- Open Access
Dynamics of pro-inflammatory and anti-inflammatory cytokine release during acute inflammation in chronic obstructive pulmonary disease: an ex vivo study
© Hackett et al. 2008
- Received: 12 December 2007
- Accepted: 29 May 2008
- Published: 29 May 2008
Exacerbations of Chronic obstructive pulmonary disease (COPD) are an important cause of the morbidity and mortality associated with the disease. Strategies to reduce exacerbation frequency are thus urgently required and depend on an understanding of the inflammatory milieu associated with exacerbation episodes. Bacterial colonisation has been shown to be related to the degree of airflow obstruction and increased exacerbation frequency. The aim of this study was to asses the kinetics of cytokine release from COPD parenchymal explants using an ex vivo model of lipopolysaccharide (LPS) induced acute inflammation.
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, FEV1 98.8 ± 4.1% of predicted) was stimulated with 100 ng/ml LPS alone or in combination with either neutralising TNFα or IL-10 antibodies and supernatant collected at 1,2,4,6,24, and 48 hr time points and analysed for IL-1β, IL-5, IL-6, CXCL8, IL-10 and TNFα using ELISA. Following culture, explants were embedded in glycol methacrylate and immunohistochemical staining was conducted to determine the cellular source of TNFα, and numbers of macrophages, neutrophils and mast cells.
In our study TNFα was the initial and predictive cytokine released followed by IL-6, CXCL8 and IL-10 in the cytokine cascade following LPS exposure. The cytokine cascade was inhibited by the neutralisation 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 predominantly expressed 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 response compared to patients with normal lung function (n = 13).
We report on a reliable ex vitro model for the investigation of acute lung inflammation and its resolution using lung parenchymal explants from COPD patients. We propose that differences in the production of both TNFα and IL-10 in COPD lung tissue following exposure to bacterial LPS may have important biological implications for both episodes of exacerbation, disease progression and amelioration.
- Chronic Obstructive Pulmonary Disease
- Chronic Obstructive Pulmonary Disease Patient
- Single Nucleotide Polymorphism
- Neutrophil Elastase
- Chronic Obstructive Pulmonary Disease Exacerbation
Chronic obstructive pulmonary disease (COPD) is a major cause of mortality world wide and is predicted to be the third-leading cause of death by 2020. COPD is defined by the American Thoracic society as a disease process involving progressive chronic airflow obstruction because of chronic bronchitis, emphysema or both. Both the emphysematous destruction of lung tissue and the enlargement of air spaces along with excessive cough and sputum productions associated with bronchitis are believed to be related to an exaggerated inflammatory response. Indeed the activation and infiltration of inflammatory cells including (CD8+) T lymphocytes, macrophages and neutrophils is a prominent feature of COPD[4, 5]. In addition to the chronic state of inflammation observed in the airway patients with COPD are also prone to periods of exacerbation of the disease which are an important cause of the morbidity and mortality found in COPD [6–8]. COPD exacerbations are caused by a variety of factors such as viruses, bacteria and common pollutants. COPD exacerbations are now being recognised as important features of the natural history of COPD, as the frequency of exacerbations is associated with the severity of disease[9, 10]. Statergies to reduce exacerbation frequency are thus urgently required and depend on an understanding of the inflammatory milieu associated with exacerbation episodes. The precise role of bacteria in COPD exacerbation has been difficult to asses due to approximately 30% of stable state COPD patients having bacterial colonisation within the airways. The most common organism isolated from COPD patients is Haemophilus Influenzae and others include streptococcus pheumoniae and Bramhemella carrarhalis. Bacterial colonisation has been shown to be related to the degree of airflow obstruction and increased exacerbation frequency[9, 12–14]. More recently Stockley and colleagues have shown that COPD exacerbations associated with purulent sputum are more likely to produce positive bacterial cultures than exacerbations where the sputum was mucoid. Additionally Sethi and collegues have shown that exacerbations associated with H. influenza and B. catarrhalis both gram negative bacteria are associated with significantly higher levels of inflammatory markers compared to pathogen-negative exacerbations.
Wedzicha and colleagues have shown that stable state COPD patients with high sputum levels of Interleukin-6 (IL-6) and CXCL8 have more numerous exacerbations, suggesting that the frequency of exacerbations is associated with increased airway inflammation[17, 18]. Cytokines such as IL-6 and CXCL8 are rarely produced individually instead they are more usually released in combination with other cytokines and mediators that are characteristic of a particular disease state. These cytokine networks exhibit great pleiotropy and redundancy to the effect that any one cytokine may be influenced by another released simultaneously. TNFα and IL-1β have been identified as key cytokines that are able to initiate inflammatory cascades during exacerbations of chronic inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease, and severe asthma [19–21]. Although it is presumed that COPD exacerbations are associated with increased airway inflammation, as in patients with asthma, there is little information on the nature of the inflammatory mediator milieu during an exacerbation, especially when studied from the onset of symptoms.
