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The Effects of Macrolides on Inflammatory Cells* FREE TO VIEW

Jun Tamaoki, MD, FCCP
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*From the First Department of Medicine, Tokyo Women’s Medical University School of Medicine Tokyo, Japan.

Correspondence to: Jun Tamaoki, MD, FCCP, First Department of Medicine, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-Cho, Shinjuku, Tokyo 162-8666, Japan; e-mail: jtamaoki@chi.twmu.ac.jp



Chest. 2004;125(2_suppl):41S-51S. doi:10.1378/chest.125.2_suppl.41S
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Bronchial epithelial damage and mucus hypersecretion are characteristic features of chronic airway inflammation that can impair mucociliary clearance and can cause recurrent or persistent respiratory infection. In response to chemoattractants produced by damaged or inflamed tissue, neutrophils move through sequential steps of recruitment, migration, accumulation, and adhesion to endothelial and bronchial epithelial cells. Neutrophils engage in bacteriocidal activity by phagocytosis, release of lysosomal enzymes, and generation of reactive oxygen species, and they synthesize and release proinflammatory cytokines. Data confirm that many macrolide antibiotics have nonbactericidal properties that include inhibiting inflammatory cell chemotaxis, cytokine synthesis, adhesion molecule expression, and reactive oxygen species production. Macrolides also can decrease airway mucus hypersecretion in patients with diffuse panbronchiolitis, chronic sinusitis, and chronic bronchitis. Macrolides accumulate in neutrophils and macrophages at significantly higher concentrations than in extracellular fluid. This article discusses the action of macrolides on neutrophil accumulation, immune complex-mediated production of nitric oxide, mucin production, and the expanded therapeutic role of macrolides as biological response modifiers.

Figures in this Article

Diffuse panbronchiolitis (DPB), a disease previously recognized almost exclusively in Japan, Korea, and China, is characterized clinically by progressive airflow limitation and recurrent respiratory tract infection, and pathologically by neutrophils and T lymphocytes in respiratory bronchiole walls out to the lung parenchyma.1Kudoh and coworkers2 first observed the clinical effects of macrolide antibiotics in patients with DPB and suggested that the efficacy might be derived from their anti-inflammatory and immunomodulatory activities. Reports of the success of macrolide therapy in symptom reduction and improved survival spurred interest in the potential benefits of their long-term application in a variety of chronic inflammatory pulmonary diseases. A number of hypotheses have been proposed to explain the pharmacologic mechanisms by which the macrolides alter the response of an immune system activated by inflammatory triggers. However, no single theory has yet to define a unified process by which the macrolides exert their protean effects as biological response modifiers. It is likely that 14-membered and 15-membered ring macrolides act in a time-dependent manner at numerous junctures along the inflammatory cascade.

Studies in vitro and in animal models have generated data to support the inhibitory effects of macrolides on neutrophil influx and chemotactic activity. Long-term treatment with macrolides has been shown to decrease levels of interleukin (IL)-8 and neutrophil counts in the BAL fluid of patients with DPB,3and to decrease concentrations of IL-8 released by eosinophils from atopic subjects.4Cultured human bronchial epithelial cells treated with erythromycin show reduced levels of IL-6, IL-8, and intercellular adhesion molecule (ICAM)-1.56 Several laboratories78 have demonstrated that macrolides may reduce pulmonary tissue damage by impairing superoxide generation by activated neutrophils.

In addition, treatment with macrolides reduces sputum production in patients with mucus hypersecretion.9Experimental evidence suggests that this is not a direct effect on mucus-producing cells, but rather may be associated with anti-inflammatory activities. Their multiple and diverse immunomodulatory actions hold promise for macrolides as chemotherapeutic agents for a variety of inflammatory disease states. However, as Figure 1 illustrates,10 much work lies ahead to attain a more complete understanding of how the immune system functions in vivo, and to identify the precise molecular target of the macrolides and their respective modulatory steps in the transduction pathway. Because proteins and peptides are generally hydrophilic, they do not readily penetrate the hydrophobic phospholipids that comprise a cell membrane. Rather, peptides and proteins usually bind to an active site of a cell-surface G protein-linked receptor and transmit their message to the receptor, which in turn conveys the message to the cell, where it is transduced into second messengers and then into gene expression. Where and how the macrolides influence this complex network of extracellular, intracellular, and intercellular communications is the subject of continuing investigation.

Neutrophils act selectively in their proinflammatory role by simultaneously integrating multiple signals derived from high-affinity integrins, from inflammatory cytokines, and from chemoattractants produced by bacteria and epithelial cells of inflamed tissues. Historically, neutrophils have been considered to be lacking in transcriptional activity. However, molecular evidence now indicates that the human neutrophil is a source of various proinflammatory cytokines, chemokines, and growth factors. Neutrophils either possess the constitutive ability or are induced to synthesize proinflammatory cytokines, although to a lesser degree than mononuclear phagocytes. The type of stimuli, including bacterial endotoxins, regulatory cytokines, and chemokines, influences the pattern of neutrophilic cytokine production.

It is known that the chemokine IL-8 is expressed in response to lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, and aggregated immune complexes. A variety of cells, including neutrophils, T lymphocytes, and epithelial cells, secrete IL-8, which in turn stimulates the recruitment and activation of neutrophils. Thus, neutrophils are both the primary source and the primary cellular target of IL-8. In addition to cytokines, neutrophils that are recruited into the airway mucosa release enzymes and superoxide anions. Neutrophil-derived proteases, including elastase and proteinase 3, stimulate the degranulation of mucin granules and the release of mucin glycoprotein by goblet cells and submucosal glands. Reactive nitrogen intermediates released from airway epithelial cells and various inflammatory cells can enhance their innate toxicity by reacting with superoxide to form peroxynitrite.

