Contribution of CD137L to Sensory Hypersensitivity in a Murine Model of Neuropathic Pain

Abstract

CD137L (4-1BBL) is a costimulatory molecule whose signaling can promote monocyte/macrophage functions; however, CD137L-mediated microglial response and its role in neuropathic pain remain unknown. We investigated CD137L following peripheral nerve injury-induced neuropathic pain using a spinal nerve L5 transection (L5Tx) murine model in both sexes. First, C57BL/6_CD137L knock-out (KO) mice displayed decreased mechanical and diminished heat hypersensitivity compared to wild-type (WT) controls, beginning on day 3 to up to day 35 post-L5Tx. Purified anti-mouse CD137L neutralizing monoclonal antibody (0.1 or 0.5 µg) was also used to identify CD137L's window of action in BALB/c mice. Anti-CD137L antibody was intrathecally administered either from day 0 (before surgery) to day 7 (early treatment), or from day 6 to 13 post-L5Tx (late treatment), and nociceptive thresholds were assessed before surgery to up to day 35 post-surgery. Early treatment with anti-CD137L reduced L5Tx-induced mechanical but not heat hypersensitivity, while later treatment did not alter either sensitivity. Pro- versus anti-inflammatory responses within the lumbar spinal cord following L5Tx were further evaluated via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) in time-course studies. Following L5Tx, female CD137L KO mice did not show increased iNOS mRNA and had reduced numbers of IL-1β⁺ cells compared to WT. At 21 d post-surgery, CD137L KO mice had higher total numbers of arginase (Arg)-1⁺ cells and Arg-1⁺ microglia. Altogether, results indicate that spinal cord CD137L contributes to the development of peripheral nerve injury-induced neuropathic pain, which may be in part mediated through CD137L's modulation of the pro- and anti-inflammatory balance within the spinal cord.

Keywords: 4-1BBL; CD137L; L5 transection; microglia; mouse; neuropathic pain.

Figure 1.

Decreased mechanical and heat hypersensitivity was observed in L5Tx CD137L KO mice. Mechanical (A) and heat (B) sensitivities in CD137L KO (gray) and WT (black) mice following a sham (open circles/dashed line) or L5Tx (triangles/solid line) surgery are shown. Data show mean ± SEM of 12 mice/group. For Hargreaves data, individual baseline responding was set to 100% within each group. Equal numbers of male and female mice were used in this experiment. Due to a lack of sex differences, results shown are collapsed across sex; *p ≤ 0.05, significantly different from corresponding baseline level on day 0; #p ≤ 0.05, significantly different from the L5Tx WT group at the same time point.

Figure 2.

Early administration of anti-CD137L antibody reversed L5Tx-induced mechanical hypersensitivity in both male and female mice. Mechanical hypersensitivity following early (days 0–7; A, B) or late (days 6–13; C, D) intrathecal administration of 0.1 μg/μl (slanted white bars) and 0.5 μg/μl (light gray bars) anti-CD137L antibody (α-CD137L; horizontal white bars) to male (A, C) and female (B, D) BALB/c mice is shown. Data show mean ± SEM of six to eight mice/group; *p ≤ 0.05, day 0 significantly different from corresponding group at all other time points tested; #p ≤ 0.05, significantly different from no injection and PBS controls groups at the same time point; §p ≤ 0.05, significantly different from all other groups at the same time point; @p ≤ 0.05, significantly different from isotype control at the same time point; bp ≤ 0.05; significantly different from PBS-treated group at same time point.

Figure 3.

Early administration of anti-CD137L antibody reversed heat hypersensitivity only in males. Heat hypersensitivity following early (days 0–7; A, B) or late (days 6–13; C, D) intrathecal administration of 0.1 μg/μl (slanted white bars) and 0.5 μg/μl (light gray bars) anti-CD137L antibody (α-CD137L; horizontal white bars) to male (A, C) and female (B, D) BALB/c mice is shown. Within each group, individual baseline responding was set as 100%. Data show mean ± SEM of six mice/group; *p ≤ 0.05, day 0 significantly different from corresponding group at day 3 to day 35; ap ≤ 0.05, significantly different from no injection control group at same time point; @p ≤ 0.05, significantly different from isotype control at same time point; &p ≤ 0.05, significantly difference between 0.1 and 0.5 μg/μl anti-CD137L antibody at same time point.

