BTS guidelines for the insertion of a chest drain D Laws, E Neville, J Duffy, on behalf of the British Thoracic Society Pleural Disease Group, a subgroup of the British Thoracic Society Standards of Care Committee ............................................................................................................................. Thorax 2003;58(Suppl II):ii53–ii59 1 BACKGROUND In current hospital practice chest drains are used in many different clinical settings and doctors in most specialities need to be capable of their safe insertion. The emergency insertion of a large bore chest drain for tension pneumothorax following trauma has been well described by the Advanced Trauma and Life Support (ATLS) recommenda- tions in their instructor’s manual 1 and there have been many general descriptions of the step by step method of chest tube insertion. 2–9 It has been shown that physicians trained in the method can safely perform tube thoracostomy with 3% early complications and 8% late. 10 In these guidelines we discuss the safe insertion of chest tubes in the controlled circumstances usually encountered by physicians. A summary of the process of chest drain insertion is shown in fig 1. 2 TRAINING • All personnel involved with insertion of chest drains should be adequately trained and supervised. [C] Before insertion of a chest drain, all operators should have been adequately trained and have completed this training appropriately. In all other circumstances, insertion should be supervised by an appropriate trainer. This is part of the SHO core curriculum training process issued by the Royal College of Physicians and trainees should be expected to describe the indications and compli- cations. Trainees should ensure each procedure is documented in their log book and signed by the trainer. With adequate instruction, the risk of complications and patient pain and anxiety can be reduced. 11 These guidelines will aid the training of junior doctors in the procedure and should be readily available for consultation by all doctors likely to be required to carry out a chest tube insertion. 3 INDICATIONS Chest tubes may be useful in many settings, some of which are listed in box 1. 4 PRE-DRAINAGE RISK ASSESSMENT • Risk of haemorrhage: where possible, any coagulopathy or platelet defect should be corrected prior to chest drain insertion but routine measurement of the platelet count and prothrombin time are only rec- ommended in patients with known risk factors. [C] • The differential diagnosis between a pneumothorax and bullous disease re- quires careful radiological assessment. Similarly it is important to differentiate between the presence of collapse and a pleural effusion when the chest radio- graph shows a unilateral “whiteout”. • Lung densely adherent to the chest wall throughout the hemithorax is an absolute contraindication to chest drain insertion. [C] • The drainage of a post pneumonectomy space should only be carried out by or after consultation with a cardiothoracic surgeon. [C] There is no published evidence that abnormal blood clotting or platelet counts affect bleeding complications of chest drain insertion. However, where possible it is obvious good practice to correct any coagulopathy or platelet defect prior to drain insertion. Routine pre-procedure checks of platelet count and/or prothrombin time are only required in those patients with known risk factors. For elective chest drain insertion, warfa- rin should be stopped and time allowed for its effects to resolve. 5 EQUIPMENT All the equipment required to insert a chest tube should be available before commencing the procedure and are listed below and illustrated in fig 2. • Sterile gloves and gown • Skin antiseptic solution, e.g. iodine or chlor- hexidine in alcohol • Sterile drapes • Gauze swabs • A selection of syringes and needles (21–25 gauge) • Local anaesthetic, e.g. lignocaine (lidocaine) 1% or 2% • Scalpel and blade • Suture (e.g. “1” silk) • Instrument for blunt dissection (e.g. curved clamp) Box 1 Indications for chest drain insertion • Pneumothorax • in any ventilated patient • tension pneumothorax after initial needle relief • persistent or recurrent pneumothorax after simple aspiration • large secondary spontaneous pneumot- horax in patients over 50 years • Malignant pleural effusion • Empyema and complicated parapneumonic pleural effusion • Traumatic haemopneumothorax • Postoperative—for example, thoracotomy, oesophagectomy, cardiac surgery See end of article for authors’ affiliations ....................... Correspondence to: Dr D Laws, Department of Thoracic Medicine, Royal Bournemouth Hospital, Castle Lane East, Bournemouth BH7 7DW, UK; diane.laws@ rbch-tr.swest.nhs.uk ....................... ii53 www.thoraxjnl.com group.bmj.com on September 26, 2011 - Published by thorax.bmj.com Downloaded from
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
BTS guidelines for the insertion of a chest drainD Laws, E Neville, J Duffy, on behalf of the British Thoracic Society Pleural DiseaseGroup, a subgroup of the British Thoracic Society Standards of Care Committee. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thorax 2003;58(Suppl II):ii53–ii59
1 BACKGROUNDIn current hospital practice chest drains are used
in many different clinical settings and doctors in
most specialities need to be capable of their safe
insertion. The emergency insertion of a large bore
chest drain for tension pneumothorax following
trauma has been well described by the Advanced
Trauma and Life Support (ATLS) recommenda-
tions in their instructor’s manual1 and there have
been many general descriptions of the step by
step method of chest tube insertion.2–9
It has been shown that physicians trained in the
method can safely perform tube thoracostomy
with 3% early complications and 8% late.10 In these
guidelines we discuss the safe insertion of chest
tubes in the controlled circumstances usually
encountered by physicians. A summary of the
process of chest drain insertion is shown in fig 1.
2 TRAINING• All personnel involved with insertion of
chest drains should be adequately trainedand supervised. [C]
Before insertion of a chest drain, all operators
should have been adequately trained and have
completed this training appropriately. In all other
circumstances, insertion should be supervised by
an appropriate trainer. This is part of the SHO core
curriculum training process issued by the Royal
College of Physicians and trainees should be
expected to describe the indications and compli-
cations. Trainees should ensure each procedure is
documented in their log book and signed by the
trainer. With adequate instruction, the risk of
complications and patient pain and anxiety can
be reduced.11
These guidelines will aid the training of junior
doctors in the procedure and should be readily
available for consultation by all doctors likely to
be required to carry out a chest tube insertion.
3 INDICATIONSChest tubes may be useful in many settings, some
of which are listed in box 1.
4 PRE-DRAINAGE RISK ASSESSMENT• Risk of haemorrhage: where possible, any
coagulopathy or platelet defect should becorrected prior to chest drain insertionbut routine measurement of the plateletcount and prothrombin time are only rec-ommended in patients with known riskfactors. [C]
• The differential diagnosis between apneumothorax and bullous disease re-quires careful radiological assessment.Similarly it is important to differentiatebetween the presence of collapse and a
pleural effusion when the chest radio-graph shows a unilateral “whiteout”.
• Lung densely adherent to the chest wallthroughout the hemithorax is an absolutecontraindication to chest drain insertion.[C]
• The drainage of a post pneumonectomyspace should only be carried out by orafter consultation with a cardiothoracicsurgeon. [C]
There is no published evidence that abnormal
blood clotting or platelet counts affect bleeding
complications of chest drain insertion. However,
where possible it is obvious good practice to
correct any coagulopathy or platelet defect prior
to drain insertion. Routine pre-procedure checks
of platelet count and/or prothrombin time are
only required in those patients with known risk
factors. For elective chest drain insertion, warfa-
rin should be stopped and time allowed for its
effects to resolve.
5 EQUIPMENTAll the equipment required to insert a chest tube
should be available before commencing the
procedure and are listed below and illustrated in
fig 2.
• Sterile gloves and gown
• Skin antiseptic solution, e.g. iodine or chlor-hexidine in alcohol
• Sterile drapes
• Gauze swabs
• A selection of syringes and needles (21–25gauge)
• Local anaesthetic, e.g. lignocaine (lidocaine)1% or 2%
• Scalpel and blade
• Suture (e.g. “1” silk)
• Instrument for blunt dissection (e.g. curvedclamp)
Box 1 Indications for chest drain insertion
• Pneumothorax• in any ventilated patient• tension pneumothorax after initial needle
relief• persistent or recurrent pneumothorax
after simple aspiration• large secondary spontaneous pneumot-
horax in patients over 50 years• Malignant pleural effusion• Empyema and complicated parapneumonic
pleural effusion• Traumatic haemopneumothorax• Postoperative—for example, thoracotomy,
oesophagectomy, cardiac surgery
See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .
be undertaken. In a study of chest tubes inserted in trauma
suites using full aseptic technique, there were no infective
complications in 80 cases.38
Studies of the use of antibiotic prophylaxis for chest tube
insertion have been performed but have failed to reach
significance because of small numbers of infectious complica-
tions. However, a meta-analysis of these studies has been per-
formed which suggested that, in the presence of chest trauma
(penetrating or blunt), the use of prophylactic antibiotics
reduces the absolute risk of empyema by 5.5–7.1% and of all
infectious complications by 12.1–13.4%.39 The use of prophy-
lactic antibiotics in trauma cases is therefore recommended.
The antibiotics used in these studies were cephalosporins or
clindamycin.
The use of prophylactic antibiotics is less clear in the event
of spontaneous pneumothorax or pleural effusion drainage as
no studies were found which addressed these circumstances.
