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

Lisette I Z Kunz, Thérèse S Lapperre, Jiska B Snoeck-Stroband, Simona E Budulac, Wim Timens, Simone van Wijngaarden, Jasmijn A Schrumpf, Klaus F Rabe, Dirkje S Postma, Peter J Sterk, Pieter S Hiemstra, Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease Study Group, H F Kauffman, D de Reus, H M Boezen, D F Jansen, J M Vonk, M D W Barentsen, W Timens, M Zeinstra-Smit, A J Luteijn, T van der Molen, G ter Veen, M M E Gosman, N H T ten Hacken, H A M Kerstjens, M S van Maaren, D S Postma, C A Veltman, A Verbokkem, I Verhage, H K Vink-Klooster, H A Thiadens, J B Snoeck-Stroband, J K Sont, J Gast-Strookman, P S Hiemstra, K Janssen, T S Lapperre, K F Rabe, A van Schadewijk, J A Schrumpf, J Smit-Bakker, P J Sterk, J Stolk, A C J A Tiré, H van der Veen, M M E Wijffels, L N A Willems, P J Sterk, T Mauad, Lisette I Z Kunz, Thérèse S Lapperre, Jiska B Snoeck-Stroband, Simona E Budulac, Wim Timens, Simone van Wijngaarden, Jasmijn A Schrumpf, Klaus F Rabe, Dirkje S Postma, Peter J Sterk, Pieter S Hiemstra, Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease Study Group, H F Kauffman, D de Reus, H M Boezen, D F Jansen, J M Vonk, M D W Barentsen, W Timens, M Zeinstra-Smit, A J Luteijn, T van der Molen, G ter Veen, M M E Gosman, N H T ten Hacken, H A M Kerstjens, M S van Maaren, D S Postma, C A Veltman, A Verbokkem, I Verhage, H K Vink-Klooster, H A Thiadens, J B Snoeck-Stroband, J K Sont, J Gast-Strookman, P S Hiemstra, K Janssen, T S Lapperre, K F Rabe, A van Schadewijk, J A Schrumpf, J Smit-Bakker, P J Sterk, J Stolk, A C J A Tiré, H van der Veen, M M E Wijffels, L N A Willems, P J Sterk, T Mauad

Abstract

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 × 10(4)/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.

Figures

Figure 1
Figure 1
Photomicrograph of membrane-bound CD163 staining on BAL and sputum cells. A BAL cytospin is shown in the left photograph and a sputum cytospin in the right photograph. Scale bar = 20 μm
Figure 2
Figure 2
BAL differential cell counts expressed as percentage and cell concentrations of COPD patients. Percentage is shown in the left panel, cell concentrations in the right panel. Open circles represent ex-smokers, closed circles represent current smokers. Horizontal bars represent medians. P-values are corrected for recovery of BAL fluid using multiple linear regression.
Figure 3
Figure 3
Sputum differential cell counts expressed as percentage and cell concentrations of COPD patients. Percentage is shown in the left panel, cell concentrations in the right panel. Open circles represent ex-smokers, closed circles represent current smokers. Horizontal bars represent medians.
Figure 4
Figure 4
The percentage and number of CD163+ macrophages in BAL and induced sputum in COPD patients. The percentage (left panel) and number of CD163+ macrophages (right panel) in BAL and induced sputum between ex-smokers (open symbols) and smokers (closed symbols) with COPD. Horizontal bars represent medians. P-values are corrected for recovery of BAL fluid using multiple linear regression.
Figure 5
Figure 5
Soluble mediators measured in BAL and induced sputum supernatants of COPD patients. Ex-smokers are represented by open symbols and smokers by closed symbols. Horizontal bars represent medians. P-values are corrected for recovery of BAL fluid using multiple linear regression.

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