Katalin Sandor

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism

Objective An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation. Methods Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin. Results Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin. Conclusions The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

Collagen antibody–induced arthritis evokes persistent pain with spinal glial involvement and transient prostaglandin dependency

OBJECTIVE Pain is one of the most debilitating symptoms reported by rheumatoid arthritis (RA) patients. While the collagen antibody-induced arthritis (CAIA) model is used for studying the effector phase of RA pathologic progression, it has not been evaluated as a model for studies of pain. Thus, this study was undertaken to examine pain-like behavior induced by anticollagen antibodies and to assess the effect of currently prescribed analgesics for RA. In addition, the involvement of spinal glia in antibody-induced pain was explored. METHODS CAIA was induced in mice by intravenous injection of a collagen antibody cocktail, followed by intraperitoneal injection of lipopolysaccharide. Disease severity was assessed by visual and histologic examination. Pain-like behavior and the antinociceptive effect of diclofenac, buprenorphine, gabapentin, pentoxifylline, and JNK-interacting protein 1 were examined in mechanical stimulation experiments. Spinal astrocyte and microglia reactivity were investigated by real-time polymerase chain reaction and immunohistochemistry. RESULTS Following the induction of CAIA, mice developed transient joint inflammation. In contrast, pain-like behavior was observed prior to, and outlasted, the visual signs of arthritis. Whereas gabapentin and buprenorphine attenuated mechanical hypersensitivity during both the inflammatory and postinflammatory phases of arthritis, diclofenac was antinociceptive only during the inflammatory phase. Spinal astrocytes and microglia displayed time-dependent signs of activation, and inhibition of glial activity reversed CAIA-induced mechanical hypersensitivity. CONCLUSION CAIA represents a multifaceted model for studies exploring the mechanisms of pain induced by inflammation in the articular joint. Our findings of a time-dependent prostaglandin and spinal glial contribution to antibody-induced pain highlight the importance of using appropriate disease models to assess joint-related pain.

Interleukin-6 Secretion by Astrocytes Is Dynamically Regulated by PI3K-mTOR-Calcium Signaling

After contusion spinal cord injury (SCI), astrocytes become reactive and form a glial scar. While this reduces spreading of the damage by containing the area of injury, it inhibits regeneration. One strategy to improve the recovery after SCI is therefore to reduce the inhibitory effect of the scar, once the acute phase of the injury has passed. The pleiotropic cytokine interleukin-6 (IL-6) is secreted immediately after injury and regulates scar formation; however, little is known about the role of IL-6 in the sub-acute phases of SCI. Interestingly, IL-6 also promotes axon regeneration, and therefore its induction in reactive astrocytes may improve regeneration after SCI. We found that IL-6 is expressed by astrocytes and neurons one week post-injury and then declines. Using primary cultures of rat astrocytes we delineated the molecular mechanisms that regulate IL-6 expression and secretion. IL-6 expression requires activation of p38 and depends on NF-κB transcriptional activity. Activation of these pathways in astrocytes occurs when the PI3K-mTOR-AKT pathway is inhibited. Furthermore, we found that an increase in cytosolic calcium concentration was necessary for IL-6 secretion. To induce IL-6 secretion in astrocytes, we used torin2 and rapamycin to block the PI3K-mTOR pathway and increase cytosolic calcium, respectively. Treating injured animals with torin2 and rapamycin for two weeks, starting two weeks after injury when the scar has been formed, lead to a modest effect on mechanical hypersensitivity, limited to the period of treatment. These data, taken together, suggest that treatment with torin2 and rapamycin induces IL-6 secretion by astrocytes and may contribute to the reduction of mechanical hypersensitivity after SCI.