In one study only one infectious complication (in the chest
tube track) occurred in a series of 39 spontaneous pneumo-
thoraces treated with chest tubes.40
12 ANAESTHESIA• Local anaesthetic should be infiltrated prior to inser-
tion of the drain. [C]
Local anaesthetic is infiltrated into the site of insertion of the
drain. A small gauge needle is used to raise a dermal bleb
before deeper infiltration of the intercostal muscles and pleu-
ral surface. A spinal needle may be required in the presence of
a thick chest wall.
Local anaesthetic such as lignocaine (up to 3 mg/kg ) is
usually infiltrated. Higher doses may result in toxic levels. The
peak concentration of lignocaine was found to be <3 µg/ml
(that is, a low risk of neurotoxic effects) in 85% of patients
given 3 mg/kg intrapleurally.41 The volume given is considered
to be more important than the dose to aid spread of the effec-
tive anaesthetic area. The use of adrenaline to aid haemostasis
and localise the anaesthesia is used in some centres but is not
evidence based.
13 INSERTION OF CHEST TUBE• Chest drain insertion should be performed without
substantial force. [C]
Insertion of a chest tube should never be performed with any
substantial force since this risks sudden chest penetration and
damage to essential intrathoracic structures. This can be
avoided either by the use of a Seldinger technique or by blunt
dissection through the chest wall and into the pleural space
before catheter insertion. Which of these approaches is appro-
priate depends on the catheter size and is discussed below.
13.1 Small bore tube (8–14 F)• Insertion of a small bore drain under image guidance
with a guidewire does not require blunt dissection.
Small bore chest tubes are usually inserted with the aid of a
guidewire by a Seldinger technique. Blunt dissection is
unnecessary as dilators are used in the insertion process. After
infiltration with local anaesthesia, a needle and syringe are
used to localise the position for insertion by the identification
of air or pleural fluid. A guidewire is then passed down the hub
of the needle, the needle is removed, and the tract enlarged
using a dilator. A small bore tube can then be passed into the
thoracic cavity along the wire. These have been successfully
used for pneumothorax, effusions, or loculated
empyemas.15 23 42
13.2 Medium bore tube (16–24 F)Medium sized chest drains may be inserted by a Seldinger
technique or by blunt dissection as outlined below. As the
incision size should afford a snug fit around the chest tube, it
is not possible to insert a finger to explore the pleura when
inserting this size of tube. Exploration with a finger is felt to be
unnecessary for the elective medical insertion of these
medium sized chest tubes.
13.3 Large bore tube (>24 F)• Blunt dissection into the pleural space must be
performed before insertion of a large bore chestdrain. [C]
13.3.1 Incision• The incision for insertion of the chest drain should be
similar to the diameter of the tube being inserted.[C]
Once the anaesthetic has taken effect an incision is made. This
should be slightly bigger than the operator’s finger and tube.
The incision should be made just above and parallel to a rib.
13.3.2 Blunt dissectionMany cases of damage to essential intrathoracic structures
have been described following the use of trocars to insert large
bore chest tubes. Blunt dissection of the subcutaneous tissue
and muscle into the pleural cavity has therefore become
universal43 and is essential. In one retrospective study only
four technical complications were seen in 447 cases using
blunt dissection.37 Using a Spencer-Wells clamp or similar, a
path is made through the chest wall by opening the clamp to
separate the muscle fibres. For a large chest drain, similar in
size to the finger, this track should be explored with a finger
through into the thoracic cavity to ensure there are no under-
lying organs that might be damaged at tube insertion.2–9 The
creation of a patent track into the pleural cavity ensures that
excessive force is not needed during drain insertion.
13.3.3 Position of tube tip• The position of the tip of the chest tube should
ideally be aimed apically for a pneumothorax orbasally for fluid. However, any tube position can beeffective at draining air or fluid and an effectivelyfunctioning drain should not be repositioned solelybecause of its radiographic position. [C]
In the case of a large bore tube, after gentle insertion through
the chest wall the trocar positioned a few centimetres from the
tube tip can afford support of the tube and so help its
positioning without incurring organ damage. A smaller clamp
can also be used to direct the tube to its desired position.1 3
If possible, the tip of the tube should be aimed apically to
drain air and basally for fluid. However, successful drainage
can still be achieved when the drain is not placed in an ideal
position,21 so effectively functioning tubes should not be
repositioned simply because of a suboptimal radiographic
appearance.
13.3.4 Securing the drain• Large and medium bore chest drain incisions should
be closed by a suture appropriate for a linear incision.[C]
• “Purse string” sutures must not be used. [C]
Two sutures are usually inserted—the first to assist later
closure of the wound after drain removal and the second, a
stay suture, to secure the drain.
The wound closure suture should be inserted before blunt
dissection. A strong suture such as “1” silk is appropriate.6 21 A
“mattress” suture or sutures across the incision are usually
employed and, whatever closure is used, the stitch must be of
a type that is appropriate for a linear incision (fig 4). Compli-
cated “purse string” sutures must not be used as they convert
ii56 Laws, Neville, Duffy
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
a linear wound into a circular one that is painful for the
patient and may leave an unsightly scar.9 A suture is not usu-
ally required for small gauge chest tubes.
The drain should be secured after insertion to prevent it
falling out. Various techniques have been described,44 but a
simple technique of anchoring the tube has not been the sub-
ject of a controlled trial. The chosen suture should be stout and
non absorbable to prevent breaking (e.g. “1” silk),6 and it
should include adequate skin and subcutaneous tissue to
ensure it is secure (fig 4).
Large amounts of tape and padding to dress the site are
unnecessary and concerns have been expressed that they may
restrict chest wall movement6 or increase moisture collection.
A transparent dressing allows the wound site to be inspected
by nursing staff for leakage or infection. An omental tag of
tape has been described2 which allows the tube to lie a little
away from the chest wall to prevent tube kinking and tension
at the insertion site (fig 5).
14 MANAGEMENT OF DRAINAGE SYSTEM14.1 Clamping drain• A bubbling chest tube should never be clamped. [C]
• Drainage of a large pleural effusion should becontrolled to prevent the potential complication ofre-expansion pulmonary oedema. [C]
• In cases of pneumothorax, clamping of the chest tubeshould usually be avoided. [B]
• If a chest tube for pneumothorax is clamped, thisshould be under the supervision of a respiratory phy-sician or thoracic surgeon, the patient should bemanaged in a specialist ward with experienced nurs-ing staff, and the patient should not leave the wardenvironment. [C]
• If a patient with a clamped drain becomes breathlessor develops subcutaneous emphysema, the drainmust be immediately unclamped and medical advicesought. [C]
There is no evidence to suggest that clamping a chest drain
prior to its removal increases success or prevents recurrence of
a pneumothorax and it may be hazardous. This is therefore
generally discouraged. Clamping a chest drain in the presence
of a continuing air leak may lead to the potentially fatal com-
plication of tension pneumothorax.3 6 9 A bubbling drain
therefore should never be clamped. However, many experi-
enced specialist physicians support the use of the clamping of
non-bubbling chest drains inserted for pneumothorax to
detect small air leaks not immediately obvious at the bedside.
By clamping the chest drain for several hours, followed by a
chest radiograph, a minor air leak may be detected, avoiding
the need for later chest drain reinsertion. In the ACCP Delphi
consensus statement45 about half the consensus group
supported clamping and half did not, and this seems similar to
the UK spread of opinion. Drain clamping is therefore not
generally recommended for safety reasons, but is acceptable
under the supervision of nursing staff who are trained in the
management of chest drains and who have instructions to
unclamp the chest drain in the event of any clinical deteriora-
tion. Patients with a clamped chest drain inserted for
pneumothorax should not leave the specialist ward area.
There have been reports of re-expansion pulmonary
oedema following rapid evacuation of large pleural effusions46
as well as in association with spontaneous pneumothorax.47 48
This has been reported to be fatal in some cases (up to 20% of
subjects in one series of 53 cases49). In the case of spontaneous
pneumothorax this is a rare complication with no cases of
re-expansion pulmonary oedema reported in two large studies
of 400 and 375 patients, respectively.50 51 It is usually associated
with delayed diagnosis and therefore awareness of its
potential occurrence is sufficient.
Milder symptoms suggestive of re-expansion oedema are
common after large volume thoracentesis in pleural effusion,
with patients experiencing discomfort and cough. It has been
suggested that the tube be clamped for 1 hour after draining
1 litre.52 While there is no evidence for actual amounts, good
practice suggests that no more than about 1.5 litres should be
drained at one time, or drainage should be slowed to about
500 ml per hour.
14.2 Closed system drainage• All chest tubes should be connected to a single flow
drainage system e.g. under water seal bottle or fluttervalve. [C]
• Use of a flutter valve system allows earlier mobilisa-tion and the potential for earlier discharge ofpatients with chest drains.
The chest tube is then attached to a drainage system which
only allows one direction of flow. This is usually the closed
underwater seal bottle in which a tube is placed under water
at a depth of approximately 3 cm with a side vent which
allows escape of air, or it may be connected to a suction
pump.2–4 7 This enables the operator to see air bubble out as the
lung re-expands in the case of pneumothorax or fluid evacua-
tion rate in empyemas, pleural effusions, or haemothorax. The
continuation of bubbling suggests a continued visceral pleural
air leak, although it may also occur in patients on suction
when the drain is partly out of the thorax and one of the tube
holes is open to the air. The respiratory swing in the fluid in
the chest tube is useful for assessing tube patency and
confirms the position of the tube in the pleural cavity. The dis-
advantages of the underwater seal system include obligatory
inpatient management, difficulty of patient mobilisation, and
the risk of knocking over the bottle.
