The C-terminus of murine S100A9 protein inhibits hyperalgesia induced by the agonist peptide of protease-activated receptor 2 (PAR2)

Abstract

Background and purpose: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2).

Experimental approach: Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods.

Key results: mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed.

Conclusions and implications: These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.

Figure 1

Effects of mS100A9p on mechanical hyperalgesia induced by trypsin in rats. Trypsin (50 μg) was incubated for 30 min at 37°C with 1 or 4 μg of mS100A9p and the combination was then injected into the rat paw. Pain threshold in response to mechanical stimulation was measured in rats before (basal measurement) and 1 h after treatment using the paw pressure test. Rats injected with saline or trypsin alone were submitted to the same protocol. Data represent mean±s.e.m. of 6–8 animals per group. (^*) Significantly different from basal measurements (P<0.05, one-way ANOVA followed by Tukey's multiple comparison post-test).

Figure 2

Effects of time of treatment with mS100A9p on mechanical nociception in rats induced by SLIGRL-NH₂. The mS100A9p was given in doses of 1, 4 or 8 μg concomitantly (a), 30 min before (b) or 1 h after (c) i.pl. injection of 10 μg of SLIGRL-NH₂. Animals were evaluated before (time 0), 1, 2, 3 and 4 h after treatment. Rats injected only with SLIGRL-NH₂ received the same total volume as the other groups. Data represent mean±s.e.m. of 6–8 animals per group. (^*) Significantly different from SLIGRL-NH₂ alone at the same time-point (P<0.05, one-way ANOVA followed by Tukey's multiple comparison post-test).

Figure 3

Effects of time of treatment with mS100A9p on thermal nociception in rats, induced by SLIGRL-NH₂. The mS100A9p was given in doses of 0.1, 1 or 20 μg concomitantly (a), 30-min before (b) or 1-h after (c) i.pl. injection of 10 μg of SLIGRL-NH₂. Animals were evaluated before (time 0), 1, 2 and 3 h after SLIGRL-NH₂ administration. Rats injected only with SLIGRL-NH₂ received the same total volume as the other groups. Data represent mean±s.e.m. of eight animals per group. (^*) Significantly different from SLIGRL-NH₂ alone at the same time-point (P<0.05), (^**) significantly different from SLIGRL-NH₂ alone at the same time-point (P<0.001), one-way ANOVA followed by Tukey's multiple comparison post-test).

Figure 4

Effects of saline, mS100A9p, SLIGRL-NH₂ or SLIGRL-NH₂ plus mS100A9p on dorsal horn expression of Egr-1. (a–e) Photomicrographs of immunostained sections of spinal cord dorsal horn from (a) naïve (control) animals, or from rats after i.pl. injection of: (b) saline; (c) 4 μg mS100A9p; (d) 10 μg SLIGRL-NH₂; (e) concomitant treatment with SLIGRL-NH₂ and mS100A9p. (f) quantitative changes in Egr-1 immunoreactivity in dorsal horn (L4 and L5) of rats. The spinal cords were collected 3 h after the treatments. Scale bar=50 μm. Data represent mean±s.e.m. of six animals per group. (^*) Significantly different from all other groups (P<0.05, one-way ANOVA followed by Tukey's multiple comparison post-test).

Figure 5

Effect of mS100A9p on calcium flux in DRG neurons. Neurons were exposed to SLIGRL (100 μM; (a)), trypsin (5 U ml⁻¹; (b)), capsaicin (100 nM; (c)) or bradykinin (10 nM; (d)) and concomitantly to mS100A9p (0.5 5, 50 or 100 μM). Neurons exposed only to SLIGRL, trypsin, capsaicin or bradykinin were considered as control groups. Calcium flux was measured by fluorescence (460–490 nm excitation and 515 nm emission) in individual cells using a Wide-Field Fluorescence Microscope. Kinetic studies of 30 pictures in 90 s were performed. Data represent mean±s.e.m. of 15–20 neurons per group. (^*) Significantly different from control groups (P<0.05), (^**) significantly different from control groups (P<0.001, one-way ANOVA followed by Tukey's multiple comparison post-test).

Figure 6

Effects of vehicle, LRGILS-NH₂, SLIGRL-NH₂, mS100A9p or SLIGRL-NH₂ plus mS100A9p on SP release by cultured DRG neurons. Data represent mean±s.e.m. of 15–20 neurons per group. (^**) Significantly different from vehicle pr LRGILS-NH₂ (P<0.001), (ψ) significantly different from SLIGRL-NH₂ alone (P<0.05, one-way ANOVA followed by Tukey's multiple comparison post-test).

Figure 7

Effect of mS100A9p on calcium signaling in HEK-293 cells. (a) Cells were activated by the exposure to 5 U of trypsin alone or by trypsin plus 50 μM of mS100A9p. Trypsin and mS100A9p were incubated either 10 min before cell exposure or at the same time (b). Cells were activated by the exposure to 50 μM of SLIGRL-NH₂ alone or by SLIGRL-NH₂ plus 50 μM of mS100A9p. SLIGRL-NH₂ and mS100A9p were incubated either 10 min before cell exposure or at the same time. Cells were monitored for fluorescence and the response to mS100A9p treatment was compared to the response of cells exposed only to the agonists (trypsin or SLIGRL-NH₂). The data are illustrative of four or more independently conducted experiments.

Figure 8

Effect of mS100A9p on calcium signaling in KNRK-PAR₂ cells. (a) Cells were activated by the exposure to 1 U of trypsin alone or by trypsin plus 50 μM of mS100A9p. Trypsin and mS100A9p were incubated either 10 min before cell exposure or at the same time (b). Cells were activated by the exposure to 50 μM of SLIGRL-NH₂ alone or by SLIGRL-NH₂ plus 50 μM of mS100A9p. SLIGRL-NH₂ and mS100A9p were incubated either 10 min before cell exposure or at the same time. Cells were monitored for fluorescence and the response to mS100A9p treatment was compared to the response of cells exposed only to the agonists (trypsin or SLIGRL-NH₂). The data are illustrative of four or more independently conducted experiments.