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Alternative polarization induces tissue factor
activation of the plasminogen activator inhibitor-1 promot- er for inflammatory induction.7 Here we show that TF inducibility with IL-4 and IL-13 was reduced after prior alternative polarization. However, prior inflammatory polarization did not alter the capability of IL-4 and IL-13 to induce TF. Previous reports suggested a dysfunctional mitochondrial phenotype after proinflammatory polariza- tion resulting in an altered capability of IL-4 to induce its target genes and preventing macrophage repolarization.33 Our results suggest that TF is an alternative activation- dependent target gene that is not affected by changes in mitochondrial function as TF production could still be induced after alternative polarization of previously proin- flammatory polarized macrophages. Furthermore, TF release seems to be delayed and in parallel to microvesicle release as both TF amount and microvesicle number were significantly different in alternatively polarized macrophages 24 h after polarization.
derived from the same donor and observed a higher grade of methylation of the TF promoter in macrophages as com- pared to the respective monocytes from the same donor. Interestingly, addition of a demethylating agent restored the capacity of macrophages to react to LPS stimulation with upregulation of TF. We, therefore, hypothesize that the difference between monocytes and macrophages in their respective ability to react towards LPS with an increase in TF could be due to epigenetic changes within the TF promoter.
Extracellular vesicles were identified because of their ability to support coagulation.48 Among such vesicles, TF- bearing ones were demonstrated to propagate thrombus formation.49 Whereas monocyte-derived, TF-positive extracellular vesicles are common in the circulation50 and monocyte-derived extracellular vesicles are associated with several pathological states including cardiovascular disease and inflammatory disorders,51 little is known about the production of TF-positive extracellular vesicles derived from macrophages. Our results indicate that macrophages show a basal production of extracellular vesicles, which can be increased by alternative activation. In addition, we found that these extracellular vesicles shed from macrophages are phosphatidylserine positive. It should be emphasized that TF it is not only regulated at the level of mRNA expression but also at the post-translational level with phosphatidylserine converting TF from a cryptic to a decrypted prothrombotic form.10,52,53 Our findings described above therefore support the notion that macrophage- derived vesicles might have the capacity to activate TF. We also show here that alternative polarization increased both the total amount of extracellular vesicles as well as the amount of TF on these extracellular vesicles significantly as compared to vesicles from unpolarized macrophages or macrophages that had undergone proinflammatory polar- ization.
Finally, our in vitro data showing that alternatively acti- vated macrophages produce TF were supported by immunohistochemical analysis of colon carcinoma sec- tions. As alternative polarization is most common in tumors, we analyzed histochemical sections of colon carci- noma, a tumor which has already been shown to have increased IL-4 expression54 and contain TF+ cells.55 To iden- tify proinflammatory macrophages we used CD80, where- as alternatively activated macrophages were identified by CD206. Tumor-associated macrophages have already been associated with IL-4 activation.56 Here we were able to identify STAT6+ macrophages within these sections of colon carcinoma, indicating IL-4 and IL-13 signaling. Furthermore, macrophages within these sections stained positive for CD206, an established marker for alternatively polarized macrophages, and for TF. We also showed that extracellular TF in part co-localized with extracellular CD206 possibly suggesting the presence of macrophage- derived extracellular vesicles in these sections. To under- stand a possible link between TF+ areas and areas positive for CD206 we evaluated tumor tissue from four different human donors. At least seven sections from each tumor were scored for the presence of CD206 or TF. We found that especially regions with high expression of TF also had high expression of CD206 and regions with low TF expres- sion were predominantly negative or low for CD206. Besides tumor tissue, atherosclerotic plaques have also been demonstrated to contain TF with TF protein localiza- tion associated with plaque macrophages.57 Interestingly,
In monocytes TF was shown to be induced by NF-κB and AP-1-dependent signaling.34 In murine macrophages an induction of TF was demonstrated after LPS stimulation.35 Human cells were also reported to retain the capacity to react to LPS or IFN-γ for a certain time while differentiating from monocytes to macrophages.36 In addition to NF-κB and AP-1, the TF promoter contains Sp1 binding sites37 and is enhanced by Pin1.38 Furthermore, in mouse macrophages loss of PARP14 was demonstrated to increase the levels of TF expression.39 In addition, PARP14 is in part regulated by STAT6 as STAT6 activation induces a switch in PARP14 from a repressor to a promoter of STAT6 signaling via the ribosylation of histone deacetylases previously recruited to IL-4 response elements.40 Of note, STAT6 is the main down- stream target activated by IL-4 and IL-13 signaling.41 In the present work we demonstrate that inhibition of either STAT6 or PARP activity abolished the induction of TF mRNA in macrophages by IL-4 and IL-13. Our findings sug- gest that macrophages upregulate TF in vivo in a STAT6- inducing environment and that TF might be a marker pro- tein for STAT6 signaling and, therefore, alternative polariza- tion in macrophages. Nonetheless, it must be emphasized that macrophages in general express TF already at baseline.
There is ample evidence to support the notion that monocytes are an important source of circulating TF. At resting conditions already around 1.5% of CD14+ mono- cytes are positive for TF.42 When monocytes are exposed to LPS TF expression is quickly and transiently upregulated in these cells.43,44 We have identified in our study a difference of TF induction between monocytes and macrophages as our results indicate that human macrophages do not react to LPS treatment with upregulation of TF but display increased TF production only after alternative polarization. This is somehow in contrast to earlier reports that suggest- ed a similar behavior of monocytes and macrophages in response to defined stimuli.45,46 However, whereas we used MCSF or GMCSF for macrophage maturation those stud- ies were performed on macrophages derived from mono- cytes in the presence of human serum without a defined macrophage maturation factor suggesting that the cells studied could be monocytes still in transition to macrophages.
Epigenetic changes within a promoter region hold the potential to alter the response of cells to certain stimuli.47 We, therefore, determined the methylation state around the NF-κB response element, which is responsible for reacting to LPS signaling, in monocytes and macrophages
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