A group of researchers from Charité – Universitätsmedizin Berlin has discovered a new mechanism of long-lasting pain relief.
The cell-signaling protein interleukin-4 induces a specific type of blood cell to produce endogenous opioids at the site of inflammation.
The researchers’ findings have been published in the JCI Insight.
The inflammatory response is mediated by a number of blood-derived immune cells.
These produce cytokines, cell-signaling proteins which either enhance or reduce inflammation and pain.
Thanks to its anti-inflammatory properties, one of these cytokines – known as interleukin-4 (IL-4) – is already being used to treat pain.
The team, led by Prof. Halina Machelska from Charité’s Department of Experimental Anesthesiology on Campus Benjamin Franklin, used an animal model of sciatic pain to study the analgesic mechanisms of IL-4.
Initially, a single injection of IL-4 near the inflamed nerve produced pain relief which only lasted for several minutes.
When repeated daily, however, injections reduced pain for up to eight days, even in the absence of further IL-4 injections.
This resulted from the IL-4-induced accumulation of M2 macrophages, a type of immune system scavenger cell which produces opioids and thereby reduces pain.
Isolated M2 macrophages (blue nuclei and red dots) produce endogenous opioids such as enkephalin (green dots). The image is credited to Machelska/ Charité.
Prof. Machelska proposes that not general inhibition of inflammation, but fostering the beneficial properties of the M2 macrophages is most promising to tackle pathological pain.
“Our findings are relevant to many immune-mediated diseases, ranging from arthritis to neurodegenerative diseases and cancer.”
The M2 macrophages were then isolated from the inflamed nerve and transferred into a different animal, where they also reduced pain. When the researchers studied the isolated cells in greater detail, they found that these cells produced various endogenous opioids, such as endorphin, enkephalin and dynorphin which activated opioid receptors at the site of inflammation.
“As these analgesic effects occur at the peripheral nerves, outside the brain, it is possible to prevent undesirable side effects such as sedation, nausea and addiction,” explains Prof. Machelska. She adds:
“These findings may offer new perspectives in our endeavors to develop alternative pain management options for patients.”
IL-4 is an antiinflammatory cytokine acting as a pleiotropic regulator of numerous immune and inflammatory processes. It is typically secreted by T helper 2 lymphocytes, mast cells, eosinophils, and basophils and plays a protective role in neurological disorders (e.g., multiple sclerosis, spinal cord injury). The beneficial actions of IL-4 are considered to result from the inhibition of the production and release of proinflammatory cytokines, chemokines, proteases, and reactive oxygen species (1, 2). Earlier studies reported analgesic actions of IL-4 to be mediated by dampening the proinflammatory cytokine response in animal models (3–5). Importantly, pathological pain, such as pain resulting from nerve injury, is associated with neuroinflammation, as immune cells, including macrophages, accumulate at the damaged nerves. Current research predominately focuses on the contribution of these cells to pain pathogenesis (6–8). However, macrophages are functionally diverse and comprise various subtypes, including classically activated proinflammatory M1 and alternatively activated antiinflammatory M2 populations. M1 macrophages are induced by proinflammatory cytokines and bacterial lipopolysaccharides and secrete various proinflammatory mediators, IL-1β, TNF, and nitric oxide (9, 10), which activate sensory neurons and exacerbate pain (6, 8, 11). In contrast, M2 macrophages release low amounts of proinflammatory molecules but higher levels of antiinflammatory mediators, such as IL-10. Furthermore, IL-4 is critical for the skewing of macrophages toward an M2 phenotype (2, 10), which was suggested to attenuate pain by elevating IL-10 expression in animals (12).
Notably, immune cells, such as lymphocytes, neutrophils, and macrophages, contain opioid peptides, Met-enkephalin (ENK), β-endorphin (END), and dynorphin A 1-17 (DYN), which upon release reduce pain in animal models and in humans (13–18). Additionally, we have recently shown that opioid peptides are secreted by IL-4–polarized M2 macrophages in vitro (19).
In this study, we demonstrate that repetitive IL-4 application at the damaged nerves produced long-lasting analgesia via endogenous opioids in a mouse model of neuropathy-induced pathological pain. Specifically, IL-4 locally induced M2 macrophages to produce opioid peptides, which via activation of peripheral opioid receptors attenuated neuropathy-triggered mechanical hypersensitivity. The analgesic effects persisted for several days after cessation of IL-4 treatment. Our results suggest that the endogenous opioid system is crucial to the action of IL-4 and M2 macrophages in pain control.
Our main finding in this study is that IL-4–induced M2 macrophages continuously produced opioids to relieve pain. Hence, IL-4 repetitively applied at the injured peripheral nerve shifted macrophages from the M1 to M2 phenotype, which produced opioid peptides (ENK, END, DYN).
