Coagulation and Fibrinolysis in Kidney Graft Rejection.

Coagulation system is currently considered an integrated part of innate immunity. Clotting activation in response to bacterial surface along with complement cascade priming represents the first line of defense against pathogens. In the last three decades, we learned that several coagulation factors, including factor II or thrombin and factor X, can interact with specific cell surface receptors activated by an unusual proteolytic mechanism and belonging to a novel class of G-protein-coupled receptors known as protease-activated receptors (PARs). PARs are expressed by a variety of cells, including monocytes, dendritic cells, and endothelial cells and may play a key role in the modulation of innate immunity and in the regulation of its interaction with the adaptive branch of the immune system. Also, the fibrinolytic system, in which activation is controlled by coagulation, can interact with innate immunity, and it is a key modulator of extracellular matrix deposition eventually leading to scarring and fibrosis. In the setting of kidney transplantation, coagulation and fibrinolytic systems have been shown to play key roles in the ischemia/reperfusion injury featuring delayed graft function and in the pathogenesis of tissue damage following acute and chronic rejection. In the present review, we aim to describe the mechanisms leading to coagulation and fibrinolysis activation in this setting and their interaction with the priming of the innate immune response and their role in kidney graft rejection.

Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells.

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4-CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.

The Role of Protease-Activated Receptors for the Development of Myocarditis: Possible Therapeutic Implications.

Protease-activated receptors (PARs) are a unique group of four G-protein coupled receptors. They are widely expressed within the cardiovascular system and the heart. PARs are activated via cleavage by serine proteases. In vitro and in vivo studies showed that the activation of PAR1 and PAR2 plays a crucial role in virus induced inflammatory diseases. The receptors enable cells to recognize pathogen-derived changes in the extracellular environment. An infection with Coxsackie-virus B3 (CVB3) can cause myocarditis. Recent studies have been shown that PAR1 signaling enhanced the antiviral innate immune response via interferon ß (IFNß) and thus limited the virus replication and cardiac damage. In contrast, PAR2 signaling decreased the antiviral innate immune response via IFNß und thus increased the virus replication, which caused severe myocarditis. Along with CVB3 other viruses such as influenza A virus (IAV) and herpes simplex virus (HSV) can induce myocarditis. The role of PAR signaling in IAV infections is contrarily discussed. During HSV infections PARs facilitate the virus infection of the host cell. These studies show that PARs might be interesting drug targets for the treatment of virus infections and inflammatory heart diseases. First studies with PAR agonists, antagonists, and serine protease inhibitors have been conducted in mice. The inhibition of thrombin the main PAR1 activating protease decreased the IFNß response and increased the virus replication in CVB3-induced myocarditis. This indicates that further studies with direct PAR agonists and antagonists are needed to determine whether PARs are useful drug targets for the therapy of virus-induced heart diseases.

[Roles of Pseudomonas aeruginosa-derived proteases as a virulence factor].

The major virulence factors produced by Pseudomonas aeruginosa include secreted proteases that damage host tissues. Of the proteases analyzed, alkaline protease (AprA) and elastase B (LasB) have been characterized extensively. Although P. aeruginosa protein database predicts the presence of several other potential proteases, little has been known about the proteases involving in the pathogenicity of this organism. In this study, we found that P. aeruginosa produces a novel large extracellular protease (LepA) distinct from known proteases such as AprA and LasB. Sequence analysis of LepA showed a molecular future of the proteins transported by the two-partner secretion pathway. We demonstrated that LepA can activate NF-kB-driven promoter through protease-activated receptor-1, -2 or -4. On the other hand, one of the functions of proteases is to hydrolyze proteins and peptides for nutrient acquisition either by degrading host enzymes or even by causing tissue damage to further the survival of the bacterium. Therefore, to investigate the role of LepA in in vivo virulence and growth of P. aeruginosa, we compared the virulence and growth of a wild-type strain and its mutant using a mouse model of acute systemic infection by P. aeruginosa. Our results suggest that LepA contributes to the in vivo virulence and growth of P. aeruginosa.

Proteinase-activated receptor-4 plays a major role in the recruitment of neutrophils induced by trypsin or carrageenan during pleurisy in mice.

