Safety and Complications of Bronchoscopy in an Adult Intensive Care Unit

Aarthi Ganesh, MBBS¹

Nirmal Singh, MBBS, MPH²

Gordon E. Carr, MD¹

¹Department of Pulmonary & Critical Care

²Department of Internal Medicine

University of Arizona

Tucson, Arizona

Abstract

Background: Bronchoscopy is a common procedure performed in adult intensive care units (ICU). However, very few studies report the safety and complications of the bronchoscopy and related procedures performed on critically ill patients. The primary aim of this study was to determine the incidence of complications following ICU bronchoscopy.

Methods: We conducted a retrospective chart review of patients admitted to an adult ICU and underwent bronchoscopy with or without bronchoalveolar lavage (BAL) and other bronchoscopic procedures. Data included patient demographics, APACHE II score, hemodynamics, comorbidities, type of ventilation and procedure performed. Data from BAL, including cellular differential and microbiology, were also collected.

Results: We identified 120 patient charts between November 2011 to March 2012. The most common procedure was bronchoscopy with BAL (62%) to evaluate for pneumonia (58%). Other procedures included transbronchial biopsy, APC and cryotherapy, balloon and stent placement, endobronchial biopsy and EBUS. Complications occurred in 18% of the patients, with hypoxia being the most common (7.5%). No deaths occurred related to the procedures. Nine percent of patients who had BAL or inspection had complications compared to 29% who underwent other procedures. Subgroup analysis conducted on patients undergoing BAL revealed significantly higher neutrophil counts (p=0.001) and higher APACHE II score (p=0.02) among those with BAL positive for bacteria and co-infection.

Conclusion: Bronchoscopy with BAL and inspection is relatively safe procedure even in critically ill patients. However, other interventional bronchoscopic procedures should be performed with caution in the ICU.

Abbreviations:

ICU: Intensive care unit

BAL: Bronchoalveolar lavage

EBUS: Endobronchial Ultrasound

APC: Argon Plasma Coagulation

SBP: Systolic Blood Pressure

CI: Confidence Interval

IP: Interventional pulmonary

MAP: Mean arterial pressure

SD: Standard deviation

CHF: Congestive heart Failure

COPD: Chronic Obstructive Pulmonary Disease

ILD: Interstitial Lung Disease

ET: Endotracheal

Introduction

Fiberoptic bronchoscopy is a commonly performed procedure in the medical intensive care unit (ICU). Prior studies have indicated that bronchoscopy is generally safe, making it a relatively low-risk procedure in appropriately selected ICU patients (1-3). Most prior studies reporting the safety of bronchoscopy were performed in early 1990s. The rates of complications or adverse events in these earlier studies ranged from 2% to 40% (2,4-6). The primary aim of this study was to assess the incidence of complications in ICU patients undergoing bronchoscopy in the contemporary era.

Methods

The study was approved by the Institutional Review Board at the University of Arizona. We conducted a retrospective chart review of patients, 18 years or older, admitted to the adult medical intensive care unit, who underwent bronchoscopy with or without bronchoalveolar lavage (BAL) and other bronchoscopic procedures from November 1, 2011 to March 31, 2012. The other bronchoscopic procedures included transbronchial biopsies, endobronchial ultrasound (EBUS) guided biopsy, argon plasma coagulation (APC) and cryotherapy, balloon dilatation with stenting, and endobronchial biopsy. We excluded patients with incomplete charts, and patients who had bronchoscopy as a part of percutaneous tracheostomy procedure. Data included patient demographics, APACHE II scores, hemodynamics, co-morbidities, type of ventilation, type of procedure performed and the complications. Sedation used in the procedures included propofol or midazolam with fentanyl for analgesia. BAL results, including cellular differential and microbiology studies, were also collected. We used pre-specified definitions to assess for complications. We defined hypotension as reduction in systolic blood pressure (SBP) by >20 mm Hg or when a patient required vasopressors to maintain a mean arterial pressure (MAP) > 60 mm Hg during or after the procedure. Hypoxia was defined by drop in saturation to < 90% or when the FiO2 requirement increased by > 20% for more than 2 hours after the procedure. Hemorrhage was indicated as per the procedure note by the bronchoscopist or when the note indicated use of epinephrine or when additional procedures needed to be performed to control the bleeding. During the procedure all the patients FiO2 was increased but was turned down to their previous ventilatory settings unless there was significant hypoxia.

