Guia Drenaje Torax

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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):ii53ii59
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) recommendations in their instructors manual1 and there have
been many general descriptions of the step by
step method of chest tube insertion.29
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 complications. 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
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
.......................
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 recommended in patients with known risk
factors. [C]
The differential diagnosis between a
pneumothorax and bullous disease requires careful radiological assessment.
Similarly it is important to differentiate
between the presence of collapse and a
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 pneumothorax in patients over 50 years
Malignant pleural effusion
Empyema and complicated parapneumonic
pleural effusion
Traumatic haemopneumothorax
Postoperativefor example, thoracotomy,
oesophagectomy, cardiac surgery
pleural effusion when the chest radiograph 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, warfarin 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 chlorhexidine in alcohol
Sterile drapes
Gauze swabs
A selection of syringes and needles (2125
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)
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Laws, Neville, Duffy
Indication to insert
chest drain
(section 3)
Consent
(section 6)
Premedication
(section 6)
Figure 2
Confirmation of site of insertion
clinically and on radiography
(section 8)
Positioning of
patient
(section 7)
Equipment required for insertion of chest drains.
Guidewire with dilators (if small tube being used)

Chest tube
Connecting tubing
Closed drainage system (including sterile water if underwater seal being used)
Dressing
Equipment may also be available in kit form.
6. CONSENT AND PREMEDICATION

Size of chest drain
(sections 10 and 13)
Aseptic technique (section 11)

Local anaesthesia (section 12)
Blunt dissection if required (section 13.3.2)
Securing drain and suture

(section 13.3.4)
Underwater seal (section 14.2)

Clamping instructions (section 14.1)
Decision re suction
(section 14.3)
Removal of drain
(section 14.5)
Figure 1 Summary of chest drain insertion process.
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Prior to commencing chest tube insertion the procedure should be explained fully to the patient and
consent recorded in accordance with national guidelines. [C]
Unless there are contraindications to its use, premedication (benzodiazepine or opioid) should be
given to reduce patient distress. [B]
Consent should be taken and recorded in keeping with
national guidelines. The General Medical Council (GMC)
guidelines for consent state that it is the responsibility of the
doctor carrying out a procedure, or an appropriately trained
individual with sufficient knowledge of a procedure, to
explain its nature and the risks associated with it. It is within
the rights of a competent individual patient to refuse such
treatment. In the case of an emergency, when the patient is
unconscious and the treatment is lifesaving, treatment may be
carried out but must be explained as soon as the patient is
sufficiently recovered to understand. If possible, an information leaflet should be given before the procedure.
Chest drain insertion has been reported to be a painful procedure with 50% of patients experiencing pain levels of 910
on a scale of 10 in one study,11 and therefore premedication
should be given. Despite the apparent common sense of this
approach, there is little established evidence of the effect from
these medications. Premedication could be an intravenous
anxiolyticfor example, midazolam 15 mg titrated to
achieve adequate sedationgiven immediately before the
procedure or an intramuscular opioid given 1 hour before,
although neither drug has been shown to be clearly superior.
Both these classes of drugs may cause respiratory depression
and patients with underlying lung disease such as COPD
should be observed as reversal agentsfor example, naloxone
or flumazenilare occasionally necessary.
While the use of atropine as part of premedication for fibreoptic bronchoscopy has been assessed, no controlled trial of its
use in chest tube insertion has been identified, although it is
advocated in some centres. Case reports of vasovagal
reactions12 and a death due to vagal stimulation following tube
insertion13 may support its use as premedication.

Figure 3 Diagram to illustrate the safe triangle.
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unsightly scarring. A more posterior position may be chosen if

suggested by the presence of a locule. While this is safe, it is
not the preferred site as it is more uncomfortable for the
patient to lie on after insertion and there is a risk of the drain
kinking.
For apical pneumothoraces the second intercostal space in
the mid clavicular line is sometimes chosen but is not recommended routinely as it may be uncomfortable for the patient
and may leave an unsightly scar. Loculated apical pneumothoraces are not uncommonly seen following thoracotomy and
may be drained using a posteriorly sited (suprascapular) apical tube.19 20 This technique should be performed by an operator experienced in this techniquefor example, a thoracic
surgeon. If the drain is to be inserted into a loculated pleural
collection, the position of insertion will be dictated by the site
of the locule as determined by imaging.
7 PATIENT POSITION
The preferred position for drain insertion is on the bed,
slightly rotated, with the arm on the side of the lesion behind
the patients head to expose the axillary area.7 9 An alternative
is for the patient to sit upright leaning over an adjacent table
with a pillow or in the lateral decubitus position.14 Insertion
should be in the safe triangle illustrated in fig 3. This is the
triangle bordered by the anterior border of the latissimus
dorsi, the lateral border of the pectoralis major muscle, a line
superior to the horizontal level of the nipple, and an apex
below the axilla.
8 CONFIRMING SITE OF DRAIN INSERTION

A chest tube should not be inserted without further
image guidance if free air or fluid cannot be aspirated
with a needle at the time of anaesthesia. [C]
Imaging should be used to select the appropriate site
for chest tube placement. [B]
A chest radiograph must be available at the time of
drain insertion except in the case of tension
pneumothorax. [C]
Immediately before the procedure the identity of the patient
should be checked and the site and side for insertion of the
chest tube confirmed by reviewing the clinical signs and the
chest radiograph. Fluoroscopy, ultrasonography, and CT scanning can all be used as adjunctive guides to the site of tube
placement.15 Before insertion, air or fluid should be aspirated;
if none is forthcoming, more complex imaging than a chest
radiograph is required.
The use of ultrasonography guided insertion is particularly
useful for empyema and effusions as the diaphragm can be
localised and the presence of loculations and pleural thickening defined.16 Using real time scanning at the time of the procedure can help to ensure that the placement is safe despite
the movement of the diaphragm during respiration. The complication rate following image guided thoracocentesis is low
with pneumothoraces occurring in approximately 3% of
cases.17 Success rates of image guided chest tube insertion are
reported to be 7186%.12 If an imaging technique is used to
indicate the site for drain insertion but the procedure is not
carried out at the time of imaging, the position of the patient
at the time must be clearly documented to aid accurate insertion when the patient returns to the ward. It is recommended
that ultrasound is used if the effusion is very small or initial
blind aspiration fails.
9 DRAIN INSERTION SITE