Rat Substrains Differ in the Magnitude of Spontaneous Locomotor Recovery and in the Development of Mechanical Hypersensitivity after Experimental Spinal Cord Injury

A number of different rodent experimental models of spinal cord injury have been used in an attempt to model the pathophysiology of human spinal cord injury. As a result, interlaboratory comparisons of the outcome measures can be difficult. Further complicating interexperiment comparisons is the fact that the rodent response to different experimental models is strain-dependent. Moreover, the literature is abundant with examples in which the same injury model and strain result in divergent functional outcomes. The objective of this research was to determine whether substrain differences influence functional outcome in experimental spinal cord injury. We induced mild contusion spinal cord injuries in three substrains of Sprague-Dawley rats purchased from three different European breeders (Scanbur, Charles River, and Harlan) and evaluated the impact of injury on spontaneous locomotor function, hypersensitivity to mechanical stimulation, and bladder function. We found that Harlan rats regained significantly more hindlimb function than Charles River and Scanbur rats. We also observed substrain differences in the recovery of the ability to empty the bladder and development of hypersensitivity to mechanical stimulation. The Harlan substrain did not show any signs of hypersensitivity in contrast to the Scanbur and Charles River substrains, which both showed transient reduction in paw withdrawal thresholds. Lastly, we found histological differences possibly explaining the observed behavioral differences. We conclude that in spite of being the same strain, there might be genetic differences that can influence outcome measures in experimental studies of spinal cord injury of Sprague-Dawley rats from different vendors.

Spinal release of tumour necrosis factor activates c-Jun N-terminal kinase and mediates inflammation-induced hypersensitivity

Mounting evidence points to individual contributions of tumour necrosis factor‐alpha (TNF) and the c‐Jun N‐terminal kinase (JNK) pathway to the induction and maintenance of various pain states. Here we explore the role of spinal TNF and JNK in carrageenan‐induced hypersensitivity. As links between TNF and JNK have been demonstrated in vitro, we investigated if TNF regulates spinal JNK activity in vivo.

Identification and quantification of neuropeptides in naïve mouse spinal cord using mass spectrometry reveals [des-Ser1]-cerebellin as a novel modulator of nociception.

Neuropeptide transmitters involved in nociceptive processes are more likely to be expressed in the dorsal than the ventral horn of the spinal cord. This study was designed to examine the relative distribution of neuropeptides between the dorsal and ventral spinal cord in naïve mice using liquid chromatography, high‐resolution mass spectrometry. We identified and relatively quantified 36 well‐characterized full‐length neuropeptides and an additional 168 not previously characterized peptides. By extraction with organic solvents we identified seven additional full‐length neuropeptides. The peptide [des‐Ser1]‐cerebellin (desCER), originating from cerebellin precursor protein 1 (CBLN1), was predominantly expressed in the dorsal horn. Immunohistochemistry showed the presence of CBLN1 immunoreactivity with a punctate cytoplasmic pattern in neuronal cell bodies throughout the spinal gray matter. The signal was stronger in the dorsal compared to the ventral horn, with most CBLN1 positive cells present in outer laminae II/III, colocalizing with calbindin, a marker for excitatory interneurons. Intrathecal injection of desCER induced a dose‐dependent mechanical hypersensitivity but not heat or cold hypersensitivity. This study provides evidence for involvement of desCER in nociception and provides a platform for continued exploration of involvement of novel neuropeptides in the regulation of nociceptive transmission.

Cardiomyopathy, oxidative stress and impaired contractility in a rheumatoid arthritis mouse model

Objectives Patients with rheumatoid arthritis (RA) display an increased risk of heart failure independent of traditional cardiovascular risk factors. To elucidate myocardial disease in RA, we have investigated molecular and cellular remodelling of the heart in an established mouse model of RA. Methods The collagen antibody-induced arthritis (CAIA) RA mouse model is characterised by joint inflammation and increased inflammatory markers in the serum. We used CAIA mice in the postinflammatory phase that resembles medically controlled RA or RA in remission. Hearts were collected for cardiomyocyte isolation, biochemistry and histology analysis. Results Hearts from mice subjected to CAIA displayed hypertrophy (heart/body weight, mean±SD: 5.9±0.8vs 5.1±0.7 mg/g, p<0.05), fibrosis and reduced left ventricular fractional shortening compared with control. Cardiomyocytes from CAIA mice showed reduced cytosolic [Ca2+]i transient amplitudes (F/F0, mean±SD: 3.0±1.2vs 3.6±1.5, p<0.05) that was linked to reductions in sarcoplasmic reticulum (SR) Ca2+ store (F/F0, mean±SD: 3.5±1.3vs 4.4±1.3, p<0.01) measured with Ca2+ imaging. This was associated to lower fractional shortening in the cardiomyocytes from the CAIA mice (%FS, mean±SD: 3.4±2.2 vs 4.6%±2.3%, p<0.05). Ca2+ handling proteins displayed oxidation-dependent posttranslational modifications that together with an increase in superoxide dismutase expression indicate a cell environment with oxidative stress. Conclusions This study shows that inflammation during active RA has long-term consequences on molecular remodelling and contractile function of the heart, which further supports that rheumatology patients should be followed for development of heart failure.

Collagen antibody-induced arthritis (CAIA) evokes transient inflammation but persistent allodynia

Background and objectives Pain is one of the most agregious symptoms reported by patients with rheumatoid arthritis (RA), a chronic disease affecting 1% of the population. RA is characterised by infiltration of inflammatory cells into the joints, synovial hypertrophy and bone erosion. Injection of CII antibodies intravenously to mice induces arthritis-like symptoms and a joint pathology that resembles human RA. While CII antibody-induced arthritis (CAIA) is a common model in the rheumatology field, it has not been evaluated as a model of arthritis-induced pain. Hence, the aim with our study was to characterise this model from a pain perspective. Material and method QB and B10.RIII mice (males, 25–35 g), were injected with collagen antibody cocktail or saline (control) intravenously on day 0 and 25 μg LPS or saline intraperitoneally on day 5. Clinical scores of arthritis (visual scoring), tactile allodynia (von Frey filaments) and locomotion (CLAMS system) were assessed for 40 days. The antinociceptive effect of diclofenac (30 mg/kg), buprenorphine (0.1 mg/kg), gabapentin (100 mg/kg), anakinra (300 mg/kg) and pentoxifylline (30 mg/kg, 30 μg intrathecally) was assessed. Lumbar spinal cords were processed for immunohistochemistry (GFAP and Iba-1, marker of astrocyte and microglia activity, respectively) and quantitative PCR (GFAP, Cd11b, pituitary adenylate cyclase-activating polypeptide (PACAP) and galanin). Results CAIA mice displayed significant increase in arthritic clinical score from days 6–18. Tactile allodynia was observed in the CAIA group throughout the study, starting prior to and outlasting reversal of the clinical score. Locomotion was significantly reduced in the postinflammatory phase in CAIA animals. Buprenorphine and gabapentin reversed CAIA-induced hypersensitivity during both the inflammatory and postinflammatory phase, while diclofenac only showed antiallodynic effect during the inflammatory phase. Surprisingly, anakinra did not display antinociceptive effect. Spinal gene and protein expression of GFAP, but not Cd11b or Iba-1, were elevated in the spinal cord in the CAIA group, as well as PACAP, a neuropeptide associated with pain modulation, both showing the most striking increase in the postinflammatory phase. Intrathecal injection of the glia-inhibitor pentoxifylline attenuated postinflammatory allodynia, pointing to potential astrocyte involvement in arthritis-associated pain. Conclusions This study demonstrates that CAIA generates robust and highly reproducible hypersensitivity making this model suitable for studies of joint pain driven by antibody-mediated inflammation, both during peak and remittent phases of the RA. Interestingly, the tactile allodynia was prostaglandin-mediated only in the inflammatory phase, indicating that arthritis-induced pain may be driven by different mechanisms dependent on stage of disease.

