November 2012 Pulmonary Case of the Month: The Wolves Are at the Door

Lewis J. Wesselius, MD

Department of Pulmonary Medicine

Mayo Clinic Arizona

Scottsdale, AZ

History of Present Illness

A 49 year old female was admitted for hypoxia, lethargy, and an abnormal chest x-ray. She was recently discharged after a 10 day outside hospital stay for a diagnosis of pneumonia treated initially with azithromycin, then clindamycin and discharged on levofloxacin. Corticosteroids given during that hospitalization and she was discharged on taper. As the steroids were tapered, she had increasing dyspnea, confusion, and lethargy. She presented to the emergency room with an abnormal CT chest x-ray and was started on meropenem, vancomycin and azithromycin, and was also given IV methylprednisolone (125 mg initial dose).

PMH, FH and SH

She had her first stroke at age 18 and walks with a cane and has some expressive aphasia. There were multiple prior episodes of pneumonia (25 in 5 years). She was diagnosed with systemic lupus erythematosis (SLE) with lupus pneumonitis (based on surgical lung biopsy) about 3-4 years prior to admission. She had a St. Jude mitral valve replacement 12 years ago and had suffered a hemorrhagic stroke presumed secondary to anticoagulation. There is also a history of nephrolithiasis and recurrent urinary tract infections and anemia with multiple prior transfusions.

Her mother died at 49 reportedly due to complications of SLE.

Physical Examination

• Temperature 37.1° C;  Blood Pressure127/75 mm Hg;  Pulse 80 beats/min; SaO2 96% on 3 LPM
• HEENT: no significant abnormalities identified
• Chest: clear to auscultation and percussion
• Cardiovascular: mechanical click, no murmur
• Extremities: trace edema

Laboratory Evaluation

• Hemoglobin 10.1 g/dL   WBC  11,900  cells/μL   Platelets 137,000 cells/μL  
• INR 2.62
• Creatinine 0.9 mg/dL  BUN 15 mmol/L
• N-terminal pro-brain natriuretic peptide (NT pro-BNP) 1,294 pg/ml
• C-reactive protein (CRP) 74.7 mg/L 
• Erythrocyte sedimentation rate (ESR) 14 mm/hr
• Drug Screen:  negative

Chest X-ray

Her chest x-ray is shown below (Figure 1).

Figure 1. Portable chest radiography at the time of admission.

Which of the following are pulmonary complications of SLE?

1. Pleuritis
2. Chronic interstitial pneumonitis
3. Acute lupus pneumonitis
4. Pulmonary hypertension
5. All of the above

Reference as: Wesselius LJ. November 2012 pulmonary case of the month: the wolves are at the door. Southwest J Pulm Crit Care 2012;5: 223-8. PDF 

October 2012 Pulmonary Case of the Month: Hemoptysis from an Uncommon Cause

Lewis J. Wesselius, MD

Department of Pulmonary Medicine

Mayo Clinic Arizona

Scottsdale, AZ

History of Present Illness

A 39 year old woman is seen with a history of cough intermittently productive of small amounts of blood or blood-tinged sputum for 4 months. She reports no other respiratory symptoms and has otherwise felt well.

PMH, FH and SH

There was no significant PMH and no prior history of lung disease. Her father has a history of Parkinson’s disease and osteosarcoma. She is a nonsmoker, does not drink alcohol, and has never abused drugs. She has 2 children and is engaged to be remarried.

Physical Examination

Her physical examination is normal.

Chest X-ray

Her chest x-ray is below (Figure 1).

Figure 1. Panel A: Frontal chest radiography. Panel B: Lateral chest radiography.

Laboratory Evaluation

Hemoglobin was 13.2 g/dL and WBC was 8400 cells/μL with a normal differential. Urinanalysis was unremarkable.

Which of the following statements regarding hemoptysis is or are true?

1. A normal chest x-ray makes a benign cause of the hemoptysis more likely
2. Most patients with lung cancer are asymptomatic
3. Hemoptysis in children is usually associated with an infection or a foreign body
4. 1 + 3
5. All of the above

Reference as: Wesselius LJ. October 2012 pulmonary case of the month: hempotypsis from an uncommon cause. Southwest J Pulm Crit Care 2012;5:169-75.  PDF

Acetylcholine Stimulation of Human Neutrophil Chemotactic Activity Is Directly Inhibited by Tiotropium Involving Gq Protein and ERK-1/2 Regulation

Makoto Kurai^1,2,3

Richard A. Robbins^1,2

Sekiya Koyama⁴

Jun Amano³

John M. Hayden¹

¹Carl T. Hayden Veterans Affairs Medical Center, Phoenix, Arizona 85012, ²Arizona Respiratory Center, University of Arizona, Tucson, Arizona 85724, ³Second Department of Surgery, Shinshu University School of Medicine, Matsumoto 390-8621, Japan, ⁴Department of Pulmonary Internal Medicine, National Hospital Organization Chushin Matsumoto Hospital, Matsumoto 399-0021, Japan

Abstract

Tiotropium, a long-acting anticholinergic, may improve chronic obstructive pulmonary disease (COPD) by mechanisms beyond bronchodilatation. We tested the hypothesis that tiotropium may act as an anti-inflammatory mediator by directly acting on and inhibiting human neutrophil chemotactic activity (NCA) that is promoted by acetylcholine (ACh) exposure.  ACh treatment increased NCA in a dose dependent manner (p < 0.001) and tiotropium pretreatment reduced ACh stimulation (dose effect; 0 to 1000 nM; p < 0.001).  Selective muscarinic receptor inhibitors demonstrated that subtype-3 (M3) receptor plays a role in NCA regulation.  In addition, NCA that was stimulated by cevimeline (M3 agonist) and pasteurella multocida toxin (PMT, M3 coupled Gq agonist). However, the increased NCA to cevimeline and PMT was reduced by tiotropium pretreatment (p < 0.001).  ACh treatment stimulated ERK-1/2 activation by promoting protein phosphorylation and tiotropium reduced this effect (p < 0.01). In addition, pretreatment of the cells with a specific MEK-1/2 kinase inhibitor reduced ACh stimulated NCA (p < 0.01). Together these results demonstrated that cholinergic stimulation of NCA is effectively inhibited by tiotropium and is governed by a mechanism involving M3 coupled Gq signaling and downstream ERK signaling. This study further demonstrates that tiotropium may act as an anti-inflammatory agent in lung disease.