Figure 4 Example of stay and closing sutures.
Figure 5 Omental tag to support the tube while allowing it to lie alittle away from the chest wall.
BTS guidelines for the insertion of a chest drain ii57
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
complication of lung penetration,63 although as long as blunt
dissection is carried out and no sharp instruments are used,
this risk is reduced.64
ACKNOWLEDGEMENTSThe authors are grateful to Dr Richard Holmes for figs 3, 4, and 5.
. . . . . . . . . . . . . . . . . . . . .Authors’ affiliationsD Laws, Department of Thoracic Medicine, Royal Bournemouth Hospital,Bournemouth BH7 7DW, UKE Neville, Respiratory Centre, St Mary’s Hospital, Portsmouth PO3 6AD,UKJ Duffy, Cardiothoracic Surgery Department, City Hospital, NottinghamNG5 1PB, UK
REFERENCES1 American College of Surgeons Committee on Trauma. In: Thoracic
trauma. Advanced Trauma Life Support program for physicians: instructormanual. Chicago: AmericanCollege of Surgeons, 1993. [IV]
2 Miller KS, Sahn SA. Review. Chest tubes. Indications, technique,management and complications. Chest 1987;91:258–64. [IV]
3 Parmar JM. How to insert a chest drain. Br J Hosp Med1989;42:231–3. [IV]
4 Treasure T, Murphy JP. Pneumothorax. Surgery 1989;75:1780–6. [IV]5 Westaby S, Brayley N. Thoracic trauma – I. BMJ 1990;330:1639–44.
[IV]6 Harriss DR, Graham TR. Management of intercostal drains. Br J Hosp
Med 1991;45:383–6. [IV]7 Iberti TJ, Stern PM. Chest tube thoracostomy. Crit Care Clin
1992;8:879–95. [IV]8 Quigley R.L. Thoracentesis and chest tube drainage. Crit Care Cli
1995;11:111–26. [IV]9 Tomlinson MA. Treasure T. Insertion of a chest drain : how to do it. Br J
Hosp Med 1997;58:248–52. [IV]10 Collop NA, Kim S, Sahn SA. Analysis of tube thoracostomy performed
by pulmonologists at a teaching hospital. Chest 1997;112:709–13. [III]11 Luketich JD, Kiss MD, Hershey J, et al. Chest tube insertion: a
prospective evaluation of pain management. Clin J Pain1998;14:152–4. [IIa]
12 Reinhold C, Illescas FF, Atri M, et al. The treatment of pleural effusionsand pneumothorax with catheters placed percutaneously under imageguidance. AJR 1989;152:1189–91. [III]
13 Ward EW, Hughes TE. Sudden death following chest tube insertion: anunusual case of vagus nerve irritation. J Trauma 1994;36:258–9. [IV]
14 Boland GW, Lee MJ, Silverman S, et al. Review. Interventional radiologyof the pleural space. Clin Radiol 1995;50:205–14. [IV]
15 Klein JS, Schultz S, Heffner JE. Intervential radiology of the chest:image-guided percutaneous drainage of pleural effusions, lung abscess,and pneumothorax. AJR 1995;164:581–8. [IV]
16 Rosenberg ER. Ultrasound in the assessment of pleural densities. Chest1983;84:283–5. [IV]
17 Harnsberger HR, Lee TG, Mukuno DH. Rapid, inexpensive real timedirected thoracocentesis. Radiology 1983;146:545–6. [IV]
18 Holden MP. Management of intercostal drainage tubes. In: Practice ofcardiothoracic surgery. Bristol: John Wright, 1982: 3. [IV]
19 Aslam PA, Hughes FA. Insertion of an apical tube. Surg Gynecol Obstet1970;130:1097. [IV]
20 Galvin IF, Gibbons JRP, Magout M, et al. Placement of an apical chesttube by a posterior approach. Br J Hosp Med 1990;44:330–1. [IV]
21 Hyde J, Sykes T, Graham T. Reducing morbidity from chest drains. BMJ1997;311:914–5. [IV]
22 Clementsen P, Evald T, Grode G, et al. Treatment of malignant pleuraleffusion : pleurodesis using a small bore catheter. A prospectiverandomized study. Respir Med 1998;92:593–6. [Ib]
24 Henderson AF, Banham SW, Moran F. Re-expansion pulmonaryoedema: a potentially serious complication of delayed diagnosis ofpneumothorax. BMJ 1985;29:593–4. [IV]
25 Thomas RJ, Sagar SM. What size pleural tube for pleural effusions(letter)? Br J Hosp Med 1990;43:184. [IV]
26 Taylor PM. Catheters smaller then 24 French gauge can be used forchest drains (letter). BMJ 1997;315:186. [IV]
27 Conces DJ, Tarver RD, Gray WC, et al. Treatment of pneumothoracesutilizing small caliber chest tubes. Chest 1988;94:55–7. [III]
28 Parker LA, Charnock GC, Delany DJ. Small bore catheter drainage andsclerotherapy for malignant pleural effusions. Cancer 1989;64:1218–21. [III]
29 Morrison MC, Mueller PR, Lee MJ, et al. Sclerotherapy of malignantpleural effusion through sonographically placed small-bore catheters. AJR1992;158:41–3. [III]
30 Goff BA, Mueller PR, Muntz HG, et al. Small chest tube drainagefollowed by bleomycin sclerosis for malignant pleural effusions. ObstetGynecol 1993;81:993–6. [III]
31 Seaton KG, Patz EF, Goodman PC. Palliative treatment of malignantpleural effusions: value of small-bore catheter thoracostomy anddoxycycline sclerotherapy. AJR 1995;164:589–91. [IIb]
32 Thompson RL, Yau JC, Donnelly RF, et al. Pleurodesis with iodized talcfor malignant effusions using pigtail catheters. Ann Pharmacother1998;32:739–42. [IIb]
33 Van Le L, Parker LA, DeMars LR, et al. Pleural effusions: outpatientmanagement of pigtail catheter chest tubes. Gynecol Oncol1994;54:215–7. [IV]
34 Matsumoto AH. Image-guided drainage of complicated pleural effusionsand adjunctive use of intrapleural urokinase. Chest 1995;108: 1190–1.[IV]
35 Moulton JS, Benkert RE, Weisiger KH, et al. Treatment of complicatedpleural fluid collections with image guided drainage and intra cavitaryurokinase. Chest 1995;108:1252–9. [III]
36 Parry GW, Morgan WE, Salama FD. Management of haemothorax. AnnR Coll Surg Engl 1996;78:325–6. [IV]
37 Millikan JS, Moore EE, Steiner E, et al. Complications of tubethoracostomy for acute trauma. Am J Surg 1980;140:738–41. [III]
38 Davis JW, MacKersie RC, Hoyt DB, et al. Randomised study ofalgorithms for discontinuing tube thoracostomy drainage. J Am Coll Surg1994;179:553–7. [Ib]
39 Fallon WF, Wears RL. Prophylactic antibiotics for the prevention ofinfectious complications including empyema following tube thoracoscopyfor trauma: results of a meta-analysis. J Trauma 1992;33:110–7. [Ia]
40 LeBlanc KA, Tucker WY. Prophylactic antibiotics and closed tubethoracostomy. Surg Gynecol Obstet 1985;160:259–63. [Ib]
41 Wooten SA, Barbarash RA, Strange C, et al. Systemic absorption oftetracycline following intrapleural instillation. Chest 1988;94:960–3.[IIa]
42 Mellor DJ. A new method of chest drain insertion. Anaesthesia1996;51:713–4. [IV]
43 Haggie JA. Management of pneumothorax: chest drain trocar is unsafeand unnecessary. BMJ 1993;307:443. [IV]
44 Rashid MA, Wikstrom T, Ortenwall P. A simple technique for anchoringchest tubes. Eur Respir J 1998;12:958–9. [IV]
45 Baumann MH, Strange C, Heffner JE, et al. Management ofspontaneous pneumothorax. An American College of Chest PhysiciansDelphi Consensus Statement. Chest 2001;119:590–602.
