercellular adhesion molecule-1 (ICAM-1) (3), which plays a vital role in the accumulation of immune effector cells to sites of local inflammation and is also known as a receptor for a major subgroup of RVs such as RV14 (4). Because glucocorticoid-induced reductions in ICAM-1 expression inhibit RV14 infection (5), macrolide antibiotics may also inhibit RV14 infection. However, the effects of erythromycin, a clinically used macrolide antibiotic, on RV infection have not been investigated, although a macrolide antibiotic and H+ATPase inhibitor, bafilomycin (6), inhibits RV infection (7, 8).

Recent reports revealed that RV enters the cytoplasm of infected cells after binding to its receptor, ICAM-1, and a low-density lipoprotein (LDL) receptor (7, 9-12). The entry of RV14 RNA into the cytoplasm of infected cells is suggested to be mediated by the destabilization from receptor binding, by endosomal acidification, or both (12). The entry of a minor subgroup of RVs, RV2, is also mediated by endosomal acidification in HeLa cells (11). However, the effects of erythromycin on the endosomal pH and on the expression of ICAM-1 and LDL receptor have not been elucidated.

Macrolide antibiotics also inhibit cytokine production in the airway epithelial cells (13). RV infection induces the production of cytokines including interleukin (IL)-1 (14, 15) through the activation of nuclear factor-kappa B (NF-kappa B) (16-19). These cytokines have proinflammatory effects (20) and may be related to the pathogenesis of RV infections. Endogenous IL-1beta is related to the ICAM-1 expression after RV infection (15). However, the effects of erythromycin on the cytokine production by RV infection have not been studied.

We therefore examined the effects of erythromycin on the production of ICAM-1, LDL receptor, and cytokines and on the endosomal pH to clarify the mechanisms responsible for the inhibition of RV infection. We also studied its effects on NF-kappa B activation, which modulates the production of ICAM-1 and cytokines (8, 16-19).

    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Viral Stocks

RV2 and RV14 stocks were prepared from patients with common colds by infecting human embryonic fibroblast cells as described (5, 15, 21) and identified with a microneutralization test using an antibody for RV2 or RV14 (22).

Detection and Titration of Viruses

The detection and titration of RVs were performed by observing the cytopathic effects of the viruses on the human embryonic fibroblast cells as previously described (5, 15, 21), and the amount of specimen required to infect 50% of the human embryonic fibroblast cells (50% tissue culture infectious dose [TCID50]) was determined.

RV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) (5, 15) and quantified with real-time quantitative RT-PCR using the Taqman technique (Roche Molecular Diagnostic Systems, Basel, Switzerland) as previously described (23-25), in which forward primer (5'-GCACTTCTGTTTCCCC-3') and reverse primer (5'-CGGACA CCCAAAGTAG-3') were designed for RV2 and RV14, and Taqman probes RV2 (5'-[FAM] CAAAAACAACTGCGATCGT TAACCGCA [TAMRA]-3') and RV14 (5'-[FAM] CGAGGTATAG GCTGTACCC ACTGCCAAAA [TAMRA]-3') were designed for RV2 and RV14, respectively.

Human Tracheal Epithelial Cell Culture and Effects of Erythromycin on Viral Infection

The tracheas used for the cell culture were obtained after death from 73 patients (age 62 ± 4 years; 35 female, 38 male). Human tracheal surface epithelial cells were cultured as described (15, 26). The cells were infected with either RV2 (105 TCID50 U/ml) or RV14 (105 TCID50 U/ml) for 60 minutes and cultured as described (15).

Measurement of ICAM-1 and LDL Receptor Expression

The mRNA and protein of ICAM-1 and LDL receptor were examined with Northern blot analysis and flow cytometry analysis as previously described (8, 15, 17).

Measurement of Cytokine Production

We measured the IL-1beta , IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha ) of the culture supernatants by specific enzyme-linked immunosorbent assays (ELISAs) before and at 1, 3, and 5 days after infection with RV2 or RV14 as previously described (15, 17).

Isolation of Nuclear Extracts and Electrophoretic Mobility Shift Assays

Extraction of nuclei, electrophoretic mobility shift assays, and supershift assays were performed as previously described (8, 16-18).

Measurement of Intracellular pH

Intracellular pH (pHi) was measured as previously described (8, 27, 28). The recovery of pHi after the addition of sodium propionate was expressed as the initial recovery rate of pHi (dpHi/dt).

