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doi:10.1136/thorax.58.suppl_2.ii532003;58;ii53-ii59Thorax

D Laws, E Neville and J DuffyBTS guidelines for the insertion of a chest drain

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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 usedin many different clinical settings and doctors in

most specialities need to be capable of their safe

insertion. The emergency insertion of a large borechest 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 bystep method of chest tube insertion.2–9

It has been shown that physicianstrainedin the

method can safely perform tube thoracostomy with 3% early complications and 8% late.10 In these

guidelines we discuss the safe insertion of chesttubes 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 operatorsshould have been adequately trained and have

completed this training appropriately. In all other

circumstances, insertion should be supervised byan appropriate trainer. This is partof theSHO 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 isdocumented in their log book and signed by the

trainer. With adequate instruction, the risk of

complications and patient pain and anxiety canbe reduced.11

These guidelines will aid the training of juniordoctors 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 pneumonectomy space should only be carried out by orafter consultation with a cardiothoracicsurgeon. [C]

There is no published evidence that abnormalblood clotting or platelet counts affect bleeding

complications of chest drain insertion. However,

where possible it is obvious good practice tocorrect any coagulopathy or platelet defect prior

to drain insertion. Routine pre-procedure checks

of platelet count and/or prothrombin time areonly required in those patients with known risk

factors. For elective chest drain insertion, warfa-

rin should be stopped and time allowed for itseffects 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 infig 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. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr D Laws, Department of Thoracic Medicine, RoyalBournemouth Hospital,Castle Lane East,Bournemouth BH7 7DW,UK; [email protected]. . . . . . . . . . . . . . . . . . . . . . .

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• Guidewire with dilators (if small tube being used)

• Chest tube

• Connecting tubing

• Closed drainage system (including sterile water if underwa-ter seal being used)

• DressingEquipment may also be available in kit form.

6. CONSENT AND PREMEDICATION

• Prior to commencing chest tube insertion the proce-dure should be explained fully to the patient andconsent recorded in accordance with national guide-lines. [C]

• Unless there are contraindications to its use, pre-medication (benzodiazepine or opioid) should begiven to reduce patient distress. [B]

Consent should be taken and recorded in keeping withnational 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 trainedindividual with sufficient knowledge of a procedure, to

explain its nature and the risks associated with it. It is withinthe 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 becarried out but must be explained as soon as the patient is

sufficiently recovered to understand. If possible, an infor-

mation leaflet should be given before the procedure.Chest drain insertion has been reported to be a painful pro-

cedure with 50% of patients experiencing pain levels of 9–10

on a scale of 10 in one study,11 and therefore premedication

should be given. Despite the apparent common sense of thisapproach, there is little established evidence of the effect from

these medications. Premedication could be an intravenous

anxiolytic—for example, midazolam 1–5 mg titrated toachieve adequate sedation—given 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 agents—for example, naloxoneor flumazenil—are occasionally necessary.

While the use of atropine as par t of premedication for fibre-optic 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 vasovagalreactions12 and a death dueto vagal stimulation following tube

insertion13 may support its use as premedication.

Figure 1 Summary of chest drain insertion process.

I n d i c a t i o n t o i n s e r t

c h e s t d r a i n

( s e c t i o n 3 )

C o n s e n t

( s e c t i o n 6 )

P r e m e d i c a t i o n

( s e c t i o n 6 )

C o n f i r m a t i o n o f s i t e o f i n s e r t i o n

c l i n i c a l l y a n d o n r a d i o g r a p h y

( s e c t i o n 8 )

P o s i t i o n i n g o f

p a t i e n t

( s e c t i o n 7 )

S i z e o f c h e s t d r a i n

( s e c t i o n s 1 0 a n d 1 3 )

A s e p t i c t e c h n i q u e ( s e c t i o n 1 1 )

L o c a l a n a e s t h e s i a ( s e c t i o n 1 2 )

B l u n t d i s s e c t i o n i f r e q u i r e d ( s e c t i o n 1 3 . 3 . 2 )

S e c u r i n g d r a i n a n d s u t u r e

( s e c t i o n 1 3 . 3 . 4 )

U n d e r w a t e r s e a l ( s e c t i o n 1 4 . 2 )

C l a m p i n g i n s t r u c t i o n s ( s e c t i o n 1 4 . 1 )

D e c i s i o n r e s u c t i o n

( s e c t i o n 1 4 . 3 )

R e m o v a l o f d r a i n

( s e c t i o n 1 4 . 5 )

Figure 2 Equipment required for insertion of chest drains.

ii54 Laws, Neville, Duffy

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7 PATIENT POSITIONThe preferred position for drain insertion is on the bed,

slightly rotated, with the arm on the side of the lesion behindthe patient’s 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 latissimusdorsi, the lateral border of the pectoralis major muscle, a line

superior to the horizontal level of the nipple, and an apexbelow the axilla.

