W. Marie Campana

The role of neuroinflammation in neuropathic pain: mechanisms and therapeutic targets

Neuroinflammation is a proinflammatory cytokine-mediated process that can be provoked by systemic tissue injury but it is most often associated with direct injury to the nervous system. It involves neural-immune interactions that activate immune cells, glial cells and neurons and can lead to the debilitating pain state known as neuropathic pain. It occurs most commonly with injury to peripheral nerves and involves axonal injury with Wallerian degeneration mediated by hematogenous macrophages. Therapy is problematic but new trials with anti-cytokine agents, cytokine receptor antibodies, cytokine-signaling inhibitors, and glial and neuron stabilizers provide hope for future success in treating neuropathic pain.

Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury.

Erythropoietin (Epo) has been shown to have potent anti‐apoptotic activity in central nervous system neurons in animal models of ischaemic injury. Recently, Epo and its receptor (EpoR) have been identified in the peripheral nervous system [Campana & Myers (2001), FASEB J., 15, 1804–1806]. Herein, we demonstrate that in painful neuropathy caused by L5 spinal nerve crush (SNC), therapy with recombinant human Epo (rhEpo) reduced dorsal root ganglion (DRG) apoptosis and pain behaviours. Quantification of both DRG neurons and satellite cells revealed that vehicle‐treated, crush‐injured DRGs had 35.5 ± 8.3% apoptotic neurons and 23.5 ± 2.36% satellite cells compared with 7.5 ± 6.3% apoptotic neurons and 6.4 ± 3.94% satellite cells in rhEpo‐treated, crush‐injured DRGs (P < 0.05). While rhEpo‐treated animals were not initially protected from mechanical allodynia associated with L5 SNC, rhEpo did significantly improve recovery rates compared to vehicle‐treated animals (P < 0.01). Systemic rhEpo therapy increased JAK2 phosphorylation, a key anti‐apoptotic signalling molecule for Epo‐induced neuroprotection, in DRGs after crush. Dual immunofluorescence demonstrated Epo‐induced JAK2‐p was associated with both neuronal and glial cells. JAK2‐p was associated with NF200‐positive large neurons and with smaller neurons. This population of small neurons did not colocalize with IB4, a marker of nonpeptidergic, glial derived growth factor‐responsive neurons. The findings link anti‐apoptosis activities of Epo/EpoR/JAK2 in DRG neurons capable of inducing protracted pain states with reductions in pain behaviours, and therefore support a role for Epo therapy in the treatment of neuropathic pain.

Erythropoietin and erythropoietin receptors in the peripheral nervous system: changes after nerve injury

Erythropoietin (Epo) is a hematopoietic factor that has recently been shown to function in the central nervous system. The presence or function of Epo or the erythropoietin receptor (EpoR) in the peripheral nervous system is unknown. Our results demonstrate the presence of both Epo and EpoR in the rat sciatic nerve. Epo was present in axons and was up‐regulated in Schwann cells after chronic constriction injury (CCI). EpoR was present in endothelial cells, cell bodies of the dorsal root ganglia (DRG), axons, and Schwann cells; it was not changed after injury. Using two different models of nerve injury, CCI and crush injury adjacent to the DRG, we demonstrated TUNEL labeling in DRG cell bodies after adjacent nerve crush, but not after CCI. TUNEL labeling in crush injury correlated with a significant reduction of EpoR in DRG as determined by morphometry and Western blotting. Immunoblotting of uninjured DRG homogenates revealed an immunoreactive band at 90 kDa and 70 kDa. After crush injury, both 70 kDa and 90 kDa proteins were reduced and the 90 kDa protein showed enhanced tyrosine phosphorylation, whereas the 70 kDa showed less. EpoR colocalized with NF200 in normal DRG, but not after crush injury to the adjacent axon. These findings demonstrate Epo and EpoR in the peripheral nervous system and their regulation after painful nerve injury.