In this study we aimed to assess the kinetics of key pro- and anti-inflammatory cytokines released from lung parenchymal explants obtained from COPD patients, using an ex vivo model of Gram negative Lipopolysaccharide (LPS) induced acute inflammation. We found that COPD disease severity was associated with an enhanced ex vivo pro-inflammatory cytokine response led by TNFα which was not ameliorated by the anti-inflammatory cytokine IL-10.
Patient characteristics for human lung tissue experiments
Patient characteristics of subjects prior to the removal of lung tissue
Normal Lung Function
FEV1/FVC > 70%
FEV1 ≥ 90%
FEV1/FVC < 70%
FEV1 ≥ 80%
FEV1/FVC < 70%
50% ≤ FEV1 < 80%
55.6 ± 4.1
69.2 ± 2.9
66.9 ± 2.8
Lung function (FEV 1 /FVC)
0.82 ± 0.02
0.63 ± 0.03
0.59 ± 0.02
FEV 1 % predicted
98.8 ± 4.1
90 ± 4.0
65.6 ± 2.4
6 current smokers
6 current smokers
8 current smokers
5 ex smokers
Preparation of human lung tissue for primary cell culture
The procedure for preparation of human lung tissue has been described previously elsewhere. Briefly, resected lung tissue was dissected free of tumour, large airways, pleura and visible blood vessels and finely chopped using dissecting scissors, into 2 mm3 fragments during several washes with Tyrode's buffer containing 0.1% sodium bicarbonate. Six explants (total weight approx. 30 mg) were incubated per well (2.0 cm2) of a 24 well plate with RPMI-1640 medium containing 1% penicillin, 1% streptomycin, and 1% gentamycin at 37°C in 5% carbon dioxide/air for 16 hours. Tissue was then either stimulated with 100 ng/ml LPS (Sigma-Aldrich, UK) or maintained in cell culture media alone for 1, 2, 4, 6, 24, or 48 hours. For neutralisation of TNFα and IL-10 bioactivity, tissue was incubated with 1 μg/ml of neutralising TNFα or IL-10 antibody or an isotype control (R&D Systems, Minneapolis, USA) for 1 hr prior to stimulation with 100 ng/ml LPS. Lung tissue fragments and supernatant were harvested at each time point and both were stored at -80°C until analysis. The tissue fragments were weighed to determine total tissue weight to normalize the levels of released cytokines.
Immunohistochemistry of human lung tissue
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)
Lung function (FEV 1 /FVC)
0.79 ± 0.02
0.62 ± 0.03
FEV 1 % predicted
99.2 ± 9.7
77.2 ± 8.5
4 current smokers
4 current smokers
64.7. ± 7.9
71.2 ± 2.0
Macrophage (CD68) cell/mm 2
2.8 ± 0.6
5.4 ± 1.4
Mast Cell (Tryptase) cell/mm 2
20.6 ± 5.5
17.1 ± 3.9
Neutrophil (Neutrophil elastase) cell/mm 2
8.1 ± 0.7
11.5 ± 3.6
Enzyme-Linked Immunosorbent Assay
The levels of each cytokine in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) and the concentration corrected for tissue weight. Human TNFα and IL-1β specific ELISA kits (limit of detection of 0.3 pg/mg of tissue and 0.1 pg/mg of tissue, respectively) were purchased from R&D Systems Europe Ltd, Abingdon, UK. Human IL-5, IL-6, CXCL8 and IL-10 were all measured using commercially available ELISA Duosets from Biosource Europe, SA (limits of detection 0.3 pg/mg of tissue, 0.28 pg/mg of tissue, 0.26 pg/mg of tissue and 0.25 pg/mg of tissue, respectively). The manufacturer's protocol was followed for each ELISA.
Lactate dehydrogenase assay
To test for tissue viability Lactate dehydrogenase (LDH) levels were measured in lung supernatant using a commercially available assay and LDH standard from Roche (Indianapolis, USA). For a positive control, lung explants were homogenised on ice using a XL10 sonicator set at an amplitude of 2 microns, for 12 cycles of 10 seconds sonication followed by 20 seconds rest, in 10% triton PBS buffer containing protease inhibitor cocktail (P2714, Sigma-Aldrich, UK). Following sonication samples were centrifuged at 15,000 g for 15 mins at 4°C, and supernatant removed for storage. The limit of detection for the assay was 1.95 ng/mg of tissue.