Figure 4.

L5Tx-induced changes in mRNA expression of inflammatory markers in CD137L KO and WT mice. Figure shows the relative mRNA expression of IL-1β (A, B), iNOS (C, D), IL-1ra (E, F), and Arg-1 (G, H) in the lumbar spinal cord of male (left panels) and female (right panels) CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars). Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. Data show mean ± SEM of six mice/group; *p ≤ 0.05, significantly different from the corresponding naïve group; +p ≤ 0.05, significantly different from the corresponding sham group at the same time point; §p ≤ 0.05, significantly different from all other groups at same time point.

Figure 5.

L5Tx-induced changes in mRNA expression of GFAP and CD11b in CD137L KO and WT mice. Figure shows the relative mRNA expression of the astrocytic marker, GFAP (A, B) and the microglial marker, CD11b (C, D) in the lumbar spinal cord of male (left panels) and female (right panels) CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars). Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. Data show mean ± SEM of six mice/group; *p ≤ 0.05, significantly different from the corresponding naïve group; +p ≤ 0.05, significantly different from the corresponding sham group at the same time point; §p ≤ 0.05, significantly different from all other groups at same time point.

Figure 6.

Microglia expressing pro-/anti-inflammatory markers. Representative confocal immunofluorescent microscopy of CD11b (red) and pro-/anti-inflammatory markers (green) within the lumbar spinal cord are shown (images are from a WT B6 mouse on day 1 post-L5Tx surgery). Merged panels show colocalization of CD11b with the corresponding inflammatory marker. Scale bar: 40 μm.

Figure 7.

L5Tx-induced changes in the total numbers of CD11b⁺ cells within the dorsal horn region of the lumbar spinal cord in CD137L KO and WT mice. Total numbers of CD11b⁺ cells in the ipsilateral (A) and contralateral (B) dorsal horn of the L5 segment of the spinal cord of CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars) are shown. Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. Data show mean ± SEM of four mice/group; *p ≤ 0.05, significantly different from the corresponding naïve group; +p ≤ 0.05, significantly different from the corresponding sham group at the same time point; #p ≤ 0.05, significantly different from the corresponding WT group at the same time point.

Figure 8.

L5Tx-induced changes in cellular expression of IL-1β, iNOS and microglial colocalization in CD137L KO and WT mice. The number of IL-1β⁺ (A, B) and iNOS⁺ (E, F) cells in the ipsilateral (A, E) and contralateral (B, F) dorsal horn of the L5 segment of the spinal cord of CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars) are shown. Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. Percentage of microglia expressing IL-1β (C, D) and iNOS (G, H) in the ipsilateral (C, G) and contralateral (D, H) dorsal horn in the L5 segment of the spinal cord of CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars) are also shown. Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. Data show mean ± SEM of four mice/group; *p ≤ 0.05, significantly different from the corresponding naïve group; +p < 0.05, significantly different from the corresponding sham group at the same time point; #p < 0.05, significantly different from the corresponding WT group at the same time point.

Figure 9.

L5Tx-induced changes in cellular expression of IL-1ra, Arg-1, and microglial colocalization in CD137L KO and WT mice. Total numbers of IL-1ra⁺ (A, B) and Arg-1⁺ (E, F) cells in the ipsilateral (A, E) and contralateral (B, F) dorsal horn of the L5 segment of the spinal cord of CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars) are shown. Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. The percentage of microglia expressing IL-1ra (C, D) and Arg-1 (G, H) cells in the ipsilateral (C, G) and contralateral (D, H) dorsal horn of the L5 segment of the spinal cord of CD137L KO (black bars) and WT (light gray bars) mice following sham (open hatched bars for WT; open slanted bars for CD137L KO) and L5Tx (closed bars) are also shown. Naïve (day 0, no surgery) mice are represented by open bars in corresponding genotype color. Data show mean ± SEM of four mice/group; *p ≤ 0.05, significantly different from the corresponding naïve group; +p ≤ 0.05, significantly different from the corresponding sham group at the same time point; #p ≤ 0.05, significantly different from the corresponding WT group at the same time point; §p ≤ 0.05, significantly different from all other groups at the same time point; $p ≤ 0.05, significantly different from all other time points except day 14.