respiratory research biomed central...copd[4,5]. in addition to the chronic state of inflamma-tion...

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Open AcceResearchDynamics of pro-inflammatory and anti-inflammatory cytokine release during acute inflammation in chronic obstructive pulmonary disease: an ex vivo studyTillie-Louise Hackett*1, Rebecca Holloway1, Stephen T Holgate2 and Jane A Warner1
Address: 1School of Biological Sciences, University of Southampton, Southampton, UK and 2Infection, Inflammation and Repair Division, Southampton General hospital, Southampton, UK

Email: Tillie-Louise Hackett* - [email protected]; Rebecca Holloway - [email protected]; Stephen T Holgate - [email protected]; Jane A Warner - [email protected]

* Corresponding author

AbstractBackground: Exacerbations of Chronic obstructive pulmonary disease (COPD) are an important causeof the morbidity and mortality associated with the disease. Strategies to reduce exacerbation frequencyare thus urgently required and depend on an understanding of the inflammatory milieu associated withexacerbation episodes. Bacterial colonisation has been shown to be related to the degree of airflowobstruction and increased exacerbation frequency. The aim of this study was to asses the kinetics ofcytokine release from COPD parenchymal explants using an ex vivo model of lipopolysaccharide (LPS)induced acute inflammation.

Methods: Lung tissue from 24 patients classified by the GOLD guidelines (7F/17M, age 67.9 ± 2.0 yrs,FEV1 76.3 ± 3.5% of predicted) and 13 subjects with normal lung function (8F,5M, age 55.6 ± 4.1 yrs, FEV198.8 ± 4.1% of predicted) was stimulated with 100 ng/ml LPS alone or in combination with eitherneutralising TNFα or IL-10 antibodies and supernatant collected at 1,2,4,6,24, and 48 hr time points andanalysed for IL-1β, IL-5, IL-6, CXCL8, IL-10 and TNFα using ELISA. Following culture, explants wereembedded in glycol methacrylate and immunohistochemical staining was conducted to determine thecellular source of TNFα, and numbers of macrophages, neutrophils and mast cells.

Results: In our study TNFα was the initial and predictive cytokine released followed by IL-6, CXCL8 andIL-10 in the cytokine cascade following LPS exposure. The cytokine cascade was inhibited by theneutralisation of the TNFα released in response to LPS and augmented by the neutralisation of the anti-inflammatory cytokine IL-10. Immunohistochemical analysis indicated that TNFα was predominantlyexpressed in macrophages and mast cells. When patients were stratified by GOLD status, GOLD I (n =11) and II (n = 13) individuals had an exaggerated TNFα responses but lacked a robust IL-10 responsecompared to patients with normal lung function (n = 13).

Conclusion: We report on a reliable ex vitro model for the investigation of acute lung inflammation andits resolution using lung parenchymal explants from COPD patients. We propose that differences in theproduction of both TNFα and IL-10 in COPD lung tissue following exposure to bacterial LPS may haveimportant biological implications for both episodes of exacerbation, disease progression and amelioration.

Published: 29 May 2008

Respiratory Research 2008, 9:47 doi:10.1186/1465-9921-9-47

Received: 12 December 2007Accepted: 29 May 2008

This article is available from: http://respiratory-research.com/content/9/1/47

© 2008 Hackett et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Respiratory Research 2008, 9:47 http://respiratory-research.com/content/9/1/47

BackgroundChronic obstructive pulmonary disease (COPD) is amajor cause of mortality world wide and is predicted to bethe third-leading cause of death by 2020[1]. COPD isdefined by the American Thoracic society as a diseaseprocess involving progressive chronic airflow obstructionbecause of chronic bronchitis, emphysema or both[2].Both the emphysematous destruction of lung tissue andthe enlargement of air spaces along with excessive coughand sputum productions associated with bronchitis arebelieved to be related to an exaggerated inflammatoryresponse[3]. Indeed the activation and infiltration ofinflammatory cells including (CD8+) T lymphocytes,macrophages and neutrophils is a prominent feature ofCOPD[4,5]. In addition to the chronic state of inflamma-tion observed in the airway patients with COPD are alsoprone to periods of exacerbation of the disease which arean important cause of the morbidity and mortality foundin COPD [6-8]. COPD exacerbations are caused by a vari-ety of factors such as viruses, bacteria and common pollut-ants. COPD exacerbations are now being recognised asimportant features of the natural history of COPD, as thefrequency of exacerbations is associated with the severityof disease[9,10]. Statergies to reduce exacerbation fre-quency are thus urgently required and depend on anunderstanding of the inflammatory milieu associatedwith exacerbation episodes. The precise role of bacteria inCOPD exacerbation has been difficult to asses due toapproximately 30% of stable state COPD patients havingbacterial colonisation within the airways[11]. The mostcommon organism isolated from COPD patients is Hae-mophilus Influenzae and others include streptococcus pheu-moniae and Bramhemella carrarhalis[11]. Bacterialcolonisation has been shown to be related to the degree ofairflow obstruction and increased exacerbation fre-quency[9,12-14]. More recently Stockley and colleagueshave shown that COPD exacerbations associated withpurulent sputum are more likely to produce positive bac-terial cultures than exacerbations where the sputum wasmucoid[15]. Additionally Sethi and collegues have shownthat exacerbations associated with H. influenza and B.catarrhalis both gram negative bacteria are associated withsignificantly higher levels of inflammatory markers com-pared to pathogen-negative exacerbations[16].

