PAR2 Knockout Mice Research Articles

Suppression of pancreatitis‐related allodynia/hyperalgesia by proteinase‐activated receptor‐2 in mice

1 Proteinase-activated receptor-2 (PAR2), a receptor activated by trypsin and tryptase, is abundantly expressed in the gastrointestinal tract including the C-fiber terminal, and might play a role in processing of visceral pain. In the present study, we examined and characterized the roles of PAR2 in pancreatitis-related abdominal hyperalgesia/allodynia in mice. 2 Caerulein, administered i.p. once, caused a small increase in abdominal sensitivity to stimulation with von Frey hairs, without causing pancreatitis, in PAR2-knockout (KO) mice, but not wild-type (WT) mice. 3 Caerulein, given hourly six times in total, caused more profound abdominal hyperalgesia/allodynia in PAR2-KO mice, as compared with WT mice, although no significant differences were detected in the severity of pancreatitis between the KO and WT animals. 4 The PAR2-activating peptide, 2-furoyl-LIGRL-NH(2), coadministered repeatedly with caerulein six times in total, abolished the caerulein-evoked abdominal hyperalgesia/allodynia in WT, but not PAR2-KO, mice. Repeated doses of 2-furoyl-LIGRL-NH(2) moderately attenuated the severity of caerulein-induced pancreatitis in WT animals. 5 Our data from experiments using PAR2-KO mice provide evidence that PAR2 functions to attenuate pancreatitis-related abdominal hyperalgesia/allodynia without affecting pancreatitis itself, although the PAR2AP applied exogenously is not only antinociceptive but also anti-inflammatory.

Colonic hyperalgesia triggered by proteinase-activated receptor-2 in mice: Involvement of endogenous bradykinin

Intracolonic (i.col.) administration of the PAR2-activating peptide (PAR2AP) SLIGRL-NH 2 slowly develops visceral hypersensitivity to i.col. capsaicin in ddY mice. Thus, we further analyzed roles of PAR2 in colonic hypersensitivity, using the novel potent PAR2AP, 2-furoyl-LIGRL-NH 2 and PAR2-knockout (KO) mice. In ddY mice, i.col. 2-furoyl-LIGRL-NH 2 produced delayed (6 h later) facilitation of capsaicin-evoked visceral nociception, an effect being much more potent than SLIGRL-NH 2. Such effects were mimicked by i.col. trypsin. In wild-type (WT), but not PAR2-KO, mice of C57BL/6 background, i.col. PAR2 agonists caused delayed facilitation of sensitivity to capsaicin. The PAR2-triggered visceral hypersensitivity was abolished by a bradykinin B2 receptor antagonist, HOE-140. Our data thus provide ultimate evidence for role of PAR2 in colonic hypersensitivity, and suggest involvement of the bradykinin-B2 pathway.

Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis

The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties of inflammation and neurodegeneration. We investigated the role of PAR2 in the pathogenesis of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. PAR2 expression was increased on astrocytes and infiltrating macrophages in human MS and murine EAE central nervous system (CNS) white matter (P < 0.05). Macrophages and astrocytes from PAR2 wild-type (WT) and knockout (KO) mice exhibited differential immune gene expression with PAR2 KO macrophages showing significantly higher interleukin 10 production after lipopolysaccharide stimulation (P < 0.001). PAR2 activation in macrophages resulted in the release of soluble oligodendrocyte cytotoxins (P < 0.01). Myelin oligodendrocyte glycoprotein–induced EAE caused more severe inflammatory gene expression in the CNS of PAR2 WT animals (P < 0.05), together with enhanced T cell proliferation and interferon γ production (P < 0.05), compared with KO littermates. Indeed, PAR2 WT animals showed markedly greater microglial activation and T lymphocyte infiltration accompanied by worsened demyelination and axonal injury in the CNS compared with their PAR2 KO littermates. Enhanced neuropathological changes were associated with a more severe progressive relapsing disease phenotype (P < 0.001) in WT animals. These findings reveal previously unreported pathogenic interactions between CNS PAR2 expression and neuroinflammation with ensuing demyelination and axonal injury.

Protease-Activated Receptor-2 (PAR-2) as a Novel Target for Modulating Immune Responses to Neo Antigens Following In Vivo Gene Transfer.