All results were normalised using the tissue weight and are expressed as the mean ± SEM. Before statistical evaluation, all results were tested for population normality and homogeneity of variance, and where applicable, a Student t test was performed. A value of P < 0.05 was accepted as significant. Differences within standard curves were analysed by ANOVA with a Tukey/Kramer post hoc correction again a value of P < 0.05 was accepted as significant. Correlations between parameters were examined for statistical significance by Spearman's correlation. Experiments were performed on each of the patients in the cohort.
Kinetics of the acute inflammatory response in human lung tissue
Cytokine cascades in the acute inflammatory response
TNFα release at 6 hours predicts subsequent cytokine levels at 24 hours
If TNFα is a key initiating step in the inflammatory cascade, then removal of TNFα should arrest or attenuate subsequent cytokine release. Pre-treatment of explants with a TNFα neutralising antibody (nTNFα Ab) for 1 hr before LPS stimulation reduced the release of IL-6 and CXCL8 back to baseline levels and the effect was still evident at 48 hrs post stimulation compared to treatment with an isotype control and LPS (figures 3D &3E). Pre-treatment with nTNFα Ab also completely abrogated the release of IL-10 up to 48 hrs after LPS stimulation (figure 3F).
Co-localisation of TNFα with macrophages and mast cells in response to LPS
IL-10, a negative regulator of TNFα production
Severity of COPD influences cytokine release
In this study, we have employed an ex vivo lung explant model to investigate the initial acute inflammatory response initiated by exposure to Gram negative bacterial cell wall component LPS in lung tissue derived from COPD patients and normal individuals. We demonstrate that lung explants obtained from COPD patients classified with mild to moderate airflow obstruction (GOLD I and II) release elevated concentrations of pro-inflammatory cytokines TNFα, IL-6 and CXCL8 in response to LPS but failed to mount an appropriate anti-inflammatory IL-10 response when compared to normal lung tissue. We suggest that these findings may have important clinical implications for the pathogenesis of COPD as dysregulated resolution of inflammation by IL-10 could account for the exaggerated inflammation observed in COPD patients during episodes of exacerbation.
The association between bacterial colonization and the development and progression of airway inflammation in COPD has been a subject of study for several years[29, 30]. Although bacteria such as H. influenzae have been associated with COPD exacerbation, early studies have provided conflicting results as to its isolation during exacerbation [12–15]. Later evidence for the role of bacteria in COPD exacerbations has come from antibiotic therapy studies. Hill and colleagues in a large COPD study showed that the airway bacterial load was related to inflammatory markers and that the bacterial species present was related to the degree of inflammation. Although the subsequent inflammatory response following a bacterial infection is considered to play a key role in the pathogenesis of COPD, the nature and sequence of the cytokine networks involved in an exacerbation have remained unexplored. The majority of clinical studies have previously concentrated on examining the acute inflammatory response during exacerbations of COPD patients using induced sputum and bronchial alveolar lavage (BAL) fluid. To our knowledge this is the first study to compare explants from patients with characterised COPD and individuals with normal lung function to investigate the kinetics of the acute inflammatory cytokine response within the distal lung towards LPS, a bacterial wall component. LPS is a widely used stimulus that acts on a number of cells within the lung through well-defined signalling cascades [32–34]. Within the literature the typical dose of LPS used in cell culture experiments and rodent models of airways disease is 1 μg/ml [35–37]. We carried out dose response curves for LPS on the tissue and deliberately chose a sub-maximal concentration of LPS 0.1 μg/ml in order to explore cytokine release and interactions on a number of cells within the lung explants.
In our model of acute inflammation in human lung tissue we found that TNFα, IL-6 and CXCL8 were released following stimulation with LPS. This model using LPS mimics the cytokine profile previously reported by several groups in COPD patients with bacterial infections. In particular Solar and colleagues showed that the presence of potentially pathogenic organisms in the bronchoaleolar lavage from COPD patients was associated with a greater degree of neutrophillia and higher TNFα levels. Indeed several studies have confirmed that higher bacterial load is associated with greater airway inflammation measured by elevated TNFα, IL-6 and CXCL8 in BAL fluid from COPD patients[13, 38]. Additionally several exacerbation studies have reported elevated levels of TNFα, IL-6 and CXCL8 in induced sputum from COPD patients admitted to hospital following an exacerbation[9, 39]. Although bacterial load was not assessed in these exacerbation studies the cytokines reported, TNFα, IL-6 and CXCL8 are the same cytokines that we observe in our acute inflammatory model using LPS. The advantage of this model over in vivo studies is that we have been able to determine the kinetic profile of release of the cytokines most reportedly elevated in COPD patients during exacerbations.