Wedzicha and colleagues have shown that stable stateCOPD patients with high sputum levels of Interleukin-6(IL-6) and CXCL8 have more numerous exacerbations,suggesting that the frequency of exacerbations is associ-ated with increased airway inflammation[17,18].Cytokines such as IL-6 and CXCL8 are rarely producedindividually instead they are more usually released incombination with other cytokines and mediators that arecharacteristic of a particular disease state. These cytokinenetworks exhibit great pleiotropy and redundancy to the

effect that any one cytokine may be influenced by anotherreleased simultaneously. TNFα and IL-1β have been iden-tified as key cytokines that are able to initiate inflamma-tory cascades during exacerbations of chronicinflammatory conditions such as rheumatoid arthritis,inflammatory bowel disease, and severe asthma [19-21].Although it is presumed that COPD exacerbations areassociated with increased airway inflammation, as inpatients with asthma, there is little information on thenature of the inflammatory mediator milieu during anexacerbation, especially when studied from the onset ofsymptoms.

In this study we aimed to assess the kinetics of key pro-and anti-inflammatory cytokines released from lungparenchymal explants obtained from COPD patients,using an ex vivo model of Gram negative Lipopolysaccha-ride (LPS) induced acute inflammation. We found thatCOPD disease severity was associated with an enhancedex vivo pro-inflammatory cytokine response led by TNFαwhich was not ameliorated by the anti-inflammatorycytokine IL-10.

MethodsPatient characteristics for human lung tissue experimentsHuman parenchymal lung tissue was obtained from 37patients (15F/21M) undergoing resection for carcinomaand 1 male undergoing surgery to remove a cyst at Guy'sHospital, London. All specimens of parenchymal tissuewere obtained from sites distant from the tumour. Thestudy was approved by the institutional ethics committeeand all volunteers gave informed consent. The Global Ini-tiative for Chronic Obstructive Pulmonary Disease(GOLD) uses a four step classification for the severity ofCOPD based on measurements of airflow limitation dur-ing forced expiration[22,23]. Each stage is determined bythe volume of air that can be forcibly exhaled in one sec-ond (FEV1) and by the ratio of FEV1 to the forced vitalcapacity (FVC); lower stages indicate less severe disease.Using the GOLD guidelines our patient cohort was strati-fied into the following groups, GOLD I (FEV1/FVC < 70%,FEV1 ≥ 80% predicted), GOLD II (FEV1/FVC < 70%, 50%≤ FEV1 < 80% predicted) and individuals with normallung function (FEV1/FVC > 70%, FEV1 ≥ 90% pre-dicted)[23]. Table 1 shows the number of patients in eachGOLD stage and their demographics which include age,gender, lung function and smoking history. For the pur-poses of this study ex-smokers were defined as individualsthat had given up smoking for ≥ 3 years to ensure forsmoking cessation. All demography data was available upto the date of surgery and none of the subjects were treatedprior with inhaled or oral corticosteroids or bronchodila-tors.



Preparation of human lung tissue for primary cell cultureThe procedure for preparation of human lung tissue hasbeen described previously elsewhere[24]. Briefly, resectedlung tissue was dissected free of tumour, large airways,pleura and visible blood vessels and finely chopped usingdissecting scissors, into 2 mm3 fragments during severalwashes with Tyrode's buffer containing 0.1% sodiumbicarbonate. Six explants (total weight approx. 30 mg)were incubated per well (2.0 cm2) of a 24 well plate withRPMI-1640 medium containing 1% penicillin, 1% strep-tomycin, and 1% gentamycin at 37°C in 5% carbon diox-ide/air for 16 hours. Tissue was then either stimulatedwith 100 ng/ml LPS (Sigma-Aldrich, UK) or maintainedin cell culture media alone for 1, 2, 4, 6, 24, or 48 hours.For neutralisation of TNFα and IL-10 bioactivity, tissuewas incubated with 1 μg/ml of neutralising TNFα or IL-10antibody or an isotype control (R&D Systems, Minneapo-lis, USA) for 1 hr prior to stimulation with 100 ng/ml LPS.Lung tissue fragments and supernatant were harvested ateach time point and both were stored at -80°C until anal-

ysis. The tissue fragments were weighed to determine totaltissue weight to normalize the levels of released cytokines.

