Axonal transport of TNF-α in painful neuropathy: distribution of ligand tracer and TNF receptors

Abstract

Tumor necrosis factor-α (TNF-α) is a key player in peripheral nerve injury. In the inflammatory chronic constriction injury (CCI) model of sciatic neuropathy, upregulation of TNF-α mRNA and protein at the site of nerve injury has been associated with pain. We now report the distribution of endogenous TNF-α protein and its receptors along normal and CCI-injured sciatic nerves, and within the corresponding lumbar dorsal root ganglia (DRG). Using Western blotting, TNF-α was found to be distinctly increased at the injury site, as well as in the axons just distal to the corresponding DRG. The TNF-α signal between the injury site and DRG (midaxonal) was induced between 2 and 5 days post-CCI, suggesting activation of TNF-α axonal transport. Endogenous TNF-α was localized in small-diameter, presumably nociceptive, and large-diameter, presumably mechanoceptive, DRG sensory neurons in both normal and CCI animals. Intraneural microinjection of biotin-labeled TNF-α showed specific axonal uptake at the injection site, as detected by avidin–biotin–peroxidase histochemistry, and confirmed by co-localization with neurobiotin tracer. In control animals, fast retrograde transport of biotinylated TNF-α to both L4 and L5 DRG neurons was apparent 6 h following injection. TNF receptors TNFRI and TNFRII co-localized with biotinylated TNF-α tracer along the nerve trunk, suggesting that TNF-α transport may be receptor-mediated. In animals with CCI neuropathy, uptake of biotinylated TNF-α by neuronal soma was inhibited. Instead, there was signal accumulation in the axons immediately distal to the DRG, and TNFRI and RII were increased at this same anatomic location. These findings highlight a dynamic process of TNF-α protein and receptor regulation throughout the peripheral neural axis that bears on both the normal function of DRG neurons and the pathogenesis of painful neuropathies.

Introduction

During the first week of peripheral nerve injury, dynamic reorganization within the damaged sensory neuron causes an increase in retrograde axonal transport of small proteins (Redshaw and Bisby, 1984, Redshaw and Bisby, 1987), including neurotrophins (DiStefano and Curtis, 1994, Curtis et al., 1998). Selective retrograde transport provides a way of introducing regeneration-modulating factors into neuronal cell bodies of the dorsal root ganglia (DRG) (Kristensson, 1984). This process of communication is dynamic, and in sensory neurons retrograde transport returns to basal levels by 7 days following injury (Griffin and Hoffman, 1984, Curtis et al., 1998). In painful chronic constriction injury (CCI) of rat sciatic nerve, many of the early genes controlling cell fate are induced at the injury site within the first week and have an important effect on local cell survival. We have hypothesized that cytokine factors such as tumor necrosis factor alpha (TNF-α) may also have a direct effect on DRG neurons via retrograde axonal transport (Myers et al., 1996).

TNF-α is a proinflammatory cytokine that is a principal modulator of the early degenerative changes during peripheral nerve injury (Myers et al., 1999). Produced primarily by endoneurial macrophages and Schwann cells (Wagner and Myers, 1996a), TNF-α protein (Shubayev and Myers, 2000) and mRNA (Taskinen et al., 2000) are expressed in a bimodal fashion within the first week following the injury, peaking at the injury site within 1 day and then at 5 days post-injury. The pathophysiologic effect of TNF-α has been confirmed by direct injection of TNF-α into the sciatic nerve, which induces painful neuropathy (Wagner and Myers, 1996b), and produces degenerative neuropathology (Redford et al., 1995, Wagner and Myers, 1996b), including endoneurial inflammation, primary demyelination and axonal degeneration.

Two distinct functional TNF-α receptors (TNFR) have been described with molecular weights of approximately 55 kDa (TNFRI, p55) and 75 kDa (TNFRII, p75). Activation of these receptors can initiate mechanisms controlling either cell death or survival (Baker and Reddy, 1998, Darnay and Aggarwal, 1997). TNFRI, a death-domain-containing receptor, transmits the apoptotic signal through caspase and JUN kinase pathways by recruitment of TRADD, FADD and RIP adaptor proteins, ultimately resulting in cell death (Pettmann and Henderson, 1998, Muzio, 1998). The survival signals are mediated via TRAF-dependent activation of nuclear factor kappa B (NF-κB) by both TNFRI and TNFRII (Baker and Reddy, 1998). TNFRI is upregulated following CCI to sciatic nerve (Shubayev and Myers, 2000), but its distribution has not been fully characterized. This study is the first to examine axonal transport of TNF-α and its receptors, which we think is related to their role in TNF-α-dependent signaling in the pathogenesis of painful neuropathy.

To assay TNF-α upregulation and mobility, we employed semiquantitative Western blot analysis of TNF-α at different levels of the neural axis from the CCI injury site in the distal sciatic nerve to the L4–5 dorsal root ganglia. We further determined if injury-induced TNF-α seen midaxonally reflected axonal transport. Rather than using the standard double-ligation experimental procedure for evaluating the direction of endogenous axonal transport (Ranish and Ochs, 1972), we studied the distribution of injected biotin-labeled TNF-α to avoid ligature-induced TNF-α production. Biotin is a small (300 Daltons), non-radioactive, easily detectable tag, which has been successfully used for TNF-α labeling of tumor cells without altering TNF-α cytotoxic activity (Moro et al., 1997, Gasparri et al., 1999). To determine the role of TNF-α receptors in TNF-α retrograde transport, we further co-localized biotinylated TNF-α with TNFRI and TNFRII within the nerve. Finally, lumbar DRG were analyzed for endogenous TNF-α ligand and receptor protein levels using immunohistochemical techniques.