Classification of the patients in our study using the GOLD guidelines for COPD diagnosis allowed us to segregate patients into those with normal lung function and those with mild (GOLD I) and moderate (GOLD II) COPD. Using this approach, we found that lung explants from patients with GOLD I and II status had an elevated TNFα and subsequent IL-6 and CXCL8 response compared to explants obtained from patients with normal lung function. Our data therefore suggests that the parenchyma tissue of an individual with COPD would respond with an enhanced inflammatory response following exposure to LPS. The relationship between the magnitude of the inflammatory response and disease severity in our study may therefore have important clinical implications. Recent findings indicate that some patients with COPD develop frequent exacerbations, and recurrent exacerbations may be associated with increased airway inflammation. Indeed Bhowmik et al., reported that COPD patients with elevated concentrations of IL-6 and CXCL8 in sputum were more likely to have frequent exacerbations, which is thought to lead to the rapid decline of lung function in these patients. In support of these findings other studies have also demonstrated a negative correlation between FEV1 and the levels of TNFα, IL-6 and CXCL8 in sputum and BAL fluid[13, 38]. These in vivo studies therefore provide biological significance to our findings that release of TNFα, IL-6 and CXCL8 from explants in vitro negatively correlates with patients lung function. Altogether the data suggests that the heightened inflammatory response in both our model and in vivo studies of exacerbations may lead to the accelerated decline in lung function observed in COPD patients and therefore has prognostic importance for the disease. In support of these finding Donaldson and colleagues have previous reported that exacerbations in moderate to severe COPD patients contribute a greater extent to the accelerated decline in FEV1 per year observed in these patients. In addition to the role of exacerbations in COPD progression the work of Hurst and colleagues has recently raised important awareness to the impact exacerbations have on systemic inflammation as they have shown that the degree of systemic inflammation observed in COPD patients is related to the extent of lower airway inflammation during exacerbation. These data bring focus to the accumulating evidence of extra pulmonary manifestations in COPD including cachexia and systemic inflammation which are observed in severe COPD patients. In our model of acute inflammation we observed with disease severity elevated release of cytokines such as IL-6 which could act systemically on the liver to promote fibrinogen production. As raised levels of plasma fibrinogen is a independent risk factor of for cardiovascular disease. Future studies using whole animal models would therefore be useful to determine the role exacerbation derived inflammatory mediators play in systemic inflammation
In our study TNFα was the initial and predictive cytokine released in the cascade following LPS exposure. Given the heterogeneity of lung tissue obtained it was of interest to characterize which cells were responsible for the TNFα release in our model. Applying immunohistochemistry to GMA sections, we found that macrophages and mast cells accounted for the majority of TNFα positive cells following LPS exposure. This finding is supported by previous data showing that endotoxins of both Gram positive and Gram negative bacteria stimulate TNFα release from both these cell types[26, 27]. Although we observed a 0.92 fold increase in the number and distribution of TNFα positive cells between GOLD I/II patients and controls this difference did not reach statistical significance. An extensive small airway study by Hogg et al has previously reported that the percentage of airways positive for macrophages and neutrophils is elevated in the moderate to severe stages of COPD. It is difficult to compare our observations due to the differences in atomical location of the tissue analysed, small airways verses parenchyma and the methodologies used and additonally mast cells were not analysed in the Hogg et al study. Our investigations have only been able to focus on a narrow window of the disease spectrum due to the nature of patients undergoing surgery and therefore we are unable to include GOLD III and IV patients. Therefore it is difficult to determine if the increase in the numbers of macrophages with increasing COPD severity is responsible for the elevated levels of TNFα observed or that macrophages and mast cells in COPD patients have an exaggerated TNFα response due to pre-sensitisation. Indeed it has been shown that pre-sensitisation with LPS promotes an exaggerated Th1 cytokine response in mouse models of allergic asthma. Future studies are therefore required to determine if pre-sensitisation of lung tissue to bacterial agents is related to the degree of inflammation observed in COPD patients.
