Naturheilkunde und klinische Pharmakologie
 


Dr. Yves Laumonnier
Dr. Tatiana Syrovets
Dr. Ladislav Burysek
Berthold Büchele, Dipl-Ing.chem. (FH)

 

Projekt 1

Characterization of the Plasmin and Lipoprotein(a) Induced Activation of
Human Primary Monocytes

Monocytes and other antigen-presenting cells play a central role in the chronic phase of inflammation and are implicated in several human diseases such as atherosclerosis, arthritis, impaired wound healing, and others. These pathological processes are generally accompanied by increased expression of proteolytic enzymes, among them the proteases thrombin and plasmin. It has been, for example, proposed that the increased expression of fibrinolytic genes in atherosclerotic plagues might have a causal effect on the development of atherosclerosis.

Blood monocyte

 

The plasmin/plasminogen system is composed of the inactive zymogen plasminogen that can be activated in vivo by two activators: t-PA and u-PA. While t-PA is believed to play the main role during fibrinolysis, u-PA is thought to be responsible for the extracellular proteolysis, for example during the inflammation. Monocytes express u-PA receptors and bind u-PA as well as plasminogen, which lead to increased plasmin generation in close proximity to the monocyte membrane. Further, contact activation taking place in inflammatory lesions leads to plasmin generation.

We have found 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, activation of MKK3/6-p38, which lead 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 IKKß inducing I κBα degradation leading to activation of NF-κB. Activation of the aforementioned transcription factors results in the induction of proinflammatory genes (chemokine monocyte chemoattractant protein-1, CD-40, tissue factor, and cytokines TNF-α and IL-1). These mediators are crucial for atherogenesis and other chronic inflammatory diseases. In addition, plasmin is a potent chemoattractant of human peripheral monocytes.

Plasmin-apoa-signaling

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 PPARγ activator ciglitazone.

We have identified and characterized a novel, endogenous mechanism of proinflammatory activation of human monocytes. This novel and important plasmin-induced signal transduction pathway have been described at the molecular level. In addition, a new target for the pharmacotherapeutic intervention has been proposed.

We intend to further characterize the plasmin-mediated gene activation by molecular biological and biochemical approaches. Gene expression in plasmin-treated monocytes will be analysed by the DNA array.


Projekt 2

Characterization of the Receptor for Plasmin on Human Peripheral Monocytes

Plasmin has been shown in this laboratory to stimulate monocyte chemotaxis through a process which involves binding to a specific receptor and proteolysis. The plasminogen binding sites on the cell surface are heterogeneous and their identity is obscure. The feedback activities of plasmin on plasminogen, plasminogen binding sites and plasminogen activators, certainly complicate the analysis. All blood cells, except erythrocytes, express binding sites for plasminogen, which could bind plasmin as well. Typical characteristics of the plasmin binding sites are broad distribution, low affinity with KD values in the range of 0.1-2.0 µM, and high density (Bmax about. 0.4-5.0 x 105/cell). Lys-plasminogen, a short form of plasminogen that can be generated by plasmin, and plasmin itself are frequently observed to have lower KD values. At the moment the identity of the receptor remains unknown. Some evidence suggests that numerous proteins, as, for example, &alpa;-enolase (Pancholi 1998, Bergmann 2001), annexin II und TIP49a on the monocytic U937 cells (Hawley et al. 2001), annexin II on the endothelial cells, macrophages and monocytes (McLeod 2003, Falcone 2001), gp330 on the kidney epithelial cells, as plasmin binding sites. Occasionally these binding sites are proposed as receptors, often in terms of membrane-associated proteolytic activity. Some of these proteins already expose lysine, to which plasmin may bind. Some receptors have to be cleaved before the lysine residue is exposed for plasminogen binding.

We will apply the yeast two hybrid screening of a human bone marrow or macrophage library for the search of plasmin binding proteins. The putative binding proteins will be characterized by sequencing. The interaction with plasmin will be confirmed by in vitro pull-down assay and co-immunoprezipitation. Antisense nucleotide approach will be use to investigate the functions of the putative plasmin receptors.

Projekt 3

Characterization of Protease-Activated Receptors (PARs)
on Human Monocytes and other Antigen-Presenting Cells

Monocytes and macrophages express receptors for another blood serine protease thrombin, which triggers release monocyte chemoattractant protein 1.

Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain, unmasking a new N-terminus acting as tethered ligand. Whereas the role of PARs in platelets is well known, their presence and function in human monocytes and other antigen-presenting cells has not been characterized. We demonstrated that human peripheral monocytes, and monocyte-derived macrophages and dendritic cells differentially express PARs. Human monocytes express mainly PAR1 and much less PAR3.

Projekt3.jpg

Differentiation of monocytes into macrophages by either macrophage colony-stimulating factor (M-CSF) or granulocyte - macrophage colony - stimulating factor (GM-CSF) elicits enhanced expression of PAR1, PAR2, and PAR3. In contrast, dendritic cells differentiated from monocytes by GM-CSF and interleukin (IL)-4 strongly downregulated PAR1, PAR2, and PAR3, both at the mRNA and the protein level. Downregulation of the PAR expression was apparently due to IL-4, because treatment of macrophages with IL-4 caused downregulation of PAR1, PAR2, and PAR3. PAR4 mRNA expression remained undetectable in any of the cell types investigated. Stimulation of PAR1, -2 and -3 with thrombin, trypsin or their respective receptor-activating peptides (PAR-AP) triggered cytosolic Ca2+ responses indicating functionally active PARs. Further, stimulation of monocytes or macrophages with thrombin or PAR1-AP, but not with any of the other PAR-AP, triggers expression of monocyte chemoattractant protein (MCP)-1 both at the mRNA and the protein level.

These data demonstrate that differentiation of human monocytes is associated with differential expression of functionally active PARs that mediate distinct regulatory functions in inflammation and atherogenesis.

We plan to further characterize the genes regulated upon activation of PAR receptors in monocytic cells using the DNA microarray approach.

Methods: To characterize the effects of plasmin, thrombin and Lp(a) on human monocytes and other antigen presenting cells we employ following methods:


Publications:

Syrovets T, Tippler B, Rieks M, Simmet Th (1997) Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic GMP-dependent pathway. Blood 89:4574-4583

Syrovets T, Thillet J, Chapman MJ, Simmet Th (1997) Lipoprotein(a) is a potent chemoattractant for human peripheral monocytes. Implications for the lipoprotein(a)-associated atherogenesis. Blood 90:2027-2036

Syrovets T, Büchele B, Gedig E, Slupsky JR, Simmet Th (2000). Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIα. Mol Pharmacol 58:71-81

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

Syrovets T, Schüle A, Jendrach M, Büchele B, Simmet Th (2002). A role for p38 MAP kinase in the inhibition of plasmin-induced monocyte activation by the PPARγ agonist ciglitazone. Thromb Haemost 88:274-281

Burysek L,Syrovets T, Simmet Th (2002) The serine protease plasmin triggers expression of MCP-1 and CD-40 in human primary monocytes via activation of p38 MAPK and JAK/STAT signaling pathway. J Biol Chem 277:33509-33517

Colognato R, Slupsky JR, Jendrach M, Burysek L, Syrovets T and Simmet Th (2003) Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells
(Blood in press, http://www.bloodjournal.org/cgi/reprint/2002-08-2497v1)



   Pharmacology of Natural Products
 Pharmacology of Inflammation
  Mechanisms of Acute Inflammmation and Antiviral Defense
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