Figure 1

(A) Enrichment of sputum macrophages by RosetteSep technique. Induced sputum samples from a healthy individual (left panel) and a COPD patient (right panel) were used to isolate the macrophage populations by using the Rosette-Sep technique. Purified cells were stained with CD66b/CD16b/CD14 and analyzed by flow cytometry. Resulting macrophage populations were regated on a forward versus side scatter plot to show the differential properties in size and granularity. In the given example, the proportion of small macrophages in the control donor is 4.9% (left) whereas the COPD patient shows an increase to 80.6% (right) of all macrophages. Shown is one example out of 11 for COPD and of 8 for controls donors. (B) Chemokine mRNA expression in controls and COPD. Purified sputum macrophages (>90%) from controls (left bar in each plot) and COPD patients (right bar) were analyzed by RT-PCR for mRNA expression for CCL2, CCL7, CCL13 and CCL22. Shown is the relative fold difference in expression of COPD patients as compared with controls that have been set as one. Values have been normalized to the expression of the house keeping gene α-enolase. Results were in fold difference 16.2 ± 20.9 for CCL2, 13.01 ± 8.2 for CCL7, 110 ± 225 for CCL13 and 12.3 ± 15.5 for CCL22. Shown are the mean values of 8 controls and 9–11 COPD patients. *P < 0.05, **P < 0.01, ***P < 0.001. The average percentage of small sputum macrophages in the two cohorts used for RT-PCR analysis was 12.9% ± 7.2% in controls and 85.9% ± 8.3% in COPD (P < 0.01).