interleukin-8 and Intervertebral-Disc-Degeneration

Embelin is a natural benzoquinone compound that displays a beneficial effect in various inflammatory-related diseases. However, the effect of embelin on degeneration of intervertebral disc (IDD), a chronic inflammatory disorder, has not been reported. This study was attempted to explore the therapeutic action of embelin on IDD in vitro. Network pharmacology analysis was performed for evaluating the link between embelin and IDD. The human nucleus pulposus cells (NPCs) were stimulated with IL-1β to induce inflammation. Cell viability of NPCs was assessed by CCK-8 assay. Western blotting was conducted to detect the expression levels of PI3K, p-PI3K, Akt, p-Akt, cleaved caspase-3, caspase-3, Bax, Bcl-2, p65 and p-p65. Apoptotic deaths of NPCs were examined by TUNEL assay. The production of COX-2, IL-6, IL-8, and TNF-α was examined by ELISA. It can be seen that 16 overlapping genes were selected from 109 possible targets of embelin and 342 possible targets of IDD. KEGG pathway enrichment analysis showed that the PI3K/Akt signaling pathway was a close link between embelin and IDD. We found that embelin dose-dependently improved the cell viability in IL-1β-stimulated NPCs. Embelin elevated the relative levels of p-PI3K/PI3K and p-Akt/Akt in IL-1β-stimulated NPCs. IL-1β induced a significant increase in apoptotic deaths of NPCs, which was attenuated by embelin treatment. IL-1β-induced alternations in expression levels of apoptotic-related proteins including cleaved caspase-3, Bax and Bcl-2 were prevented by embelin treatment. Pretreatment with LY294002 (an inhibitor of PI3K) reversed the inhibitory effect of embelin on IL-1β-induced apoptosis in NPCs. Embelin treatment caused inhibitory effects on the IL-1β-stimulated production of COX-2, IL-6, IL-8, and TNF-α, which were abolished by LY294002 treatment. Furthermore, embelin treatment prevented IL-1β-induced phosphorylation of p65 in NPCs, while LY294002 elevated the embelin-caused decrease in p-p65/p65 level. Overall, embelin protected human NPCs against IL-1β-stimulated apoptosis and inflammation by regulating the PI3K/Akt signaling pathway. These findings provided new ideas for the clinical usage of embelin in the prevention and treatment of IDD.

Topics: Apoptosis; bcl-2-Associated X Protein; Benzoquinones; Caspase 3; Cells, Cultured; Cyclooxygenase 2; Humans; Inflammation; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; Nucleus Pulposus; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Necrosis Factor-alpha

Behçet's disease (BD) is a chronic systemic vasculitis with a wide range of clinical findings. It has both autoinflammatory and autoimmune features and manifests with recurrent inflammatory attacks involving the innate immune system. Recently, autoinflammation has started to take place in the pathogenesis of intervertebral disc degeneration. The aim of this study is to evaluate the relationship between intervertebral disc degeneration and BD. We evaluated patients with BD who suffered neck or low back pain in the last 1 year. Eighty four patients underwent musculoskeletal system examination with MRI imaging of the cervical and lumbar vertebrae, and serum levels of IL6, IL8, and TNF-α were determined. The mean age was 47.7 ± 11.5 (range 20-68) years. Cervical and/or lumbar herniation was detected in the MRI imaging of 65 (77.3%) out of 84 patients. The mean IL8 levels of the group with pain and disc herniation and the group with pain and bulging were statistically significantly higher than the other groups (p = 0.007; p = 0.045, respectively). Chronic inflammation in BD may cause disc degeneration and radicular pain to begin and progress earlier in patients.

Topics: Adult; Aged; Behcet Syndrome; Humans; Interleukin-8; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Low Back Pain; Magnetic Resonance Imaging; Middle Aged; Young Adult

The degeneration of an intervertebral disc (IVD) is a major cause of lower back pain. IVD degeneration is characterized by the abnormal expression of inflammatory cytokines and matrix degradation enzymes secreted by IVD cells. In addition, macrophage-mediated inflammation is strongly associated with IVD degeneration. However, the precise pathomechanisms of macrophage-mediated inflammation in IVD are still unknown. In this study, we developed a microfluidic platform integrated with an electrical stimulation (ES) array to investigate macrophage-mediated inflammation in human nucleus pulposus (NP). This platform provides multiple cocultures of different cell types with ES. We observed macrophage-mediated inflammation and considerable migration properties via upregulated expression of interleukin (IL)-6 (p < 0.001), IL-8 (p < 0.05), matrix metalloproteinase (MMP)-1 (p < 0.05), and MMP-3 (p < 0.05) in human NP cells cocultured with macrophages. We also confirmed the inhibitory effects of ES at 10 μA due to the production of IL-6 (p < 0.05) and IL-8 (p < 0.01) under these conditions. Our findings indicate that ES positively affects degenerative inflammation in diverse diseases. Accordingly, the microfluidic electroceutical platform can serve as a degenerative IVD inflammation in vitro model and provide a therapeutic strategy for electroceuticals.