The safety of several strategies using novel adeno-associated viral (AAV) vectors (serotypes-1,-5,-7,and -8) to skeletal muscle of large animals have been hampered by the immune response to the transgene ( Blood 103: 85, 3303, and 3330). Dendritic cells (DC) are potent antigen-presenting cells that govern the immune responses. The involvement of proteases is critical to DC antigen processing and presentation. Proteases' effects on cells are mediated by protease-activated receptor (PAR) a group of four G-protein-coupled receptors. Bone marrow-derived DC from PAR-2 knockout mice do not spontaneously develop to mature state and only do so upon stimulations of inflammatory signals, therefore, PAR-2 proved critical for DC activation. Work with selective PAR-2 agonists and knockout animals suggests a in vivo contribution of this receptor to several chronic inflammatory diseases and tumor development. In fact, small peptides functioning as PAR-2 antagonists are now being tested as anti-angiogenic agents. We sought to determine whether inhibiting specific targets on the T-cell activation cascade would provide a strategy to prevent the development of antibody following IM injection of AAV. To do this we evaluated the risk of inhibitior formation to clotting factor IX (FIX) in PAR-2 deficient mice compared with heterozygous (+/−) or normal (+/+) genotypes. Mice received IM injection of AAV-1 encoding human FIX (hFIX) at two different doses (low and high cohorts). FIX and antibody levels were monitored weekly. PAR-2(−/ −) mice(n=4) given the low dose exhibited circulating FIX levels of 500± 99ng/ml which remained stable for the duration of the experiment(10 weeks), and no antibody for FIX was detected. In three PAR-2(+/+) mice, FIX expression was transiently detected at week 2 followed by a period of 4 weeks with undetectable levels. This was due to the formation of FIX specific IgG-1 antibody, which results from activation of CD4+ T-cell mainly of Th2 subset, that peaked at week 6. These antibodies inhibit FIX clotting activity as determined by a functional assay at titre of 3–5.5BU (Bethesda Unit) in all 3 mice. High dose AAV-1 treated PAR-2(−/ −) mice(n=6) resulted in continuous expression of FIX(1,500±353ng/ml) in the absence of FIX antibody. In contrast, inhibitors of FIX were detected in all mice(5 per group) PAR-2(+/−) or PAR-2(+/+). All animals develop IgG-1 antibodies to FIX through 10 weeks of observation. At week 6 post-injection, FIX inhibitors were detected in 3/10 mice at levels of ~1 BU. Continuous follow-up showed that a decrease in antibody titers to FIX was associated with slowly increasing FIX antigen. In addition, splenocytes from PAR-2 (+/+) mice exhibit significant increase (p<0.05) in antigen-specific IFN-γ secretion detected by ELISpot whereas in PAR-2 (−/ −) mice no response was detected. Furthermore, in PAR-1(−/ −) and PAR-1(+/+) mice IM injected with AAV-1, FIX inhibitor was detected in all animals (n=4/group). This demonstrates a PAR-2-specific immunomodulatory mechanism. In summary, these data suggest that pharmacological inhibition of PAR-2 may provide a novel strategy to evade the immune response to the transgene in a variety of gene-based strategies.

Therapeutic Promise of Proteinase-Activated Receptor-2 Antagonism in Joint Inflammation

Biological therapies such as tumor necrosis factor-alpha inhibitors have advanced the treatment of rheumatoid arthritis, but one-third of patients do not respond to such therapy. Furthermore, these inhibitors are now usually administered in combination with conventional disease-modifying antirheumatic drugs, suggesting they have not achieved their early promise. This study investigates a novel therapeutic target, proteinase-activated receptor (PAR)-2, in joint inflammation. Intra-articular carrageenan/kaolin (C/K) injection in mice resulted in joint swelling that was associated with synovial PAR2 up-regulation. Inhibiting receptor up-regulation using small interfering RNA technology, as confirmed by immunoblotting, substantially reduced the inflammatory response in the joint. Serine proteinase-induced joint swelling was mediated primarily via PAR2 activation, since the response to exogenous application of trypsin and tryptase was absent in PAR2 knockout mice. Furthermore, serine proteinase inhibitors were effective anti-inflammatory agents in this model. Disrupting proteolytic activation of PAR2 using antiserum (B5) directed to the receptor cleavage/activation site also attenuated C/K-induced inflammation, as did the similarly targeted PAR2 monoclonal antibody SAM-11. Finally, we report the activity of a novel small molecule PAR2 antagonist, N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068), that dose dependently attenuated joint inflammation. Our findings represent a major advance in collectively identifying PAR2 as a novel target for the future treatment of arthritis.

Protease-Activated Receptor-2 Activation Induces Acute Lung Inflammation by Neuropeptide-Dependent Mechanisms

Protease-activated receptors (PARs) and tachykinin-immunoreactive fibers are located in the lung as sentries to respond to a variety of pathological stimuli. The effects of PAR activation on the lung have not been adequately studied. We report on the effects of instilling PAR-activating peptides (PAR-APs, including PAR1-, PAR2-, and PAR4-AP) into the lungs of ventilated or spontaneously breathing mice. PAR2-AP, but not PAR1-AP or PAR4-AP, caused a sharp increase in lung endothelial and epithelial permeability to protein, extravascular lung water, and airway tone. No responses to PAR2-AP were detected in PAR2 knockout mice. In bronchoalveolar lavage, PAR2 activation caused 8- and 5-fold increase in MIP-2 and substance P levels, respectively, and a 12-fold increase in the number of neutrophils. Ablation of sensory neurons (by capsaicin) markedly decreased the PAR2-mediated airway constriction, and virtually abolished PAR2-mediated pulmonary inflammation and edema, as did blockade of NK1 or NK2 receptors. Thus, PAR2 activation in the lung induces airway constriction, lung inflammation, and protein-rich pulmonary edema. These effects were either partly or completely neuropeptide dependent, suggesting that PAR2 can cause lung inflammation by a neurogenic mechanism.

Thrombin Is a Potent Inducer of Connective Tissue Growth Factor Production via Proteolytic Activation of Protease-activated Receptor-1

The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and pro-fibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor beta (TGF-beta) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-beta-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-beta(1). Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression.