Regulation of peripheral inflammation by spinal p38 MAP kinase in rats

Abstract

Background: Somatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP) kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.

Methods and findings: Selective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT) catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and -6 and matrix metalloproteinase (MMP3) gene expression. Activation of p38 required tumor necrosis factor alpha (TNFalpha) in the nervous system because IT etanercept (a TNF inhibitor) given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.

Conclusions: These data suggest that peripheral inflammation is sensed by the central nervous system (CNS), which subsequently activates stress-induced kinases in the spinal cord via a TNFalpha-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses.

Figure 1. Phosphorylation of p38 MAP Kinase in the Spinal Cord

(A) Western blot analysis of p38, P-p38, and COX2 in the spinal cord of rats with adjuvant arthritis. Rats were sacrificed from day (D) 0 through day 17. Note faint P-p38 staining on days 8 to day 17. GAPDH normalized expression = 0.22 ± 0.02 for prearthritis (days 0–5) and 0.32 ± 0.3 postarthritis (days 8–17; p = 0.0255). (B) Low-power view showing P-p38 expression in adjuvant immunized rats, especially in lamina II (CST, corticospinal tract). (C–E) High power view of P-p38 (C), OX-42 (D), and merged view (E) demonstrating expression of P-p38 in microglia of adjuvant immunized rats. (F–H) High-power view of P-p38 (F), NeuN (G), and merged view (H) demonstrating scattered neurons expressing P-p38 in adjuvant immunized rats. (I) and (J) show higher power view of (E) and (H), respectively. Arrow indicates positively staining cells in all photomicrographs.

Figure 2. Effect of IT p38 Inhibitor on Paw Swelling in Adjuvant Arthritis

Rats were immunized with CFA on day 0 and treated daily with IT p38 inhibitors (SB203580 at 8 μg/d), SC p38 inhibitor (SB203580 at 8 μg/d), or IT saline beginning on days 8–20. Paw swelling was measured by water displacement plethysmometry. The IT p38 inhibitor significantly decreased paw swelling (*p = 0.001 by ANOVA and Dunnett's post-ANOVA test; n = 5–6/group). This graph presents data from one of three separate experiments with similar results.

Figure 3. Effect of IT p38 Inhibitor on Joint Damage and Gene Expression in Adjuvant Arthritis

Rats were immunized with CFA on day 0 and treated daily with IT SB203580 (8 μg) or IT saline beginning on days 8–20. (A) Radiographs of the hind paws were evaluated using a scoring system that demonstrated decreased damage in the SB203580-treated group compared with control rats. *p = 0.036 for IT SB203580 (n = 6/group). Similar results were observed in a separate experiment with treatment beginning on day 8 (n = 5–6/group). (B) Representative radiographs illustrate decreased joint destruction in the treated group. (C) Representative hematoxylin and eosin-stained sections demonstrate decreased erosions, cartilage damage, and synovial inflammation in the IT SB203580-treated group. (D) Quantitative real-time PCR was performed on the hind paws as described in Methods. Normal rat joints are also shown as a control. IT SB203580 significantly decreased IL-1 (*p = 0.025), IL-6 (**p = 0.006), and MMP3 (+p = 0.001) gene expression (n = 5–6/group); TNFα gene expression was modestly reduced, but did not reach statistical significance (^p = 0.089).

Figure 4. Effect of IT Etanercept on Adjuvant Arthritis

Rats were immunized with CFA on day 0 and treated with IT etanercept (100 or 300 μg q.o.d.) beginning on day 1. (A) IT etanercept (Etan) significantly decreased paw swelling (*p = 0.001 for IT saline compared with either IT etanercept group) (n = 6/group). (B) IT etanercept significantly decreased joint damage (by radiographic score; *p = 0.026 for IT saline compared with either IT etanercept group) (n = 6/group). (C) Similar doses of etanercept given systemically (SC) beginning on day 1 had minimal effect on paw swelling (p = 0.398 for IT saline compared with either etanercept group) (n = 6/group). (D and E) IT etanercept decreased spinal P-p38 staining in adjuvant arthritis. Rats were immunized on day 0 and injected IT with 300 μg of etanercept or vehicle on days 7 and 10. They were sacrificed on day 11 and P-p38 was determined by Western blot (D) and immunofluorescence (E). Western blot data were normalized to GAPDH and demonstrated significantly lower levels of P-p38 after IT etanercept treatment (n = 6 for etanercept, 4 for saline, and 2 for naïve). *p = 0.023 compared with IT saline.