There is some disagreement as to the effects of macrolide antibiotics on neutrophil migration. To date, the inhibitory effects in vitro11and in vivo12have been described, while others have reported either no effect in vitro13or stimulatory effects in vitro and in vivo.14 The reason for this controversy is unknown but might be due to differences in species and/or experimental conditions. Kawasaki et al15 conducted an in vitro study of the potential effects of roxithromycin on neutrophil adhesion to bronchial epithelial cells and the role of ICAM-1. The pretreatment of neutrophils with N-formyl-methionyl-leucyl-phenylalanine, but not with LPS, significantly enhanced adhesion. The stimulation of human bronchial epithelial cells with interferon (IFN)-γ and TNF-α also up-regulated neutrophil adhesion to the epithelial cells. When the neutrophils had been pretreated with N-formyl-methionyl-leucyl-phenylalanine, roxithromycin inhibited neutrophil adhesion to cultured epithelial cells in a concentration-dependent fashion between 1 and 25 μg/mL. However, roxithromycin exhibited an attenuated inhibitory effect on the adhesion of naïve (ie, untreated) neutrophils that achieved statistical significance only at a concentration of 25 μg/mL. The results from this study suggest that roxithromycin may decrease the number of neutrophils in BAL fluid from patients with airway inflammation in a process that differs from its antibiotic activity. Kawasaki and colleagues,15 then employed a cell enzyme-linked immunosorbent assay method to evaluate the role of ICAM-1 in neutrophil recruitment into the airways. A human bronchial epithelial cell line (Bet-1A) was cultured and treated with IFN-γ with or without roxithromycin for 18 h, followed by the addition of antihuman ICAM-1 monoclonal antibody. IFN-γ up-regulated the expression of ICAM-1 on the epithelial cell surfaces, but roxithromycin significantly decreased the magnitude of its expression, again in a concentration-dependent manner. These findings suggest that the erythromycin A derivatives (in this case, roxithromycin) indirectly modulate the recruitment of neutrophils to inflamed sites by suppressing the expression of ICAM-1.

Our laboratory has shown that LPS up-regulates the expression of ICAM-1 when added to cultured rat tracheal epithelial cells, and that the preincubation of cells with clarithromycin significantly reduced ICAM-1 expression in a concentration-dependent manner. In contrast, even high concentrations of amoxicillin, cefaclor, or amikacin did not demonstrate this effect (Fig 2 ; and K. Isuno, J. Tamaoki, M. Kondo, A. Nagai; unpublished observation; May 2001), suggesting that the effect might be specific for macrolide antibiotics. Therefore, such inhibition of adhesion molecule expression may explain, at least in part, the mechanism of macrolide-induced attenuation of neutrophil accumulation into the airways.

We then studied the effects of macrolides on LPS-induced neutrophil infiltration into the guinea pig airways in vivo.16 The inhalation of LPS from Escherichia coli caused a time-dependent increase in the number of neutrophils in the tracheal mucosa. The mean (± SE) number of neutrophils significantly increased from 4 ± 2 to 19 ± 3 cells per 100 epithelial cells at 1 h after LPS inhalation, and reached a maximal mean value of 42 ± 5 cells per 100 epithelial cells at 3 h. Pretreatment with oral clarithromycin (10 mg/kg) inhibited the LPS-induced neutrophil recruitment at 3, 6, and 9 h after LPS, but pretreatment with amoxicillin (30 mg/kg) did not (Fig 3 ).

As a primary anti-inflammatory activity, the macrolides appear to target nuclear transcriptional regulation. The stimulation of cells with various cytokines (eg, IL-8 and TNF-α) induces and activates a number of nuclear DNA-binding proteins, which in turn trigger the transcriptional process to initiate and amplify the inflammatory response.17

Nuclear factor-κB (NF-κB) is a protein that is essential for the transcription of genes that encode a number of proinflammatory molecules that participate in the acute inflammatory responses, including TNF-α, ICAM-1, inducible nitric oxide synthase (iNOS), IL-6, and IL-8. NF-κB is composed of two proteins, p50 and p65 (Rel-A). Although both protein subunits contact DNA, only the C-terminal end of Rel-A interacts with the nuclear transcription apparatus. Through an interaction with the inhibitor of NF-κB (I-κB), NF-κB is sequestered in the cytoplasm of quiescent cells (ie, cells that are not responding to an inflammatory assault). On cell stimulation, I-κBα is phosphorylated and degraded by the enzyme 26S proteasome. This interaction unmasks the nuclear localization signals on NF-κB that permit the transcription factor to be transported from the cytoplasm to the cell nucleus. Activated NF-κB binds to the promoter region of I-κBα, thereby up-regulating I-κBα messenger RNA.18

Krunkosky and colleagues19 used human recombinant TNF-α in a series of elegant experiments to examine the intracellular signaling pathways that mediate TNF-α-induced activation of NF-κB and that lead to enhanced ICAM-1 expression on normal human bronchial epithelial (NHBE) cells. They found that exposing these cells to TNF-α enhanced both ICAM-1 messenger RNA levels and the surface expression of ICAM-1. Additionally, incubation of NHBE cells with a neutralizing monoclonal antibody against TNF-α receptor attenuated the TNF-α-induced ICAM-1 expression, indicating that the observed effect of TNF-α requires binding to a specific receptor on the cell surface. When coupled with TNF-α, the transmembrane receptor activation up-regulates the expression of a variety of genes via intracellular signaling mechanisms.

To test the hypothesis that macrolides modulate inflammation by inhibiting NF-κB activation, Ichiyama and colleagues20 analyzed nuclear and cytoplasmic extracts from the following four human cell lines stimulated by TNF-α: a human monocytic leukemia cell line (U-937); Jurkat cells (a T-cell line); a pulmonary epithelial cell line (A549); and peripheral blood mononuclear cells. As shown in Figure 4 , the pretreatment of U-937, A549, and Jurkat cells with clarithromycin inhibited NF-κB activation in a concentration-dependent manner. These findings suggest that clarithromycin modulates inflammatory activity in pulmonary epithelial cells and peripheral blood monocytes via a fundamental event or process, quite possibly at the level of gene transcription for proinflammatory cytokines.

The observations from this study corresponded with those of Aoki and Kao,17 who reported that erythromycin inhibited NF-κB activation in Jurkat T cells stimulated with phorbol 12-myrisate 13-acetate and calcium ionophore. These investigators demonstrated that erythromycin inhibited IL-8 protein expression, but not IL-2 protein expression, at the transcriptional level. This action could be attributed to the inhibition of the induction of IL-8, NF-κB, and DNA-binding activities by erythromycin. Erythromycin readily diffuses into intracellular fluids and may inhibit NF-κB activation by interfering with the generation of reactive oxygen intermediates that mediate the activation of nuclear transcription factors such as NF-κB.

The gaseous molecule nitric oxide (NO) is a second messenger that is produced by NO synthase (NOS), which exists in both constitutive NOS and iNOS isoforms. NOS catalyzes the conversion of L-arginine to L-citrulline and NO. NO participates in a number of normal physiologic processes and also acts as an inflammatory mediator, thereby playing a role in the pathogenesis of lung inflammation and injury.21 Several cell types, including neutrophils, airway epithelial cells, and pulmonary macrophages, synthesize NO, the effects of which contribute to the regulation of airway smooth muscle tone, microvascular permeability, and host defense. In addition, events such as Gram-negative bacteremia that activate the release of proinflammatory mediators and cytokines enhance the release of NO and its derivative, peroxynitrite, causing pulmonary injury.