50 Mills M, Balsch B. Spontaneous pneumothorax : a series of 400 cases.Ann Thorac Surg 1965;1:286. [IV]
51 Brooks J. Open thoracotomy in the management of spontaneouspneumothorax. Ann Surg 1973;177:798. [IV]
52 Hall M, Jones A. Clamping may be appropriate to prevent discomfortand reduce risk of oedema (letter). BMJ 1997;315:313. [IV]
53 Roegela M, Roeggla G, Muellner M, et al. The cost of treatment ofspontaneous pneumothorax with the thoracic vent compared withconventional thoracic drainage (letter). Chest 1996;110:303. [Ib]
Background: A common pathological feature of chronic inflammatory airway diseases such as asthmaand chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominantmucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinicaltrials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD andasthma. However, their direct effects on mucin expression have been scarcely investigated.Methods: MUC5AC mRNA and protein expression were examined in cultured human airway epithelialcells (A549) and in human isolated bronchial tissue stimulated with epidermal growth factor (EGF; 25 ng/ml). MUC5AC mRNA was measured by real time RT-PCR and MUC5AC protein by ELISA (cell lysates andtissue homogenates), Western blotting (tissue homogenates) and immunohistochemistry.Results: EGF increased MUC5AC mRNA and protein expression in A549 cells. PDE4 inhibitors produceda concentration dependent inhibition of the EGF induced MUC5AC mRNA and protein expression withpotency values (2log IC50): roflumilast (,7.5) . rolipram (,6.5) . cilomilast (,5.5). Roflumilast alsoinhibited the EGF induced expression of phosphotyrosine proteins, EGF receptor, and phospho-p38- andp44/42-MAPK measured by Western blot analysis in A549 cells. In human isolated bronchus, EGFinduced MUC5AC mRNA and protein expression was inhibited by roflumilast (1 mM) as well as theMUC5AC positive staining shown by immunohistochemistry.Conclusion: Selective PDE4 inhibition is effective in decreasing EGF induced MUC5AC expression inhuman airway epithelial cells. This effect may contribute to the clinical efficacy of this new drug category inmucus hypersecretory diseases.
Mucus hypersecretion is an important feature ofchronic inflammatory airway diseases such as chronicobstructive pulmonary disease (COPD) and asthma,
and contributes to their morbidity and mortality.1 2 MUC5ACis the predominant mucin gene expressed in healthy humanairway epithelial cells and its expression is augmented inasthmatic1 and COPD patients,3 yet MUC5B upregulation is asignificant component of airway mucus in asthma4 andCOPD.5 Mucin MUC5AC expression in response to manydifferent stimuli appears regulated by an epidermal growthfactor receptor (EGFR) signalling cascade.6 Although sparsein healthy adult human airways, EGFR expression isupregulated by proinflammatory cytokines and in chronicairway diseases such as asthma, suggesting that it may havea role in the pathogenesis of mucus hypersecretion in theseconditions.1 7
Cyclic AMP (cAMP) is an important second messengerdetermining many aspects of cellular function through theactivation of protein kinase A (PKA). This cyclic nucleotide isinactivated by phosphodiesterases (PDEs). Many distinctforms of PDEs have been described, but PDE4 appears to bethe major PDE isoenzyme involved in the regulation of cAMPmediated functions in airway inflammatory and structuralcells.8 In vitro and in vivo studies have established thatselective PDE4 inhibitors suppress the activity of manyproinflammatory and immune cells, indicating that theymay be effective in the treatment of airway inflammatorydiseases. Indeed, oral PDE4 inhibitors are in phase II/IIIclinical trials for COPD and asthma.8 Recent work has shownthat rolipram, the archetypal PDE4 inhibitor, markedlydecreased goblet cell hyperplasia in animal models ofsecondary allergen challenge and chronic lipopolysaccharide
exposure.9 10 This effect of rolipram was attributed to itsknown ability to reduce the release of inflammatorymediators which activate goblet cells. However, the directeffects of PDE4 inhibitors on mucin gene expression andproduction by airway epithelial cells have not so far beeninvestigated to our knowledge.Normal human airway epithelial cells as well as the human
pulmonary epithelial A549 cells predominantly express PDE4with lesser activity of other PDEs;11 12 epithelial PDE4 activitymay therefore be an important target for monoselective PDE4inhibitors in the control of those inflammatory mediatorsproduced by these cells. Furthermore, the functioning of thecAMP/PKA pathway appears to be linked to that of theextracellular signal regulated kinase (ERK)/mitogen acti-vated protein kinase (MAPK) pathway, the downstreamsignalling of the EGFR.13
The aim of this study was to examine the effects of PDE4inhibition on the MUC5AC mucin gene expression andproduction triggered by the activation of the EGFR with oneof its endogenous ligands, the epidermal growth factor(EGF), in cultured human airway epithelial cells (A549 cells)and in human isolated bronchus.
METHODSPreparations and chemicalsThe human pulmonary epithelial cancer cell line (A549) waspurchased from ATCC (American Type Culture Collection;Rockville, MD, USA). This cell line has previously beenshown to be appropriate for studies of MUC5AC mRNA andprotein expression.14 A549 cells were grown on 24-wellcultured plates for MUC5AC mRNA experiments or T25flasks for MUC5AC protein experiments (Corning, NY, USA)
144
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
in Roswell Park Memorial Institute (RPMI) 1640 mediumcontaining 10% endotoxin-free fetal calf serum (FCS),10 mM HEPES, L-glutamine (4 mM), and standard anti-microbials.Human lung tissue was obtained from patients (five men,
one woman) of mean age 59 years (range 48–69) who hadundergone surgery for lung carcinoma as previously out-lined.15 Experiments were approved by the local ethicscommittee and informed consent was obtained. At the timeof operation all patients were active smokers but lungfunction was within normal limits by spirometry. None ofthe patients was being chronically treated with theophylline,b-adrenoceptor agonists, corticosteroids, or anticholinergicdrugs. Bronchial tissue fragments (,363 mm) were placedin a 24-well plate (3–4 fragments per well) with 1 ml RPMI1640 medium added to each well and left for 30 minutes at37 C before use. A similar preparation has previously beenshown to be appropriate for measuring MUC5AC mucinproduction from goblet cells in the epithelial layer.16
Rolipram, cilomilast, and roflumilast were synthesised atAltana Pharma (Konstanz, Germany). Dibutyryl-cAMP, for-skolin, and human recombinant epidermal growth factorwere from Sigma-Aldrich (Madrid, Spain). H-89, SB202190,PD98059, tyrphostin A46 and AG1478 were from Calbiochem(Nottingham, UK). Sp-5,6-DCl-cBIMPS was from Biolog LifeScience Institute (Bremen, Germany). Stock solutions wereprepared in water for H89 and dibutyryl-cAMP or in dimethylsulfoxide (DMSO) for the other compounds except EGFwhich was reconstituted as a stock solution of 50 mg/ml in10 mM acetic acid and 0.1% bovine serum albumin (BSA) asrecommended by the supplier. Drugs were further dilutedinto buffer solutions. The DMSO final concentration in theassay solutions was 0.1% (v/v). Water purified on a Milli-Q(Millipore Iberica, Madrid, Spain) system was used through-out.
Experimental protocolIn preliminary experiments with A549 cells the MUC5ACexpression in response to EGF stimulation was determined at3, 12, 18 and 24 hours. Peak responses were observed at 18–24 hours for MUC5AC mRNA and at 24 hours for MUC5ACprotein; an incubation time of 24 hours was thereforeselected in further experiments. Also, 25 ng/ml EGF wasselected as a near maximal response from pilot experimentswith EGF (5–50 ng/ml). The selected EGF concentration andtime of observation are within the values reported by othersin cultured airway epithelial cells.6 17 18 For human isolatedbronchus, MUC5AC responses to EGF stimulation were
studied at 0.5, 1, 3, 12 and 24 hours. In inhibition studiesA549 cells and human bronchus were pretreated with drugsor their vehicles for 15 minutes before stimulation with EGFand remained until termination of experiments. When used,antagonists were added 15 minutes before the correspondingdrug and remained for the rest of the experiment.