BACKGROUND/AIMS: The activation of proteinase-activated receptors (PARs) has been implicated in the development of important hallmarks of inflammation, including in vivo leukocyte recruitment. Here, we examined the effects of aprotinin, a potent inhibitor of trypsin proteinase and the kallikrein-kinin system, and the PAR-4 antagonist YPGKF-NH(2) (tcY-NH(2)) on neutrophil recruitment in response to carrageenan and trypsin in the pleural cavity of mice. METHODS: BALB/c mice were intrapleurally injected with trypsin or PAR-4-activating peptide AY-NH(2), pretreated with aprotinin or tcY-NH(2) (1 µg/cavity) prior to an intrapleural injection of trypsin or carrageenan, or pretreated with leukotriene B(4) antagonist U-75302 (3 µg/cavity) prior to a trypsin injection. The number of infiltrating neutrophils was evaluated after 4 h. RESULTS: PAR-4-activating peptide AY-NH(2) and trypsin-induced neutrophil recruitment was inhibited by aprotinin, tcY-NH(2) or U-75302. Aprotinin and tcY-NH(2) also inhibited neutrophil recruitment induced by carrageenan. CONCLUSION: These data suggest a key role for PAR-4 in mediating neutrophil recruitment in a mouse model of pleurisy induced by the activity of trypsin or trypsin-like enzymes.

The roles of thrombin and protease-activated receptors in inflammation.

Inflammation and coagulation constitute two host defence systems with complementary physiological roles in limiting tissue damage, restoring homeostasis and eliminating invading pathogens, functions reliant on effective regulation of both processes at a variety of levels. Dysfunctional activation or regulation of either pathway may lead to pathology and contribute to human diseases as diverse as myocardial infarction and septic shock. The serine protease thrombin, a key protein in the coagulation pathway, can activate cellular signalling directly via proteolytic cleavage of the N-terminal domain of a family of G protein-coupled receptors or indirectly through the generation of molecules such as activated protein C. These events transmit signals to many cell types and can elicit the production of various pro-inflammatory mediators such as cytokines, chemokines and growth factors, thereby influencing cell activation, differentiation, survival and migration. This review discusses recent progress in understanding how thrombin and protease-activated receptors influence biological processes, highlighting the detrimental and protective cellular effects of thrombin and its signalling pathways.

The role of innate immunity activation in house dust mite allergy.

House dust mite (HDM) allergy is a frequent inflammatory disease found worldwide. Although allergen-specific CD4(+) Th2 cells orchestrate the HDM allergic response, notably through induction of IgE directed towards mite allergens, recent studies have demonstrated that innate immunity activation also plays a critical role in HDM-induced allergy pathogenesis. HDM allergens can not only be considered proteins that induce adaptive Th2-biased responses in susceptible subjects but also as strong activators of innate immune cells, including skin keratinocytes and airway epithelial cells. The contribution of microbial adjuvant factors, derived from HDM carriers or the environment, is also essential in such cell stimulation. This review highlights how HDM allergens, together with microbial compounds, promote allergic responses through pattern recognition receptor-dependent pathways.

Differential expression of protease-activated receptors in monocytes from patients with primary antiphospholipid syndrome.

OBJECTIVE: To investigate the expression of protease-activated receptors (PARs), their potential regulation by anticardiolipin antibodies (aCL), and their association with the expression of other molecules relevant to thrombosis in monocytes obtained from 62 patients with primary antiphospholipid syndrome (APS). METHODS: Monocytes were isolated from peripheral blood mononuclear cells by magnetic depletion of nonmonocytes. Expression of tissue factor (TF) and PARs 1-4 genes was measured by quantitative real-time reverse transcription-polymerase chain reaction. Cell surface TF and PARs 1-4 expression was analyzed by flow cytometry. For in vitro studies, purified normal monocytes were incubated with purified APS patient IgG, normal human serum IgG, or lipopolysaccharide, in the presence or absence of specific monoclonal antibodies anti-PAR-1 (ATAP2) or anti-PAR-2 (SAM11) to test the effect of blocking the active site of PAR-1 or PAR-2. RESULTS: Analysis of both mRNA and protein for the 4 PARs revealed significantly increased expression of PAR-2 as compared with the control groups. PAR-1 was significantly overexpressed in APS patients with thrombosis and in the control patients with thrombosis but without APS. PAR-3 expression was not significantly altered. PAR-4 expression was absent in all groups analyzed. In addition, we demonstrated a correlation between the levels of PAR-2 and the titers of IgG aCL, as well as parallel behavior of TF and PAR-2 expression. In vitro, IgG from APS patients significantly increased monocyte expression of PAR-1 and PAR-2. Inhibition studies suggested that there was direct cross-talk between TF and PAR-2, such that inhibition of PAR-2 prevented the aCL-induced expression of TF. CONCLUSION: These results provide the first demonstration of increased expression of PARs in monocytes from patients with APS. Thus, PAR antagonists might have therapeutic potential as antithrombotic agents in APS.

