Aims. To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. Methods. We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by flow cytometry as T-regs. Their correlations were calculated and the changes after anti-TNF-α therapy were compared. Results. The frequency of T-regs in PBMCs was positively correlated to ESR and CRP in AS (r = 0.35 and 0.43; p = 0.032 and 0.027, respectively), and there was also a significant correlation between serum level of TNF-α and CRP (p = 0.041). The frequency of T-regs in PBMCs positively correlated to serum levels of TNF-α, IL-10, and TGF-β, while IL-2, IL-4, and IFN-γ showed opposite results. After anti-TNF-α treatment, there were significantly lower serum levels of TNF-α, IL-10, TGF-β, and frequency of T-regs in PBMCs among these AS patients (p = 0.026, 0.032, 0.029, and 0.037, respectively). Conclusion. In AS patients, proinflammatory cytokine may give positive feedback to induce more T-reg production and anti-inflammatory cytokine secretion to suppress this inflammatory status, and they can be reversed by anti-TNF-α therapy. However, the detailed interactions among T-regs and complex cytokine networks in autoinflammatory diseases still need more studies and further functional assay. Cite this article: Bone Joint Res 2023;12(2):133–137

Introduction and Objective. Total joint replacement is indicated for osteoarthritis where conservative treatment has failed, and in the UK the number of patients requiring hip and knee replacements is set to increase with an ageing population. Survival of total hip replacements is around 85% at 20 years with the most common reason for revision being aseptic loosening of the implant secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can also cause pseudotumour formation. As revision surgery is associated with higher morbidity, mortality, infection rates, venous thromboembolism, resource demand and poorer subsequent function it is important to understand the mechanisms underlying the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4), an innate immune receptor, has been demonstrated to mediate deleterious immune responses by the Tyson-Capper research group, including inflammatory cytokine interleukin-8 (IL-8) secretion. Statin use in epidemiological studies has been associated with reduced overall risk of revision surgery after hip replacement. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses which can lead to osteolysis and pseudotumour formation. As literature from cardiological investigations demonstrate that statins can reduce the expression and responsiveness of TLR4, this could be an exciting mechanism to exploit to reduce the host immune response to orthopaedic wear debris, thereby improving implant survival by reducing immune mediated osteolysis. This ongoing study investigates simvastatin's effect on cobalt ion-mediated changes in gene and protein expression of interleukin-8 and soluble-ICAM-1 (sICAM-1) which is an angiogenic factor implicated in pseudotumour formation. Materials and Methods. TLR4-expressing human monocyte/macrophage THP-1 cells were pre-incubated with 50μM simvastatin for 2-hours or a vehicle control, before being exposed to exposed to 0.75mM cobalt chloride, in addition to a further 24-hour co-incubation with 50μM simvastatin or vehicle control. IL-8 protein and sICAM-1 secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Pre-treatment with simvastatin significantly reduced cobalt-mediated IL-8 protein secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells (p-value<0.0001). Work will be undertaken to determine changes in gene expression, the role of TLR4 in these responses and the effect of simvastatin on additional inflammatory markers. Conclusions. Simvastatin significantly reduces cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving aseptic loosening and pseudotumour formation

To identify the differences in inflammatory profiles between hip OA, knee OA and non-OA control cohorts and investigate the association between cytokine expression and clinical outcome measurements, specifically pain. A total of 250 individuals were recruited in three cohorts (100 knee OA, 50 hip OA, 100 control). Serum was collected and inflammatory profiles analysed using the Multiplex Human Cytokine Panel (Millipore) on the Luminex 100 platform (Luminex Corp., Austin, TX). The pain, physical function and activity limitations of hip OA cohort were scored using the WOMAC, SF-36, HHS and UCLA scores. All cytokine levels were compared between cohorts individually using Mann–Whitney–Wilcoxon (MWW) test with Bonferroni multiple comparison correction. Within hip OA cohorts, the effect of hip alignment (impingement and dysplasia) and radiographic grade (Kellgren and Lawrence grade, K/L grade) on cytokine levels were accessed by MWW test. Spearman's rank correlation test used to assess the association between cytokines and pain levels. The three cohorts showed distinct inflammatory profiles. Specifically, EGF, FGF-2, MCP-3, MIP-1a, IL-8 were significant different between knee and hip OA; FGF-2, GRO, IL-8, MCP-1, VEGF were significant different between hip OA and control; Eotaxin, GRO, MCP-1, MIP-1b, VEGF were significant different between knee OA and control (p-value < 0.0012). For hip OA cohorts, cytokines do not differ between K/L grade three and K/L grade four or between patients that displayed either impingement or dysplasia. Three cytokines were significant associated with pain: IL-6 (p-value = 0.045), MDC (p-value = 0.032) and IP-10 (p-value = 0.038). We have demonstrated that differences in serum inflammatory profiles exist between hip and knee OA patients. These differences suggest that OA may include different inflammatory subtypes according to affected joints. We also identified that the cytokine IL-6, MDC and IP-10 are associated with pain level in hip OA patients. These cytokines might help explain the inconsistent of presentation of pain with radiographical severity of OA joints. Future studies are needed to validate our findings and then to understand the following questions: (1) how differently affected joints are reflected in systematic biomarkers; (2) how these cytokines are biologically involved in the OA pain pathway

