Hemokinin-1 as a Mediator of Arthritis-Related Pain via Direct Activation of Primary Sensory Neurons

Abstract

The tachykinin hemokinin-1 (HK-1) is involved in immune cell development and inflammation, but little is known about its function in pain. It acts through the NK1 tachykinin receptor, but several effects are mediated by a yet unidentified target. Therefore, we investigated the role and mechanism of action of HK-1 in arthritis models of distinct mechanisms with special emphasis on pain. Arthritis was induced by i.p. K/BxN serum (passive transfer of inflammatory cytokines, autoantibodies), intra-articular mast cell tryptase or Complete Freund's Adjuvant (CFA, active immunization) in wild type, HK-1- and NK1-deficient mice. Mechanical- and heat hyperalgesia determined by dynamic plantar esthesiometry and increasing temperature hot plate, respectively, swelling measured by plethysmometry or micrometry were significantly reduced in HK-1-deleted, but not NK1-deficient mice in all models. K/BxN serum-induced histopathological changes (day 14) were also decreased, but early myeloperoxidase activity detected by luminescent in vivo imaging increased in HK-1-deleted mice similarly to the CFA model. However, vasodilation and plasma protein extravasation determined by laser Speckle and fluorescent imaging, respectively, were not altered by HK-1 deficiency in any models. HK-1 induced Ca²⁺-influx in primary sensory neurons, which was also seen in NK1-deficient cells and after pertussis toxin-pretreatment, but not in extracellular Ca²⁺-free medium. These are the first results showing that HK-1 mediates arthritic pain and cellular, but not vascular inflammatory mechanisms, independently of NK1 activation. HK-1 activates primary sensory neurons presumably via Ca²⁺ channel-linked receptor. Identifying its target opens new directions to understand joint pain leading to novel therapeutic opportunities.

Keywords: arthritic pain; experimental arthritis; in vivo optical imaging; neuroinflammation; primary sensory neuron; tachykinin.

FIGURE 1

Change in mechanonociceptive threshold (A), heat threshold (B), paw volume (C), cold tolerance (D), body weight (E) and time spent on grid (F) in K/BxN serum-induced arthritis of wild type (WT) and hemokinin-1-deficient (Tac4 ^−/−) mice in comparison with BxN serum-treated non-arthritic groups (n = 4–10 per group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. arthritic WT, repeated measures two-way ANOVA + Bonferroni’s post test).

FIGURE 2

Quantitative evaluation of MPO-activity (A) and IR-676 dye extravasation (B) in K/BxN arthritogenic serum-treated wild type (WT) and hemokinin-1-deficient (Tac4 ^−/−) mice. Representative images of MPO-activity (C). Box plots demonstrate medians with the upper and lower quartiles and all individual data points; n = 6–12 per group; *p < 0.05 vs. WT, ##p < 0.01, ###p < 0.001 vs. control (one-way ANOVA + Bonferroni’s post test).

FIGURE 3

Representative histopathological pictures of safranin O-stained tibiotarsal joint showing ×100 and ×200 magnifications with 500 and 200 µm scale bars, respectively. ti = tibia, ta = tarsus, s = synovium (A). Semiquantitative histopathological score of K/BxN serum treated wild type (WT) and hemokinin-deficient (Tac4 ^−/−) arthritic mice in comparison with non-arthritic controls. Box plots demonstrate the medians with upper and lower quartiles of n = three to five per group (*p < 0.05 vs. WT, Mann-Whitney test) (B).

FIGURE 4

Changes in mechanonociceptive threshold (A), knee diameter (B) and blood flow (C) in mast cell tryptase-induced acute arthritis of wild type (WT) and hemokinin-1-deficient (Tac4 ^−/−) mice. Data points demonstrate the means ± SEM of n = 5–10 per group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT (repeated measures two-way ANOVA + Bonferroni’s post test).

FIGURE 5

Changes in mechanonociceptive threshold (A), AP knee diameter (B), and MPO-activity (C) in CFA-induced acute arthritis of WT and HK-1-deficient (Tac4 ^−/−) mice. Data points demonstrate the means ± SEM (A), (B) or medians with upper and lower quartiles of n = 8–15 per group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT, ###p < 0.001 vs. control (repeated measures two-way ANOVA + Bonferroni’s post test; for MPO activity: one-way ANOVA + Bonferroni’s post test).

FIGURE 6

Effect of HK-1 and SP in cultured primary sensory neurons. Change in the fluorescence ratio (R = F340/F380) in HK-1-sensitive cells (•) is presented. ●: HK-1 and PTX co-administration, ♦: fluorescence signal in neurons from NK1R ^−/− animals, ○: HK-1 and CP99994 NK1 receptor antagonist administration, □: HK-1 and AMG9810 TRPV1 receptor antagonist, ■: HK-1 and HC 030031 TRPA1 receptor antagonist. No signal was detected in Ca²⁺-free extracellular solution. N = 19–49 cells/group (A). The percentage of responsive cells to HK-1, HK-1+PTX, HK-1 in Ca²⁺-free condition, HK-1+NK1 receptor antagonist CP99994 and HK-1 in TG from NK1R ^−/− animals, HK-1 + TRPV1 receptor antagonist AMG9810 and HK-1 + TRPA1 receptor antagonist HC 030031 is presented. Ca²⁺-responses are presented in % of total number of examined neurons. N = 19–49 cells per group (B). Original Ca²⁺-imaging registrations after HK-1 administration. Increases of R = 340/380 fluorescence in fura-2 loaded cultured TG neurons (C). Original Ca²⁺-imaging registrations after HK-1 administration in TG neurons from NK1 gene deficient mice (D). Original Ca²⁺-imaging registrations after HK-1 administration in Ca²⁺-free solution (E). Effect of HK-1 on capsaicin-induced Ca²⁺-influx. Increases of R = 340/380 fluorescence in fura-2 loaded neurons are presented, **p < 0.01, NS (vs. caps1, one-way ANOVA with Bonferroni’s multiple comparison post hoc test, n = 16) (F). Effect of HK-1 on capsaicin-induced Ca²⁺-influx. Original Ca²⁺-imaging registrations after capsaicin and HK-1 administration (G). Effect of SP on capsaicin-induced Ca²⁺-influx. Increases of R = 340/380 fluorescence in fura-2 loaded neurons are presented, ***p < 0.001, NS (vs. caps1, one-way ANOVA with Bonferroni’s multiple comparison post hoc test, n = 17) (H). Effect of SP on capsaicin-induced Ca²⁺-influx. Original Ca²⁺-imaging registrations after capsaicin and SP administration (I).