The most common position for chest tube insertion is in the
mid axillary line,29 through the safe triangle18 illustrated in
fig 3 and described above. This position minimises risk to
underlying structures such as the internal mammary artery
and avoids damage to muscle and breast tissue resulting in
10 DRAIN SIZE
Small bore drains are recommended as they are more
comfortable than larger bore tubes [B] but there is
no evidence that either is therapeutically superior.
Large bore drains are recommended for drainage of
acute haemothorax to monitor further blood loss. [C]
The use of large bore drains has previously been
recommended6 8 21 as it was felt that there was an increase in
the frequency of drain blockage, particularly by thick
malignant or infected fluid. The majority of physicians now
use smaller catheters (1014 French (F)) and studies have
shown that these are often as effective as larger bore tubes22
and are more comfortable and better tolerated by the
patient.23 There remains intense debate about the optimum
size of drainage catheter2426 and no large randomised trials
directly comparing small and large bore tubes have been performed.
In pneumothoraces 9 F catheters have been used with success rates of up to 87%, although in a few patients the air leak
seems to exceed the capacity of this small catheter.27 In the
event of failure to drain a pneumothorax due to excessive air
leakage, it is recommended that a larger bore tube be inserted.
There is no evidence to suggest that surgical emphysema rates
vary between the size of drains. Ultrasonographically guided
insertion of pigtail catheters for treatment of malignant pleural effusions for sclerotherapy has been particularly well studied with good effect.2832 The use of small bore pigtail catheters
has allowed outpatient treatment of malignant pleural
effusions which have not responded to chemotherapy.33
Empyemas are often successfully drained with ultrasonically
placed small bore tubes with the aid of thrombolytic
agents.34 35
In the case of acute haemothorax, however, large bore tubes
(2830 F minimum) continue to be recommended for their
dual role of drainage of the thoracic cavity and assessment of
continuing blood loss.36
11 ASEPTIC TECHNIQUE
Aseptic technique should be employed during catheter insertion. [C]
Prophylactic antibiotics should be given in trauma
cases. [A]
As a chest drain may potentially be in place for a number of
days, aseptic technique is essential to avoid wound site infection or secondary empyema. Although this is uncommon,
estimations of the empyema rate following drain insertions
for trauma are approximately 2.4%.37 While the full sterile
technique afforded by a surgical theatre is usually unnecessary, sterile gloves, gown, equipment and the use of sterile
towels after effective skin cleansing using iodine or chlorhexidine are recommended. A large area of skin cleansing should
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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 complications. However, a meta-analysis of these studies has been performed which suggested that, in the presence of chest trauma
(penetrating or blunt), the use of prophylactic antibiotics
reduces the absolute risk of empyema by 5.57.1% and of all
infectious complications by 12.113.4%.39 The use of prophylactic 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 pneumothoraces treated with chest tubes.40
12 ANAESTHESIA
Local anaesthetic should be infiltrated prior to insertion 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 pleural 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 g/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 effective 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 appropriate depends on the catheter size and is discussed below.
13.1 Small bore tube (814 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 (1624 F)
Medium sized chest drains may be inserted by a Seldinger
technique or by blunt dissection as outlined below. As the
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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 chest
drain. [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 operators finger and tube.
The incision should be made just above and parallel to a rib.
13.3.2 Blunt dissection

Many 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 underlying organs that might be damaged at tube insertion.29 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 or
basally for fluid. However, any tube position can be
effective at draining air or fluid and an effectively
functioning drain should not be repositioned solely
because 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 insertedthe 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). Complicated purse string sutures must not be used as they convert

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If a patient with a clamped drain becomes breathless

or develops subcutaneous emphysema, the drain
must be immediately unclamped and medical advice
sought. [C]
Figure 4 Example of stay and closing sutures.
a linear wound into a circular one that is painful for the

patient and may leave an unsightly scar.9 A suture is not usually 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 subject 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 SYSTEM

14.1 Clamping drain
A bubbling chest tube should never be clamped. [C]
Drainage of a large pleural effusion should be
controlled to prevent the potential complication of
re-expansion pulmonary oedema. [C]
In cases of pneumothorax, clamping of the chest tube
should usually be avoided. [B]
If a chest tube for pneumothorax is clamped, this
should be under the supervision of a respiratory physician or thoracic surgeon, the patient should be
managed in a specialist ward with experienced nursing staff, and the patient should not leave the ward
environment. [C]
Figure 5 Omental tag to support the tube while allowing it to lie a

little away from the chest wall.
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 complication of tension pneumothorax.3 6 9 A bubbling drain
therefore should never be clamped. However, many experienced 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 deterioration. 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 flutter
valve. [C]
Use of a flutter valve system allows earlier mobilisation and the potential for earlier discharge of
patients 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.24 7 This enables the operator to see air bubble out as the
lung re-expands in the case of pneumothorax or fluid evacuation 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 disadvantages of the underwater seal system include obligatory
inpatient management, difficulty of patient mobilisation, and
the risk of knocking over the bottle.
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The use of integral Heimlich flutter valves has been

advocated in patients with pneumothoraces, especially as they
permit ambulatory or even outpatient management which has
been associated with a 8595% success rate.53 54 In 176 cases of
pneumothorax treated with small chest tubes and a Heimlich
flutter valve there were only eight failures (hospital admissions for problems with tube function or placement). The
mean length of inpatient stay has been quoted at 5 hours with
a thoracic vent and 144 hours with an underwater seal, with a
cost saving US$5660.53 Case reports of incorrect use (wrong
direction of flow) of such valves have been described, however,
with tension pneumothorax as a result.55 Flutter valves cannot
be used with fluid drainage as they tend to become blocked.
However, in the UK a similar short hospital stay is achieved by
initial aspiration of pneumothoraces (see guidelines on pneumothorax, page ii39).
The use of a drainage bag with an incorporated flutter valve
and vented outlet has been successfully used
postoperatively.56 57 A randomised trial of 119 cases following
elective thoracotomy compared the use of an underwater seal
with the flutter bag and found no difference in drainage volumes, requirement for suction, or complications with the
added advantage of earlier mobilisation with drainage bags.57
In cases of malignant pleural effusion drainage a closed
system using a drainage bag or aspiration via a three way tap
has been described to aid palliation and outpatient
management.33 One report of a modified urinary collecting
bag for prolonged underwater chest drainage has been
described for use with empyemas, bronchopulmonary fistula,
and pneumothorax associated with emphysema with no complications in the 12 patients studied.58
14.3 Suction
When chest drain suction is required, a high volume/
low pressure system should be used. [C]
When suction is required, the patient must be nursed
by appropriately trained staff. [C]
The use of high volume/low pressure suction pumps has been
advocated in cases of non-resolving pneumothorax or following chemical pleurodesis,6 but there is no evidence to support
its routine use in the initial treatment of spontaneous
pneumothorax.59 60 If suction is required, this may be
performed via the underwater seal at a level of 1020 cm H2O.
A high volume pump (e.g. Vernon-Thompson) is required to
cope with a large leak. A low volume pump (e.g. Roberts
pump) is inappropriate as it is unable to cope with the rapid
flow, thereby effecting a situation similar to clamping and
risking formation of a tension pneumothorax. A wall suction
adaptor may also be effective, although chest drains must not
be connected directly to the high negative pressure available
from wall suction.
In the management of pleural infection, the use of suction
is less clear. Most studies are observational and have used suction applied via the chest tube after flushing to prevent blocking and have reported success, but this has not been compared
with cases without suction. This is discussed further in the
guideline on pleural infection (page ii18).
There is no evidence that briefly disconnecting a drain from
suction used for spontaneous pneumothorax or pleural
effusion is disadvantageous. Therefore, as long as adequate
instruction is given to patient, portering and nursing staff
with regard to keeping the underwater seal bottle below the
level of the chest, it is acceptable to stop suction for short periods such as for radiography.
14.4 Ward instructions
Patients with chest tubes should be managed on
specialist wards by staff who are trained in chest
drain management. [C]
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Audit points
The presence and use of an appropriate nursing chest drain
observation chart should be noted.
The frequency of chest drain complications should be
recorded.
The use of premedication and analgesics and patient pain
scores relating to chest drain insertion should be recorded.
The duration of chest tube drainage should be recorded.
A chest radiograph should be performed after insertion of a chest drain. [C]

