The following is Part 3 of a three-part series of selected talks from the 34th Annual Scientific Meeting of the American Pain Society (APS) held May 13-16, 2015, in Palm Springs, California, US. Also see Part 1 and Part 2.
After an injury, pain usually subsides as damaged tissue heals, but other times, pain mysteriously becomes chronic. “Clinical experience suggests that the transition from acute to chronic pain is not arbitrary,” said Jon Levine, University of California, San Francisco, US, at a symposium exploring peripheral nervous system mechanisms that contribute to chronic pain. “The transition is associated with a disconnection of pain generation from the initial tissue injury and sometimes with loss of responsiveness to therapies that were initially effective.”
Levine pioneered a model of chronic pain called hyperalgesic priming, in which rats receive an inflammatory insult or nerve injury, inducing pain that resolves after a few days. Days or weeks after this “priming,” painful inflammatory substances such as prostaglandin E2 (PGE2) delivered at the injury site evoked long-lasting hypersensitivity to noxious stimuli. “If you did this testing even months after this injury, you would still see the hyperalgesia,” Levine said. The model has been used to investigate central as well as peripheral mechanisms that contribute to priming (see PRF related news story).
Separate mechanisms seem to control the induction of priming, expression of hyperalgesia after priming, and maintenance of priming. To better understand the transition to chronic pain, Levine said, “we need to separate these phenomena very clearly.” Levine presented work from two recently published papers in the Journal of Neuroscience tackling this issue (Ferrari et al., 2015a; Ferrari et al., 2015b).
Levine and others had previously shown (Parada et al., 2003) that hyperalgesia in primed animals depends on the novel protein kinase Cε (PKCε), but its activity could not explain the long-lasting maintenance of priming. “When we inhibit PKCε after priming, we don’t see hyperalgesia, but it comes on again after we take away the inhibitor,” Levine said.
The priming process takes about three days after the initial injury—hyperalgesia cannot be evoked before that. “We hypothesized that something was being transported to the cell body in the DRG [dorsal root ganglia] and then back out to the peripheral terminal—that’s why it took 72 hours,” said Levine. When Levine and colleagues injected the DRG with a stable analog of cyclic adenosine monophosphate (cAMP), a second messenger that promotes gene transcription, priming was established more quickly. DRG treatment with antisense oligonucleotides aimed at messenger RNA (mRNA) for the nuclear transcription factor cAMP response-element binding protein (CREB) prevented priming, indicating that priming required CREB-dependent gene transcription at the cell body. In contrast, delivery of a potent, membrane-permeable form of cAMP in the periphery brought on hyperalgesia but failed to establish priming. These findings suggested that either the mRNA or the protein product of CREB-dependent transcription was transported back to the terminal.
Did priming also require protein translation? Yes, but at peripheral terminals, not the cell body. Injection of a protein translation blocker at peripheral terminals—but not in the DRG—prevented hyperalgesia, suggesting that mRNA, rather than protein, was being transported down the axon to the periphery. Translation depended on cytoplasmic polyadenylation element-binding protein (CPEB), which promotes local protein translation. A treatment that prevented CPEB-mediated translation at peripheral terminals actually reversed priming, suggesting that maintenance of the primed state relies on that translation—a process that might one day be targeted to reverse chronic pain. Levine postulates that either the mRNA for CPEB or the protein itself is transported from the DRG to peripheral terminals, accounting for the required three-day delay between priming and hyperalgesia.
Although Levine’s work demonstrates that distinct processes at the cell body and nerve terminals mediate various phases of hyperalgesic priming, many questions remain. “Our major effort now is to understand the nature of the molecule that comes from peripheral terminals to activate CREB in cell bodies,” Levine said. The real question at the root of this work, said Levine, is “why do some people get chronic pain and others don't? We would like to understand resilience to chronic pain.”
Cobi Heijnen and Annemieke Kavelaars, both at University of Texas MD Anderson Cancer Center, Houston, US, presented evidence for the increasingly recognized role of the peripheral immune system in the transition to chronic pain.
