Neutrophils and the kallikrein-kinin system in proteinase-activated receptor 4-mediated inflammation in rodents

Abstract

1 We evaluated a potential role for proteinase-activated receptor 4 (PAR(4)) in a rodent paw inflammation model, with a focus on two main features of inflammation: (1) oedema and (2) granulocyte recruitment. 2 A PAR(4) antagonist (Pepducin P4pal-10; palmitoyl-SGRRYGHALR-NH(2)) reduced both the oedema and granulocyte recruitment induced by a localized administration of carrageenan in the rat hind paw, pointing to a key role for PAR(4) in this inflammation model. 3 Further, intraplantar injection in the mouse hind paw of a PAR(4) agonist (AYPGKF-NH(2)), but not its standard PAR(4)-inactive peptide control (YAPGKF-NH(2)), caused an inflammatory reaction characterized by oedema (increased paw thickness) and granulocyte recruitment (increased paw myeloperoxidase activity). The PAR(4) agonist-induced effects were inhibited in mice pretreated with pepducin P4pal10. 4 These PAR(4) agonist-mediated effects were not affected by pretreatment with inhibitors of either NO production or prostaglandin release (L-NAME and indomethacin, respectively). 5 However, selective immuno-depletion of neutrophils significantly reduced PAR(4) agonist-induced oedema formation. 6 Moreover, AYPGKF-NH(2)-induced oedema was also reduced by pretreatment with either a kinin B(2) receptor antagonist (icatibant) or a tissue or plasma kallikrein inhibitor (FE999024 and FE999026, respectively), but not with a kinin B(1) receptor antagonist (SSR240612). 7 We conclude: (1) that PAR(4) plays an important role in the inflammatory response as it mediates some of the hallmarks of inflammation and (2) that PAR(4)-mediated oedema is dependent on the recruitment of neutrophils and components of the kallikrein-kinin system.

Figure 1

Effect of a PAR₄ antagonist on a general inflammatory reaction induced by carrageenan in the rat paw. Pepducin P4pal10 (0.5 mg kg⁻¹)-dependent reduction of the carrageenan (200 μg)-mediated oedema formation (a) and granulocyte recruitment (b). Saline was used as a control for both the pepducin P4pal10 and carrageenan injections. Values are mean±s.e.m. of n=8 per group. ^*Significantly different from the vehicle+carrageenan group, P<0.05.

Figure 2

Direct inflammatory effects of PAR₄ activation in the mouse paw. The oedema formation (a) and granulocyte recruitment (b) were evaluated following an intraplantar injection of the PAR₄-AP, AYPGKF-NH₂ (50 μg), the control inactive peptide, YAPGKF-NH₂ (50 μg), or the saline vehicle. The effect of a pepducin P4pal10 (0.25 mg kg⁻¹) pretreatment was also evaluated against the oedema formation (c) and granulocyte recruitment (d) induced by AYPGKF-NH₂ (50 μg) and YAPGKF-NH₂ (50 μg). Values are mean±s.e.m. of n=5–8 per group. ^*Significantly different from the saline vehicle or the control peptide group (a and b), P<0.05; ^#significantly different from the vehicle+AYPGKF-NH₂ group (c and d), P<0.05.

Figure 3

Noninvolvement of NO and prostanoids in PAR₄-mediated oedema formation and granulocyte recruitment in the mouse paw. Effect of indomethacin (cyclooxygenases inhibitor, 5 mg kg⁻¹) or L-NAME (nitric oxide synthases inhibitor, 25 mg kg⁻¹) on oedema formation (a) and on granulocyte recruitment (b) observed following PAR₄ activation with AYPGKF-NH₂ (50 μg). Values are mean±s.e.m. of n=8–9 per group. No statistically significant difference was observed between groups.

Figure 4

Role for neutrophils in the PAR₄-mediated oedema formation in the mouse paw. A granulocyte-depleting antibody (anti-mouse Ly-6G antibody clone RB6-8C5, 125 μg) causes a marked reduction of myeloperoxidase, indicative of neutropenia (b) as well as reducing the oedema (a) mediated by AYPGKF-NH₂ (50 μg) whereas a control antibody (rat IgG2bκ antibody, 125 μg) does not. Values are mean±s.e.m. of n=8 per group. ^*Significantly different from the vehicle+AYPGKF-NH₂ group, P<0.05.

Figure 5

Role for the tissue and plasma kallikreins in PAR₄-mediated oedema formation in the mouse paw. Effect of FE999024 (tissue kallikrein inhibitor, 60 μmol kg⁻¹) or FE999026 (plasma kallikrein inhibitor, 60 μmol kg⁻¹) on the oedema formation (a) and granulocyte recruitment (b) observed following PAR₄ activation with AYPGKF-NH₂ (50 μg). Values are mean±s.e.m. of n=8 per group. ^*Significantly different from the vehicle+AYPGKF-NH₂ group, P<0.05.

Figure 6

Role for the kinin B₂ receptor, but not the B₁, in PAR₄-mediated oedema formation in the mouse paw. Effect of SSR240612 (kinin B₁ receptor antagonist, 10 mg kg⁻¹) or of icatibant (kinin B₂ receptor antagonist, 50 μg kg⁻¹) on the oedema formation (a and b, respectively) and granulocyte recruitment (c) observed following PAR₄ activation with AYPGKF-NH₂ (50 μg). Values are mean±s.e.m. of n=8 per group. ^*Significantly different from the vehicle+AYPGKF-NH₂ group, P<0.05; ^#significantly different from either the vehicle+saline or the icatibant+saline groups, P<0.05.

Figure 7

The PAR₄-AP, AYPGKF-NH₂, does not activate directly the kinin B₂ receptor. Calcium signal emission was recorded at 530 nm in KNRK cells following stimulation with bradykinin (10 nM) or AYPGKF-NH₂ (200 μM). Some cells were pretreated with icatibant (kinin B₂ receptor antagonist; 30 nM). Values are mean±s.e.m. of n=3 experiments and are expressed as the percentage of the fluorescence peak height yielded by the addition of 2 μM calcium ionophore (A23187). ^*Significantly different from the bradykinin group, P<0.05.

Figure 8

Proposed model for the mechanisms of action of PAR₄-mediated oedema formation. In the blood vessel depicted schematically, we propose that PAR₄ activation could occur both on endothelial cells and neutrophils to affect vascular permeability (1). Activated neutrophils would release tissue kallikrein activity (2). Tissue kallikrein would then cleave kininogens found at the surface of neutrophils and endothelial cells to produce active kinins (3). These kinins would in turn bind to and activate endothelial B₂ receptors to increase vascular permeability, resulting in oedema and the migration of neutrophils into the tissue (4). With the breach of endothelial integrity, plasma kallikrein could then add to the response following activation of the contact system (5). TK, tissue kallikrein; PK, plasma kallikrein; BK, bradykinin.