Effect of pro‐inflammatory and immunoregulatory cytokines on human tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1071–1077, 2010     

Combined effects of TNF‐α, IL‐1β, and HIF‐1α on MMP‐2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl₂‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082). © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1008–1014, 2011     

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF‐α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC‐seeded PLG scaffold, or TNF‐α‐primed MSC‐seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post‐injury. In vivo, MSCs, regardless of priming, increased IL‐10 production and reduced the inflammatory factor, IL‐1α. Primed MSCs reduced IL‐12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti‐inflammatory factor IL‐4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post‐injury. Taken together delivery of TNF‐α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC‐induced healing response. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269–280, 2017.

     

Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

Quantitative flow cytometry to measure the TNF‐α and IL‐2 system after heart transplantation

Abstract After heart transplantation a high incidence of infections and malignancies is found. Not only immunosuppression, but also intrinsic cytokine systems with some unbalance, e.g. TNF‐α and IL‐2, can result in impaired immune competence and may have a role in these complications. The aim of this study was to assess the activity of the TNF‐α and IL‐2 systems after heart transplantation. In peripheral blood we measured expression of activation markers of TNF‐α (TNF‐R2) and IL‐2 (IL‐2Rα, IL‐2Rβ‐chain) on monocytes and lymphocytes using quantitative flow‐cytometric analysis. TNF‐R2 expression was significantly enhanced on monocytes and lymphocytes in patients after heart transplantation, while the expression of IL‐2Rα and IL‐2Rβ was not elevated. Increased TNF‐R2 expression in peripheral blood after heart transplantation reflects an activated TNF‐α system, leading to high levels of actives TNF‐R, which impairs TNF‐α bioavailibility and consequently leads to immune incompetence.     

The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin‐1β in human chondrocytes

In this study, we examined the effects of overexpression of SIRT1 on IL‐1β‐induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL‐1β. SIRT1 was also inhibited by siRNA under stimulation with IL‐1β. Gene expression changes were examined by real‐time PCR. The interaction of SIRT1 and p65 (NF‐κB) were examined by Western blotting. SIRT1, MMP‐13, and ADAMTS‐5 expressions in human cartilage were examined by immunohistochemistry. IL‐1β stimulation significantly up‐regulated MMP‐1, 2, 9, and 13 and ADAMTS‐5. Overexpression of SIRT1 significantly inhibited the up‐regulation of those genes caused by IL‐1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL‐1β‐induced acetylation of p65. SIRT1 expression was clearly detected in the non‐OA cartilage while MMP‐13 and ADAMTS‐5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP‐13 and ADAMTS‐5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL‐1β‐induced expressions of cartilage‐degrading enzymes partially through the modulation of the NF‐κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 531–537, 2013     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

Expression of interleukin‐1β, cyclooxygenase‐2, and prostaglandin E2 in a rotator cuff tear in rabbits

We investigated the specific factors related to shoulder pain due to a rotator cuff tear using a model in rabbits. A rotator cuff tear was surgically created, and the expression of interleukin‐1β (IL‐1β), prostaglandin E2 (PGE2), and cyclooxygenase‐2 (COX‐2) was analyzed. In the supernatant of the tissue culture of the torn tendon, IL‐1β production was detected. The amount of IL‐1β was highest 1 day after injury, and then decreased gradually to 21 days. PGE2, the mediator of pain and the product of COX‐2, was also detected in the supernatant of the tissue culture. The production of PGE2 significantly increased to 7 days after injury, and then decreased to 21 days. RT‐PCR analysis confirmed the mRNA expression of IL‐1β and COX‐2 in the torn tendon. Immunohistochemical study demonstrated that cells in the tendon stump were immunopositive for IL‐1β and COX‐2. Furthermore, in the affected joint, articular chondrocytes in the remote area from the tear expressed COX‐2 strongly. When the rotator cuff is torn, IL‐1β is produced in the torn tendon, and stimulates the expression of COX‐2 in not only the torn tendon but also in articular chondrocytes. The COX‐2 then produces PGE2, which would mediate shoulder pain. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:92–97, 2007     

TNFα From Classically Activated Macrophages Accentuates Epithelial to Mesenchymal Transition in Obliterative Bronchiolitis

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL‐1β and IL‐8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL‐1β than alternatively activated macrophages and dramatically accentuate TGF‐β1‐driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL‐1β, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti‐TNFα treatment improved forced expiratory volume in 1 second and 6‐min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.     