Measurement of Changes in Acidic Endosome Distribution

The distribution of acidic endosomes in the cells was measured as previously described with a dye, LysoSensor DND-189 (Molecular Probes, Eugene, OR) (8, 29).

Erythromycin Treatment

To examine the effects of erythromycin on RV infection and the cell functions described previously, cells were treated with erythromycin (10 µM) (30) from 3 days before RV infection until the end of the experiments (5), unless described otherwise. To examine the effects of erythromycin on the cells' susceptibility to RV infection, the cells were pretreated for 3 days before RV infection (5, 14).

Statistical Analysis

Results are expressed as means ± SEM. Statistical analysis was performed using two-way repeated measures analysis of variance. Subsequent post hoc analysis was made using Bonferroni's method. For all analyses, values of p < 0.05 were assumed to be significant; n refers to the number of donors from whom cultured cells were used.

    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Erythromycin on RV Infection of Human Tracheal Epithelial Cells

Exposing confluent human tracheal epithelial cell monolayers to RV2 and RV14 (105 TCID50 U/ml) consistently led to infection. To measure the time course of viral release during the first 24 hours, we used four separate cultures. We collected the culture supernatants at 1, 6, 12, or 24 hours after RV infection. No detectable virus was revealed at 1 hour after infection. Both RV2 and RV14 were detected in culture medium 6 hours after infection, and the viral content progressively increased between 6 and 24 hours after infection (Figures 1A and 1B). To examine the effects of erythromycin on the viral titers, the cells were treated with 10 µM of erythromycin (30) or vehicle (ethanol, 0.1%) from 3 days before RV infection until the end of the experiments after RV infection. To measure the viral release from 1-3, 3-5, or 5-7 days, cells in the tubes were rinsed with phosphate buffered saline and fresh medium containing erythromycin or vehicle was added at 24 hours after RV infection. The whole volume of the medium was taken for the measurement of the viral content, and the same volume of fresh medium containing erythromycin or vehicle was replaced at Days 3 and 5, and the whole volume of the medium was taken at Day 7. Evidence of continuous viral production was obtained by demonstrating that the viral titers of supernatants collected during 1-3, 3-5, and 5-7 days after infection each contained significant amounts of RV2 or RV14 (Figures 1C and 1D) (see Figure E1 in the online data supplement). The viral titer levels in the supernatants increased significantly with time during the first 24 hours (p < 0.05 in each case by analysis of variance), then remained constant thereafter. Treatment of the cells with erythromycin significantly decreased the viral titers of RV14 in the supernatants from 12 hours after infection (Figures 1A and 1C). In contrast, erythromycin did not decrease the viral titers of RV2 24 hours after infection, but significant decreases in RV2 titers were observed 3 days after infection (Figures 1B and 1D) (Figure E1). The inhibitory effects of erythromycin on RV14 infection were concentration dependent and the maximum effect was obtained at 10 µM (Figure E2). To examine whether erythromycin is toxic to RV, we incubated RV14 with erythromycin (10 µM) or vehicle for 24 hours at 33° C and washed the RV14 by ultracentrifugation with sucrose gradient (31). The virus titers in the supernatants of the cells infected with RV14 after treatment with erythromycin were not different from those infected with RV14 after treatment with the vehicle alone (see online data supplement).


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  		Figure 1.   Viral titers in supernatants of human tracheal epithelial cells obtained at different times after exposure to 105 TCID50 U/ml of RV14 (A and C ) or RV2 (B and D) in the presence (closed circles) or absence (open circles) of erythromycin (10 µM). Viral titers in supernatants collected sequentially during the first 24 hours after infection (A and B). Viral titers in supernatants collected during 1- 3, 3-5, 5-7 days after infection (C and D). The cells were treated with erythromycin or vehicle (ethanol, 0.1%) from 3 days before RV infection until the end of the experiments after RV infection. Viral content in the supernatant is expressed as TCID50 U/ml. Results represent as means ± SEM from seven samples. Significant differences from viral infection alone are indicated by **p < 0.01.

