C. Bernini

Thrombin‐mediated IL‐10 up‐regulation involves protease‐activated receptor (PAR)‐1 expression in human mononuclear leukocytes

Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease‐activated receptors (PARs). Interleukin (IL)‐10, besides its anti‐inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL‐12 production in human mononuclear cells and that such inhibition is accompanied by IL‐10 up‐regulation. To our knowledge, there are no data available to show that thrombin mediates IL‐10 production by its interactions with PAR‐1. We here report that human α‐thrombin enhances IL‐10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up‐regulation requies PAR‐1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR‐1 agonist peptides, such as SFLLRN, results in a significant increase of IL‐10 production. PAR‐1 expression is required for thrombin‐induced IL‐10 production, as shown by experiments performed with antisense or sense PAR‐1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac‐6, results in an increased IL‐10 production. This suggests that the observed IL‐10 up‐regulation may be the result of a direct interaction with monocytes. The observation that thrombin‐mediated up‐regulation of IL‐10 may require the expression of the PAR‐1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses.

Regulation of Angiogenesis by Th1- and Th2-Type Cytokines

Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Such biological processes occur in several physiological conditions, such as embryo development and wound healing, as well as in pathological conditions, including tumours and diabetic retinopathy. T lymphocytes, neutrophils and monocytes fully participate in the angiogenic process by secreting cytokines that may control endothelial cell (EC) proliferation, their survival and apoptosis, as well as their migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. This balance is conceptually very similar to that of the Th1/Th2 cells that modulate an appropriate and specific immune response. Th1 or Th2 cytokines may control angiogenesis directly, by acting on cell growth and differentiation, indirectly by inducing the release of other cytokines in the microenvironment, and by modulating the expression of specific receptors, involved in the control of angiogenic processes, such as EC proliferation and migration. In this review we will mainly discuss the role of Th1- and Th2-type cytokines in the angiogenic process, emphasizing the complexity of the cytokine and leukocyte/EC network, and highlighting the care that needs to be taken when designing new therapeutic interventions involving Th1 and Th2 cytokines.

Inhibition of interleukin‐12 expression by α‐thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses

In addition to its central role in blood coagulation and hemostasis, human α‐thrombin is a powerful regulator of inflammatory responses and is known to affect cell‐mediated immunity. Interleukin (IL)‐12 is a strong promoter of the development of Th1‐type lymphocytes and its downregulation implies a positive feedback mechanism for development of Th2 responses. We have previously shown that thrombin enhances the release of IL‐6, a Th2‐related cytokine, in human peripheral blood mononuclear cells (PBMC). Here we show that thrombin downregulates IL‐12 production at both protein and mRNA levels in human PBMC. The inhibition of IL‐12 production was accompanied by an enhanced release of IL‐10, which inhibits Th1‐related processes and promotes Th2‐type responses. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, reveals that this downregulation is thrombin‐specific and requires thrombin proteolytic activity. In addition, activation of coagulation inhibits IL‐12 production in whole blood cultures, confirming the tight relationship between the coagulation pathway, where thrombin is a key enzyme, and inflammation. Decreased IL‐12 production appears to be related also to IL‐10 production, since the addition of an anti‐IL‐10 monoclonal antibody to thrombin‐treated PBMC resulted in a partial restoration of IL‐12 production. In conclusion, the observation that thrombin significantly affects the production of IL‐12, as well as of IL‐10, implies a concerted role orchestrated by thrombin in PBMC that could be crucial to effective immunity and inflammation.

The thrombin peptide, TP508, enhances cytokine release and activates signaling events.

The thrombin peptide, TP508, accelerates tissue repair and initiates a cascade of cellular events. We have previously shown that alpha-thrombin induces cytokine expression in human mononuclear cells. We, therefore, investigated the possibility that TP508 might activate cytokine production and intracellular signaling pathways associated with cytokine activation. Our results show that TP508 induces cytokine expression in human mononuclear cells. TP508 treatment enhances extracellular signal-regulated kinase (Erk1/2) activities in U937 cells, as well as Erk1/2 and p38 activation in Jurkat T cells. These data support the hypothesis that TP508 may accelerate tissue repair through the activation of the inflammatory response.