Topics: Cells, Cultured; Electric Stimulation; Humans; Inflammation; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; Microfluidics; Nucleus Pulposus

Degeneration of the intervertebral disc (IVD) is a major contributor to low back pain (LBP). IVD degeneration is characterized by abnormal production of inflammatory cytokines secreted by IVD cells. Although the underlying molecular mechanisms of LBP have not been elucidated, increasing evidence suggests that LBP is associated particularly with microglia in IVD tissues and the peridiscal space, aggravating the cascade of degenerative events. In this study, we implemented our microfluidic chemotaxis platform to investigate microglial inflammation in response to our reconstituted degenerative IVD models. The IVD models were constructed by stimulating human nucleus pulposus (NP) cells with interleukin-1β and producing interleukin-6 (129.93 folds), interleukin-8 (18.31 folds), C-C motif chemokine ligand-2 (CCL-2) (6.12 folds), and CCL-5 (5.68 folds). We measured microglial chemotaxis (p < 0.05) toward the conditioned media of the IVD models. In addition, we observed considerable activation of neurodegenerative and deactivation of protective microglia via upregulated expression of CD11b (p < 0.001) and down-regulation of CD206 protein (p < 0.001) by soluble factors from IVD models. This, in turn, enhances the inflammatory milieu in IVD tissues, causing matrix degradation and cellular damage. Our findings indicate that degenerative IVD may induce degenerative microglial proinflammation, leading to LBP development.

Topics: Culture Media, Conditioned; Cytokines; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Ligands; Low Back Pain; Microglia

Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), a secreted protein associated with inflammation, is believed to possess momentous and multiple anti-inflammatory and tissue-protective properties. However, the role and potential mechanism of TSG-6 in cervical disk degeneration (CDD) are still not clear. Hence, we aimed to explore the effect of TSG-6 on CDD.. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or enzyme-linked immunosorbent assay was applied to detect the expression level of TSG-6 and IL-1β in normal and degenerated nucleus pulposus (NP) tissues. Then, qRT-PCR and western blot were adopted to test the TSG-6 protein expression after IL-1β treatment (10 ng/mL) in human NP cells (HNPCs). After over-expressing TSG-6, qRT-PCR was also utilized to evaluate the expression of TNF-α, IL-8, and IL-6 and the synthesis of sulfated glycosaminoglycans (sGAGs), western blot to check the expression of extracellular matrix (ECM) proteins [collagen II, aggrecan, and matrix metalloproteinase-3 (MMP-3)], pain-related molecules (CGRP, calcitonin gene-related peptide; NGF, nerve growth factor; SP, substance P), and PI3K/Akt signaling pathway-related proteins.. Briefly speaking, TSG-6 and IL-1β expression levels were significantly increased in CDD patient tissues; and IL-1β treatment could significantly increase TSG-6 expression in HNPCs. Further research revealed that, in addition to greatly promoting sGAGs synthesis, TSG-6 over-expression also inhibited TNF-α, IL-8, and IL-6 expression and ECM degradation in IL-1β-induced HNPCs. (The collagen II and aggrecan expression was up-regulated and MMP-3 expression was down-regulated.) Furthermore, over-expression of TSG-6 could decrease the levels of CGRP, NGF, and SP protein expression and activate the PI3K/Akt signaling pathway in IL-1β-treated HNPCs.. TSG-6 inhibits inflammatory responses, ECM degradation, and expression of pain-related molecules in IL-1β-induced HNPCs by activating the PI3K/Akt signaling pathway.

Topics: Aggrecans; Calcitonin Gene-Related Peptide; Cells, Cultured; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; Matrix Metalloproteinase 3; Nerve Growth Factor; Nucleus Pulposus; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Necrosis Factor-alpha

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

Topics: Animals; Annulus Fibrosus; Cattle; Culture Media, Conditioned; Gene Expression Regulation; Inflammation; Injections; Interleukin-8; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Models, Biological; Nucleus Pulposus; Organ Culture Techniques; Tumor Necrosis Factor-alpha

Intervertebral disc (IVD) degeneration with chronic low back pain is associated with neo-vascularisation into the deeper IVD regions. During this process, endothelial cells (ECs), which are primarily responsible for angiogenesis, interact with the adjacent annulus fibrosus (AF) cells, which are the first line of defence against the invasion of vascular structures into deeper IVD regions. However, the accumulation of inflammatory and catabolic enzymes that results from this interaction promotes matrix degradation and an inflammatory response. Thus, regulating the production of these mediators and catabolic enzymes could ameliorate IVD degeneration. Photobiomodulation (PBM) therapy is a non-invasive stimulation known to have biologically beneficial effects on wound healing, tissue repair, and inflammation. Here, we examined the effects of PBM, administered at various wavelengths (645, 525, and 465 nm) and doses (16, 32, and 64 J/cm

Topics: Adult; Annulus Fibrosus; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Radiation; Endothelial Cells; Extracellular Matrix; Female; Gene Expression Regulation; Humans; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; Low-Level Light Therapy; Male; Matrix Metalloproteinases; Middle Aged; Models, Biological; Neovascularization, Pathologic