Patients should be managed on a ward familiar with chest
tubes. Instruction to and appropriate training of the nursing
staff is imperative. If an underwater seal is used, instructions
must be given to keep the bottle below the insertion site at all
times, to keep it upright, and to ensure that adequate water is
in the system to cover the end of the tube.9 Daily reassessment
of the amount of drainage/bubbling and the presence of respiratory swing should be documented, preferably on a dedicated
chest drain chart. Instruction with regard to chest drain
clamping must be given and recorded.61
Patients should be encouraged to take responsibility for
their chest tube and drainage system. They should be taught
to keep the underwater seal bottle below the level of their
chest and to report any problems such as pulling on the drain
insertion site. Educational material (e.g. leaflets) should be
available on the ward for patients and nursing staff.
A chest radiograph should be performed to assess tube
position, exclude complications such as pneumothorax or surgical emphysema, and assess the success of the procedure in
the volume of fluid drainage or pneumothorax resolution.
Concern has previously been expressed in cases where the
tube enters the lung fissure. In a study of 66 patients with
chest tubes inserted for acute chest trauma, 58% of which
were located within a pulmonary fissure,62 no difference in
outcome was seen between these cases and those in whom the
tube was located outside the fissures.
14.5 Removal of the chest tube
In cases of pneumothorax, the chest tube should not
be clamped at the time of its removal. [B]
In cases of pneumothorax, there is no evidence that clamping
a chest drain at the time of its removal is beneficial.60
The chest tube should be removed either while the patient
performs Valsalvas manoeuvre or during expiration with a
brisk firm movement while an assistant ties the previously
placed closure suture.24 7 8 The timing of removal is dependent
on the original reason for insertion and clinical progress (see
guidelines for management of pneumothorax (page ii39),
malignant pleural effusions (page ii29), and pleural infections
(page ii18)).
In the case of pneumothorax, the drain should not usually
be removed until bubbling has ceased and chest radiography
demonstrates lung reinflation.4 Clamping of the drain before
removal is generally unnecessary. In one study the removal of
chest tubes after continuous suction was compared with the
removal after a period of disconnection from suction to an
underwater seal. No significant difference was seen between
these two methods with only two of 80 cases (2.5%) requiring
reinsertion of a chest tube.38
15 PATIENTS REQUIRING ASSISTED VENTILATION

During the insertion of a chest tube in a patient on a high
pressure ventilator (especially with positive end expiratory
pressure (PEEP), it is essential to disconnect from the ventilator at the time of insertion to avoid the potentially serious

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
ACKNOWLEDGEMENTS
The authors are grateful to Dr Richard Holmes for figs 3, 4, and 5.
.....................
Authors affiliations
D Laws, Department of Thoracic Medicine, Royal Bournemouth Hospital,
Bournemouth BH7 7DW, UK
E Neville, Respiratory Centre, St Marys Hospital, Portsmouth PO3 6AD,
UK
J Duffy, Cardiothoracic Surgery Department, City Hospital, Nottingham
NG5 1PB, UK
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www.thoraxjnl.com

144
AIRWAY BIOLOGY
Phosphodiesterase 4 inhibition decreases MUC5AC

expression induced by epidermal growth factor in human
airway epithelial cells
M Mata, B Sarria, A Buenestado, J Cortijo, M Cerda, E J Morcillo
...............................................................................................................................
Thorax 2005;60:144152. doi: 10.1136/thx.2004.025692
See end of article for

authors affiliations
.......................
Correspondence to:
Dr E J Morcillo,
Department of
Pharmacology, Faculty of
Medicine, Av. Blasco
Ibanez 15, E-46010
Valencia, Spain; Esteban.
Morcillo@uv.es
Received 29 March 2004
Accepted
3 November 2004
.......................
Background: A common pathological feature of chronic inflammatory airway diseases such as asthma
and chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominant
mucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinical
trials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD and
asthma. However, their direct effects on mucin expression have been scarcely investigated.
Methods: MUC5AC mRNA and protein expression were examined in cultured human airway epithelial
cells (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 and
tissue homogenates), Western blotting (tissue homogenates) and immunohistochemistry.
Results: EGF increased MUC5AC mRNA and protein expression in A549 cells. PDE4 inhibitors produced
a concentration dependent inhibition of the EGF induced MUC5AC mRNA and protein expression with
potency values (2log IC50): roflumilast (,7.5) . rolipram (,6.5) . cilomilast (,5.5). Roflumilast also
inhibited the EGF induced expression of phosphotyrosine proteins, EGF receptor, and phospho-p38- and
p44/42-MAPK measured by Western blot analysis in A549 cells. In human isolated bronchus, EGF
induced MUC5AC mRNA and protein expression was inhibited by roflumilast (1 mM) as well as the
MUC5AC positive staining shown by immunohistochemistry.
Conclusion: Selective PDE4 inhibition is effective in decreasing EGF induced MUC5AC expression in
human airway epithelial cells. This effect may contribute to the clinical efficacy of this new drug category in
mucus hypersecretory diseases.
ucus hypersecretion is an important feature of

chronic inflammatory airway diseases such as chronic
obstructive pulmonary disease (COPD) and asthma,
and contributes to their morbidity and mortality.1 2 MUC5AC
is the predominant mucin gene expressed in healthy human
airway epithelial cells and its expression is augmented in
asthmatic1 and COPD patients,3 yet MUC5B upregulation is a
significant component of airway mucus in asthma4 and
COPD.5 Mucin MUC5AC expression in response to many
different stimuli appears regulated by an epidermal growth
factor receptor (EGFR) signalling cascade.6 Although sparse
in healthy adult human airways, EGFR expression is
upregulated by proinflammatory cytokines and in chronic
airway diseases such as asthma, suggesting that it may have
a role in the pathogenesis of mucus hypersecretion in these
conditions.1 7
Cyclic AMP (cAMP) is an important second messenger
determining many aspects of cellular function through the
activation of protein kinase A (PKA). This cyclic nucleotide is
inactivated by phosphodiesterases (PDEs). Many distinct
forms of PDEs have been described, but PDE4 appears to be
the major PDE isoenzyme involved in the regulation of cAMP
mediated functions in airway inflammatory and structural
cells.8 In vitro and in vivo studies have established that
selective PDE4 inhibitors suppress the activity of many
proinflammatory and immune cells, indicating that they
may be effective in the treatment of airway inflammatory
diseases. Indeed, oral PDE4 inhibitors are in phase II/III
clinical trials for COPD and asthma.8 Recent work has shown
that rolipram, the archetypal PDE4 inhibitor, markedly
decreased goblet cell hyperplasia in animal models of
secondary allergen challenge and chronic lipopolysaccharide
www.thoraxjnl.com
exposure.9 10 This effect of rolipram was attributed to its