A variety of viral and bacterial infections can induce antigen-antibody immune complex hypersensitivity reactions.22 A moderate excess of antigen can activate the C′-neutrophil-macrophage-mediated mechanisms of increased vascular permeability, neutrophil recruitment, and release of inflammatory mediators from neutrophils and macrophages. The deposition of immune complexes initiates the release of proteases, cationic proteins, TNF-α, IL-1, and IL-6 that leads to inflammation and tissue injury. TNF-α activates endothelial cells, monocytes, and macrophages, and releases cytokines and mediators of the inflammatory response, including acute phase proteins, IL-4, IL-8, IL-10, and cyclooxygenase products. In the presence of IL-1, high levels of IL-6 have been associated with death in patients with septic shock.

Prior work has established that immune complexes can stimulate both type II alveolar epithelial cells and pulmonary alveolar macrophages to produce NO, and that the cytokines IL-1β and TNF-α may contribute to immune complex-induced NO generation by pulmonary alveolar macrophages.23 We sought to determine the effects of macrolide antibiotics on immune complex-induced lung injury and to elucidate whether those effects are associated with NO generation. Groups of pathogen-free rats received 50 mg/kg erythromycin, josamycin, amoxicillin, cefaclor, or vehicle (0.9% saline solution) 3 h prior to IgG immune complex (IgG-ICx)-induced lung injury. In the control group, exhaled NO concentration remained unchanged from baseline values for at least 36 h. The exhaled NO concentration increased threefold at 6 h following the intrapulmonary deposition of IgG-ICx, and achieved a plateau 6 h later (Fig 5 ). This delayed response may reflect the induction of iNOS within cells of the lower respiratory tract. Erythromycin in a 50 mg/kg dose significantly reduced the IgG-ICx-induced increase in exhaled NO concentration. The histopathologic features of IgG-ICx-induced lung injury included marginated neutrophils in blood vessels, and increased RBCs, macrophages, fibrin strands, and neutrophils in alveolar spaces. Unlike amoxicillin or cefaclor, pretreatment with either erythromycin or josamycin reduced neutrophil accumulation within the alveolar spaces.

The in vitro incubation of pulmonary alveolar macrophages with IgG-ICx was associated with increased concentrations of IL-1β and TNF-α in the culture medium. Coincubation with erythromycin significantly inhibited the release of these cytokines, whereas equal concentrations of amoxicillin failed to produce similar effects. Then, to directly evaluate the release of NO from pulmonary alveolar macrophages, cells were incubated for 24 h with saline solution with or without L-arginine or IgG-ICx. An NO-selective electrode detected no baseline current from the control medium containing L-arginine or IgG-ICx. However, when L-arginine was added to pulmonary alveolar macrophages that had been incubated with IgG-ICx, a significant electrical response occurred. Similar effects were observed with the addition of IL-β and TNF-α to the pulmonary alveolar macrophage cell system. Once the electrical response attained a plateau, aminoguanidine, a specific inhibitor of NO synthesis mediated through iNOS, was added to the medium of IgG-ICx-treated pulmonary alveolar macrophages. The addition of aminoguanidine precipitated a rapid decline in electrical current, suggesting that the measured response was associated with iNOS. Furthermore, all macrolides introduced into the medium (ie, erythromycin, clarithromycin, roxithromycin, and josamycin), but not amoxicillin, exerted a dose-dependent inhibitory effect on the IgG-ICx-induced electrical current, but produced no effect on the electrical current induced by IL-1β and TNF-α (Fig 6 ). Therefore, the effect of macrolides on NO release may be mediated by inhibition of the production of these particular cytokines that up-regulate iNOS activity. This was confirmed by evaluating the effect of macrolides on iNOS gene expression. Unlike amoxicillin or cefaclor, coincubation with all of the macrolides tested inhibited IgG-ICx-mediated induction of iNOS messenger RNA (Fig 7 ).

The results from this study strongly indicate that macrolide antibiotics inhibit iNOS gene expression and NO release stimulated by immune complex. Macrolides also inhibit neutrophil accumulation in pulmonary alveoli, which might be explained by their inhibitory effects on the expression of ICAM-1 and the release of neutrophil chemoattractant chemokines such as IL-8.

LPS endotoxin comprises a major portion of the cell walls of Gram-negative bacteria and interacts with a variety of cell types, including neutrophils, basophils, and monocytes. Studies have demonstrated that LPS increases microvascular permeability, neutrophil chemotaxis, accumulation into the airway wall and, hence, neutrophilic airway inflammation.

To determine the effects of LPS on airway goblet cell secretion, Tamaoki and colleagues16 pretreated pathogen-free guinea pigs with single daily doses of clarithromycin, erythromycin, amoxicillin, or cefaclor for 1 week prior to exposure with nebulized LPS. The study found that a time-dependent increase in goblet cell mucus discharge occurred with the inhalation of LPS and that this response reached a plateau 3 h after exposure. The oral administration of clarithromycin or erythromycin inhibited the effects of LPS on goblet cell mucus secretion in a dose-dependent manner. In contrast to the inhibitory effects of the two macrolides, pretreatment with either amoxicillin or cefaclor did not influence LPS-induced goblet cell mucus secretion.

Similar to the results obtained with aerosolized LPS on goblet cell discharge in the guinea pig model, the inhalation of IL-8 was observed to produce a time-dependent increase in goblet cell secretion, with a maximal response at 6 h after exposure.24 In a separate rodent model, IL-8 stimulated mucus secretion by tracheal goblet cells, an event that could figure prominently in airway hypersecretion. Increased numbers of neutrophils in the tracheal mucosa coincided with the time of mucus discharge, suggesting that neutrophils may mediate airway goblet cell secretion induced by IL-8. Pretreatment with single daily doses of roxithromycin or erythromycin for 1 week inhibited the cytokine-induced goblet cell secretion and neutrophil recruitment, whereas amoxicillin and cefaclor had no effect.