Immunohistochemistry of human lung tissueFor the last 18 individuals recruited in the study the lungexplants collected (6 per experimental condition) wereembedded in glycol methacrylate (GMA), following stim-ulation with LPS or cell culture media alone for 1 or 6 hrs,as described above. The patient demographics whichinclude age, gender, lung function, GOLD stage andsmoking history as well as the mean number of macro-phages, mast cells and lymphocytes counted for eachgroup determined by lung function are given in table 2. Todetermine the cell types responsible for TNFα release inresponse to LPS, immunohistochemical staining of thesamples was conducted as previously described[25].Briefly, serial sections of 2 μM were stained immunohisto-chemically using the streptavidin biotin-peroxidase detec-tion system and murine monoclonal antibodies directedto either human TNFα (1:100, clone 2B3A6A2, Biosource,SA), CD68 (1:200, clone PG-M1, DAKO), mast cell tryp-

Table 1: Patient characteristics of subjects prior to the removal of lung tissue

Classification Normal Lung Function GOLD I GOLD IIFEV1/FVC > 70%

FEV1 ≥ 90%predicted

FEV1/FVC < 70%FEV1 ≥ 80%predicted

FEV1/FVC < 70%50% ≤ FEV1 < 80%

predicted

No. subjects 13 11 13Age 55.6 ± 4.1 69.2 ± 2.9 66.9 ± 2.8

Gender 8 F5 M

3 F8 M

4 F9 M

Lung function (FEV1/FVC) 0.82 ± 0.02 0.63 ± 0.03 0.59 ± 0.02FEV1 % predicted 98.8 ± 4.1 90 ± 4.0 65.6 ± 2.4Smoking status 6 current smokers 6 current smokers 8 current smokers

4 ex-smokers 5 ex-smokers 5 ex smokers3 non-smokers

Tissue samples were taken from 37 patients. Patient details including age, gender, lung function given as the ratio of air that can be forcibly exhaled in one second (FEV1) to the forced vital capacity (FVC), FEV1/FVC and FEV1% predicted and smoking status are listed as the mean ± SEM.

Table 2: Numbers of Macrophages, Mast cells and Neutrophils in lung tissue from COPD patients and individuals with normal lung function

Normal lung Function (4M/6F) GOLD I/II (6M/4F) p Value

Lung function (FEV1/FVC) 0.79 ± 0.02 0.62 ± 0.03 0.05FEV1 % predicted 99.2 ± 9.7 77.2 ± 8.5 0.05Smoking status 4 current smokers 4 current smokers

3 ex-smokers 5 ex-smokers2 non-smokers

Age 64.7. ± 7.9 71.2 ± 2.0 0.07Macrophage (CD68) cell/mm2 2.8 ± 0.6 5.4 ± 1.4 0.10Mast Cell (Tryptase) cell/mm2 20.6 ± 5.5 17.1 ± 3.9 0.23

Neutrophil (Neutrophil elastase) cell/mm2 8.1 ± 0.7 11.5 ± 3.6 0.15

Patient data including lung function given as the ratio of air that can be forcibly exhaled in one second (FEV1) to the forced vital capacity (FVC), FEV1/FVC and FEV1% predicted, smoking status, age and gender are listed as the mean ± SEM. Cell numbers are listed as the mean number of cells/mm2, ± SEM and the p value obtained when comparing the each factor between the two groups is given, p < 0.05 was considered statistically significant.



tase (1:1000, clone AA1, DAKO) or neutrophil elastase(1:500, clone NP57, DAKO). Control sections were incu-bated with isotype-matched immunoglobulins. The previ-ously described camera-lucida technique was used todetermine which cells per mm2 of alveolar tissue co-local-ised with TNFα positive staining on the serial sec-tions[26].

Enzyme-Linked Immunosorbent AssayThe levels of each cytokine in the supernatant were meas-ured by enzyme-linked immunosorbent assay (ELISA)and the concentration corrected for tissue weight. HumanTNFα and IL-1β specific ELISA kits (limit of detection of0.3 pg/mg of tissue and 0.1 pg/mg of tissue, respectively)were purchased from R&D Systems Europe Ltd, Abing-don, UK. Human IL-5, IL-6, CXCL8 and IL-10 were allmeasured using commercially available ELISA Duosetsfrom Biosource Europe, SA (limits of detection 0.3 pg/mgof tissue, 0.28 pg/mg of tissue, 0.26 pg/mg of tissue and0.25 pg/mg of tissue, respectively). The manufacturer'sprotocol was followed for each ELISA.

Lactate dehydrogenase assayTo test for tissue viability Lactate dehydrogenase (LDH)levels were measured in lung supernatant using a com-mercially available assay and LDH standard from Roche(Indianapolis, USA). For a positive control, lung explantswere homogenised on ice using a XL10 sonicator set at anamplitude of 2 microns, for 12 cycles of 10 seconds soni-cation followed by 20 seconds rest, in 10% triton PBSbuffer containing protease inhibitor cocktail (P2714,Sigma-Aldrich, UK). Following sonication samples werecentrifuged at 15,000 g for 15 mins at 4°C, and superna-tant removed for storage. The limit of detection for theassay was 1.95 ng/mg of tissue.

Statistical AnalysisAll results were normalised using the tissue weight and areexpressed as the mean ± SEM. Before statistical evaluation,all results were tested for population normality andhomogeneity of variance, and where applicable, a Studentt test was performed. A value of P < 0.05 was accepted assignificant. Differences within standard curves were ana-lysed by ANOVA with a Tukey/Kramer post hoc correctionagain a value of P < 0.05 was accepted as significant. Cor-relations between parameters were examined for statisti-cal significance by Spearman's correlation. Experimentswere performed on each of the patients in the cohort.