Abbreviation List

• Ach – acetylcholine
• ANOVA – analysis of variance
• AS - complement activated serum
• BCA - bicinchoninic acid
• ChAT - choline acetyltranferase
• COPD – chronic obstructive pulmonary disease
• ERK - extracellular-signal-regulated kinases
• GAPDH - glyceraldehyde-3-phosphate dehydrogenase
• LPS – lipopolysaccharide
• M3 – muscarinic subtype 3 receptor
• MEK - mitogen-activated protein/extracellular signal-regulated kinase
• NCA – neutrophil chemotactic activity
• PMT - pasteurella multocida toxin
• rhIL-8 - recombinant human interleukin-8
• RIPA - radioimmunoprecipitaion assay
• SEM – standard error of mean
• TBST - tris-buffered saline and tween 20

Introduction

Anticholinergic therapy has been regarded as a first choice bronchodilator in the management of stable chronic obstructive pulmonary disease (COPD) (1).  The agents included within this class of therapeutics effectively reverse the stimulation of parasympathetic produced acetylcholine (ACh) on smooth muscle airway contraction. Parasympathetic activity is increased with airway inflammation, and in regards to COPD, is an important mechanism because vagal tone appears to be one of the only reversible components of airflow restriction (2).  Besides bronchoconstriction, ACh may also be involved in airway remodeling and other pathophysiogical mechanisms that are important in the propagation of lung disease (1,3-8). Recently it has been suggested that ACh may be expressed in the lung independent of a parasympathetic mechanism. In support of this notion, ACh synthesizing enzyme (choline acetyl transferase) has been found to be ubiquitously expressed in the airways, pulmonary epithelial cells, and immune cells such as neutrophils and monocytes (9-12). In addition, these cells also appear to express functional muscarinic receptors (9-11).  Interestingly, the expression and function of certain muscarinic receptors in neutrophils may be increased in COPD (13), thus suggesting increase bioactivity associated with enhanced lung inflammation. We and others have previously demonstrated that ACh may also stimulate resident lung cells to release chemotactic factors and subsequently these factors can induce pro-inflammatory chemotaxis indirectly in vitro (3,4,8,9). 

It has been recently reported that outcomes of COPD are improved by inhalation of cholinergic inhibitors, and tiotropium (tiotropium bromide, Spiriva®; Boehringer Ingelheim, Ingelheim, Germany) demonstrates the greatest improvements in COPD because of its long-acting, once daily administered, anticholinergic capability (1).  Although tiotropium predominantly functions as a bronchodilator, it has also been shown to inhibit ACh-induced proliferation of fibroblasts and myofibroblasts (16), and inhibit the release of chemotactic factors from cultured lung epithelial cells, fibroblasts and alveolar macrophages in vitro (3,4). Taken together these results suggest a plausible beneficial role of tiotropium on airway remodeling and action as an anti-inflammatory agent in chronic airway disease.

We have previously reported that supernatants from macrophages that were treated with tiotropium prior to a challenge with lipopolysaccharide (LPS) greatly reduced the subsequent stimulation of NCA and this result did not occur by inhibited release of chemotactic factors (17). Based on these results, we postulated that tiotropium from the test media may actually passively diffuse through the pores of the filter that separates the chambers of the microchemotaxis unit and possibly interact directly with the neutrophils. In this study, we tested the hypothesis that tiotropium may act as an anti-inflammatory agent by directly interacting on neutrophils and inhibiting their chemotactic capability.

It has been well established that infiltration of neutrophils and the modulation of their activity play an important role in propagating and governing inflammation in a variety of lung diseases such as COPD (18).  In addition, muscarinic receptor G-protein coupled signal transduction (19) and downstream ERK-1/2 activity (20-22) may also play an important regulatory role in controlling the migration of neutrophils.  In this study, we further demonstrate that tiotropium may inhibit NCA, in part, through the regulation of muscarinic receptor coupled Gq-protein and ERK-1/2 mediated signal transduction (Figure 1).

Figure 1. Putative mechanism of tiotropium effect on neutrophil chemotaxis. Acetylcholine (ACh) either exogenously released or acting in a paracrine fashion stimulates the muscarinic type 3 (M3) receptor. This subsequently activates Gq protein which activates extracellular-signal-regulated kinases (ERK) 1/2. ERK 1/2 translocates into the nucleus activating various transcription factors which result in cell migration. Tiotropium decreases chemotaxis by inhibiting the binding of ACh to the M3 receptor.

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 Human Blood Neutrophils and Experimental Models

Human primary neutrophils were isolated and purified from heparinized plasma obtained from normal healthy individuals by the method of Böyum (23). The purified neutrophils were exposed to ACh (sodium acetylcholine, Sigma-Aldrich) up to 60 min prior to chemotaxis.  For most experiments, cells were also pretreated with the various factors listed below for 30 min prior before selected agonist treatment. Inclusive of these agents are tiotropium bromide (Boehringer-Ingelheim); muscarinic (M) receptor antagonists including  pirenzepine dihydrochloride  (M₁; Sigma-Aldrich); gallamine trithiodide M₂ (M₂; Sigma-Aldrich); 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M₃; Sigma-Aldrich); M₃ receptor agonist cevimeline HCl (EVOXAC^®, Daiichi Sankyo, Inc., Parsippany, NJ); selective G-protein agonists(G_q, Pasteurella multocida toxin [PMT], EMD Biosciences Inc., San Diego, CA, G_o, mastoparan, Biomol International, Plymouth Meeting, PA) and a specific mitogen-activated protein/extracellular signal-regulated kinase (MEK)-1/2 inhibitor (U0126; Sigma-Aldrich). 

Neutrophil Chemotaxis Analysis

The chemotaxis assay was performed in a 48-well microchemotaxis chamber (NeuroProbe Inc., Cabin John, MD) using previously described methods (14).   Either recombinant human interleukin-8 (rhIL-8, Sigma-Aldrich) or complement activated serum (AS) were used as the chemoattractants. Neutrophil viability was assessed and not altered by tiotropium.

Western Blot Analysis

The examination of corresponding regulation of extracellular signal-regulated kinase (ERK)-1/2 proteins by ACh and tiotropium in neutrophils was performed by Western Blot analyses. Both phosphorylated (p) and total (t) ERK-1/2 proteins were examined. Rabbit monoclonal antibodies directed against human pERK-1/2, tERK-1/2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins were purchased from Cell Signaling Technology (Beverly, MA).