Mucins are macromolecular glycoproteins that impart viscoelastic properties to mucus. Airway mucus protects the epithelial surface from injury and facilitates the removal of bacterial, cellular, and particulate debris from the lung. Goblet cells and submucosal glands secrete mucus, and ciliated epithelial cells clear these secretions by a mucociliary transport system, which propels mucus through the airways toward the nose and throat. Mucins also play a principal role in the pathogenesis of lung diseases that involve chronic inflammation of the airways, impaired pulmonary function, and increased susceptibility to infection, such as asthma, chronic bronchitis, and cystic fibrosis. This adhesive glycoprotein gel is a polymer network of carbohydrates, proteins, and a small amount of sulfate. The elasticity and the viscosity of mucus can be attributed largely to high-density mucin O-glycoproteins.25

To date, a number of mucin genes have been identified, and in situ messenger RNA hybridization techniques, employing gene-specific oligonucleotides or riboprobes, have permitted the cell-specific localization of mucin gene expression in both the digestive and respiratory tracts. In an attempt to discern which mucin genes are expressed during the differentiation of the human pulmonary epithelium, Reid and colleagues26 performed in situ hybridization on fetal and adult lung tissue, and they identified MUC4, MUC5AC, and MUC5B as the major constituents of airway mucus in the developing lung. MUC4 was detected only in the tracheal epithelium, and in the epithelium lining the bronchi and larger bronchioles. MUC5AC expression was restricted to individual goblet cells in the airway epithelium and was not observed until later in the second trimester, around 17 to 18 weeks gestational age. MUC5AC messenger RNA was localized to tracheal goblet cells with no expression in the small bronchioles or distal lung.

Neutrophil elastase, a serine protease that is released from lysed neutrophils, induces mucin production and secretion. Neutrophil elastase also regulates the expression of IL-8 and ICAM-1 messenger RNA. Voynow and colleagues27 used two distinct cell lines to induce mucociliary differentiation (A549 and NHBE cells). A549 cells express both MUC5AC messenger RNA and glycoprotein. This cell line has been used for investigating iNOS expression and NF-κB-mediated gene regulation. The exposure of confluent A549 and NHBE cells in culture to human neutrophil elastase increased MUC5AC messenger RNA levels in a dose-dependent and time-dependent manner. Compared with vehicle alone, exposing A549 cells to neutrophil elastase increased MUC5AC messenger RNA levels sixfold. After 4 h of exposure, MUC5AC transcript levels began to rise and continued to increase for up to 24 h, approaching levels that are twofold to fourfold higher than those found in NHBE cells not exposed to human neutrophil elastase. They also showed that inactivated neutrophil elastase produced no effect on MUC5AC gene expression, that serine protease activity regulated this gene expression, and that in A549 cells neutrophil elastase increased MUC5AC messenger RNA levels by increasing messenger RNA stability. As cytokines such as TNF-α can promote the expression of mucin genes, the combination of neutrophil elastase and these proinflammatory mediators may act in synergy to up-regulate the expression of mucin genes with a resultant increase in mucin production.

Recently, our group studied MUC5AC messenger RNA expression in a human bronchial epithelial cell line (NCI-H292 cells) by Northern blot and in situ hybridization, and found that LPS up-regulated MUC5AC gene expression and protein expression (Fig 8 ). The stimulation of cells with LPS or transforming growth factor (TGF)-α also induced phosphorylation of the transcription factor I-κBα (K. Takeyama, J. Tamaoki, E. Tagaya, A. Nagai; unpublished observation; January 10, 2003). The addition of erythromycin or clarithromycin inhibited LPS-induced MUC5AC gene expression and attenuated TGF-α-induced and LPS-induced phosphorylation of I-κBα. Thus, LPS or TGF-α up-regulates mucin production in this cell system, and macrolides appear to inhibit the augmented response at the levels of phosphorylation of transcription factors.

In an earlier study,9 we assessed the effects of long-term treatment with oral clarithromycin on sputum production and rheologic properties in a group of patients with chronic bronchitis, bronchiectasis, or DPB. Patients in whom chronic bronchitis was diagnosed conformed to the World Health Organization definition of the disease and were current or ex-smokers. CT scans of the chest confirmed the presence of bronchiectasis, and transbronchial lung biopsy specimens established the diagnosis of DPB. All patients had expectorated > 30 g sputum per day for at least 2 weeks prior to study entry. Thirty-one patients, aged 33 to 77 years, enrolled in a double-blind, parallel-group, placebo-controlled 10-week trial of clarithromycin vs placebo, in equivalent doses of 100 mg bid. During a 2-week run-in period, no antibiotics, mucolytic agents, corticosteroids, or anticholinergics were administered. Initially, all study participants were hospitalized for 48 h, during which time they collected and weighed baseline sputum samples. Patients repeated this process daily during both the run-in and the active treatment periods. We determined alterations in the elastic modulus and dynamic viscosity properties of sputum by microrheologic measurements of collected specimens. We also performed sputum culture to assess any changes in the bacterial density of the expectorant.

During the 8-week trial period, the daily quantity of sputum produced by the placebo group did not vary appreciably from baseline. In contrast, at 6 weeks those patients randomized to clarithromycin had less daily sputum production, an outcome that was sustained after 8 weeks of treatment (Fig 9 ). Although patients with DPB produced comparatively greater amounts of sputum than the groups with chronic bronchitis or bronchiectasis, clarithromycin demonstrated similar efficacy in all three disease states. While twice-daily therapy with clarithromycin did not affect sputum viscoelasticity prior to 4 weeks of completed treatment, by week 8, therapy with low-dose clarithromycin increased the elastic modulus but did not alter the dynamic viscosity of the samples. Neither placebo nor clarithromycin altered the total number of bacterial colony-forming units or the type of microbial flora in sputum. Thus, the effectiveness of clarithromycin in suppressing sputum production was not correlated with colony counts. This suggests that the clarithromycin-induced reduction in expectorated sputum volume is probably due to the inhibition of inflammatory cells and/or secretory cells.

In a more recent, related study, Tagaya et al28 evaluated a variety of antibiotics for their effects on sputum production, epithelial chloride secretion, and water transport across airway mucosa in a group of subjects with chronic airway hypersecretion. Forty-five patients with chronic bronchitis or bronchiectasis were enrolled in a double-blind, parallel-group, 7-day trial of oral clarithromycin, 200 mg bid, vs oral amoxicillin, 500 mg tid, or cefaclor, 250 mg tid. Before starting the study, all patients were free of respiratory infection and had been expectorating > 20 g sputum per day. Sputum samples were weighed, the percentage of solid composition was calculated from the ratio of wet to dry weight, and the concentrations of chloride in the supernatant of the centrifuged sputum specimens were measured by a chloridometer.