ResultsKinetics of the acute inflammatory response in human lung tissueRelease of the pro-inflammatory cytokine TNFα was sig-nificantly higher in the LPS stimulated tissue as early as 1hr, continued to rise at 2 and 4 hrs, and peaked at 6 hrs

(mean = 17.4 ± 1.5 pg/mg of tissue) compared to undetec-table levels in the non-stimulated controls (Figure 1A).Release of TNFα from LPS-stimulated tissue was dose-dependant within the range of 0.1–1000 ng/ml, with amaximal response at 1000 ng/ml therefore, in subsequentexperiments, we used a sub-maximal LPS dose of 100 ng/ml. Over a 48 hr time period there was no change in thelevels of LDH in supernatants from LPS stimulated tissue,compared to buffer, indicating the absence of cytotoxiceffects. While LPS can potentially activate a range of differ-ent cell types, not all pro-inflammatory cytokines werereleased. Figure 1B shows that when the tissue was stimu-lated with LPS or buffer for 48 hrs there was no statisticalsignificant difference in the levels of IL-1β released.

Cytokine cascades in the acute inflammatory responseAs shown in figure 2A the maximal increase of IL-6occurred later than TNFα, peaking at 48 hrs (mean =685.7 ± 189 pg/mg of tissue) compared to tissue chal-lenged with buffer alone (mean = 238.3 ± 50 pg/mg of tis-sue, P < 0.05). The release of the chemokine CXCL8followed a similar pattern to IL-6, with a maximalresponse occurring at 24 hrs (mean = 1490.4 ± 394 pg/mgof tissue) versus tissue challenged with buffer (mean =692.3 ± 251 pg/mg of tissue, P < 0.05) (figure 2B). The lev-els of anti-inflammatory cytokine IL-10 were still increas-ing between 24 hrs and 48 hrs (mean = 15.2 ± 2.4 pg/mgof tissue) compared to undetectable levels in the tissuechallenged with buffer (P < 0.05, figure 2C). In contrast,IL-5 was not released in response to LPS (figure 2D).

TNFα release at 6 hours predicts subsequent cytokine levels at 24 hoursThe kinetic data indicated that a succession of cytokinesare released in response to LPS, with TNFα reaching max-imal release first. We examined the relationship betweenthe levels of TNFα released at 6 hrs and the levels of theother cytokines measured at 24 hrs (Figures 3A, B and3C). The resultant data indicated that the amount of TNFαreleased at 6 hrs could be used to predict IL-6, CXCL8 andIL-10 release at 24 hrs.

If TNFα is a key initiating step in the inflammatory cas-cade, then removal of TNFα should arrest or attenuatesubsequent cytokine release. Pre-treatment of explantswith a TNFα neutralising antibody (nTNFα Ab) for 1 hrbefore LPS stimulation reduced the release of IL-6 andCXCL8 back to baseline levels and the effect was still evi-dent at 48 hrs post stimulation compared to treatmentwith an isotype control and LPS (figures 3D &3E). Pre-treatment with nTNFα Ab also completely abrogated therelease of IL-10 up to 48 hrs after LPS stimulation (figure3F).



Co-localisation of TNFα with macrophages and mast cells in response to LPSAs we demonstrated in figure 1A that the release of TNFαwas statistically elevated after 1 hour of LPS exposure itwas important to determine which cell/cells were respon-sible for this early TNFα release. The cellular source ofTNFα was analysed in 18 subjects (9F/9M) of the studyconsisting of 8 current, 7 ex and 3 non-smokers with arange of lung functions (FEV1 % predicted 55 – 92%). Todetermine the inflammatory cell types responsible forTNFα release, serial sections were stained for TNFα andone of the following cell markers: neutrophil elastase(neutrophils), CD68 (macrophages), or mast cell tryptase(mast cells). All sections stained positively for varyingamounts of neutrophil elastase, CD68 and mast cell tryp-tase. Figure 4 shows a representative section of lung paren-chyma from a 65-year-old female smoker (FEV1 83%predicted), immuno-stained with anti-TNFα monoclonalantibody after 1 hr exposure to LPS (see figure 4A and 4C)and the serial sections stained for CD68 (see figure 4B)and mast cell tryptase (see figure 4D). Co-localisation ofTNFα occurred in association with macrophages and mastcells after 1 hr of exposure to LPS, and was consistent forall individuals studied. TNFα did not co-localise with neu-trophil elastase staining. We also analysed tissue follow-ing 6 hours of LPS exposure however we found nodifference in the cellular sources of TNF alpha. As shown

in table 2, within the parenchymal tissue collected wefound no statistically significant differences in the num-bers and distribution of macrophages, mast cells or neu-trophils within the tissue obtained from GOLDI/IIpatients compared to individuals with normal lung func-tion.