For the ACh time-course experiment, neutrophils (1 x 10⁷) were treated with 100 μM ACh for period ranging from 0 to 60 min of exposure. After establishing the maximal time effect (~15-20 min), subsequent experiments were conducted examining the effect of a 30 min tiotropium (100 nM) pretreatment on ACh challenged ERK-1/2 protein expression.

Neutrophils were lysed with ice-cold radioimmunoprecipitaion assay (RIPA) buffer including a proteolytic inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) as per the manufacturer’s instructions. Total protein concentration of the lysates was determined by the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Rockford IL). Protein concentrations were then adjusted to 40 µg in a standardized volume before addition of 2x sample buffer (Invitrogen, Grand Island, NY) and heating for 5 min at 85˚C.  Cell proteins were then separated by electrophoresis on 4-20% tris-glycine acrylamide gels (Invitrogen, Grand Island, NY) and transferred to membranes (HCL-hybond, GE Healthcare, Piscataway, NJ) by electroblotting at 25 volts overnight at 4^oC.  The membranes were then pretreated with 1x tris-buffered saline and tween 20 (TBS-T) plus 5% non-fat dried milk for at least 2 hours at room temperature before exposure to the primary antibodies (1:2000) as per manufacturer’s suggestion overnight at 4^oC. After subsequent washing with TBS-T a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2000) was added for at least 1 h at room temperature.  

After multiple washings, the membranes were exposed to peroxidase substrate for enhanced chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific, Rockford, IL) for 5 min. Membranes were wrapped and placed against autoradiograph film (Hyperfilm ECL; GE Healthcare, Piscataway, NJ) and developed (up to 30 min). The resulting protein bands were quantified by densitometry (Personal Densitometer SI, Image Quant ver. 5, Molecular Dynamics, GE Healthcare Biosciences Corp.).

Statistical Analyses

Unless stated otherwise data are means ± SEM resulting from at least 3 individual experiments. Data were analyzed by one-way ANOVA followed by selected post-hoc Neuman-Keuls tests. p < 0.05 was considered significant.

Results

Stimulation of neutrophil chemotactic activity by cholinergic challenge

Neutrophils were pretreated with varied concentrations of ACh ranging between 1-100 µM prior to exposure to two different chemotactic agents including rhIL-8 (500 ng/ml) and AS.  As demonstrated in Figure 2, ACh treatment stimulated NCA in a dose dependent manner for both IL-8 and AS (p < 0.001). Similarly, at the 1 or 10 µM level ACh stimulated NCA when exposed to either IL-8 or AS, respectively. Moreover, the maximal level of stimulation by ACh was attained when the cells were treated with 100 µM ACh (Figure 2).  As reported previously, this concentration of ACh provided maximal effects in other cell types (13,15).  Beyond the dose effect studies, we also tested the effect of duration of ACh exposure (15 to 60 min) on NCA and found a significant stimulatory effect to occur within 60 min of exposure (data not shown).

Figure 2.  The effect of acetylchoine (ACh) stimulation on neutrophil chemotaxis. Neutrophils were treated with varied concentrations of ACh for 60 min prior to exposure to rhIL-8 (closed diamond) or complement activated serum (open diamond).  Neutrophil chemotactic activity (NCA) is on the ordinate and the concentration of ACh is on the abscissa. Values are expressed as means ± SEM.  For each experiment a significant dose effect was demonstrated (ANOVA, p < 0.0001; 15 observations per experiment). *p < 0.05, **p < 0.001 means differed as compared with those from non-treated controls.

Tiotropium pretreatment inhibited cholinergic stimulation of neutrophils

Neutrophils were pretreated for 30 min with varied concentrations of tiotropium ranging between 0.1 to 1000 nM prior to exposure to ACh.  Tiotropium pretreatment significantly reversed the stimulatory effect of effect of ACh on NCA at concentrations ranging greater than 1 nM. A dose dependent was observed with the maximum reduction approaching 45% (p<0.001) at levels beyond 10 nM (Figure 3).

Figure 3.  The effect of tiotropium on ACh-stimulated neutrophil chemotaxis.  Neutrophils were treated with tiotropium at various concentrations (0.1 to 1000 nM) for 30 min prior to treatment to ACh for an additional 60 min and exposure to rhIL-8 as the chemoattractant. Values are expressed as means ± SEM.  A treatment effect was demonstrated by one-way ANOVA (p < 0.0001) for three independent experiments.  ^#p < 0.01 means differed compared with non-treated controls; *p < 0.05, **p < 0.001 means differed compared with those from ACh-stimulated neutrophils.

The effect of selective muscarinic receptor antagonists on cholinergic stimulation of neutrophil chemotaxis.

It has been recently demonstrated that neutrophils express muscarinic receptors sub-types 1 through 3 (10,11) and tiotropium can interact amongst these receptors as an antagonist with varying affinities (M₃>M₁>M₂) (1).  Thus, we examined the effect of a variety of muscarinic receptor antagonists with specificity to the varied receptors including pirenzepine dihydrochloride (M₁), gallamine trithiodide (M₂) and 4-DAMP (M₃). Neutrophils were pretreated with these muscarinic antagonists at the varied concentrations (0.1 – 1000 nM) for 30 min prior to exposure to 100 µM ACh.

As demonstrated in Figure 4C, 4-DAMP significantly inhibited the increase of NCA that resulted from ACh treatment (32% decrease; p<0.05) although this effect was not as robust as those of tiotropium demonstrated in Figure 3.  In contrast to 4-DAMP, gallamine pretreatment did not alter NCA that was stimulated by ACh treatment.  Although not significant, an inhibitory trend was observed by pirenzepine pretreatment on cholinergic stimulation of NCA (Figure 4A).

Figure 4.  The effect of various muscarinic (M) receptor antagonists on ACh-stimulated neutrophil chemotaxis.  Neutrophils were treated with pirenzepine (M₁ inhibitor; figure 3A), gallamine (M₂ inhibitor; figure 3B) and 4-DAMP (M₃ inhibitor; figure 3C) at various concentrations (0.1 – 1000 nM) for 30 min prior to treatment with ACh and exposure to rhIL-8.  Values are expressed as means ± SEM.  Treatment effects were displayed by ANOVA for pirenzepine (p < 0.03; n = 5), gallamine (p < 0.02; n = 3) and 4-DAMP (p < 0.01; n = 3) experiments. ^#p < 0.05 means differed as compared with non-treated controls.  *p< 0.05 means differed compared with those from ACh-stimulated cells.

