Antibiotics as Anti-inflammatories in Pulmonary Diseases

Richard A. Robbins, MD

Phoenix Pulmonary and Critical Care Research and Education Foundation

Gilbert, AZ USA

Abstract

The currently available evidence for the use of chronic antibiotic therapy, principally macrolides and tetracyclines, as anti-inflammatory therapy in pulmonary disorders is reviewed. Historically, treatment of a number of chronic diseases with tetracyclines showed modest benefits but reports of the successful treatment of diffuse panbronchiolitis with erythromycin stimulated research in other lung diseases as well as shifting the focus from tetracyclines to macrolides. Chronic macrolide therapy is now recommended for patients with frequent exacerbations of cystic fibrosis and COPD and considerable evidence exists for potential benefits in asthma. There is also evidence of macrolide efficacy in the prevention of obliterative bronchiolitis after lung transplantation. Small trials have suggested possible benefit of macrolides in IPF. Taken together these suggest a potential for antibiotics, particularly macrolides, in some pulmonary inflammatory disorders.

History

Based on responses to antibiotics the concept arose over 70 years ago that several common diseases might have an infectious origin. In 1949, Thomas McPherson Brown reported favorable results of tetracycline treatment for rheumatoid arthritis patients at the 7th International Congress on Rheumatic Diseases (1). It was hypothesized these effects were due to a mycoplasma infection. However, the beneficial effects of cortisone in the treatment of arthritis were described at the same meeting. The effect of tetracycline paled beside that of steroids, and the salutary effects of antibiotics on rheumatoid arthritis were largely ignored.

Acne rosacea is a common, chronic dermatologic condition, whose cause remains unknown. Tetracyclines were the first systemic drugs used in the treatment of rosacea, and have been the mainstay therapy for more than 50 years (2). More recently, sub-antimicrobial doses of tetracyclines have been shown to be effective in rosacea presumably through an anti-inflammatory effect (3). Dermatitis herpetiformis is a disease now thought to be secondary to gluten sensitivity. However, this disorder has been treated with dapsone for over 60 years despite its non-infectious origin (4).

Tetracyclines have long been used for periodontal disease with clinical benefit presumed to be from their antimicrobial properties. However, as early as 1983, Golub (5) proposed that tetracyclines might have a beneficial effect by modifying inflammation. Now the tetracyclines are thought to exert their beneficial effects by anti-inflammatory effects, anti-collagenase effects, and a reduction in bone loss (6).

In 1959 the late Neil Cherniack published a double-blind study of 67 patients with chronic bronchitis or bronchiectasis treated with tetracycline, penicillin, a combination of oleandomycin and penicillin, or placebo for 3 to 22 months (7). Patients who received tetracycline had significantly fewer lower respiratory illnesses than those treated with placebos or penicillin. The average duration of these illnesses was also shorter in patients treated with tetracycline.

The anti-inflammatory effects of the macrolides were brought to light because of their effects on an uncommon pulmonary disease, diffuse panbronchiolitis (DPB). DPB is a rare disease seen in Japan and characterized by a chronic inflammatory neutrophilic inflammation of the airways, DPB has a 5-year survival rate of just 63% but only 8% when patients’ airways became colonized with Pseudomonas aeruginosa (8). However, in the early 1980s it was discovered that chronic treatment with erythromycin resulted in dramatically improved 5-year survival to 92% (8). This improvement occurred despite a failure to eliminate the bacterial colonization and was associated with a dramatic decrease in the accumulation of airway neutrophils (8,9). Interestingly, the effect on neutrophilic inflammation was found to be a nonspecific effect of the macrolides. Other macrolides (clarithromycin, roxithromycin and azithromycin) produced a similar suppression of the neutrophilic inflammation (10).

Gradually, with a better understanding of the pathogenesis of these common disease and basic studies examining anti-inflammatory effects, the macrolides and tetracyclines were recognized as anti-inflammatories. Inflammation is proposed to play a role in the pathogenesis of a number of pulmonary disorders. The encouraging results of the above suggested that macrolides and tetracyclines might be beneficial in pulmonary inflammatory conditions. Studies have examined a number of disorders including cystic fibrosis, chronic obstructive pulmonary disease, bronchiectasis, and asthma.

Anti-inflammatory Mechanisms of Action

Macrolides and tetracyclines exert their antibacterial effects by inhibiting bacterial protein synthesis. Although the anti-inflammatory mechanisms of action of the tetracyclines and macrolides are likely multiple, one important mechanism by both is a reduction in production of a multitude of pro-inflammatory cytokines. Most of these cytokines are regulated at the transcriptional level through proteins such as nuclear factor-κβ (NF- κβ), activator protein-1 (AP-1) and/or p38 mitogen-activated protein kinases (p38 MAPK). Although the studies have varied depending on the in vitro systems examined, most have described a shortening of the half-life of pro-inflammatory cytokine mRNA usually through effect on one or more of the transcriptional control proteins (10-13).