Statistical analysis was performed using STATA/IC 13.1 (StataCorp LP, Texas). Numerical variables are expressed as mean ± standard deviation (SD). Ninety-five percent confidence intervals (CIs) were calculated where appropriate. Univariate comparisons between patients who did and did not develop complications were calculated using a χ2 test or Fischer's exact test for categorical variables and a 2-sample t test for continuous variables applying central limit theorem. All statistical testing was two-tailed with significance level set at the alpha level of ≤0.05.

Results

We identified 140 patients who underwent ICU bronchoscopy during the study period. Eighteen patients were excluded due to incomplete information. Two charts were excluded as the bronchoscopy was performed for percutaneous tracheostomy. Table 1 shows the baseline characteristics of patients undergoing ICU bronchoscopy.

Table 1. Baseline Characteristics of Patients Prior to Bronchoscopy

Key: CAD: Coronary Artery Disease

        CHF: Congestive Heart Failure

        COPD: Chronic obstructive pulmonary disease

        FiO2: Oxygen required

        ILD: Interstitial Lung Disease

        MAP: Mean arterial pressure

        NM Disease: Neuromuscular disease

Sixty-nine percent of the patients were male and average age was 52 ± 16 years. The average APACHE II score was 18 ± 6 with a median of 18 and 88% of the patients were intubated and mechanically ventilated. The mean percentage oxygen (FiO2) requirement in the patients prior to the procedure was 63% ± 26. Sixty-three percent of the patients were immunocompromised, likely related to the large proportion of lung transplant recipients in our study population. Fifty-four percent also had chronic lung disease including chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). Other common co-morbidities included cardiovascular disease including congestive heart failure (CHF) and arrhythmias, malignancy and neuromuscular diseases. Table II shows the indications for ICU bronchoscopy. The most common indication for the procedure was to evaluate for pneumonia or infiltrate in 87 cases (72%), followed by atelectasis/ collapse/ secretions in 19 cases (15.8%) (Table 2).

Table 2. Indications For Procedures

Other indications included tracheal or airway diseases, which included tracheal stenosis, upper airway obstruction, tracheal mass and bronchopleural fistula in 11 (8%) and hemoptysis (2%). The most common procedures performed were bronchoscopy with BAL in 75 (62%) and inspection in 31 (26%) (Table 3).

Table 3. Procedures

Key:  APC: Argon plasma coagulation

         BAL: Bronchoalveolar lavage

         Cryo: Cryotherapy

         EBUS: Endobronchial ultrasound

         ET: Endotracheal tube

Other procedures included transbronchial biopsy, APC and cryotherapy, balloon and stent placement, endobronchial biopsy and EBUS.

Table 4 shows the complications resulting from ICU bronchoscopy in this study population.

Table 4. Complications

Twenty two complications occurred during or within 2 hours after the procedure (18%), with hypoxia being the most common (7.5%). Hypoxia in two patients occurred secondary to hemorrhage. Pneumothorax was seen in one patient who underwent transbronchial biopsy with no fluoroscopic guidance. Hypotension which needed treatment with fluids or vasopressors occurred in 5.8% and hemorrhage in 3.3%. Hemorrhage was unrelated to coagulopathy in the patients. Significant bradycardia requiring treatment with atropine occurred in one patient. No deaths were reported related to the procedures. None of the procedures had to be terminated secondary to the complications. More adverse events were seen among the patients who underwent other bronchoscopic procedures (29%) than those undergoing BAL or inspection only (9%), though this was not statistically significant (p = 0.07).

As depicted in Table 5, none of the complications were significantly affected by the underlying comorbidities or the APACHE scores.

