The researchers believe that the finding, which is published in the Journal of Experimental Medicine, can represent a general mechanism in autoimmunity and that the results can facilitate the development of new ways of reducing non-inflammatory pain caused by rheumatoid arthritis and other autoimmune diseases.
“We all know that inflammation is painful,” says Camilla Svensson, professor at the Department of Physiology and Pharmacology, Karolinska Institutet.
“But pain can appear before any sign of inflammation in the joints and can remain a problem after it has healed. Our aim was to find possible mechanisms to explain that.”
Rheumatoid arthritis is an autoimmune disease that occurs when immune cells attack the cartilage and bone of the joints.
The disease affects roughly one percent of the Swedish population.
A common early symptom is joint pain, but even before that, the body has started to produce immune antibodies against proteins in the joint.
Researchers at Karolinska Institutet have now studied how these autoantibodies can generate pain.
After injecting cartilage-binding autoantibodies into mice, which served as a model for human rheumatoid arthritis, the researchers found that the mice became more sensitive to pain even before they could observe any signs of inflammation in the joints.
Antibodies that had been designed not to activate immune cells and trigger inflammation also induced pain-like behavior in the mice, suggesting increased pain sensitivity in the joints.
The researchers found that the antibodies that caused the behavioral change form so called immune complexes, comprising clusters of antibodies and cartilage proteins in the joints.
These complexes activate pain cells via so-called Fc-gamma receptors, which the researchers discovered were present on pain neurons in the tissue.
When they cultivated pain neurons from the mice, the researchers found that the cells were activated when coming into contact with the antibody complexes.
The process was dependent upon the Fc-gamma receptors on the neurons but not on the presence of immune cells.
Antibodies in complex can thus act as pain-generating molecules in themselves, independently of the activity of the immune cells, as Camilla Svensson, one of the study’s two corresponding authors explains:
“Antibodies in these immune complexes can activate the pain neurons directly, and not, as previously thought, as a result of the destructive joint inflammation,” she says.
“The antibodies can affect the pain neurons also in conditions without any distinct tissue damage or inflammation.”
Although the study was conducted in mice, the researchers show that human pain neurons also have antibody receptors that are functionally similar to those they found on the mouse pain neurons, which leads them to believe that their findings are also relevant to humans.
The results can explain the early pain symptoms in rheumatoid arthritis patients.
However, joint and muscle pain are also common symptoms of other autoimmune diseases, and since this newly discovered mechanism operates through the constant part – the shaft – of the autoantibody, the researchers believe that it can explain non-inflammatory pain caused by other autoimmune diseases too.
“We think that this can be a general pain mechanism in effectively all autoimmune diseases in which these kinds of immune complex form locally in tissue,” says Professor Svensson.
More detailed study of what happens in the nerve cell when the antibody complex binds to the receptor could also lead to new targets for reducing the neuronal activity.
“By learning more about the molecular mechanisms of antibody-mediated pain we hope to lay the groundwork for a new way of reducing pain caused by rheumatoid arthritis and other autoimmune diseases,” says Rikard Holmdahl, professor at the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, and the study’s other corresponding author.
The study was financed with grants from the Swedish Research Council, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, the Ragnar Söderberg Foundation, the Torsten Söderberg Foundation, the Åke Wiberg Foundation, the Alfred Österlund Foundation, the Krapperup Foundation, the King Gustaf V 80-year Foundation, the Swedish Rheumatism Association, Hansa Medical AB, the Royal Physiographic Society, Karolinska Institutet’s funds, the Canadian Institutes of Health Research and the Guangdong province of China. The financiers have had no influence on the paper or the decision to publish it.
One of the researchers holds a patent on the use of a type of antibody used in the study and a royalty agreement with Hansa Medical, which also holds a patent on a particular application of the antibodies.
Autoantibodies and pain
The immune system is intricately linked to chronic pain.
When an injury or illness occurs, immune cells release substances that fight off infection and promote healing.
They also sensitize pain neurons to promote tissue-protective behaviors (Pinho-Ribeiro et al., 2017).