known ability to reduce the release of inflammatory
mediators which activate goblet cells. However, the direct
effects of PDE4 inhibitors on mucin gene expression and
production by airway epithelial cells have not so far been
investigated to our knowledge.
Normal human airway epithelial cells as well as the human
pulmonary epithelial A549 cells predominantly express PDE4
with lesser activity of other PDEs;11 12 epithelial PDE4 activity
may therefore be an important target for monoselective PDE4
inhibitors in the control of those inflammatory mediators
produced by these cells. Furthermore, the functioning of the
cAMP/PKA pathway appears to be linked to that of the
extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway, the downstream
signalling of the EGFR.13
The aim of this study was to examine the effects of PDE4
inhibition on the MUC5AC mucin gene expression and
production triggered by the activation of the EGFR with one
of its endogenous ligands, the epidermal growth factor
(EGF), in cultured human airway epithelial cells (A549 cells)
and in human isolated bronchus.
METHODS
Preparations and chemicals
The human pulmonary epithelial cancer cell line (A549) was
purchased from ATCC (American Type Culture Collection;
Rockville, MD, USA). This cell line has previously been
shown to be appropriate for studies of MUC5AC mRNA and
protein expression.14 A549 cells were grown on 24-well
cultured plates for MUC5AC mRNA experiments or T25
flasks for MUC5AC protein experiments (Corning, NY, USA)

PDE4 inhibition and MUC5AC expression in airway epithelial cells
Table 1
Gene
MUC5AC
GAPDH
145
Primers and probes for real time quantitative RT-PCR

Primers and
probes
Sequence
Product size
(bp)
GenBank
accession no
Forward
Reverse
TaqMan probe
59-TGTTCTATGAGGGCTGCGTCT-39
59-ATGTCGTGGGACGCACAGA-39
59-TGACCGGTGCCACATGACGGA-39
102
U06711
Forward
Reverse
TaqMan probe
59-AGTGGATATTGTTGCCATCA-39
59-GAAGATGGTGATGGGATTTC-39
59-CAAGCTTCCCGTTCTCAGCC-39
146
BCO14085
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 and
the 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 the
validation of the DDCt method, the Ct values for target (MUC5AC) and reference (GAPDH) genes were measured
at different input amounts of total RNA (2.34300 ng); DCt values (target v reference) were then plotted against log
total RNA and the absolute value of the slope was found to be 0.008 (i.e. ,0.1), indicating similar efficiency of the
two systems.
in Roswell Park Memorial Institute (RPMI) 1640 medium

containing 10% endotoxin-free fetal calf serum (FCS),
10 mM HEPES, L-glutamine (4 mM), and standard antimicrobials.
Human lung tissue was obtained from patients (five men,
one woman) of mean age 59 years (range 4869) who had
undergone surgery for lung carcinoma as previously outlined.15 Experiments were approved by the local ethics
committee and informed consent was obtained. At the time
of operation all patients were active smokers but lung
function was within normal limits by spirometry. None of
the patients was being chronically treated with theophylline,
b-adrenoceptor agonists, corticosteroids, or anticholinergic
drugs. Bronchial tissue fragments (,3 6 3 mm) were placed
in a 24-well plate (34 fragments per well) with 1 ml RPMI
1640 medium added to each well and left for 30 minutes at
37C before use. A similar preparation has previously been
shown to be appropriate for measuring MUC5AC mucin
production from goblet cells in the epithelial layer.16
Rolipram, cilomilast, and roflumilast were synthesised at
Altana Pharma (Konstanz, Germany). Dibutyryl-cAMP, forskolin, and human recombinant epidermal growth factor
were 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 Life
Science Institute (Bremen, Germany). Stock solutions were
prepared in water for H89 and dibutyryl-cAMP or in dimethyl
sulfoxide (DMSO) for the other compounds except EGF
which was reconstituted as a stock solution of 50 mg/ml in
10 mM acetic acid and 0.1% bovine serum albumin (BSA) as
recommended by the supplier. Drugs were further diluted
into buffer solutions. The DMSO final concentration in the
assay solutions was 0.1% (v/v). Water purified on a Milli-Q
(Millipore Iberica, Madrid, Spain) system was used throughout.
Experimental protocol
In preliminary experiments with A549 cells the MUC5AC
expression in response to EGF stimulation was determined at
3, 12, 18 and 24 hours. Peak responses were observed at 18
24 hours for MUC5AC mRNA and at 24 hours for MUC5AC
protein; an incubation time of 24 hours was therefore
selected in further experiments. Also, 25 ng/ml EGF was
selected as a near maximal response from pilot experiments
with EGF (550 ng/ml). The selected EGF concentration and
time of observation are within the values reported by others
in cultured airway epithelial cells.6 17 18 For human isolated
bronchus, MUC5AC responses to EGF stimulation were
studied at 0.5, 1, 3, 12 and 24 hours. In inhibition studies

A549 cells and human bronchus were pretreated with drugs
or their vehicles for 15 minutes before stimulation with EGF
and remained until termination of experiments. When used,
antagonists were added 15 minutes before the corresponding
drug and remained for the rest of the experiment.
Mucin MUC5AC expression

The mucin MUC5AC mRNA transcripts were measured by
real time quantitative RT-PCR as previously described.19 The
method used for obtaining quantitative data of relative gene
expression, the comparative Ct (DDCt) method, was as
described by the manufacturer (PE-ABI PRISM 7700
Sequence Detection System; Perkin-Elmer Applied
Biosystems,
Perkin-Elmer
Corporation,
CA,
USA).
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was
chosen as the endogenous control gene. Total RNA was
extracted using TriPure isolation reagent (Roche, IN, USA).
The PCR primers and probes for human MUC5AC and
human GAPDH were designed using the Primer Express (PE
Biosystems, Morrisville, NC, USA) according to the published
human MUC5AC and GAPDH cDNA sequences (table 1).
MUC5AC protein in A549 cells and human bronchial
tissues was measured by enzyme linked immunosorbent
assay (ELISA) as outlined previously.6 In brief, for cell lysates,
A549 cells cultured in T25 flasks were trypsinised, washed in
PBS, centrifuged (5 minutes, 300 g, 4C), and resuspended in
five 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 mM
phenylmethylsulphonyl fluoride (PMSF), 1 mM dithiotreitol
(DTT), 2 mg/ml leupeptin, 5 mg/ml aprotinin, 5 mg/ml pepstatin), vortexed for 20 seconds, sonicated, and centrifuged
(30 minutes, 13 000 g, 4C). Human bronchial tissues were
homogenised in five volumes of ice cold lysis buffer (50 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 2 mM MgCl2, 1 mM phenylmethylsulphonyl fluoride, 1 mM dithiotreitol, 2 mg/ml leupeptin, 5 mg/ml aprotinin, and 5 mg/ml pepstatin) and
centrifuged (35 minutes, 13 000 g, 4C). The total protein in
cell and tissue samples was estimated using the Bradford
assay.20 Samples were stored at 280C.
For ELISA, 100 mg total protein was incubated with
bicarbonate-carbonate buffer at 40C in a 96-well plate until
dry. Plates were washed with PBS and blocked with 2% BSA
(fraction V; Sigma, St Louis, MO, USA) for 1 hour at room
temperature. After three washes, plates were incubated with
50 ml mouse monoclonal antibody (mAb) to MUC5AC (clone
45M1, 1:100; Neomarkers, Fremont, CA, USA; according to
the supplier, this mAb recognises the peptide core of mucin
www.thoraxjnl.com