Table 5. Patient Characteristics Stratified by Complications

Key: BAL: Bronchoalveolar lavage

       MAP: Mean Arterial Pressure

Complications were not significantly associated with the amount of oxygen required (FiO2) and the mode of ventilation which the patients were on prior to the procedure. Similarly, neither the mean arterial pressure before the procedure or coagulopathy influenced the rate of complications. Hospital mortality was not different in the group with or without complications.

Figure 1 and Table 6 show the BAL cell differential.

Figure 1. BAL differential in culture with normal respiratory flora (0), bacteria (1), Viral (2), Fungal (3) and Co-infection (4). Each bar represents the differential in percentage.

Key: BAL: Bronchoalveolar lavage

          BAL N: Neutrophil count in BAL (in percentage)

          BAL L: Lymphocyte count in BAL (in percentage)

          BAL M: Macrophages count in BAL (in percentage)

          BAL E: Eosinophils count in BAL (in percentage)

Table 6. Bronchoalveolar Lavage Differential

Patients found to have bacterial pneumonia or mixed viral and bacterial infection had significantly higher neutrophil counts (mean BAL neutrophil count 82% for bacterial infection, and 80% for co-infections) than other patients (p=0.001) (Figure 2).

Figure 2. Neutrophil predominance in bacterial pneumonia. KEY: BAL-N: Bronchoalveolar lavage, neutrophil differential (in percentage).

These patients also had a higher APACHE II score (p=0.02). Hospital mortality was higher among those with BAL positive for bacteria (p= 0.012). Mortality was also significantly higher among patients with underlying malignancy (p= 0.002).

Discussion

In our study of 120 ICU bronchoscopies, we found a complication rate of 18%. No deaths were observed in this study. Hypoxia was the most common adverse event in our study, occurring in 9 procedures (7.5%) as has been noticed in the previous studies. Introduction of a bronchoscope through an endotracheal (ET) tube is known to cause airway obstruction resulting in increasing intra-tracheal pressures and variation in respiratory physiology (6). Almost all the patients who were mechanically ventilated had a size 7.5 - 8.5 ET tube or had tracheostomy in place. As in prior studies, BAL performed for evaluation of pneumonia and atelectasis were the two most common indications of the procedure (72% and 15.8% respectively) in our study (1-7). Even though bronchoscopy has not shown to be routinely superior to chest physiotherapy, certain subset of patient population may benefit from it (3,8,9). Improvement in oxygenation has been shown to occur in certain earlier studies (10,11).

Hypotension is also a known complication occurring during bronchoscopy. Our study had 7 events (5.8%) of hypotension needing vasopressor or fluid infusion. This was likely related to the sedation. Hypertension was observed in one case and bradycardia requiring treatment was seen in one. Cardiovascular abnormalities associated with bronchoscopy is generally related to the sympathetic surge happening during the procedure and the hypoxia (12-14). Per earlier studies, the complication rate of transbronchial biopsies in mechanically ventilated patients range between 0-15% (15,16,17). But it is relatively safe in comparison to open lung biopsy.

With the advent of newer technology, there has been an increase in the number of other bronchoscopic interventional pulmonary (IP) procedures, including endobronchial ablative therapies such as APC and cryotherapy. Endobronchial lesions occupying more than 50% of the airway lumen can alter the airway physiology and result in hypoxia, ventilation perfusion mismatch and hence respiratory failure. Use of ablative therapies can potentially reverse this (18). APC has been an useful tool to remove endobronchial lesions and relieve obstruction. It has been shown to be efficient and relatively safe in outpatient setting, but APC on mechanically ventilated patients has not been very well studied (19). APC in mechanically ventilated patient requires decrease in the FiO2 to less than or equal to 40%. Complications related to IP procedures performed specifically in patients requiring mechanical ventilation are difficult to assess  from the available literature (20). However, given the complexity of these cases and underlying illness, usually the complications are minor. In our study, interventional bronchoscopy procedures like APC, cryotherapy was to relieve airway obstruction which was the cause of mechanical ventilation. In our study, APC case was associated with hemorrhage. The balloon dilatation and stenting which was performed for a case of tracheal stenosis arising from malignancy. This was not associated with any complications related to the procedure in our study. Further study is needed to refine our understanding of the risks of advanced bronchoscopic techniques in ICU patients.