Cystic Fibrosis

A major step in the use of antibiotics as anti-inflammatories occurred with the introduction of macrolides as adjunctive therapy in cystic fibrosis in 2003. Like diffuse panbronchiolitis, airways of cystic fibrosis patients show chronic inflammation with neutrophils which are often infected with Pseudomonas aeruginosa. Saiman et al. (14) conducted a multicenter, randomized, double-blind, placebo-controlled trial of azithromycin in cystic fibrosis patients infected with Pseudomonas. They found a reduction in exacerbations and greater weight gain in those treated with azithromycin compared to control. Following several confirming studies, cystic fibrosis patients are now commonly treated with macrolide antibiotics, especially when infected with Pseudomonas (15).

Tetracyclines have been less commonly used probably because of the staining of teeth and bone in younger, growing children. However, a recent small trial of 19 adult cystic fibrosis treated with chronic doxycycline showed an improvement in FEV1 and an increase in time to the next exacerbation compared to 20 placebo-treated controls (16). This might suggest an alternative in older patients or those at high risk for side effects from macrolides.

Non-CF Bronchiectasis

Long-term treatment with antibiotics has been recommended in patients with bronchiectasis and frequent exacerbations (17). This is based on studies showing decreased rates of exacerbations and some improvement in quality of life. It is not clear whether this effect is due to the antibacterial or anti-inflammatory properties of macrolides. In addition to Cherniak’s tetracycline trial which included bronchiectatics (7), an early MRC trial in 1957 showed that long-term twice weekly oxytetracycline over 1 year led to reduced sputum purulence, fewer days confined to bed and fewer days off work (18). Later trials in non-CF bronchiectasis have been done primarily with azithromycin and it is noted that there is an increased risk of macrolide-resistant organisms developing in these patients, as well as other risks associated with macrolide therapy including ototoxicity and QT prolongation (19).

Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is one of the most expensive diseases to treat (20). A number of studies examining costs of COPD have shown that exacerbations, especially those resulting in hospitalization, account for the majority of costs (21,22). Although treatment with glucocorticoids, long-acting beta2-agonists, and long-acting muscarinic antagonists reduce the frequency of acute exacerbations, COPD patients receiving all three of these medications still average 1.4 acute exacerbations per year (23). Beginning in the early 2000’s there were a number of studies that reported an improvement in COPD exacerbations with macrolides (24-27). This culminated in a large, NIH-sponsored, randomized, placebo-controlled, multi-center trial demonstrating that azithromycin decreased COPD exacerbations by about 20% (28).

However, despite overwhelming data that macrolides modestly reduce COPD exacerbations and professional society recommendations for macrolide use in COPD patients at high risk for COPD exacerbations, adoption of chronic therapy with macrolides in COPD has been slow (29). The major reason appears to be concerns over side effects (29). Although azithromycin is well tolerated in the majority of patients, the drug can have serious adverse effects as noted in the trials in non-CF bronchiectasis including hearing loss and QT prolongation (29). The latter is especially concerning given that within less than one year of publication of the azithromycin NIH trial in the New England Journal of Medicine, a large trial the same reported a near 3-fold increase in mortality in patients receiving macrolides (30).

Despite early trials demonstrating efficacy in decreasing COPD exacerbations, tetracyclines have received little attention compared to macrolides. In addition to Cherniak’s study (7) there is a confirming report by Norman in 1962 (31). Tetracyclines might represent an alternative to macrolides in patients at high risk for complications from the macrolides.

Asthma

Asthma, like cystic fibrosis and COPD, is an inflammatory airway disease although usually characterized by eosinophilic inflammation. Studies suggesting macrolides might be useful as anti-inflammatories in asthma go back as far as 1970 (32). After the initial study by Itkin and Menzel (32), few studies were performed until the 2000’s. However, a 1993 study from National Jewish suggested troleandomycin might be useful as a steroid-sparing agent in children with asthma and two Japanese studies published in 1999 and 2000 with roxithromycin and clarithromycin both gave positive results in small numbers of patients (33-35).

In studies whose logic is reminiscent of Thomas McPherson Brown’s concept of mycoplasma infection in rheumatoid arthritis, Kraft et al. (36) investigated chronic chlamydia and mycoplasma infection in asthma and the response to macrolide therapy. In 2002 they reported that clarithromycin treatment increased FEV1 in asthmatics but only in those with evidence of C. pneumoniae or M. pneumoniae infection by PCR in upper and lower airway samples. Sutherland and co-workers (37) also showed improvement in airway hyper-responsiveness with clarithromycin therapy but in both PCR-positive and negative groups. The difference likely resides in identifying and chronic chlamydia and mycoplasma infection. A positive PCR does not necessarily equate to chronic infection and the serologic results from different assays are variable complicating these studies (38,39).

A number of studies have been conducted since Kraft’s investigation examining clarithromycin or azithromycin and assessing various clinical responses and inflammatory parameters in asthma (40-47). These studies have been inconsistent with some showing benefits while others did not. A Cochrane review in 2005 by Richeldi et al. (48) and a review article in 2014 by Wong et al. (49) both concluded that insufficient data existed to recommend chronic macrolide therapy in asthma.

