Ladislav Burysek

The serine protease plasmin triggers expression of MCP-1 and CD40 in human primary monocytes via activation of p38 MAPK and janus kinase (JAK)/STAT signaling pathways.

The mechanism of proinflammatory activation of human monocytes by plasmin is unknown. Here we demonstrate that in human primary monocytes, plasmin stimulates mitogen-activated protein kinase (MAPK) signaling via phosphorylation of MAPK kinase 3/6 (MKK3/6) and p38 MAPK that triggers subsequent DNA binding of transcription factor activator protein-1 (AP-1). The AP-1 complex contained phosphorylated c-Jun and ATF2, and its DNA binding activity was blocked by the p38 MAPK inhibitor SB203580. In addition, plasmin elicits Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, as detected by phosphorylation of JAK1 tyrosine kinase and STAT1 and STAT3 proteins. Plasmin-induced DNA binding of STAT1 and STAT3 was blocked by SB203580 and AG490, inhibitors of p38 MAPK and JAK, respectively, but not by U0126, an inhibitor of MKK1/2. DNA binding of NF-κB remained unaffected by any of these inhibitors. The plasmin-induced signaling led to expression of monocyte chemoattractant protein-1 (MCP-1) and CD40, which required activation of both p38 MAPK and JAK/STAT signaling pathways. Additionally, signaling through both p38 MAPK and JAK is involved in the plasmin-mediated monocyte migration, whereas the formylmethionylleucylphenylalanine-induced chemotaxis remained unaffected. Taken together, our data demonstrate a novel function of the serine protease plasmin in a proinflammatory signaling network.

Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration

CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane‐anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up‐regulated in endothelial cells by proinflammatory cytokines such as IL‐1 or TNF‐α. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time‐ and concentration‐dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT‐PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease‐activated receptor 1 (PAR1) as demonstrated by the use of PAR1‐activating peptide and the PAR1‐specific antagonist SCH 79797. The thrombin‐induced CX3CL1 expression was NF‐κB‐dependent, as shown by EMSA, ELISA, and by inhibition of the NF‐κB signaling pathway by the IκB kinase inhibitor acety‐11‐keto‐β‐boswellic acid or by transient overexpression of a transdominant‐negative form of IκBα. Upon cocultivation of human monocytes with HUVEC, the thrombin‐dependent induction of membrane‐anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP‐1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin‐induced monocyte/endothelial cell cross‐talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas.

Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1α and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation.