Characterization of inflammation in COPD : clinical and experimental approach

Research output: ThesisDoctoral ThesisInternal

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Abstract

Chronic inflammation is an important feature of COPD. This inflammatory response is not restricted to the local compartment - including airways, lung parenchyma, and pulmonary vasculature - but is also present in the circulation. However, the origin of the systemic inflammation present in COPD patients remains still to be elucidated. In Chapter 3 and 4, we investigated the relationship between the local inflammatory response in the respiratory organ and systemic inflammation in patients with mild-to-moderate COPD. We assessed airway inflammation using induction of sputum by inhalation of hypertonic saline, which is a safe, non-invasive, and reproducible method to determine cell counts and protein concentrations in the airway compartment of subjects.
In Chapter 3, levels of sTNF-receptors, TNF-a, and IL-8 in induced sputum and plasma were compared between COPD patients and healthy smokers. COPD patients appeared to have significantly higher percentages of neutrophils and levels of sTNF-receptors and IL-8 in induced sputum as compared to healthy smokers. These parameters highly correlated with each other and with the degree of airflow limitation. In contrast, sputum TNF-a levels were similar in both groups. When comparing levels of inflammatory mediators in sputum and plasma, no direct correlations were found. In addition, sputum levels of sTNF-receptors were related with degree of airflow limitation in the COPD group, whereas plasma levels were not. These observations suggests that the systemic inflammatory response in COPD is not due to an overflow of inflammatory mediators from the local compartment, but that the inflammatory processes in the local and systemic compartment are differently regulated.
In Chapter 4, levels of three major acute-phase reactants -AGP, CRP, and LBP- were assessed in induced sputum and plasma of COPD patients and healthy smokers. Sputum levels of AGP and LBP were significantly increased in COPD patients versus healthy smokers, and inversely correlated with the FEV1 values in COPD. In contrast, sputum CRP was not different between the study groups. In plasma, LBP was markedly elevated in COPD patients versus healthy smokers, whereas circulating levels of AGP and CRP tended to be increased in COPD. In contrast to sputum, AGP and LBP in plasma were positively related to the FEV1 values. Exploring the relationship between local and systemic acute-phase response, no direct relations, which suggests that acute-phase reactants in sputum may be due to local production rather than leakage from the systemic compartment.


FURTHER CHARACTERIZATION THE LOCAL INFLAMMATORY RESPONSE IN COPD PATIENTS

Increased levels of several cytokines and chemokines have been found in association with COPD. However, overall fundamental questions regarding the specific role inflammatory mediators and mechanisms of inflammatory cell recruitment in COPD are still remaining because our knowledge about chronic inflammation is still fragmentary. In order to get insight in the underlying mechanisms of the chronic inflammation in COPD, we further characterized the local inflammatory response.
As indicated above, COPD patients had levels of sTNF-receptors, in particular sTNF-R55, in induced sputum as compared to healthy smokers (Chapter 3). In addition, increased levels of AGP and LBP were demonstrated in COPD sputum as compared to controls (Chapter 4). Our data on smoking cessation (Chapter 3) suggest an ongoing inflammation in airways and circulation of COPD patients after smoking cessation.
Leptin, initially discovered as a regulator of food intake and energy expenditure, is emerging as a pleiotropic cytokine involved in immune and inflammatory responses. In Chapter 5, we focused on the role of leptin as proinflammatory mediator in the local inflammatory response in COPD. Leptin was detectable in induced sputum of male COPD patients. Sputum leptin showed a strong positive correlation with sputum levels of CRP and TNF-a, whereas leptin was inversely related to sTNF-receptors in sputum. These correlations may indicate that leptin is involved in the local inflammatory response in COPD.
In Chapter 6, we paid attention to on another aspect of the local inflammatory response, that is the proteinase-anti-proteinase imbalance in COPD. MMPs are suggested to play a critical role in the development and progression of COPD, but quantitative data on MMP activity in airways are lacking. Using specific immuno-capture assays that allow determination of both active and potentially activatable (total) MMPs, we compared activity of MMP-1, MMP-8, and MMP-9 in sputum of COPD patients and healthy smokers. COPD patients appeared to have increased sputum activity of MMP-8 and MMP-9, whereas MMP-1 activity was not increased. MMP-8 and MMP-9 (active and total) were strongly correlated, and were related to the absolute number of neutrophils in both study groups. In addition, an inverse correlation between MMP-8 and MMP-9 activity in sputum and airflow obstruction (FEV1) was found in the combined study groups. Immunohistochemistry demonstrated strong positive staining for MMP-9 in both alveolar macrophages and neutrophils, whereas specific staining for MMP-8 was exclusively observed in neutrophils. The presence of increased MMP-8 and MMP-9 activity in the airways of COPD patients supports the concept of an impaired proteinase-anti-proteinase imbalance in COPD.