Protective role of IL‐1β against post‐arthroplasty Staphylococcus aureus infection

MyD88 is an adapter molecule that is used by both IL‐1R and TLR family members to initiate downstream signaling and promote immune responses. Given that IL‐1β is induced after Staphylococcus aureus infections and TLR2 is activated by S. aureus lipopeptides, we hypothesized that IL‐1β and TLR2 contribute to MyD88‐dependent protective immune responses against post‐arthroplasty S. aureus infections. To test this hypothesis, we used a mouse model of a post‐arthroplasty S. aureus infection to compare the bacterial burden, biofilm formation and neutrophil recruitment in IL‐1β‐deficient, TLR2‐deficient and wild‐type (wt) mice. By using in vivo bioluminescence imaging, we found that the bacterial burden in IL‐1β‐deficient mice was 26‐fold higher at 1 day after infection and remained 3‐ to 10‐fold greater than wt mice through day 42. In contrast, the bacterial burden in TLR2‐deficient mice did not differ from wt mice. In addition, implants harvested from IL‐1β‐deficient mice had more biofilm formation and 14‐fold higher adherent bacteria compared with those from wt mice. Finally, IL‐1β‐deficient mice had ～50% decreased neutrophil recruitment to the infected postoperative joints than wt mice. Taken together, these findings suggest a mechanism by which IL‐1β induces neutrophil recruitment to help control the bacterial burden and the ensuing biofilm formation in a post‐surgical joint. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1621–1626, 2011     

Increased IL‐1β expression and myofibroblast recruitment in subacromial bursa is associated with rotator cuff lesions with shoulder stiffness

We evaluated whether proinflammatory cytokine expression and myofibroblast recruitment in subacromial bursa was linked to rotator cuff lesions with shoulder stiffness. We analyzed expressions of IL‐1β, IL‐6, and TNF‐α in subacromial bursa and joint fluid collected from 14 patients with cuff tears with stiffness as a study group (Group I) and 14 patients with rotator cuff tears without shoulder stiffness as a control group (Group II) using real‐time RT‐PCR, immunohistochemistry, and ELISA. Myofibroblast apoptosis in subacromial bursa was analyzed using terminal deoxynucleotidyl transferase ‐mediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) and α‐smooth muscle actin immunofluorescence staining. Shoulder function was evaluated using the Constant score. Group I had higher mRNA expression (p < 0.001) and immunoreactivities (p < 0.001) of IL‐1β. They also had higher levels of IL‐1β, IL‐6, and TNF‐α in joint fluid. Increased IL‐1β mRNA expression in the subacromial bursa and IL‐1β levels in joint fluid were correlated with a preoperative deficit in shoulder motion (p < 0.001) and preoperative Constant scores (p < 0.001). Immunofluorescence observations showed that Group I subjects had more myofibroblasts (p < 0.001) than Group II. In Group II, a significant correlation was found between apoptotic myofibroblasts and total myofibroblasts (p = 0.002), but not in Group I (p = 0.510). Increased expression of IL‐1β and myofibroblast recruitment in the subacromial bursa in rotator cuff lesions are linked to shoulder stiffness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1090–1097, 2008     

Intra‐articular resveratrol injection prevents osteoarthritis progression in a mouse model by activating SIRT1 and thereby silencing HIF‐2α