The adaptive immune system produces antibodies that tag invading pathogens so they can be identified and destroyed.
However, with autoimmune disorders, the immune system mistakes proteins of the host organism for those pathogens and produces antibodies against them.
These “self-antibodies,” or autoantibodies, lead to a range of symptoms depending on the protein they target.
But the antibody contribution to pain has often been overlooked (see PRF related news story).
“Antibodies and pain have been relatively ignored.
There’s a lot of literature about how macrophages or microglia are involved in pain, but not so much on how antibody-mediated disorders can promote pain,” explains Bennett.
For some autoimmune disorders such as Morvan’s syndrome or neuromyotonia, both of which feature hyperexcitability of peripheral nerves and thus spontaneous muscular activity, the body produces autoantibodies against CASPR2, a protein that localizes in specialized domains of myelinated neurons.
Although these disorders present with their own unique set of symptoms, neuropathic pain is common among them (Klein et al., 2012).
CASPR2 localizes a voltage-gated potassium channel called Kv1 to the juxtaparanode of myelinated axons, where the channel remains electrically inactive.
Following injury, however, the channels are relocated to the paranode, where they can suppress hyperexcitability (see figure below). In the current paper, the authors found that both CASPR2 and Kv1 are also expressed in the cell bodies of sensory neurons.
“These potassium channels essentially put the brake on neuronal excitability, and in their absence neurons become hyperexcitable,” explains Bennett.
When Bennett first moved to the University of Oxford, he had a conversation with coauthor Angela Vincent about a unique observation she made in her patients.
“She told me that, of her patients with CASPR2 autoantibodies, many seemed to experience pain,” he said.
This led the two to wonder if these patients experienced neuropathic pain because the CASPR2 autoantibodies caused mislocalization of Kv1 channels, resulting in hyperexcitability of sensory neurons.
CASPR2 antibodies are pathogenic
The group first tested whether CASPR2 autoantibodies (CASPR2-Abs) could actually cause pain.
“We used a classic passive immunization paradigm where you take an antibody you think is pathogenic and put it into an animal,” according to Bennett.
The researchers isolated antibodies from two patients who had high serum levels of CASPR2-Abs and were experiencing neuropathic pain.
“We chose these patients because neuropathic pain was a big component of their clinical presentation,” said Bennett.
First author John Dawes and colleagues treated wild-type mice daily with purified CASPR2-Abs from either patient 1 for 14 days or from patient 2 for 22 days, while assessing mechanical sensitivity with von Frey hairs.
“We were looking for any indication these antibodies were pathogenic and causal of pain in the patients,” according to Dawes.
Mice treated with CASPR2-Abs from patient 1 developed mechanical hypersensitivity by day 11, and animals treated with CASPR2-Abs from patient 2 developed it by day 15.
To control for nonspecific effects, the investigators treated other sets of mice with antibodies from healthy donors who lacked CASPR2-Abs and confirmed that no hypersensitivity was present.
These experiments showed that an autoimmune, peripheral neuropathic pain disorder could be passively transferred.
“We show for the first time that these antibodies can be directly pathogenic and cause pain” in mice, said Dawes.
Proposed Mechanism of Action of CASPR2-Ab Induced Pain. (A) VGKCCs [voltage-gated potassium channel complexes; CASPR2 is one component of these complexes] are localized in the juxtaparanodal region of myelinated fibers under normal conditions. (B)
Medium-sized DRG neurons extending Aδ fibers into the periphery where their terminals contain low-threshold D-hair mechanoreceptors are displayed in red. (C) CASPR2-Abs binding to the soma of medium-sized DRG neurons results in decreased membrane-bound VGKCCs, as well as redistribution along internal segments, resulting in hyperexcitable Aδ fibers. Reprinted from review by Hunt et al., 2018, with permission from Elsevier.
No inflammation or nerve injury, but still pain
It’s possible that introduction of a foreign antibody caused neuroinflammation that sensitized the mice.
To rule this out, the team examined the peripheral and central nervous systems for signs of inflammation.