THE EFFECTS OF ACUTE AND CHRONIC LPS EXPOSURE TO THE LUNG IN MICE

Lipopolysaccharide (LPS) is ubiquitously present as contaminant on airborne particles, including air pollution and organic dusts. Chronic exposure to significant levels of LPS was reported to be associated with the development and/or progression of many types of lung diseases, including COPD. Recently, bioactive LPS was demonstrated to be present at high levels in cigarette smoke. Since long-term cigarette smoking is the major environmental factor that predisposes patients to COPD, LPS present in cigarette smoke may be a candidate to trigger smoking-induced accumulation of macrophages, lymphocytes, and neutrophils in respiratory bronchioles, alveolar ducts, and alveoli in susceptible cigarette smokers. Therefore, we performed animal studies to investigate the effects of acute and long-term LPS exposure to the lung in vivo in more detail.
To study the effects of acute LPS on inflammation and tissue injury, we made use of a well-characterized murine model. Single intratracheal instillation of 5 µg LPS resulted in a rapid but transient inflammatory response, characterized by elevated TNF-a expression and a profound infiltration of neutrophils from the pulmonary vascular bed into the respiratory air spaces (Chapter 7). In addition, local LPS exposure appeared to result in early apoptosis of bronchial epithelial cells independent of infiltrating neutrophils and TNF-a. This indicates that exposure of the lung to LPS not only induces pulmonary inflammation, but may also be directly involved in rapid airway injury.
Expression of acute-phase reactants (APRs) at the site of LPS exposure may enhance the early innate immune response to injury and infection. Therefore, the effects of local LPS exposure on both pulmonary and hepatic expression of four major acute-phase proteins - SAP, AGP, LBP, and a1-AT - were assessed (Chapter 8). Local LPS exposure in mice resulted in a rapid increase of mRNA expression for three major APRs (a1-AT, AGP, and LBP) in the lung. In addition, exposure to LPS in the pulmonary compartment resulted in elevated levels of AGP, LBP, and SAP in the circulation, which was consistent with the APR expression pattern in the liver. Furthermore, the increased plasma IL-6 levels after local LPS exposure suggest that IL-6 acts as mediator between the pulmonary and hepatic system. As local production of APRs results in higher concentrations in the intra-alveolar compartment and shortens the time path, pulmonary expression of APRs may be more efficient than extravasation of serum APRs to control the early phase of innate immunity to injury and infection.
In Chapter 9, the inflammatory and pathological effects of long-term LPS exposure to the lung were investigated in detail. To this end, a novel murine model in which mice were exposed to repeated intratracheal instillation of E.coli LPS was developed. Long-term LPS exposure was shown to result in chronic inflammation, characterized by peribronchial and perivascular lymphocytic aggregates (CD4+, CD8+, and CD19+), parenchymal accumulation of macrophages and CD8+ T cells, and altered cytokine expression (TNF-a, IL-18 and IFN-g). The absence of neutrophils indicates that neutrophil infiltration is rather a transient process triggered by each LPS exposure instead of being a chronic factor. In addition, long-term LPS exposure resulted in significant airway and alveolar alterations such as increased mucus cell metaplasia in larger airways, thickening of the airway smooth muscle layer, and irreversible alveolar enlargement without proliferation of alveolar epithelial cells. Interestingly, the observed inflammatory and pathological changes in mice exposed to repeated local LPS exposure mimic important features observed in human subjects with chronic pulmonary inflammatory disorders, especially COPD. Therefore, this murine model could be applicable to dissect the role of inflammation in the pathogenesis of these disease conditions.

Chapter 10 comprises the general discussion of the thesis and gives implications for future investigations.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Maastricht University
Supervisors/Advisors
  • Wouters, Emiel, Supervisor
  • Buurman, Willem, Supervisor
  • Dentener, Mieke, Co-Supervisor
Award date2 May 2003
Place of PublicationMaastricht
Publisher
Print ISBNs90-9016781-1
DOIs
Publication statusPublished - 1 Jan 2003

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