We investigated the feasibility of the intra‐articular injection of resveratrol for preventing the progression of existing cartilage degeneration in a mouse model of osteoarthritis (OA). The effects of resveratrol on the expression of silent information regulator 2 type 1 (SIRT1), hypoxia‐inducible factor‐2α (HIF‐2α) and catabolic factors in OA cartilage was explored. OA was induced in the mouse knee via destabilization of the medial meniscus (DMM). Resveratrol was injected weekly into the operated knee beginning 4 weeks after surgery. The OA phenotype was evaluated via histological and immunohistochemical analyses at 8 weeks after DMM. Western blot analysis was performed to identify whether resveratrol modulated the interleukin (IL)‐1β‐induced expression of HIF‐2α in human chondrocytes. Histologically, resveratrol treatment preserved the structural homeostasis of the articular cartilage and the subchondral bone. Following resveratrol injection, the expression of collagen type II was retained, but the expression of inducible nitric oxide synthase and matrix metalloproteinase‐13 was reduced in OA cartilage. Moreover, the administration of resveratrol significantly induced the activation of SIRT1 and the inhibition of HIF‐2α expression in mouse OA cartilage and in IL‐1β‐treated human chondrocytes. These findings indicate that the intra‐articular injection of resveratrol significantly prevents the destruction of OA cartilage by activating SIRT1 and thereby suppressing the expression of HIF‐2α and catabolic factors. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1061–1070, 2015.     

Interleukin‐1β induces angiogenesis and innervation in human intervertebral disc degeneration

Degenerative disorders of the intervertebral discs (IVDs) are generally characterized by enhanced matrix degradation, angiogenesis, innervation, and increased expression of catabolic cytokines. In this study, we investigated the effects of inflammatory cytokines, IL‐1β, and TNF‐α, on the expression of an angiogenic factor, vascular endothelial growth factor (VEGF), and neurotrophic factors, nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF), in human IVD degeneration. IL‐1β and TNF‐α stimulated the gene expression of VEGF, NGF, and BDNF in nucleus pulposus (NP) cells isolated from patient tissues. Immunohistochemical results demonstrated a positive correlation between IL‐1β and VEGF/NGF/BDNF expression in human IVD tissues. RNA expression analysis of patient tissues also identified positive correlations between VEGF and platelet endothelial cell adhesion molecule‐1 (PECAM‐1) and between NGF/BDNF and protein gene product 9.5 (PGP9.5). Our findings suggest that IL‐1β is generated during IVD degeneration, which stimulates the expression of VEGF, NGF, and BDNF, resulting in angiogenesis and innervation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:265–269, 2011     

Hyaluronan inhibits matrix metalloproteinase‐13 in human arthritic chondrocytes via CD44 and P38

We investigated the effects of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated matrix metalloproteinase (MMP)‐13 production in human chondrocytes from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Secreted levels of MMP‐13 in conditioned media were detected by immunoblotting, while intracellular MMP‐13 synthesis in articular cartilage was evaluated by immunofluorescence microscopic analysis. Mitogen‐activated protein kinases (MAPKs), p38, extracellular signal‐regulated kinases (ERK), and c‐jun NH2‐terminal kinase (JNK) were assessed by Western blotting. IL‐1β (2 ng/ml) stimulates the secretion of MMP‐13 in both OA and RA chondrocytes. Inhibition studies using specific MAPK inhibitors revealed that IL‐1β induced MMP‐13 via p38 in both OA and RA chondrocytes. HA down‐regulates IL‐1β‐stimulated MMP‐13 and phosphorylated p38 (p‐p38) in a dose‐dependent manner (0.1, 1, 2, and 4 mg/ml). When used at 4 mg/ml, HA inhibits p‐p38 phosphorylation by more than 60%. In response to IL‐1β, RA chondrocytes express a higher level of p‐p38 than that of OA chondrocytes. Inhibition of CD44, using a blocking antibody, significantly reversed the inhibitory effect of HA on both MMP‐13 and p‐p38. Our study clearly shows that HA inhibits IL‐1β‐induced MMP‐13 via its principal receptor, CD44, and subsequent intracellular p38 MAPK signaling in OA and RA chondrocytes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:258–264, 2011     