The inconsistency in these results might be explained by the small patient numbers and because various phenotypes of asthma were included. Brusselle et al. (47) reported that azithromycin treatment significantly reduced exacerbation rates only in patients with severe neutrophilic asthma compared with placebo. However, neutrophilic asthma has been associated with increased bacterial load confusing whether benefits are due to an anti-inflammatory or an antibiotic effect (50). Furthermore, clarithromycin reduces neutrophil numbers in patients with severe asthma and it has been suggested that those patients with a neutrophilic phenotype might respond better to the anti-inflammatory effects of macrolide therapy (44,51).

A recent well-done recent study from Australia might tip the balance in favor of chronic macrolide therapy in difficult-to-control asthma. Gibson et al. (52) performed a randomized, double-blind, placebo controlled parallel group trial to determine whether oral azithromycin decreases the frequency of asthma exacerbations in 420 adults with symptomatic asthma despite current use of inhaled corticosteroid and a long-acting bronchodilator. Patients were randomly assigned to receive azithromycin 500 mg or placebo three times per week for 48 weeks. Azithromycin reduced asthma exacerbations by nearly half and significantly improved asthma-related quality of life.

Tetracyclines as anti-inflammatories in asthma have received much less attention than the macrolides. In 2008 Daoud et al. (53) reported that minocycline allowed for a reduction in steroid dose in asthmatics who were steroid-dependent. A study from India demonstrated an improvement in post bronchodilator FEV1, the FVC, and the FEF (25-75) in asthmatics treated with doxycycline (54).

Obliterative Bronchiolitis

Obliterative bronchiolitis (OB) has historically gone by a variety of terms including bronchiolitis obliterans, bronchiolitis obliterans with organizing pneumonia (BOOP) and, more recently, cryptogenic organizing pneumonia (COP) although some now separate OB as a separate entity (55). Histologically OB is very similar to diffuse panbronchiolitis, and in fact, panbronchiolitis has been grouped with OB (55). The OB histological pattern is now most commonly seen after lung transplantation or hematopoietic stem-cell transplantation (HSCT). However, OB can be seen with autoimmune disease, particularly rheumatoid arthritis; exposure to inhalational toxins such as sulfur dioxide, hydrogen sulfide, nitrogen oxides, and fly ash; and as an unusual complication following infection with adenovirus, measles virus, or mycoplasma (55).

The treatment of OB is usually corticosteroids or other immunosuppressants (55). However, since OB can result in death or decreased respiratory function, studies with adjunctive therapy or prevention of OB have been of interest. Azithromycin has resulted in improved pulmonary function in approximately 50% of lung-transplant recipients with obliterative bronchiolitis (56,57). A retrospective analysis indicated that the administration of azithromycin in patients with obliterative bronchiolitis after lung transplantation is associated with improved survival (58). Studies examining azithromycin after HSCT were done given the beneficial effects after lung transplantation. Surprisingly, the results were completely different. In a randomized clinical trial that included 465 patients, 2-year airflow decline-free survival was significantly worse for the azithromycin group than for the placebo group (59). The trial was terminated early for a significant increased risk in the azithromycin group of hematological relapses. The FDA recently issued a warning against using chronic azithromycin therapy in HSCT.

There is a paucity of data on treatment of OB with macrolides in non-transplant conditions. In 1993, Ichikawa et al. (60) used erythromycin for 3-4 months in six patients with a diagnosis of bronchiolitis obliterans OP confirmed on histological examination. All improved by the completion of therapy. However, a recent trial of azithromycin in eight patients with post-infectious OB did not produce an improvement in pulmonary function parameters (61). No studies were identified using tetracyclines as therapy in OB.

Cryptogenic Organizing Pneumonia

This entity, which was formerly known as bronchiolitis with organizing pneumonia (BOOP) can involve small airways, but also involves alveolar ducts and alveoli and can present as patchy peripheral opacities (62). It is considered an inflammatory disease which is usually very responsive to corticosteroid therapy, but may relapse when steroid therapy is withdrawn (63). There are several reports now that cryptogenic organizing pneumonia responds to treatment with macrolide and suggest that long term suppression with macrolides can avoid side effects associated with long term steroid therapy (63).

Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a condition that has also been associated with neutrophils but with inflammation in the alveoli rather than the airways. With the introduction of nintedanib and pirfenidone and the realization that corticosteroids are of no benefit, the management of IPF has dramatically changed over the past decade (64). A recent publication done during the course of the shift in IPF therapy suggests that azithromycin added to conventional reduced the incidence of acute exacerbations (65). However, these retrospective results need to be interpreted with caution, since as noted above “conventional therapy” for IPF has changed profoundly. For example, many of the patients included in this study were subjected to corticosteroid therapy or other immunosuppressive agents, both of which are no longer recommended in IPF treatment (65). A similar study was performed by Kawamura et al. (66) performed from 2005-16. This single-center retrospective study of patients with IPF demonstrated that treatment of 38 consecutive patients with azithromycin (500 mg/day) for 5 days led to increased survival compared to 47 historical controls treated with a fluoroquinolone-based regimen.

