Recombinant IL-1Ra Stimulation of Native NP Cells Inhibits a Multitude of Pathological Features (original) (raw)
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European Journal of Pain, 2011
Neuropathic pain is a chronic pain state resulting from peripheral nerve injury, characterized by hyperalgesia and allodynia. We have reported that mice with genetic impairment of IL-1 signaling display attenuated neuropathic pain behavior and ectopic neuronal activity. In order to substantiate the role of IL-1 in neuropathic pain, WT mice were implanted subcutaneously with osmotic micropumps containing either IL-1ra or vehicle. Two days following the implantation, two models of neuropathic pain were used; partial nerve injury (spinal nerve transection, SNT), or complete nerve cut (spinal neuroma model). Mechanosensitivity was assessed seven consecutive days following SNT, and on day 7 recordings of spontaneous ectopic activity were performed. In the spinal nerve neuroma model, autotomy scores were recorded up to 35 days. Vehicle-treated mice developed significant allodynia and autotomy, and clear ectopic activity (4.1 ± 1.1% of the axons); whereas IL-1ra-treated mice did not display allodynic response, displayed delayed onset of autotomy and markedly reduced severity of autotomy scores, and displayed reduced spontaneous activity (0.8 ± 0.4% of the axons). To test whether IL-1 is involved in maintenance of mechanical allodynia, a separate group of WT mice was treated with a single injection of either saline or IL-1ra four days following SNT, after the allodynic response was already manifested. Whereas salinetreated mice displayed robust allodynia, acute IL-1ra treatment induced long-lasting attenuation of the allodynic response. The results support our hypothesis that IL-1 signaling plays an important role in neuropathic pain and in the ectopic neuronal activity that underling its development.
Interleukin-1 activity in lesioned peripheral nerve
Journal of Neuroimmunology, 1992
The cytokine interleukin-1 (IL-1) is involved in a wide range of inflammatory and immune responses. As such, IL-1 could play a role in peripheral nerve repair mechanisms. Specifically, by its already established properties as a regulator of nerve growth factor (NGF) synthesis, and as a chemotactant to macrophages. We examined, therefore, IL-1 production in injured mouse peripheral nerve. Injured nerve segments were incubated in serum free medium to produce conditioned medium (CM) that was then tested for IL-1 activity in a thymocyte proliferation assay. CM induced thymocyte proliferation in a dose-dependent manner. Proliferation was inhibited by the M20 IL-1 inhibitor, the IL-1 receptor antagonist, and antisera raised against recombinant mouse IL-la. Inhibitions produced by these three specific inhibitors of IL-l-induced thymocyte proliferation strongly suggest that proliferation induced by CM was mediated largely by IL-1 secreted by non-neuronal cells residing in the damaged nerve. IL-1 activity was detected within hours after lesion, and 1 week thereafter. The rapid and prolonged production of IL-1 indicates that IL-l-dependent mechanisms can play roles in the response of the peripheral nerve to injury: degeneration and regeneration. The regulation of NGF synthesis, and the recruitment of white blood cells, macrophages in particular, from blood into the damaged nerve tissue, are two such mechanisms.