8 CONFIRMING SITE OF DRAIN INSERTION

• A chest tube should not be inserted without furtherimage 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 sitefor chest tube placement. [B]

• A chest radiograph must be available at the time of drain insertion except in the case of tensionpneumothorax. [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 thechest radiograph. Fluoroscopy, ultrasonography, and CT scan-

ning can all be used as adjunctive guides to the site of tubeplacement.15 Before insertion, air or fluid should be aspirated;

if none is forthcoming, more complex imaging than a chestradiograph is required.

The use of ultrasonography guided insertion is particularly

useful for empyema and effusions as the diaphragm can belocalised and the presence of loculations and pleural thicken-

ing defined.16 Using real time scanning at the time of the pro-

cedure can help to ensure that the placement is safe despitethe movement of the diaphragm during respiration. The com-

plication 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 71–86%.12 If an imaging technique is used to

indicate the site for drain insertion but the procedure is notcarried out at the time of imaging, the position of the patient

at the time must be clearly documented to aid accurate inser-tion when the patient returns to the ward. It is recommended

that ultrasound is used if the effusion is very small or initialblind aspiration fails.

9 DRAIN INSERTION SITEThe most common position for chest tube insertion is in the

mid axillary line,2–9 through the “safe triangle”18 illustrated infig 3 and described above. This position minimises risk to

underlying structures such as the internal mammary arteryand avoids damage to muscle and breast tissue resulting in

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 thepatient to lie on after insertion and there is a risk of the drain

kinking.

For apical pneumothoraces the second intercostal space inthe mid clavicular line is sometimes chosen but is not recom-

mended routinely as it may be uncomfortable for the patientand may leave an unsightly scar. Loculated apical pneumotho-

races are not uncommonly seen following thoracotomy and

may be drained using a posteriorly sited (suprascapular) api-cal tube.19 20 This technique should be performed by an opera-

tor experienced in this technique—for example, a thoracicsurgeon. 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.

10 DRAIN SIZE

• Small bore drains are recommended as they are morecomfortable than larger bore tubes [B] but there isno 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

recommended

6 8 21

as it was felt that there was an increase inthe frequency of drain blockage, particularly by thick

malignant or infected fluid. The majority of physicians nowuse smaller catheters (10–14 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 catheter24–26 and no large randomised trialsdirectly comparing small and large bore tubes have been per-

formed.

In pneumothoraces 9 F catheters have been used with suc-cess rates of up to 87%, although in a few patients the air leak

seems to exceed the capacity of this small catheter.27 In theevent 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 guidedinsertion of pigtail catheters for treatment of malignant pleu-

ral effusions for sclerotherapy has been particularly well stud-ied with good effect.28–32 The use of small bore pigtail catheters

has allowed outpatient treatment of malignant pleuraleffusions which have not responded to chemotherapy.33

Empyemas are often successfully drained with ultrasonically

placed small bore tubes with the aid of thrombolyticagents.34 35

In the case of acute haemothorax, however, large bore tubes(28–30 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 cath-eter insertion. [C]

• Prophylactic antibiotics should be given in traumacases. [A]

As a chest drain may potentially be in place for a number of days, aseptic technique is essential to avoid wound site infec-

tion or secondary empyema. Although this is uncommon,

estimations of the empyema rate following drain insertionsfor trauma are approximately 2.4%.37 While the full sterile

technique afforded by a surgical theatre is usually unneces-sary, sterile gloves, gown, equipment and the use of sterile

towels after effective skin cleansing using iodine or chlorhex-

idine are recommended. A large area of skin cleansing should

Figure 3 Diagram to illustrate the “safe triangle”.

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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 reachsignificance 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 orclindamycin.

The use of prophylactic antibiotics is less clear in the event

of spontaneous pneumothorax or pleural effusion drainage asno 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 thedrain. 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. Thepeak concentration of lignocaine was found to be <3 µg/ml

(that is, a low risk of neurotoxic effects) in 85% of patientsgiven 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 haemostasisand localise the anaesthesia is used in some centres but is not

evidence based.

13 INSERTION OF CHEST TUBE

• Chest drain insertion should be performed withoutsubstantial 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 beavoided 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 isunnecessary as dilators are used in the insertion process. Afterinfiltration with local anaesthesia, a needle and syringe are

used to localise the position for insertion by the identification

of airor pleural fluid.A guidewireis then passeddown thehubof 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 successfullyused 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 beunnecessary 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 beperformed before insertion of a large bore chestdrain. [C]

13.3.1 Incision

• The incision for insertion of the chest drain should besimilar 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 largebore 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 usingblunt dissection.37 Using a Spencer-Wells clamp or similar, a

path is made through the chest wall by opening the clamp toseparate 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 thatexcessive force is not needed during drain insertion.

13.3.3 Position of tube tip

• The position of the tip of the chest tube shouldideally be aimed apically for a pneumothorax orbasally for fluid. However, any tube position can beeffective 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 throughthechest wall thetrocar positioned a fewcentimetres from the

tube tip can afford support of the tube and so help its

positioning without incurring organ damage. A smaller clampcan also be used to direct the tube to its desired position. 1 3

If possible, the tip of the tube should be aimed apically todrain 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 berepositioned simply because of a suboptimal radiographic

appearance.

13.3.4 Securing the drain

• Large and medium bore chest drain incisions shouldbe 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 laterclosure of the wound after drain removal and the second, a

stay suture, to secure the drain.

The wound closure suture should be inserted before bluntdissection. A strong suture such as “1” silk is appropriate.6 21 A

“mattress” suture or sutures across the incision are usuallyemployed 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


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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 asimple 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 toensure it is secure (fig 4).

Large amounts of tape and padding to dress the site areunnecessary 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 littleaway 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 be

controlled to prevent the potential complication of re-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 drainprior 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 presenceof a continuing air leak may lead to the potentially fatal com-

plication of tension pneumothorax.3 6 9 A bubbling draintherefore should never be clamped. However, many experi-

enced specialist physicians support the use of the clamping of

non-bubbling chest drains inserted for pneumothorax todetect 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, avoidingthe need for later chest drain reinsertion. In the ACCP Delphi

consensus statement45 about half the consensus groupsupported clamping and half didnot, and this seems similar to

the UK spread of opinion. Drain clamping is therefore not

generally recommended for safety reasons, but is acceptableunder 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 effusions 46

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 spontaneouspneumothorax 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 arecommon after large volume thoracentesis in pleural effusion,

with patients experiencing discomfort and cough. It has beensuggested 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 bedrained 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 mobilisa-tion 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 closedunderwater seal bottle in which a tube is placed under water

at a depth of approximately 3 cm with a side vent whichallows 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 pleuralair 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 inthe 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 obligatoryinpatient 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.


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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 hasbeen associated with a 85–95% success rate.53 54 In 176 cases of

pneumothorax treated with small chest tubes and a Heimlichflutter valve there were only eight failures (hospital admis-

sions for problems with tube function or placement). The

mean length of inpatient stay has been quoted at 5 hours witha 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 pneu-

mothorax, 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 followingelective thoracotomy compared the use of an underwater seal

with the flutter bag and found no difference in drainage vol-umes, requirement for suction, or complications with the

added advantage of earlier mobilisation with drainage bags. 57

In cases of malignant pleural effusion drainage a closedsystem using a drainage bag or aspiration via a three way tap

has been described to aid palliation and outpatientmanagement.33 One report of a modified urinary collectingbag for prolonged underwater chest drainage has been

described for use with empyemas, bronchopulmonary fistula,and pneumothorax associated with emphysema with no com-

plications 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 nursedby appropriately trained staff. [C]

The use of high volume/low pressure suction pumps has been

advocated in cases of non-resolving pneumothorax or follow-ing chemical pleurodesis,6 but there is no evidence to support

its routine use in the initial treatment of spontaneouspneumothorax.59 60 If suction is required, this may beperformed via the underwater seal at a level of 10–20 cm H 2O.