A trial with minocycline in IPF was registered at clinicaltrials.gov but results were apparently never published (67). A small trial in 6 IPF patients treated with doxycycline for 24 weeks showed significant improvement in 6-minute walk time, St. George’s Respiratory Questionnaire, FVC, and quality of life compared to 6 controls (68).

Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease that lead to progressive cystic destruction of the lungs. A recent study with doxycycline in LAM patients produced no effect upon vital capacity, gas transfer, shuttle walk distance or quality of life (69). The authors concluded that it is unlikely that doxycycline has a useful effect in LAM.

Summary

Macrolides are clinically useful in reducing exacerbations of cystic fibrosis, chronic obstructive pulmonary disease, bronchiolitis obliterans after lung transplantation, and possibly asthma. Tetracyclines might be considered as a substitute in some situations.

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Cite as: Robbins RA. Antibiotics as anti-inflammatories in pulmonary diseases. Southwest J Pulm Crit Care. 2018;17(3):97-107. doi: https://doi.org/10.13175/swjpcc104-18 PDF 

Tiotropium Bromide Inhibits Human Monocyte Chemotaxis

Makoto Kurai MD (mkurai08@shinshu-u.ac.jp)^{1, 2, 3}

Richard A. Robbins MD (rickrobbins@cox.net)^{1, 2, 5}

Sekiya Koyama MD (syjskoyama@go.tvm.ne.jp) ⁴

Jun Amano MD, PhD (junamano@shinshu-u.ac.jp) ³

John M. Hayden PhD (John.Hayden2@va.gov)¹

¹Carl T. Hayden VA Medical Center, Phoenix, AZ, 85012, USA

²Arizona Respiratory Center, University of Arizona, Tucson, AZ, 85724, USA

³The Second Department of Surgery,

Shinshu University School of Medicine, Matsumoto 390-8621, Japan

⁴The Department of Pulmonary Internal Medicine,

National Hospital Organization Chushin Matsumoto Hospital, Matsumoto 390-0021, Japan

⁵Phoenix Pulmonary and Critical Care Research and Education Foundation, Gilbert, AZ 85295, USA

Abstract

Tiotropium bromide (Spiriva®) is used as a bronchodilator in chronic obstructive pulmonary disease (COPD).  However, clinical evidence suggests that tiotropium bromide may improve COPD by mechanisms beyond bronchodilation.  We hypothesized that tiotropium bromide may act as an anti-inflammatory agent by inhibiting monocyte chemotaxis, a process that plays an important role in the lung inflammation of COPD.  To test this hypothesis monocytes were pretreated with tiotropium bromide prior to exposure to chemotactic agents and monocyte chemotactic activity (MCA) was evaluated with a blind chamber technique.  Tiotropium bromide inhibited MCA in a dose- and time- dependent manner (respectively, p< 0.01) by directly acting on the monocyte. Acetylcholine (ACh) challenge increased MCA (p< 0.01), and tiotropium bromide effectively reduced (p< 0.01) the increase in MCA by ACh. The inhibition of MCA by tiotropium bromide was reversed by a muscarinic type 3 (M₃)-muscarinic receptor antagonist (p< 0.01), and was not effected by an M₂ receptor antagonist.  Furthermore, a selective M₃ receptor agonist, cevimeline, and G_q protein stimulator, Pasteurella multocida toxin, significantly increased MCA (P < 0.01), and tiotropium bromide pretreatment reduced (p< 0.01) the increase in MCA induced by these agents. These results suggest that tiotropium might regulate monocyte chemotaxis, in part, by interfering with M₃-muscarinic receptor coupled G_q protein signal transduction. These results provide new insight that an anti-cholinergic therapeutic may provide anti-inflammatory action in the pulmonary system.  

Introduction

Tiotropium bromide is a novel long-acting, inhaled, anticholinergic agent that is used as a treatment for chronic obstructive pulmonary disease (COPD). It has reported to have beneficial effects on the pulmonary function compared to other anticholinergic (short-acting) and beta-2 adrenergic agents. Although tiotropium predominantly functions as a bronchodilator, it reduces the development of acute exacerbations in COPD (1,2). These effects suggest that tiotropium might possess some function as an anti-inflammatory agent in addition to a bronchodilator (3-6). Tiotropium also has the potential to inhibit acetylcholine-induced proliferation of fibroblasts and myofibroblasts (7), further suggesting plausible beneficial influences on airway remodeling in COPD patients.

Acetylcholine (ACh) participates in the control of airway tone, which is an important factor contributing to the airway obstruction in the airway diseases (8). Anticholinergic agents effectively reverse the parasympathetic nerve stimulation and attenuate the smooth muscle contraction in the airway. This effect is especially important in regard to COPD because the  parasympathetic nerve stimulation is augmented in the airway inflammation (9). Recent studies have demonstrated that ACh participates in the inflammatory processes through the release of chemotactic factors from alveolar macrophages and bronchial epithelial cells which subsequently promote inflammatory cell infiltration (10,11). Moreover, ACh treatment induces the proliferation of fibroblasts and myofibroblasts (7).  Recently, it has also been reported that ACh synthesizing enzyme, choline acetyltransferase, is ubiquitously expressed in the airway cells (12), and that lung epithelial cells and pulmonary inflammatory cells can produce ACh and express functional muscarinic receptors (13). Thus, ACh may be involved in the airway inflammation, remodeling, and other pathophysiological phenomena acting in an autocrine and paracrine fashion (13,14).

