Effect of tryptase inhibition on joint inflammation: a pharmacological and lentivirus-mediated gene transfer study.

BACKGROUND: Increasing evidences indicate that an unbalance between tryptases and their endogenous inhibitors, leading to an increased proteolytic activity, is implicated in the pathophysiology of rheumatoid arthritis. The aim of the present study was to evaluate the impact of tryptase inhibition on experimental arthritis. METHODS: Analysis of gene expression and regulation in the mouse knee joint was performed by RT-qPCR and in situ hybridization. Arthritis was induced in male C57BL/6 mice with mBSA/IL-1ß. Tryptase was inhibited by two approaches: a lentivirus-mediated heterologous expression of the human endogenous tryptase inhibitor, sperm-associated antigen 11B isoform C (hSPAG11B/C), or a chronic treatment with the synthetic tryptase inhibitor APC366. Several inflammatory parameters were evaluated, such as oedema formation, histopathology, production of IL-1ß, -6, -17A and CXCL1/KC, myeloperoxidase and tryptase-like activities. RESULTS: Spag11c was constitutively expressed in chondrocytes and cells from the synovial membrane in mice, but its expression did not change 7 days after the induction of arthritis, while tryptase expression and activity were upregulated. The intra-articular transduction of animals with the lentivirus phSPAG11B/C or the treatment with APC366 inhibited the increase of tryptase-like activity, the late phase of oedema formation, the production of IL-6 and CXCL1/KC. In contrast, neutrophil infiltration, degeneration of hyaline cartilage and erosion of subchondral bone were not affected. CONCLUSIONS: Tryptase inhibition was effective in inhibiting some inflammatory parameters associated to mBSA/IL-1ß-induced arthritis, notably late phase oedema formation and IL-6 production, but not neutrophil infiltration and joint degeneration. These results suggest that the therapeutic application of tryptase inhibitors to rheumatoid arthritis would be restrained to palliative care, but not as disease-modifying drugs. Finally, this study highlighted lentivirus-based gene delivery as an instrumental tool to study the relevance of target genes in synovial joint physiology and disease.

Proteinase-activated receptor 2 blockade impairs CCL11- or allergen-induced eosinophil recruitment in experimental pleurisy.

Although proteinase-activated receptor (PAR)-2 has been implicated in inflammatory diseases, its role in regulating eosinophil recruitment in response to chemoattractants remains unclear. Here, we investigated the role of PAR-2 and PAR-2-activating Mast Cell (MC) tryptase on chemokine C-C motif ligand (CCL)11- and antigen-induced eosinophil recruitment to the pleural cavity of BALB/c mice. The PAR-2-activating peptide H-Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL-NH2) induced eosinophil recruitment whereas PAR-2 blockade inhibited ovalbumin (OVA)- or CCL11-induced eosinophil recruitment. Moreover, OVA and CCL11 induced PAR-2 expression in pleural leukocytes, and the MC tryptase inhibitor APC 366 ([N-(1-hydroxy-2-napthoyl)-l-arginyl-l-prolinamide hydrochloride]) abolished CCL11-induced eosinophil recruitment. These results suggest a pro inflammatory effect of PAR-2 and support a role for MC tryptase mediating eosinophil migration via PAR-2 signaling. Taken together, our results suggest that PAR-2 activation through endogenous MC tryptase activity could be required, at least partially, to mediate CCL11-induced eosinophil migration.

Critical role of protease-activated receptor 2 activation by mast cell tryptase in the development of postoperative pain.

BACKGROUND: Studies have indicated that nearly half of all surgical patients still have inadequate pain relief. Thus, it is crucial to understand the mechanisms involved in postoperative pain in order to better treat it. Thus, the aim of this study was to investigate the involvement of mast cell degranulation, tryptase and its substrate, the protease-activated receptor 2, in a model of postoperative pain in mice. METHODS: We evaluated the effect of the compound 48/80 (to cause mast cell mediator depletion), cromoglycate or ketotifen (mast cell stabilizers), gabexate (tryptase inhibitor) or N3-methylbutyryl-N-6-aminohexanoyl-piperazine (protease-activated receptor 2 antagonist) in a postoperative pain model in mice (n = 5-10). Mast cell degranulation and tryptase activity were also evaluated in the operated tissue (n= 5-8). RESULTS: The pre-treatment with compound 48/80 or ketotifen was able to prevent nociception throughout the postoperative hyperalgesia course (until 5 days after surgery), whereas cromoglycate presented a shorter effect (until 1 day). Gabexate or N3-methylbutyryl-N-6-aminohexanoyl-piperazine also produced a short-lasting effect in preventing postoperative nociception. However, neither gabexate, N3-methylbutyryl-N-6-aminohexanoyl-piperazine nor cromoglycate was capable of reversing nociception when administered after incision. Surgery led to early mast cell degranulation on the incised tissue and increased tryptase activity in tissue perfusates. Cromoglycate fully prevented the tryptase release in the perfusate and the compound 48/80 substantially reduced tryptase activity in the incised tissue. CONCLUSION: Thus, the mast cell degranulation with the subsequent release of tryptase and protease-activated receptor 2 activation are potential targets for the development of novel therapies to prevent, but not reverse, postoperative pain.

Nafamostat mesilate, a potent tryptase inhibitor, modulates periodontitis in rats.

Previous reports have demonstrated increased tryptase-like proteolytic activity in the crevicular fluid of patients with periodontal disease. In the present study, we have investigated the effect of tryptase inhibition with nafamostat mesilate (NM, 6-amino-2-naphtlyl p-guanidinobenzoate dimethansulfonate) on the development of experimental periodontitis in rats. Eighty (80) male Wistar rats were randomly separated into four groups: Control group, NM group (daily 0.1 mg/kg body weight of NM, i.p.), Ligature group (ligature placed at lower right first molars), and NM+Ligature group. The amount of alveolar bone loss (ABL) around the mesial root surface of the first mandibulary molar, as well as the myeloperoxidase (MPO) activity, and total proteolytic activity [N-benzoyl-L: -arginine-p-nitroanilide (BApNA) substrate] were determined at 7 and 14 days. NM led to significantly (p < 0.05) decreased ABL in animals subjected to ligature-induced periodontitis. Tryptase inhibition prevented the onset of significant ABL at 7 days of experiment (0.44 ± 0.16 and 0.60 ± 0.22, p > 0.05, NM+Ligature and Control, respectively) and significantly decreased the ABL at 14 days (0.97 ± 0.17 versus 1.82 ± 0.26, p < 0.001, NM+Ligature versus Ligature, respectively). In addition, NM significantly decreased MPO and total proteolytic activity at 14 days (p < 0.05). These data provided evidence that tryptase inhibition with NM attenuates gingival granulocyte infiltration and ABL in an experimental model of periodontitis in rats.