Tiotropium bromide effects NCA by altering M₃ receptor G_q-protein coupling

As suggested by results of the muscarinic receptor antagonists above, the M₃ receptor seems to play a prominent role in the regulation of cholinergic induction of NCA. To confirm this role, we examined the effect of the specific M₃ receptor agonist cevimeline on NCA.  Neutrophils were pretreated with tiotropium (30 min) prior to exposure to 300 µM cevimeline for an additional 30 min.  As seen in Figure 4A, NCA was promoted by cevimeline treatment when exposed to rhIL-8 (~41% increase as compared to controls; p < 0.001).  Similar to the response demonstrated in the ACh series of experiments, tiotropium pre-treatment significantly reversed the stimulatory effect of cevimeline (~40% decrease, p < 0.001) on NCA to a level that was similar to non-treated control neutrophils (Figure 5A). 

  

Figure 5. Tiotropium inhibited the stimulatory effect of cevimeline (M₃ receptor agonist) and pasteurella multocida toxin (PMT; G_q signaling stimulator) on NCA. Neutrophils were pre-treated with tiotropium for 30 min before the addition of cevimeline (Figure 4A) or PMT (Figure 4B) for an additional 30 min and exposure to rhIL-8. Values are expressed as means ± SEM. Treatment effects were displayed for both series of experiments (ANOVA; p < 0.0001, n=3). ^#p < 0.001 means differed as compared with non-treated neutrophils. *p < 0.001 means differed as compared with those from cevimeline- and PMT- stimulated cells.

G-proteins are important regulators in chemokine and complement mediated chemotaxis, and are early-stage regulatory components coupled to muscarinic receptor function (19,24,25). To test whether M₃ receptor coupled G-protein pathway is involved in the regulation of cholinergic stimulation of NCA, we treated neutrophils with a potent G_q agonist (Pasteurella multocida toxin; PMT) (26). As demonstrated in Figure 5B, PMT treatment effectively stimulated NCA (~32% increase; p < 0.001) when compared to non-treated controls. In addition, when the neutrophils were pretreated with tiotropium for 30 min prior to PMT stimulation, NCA was markedly reduced by 38% (p < 0.001) as compared to PMT treatment alone (Figure 5B). To further examine the specificity of this event we treated neutrophils with mastoparan, an agonist of the G_o proteins coupled to the M₂ and M₄ receptor function. In contrast to PMT, mastoparan treatment did not influence NCA (data not shown).

 Cholinergic activation of ERK-1/2 in neutrophils is inhibited by tiotropium treatment.

It has been previously established that ERK-1/2 protein activation provides a pivotal regulatory role on neutrophil chemotaxis (22,27,28) and it is a downstream signaling pathway that is influenced by G-proteins (29-31). Thus, we examined the effect of ACh activation (100 µM) on ERK-1/2 signaling in neutrophils and began with examining the effect of time of cholinergic exposure (0 to 60 min) on ERK-1/2 protein expression. As seen in Figure 5, ACh treatment activated pERK-1/2 expression but did not alter the level of tERK-1/2 proteins in the cells. The stimulation of pERK-1/2 reached the maximal effect within 15-30 min of exposure to ACh, and began to decrease after 45 min of exposure (Fig 6). Similar reductions on pERK-1/2 expression were demonstrated in experiments where neutrophils were treated with ACh for longer periods (>60 min; data not shown).

Figure 6. The effect of time of exposure of ACh on ERK-1/2 protein activation in neutrophils. Cells were treated with 100 μM ACh for various times from 0 through 45 min of exposure. Total cell proteins were isolated and examined for phosphorylated (p) and total (t) ERK-1/2 expression assessed by Western-blot (Figure 5A). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assessed as a loading control. The corresponding mean ratio of pERK-1/2: tERK-1/2 resulting from densitometric scans is demonstrated in figure 6B.

A further series of experiments (n=4) were conducted to examine the effect of tiotropium on the inhibition of cholinergic stimulation of ERK-1/2. Neutrophils were pretreated with 100 nM tiotropium for 30 min prior to exposure to 100 µM ACh for 15 min. As seen in Figure 7, ACh treatment increased the activation of ERK proteins (pERK/tERK ratio = 0.85 for ACh vs. 0.52 in non-treated control cells; p < 0.01) and tiotropium pretreatment markedly reversed this effect and where expression was reduced to control cell levels (Figure 7).

Figure 7. The effect of tiotropium on ACh stimulated ERK-1/2 protein activation in neutrophils. Cells were treated with 100 nM of tiotropium prior to expose to 100 μM of ACh for 15 min. Total cell proteins were isolated and pERK-1/2, tERK-1/2 and GAPDH expression was determined by Western-blot. A representative experiment is shown in Figure 6A and ERK-1/2 activation expressed as pERK1/2: tERK-1/2 is demonstrated in Figure 6B. Values are expressed as means ± SEM. A treatment effect was demonstrated by ANOVA (p < 0.005; n=4 experiments). ^#p < 0.01 means differ as compared with non-treated neutrophils; * p < 0.01 means differ as compared to those from ACh stimulated cells.

Cholinergic stimulation of NCA is reduced by an inhibitor of ERK-1/2 phosphorylation

Based of the aforementioned results on pERK-1/2 expression activation of ERK-1/2 by phosphorylation may govern NCA. Neutrophils were pretreated with U0126 (a specific MEK-1/2 kinase inhibitor) for 30 min prior to exposure to ACh (100µM) for an additional 60 min. As seen in Figure 8, U0126 pretreatment strongly inhibited (p < 0.01) the increase of NCA by ACh treatment to levels similar to non-treated control cells.

Figure 8. Neutrophil chemotactic activity that is stimulated by ACh is inhibited by an antagonist of ERK-1/2 phosphorylation. Cells were pretreated with a specific inhibitor of MEK-1 and -2 (U0126; 10 µM) for 30 min prior to the addition of 100 µM ACh for an additional 30 min before assessing NCA as described above. Values are expressed as means ± SEM. A treatment effect was demonstrated by ANOVA (p < 0.001) resulting from three independent experiments. ^#p < 0.001 means differed as compared with non-treated neutrophils. *p< 0.01 means differed compared with those from ACh stimulated cells.