Aim. The aim of this prospective comparative study was to evaluate the serum levels of different cytokines in patients underwent total knee replacement (TKR) and received allogeneic blood transfusion, post-operative auto-transfusion or not transfused. Material and Methods. This was a prospective non-randomized comparative study in 248 patients underwent TKR. Patient's demographic and clinical data including age, gender, body mass index (BMI), preoperative Hb value, complications were documented. The serum levels of IL-1b, IL-6, IL-8, IL-10, and TNF were measure pre-operatively, the 1st, 2nd, 3rd and 5th post-operative day. Patients were categorized in three groups; in Group 0 patients received no blood transfusion, in Group 1 patients received post-operative auto-transfusion and in Group 2 allogeneic blood transfusion was applied. Statistical analysis of the results was performed using repeated measures ANOVA. Results. Significant changes were observed in cytokines levels in Groups 1 and 2. In Group 1 (auto-transfusion) the levels of all cytokines significantly increased the 1st postoperative day, remaining above the pre-operative levels even the 5th post-operative day. In Group 2 (allogenic transfusion), although the levels of IL-6, IL-8 and IL-10 were also significantly increased the 1st postoperative day, they gradually returned to the per-operative levels by the 5th post-operative day. In Group 0 (no transfusion) the only significant increase was observed in IL-6 between pre-operative and 1st and 3rd day values. Furthermore, the area under the curve (AUC) of IL-1b, IL-6, IL-8 and IL-10 levels in Group 1 and AUC of IL-6, IL-8 and IL-10 levels in Group 2, were significantly higher compared to Group 0. There was no significant difference in post-operative patient's complications. Conclusion. According to the results of this study significant elevation of cytokine values were observed during the first five post-operative days in patients received blood transfusion after TKR. These changes were more pronounced in the auto-transfusion group

Aims. Insufficient treatment response in rheumatoid arthritis (RA) patients requires novel treatment strategies to halt disease progression. The potential benefit of combination of cytokine-inhibitors in RA is still unclear and needs further investigation. To explore the impact of combined deficiency of two major cytokines, namely interleukin (IL)-1 and IL-6, in this study double deficient mice for IL-1αβ and IL-6 were investigated in different tumour necrosis factor (TNF)-driven inflammatory bone disorders, namely peripheral arthritis and sacroiliitis, as well as systemic bone loss. Methods. Disease course, histopathological features of arthritis, and micro-CT (µCT) bone analysis of local and systemic bone loss were assessed in 15-week-old IL1-/-IL6-/-hTNFtg in comparison to IL1-/-hTNFtg, IL6-/-hTNFtg, and hTNFtg mice. µCT bone analysis of single deficient and wild-type mice was also performed. Results. Combined deficiency of IL-1/IL-6 markedly ameliorated TNF-mediated arthritis and bilateral sacroiliitis, but without additive benefits compared to single IL-1 deficiency. This finding confirms the important role of IL-1 and the marginal role of IL-6 in TNF-driven pathways of local joint damage, but questions the efficacy of potential combinatorial therapies of IL-1 and IL-6 in treatment of RA. In contrast, combined deficiency of IL-1/IL-6 led to an additive protective effect on TNF-driven systemic bone loss compared to single IL-1 and IL-6 deficiency. This finding clearly indicates a common contribution of both IL-1 and IL-6 in TNF-driven systemic bone loss, and points to a discrepancy of cytokine dependency in local and systemic TNF-driven mechanisms of inflammatory arthritis. Conclusion. Combinatorial treatments in RA might provide different benefits to inflammatory local arthritis and systemic comorbidities. Cite this article: Bone Joint Res 2022;11(7):484–493

