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Alternative polarization induces tissue factor
tissue.1 Some tissue macrophages and precursors are already established embryonically in the yolk sac and fetal liver before the onset of definitive hematopoiesis.2 Upon inflammation, the pool of resident macrophages gets quickly replaced by macrophages derived from circulating monocytes.3
In order to be able to respond to multiple tasks, macrophages can adapt to the environment when exposed to specific cues. This macrophage polarization can be simulated in vitro using lipopolysaccharide (LPS) and interferon (IFN)-γ stimulation for a proinflammatory subset termed classical activation and stimulation with interleukin (IL)-4 and IL-13 for an alternative polarization phenotype.4 Upon polarization, macrophages react to the respective stimulus with the expression of a distinct phe- notype. Classical polarization is usually associated with a proinflammatory response, including the increased pro- duction of tumor necrosis factor (TNF)-α, IL-1, and IL-6.5 Functionally, proinflammatory polarization leads to potent effector cells that kill intracellular micro-organisms and tumor cells.6 In addition, these cells are present dur- ing early wound healing and proinflammatory macrophages are characterized by a pronounced ability to degrade tissue.7 In contrast, alternatively activated macrophages are characterized by increased expression of IL-10 and of scavenger receptors. Besides scavenging debris, promoting angiogenesis, tissue remodeling and repair, alternatively activated macrophages are able to fine tune inflammatory responses.8 In vivo, distinct macrophage subtypes are more subtle. Nevertheless, in vitro polarization phenotypes are present in in vivo situa- tions.4 Macrophages resembling classical macrophages can be found in environments with bacterial infection or in inflammatory pathologies, whereas alternatively polar- ized macrophages are prominent in cancer and in diseases with a Th2 cytokine signature.9
An inflammatory milieu is usually accompanied by an increased procoagulatory risk. This risk increase is due to the close link between inflammation and tissue factor (TF) induction. TF is the primary activator of the coagulation cascade.10 TF-bearing monocytes are essential for the initi- ation of coagulation11 and TF expression in monocytes is triggered strongly by inflammatory mediators including LPS.10 Nonetheless, whereas TF expression in human monocytes is well studied, data on TF production in human macrophages, especially under different polariza- tion conditions, are scarce. TF is a transmembrane protein that functions as a high affinity receptor for factor VII and the activated form of this clotting factor.10 Especially in cir- culation, TF is present on circulating extracellular vesicles. In pathological conditions, high levels of procoagulant, TF- exposing particles can be found in the circulation.12 Interestingly, even though TF and inflammation are closely connected, cancer and hence a Th2-promoting environ- ment were also found to be associated with increased TF- bearing extracellular vesicles.13 Extracellular vesicles are membrane-enclosed structures of different sizes.14 TF is mainly associated with vesicles within the microvesicle fraction of extracellular vesicles which range in size from 200-1000 nm.15 These vesicles form through an active bud- ding mechanism from the parental cell and, therefore, include membrane-linked proteins.16 In addition to TF those vesicles can have phosphatidylserine exposed on the surface which, in turn, can both support coagulation17 and activate TF.10
In order to understand a possible role of macrophages in fostering a procoagulatory environment in different disease states, we investigated the contribution of macrophages and their respective polarization to the production and release of TF and of extracellular vesicles.
Methods
Full details of the experimental setups and conditions are avail- able in the Online Supplement.
Generation of human monocytes and macrophages
Human macrophages were derived from peripheral blood monocytes from leukapheresis chambers, as described previously, obtained from healthy platelet donors according to recommenda- tions of the ethical board of the Medical University of Vienna including informed consent (approval number 1575/2014).7,18 Human macrophages were always prepared from fresh blood. Macrophages from 75 healthy volunteers were used in the course of the study.
Flow cytometry
Extracellular vesicles were analyzed using flow cytometry on a Cytoflex (Beckman Coulter, CA, USA) or AttuneNXT (Thermo Fisher, MA, USA) flow cytometer. Values are given as events/mL. Microvesicles were defined as being between 200 nm and 900 nm according to size-specific fluorescence beads (Megamix Plus, Biocytex, France). Human and mouse monocytes were investigat- ed for membrane-bound TF using a specific antibody (Thermo Fisher [MA, USA] for human TF, Biotechne [MN, USA] for mouse TF) as published previously.19
Enzyme-linked immunosorbent assay of extracellular vesicles
We used a commercially available enzyme-linked immunosor- bent assay (ELISA) kit (Hyphen Biomed, France) to determine the concentration of annexin V+ extracellular vesicles in the circulation according to the manufacturer’s instructions.
RNA isolation and quantitative polymerase chain reaction analysis
Detailed information about RNA isolation and quantitative polymerase chain reactions are available in the Online Supplement.
Protein determination
TF was determined by a commercially available ELISA (Biotechne, MN, USA) as suggested by the manufacturer on extra- cellular vesicles and in cell lysates. Extracellular vesicles were iso- lated by centrifugation at 18,000 g for 20 min at 4°C.
Tissue factor activity assay
Extracellular vesicle-associated TF activity was measured as previously described.20 A detailed protocol is available in the Online Supplement.
Immunohistochemistry and fluorescence microscopy
Immunohistochemical staining was performed on cryopre- served sections of TissueTek (Agilent, CA, USA)-embedded colon carcinoma tissue (4 patients) or on paraffin-embedded atheroscle- rotic tissue sections, as published previously.7,21 Specimens were collected according to the recommendations of the institutional ethics board including informed consent. Plaque tissue was derived from patients undergoing carotid endarterectomy (mean age 71±6.4 years, 72% male, 27% symptomatic, n=16).
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