Procedures like EBUS are usually not done in critically ill patients. There are no studies which have looked into the use of and complications of performing EBUS in critically ill patients. Bhaskar et al. (21) report the use of esophageal access for mediastinal sampling through EBUS in ICU patients for the reason of causing hypoxia and changes in airway physiology with the EBUS scope in airway. Our study had one patient who had an EBUS for lung mass and this was not associated with any complications.

Subgroup analysis in our study showed the presence of neutrophilic predominance with neutrophil count of >80% in the BAL differential in patients diagnosed with bacterial infections and co-infections compared to those with viral/ fungal or mixed flora (p=0.001). This was similar to results from earlier studies (22,23). Neutrophilic pleocytosis in BAL fluid is frequently found in patients with pneumonia. As the neutrophil count is higher in bacterial pneumonia, it can indicate towards a differential of bacterial pneumonia even prior to the final microbiology results. Hence BAL differential may be complimentary to final culture results and maybe helpful to initiate or discontinue antibiotics in critically ill patients. Mortality among critically ill patients with bacterial pneumonia was higher compared to others (p=0.012). These patients tend to be sicker with higher APACHE II scores.

The weaknesses of the study includes the fact that it was retrospective chart review. The total number is small, and the number of the IP procedures performed is even smaller. Hence it is important that more studies should be conducted looking into the safety and complications of IP procedures in critically ill patients.

Conclusion

Our study looked into the fiberoptic bronchoscopy with BAL and inspection as well as other therapeutic procedures done in the critically ill patients. It indicates that even in critically ill patients, bronchoscopy with inspection and BAL is safe. Other interventional pulmonary procedures may have more complications. Even though the number of IP procedures performed in the study is low, the evidence of slightly more number of complications with these procedures indicates the need for caution before attempting them in the critically ill patients.