Mucin MUC5AC expressionThe mucin MUC5AC mRNA transcripts were measured byreal time quantitative RT-PCR as previously described.19 Themethod used for obtaining quantitative data of relative geneexpression, the comparative Ct (DDCt) method, was asdescribed by the manufacturer (PE-ABI PRISM 7700Sequence Detection System; Perkin-Elmer AppliedBiosystems, Perkin-Elmer Corporation, CA, USA).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) waschosen as the endogenous control gene. Total RNA wasextracted using TriPure isolation reagent (Roche, IN, USA).The PCR primers and probes for human MUC5AC andhuman GAPDH were designed using the Primer Express (PEBiosystems, Morrisville, NC, USA) according to the publishedhuman MUC5AC and GAPDH cDNA sequences (table 1).MUC5AC protein in A549 cells and human bronchial
tissues was measured by enzyme linked immunosorbentassay (ELISA) as outlined previously.6 In brief, for cell lysates,A549 cells cultured in T25 flasks were trypsinised, washed inPBS, centrifuged (5 minutes, 300 g, 4 C), and resuspended infive volumes of ice cold lysis buffer (50 mM Tris-HCl, pH 7.4,1% SDS, 50 mM NaCl, 2 mM EDTA, 1 mM MgCl2, 1 mMphenylmethylsulphonyl fluoride (PMSF), 1 mM dithiotreitol(DTT), 2 mg/ml leupeptin, 5 mg/ml aprotinin, 5 mg/ml pep-statin), vortexed for 20 seconds, sonicated, and centrifuged(30 minutes, 13 000 g, 4 C). Human bronchial tissues werehomogenised in five volumes of ice cold lysis buffer (50 mMTris-HCl, pH 7.4, 1 mM EDTA, 2 mM MgCl2, 1 mM phenyl-methylsulphonyl fluoride, 1 mM dithiotreitol, 2 mg/ml leu-peptin, 5 mg/ml aprotinin, and 5 mg/ml pepstatin) andcentrifuged (35 minutes, 13 000 g, 4 C). The total protein incell and tissue samples was estimated using the Bradfordassay.20 Samples were stored at 280 C.For ELISA, 100 mg total protein was incubated with
bicarbonate-carbonate buffer at 40 C in a 96-well plate untildry. Plates were washed with PBS and blocked with 2% BSA(fraction V; Sigma, St Louis, MO, USA) for 1 hour at roomtemperature. After three washes, plates were incubated with50 ml mouse monoclonal antibody (mAb) to MUC5AC (clone45M1, 1:100; Neomarkers, Fremont, CA, USA; according tothe supplier, this mAb recognises the peptide core of mucin
Table 1 Primers and probes for real time quantitative RT-PCR
bp, base pairs.For MUC5AC, reverse transcription of RNA to generate cDNA was performed with Taqman RT reagents (ref.N808-0234; Applied Biosystems, NJ, USA) and the PCR was performed with TaqMan Universal PCR Master Mix(ref. 4304437; Applied Biosystems). The specificity of PCR primers was tested under normal PCR conditions andthe products of the reaction were electrophoresed into a 2.5% NusieveH GTGH agarose gel (BMA, Rockland, ME,USA). One single band with the expected molecular size was observed for MUC5AC and GAPDH. For thevalidation of the DDCt method, the Ct values for target (MUC5AC) and reference (GAPDH) genes were measuredat different input amounts of total RNA (2.34–300 ng); DCt values (target v reference) were then plotted against logtotal RNA and the absolute value of the slope was found to be 0.008 (i.e. ,0.1), indicating similar efficiency of thetwo systems.
PDE4 inhibition and MUC5AC expression in airway epithelial cells 145
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
5AC and has no cross reactivity with other mucins). After1 hour the plates were washed with PBS and then incubatedwith 100 ml horseradish peroxidase-goat anti-mouse IgGconjugated (1:10 000). The colour reaction was developed
with TMB peroxidase solution (Sigma) and stopped with 1 MH2SO4. Absorbance was read at 450 nm.In addition, Western blot analysis of MUC5AC was carried
out in human bronchial homogenates as previously
�
�
�
�
������
��� �����
����������
�
�
�
�
������
��� �����
����������
�
�
�
�
������
��� �����
������� ����
�
�
�
�
������
��� �����
������� ����
���� �� ���� ��� ������
�! ����" �! ���" �! ������" �!
���� �� ��! �! ��!" �!
#
#
#
#
# #
$ $ $ $
$ $
Figure 1 Relative quantitation of MUC5AC mRNA and protein levels in A549 cells unstimulated (control) or stimulated with epidermal growth factor(EGF; 25 ng/ml, 24 hours incubation) in the absence or presence of selective inhibitors of EGF receptor tyrosine kinase activity (tyrphostin A46 andAG1478; upper panels) or a selective phosphodiesterase 4 inhibitor (roflumilast; lower panels). Incubation with DMSO (0.1% v/v) was withoutsignificant effect on MUC5AC expression in the absence and presence of EGF (upper panel). The EGF induced increase in MUC5AC expression wasabolished by pre-incubation with EGF receptor tyrosine kinase inhibitors (A46 100 mM or AG1478 3 mM) or roflumilast (1 mM). MUC5AC mRNA wasdetermined using real time RT-PCR by the DDCt method; columns show the fold increase in expression of MUC5AC relative to GAPDH values as mean(SE) of the 2-DDCt values of three independent experiments. MUC5AC protein was determined by enzyme linked immunosorbent assay (ELISA); columnsshow the fold increase from control levels as mean (SE) values of three independent experiments. *p,0.05 v control; �p,0.05 v EGF.
���
��
��
��
��
� ��
��
��
��
��
�� ��� ��
��
��
��
��
��
�� ��� ��
��
��
��
���
�
�����
����
� �
�
���
��
��
��
��
�
��
��
��
��
����
�����
����
� �
�
�� �����!"�
�����!�
�������!"�
Figure 2 Concentration-response curves for inhibition by the selective PDE4 inhibitors roflumilast, cilomilast and rolipram of the epidermal growthfactor (25 ng/ml; 24 hours incubation) induced expression of MUC5AC mRNA (left panel) and protein (right panel) in A549 cells. MUC5AC mRNAand protein were determined as indicated in fig 1. Points are mean (SE) values of three to five independent experiments. The corresponding IC50 valuefor each PDE4 inhibitor is shown in the Results section.
146 Mata, Sarria , Buenestado, et al
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
reported.19 In brief, aliquots of supernatants from 13 000 gcentrifugation of the tissue homogenate containing 25 mgtotal protein were suspended in SDS sample buffer andboiled for 5 minutes. Proteins were separated by SDS-PAGEelectrophoresis in 8% acrylamide-bisacrylamide (80:1). Theresulting gel was equilibrated in the transfer buffer: 25 mMTris-HCl, 192 mM glycine, and 20% (v/v) methanol, pH 8.3.The proteins were then transferred electrophoretically tonitrocellulose membranes which were incubated with 5% fat-free skimmed milk in phosphate buffered saline (PBS)containing 0.5% BSA and 0.05% Tween 20 for 1 hour, andincubated with mAb to MUC5AC (clone 45M1, 1:500,NeoMarkers) for 2 hours at room temperature. Boundantibody was visualised according to standard protocols forthe avidin-biotin-alkaline phosphatase complex method(ABC kit; Vector Laboratories, Burlingame, CA, USA).For MUC5AC immunocytochemical staining, A549 cells
were fixed and stained as previously outlined.17 For MUC5ACimmunohistochemical analysis of human bronchus, speci-mens were fixed, cut into sections, stained with haematox-ylin-eosin and periodic acid-Schiff (PAS) reagent (tovisualise goblet cells), and incubated with mouse monoclonalantibody to MUC5AC (clone 45M1, 1:100; NeoMarkers,Fremont, CA) as previously reported.1
Western blotting of EGFR, phospho-p38 MAPK,phospho-p44/42 MAPK and phosphotyrosineA549 cells were prepared for Western blot analysis asindicated above, and preparations were incubated with eitherEGFR mouse mAb (Ab-12, cocktail R19/48, Neomarkers, CA,USA), phospho-p38 MAPK (Thr180/Tyr182) mAb (28B10;Cell Signaling Technology, Beverly, MA, USA), phospho-p44/42 MAPK (Thr202/Tyr204) mAb (20G11; Cell SignalingTechnology), or anti-phosphotyrosine mAb (clone PY20;ICN Biomedical Inc, Aurora, OH, USA) according to themanufacturers’ instructions. Expression of EGFR and phos-photyrosine was measured at 24 hours and expression ofphospho-p38 MAPK and phospho-p44/42 MAPK at 5, 15, 30and 60 minutes of EGF (25 ng/ml) exposure. According to
the supplier information, these mAbs are highly selective anddo not appreciably cross react with the correspondingconfounding targets.
Measurement of cAMP accumulationFormation of cAMP was measured as previously outlined.21
Cultured A549 cells were exposed to EGF or vehicle in theabsence or presence of roflumilast for the indicated times,and the cAMP content was quantified using an enzymeimmunoassay kit according to the assay protocol provided bythe manufacturer (RPN225; Amersham Life Sciences, UK).
Cytotoxicity assessmentTo exclude the presence of non-selective detrimental effectsof the compounds studied, the percentage of lactate���� ���� �
�� ��� ����
���� ��� �
�� ��� ��������
���� ���� �� �
�����
�����
����������
����
���
��
!�
��"�
��"�
��"�
��"�
� # $� # $
� # $
� # $
Figure 3 Western analysis of the cellular proteins with PY20 anti-phosphotyrosine antibody, anti-EGFR antibody, and phospho-p38 andphospho-p44/42 MAPK antibodies in A549 cell lysates as indicated.Control levels are shown in lane A and the activation produced by EGF(25 ng/ml) is shown in lane B. Pretreatment with roflumilast (1 mM; laneC) reduced the EGF induced response. The duration of EGF exposurewas 24 hours for anti-phosphotyrosine and EGFR experiments and15 minutes for phospho-p38 and phospho-p44/42 MAPK experiments.Data presented are representative of three separate experiments.
�
��
���
���
���
���
���
���
���
���
��� �
����� ��
����������
������ ����������� ���
!"# ��������$!"# ��� ���$!"#
Figure 4 Relative quantitation of MUC5AC mRNA in A549 cellsunstimulated (control) or stimulated with epidermal growth factor (EGF;25 ng/ml, 24 hours incubation) in the absence or presence of selectiveinhibitors of p38-MAPK activity (SB202190) and p44/42 MAPK(PD98059). The EGF induced increase in MUC5AC expression wasabolished by pre-incubation with SB202190 (3 mM) or PD98059(10 mM). MUC5AC mRNA was determined using real time RT-PCR bythe DDCt method; columns show the fold increase in expression ofMUC5AC relative to GAPDH values as mean (SE) of the 2-DDCt values ofthree independent experiments; columns show the fold increase fromcontrol levels as mean (SE) values of three independent experiments.*p,0.05 v control; �p,0.05 v EGF.