146
Mata, Sarria , Buenestado, et al
Control
DMSO
A46
AG1478
EGF
DMSO+EGF
A46+EGF
AG1478+EGF
Relative expression
MUC5AC protein
Relative expression
MUC5AC mRNA
Control
ROF
EGF
ROF+EGF
Relative expression
MUC5AC protein
Relative expression
MUC5AC mRNA
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 and
AG1478; upper panels) or a selective phosphodiesterase 4 inhibitor (roflumilast; lower panels). Incubation with DMSO (0.1% v/v) was without
significant effect on MUC5AC expression in the absence and presence of EGF (upper panel). The EGF induced increase in MUC5AC expression was
abolished by pre-incubation with EGF receptor tyrosine kinase inhibitors (A46 100 mM or AG1478 3 mM) or roflumilast (1 mM). MUC5AC mRNA was
determined 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); columns
show the fold increase from control levels as mean (SE) values of three independent experiments. *p,0.05 v control; p,0.05 v EGF.
5AC and has no cross reactivity with other mucins). After

1 hour the plates were washed with PBS and then incubated
with 100 ml horseradish peroxidase-goat anti-mouse IgG
conjugated (1:10 000). The colour reaction was developed
with TMB peroxidase solution (Sigma) and stopped with 1 M

H2SO4. Absorbance was read at 450 nm.
In addition, Western blot analysis of MUC5AC was carried
out in human bronchial homogenates as previously
Roflumilast
Rolipram
Cilomilast
80
80
MUC5AC protein
inhibition (%)
100
MUC5AC mRNA
inhibition (%)
100
60
40
20
0
60
40
20
_8
_7
_6
Drug (log M)
_5
_4
_8
_7
_6
Drug (log M)
_5
_4
Figure 2 Concentration-response curves for inhibition by the selective PDE4 inhibitors roflumilast, cilomilast and rolipram of the epidermal growth
factor (25 ng/ml; 24 hours incubation) induced expression of MUC5AC mRNA (left panel) and protein (right panel) in A549 cells. MUC5AC mRNA
and protein were determined as indicated in fig 1. Points are mean (SE) values of three to five independent experiments. The corresponding IC50 value
for each PDE4 inhibitor is shown in the Results section.
www.thoraxjnl.com

Western blotting of EGFR, phospho-p38 MAPK,

phospho-p44/42 MAPK and phosphotyrosine
A549 cells were prepared for Western blot analysis as
indicated above, and preparations were incubated with either
EGFR 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 Signaling
Technology), or anti-phosphotyrosine mAb (clone PY20;
ICN Biomedical Inc, Aurora, OH, USA) according to the
manufacturers instructions. Expression of EGFR and phosphotyrosine was measured at 24 hours and expression of
phospho-p38 MAPK and phospho-p44/42 MAPK at 5, 15, 30
and 60 minutes of EGF (25 ng/ml) exposure. According to
C
202
C
EGFR (170 kD)
115
Control
PD98059
SB202190
EGF
SB202190+EGF
PD98059+EGF
MUC5AC mRNA
3.0
2.5
2.0
0.5
0.0
Figure 4 Relative quantitation of MUC5AC mRNA 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 p38-MAPK activity (SB202190) and p44/42 MAPK
(PD98059). The EGF induced increase in MUC5AC expression was
abolished by pre-incubation with SB202190 (3 mM) or PD98059
(10 mM). MUC5AC mRNA was determined 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; columns show the fold increase from
control levels as mean (SE) values of three independent experiments.
*p,0.05 v control; p,0.05 v EGF.
the supplier information, these mAbs are highly selective and

do not appreciably cross react with the corresponding
confounding targets.
Measurement of cAMP accumulation
Formation of cAMP was measured as previously outlined.21
Cultured A549 cells were exposed to EGF or vehicle in the
absence or presence of roflumilast for the indicated times,
and the cAMP content was quantified using an enzyme
immunoassay kit according to the assay protocol provided by
the manufacturer (RPN225; Amersham Life Sciences, UK).
Cytotoxicity assessment
To exclude the presence of non-selective detrimental effects
of the compounds studied, the percentage of lactate
Phospho-p38
MAPK (43 kD)
28.2
-actin
-actin
Phospho-p44/p42
MAPK (42, 44 kD)
Cyclic AMP (tmol/well)
34.7
21.1
1.0
1250
PY20
1.5
-actin
93
48.2
3.5
Relative expression
reported.19 In brief, aliquots of supernatants from 13 000 g