Discussion

Previous clinical results have suggested that tiotropium inhalation provides beneficial clinical outcomes in COPD that may result from modulating mechanisms beyond bronchodilatation (1,10). An intriguing suggestion has been that anticholinergic therapy may act as an anti-inflammatory. The mechanism(s) by which this action occurs has not been fully elucidated; however, recent in vitro studies have suggested that tiotropium may indirectly influence neutrophil chemotaxis by inhibiting the release of chemotactic factors by resident lung cells that would subsequently promote NCA (3,4). In a model using U937 macrophages, we previously reported that NCA was decreased from supernatants that were obtained from LPS-challenged cells treated with tiotropium and that this result did not occur from a reduction in corresponding chemotactic factor expression measured in the supernatants (17). Specifically, we found that heightened levels of IL-8 did not correlate (r = 0.38; p > 0.13) with the reduction in NCA upon tiotropium treatment (0.1 to 1000 nM). Similar effects were also shown regarding LTB₄ analyses (17). Based on these results, we formulated the hypothesis that tiotropium contained in the supernatants may actually interact with the neutrophils and influence their activity directly.

Current concepts suggest that an influx of neutrophils is important in the pathogenesis of COPD (18). These neutrophils release proteases and toxic oxygen radicals that contribute to the inflammation seen in COPD. It is this inflammation that results in the emphysema and airway remodeling that causes the structural changes in COPD that lead to the clinical symptoms of breathlessness and/or cough. Previous studies in animal models of COPD have shown that tiotropium is anti-inflammatory (5,32). More recent studies in humans suggest that tiotropium reduces neutrophil chemotaxis (33). Migration of neutrophils from COPD patients are also decreased by tiotropium similarly to the normal human neutrophils used in this study (34). The present studies are consistent with these results and support an anti-inflammatory role for tiotropium in COPD.

It has not been established to date that cholinergic stimulation may directly affect NCA in vitro. In the present study, we report that exogenous ACh pretreatment enhanced NCA when the cells were exposed to differing chemotactic agents. In addition, we found that tiotropium treatment prior to ACh exposure very effectively reduced stimulated NCA. The bioactive concentrations of tiotropium that were used in this study initially ranged from 10 -1000 nM and the lower bioactive responsive doses were similar to those previously reported to affect human lung fibroblast proliferation (35), fibroblast differentiation (16), and the release of chemotactic factors from epithelial cells, fibroblasts and alveolar macrophages in vitro (3,4). In order to elicit a robust effect on NCA, we opted to use a dose of 100 nM of tiotropium throughout the study. At this level, tiotropium was non-toxic and remained below the estimated maximum concentration of ~2000 nM to be present in the lung epithelial lining fluid after inhalation of the drug (36).

There is increasing evidence that signaling from extraneuronally produced ACh may play an important role in regulation of lung inflammation (1,9). ACh may enhance proinflammatory cell chemotaxis indirectly by stimulating resident lung cells to release chemotactic factors (3,4,14,15). Recently, choline acetyltranferase (ChAT) has been localized in human blood and skin derived neutrophils; however, to date there have been no studies establishing ChAT expression in pulmonary neutrophils (10). However, a recent report by Neumann et al. (37) demonstrated that mononuclear cells (T cells and monocytes) expressed ~0.36 pmol ACh/10⁶ cells, whereas granulocytes (containing predominantly neutrophils) expressed considerably less concentration of ACh although their synthetic capacity was greater than CD3⁺ cells. Thus, it remains to be established whether pulmonary neutrophils may produce Ach, especially under conditions of inflammation. It also remains to be established whether neutrophils produce sufficient ACh to regulate a cholinergic response in an autocrine manner.

It has also been reported that neutrophils express muscarinic receptors (9.10,13,38). Interestingly, the expression of muscarinic receptors is modulated in neutrophils in COPD, particularly the M₃ receptors which are more highly expressed under this condition (13). In this study we demonstrated that neutrophils may react to exogenous cholinergic stimulation thus suggesting that paracrine cholinergic stimulation may be a viable mechanism of control of neutrophil activity associated with inflammation.

In an early attempt to characterize the muscarinic receptor(s) involved in cholinergic regulation of NCA we used a panel of selective antagonists and tested their reactivity against ACh stimulation. To accomplish this objective we pretreated neutrophils with pirenzepine, gallamine and 4-DAMP prior to cholinergic treatment. Our results demonstrated that only the inhibitor 4-DAMP significantly reversed the effect of ACh on NCA. These results further confirm that anti-inflammatory control may entail the antagonism of the muscarinic type-3 receptor. This is comparable to our previous studies that have demonstrated that ACh may promote chemotactic factor release from resident lung cells by influencing M₃ receptors (4,14,15).

We further treated neutrophils with cevimeline, a M₃ receptor agonist (39), and found that this compound markedly increased NCA. When neutrophils were treated with PMT, a G_q agonist (27,40), it stimulated their activity and to a level comparable to those of cevimeline. Moreover, tiotropium pretreatment dramatically inhibited PMT stimulated NCA. Taken together, these results suggest that tiotropium may interact with the M₃ receptor and possibly modulate early G_q mediated signaling cues affecting NCA by cholinergic treatment.

The M₃ receptors have the capacity to activate multiple signaling pathways in various cell types. For example, it has been established that the M₃ receptor and G_q protein pathway is involved in airway smooth muscle contraction and may function by regulating PLC, inositol 1,4,5-triphosphate (IP₃) and intracellular Ca²⁺ signaling (41). In addition, it has been shown that G_q-deficient neutrophils possess deficient calcium signaling and defective chemotactic responsiveness (42). Furthermore it has been reported that ERK activation is associated with G_q-protein stimulation (29,30) and ERK signaling is an important integral regulator of NCA (22,27,28). In this study, we find that ACh treatment enhanced neutrophil ERK-1/2 protein phosphorylation but not total ERK1/2 expression. In addition, the pretreatment of the cells with tiotropium reversed this activity. Similarly, Profita et al. (4) demonstrated that ACh mediated release of IL-8 in human bronchial epithelial cells may be regulated in part by an ERK-dependent mechanism.

In summary, these data support the role of cholinergic stimulation on NCA an important inflammatory process contributing to pulmonary disease. This study also demonstrated an alternative anti-inflammatory role of tiotropium on directly reducing chemotactic activity by inhibiting, in part, G_q protein and ERK activation in neutrophils. Furthermore, this action was independent of type or concentration of chemotactic factor. This present study may provide some insight into the recently reported discordance between significant reductions in total exacerbation compared with no reduction in proinflammatory marker (including IL-8) concentration in sputa from COPD patients treated with tiotropium (43). The inhibition of neutrophil migration is one effect which may contribute to the anti-inflammatory effects of anticholinergics and may explain, at least in part, the reduction in exacerbations of COPD seen with tiotropium.