�
�
�
���
���
���
����
����
����
�� ��������
���
����������
����
��������
�����
�����
�����
�����
������
Figure 5 Cyclic AMP levels in A549 cells exposed to EGF (E, 25 ng/ml)for different times (5 minutes, 30 minutes and 24 hours, as indicated) inthe absence (control, C) or presence of roflumilast (R, 1 mM). Asignificant increase was detected for roflumilast alone and in thepresence of EGF only at 5 minutes. Columns are mean (SE) of three tofive independent experiments. *p,0.05 from C and E.
PDE4 inhibition and MUC5AC expression in airway epithelial cells 147
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
dehydrogenase (LDH) release was assessed using a commer-cially available colorimetric assay (Sigma) according to themanufacturer’s instructions. Cell culture supernatants andcell lysates were collected and assessed for LDH content. Thepercentage of LDH release was calculated by taking the ratioof LDH in supernatants of experimental wells to the LDH incontrol supernantants plus cells lysates times 100.
Statistical analysisData are expressed as mean (SE) of n experiments. Inconcentration-response experiments the 2log inhibitoryconcentration 50% (IC50) was calculated by non-linearregression to express compound potency (GraphPadSoftware Inc, San Diego, USA). Statistical analysis wascarried out by analysis of variance followed by appropriatepost hoc tests including Bonferroni correction. Significancewas accepted as p,0.05.
RESULTSCytotoxicity studies and drug vehicle effectsNone of the compounds at their maximal concentrationsused showed any significant cytotoxicity (values for LDHrelease were below 5%).DMSO (0.1% v/v) did not alter the MUC5AC mRNA and
protein expression in the absence and presence of EGF 25 ng/ml (fig 1).
Effect of PDE4 inhibition on EGF induced MUC5ACexpression and EGFR signalling cascade in A549 cellsEGF (25 ng/ml; 24 hours incubation) increased MUC5ACgene expression and protein production in A549 cells (fig 1).This finding was confirmed by immunocytochemical stainingfor MUC5AC (not shown). The dependency of this responseon the tyrosine kinase activity of the EGFR was confirmed byinhibition of the EGF induced increase in MUC5AC mRNA
���
���
���
���
���
������
�� ������
����������
������ ����� ��� µ������ � µ��
!"#$��� ���� µ��
!"#$���%&'�
&'� ��� �()��� ����%&'�
�����%&'�
*
*
**
+ ++
,
* ,
+
+
���
���
���
���
���
������
�� ������
����������
������ -./ �� µ����� �� µ��
&'� ��� �()��� ���%&'� -./%���%&'�
���
���
���
���
������
�� ������
������ ����
������ -./ �� µ����� �� µ��
&'� ��� �()��� ���%&'� -./%���%&'�
���
���
���
���
������
�� ������
������ ����
*
++
+
������ ����� ��� µ������ � µ��
!"#$��� ���� µ��
!"#$���%&'�
&'� ��� �()��� ����%&'�
�����%&'�
Figure 6 Relative quantitation of MUC5AC mRNA and protein levels in A549 cells unstimulated (control) or stimulated with epidermal growth factor(EGF) in the absence or presence of roflumilast (upper panels) or drugs acting through the cAMP/PKA pathway (lower panels). Roflumilast (ROF; 1 mM)had no direct effect on MUC5AC expression but abolished the EGF (25 ng/ml) induced increase in MUC5AC expression. This inhibitory effect ofroflumilast was reversed in the presence of H89 (5 mM), a PKA inhibitor. To further explore the influence of the cAMP/PKA pathway in the EGF inducedenhancement of MUC5AC expression, the activity of forskolin (FORS; 3 mM), a direct activator of adenylyl cyclase, Sp-5,6-DCl-cBIMPS (BIMPS;10 mM), a direct activator of PKA, and db-cAMP (100 mM), a cell permeable analogue of cAMP, were tested. None of these compounds altered thebasal MUC5AC expression at the concentrations tested, but each inhibited the EGF induced overexpression of MUC5AC mRNA and protein. MUC5ACmRNA was determined using real-time RT-PCR by the DDCt method; columns show the fold increase in expression of MUC5AC relative to GAPDHvalues as mean (SE) of the 2-DDCt values of three independent experiments. MUC5AC protein was determined by enzyme linked immunosorbent assay(ELISA); columns show the fold increase from control levels as mean (SE) values of three to six independent experiments. *p,0.05 v control; `p,0.05 vEGF alone.
148 Mata, Sarria , Buenestado, et al
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
and protein in the presence of two different selectiveinhibitors of EGFR tyrosine kinase (tyrphostin A46 andAG1478, fig 1).3 18 22
Roflumilast (1 mM), a PDE4 inhibitor, did not change basalMUC5AC expression but prevented the increase in MUC5ACmRNA and protein production in response to EGF (fig 1). Therelationship between the suppression of EGF inducedMUC5AC expression and the PDE4 inhibition was furtherexplored by examining the inhibitory effects of otherstructurally unrelated PDE4 inhibitors and by exploring theirconcentration dependency. The increase in MUC5AC mRNAand protein by EGF was inhibited in a concentration-relatedfashion by pretreatment of cells with the PDE4 inhibitorsroflumilast, cilomilast, and rolipram (fig 2). The rank order ofpotencies (2log IC50 values) was roflumilast (7.59 (0.27)) .rolipram (6.66 (0.26)). cilomilast (5.58 (0.23)) for MUC5ACmRNA, and roflumilast (7.37 (0.12)) . rolipram (6.17(0.16)) . cilomilast (5.27 (0.10)) for MUC5AC protein. Afully active concentration of roflumilast (1 mM) was selectedfor additional experiments.Addition of EGF (25 ng/ml; 24 hours incubation) to A549
cells resulted in the phosphorylation of the tyrosine residuesof different intracellular proteins and the augmentedexpression of the EGFR, as shown by Western blot analysisof cell lysates with the corresponding specific antibodies(fig 3). Expression of phospho-p38 MAPK and phospho-p44/42 MAPK reached peak values after 15 minutes of exposureto EGF (25 ng/ml). Treatment with roflumilast (1 mM)abolished these EGF induced responses (fig 3). The func-tional requirement for p38 MAPK and for p44/42 MAPK inthe EGF induced augmentation of MUC5AC mRNA wasshown by using their respective selective inhibitors SB202190and PD98059 (fig 4).3 18 23
Relationship between inhibition of EGF inducedMUC5AC expression by PDE4 inhibitors and thecAMP/PKA pathway in A549 cellsWe then examined whether the inhibitory effect of roflumi-last on the overexpression of MUC5AC promoted by EGF wasrelated to its ability to inhibit PDE4, thus increasing cAMPand subsequently activating PKA. EGF alone failed to alterthe cellular content of cAMP significantly. Roflumilast(1 mM) produced an early (peak at 5 minutes) and transientincrease in the cAMP content of A549 cells (fig 5). Theinhibitory effect of roflumilast on the EGF induced MUC5ACresponse was reversed in the presence of H-89 (5 mM), aninhibitor of PKA,24 thus reinforcing the view of a mechanism
of action for roflumilast related to the cAMP/PKA pathway(fig 6).To establish the ability of the cAMP/PKA pathway to
interfere with the EGF induced overexpression of MUC5ACwe showed that forskolin (10 mM), a direct activator of
�
�
��
�
�
�
�� � � � � �� ��
�
�
�
�
��� �� � �������� �
������
������ �
���� ���! �
������
������ �
���� ��� ����
Figure 7 Time course of the relative expression of MUC5AC mRNAand protein in human isolated bronchus. The peak expression forMUC5AC mRNA was observed 1 hour after stimulation with EGF, thuspreceding the peak expression of MUC5AC protein at 3 hours.MUC5AC mRNA was determined using real time RT-PCR by the DDCt
method; points show the fold increase in expression of MUC5AC relativeto GAPDH values as mean (SE) of the 2-DDCt values. MUC5AC proteinwas determined in tissue by ELISA; points are mean (SE) of bronchialtissues. Data were obtained from three to five different patients.*p,0.05 v basal values.