British Journal of Pharmacology, 1995
Peripheral inflammation is associated with the local production of neuroactive inflammatory cytokines and growth factors. These may contribute to inflammatory pain and hyperalgesia by directly or indirectly altering the function or chemical phenotype of responsive primary sensory neurones. 2 To investigate this, inflammation was produced by the intraplantar injection of complete Freund's adjuvant (CFA) in adult rats. This resulted in a significant elevation in interleukin-l[ (IL-1[) and nerve growth factor (NGF) levels in the inflamed tissue and of the peptides, substance P and calcitonin generelated peptide (CGRP) in the L4 dorsal root ganglion 48 h post CFA injection. 3 The effects of a steroidal (dexamethasone) and a non-steroidal (indomethacin) anti-inflammatory drug on the levels of NGF and IL-IO in inflamed tissue were investigated and compared with alterations in behavioural hyperalgesia and neuropeptide expression in sensory neurones. 4 Systemic dexamethasone (120 jig kg-' per day starting the day before the CFA injection) had no effect on the inflammatory hyperalgesia. When the dose was administered 3 times daily, a reduction in mechanical and to a lesser extent thermal sensitivity occurred. Indomethacin at 2 mg kg-' daily (i.p.) had no effect on the hyperalgesia and a dose of 4 mg kg~' daily was required to reduce significantly mechanical and thermal hypersensitivity. 5 The increase in NGF produced by the CFA inflammation was prevented by both dexamethasone and indomethacin, but only at the higher dose levels. Dexamethasone at the lower and higher dose regimes diminished the upregulation of IL-1i whereas indomethacin had an effect only at the higher dose. 6 The increase in SP and CGRP levels produced by the CFA inflammation was prevented by dexamethasone and indomethacin at the lower and higher dose regimes. 7 Intraplantar injections of IL-1[ (0.01, 0.1 and 1 ng) produced a brief (6 h) thermal hyperalgesia and an elevation in cutaneous NGF levels which was prevented by pretreatment with human recombinant ILl receptor antagonist (IL-1 ra) (0.625 gg, i.v.). The thermal hyperalgesia but not the NGF elevation produced by intraplantar IL-1[ (1 ng) was prevented by administration of a polyclonal neutralizing anti-NGF serum. 8 ILl ra significantly reduced the mechanical hyperalgesia produced by CFA for 6 h after administration as well as the CFA-induced elevation in NGF levels. Anti-NGF pretreatment substantially reduced CFA-induced mechanical and thermal hyperalgesia without reducing the elevation in IL-1,B. 9 Intraplantar NGF (0.02, 0.2 and 2 Jg) injections produced a short lasting thermal and mechanical hyperalgesia but did not change IL-1i levels in the hindpaw skin. 10 Our results demonstrate that IL-13 contributes to the upregulation of NGF during inflammation and that NGF has a major role in the production of inflammatory pain hypersensitivity.
The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration
Arthritis research & therapy, 2005
In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1alpha, IL-1beta, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1beta-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1beta-converting enzyme. In addition, immunopositivity for these proteins, with the exception ...
International Journal of Experimental Pathology, 2006
Conventional therapies for low back pain (LBP) are purely symptomatic and do not target the cause of LBP, which in approximately 40% of cases is caused by degeneration of the intervertebral disc (DIVD). Targeting therapies to inhibit the process of degeneration would be a potentially valuable treatment for LBP. There is increasing evidence for a role for IL-1 in DIVD. A natural inhibitor of IL-1 exists, IL-1Ra, which would be an ideal molecular target for inhibiting IL-1-mediated effects involved in DIVD and LBP. In this study, the feasibility of ex vivo gene transfer of IL-1Ra to the IVD was investigated. Monolayer and alginate cultures of normal and degenerate human intervertebral disc (IVD) cells were infected with an adenoviral vector carrying the IL-1Ra gene (Ad-IL-1Ra) and protein production measured using an enzyme-linked immunosorbent assay. The ability of these infected cells to inhibit the effects of IL-1 was also investigated. In addition, normal and degenerate IVD cells infected with Ad-IL-1Ra were injected into degenerate disc tissue explants and IL-1Ra production in these discs was assessed. This demonstrated that both nucleus pulposus and annulus fibrosus cells infected with Ad-IL-1Ra produced elevated levels of IL-1Ra for prolonged time periods, and these infected cells were resistant to IL-1. When the infected cells were injected into disc explants, IL-1Ra protein expression was increased which was maintained for 2 weeks of investigation. This in vitro study has shown that the use of ex vivo gene transfer to degenerate disc tissue is a feasible therapy for the inhibition of IL-1-mediated events during disc degeneration.
European Cells and Materials
Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)-and endplate (EPCs)-derived cells to interleukin (IL)-1β treatment, through largescale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.
Journal of Experimental Orthopaedics, 2014
Background Inflammatory cytokines such as interleukin-1 beta (IL-1β) contribute to the progression of intervertebral disc degeneration. Previously we demonstrated, in vitro, that by delivering interleukin-1 receptor antagonist (IL-1ra) from poly(lactic co-glycolic acid) (PLGA) microspheres, we could attenuate the degradative effects of IL-1β on the nucleus pulposus (NP) for up to 20 days. The objective of this study was to undertake a preliminary investigation into whether microspheres could be successfully delivered to and retained in the disc in vivo, and whether IL-1ra released from those microspheres remained biologically active. For retention studies, fluorescently-labeled microspheres were delivered to the NPs of rat caudal discs. Rats were sacrificed at time points up to 56 days, and microspheres were localized using fluorescent microscopy. To investigate whether IL-1ra microspheres could effectively inhibit the effects of IL-1β in vivo, four disc levels were allocated to the...