TNFα-induced MMP-9 promotes macrophage recruitment into injured peripheral nerve

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced hours after injury to peripheral nerve. This study shows that MMP-9 gene deletion and neutralization with MMP-9 antibody reduce macrophage content in injured wild-type nerves. In mice with delayed Wallerian degeneration (WldS), MMP-9 and tumor necrosis factor alpha (TNFalpha) decline in association with the reduced macrophage recruitment to injured nerve that characterizes this strain of mice. We further determined that TNFalpha acts as an MMP-9 inducer by establishing increased MMP-9 levels after TNFalpha injection in rat sciatic nerve in vivo and primary Schwann cells in vitro. We found reduced MMP-9 expression in crushed TNFalpha knockout nerves that was rescued with exogenous TNFalpha. Finally, local application of MMP-9 on TNFalpha-/- nerves increased macrophage recruitment to the lesion. These data suggest that TNFalpha lies upstream of MMP-9 in the pathway of macrophage recruitment to injured peripheral nerve.

The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of schwann cells by binding to low-density lipoprotein receptor-related protein.

Low-density lipoprotein receptor-related protein (LRP-1) is an endocytic receptor for diverse proteins, including matrix metalloproteinase-9 (MMP-9), and a cell-signaling receptor. In the peripheral nervous system (PNS), LRP-1 is robustly expressed by Schwann cells only after injury. Herein, we demonstrate that MMP-9 activates extracellular-signal-regulated kinase (ERK1/2) and Akt in Schwann cells in culture. MMP-9 also promotes Schwann cell migration. These activities require LRP-1. MMP-9-induced cell signaling and migration were blocked by inhibiting MMP-9-binding to LRP-1 with receptor-associated protein (RAP) or by LRP-1 gene silencing. The effects of MMP-9 on Schwann cell migration also were inhibited by blocking the cell-signaling response. An antibody targeting the hemopexin domain of MMP-9, which mediates the interaction with LRP-1, blocked MMP-9-induced cell signaling and migration. Furthermore, a novel glutathione-S-transferase fusion protein (MMP-9-PEX), which includes only the hemopexin domain of MMP-9, replicated the activities of intact MMP-9, activating Schwann cell signaling and migration by an LRP-1-dependent pathway. Constitutively active MEK1 promoted Schwann cell migration; in these cells, MMP-9-PEX had no further effect, indicating that ERK1/2 activation is sufficient to explain the effects of MMP-9-PEX on Schwann cell migration. Injection of MMP-9-PEX into sciatic nerves, 24 h after crush injury, robustly increased phosphorylation of ERK1/2 and Akt. This response was inhibited by RAP. MMP-9-PEX failed to activate cell signaling in uninjured nerves, consistent with the observation that Schwann cells express LRP-1 at significant levels only after nerve injury. These results establish LRP-1 as a cell-signaling receptor for MMP-9, which may be significant in regulating Schwann cell migration and physiology in PNS injury.

Erythropoietin Promotes MCF-7 Breast Cancer Cell Migration by an ERK/Mitogen-activated Protein Kinase-dependent Pathway and Is Primarily Responsible for the Increase in Migration Observed in Hypoxia

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O2) for 3 h, the Epo mRNA level increased 2.4 ± 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 ± 0.1-fold. Soluble EpoR and Epo-neutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration.

Phosphatidylinositol 3‐kinase and Akt protein kinase mediate IGF‐I‐ and prosaptide‐induced survival in schwann cells

Withdrawal of trophic factors necessary for Schwann cell survival regulates Schwann cell number during development and after nerve injury. In the present study, we identified signaling pathways involved in Schwann cell survival by prosaposin, prosaptides (peptides incorporating the neurotrophic sequence of prosaposin), and insulinlike growth factor‐I (IGF‐I). When postnatal Schwann cells were placed in low serum medium, cells underwent abrupt shrinkage, condensation of nuclei occurred, and smooth rounded apoptotic bodies appeared. Dose–response studies of cell death, measured by lactate dehydrogenase (LDH) release, demonstrated that both prosaptide TX14(A) and IGF‐I dose dependently reduced cell death in primary Schwann cells. Histone‐associated DNA fragmentation enzyme‐linked immunosorbent assay, showed a 10‐ and 14‐fold increase in apoptosis after 4 and 24 hr in low serum medium, respectively, that was reduced by prosaposin, TX14(A), or IGF‐I. Phosphatidylinositol 3‐kinase (PI3K) inhibitors, wortmannin or LY294002, blocked the survival effects of both TX14(A) and IGF‐I. In contrast, only TX14(A) anti‐apoptotic activity was blocked by the MEK inhbitor, PD98059, although TX14(A) and IGF‐I are potent activators of extracellular regulated kinases in Schwann cells. Phosphorylation of the PI3K signaling target, Akt, was measured; TX14(A) and IGF‐I increased Akt activity by 12‐fold and 22‐fold, respectively, that was inhibited by LY294002. These findings indicate that prosaposin and IGF‐I use the PI3K/Akt pathway to induce survival of Schwann cells. J. Neurosci. Res. 57:332–341, 1999.