Acknowledgements

This study was funded by 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..

References

1. Restrepo RD. Use of inhaled anticholinergic agents in obstructive airway disease. Respir Care 2007;52:833-851.
2. Gross NJ, Skorodin MS. Role of the parasympathetic system in airway obstruction due to emphysema. New Engl J Med 1984;311:421-425.
3. Bühling F, Lieder N, Kühlmann UC, Waldburg N, Welte T. Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro. Respir Med 2007;101:2386-94.
4. Profita M, Bonanno A, Siena L, Ferraro M, Montalbano AM, Pompeo F, Riccobono L, Pieper MP, Gjomarkaj M. Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism. Eur J Pharmacol 2008;582:145-53.
5. Wollin L, Pieper MP. Tiotropium bromide exerts anti-inflammatory activity in a cigarette smoke mouse model of COPD. Pulm Pharmacol Ther 2010;23:345-54.
6. Profita M, Bonanno A, Montalbano AM, Ferraro M, Siena L, Bruno A, Girbino S, Albano GD, Casarosa P, Pieper MP, Gjomarkaj M. Cigarette smoke extract activates human bronchial epithelial cells affecting non-neuronal cholinergic system signaling in vitro. Life Sci 2011;89:36-43.
7. Profita M, Riccobono L, Montalbano AM, Bonanno A, Ferraro M, Albano GD, Gerbino S, Casarosa P, Pieper MP, Gjomarkaj M. In vitro anticholinergic drugs affect CD8+ peripheral blood T-cells apoptosis in COPD. Immunobiology 2012;217:345-53.
8. Profita M, Bonanno A, Montalbano AM, Albano GD, Riccobono L, Siena L, Ferraro M, Casarosa P, Pieper MP, Gjomarkaj M. β(2) long-acting and anticholinergic drugs control TGF-β1-mediated neutrophilic inflammation in COPD. Biochim Biophys Acta 2012;1822:1079-89.
9. Gosens R, Zaagsma J, Meurs H, Halayko AJ. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res 2006;7:73.
10. Gwilt CR, Donnelly LE, Rogers DF. The non-neuronal cholinergic system in the airways: an unappreciated regulatory role in pulmonary inflammation? Pharmacol Ther 2007;115:208-222.
11. Racke K, Juergens UR, Matthiesen S. Control by cholinergic mechanisms. Eur J Pharmacol 2006;533:57-68.
12. Wessler IK, Kirkpatrick CJ. The non-neuronal cholinergic system: an emerging drug target in the airways. Pulm Pharmacol Therap 2001;14:423-434.
13. Profita M, Giorgi RD, Sala A, Bonanno A, Riccobono L, Mirabella F, Gjomarkaj M, Bonsignore G, Bousquet J, Vignola AM. Muscarinic receptors, leukotriene B4 production and neutrophilic inflammation in COPD patients. Allergy 2005;60:1361-1369.
14. Koyama S, Rennard SI, Robbins RA. Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity. Am J Physiol 1992;262:L466-471.
15. Sato E, Koyama S, Okubo Y, Kubo K, Sekiguchi M. Acetylcholine stimulates alveolar macrophages to release inflammatory cell chemotactic activity. Am J Physiol 1998;274:L970-979.
16. Pieper MP, Chaudhary NI, Park JE. Acetylcholine-induced proliferation of fibroblasts and myofibroblasts in vitro is inhibited by tiotropium bromide. Life Sci 2007;80:2270-2273.
17. Rempe S, Robbins RA, Hoyt JC, Kurai M, Koyama S, Hayden JM. Tiotropium inhibits neutrophil chemotaxis [abstract]. Am J Resp Crit Care Med 2007;175:A493.
18. Niggli V. Signaling to migration in neutrophils: importance of localized pathways. Int J Biochem Cell Biol 2003;35:1619-1638.
19. Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol 2008;8:183-92.
20. Ottonello L, Montecucco F, Bertolotto M, Arduino N, Mancini M, Corcione A, Pistoia V, Dallegri F. CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2. Cell Signal 2005;17:355-63.
21. Fuhler GM, Knol GJ, Drayer AL, Vellenga E. Impaired interleukin-8- and GROalpha-induced phosphorylation of extracellular signal-regulated kinase result in decreased migration of neutrophils from patients with myelodysplasia. J Leukoc Biol 2005;77:257-66.
22. Hii CS, Anson DS, Costabile M, Mukaro V, Dunning K, Ferrante A.Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response. J Biol Chem 2004;279:49825-34.
23. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest 1968;97:77-89.
24. Honda Z, Takano T, Hirose N, Suzuki T, Muto A, Kume S, Mikoshiba K, Itoh K, Shimizu T. Gq pathway desensitizes chemotactic receptor-induced calcium signaling via inositol trisphosphate receptor down-regulation. J Biol Chem 1995;270:4840-4844.
25. Wess J. Molecular basis of receptor/G-protein-coupling selectivity. Pharmacol Therap 1998;80:231-64.
26. Wilson BA, Ho M. Pasteurella multocida toxin as a tool for studying Gq signal transduction. Rev Physiol Biochem Pharmacol 2004;152:93-109.
27. Van Lint J, Van Damme J, Billiau A, Merlevede W, Vandenheede JR. Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils. Mol Cell Biochem 1993;127-128:171-7.
28. Coxon PY, Rane MJ, Uriarte S, Powell DW, Singh S, Butt W, Chen Q, McLeish KR. MAPK-activated protein kinase-2 participates in p38 MAPK-dependent and ERK-dependent functions in human neutrophils. Cell Signal 2003;15:993-1001.
29. Budd DC, Rae A, Tobin AB. Activation of the mitogen-activated protein kinase pathway by a Gq/11-coupled muscarinic receptor is independent of receptor internalization.J Biol Chem 1999;274:12355-60.
30. Budd DC, Willars GB, McDonald JE, Tobin AB. Phosphorylation of the Gq/11-coupled m3-muscarinic receptor is involved in receptor activation of the ERK-1/2 mitogen-activated protein kinase pathway. J Biol Chem 2001;276:4581-7.
31. Osmond RI, Sheehan A, Borowicz R, Barnett E, Harvey G, Turner C, Brown A, Crouch MF, Dyer AR.GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors. J Biomol Screen 2005;10:730-7.
32. Pera T, Zuidhof A, Valadas J, Smit M, Schoemaker RG, Gosens R, Maarsingh H, Zaagsma J, Meurs H. Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD. Eur Respir J 2011;38:789-96.
33. Vacca G, Randerath WJ, Gillissen A. Inhibition of granulocyte migration by tiotropium bromide. Respir Res 2011;12:24.
34. Santus P, Buccellati C, Centanni S, Fumagalli F, Busatto P, Blasi F, Sala A. Bronchodilators modulate inflammation in chronic obstructive pulmonary disease subjects. Pharmacol Res. 2012;66:343-8.
35. Matthiesen S, Bahulayan A, Kempkens S, Haag S, Fuhrmann M, Stichnote C, Juergens UR, Racke K. Muscarinic receptors mediate stimulation of human lung fibroblast proliferation. Am J Respir Cell Mol Biol 2006;35:621-7.
36. Disse B, Speck GA, Rominger KL, Witek TJ, Jr., Hammer R. Tiotropium (Spiriva): mechanistical considerations and clinical profile in obstructive lung disease. Life Sci 1999;64:457-64.
37. Neumann S, Razen M, Habermehl P, Meyer CU, Zepp F, Kirkpatrick CJ, Wessler I. The non-neuronal cholinergic system in peripheral blood cells: effects of nicotinic and muscarinic receptor antagonists on phagocytosis, respiratory burst and migration. Life Sci 2007;80:2361-4.
38. Bany U, Gajewski M, Ksiezopolska-Pietrzak K, Jozwicka M, Klimczak E, Ryzewski J, Chwalinska-Sadowska H, Maslinski W. Expression of mRNA encoding muscarinic receptor subtypes in neutrophils of patients with rheumatoid arthritis. Ann NY Acad Sci 1999;876:301-304.
39. Weber J, Keating GM. Cevimeline. Drugs 2008;68:1691-8.
40. Orth JH, Lang S, Taniguchi M, Aktories K. Pasteurella multocida toxin-induced activation of RhoA is mediated via two families of G{alpha} proteins, G{alpha}q and G{alpha}12/13. J Biol Chem 2005;280:36701-7.
41. McGraw DW, Elwing JM, Fogel KM, Wang WC, Glinka CB, Mihlbachler KA, Rothenberg ME, Liggett SB. Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation. J Clin Invest 2007;117:1391-8.
42. Shi G, Partida-Sánchez S, Misra RS, Tighe M, Borchers MT, Lee JJ, Simon MI, Lund FE. Identification of an alternative G{alpha}q-dependent chemokine receptor signal transduction pathway in dendritic cells and granulocytes. J Exp Med 2007;204:2705-18.
43. Powrie DJ, Wilkinson TM, Donaldson GC, Jones P, Scrine K, Viel K, Kesten S, Wedzicha JA. Effect of tiotropium on sputum and serum inflammatory markers and exacerbations in COPD. Eur Respir J 2007;30:472-8.

Reference as: Kurai M, Robbins RA, Koyama S, Amano J, Hayden JM. Acetylcholine stimulation of human neutrophil chemotactic activity is directly inhibited by tiotropium involving Gq and ERK-1/2 regulation. Southwest J Pulm Crit Care 2012:5:152-68. (Click here for a PDF version)

September 2012 Pulmonary Case of the Month: The War on Drugs

Sudheer Penupolu, MD

Philip J. Lyng, MD

Lewis J. Wesselius, MD

Department of Pulmonary Medicine

Mayo Clinic Arizona

Scottsdale, AZ

History of Present Illness

A 69 year old woman was seen with a three day history of nonproductive cough and shortness of breath.

PMH, SH and FH

She has a past history of atrial fibrillation and hypothyroidism.

Her present medications include:

• Diltiazem
• Amiodarone
• Aspirin
• Levothyroxine
• Multi vitamins

There is a 20 pack-year smoking history but she quit in 1998. She is employed as a law school professor.

Physical Examination

Her physical examination is normal.

Chest X-ray

Her admission chest x-ray is shown in Figure 1.

An electrocardiogram showed normal sinus rhythm.

Which of the following is the most likely cause of the patient’s clinical picture?

1. Viral bronchitis
2. Exacerbation of COPD
3. Pneumonia
4. Congestive heart failure
5. Drug reaction

Reference as: Penupolu S, Lyng PJ, Wesselius LJ. September 2012 pulmonary case of the month: the war on drugs. Southwest J Pulm Crit Care 2012;5:107-14. (Click here for a PDF version of the article)

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..

