Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.

Mast cell tryptase controls paracellular permeability of the intestine: role of protease-activated receptor 2 and beta-arrestins

Tight junctions between intestinal epithelial cells prevent ingress of luminal macromolecules and bacteria and protect against inflammation and infection. During stress and inflammation, mast cells mediate increased mucosal permeability by unknown mechanisms. We hypothesized that mast cell tryptase cleaves protease-activated receptor 2 (PAR2) on colonocytes to increase paracellular permeability. Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i. Supernatant from degranulated mast cells increased [Ca2+]i in colonocytes, which was prevented by a tryptase inhibitor, and desensitized responses to PAR2 agonist, suggesting PAR2 cleavage. When applied to the basolateral surface of colonocytes, PAR2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of tight junction ZO-1 and occludin and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increased paracellular permeability of colonocytes. This was prevented by a tryptase inhibitor. We determined the role of ERK1/2 and of beta-arrestins, which recruit ERK1/2 to PAR2 in endosomes and retain ERK1/2 in the cytosol, on PAR2-mediated alterations in permeability. An ERK1/2 inhibitor abolished the effects of PAR2 agonist on permeability and redistribution of F-actin. Down-regulation of beta-arrestins with small interfering RNA inhibited PAR2-induced activation of ERK1/2 and suppressed PAR2-induced changes in permeability. Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. PAR2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation.

A defined intestinal colonization microbiota for gnotobiotic pigs

Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.

Dendritic cells interact with CD4 T cells in intestinal mucosa

Absence of lymph nodes in nonmammalian species, expression of MHCII by APCs in the periphery, and the recent findings that T cells can change their polarization status after presentation in the lymph nodes imply a role for MHCII-mediated presentation outside the organized lymphoid tissue. This study shows that MHCII+ ECs and DCs from the intestinal mucosa of the pig can present antigen to T cells in vitro. In vivo, APCs colocalize with T cells in pig and mouse intestinal mucosa. In the pig, endothelium is involved in these interactions in neonates but not in adults, indicating different roles for stromal and professional APCs in the neonate compared with the adult. The ratio of expression of DQ and DR MHCII locus products was lower on ECs than on other mucosal APCs, indicating that the two types of cells present different peptide sets. Adult nonendothelial APCs expressed a higher ratio of DQ/DR than in neonates. These results suggest that mucosal DCs can present antigen locally to primed T cells and that stromal APCs are recruited to these interactions in some cases. This raises the possibility that local presentation may influence T cell responses at the effector stage after initial presentation in the lymph node.

Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Cigarette smoke-induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.