Low‐level laser therapy in collagenase‐induced Achilles tendinitis in rats: Analyses of biochemical and biomechanical aspects

NSAIDs are widely prescribed and used over the years to treat tendon injuries despite its well‐known long‐term side effects. In the last years several animal and human trials have shown that low‐level laser therapy (LLLT) presents modulatory effects on inflammatory markers, however the mechanisms involved are not fully understood. The aim of this study was to evaluate the short‐term effects of LLLT or sodium diclofenac treatments on biochemical markers and biomechanical properties of inflamed Achilles tendons. Wistar rats Achilles tendons (n = 6/group) were injected with saline (control) or collagenase at peritendinous area of Achilles tendons. After 1 h animals were treated with two different doses of LLLT (810 nm, 1 and 3 J) at the sites of the injections, or with intramuscular sodium diclofenac. Regarding biochemical analyses, LLLT significantly decreased (p < 0.05) COX‐2, TNF‐α, MMP‐3, MMP‐9, and MMP‐13 gene expression, as well as prostaglandin E₂ (PGE₂) production when compared to collagenase group. Interestingly, diclofenac treatment only decreased PGE₂ levels. Biomechanical properties were preserved in the laser‐treated groups when compared to collagenase and diclofenac groups. We conclude that LLLT was able to reduce tendon inflammation and to preserve tendon resistance and elasticity. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1945–1951, 2012     

Interleukin‐17 synergizes with IFNγ or TNFα to promote inflammatory mediator release and intercellular adhesion molecule‐1 (ICAM‐1) expression in human intervertebral disc cells

Interleukin‐17 (IL‐17) is a cytokine recently shown to be elevated, along with interferon‐γ (IFNγ) and tumor necrosis factor (TNFα), in degenerated and herniated intervertebral disc (IVD) tissues, suggesting a role for these cytokines in intervertebral disc disease. The objective of our study was to investigate the involvement of IL‐17 and costimulants IFNγ and TNFα in intervertebral disc pathology. Cells were isolated from anulus fibrosus and nucleus pulposus tissues of patients undergoing surgery for intervertebral disc degeneration or scoliosis. The production of inflammatory mediators, nitric oxide (NOx), prostaglandin E2 (PGE2) and interleukin‐6 (IL‐6), as well as intercellular adhesion molecule (ICAM‐1) expression, were quantified for cultured cells following exposure to IL‐17, IFNγ, and TNFα. Intervertebral disc cells exposed to IL‐17, IFNγ, or TNFα showed a remarkable increase in inflammatory mediator release and ICAM‐1 expression (GLM and ANOVA, p < 0.05). Addition of IFNγ or TNFα to IL‐17 demonstrated a synergistic increase in inflammatory mediator release, and a marked increase in ICAM‐1 expression. These findings suggest that IVD cells not only respond with a catabolic phenotype to IL‐17 and costimulants IFNγ and TNFα, but also express surface ligands with consequent potential to recruit additional lymphocytes and immune cells to the IVD microenvironment. IL‐17 may be an important regulator of inflammation in the IVD pathologies. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:1–7, 2011     

Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL‐1β and p38 MAPK signal

Nucleus pulposus cells (NPCs) from degenerating disks produce catabolic and inflammatory factors, including interleukin (IL)‐1, nitric oxide (NO), prostaglandin E2 (PGE‐2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disk. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O₂ at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time quantitative polymerase chain reaction (Q‐PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL‐1β, NO, PGE‐2, MMP‐3, and TIMP‐1 in the conditioned media were quantified by enzyme‐linked immunosorbent assay (ELISA). Our data showed that HBO treatment decreased expression of IL‐1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE‐2, and MMP‐3, and increased TIMP‐1 expression in NPCs as compared with the atmospheric treatment. These results support the hypothesis that IL‐1β and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disk degeneration, and support our proposal that HBO treatment‐induced increase of the anabolic factor (TIMP‐1)/catabolic factor (MMP‐3) ratio may provide a therapeutic approach to slow the course of intervertebral disk degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:14–19, 2011