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.     

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.     

Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD

Background

Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD.

Methods

114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV₁ 63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163⁺ macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants.

Results

Ex-smokers with COPD had a higher percentage, but lower number of CD163⁺ macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 ×10⁴/ml, p = 0.001 respectively). The percentage CD163⁺ M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163⁺ BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum.

Conclusions

Our data suggest that smoking cessation partially changes the macrophage polarization in vivo in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.     

Patterns of airway inflammation and MMP-12 expression in smokers and ex-smokers with COPD

Background

Smoking activates and recruits inflammatory cells and proteases to the airways. Matrix metalloproteinase (MMP)-12 may be a key mediator in smoke induced emphysema. However, the influence of smoking and its cessation on airway inflammation and MMP-12 expression during COPD is still unknown. We aimed to analyse airway inflammatory cell patterns in induced sputum (IS) and bronchoalveolar lavage (BAL) from COPD patients who are active smokers and who have ceased smoking >2 years ago.

Methods

39 COPD outpatients – smokers (n = 22) and ex-smokers (n = 17) were studied. 8 'healthy' smokers and 11 healthy never-smokers were tested as the control groups. IS and BAL samples were obtained for differential and MMP-12⁺-macrophages count analysis.

Results

The number of IS neutrophils was higher in both COPD groups compared to both controls. The amount of BAL neutrophils was higher in COPD smokers compared to healthy never-smokers. The number of BAL MMP-12⁺-macrophages was higher in COPD smokers (1.6 ± 0.3 × 10⁶/ml) compared to COPD ex-smokers, 'healthy' smokers and healthy never-smokers (0.9 ± 0.4, 0.4 ± 0.2, 0.2 ± 0.1 × 10⁶/ml respectively, p < 0.05).

Conclusion

The lower amount of BAL neutrophils in COPD ex-smokers, compared to COPD smokers, suggests positive alterations in alveolar compartment after smoking cessation. Smoking and disease itself may stimulate MMP-12 expression in airway compartments (IS and BAL) from COPD patients.     

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.     

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.     

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.     

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.     

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD

Background

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids.

Methods

Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry.

Results

There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10^-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all).

Conclusions

Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.     

Cytotoxic lymphocytes in COPD airways: increased NK cells associated with disease,iNKT and NKT-like cells with current smoking

Background

Cytotoxic lymphocytes are increased in the airways of COPD patients. Whether this increase is driven primarily by the disease or by smoking is not clear, nor whether it correlates with the rate of decline in lung function.

Methods

Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study according to pre-determined criteria; 12 with COPD and a rapid decline in lung function (loss of FEV₁?≥?60?ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV₁?≤?30?ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry.

Results

In BAL fluid, the proportions of NK, iNKT and NKT-like cells all increased with pack-years. Within the COPD group, NK cells – but not iNKT or NKT-like cells – were significantly elevated also in subjects that had quit smoking. In contrast, current smoking was associated with a marked increase in iNKT and NKT-like cells but not in NK cells. Rate of lung function decline did not significantly affect any of the results.

Conclusions

In summary, increased proportions of NK cells in BAL fluid were associated with COPD; iNKT and NKT-like cells with current smoking but not with COPD. Interestingly, NK cell percentages did not normalize in COPD subjects that had quit smoking, indicating that these cells might play a role in the continued disease progression seen in COPD even after smoking cessation.

Trial registration

Clinicaltrials.gov identifier NCT02729220.

     

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

Background

It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.

Methods

We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺ T lymphocytes by real-time PCR.

Results

Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.

Conclusion

Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.     

The IL-10 and IFN-γ pathways are essential to the potent immunosuppressive activity of cultured CD8+ NKT-like cells

Background  

CD8⁺ NKT-like cells are naturally occurring but rare T cells that express both T cell and natural killer cell markers. These cells may play key roles in establishing tolerance to self-antigens; however, their mechanism of action and molecular profiles are poorly characterized due to their low frequencies. We developed an efficient in vitro protocol to produce CD8⁺ T cells that express natural killer cell markers (CD8⁺ NKT-like cells) and extensively characterized their functional and molecular phenotypes using a variety of techniques.     

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.     

Variability in the precursor proteins of collagen I and III in different stages of COPD

Background

Levels of precursor proteins of collagen I and III are increased in fibrotic pulmonary diseases. This study determined whether the expression of precursors of type I and III collagen proteins would be increased in small and large airways of COPD patients in various stages of the disease reflecting fibrogenesis.

Methods

The levels of precursor proteins of collagen I and III were studied by immunohistochemistry and quantified by image analysis in lung tissue of 16 non-smokers, 20 smokers with normal lung function, 20 smokers with stage I-II COPD and 8 ex-smokers with stage IV COPD.

Results

In large airways, the subepithelial layer which was positive for precursor proteins of collagen I and III was thicker in smokers and in stage I-II COPD compared to non-smokers. Large airways in stage IV COPD showed reduced expression of precursor protein of collagen I whereas precursor of collagen III was increased. The amount of precursor protein of collagen III was increased in small airways of smokers and stage I-II COPD but reduced in stage IV COPD.

Conclusions

Precursor proteins of collagen I and III revealed different expression profiles in large and small airways in various stages of COPD. Smoking enhanced expression of both precursors in large airways with a positive correlation with pack-years.     