�
�
�
�
�
�
�
�
�
�
�
�
��� � �� ��
��� ���
� ����
�����������������
������������
�����������������
������ �!�
������
�"#
�$#
�"#��$#
Figure 8 Relative quantitation of MUC5AC mRNA (upper panel) andprotein levels (middle panel) in human bronchus unstimulated (control)or stimulated with epidermal growth factor (EGF; 25 ng/ml) in theabsence or presence of roflumilast (ROF, 1 mM). Exposure time was1 hour for MUC5AC mRNA determination and 3 hours for MUC5ACprotein measurements. The EGF induced increase in MUC5ACexpression was abolished by roflumilast. MUC5AC mRNA wasdetermined using real time RT-PCR by the DDCt method; columns showthe fold increase in expression of MUC5AC relative to GAPDH values asmean (SE) of the 2-DDCt values of three independent experiments.MUC5AC protein was determined by enzyme linked immunosorbentassay (ELISA); columns show the fold increase from control levels asmean (SE) values of three independent experiments. *p,0.05 v control;�p,0.05 v EGF. The lower panel shows MUC5AC protein in humanbronchus determined by Western blotting with anti-MUC5ACmonoclonal antibody. A representative experiment of three independentexperiments is shown for the same experimental groups (C, control; E,EGF; R+E, roflumilast+EGF). Molecular weight marker is shown on theleft (213 kDa). The immunostained band of high molecular weight wasaugmented in EGF exposed samples and markedly diminished inroflumilast treated preparations.
PDE4 inhibition and MUC5AC expression in airway epithelial cells 149
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
adenylyl cyclase,24 db-cAMP (100 mM), a membrane perme-able analogue of cAMP,25 and Sp-5,6-DCl-cBIMPS (100 mM),an activator of PKA26—while not altering the control level ofMUC5AC expression—were impeding the enhanced expres-sion of MUC5AC elicited by EGF (fig 6).
Effect of PDE4 inhibition on EGF induced MUC5ACexpression in human isolated bronchusSince A549 cells are a cancer cell line, the results obtainedwith these cells may differ from responses of normal airwayepithelium. Additional experiments were therefore per-formed using human isolated bronchial tissue. In thispreparation EGF (25 ng/ml) augmented the MUC5ACmRNA and protein expression with peak values reached at1 hour and 3 hours after EGF exposure, respectively (fig 7).These effects of EGF were suppressed in the presence oftyrphostin A46 (not shown). Roflumilast (1 mM) preventedthe EGF induced overexpression of MUC5AC (fig 8).Immunohistochemistry experiments showed that
MUC5AC immunoreactivity was localised in goblet cells thatwere stained with PAS (fig 9). The MUC5AC positive stainingin airway epithelium was increased in EGF exposed prepara-tions, and this augmentation was reduced in roflumilasttreated tissues.
DISCUSSIONIn this study we found that PDE4 inhibition abolished theEGF induced augmentation of MUC5AC mRNA and proteinexpression in cultured human airway epithelial cells and inhuman bronchial tissue in vitro. To our knowledge, this is the
first report of a direct inhibitory effect on mucin productionof PDE4 inhibitors, a new class of drugs with potentialtherapeutic interest in the treatment of COPD and asthma—diseases in which mucus hypersecretion is consideredpathologically relevant.
EGF activates EGFR signalling cascade and MUC5ACexpression in A549 cellsThe EGFR signalling cascade is important for regulatingMUC5AC mucin gene expression and protein production byairway epithelial cells,6 and both the EGFR and the MUC5ACexpression are upregulated in chronic airway diseases such asasthma and COPD.1 3 7 The EGFR signalling pathway trans-lates into increased MUC5AC expression, the activationproduced by many different stimuli including oxidativestress, neutrophil elastase, tobacco smoke, bacterial and viralproducts, and inflammatory cytokines.17 18 27 In this study wehave selected EGF, an endogenous ligand of the EGFR, as adirect activator of this pathway based on previous studies incultured human airway epithelial NCI-H292 cells.6 18
We confirmed that A549 cells have a constitutive expres-sion of EGFR28 as shown by the faint band observed inWestern blot analysis with anti-EGFR mAb in the controlgroup (fig 3). The activation of the EGFR system results in anincrease of about twofold in MUC5AC mRNA and proteinexpression as shown by ELISA data obtained after 24 hoursof incubation with EGF. Immunocytochemistry of A549 cellsconfirmed this finding. The increase in MUC5AC mRNAand protein at 24 hours is within the time dependencyshown in cultured human airway epithelial cells for MUC5AC
Figure 9 Photomicrographs of representative histological sections from human bronchial tissue unstimulated (A, B, C) or stimulated with EGF (25 ng/ml) in the absence (D, E, F) or presence (G, H, I) of roflumilast (1 mM). Sections show haematoxylin-eosin (A, D, G) or periodic acid-Schiff (PAS; B, E, H)staining or immunohistochemical staining of MUC5AC (C, F, I). Mucin stores in goblet cells appear as purple staining (B, E, H). MUC5ACimmunoreactivity was observed as brown staining in goblet cells (C, F, I). Ciliated cells showed no staining for MUC5AC. The sections demonstrateincreased PAS and MUC5AC staining in the tissues exposed to EGF, and roflumilast prevented this augmentation. Original magnification6400 (exceptpanel E: 6250). Goblet cells are indicated by thick arrows and ciliated cells as thin arrows.
150 Mata, Sarria , Buenestado, et al
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
production elicited with various stimuli activating EGFRincluding EGF.6 17 18
Consistent with the notion that the overexpression ofMUC5AC is the consequence of the activation of the EGFRsignalling cascade, we also found that preincubation withEGFR tyrosine kinase inhibitors prevented the EGF inducedaugmentation of the MUC5AC mRNA expression and proteinproduction (fig 1). EGF therefore increases the protein-tyrosine kinase activity of its receptor and thereby activatesother kinase cascades such as MAPKs including p38 and p44/42 MAPKs.29 As expected, we found an early activation ofp38- and p44/42-MAPK as well as phosphorylation oftyrosine residues of different cell proteins and upregulationof the EGFR after exposure to EGF for 24 hours (fig 3).Furthermore, inhibition of p38-and p44/42-MAPKs with theselective inhibitors SB20202190 and PD98059 abrogated theEGF induced MUC5AC mRNA expression.
PDE4 inhibitors suppress the EGF induced MUC5ACexpression in A549 cells by activating the cAMP/PKApathwayThere is evidence to indicate that the functioning of thecAMP/PKA pathway is linked with that of the ERK/MAPKpathway. Thus, agents that increase the intracellular cAMPconcentration block growth factor stimulated ERK activationin a number of cell types by inhibiting the activation of Rafproteins.13 30 In fact, PDE4 isoenzymes may provide a pivotalpoint for integrating cAMP and ERK signal transduction incells.31 The known relevance of PDE4 isoenzyme activity inthe regulation of cAMP levels in human airway epithelialcells, including A549 cells,11 12 prompted us to investigate theeffects of monoselective PDE4 inhibitors on the EGF inducedMUC5AC expression and related events occurring in A549cells.We found that three different structurally unrelated PDE4
inhibitors—the archetypal PDE4 inhibitor rolipram and thesecond generation PDE4 inhibitors cilomilast and roflumi-last—produced concentration dependent inhibitions of theEGF induced MUC5AC mRNA and protein expression. Thepotency order of their activities (expressed as –log IC50
values) was roflumilast (,7.5) . rolipram (,6.5) .
cilomilast (,5.5). These differences in potencies are consis-tent with results obtained in other in vitro human cellsystems, yet variation may exist depending on the stimulusand the cell type studied.32 Since roflumilast (1 mM)suppressed both MUC5AC mRNA and protein production inresponse to EGF, this concentration was selected for furtherstudies.The inhibitory action of roflumilast appears to be exerted at
different levels of the EGFR signalling cascade. Thus, weshowed that roflumilast (1 mM) markedly inhibited the earlyphospho-p38 MAPK expression as well as the phosphoryla-tion of tyrosine residues of proteins and the overexpression ofEGFR in response to EGF stimulation measured at 24 hoursEGF exposure.The inhibitory effects of roflumilast on the EGFR cascade
events leading to enhanced MUC5AC expression are probablyrelated to the activation of the cAMP/PKA pathway since thisselective PDE4 inhibitor elicited a transient early increase incAMP levels in A549 cells, and its inhibitory effects onMUC5AC expression were reversed by preincubation with H-89, an inhibitor of PKA activity.24 Furthermore, forskolin (adirect activator of adenylyl cyclase),24 db-cAMP (a membranepermeant analogue of cAMP),25 and Sp-5,6-DCl-cBIMPS (aspecific activator of PKA)26 prevented the enhanced expres-sion of MUC5AC elicited by EGF (fig 6), thus supporting thenotion that the activation of the cAMP/PKA pathway iseffective in exerting an inhibitory influence on the EGFRcascade leading to MUC5AC expression in A549 cells.