Notochordal cells (NCs) reside in the core of the healthy disc and produce soluble factors that can stimulate nucleus pulposus cells (NPCs). These NC-derived factors may be applied in intervertebral disc regeneration for treatment of low-back pain. However, identification of the active soluble factors is challenging. Therefore a novel approach to directly use porcine NC-rich NP matrix (NCM) is introduced. We explored porcine NCM's anabolic effects on bovine NPCs harvested from caudal discs of adolescent and adult (2-2.5 vs. 4-6 year old) cows. NC-conditioned medium (NCCM) and NCM were produced from porcine NC-rich NP tissue. Bovine NPCs were cultured in alginate beads for 4 weeks in base medium (BM), NCCM, and NCM to investigate NCM's regenerative potential. Porcine NCM increased glycosaminoglycan (GAG) content of both adolescent and adult bovine NPCs. This was through increased proliferation of adolescent bovine NPCs, whereas in adult bovine NPCs, it was mostly through increased GAG production per NPC. Furthermore, adolescent bovine NPCs were cultured in BM and porcine NCM treated with interleukin (IL)-1β to investigate NCM's potential in an inflammatory environment. Addition of IL-1β enhanced

Topics: Animals; Cattle; Cells, Cultured; Culture Media, Conditioned; Extracellular Matrix; Interleukin-1beta; Interleukin-8; Intervertebral Disc Degeneration; Notochord; Nucleus Pulposus; Swine

The aim of the study was to identify inflammatory cytokines/chemokines associated with neuroinflammation and periphery-to-CNS inflammatory cross-talk in degenerative disc disease (DDD) and lumbar disc herniation (LDH), common causes of low back pain (LBP). A secondary aim was to investigate the associations between cytokines and symptom severity.. In total, 40 DDD and 40 LDH patients were recruited from a surgical waiting list, as well as 39 healthy controls (HC) and 40 cerebrospinal fluid (CSF) controls. The subjects completed questionnaires and pressure algometry was performed at the lumbar spine and forearm. The CSF, serum and disc tissues were collected during surgery. Inflammatory mediators TNF, INFg, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and MCP1 were analysed by immunoassay (Meso Scale Discovery) and quantitative real-time polymerase chain reaction (qPCR) was used for analysis of IL-6, IL-8, MCP1 and TSPO expression in intervertebral discs (IVDs).. In the LDH group, we found elevated IL-8 concentrations in CSF indicating neuroinflammation, while IL-8 and MCP1 concentrations in serum were lower compared to HC. The IVD expression of IL-6, IL-8 and TSPO was lower in LDH patients compared to DDD. LDH patients had a positive correlation between IL-8 concentrations in CSF and serum and IL-8 in CSF was associated with higher pain intensity and increased spinal pressure pain sensitivity. The MCP1 concentration in serum was associated with higher global pain ratings and increased spinal pressure pain sensitivity, while IL-6 serum concentration correlated with the intensity of the neuropathic pain component (leg pain) in LDH patients. IVD expression of TSPO in LDH patients was associated with increased intensity of back pain. No differences were found in cytokine CSF concentrations between DDD patients and CSF controls, but DDD patients had lower IL-8 and MCP1 serum concentrations than HC. In female DDD patients, IL-8 and MCP1 concentrations in serum were associated with increased intensity of back pain.. Our results suggest that neuroinflammation mediated by elevated IL-8 concentrations in CSF and IL-8 mediated periphery-to-CNS inflammatory cross-talk contributes to pain in LDH patients and suggest a link between TSPO expression in discs and low back pain.

Topics: Adult; Aged; Chemokines; Cytokines; Female; Humans; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Low Back Pain; Lumbar Vertebrae; Male; Middle Aged; Neuroimmunomodulation; Pain; Receptors, GABA

Latent infection of Propionibacterium acnes was considered as a new pathogeny for low back pain (LBP); however, there is no credible animal evidence or mechanism hypothesis. This study proved that P. acnes is a causative pathogen of bacteria-induced LBP and investigated its underlying mechanism. For this, P. acnes was firstly identified in patients' degenerated intervertebral disc (IVDs) samples. The results of patients' Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ), Japanese Orthopaedic Association (JOA), and Oswestry Disability Index (ODI) scores indicated that P. acnes-positive patients showed more severe LBP and physical disability. Then, a P. acnes-inoculated lumbar IVDs model was established in rats. The results of paw/foot withdrawal threshold and qRT-PCR indicated that P. acnes-inoculated rats had obvious LBP in behavioral evaluation and over-expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in IVDs. Subsequently, enzyme-linked immunosorbent assay (ELISA) results demonstrated that increased expression of IL-8 or CINC-1 (the homolog of IL-8 in rats) in the P. acnes-positive IVDs of human and rats. The CINC-1 injected animal model proved that the cytokines were able to induce LBP. Finally, the co-culture experiments showed that nucleus pulposus cells (NPCs) were able to respond to P. acnes and secreted IL-8/CINC-1 via TLR-2/NF-κB p65 pathway. In conclusion, P. acnes had strong association with LBP by stimulating NPCs to secrete pro-algesic factor of IL-8/CINC-1 via TLR2/NF-κBp65 pathway. The finding may provide a promising alternative therapy strategy for LBP in clinical. KEY MESSAGES: Patients with P. acnes-positive IVDs tended to have more severe LBP, physical disability, and increased IL-8 expressions. P. acnes can induce LBP via IL-8/CINC-1 in IVDs. P. acnes stimulate the NPCs to secrete pro-algesic factor of IL-8/CINC-1 via TLR2/NF-κBp65 pathway.