IL-10, a negative regulator of TNFα productionIL-10 has been shown to act as a negative regulator ofTNFα production [27,28]. We were therefore interested instudying the effects of IL-10 and whether it was able toregulate the release of TNFα. Pre-treatment with an IL-10neutralising antibody (nIL-10Ab) for 1 hour before LPSstimulation augmented the release of TNFα (figure 5A),particularly at the later time points where we previouslyobserved maximal IL-10 release (figure 2C). Since neutral-ising the activity of IL-10 resulted in augmented release ofTNFα, we next examined if there was a similar increase inthe release of any other cytokines involved in the inflam-matory cascade. Pre-treatment with nIL-10 Ab alsoresulted in a significantly augmented release of both IL-6and CXCL8 at 24 hrs, which was maintained for at least 48hrs (figures 5B &5C).

Severity of COPD influences cytokine releaseWe observed large variation in cytokine release betweenindividuals and therefore sought to assess if there was an

Kinetics of the acute inflammatory response in human lung tissueFigure 1Kinetics of the acute inflammatory response in human lung tissue. Human lung tissue (n = 37) was stimulated with 100 ng/ml LPS (filled circles) or buffer control (open circles). The release of (A) TNFα and (B) IL-1β into the supernatant was measured by commercial ELISA. Values shown are the mean ± SEM and are expressed as pg/mg of tissue, * indicates a p value < 0.05.

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association between lung function and cytokine release inour explant model. Patients were classified into the fol-lowing groups, normal lung function (n = 13), GOLD I (n= 11) and GOLD II (n = 13) using the GOLD guide-lines[23]. We observed that all patients showed a similarlevel of TNFα release up to the 6 hr time point, howeverat 24 hrs, TNFα release continued to increase in individu-

als classified as GOLD I and GOLD II (mean = 24.7 ± 3.3and 27.6 ± 4.2 pg/mg of tissue respectively), when com-pared to patients with normal lung function (mean = 13.7± 2.1 pg/ml of tissue, P < 0.05; figure 6A). By 48 hrs TNFαrelease plateaued in all groups. Release of IL-6 and CXCL8followed a similar pattern to that observed for TNFα withGOLD II explants releasing elevated levels of these medi-

Cytokine cascades in the acute inflammatory responseFigure 2Cytokine cascades in the acute inflammatory response. Human lung tissue (n = 37) was stimulated with 100 ng/ml LPS (filled circles) or buffer control (open circles). The supernatants were analysed for (2A) IL-6, (2B) CXCL8, (2C) IL-10 and (2D) IL-5 using commercially available ELISAs. For all values are the mean ± SEM and are expressed as pg/mg tissue. * indicates p < 0.05.

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TNFα, the key cytokine in the inflammatory responseFigure 3TNFα, the key cytokine in the inflammatory response. Data from figures 1A and 2A, B, and 2C were re-plotted to ana-lyse the relationship between TNFα release at 6 hrs and IL-6 (3A), CXCL8 (3B) and IL-10 (3C) release at 24 hrs. Data was ana-lysed using Spearman rank correlation, the values given are the Rho and p < 0.05. To confirm the role of TNFα in the cytokine cascade human lung tissue (n = 37) was pre-treated with neutralising TNFα antibody (nTNFαAb) (grey circles) or an isotype control (open circles) for 1 hr and then stimulated with 100 ng/ml LPS (filled circles). The supernatants were analysed for IL-6 (3D), CXCL8 (3E), and IL-10 (3F) using commercial ELISAs. For all values given are the mean ± SEM and are expressed as pg/mg of tissue * indicates a P value < 0.05.



Co-localisation of TNFα with macrophages in the lung parenchymaFigure 4Co-localisation of TNFα with macrophages in the lung parenchyma. Lung tissue was obtained from a 65 year-old female smoker (GOLD 1) stimulated with LPS for 1 hour. The tissue was then embedded and sequential sections of the lung parenchyma stained with monoclonal antibodies for TNFα (figure 4A and 4C) and CD68 (4B) and mast cell tryptase (4D). Staining specificity was determined by IgG1 isotype antibody controls 1:200 (4E) and 1:1000 (4F) for CD68 and mast cell tryp-tase respectively. Bars represents 10 μm, positive cells are stained red.


ators at 24 and 48 hrs (figures 6C and 6D). In contrast, IL-10 release from GOLD I (mean = 8.5 ± 2.7 pg/mg of tis-

sue) and GOLD II patients (mean = 7.8 ± 1.8 pg/mg of tis-sue) was actually lower compared to patients with normallung function (mean = 17.9 ± 3.1 pg/mg of tissue, P <0.05) (see figure 6B). Importantly for all of the patientdemographic data collected including age, gender, andsmoking status these data did not influence cytokinerelease in response to LPS.