If TNFα is a key cytokine in acute airway inflammation then neutralising its biological activity could provide an important therapeutic treatment if given early enough after a COPD exacerbation. Indeed, in our model inhibition of TNFα activity prevented the release of IL-6, CXCL8 and IL-10 following LPS exposure. Blockade of TNFα activity using monoclonal antibodies or the soluble TNFα receptor has been used as an effective therapy in rheumatoid arthritis, inflammatory bowel disease and severe asthma [19–21]. However published reports of two clinical trials which examined the effects of the chimeric monoclonal TNFα antibody infliximab (Remicade) in COPD patients found no improvement in symptoms, lung function or reduction of inflammation in induced sputum[45, 46]. The failure of anti-TNFα therapies may reflect the fact that COPD is a highly complex inflammatory disease in which many mediators are involved. However, the substantial increase in TNFα production following LPS exposure in our model and in vivo exacerbation studies suggests that the role of TNFα may be more predominant in acute inflammatory episodes rather than in the chronic disease process. Therefore future studies maybe better focused on the roles of anti-TNF therapies in preventing or modifying the severity of acute exacerbations.
Several studies have shown that IL-10 acts as a classical negative feedback inhibitor on TNFα release from macrophages[27, 47]. In support of this mechanism of action, we report that neutralization of IL-10 activity significantly augmented LPS stimulated TNFα release from lung explants. Release of IL-6 and CXCL8 were also shown to be augmented following IL-10 inhibition, although this was likely a direct result of the increased levels of TNFα. We also show that IL-10 release was completely abolished by neutralisation of the initial cytokine in the cascade, TNFα. This supports a role for a delicate cytokine balance between pro-inflammatory TNFα and anti-inflammatory IL-10 in both resolution of inflammation and normal homeostasis of the lung. Our finding that lung tissue from GOLD I and GOLD II COPD patients releases decreased levels of IL-10 in LPS derived acute inflammation compared to patients with normal lung function has potential important pathophysiologic relevance. In support of our finding Takanashi et al have also reported evidence of IL-10 disregulation in COPD as they demonstrated that the level of IL-10 in sputum from COPD patients is decreased in comparison with healthy non-smokers. As decreased expression of the anti-inflammatory mediator IL-10 could lead to the enhanced TNFα released observed in the COPD explants in this study. This raises important questions as to the balance of pro and anti-inflammatory mediators released within the lung during exacerbations and their cause or effect relationship to the inflammatory profile observed in COPD. One possible mechanism for altered IL-10 gene expression could be single nucleotide polymorphisms (SNP) within the gene. To date no consensus has been reached regarding any IL-10 SNP in the progression of COPD. Alternatively IL-10 gene expression could be altered epigenetically due to environmental insults such as cigarette smoke or the oxidants released in response to smoke exposure. Future studies will hopefully provide more information as to the mechanisms and outcomes involved in these modifications and their role in disease progression. As therapeutic approaches aimed at preventing the inflammatory cascade in COPD are currently focused on pro-inflammatory mediators, anti-inflammatory interventions could therefore be equally if not more important. Since IL-10 is able to ameliorate the release of TNFα in acute inflammation, therapeutic strategies which enhance the endogenous release or activity of IL-10 could be used to dampen TNFα responses without compromising the immune system, providing important targets as new therapeutic strategies for a major clinical unmet need.
Due to the nature of COPD exacerbations it is technically difficult to investigate the kinetics of acute inflammatory events within the lung following admission of patients to hospital. In this ex vivo lung explant model, we have been able to interrogate further the acute inflammatory profile in terms of the tissue's response to LPS. The use of lung explants has several advantages over isolated cell cultures, including preservation of normal tissue architecture and cellular interactions. In addition, explants can be manipulated to dissect the role of various resident cells and specific cytokines they release using neutralizing antibodies. Using this model we have been able to clarify the intrinsic response of resident cells within the lung tissue following LPS exposure and eliminate the contribution of cytokine release from circulating cells. Therefore the model also has some disadvantages as it does not entirely mimic the in vivo situation as we have not studied the role of recruited inflammatory cells following LPS exposure. Another disadvantage is the fact that lung explants are extremely heterogenous between individuals especially COPD patients, and we have tried to account for this by selecting 6 explants randomly per experimental condition. Additionally all of the explants used were dissected free of small airways and therefore the model does not represent the contribution of small airways following LPS exposure. Other causes of COPD exacerbations include viruses and common pollutants; the role of bacterial-viral or bacterial-pollutant interactions may exist and have not been investigated in this study.
In summary, we report on a reliable ex vitro model for the investigation of acute lung inflammation and its resolution using lung parenchymal explants from COPD patients. Using this model, we propose that differences in the production of both TNFα and IL-10 in COPD lung tissue following exposure to bacterial endotoxin LPS may have important biological implications for both episodes of exacerbation, disease progression and amelioration. Thus further work is required to determine the role of bacterial colonization, exacerbations and airway inflammation in the pathogenesis of COPD.
The 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. Summers for their critical evaluation of this manuscript. This work was supported by Sosei plc.
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