Faculty Bibliography

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH2) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

Proteinase-activated receptor (PAR)-2 is cleaved within its aminoterminal extracellular domain by serine proteinases such as trypsin, unmasking a new aminoterminus starting with the sequence SLIGKV, which binds intramolecularly and activates the receptor. PAR-2 has been reported to be involved in inflammation within the lungs. We show that PAR-2 is expressed not only by human alveolar (A549), but also by bronchial (16HBE) epithelial cell lines, using RT-PCR and flow cytometry with a PAR-2 antibody whose epitope maps over the trypsin cleavage site. PAR-2 activation by trypsin and by the activating peptide SLIGKV-NH(2) leads to intracellular calcium mobilization in both lung epithelial cells. During lung inflammation, airspaces are burdened by neutrophils that release elastase and cathepsin G, two serine proteinases. We demonstrate that these proteinases do not activate PAR-2, but rather disarm the receptor, preventing activation by trypsin but not by SLIGKV-NH(2). Preincubation of a PAR-2-transfected cell line, as well as 16HBE and A549 cells, with either proteinase led to the disappearance of the cleavage/activation epitope recognized by the PAR-2 antibody. We hypothesize that elastase and cathepsin G disarm PAR-2 by proteolysis of the extracellular domain downstream from the trypsin cleavage/activation site, while leaving unmodified the SLIGKV-NH(2)-binding site. These findings suggest that the neutrophil serine proteinases may play a role in PAR-2-mediated lung inflammation.

Protease-activated receptor 2 (PAR2) is the second member of a new subfamily of G-protein coupled receptors: the protease-activated receptors (PARs). At present, four different PARs have been cloned and all of them share the same basic mechanism of activation. A serine protease cleaves the extended, extracellular N-terminus of the receptor at a specific site within the protein chain to expose an N-terminal tethered ligand domain, which binds to and activates the cleaved receptor. In this manner, trypsin and mast cell beta-tryptase activate PAR2. PARs are single use receptors because proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes. Thus, PARs play important roles in emergency situations, such as trauma and inflammation. Emerging evidence indicates that PAR2 is involved in the cardiovascular, pulmonary and gastrointestinal systems, where it controls inflammation and nociception. Work with selective agonists and knockout animals suggests a contribution of PAR2 to certain inflammatory diseases. Therefore, selective antagonists or agonists of these receptors may be useful therapeutic agents for the treatment of human diseases.

PURPOSE: The objective of this study was to examine whether HCECs express functional proteinase-activated receptor (PAR)-1 and -2 and evaluate the effects of receptor activation on corneal epithelial cell proinflammatory cytokine production. METHODS: Expression of PAR-1 and -2 mRNAs was determined by RT-PCR in cultured primary human corneal epithelial cells (HCECs) and the human corneal epithelial cell line HCE-T. Localization of PAR-1 and -2 in whole normal human corneas was determined by immunofluorescence with PAR-1 and -2 antibodies. The functional competence of PAR-1 and -2 in corneal epithelial cells was assessed by measuring the rapid induction of intracellular [Ca(2+)] in response to thrombin, trypsin, and specific receptor-activating peptides derived from the tethered ligands of the PAR receptors. HCE-T expression of cytokines (IL-6, IL-8, and TNFalpha) in response to activation of PAR-1 and -2 was measured by quantitative RT-PCR and ELISA. RESULTS: Functional PAR-1 and -2 were expressed in both HCECs and HCE-T cells. Immunoreactivity for PAR-1 and -2 was detected in the outer epithelial layer of the cornea in whole human corneal sections. Activation of PAR-1 and -2 led to upregulation in HCE-T cells of both expression of mRNA and secretion of the proinflammatory cytokines IL-6, IL-8, and TNFalpha. CONCLUSIONS: The results show for the first time that functional PAR-1 and -2 are present in human cornea. Activation of these receptors results in the production of various corneal epithelial cell proinflammatory cytokines. These observations indicate that PAR-1 and -2 may play an important role in modulating corneal inflammatory and wound-healing responses. These receptors may be useful therapeutic targets in several corneal disease processes

Cutaneous sensory nerves mediate inflammation and wound healing by releasing neuropeptides, such as substance P, which stimulates pro-inflammatory responses by keratinocytes, fibroblasts, and endothelial cells. The cell surface enzyme, neutral endopeptidase, degrades substance P, thereby regulating its biologic actions. We hypothesized that neutral endopeptidase enzymatic activity is increased in chronic wounds and skin from subjects with diabetes. We compared cutaneous neutral endopeptidase expression and enzymatic activity between normal controls and diabetic subjects with neuropathy and chronic wounds. Skin samples from subjects with diabetes were taken at the time of amputation for nonhealing ulcers. Skin taken from the ulcer margin, 1 cm from the ulcer (adjacent), and from the most proximal region of the amputated leg were studied. Skin biopsies from the leg of healthy control subjects were also studied. Neutral endopeptidase was localized by immunohistochemistry in all tissue sections. Neutral endopeptidase activity was measured using a fluorimetric assay. The median neutral endopeptidase activity of the ulcer margin was 1.21 x higher (p>0.2) than adjacent skin, 5.26 (p<0.001) than proximal skin, and 15.22 x higher (p<0.001) than control skin. Adjacent skin had a median neutral endopeptidase activity 4.34 x higher (p<0.001) than proximal skin and 12.58 x higher (p<0.001) than control skin. The median neutral endopeptidase activity of proximal skin was 2.90 x higher (p<0.001) than control skin. This elevated neutral endopeptidase activity in the skin and chronic ulcers of subjects with diabetes combined with peripheral neuropathy may contribute to deficient neuroinflammatory signaling and may impair wound healing in subjects with diabetes.