References

1. Barrett CR Jr. Flexible fiberoptic bronchoscopy in the critically ill patient. Methodology and indications. Chest. 1978;73(5 Suppl):746-9. [CrossRef] [PubMed]
2. Hertz MI, Woodward ME, Gross CR, Swart M, Marcy TW, Bitterman PB. Safety of bronchoalveolar lavage in the critically ill, mechanically ventilated patient. Crit Care Med. 1991;19(12):1526-32. [CrossRef] [PubMed]
3. Olopade CO, Prakash UB. Bronchoscopy in the critical-care unit. Mayo Clin Proc. 1989;64(10):1255-63. [CrossRef] [PubMed]
4. Steinberg KP, Mitchell DR, Maunder RJ, Milberg JA, Whitcomb ME, Hudson LD. Safety of bronchoalveolar lavage in patients with adult respiratory distress syndrome. Am Rev Respir Dis. 1993;148(3):556-61. [CrossRef] [PubMed]
5. Prebil SE, Andrews J, Cribbs SK, Martin GS, Esper A. Safety of research bronchoscopy in critically ill patients. J Crit Care. 2014;29(6):961-4. [CrossRef] [PubMed]
6. Guerreiro da Cunha Fragoso E, Gonçalves JM. Role of fiberoptic bronchoscopy in intensive care unit: current practice. J Bronchology Interv Pulmonol. 2011;18(1):69-83. [CrossRef] [PubMed]
7. Raoof S, Mehrishi S, Prakash UB. Role of bronchoscopy in modern medical intensive care unit. Clin Chest Med. 2001;22(2):241-61, vii. [CrossRef] [PubMed]
8. Kreider ME, Lipson DA. Bronchoscopy for atelectasis in the ICU: a case report and review of the literature. Chest. 2003;124(1):344-50. [CrossRef] [PubMed]
9. Marini JJ, Pierson DJ, Hudson LD. Acute lobar atelectasis: a prospective comparison of fiberoptic bronchoscopy and respiratory therapy. Am Rev Respir Dis. 1979;119(6):971-8. [PubMed]
10. Snow N, Lucas AE. Bronchoscopy in the critically ill surgical patient. Am Surg. 1984;50(8):441-5. [PubMed]
11. Stevens RP, Lillington GA, Parsons GH. Fiberoptic bronchoscopy in the intensive care unit. Heart Lung. 1981;10(6):1037-45. [PubMed]
12. Katz AS, Michelson EL, Stawicki J, Holford FD. Cardiac arrhythmias. Frequency during fiberoptic bronchoscopy and correlation with hypoxemia. Arch Intern Med. 1981;141(5):603-6. [CrossRef] [PubMed]
13. Lindholm CE, Ollman B, Snyder JV, Millen EG, Grenvik A. Cardiorespiratory effects of flexible fiberoptic bronchoscopy in critically ill patients. Chest. 1978;74(4):362-8. [CrossRef] [PubMed]
14. Trouillet JL, Guiguet M, Gibert C, Fagon JY, Dreyfuss D, Blanchet F, Chastre J. Fiberoptic bronchoscopy in ventilated patients. Evaluation of cardiopulmonary risk under midazolam sedation. Chest. 1990;97(4):927-33. [CrossRef] [PubMed]
15. Bulpa PA, Dive AM, Mertens L, Delos MA, Jamart J, Evrard PA, Gonzalez MR, Installé EJ. Combined bronchoalveolar lavage and transbronchial lung biopsy: safety and yield in ventilated patients. Eur Respir J. 2003;21(3):489-94. [CrossRef] [PubMed]
16. O'Brien JD, Ettinger NA, Shevlin D, Kollef MH. Safety and yield of transbronchial biopsy in mechanically ventilated patients. Crit Care Med. 1997;25(3):440-6. [CrossRef] [PubMed]
17. Casal RF, Ost DE, Eapen GA. Flexible bronchoscopy. Clin Chest Med. 2013;34(3):341-52. [CrossRef] [PubMed]
18. Seaman JC, Musani AI. Endobronchial ablative therapies. Clin Chest Med. 2013;34(3):417-25. [CrossRef] [PubMed]
19. Morice RC, Ece T, Ece F, Keus L. Endobronchial argon plasma coagulation for treatment of hemoptysis and neoplastic airway obstruction. Chest. 2001;119(3):781-7. [CrossRef] [PubMed]
20. Boyd M, Rubio E. The utility of interventional pulmonary procedures in liberating patients with malignancy-associated central airway obstruction from mechanical ventilation. Lung. 2012;190(5):471-6. [CrossRef] [PubMed]
21. Bhaskar N, Shweihat YR, Bartter T. The intubated patient with mediastinal disease--a role for esophageal access using the endobronchial ultrasound bronchoscope. J Intensive Care Med. 2014;29(1):43-6. [CrossRef] [PubMed]
22. Stolz D, Stulz A, Müller B, Gratwohl A, Tamm M. BAL neutrophils, serum procalcitonin, and C-reactive protein to predict bacterial infection in the immunocompromised host. Chest. 2007;132(2):504-14. [CrossRef] [PubMed]
23. Choi SH, Hong SB, Hong HL, Kim SH, Huh JW, Sung H, Lee SO, Kim MN, Jeong JY, Lim CM, Kim YS, Woo JH, Koh Y. Usefulness of cellular analysis of bronchoalveolar lavage fluid for predicting the etiology of pneumonia in critically ill patients. PLoS One. 2014;9(5):e97346. [CrossRef] [PubMed]

Cite as: Ganesh A, Singh N, Carr GE. Safety and complications of bronchoscopy in an adult intensive care unit. Southwest J Pulm Crit Care. 2015;11(4):156-66. doi: http://dx.doi.org/10.13175/swjpcc106-15 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)