Spinal injection of newly identified cerebellin-1 and cerebellin-2 peptides induce mechanical hypersensitivity in mice

By screening for neuropeptides in the mouse spinal cord using mass spectrometry (MS), we have previously demonstrated that one of the 78 peptides that is expressed predominantly (> 6-fold) in the dorsal horn compared to the ventral spinal cord is the atypical peptide desCER [des-Ser1]-cerebellin, which originates from the precursor protein cerebellin 1 (CBLN1). Furthermore, we found that intrathecal injection of desCER induces mechanical hypersensitivity in a dose dependent manner. The current study was designed to further investigate the relative expression of other CBLN derived peptides in the spinal cord and to examine whether they share similar nociceptive properties. In addition to the peptides cerebellin (CER) and desCER we identified and relatively quantified nine novel peptides originating from cerebellin precursor proteins CBLN1 (two peptides), CBLN2 (three peptides) and CBLN4 (four peptides). Ten out of eleven peptides displayed statistically significantly (p < 0.05) higher expression levels (200-350%) in the dorsal horn compared to the ventral horn. Intrathecal injection of three of the four CBLN1 and two of the three CBLN2 derived peptides induced mechanical hypersensitivity in response to von Frey filament testing in mice during the first 6 h post-injection compared to saline injected mice, while none of the four CBLN4 derived peptides altered withdrawal thresholds. This study demonstrates that high performance MS is an effective tool for detecting novel neuropeptides in CNS tissues. We show the presence of nine novel atypical peptides originating from CBLN1, CBLN2 and CBLN4 precursor proteins in the mouse dorsal horn, whereof five peptides induce pain-like behavior upon intrathecal injection. Further studies are required to investigate the mechanisms by which CBLN1 and CBLN2 derived peptides facilitate nociceptive signal transmission.

Collagen antibody-induced arthritis (CAIA) leads to evoked and ongoing persistent pain-like behavior, but transient joint inflammation

Backgroundand objectives Joint pain is one of the most common types of persistent pain, and it is frequently reported to be a bothersome symptom by rheumatoid arthritis (RA) patients. RA is a chronic autoimmune inflammatory disease characterised by joint swelling, stiffness, and cartilage and bone erosion. Currently there are few effective treatments for persistent pain conditions, thus, it is important to increase our understanding of chronic pain mechanisms. The collagen type II antibody-induced arthritis (CAIA) model is commonly used in the rheumatology field. This model is based on injection of monoclonal antibodies against CII, which induces arthritis-like symptoms and a joint pathology that resembles human RA. However, CAIA has not been evaluated as a model of arthritis-induced pain. Hence, our aim was to characterise CAIA from a pain perspective. Materials and methods B10.RIII male mice were injected intravenously with collagen antibody cocktail (CAIA group) or saline on day 0 (control group). The CAIA group received lipopolysaccharide (LPS) and the control group saline intraperitoneally on day 5. Another group received intravenous saline followed by intraperitoneal LPS (LPS group). The degree of arthritis was assessed by visual scoring of the paws and evoked nociception by von Frey filament testing for 70 days. Ongoing pain-like behaviour (locomotion) was monitored for 48 h at two occasions and the antinociceptive effect on locomotion of the opioid buprenorphine (0.1 mg/kg intraperitoneal) was investigated. Spinal mRNA levels of inflammatory mediators were measured by real-time quantitative PCR. Results CAIA mice displayed transient signs of arthritis, while mechanical hypersensitivity was observed prior to and outlasting reversal of joint inflammation. The LPS group showed reduced nociceptive thresholds only the day after LPS injection. Locomotion was reduced both during and after inflammation in the CAIA group, which was reversed by buprenorphine in both phases. Interestingly, the mRNA level of IL-1β was elevated in the spinal cord during both phases, while other inflammatory mediators, such as tumour necrosis factor and high mobility group box 1 (HMGB1) were elevated only during the post-inflammatory phase. Conclusions This study demonstrates that CAIA generates robust and highly reproducible hypersensitivity, making this model suitable for studies of joint pain driven by antibody-mediated inflammation, both during peak and remittent phases of RA. Our findings also indicate that peripheral joint inflammation drives time-dependent spinal cytokine synthesis.