Perspectives on the etiology of chronic rhinosinusitis.

PURPOSE OF REVIEW: This article reviews recent insights surrounding the etiology and pathogenesis of chronic rhinosinusitis. In particular, we highlight the increasing recognition of host-mediated mechanisms in driving mucosal inflammation. RECENT FINDINGS: Published differences between epithelium from patients with chronic rhinosinusitis and normal controls can be classified into several broad categories. Alterations are reported in the various components of the epithelial innate immune system including epithelial-expressed pattern-recognition receptors (PRRs) and the levels of antimicrobial innate immune effector molecules. Other studies demonstrate differences in the proteins involved in maintaining epithelial barrier integrity. Finally, recent studies show in chronic rhinosinusitis that epithelial-derived cytokines, chemokines and inducible surface proteins are involved in recruiting and activating cells of the adaptive immune system. CONCLUSION: The sinonasal epithelium provides a mechanical and innate immune barrier to a diverse array of environmental agents. This barrier also plays a key role in regulating the acquired mucosal immune response in the nose. Recent studies suggest that defects in this barrier may foster development of chronic sinonasal inflammation in response to environmental agents, and pathogenic or commensal organisms. The ability to dissect and analyze defects in the inflammatory response in rhinosinusitis may help identify novel targets for drug development.

Protease signaling to G protein-coupled receptors: implications for inflammation and pain.

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR(1), PAR(2), and PAR(4) either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR(2) agonists induce hyperalgesia and allodynia, and PAR(2) activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR(1) or PAR(4) increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.

Proteinase-activated receptors (PARs): crossroads between innate immunity and coagulation.

Coagulation cascade and innate immunity are intimately linked in their endeavor to organize the body's response to injury. Protease-activated receptors (PARs) are important mediators of inflammatory response that can be activated by proteases of the coagulation cascade. Their recent discovery has shed new light on the crosstalk between coagulation and innate immunity. Recent studies have investigated the physiological relevance of PARs in the context of immunity and vascular injury, suggesting that these receptors could be used as therapeutic targets for the treatment of pathologies related to innate immunity, endothelial functions and coagulation processes.

Innate immune responses to environmental allergens.

Aero-allergens, including plant pollens, house dust mite particles, fungal spores, and mycelium fragments, are continuously inhaled and deposited on the airway mucosa. These particles and their soluble components actively interact with innate recognition systems present in the mucosal layer (e.g., surfactant proteins) and with various receptors present on a diversity of cells in the airways. Deposited particles are first removed by active transportation, the rate of which is either enhanced or inhibited by components present in the inhaled substances. Cleaning further depends on innate recognition, beginning with (a) soluble factors released into the mucosal surface layer that bind different bio-organic components; (b) innate receptors on phagocytic cells, alveolar macrophages, and dendritic monocytes; and (c) innate receptors on airway epithelial cells. Different innate receptor families (Toll-like receptors [TLRs], nucleotide-binding oligomerization domain receptors, and protein-activated receptors [PARs]) have been demonstrated on airway cells, including alveolar macrophages, monocytes, dendritic cells, and airway tissue cells (e.g., epithelial cells and mast cells). However, although the functional role of these receptors has been studied for infectious diseases, the functional role for reaction of airways to inhaled bio-organic substances, including allergens, is largely unexplored. Indirect evidence for functional interactions has come from in vivo animal studies, as well as in vitro studies with monocytes, macrophages, and epithelial cells, which have demonstrated release of cytokines and chemokines after contact with allergens such as house dust mite, cat, pollen, and fungi. Most allergens show direct activation of airway epithelial cells, suggesting a role for the innate receptors. However, the role of TLRs, PARs, and other receptors was studied for only a limited number of allergens. Current studies indicate synergistic interactions between members of the same receptor family (TLRs) as well as synergistic interactions between members of different families (TLRs, PARs, and nucleotide-binding oligomerization domain receptors), modulating responses into feed-forward or inhibitory actions. Study of these synergistic interactions and their genetic variations will provide insight regarding how the innate immune system determines the inflammatory reactions of the airways and the outcome of the T-helper-1- and T-helper-2-type responses to inhaled allergens.

