Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2)

Abstract

Background: Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2.

Objectives: To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model.

Methods: We have injected recombinant human βII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated.

Results: Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH₂ ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice.

Conclusions: Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.

Keywords: allergy; inflammation; mast cells/basophils; neutrophils; transgenic/knockout mice.

Figure 1

Eosin/methylene blue‐stained cytocentrifuge preparations of cells recovered by peritoneal lavage from C57BL/6 mice 24 hours following injection with (A) tryptase or (B) saline. Examples of neutrophils (N), eosinophils (E), macrophages (M), lymphocytes (L) and mast cells (MC) are indicated. Numbers are indicated of (C) nucleated cells, (D) neutrophils, (E) eosinophils, (F) macrophages, (G) lymphocytes and (H) mast cells recovered from the peritoneum of BALB/c, C57BL/6 PAR2^+/+ and C57BL/6 PAR2^−/− mice 24 hours following injection of tryptase (Tryp; 0.5 μg/mouse). Saline‐injected mice (Sal). Also shown are (I) neutrophil numbers in peritoneal lavage fluid from PAR2^−/− and PAR2^+/+ mice 24 hours following injection of tryptase, heated tryptase, tryptase pre‐incubated with the selective inhibitor, the inhibitor alone and the saline vehicle. N = 5‐13 mice per group. Median values and interquartile range are shown.*P < .05, **P < .005, (Mann‐Whitney U test). n.s., not significant

Figure 2

A, Gelatin zymography showing the presence of MMP2 and MMP9 in a standard (Std) supernatant from the HT1080 fibrosarcoma cell line, and supernatants from peritoneal lavage fluid from mice 24 hours after injection of saline alone, or tryptase (mice 1 to 3). B, MMP2 and (C) MMP9 activity in peritoneal lavage fluid from PAR2^−/− and PAR2^+/+ mice 6, 12 and 24 hours following intraperitoneal injection of tryptase (Tryp; 0.5 μg/mouse). Median values and interquartile range are shown. *P < .05 **P < .005 ***P < .0001, (Mann‐Whitney U test). n.s., not significant

Figure 3

A, Gelatin zymography illustrated for neutrophil‐rich and neutrophil‐depleted peritoneal lavage cell populations isolated from 8 mice 24 hours following tryptase injection. Culture supernatants were incubated for 1 hours with tryptase at 13 μg mL⁻¹ (40 mU mL⁻¹), heat‐inactivated tryptase (of the same original concentration) or medium alone. Bands for molecular weight (MW) markers are also shown. B, Relative MMP9 activity in culture supernatants of neutrophil‐rich and neutrophil‐depleted cell populations maintained in culture for 1, 6 or 24 hours (n = 6 for each group). Median and interquartile range are indicated. *P < .05, **P < .005, (Mann‐Whitney U test) compared with activity in the neutrophil‐depleted cell population