Topics: Animals; Cells, Cultured; Chemokine CXCL1; Gram-Positive Bacterial Infections; Host-Pathogen Interactions; Humans; Interleukin-8; Intervertebral Disc Degeneration; Low Back Pain; Nucleus Pulposus; Propionibacterium acnes; Rats; Signal Transduction; Toll-Like Receptor 2; Transcription Factor RelA

Low back pain (LBP) is the leading global cause of disability and is associated with intervertebral disc degeneration (DD) in some individuals. However, many adults have DD without LBP. Understanding why DD is painful in some and not others may unmask novel therapies for chronic LBP. The objectives of this study were to a) identify factors in human cerebrospinal fluid (CSF) associated with chronic LBP and b) examine their therapeutic utility in a proof-of-concept pre-clinical study.. Pain-free human subjects without DD, pain-free human subjects with DD, and patients with chronic LBP linked to DD were recruited and lumbar MRIs, pain and disability levels were obtained. CSF was collected and analyzed by multiplex cytokine assay. Interleukin-8 (IL-8) expression was confirmed by ELISA in CSF and in intervertebral discs. The SPARC-null mouse model of progressive, age-dependent DD and chronic LBP was used for pre-clinical validation. Male SPARC-null and control mice received systemic Reparixin, a CXCR1/2 (receptors for IL-8 and murine analogues) inhibitor, for 8 weeks. Behavioral signs of axial discomfort and radiating pain were assessed. Following completion of the study, discs were excised and cultured, and conditioned media was evaluated with a protein array.. IL-8 was elevated in CSF of chronic LBP patients with DD compared to pain-free subjects with or without DD. Chronic inhibition with reparixin alleviated low back pain behaviors and attenuated disc inflammation in SPARC-null mice.. These studies suggest that the IL-8 signaling pathway is a viable therapy for chronic LBP. FUND: Supported by NIH, MMF, CIHR and FRQS.

Topics: Adult; Animals; Cytokines; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Interleukin-8; Intervertebral Disc Degeneration; Low Back Pain; Magnetic Resonance Imaging; Male; Mice; Mice, Knockout; Middle Aged; Osteonectin; Signal Transduction; Sulfonamides

The intervertebral disc (IVD) is an avascular structure, and is therefore stable under hypoxic conditions. Previous studies have demonstrated that hypoxia might be related to symptomatic degenerative disc diseases (DDDs); however, the pathomechanism is still poorly understood.. To identify the effect of hypoxia on the production of inflammatory mediators, angiogenic factors, and extracellular matrix-regulating enzymes of IVD cells during inflammatory reactions.. Human nucleus pulposus (NP) and annulus fibrosus (AF) cells harvested during surgery for DDDs were cultured in macrophage conditioned media or interleukin (IL)-1β-stimulated media under hypoxic (2%) and normoxic (21%) conditions. Hypoxia-inducible factor-1α transcription factor activation was analyzed by western blotting. IL-6, IL-8, vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), matrix metalloproteinase (MMP)-1, MMP-3, tissue inhibitor of metalloprotease (TIMP)-1, and TIMP-2 in conditioned media were measured by an enzyme-linked immunosorbent assay.. NP cells expressed higher hypoxia-inducible factor-1α in the IL-1β-stimulated group under hypoxic condition. MMP-1 was significantly increased in the AF cells under hypoxic condition; TIMP-1 and TIMP-2 were significantly decreased in both naïve NP and AF cells during hypoxia. Both cells in macrophage conditioned media significantly diminished the production of IL-6 and VCAM, while VEGF significantly increased during hypoxia. After 1 ng/mL IL-1β stimulation, IL-8, VEGF, MMP-1, and MMP-3 were significantly increased in both cell types during hypoxia, while VCAM, TIMP-1, and TIMP-2 were decreased.. We found that hypoxia can enhance the angiogenic ability of IVD during inflammatory reactions, and cause progress in development of DDD via extracellular matrix regulation in this in vitro study.

Topics: Blotting, Western; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Macrophages; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Neovascularization, Pathologic; Tissue Inhibitor of Metalloproteinase-3; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

The aim of this study was to investigate whether repopulating the degenerating intervertebral disk (IVD) with articular chondrocytes will decrease inflammation in the degenerating rabbit IVD.. This was a biologic study in a rabbit IVD-injury model in vivo. Dual cell tracking methods (infrared dye labeling and adenovirus transduction) were used to demonstrate the viability of allogeneic articular chondrocytes injected into degenerating rabbit IVDs. Interleukin 8 gene expression was determined via real-time polymerase chain reaction. Infiltrating inflammatory cells (macrophages, T cells, or neutrophils) were examined with immunohistochemistry. The IVDs were also examined by routine histology.. Articular chondrocytes labeled with infrared dye were detected in the degenerating IVDs at both 2 and 8 wks after injection. At the 2-wk time point, interleukin 8 gene expression was comparable in IVDs injected with chondrocytes and in intact disks as control (P = 0.647), whereas its expression in IVDs injected with saline increased 50-fold (P = 0.028). Transgene expression of red fluorescent protein, β-galactosidase, and human bone morphogenetic protein 7 diminished at 8 wks after injection. IVDs injected with chondrocytes overexpressing human bone morphogenetic protein 7 did not show lower interleukin 8 gene expression or improved histology. Macrophages were consistently detected by immunohistochemistry in the cartilage formed around the needle insertion sites in both the saline and chondrocyte groups, whereas neither T cells nor neutrophils were detected.. Allogeneic rabbit articular chondrocyte survived in the degenerating rabbit IVDs for at least 8 wks. Cell treatment resulted in reduced IVD inflammation but did not significantly improve IVD structure.