DiscussionIn this study, we have employed an ex vivo lung explantmodel to investigate the initial acute inflammatoryresponse initiated by exposure to Gram negative bacterialcell wall component LPS in lung tissue derived fromCOPD patients and normal individuals. We demonstratethat lung explants obtained from COPD patients classi-fied with mild to moderate airflow obstruction (GOLD Iand II) release elevated concentrations of pro-inflamma-tory cytokines TNFα, IL-6 and CXCL8 in response to LPSbut failed to mount an appropriate anti-inflammatory IL-10 response when compared to normal lung tissue. Wesuggest that these findings may have important clinicalimplications for the pathogenesis of COPD as dysregu-lated resolution of inflammation by IL-10 could accountfor the exaggerated inflammation observed in COPDpatients during episodes of exacerbation.

The association between bacterial colonization and thedevelopment and progression of airway inflammation inCOPD has been a subject of study for several years[29,30].Although bacteria such as H. influenzae have been associ-ated with COPD exacerbation, early studies have providedconflicting results as to its isolation during exacerbation[12-15]. Later evidence for the role of bacteria in COPDexacerbations has come from antibiotic therapy studies.Hill and colleagues in a large COPD study showed thatthe airway bacterial load was related to inflammatorymarkers and that the bacterial species present was relatedto the degree of inflammation[31]. Although the subse-quent inflammatory response following a bacterial infec-tion is considered to play a key role in the pathogenesis ofCOPD, the nature and sequence of the cytokine networksinvolved in an exacerbation have remained unexplored.The majority of clinical studies have previously concen-trated on examining the acute inflammatory responseduring exacerbations of COPD patients using inducedsputum and bronchial alveolar lavage (BAL) fluid. To ourknowledge this is the first study to compare explants frompatients with characterised COPD and individuals withnormal lung function to investigate the kinetics of theacute inflammatory cytokine response within the distallung towards LPS, a bacterial wall component. LPS is awidely used stimulus that acts on a number of cells withinthe lung through well-defined signalling cascades [32-34].Within the literature the typical dose of LPS used in cellculture experiments and rodent models of airways disease

IL-10, a negative regulator of TNFα productionFigure 5IL-10, a negative regulator of TNFα production. Human lung tissue (n = 37) was pre-treated with neutralising IL-10 antibody (nIL-10Ab) (grey circles) or an isotype control (open circles) for 1 hr and then stimulated with 100 ng/ml LPS (filled circles). The supernatants were analysed for (A) TNFα, (B) IL-6, and (C) CXCL8 using commercially available ELISAs. Values given are the mean ± SEM and are expressed as pg/mg of tissue, * indicates a P value < 0.05.

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is 1 μg/ml [35-37]. We carried out dose response curvesfor LPS on the tissue and deliberately chose a sub-maxi-mal concentration of LPS 0.1 μg/ml in order to explorecytokine release and interactions on a number of cellswithin the lung explants.

In our model of acute inflammation in human lung tissuewe found that TNFα, IL-6 and CXCL8 were released fol-lowing stimulation with LPS. This model using LPS mim-ics the cytokine profile previously reported by severalgroups in COPD patients with bacterial infections. In par-ticular Solar and colleagues showed that the presence ofpotentially pathogenic organisms in the bronchoaleolarlavage from COPD patients was associated with a greaterdegree of neutrophillia and higher TNFα levels[13].

Indeed several studies have confirmed that higher bacte-rial load is associated with greater airway inflammationmeasured by elevated TNFα, IL-6 and CXCL8 in BAL fluidfrom COPD patients[13,38]. Additionally several exacer-bation studies have reported elevated levels of TNFα, IL-6and CXCL8 in induced sputum from COPD patientsadmitted to hospital following an exacerbation[9,39].Although bacterial load was not assessed in these exacer-bation studies the cytokines reported, TNFα, IL-6 andCXCL8 are the same cytokines that we observe in ouracute inflammatory model using LPS. The advantage ofthis model over in vivo studies is that we have been able todetermine the kinetic profile of release of the cytokinesmost reportedly elevated in COPD patients during exacer-bations.

Severity of COPD influences cytokine releaseFigure 6Severity of COPD influences cytokine release. Using the GOLD guidelines the 37 individuals in this study were classified as GOLD I (grey circles) and GOLD II (filled circles) or subjects with normal lung function (open circles). Data from figures 1A, 2A, 2B and 2C were then re-analysed to determine the kinetics of (A) TNFα, (B) IL-10, (C) IL-6 and (D) CXCL8 release from the lung tissue of the patients in the three classified groups. Values given are the mean ± SEM and are expressed as pg/mg of tis-sue, † indicates P < 0.05 for both GOLD I and GOLD II compared to GOLD 0, and * indicates P < 0.05 for GOLD II compared to GOLD 0.



