Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages
Arteriosclerosis, Thrombosis and Vascular Biology
Objective-Fibrinolytic activity is upregulated in atherosclerotic lesions, yet little is known about the role of plasmin in macrophage function. We postulated a direct effect of plasmin on human monocyte-derived macrophages. Methods and Results-Plasmin activates macrophages via the annexin A2 heterotetramer composed of annexin A2 and S100A10 with subsequent stimulation of Janus kinase JAK1/TYK2 signaling. JAK1/TYK2 leads to STAT3 activation, Akt-dependent NF-B activation, and phosphorylation of
... phosphorylation of extracellular signal-regulated kinase 1/2 and mitogen-activated kinase p38. These signaling pathways trigger nuclear translocation of STAT3 and p65 transcription factors and the induction of the proinflammatory cytokines tumor necrosis factor-␣ and IL-6. Inhibitors of JAK, p38, and NF-B revealed that these signaling pathways are indispensable for the plasmin-mediated tumor necrosis factor-␣ and IL-6 induction. By contrast, the extracellular signal-regulated kinase 1/2 activation is essential only for the IL-6 expression. The activation clearly depends on the proteolytic activity of plasmin, which cleaves the A2 subunit of the annexin A2 heterotetramer. Downregulation of each of the receptor subunits by antisense oligodeoxynucleotides abolished the plasmin-induced expression of proinflammatory cytokines stressing the crucial role the annexin A2 heterotetramer. Conclusions-Plasmin generated at sites of inflammation such as atherosclerotic lesions will trigger cytokine expression in human macrophages. (Arterioscler Thromb Vasc Biol. 2007;27:1-2.) On this background, we hypothesized that plasmin generated in atherosclerotic lesion could induce macrophage activation. Proteins were analyzed by Western immunoblot and flow cytometry 11 ; cytokine levels were quantified with enzyme-linked immunosorbent assays. Reverse Transcription and Polymerase Chain Reaction Analysis Cytokine mRNA was quantified by semi-quantitative reversetranscription polymerase chain reaction from total RNA of 1ϫ10 6 macrophages. For in vitro knockdown phosphorothioate-modified oligodeoxynucleotides (ODN; Thermo Hybaid, Ulm, Germany) were applied to macrophage cultures. 11 STAT and NF-B Family Transcription Factor Assays Activations of STAT1␣, 3, 5A, 5B, and p65, c-Rel, RelB were determined with DNA-binding TransAM enzyme-linked immunosorbent assays for STAT and NF-B family transcription factors in nuclear extracts from stimulated macrophages. 9,12 Statistical Analysis MeansϮSEM are shown. Probabilities calculated with the Newman-Keuls test were considered significant for PϽ0.05.