Basement membrane and vascular remodelling in smokers and chronic obstructive pulmonary disease: a cross-sectional study

Background

Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD.

Methods

To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects.

Results

Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02).

Conclusions

Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.     

The role of uPAR in epithelial-mesenchymal transition in small airway epithelium of patients with chronic obstructive pulmonary disease

Background

Epithelial-mesenchymal transition (EMT) plays a crucial role in small airway fibrosis of patients with chronic obstructive pulmonary disease (COPD). Increasing evidence suggests that the urokinase plasminogen activator receptor (uPAR) is involved in the pathogenesis of COPD. Increased uPAR expression has been implicated in the promotion of EMT in numerous cancers; however the role of uPAR in EMT in small airway epithelial cells of patients with COPD remains unclear. In this study, we investigated the degree of EMT and uPAR expression in lung epithelium of COPD patients, and verified the effect of uPAR on cigarette smoke extract (CSE)-induced EMT in vitro.

Methods

The expression of EMT biomarkers and uPAR was assessed in lung epithelium specimens from non-smokers (n = 25), smokers (n = 25) and non-smokers with COPD (n = 10) and smokers with COPD (n = 18). The role of uPAR on CSE-induced EMT in human small airway epithelial cells (HSAEpiCs) was assessed by silencing uPAR expression in vitro.

Results

Markers of active EMT and uPAR expression were significantly increased in the small airway epithelium of patients with COPD compared with controls. We also observed a significant correlation between uPAR and vimentin expression in the small airway epithelium. In vitro, CSE-induced EMT in HSAEpiCs was associated with high expression of uPAR, and targeted silencing of uPAR using shRNA inhibited CSE-induced EMT. Finally, we demonstrate that the PI3K/Akt signaling pathway is required for uPAR-mediated EMT in HSAEpiCs.

Conclusions

A uPAR-dependent signaling pathway is required for CSE-induced EMT, which contributes to small airway fibrosis in COPD. We propose that increased uPAR expression in the small airway epithelium of patients with COPD participates in an active EMT process.     

Cytotoxic T cells expressing the co-stimulatory receptor NKG2 D are increased in cigarette smoking and COPD

Background

A suggested role for T cells in COPD pathogenesis is based on associations between increased lung cytotoxic T lymphocyte (CD8⁺) numbers and airflow limitation. CD69 is an early T cell activation marker. Natural Killer cell group 2 D (NKG2D) receptors are co-stimulatory molecules induced on CD8⁺ T cells upon activation. The activating function of NKG2 D is triggered by binding to MHC class 1 chain-related (MIC) molecules A and B, expressed on surface of stressed epithelial cells. The aim of this study was to evaluate the expression of MIC A and B in the bronchial epithelium and NKG2 D and CD69 on BAL lymphocytes in subjects with COPD, compared to smokers with normal lung function and healthy never-smokers.

Methods

Bronchoscopy with airway lavages and endobronchial mucosal biopsy sampling was performed in 35 patients with COPD, 21 healthy never-smokers and 16 smokers with normal lung function. Biopsies were immunohistochemically stained and BAL lymphocyte subsets were determined using flow cytometry.

Results

Epithelial CD3⁺ lymphocytes in bronchial biopsies were increased in both smokers with normal lung function and in COPD patients, compared to never-smokers. Epithelial CD8⁺ lymphocyte numbers were higher in the COPD group compared to never-smoking controls. Among gated CD3⁺cells in BAL, the percentage of CD8⁺ NKG2D⁺ cells was enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. The percentage of CD8⁺ CD69⁺ cells and cell surface expression of CD69 were enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. No changes in the expression of MIC A or MIC B in the airway epithelium could be detected between the groups, whereas significantly decreased soluble MICB was detected in bronchial wash from smokers with normal lung function, compared to never-smokers.

Conclusions

In COPD, we found increased numbers of cytotoxic T cells in both bronchial epithelium and airway lumen. Further, the proportions of CD69- and NKG2D-expressing cytotoxic T cells in BAL fluid were enhanced in both subjects with COPD and smokers with normal lung function and increased expression of CD69 was found on CD8⁺ cells, indicating the cigarette smoke exposure-induced expansion of activated cytotoxic T cells, which potentially can respond to stressed epithelial cells.     

T lymphocyte insensitivity to corticosteroids in chronic obstructive pulmonary disease

Background

There are increased numbers of activated lymphocytes in the lungs of chronic obstructive pulmonary disease (COPD) patients. The clinical benefits of corticosteroids in COPD patients are limited. Our hypothesis is that lymphocytes play a role in this corticosteroid insensitivity.

Objectives

To investigate the effects of the corticosteroid dexamethasone on lung lymphocyte cytokine production from patients with COPD compared to controls.