The study protocol defined responders as patients who reduced their sputum volume by > 30% of baseline values. Compared with nonresponders, the sputum of responders tended to contain a higher chloride content and a lower percentage of solids. A 7-day course of clarithromycin significantly reduced sputum production, whereas amoxicillin and cefaclor had no effect. Furthermore, in the responder group, treatment with clarithromycin decreased chloride concentrations and increased the percentage of solid composition. Among nonresponders, clarithromycin did not affect chloride levels or the percentage of solids. In this trial, the baseline sputum samples from the responder group contained a higher chloride content and a lower percentage of solids. These values changed as the sputum volume declined with clarithromycin therapy, suggesting that macrolides may exert their antisecretory actions on more hydrated, “watery” sputum by inhibiting airway epithelial chloride secretion and the concomitant secretion of water toward the lumen. This notion is compatible with previous in vitro findings that macrolides can inhibit chloride channels in airway epithelial cells and glandular cells.2930

Investigations into the biological response-modifying properties of the macrolides have uncovered a variety of salutary effects and plausible mechanisms of action by which the extrabactericidal properties of macrolides impact the immune system. Macrolides appear to modulate inflammatory activity in airway epithelial cells by inhibiting NF-κB activation that leads to IL-8 production and enhanced neutrophil accumulation. Additionally, in vitro studies have suggested that macrolides inhibit the expression of ICAM, thereby also modulating the recruitment of neutrophils to inflamed sites. The extraribosomal effects of macrolides reduce the number of neutrophils in the BAL fluid from patients with neutrophilic, inflammatory airway diseases. In such diseases as DPB, cystic fibrosis, acute exacerbation of chronic bronchitis, and COPD, macrolides also may attenuate mucus hypersecretion by goblet cells as an indirect consequence of inhibiting neutrophil migration, activation, and accumulation.

The function of macrolide antibiotics as biological response modifiers must be explained in the context of their integrated effects on the activation of nuclear transcription factors, the gene expression of inflammatory mediators, and specific cellular targets. A thorough understanding of the biological properties of macrolides in the host either to disrupt or to redirect signaling pathways that lead to prolonged inflammation and airway hypersecretion should establish a firm foundation on which to construct novel therapies that effectively prevent or ameliorate both the symptoms and the long-term morbidity of chronic respiratory illnesses.

The American College of Chest Physicians designates this continuing medical education activity for 1 credit hour in category 1 of the Physician’s Recognition Award of the American Medical Association. To obtain credit, please complete the question form at www.chestnet.org. Credit can be obtained ONLY through our online process.

  1. Which of the following drugs does not inhibit the release of NO?

    1. Clarithromycin

    2. Roxithromycin

    3. Erythromycin

    4. Amoxicillin

    5. Josamycin

  2. Proposed mechanisms of action of the macrolides for treatment of chronic diseases of the airways include all of the following except:

    1. Inhibition of inflammatory cell chemotaxis

    2. Inhibition of cytokine production

    3. Stimulation of adhesion cell molecule expression

    4. Inhibition of reactive oxygen species production

    5. Inhibition of mucus hypersecretion

  3. Macrolides affect the accumulation of neutrophils by all of the following except:

    1. Inhibition of ICAM-1 expression

    2. Decreased levels of IFN-γ

    3. Inhibition of neutrophil adhesion to bronchial epithelial cells

    4. Decreased levels of IL-8

    5. Increased levels of IL-6

  4. Which of the following inhibits the expression of ICAM-1 and neutrophil adhesion to bronchial epithelial cells?

    1. Amoxicillin

    2. Cefaclor

    3. Clarithromycin

    4. Amikacin

    5. Ampicillin

  5. Clarithromycin was shown to effect mucus hypersecretion by all of the following except:

    1. Decreased concentrations of chloride in sputum samples

    2. Decreased colony counts

    3. Increased percentage of solid composition in sputum samples

    4. Reduction of sputum volume

    5. Increased elastic modulus of sputum

Abbreviations: DPB = diffuse panbronchiolitis; ICAM = intercellular adhesion molecule; IFN = interferon; IgG-ICx = IgG immune complex; I-κB = inhibitor of NF-κB; IL = interleukin; iNOS = inducible nitric oxide synthase; LPS = lipopolysaccharide; NF-κB = nuclear factor-κB; NHBE = normal human bronchial epithelium; NO = nitric oxide; NOS = nitric oxide synthase; TNF = tumor necrosis factor

Learning objectives:

  1. To recognize the proposed mechanisms of action of macrolides for the treatment of chronic diseases of the airways.

  2. To understand the mechanisms by which macrolides affect the accumulation of neutrophils in chronic diseases of the airways.

  3. To understand the effects of macrolides on NO-induced lung inflammation and injury.

  4. To understand the effects of macrolides on airway goblet cell secretion.

  5. To understand the effects of macrolides on mucin production.

Neither Dr. Tamaoki nor the department(s) with which he is affiliated have received something of value (ie, any item, payment, or service valued in excess of $750.00) from a commercial or other party related directly or indirectly to the subject of this submission.