Topics: Allografts; Animals; Bone Morphogenetic Protein 7; Cell Transplantation; Chondrocytes; Disease Models, Animal; Down-Regulation; Gene Expression Regulation; Graft Rejection; Graft Survival; Humans; Interleukin-8; Intervertebral Disc Degeneration; Rabbits; Random Allocation; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators.. Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media.. Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture.. AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration.

Topics: Adult; Capillaries; Cell Survival; Cells, Cultured; Collagen Type II; Culture Media, Conditioned; Endothelial Cells; Endothelium, Vascular; Extracellular Matrix; Female; Humans; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Metalloproteases; Middle Aged; Neovascularization, Pathologic; Nerve Growth Factor; Platelet-Derived Growth Factor; Vascular Endothelial Growth Factor A

The burst fracture of a vertebra is the result of a complex loading procedure and is often associated with intervertebral disc (IVD) degeneration. Likewise, the presumed etiologies are (i) the structural perturbation of the IVD/end plate, (ii) the impact of loading energy alone, and (iii) the depressurization of the nucleus pulposus.. To describe the pathogenesis of post-traumatic disc degeneration (DD) by comparing the severity and patterns of degeneration with different injury models.. New data from an in vitro organ culture study are compared with the previous work on the same model system.. To investigate in detail the contribution of each factor (i-iii) to DD, we extended our previous work to compare three different segmental trauma processes in a rabbit full-organ in vitro model: burst fracture (Group A, etiologies i-iii), equienergetic loading without a fracture (Group B, ii), and endplate puncturing (Group C, iii). DD markers (apoptosis, necrosis, matrix remodeling, inflammation) were monitored up to 28 days posttrauma. Gene transcription data were subjected to principal component analysis and agglomerative hierarchical clustering to identify and compare pathologic patterns.. Only Group A showed the full profile of DD: reduced glycosaminoglycan content, increased caspase-3/7 and lactate dehydrogenase (LDH) activity, and elevated messenger RNA of catabolic (matrix metalloproteinase-1, -3, -13) and proinflammatory (tumor necrosis factor-alpha, interleukin [IL]-6, IL-8, and monocyte chemotactic protein-1) genes. In Group B, only catabolic and proinflammatory genes were slightly upregulated. In Group C, LDH but not caspase-3/7 activity was increased. Catabolic and proinflammatory genes were upregulated, although less compared with Group A. Principal component analysis revealed different transcription patterns for Group C.. The structural perturbation of the end plate/IVD, but not the loading energy or nuclear depressurization, promotes DD. In addition, end-plate puncturing triggers a different pathogenesis, consistent with a more continuous matrix remodeling process.

Topics: Animals; Apoptosis; Caspase 3; Caspase 7; Chemokine CCL2; Glycosaminoglycans; Inflammation; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; L-Lactate Dehydrogenase; Matrix Metalloproteinase 1; Rabbits; Spinal Fractures; Spinal Injuries; Tumor Necrosis Factor-alpha; Up-Regulation

In the present study, the influence of cytokines on 1-year recovery in lumbar radicular pain was examined.. In total, 110 patients with symptomatic lumbar disc herniation were followed for 1 year. Uni- and multivariate linear regression was used to assess the influence of interleukin (IL)-6, IL-8, disc degeneration and endplate changes (Modic changes) on the changes in the Oswestry Disability Index (ODI change; primary outcome) and visual analogue scale (VAS) for low back pain (LBP) and leg pain (secondary outcomes).. Less favourable ODI outcome correlated with higher serum IL-6 levels (B = -3.41, 95% CI -5.52 to -1.30, p = 0.002), non-surgical treatment (B = -7.03, 95% CI 1.21 to 12.84, p = 0.018), higher baseline back pain intensity (B = -2.28, 95% CI -3.21 to -1.35, p < 0.001) and low educational level (B = -5.57, 95% CI 0.66 to 10.47, p = 0.027). High VAS for LBP and leg pain at 1 year was associated with high levels of serum IL-6, higher back pain intensity and longer duration of lumbar radicular pain at baseline.. High serum IL-6 levels, but not disc degeneration or Modic changes, were associated with less favourable recovery in patients with lumbar radicular pain. Intense initial back pain, non-surgical treatment, lower educational level and longer duration of radicular pain before treatment also correlated with a slower recovery the first year after disc herniation.