PDE4 inhibition attenuates EGF induced MUC5ACexpression in human airways in vitroThe inhibitory effects resulting from PDE4 inhibition withroflumilast in cultured A549 cells may not necessarily berepresentative of the responses of the epithelial cells in thehuman airways. MUC5AC expression was therefore alsoexamined in human isolated bronchus, a preparation thathas previously been shown to have a basal secretion of mucinMUC5AC produced principally by goblet cells.16 In the humanairways in vitro, MUC5AC mRNA expression reached a peakat 1 hour after stimulation with EGF, while peak MUC5ACprotein production in tissue and medium was observed at3 hours (fig 7). This represents faster kinetics of MUC5ACexpression than in cultured A549 cells, but we have notinvestigated the reason for this difference. Pretreatment withroflumilast (1 mM) markedly inhibited this augmentedexpression of MUC5AC induced by EGF activation, indicatingthat the direct inhibitory effects produced by this PDE4inhibitor in cultured A549 cells are reproducible in intactairway epithelial cells. Immunohistochemical analysis ofhuman bronchial tissues confirmed that EGF exposureresulted in an augmented expression of MUC5AC positivestained cells in airway epithelium and treatment withroflumilast effectively prevented this EGF induced over-expression of MUC5AC (fig 9).In summary, the results of this study indicate that putative
PDE4 inhibitors, in addition to their established inhibitoryeffects on the airway inflammatory cells,9 10 may also exertdirect effects on human airway epithelial cells inhibiting theMUC5AC expression that follows the activation of the EGFRsignalling cascade. These findings may be of added value toresults from recent phase II/III clinical trials which suggest atherapeutic benefit for PDE4 inhibitors in mucus hyperse-cretory diseases such as COPD and asthma.8
ACKNOWLEDGEMENTSThe authors are indebted to the teams of the Services of ThoracicSurgery and Pathology of the University Clinic Hospital and ‘La Fe’University Hospital of Valencia (Spain) for making the human lungtissue available to us, and to Altana Pharma for the gift ofphosphodiesterase 4 inhibitors. The technical assistance of PedroSantamaria and Dora Martı is also gratefully acknowledged.
M Mata, B Sarria, A Buenestado, J Cortijo, E J Morcillo, Department ofPharmacology, Faculty of Medicine, University of Valencia, Valencia,SpainJ Cortijo, Research Foundation, University General Hospital, Universityof Valencia, Valencia, SpainM Cerda, Department of Pathology, Faculty of Medicine, University ofValencia, Valencia, Spain
This work was supported by grants SAF2002-04667 and SAF2003-07206-C02-01 from CICYT (Ministry of Science and Technology,Spanish Government) and Research Groups-03/166 funding fromRegional Government (Generalitat Valenciana).
3 Gensch E, Gallup M, Sucher A, et al. Tobacco smoke control of mucinproduction in lung cells requires oxygen radicals AP-1 and JNK. J Biol Chem2004;279:39085–93.
4 Groneberg DA, Eynott PR, Lim S, et al. Expression of respiratory mucins infatal status asthmaticus and mild asthma. Histopathology 2002;40:367–73.
5 Kirkham S, Sheehan JK, Knight D, et al. Heterogeneity of airway mucus:variations in the amounts and glycoforms of the major oligomeric mucinsMUC5AC and MUC5B. Biochem J 2002;361:537–46.
6 Takeyama K, Dabbagh K, Lee HM, et al. Epidermal growth factor systemregulates mucin production in airways. Proc Natl Acad Sci USA1999;96:3081–6.
PDE4 inhibition and MUC5AC expression in airway epithelial cells 151
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from
7 Polosa R, Puddicombe SM, Krishna MT, et al. Expression of c-erbB receptorsand ligands in the bronchial epithelium of asthmatic subjects. J Allergy ClinImmunol 2002;109:75–81.
8 Giembycz MA. Development status of second generation PDE4 inhibitorsfor asthma and COPD: the story so far. Monaldi Arch Chest Dis2002;57:48–64.
9 Kanehiro A, Ikemura T, Makela MJ, et al. Inhibition of phosphodiesterase 4attenuates airway hyperresponsiveness and airway inflammation in a modelof secondary allergen challenge. Am J Respir Crit Care Med2001;163:173–84.
10 Toward TJ, Broadley KJ. Goblet cell hyperplasia, airway function, andleukocyte infiltration after chronic lipopolysaccharide exposure in consciousguinea pigs: effects of rolipram and dexamethasone. J Pharmacol Exp Ther2002;302:814–21.
11 Dent G, White SR, Tenor H, et al. Cyclic nucleotide phosphodiesterase inhuman bronchial epithelial cells: characterization of isoenzymes andfunctional effects of PDE inhibitors. Pulm Pharmacol Ther 1998;11:47–56.
12 Fuhrmann M, Jahn H-U, Seybold J, et al. Identification and function of cyclicnucleotide phosphodiesterase isoenzymes in airway epithelial cells.Am J Respir Cell Mol Biol 1999;20:292–302.
13 Sevetson BR, Kong X, Lawrence JC Jr. Increasing cAMP attenuates activationof mitogen-activated protein kinase. Proc Natl Acad Sci USA1993;90:10305–9.
14 Berger JT, Voynow JA, Peters KW, et al. Respiratory carcinoma cell lines.MUC genes and glycoconjugates. Am J Respir Cell Mol Biol1999;20:500–10.
15 Sarria B, Naline E, Zhang Y, et al. Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus. Am J Physiol2002;283:L1125–32.
16 Labat C, Bara J, Gascard JP, et al. M1/MUC5AC mucin released by humanairways in vitro. Eur Respir J 1999;14:390–5.
17 Takeyama K, Dabbagh K, Shim JJ, et al. Oxidative stress causes mucinsynthesis via transactivation of epidermal growth factor receptor: role ofneutrophils. J Immunol 2000;164:1546–52.
19 Mata A, Ruız A, Cerda M, et al. Oral N-acetylcysteine reduces bleomycin-induced lung damage and mucin Muc5ac expression in rats. Eur Respir J2003;22:900–5.
20 Bradford M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. AnalBiochem 1976;72:248–54.
21 Cortijo J, Villagrasa V, Pons R, et al. Bronchodilator and anti-inflammatoryactivities of glaucine: in vitro studies in human airway smooth muscle andpolymorphonuclear leukocytes. Br J Pharmacol 1999;127:1641–51.
22 Crump CM, Williams JL, Stephens DJ, et al. Inhibition of the interactionbetween tyrosine-based motifs and the medium chain subunit of the AP-2adaptor complex by specific tyrphostins. J Biol Chem 1998;273:28073–7.
23 Kawaguchi M, Kokubu F, Matsukura S, et al. Induction of C-X-C chemokines,growth-related oncogene a expression, and epithelial cell-derived neutrophil-activating protein-78 by ML-1 (interleukin-17F) involves activation of raf1-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase1/2 pathway. J Pharmacol Exp Ther 2003;307:1213–20.
24 Chijiwa T, Mishima A, Hagiwara M, et al. Inhibition of forskolin-inducedoutgrowth and portein phosphorylation by a newly synthesized selectiveinhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC 12DPheochromocytoma cells. J Biol Chem 1990;265:5267–72.
25 Pedrosa R, Gomes P, Soares-da-Silva P. Distinct signalling cascadesdownstream to Gsa coupled dopamine D1-like NHE3 inhibition in rat andopossum renal epithelial cells. Cell Physiol Biochem 2004;14:91–100.
26 Sandberg M, Butt E, Nolte C, et al. Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-39,59-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependentprotein kinase in cell extracts and intact cells. Biochem J 1991;279:521–7.
27 Basbaum C, Li D, Gensch E, et al. Mechanisms by which Gram-positivebacteria and tobacco smoke stimulate mucin induction through the epidermalgrowth factor receptor (EGFR). In: Chadwick DJ, Goode JA, eds. Mucushypersecretion in respiratory disease. Chichester: John Wiley & Sons,2002:171–6.
28 Hauck CR, Sieg DJ, Hsia DA, et al. Inhibition of focal adhesion kinaseexpression or activity disrupts epidermal growth factor-stimulated signalingpromoting the migration of invasive human carcinoma cells. Cancer Res2001;61:7079–90.
29 Wetzker R, Bohmer F-D. Transactivation joins multiple tracks to the ERK/MAPK cascade. Nature Rev Mol Cell Biol 2003;4:651–7.
30 Piiper A, Gebhardt R, Kronenberger B, et al. Pertussis toxin inhibitscholecystokinin- and epidermal growth factor-induced mitogen-activatedprotein kinase activation by disinhibition of the cAMP signaling pathway andinhibition of c-Raf-1. Mol Pharmacol 2000;58:608–13.
31 Baillie GS, MacKenzie SJ, McPhee I, et al. Sub-family selective actions in theability of Erk2 MAP kinase to phosphorylate and regulate the activity of PDE4cyclic AMP-specific phosphodiesterases. Br J Pharmacol 2000;131:811–9.
32 Hatzelmann A, Schudt C. Anti-inflammatory and immunomodulatorypotential of the novel PDE4 inhibitor roflumilast in vitro. J Pharmacol Exp Ther2001;297:267–79.
Correction
It has been brought to our attention that there is an error in figure 3 on page ii55 of the PleuralDisease Guideline available at www.brit-thoracic.org.uk/docs/PleuralDiseaseChestDrain/pdf. Below is a corrected diagram illustrating the ‘‘safe triangle’’ for a chest drain. Thepublishers apologise for this error.
152 Mata, Sarria , Buenestado, et al
www.thoraxjnl.com
group.bmj.com on September 26, 2011 - Published by thorax.bmj.comDownloaded from