Proteinase-activated receptor 2 activation in the airways enhances antigen-mediated airway inflammation and airway hyperresponsiveness through different pathways.

BACKGROUND: Serine proteinases such as mast cell tryptase, trypsin-like enzymes, and certain allergens are important in the pathogenesis of asthma. These proteinases can activate the proteinase-activated receptor (PAR)-2, which has been shown to be upregulated in the airways of patients with asthma. OBJECTIVE: The purpose of this study was to investigate PAR-2 activation in the airways during allergen challenge and its effects on the 2 principle features of asthma, airway inflammation and airway hyperresponsiveness (AHR). METHODS: Proteinase-activated receptor 2 activating peptide SLIGRL-NH2 (PAR-2 activating peptide [ap]) or control peptide LSIGRL-NH2 (PAR-2 control peptide [cp]) was administered alone or in conjunction with ovalbumin intranasally to mice, and AHR and airway inflammation were evaluated. RESULTS: PAR2ap did not induce AHR or airway inflammation in ovalbumin-sensitized mice that had not been challenged with ovalbumin. When administered with ovalbumin, PAR-2ap enhanced AHR and airway inflammation compared with ovalbumin administered alone or with PAR-2cp. The enhanced AHR persisted for 5 days, whereas the enhancement to airway inflammation dissipated. Mice administered PAR-2ap alone during the 5 days after the final antigen challenge demonstrated an additional enhancement to airway inflammation compared with the control animals. PAR-2ap administered with allergen increased TNF and IL-5 mRNA in lung tissue and IL-13 and TNF in bronchoalveolar lavage fluid. CONCLUSION: Exogenous PAR-2 activation in parallel with allergen challenge enhances allergen-mediated AHR and airway inflammation through distinct mechanisms. PAR-2 activation can also enhance established airway inflammation even when dissociated from exposure to allergen. Therefore, PAR-2 activation may play a pathogenic role in the development of AHR and airway inflammation.

Regulated expression of platelet factor 4 in human monocytes--role of PARs as a quantitatively important monocyte activation pathway.

Human mononuclear phagocytes have recently been shown to express constitutively and even more so, upon stimulation with bacteria, fungi, lipopolysaccharide (LPS), zymosan, or thrombin platelet basic protein (PBP). This CXC chemokine as well as platelet factor 4 (PF4), which is located genomically at a short distance from the PBP, were previously considered to be specific markers for the megakaryocyte cell lineage. Both chemokines have signaling and antimicrobial activity. In the present studies, transcriptional and expressional regulation of PF4 and related chemokines was studied in human monocytes. As shown by quantitative mRNA analysis, Western blots, radioimmunoprecipitation of cell extracts, and immunofluorescence and quantitatively with enzyme-linked immunosorbent assay, human monocytes express PF4 in the same order of magnitude as the known, regulated CXC chemokine interleukin (IL)-8. Expression of PF4 is up-regulated at the mRNA and protein level by thrombin and mediated by proteinase-activated receptors (PARs), resulting in a 32- to 128-fold higher mRNA level and leading to an up-to-sixfold increase of the peptide concentration in monocyte culture supernatants. Thrombin and the synthetic ligand of PAR-1 and PAR-2, SFLLRN, also induced comparable increases in the levels of mRNA for PBP, IL-8, regulated on activation, normal T expressed and secreted (RANTES), monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha and increased synthesis of these chemokines as shown by immunofluorescence or a quantitative immunobead-based method. The induction of increased mRNA levels for all chemokines by SFLLRN was unsurpassed by LPS, zymosan, interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-1. Activation of monocytes through PARs represents an alternate activation mechanism, independent from IFN-gamma, TNF-alpha, or other signaling pathways.