Topics: Adult; Cohort Studies; Educational Status; Female; Humans; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Linear Models; Low Back Pain; Magnetic Resonance Imaging; Male; Middle Aged; Orthopedic Procedures; Physical Therapy Modalities; Prognosis; Prospective Studies; Radiculopathy; Time-to-Treatment; Treatment Outcome

Plasma-mediated radiofrequency-based ablation (coblation) is an electrosurgical technique currently used for tissue removal in a wide range of surgical applications, including lumbar microdiscectomy. In vitro and in vivo studies have shown the technique to alter the expression of inflammatory cytokines in the disc, increasing the levels of interleukin-8 (IL-8), which may promote maturation and remodeling of the disc matrix.. To better understand the effect of coblation treatment, this study characterizes the temporal and spatial pattern of healing after stab injury to the rabbit intervertebral disc, with and without plasma-mediated radiofrequency treatment.. A total of 23 New Zealand white rabbits.. Annular and nuclear stab injuries.. Sandwich enzyme-linked immunosorbent assay evaluated the concentrations of cytokines tumor necrosis factor-α, IL-1β, and IL-8. Histopathologic evaluations were performed on whole discs and end plates. Tissue sections were stained with Safranin-O to evaluate nucleus pulposus and annulus fibrosus proteoglycan content and with Alcian blue for extracellular proteoglycan content. Intradiscal leakage pressure was evaluated by injecting methylene blue dye into the nucleus.. Animals underwent annular and nuclear stab injuries on three consecutive lumbar discs (L2-L3 to L4-L5). The three levels were randomly assigned into one of the three groups for treatment with a plasma-mediated radiofrequency ablation device (TOPAZ; ArthroCare Corp., Austin, TX, USA): active treatment of the nucleus only (SN); active treatment of both nucleus and annulus (SNA); sham treatment. Unstabbed/untreated discs from L5-L6 (n=5) served as normal controls. Animals were euthanized at 4, 8, and 28 days postsurgery.. Tumor necrosis factor-α was detected in sham discs at 4 and 8 days, but not in coblation groups (SN or SNA); IL-1β was below detection in all three treatment groups. Interleukin-8 levels increased in all treatment groups at 4 and 8 days compared with normal control, peaking at 4th day for sham and SN groups and 8th day (p>.3) for the SNA group (a 2.5-fold increase). Pressure measurements revealed higher leakage in the SN group, but no statistically significant differences. Histopathology showed higher proteoglycan production by 28 days in the SNA and SN groups compared with sham. All three treatment groups showed ruptured annular fibers from the stab injury, but maintained the overall architecture. Remnants of notochordal tissue within the nucleus were evident in all treatment groups at 4 and 8 days, but were only found in sham group by 28 days. At this time, unlike the normal or sham controls, the nucleus of SN and SNA discs had fibrocartilaginous tissue with chondrocyte-like cells. Significant differences in the disc architecture grade were only noted when comparing normal controls with other groups by 28 days (p<.001).. Plasma-mediated radiofrequency ablation appears to have an anabolic effect on disc cells, stimulating proteoglycan and IL-8 production and maintaining annulus architecture. Coblation treatment appears to reduce cellular response to proinflammatory stimuli and restore overall disc architecture that may prove beneficial in a number of degenerative disc paradigms. Further studies are encouraged to investigate the therapeutic effect of the technique.

Topics: Animals; Catheter Ablation; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Lumbar Vertebrae; Proteoglycans; Rabbits; Tumor Necrosis Factor-alpha

Conditioned media (CM) of cartilaginous endplates (CEPs) of intervertebral discs were analyzed in a bioassay with regard to their influence on matrix turnover and inflammatory factors on nucleus pulposus (NP) cells of the same patient. CEP tissue underwent further histological and ultrastructural analysis.. To identify possible interactions between the CEP and the disc via molecular factors that may influence disc matrix degradation and to determine degenerative changes of CEP tissue.. Impaired endplate perme-ability due to degeneration and calcification is considered to be a key contributor to disc degeneration. An upregulation of metalloproteinases and inflammatory cytokines has been observed in degenerated intervertebral discs. Possibly, the CEP contributes to the regulation of disc matrix degradation via molecular interactions with the disc tissue.. CEP and NP cells from the same patients (n = 6) were investigated in a bioassay with regard to their influence on matrix turnover and inflammatory factors. We determined gene expression of NP cells in alginate beads that were exposed to CM of CEP punches (CEP-CM) from the same patients. The CEP-CMs were analyzed by protein array for inflammatory cytokines. Further CEP samples underwent histological (n = 15) and ultrastructural analysis (n = 8) to determine alterations of cell and matrix structure.. NP cells exposed to their donor-corresponding CEP-CM significantly upregulated interleukins (IL-6, IL-8) and matrix metalloproteinase (MMP-3, MMP-13) expression, and significantly decreased aggrecan and collagen type 2 expression. Proinflammatory cytokines were identified in the CEP-CM. The occurrence of apoptotic cells and degraded matrix fragments varied strongly between donors.. Our results indicate interactions between the CEP and the NP tissue via molecular factors that upregulate matrix degrading enzymes and inflammatory cytokines and thereby influence the pathophysiology of disc degeneration. Ongoing investigations will further identify the regulative role of potential molecular factors that are responsible for these degenerative alterations.. N/A.