References

1. Casaburi R, Mahler DA, Jones PW, Wanner A, San PG, ZuWallack RL, Menjoge SS, Serby CW, Witek T Jr. A long-term evaluation of once-daily inhaled tiotropium in chronic obstructive pulmonary disease. Eur Respir J 2002;19:217-224.
2. Vincken W, van Noord JA, Greefhorst AP, Bantje TA, Kesten S, Korducki L, Cornelissen PJ. Improved health outcomes in patients with COPD during 1 yr's treatment with tiotropium. Eur Respir J 2002;19:209-216.
3. Wollin L, Pieper MP. Tiotropium bromide exerts anti-inflammatory activity in a cigarette smoke mouse model of COPD. Pulm Pharmacol Ther 2010;23:345-54.
4. Profita M, Bonanno A, Montalbano AM, Ferraro M, Siena L, Bruno A, Girbino S, Albano GD, Casarosa P, Pieper MP, Gjomarkaj M. Cigarette smoke extract activates human bronchial epithelial cells affecting non-neuronal cholinergic system signaling in vitro. Life Sci 2011;89:36-43.
5. Profita M, Riccobono L, Montalbano AM, Bonanno A, Ferraro M, Albano GD, Gerbino S, Casarosa P, Pieper MP, Gjomarkaj M. In vitro anticholinergic drugs affect CD8+ peripheral blood T-cells apoptosis in COPD. Immunobiology 2012;217:345-53.
6. Profita M, Bonanno A, Montalbano AM, Albano GD, Riccobono L, Siena L, Ferraro M, Casarosa P, Pieper MP, Gjomarkaj M. β(2) long-acting and anticholinergic drugs control TGF-β1-mediated neutrophilic inflammation in COPD. Biochim Biophys Acta 2012;1822:1079-89.
7. Pieper MP, Chaudhary NI, Park JE. Acetylcholine-induced proliferation of fibroblasts and myofibroblasts in vitro is inhibited by tiotropium bromide. Life Sci 2007;80:2270-2273.
8. Barnes NC, Evans J, Zakrzewski JT, Piper PJ, Costello JF. A critical evaluation of the clinical relevance of leukotrienes in bronchial asthma. Agents Actions 1989;Suppl 28:123-133.
9. Gross NJ, Skorodin MS. Role of the parasympathetic system in airway obstruction due to emphysema. N Engl J Med 1984;311:421-425.
10. Koyama S, Sato E, Nomura H, Kubo K, Nagai S, Izumi T. Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity. J Appl Physiol 1998;84:1528-1534.
11. Sato E, Koyama S, Okubo Y, Kubo K, Sekiguchi M. Acetylcholine stimulates alveolar macrophages to release inflammatory cell chemotactic activity. Am J Physiol 1998;274:L970-979.
12. Wessler IK, Kirkpatrick CJ. The non-neuronal cholinergic system: an emerging drug target in the airways. Pulm Pharmacol Ther 2001;14:423-434.
13. Gosens R, Zaagsma J, Meurs H, Halayko AJ. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res 2006;7:73.
14. Racke K, Juergens UR, Matthiesen S. Control by cholinergic mechanisms. Eur J Pharmacol 2006;533:57-68.
15. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of nonnuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968;97:77-89.
16. Harvath L, Falk W, Leonard EJ. Rapid quantitation of neutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly. J Immunol Methods 1980;37:39-45.
17. Sato E, Simpson KL, Grisham MB, Koyama S, Robbins RA. Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro. Am J Physiol 1999;277:L543-549.
18. Bagley KC, Abdelwahab SF, Tuskan RG, Lewis GK. Pasteurella multocida toxin activates human monocyte-derived and murine bone marrow-derived dendritic cells in vitro but suppresses antibody production in vivo. Infect Immun 2005;73:413-421.
19. Essler M, Hermann K, Amano M, Kaibuchi K, Heesemann J, Weber PC, Aepfelbacher M. Pasteurella multocida toxin increases endothelial permeability via Rho kinase and myosin light chain phosphatase. J Immunol 1998;161:5640-5646.
20. Disse B, Speck GA, Rominger KL, Witek TJ Jr, Hammer R. Tiotropium (Spiriva): mechanistical considerations and clinical profile in obstructive lung disease. Life Sci 1999;64:457-464.
21. Matthiesen S, Bahulayan A, Kempkens S, Haag S, Fuhrmann M, Stichnote C, Juergens UR, Racke K. Muscarinic receptors mediate stimulation of human lung fibroblast proliferation. Am J Respir Cell Mol Biol 2006;35:621-627.
22. Adamson IY, Bowden DH. Role of monocytes and interstitial cells in the generation of alveolar macrophages II. Kinetic studies after carbon loading. Lab Invest 1980;42:518-524.
23. Blusse van Oud Alblas A, van der Linden-Schrever B, Van Furth R. Origin and kinetics of pulmonary macrophages during an inflammatory reaction induced by intra-alveolar administration of aerosolized heat-killed BCG. Am Rev Respir Dis 1983;128:276-281.
24. Fels AO, Cohn ZA. The alveolar macrophage. J Appl Physiol 1986;60:353-369.
25. Nathan CF. Secretory products of macrophages. J Clin Invest 1987;79:319-326.
26. Sibille Y, Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Rev Respir Dis 1990;141:471-501.
27. Fujii T, Kawashima K. An independent non-neuronal cholinergic system in lymphocytes. Jpn J Pharmacol 2001;85:11-15.
28. Strom TB, Deisseroth A, Morganroth J, Carpenter CB, Merrill JP. Alteration of the cytotoxic action of sensitized lymphocytes by cholinergic agents and activators of adenylate cyclase. Proc Natl Acad Sci USA 1972;69:2995-2999.
29. Fujii T, Kawashima K. Ca2+ oscillation and c-fos gene expression induced via muscarinic acetylcholine receptor in human T- and B-cell lines. Naunyn Schmiedebergs Arch Pharmacol 2000;362:14-21.
30. Fujii T, Kawashima K. Calcium signaling and c-Fos gene expression via M3 muscarinic acetylcholine receptors in human T- and B-cells. Jpn J Pharmacol 2000;84:124-132.
31. Kawashima K, Fujii T. Extraneuronal cholinergic system in lymphocytes. Pharmacol Ther 2000;86:29-48.
32. Kirkpatrick CJ, Bittinger F, Nozadze K, Wessler I. Expression and function of the non-neuronal cholinergic system in endothelial cells. Life Sci 2003;72:2111-2116.
33. Wessler I, Kilbinger H, Bittinger F, Unger R, Kirkpatrick CJ. The non-neuronal cholinergic system in humans: expression, function and pathophysiology. Life Sci 2003;72:2055-2061.
34. Caulfield MP Birdsall NJ. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 1998;50:279-290.
35. Bany U, Ryzewski J, Maslinski W. Relative amounts of mRNA encoding four subtypes of muscarinic receptors (m2-m5) in human peripheral blood mononuclear cells. J Neuroimmunol 1999;97:191-195.
36. Costa P, Auger CB, Traver DJ, Costa LG. Identification of m3, m4 and m5 subtypes of muscarinic receptor mRNA in human blood mononuclear cells. J Neuroimmunol 1995;60:45-51.
37. Costa P, Traver DJ, Auger CB, Costa LG. Expression of cholinergic muscarinic receptor subtypes mRNA in rat blood mononuclear cells. Immunopharmacology 1994;28:113-123.
38. Hellstrom-Lindahl E, Nordberg A. Muscarinic receptor subtypes in subpopulations of human blood mononuclear cells as analyzed by RT-PCR technique. J Neuroimmunol 1996;68:139-144.
39. Profita M, Giorgi RD, Sala A, Bonanno A, Riccobono L, Mirabella F, Gjomarkaj M, Bonsignore G, Bousquet J,Vignola AM. Muscarinic receptors, leukotriene B4 production and neutrophilic inflammation in COPD patients. Allergy 2005;60:1361-1369.
40. Han SJ, Hamdan FF, Kim SK, Jacobson KA, Bloodworth LM, Li B, Wess J. Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor. J Biol Chem 2005;280:34849-34858.
41. Ghanouni P, Steenhuis JJ, Farrens DL, Kobilka BK. Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor. Proc Natl Acad Sci U S A 2001;98:5997-6002.
42. Jensen AD, Guarnieri F, Rasmussen SG, Asmar F, Ballesteros JA, Gether U. Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta 2 adrenergic receptor mapped by site-selective fluorescent labeling. J Biol Chem 2001;276:9279-9290.

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).