In the present study, we evaluated tiotropium as an anti-inflammatory agent in monocyte chemotaxis. Moreover, we assessed key intracellular mechanisms that may regulate the inhibition of tiotropium-induced monocyte chemotaxis. We found that muscarinic receptor coupled G-protein signal transduction plays a role in the migration of monocyte. The results demonstrated that tiotropium inhibited the capability of monocytes to migrate to the chemotactic factor MCP-1, at least partly, by modulating muscarinic type 3 (M₃) receptor coupled G_q protein signal transduction. These data suggest that tiotropium may play an anti-inflammatory role by inhibiting monocyte chemotaxis.

Materials and Methods

This study was conducted with the approval from the Research and Development and Institutional Review Board Committees of the Carl T. Hayden Veteran’s Affairs Medical Center, Phoenix, Arizona.

Purification of peripheral blood monocytes and the monocyte chemotaxis assay.  Mononuclear cells for the chemotaxis assay were obtained from normal human volunteers by Ficoll-Hypaque density centrifugation to separate red blood cells and neutrophils from mononuclear cells (15). The enriched population of monocytes isolated by this method routinely display >98% viability as assessed by trypan blue exclusion (Sigma-Aldrich, St. Louis, MO). The cells were suspended in HBSS (Invitrogen, Carlsbad, CA) containing 2% bovine serum albumin (BSA, Sigma-Aldrich) at pH 7.5 to give a final concentration of 5 x 10⁶ cells/ml. The suspension was then used for the monocyte chemotaxis assay.              

The monocyte chemotaxis assay was performed by a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD) as described previously (16). Briefly, 25 μL of a solution containing 100 ng/ml of recombinant human monocyte chemoattractant protein (MCP-1; Sigma-Aldrich) was placed into the lower wells, and a 10 μm thick polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA) with a pore size of 5 μm was placed over the bottom chamber. The concentration of MCP-1 used in this study was established previously (17). The silicon gasket and top pieces of the chamber were applied, and 50 μL of the cell suspension described above was placed into the top wells above the filter. The chambers were incubated in humidified air in 5% CO2 at 37° C for 90 min. After incubation, the chamber was disassembled, and non-migrated cells were wiped away from the filter. The filter was then immersed in methanol for 5 min, stained with Diff-Quik (American Scientific Product, McGraw Park, IL), and mounted on a glass slide. Cells that completely migrated through the filter were counted by using light microscopy (1000x) in at least 10 random high-power fields (HPF) per well.

Effects of tiotropium on MCP-1 - induced MCA. To evaluate the dose-dependent effects of tiotropium, monocytes were treated with 0, 0.01, 0.1, 1.0, 10, 100, 1000 nM for 30 min at 37º C in a humidified 5% CO2 atmosphere prior to MCA assay. After this interim, the cells were transferred to the chemotaxis chamber and exposed to the chemotactic agent for an additional 90 min in the environment described above.

To assess the time-dependent effects of tiotropium, monocytes were treated with 1000 nM tiotropium (maximal responsive dose) for varying times (0, 15, 30, 60, 90, 120 min) prior to MCA assay, and then they were transferred to the chemotaxis chamber and exposed to MCP-1 for additional 90 min.

The viability of monocytes after tiotropium exposure was evaluated by examining cell morphology and measuring lactate dehydrogenase (LDH) activity in the supernatant fluids. LDH activity was assessed by use of a commercially available kit (Tox-7; Sigma-Aldrich) according to the manufacture’s instructions. Dose- (up to 1000 nM) and time- (up to 3 hours) effects of tiotropium exposure on LDH activity in the supernatant fluids were not significant in these experiments (p > 0.3; data not shown).  Based on observations of cellular morphology and LDH activity, no toxicity was observed with the concentration or time of exposure of tiotropium used in this study.

Effects of ACh on MCP-1 induced MCA and reversal by tiotropium. Since tiotropium inhibited MCP-1 induced MCA, we determined whether ACh directly interacted with the monocytes and increased their activity in vitro. Monocytes were treated with 100 μM ACh (Sigma-Aldrich) for 60 min at 37° C prior to transfer to the chemotaxis chamber and exposure to MCP-1 for an additional 90 min. The concentration of ACh used in this study was based on a dose that was established previously (8).  Furthermore, to test the effects of tiotropium on MCA that was augmented by ACh, monocytes were treated with 1000 nM of tiotropium for 30 min at 37° C prior to ACh challenge. After this interim of exposure, the chemotaxis assays were performed as described above.

Effects of muscarinic receptor antagonists on MCP-1 induced MCA and abolishment of   tiotropium effects. To determine which muscarinic receptor is involved with the regulation of MCA by tiotropium, gallamine (Sigma-Aldrich) an M₂-receptor antagonist and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (Sigma-Aldrich) an M₃-receptor antagonist were used at the concentration of 300 µM, respectively, according to previous reports (10,11). Monocytes were exposed to these agents for 30 min before treatment with 1000 nM tiotropium for an additional 30 min. At this time the cells were transferred to the chemotaxis chamber as described above.

Effects of M₃-receptor agonists on MCP-1 induced MCA. Because M₃-receptor seemed to be involved with the ACh modulation, we evaluated   whether tiotropium affected the reaction which was mediated through the M₃-receptor agonist. A selective M₃-receptor agonist, cevimeline HCl (EVOXAC^®, Daiichi Sankyo, Inc., Parsippany, NJ), was used at the concentration of 300 μM. Monocytes were pretreated with 1000 nM tiotropium for 30 min at 37° C before the addition of cevimeline for an additional 30 min at 37° C prior to conducting the MCA assay.

Role of muscarinic receptor coupled Gq protein in MCA. Because M₃ receptors are reported to couple with G_q proteins, we evaluated the effects of G_q protein stimulation on MCA.  Pasteurella multocida toxin (PMT) was used as a G_q protein stimulator at the concentration of 100 ng/ml as previously reported (18,19). Monocytes were pretreated with 1000 nM tiotropium for 30 min before exposure to PMT for an addition 30 min at 37º C. The cells then were transferred to the microwell chambers and exposed to MCP-1 as described above.

Statistical Analyses. Unless stated otherwise all the results presented are expressed as means ± SEM from at least 3 individual experiments. Data were analyzed by one-way ANOVA, followed by selected post-hoc Neuman-Keuls multiple comparison tests. In selected experiments, Dunnett’s test was used to examine treatment effects compared to non-treated controls. In all cases, p < 0.05 was considered significant.

Results

Tiotropium inhibition of MCP-1 induced MCA.  Tiotropium significantly inhibited MCP-1-induced MCA in a dose-dependent manner (Figure 1A, p< 0.005). The lowest dose to inhibit MCA was 10 nM (p< 0.05) and the greatest inhibition (~40%; p< 0.01) occurred with a dose of 1000 nM tiotropium. Tiotropium also significantly inhibited MCP-1-induced MCA in a time-dependent manner (Figure 1B, p< 0.001). MCA was inhibited significantly after 60 min (p< 0.01) and reached a plateau after 90 min of exposure to 1000 nM tiotropium.

Figure 1. Panel A: A dose-dependent inhibition of monocyte chemoattractant protein (MCP-1) induced MCA by tiotropium. Monocytes were treated with tiotropium for 30 min before the MCA assay (n = 3). Chemotactic activity is on the ordinate and the concentration of tiotropium is on the abscissa. Values are expressed as means ± SEM. Dose effect by ANOVA (p < 0.005). *p< 0.05, **p< 0.01 means differ compared with non-tiotropium treated controls.  Panel B:  A time-dependent inhibition of MCP-1 induced MCA by tiotropium. Monocytes were treated with 1000 nM tiotropium under all conditions (n = 3). Chemotactic activity is on the ordinate and the duration of tiotropium exposure is on the abscissa. Values are expressed as means ± SEM. Time effect by ANOVA (p< 0.001). *p< 0.01 means differ compared to non-treated controls.

ACh augmentation of MCP-1 induced MCA. ACh challenge of the monocytes increased their MCA ~1.2-fold (Figure 2; p< 0.01).  In addition, pretreatment of the monocytes with tiotropium inhibited (p< 0.01) MCA that was stimulated by ACh to a level that was lower than non-treated controls (Figure 2).

Figure 2. ACh augments MCA induced by MCP-1 exposure. Monocytes were treated with 1000 nM tiotropium for 30 min prior to exposure of 100 μM Ach (n = 3). Chemotactic activity is on the ordinate and the experimental groups are on the abscissa. Values are expressed as means ± SEM. a vs. b; a vs. c means differ (p< 0.01); c vs. d means differ (p < 0.001).

Muscaric receptor type 3 inhibitor reversed the effects of tiotropium.  4-DAMP pretreatment of the monocytes reversed the inhibitory effect of tiotropium on MCP-1 induced MCA (Figure 3). In contrast to 4-DAMP, the pretreatment of gallamine did not alter the reduction of MCA caused by anticholinergic treatment. As shown the figure 3, both gallamine and 4-DAMP that was administered individually did not affect the chemotaxis of monocytes when exposed to MCP-1.

Figure 3. Muscarinic receptor type-3 inhibitor reversed the effects of tiotropium.  Monocytes were pre-exposed to gallamine (M₂ receptor inhibitor) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M₃ receptor inhibitor) at the concentration of 300 µM for 30 min before treatment with 1000 nM tiotropium for an additional 30 min (n = 5). Chemotactic activity is on the ordinate and the experimental groups are on the abscissa. Values are expressed as means ± SEM. a vs. b means differ (p<0.01).