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.

Proteinase-activated receptor (PAR)-2, a G-protein-coupled receptor for trypsin and mast cell tryptase, is highly expressed in the intestine. Luminal trypsin and tryptase are elevated in the colon of inflammatory bowel disease patients. We hypothesized that luminal proteinases activate PAR-2 and induce colonic inflammation. Mice received intracolonically PAR-2 agonists (trypsin, tryptase, and a selective PAR-2-activating peptide) or control drugs (boiled enzymes, inactive peptide) and inflammatory parameters were followed at various times after this treatment. Colonic administration of PAR-2 agonists up-regulated PAR-2 expression and induced an inflammatory reaction characterized by granulocyte infiltration, increased wall thickness, tissue damage, and elevated T-helper cell type 1 cytokine. The inflammation was maximal between 4 and 6 hours and was resolved 48 hours after the intracolonic administration. PAR-2 activation also increased paracellular permeability of the colon and induced bacterial trans-location into peritoneal organs. These proinflammatory and pathophysiological changes observed in wild-type mice were not detected in PAR-2-deficient mice. Luminal proteinases activate PAR-2 in the mouse colon to induce inflammation and disrupt the integrity of the intestinal barrier. Because trypsin and tryptase are found at high levels in the colon lumen of patients with Crohn's disease or ulcerative colitis, our data may bear directly on the pathophysiology of human inflammatory bowel diseases.

Background. Patients with diabetic sensory neuropathy have significant risk of chronic ulcers. Insufficient nerve-derived mediators such as substance P (SP) may contribute to the impaired response to injury. Mutant diabetic mice (db/db), which develop neuropathy and have delayed healing, may provide a model to study the role of nerves in cutaneous injury.Methods. Skin from human chronic nonhealing ulcers and age-matched control skin was immunohistochemically evaluated for nerves. Nerve counts were also compared in murine diabetic (C57BL/KsJ-m+/+ Lepr(db); db/db) and nondiabetic (db/-) skin. Excisional wounds on the backs of db/db and db/- mice were grouped as: (a) untreated db/- mice; (b) untreated db/db mice; (c) db/db mice with polyethylene glycol (PEG); (d) db/db mice with PEG and SP 10(-9) M; or (e) db/db mice with PEG and SP 10(-6) M.Results. We demonstrated fewer nerves in the epidermis and papillary dermis of skin from human subjects with diabetes. Likewise, db/db murine skin had significantly fewer epidermal nerves than nondiabetic littermates. We confirmed increased healing times in db/db mice (51.7 days) compared to db/- littermates (19.8 days; P </= 0.001). SP 10(-6) M (44 days; P = 0.02) and SP 10(-9) M (45 days; P = 0.03) shortened time to closure compared to PEG treatment alone (68 days). Since there was no difference in the percentage contraction in these treatment groups, SP may favorably promote wound epithelization.Conclusions. Our data support the use of db/db murine excisional wounds to evaluate the role of nerves in healing. We have demonstrated that exogenous SP improves wound healing kinetics in an animal model.

INTRODUCTION AND AIMS/OBJECTIVE:It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus. METHODOLOGY/METHODS:Canine CCK-58 was purified from intestinal extracts and CCK-8 was purchased. Their amounts were determined by amino acid analysis. The effect of tertiary structure on receptor binding at CCK-A receptors and CCK-B receptors was evaluated using membrane preparations from mouse pancreas and brain. The influence of C-terminal tertiary structure on stability to enzymatic digestion was evaluated by reacting CCK-8 and CCK-58 with endopeptidase 24:11. RESULTS:CCK-58 was three times more potent than CCK-8 for binding mouse pancreatic membrane CCK-A receptors and equipotent to CCK-8 for binding mouse brain CCK-B receptors. CCK-8 was readily digested by endopeptidase 24:11, whereas CCK-58 was not. CONCLUSIONS:The results strongly support the hypothesis that differences in tertiary structure of the carboxyl terminus of CCK-8 and CCK-58 influence receptor binding and stability to enzymatic digestion.

BACKGROUND:Genetic deletion of neutral endopeptidase (NEP), a cell-surface metalloprotease that degrades proinflammatory peptides, exacerbates lung injury induced by pancreatic elastase in a model of pancreatitis-associated lung injury. We tested 3 hypotheses: (1) genetic deletion of NEP prolongs lung recovery after elastase injections; (2) elastase-mediated lung injury is associated with down-regulation of NEP; and (3) pretreatment of NEP (-/-) and (+/+) animals with recombinant human NEP (rhNEP) reduces pulmonary damage in this model. METHODS:NEP (+/+) or (-/-) mice were injected with pancreatic elastase (0.085 U/g/dose intraperitoneally) or saline carrier at t = 0 hours and t = 1 hour. Some mice were pretreated with rhNEP (3 mg/kg intraperitoneally). Serum elastase, lung histologic score, myeloperoxidase, and NEP activities were measured at 4, 8, or 12 hours. RESULTS:NEP (-/-) mice had worse pulmonary inflammation at 4 and 8 hours versus (+/+) mice. Lung NEP activity was similar in elastase-treated and control (+/+) animals. Pretreatment with rhNEP reduced myeloperoxidase and improved histology at 4 hours in NEP (-/-) and (+/+) mice. CONCLUSIONS:Pancreatic elastase induces lung injury that is worse and prolonged in NEP (-/-) mice. Pretreatment with rhNEP ameliorates this injury. Thus, upregulation of NEP is a potential therapeutic approach for pancreatitis-associated lung injury.