Differential activation of killer cells in the circulation and the lung: a study of current smoking status and chronic obstructive pulmonary disease (COPD).

BACKGROUND: CD8(+) T-lymphocytes, natural killer T-like cells (NKT-like cells, CD56(+)CD3(+)) and natural killer cells (NK cells, CD56(+)CD3(-)) are the three main classes of human killer cells and they are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Activation of these cells can initiate immune responses by virtue of their production of inflammatory cytokines and chemokines that cause lung tissue damage, mucus hypersecretion and emphysema. The objective of the current study was to investigate the activation levels of human killer cells in healthy non-smokers, healthy smokers, ex-smokers with COPD and current smokers with COPD, in both peripheral blood and induced sputum. METHODS/PRINCIPAL FINDINGS: After informed consent, 124 participants were recruited into the study and peripheral blood or induced sputum was taken. The activation states and receptor expression of killer cells were measured by flow cytometry. In peripheral blood, current smokers, regardless of disease state, have the highest proportion of activated CD8(+) T-lymphocytes, NKT-like cells and NK cells compared with ex-smokers with COPD and healthy non-smokers. Furthermore, CD8(+) T-lymphocyte and NK cell activation is positively correlated with the number of cigarettes currently smoked. Conversely, in induced sputum, the proportion of activated killer cells was related to disease state rather than current smoking status, with current and ex-smokers with COPD having significantly higher rates of activation than healthy smokers and healthy non-smokers. CONCLUSIONS: A differential effect in systemic and lung activation of killer cells in COPD is evident. Systemic activation appears to be related to current smoking whereas lung activation is related to the presence or absence of COPD, irrespective of current smoking status. These findings suggest that modulating killer cell activation may be a new target for the treatment of COPD.

Standards of care for occupational asthma: an update.

BACKGROUND: The British Thoracic Society (BTS) Standards of Care (SoC) Committee produced a standard of care for occupational asthma (OA) in 2008, based on a systematic evidence review performed in 2004 by the British Occupational Health Research Foundation (BOHRF). METHODS: BOHRF updated the evidence base from 2004-2009 in 2010. RESULTS: This article summarises the changes in evidence and is aimed at physicians, nurses and other healthcare professionals in primary and secondary care, occupational health and public health and at employers, workers and their health, safety and other representatives. CONCLUSIONS: Various recommendations and evidence ratings have changed in the management of asthma that may have an occupational cause.

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.

Regulation in chronic obstructive pulmonary disease: the role of regulatory T-cells and Th17 cells.

COPD (chronic obstructive pulmonary disease) is an inflammatory disorder of the airways, which is associated with irreversible airway obstruction. The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the central airways (chronic bronchitis) and inflammation of the peripheral airways (respiratory bronchiolitis). Tobacco smoking is established as the main aetiological factor for COPD. A maladaptive modulation of inflammatory responses to inhalation of noxious particles and gases is generally accepted as being a key central pathogenic process; however, the precise regulatory mechanisms of the disease are poorly understood. Two cell types are known to be important in immune regulation, namely regulatory T-cells and the newly identified Th17 (T-helper 17) cells. Both types of cells are subsets of CD4 T-lymphocytes and modulate the immune response through secretion of cytokines, for example IL (interleukin)-10 and IL-17 respectively. The present review will begin by describing the current understanding of inflammatory cell involvement in the disease process, and then focus on the possible role of subsets of regulatory and helper T-cells in COPD.

The trainee-led clinicopathological conference: an innovative method for educating medical trainees.

Adult learning theory states that adults learn by setting their own goals and making their learning applicable to the real world. Postgraduate training in medicine is now regulated by the 'gold guide', which emphasises the importance of trainee-led education. A new method of delivering education that allows trainees to develop generic skills as well as knowledge, the trainee-led clinicopathological conference, will be discussed. This method embraces the principles of adult learning theory and has been well received by trainees.

Quantitative validation and comparison of multiplex cytokine kits.

The focus of biomarker studies is shifting toward deciphering patterns of biomolecules as they provide a more comprehensive depiction of disease than individual biomarkers. Multiplexing technologies are crucial in deciphering such patterns, but it is essential that they are validated for reproducibility and precision to ensure accurate protein identification. Here the authors examine such properties in Cytokine Bead Array (CBA) and Luminex kits and compare concentration measurements to those obtained using enzyme-linked immunosorbent assay (ELISA). Luminex kits were found to be highly reproducible and reliable; however, CBA kits were not due to aberrant standards. Absolute cytokine concentrations were dependent on the detection kit, but correlations with ELISA were good for all technologies.

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.

Systems biology coupled with label-free high-throughput detection as a novel approach for diagnosis of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a treatable and preventable disease state, characterised by progressive airflow limitation that is not fully reversible. Although COPD is primarily a disease of the lungs there is now an appreciation that many of the manifestations of disease are outside the lung, leading to the notion that COPD is a systemic disease. Currently, diagnosis of COPD relies on largely descriptive measures to enable classification, such as symptoms and lung function. Here the limitations of existing diagnostic strategies of COPD are discussed and systems biology approaches to diagnosis that build upon current molecular knowledge of the disease are described. These approaches rely on new 'label-free' sensing technologies, such as high-throughput surface plasmon resonance (SPR), that we also describe.

