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ii53

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
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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 g 1.

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

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.

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 g 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)

3 INDICATIONS
Chest tubes may be useful in many settings, some of which are listed in box 1.

4 PRE-DRAINAGE RISK ASSESSMENT


Risk of haemorrhage: where possible, any coagulopathy or platelet defect should be corrected prior to chest drain insertion but routine measurement of the platelet count and prothrombin time are only 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

See end of article for authors affiliations

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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

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ii54 Laws, Neville, Duffy

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6. CONSENT AND PREMEDICATION


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Figure 1 Summary of chest drain insertion process.

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 sufcient 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 sufciently recovered to understand. If possible, an information leaet 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 umazenilare occasionally necessary. While the use of atropine as part of premedication for breoptic bronchoscopy has been assessed, no controlled trial of its use in chest tube insertion has been identied, 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.

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BTS guidelines for the insertion of a chest drain ii55

Figure 3 Diagram to illustrate the safe triangle.

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 g 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.

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 uid. 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

8 CONFIRMING SITE OF DRAIN INSERTION


A chest tube should not be inserted without further image guidance if free air or uid 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 conrmed 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 uid 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 dened.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.

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

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 g 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

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ii56 Laws, Neville, Duffy

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 signicance 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

incision size should afford a snug t around the chest tube, it is not possible to insert a nger to explore the pleura when inserting this size of tube. Exploration with a nger 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 nger 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 bres. For a large chest drain, similar in size to the nger, this track should be explored with a nger 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.

12 ANAESTHESIA
Local anaesthetic should be inltrated prior to insertion of the drain. [C] Local anaesthetic is inltrated into the site of insertion of the drain. A small gauge needle is used to raise a dermal bleb before deeper inltration 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 inltrated. 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.

13.3.3 Position of tube tip


The position of the tip of the chest tube should ideally be aimed apically for a pneumothorax or basally for uid. However, any tube position can be effective at draining air or uid 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 uid. 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 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 inltration with local anaesthesia, a needle and syringe are used to localise the position for insertion by the identication of air or pleural uid. 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

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 rst 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 (g 4). Complicated purse string sutures must not be used as they convert

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BTS guidelines for the insertion of a chest drain ii57

If a patient with a clamped drain becomes breathless or develops subcutaneous emphysema, the drain must be immediately unclamped and medical advice sought. [C] There is no evidence to suggest that clamping a chest drain prior to its removal increases success or prevents recurrence of a pneumothorax and it may be hazardous. This is therefore generally discouraged. Clamping a chest drain in the presence of a continuing air leak may lead to the potentially fatal 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 sufcient. 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 ow drainage system e.g. under water seal bottle or utter valve. [C] Use of a utter 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 ow. 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 uid 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 uid in the chest tube is useful for assessing tube patency and conrms the position of the tube in the pleural cavity. The disadvantages of the underwater seal system include obligatory inpatient management, difculty of patient mobilisation, and the risk of knocking over the bottle.

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 (g 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 (g 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.

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ii58 Laws, Neville, Duffy

The use of integral Heimlich utter 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 utter 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 ow) of such valves have been described, however, with tension pneumothorax as a result.55 Flutter valves cannot be used with uid 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 utter 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 utter 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 modied urinary collecting bag for prolonged underwater chest drainage has been described for use with empyemas, bronchopulmonary stula, 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 ow, 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 ushing 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 briey 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]

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. leaets) 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 uid drainage or pneumothorax resolution. Concern has previously been expressed in cases where the tube enters the lung ssure. In a study of 66 patients with chest tubes inserted for acute chest trauma, 58% of which were located within a pulmonary ssure,62 no difference in outcome was seen between these cases and those in whom the tube was located outside the ssures. 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 benecial.60 The chest tube should be removed either while the patient performs Valsalvas manoeuvre or during expiration with a brisk rm 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 reination.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 signicant 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