Topics: Adult; Aged; Aggrecans; Cartilage; Cells, Cultured; Female; Humans; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Middle Aged

In canine intervertebral disc (IVD) disease, a useful animal model, only little is known about the inflammatory response in the epidural space.. To determine messenger RNA (mRNA) expressions of selected cytokines, chemokines, and matrix metalloproteinases (MMPs) qualitatively and semiquantitatively over the course of the disease and to correlate results to neurologic status and outcome.. Prospective study using extruded IVD material of dogs with thoracolumbar IVD extrusion.. Seventy affected and 13 control (24 samples) dogs.. Duration of neurologic signs, pretreatment, neurologic grade, severity of pain, and outcome were recorded. After diagnostic imaging, decompressive surgery was performed.. Messenger RNA expressions of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF), interferon (IFN)γ, MMP-2, MMP-9, chemokine ligand (CCL)2, CCL3, and three housekeeping genes was determined in the collected epidural material by Panomics 2.0 QuantiGene Plex technology. Relative mRNA expression and fold changes were calculated. Relative mRNA expression was correlated statistically to clinical parameters.. Fold changes of TNF, IL-1β, IL-2, IL-4, IL-6, IL-10, IFNγ, and CCL3 were clearly downregulated in all stages of the disease. MMP-9 was downregulated in the acute stage and upregulated in the subacute and chronic phase. Interleukin-8 was upregulated in acute cases. MMP-2 showed mild and CCL2 strong upregulation over the whole course of the disease. In dogs with severe pain, CCL3 and IFNγ were significantly higher compared with dogs without pain (p=.017/.020). Dogs pretreated with nonsteroidal anti-inflammatory drugs revealed significantly lower mRNA expression of IL-8 (p=.017).. The high CCL2 levels and upregulated MMPs combined with downregulated T-cell cytokines and suppressed pro-inflammatory genes in extruded canine disc material indicate that the epidural reaction is dominated by infiltrating monocytes differentiating into macrophages with tissue remodeling functions. These results will help to understand the pathogenic processes representing the basis for novel therapeutic approaches. The canine IVD disease model will be rewarding in this process.

Topics: Animals; Chemokine CXCL2; Decompression, Surgical; Disease Models, Animal; Dogs; Epidural Space; Female; Interleukin-1beta; Interleukin-8; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; RNA, Messenger; Tumor Necrosis Factor-alpha

Although inflammatory processes play an essential role in painful intervertebral disc (IVD) degeneration, the underlying regulatory mechanisms are not well understood. This study was designed to investigate the expression, regulation and importance of specific toll-like receptors (TLRs)--which have been shown to play an essential role e.g. in osteoarthritis--during degenerative disc disease.. The expression of TLRs in human IVDs was measured in isolated cells as well as in normal or degenerated IVD tissue. The role of IL-1β or TNF-α in regulating TLRs (expression/activation) as well as in regulating activity of down-stream pathways (NF-κB) and expression of inflammation-related genes (IL-6, IL-8, HSP60, HSP70, HMGB1) was analyzed.. Expression of TLR1/2/3/4/5/6/9/10 was detected in isolated human IVD cells, with TLR1/2/4/6 being dependent on the degree of IVD degeneration. Stimulation with IL-1β or TNF-α moderately increased TLR1/TLR4 mRNA expression (TNF-α only), and strongly increased TLR2 mRNA expression (IL-1β/TNF-α), with the latter being confirmed on the protein level. Stimulation with IL-1β, TNF-α or Pam3CSK4 (a TLR2-ligand) stimulated IL-6 and IL-8, which was inhibited by a TLR2 neutralizing antibody for Pam3CSK4; IL-1β and TNF-α caused NF-κB activation. HSP60, HSP70 and HMGB1 did not increase IL-6 or IL-8 and were not regulated by IL-1β/TNF-α.. We provide evidence that several TLRs are expressed in human IVD cells, with TLR2 possibly playing the most crucial role. As TLRs mediate catabolic and inflammatory processes, increased levels of TLRs may lead to aggravated disc degeneration, chronic inflammation and pain development. Especially with the identification of more endogenous TLR ligands, targeting these receptors may hold therapeutic promise.

Topics: Cells, Cultured; Chaperonin 60; Gene Expression Regulation; HMGB1 Protein; HSP70 Heat-Shock Proteins; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Lipopeptides; Mitochondrial Proteins; NF-kappa B; Osteoarthritis; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Laboratory study.. To evaluate expression of chemokine regulated and normal T cell expressed and secreted (RANTES)/C-C motif ligand 5 (CCL5) and interleukins in intervertebral discs (IVDs) specimens from patients with discogram-proven painful degeneration.. Discogenic back pain results in tremendous costs related to treatment and lost productivity. The relationship between inflammation, degeneration (IVD), and cytokine upregulation is well established, but other mediators of the inflammatory cascade are not well characterized.. Painful IVDs were taken from 18 patients undergoing surgery for discogenic pain with positive preoperative discogram. Painless control tissue was taken at autopsy from patients without back pain/spinal pathology or spinal levels with negative discograms resected for deformity.Quantitative real time polymerase chain reaction (qRT-PCR) was performed to evaluate RANTES, IL-1β, IL-6, and IL-8 expression in painful and control discs. RANTES and interleukin expression were analyzed on the basis of Pfirrmann grade.Disc cells were cultured in alginate beads using 2 groups: an untreated group and a group treated with 10 ng/mL IL-1β, 10 ng/mL TNF-α, and 1% fetal bovine serum to induce a degenerative phenotype.. Nine painless IVD specimens and 7 painful IVD specimens were collected. RANTES expression demonstrated a 3.60-fold increase in painful discs versus painless discs, a significant difference (P = 0.049). IL-1β expression demonstrated significantly higher expression in painful discs (P = 0.03). RANTES expression data demonstrated significant upregulation with increasing Pfirrmann grade (P = 0.045). RANTES expression correlated significantly with IL-1β expression (ρ = 0.67, P < 0.0001). RANTES expression increased more than 200-fold in the alginate culture model in cells treated with IL-1β/TNF-α, 1% fetal bovine serum (P < 0.001).. RANTES and IL-1β expression was significantly elevated in painful IVDs after careful selection of painless versus painful IVD tissue. RANTES expression was found to correlate significantly with expression of IL-1β. RANTES was upregulated by IL-1β/TNF-α/1% fetal bovine serum an in vitro treatment to induce a degenerative phenotype.