Killer cells in chronic obstructive pulmonary disease.

COPD (chronic obstructive pulmonary disease) is a treatable and preventable disease state, characterized by progressive airflow limitation that is not fully reversible. It is a current and growing cause of mortality and morbidity worldwide, with the WHO (World Health Organization) projecting that total deaths attributed to COPD will increase by more than 30% in the next 10 years. The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the central airways (chronic bronchitis) and inflammation of the peripheral airways (respiratory bronchiolitis). The destructive changes and tissue remodelling observed in COPD are a result of complex interactions between cells of the innate and adaptive immune systems. The focus of the present review is directed towards the role of CD8(+) T-lymphocytes, NK (natural killer) cells and NKT cells (NK T-cells). These three classes of killer cell could all play an important part in the pathogenesis of COPD. The observed damage to the pulmonary tissue could be caused in three ways: (i) direct cytotoxic effect against the lung epithelium mediated by the activities of perforin and granzymes, (ii) FasL (Fas ligand)-induced apoptosis and/or (iii) cytokine and chemokine release. The present review considers the role of these killer cells in COPD.

MMP-9 protein level does not reflect overall MMP activity in the airways of patients with COPD.

BACKGROUND: Animal and human studies have implicated an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the pathogenesis of chronic obstructive pulmonary disease (COPD). MMP-9 protein is increased in COPD and we hypothesized that total MMP activity would be raised although this has not previously been measured. METHODS: Using fluorescence and biotin labelled MMP assays, RT-PCR, western blotting and enzyme-linked immunosorbent assay we examined total MMP activity, specific gelatinase, elastase, collagenase activity, TIMP-1 and TIMP-2 in induced sputum from smokers with COPD and smokers without COPD. RESULTS: Induced sputum was obtained from 15 smokers with COPD and 14 smokers without COPD. MMP-9 levels were higher in those with COPD compared with controls (p<0.05). Total MMP activity, specific gelatinase, collagenase and elastase activities were not higher in COPD patients. In addition, reduced MMP activity was correlated with increasing airflow obstruction in COPD (p=0.016). CONCLUSION: MMP-9 protein but not MMP activity was higher in sputum of COPD patients compared with controls. These results suggest that MMP-9 levels may not reflect the overall MMP activity in the airways of patients with COPD suggesting a complex relationship between MMP-9 protein and activity. Further studies of MMPs in COPD should comprise activity measures in addition to protein levels.

Pulmonary expression of leukemia inhibitory factor induces B cell hyperplasia and confers protection in hyperoxia.

Leukemia inhibitory factor (LIF) is produced by a large number of pulmonary cells in response to diverse stimuli. Exaggerated levels of LIF have also been detected in the adult respiratory distress syndrome and other disorders. The biologic effects of LIF in the lung, however, have not been elucidated. To define the respiratory effects of LIF, we generated transgenic mice in which human LIF was selectively targeted to the mature lung. In these mice, transgene activation caused an impressive increase in bronchoalveolar lavage (BAL) cellularity with a significant increase in BAL and tissue B lymphocytes. LIF also conferred protection in 100% O2 where it decreased alveolar-capillary protein leak and enhanced survival. This protective effect was associated with the induction of interleukin (IL)-6 mRNA and protein. LIF transgenic mice with a null mutation in IL-6 were more sensitive to the toxic effects of 100% O2 than LIF-transgenic animals with a wild-type IL-6 locus. These studies demonstrate that LIF induces B cell hyperplasia and confers protection in hyperoxic acute lung injury. They also demonstrate that LIF induces IL-6 and that the protective effects of LIF are mediated, in part, via this inductive event. LIF may be an important regulator of B cell-mediated responses and oxidant injury in the lung.

Frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study.

BACKGROUND: Rhinovirus infections cause exacerbations of asthma. We postulated that people with asthma are more susceptible to rhinovirus infection than people without the disease and compared the susceptibility of these groups. METHODS: We recruited 76 cohabiting couples. One person in every couple had atopic asthma and one was healthy. Participants completed daily diary cards of upper-respiratory-tract (URT) and lower-respiratory-tract (LRT) symptoms and measured peak expiratory flow twice daily. Every 2 weeks nasal aspirates were taken and examined for rhinovirus. Mixed models were used to compare risks of infection between groups. We also compared the severity and duration of infections. FINDINGS: We analysed 753 samples. Rhinovirus was detected in 10.1% (38/378) of samples from participants with asthma and 8.5% (32/375) of samples from healthy participants. After adjustment for confounding factors, asthma did not significantly increase risk of infection (odds ratio 1.15, 95% CI 0.71-1.87). Groups did not differ in frequency, severity, or duration of URT infections or symptoms associated with rhinovirus infection. First rhinovirus infection was associated more frequently with LRT infection in participants with asthma than in healthy individuals (12 of 28 infections vs four of 23, respectively, p=0.051). Symptoms of LRT associated with rhinovirus infection were significantly more severe (p=0.001) and longer-lasting in participants with asthma than in healthy participants (p=0.005). INTERPRETATION: People with atopic asthma are not at greater risk of rhinovirus infection than healthy individuals but suffer from more frequent LRT infections and have more severe and longer-lasting LRT symptoms.