Pharmacology of Inflammation

As to inflammation, we are specifically interested in molecular mechanisms of proinflammatory signaling relevant for chronic inflammatory diseases such as atherosclerosis. In this context, we primarily focus on the role of proteolytic signaling elicited by serine proteases such as thrombin and plasmin. These studies should contribute to a better understanding of the molecular mechanisms leading to chronification of the inflammatory process and will therefore contribute to the identification of novel therapeutic drug targets. Subsequently, some examples are given.

Plasmin Triggers Chemotaxis of Monocyte-Derived Dendritic Cells through an Akt2-Dependent Pathway and Promotes a T Helper Type 1 Response

The serine protease plasmin is generated from its zymogen plasminogen by either tPA or uPA, which activate plasminogen during fibrinolysis and inflammation, respectively. The expression of fibrinolytic genes is increased in the atherosclerotic aorta. Indeed, the upregulated fibrinolytic activity correlates with the severity of coronary lesions. Although, so far the presence of proteases in atherosclerosis was mainly regarded in terms of plaque destabilization, plasmin may also induce intracellular signaling and modulate immune and inflammatory responses as we have shown.Dendritic cells (DCs) accumulate in atherosclerotic arteries, where they can modulate atherogenesis. We investigated whether plasmin might alter the function of human DCs. Our studies revealed that stimulation of monocyte-derived DCs with plasmin elicits a time-dependent actin polymerization and chemotaxis comparable to that triggered by the standard chemoattractant FMLP. Plasmin triggered rapid activation of Akt and MAPKs, followed by phosphorylation of the regulatory myosin light chain and chemotaxis. For the chemotactic DC migration, the activation of Akt as well as the p38 and ERK1/2 MAPK was indispensable as shown by pharmacological inhibitors. DCs express Akt1 and Akt2, but not Akt3. Yet in DCs, plasmin activates exclusively Akt2 via a p38 MAPK-dependent pathway. Accordingly, knockdown of Akt2 with shRNA, but not of Akt1, blocked the plasmin-induced ERK1/2 activation and the chemotactic response. Moreover, plasmin-stimulated DCs induced polarization of CD4+ T cells towards the IFN-g-producing, proinflammatory Th1 phenotype. Consistent with a role for DCs and adaptive immune response in atherogenesis, we demonstrated DCs in human atherosclerotic vessels and showed that plasmin is abundant in human atherosclerotic lesions, where it colocalizes with DCs.
Thus, plasmin generation in the atherosclerotic vessel wall might contribute to accumulation of DCs, activation of the adaptive immune response and aggravation of atherosclerosis.

Plasmin Is an Activator of Human Monocytes

In human monocytes, we detected that the effects of plasmin are not restricted to its role in fibrinolysis, but that plasmin is a potent proinflammatory activator of human monocytes. In monocytes, plasmin, but not proteolytically inactive plasminogen, triggers a signal cascade including a G protein as well as protein kinase C and G-kinase, which lead to activation of JAK1, followed by phosphorylation of STAT1 and STAT3 transcription factors. Moreover, activation of MKK3/6-p38 MAPK leads to additional phosphorylation of STAT1 and STAT3 on serines and also of ATF2, a member of the AP-1 transcription factor family.
 

In addition, plasmin activates IKKb inducing IkBa degradation resulting in activation of NF-kB. Activation of the aforementioned transcription factors converges in the induction of proinflammatory genes (monocyte chemoattractant protein-1, CD-40, tissue factor, and cytokines TNF-a and IL-1). These mediators are crucial for atherogenesis and other chronic inflammatory diseases. In addition, we identified plasmin as a potent chemoattractant
 

These data point to a general proinflammatory plasmin-mediated activation of human monocytes, which range from chemotaxis to the expression of proinflammatory mediators. We have also shown that the plasmin-induced monocyte activation can be pharmacologically inhibited by the PPARg activator ciglitazone.