Inhibition of p38 MAP kinase activity enhances axonal regeneration

Tumor necrosis factor alpha (TNF)-induced cellular signaling through the p38 mitogen-activated protein kinase (p38 MAPK) pathway plays a critical role in Wallerian degeneration and subsequent regeneration, processes that depend on Schwann cell (SC) activity. TNF dose-dependently induces Schwann cell and macrophage activation in vivo and apoptosis in primary SC cultures in vitro, while inhibition of p38 MAPK is thought to block these cellular processes. We show with Western blots that after sciatic nerve crush injury, phosphorylated p38 (p-p38) MAPK is significantly increased (P < 0.01) in distal nerve segments. In tissue sections, p38 co-localized immunohistochemically with activated Schwann cells (GFAP) and to a lesser degree with macrophages (ED-1). In other experiments, animals were gavaged with Scios SD-169 (10 or 30 mg/kg) or excipient (PEG300) 1 day before and daily after crush injury to the sciatic nerve. SD-169 is a proprietary oral inhibitor of p38 MAPK activity. The rate of axonal regeneration was determined by the functional pinch test and was significantly increased in treated animals 8 days after crush injury (P < 0.05; 30 mg/kg dose). In SD-169-treated animals with nerve transection, nerve fibers regenerating through a silicone chamber were morphologically more mature than untreated nerves when observed 28 days after transection. TNF immunofluorescence of distal nerve segments after crush injury suggested that SD-169 reduced SC TNF protein. In support of these findings, SD-169 significantly reduced (P < 0.05) TNF-mediated primary SC death in culture experiments. We conclude that inhibition of p38 activity promotes axonal regeneration through interactions with SC signaling and TNF activity.

Schwann cells express erythropoietin receptor and represent a major target for Epo in peripheral nerve injury.

Erythropoietin (Epo) expresses potent neuroprotective activity in the peripheral nervous system; however, the underlying mechanism remains incompletely understood. In this study, we demonstrate that Epo is upregulated in sciatic nerve after chronic constriction injury (CCI) and crush injury in rats, largely due to local Schwann cell production. In uninjured and injured nerves, Schwann cells also express Epo receptor (EpoR), and its expression is increased during Wallerian degeneration. CCI increased the number of Schwann cells at the injury site and the number was further increased by exogenously administered recombinant human Epo (rhEpo). To explore the activity of Epo in Schwann cells, primary cultures were established. These cells expressed cell‐surface Epo receptors, with masses of 71 and 62 kDa, as determined by surface protein biotinylation and affinity precipitation. The 71‐kDa species was rapidly but transiently tyrosine‐phosphorylated in response to rhEpo. ERK/MAP kinase was also activated in rhEpo‐treated Schwann cells; this response was blocked by pharmacologic antagonism of JAK‐2. RhEpo promoted Schwann cell proliferation, as determined by BrdU incorporation. Cell proliferation was ERK/MAP kinase‐dependent. These results support a model in which Schwann cells are a major target for Epo in injured peripheral nerves, perhaps within the context of an autocrine signaling pathway. EpoR‐induced cell signaling and Schwann cell proliferation may protect injured peripheral nerves and promote regeneration.

Erythropoietin reduces Schwann cell TNF‐α, Wallerian degeneration and pain‐related behaviors after peripheral nerve injury

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain‐like behaviors. These effects are mediated in large part by pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNF‐α). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF‐α has been implicated in the development of pain‐related behaviors, we measured TNF‐α mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF‐α mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF‐α in Schwann cells, axoplasm and macrophages. In rhEpo‐treated animals, TNF‐α immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF‐α in CCI by blocking expression of TNF‐α in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF‐α expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF‐αin vitro. These results indicated that rhEpo regulates TNF‐α by multiple mechanisms; rhEpo regulates TNF‐α mRNA expression by Schwann cells but also may directly counteract TNF‐α signaling pathways that lead to injury, chronic pain and/or death.