Cell viability was consistently greater than 96% in the RV-infected culture and the erythromycin-treated culture, and RV14 infection did not alter the amount of LDH in the supernatants measured as previously described (see online data supplement) (15). To examine whether RV infection or erythromycin induced cytotoxic effects on the cultured cells and caused cell detachment (32) from the tubes after the cells made a confluent sheet, we counted the cell numbers after RV infection and after the treatment with erythromycin. RV2 infection, RV14 infection, or erythromycin treatment did not have any effect on the cell numbers (see online data supplement). We infected the epithelial cells with the same titers of RV (105 TCID50 U/ml). Therefore, the infection load was kept constant between the experimental and control samples. We routinely ruled out contamination with Mycoplasma pneumoniae and Chlamydia pneumoniae in the supernatants of the cultured cells using PCR techniques (33, 34). We confirmed the presence of beating cilia on the epithelial cells from the beginning of the cell culture to the end of the experiments as reported previously (5), and a dome formation when the cells made confluent cell sheets on Days 5-7 of culture, as described by Widdicombe and coworkers (35).

Effects of Erythromycin on Viral RNA by PCR

Further evidence of the inhibitory effects of erythromycin on infection by RV2 and RV14 and viral replication in human tracheal epithelial cells was provided by PCR analysis. The RNA extraction was performed at 0, 8, 24, 72, or 120 hours after RV infection. A product band was observable in RNA extracted from cells 8 hours after infection followed by a progressive increase in the intensity of the product band of viral RNA until 3 days after infection. Erythromycin (10 µM) decreased the intensity of the product band of RV14 from 8 hours after infection. In contrast, erythromycin did not decrease the intensity of the product band of RV2 at 8 or 24 hours after infection, but significant decreases in the RV2 product band intensity were observed 3 days after infection (Figure E3).

Effects of Erythromycin on Susceptibility to RV14 Infection

To examine the effects of erythromycin on the susceptibility to infection by RV2 and RV14, the human tracheal epithelial cells were treated with erythromycin (10 µM) from 3 days before infection with RV2 or RV14 until just before infection with RV2 or RV14. The cells were then exposed to serial 10-fold dilutions of RV2 or RV14 for 1 hour at 33° C. The presence of RV in the supernatants collected for 1-3 days after infection was determined with the human embryonic fibroblast cell assay described previously to assess whether infection occurred at each dose of RV used. Treatment of the cells with erythromycin decreased the susceptibility of the cells to infection by RV2 and RV14 (Figure 2) (Figure E4). The minimum dose of RV14 necessary to cause infection in the cells treated with erythromycin was significantly higher than that of RV2 (Figure 2) (Figure E4).


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  		Figure 2.   The susceptibility to infection by RV2 or RV14 in human tracheal epithelial cells after treatment with and without erythromycin (EM, 10 µM). To examine the effects of erythromycin on the susceptibility to RV infection, the cells were pretreated with erythromycin or vehicle from 3 days before infection by RV2 or RV14 until just before infection by RV2 or RV14. The viral release was measured in supernatants collected over 1-3 days after infection by RV2 or RV14. Results are means ± SEM from seven samples.

Effects of Erythromycin on Expression of ICAM-1 and LDL Receptor

To examine the effects of erythromycin on the expression of ICAM-1 and LDL receptor, the human tracheal epithelial cells were treated with erythromycin (10 µM) or vehicle from 3 days before RV infection until the assay of the expression after RV infection. The mRNA was extracted at 0, 8, 24, or 72 hours after RV infection. RV14 infection increased ICAM-1 mRNA with time in the absence of erythromycin. Erythromycin inhibited the increases in ICAM-1 mRNA induced by RV14 infection as well as the baseline ICAM-1 mRNA expression. Likewise, erythromycin inhibited the increases in LDL receptor mRNA induced by RV2 infection and significantly decreased LDL receptor mRNA 3 days after RV2 infection (Figure E5). In contrast to ICAM-1 mRNA, erythromycin did not reduce the baseline LDL receptor mRNA expression.

The expression of ICAM-1 was also assayed by flow cytometric analysis at 3 days after RV14 infection. The epithelial cells 3 days after RV14 infection were shown to increase in ICAM-1-specific fluorescence intensity compared with those 3 days after sham exposure (Figures 3A, 3B, and 3E). Erythromycin decreased the baseline ICAM-1-specific fluorescence intensity (Figures 3C and 3E) and prevented the increases in the intensity that would otherwise be induced by RV14 infection (Figures 3D and 3E) (Figure E6).


