Antinociception by the anti-oxidized phospholipid antibody E06

Abstract

Background and Purpose: ROS and their downstream molecules such as oxidized phospholipids (OxPL) and 4‐hydroxynonenal activate TRPA1 and TRPV1 (vanilloid 1) cation channels in vivo and in vitro shaping thermal and mechanical hypersensitivity in inflammatory pain. E06/T15 is a monoclonal autoantibody against oxidized phosphatidylcholine (OxPC) used in diagnostics in arteriosclerosis. Recently, we provided evidence that E06 also ameliorates inflammatory pain. Here, we studied E06 for local treatment against hypersensitivity evoked by endogenous and exogenous agonists of TRPA1 or TRPV1 channels.

Experimental Approach: We utilized a combination of reflexive and complex behavioural pain measurements, live‐cell calcium imaging and OxPC‐binding assays. The lipid peroxidation metabolite 4‐hydroxynonenal, hydrogen peroxide as a source of ROS, allyl isothiocyanate and capsaicin were used to activate their respective receptors.

Key Results: All irritants induced thermal and mechanical hypersensitivity, spontaneous nocifensive and affective‐motivational behaviour, as well as calcium influx in HEK_TRPA1‐ or HEK_TRPV1‐cells and dorsal root ganglion neurons. E06 prevented prolonged mechanical hypersensitivity induced by all irritants except for H₂O₂. E06 did not alter immediate irritant‐induced nocifensive or affective motivational behaviour. In vitro, E06 blocked only 4‐hydroxynonenal‐induced calcium influx although this compound did not bind to E06. After 1–3 h, all tested irritants elicited formation of OxPC in paw tissue.

Conclusions and Implications: E06 ameliorates not only inflammatory pain but also prolonged hypersensitivity due to formation of OxPC. This supports the view that neutralizing certain OxPL as endogenous activators of TRPA1 or TRPV1 channels may be valuable for pain therapy.

Figure 1

Sensitivity of 4‐HNE, AITC or capsaicin‐induced mechanical hypersensitivity to block by E06. Male Wistar rats were intraplantarly injected with either 6 μmol 4‐HNE (A), 19.2 μmol H₂O₂ (B), 10 μmol AITC (C) or 245 nmol capsaicin (D) together with E06 (2 μg), IgM isotype control (2 μg) or saline. Mechanical hypersensitivity (paw pressure thresholds) was measured before injection and 1, 3 and 6 h after injection. Data are shown as mean ± SEM. 4‐HNE (N = 6), H₂O₂ (N = 7), AITC (N = 6), capsaicin (N = 6), *P < 0.05, significantly different from IgM isotype or saline; two‐way RM ANOVA, post hoc Holm–Sidak test.

Figure 2

Thermal hypersensitivity induced by 4‐HNE, H₂O₂, AITC or capsaicin‐is unchnged after E06 treatment. Thermal hypersensitivity (paw withdrawal threshold) was measured in male Wistar rats after intraplantar co‐application of either 2 μg E06, 2 μg IgM isotype or saline with 6 μmol 4‐HNE (A), 19.2 μmol H₂O₂ (B), 10 μmol AITC (C) or 245 nmol capsaicin (D) and simultaneously, 2 μg E06, 2 μg IgM isotype or saline in a separate injection. Paw withdrawal thresholds were recorded before injection (baseline) and 1 h, 3 h and 6 h after injections. Data are shown as mean paw withdrawal threshold for each treatment group ± SEM. 4‐HNE (N = 6), H₂O₂ (N = 6), AITC + E06 (N = 6), AITC + IgM (N = 5), AITC + saline (N = 5), capsaicin (N = 6). No significant effect of E06; two‐way RM ANOVA.

Figure 3

Resistance of pungent‐induced spontaneous nocifensive behaviour to E06 blockade. Duration of spontaneous nocifensive behaviour in male Wistar rats were recorded after intraplantar administration of 6 μmol 4‐HNE (A), 19.2 μmol H₂O₂ (B), 10 μmol AITC (C) or 245 nmol capsaicin (D) pre‐incubated with either saline, 2 μg E06 mAb or 2 μg IgM. Data are shown as cumulative duration of spontaneous nocifensive behaviour within 10 min after injection for each animal with the mean (black line) and the median (grey line). 4‐HNE (N = 10), H₂O₂ (N = 7), AITC (N = 5), capsaicin (N = 5). No significant effect of E06; one‐way ANOVA with post hoc Bonferroni test.