Topics: Adult; Aged; Animals; Cattle; Cells, Cultured; Chemokine CCL5; Cohort Studies; Culture Media; Fetal Blood; Gene Expression; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Middle Aged; Pain; Reverse Transcriptase Polymerase Chain Reaction; Serum; Tumor Necrosis Factor-alpha; Up-Regulation

Human annulus fibrosus (AF) cells were stimulated in vitro with interleukin (IL)-1β and exposed to biphasic electrical currents.. To identify the effect of biphasic electrical currents on the production of the extracellular matrix-modifying enzymes and inflammatory mediators in IL-1β-stimulated AF cells.. Symptomatic disc degeneration is an important cause of chronic intractable lumbar pain and is associated with macrophage-mediated inflammation in the AF. The inflammatory reaction relationship has not been studied in the AF.. Human AF cells were treated with 1 ng/mL IL-1β and cultured in a microcurrent generating chamber system. The levels of matrix metalloproteinase (MMP)-1, MMP-3, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, IL-6, IL-8, vascular endothelial growth factor (VEGF), insulin-like growth factor, and nitric oxide (NO) were measured. Expression of cyclooxygenase 2 and type I collagen mRNA was analyzed.. Compared with unstimulated cells, IL-1β-stimulated AF cells produced significantly higher levels of MMP-1, MMP-3, IL-6, IL-8, NO, and VEGF, and lower levels of TIMP-1 and TIMP-2. Exposure to a 250-mV/mm field induced time-dependent increases in IL-6, NO, MMP-1, TIMP-1, VEGF, and insulin-like growth factor-1 production. The cells exposed to 500-mV/mm field produced significantly less MMP-1, TIMP-1, IL-6, and VEGF than unexposed cells (MMP-1, 17.2 ± 4.7 ng/mL vs. 27.3 ± 3.9 ng/mL, P< 0.05; TIMP-1, 12.4 ± 3.3 ng/mL vs. 22.3 ± 2.1 ng/mL, P< 0.02; IL-6, 2.5 ± 0.9 ng/mL vs. 6.39 ± 1.90 ng/mL, P< 0.05; and VEGF, 0.1 ± 0.04 ng/mL vs. 0.44 ± 0.15 ng/mL, P< 0.03). NO production was markedly increased at 500 mV/mm (P< 0.0001).. We showed that exposure of IL-1β-stimulated AF cells to a 500 mV/mm inhibited MMP-1, IL-6, VEGF, and TIMP-1 production. The results suggest that biphasic electrical current stimulation may have efficacy in diminishing symptomatic disc degeneration.. N/A.

Topics: Cells, Cultured; Collagen Type I; Cyclooxygenase 2; Electric Stimulation; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Nitric Oxide; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Vascular Endothelial Growth Factor A

STUDY DESIGN.: Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1β or TNF-α. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. OBJECTIVE.: To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-α actions in these cells. SUMMARY OF BACKGROUND DATA.: Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. METHODS.: NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1β or TNF-α. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. RESULTS.: IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-α stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-α. MMP-13 induction by TNF-α was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-α fourfold. CONCLUSION.: The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-α, thus, helping preserve disc composition, structure, and function.

Topics: Adolescent; Adult; Aged; Aggrecans; Cells, Cultured; Collagen; Cyclooxygenase 2; Drug Synergism; Female; Gene Expression; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Matrix Metalloproteinase 3; Middle Aged; Proteoglycans; Receptors, Interleukin-6; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Young Adult

This study was conducted to investigate the expression of cytokines and growth factors in disc specimens obtained from patients with herniated nucleus pulposus (HNP) and degenerated disc disease (DDD).. MRI and Western blot analyses were performed to evaluate the levels of disc degeneration and the expression levels of cytokines and growth factors.. The levels of TNF-alpha and IL-8 were significantly greater in the DDD group than in the HNP group, but no statistical differences were observed in the expression of IL-1beta, IL-6 and IL-12 between the HNP and DDD groups. In addition, the expression of TGF beta, VEGF and NGF was significantly higher in the DDD group than in the HNP group.. The greater levels of cytokine and growth factor expression in the DDD group than in the HNP explain why discogenic patients usually have more severe back pain than patients with herniated discs.

Topics: Adult; Aged; Cytokines; Female; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-12; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Low Back Pain; Male; Middle Aged; Nerve Growth Factor; Spinal Diseases; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A