centrifugation of the tissue homogenate containing 25 mg
total protein were suspended in SDS sample buffer and
boiled for 5 minutes. Proteins were separated by SDS-PAGE
electrophoresis in 8% acrylamide-bisacrylamide (80:1). The
resulting gel was equilibrated in the transfer buffer: 25 mM
Tris-HCl, 192 mM glycine, and 20% (v/v) methanol, pH 8.3.
The proteins were then transferred electrophoretically to
nitrocellulose membranes which were incubated with 5% fatfree skimmed milk in phosphate buffered saline (PBS)
containing 0.5% BSA and 0.05% Tween 20 for 1 hour, and
incubated with mAb to MUC5AC (clone 45M1, 1:500,
NeoMarkers) for 2 hours at room temperature. Bound
antibody was visualised according to standard protocols for
the 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 MUC5AC
immunohistochemical analysis of human bronchus, specimens were fixed, cut into sections, stained with haematoxylin-eosin and periodic acid-Schiff (PAS) reagent (to
visualise goblet cells), and incubated with mouse monoclonal
antibody to MUC5AC (clone 45M1, 1:100; NeoMarkers,
Fremont, CA) as previously reported.1
147
1000
750
500
250
-actin
0
Figure 3 Western analysis of the cellular proteins with PY20 antiphosphotyrosine antibody, anti-EGFR antibody, and phospho-p38 and
phospho-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; lane
C) reduced the EGF induced response. The duration of EGF exposure
was 24 hours for anti-phosphotyrosine and EGFR experiments and
15 minutes for phospho-p38 and phospho-p44/42 MAPK experiments.
Data presented are representative of three separate experiments.
5 E5 R5 E5
R+
0
0
0
0
3 E3 R3 E3
R+
4h 4h 4h 4h
2 E2 R2 E2
R+
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) in
the absence (control, C) or presence of roflumilast (R, 1 mM). A
significant increase was detected for roflumilast alone and in the
presence of EGF only at 5 minutes. Columns are mean (SE) of three to
five independent experiments. *p,0.05 from C and E.
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148
dehydrogenase (LDH) release was assessed using a commercially available colorimetric assay (Sigma) according to the
manufacturers instructions. Cell culture supernatants and
cell lysates were collected and assessed for LDH content. The
percentage of LDH release was calculated by taking the ratio
of LDH in supernatants of experimental wells to the LDH in
control supernantants plus cells lysates times 100.
Statistical analysis
Data are expressed as mean (SE) of n experiments. In
concentration-response experiments the 2log inhibitory
concentration 50% (IC50) was calculated by non-linear
regression to express compound potency (GraphPad
Software Inc, San Diego, USA). Statistical analysis was
carried out by analysis of variance followed by appropriate
post hoc tests including Bonferroni correction. Significance
was accepted as p,0.05.
RESULTS
Cytotoxicity studies and drug vehicle effects
None of the compounds at their maximal concentrations
used showed any significant cytotoxicity (values for LDH
release 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 MUC5AC
expression and EGFR signalling cascade in A549 cells
EGF (25 ng/ml; 24 hours incubation) increased MUC5AC
gene expression and protein production in A549 cells (fig 1).
This finding was confirmed by immunocytochemical staining
for MUC5AC (not shown). The dependency of this response
on the tyrosine kinase activity of the EGFR was confirmed by
inhibition of the EGF induced increase in MUC5AC mRNA
Control
ROF (1 M)
H89 (5 M)
Control
ROF (1 M)
H89 (5 M)
EGF (25 ng/ml)
ROF+EGF
H89+ROF+EGF
EGF (25 ng/ml)
ROF+EGF
H89+ROF+EGF
1.5
1.0
1.5
Relative expression
MUC5AC protein
Relative expression
MUC5AC mRNA
2.0
0.5
0.0
1.0
0.5
0.0
Control
FORS (3 M)
BIMPS (10 M)
Control
FORS (3 M)
BIMPS (10 M)
db-cAMP (100 M)
EGF (25 ng/ml)
FORS+EGF
db-cAMP (100 M)
EGF (25 ng/ml)
FORS+EGF
BIMPS+EGF
db-cAMP+EGF
BIMPS+EGF
db-cAMP+EGF
2.0
*
1.5
1.5
1.0
0.5
0.0
Relative expression
MUC5AC protein
Relative expression
MUC5AC mRNA
1.0
0.5
0.0
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 of
roflumilast 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 induced
enhancement 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 the
basal MUC5AC expression at the concentrations tested, but each inhibited the EGF induced overexpression of MUC5AC mRNA and protein. MUC5AC
mRNA was determined 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); 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 v
EGF alone.
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0
0
12
24
Relative expression
MUC5AC mRHA
Relative expression
MUC5AC protein ( )
Relative expression
MUC5AC mRNA ( )
149
Time (h) of incubation
and protein in the presence of two different selective

inhibitors of EGFR tyrosine kinase (tyrphostin A46 and
AG1478, fig 1).3 18 22
Roflumilast (1 mM), a PDE4 inhibitor, did not change basal
MUC5AC expression but prevented the increase in MUC5AC
mRNA and protein production in response to EGF (fig 1). The
relationship between the suppression of EGF induced
MUC5AC expression and the PDE4 inhibition was further
explored by examining the inhibitory effects of other
structurally unrelated PDE4 inhibitors and by exploring their
concentration dependency. The increase in MUC5AC mRNA
and protein by EGF was inhibited in a concentration-related
fashion by pretreatment of cells with the PDE4 inhibitors
roflumilast, cilomilast, and rolipram (fig 2). The rank order of
potencies (2log IC50 values) was roflumilast (7.59 (0.27)) .
rolipram (6.66 (0.26)) . cilomilast (5.58 (0.23)) for MUC5AC
mRNA, and roflumilast (7.37 (0.12)) . rolipram (6.17
(0.16)) . cilomilast (5.27 (0.10)) for MUC5AC protein. A
fully active concentration of roflumilast (1 mM) was selected
for additional experiments.
Addition of EGF (25 ng/ml; 24 hours incubation) to A549
cells resulted in the phosphorylation of the tyrosine residues
of different intracellular proteins and the augmented
expression of the EGFR, as shown by Western blot analysis
of 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 exposure
to EGF (25 ng/ml). Treatment with roflumilast (1 mM)
abolished these EGF induced responses (fig 3). The functional requirement for p38 MAPK and for p44/42 MAPK in
the EGF induced augmentation of MUC5AC mRNA was
shown by using their respective selective inhibitors SB202190
and PD98059 (fig 4).3 18 23
Relationship between inhibition of EGF induced
MUC5AC expression by PDE4 inhibitors and the
cAMP/PKA pathway in A549 cells
We then examined whether the inhibitory effect of roflumilast on the overexpression of MUC5AC promoted by EGF was
related to its ability to inhibit PDE4, thus increasing cAMP
and subsequently activating PKA. EGF alone failed to alter
the cellular content of cAMP significantly. Roflumilast
(1 mM) produced an early (peak at 5 minutes) and transient
increase in the cAMP content of A549 cells (fig 5). The
inhibitory effect of roflumilast on the EGF induced MUC5AC
response was reversed in the presence of H-89 (5 mM), an
inhibitor of PKA,24 thus reinforcing the view of a mechanism
0
3
Relative expression
MUC5AC protein
Figure 7 Time course of the relative expression of MUC5AC mRNA