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  		Figure 3.   Flow cytometry analysis demonstrating increases in ICAM-1 expression in human tracheal epithelial cells 3 days after RV14 (B) or sham (A) infection and inhibition of ICAM-1 expression by erythromycin 3 days after RV14 (D) or sham (C) infection (EM, 10 µM). (E ) Effects of erythromycin (EM, 10 µM) on ICAM-1 fluorescence intensity in human tracheal epithelial cells 3 days after RV14 or sham infection. Results are means ± SEM from seven samples. Significant differences from sham infection values are indicated by **p < 0.01. Significant differences from RV14 infection alone are indicated by ++p < 0.01. To examine the effects of erythromycin on ICAM-1 protein expression, the human tracheal epithelial cells were treated with erythromycin or vehicle from 3 days before RV infection to 3 days after RV infection.

Effects of Erythromycin on Cytokine Production

To examine the effects of erythromycin on cytokine production after RV infection, the human tracheal epithelial cells were treated with erythromycin (10 µM) or vehicle from 3 days before RV infection until the collection of the supernatants after RV infection. The secretion of IL-beta , IL-6, IL-8, and TNF-alpha all increased in response to both RV2 and RV14. Furthermore, erythromycin inhibited the baseline and RV infection-induced production of all of these cytokines both during the maximal production of each cytokine (Figure 4) and at other times after infection (Figure E7), although the potency of the inhibitory effects of erythromycin was higher for IL-1beta , IL-6, and IL-8 than for TNF-alpha . UV-irradiated RV14 did not increase the maximal production of IL-1beta , IL-6, IL-8, and TNF-alpha (see online data supplement). Of the cytokines measured, interferon-alpha , interferon-beta , or interferon-gamma in the supernatants were under the limit of detection of the assay, and RV14 infection did not alter IL-1alpha production (see online data supplement).


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  		Figure 4.   Effect of erythromycin (closed bars; 10 µM) on the release of cytokines IL-1beta , IL-6, and IL-8, and TNF-alpha into supernatants after RV2 (A) and RV14 (B) infection. Effects of erythromycin were examined during maximal production of each cytokine after RV2 or RV14 infection (1 day after RVs infection in IL-6 and IL-8, and 3 days after RVs infection in IL-1beta and TNF-alpha ). Results represent means ± SEM from seven samples. Significant differences from infection by RV2 or RV14 alone (open bars) are indicated by *p < 0.05 and **p < 0.01.

Effects of Anti-Human IL-1beta on ICAM-1 Expression

Monoclonal mouse anti-human IL-1beta (10 µg/ml) significantly inhibited the ICAM-1 mRNA expression induced by RV14 infection in the human tracheal epithelial cells. In contrast, neither monoclonal mouse anti-human TNF-alpha (10 µg/ml) nor the mouse IgG1 control monoclonal antibody (10 µg/ml) altered the ICAM-1 mRNA expression (see online data supplement).

NF-kappa B DNA Binding Activity in Human Tracheal Epithelial Cells

To examine the effects of erythromycin on NF-kappa B DNA-binding activity, the human tracheal epithelial cells were treated with erythromycin (10 µM) or vehicle from 3 days before RV14 infection until the end of the experiments. NF-kappa B DNA-binding activity was examined before and at 30, 60, and 120 minutes after RV14 infection. The baseline intensity of NF-kappa B-binding activity was constant, and increased activation of NF-kappa B was present in the cells from 30 minutes after RV14 infection. Erythromycin reduced the increased activation of NF-kappa B by RV14 infection as well as the baseline intensity of NF-kappa B binding activity (Figure E8). The specificity of the NF-kappa B binding was confirmed by supershift electrophoretic mobility shift assay, in which antibodies to the p50 or p65 subunit of NF-kappa B ablated the NF-kappa B bands (see online data supplement).

Effects of Erythromycin on pHi

Erythromycin (10 µM), administered from 5 minutes before until the end of the pHi measurement, inhibited the alkalinization of the cells after adding sodium propionate without changes in the baseline pHi. The initial recovery rate after adding sodium propionate to the cells treated with erythromycin was significantly lower than that of cells treated with the vehicle alone (see online data supplement). The inhibitory effect of erythromycin was concentration dependent and the maximum effect was obtained at 10 µM (see online data supplement).