The opioids activated peripheral opioid receptors (δ, μ, κ) and ameliorated nerve injury-triggered mechanical hypersensitivity, beyond the discontinuation of IL-4 treatment.
Specifically, IL-4 primarily increased F4/80+ macrophage counts at damaged nerves, and these cells expressed low mRNA levels of proinflammatory markers (Il-1β, Tnf, iNos) and enhanced mRNA levels of antiinflammatory markers (Il-10, Arg-1, Ym1), supporting their M2 status. Concurrently, IL-4–induced macrophages expressed elevated levels of opioid peptides (ENK, END, DYN) and mRNAs of their precursors (Penk, Pomc, Pdyn).
Single-cell analysis revealed the higher percentage of macrophages coexpressing Arg-1 and Penk mRNAs after IL-4 treatment, proving that opioids were produced by M2 cells. Adoptive transfer of these cells diminished mechanical hypersensitivity in recipient mice, directly showing their analgesic actions. Persistent IL-4–induced analgesia was indeed opioid-dependent, since it was abolished by opioid peptide antibodies and opioid receptor antagonists.
Upon injury, blood monocyte-derived macrophages are recruited to the damaged tissue, and by secretion of proinflammatory mediators, including proinflammatory cytokines, contribute to the generation of pain (7, 21–23).
Accordingly, we detected higher mRNA levels of proinflammatory mediators (Il-1β, Tnf, iNos) than antiinflammatory markers (Il-10, Arg-1, Ym1) in F4/80+ macrophages isolated from injured nerves of control (vehicle-treated) mice (on day 22 after CCI), indicating their M1 status. Concomitantly, we measured low levels of opioid peptides (ENK, END, DYN) and mRNAs of their precursors (Penk, Pomc, Pdyn).
Thus, as all these effects correlated with the CCI-induced hypersensitivity, not only classical pro/antiinflammatory molecule imbalance, but also endogenous opioid deficiency in macrophages, appear relevant to pain pathogenesis. Hence, strategies predominately based on dampening the proinflammatory properties of macrophages might be insufficient to inhibit pain.
We used IL-4 as a therapeutic agent and did not aim to mimic its endogenous levels, since they are apparently insufficient to resolve pain, as the CCI-triggered hypersensitivity persisted in the control (vehicle-treated) group throughout the whole time course, up to day 26 after CCI. Using flow cytometry, we found that IL-4 elevated numbers of immune cells at the injured nerves, which could result from the IL-induced immune cell extravasation and/or proliferation (31–33).
Importantly, F4/80+ macrophages were the predominant cell population after IL-4 treatment. Previous studies found a correlation between IL-4–induced analgesia and decreased expression of Il-1β and Tnf, or increased expression of IL-10 and M2 markers (Arg-1, Cd206).
Clearly, these conditions do not reflect the in vivo IL-4 effects, and macrophages were not directly examined in those studies. In contrast, we have tested fresh, uncultured F4/80+ macrophages isolated by IMS from injured nerves and found that after in vivo IL-4 treatment, they downregulated mRNAs of M1 markers (Il-1β, Tnf, iNos) and upregulated mRNAs of M2 markers (Il-10, Arg-1, Ym1).
Importantly, these M2 macrophages synthesized higher amounts of opioid peptides than M1 cells. This was evidenced by elevated mRNA levels of opioid peptide precursors (Penk, Pomc, Pdyn) measured by qRT-PCR and confirmed by single-cell FISH, which showed Penk and Arg-1 mRNA coexpression. The M2 macrophages also contained elevated levels of ENK, END, and DYN proteins determined by enzyme immunoassays.
A typical pathway of IL-4–induced M2 macrophage polarization involves activation of the transcription factor signal transducer and activator of transcription 6, which enhances transcription of M2-associated genes and decreases transcription of M1-associated genes (1, 2, 9). It will thus be interesting to examine whether this pathway is also involved in the IL-4–induced upregulation of opioid peptides in M2 macrophages.
Furthermore, the F4/80+ macrophages isolated from injured nerves of IL-4–treated, but not vehicle-treated, donor mice diminished mechanical hypersensitivity after adoptive transfer at damaged nerves in recipient mice, clearly showing that M2 cells account for IL-4–induced analgesia.
The longer-lasting analgesia after the second macrophage injection is in line with our previous study, which showed that after the first injection of in vitro–polarized M2 macrophages, only small proportion of cells remained at the nerves of donor mice, possibly due to their removal by endogenous cells, and the second injection was more efficient (19).
Moreover, the reduction of IL-4–induced analgesia by opioid peptide antibodies and opioid receptor antagonists applied at the CCI site clearly demonstrates that the local endogenous opioid system is essential for pain control by IL-4 and M2 macrophages. All 3 opioid peptides (ENK, END, DYN) and receptors (δ, μ, κ) are involved, since the selective blockade of each was equally effective.
Additionally, the inhibition of IL-4–induced analgesia by NLXM, an opioid receptor antagonist with limited blood-brain barrier permeability (34), suggests that the effects were mediated by peripheral opioid receptors.
This has important implications because in contrast to opioid receptors in the brain, the activation of peripheral opioid receptors is devoid of serious side effects such as respiratory arrest, sedation, and addiction (35, 36). Interestingly, all these opioid effects occurred both 24 hours and 5 days after discontinuation of IL-4 treatment; of note, the analgesia was equally efficient and did not diminish even at the latter time point.
Since this persistent analgesia was not attenuated by IL-4Rα antibody, it was apparently independent of IL-4Rα, regardless whether they are expressed on immune cells or neurons (37–39), but appears to involve constitutive release of opioid peptides from M2 macrophages. This is in line with our earlier in vitro experiments, in which extracellular levels of opioid peptides were measured (18 hours) after the removal of IL-4 from the medium (19).
Conversely, the short-lasting (5 minutes), IL-4–induced analgesia clearly involved IL-4Rα, since it was diminished by IL-4Rα antibody coinjected with IL-4 (day 21 after CCI). This suggests an acute direct action of IL-4 via IL-4Rα, possibly involving the release of opioids, which will be addressed in a follow-up study.
These findings suggest that when macrophages are polarized by IL-4 into the M2 phenotype, they can continuously use opioids to ameliorate nerve injury-triggered mechanical pain.
The involvement of δ-, μ- and κ-opioid receptors at the CCI site in analgesia, mediated by IL-4–induced M2 macrophages, is supported by studies showing that all 3 receptors were detected in sensory fibers (16) and μ and δ receptors were upregulated (40–42) at the nerve injury site. The CCI predominantly results in the degeneration of Aβ fibers (43), and thus the main remaining sensory fibers are C and Aδ fibers.
Both fiber types express opioid receptors (44–47) and transmit mechanical and heat stimuli (48, 49). Consistently, exogenous opioid receptor agonists applied at the CCI site attenuated mechanical and heat hypersensitivity (40, 46, 50–52).
However, here we found that endogenous opioid peptides, derived from IL-4–induced M2 macrophages, attenuated mechanical but not heat hypersensitivity. This is in agreement with our previous study, in which heat hypersensitivity was attenuated by perineurally applied exogenous opioids, but not by opioid peptides, including ENK, END, and DYN (53).
Even though both exogenous and endogenous opioids activate the same opioid receptors, it is possible that these ligands initiate different downstream mechanisms to interact with various pain modality-dependent ion channels or intracellular pathways (36, 53), and further studies are required.
Interestingly, other studies found that M2 macrophages polarized by morphine or peroxisome proliferator-activated receptor-γ agonist reduced mechanical but not heat hypersensitivity in models of inflammatory and postoperative pain (28, 54).
Additionally, IL-4–knockout mice displayed enhanced, acute mechanical but unaltered heat sensitivity (55), although the reasons for these differences were not provided. Thus, as the role of immune cells in the modulation of different pain modalities remains to be clarified, it appears that opioid-dependent actions of IL-4 and M2 macrophages are mainly beneficial in diminishing mechanical hypersensitivity. Because we used male mice, it will also be important to examine females, since the involvement of macrophages in pain may be sex dependent (56).
In conclusion, in this project we explored pain-inhibiting actions of macrophages in response to IL-4 treatment. This is a timely area of research because the lack of and the need for such studies have been increasingly recognized (8, 57).
We propose that fostering the beneficial effects of intrinsic M2 macrophages is more promising than the general inhibition of neuroinflammation for tackling pathological pain. The actions of IL-4 are believed to be mediated primarily by the inhibition of proinflammatory mediators (2, 58). Yet, here we provide evidence that the endogenous opioid system is essential to the actions of IL-4 and M2 macrophages in pain control.
Interestingly, IL-4 blood levels were reduced in patients with chronic widespread pain (including fibromyalgia and complex regional pain syndrome type 1) compared with healthy volunteers (59) and tended to be lower in patients with painful peripheral neuropathies than patients with painless peripheral neuropathies (60).
It might thus be interesting to test IL-4 for pain treatment in clinical trials. Notably, IL-4 therapy (for 6 weeks or 3 months) was well tolerated and diminished inflammation in patients with psoriasis (61, 62). Together, our findings may have wide implications, since IL-4 and M2 cells play a role in inflammatory and neurodegenerative diseases (2, 9, 10, 63).
The opioid system may be particularly beneficial in peripheral neuroinflammatory conditions, since the activation of peripheral opioid receptors can provide analgesia without centrally mediated adverse effects (35, 36).
Nonetheless, our study does not preclude the search for strategies targeting other macrophage-derived mediators, including antiinflammatory cytokines and specialized proresolving mediators.
Reference information: JCI Insight. 2020;5(4):e133093.https://doi.org/10.1172/jci.insight.133093.