IL-1ra alleviates inflammatory hyperalgesia through preventing phosphorylation of NMDA receptor NR-1 subunit in rats

Abstract

Although it has been shown that pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) facilitate perception of noxious inputs at the spinal level, the mechanisms have not been understood. This study determined the cell type that produces IL-1beta, the co-localization of IL-1 receptor type I (IL-1RI) and Fos and NR1 in the spinal cord, and the effects of IL-1 receptor antagonist (IL-1ra) on NR1 phosphorylation and hyperalgesia in a rat model of inflammatory pain. Phosphorylation of NR1, an essential subunit of the NMDA receptor (NMDAR), is known to modulate NMDAR activity and facilitate pain. Hyperalgesia was induced by injecting complete Freund's adjuvant (CFA, 0.08ml, 40microg Mycobacterium tuberculosis) into one hind paw of each rat. Paw withdrawal latency (PWL) was tested before CFA (-48h) for baseline and 2 and 24h after CFA to assess hyperalgesia. IL-1ra was given (i.t.) 24h before CFA to block the action of basal IL-1beta and 2h prior to each of two PWL tests to block CFA-induced IL-1beta. Spinal cords were removed for double immunostaining of IL-1beta/neuronal marker and IL-1beta/glial cell markers, IL-1RI/Fos and IL-1RI/NR1, and for Western blot to measure NR1 phosphorylation. The data showed that: (1) astrocytes produce IL-1beta, (2) IL-1RI is localized in Fos- and NR1-immunoreactive neurons within the spinal dorsal horn, and (3) IL-1ra at 0.01mg/rat significantly increased PWL (P<0.05) and inhibited NR1 phosphorylation compared to saline control. The results suggest that spinal IL-1beta is produced by astrocytes and enhances NR1 phosphorylation to facilitate inflammatory pain.

Figures

Fig. 1

Photomicrographs showing IL-1β expression and co-localization of IL-1β and GFAP in ipsilateral lumbar spinal superficial laminae 24 h after CFA injection into one hind paw. Sections were double-labeled with anti-IL-1β (green) and anti-GFAP, anti-OX-42 or anti-NeuN (red). The first column is IL-1β immunostaining; the second column is immunostaining of the astrocyte marker GFAP, microglia marker OX-42, and neuron marker NeuN. The third column shows the merged graphs of IL-1β with each. Note that IL-1β is localized in astrocytes, the yellow cells in a’’.

Fig. 2

Values of IL-1β immunoreactive astrocytes in the spinal cord (Mean ± SE). A: Representative photographs showing IL-1β immunoreactive cells in superficial laminae of spinal cord 24 hr after CFA (left) or saline (right) injection. Note that CFA injection induced more IL-1β compared to saline injection. B: The number of IL-1β-immunoreactive astrocytes was significantly increased in laminae I–II at 2 and 24 h post-CFA compared to that of the contralateral side and of saline-injected rats. C-D: It also significantly increased 24 h post-CFA in both ipsilateral and contralateral spinal laminae V–VI and lamina X compared to that of saline-injected rats. * P<0.05 vs saline-injected rats; #P<0.05 vs contralateral side.

Fig. 3

Representative photograph showing the colocalization of immunoreactive IL-1RI and Fos in superficial laminae (A) and lamina V (B) of the spinal dorsal horn. Note that the immunostaining product for IL-1RI is localized in cytoplasm and dendrites, while that associated with Fos is present in the nucleus. Arrows indicate doubled-labeled IL-1RI/ Fos neurons. Bars = 50 μm.

Fig. 4

Micrographs showing co-localization of NR1 and IL-1RI in lumbar spinal dorsal horn neurons. Sections were double-labeled with anti-NR1 (green) and anti-IL-1RI (red). a: NR1-immunoreactive neurons in laminae I-II; b: IL-1RI-immunoreactive neurons in laminae I–II; c: Merged graphs of a and b. Arrow indicates double-labeled NR1/IL-1RI neuron (yellow); Scale bars represent 40 μm.

Fig. 5

Effects of IL-1ra on CFA-induced hyperalgesia. IL-1ra at 0.01 (n=7) mg/rat (i.t.) significantly increased PWL compared to vehicle control (n=7). *P

Fig. 6

A: Effects of IL-1ra on spinal cord NR1 phosphorylation. B: The values of NR1 phosphorylation in saline-injected rats were arbitrarily set at 100%. Each bar is expressed as a percentage of the saline-injected rats. CFA induced significant NR1 phosphorylation compared to saline-injected control (vehicla vs saline-injected). IL-1ra at 0.01 (n=7) mg/rat (i.t.) significantly inhibited spinal cord NR1 phosphorylation compared to vehicle control (n=7). **P<0.01 compared to saline-injected control and 0.01 mg group.