Figure Jump LinkFigure 1. Macrolides and inflammation. Proposed immunomodulatory activities induced by macrolides (derived from in vitro and ex vivo data). Figure from Labro,10 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 2. Effects of macrolides on airway epithelial ICAM-1 expression. The rat tracheal epithelial cells were incubated with LPS (10−5 mol/L) in the absence and presence of various concentrations of clarithromycin (CAM; 0.1 to 100 μmol/L) or high concentrations of amoxicillin (AMPC), cefaclor (CCL), or amikacin (AMK) at 100 μmol/L. Data given as the mean ± SE (n = 6 for each column). ** = p < 0.01 (significantly different from values for LPS alone).Grahic Jump Location
Figure Jump LinkFigure 3. Time course of the effects of antibiotics on LPS (5 mg/kg)-induced recruitment of neutrophils into the guinea pig tracheal mucosa. The guinea pigs were given inhaled saline or LPS, and some animals were treated for 1 week with oral clarithromycin (10 mg/kg) or amoxicillin (30 mg/kg), and then given inhaled LPS. Responses are expressed as the number of neutrophils per 100 epithelial cells in the tracheal mucosa. Data are given as the mean ± SE (n = 9 for each point). * = p < 0.05; *** = p < 0.001 (both significantly different from control values of the saline solution group). † = p < 0.05; †† = p < 0.01 (both significantly different from the corresponding values for LPS alone). See Figure 2 for abbreviations not used in the text. Figure from Tamaoki et al,16 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 4. Representative Western blot of nuclear extracts of U-937, Jurkat, and A549 cells revealing that pretreatment with clarithromycin inhibited NF-κB activation induced by TNF-α in a concentration-dependent manner. Figure from Ichiyama et al,20 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 5. Time course of exhaled NO concentration in rats. The animals were given 50 mg/kg erythromycin (EM), josamycin (JM), amoxicillin, cefaclor, or vehicle alone (0.9% sterile saline solution). After intrapulmonary deposition of IgG-ICx at time 0, the concentration of NO in exhaled air was measured by a chemiluminescence analyzer. In the control group, the animals were not subjected to IgG-ICx. Data are given as the mean ± SE (n = 12 for each point). See Figure 2 for abbreviations not used in the text. Figure from Tamaoki et al,23 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 6. Representative tracing of the current detected by an NO-selective electrode in Roswell Park Memorial Institute medium containing rat pulmonary alveolar macrophages. After equilibration, L-arginine (L-Arg; 10−3 mol/L) was added to the medium (arrows). Top, A: Response of electrical current in the medium containing unstimulated cells. Top middle, B: Macrophages were incubated for 24 h with a mixture (Cytomix) of IL-1β (1,500 pg/mL) and TNF-α (3,000 pg/mL). Macrophages were incubated for 24 h with Cytomix in the presence of erythromycin (bottom middle, C) [10−4 mol/L] or roxithromycin (bottom, D) [RXM; 10−4 mol/L]. See Figure 5 for abbreviations not used in the text. Figure from Tamaoki et al,23 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 7. Effects of macrolides on IgG-ICx-induced iNOS messenger RNA expression by rat pulmonary alveolar macrophages. The cells were stimulated with IgG-ICx (3 μg/mL) in the absence (B) or presence of erythromycin (C), roxithromycin (D), and josamycin (E) at a concentration of 10−4 mol/L, and dexamethasone (F) at 10−7 mol/L. In the control experiment, macrophages were incubated with saline solution alone (A). Total RNA was isolated after a 24-h incubation period, and was analyzed by Northern blotting with iNOS and β-actin complementary DNA probes. Figure from Tamaoki et al,23 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 8. Effects of clarithromycin on LPS-induced MUC5AC messenger RNA expression in NCI-H292 cells. The cells were incubated with LPS (10−5 mol/L) for 72 h in the absence or presence of clarithromycin (10−4 mol/L). Upper panels: Northern blot; lower panels: in situ hybridization. See Figure 2 for abbreviations not used in the text.Grahic Jump Location
Figure Jump LinkFigure 9. Changes in sputum volume in patients with chronic bronchitis, bronchiectasis, or DPB. The administration of clarithromycin (•) or placebo (○) was conducted for 8 weeks, after a 2-week run-in period. Values are given as the mean ± SE (clarithromycin group, n = 16; placebo group, n = 15). *** = p < 0.001 (significantly different from values for placebo). See Figure 2 for abbreviations not used in the text. Figure from Tamaoki et al,9 and used with permission.Grahic Jump Location
Tamaoki, J (1997) Interstitial, inflammatory, and occupational lung disease: diffuse panbronchiolitis.Clin Pulm Med4,324-329
 
Kudoh, S, Uetake, T, Hagiwara, K, et al Clinical effects of low-dose long-term erythromycin chemotherapy on diffuse panbronchiolitis.Nippon Kyobu Shikkan Gakkai Zasshi1987;25,632-642
 
Sakito, O, Kadota, J, Kohno, S, et al Interleukin 1 beta, tumor necrosis factor alpha, and interleukin 8 in bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis: a potential mechanism of macrolide therapy.Respiration1996;63,42-48
 
Kohyama, T, Takizawa, H, Kawasaki, S, et al Fourteen-member macrolides inhibit interleukin-8 release by human eosinophils from atopic donors.Antimicrob Agents Chemother1999;43,907-911
 
Takizawa, H, Desaki, M, Ohtoshi, T, et al Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells.Am J Respir Crit Care Med1997;156,266-271
 
Khair, OA, Devalia, JL, Abdelaziz, MM, et al Effect of erythromycin onHaemophilus influenzaeendotoxin-induced release of IL-6, IL-8 and sICAM-1 by cultured human bronchial epithelial cells.Eur Respir J1995;8,1451-1457
 
Mitsuyama, T, Furuno, T, Hidaka, K, et al Inhibition by erythromycin of human pulmonary artery endothelial cell injury induced by human neutrophils.Respiration1997;64,206-210
 
Kadir, T, Izzetin, FV, Cevikbas, A, et al In vitroeffects of clarithromycin on human polymorphonuclear leukocyte functions.Chemotherapy2000;46,198-203
 
Tamaoki, J, Takeyama, K, Tagaya, E, et al Effect of clarithromycin on sputum production and its rheological properties in chronic respiratory tract infections.Antimicrob Agents Chemother1995;39,1688-1690
 
Labro, MT Anti-inflammatory activity of macrolides: a new therapeutic potential?J Antimicrob Chemother1998;41(suppl),37-46
 
Esterly, NB, Furey, NL, Flanagan, LE The effect of antimicrobial agents on leukocyte chemotaxis.J Invest Dermatol1978;70,51-55
 
Nelson, S, Summer, WR, Terry, PB, et al Erythromycin-induced suppression of pulmonary antibacterial defenses: a potential mechanism of superinfection in the lung.Am Rev Respir Dis1987;136,1207-1212
 
Forsgren, A, Schimeling, D Effect of antibiotics on chemotaxis of human leukocytes.Antimicrob Agents Chemother1977;11,580-584
 
Fernades, AC, Anderson, R, Theron, AJ, et al Enhancement of human polymorphonuclear leukocyte motility by erythromycinin vitroandin vivo.S Afr Med J1983;66,173-177
 
Kawasaki, S, Takizawa, H, Takayuki, O, et al Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro.Antimicrob Agents Chemother1998;42,1499-1502
 
Tamaoki, J, Takeyama, K, Yamawaki, I, et al Lipopolysaccharide-induced goblet cell hypersecretion in the guinea pig trachea: inhibition by macrolides.Am J Physiol1997;272,L15-L19
 
Aoki, Y, Kao, PN Erythromycin inhibits transcriptional activation of NF-κB but not NFAT, through calcineurin-independent signaling in T cells.Antimicrob Agents Chemother1999;43,2678-2684
 