Methods

Cultured airway lymphocytes obtained by bronchoscopy from healthy non-smokers (HNS), smokers (S) and COPD patients were stimulated with phytohaemagglutinin (PHA) & phorbol myristate acetate (PMA), +/- dexamethasone. Supernatants were assayed for interleukin (IL)-2 and interferon (IFN)γ. Immunofluoresence was used to analyse changes in CD8 glucocorticoid receptor (GRα and GRβ) expression.

Results

The inhibition of PHA/PMA stimulated IFNγ production by dexamethasone was reduced in COPD patients compared to HNS (p < 0.05 at concentrations from 0.1-1 μM). There was also a significant reduction (p < 0.05) in the mean inhibitory effect at 1 μM in COPD patients (54.1%) compared to smokers (72.1%), and in smokers compared to HNS (85.5%). There was a numerically reduced effect of dexamethasone on IL-2 production that did not reach statistical significance. There was no difference in GRα and GRβ expression in follicular CD8 cells between COPD patients (50.9% and 30.4% respectively) and smokers (52.9% and 29.7% respectively).

Conclusions

IFNγ production from COPD airway lymphocytes is corticosteroid insensitive. This phenomenon may be important in the poor clinical response often observed with corticosteroids.     

Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD

Background

Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface.

Method

Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers.

Results

In smokers with normal lung function, the expression of CD25⁺CD4⁺ was increased, whereas the proportions of FoxP3⁺ and CD127⁺ were unchanged compared to never-smokers. Among CD4⁺ cells expressing high levels of CD25, the proportion of FoxP3⁺ cells was decreased and the percentage of CD127⁺ was increased in smokers with normal lung function. CD4⁺CD25⁺ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers.

Conclusion

The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25⁺ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development.     

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

Backround

Cigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR''s). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers.

Methods

The study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative.

Results

The expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients.

Conclusion

Our data suggest a smoke related change in the phenotype of AM''s and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.     

Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients

Background

We have previously shown that NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells are reduced in both numbers and cytotoxicity in peripheral blood. The aim of the present study was to investigate their numbers and function within induced sputum.

Methods

Induced sputum cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD56⁺ cells (NK and NKT-like cells) were used in an LDH release assay to determine cytotoxicity.

Results

The proportion of NK cells and NKT-like cells in smokers with COPD (COPD subjects) was significantly higher (12.7% and 3%, respectively) than in healthy smokers (smokers) (5.7%, p < 0.01; 1%, p < 0.001) and non-smoking healthy subjects (HNS) (4.2%, p < 0.001; 0.8%, p < 0.01). The proportions of NK cells and NKT-like cells expressing both perforin and granzyme B were also significantly higher in COPD subjects compared to smokers and HNS. CD56⁺ cells from COPD subjects were significantly more cytotoxic (1414 biological lytic activity) than those from smokers (142.5; p < 0.01) and HNS (3.8; p < 0.001) and were inversely correlated to FEV₁. (r = -0.75; p = 0.0098).

Conclusion

We have shown an increased proportion of NK and NKT-like cells in the induced sputum of COPD subjects and have demonstrated that these cells are significantly more cytotoxic in COPD subjects than smokers and HNS.     

Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study

Background

The aim of the study was to investigate how the expression of adhesion molecules changes as neutrophils migrate from the circulation to the lung and if these changes differ between non-smoking subjects and smokers with and without COPD.

Methods

Non-smoking healthy subjects (n=22), smokers without (n=21) and with COPD (n=18) were included. Neutrophils from peripheral blood, sputum and bronchial biopsies were analysed for cell surface expression of adhesion molecules (CD11b, CD62L, CD162). Serum, sputum supernatant and BAL-fluid were analysed for soluble adhesion molecules (ICAM-1, -3, E-selectin, P-selectin, VCAM-1, PECAM-1).

Results

Expression of CD11b was increased on circulating neutrophils from smokers with COPD. It was also increased on sputum neutrophils in both smokers groups, but not in non-smokers, as compared to circulating neutrophils.Serum ICAM-1 was higher in the COPD group compared to the other two groups (p<0.05) and PECAM-1 was lower in smokers without COPD than in non-smoking controls and the COPD group (p<0.05). In BAL-fluid ICAM-1 was lower in the COPD group than in the other groups (p<0.05).

Conclusions

Thus, our data strongly support the involvement of a systemic component in COPD and demonstrate that in smokers neutrophils are activated to a greater extent at the point of transition from the circulation into the lungs than in non-smokers.     

Circulating Progenitor Cells and Vascular Dysfunction in Chronic Obstructive Pulmonary Disease

Background

In chronic obstructive pulmonary disease (COPD), decreased progenitor cells and impairment of systemic vascular function have been suggested to confer higher cardiovascular risk. The origin of these changes and their relationship with alterations in the pulmonary circulation are unknown.