Figure 4

E06 did not block the affective motivational behaviours due to intraplantar irritants. Affective motivational behaviour following intraplantar injection of 6 μmol 4‐HNE (A), 19.2 μmol H₂O₂ (B), 10 μmol AITC (C) or 245 nmol capsaicin (D) pre‐incubated with either saline, 2 μg IgM isotype or 2 μg E06 mAb into rat's hind paw was measured. Data are shown as cumulative duration of affective motivational behaviours within 10 min after injection for each animal with the mean (black line) and the median (grey line). 4‐HNE (N = 10), H₂O₂ (N = 7), AITC (N = 5), capsaicin (N = 5). No significant effect of E06; one‐way ANOVA with post hoc Bonferroni test.

Figure 5

E06‐blocked activation of TRPA1 channels by the oxidized lipid metabolite 4‐HNE but not that induced by the ROS source H₂O₂. Ratiometric calcium imaging (ratio F(340/380)) of Fura‐2/AM‐loaded HEK‐293_hTRPA1 cells upon stimulation with 4‐HNE (30 μM; A–E) or H₂O₂ (10 μM; F–J). Either E06 (3 ng·μL⁻¹; A, F) or isotype control antibody IgM (3 ng·μL⁻¹; B, G) were pre‐incubated before application and compared to controls (C, H). Grey lines depict 100 individual cells; the black line represents the mean; grey lines indicate the median. (D, E, I, J) Summary graphs of number of reacting cells (D, I) and AUC (mean; E, J). Number of experiments: 4‐HNE (N = 9), 4‐HNE + E06 (N = 7), 4‐HNE + IgM (N = 5), H₂O₂ (N = 7), H₂O₂ + E06 (N = 6), H₂O₂ + IgM (N = 5), *P < 0.05, significantly different as indicated; one‐way ANOVA post hoc Bonferroni test.

Figure 6

No effects of E06 on the responses of HEK‐293 cells to the exogenous TRPV1 channel agonist, capsaicin. Representative calcium imaging experiments on HEK‐293 cells stably transfected with TRPV1 channels (A, B). Stimulation of TRPV1 channels with capsaicin co‐incubated with E06 (A) or capsaicin alone (B). Grey lines: 100 single cells, black line: average. (C, D) Summary graphs comparing the calcium responses expressed as number of reacting cells (C) and AUC (D) are displayed. Number of experiments: capsaicin (N = 6), capsaicin + E06 (N = 5). No significant effect of E06; two sample t‐test.

Figure 7

Blockade of 4‐HNE‐evoked calcium influx in murine DRGs by E06. Single cell calcium traces obtained by ratiometric calcium imaging (ratio F(340/380)) of fura‐2/AM‐loaded DRG neurons upon stimulation with a mixture of 4‐HNE (30 μM; A) or H₂O₂ (10 μM; D) and E06 (3 ng·μL⁻¹). Application of AITC (10 μM), capsaicin (1 μM) and potassium (40 mM) served as internal control. Grey lines: 45–50 single cells, black line: average. (B, C, E, F) Summary graphs compare number of reacting cells and AUC of the interval between 120 and 540 s. Number of experiments: 4‐HNE (N = 5), 4‐HNE + E06 (N = 5), 4‐HNE + IgM (N = 5), H₂O₂ (N = 5), H₂O₂ + E06 (N = 5), H₂O₂ + IgM (N = 5). *P < 0.05. significantly different as indicated; in (B), Kruskal–Wallis ANOVA with post hoc Dunn's test; in (C, E, F), one‐way ANOVA with post hoc Bonferroni test.

Figure 8

E06 does not bind to 4‐HNE or 4‐HNE‐BSA in vitro. 4‐HNE or 4‐HNE coupled to BSA in different concentrations as outlined were incubated together with E06 in two different binding assays. Fluorescent E06 dose‐dependently bound to synthetic OxPAPC as a positive control but not to 4‐HNE and 4‐HNE protein adduct in the competitive (A) and direct (B) binding assays. Representative graphs of six individual experiments are displayed.

Figure 9

Accumulation of OxPLs in rat paws treated with agonists of TRPA1 or TRPV1 channels. E06‐reactive lipids from hind paws were quantified in comparison to OxPAPC. Male Wistar rats were intraplantarly treated with either H₂O₂, 4‐HNE, AITC, capsaicin or saline. Lipids were extracted from the paws ex vivo after the indicated time points. Using a competitive binding assay, there were significantly more E06 reactive lipids in rat paws 3 h after H₂O₂, 4‐HNE and AITC and 1 h after capsaicin treatment, compared with those in saline treated paws. Data are shown as E06‐reactive lipids·per mg of paw tissue (wet weight) for each animal with the mean (black line) and the median (grey line). H₂O₂ (N = 6), 4‐HNE (N = 7), AITC (N = 5), capsaicin (N = 6), saline (N = 6). *P < 0.05, significantly different from saline; one‐way ANOVA, post hoc Holm–Sidak test.