154 Chapter 5 Granulocytes in burn tissue consist mainly of activated mature neutrophils IHC analysis of myeloperoxidase (MPO) expression, an enzyme abundantly present in azurophilic granules of neutrophils [16], showed an immediate increase in neutrophil numbers in burn tissue at PBW 1, and an even larger increase from PBW 2 onward (Figures 2A,B). This was confirmed by FCM analysis of neutrophils (CD15+CD16+ granulocytes) (Figure 2C). Eosinophils (CD9+CD15+CD16¯ granulocytes) were increased at PBW 2-3, but to a lesser extent (Supplementary Figure 2C). In both healthy skin and burn tissue neutrophils were almost exclusively CD10+, a marker that is associated with neutrophil maturation [17] (Figure 2D). Only in burn tissue from PBW 1 there was a slight increase in immature (CD10¯) neutrophils. Activation markers CD11b and CD66b were upregulated in neutrophils at PBW 2-3 (Figures 2E,F). Self-organizing map clustering of flow data (FlowSOM) using Cytobank displayed cell populations (nodes) and clusters based on marker expression in an unsupervised manner (Figure 2G). This analysis highlights some of the burn-specific changes that occur in wound neutrophils. Burn injury caused significant differences in the percentage of neutrophils per cluster (Figure 2H). CD11blowCD14+CD66b¯ neutrophils (cluster 1) were decreased early after burn injury, while CD11b+CD66blow neutrophils (cluster 2) were increased. Although CD11bhighCD66b+ neutrophils (cluster 3) seemed more represented in burn tissue than in healthy skin, no significant difference was found. A small population of CD16low neutrophils (cluster 4) was significantly increased at PBW 1 and the percentage of CD16lowCD14+ neutrophils (cluster 5) was significantly increased at PBW 4.
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