There was no increase in markers for neutrophils, macrophages, lymphocytes, or inflammatory cytokines within the dorsal root ganglion (DRG), and no elevation in markers for neutrophils, lymphocytes, or activated astrocytes in the spinal cord.
Critically, the researchers found no trace of CASPR2-Abs in the spinal cord, suggesting they didn’t pass through the blood-brain barrier. Instead, CASPR2-Abs coated the cell bodies of DRG neurons.
“We then wanted to see whether the antibodies caused damage to the peripheral nervous system, which could also cause a general neuropathy,” said Dawes.
Using intra-epidermal nerve fiber density in the paw as a measure of nerve damage and examining peripheral nerve structure with electron microscopy, they found no abnormalities in the nerves of antibody-treated mice.
However, what they did see in these animals, here with immunostaining, was a decrease in both CASPR2 protein and Kv1 along the sciatic nerve.
“With no inflammation or nerve damage,” said Dawes, “it seemed likely that CASPR2-Abs were instead working through a novel mechanism involving Kv1 channels.”
Next, the team obtained mice genetically engineered to lack full-length CASPR2. Instead, these mice only expressed a version of the protein missing most of its extracellular portion, a modification that would interfere with its ability to interact with Kv1 channels.
Similar to the mice treated with patient-derived CASPR2-Abs, these animals were hypersensitive to mechanical stimulation. They also featured enhanced pain behaviors in response to hind paw injection of either capsaicin or formalin, chemicals that result in inflammation and activate pain neurons.
Releasing the brakes
The team also looked at how the loss of full-length CASPR2 and a decrease in Kv1 expression affected sensory neuron excitability in a series of electrophysiological and imaging experiments.
Using in vivo calcium imaging to directly observe cell body activity of sensory neurons in anesthetized animals while mechanically stimulating the hind paw, they found hyperexcitability in small- and medium-diameter neurons, which transmit nociceptive signals.
The researchers explored physiological changes in the cell bodies of cultured DRG neurons from mice lacking full-length CASPR2 using patch-clamp electrophysiology.
Here, too, small- and medium-diameter neurons showed increased excitability. Use of a potassium channel blocker revealed that this hyper-responsiveness was likely due to the loss of potassium channel function.
The group then turned to in vivo extracellular recordings of spinal cord neurons in anesthetized mice to examine changes of sensory neuron integration into the spinal cord. This, too, revealed hyperexcitability in response to mechanical stimulation of the paw with von Frey hairs.
Next, use of a tibial nerve-skin ex vivo preparation allowed for recording from the axons of tibial nerve fibers during mechanical stimulation of the paw.
Considering known electrophysiological properties and using various mechanical stimulation parameters to categorize sensory neuron subtypes, the authors found that only D-hair afferents were hyperexcitable when recording at the axon; these afferents are a type of low-threshold, A-delta mechanoreceptor that forms lanceolate endings around hair follicles in the skin and play a role in pain under pathological conditions.
“We do see hyperexcitability in other cell types when recording from the cell body, so I suspect that potassium channels are having a different effect depending on the compartment of the neuron you look at,” said Bennett.
Finally, the investigators tested whether treatment with CASPR2-Abs could cause changes in neuronal excitability similar to those seen in mice lacking full-length CASPR2 protein.
Patch-clamp electrophysiology at the level of the cell body showed that treating cultured sensory neurons from normal mice with patient-derived, CASPR2-Ab-containing plasma for 24 hours also increased neuronal excitability and decreased Kv1 expression.
Given that CASPR2-Abs bound the cell bodies of DRG neurons, the team concluded that CASPR2-Abs likely enhanced pain by regulating Kv1 trafficking to the cell bodies and axons of sensory neurons.
More information: Alex Bersellini Farinotti et al. Cartilage-binding antibodies induce pain through immune complex–mediated activation of neurons, The Journal of Experimental Medicine (2019). DOI: 10.1084/jem.20181657
Journal information: Journal of Experimental Medicine
Provided by Karolinska Institutet