Tryptase as a PAR-2 activator in joint inflammation

Protease-activated receptor-2 (PAR-2) is one of a family of G-protein coupled transmembrane receptors activated by proteolytic release of a 'tethered' ligand. We previously reported this receptor has a pivotal role in chronic joint inflammation using a PAR-2 'knockout' mouse [1], but the serine protease responsible for its activation remains uncertain.

We investigated whether β-tryptase has proinflammatory actions in the mouse knee and specifically whether a tryptase inhibitor can modulate experimentally induced chronic joint inflammation.

Five micrograms of human β-tryptase was injected into the knee joint cavity of two groups of anaesthetized (Halothane/O₂/N₂O) mice (30 g): wild-type (PAR-2^+/+) C57BL/6J mice and PAR-2 gene disrupted mice (PAR-2^-/-). Joint swelling was assessed by comparing caliper measurements of knee joint diameter pre-injection and post-injection. Chronic monoarthritis was induced using the same anaesthetic regime in separate wild-type mice by intra-articular and peri-articular injection of Freunds complete adjuvant (FCA) (in 5% methycellulose). In a parallel group of mice, 50 μg of the tryptase inhibitor 4-amidino phenyl pyruvic acid (APPA) was co-administered with the FCA/methycellulose emulsion. Joint diameter was measured over 10 days.

Intra-articular injection of β-tryptase resulted in rapid joint swelling in wild-type mice that was completely abrogated in PAR-2^-/- mice (Fig. 1a), suggesting that tryptase-mediated inflammatory actions require functional PAR-2. Tryptase plays an important role in mediating chronic inflammation as APPA co-administration substantially inhibited FCA-induced joint swelling (Fig. 1b). The present study extends our previous finding [1] that PAR-2 plays a key role in mediating chronic joint inflammation, by demonstrating that tryptase may be a crucial activating protease required for such PAR-2-mediated actions.

(a) Six hours after intra-articular injection of β-tryptase, knee joint swelling is evident in wild-type (PAR-2^+/+) mice but is virtually absent in PAR-2 gene disrupted (PAR-2^-/-) mice (P < 0.001, n = 4). (b) Knee joint swelling 10 days after Freunds complete adjuvant (FCA) treatment is significantly inhibited by co-administration of 4-amidino phenyl pyruvic acid (APPA) with FCA (P = 0.014, n = 4).

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Affiliations

1. Centre for Rheumatic Diseases, University of Glasgow, UK
   • EB Kelso
   •  & WR Ferrell
2. Biological Sciences, University of Paisley, UK
   • L Dunning
   •  & JC Lockhart
3. Department of Physiology & Pharmacology, University of Strathclyde, Glasgow, UK
   • R Plevin
4. Department Clinical Biochemistry, University of Munich, Germany
   • CP Sommerhoff

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