Tiotropium inhibition of a specific muscarinic type 3 receptor agonist induced MCA. Cevimeline (300 µM) treatment increased MCP-1 induced MCA as compared to nontreated controls (Figure 4, ~1.4-fold; p < 0.01). This effect was greater than that displayed by individual ACh treatment (Figure 2). In addition, tiotropium abolished the increase in MCA that was augmented by cevimeline treatment (Figure 4, p< 0.01).

Figure 4. Tiotropium inhibited MCA induced by muscarinic type-3 receptor agonist treatment. Monocytes were treated with 1000 nM tiotropium for 30 minutes prior to exposure of 300 µM cevimeline for an additional 30 minutes (n = 4). Chemotactic activity is on the ordinate and the experimental groups are on the abscissa. Values are expressed as means ± SEM. a vs.b; a vs. c; c vs. d means differ (p< 0.01).

Tiotropium inhibition of G_q protein agonist induced MCA.  MCA was significantly increased (1.3-fold; p< 0.01) in response to 100 ng/ml PMT as compared to non-treated controls (Figure 5). Similar to cevimeline, this effect was higher than that of ACh administered alone.  In combination with PMT, tiotropium abolished the increase in MCA to a level that was similar to tiotropium treatment alone (Figure 5, p< 0.01). 

Figure 5. Tiotropium inhibited MCA induced by G_q protein agonist treatment. Monocytes were treated with 1000 nM tiotropium for 30 min prior to exposure of 100 ng/ml Pasteurella multocida toxin (PMT) for an additional 30 min (n = 6).  Chemotactic activity is on the ordinate and the both experimental groups are on the abscissa. Values are expressed as means ± SEM. a vs.b; a vs.c; c vs.d means differ (p< 0.001).

Discussion

In the present study, we demonstrated that tiotropium directly interacted with the monocyte and inhibited MCP-1 induced MCA.  The exogenous addition of ACh increased MCP-1-induced MCA, and tiotropium reversed the increase in MCP-1 induced MCA by ACh.  Interestingly, 4-DAMP, a M₃-receptor antagonist, abolished the effect of tiotropium. Furthermore, cevimeline, a M₃-receptor agonist, and PMT, a G_q protein stimulator, increased MCP-1 induced MCA, and tiotropium pretreatment reversed the increase in MCA by these agents.  These data may suggest that tiotropium directly interacts with monocytes and inhibits their capability to migrate to chemotactic agents by interfering with M₃ receptor coupled G_q protein signal transduction.

The initial concentrations used in this study demonstrated a reduction in MCA from tiotropium ranging from 10 through 1000 nM. The lower doses of tiotropium demonstrating a response in this study are within range to those affecting human lung fibroblast proliferation (7) and fibroblast differentiation (20).  In order to elicit a robust effect on MCA, we opted to use the highest responsive dose (1000 nM) of tiotropium bromide throughout the study. At this level, tiotropium bromide was non-toxic and remained below the estimated maximum concentration of ~2 μM to be present at the lung epithelial lining fluid after inhalation of the drug (21).

Tiotropium has been shown to possess anti-inflammatory effects (1,2,9,13).  Although the recruitment of peripheral blood monocytes to the lung is essential for innate lung immunity, it is also involved in the generation and propagation of an inflammatory response. The excessive migration of blood monocytes to the lung tissue can lead to increased number of alveolar macrophages (22,23), leading to the lung tissue injury via excessive elaboration of inflammatory cytokines, eicosanoids, proteolytic enzymes, and oxygen radicals (24-26). In this context, it might be important to suppress the excessive migration of monocytes to the site of inflammation during acute and chronic inflammation. In the present study, we demonstrated that tiotropium inhibited MCP-1 induced MCA. This result suggests that tiotropium may provide an anti-inflammatory action by inhibiting the monocytes capability to migrate to chemotactic agents that are produced at heightened levels under inflammatory conditions in the lung.

Recently, accumulating evidence demonstrates that acetylcholine and its synthesizing enzyme choline acetyltransferase (ChAT) are present not only in airway nerves, but also in various cells such as airway epithelial cells, endothelial cells, smooth muscle cells, lymphocytes, macrophages, mast cells, eosinophils and neutrophils (11). Furthermore, most inflammatory cells express functional muscarinic receptors (10,11,27,28). Muscarinic receptor agonists increase cytosolic Ca²⁺ and c-fos mRNA expression both in human T- and B- cell lines in an atropine-sensitive manner (29,30).  These findings may suggest that ACh may regulate inflammatory processes in a paracrine and/or autocrine fashion(s) in inflammatory cells (29, 31-33). In the present study, ACh directly interacted with monocytes and stimulated their chemotactic activity. This observation is consistent with the above concept that ACh can regulate inflammatory processes. We demonstrated that tiotropium inhibited the increase in MCA which was induced by ACh. Moreover, tiotropium attenuated MCP-1 induced MCA in the absence of ACh.  Several reports (12,29) suggest that ACh can be generated by ChAT that is localized in monocytes and regulate inflammatory processes in an autocrine and/or paracrine fashion(s). Based on these reports, we postulated that ACh can be generated within the monocyte in response to exterior stimuli and that tiotropium may modulate MCA that is augmented by intrinsic ACh.

