241 General Discussion To study the precise role of neutrophils during burn injury, neutrophils can be isolated from patient blood or wound tissue and studied in functional assays. This could provide more detail about their inflammatory state and anti-bacterial potential at the different stages of wound healing. The in vitro skin models we described in Chapter 7 might be useful to study the effect of burn injury on cell phenotype and cytokine expression and the effect of neutrophils on re-epithelization. However, because neutrophils are shortlived cells [62,63] and difficult to culture in vitro, multiple administrations of different batches of neutrophils to the skin models will be required to study effects over a longer period of time. Nevertheless, in vitro culturing of neutrophils has been demonstrated before in chemotaxis assays [64]. Several studies have even shown that the lifespan of neutrophils can be increased by specific culture conditions [65–67]. Another opportunity to supplement skin models with neutrophils is the use of the HL-60 promyeoloblast cell line that can easily be differentiated to neutrophil-like cells [68]. Experimental skin models in which neutrophils from burn patients at different phases of wound healing are incorporated could be used to determine the exact role of neutrophils in burninduced inflammation. It would be interesting to see how immature neutrophils behave compared to mature neutrophils. Moreover, such models could be used to test the effect of manipulation of neutrophil behavior on wound healing in a pre-clinical setting. BURN INJURY CAUSES HIGH LEVELS OF CLASSICAL MONOCYTES IN BLOOD AND ACCUMULATION OF MACROPHAGES WITH AFFECTED DIFFERENTIATION IN WOUND TISSUE Macrophages are, similar to neutrophils, early responders to burn injury to ensure the removal of invading pathogens and damaged tissue [69]. When circulating monocytes migrate into tissues they differentiate into dendritic cells or macrophages [70]. After trauma, the bone marrow will release its reservoir of monocytes into the bloodstream to compensate for monocytes that enter tissues [71]. In this thesis, we showed that the number of blood monocytes is highly increased for several weeks after burn injury in both animals (Chapter 2) and patients (Chapter 4). Monocytes are progenitors to both macrophages with a pro-inflammatory phenotype (i.e. M1) and macrophages that are supporters of wound healing processes (i.e. M2). Studies have suggested that classical monocytes are more likely to differentiate into M1-like macrophages, while intermediate and non-classical monocytes are progenitors to M2-like macrophages [72–74]. Analysis of CD14 and CD16 expression on monocytes in patient blood (Chapter 4) and burn tissue (Chapter 5) revealed that the classical monocyte was the most prevalent subtype and that their numbers were increased compared to healthy subjects. High numbers of CD14highCD16¯ (classical) monocytic cells were also detected in burn tissue. This could be an indication of enhanced M1 macrophage activity in the burn wound. The increased 8
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