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.     

Cigarette Smoke Disturbs the Survival of CD8+ Tc/Tregs Partially through Muscarinic Receptors-Dependent Mechanisms in Chronic Obstructive Pulmonary Disease

Background

CD8⁺ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8⁺ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated.

Methods

Circulating CD8⁺ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8⁺ Tc/Tregs.

Results

While COPD patients have elevated circulating percentage of CD8⁺ T cells, healthy smokers have higher frequency of CD8⁺ Tregs. Elevated percentages of CD8⁺ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8⁺ T cells, while facilitated both the proliferation and apoptosis of CD8⁺ Tregs. Notably, the effects of CSE on CD8⁺ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8⁺ Tregs.

Conclusion

The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation.     

Relationship between the anti-inflammatory properties of salmeterol/fluticasone and the expression of CD4+CD25+Foxp3+ regulatory T cells in COPD

Background

Salmeterol and fluticasone combination (SFC) has anti-inflammatory effects and improves clinical symptoms in patients with chronic obstructive pulmonary disease (COPD). However, the anti-inflammatory mechanism of SFC remains unclear. In this study, we investigated the inflammatory responses of COPD, as well as the relationship of the inflammatory factors with the levels of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Foxp3⁺Tregs) after SFC therapy.

Methods

Twenty-one patients with moderate or severe COPD received treatment with 50/500 μg of SFC twice a day for 12 weeks. Before and after treatment, the patients were evaluated using the Modified Medical Research Council (MMRC) dyspnea scale and by conducting a 6-min walk test. The number of neutrophils, monocytes and lymphocytes in induced sputum were counted. Levels of cytokines, including pre-inflammatory IL-8, TNF-α, IL-17A and cytokine IL-10, in the sputum supernatant and peripheral blood were measured by ELISA. The proportion of Foxp3⁺Tregs in the total CD4⁺ T cell of the peripheral blood was determined by flow cytometry. The relationship between IL-17A levels and the percentage of Foxp3⁺Tregs was analyzed by statistical analysis.

Results

After treatment with SFC, the forced expiratory volume in 1 s as a percentage of predicted values (FEV1%) and the 6-min walk distance in the COPD patients significantly increased, while dyspnea scores decreased. The total number of cells, neutrophils, and the percentage of neutrophils in induced sputum reduced notably, while the proportion of monocytes was significantly increased. Levels of the inflammatory cytokines IL-8, TNF-α, and IL-17A in the sputum supernatant and in the blood were markedly lowered, while IL-10 levels were unchanged. The proportion of Foxp3⁺Tregs in the total CD4⁺T cell population in the peripheral blood was drastically higher than that before treatment. The level of IL-17A was negatively correlated with the proportion of Foxp3⁺Tregs in CD4⁺T cells.

Conclusion

SFC can reduce the levels of inflammatory factors and improve symptoms of COPD. The levels of inflammatory factors are associated with the variation of Foxp3⁺Tregs in COPD.

Trial registration

This study was registered with http://www.chictr.org (Chinese Clinical Trial Register) as follows: ChiCTR-TNC-10001270     

Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

Background

Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation.

Methods

114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV₁ 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue⁺) area (%), proliferating (Ki-67⁺) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies.

Results

Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001).

Conclusion

Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years).

Trial registration

NCT00158847     

Cigarette Smoking Promotes Inflammation in Patients with COPD by Affecting the Polarization and Survival of Th/Tregs through Up-Regulation of Muscarinic Receptor 3 and 5 Expression

Background

CD4⁺ T cells in the lung are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), although CD4⁺ T cell subsets and the direct effect of smoking on these cells, especially the expression of MRs, have not been comprehensively examined.

Methods

First, circulating CD4⁺ T cell subsets in healthy nonsmokers, patients with SCOPD and patients with AECOPD were evaluated by flow cytometry. Then, differentiation experiments were carried out using RT-PCR, and Ki-67/Annexin V antibodies were used to measure proliferation and apoptosis. We also explored the impact of CSE on the differentiation and survival of CD4⁺Th/Tregs and examined the expression of MRs in healthy nonsmokers and patients with SCOPD.

Results

We found the percentages of circulating Th1 and Th17 cells were increased in patients with AECOPD, while the percentage of Th2 cells was decreased in patients with SCOPD. The percentages of Th10 cells were decreased in both patients with SCOPD and patients with AECOPD, while the percentages of Tregs were increased. In addition, the percentages of CD4⁺α-7⁺ T cells were decreased in patients with SCOPD and patients with AECOPD. However, only the decrease observed in patients with AECOPD was significant. In vitro studies also revealed MR expression affected the polarization of T cells, with different CD4⁺ T cell subtypes acquiring different MR expression profiles. The addition of CSE facilitated CD4⁺ T cell polarization towards pro-inflammatory subsets (Th1 and Th17) and affected the survival of CD4⁺ T cells and Treg cells by up-regulating the expression of MR3 and 5, resulting in an imbalance of CD4⁺ T cell subsets.

Conclusions

Our findings suggest an imbalance of circulating CD4⁺ T cell subsets is involved in COPD pathogenesis in smokers. Cigarette smoking may contribute to this imbalance by affecting the polarization and survival of Th/Tregs through the up-regulation of MR3 and MR5.