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BTS guidelines for the insertion of a chest drain ii59
30 Goff BA, Mueller PR, Muntz HG, et al. Small chest tube drainage followed by bleomycin sclerosis for malignant pleural effusions. Obstet Gynecol 1993;81:9936. [III] 31 Seaton KG, Patz EF, Goodman PC. Palliative treatment of malignant pleural effusions: value of small-bore catheter thoracostomy and doxycycline sclerotherapy. AJR 1995;164:58991. [IIb] 32 Thompson RL, Yau JC, Donnelly RF, et al. Pleurodesis with iodized talc for malignant effusions using pigtail catheters. Ann Pharmacother 1998;32:73942. [IIb] 33 Van Le L, Parker LA, DeMars LR, et al. Pleural effusions: outpatient management of pigtail catheter chest tubes. Gynecol Oncol 1994;54:2157. [IV] 34 Matsumoto AH. Image-guided drainage of complicated pleural effusions and adjunctive use of intrapleural urokinase. Chest 1995;108: 11901. [IV] 35 Moulton JS, Benkert RE, Weisiger KH, et al. Treatment of complicated pleural fluid collections with image guided drainage and intra cavitary urokinase. Chest 1995;108:12529. [III] 36 Parry GW, Morgan WE, Salama FD. Management of haemothorax. Ann R Coll Surg Engl 1996;78:3256. [IV] 37 Millikan JS, Moore EE, Steiner E, et al. Complications of tube thoracostomy for acute trauma. Am J Surg 1980;140:73841. [III] 38 Davis JW, MacKersie RC, Hoyt DB, et al. Randomised study of algorithms for discontinuing tube thoracostomy drainage. J Am Coll Surg 1994;179:5537. [Ib] 39 Fallon WF, Wears RL. Prophylactic antibiotics for the prevention of infectious complications including empyema following tube thoracoscopy for trauma: results of a meta-analysis. J Trauma 1992;33:1107. [Ia] 40 LeBlanc KA, Tucker WY. Prophylactic antibiotics and closed tube thoracostomy. Surg Gynecol Obstet 1985;160:25963. [Ib] 41 Wooten SA, Barbarash RA, Strange C, et al. Systemic absorption of tetracycline following intrapleural instillation. Chest 1988;94:9603. [IIa] 42 Mellor DJ. A new method of chest drain insertion. Anaesthesia 1996;51:7134. [IV] 43 Haggie JA. Management of pneumothorax: chest drain trocar is unsafe and unnecessary. BMJ 1993;307:443. [IV] 44 Rashid MA, Wikstrom T, Ortenwall P. A simple technique for anchoring chest tubes. Eur Respir J 1998;12:9589. [IV] 45 Baumann MH, Strange C, Heffner JE, et al. Management of spontaneous pneumothorax. An American College of Chest Physicians Delphi Consensus Statement. Chest 2001;119:590602. 46 Trapnell DH, Thurston JGB. Unilateral pulmonary oedema after pleural aspiration. Lancet 1970;i:13679. [IV] 47 Rozenman J,Yellin A, Simansky DA, et al. Re-expansion pulmonary oedema following pneumothorax. Respir Med 1996;90:2358. [IV] 48 Henderson AK. Further advice on inserting a chest drain (letter and reply). 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;45:3405. [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 spontaneous pneumothorax. Ann Surg 1973;177:798. [IV] 52 Hall M, Jones A. Clamping may be appropriate to prevent discomfort and 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 with conventional thoracic drainage (letter). Chest 1996;110:303. [Ib] 54 Ponn RB, Siverman HJ, Federico JA. Outpatient chest tube management. Ann Thorac Surg 1997;64:143740. [III] 55 Mainini SE, Johnson FE. Tension pneumothorax complicating small-caliber chest tube insertion. Chest 1990; 97:75960. [IV] 56 Matthews HR, Mcguigan JA. Closed chest drainage without an underwater seal. Thorax 1988;41:804P. [IV] 57 Graham ANJ, Cosgrove AP, Gibbons JRP, et al. Randomised clinical trial of chest drainage systems. Thorax 1992;47:4612. [Ib] 58 Bar-El Y, Leiberman Y, Yellin A. Modified urinary collecting bag for prolonged underwater chest drainage. Ann Thorac Surg 1992;54:9956. [IIb] 59 Sharma TN, Agrihotri SP, Jain NK, et al . Intercostal tube thoracostomy in pneumothorax : factors influencing re-expansion of lung. Ind J Chest Dis Allied Sci 1988;30:325. [III] 60 So SY, Yu DY. Catheter drainage of spontaneous pneumothorax: suction or no suction, early or late removal? Thorax 1982;37:468. [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 and function. AJR 1994;163:133942. [IIa] 63 Peek GJ, Firmin RK, Arsiwala S. Chest tube insertion in the ventilated patient. Injury 1995;26:4256. [IV] 64 Main A. As few sharp objects as possible should be used on entering pleural space (letter). BMJ 1998;316:68. [IV]