Thus, we have identified and characterized a novel, endogenous mechanism that triggers an overall proinflammatory activation of human monocytes. This novel plasmin-mediated signal transduction pathway has been described at the molecular level and a new target for pharmacotherapeutic intervention has been proposed.

 

Mature Dendritic Cells Express Functional Thrombin Receptors Triggering Chemotaxis and CCL18/PARC Chemokine Induction

Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) was scarce. We detected that monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-a or CD40L, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicited actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-a-matured DC. The thrombin-induced migration was a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase 1 (ROCK1) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (MLC2). The ERK1/2- and ROCK1-mediated phosphorylation of MLC2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/PARC, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83+ DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. Therefore, it was concluded that the thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.

 

Methods

  • cDNA cloning
  • Transformation
  • Protein expression
  • Western blotting with immunostaining
  • Northern blot analysis
  • In vivo kinase assay
  • Co-immunoprecipitation
  • Electrophoretic mobility shift assay (EMSA)
  • DNA footprinting
  • RT-PCR
  • Chemotaxis analysis
  • ELISA
  • Flow cytometric analysis of the cell surface proteins
  • Cell cycle analysis by FACS
  • Laser scanning microscopy
  • Fluorescence microscopy
  • Peptide cross-linking
  • Isolation of primary cells by density gradient centrifugation
  • Surface plasmon resonance
  • Analysis of the gene promoter regulation by gene reporter assays
  • Yeast two hybrid screening
  • In vitro knockdown by antisense oligodeoxynucleotide treatment
  • DNA chip array

 

Publications (selected)

Li X, Syrovets T, Genze F, Pitterle K, Oberhuber A, Orend KH, Simmet Th. Plasmin triggers chemotaxis in monocyte-derived dendritic cells through Akt2-dependent pathways and promotes a T-helper type-1 response. Arterioscler Thromb Vasc Biol. 2010; 30:582-90

Li X, Syrovets T, Paskas S, Laumonnier Y, Simmet Th. Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction. J Immunol. 2008; 181:1215-23

Popovic M, Laumonnier Y, Burysek L, Syrovets T, Simmet Th. Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration. J Leukoc Biol. 2008; 84:215-23

Li Q, Laumonnier Y, Syrovets T, Simmet Th. Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages. Arterioscler Thromb Vasc Biol. 2007; 27:1383-9

Laumonnier Y, Syrovets T, Burysek L, Simmet Th. Identification of the annexin II heterotetramer as a receptor for the plasmin-induced signaling in human peripheral monocytes. Blood. 2006; 107:3342-9

Syrovets T, Simmet Th. Novel aspects and new roles for the serine protease plasmin. Cell Mol Life Sci. 2003; 61:873-85

Colognato R, Slupsky JR, Jendrach M, Burysek L, Syrovets T, Simmet Th. Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells. Blood. 2003; 102:2645-52

Burysek L, Syrovets T, Simmet Th. 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. J Biol Chem. 2002; 277:33509-17

Syrovets T, Schüle A, Jendrach M, Büchele B, Simmet Th. Ciglitazone inhibits plasmin-induced proinflammatory monocyte activation via modulation of p38 MAP kinase activity. Thromb Haemost. 2002; 88:274-81

Syrovets T, Jendrach M, Rohwedder A, Schüle A, Simmet Th. Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKb-mediated NF-kB activation. Blood. 2001; 97:3941-50.

Syrovets T, Thillet J, Chapman MJ, Simmet Th. Lipoprotein(a) is a potent chemoattractant for human peripheral monocytes. Implications for the lipoprotein(a)-associated atherogenesis. Blood. 1997; 90:2027-36

Syrovets T, Tippler B, Rieks M, Simmet Th. Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic GMP-dependent pathway. Blood. 1997; 89:4574-83

Weide I, Tippler B, Syrovets T, Simmet Th. Plasmin is a specific stimulus of the 5-lipoxygenase pathway of human peripheral monocytes. Thromb Haemost. 1996; 76:561-8