A high volume pump (e.g. Vernon-Thompson) is required to

cope with a large leak. A low volume pump (e.g. Robertspump) is inappropriate as it is unable to cope with the rapid

flow, thereby effecting a situation similar to clamping andrisking 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 availablefrom wall suction.

In the management of pleural infection, the use of suction

is less clear. Most studies are observational and have used suc-tion applied via the chest tube after flushing to prevent block-

ing and have reported success, but this has not been compared

with cases without suction. This is discussed further in theguideline on pleural infection (page ii18).

There is no evidence that briefly disconnecting a drain from

suction used for spontaneous pneumothorax or pleuraleffusion 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 peri-

ods such as for radiography.

14.4 Ward instructions

• Patients with chest tubes should be managed onspecialist wards by staff who are trained in chestdrain management. [C]

• A chest radiograph should be performed after inser-tion of a chest drain. [C]

Patients should be managed on a ward familiar with chesttubes. 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 alltimes, 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 respi-

ratory swing should be documented, preferably on a dedicatedchest 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 theirchest 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 sur-

gical emphysema, and assess the success of the procedure inthe volume of fluid drainage or pneumothorax resolution.

Concern has previously been expressed in cases where thetube 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 clampinga chest drain at the time of its removal is beneficial. 60

The chest tube should be removed either while the patient

performs Valsalva’s manoeuvre or during expiration with abrisk firm movement while an assistant ties the previously

placed closure suture.2– 4 7 8 The timing of removal is dependenton 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 beforeremoval 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 betweenthese two methods with only two of 80 cases (2.5%) requiring

reinsertion of a chest tube.38

15 PATIENTS REQUIRING ASSISTED VENTILATIONDuring 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 ventila-tor at the time of insertion to avoid the potentially serious

Audit points

• The presence and use of an appropriate nursing chest drainobservation chart should be noted.

• The frequency of chest drain complications should berecorded.

• The use of premedication and analgesics and patient painscores relating to chest drain insertion should be recorded.

• The duration of chest tube drainage should be recorded.


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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

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3 Parmar JM. How to insert a chest drain. Br J Hosp Med 1989;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

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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

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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. Chest 1983;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 of cardiothoracic surgery . Bristol: John Wright, 1982: 3. [IV]

19 Aslam PA , Hughes FA. Insertion of an apical tube. Surg Gynecol Obstet 1970;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. BMJ 1997;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]

23 Patz EF, Goodman PC, Erasmus JJ. Percutaneous drainage of pleuralcollections. J Thorac Imaging 1998;13:83–92. [IV]

24 Henderson AF, Banham SW, Moran F. Re-expansion pulmonaryoedema: a potentially serious complication of delayed diagnosis of pneumothorax. 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 for

chest drains (letter). BMJ 1997;315:186. [IV]27 Conces DJ, Tarver RD, Gray WC, et al . Treatment of pneumothoraces

utilizing small caliber chest tubes. Chest 1988;94:55–7. [III]28 Parker LA , Charnock GC, Delany DJ. Small bore catheter drainage and

sclerotherapy 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. AJR 1992;158:41–3. [III]

30 Goff BA , Mueller PR, Muntz HG, et al . Small chest tube drainagefollowed by bleomycin sclerosis for malignant pleural effusions. Obstet Gynecol 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 Pharmacother 1998;32:739–42. [IIb]

33 Van Le L, Parker LA, DeMars LR, et al . Pleural effusions: outpatientmanagement of pigtail catheter chest tubes. Gynecol Oncol 1994;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 of algorithms for discontinuing tube thoracostomy drainage. J Am Coll Surg1994;179:553–7. [Ib]

39 Fallon WF, Wears RL. Prophylactic antibiotics for the prevention of infectious 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 of tetracycline 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 of spontaneous pneumothorax. An American College of Chest PhysiciansDelphi Consensus Statement. Chest 2001;119:590–602.

