The differential contribution of tumour necrosis factor to thermal and mechanical hyperalgesia during chronic inflammation

Abstract

Therapies directed against tumour necrosis factor (TNF) are effective for the treatment of rheumatoid arthritis and reduce pain scores in this condition. In this study, we sought to explore mechanisms by which TNF contributes to inflammatory pain in an experimental model of arthritis. The effects of an anti-TNF agent, etanercept, on behavioural pain responses arising from rat monoarthritis induced by complete Freund's adjuvant were assessed and compared with expression of TNF receptors (TNFRs) by dorsal root ganglion (DRG) cells at corresponding time points. Etanercept had no effect on evoked pain responses in normal animals but exerted a differential effect on the thermal and mechanical hyperalgesia associated with rat arthritis induced by complete Freund's adjuvant (CFA). Joint inflammation was associated with increased TNFR1 and TNFR2 expression on DRG cells, which was maintained throughout the time course of the model. TNFR1 expression was increased in neuronal cells of the DRG bilaterally after arthritis induction. In contrast, TNFR2 expression occurred exclusively on non-neuronal cells of the macrophage-monocyte lineage, with cell numbers increasing in a TNF-dependent fashion during CFA-induced arthritis. A strong correlation was observed between numbers of macrophages and the development of mechanical hyperalgesia in CFA-induced arthritis. These results highlight the potential for TNF to play a vital role in inflammatory hyperalgesia, both by a direct action on neurons via TNFR1 and by facilitating the accumulation of macrophages in the DRG via a TNFR2-mediated pathway.

Figure 1

Etanercept treatment attenuates mechanical and thermal hyperalgesia, with little effect on swelling and histological damage. (a) Prearthritic etanercept treatment in rats with arthritis induced by complete Freund's adjuvant (CFA) reduced paw swelling significantly, by 10%, until 4 days after inflammation induction (n = 4). Thermal (b) and mechanical (c) hyperalgesia were assessed throughout the study. (b)Both prearthritic and postarthritic etanercept therapy abolished thermal hyperalgesia 7 days after inflammation (n = 4). (c) Prearthritic etanercept treatment reduced mechanical hyperalgesia significantly throughout the period studied, and postarthritic treatment reduced hyperalgesia to a lesser extent. (d) Joints were stained with haematoxylin and eosin and were assessed on a 4-point scale (where 0 = absent, 1 = mild, 2 = moderate, 3 = severe) for severity of inflammatory infiltrate, bone necrosis, and cartilage damage. CFA induced inflammatory infiltration 3 days (not shown) and 7 days (d) after CFA, while bone and cartilage were largely preserved. We found no reduction in histological score with either etanercept treatment regime. *P < 0.05; ***P < 0.005 in comparison with controls.

Figure 2

TNFR1 mRNA is increased following inflammation in the dorsal root ganglion (DRG). (a) Real-time RT-PCR showed a threefold increase in mRNA of tumour necrosis receptor type 1 (TNFR1) (expressed as the ratio of TNFR1 mRNA to 18S mRNA) in the DRG following the induction of inflammation by complete Freund's adjuvant (CFA) (n = 4). The increase was bilateral, being observed on both the ipsilateral (black bars) and the contralateral (white bars) DRGs. (b) In situ mRNA hybridisation showed no TNFR1 detection in the naive DRG. (c) In situ hybridisation for TNFR1 in the ipsilateral DRG 7 days after inflammation showed receptor expression in neuronal cells. *P < 0.05, **P < 0.01.

Figure 3

TNFR2 mRNA is increased following inflammation in the dorsal root ganglion (DRG). (a) Real-time RT-PCR showed an increase in mRNA of tumour necrosis factor receptor type 2 (TNFR2) (expressed as the ratio of TNFR2 mRNA to 18S mRNA) in the DRG ipsilateral to inflammation (n = 4), reaching five times its original level by 7 days. Contralateral TNFR2 levels increased significantly 7 days after inflammation. (b) In situ mRNA hybridisation in the naive DRG detected no TNFR2 mRNA. (c) In situ hybridisation detected TNFR2 mRNA in small cells of the DRG surrounding neuronal somata in the ipsilateral DRG 7 days after inflammation. (d) High-power magnification of TNFR2-labelled cells in the DRG, showing a perisomal distribution. *P < 0.05, **P < 0.01. CFA, complete Freund's adjuvant.

Figure 4

Monitoring expression of TNFR2 with anti-TNFR2 single-chain variable fragment (ScFv). (a) Immunoprecipitation of His-tagged tumour necrosis factor types 1 and 2 (TNFR1 and TNFR2) with anti-His antibody (left panel), and anti-TNFR2 single-chain variable fragment (ScFv) (right panel). The ScFv precipitated TNFR2, at ~75 KDa, but not TNFR1, at ~55 KDa, while the anti-His antibody precipitated both receptors, indicating specificity of the ScFv. Double immunohistochemistry against TNFR2-transfected 293T cells with anti-His antibody (b), and ScFv plus anti-myc-CY-3 (c) indicates that the selected ScFv specifically binds toTNFR2. (d) Immunohistochemistry with anti-TNFR2 ScFv in the dorsal root ganglion 7 days after inflammation ipsilateral to injection. TNFR2 colocalisation was observed with the macrophage marker, ED1 (e) but not with glial fibrillary acidic protein (GFAP) (f), indicating expression of TNFR2 by macrophages following inflammation.

Figure 5

Prearthritic treatment with etanercept reduces postarthritic macrophage accumulation in the dorsal root ganglion (DRG). (a) Inflammation due to complete Freund's adjuvant (CFA) induced a 10-fold increase in perineuronal macrophages (n = 4) by 7 days after inflammation. Infiltration of macrophages was restricted to the ipsilateral side, except for 7 days after inflammation, when a threefold increase in perineuronal macrophages was observed in the contralateral DRG. Nerve damage was assessed through double immunohistochemistry using the macrophage marker, ED1 (b) and a neuropathic marker, ATF-3 (activating transcription factor-3) (c). A maximum of two ATF-3-positive cells was detected per DRG, indicating that little nerve damage occurs in this model. (d) Prearthritic treatment with etanercept reduced macrophage numbers in the ipsilateral DRG by 75% 7 days after inflammation in comparison with controls treated with denatured etanercept (n = 4). No differences in macrophage numbers were detected with etanercept treatment commencing 3 days after inflammation induction. (e) A significant correlation between macrophage numbers in the DRG and mechanical hyperalgesia was observed 7 days after inflammation (n = 12). All treatment groups and controls were plotted. R² = 0.667, P < 0.01. *P < 0.05,**P < 0.01, ***P < 0.005.

Figure 6

Actions of tumour necrosis factor (TNF) on hyperalgesia in health and in chronic inflammation. In the naive dorsal root ganglion (DRG), TNFα acts on peripheral cells to induce a proinflammatory cascade resulting in the release of mediators, such as prostaglandins, that activate nociceptive neurons, resulting in pain. After chronic inflammation, tumour necrosis factor receptor type 1(TNFR1) is up-regulated on DRG neurons, while TNFR2 is expressed by infiltrating macrophages. TNF can directly modulate neuronal function and act on peripheral cells and DRG macrophages to induce inflammatory mediators that can modulate neuronal function. This results in exaggerated pain.