Effects of Erythromycin on the Acidification of Endosomes

The effects of erythromycin on the changes in the distribution of acidic endosomes were examined from 100 seconds before until 300 seconds after the treatment with erythromycin (10 µM) or vehicle. Acidic endosomes in human tracheal epithelial cells were stained green with LysoSensor DND-189. Green fluorescence from acidic endosomes was observed in a granular pattern in the cytoplasm. Erythromycin decreased the number and the fluorescence intensity of acidic endosomes with green fluorescence in the cells with time (Figure E9).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have shown that a macrolide antibiotic, erythromycin, reduced the viral titers in the supernatants and viral RNA of a major subgroup of RVs, RV14, in cultured human tracheal epithelial cells. Pretreatment with erythromycin inhibited the expression of the protein and mRNA of ICAM-1, the receptor for the major RVs (4). Because erythromycin reduced the susceptibility to RV14, as also observed in human tracheal epithelial cells treated with glucocorticoids (5), erythromycin may inhibit RV14 infection at least in part by reducing the production of its receptor, ICAM-1. Furthermore, erythromycin reduced the number of acidic endosomes in the cultured human tracheal epithelial cells as previously demonstrated in epithelial cells treated with another macrolide antibiotic, bafilomycin A1 (8). Because erythromycin exposure did not affect the ability of RV14 to infect epithelial cells, erythromycin may act by inhibiting RV14 RNA entry across acidic endosomes as demonstrated in HeLa cells and human tracheal epithelial cells treated with bafilomycin A1 (7, 8). Likewise, erythromycin reduced the viral titers in the supernatants and the viral RNA of a minor subgroup of RVs, RV2, in the cultured human tracheal epithelial cells. Because pretreatment with erythromycin did not affect the expression of LDL receptor mRNA, the receptor for minor RV (36), erythromycin may not affect the RV2 binding to its receptor, LDL receptor. In contrast, erythromycin decreased the number of acidic endosomes in the cytoplasm of human tracheal epithelial cells. Therefore, erythromycin may inhibit RV2 infection partly through the inhibition of the entry of RV2 RNA from acidic endosomes to the cytoplasm.

The epithelial cells in human airways express ICAM-1 on their surface, which is the site of attachment for 90% of the approximately 100 RV serotypes (4, 37). ICAM-1 interacts physiologically with leukocyte function-associated antigen-1, expressed on leukocytes, and thus plays a vital role in the recruitment and migration of immune effector cells to sites of local inflammation. Recent studies (15, 18) have shown that RV infection upregulates ICAM-1 expression on the airway epithelial cells, an effect that would facilitate viral cell attachment and entry. The increases in the expression of protein and mRNA of ICAM-1 on human tracheal epithelial cells induced by RV14 infection in the present study are in accord with those in previous studies (15, 18). In contrast to ICAM-1 expression, the inhibitory effects of erythromycin on increases in LDL receptor mRNA expression induced by RV2 were small in the cultured human tracheal epithelial cells. The differences in the inhibitory effects of erythromycin on ICAM-1 and LDL receptor may explain the differences in the erythromycin-induced inhibitory effects on RV14 and RV2 infection.

In the present study, we observed that infection of RV2 and RV14 increased the production of IL-1beta , IL-6, IL-8, and TNF-alpha in the cultured human tracheal epithelial cells and that erythromycin reduced the baseline and RV infection-induced increases in cytokine production. These cytokines induce the growth and differentiation of T and B lymphocytes, the production of other cytokines, prostaglandin E2 synthesis, and degranulation from neutrophils (20). Furthermore, these cytokines are known to mediate a wide variety of proinflammatory and immunoregulatory effects (20) and are suggested to play an important role in the pathogenesis of RV infections. Therefore, erythromycin may inhibit the airway inflammation as well as RV infection.