Arthritis Research & Therapy, 2007
Data implicate IL-1 in the altered matrix biology that characterizes human intervertebral disc (IVD) degeneration. In the current study we investigated the enzymic mechanism by which IL-1 induces matrix degradation in degeneration of the human IVD, and whether the IL-1 inhibitor IL-1 receptor antagonist (IL-1Ra) will inhibit degradation. A combination of in situ zymography (ISZ) and immunohistochemistry was used to examine the effects of IL-1 and IL-1Ra on matrix degradation and metal-dependent protease (MDP) expression in explants of nondegenerate and degenerate human IVDs. ISZ employed three substrates (gelatin, collagen, casein) and different challenges (IL-1β, IL-1Ra and enzyme inhibitors). Immunohistochemistry was undertaken for MDPs. In addition, IL-1Ra was introduced into degenerate IVD explants using genetically engineered constructs. The novel findings from this study are: IL-1Ra delivered directly onto explants of degenerate IVDs eliminates matrix degradation as assessed by multi-substrate ISZ; there is a direct relationship between matrix degradation assessed by ISZ and MDP expression defined by immunohistochemistry; single injections of IVD cells engineered to over-express IL-1Ra significantly inhibit MDP expression for two weeks. Our findings show that IL-1 is a key cytokine driving matrix degradation in the degenerate IVD. Furthermore, IL-1Ra delivered directly or by gene therapy inhibits IVD matrix degradation. IL-1Ra could be used therapeutically to inhibit degeneration of the IVD.
European Cells and Materials, 2011
Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1β) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1β upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model system to investigate this. Bovine NP cells were cultured in agarose for 49 days in a defined medium containing transforming growth factor-beta 3, after which both mechanical properties and composition were evaluated and compared to native NP. The mRNA levels of NP cell markers were compared to those of freshly isolated NP cells. Glycosaminoglycan (GAG) content, aggregate modulus and hydraulic permeability of mature constructs were similar to native NP, and aggrecan and SOX9 mRNA levels were not signifi cantly different from freshly isolated cells. To investigate direct links between IL-1β and biomechanical changes, mature agarose constructs were treated with IL-1β, and effects on biomechanical properties, extracellular matrix composition and mRNA levels were quantifi ed. IL-1β treatment resulted in upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 4, matrix metalloproteinase-13 and inducible nitric oxide sythase, decreased GAG and modulus, and increased permeability. To evaluate the model as a test platform for therapeutic intervention, cotreatment with IL-1β and IL-1 receptor antagonist (IL-1ra) was evaluated. IL-1ra signifi cantly attenuated degradative changes induced by IL-1β. These results suggest that this in vitro model represents a reliable and cost-effective platform for evaluating new therapies for disc degeneration.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011
IL-1β and TNF are potential targets in the management of neuropathic pain after injury. However, the importance of the IL-1 and TNF systems for peripheral nerve regeneration and the mechanisms by which these cytokines mediate effects are to be fully elucidated. Here, we demonstrate that mRNA and protein levels of IL-1β and TNF are rapidly upregulated in the injured mouse sciatic nerve. Mice lacking both IL-1β and TNF, or both IL-1 type 1 receptor (IL-1R1) and TNF type 1 receptor (TNFR1), showed reduced nociceptive sensitivity (mechanical allodynia) compared with wild-type littermates after injury. Microinjecting recombinant IL-1β or TNF at the site of sciatic nerve injury in IL-1β- and TNF-knock-out mice restored mechanical pain thresholds back to levels observed in injured wild-type mice. Importantly, recovery of sciatic nerve function was impaired in IL-1β-, TNF-, and IL-1β/TNF-knock-out mice. Notably, the infiltration of neutrophils was almost completely prevented in the sciatic ...