Heijnen and Kavelaars had previously established a role in pain for G protein receptor kinase 2 (GRK2), an enzyme that phosphorylates and inactivates active G proteins (Eijkelkamp et al., 2010). Heijnen presented the team’s published work investigating GRK2 in peripheral monocytes. Mice with heterozygous global deletion of the gene encoding GRK2—so that they produced only about half the normal amount of enzyme—developed long-lasting thermal and mechanical hyperalgesia after a single inflammatory paw injection with carrageenan, which normally resolves within a couple of days. A similar prolonged hyperalgesia developed in normal mice that were depleted of peripheral monocytes. When monocytes alone were deficient in GRK2, the mice still developed prolonged hyperalgesia, suggesting that immune cells need to have GRK2 to help resolve pain (Willemen et al., 2010). “Peripheral monocytes are important for tuning the system—for determining whether or not pain becomes chronic,” Heijnen said.
Heijnen hypothesized that monocytes might help resolve prolonged pain by their anti-inflammatory actions. The researchers showed that pain hypersensitivity in mice heterozygous for GRK2 could be reversed if they treated the mice with monocytes isolated from bone marrow of a wild-type mouse, but not from a mouse incapable of producing the anti-inflammatory agent interleukin 10 (IL-10; Willemen et al., 2014).
Heijnen then presented unpublished data showing that monocytes are not the only peripheral immune cells involved in chronic pain. “All of a sudden, it seems, you also need peripheral T cells to recover,” Heijnen said. Using a model of chemotherapy (paclitaxel)-induced peripheral neuropathy (CIPN), she found that mice lacking recombination-activation gene 1 (Rag-1), an enzyme necessary for T cell function, did not recover from treatment with paclitaxel the way wild-type mice did. Preliminary experiments indicate that CD8-positive T cells in particular aid in pain resolution by somehow interacting with the IL-10 pathway. “T cells are regulating pain resolution,” said Heijnen, “but they do not need to make IL-10; they set the scene for other cells to make it.”
Heijnen believes that measures of monocyte and T cell function as well as GRK2 levels could potentially be used as pain biomarkers. “I would love to look at those measures in patients, because I think we have peripheral markers that might predict their pain state,” Heijnen said. Heijnen is now exploring the memory response of T cells to injury in hopes that it might be exploited to develop a vaccine-like treatment to prevent or reverse chronic pain.
Kavelaars then presented a parallel line of published and new work investigating the role of GRK2 in sensory afferent neurons. The researchers knew that GRK2 worked to keep inflammation in check, and that it was downregulated in leukocytes during inflammatory conditions (e.g., see Vroon et al., 2005). “We thought, if GRK2 is so important for dampening inflammatory signaling and is decreased in various cells during inflammation, it might also be important for pain regulation in neurons,” she said.
The team showed previously that mice heterozygous for GRK2 specifically in sensory neurons (SNS-GRK2+/-) displayed prolonged hyperalgesia in response to PGE2 (Eijkelkamp et al., 2010). In wild-type mice, PGE2 induces hyperalgesia by signaling through cAMP to protein kinase A (PKA). But the hyperalgesia in SNS-GRK2+/- mice depended on activation of an alternative signaling protein called exchange protein directly activated by cAMP 1 (Epac1), which signaled, in turn, to PKCε. “Signaling events that work through cAMP in the nociceptor determine duration of the pain response,” said Kavelaars.
Using Levine’s hyperalgesic priming model, Kavelaars next showed that priming with either carrageenan or with PKCε created an imbalance in nociceptor levels of GRK2 and Epac1 (Wang et al., 2013). “In the primed situation, it doesn't matter which protein changes, but as long as that intricate balance is disturbed, you get an extended pain response,” Kavelaars said. “If you bring them into balance, you normalize the response.”
How does the imbalance lead to prolonged pain? Kavelaars hypothesized that GRK acts as a tonic inhibitor of Epac1 activity. “We now know that Epac1 responds to cAMP downstream of algogens, which brings Epac to the plasma membrane where it activates the GTPase Rap1 and, further downstream, PKCε, leading to hyperalgesia.” She presented unpublished work showing that GRK inhibits Epac signaling by phosphorylating the protein, which keeps Epac out of the plasma membrane.
Work from another member of the group showed previously that Epac1 signals to Piezo2, a mechanically sensitive ion channel found in sensory neurons (Eijkelkamp et al., 2013). Kavelaars is currently building on that work to determine how Piezo2, Epac1, and GRK2 interact to regulate neuronal sensitization. Kavelaars suggested that an Epac inhibitor might have translational potential to alleviate chronic pain.
Image credit: American Pain Society
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.