Five different subtypes (M₁-M₅) of muscarinic receptor have been identified (34). Although muscarinic receptors are expressed in various inflammatory cells, the expression of each subtype seemed to be variable within each individual inflammatory cell. Although Fujii et al. (27) reported that all 5 classes of muscarinic receptors have been detected by RT-PCR in mononuclear cells, Bany et al. (35) reported that M₂-M₅ but not M₁ mRNA were detected by RT-PCR. Furthermore, Costa et al. (36,37) and Hellstom-Lindahl et al. (38) reported that only M₃-M₅, but not other subtypes, were detected by RT-PCR. Therefore, the expression profile of muscarinic receptors seemed to be variable upon experimental conditions.  Based on these reports, we investigated whether the effect of gallamine, a M₂-receptor antagonist, and 4-DAMP, a M₃-receptor antagonist, may alter the effect of tiotropium on MCA. We did not investigate other muscarinic antagonists because of the reports suggesting that the other muscarinic receptors were not strongly expressed in monocytes.  Our results indicated that tiotropium may act by binding to M₃ receptor.  Profita et al. (39) also demonstrated that M₃ receptors were observed in human blood monocytes by immunohistochemical detection. In addition, Sato et al. (11) suggested that ACh stimulated the bovine alveolar macrophages via M₃ receptor to release factors promoting monocyte chemotactic activity. Furthermore, Fujii et al. (30) reported that the muscarinic receptor agonist, oxotremorine-M, increased c-fos mRNA expression in human T- and B- cell lines via the M₃ muscarinic receptor because this effect could be blocked by 4-DAMP. Taken together, these reports suggest that ACh might be acting predominantly by M₃ receptor regulation in mononuclear cells. Our results also demonstrate that 4-DAMP inhibited the effect of suppression of MCA by tiotropium. Therefore, it is feasible that ACh can be generated by monocytes themselves and act in an autocrine and paracrine manner via M₃ muscarinic receptor interaction and that tiotropium may inhibit this reaction.

Recently, Han et al. (40) reported that a conformational change of the M₃ receptors may occur in the immediate vicinity of the binding site of a G-protein coupled receptor (GPCR) activated by diffusible ligands such as the muscarinic agonist. Furthermore, several studies suggested that  similar conformation changes occur in GPCR’s activated by diffusible ligands such as the beta 2-adrenergic receptor (41, 42). According to this context, the conformational change within the M₃ receptor might occur by a muscarinic antagonist such as 4-DAMP in monocytes. It may be possible that a conformational change in the M₃ receptor, as produced by 4-DAMP interaction, may alter the original binding site for tiotropium and thus reverse its affect on MCA.

Muscarinic receptors belong to the large family of G-protein coupled receptors (GPCR). The accumulating data has demonstrated that the “odd-numbered” muscarinic receptors (M₁, M₃, M₅) couple preferentially to G-proteins of the G_q family, whereas the “even-numbered” receptors (M₂, M₄) prefer G-proteins belonging to the G_i/o family (10).  Our result and several other reports demonstrate that the most important receptor on monocytes seem to be mediated via the M₃ muscarinic receptor.  Therefore, we examined the effect of a selective M₃ muscarinic receptor agonist, cevimeline, and a selective G_q protein stimulator, PMT, on MCA. The results from these experiments demonstrated that both cevimeline and PMT were directly acting on monocytes and augmented MCP-1 induced MCA.  In addition, tiotropium abolished the increase in MCA that were induced by these agents.  These data may suggest that tiotropium may inhibit MCA via M₃ muscarinic receptor and G_q protein signaling.  Although it has been indicated that tiotropium may inhibit MCA via M₃ receptor coupled G_q protein signaling, it remains unknown as to which intracellular signaling pathways beyond G-protein induction may be important in the regulation of MCA. The effects of tiotropium in the regulation of these intracellular mechanisms in monocytes remain as an important issue to be elucidated with future research. 

In conclusion, tiotropium directly interacts with monocytes and inhibits their capability to migrate to chemotactic agents. The results in the present study provide new insight into mechanisms by which tiotropium may act as an anti-inflammatory agent in the pulmonary system. The reduction in monocyte migration may be one mechanism explaining the reduction in COPD exacerbations seen with tiotropium treatment in COPD.

Acknowledgements

Supported by a grant from Boehringer Ingelheim and the Phoenix Pulmonary and Critical Care Research and Education Foundation and the Department of Veterans Affairs. The contents do not represent the views of the Department of Veterans Affairs or the United States Government..

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Conflict of Interest Statement: M.K., S.R., R.A.R., S.K., and J.A.H. do not have any financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.A.H. received a research grant ($49,900) from Boehringer-Ingelheim (2006-7) to conduct this study.

Reference as: Kurai M, Robbins RA, Koyama S, Amano J, Hayden JM. Tiotropium bromide inhibits human monocyte chemotaxis. Southwest J Pulm Crit Care 2012;5:86-99. (Click here for a PDF version of the manuscript).