Objectives

To investigate whether changes in the number of circulating hematopoietic progenitor cells are associated with pulmonary hypertension or changes in endothelial function.

Methods

62 COPD patients and 35 controls (18 non-smokers and 17 smokers) without cardiovascular risk factors other than cigarette smoking were studied. The number of circulating progenitors was measured as CD45⁺CD34⁺CD133⁺ labeled cells by flow cytometry. Endothelial function was assessed by flow-mediated dilation. Markers of inflammation and angiogenesis were also measured in all subjects.

Results

Compared with controls, the number of circulating progenitor cells was reduced in COPD patients. Progenitor cells did not differ between control smokers and non-smokers. COPD patients with pulmonary hypertension showed greater number of progenitor cells than those without pulmonary hypertension. Systemic endothelial function was worse in both control smokers and COPD patients. Interleukin-6, fibrinogen, high sensitivity C-reactive protein, vascular endothelial growth factor and tumor necrosis factor were increased in COPD. In COPD patients, the number of circulating progenitor cells was inversely related to the flow-mediated dilation of systemic arteries.

Conclusions

Pulmonary and systemic vascular impairment in COPD is associated with cigarette smoking but not with the reduced number of circulating hematopoietic progenitors. The latter appears to be a consequence of the disease itself not related to smoking habit.     

Impact of smoking on dendritic cell phenotypes in the airway lumen of patients with COPD

Background

Myeloid dendritic cells (DCs) are increased in the airway wall of patients with chronic obstructive pulmonary disease (COPD), and postulated to play a crucial role in COPD. However, DC phenotypes in COPD are poorly understood.

Methods

Function-associated surface molecules on bronchoalveolar lavage fluid (BALF) DCs were analyzed using flow cytometry in current smokers with COPD, in former smokers with COPD and in never-smoking controls.

Results

Myeloid DCs of current smokers with COPD displayed a significantly increased expression of receptors for antigen recognition such as BDCA-1 or Langerin, as compared with never-smoking controls. In contrast, former smokers with COPD displayed a significantly decreased expression of these receptors, as compared with never-smoking controls. A significantly reduced expression of the maturation marker CD83 on myeloid DCs was found in current smokers with COPD, but not in former smokers with COPD. The chemokine receptor CCR5 on myeloid DCs, which is also important for the uptake and procession of microbial antigens, was strongly reduced in all patients with COPD, independently of the smoking status.

Conclusion

COPD is characterized by a strongly reduced CCR5 expression on myeloid DCs in the airway lumen, which might hamper DC interactions with microbial antigens. Further studies are needed to better understand the role of CCR5 in the pathophysiology and microbiology of COPD.     

The effects of corticosteroids on COPD lung macrophages: a pooled analysis

Background

There is large variation in the therapeutic response to inhaled corticosteroids (ICS) in COPD patients. We present a pooled analysis of our previous studies investigating the effects of corticosteroids on lung macrophages, in order to robustly determine whether corticosteroid sensitivity in COPD cells is reduced compared to controls, and also to evaluate the degree of between individual variation in drug response.

Methods

Data from 20 never smokers (NS), 27 smokers (S) and 45 COPD patients was used. Lung macropahges had been stimulated with lipopolysaccharide (LPS), with or without the corticosteroid dexamethasone, and tumour necrosis factor (TNF)-α, interleukin (IL)-6 and chemokine C-X-C motif ligand (CXCL) 8 production was measured.

Results

There was no difference in the anti-inflammatory effects of corticosteroids when comparing group mean data of COPD patients versus controls. The inhibition of TNF-α and IL-6 was greater than CXCL8. The effects of corticosteroids varied considerably between subjects, particularly at lower corticosteroid concentrations.

Conclusions

We confirm that overall corticosteroid sensitivity in COPD lung macrophages is not reduced compared to controls. The varied effect of corticosteroids between subjects suggests that some individuals have an inherently poor corticosteroid response. The limited suppression of lung macrophage derived CXCL8 may promote neutrophilic inflammation in COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0260-0) contains supplementary material, which is available to authorized users.     

Lung CD8+ T cells in COPD have increased expression of bacterial TLRs

Background

Toll-like receptors (TLRs) on T cells can modulate their responses, however, the extent and significance of TLR expression by lung T cells, NK cells, or NKT cells in chronic obstructive pulmonary disease (COPD) is unknown.

Methods

Lung tissue collected from clinically-indicated resections (n = 34) was used either: (a) to compare the expression of TLR1, TLR2, TLR2/1, TLR3, TLR4, TLR5, TLR6 and TLR9 on lung CD8+ T cells, CD4+ T cells, NK cells and NKT cells from smokers with or without COPD; or (b) to isolate CD8+ T cells for culture with anti-CD3ε without or with various TLR ligands. We measured protein expression of IFN-γ, TNF-α, IL-13, perforin, granzyme A, granzyme B, soluble FasL, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL9 in supernatants.