In the present study, increased activation of NF-kappa B was present in cells from 0.5 hours after RV14 infection, and erythromycin prevented it. The time course of NF-kappa B activation was consistent with those in previous reports on airway epithelial cells infected with RV14 or RV16 (16, 18). NF-kappa B increases the expression of genes for many cytokines such as IL-1beta , IL-6, IL-8, and TNF-alpha , enzymes, and adhesion molecules including ICAM-1 (16-19). Therefore, the reduction of cytokines and ICAM-1 before and after RV infection might be mediated via the erythromycin-reduced activation of NF-kappa B. Acidification of a variety of intracellular compartments in the cells is required to activate the secreted lysosomal enzymes and to facilitate protein processing (38). These mechanisms may also relate to the reduced ICAM-1 and cytokine production. Erythromycin more potently inhibited the production of IL-1beta , IL-6, and IL-8 than that of TNF-alpha . Although the reason is uncertain, the production of TNF-alpha is modulated by factors other than NF-kappa B, such as tyrosine kinase and polyunsaturated fatty acid (39, 40), which may not be influenced by erythromycin. Further studies are needed to clarify the precise mechanisms.

The intracellular pH is regulated by ion transport mechanisms across various antiporters and exchangers such as Na+/H+ antiporters (NHEs), H+-ATPase (V-ATPase) and Cl--HCO3- exchangers in the cell membrane (41, 42), and the endosomal pH is suggested to be regulated by V-ATPase (38). In the present study, erythromycin reduced the intracellular pH and increased the endosomal pH. Although we have no data to demonstrate whether erythromycin inhibits V-ATPase, NHEs, or Cl--HCO3- exchangers, another macrolide antibiotic and a V-ATPase inhibitor, bafilomycin (6), increased the endosomal pH in epithelial cells (8), suggesting the possibility that erythromycin may have an inhibitory effect on V-ATPase. However, the human tracheal epithelial cells of that study were grown in a submersed condition in culture medium, not in an air-liquid interface condition as in the present study. Therefore, because the expression of ion pumps in the epithelial cells may not have been physiologic, such a system may have limitations in terms of the clinical relevance, although the cells expressed beating cilia, could form tight junctions and make a dome formation caused by active fluid absorption as shown by Widdicombe and coworkers (35).

Recent reports have revealed the mechanisms of RV entry into the cytoplasm of infected cells (7, 9-12). RV14 forms RV-soluble ICAM-1 complexes, and these complexes can release viral RNA (12). Furthermore, RV14 releases RNA after exposure to an acidic pH (12), and infection of HeLa cells and human tracheal epithelial cells by RV14 is inhibited by bafilomycin (7, 8). Therefore, the entry of RV14 RNA into the cytoplasm of the infected cells appears to be mediated by the destabilization by receptor binding, by endosomal acidification, or both (12). The entry of RV2 is also mediated by endosomal acidification in HeLa cells (11). The inhibitory effects of erythromycin on infection by RV2 and RV14 and its effects on the endosomal pH in the present study are consistent with those of bafilomycin in previous studies (7, 8, 11).

In summary, this is the first report that the macrolide antibiotic erythromycin inhibits infection by RV14 and decreases the susceptibility of cultured human tracheal epithelial cells to RV14 infection, probably through the inhibition of ICAM-1 expression and endosomal acidification. Erythromycin also inhibited infection by a minor subgroup of RVs, RV2, in the human airway epithelial cells through the inhibition of RNA entry into the acidic endosomes. Furthermore, erythromycin reduced the production of proinflammatory cytokines and inhibited the activation of NF-kappa B. These findings suggest that erythromycin may inhibit the infection of both major and minor subgroups of RVs and modulate the inflammatory responses in the airway epithelial cells after RV infection, as reported in mice lung infected with influenza virus (43). The reduced production of cytokines and ICAM-1 may have been partly due to the inhibition of activated NF-kappa B. Although erythromycin has been clinically used for the treatment of patients with bacterial infection, these findings showed the important possibility of its clinical application for preventing RV infection and treating the airway inflammation caused by RV infection. However, Abisheganaden and associates (44) reported no significant difference in the severity of cold symptoms caused by experimental RV16 infection between healthy subjects treated with a macrolide antibiotic, clarithromycin, and those treated with an antibiotic, trimethoprim-sulfamethoxazole. Further studies are needed to clarify the clinical usefulness of macrolide antibiotics for RV infection.

    Footnotes

Correspondence and requests for reprints should be addressed to Hidetada Sasaki, M.D., Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

(Received in original form March 20, 2001 and accepted in revised form January 11, 2002).

This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Acknowledgments: The authors thank Mr. Brent Bell for reading the manuscript and Mr. Akira Ohmi, Ms. Michiko Okamoto, and Ms. Fusako Chiba for technical assistance.
    References
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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