and protein in human isolated bronchus. The peak expression for
MUC5AC mRNA was observed 1 hour after stimulation with EGF, thus
preceding 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 relative
to GAPDH values as mean (SE) of the 2-DDCt values. MUC5AC protein
was determined in tissue by ELISA; points are mean (SE) of bronchial
tissues. Data were obtained from three to five different patients.
*p,0.05 v basal values.
Control
ROF
EGF
ROF+EGF
*
R+E
Top of the gel
213 kDa
Figure 8 Relative quantitation of MUC5AC mRNA (upper panel) and
protein levels (middle panel) in human bronchus unstimulated (control)
or stimulated with epidermal growth factor (EGF; 25 ng/ml) in the
absence or presence of roflumilast (ROF, 1 mM). Exposure time was
1 hour for MUC5AC mRNA determination and 3 hours for MUC5AC
protein measurements. The EGF induced increase in MUC5AC
expression was abolished by roflumilast. MUC5AC mRNA was
determined 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); columns show the fold increase from control levels as
mean (SE) values of three independent experiments. *p,0.05 v control;
p,0.05 v EGF. The lower panel shows MUC5AC protein in human
bronchus determined by Western blotting with anti-MUC5AC
monoclonal antibody. A representative experiment of three independent
experiments is shown for the same experimental groups (C, control; E,
EGF; R+E, roflumilast+EGF). Molecular weight marker is shown on the
left (213 kDa). The immunostained band of high molecular weight was
augmented in EGF exposed samples and markedly diminished in
roflumilast treated preparations.
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 MUC5AC
we showed that forskolin (10 mM), a direct activator of
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150
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). MUC5AC
immunoreactivity was observed as brown staining in goblet cells (C, F, I). Ciliated cells showed no staining for MUC5AC. The sections demonstrate
increased PAS and MUC5AC staining in the tissues exposed to EGF, and roflumilast prevented this augmentation. Original magnification 6400 (except
panel E: 6250). Goblet cells are indicated by thick arrows and ciliated cells as thin arrows.
adenylyl cyclase,24 db-cAMP (100 mM), a membrane permeable analogue of cAMP,25 and Sp-5,6-DCl-cBIMPS (100 mM),
an activator of PKA26while not altering the control level of
MUC5AC expressionwere impeding the enhanced expression of MUC5AC elicited by EGF (fig 6).
first report of a direct inhibitory effect on mucin production

of PDE4 inhibitors, a new class of drugs with potential
therapeutic interest in the treatment of COPD and asthma
diseases in which mucus hypersecretion is considered
pathologically relevant.
Effect of PDE4 inhibition on EGF induced MUC5AC

expression in human isolated bronchus
Since A549 cells are a cancer cell line, the results obtained
with these cells may differ from responses of normal airway
epithelium. Additional experiments were therefore performed using human isolated bronchial tissue. In this
preparation EGF (25 ng/ml) augmented the MUC5AC
mRNA and protein expression with peak values reached at
1 hour and 3 hours after EGF exposure, respectively (fig 7).
These effects of EGF were suppressed in the presence of
tyrphostin A46 (not shown). Roflumilast (1 mM) prevented
the EGF induced overexpression of MUC5AC (fig 8).
Immunohistochemistry
experiments
showed
that
MUC5AC immunoreactivity was localised in goblet cells that
were stained with PAS (fig 9). The MUC5AC positive staining
in airway epithelium was increased in EGF exposed preparations, and this augmentation was reduced in roflumilast
treated tissues.
EGF activates EGFR signalling cascade and MUC5AC

expression in A549 cells
The EGFR signalling cascade is important for regulating
MUC5AC mucin gene expression and protein production by
airway epithelial cells,6 and both the EGFR and the MUC5AC
expression are upregulated in chronic airway diseases such as
asthma and COPD.1 3 7 The EGFR signalling pathway translates into increased MUC5AC expression, the activation
produced by many different stimuli including oxidative
stress, neutrophil elastase, tobacco smoke, bacterial and viral
products, and inflammatory cytokines.17 18 27 In this study we
have selected EGF, an endogenous ligand of the EGFR, as a
direct activator of this pathway based on previous studies in
cultured human airway epithelial NCI-H292 cells.6 18
We confirmed that A549 cells have a constitutive expression of EGFR28 as shown by the faint band observed in
Western blot analysis with anti-EGFR mAb in the control
group (fig 3). The activation of the EGFR system results in an
increase of about twofold in MUC5AC mRNA and protein
expression as shown by ELISA data obtained after 24 hours
of incubation with EGF. Immunocytochemistry of A549 cells
confirmed this finding. The increase in MUC5AC mRNA
and protein at 24 hours is within the time dependency
shown in cultured human airway epithelial cells for MUC5AC
DISCUSSION
In this study we found that PDE4 inhibition abolished the
EGF induced augmentation of MUC5AC mRNA and protein
expression in cultured human airway epithelial cells and in
human bronchial tissue in vitro. To our knowledge, this is the
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production elicited with various stimuli activating EGFR

including EGF.6 17 18
Consistent with the notion that the overexpression of
MUC5AC is the consequence of the activation of the EGFR
signalling cascade, we also found that preincubation with
EGFR tyrosine kinase inhibitors prevented the EGF induced
augmentation of the MUC5AC mRNA expression and protein
production (fig 1). EGF therefore increases the proteintyrosine kinase activity of its receptor and thereby activates
other kinase cascades such as MAPKs including p38 and p44/
42 MAPKs.29 As expected, we found an early activation of
p38- and p44/42-MAPK as well as phosphorylation of
tyrosine residues of different cell proteins and upregulation
of the EGFR after exposure to EGF for 24 hours (fig 3).
Furthermore, inhibition of p38-and p44/42-MAPKs with the
selective inhibitors SB20202190 and PD98059 abrogated the
EGF induced MUC5AC mRNA expression.
PDE4 inhibitors suppress the EGF induced MUC5AC

expression in A549 cells by activating the cAMP/PKA
pathway
There is evidence to indicate that the functioning of the
cAMP/PKA pathway is linked with that of the ERK/MAPK
pathway. Thus, agents that increase the intracellular cAMP
concentration block growth factor stimulated ERK activation
in a number of cell types by inhibiting the activation of Raf
proteins.13 30 In fact, PDE4 isoenzymes may provide a pivotal
point for integrating cAMP and ERK signal transduction in
cells.31 The known relevance of PDE4 isoenzyme activity in
the regulation of cAMP levels in human airway epithelial
cells, including A549 cells,11 12 prompted us to investigate the
effects of monoselective PDE4 inhibitors on the EGF induced
MUC5AC expression and related events occurring in A549
cells.
We found that three different structurally unrelated PDE4
inhibitorsthe archetypal PDE4 inhibitor rolipram and the
second generation PDE4 inhibitors cilomilast and roflumilastproduced concentration dependent inhibitions of the
EGF induced MUC5AC mRNA and protein expression. The
potency order of their activities (expressed as log IC50
values) was roflumilast (,7.5) . rolipram (,6.5) .
cilomilast (,5.5). These differences in potencies are consistent with results obtained in other in vitro human cell
systems, yet variation may exist depending on the stimulus
and the cell type studied.32 Since roflumilast (1 mM)
suppressed both MUC5AC mRNA and protein production in
response to EGF, this concentration was selected for further
studies.
The inhibitory action of roflumilast appears to be exerted at
different levels of the EGFR signalling cascade. Thus, we
showed that roflumilast (1 mM) markedly inhibited the early
phospho-p38 MAPK expression as well as the phosphorylation of tyrosine residues of proteins and the overexpression of
EGFR in response to EGF stimulation measured at 24 hours
EGF exposure.
The inhibitory effects of roflumilast on the EGFR cascade
events leading to enhanced MUC5AC expression are probably
related to the activation of the cAMP/PKA pathway since this
selective PDE4 inhibitor elicited a transient early increase in
cAMP levels in A549 cells, and its inhibitory effects on
MUC5AC expression were reversed by preincubation with H89, an inhibitor of PKA activity.24 Furthermore, forskolin (a
direct activator of adenylyl cyclase),24 db-cAMP (a membrane
permeant analogue of cAMP),25 and Sp-5,6-DCl-cBIMPS (a
specific activator of PKA)26 prevented the enhanced expression of MUC5AC elicited by EGF (fig 6), thus supporting the
notion that the activation of the cAMP/PKA pathway is
effective in exerting an inhibitory influence on the EGFR
cascade leading to MUC5AC expression in A549 cells.
151
PDE4 inhibition attenuates EGF induced MUC5AC