Results

All the lung subsets analyzed demonstrated low levels of specific TLR expression, but the percentage of CD8+ T cells expressing TLR1, TLR2, TLR4, TLR6 and TLR2/1 was significantly increased in COPD subjects relative to those without COPD. In contrast, from the same subjects, only TLR2/1 and TLR2 on lung CD4+ T cells and CD8+ NKT cells, respectively, showed a significant increase in COPD and there was no difference in TLR expression on lung CD56+ NK cells. Production of the Tc1 cytokines IFN-γ and TNF-α by lung CD8+ T cells were significantly increased via co-stimulation by Pam3CSK4, a specific TLR2/1 ligand, but not by other agonists. Furthermore, this increase in cytokine production was specific to lung CD8+ T cells from patients with COPD as compared to lung CD8+ T cells from smokers without COPD.

Conclusions

These data suggest that as lung function worsens in COPD, the auto-aggressive behavior of lung CD8+ T cells could increase in response to microbial TLR ligands, specifically ligands against TLR2/1.     

Imbalance of dendritic cell co-stimulation in COPD

Background

Dendritic cells (DCs) control immunity and play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the expression of function-associated surface molecules on circulating DCs in COPD is unknown.

Methods

Four-colour flow cytometry was used to compare blood DC surface molecules of 54 patients with COPD (median age: 59 years; median FEV₁: 38% predicted, median CAT score: 24) with two age-matched control groups with normal lung function: 21 current smokers and 21 never-smokers.

Results

Concentrations of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) and the mDC/pDC ratio did not differ between the groups. The increased expression of BDCA-1, BDCA-3, CD86 and CCR5 on mDCs in patients with COPD did not significantly differ from smokers with normal lung function. In contrast, COPD was specifically characterised by a decreased expression of the anti-inflammatory co-stimulatory molecule PD-L1 on pDCs and an increased expression of the pro-inflammatory co-stimulatory molecule OX40 ligand (OX40L) on mDCs. These changes were not confined to patients with elevated systemic inflammation markers (leukocytes, c-reactive protein, interleukin-6, fibrinogen). The ratio of OX40L to PD-L1 expression (OX40L/PD-L1 ratio), a quantitative measure of imbalanced DC co-stimulation, correlated with the severity of pulmonary emphysema in patients with COPD.

Conclusion

An imbalance of DC co-stimulation might contribute to the pathogenesis of COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0174-x) contains supplementary material, which is available to authorized users.     

Lipocalin 2 is protective against E. coli pneumonia

Background

Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD.

Methods

To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects.

Results

Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02).

Conclusions

Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.     

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background

Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms.

Methods

The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13).

Results

In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter.

Conclusions

This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.     

Altered effector function of peripheral cytotoxic cells in COPD

Background

There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8⁺ T lymphocytes, natural killer (NK; CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results

The proportion of peripheral blood NKT-like (CD56⁺CD3⁺) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56⁺CD3^-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56⁺CD3⁺) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells from smokers and HNS.

Conclusion

In this study, we show that the relative numbers of peripheral blood NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.     

An investigation of the resolution of inflammation (catabasis) in COPD

Background

Chronic Obstructive Pulmonary Disease (COPD) is characterized by an enhanced inflammatory response to smoking that persists despite quitting. The resolution of inflammation (catabasis) is a complex and highly regulated process where tissue resident macrophages play a key role since they phagocytose apoptotic cells (efferocytosis), preventing their secondary necrosis and the spill-over of their pro-inflammatory cytoplasmic content, and release pro-resolution and tissue repair molecules, such as TGFβ, VEGF and HGF. Because inflammation does not resolve in COPD, we hypothesized that catabasis may be abnormal in these patients.

Methods

To explore this hypothesis, we studied lung tissue samples obtained at surgery from 21 COPD patients, 22 smokers with normal spirometry and 13 non-smokers controls. In these samples we used: (1) immunohistochemistry to assess the expression of CD44, CD36, VEGF and TGFβ in lung macrophages; (2) real time PCR to determine HGF, PPARγ, TGFβ, VEGF and MMP-9 gene expression; and, (3) ELISA to quantify lipoxin A4, a lipid mediator of catabasis.

Results

We found that current and former smokers with COPD showed: (1) more inflammation (higher MMP-9 expression); (2) reduced macrophage surface expression of CD44, a key efferocytosis receptor; and, (3) similar levels of TGFβ, VEGF, HGF, PPARγ, and lipoxin A4 than smokers with normal spirometry, despite the presence of inflammation and disease.

Conclusions

These results identify several potential abnormalities of catabasis in patients with COPD.