Venkatakrishnan, A, Stecenko, AA, King, G, et al Exaggerated activation of nuclear factor-κB and altered IκB-α processing in cystic fibrosis bronchial epithelial cells.Am J Respir Cell Mol Biol2000;23,396-403
 
Krunkosky, TM, Fischer, BM, Martin, LD, et al Effects of TNF-α on expression of ICAM-1 in human airway epithelial cellsin vitro.Am J Respir Cell Mol Biol2000;22,685-692
 
Ichiyama, T, Nishikawa, M, Yoshitomi, T, et al Clarithromycin inhibits NF-κB activation in human peripheral blood mononuclear cells and pulmonary epithelial cells.Antimicrob Agents Chemother2001;45,44-47
 
Fujii, Y, Goldberg, P, Hussain, SNA Contribution of macrophages to pulmonary nitric oxide production in septic shock.Am J Respir Crit Care Med1998;157,1645-1651
 
Kennedy, NJ, Duncan, AW Acute meningococcaemia: recent advances in management (with particular reference to children).Anaesth Intensive Care1996;24,197-216
 
Tamaoki, J, Kondo, M, Kohri, K, et al Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.J Immunol1999;163,2909-2915
 
Tamaoki, J, Nakata, J, Tagaya, E, et al Effects of roxithromycin and erythromycin on interleukin 8-induced neutrophil recruitment and goblet cell secretion in guinea pig tracheas.Antimicrob Agents Chemother1996;40,1726-1728
 
Rubin, BK Physiology of airway mucus clearance.Respir Care2002;47,761-768
 
Reid, CJ, Gould, S, Harris, A Developmental expression of mucin genes in the human respiratory tract.Am J Respir Cell Mol Biol1997;17,592-598
 
Voynow, JA, Young, LR, Wang, Y, et al Neutrophil elastase increasesMUC5ACmRNA and protein expression in respiratory epithelial cells.Am J Physiol1999;276,L835-L843
 
Tagaya, E, Tamaoki, J, Kondo, M, et al Effect of a short course of clarithromycin therapy on sputum production in patients with chronic airway hypersecretion.Chest2002;122,213-218
 
Tamaoki, J, Isono, K, Sakai, N, et al Rythromycin inhibits Cl secretion across canine tracheal epithelial cells.Eur Respir J1992;5,234-238
 
Ikeda, K, Wu, D, Takasaka, T Inhibition of acetylcholine-evoked Cl−currents by 14-membered macrolide antibiotics in isolated acinar cells of the guinea pig nasal gland.Am J Respir Cell Mol Biol1995;13,449-454
 

Figures

Figure Jump LinkFigure 1. Macrolides and inflammation. Proposed immunomodulatory activities induced by macrolides (derived from in vitro and ex vivo data). Figure from Labro,10 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 2. Effects of macrolides on airway epithelial ICAM-1 expression. The rat tracheal epithelial cells were incubated with LPS (10−5 mol/L) in the absence and presence of various concentrations of clarithromycin (CAM; 0.1 to 100 μmol/L) or high concentrations of amoxicillin (AMPC), cefaclor (CCL), or amikacin (AMK) at 100 μmol/L. Data given as the mean ± SE (n = 6 for each column). ** = p < 0.01 (significantly different from values for LPS alone).Grahic Jump Location
Figure Jump LinkFigure 3. Time course of the effects of antibiotics on LPS (5 mg/kg)-induced recruitment of neutrophils into the guinea pig tracheal mucosa. The guinea pigs were given inhaled saline or LPS, and some animals were treated for 1 week with oral clarithromycin (10 mg/kg) or amoxicillin (30 mg/kg), and then given inhaled LPS. Responses are expressed as the number of neutrophils per 100 epithelial cells in the tracheal mucosa. Data are given as the mean ± SE (n = 9 for each point). * = p < 0.05; *** = p < 0.001 (both significantly different from control values of the saline solution group). † = p < 0.05; †† = p < 0.01 (both significantly different from the corresponding values for LPS alone). See Figure 2 for abbreviations not used in the text. Figure from Tamaoki et al,16 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 4. Representative Western blot of nuclear extracts of U-937, Jurkat, and A549 cells revealing that pretreatment with clarithromycin inhibited NF-κB activation induced by TNF-α in a concentration-dependent manner. Figure from Ichiyama et al,20 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 5. Time course of exhaled NO concentration in rats. The animals were given 50 mg/kg erythromycin (EM), josamycin (JM), amoxicillin, cefaclor, or vehicle alone (0.9% sterile saline solution). After intrapulmonary deposition of IgG-ICx at time 0, the concentration of NO in exhaled air was measured by a chemiluminescence analyzer. In the control group, the animals were not subjected to IgG-ICx. Data are given as the mean ± SE (n = 12 for each point). See Figure 2 for abbreviations not used in the text. Figure from Tamaoki et al,23 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 6. Representative tracing of the current detected by an NO-selective electrode in Roswell Park Memorial Institute medium containing rat pulmonary alveolar macrophages. After equilibration, L-arginine (L-Arg; 10−3 mol/L) was added to the medium (arrows). Top, A: Response of electrical current in the medium containing unstimulated cells. Top middle, B: Macrophages were incubated for 24 h with a mixture (Cytomix) of IL-1β (1,500 pg/mL) and TNF-α (3,000 pg/mL). Macrophages were incubated for 24 h with Cytomix in the presence of erythromycin (bottom middle, C) [10−4 mol/L] or roxithromycin (bottom, D) [RXM; 10−4 mol/L]. See Figure 5 for abbreviations not used in the text. Figure from Tamaoki et al,23 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 7. Effects of macrolides on IgG-ICx-induced iNOS messenger RNA expression by rat pulmonary alveolar macrophages. The cells were stimulated with IgG-ICx (3 μg/mL) in the absence (B) or presence of erythromycin (C), roxithromycin (D), and josamycin (E) at a concentration of 10−4 mol/L, and dexamethasone (F) at 10−7 mol/L. In the control experiment, macrophages were incubated with saline solution alone (A). Total RNA was isolated after a 24-h incubation period, and was analyzed by Northern blotting with iNOS and β-actin complementary DNA probes. Figure from Tamaoki et al,23 and used with permission.Grahic Jump Location
Figure Jump LinkFigure 8. Effects of clarithromycin on LPS-induced MUC5AC messenger RNA expression in NCI-H292 cells. The cells were incubated with LPS (10−5 mol/L) for 72 h in the absence or presence of clarithromycin (10−4 mol/L). Upper panels: Northern blot; lower panels: in situ hybridization. See Figure 2 for abbreviations not used in the text.Grahic Jump Location
Figure Jump LinkFigure 9. Changes in sputum volume in patients with chronic bronchitis, bronchiectasis, or DPB. The administration of clarithromycin (•) or placebo (○) was conducted for 8 weeks, after a 2-week run-in period. Values are given as the mean ± SE (clarithromycin group, n = 16; placebo group, n = 15). *** = p < 0.001 (significantly different from values for placebo). See Figure 2 for abbreviations not used in the text. Figure from Tamaoki et al,9 and used with permission.Grahic Jump Location

