Non-invasive biomarkers and pulmonary function in smokers.

Limited information exists regarding measurement, reproducibility and interrelationships of non-invasive biomarkers in smokers. We compared exhaled breath condensate (EBC) leukotriene B4 (LTB4) and 8-isoprostane, exhaled nitric oxide, induced sputum, spirometry, plethysmography, impulse oscillometry and methacholine reactivity in 18 smokers and 10 non-smokers. We assessed the relationships between these measurements and within-subject reproducibility of EBC biomarkers in smokers. Compared to non-smokers, smokers had significantly lower MMEF % predicted (mean 64.1 vs 77.7, p = 0.003), FEV1/FVC (mean 76.2 vs 79.8 p = 0.05), specific conductance (geometric mean 1.2 vs 1.6, p = 0.02), higher resonant frequency (mean 15.5 vs 9.9, p = 0.01) and higher EBC 8-isoprostane (geometric mean 49.9 vs 8.9 pg/ml p = 0.001). Median EBC pH values were similar, but a subgroup of smokers had airway acidification (pH < 7.2) not observed in non-smokers. Smokers had predominant sputum neutrophilia (mean 68.5%). Repeated EBC measurements showed no significant differences between group means, but Bland Altman analysis showed large individual variability. EBC 8-isoprostane correlated with EBC LTB4 (r = 0.78, p = 0.0001). Sputum supernatant IL-8 correlated with total neutrophil count per gram of sputum (r = 0.52, p = 0.04) and with EBC pH (r = -0.59, p = 0.02). In conclusion, smokers had evidence of small airway dysfunction, increased airway resistance, reduced lung compliance, airway neutrophilia and oxidative stress.

A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist.

RATIONALE: Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alphaMbeta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors. METHODS: We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses. RESULTS: In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p=0.025), and a trend for lower expression of CD11b and shape change in response to CXCL1 (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50=260.7nM) and shape change (IC50=310.5nM) in COPD patients. CONCLUSIONS: Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists.

Use of plasma biomarkers at exacerbation of chronic obstructive pulmonary disease.

IMPACT: This study explores the use of measuring plasma biomarkers at exacerbation of chronic obstructive pulmonary disease (COPD), providing insight into the underlying pathogenesis of these important events. RATIONALE: The use of measuring C-reactive protein (CRP) to confirm exacerbation, or to assess exacerbation severity, in COPD is unclear. Furthermore, it is not known whether there may be more useful systemic biomarkers. OBJECTIVE: To assess the use of plasma biomarkers in confirming exacerbation and predicting exacerbation severity. METHODS: We assessed 36 biomarkers in 90 paired baseline and exacerbation plasma samples from 90 patients with COPD. The diagnosis of exacerbation fulfilled both health care use and symptom-based criteria. Biomarker concentrations were related to clinical indices of exacerbation severity. Interrelationships between biomarkers were examined to gain information on mechanisms of systemic inflammation at exacerbation of COPD. MEASUREMENTS AND MAIN RESULTS: To confirm the diagnosis of exacerbation, the most selective biomarker was CRP. However, this was neither sufficiently sensitive nor specific alone (area under the receiver operating characteristic curve [AUC], 0.73; 95% confidence interval, 0.66-0.80). The combination of CRP with any one increased major exacerbation symptom recorded by the patient on that day (dyspnea, sputum volume, or sputum purulence) significantly increased the AUC to 0.88 (95% confidence interval, 0.82-0.93; p<0.0001). There were no significant relationships between biomarker concentrations and clinical indices of exacerbation severity. Interrelationships between biomarkers suggest that the acute-phase response is related, separately, to monocytic and lymphocytic-neutrophilic pathways. CONCLUSIONS: Plasma CRP concentration, in the presence of a major exacerbation symptom, is useful in the confirmation of COPD exacerbation. Systemic biomarkers were not helpful in predicting exacerbation severity. The acute-phase response at exacerbation was most strongly related to indices of monocyte function.

Validation of the human ozone challenge model as a tool for assessing anti-inflammatory drugs in early development.

This study aimed to test the utility of the ozone challenge model for profiling novel compounds designed to reduce airway inflammation. The authors used a randomized, double-dummy, double-blind, placebo-controlled 3-period crossover design alternating single orally inhaled doses of fluticasone propionate (inhaled corticosteroids, 2 mg), oral prednisolone (oral corticosteroids, 50 mg), or matched placebo. At a 2-week interval, 18 healthy ozone responders (>10% increase in sputum neutrophils) underwent a 3-hour ozone (250 ppb)/intermittent exercise challenge starting 1 hour after drug treatment. Airway inflammation was assessed at 2 hours (breath condensate) and 3 hours (induced sputum) after ozone challenge. Compared to placebo, pretreatment with inhaled corticosteroids or oral corticosteroids resulted in a significant reduction (mean [95% confidence interval]) of sputum neutrophils by 62% (35%, 77%) and 64% (39%, 79%) and of sputum supernatant myeloperoxidase by 55% (41%, 66%) and 42% (25%, 56%), respectively. The authors conclude that an optimized ozone challenge model (including ozone responders and ensuring adequate drug levels during exposure) may be useful for testing novel anti-inflammatory compounds in early development.