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 gs 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

REFERENCES
1 American College of Surgeons Committee on Trauma. In: Thoracic trauma. Advanced Trauma Life Support program for physicians: instructor manual. Chicago: AmericanCollege of Surgeons, 1993. [IV] 2 Miller KS, Sahn SA. Review. Chest tubes. Indications, technique, management and complications. Chest 1987;91:25864. [IV] 3 Parmar JM. How to insert a chest drain. Br J Hosp Med 1989;42:2313. [IV] 4 Treasure T, Murphy JP. Pneumothorax. Surgery 1989;75:17806. [IV] 5 Westaby S, Brayley N. Thoracic trauma I. BMJ 1990;330:163944. [IV] 6 Harriss DR, Graham TR. Management of intercostal drains. Br J Hosp Med 1991;45:3836. [IV] 7 Iberti TJ, Stern PM. Chest tube thoracostomy. Crit Care Clin 1992;8:87995. [IV] 8 Quigley R.L. Thoracentesis and chest tube drainage. Crit Care Cli 1995;11:11126. [IV] 9 Tomlinson MA. Treasure T. Insertion of a chest drain : how to do it. Br J Hosp Med 1997;58:24852. [IV] 10 Collop NA, Kim S, Sahn SA. Analysis of tube thoracostomy performed by pulmonologists at a teaching hospital. Chest 1997;112:70913. [III] 11 Luketich JD, Kiss MD, Hershey J, et al. Chest tube insertion: a prospective evaluation of pain management. Clin J Pain 1998;14:1524. [IIa] 12 Reinhold C, Illescas FF, Atri M, et al. The treatment of pleural effusions and pneumothorax with catheters placed percutaneously under image guidance. AJR 1989;152:118991. [III] 13 Ward EW, Hughes TE. Sudden death following chest tube insertion: an unusual case of vagus nerve irritation. J Trauma 1994;36:2589. [IV] 14 Boland GW, Lee MJ, Silverman S, et al. Review. Interventional radiology of the pleural space. Clin Radiol 1995;50:20514. [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;164:5818. [IV] 16 Rosenberg ER. Ultrasound in the assessment of pleural densities. Chest 1983;84:2835. [IV] 17 Harnsberger HR, Lee TG, Mukuno DH. Rapid, inexpensive real time directed thoracocentesis. Radiology 1983;146:5456. [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 chest tube by a posterior approach. Br J Hosp Med 1990;44:3301. [IV] 21 Hyde J, Sykes T, Graham T. Reducing morbidity from chest drains. BMJ 1997;311:9145. [IV] 22 Clementsen P, Evald T, Grode G, et al. Treatment of malignant pleural effusion : pleurodesis using a small bore catheter. A prospective randomized study. Respir Med 1998;92:5936. [Ib] 23 Patz EF, Goodman PC, Erasmus JJ. Percutaneous drainage of pleural collections. J Thorac Imaging 1998;13:8392. [IV] 24 Henderson AF, Banham SW, Moran F. Re-expansion pulmonary oedema: a potentially serious complication of delayed diagnosis of pneumothorax. BMJ 1985;29:5934. [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;94:557. [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 malignant pleural effusion through sonographically placed small-bore catheters. AJR 1992;158:413. [III]

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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

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)

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Table 1
Gene MUC5AC

Primers and probes for real time quantitative RT-PCR


Primers and probes Forward Reverse TaqMan probe Forward Reverse TaqMan probe Sequence 59-TGTTCTATGAGGGCTGCGTCT-39 59-ATGTCGTGGGACGCACAGA-39 59-TGACCGGTGCCACATGACGGA-39 59-AGTGGATATTGTTGCCATCA-39 59-GAAGATGGTGATGGGATTTC-39 59-CAAGCTTCCCGTTCTCAGCC-39 Product size (bp) 102 GenBank accession no U06711

GAPDH

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 37 C 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.

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.

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

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, 4 C), 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, 4 C). 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, 4 C). The total protein in cell and tissue samples was estimated using the Bradford assay.20 Samples were stored at 280 C. For ELISA, 100 mg total protein was incubated with bicarbonate-carbonate buffer at 40 C in a 96-well plate 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

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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 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

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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.

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PDE4 inhibition and MUC5AC expression in airway epithelial cells

147

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 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

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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
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Mata, Sarria , Buenestado, et al

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

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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 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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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.

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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

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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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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). 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.

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. 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.

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


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