Sphingosine 1-phosphate mediates hyperalgesia via a neutrophil-dependent mechanism

Abstract

Novel classes of pain-relieving molecules are needed to fill the void between non-steroidal anti-inflammatory agents and narcotics. We have recently shown that intraplantar administration of sphingosine 1-phosphate (S1P) in rats causes peripheral sensitization and hyperalgesia through the S1P(1) receptor subtype (S1PR(1)): the mechanism(s) involved are largely unknown and were thus explored in the present study. Intraplantar injection of carrageenan in rats led to a time-dependent development of thermal hyperalgesia that was associated with pronounced edema and infiltration of neutrophils in paw tissues. Inhibition of 1) S1P formation with SK-I, a sphingosine kinase inhibitor, 2) S1P bioavailability with the S1P blocking antibody Sphingomab, LT1002 (but not its negative control, LT1017) or 3) S1P actions through S1PR(1) with the selective S1PR(1) antagonist, W146 (but not its inactive enantiomer, W140) blocked thermal hyperalgesia and infiltration of neutrophils. Taken together, these findings identify S1P as an important contributor to inflammatory pain acting through S1PR(1) to elicit hyperalgesia in a neutrophil-dependant manner. In addition and in further support, we demonstrate that the development of thermal hyperalgesia following intraplantar injection of S1P or SEW2871 (an S1PR(1) agonist) was also associated with neutrophilic infiltration in paw tissues as these events were attenuated by fucoidan, an inhibitor of neutrophilic infiltration. Importantly, FTY720, an FDA-approved S1P receptor modulator known to block S1P-S1PR(1) signaling, attenuated carrageenan-induced thermal hyperalgesia and associated neutrophil infiltration. Targeting the S1P/S1PR(1) axis opens a therapeutic strategy for the development of novel non-narcotic anti-hyperalgesic agents.

Figure 1. Carrageenan injection leads to an increase in neutrophil infiltration that is attenuated by fucoidan.

A–C) Intraplantar injection of carrageenan (1%) led to a time-dependent development of thermal hyperalgesia that was accompanied by an increase in paw volume and an increase in myeloperoxidase activity. All were blocked by fucoidan (40 mg/kg). D) The increased myeloperoxidase activity in response to carrageenan injection correlated with pathological changes as well as a marked increase in neutrophilic infiltration as indicated by H&E staining. Fucoidan (40 mg/kg) attenuated this response. Results are expressed as mean ± SEM for 6 rats and analyzed by student's unpaired t test for paw volume, two-way repeated measures ANOVA with Bonferroni post hoc test for behavior and one-way ANOVA with Dunnett's post hoc test for MPO, where *P<0.01, **P<0.001 vs. carrageenan; † P<0.01, †† P<0.001 vs. vehicle.

Figure 2. Carrageenan-induced thermal hyperalgesia is blocked by SK-I.

Intraplantar injection of carrageenan (1%) led to a time-dependent development of thermal hyperalgesia that was attenuated in a dose-dependent manner by intraplantar injection of SK-I (250 ng, 500 ng, or 1000 ng; n = 6). Results are expressed as mean ± SEM and analyzed by two-way repeated measures ANOVA with Bonferroni post hoc test where *P<0.01, **P<0.001 vs. carrageenan.

Figure 3. Inhibition of S1P attenuates carrageenan-induced thermal hyperalgesia and the recruitment of neutrophils.

A) Intraplantar injection of LT1002 (484 μg, n = 6) but not of LT1017 (572 μg; isotype control, n = 6) attenuated carrageenan-induced thermal hyperalgesia. B) Intraplantar injection of carrageenan led to an increase in neutrophilic recruitment as evidenced by increased levels of MPO activity in paw tissues and this was blocked by LT1002 but not LT1017. Results are expressed as mean ± SEM and analyzed by two-way repeated measures ANOVA with Bonferroni post hoc test for behavior and one-way ANOVA with Dunnett's post hoc test for MPO, where *P<0.01, **P<0.001 vs. carrageenan; † P<0.05, †† P<0.001 vs. vehicle.

Figure 4. Blockade of S1PR₁ inhibits carrageenan-induced thermal hyperalgesia and the recruitment of neutrophils.

A) Intraplantar injection of carrageenan led to a time-dependent development of thermal hyperalgesia that was blocked in a dose-dependent manner by the selective S1PR₁ antagonist, W146 (0.3–1.2 μg, n = 6) but not by its inactive S-enantiomer, W140 (1.2 µg, n = 6). B) W146 (1.2 µg, n = 6) but not W140 (1.2 µg, n = 6) attenuated neutrophilic recruitment. Results are expressed as mean ± SEM and analyzed by two-way repeated measures ANOVA with Bonferroni post hoc test for behavior and one-way ANOVA with Dunnett's post hoc test for MPO, where *P<0.05, **P<0.001 vs. carrageenan; † P<0.01, †† P<0.001 vs. vehicle.

Figure 5. Fucoidan blocks S1P-induced thermal hyperalgesia.

Intraplantar injection of A) S1P (0.3 μg) or B) the S1PR₁ agonist, SEW2871 (0.3 μg), led to a time-dependent development of thermal hyperalgesia that was attenuated by pretreatment with fucoidan (40 mg/kg, i.p.). Results are expressed as mean ± SEM for 6 rats and analyzed by two-way repeated measures ANOVA with Bonferroni post hoc test where *P<0.05, **P<0.001 vs. carrageenan; † P<0.01, †† P<0.001 vs. vehicle.

Figure 6. FTY720 attenuates carrageenan-induced thermal hyperalgesia and neutrophilic recruitment.

Intraplantar injection of carrageenan (1%) led to a time- dependent development of thermal hyperalgesia. A) Pretreatment with the non-selective functional antagonist, FTY720 (0.1 mg/kg – 1.0 mg/kg, n = 7) attenuated the development of thermal hyperalgesia in response to carrageenan in a dose-dependent manner. B) FTY720 (1 mg/kg, n = 7) attenuated carrageenan-induced increases in neutrophilic recruitment. Results are expressed as mean ± SEM and analyzed by two-way repeated measures ANOVA with Bonferroni post hoc test for behavior and one-way ANOVA with Dunnetts post hoc test for MPO, where *P<0.05, **P<0.001 vs. carrageenan; † P<0.01 †† P<0.001 vs. vehicle.

Figure 7. Schematic of proposed mechanisms behind S1P-mediated hyperalgesia.

Carrageenan injection leads to the activation of sphingosine kinase enzymes favoring the conversion of sphingosine to bioactive S1P. S1P then goes on to activate S1PR₁, initiating neutrophilic recruitment to the site of injury. Once there, neutrophils release several mediators known to sensitize nociceptors which induce peripheral sensitization and hyperalgesia.