expression in human airways in vitro
The inhibitory effects resulting from PDE4 inhibition with
roflumilast in cultured A549 cells may not necessarily be
representative of the responses of the epithelial cells in the
human airways. MUC5AC expression was therefore also
examined in human isolated bronchus, a preparation that
has previously been shown to have a basal secretion of mucin
MUC5AC produced principally by goblet cells.16 In the human
airways in vitro, MUC5AC mRNA expression reached a peak
at 1 hour after stimulation with EGF, while peak MUC5AC
protein production in tissue and medium was observed at
3 hours (fig 7). This represents faster kinetics of MUC5AC
expression than in cultured A549 cells, but we have not
investigated the reason for this difference. Pretreatment with
roflumilast (1 mM) markedly inhibited this augmented
expression of MUC5AC induced by EGF activation, indicating
that the direct inhibitory effects produced by this PDE4
inhibitor in cultured A549 cells are reproducible in intact
airway epithelial cells. Immunohistochemical analysis of
human bronchial tissues confirmed that EGF exposure
resulted in an augmented expression of MUC5AC positive
stained cells in airway epithelium and treatment with
roflumilast effectively prevented this EGF induced overexpression of MUC5AC (fig 9).
In summary, the results of this study indicate that putative
PDE4 inhibitors, in addition to their established inhibitory
effects on the airway inflammatory cells,9 10 may also exert
direct effects on human airway epithelial cells inhibiting the
MUC5AC expression that follows the activation of the EGFR
signalling cascade. These findings may be of added value to
results from recent phase II/III clinical trials which suggest a
therapeutic benefit for PDE4 inhibitors in mucus hypersecretory diseases such as COPD and asthma.8
ACKNOWLEDGEMENTS
The authors are indebted to the teams of the Services of Thoracic
Surgery and Pathology of the University Clinic Hospital and La Fe
University Hospital of Valencia (Spain) for making the human lung
tissue available to us, and to Altana Pharma for the gift of
phosphodiesterase 4 inhibitors. The technical assistance of Pedro
Santamaria and Dora Mart is also gratefully acknowledged.
.....................
Authors affiliations
M Mata, B Sarria, A Buenestado, J Cortijo, E J Morcillo, Department of

Pharmacology, Faculty of Medicine, University of Valencia, Valencia,
Spain
J Cortijo, Research Foundation, University General Hospital, University
of Valencia, Valencia, Spain
M Cerda, Department of Pathology, Faculty of Medicine, University of
Valencia, Valencia, Spain
This work was supported by grants SAF2002-04667 and SAF200307206-C02-01 from CICYT (Ministry of Science and Technology,
Spanish Government) and Research Groups-03/166 funding from
Regional Government (Generalitat Valenciana).
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Correction
It has been brought to our attention that there is an error in figure 3 on page ii55 of the Pleural
Disease Guideline available at www.brit-thoracic.org.uk/docs/PleuralDiseaseChestDrain/
pdf. Below is a corrected diagram illustrating the safe triangle for a chest drain. The
publishers apologise for this error.
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BTS guidelines for the insertion of a chest

drain
D Laws, E Neville and J Duffy
Thorax 2003 58: ii53-ii59
doi: 10.1136/thorax.58.suppl_2.ii53
Updated information and services can be found at:

http://thorax.bmj.com/content/58/suppl_2/ii53.full.html
These include:
References
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Downloaded from thorax.bmj.com on February 17, 2012 - Published by group.bmj.

BTS guidelines for the insertion of a chest drain

Thorax 2003;58(Suppl II):ii53ii59

4 PRE-DRAINAGE RISK ASSESSMENT

See end of article for

Risk of haemorrhage: where possible, any

Box 1 Indications for chest drain insertion

in any ventilated patient

pleural effusion when the chest radiograph shows a unilateral whiteout.

Downloaded from thorax.bmj.com on February 17, 2012 - Published by group.bmj.com

Laws, Neville, Duffy

Equipment required for insertion of chest drains.

Guidewire with dilators (if small tube being used)

6. CONSENT AND PREMEDICATION

Aseptic technique (section 11)

Securing drain and suture

Underwater seal (section 14.2)

Figure 1 Summary of chest drain insertion process.

Downloaded from thorax.bmj.com on February 17, 2012 - Published by group.bmj.com

Figure 3 Diagram to illustrate the safe triangle.

unsightly scarring. A more posterior position may be chosen if

8 CONFIRMING SITE OF DRAIN INSERTION

9 DRAIN INSERTION SITE

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be undertaken. In a study of chest tubes inserted in trauma

13 INSERTION OF CHEST TUBE

Laws, Neville, Duffy

13.3.2 Blunt dissection

13.3.3 Position of tube tip

13.3.4 Securing the drain

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If a patient with a clamped drain becomes breathless

Figure 4 Example of stay and closing sutures.

a linear wound into a circular one that is painful for the

14 MANAGEMENT OF DRAINAGE SYSTEM

Figure 5 Omental tag to support the tube while allowing it to lie a

There is no evidence to suggest that clamping a chest drain

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The use of integral Heimlich flutter valves has been

Laws, Neville, Duffy

A chest radiograph should be performed after insertion of a chest drain. [C]

15 PATIENTS REQUIRING ASSISTED VENTILATION

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complication of lung penetration,63 although as long as blunt

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Phosphodiesterase 4 inhibition decreases MUC5AC

See end of article for

ucus hypersecretion is an important feature of

exposure.9 10 This effect of rolipram was attributed to its

Downloaded from thorax.bmj.com on February 17, 2012 - Published by group.bmj.com

Primers and probes for real time quantitative RT-PCR

bp, base pairs.

in Roswell Park Memorial Institute (RPMI) 1640 medium

studied at 0.5, 1, 3, 12 and 24 hours. In inhibition studies

Mucin MUC5AC expression

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Mata, Sarria , Buenestado, et al

5AC and has no cross reactivity with other mucins). After

with TMB peroxidase solution (Sigma) and stopped with 1 M

Downloaded from thorax.bmj.com on February 17, 2012 - Published by group.bmj.com

Western blotting of EGFR, phospho-p38 MAPK,

Figure 4 Relative quantitation of MUC5AC mRNA in A549 cells

the supplier information, these mAbs are highly selective and