Tables

References

Tamaoki, J (1997) Interstitial, inflammatory, and occupational lung disease: diffuse panbronchiolitis.Clin Pulm Med4,324-329
 
Kudoh, S, Uetake, T, Hagiwara, K, et al Clinical effects of low-dose long-term erythromycin chemotherapy on diffuse panbronchiolitis.Nippon Kyobu Shikkan Gakkai Zasshi1987;25,632-642
 
Sakito, O, Kadota, J, Kohno, S, et al Interleukin 1 beta, tumor necrosis factor alpha, and interleukin 8 in bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis: a potential mechanism of macrolide therapy.Respiration1996;63,42-48
 
Kohyama, T, Takizawa, H, Kawasaki, S, et al Fourteen-member macrolides inhibit interleukin-8 release by human eosinophils from atopic donors.Antimicrob Agents Chemother1999;43,907-911
 
Takizawa, H, Desaki, M, Ohtoshi, T, et al Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells.Am J Respir Crit Care Med1997;156,266-271
 
Khair, OA, Devalia, JL, Abdelaziz, MM, et al Effect of erythromycin onHaemophilus influenzaeendotoxin-induced release of IL-6, IL-8 and sICAM-1 by cultured human bronchial epithelial cells.Eur Respir J1995;8,1451-1457
 
Mitsuyama, T, Furuno, T, Hidaka, K, et al Inhibition by erythromycin of human pulmonary artery endothelial cell injury induced by human neutrophils.Respiration1997;64,206-210
 
Kadir, T, Izzetin, FV, Cevikbas, A, et al In vitroeffects of clarithromycin on human polymorphonuclear leukocyte functions.Chemotherapy2000;46,198-203
 
Tamaoki, J, Takeyama, K, Tagaya, E, et al Effect of clarithromycin on sputum production and its rheological properties in chronic respiratory tract infections.Antimicrob Agents Chemother1995;39,1688-1690
 
Labro, MT Anti-inflammatory activity of macrolides: a new therapeutic potential?J Antimicrob Chemother1998;41(suppl),37-46
 
Esterly, NB, Furey, NL, Flanagan, LE The effect of antimicrobial agents on leukocyte chemotaxis.J Invest Dermatol1978;70,51-55
 
Nelson, S, Summer, WR, Terry, PB, et al Erythromycin-induced suppression of pulmonary antibacterial defenses: a potential mechanism of superinfection in the lung.Am Rev Respir Dis1987;136,1207-1212
 
Forsgren, A, Schimeling, D Effect of antibiotics on chemotaxis of human leukocytes.Antimicrob Agents Chemother1977;11,580-584
 
Fernades, AC, Anderson, R, Theron, AJ, et al Enhancement of human polymorphonuclear leukocyte motility by erythromycinin vitroandin vivo.S Afr Med J1983;66,173-177
 
Kawasaki, S, Takizawa, H, Takayuki, O, et al Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro.Antimicrob Agents Chemother1998;42,1499-1502
 
Tamaoki, J, Takeyama, K, Yamawaki, I, et al Lipopolysaccharide-induced goblet cell hypersecretion in the guinea pig trachea: inhibition by macrolides.Am J Physiol1997;272,L15-L19
 
Aoki, Y, Kao, PN Erythromycin inhibits transcriptional activation of NF-κB but not NFAT, through calcineurin-independent signaling in T cells.Antimicrob Agents Chemother1999;43,2678-2684
 
Venkatakrishnan, A, Stecenko, AA, King, G, et al Exaggerated activation of nuclear factor-κB and altered IκB-α processing in cystic fibrosis bronchial epithelial cells.Am J Respir Cell Mol Biol2000;23,396-403
 
Krunkosky, TM, Fischer, BM, Martin, LD, et al Effects of TNF-α on expression of ICAM-1 in human airway epithelial cellsin vitro.Am J Respir Cell Mol Biol2000;22,685-692
 
Ichiyama, T, Nishikawa, M, Yoshitomi, T, et al Clarithromycin inhibits NF-κB activation in human peripheral blood mononuclear cells and pulmonary epithelial cells.Antimicrob Agents Chemother2001;45,44-47
 
Fujii, Y, Goldberg, P, Hussain, SNA Contribution of macrophages to pulmonary nitric oxide production in septic shock.Am J Respir Crit Care Med1998;157,1645-1651
 
Kennedy, NJ, Duncan, AW Acute meningococcaemia: recent advances in management (with particular reference to children).Anaesth Intensive Care1996;24,197-216
 
Tamaoki, J, Kondo, M, Kohri, K, et al Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.J Immunol1999;163,2909-2915
 
Tamaoki, J, Nakata, J, Tagaya, E, et al Effects of roxithromycin and erythromycin on interleukin 8-induced neutrophil recruitment and goblet cell secretion in guinea pig tracheas.Antimicrob Agents Chemother1996;40,1726-1728
 
Rubin, BK Physiology of airway mucus clearance.Respir Care2002;47,761-768
 
Reid, CJ, Gould, S, Harris, A Developmental expression of mucin genes in the human respiratory tract.Am J Respir Cell Mol Biol1997;17,592-598
 
Voynow, JA, Young, LR, Wang, Y, et al Neutrophil elastase increasesMUC5ACmRNA and protein expression in respiratory epithelial cells.Am J Physiol1999;276,L835-L843
 
Tagaya, E, Tamaoki, J, Kondo, M, et al Effect of a short course of clarithromycin therapy on sputum production in patients with chronic airway hypersecretion.Chest2002;122,213-218
 
Tamaoki, J, Isono, K, Sakai, N, et al Rythromycin inhibits Cl secretion across canine tracheal epithelial cells.Eur Respir J1992;5,234-238
 
Ikeda, K, Wu, D, Takasaka, T Inhibition of acetylcholine-evoked Cl−currents by 14-membered macrolide antibiotics in isolated acinar cells of the guinea pig nasal gland.Am J Respir Cell Mol Biol1995;13,449-454
 
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