46 Trapnell DH, Thurston JGB. Unilateral pulmonary oedema after pleuralaspiration. Lancet 1970;i:1367–9. [IV]

47 Rozenman J,Yellin A, Simansky DA, et al . Re-expansion pulmonaryoedema following pneumothorax. Respir Med 1996;90:235–8. [IV]

48 Henderson AK . Further advice on inserting a chest drain (letter andreply). Br J Hosp Med 1990;43:82. [IV]

49 Mafhood S, Hix WR, Aaron BI, et al . Re-expansion pulmonary oedema.Ann Thorac Surg 1988;45:340–5. [IV]

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 of spontaneous pneumothorax with the thoracic vent compared withconventional thoracic drainage (letter). Chest 1996;110:303. [Ib]

54 Ponn RB, Siverman HJ, Federico JA. Outpatient chest tube management.Ann Thorac Surg 1997;64:1437–40. [III]

55 Mainini SE, Johnson FE. Tension pneumothorax complicatingsmall-caliber chest tube insertion. Chest 1990; 97:759–60. [IV]

56 Matthews HR, Mcguigan JA. Closed chest drainage without anunderwater seal. Thorax 1988;41:804P. [IV]

57 Graham ANJ, Cosgrove AP, Gibbons JRP, et al . Randomised clinicaltrial of chest drainage systems. Thorax 1992;47:461–2. [Ib]

58 Bar-El Y , Leiberman Y, Yellin A. Modified urinary collecting bag forprolonged underwater chest drainage. Ann Thorac Surg1992;54:995–6. [IIb]

59 Sharma TN, Agrihotri SP, Jain NK, et al . Intercostal tube thoracostomyin pneumothorax : factors influencing re-expansion of lung. Ind J Chest Dis Allied Sci 1988;30:32–5. [III]

60 So SY , Yu DY. Catheter drainage of spontaneous pneumothorax: suctionor no suction, early or late removal? Thorax 1982;37:46–8. [Ib]

61 Williams T. To clamp or not to clamp. Nursing Times 1992;88:33. [IV]62 Curtin JJ, Goodman LR, Quebberman EJ, et al . Thoracostomy tubes after

acute chest injury: relationship between location in a pleural fissure andfunction. AJR 1994;163:1339–42. [IIa]

63 Peek GJ, Firmin RK, Arsiwala S. Chest tube insertion in the ventilatedpatient. Injury 1995;26:425–6. [IV]

64 Main A . As few sharp objects as possible should be used on enteringpleural space (letter). BMJ 1998;316:68. [IV]


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AIRWAY BIOLOGY

Phosphodiesterase 4 inhibition decreases MUC5ACexpression induced by epidermal growth factor in humanairway epithelial cells

M Mata, B Sarria, A Buenestado, J Cortijo, M Cerda, E J Morcillo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article for authors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr E J Morcillo,

Department of Pharmacology, Faculty of Medicine, Av. BlascoIbanez 15, E-46010

Valencia, Spain; [email protected]

Received 29 March 2004 Accepted3 November 2004. . . . . . . . . . . . . . . . . . . . . . .

Thorax 2005;60:144–152. doi: 10.1136/thx.2004.025692

Background: A common pathological feature of chronic inflammatory airway diseases such as asthmaand 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 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 produced

a 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 of chronic 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 is

inactivated 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 of secondary 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 and

production by airway epithelial cells have not so far beeninvestigated to our knowledge.

Normal human airway epithelial cells as well as the humanpulmonary epithelial A549 cells predominantly express PDE4 with 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 activated 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 one

of 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

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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 of the 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 at

Altana Pharma (Konstanz, Germany). Dibutyryl-cAMP, for-skolin, 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 LifeScience Institute (Bremen, Germany). Stock solutions wereprepared in water for H89 and dibutyryl-cAMP or in dimethylsulfoxide (DMSO) for the other compounds except EGF which 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 experiments with EGF (5–50 ng/ml). The selected EGF concentration andtime of observation are within the values reported by othersin cultured airway epithelial cells.6 1 7 1 8 For human isolatedbronchus, 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 drugsor their vehicles for 15 minutes before stimulation with EGFand 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 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 C t (DDC t) 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 (PE

Biosystems, Morrisville, NC, USA) according to the publishedhuman MUC5AC and GAPDH cDNA sequences (table 1).

