Results
Characterization of Participants
Table 1 presents the main demographic, clinical and functional characteristics of participants. Age was similar in all groups except that current smokers with normal spirometry were slightly younger. Most participants were males, except for never smokers where females predominate. Cumulative smoking exposure was similar between all smoker groups. All former smokers quit smoking more than 5 years before tissue sampling. As expected, spirometry was abnormal in patients with COPD and within the normal range in the other two groups. Four COPD patients had GOLD grade I and 17 GOLD grade II airflow limitation.
Immunohistochemistry
The pattern of macrophage immunostaining was predominantly membranous for anti-CD44 and anti-CD36 antibodies, whereas it was predominantly cytoplasmic for anti-VEGF and anti-TGFβ (Figure 1), as expected because of their respective cellular localization. Figure 2 presents the individual and mean (bars) values of the proportion of macrophages with intense immunostaining for CD44, CD36, VEGF and TGFβ in all participants. Independently of their smoking status (current vs. former), CD44 expression was lower in patients with COPD than in smokers and ex-smokers with normal spirometry and non-smokers (p<0.0001); by contrast, macrophage immunostaining of CD36, VEGF and TGFβ was similar in all groups (Figure 2). CD44 expression was not related to airflow limitation or treatment with inhaled steroids.
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Figure 2.
Individual and mean (bars) values of the proportion of macrophages with intense staining for CD44 , CD36, VEGF and TGFβ in patients with COPD, smokers with normal spirometry and non-smokers. (S = current smokers; EX-S = former smokers; NS = non-smokers). For further explanations, see text.
Lung Tissue Gene Expression
Figure 3 presents the individual and mean (bars) values of HGF, PPARγ, TGFβ and VEGF expression in lung tissue samples. We did not observe significant differences between groups for any of these markers. Figure 3 also shows that the lung expression of MMP-9, a marker of tissue inflammation was, as expected, significantly higher in patients with COPD, both current and former smokers, than in controls. Additional file 1: Table S1 (on-line supplement) presents a correlation matrix between HGF, PPARγ, TGFβ, VEGF and MMP-9 expression in lung tissue, and the percentage of macrophages with intense staining of CD36, VEGF, TGFβ, and CD44, as well as FEV1 values in COPD patients. The same information is presented graphically (heat-map) in Figure 4 whereas Additional file 1: Table S2 (on-line supplement) presents the individual Z-scores en each patient for each inflammatory marker determined in pulmonary macrophages (M) and lung tissue extracts. It is of note that lung tissue markers (Figure 4, bottom right corner of the heat-map) were much better correlated among them than with macrophage markers, which were not particularly well correlated either among themselves (Figure 4, top left corner of the heat-map; see also Additional file 1: Table S1 on-line supplement).
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Figure 3.
Individual and mean (bars) values of HGF, PPARγ, TGFβ, VEGF and MMP9 gene expression in lung tissue specimens, in patients with COPD, smokers with normal spirometry and non-smokers. (S = current smokers; EX-S = former smokers; NS = non-smokers) ** p < 0.01. For further explanations, see text.
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Figure 4.
Spearman correlations heat map. Colours in each cell indicate the strength of the relationship, from white (no relationship) to dark red (strong relationship). The exact Rho correlation coefficient and corresponding p values are presented in Additional file 1: Table S1 (on-line supplement). For further explanations, see text.
Pulmonary Concentration of Lipoxin A4
The concentration of lipoxin A4 in lung tissue homogenates was similar in all groups studied (Figure 5).
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Figure 5.
Individual and mean (bars) values of lipoxin A4 (LXA4) concentration in lung tissue homogenates in patients with COPD, smokers with normal spirometry and non-smokers (S = smokers; EX-S = ex-smokers; NS = non-smokers). For further explanations, see text.