Classification of the patients in our study using the GOLDguidelines for COPD diagnosis allowed us to segregatepatients into those with normal lung function and thosewith mild (GOLD I) and moderate (GOLD II) COPD[23].Using this approach, we found that lung explants frompatients with GOLD I and II status had an elevated TNFαand subsequent IL-6 and CXCL8 response compared toexplants obtained from patients with normal lung func-tion. Our data therefore suggests that the parenchyma tis-sue of an individual with COPD would respond with anenhanced inflammatory response following exposure toLPS. The relationship between the magnitude of theinflammatory response and disease severity in our studymay therefore have important clinical implications.Recent findings indicate that some patients with COPDdevelop frequent exacerbations, and recurrent exacerba-tions may be associated with increased airway inflamma-tion. Indeed Bhowmik et al.,[17] reported that COPDpatients with elevated concentrations of IL-6 and CXCL8in sputum were more likely to have frequent exacerba-tions, which is thought to lead to the rapid decline of lungfunction in these patients. In support of these findingsother studies have also demonstrated a negative correla-tion between FEV1 and the levels of TNFα, IL-6 andCXCL8 in sputum[39] and BAL fluid[13,38]. These in vivostudies therefore provide biological significance to ourfindings that release of TNFα, IL-6 and CXCL8 fromexplants in vitro negatively correlates with patients lungfunction. Altogether the data suggests that the heightenedinflammatory response in both our model and in vivostudies of exacerbations may lead to the accelerateddecline in lung function observed in COPD patients andtherefore has prognostic importance for the disease. Insupport of these finding Donaldson and colleagues haveprevious reported that exacerbations in moderate tosevere COPD patients contribute a greater extent to theaccelerated decline in FEV1 per year observed in thesepatients[40]. In addition to the role of exacerbations inCOPD progression the work of Hurst and colleagues hasrecently raised important awareness to the impact exacer-bations have on systemic inflammation as they haveshown that the degree of systemic inflammation observedin COPD patients is related to the extent of lower airwayinflammation during exacerbation[41]. These data bringfocus to the accumulating evidence of extra pulmonarymanifestations in COPD including cachexia and systemicinflammation which are observed in severe COPDpatients. In our model of acute inflammation we observedwith disease severity elevated release of cytokines such asIL-6 which could act systemically on the liver to promotefibrinogen production. As raised levels of plasma fibrino-gen is a independent risk factor of for cardiovascular dis-ease[42]. Future studies using whole animal modelswould therefore be useful to determine the role exacerba-

tion derived inflammatory mediators play in systemicinflammation

In our study TNFα was the initial and predictive cytokinereleased in the cascade following LPS exposure. Given theheterogeneity of lung tissue obtained it was of interest tocharacterize which cells were responsible for the TNFαrelease in our model. Applying immunohistochemistry toGMA sections, we found that macrophages and mast cellsaccounted for the majority of TNFα positive cells follow-ing LPS exposure. This finding is supported by previousdata showing that endotoxins of both Gram positive andGram negative bacteria stimulate TNFα release from boththese cell types[26,27]. Although we observed a 0.92 foldincrease in the number and distribution of TNFα positivecells between GOLD I/II patients and controls this differ-ence did not reach statistical significance. An extensivesmall airway study by Hogg et al[43] has previouslyreported that the percentage of airways positive for macro-phages and neutrophils is elevated in the moderate tosevere stages of COPD. It is difficult to compare our obser-vations due to the differences in atomical location of thetissue analysed, small airways verses parenchyma and themethodologies used and additonally mast cells were notanalysed in the Hogg et al study. Our investigations haveonly been able to focus on a narrow window of the diseasespectrum due to the nature of patients undergoing surgeryand therefore we are unable to include GOLD III and IVpatients. Therefore it is difficult to determine if theincrease in the numbers of macrophages with increasingCOPD severity is responsible for the elevated levels ofTNFα observed or that macrophages and mast cells inCOPD patients have an exaggerated TNFα response due topre-sensitisation. Indeed it has been shown that pre-sensi-tisation with LPS promotes an exaggerated Th1 cytokineresponse in mouse models of allergic asthma[44]. Futurestudies are therefore required to determine if pre-sensitisa-tion of lung tissue to bacterial agents is related to thedegree of inflammation observed in COPD patients[31].

If TNFα is a key cytokine in acute airway inflammationthen neutralising its biological activity could provide animportant therapeutic treatment if given early enoughafter a COPD exacerbation. Indeed, in our model inhibi-tion of TNFα activity prevented the release of IL-6, CXCL8and IL-10 following LPS exposure. Blockade of TNFαactivity using monoclonal antibodies or the soluble TNFαreceptor has been used as an effective therapy in rheuma-toid arthritis, inflammatory bowel disease and severeasthma [19-21]. However published reports of two clini-cal trials which examined the effects of the chimeric mon-oclonal TNFα antibody infliximab (Remicade) in COPDpatients found no improvement in symptoms, lung func-tion or reduction of inflammation in induced spu-tum[45,46]. The failure of anti-TNFα therapies may reflect



the fact that COPD is a highly complex inflammatory dis-ease in which many mediators are involved. However, thesubstantial increase in TNFα production following LPSexposure in our model and in vivo exacerbation studiessuggests that the role of TNFα may be more predominantin acute inflammatory episodes rather than in the chronicdisease process. Therefore future studies maybe betterfocused on the roles of anti-TNF therapies in preventing ormodifying the severity of acute exacerbations.