MUC5AC protein in A549 cells and human bronchialtissues 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 withbicarbonate-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

GenePrimers andprobes Sequence

Product size(bp)

GenBank accession no

MUC5AC Forward 59-TGTTCTATGAGGGCTGCGTCT-3 9Reverse 59-ATGTCGTGGGACGCACAGA-3 9 102 U06711TaqMan probe 59-TGACCGGTGCCACATGACGGA-3 9

GAPDH Forward 59-AGTGGATATTGTTGCCATCA-3 9Reverse 59-GAAGATGGTGATGGGATTTC-3 9 146 BCO14085TaqMan probe 59-CAAGCTTCCCGTTCTCAGCC-3 9

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% Nusieve H 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 DDC t method, the C t values for target (MUC5AC) and reference (GAPDH) genes were measuredat different input amounts of total RNA (2.34–300 ng); DC t 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

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5AC and has no cross reactivity with other mucins). After1 hour the plates were washed with PBS and then incubated with 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

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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 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 DDC t 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; Àp,0.05 v EGF.

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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 mRNA and 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

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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 haematoxylin-eosin and periodic acid-Schiff (PAS) reagent (to visualise goblet cells), and incubated with mouse monoclonalantibody to MUC5AC (clone 45M1, 1:100; NeoMarkers,Fremont, CA) as previously reported.1

We st er n bl ot ti ng of EG FR , ph os ph o- p3 8 MA PK ,phospho-p44/42 MAPK and phosphotyrosine A549 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 of phospho-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 assessment To exclude the presence of non-selective detrimental effectsof the compounds studied, the percentage of lactateE G F R ( 1 7 0 k D )

P h o s p h o - p 3 8

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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 exposure

was 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.

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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 by the DDC t 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 fromcontrol levels as mean (SE) values of three independent experiments.*p,0.05 v control; Àp,0.05 v EGF.

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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). A significant 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.


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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. In

concentration-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. Significance was 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 andprotein 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 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

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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 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 DDC t 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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and protein in the presence of two different selectiveinhibitors of EGFR tyrosine kinase (tyrphostin A46 and AG1478, fig 1).3 1 8 2 2

Roflumilast (1m

M), 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 mRNA and 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 of potencies (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. 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 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 1 8 2 3

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 tointerfere with the EGF induced overexpression of MUC5AC we showed that forskolin (10 mM), a direct activator of

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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, thuspreceding the peak expression of MUC5AC protein at 3 hours.MUC5AC mRNA was determined using real time RT-PCR by the DDC t method; points show the fold increase in expression of MUC5AC relativeto GAPDH values as mean (SE) of the 2 -DDCt values. MUC5AC protein

was 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.

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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 DDC t method; columns show the 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 immunosorbent assay (ELISA); columns show the fold increase from control levels asmean (SE) values of three independent experiments. *p,0.05 v control;Àp,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 independent experiments 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.


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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 PKA 26

—while not altering the control level of MUC5AC 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 obtained with 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 of tyrphostin A46 (not shown). Roflumilast (1 mM) prevented

the EGF induced overexpression of MUC5AC (fig 8).Immunohistochemistry experiments showed thatMUC5AC immunoreactivity was localised in goblet cells that were 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 potential

therapeutic 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 we

have 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 EGFR 28 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 mRNA and 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 (except panel E:6250). Goblet cells are indicated by thick arrows and ciliated cells as thin arrows.


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production elicited with various stimuli activating EGFR including EGF.6 1 7 1 8

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 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 of p38- and p44/42-MAPK as well as phosphorylation of tyrosine 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/PKA pathway There is evidence to indicate that the functioning of thecAMP/PKA pathway is linked with that of the ERK/MAPK pathway. Thus, agents that increase the intracellular cAMPconcentration 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 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 PDE4inhibitors—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 atdifferent 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 of EGFR 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 EGFR cascade 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 peak

at 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 of human 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 putativePDE4 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 EGFR signalling 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

AC KN OW LE DG EM EN TSThe 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 of phosphodiesterase 4 inhibitors. The technical assistance of PedroSantamaria and Dora Martı 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, SpainM Cerda, Department of Pathology, Faculty of Medicine, University of

Valencia, 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).

REFERENCES1 Takeyama K , Fahy JV, Nadel JA. Relationship of epidermal growth factor receptors to goblet cell production in human bronchi. Am J Respir Crit Care Med 2001;163:511–6.

2 Leikauf GD, Borchers MT, Prows DR, et al. Mucin apoprotein expression inCOPD. Chest 2002;121:166–82S.

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 amoun ts 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 USA 1999;96:3081–6.


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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 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.

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