Several studies have shown that IL-10 acts as a classicalnegative feedback inhibitor on TNFα release from macro-phages[27,47]. In support of this mechanism of action,we report that neutralization of IL-10 activity significantlyaugmented LPS stimulated TNFα release from lungexplants. Release of IL-6 and CXCL8 were also shown tobe augmented following IL-10 inhibition, although thiswas likely a direct result of the increased levels of TNFα.We also show that IL-10 release was completely abolishedby neutralisation of the initial cytokine in the cascade,TNFα. This supports a role for a delicate cytokine balancebetween pro-inflammatory TNFα and anti-inflammatoryIL-10 in both resolution of inflammation and normalhomeostasis of the lung. Our finding that lung tissue fromGOLD I and GOLD II COPD patients releases decreasedlevels of IL-10 in LPS derived acute inflammation com-pared to patients with normal lung function has potentialimportant pathophysiologic relevance. In support of ourfinding Takanashi et al[48] have also reported evidence ofIL-10 disregulation in COPD as they demonstrated thatthe level of IL-10 in sputum from COPD patients isdecreased in comparison with healthy non-smokers. Asdecreased expression of the anti-inflammatory mediatorIL-10 could lead to the enhanced TNFα released observedin the COPD explants in this study. This raises importantquestions as to the balance of pro and anti-inflammatorymediators released within the lung during exacerbationsand their cause or effect relationship to the inflammatoryprofile observed in COPD. One possible mechanism foraltered IL-10 gene expression could be single nucleotidepolymorphisms (SNP) within the gene. To date no con-sensus has been reached regarding any IL-10 SNP in theprogression of COPD. Alternatively IL-10 gene expressioncould be altered epigenetically due to environmentalinsults such as cigarette smoke or the oxidants released inresponse to smoke exposure. Future studies will hopefullyprovide more information as to the mechanisms and out-comes involved in these modifications and their role indisease progression. As therapeutic approaches aimed atpreventing the inflammatory cascade in COPD are cur-rently focused on pro-inflammatory mediators, anti-inflammatory interventions could therefore be equally ifnot more important. Since IL-10 is able to ameliorate therelease of TNFα in acute inflammation, therapeutic strate-gies which enhance the endogenous release or activity of

IL-10 could be used to dampen TNFα responses withoutcompromising the immune system, providing importanttargets as new therapeutic strategies for a major clinicalunmet need.

Due to the nature of COPD exacerbations it is technicallydifficult to investigate the kinetics of acute inflammatoryevents within the lung following admission of patients tohospital. In this ex vivo lung explant model, we have beenable to interrogate further the acute inflammatory profilein terms of the tissue's response to LPS. The use of lungexplants has several advantages over isolated cell cultures,including preservation of normal tissue architecture andcellular interactions. In addition, explants can be manip-ulated to dissect the role of various resident cells and spe-cific cytokines they release using neutralizing antibodies.Using this model we have been able to clarify the intrinsicresponse of resident cells within the lung tissue followingLPS exposure and eliminate the contribution of cytokinerelease from circulating cells. Therefore the model also hassome disadvantages as it does not entirely mimic the invivo situation as we have not studied the role of recruitedinflammatory cells following LPS exposure. Another dis-advantage is the fact that lung explants are extremely het-erogenous between individuals especially COPD patients,and we have tried to account for this by selecting 6explants randomly per experimental condition. Addition-ally all of the explants used were dissected free of smallairways and therefore the model does not represent thecontribution of small airways following LPS exposure.Other causes of COPD exacerbations include viruses andcommon pollutants; the role of bacterial-viral or bacterial-pollutant interactions may exist and have not been inves-tigated in this study.

ConclusionIn summary, we report on a reliable ex vitro model for theinvestigation of acute lung inflammation and its resolu-tion using lung parenchymal explants from COPDpatients. Using this model, we propose that differences inthe production of both TNFα and IL-10 in COPD lung tis-sue following exposure to bacterial endotoxin LPS mayhave important biological implications for both episodesof exacerbation, disease progression and amelioration.Thus further work is required to determine the role of bac-terial colonization, exacerbations and airway inflamma-tion in the pathogenesis of COPD.

Competing interestsProfessor ST Holgate has received research funding fromCelltech, Wyeth and Centercor in relation to the potentialrole of TNFa in severe asthma and has consulted withthese 3 companies and UCB over the clinical trials of anti-TNF therapy in asthma



Authors' contributionsTLH carried out the tissue culture studies, immunoassays,immunohistochemistry, performed the statistical analysisand drafted the manuscript, RH participated with theimmmunohistochemistry, STH participated in the designof the study and helped draft the manuscript, JAW con-ceived of the study, participated in its design, coordina-tion and helped draft the manuscript. All authors read andapproved the final manuscript.

AcknowledgementsThe authors thank Prof. T Treasure and the cardiothoracic team at Guy's hospital. Members of Dr S Hurst's asthma and allergy laboratory for help with collection of lung tissue, Dr S. Wilson for her invaluable advice and expertise with the immunohistochemistry, Prof. P. Paré and Dr C. Sum-mers for their critical evaluation of this manuscript. This work was sup-ported by Sosei plc.

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respiratory research biomed central...copd[4,5]. in addition to the chronic state of inflamma-tion...

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