BACKGROUND. Although osteoarthritis (OA) is not an inflammatory arthritis, a characteristic feature of OA is increased production of pro-inflammatory cytokines, such as interleukin 1beta (IL-1b), by articular chondrocytes. In fact, the degree of articular inflammation is often associated with disease progression; indicating that this process probably contributes to articular damage. Suppressor of cytokine signalling (SOCS) proteins are, as the name suggests, inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1-SOCS7 and CIS-1 (cytokine-inducible SH2-domain-1 with similar structure to the other SOCS proteins) have been identified, of which, SOCS1-3 and CIS-1 are the best characterised. Reduced expression of SOCS proteins would be predicted to result in increased cytokine responsiveness and thereby could contribute to OA pathology. OBJECTIVES. 1) To compare the expression of SOCS1-3 and CIS-1 in normal and OA human articular chondrocytes and 2) to analyze the effects of IL-1b on SOCS1-3 and CIS-1 mRNA expression. METHODS. Femoral heads were obtained after joint replacement surgery due to OA or following a fracture of the neck of femur (#NOF). The latter patients typically suffer from osteoporosis and their cartilage is widely used as a suitable non-OA control. Chondrocytes from the surface layer of OA or the deep zone of #NOF cartilage were isolated by sequential treatment with trypsin, hyaluronidase and collagenase B. Total RNA was extracted using the Qiagen AllPrep RNA mini kit. Primers were designed for SOCS1-3 and CIS-1 and relative gene expression was determined by real-time RT-PCR (Sybr-green method), normalized to GAPDH. Alternatively, non-OA chondrocytes were isolated from #NOF cartilage and cultured in DMEM/F12/5% FCS, with or without IL-1b plus oncostatin M (OSM, both 2.5 ng/ml) for 3-5 weeks until confluent. Total RNA was extracted as above and the effect of IL-1b on gene expression was determined by qPCR. RESULTS. Expression of SOCS1 and SOCS3 was similar in OA and #NOF chondrocytes. However the expression of SOCS2 and CIS-1 was reduced 13-fold in OA samples compared to #NOF samples (n=5, each group). Expression of one OA patient was set to =1 to determine relative expression. In #NOF chondrocytes, relative expression of SOCS2 was 24.5±20.4 versus 2.5±1.9 in OA chondrocytes and for CIS-1 the values were 26.5±19.5 versus 2.5±1.8 (p<0.01). The in vitro experiments showed that treatment of control #NOF chondrocytes with IL-1b+OSM mimicked the situation in OA: Expression of SOCS2 in cytokine-treated cultures was only 15±10% of that in control cultures and CIS-1 expression was reduced to 16±13% (P<0.01). CONCLUSIONS. To the authors' knowledge this is the first demonstration of reduced expression of SOCS2 and CIS-1, but not SOCS1 or SOCS3, in osteoarthritis. As the SOCS proteins are attenuators of cytokine-mediated processes, reduced expression would be expected to accelerate any inflammatory processes. The fact that IL-1b itself reduces the expression of its suppressors of signalling suggests a potentially damaging positive feedback mechanism that could play a role in OA pathology

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway

Patients with a femoral shaft fracture requiring intra-medullary nailing were recruited to investigate if the femoral canal could be a potential source of inflammatory cytokines, previously implicated in the pathogenesis of life-threatening inflammatory complications. Femoral and peripheral blood samples were obtained at the time of surgery from patients with a femoral shaft fracture requiring intramedullary nailing. The local femoral intramedullary and peripheral release of a group of ten Th1 and Th2 cytokines concentrations (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, TNF-a and IFN-g) after femoral shaft fracture and intramedullary reaming, if performed, was measured using a Human Cytokine Antibody 10-plex Bead Kit. A control group of patients(n=3) undergoing hip replacement was established to allow comparison with the normal femoral intramedullary cytokine environment. 21 patients with a femoral shaft fracture were recruited. Femoral shaft fracture caused a significant increase in the local femoral concentrations of IL-6 (median 3967pg/ml; range 128–25,689pg/ml) and IL-8 (median 238pg/ml; range 8–8,288pg/ml) compared to the femoral control group(p=0.0005 and p=0.001 respectively). No significant local femoral release of the other cytokines was demonstrated. In the patients who underwent intramedullary reaming of the femoral canal (n=6), a further significant local release of IL-6 (median post-ream 15,903pg/ml; range 1,854–44,922pg/ml) and IL-8 (median post-ream 1,443pg/ml; range 493–3,734pg/ml) was demonstrated (p=0.01 and p=0.03 respectively), thus showing that intramedullary reaming can cause a significant local inflammatory response. Femoral shaft fracture produces a local inflammatory response releasing large amounts of the cytokines IL-6 and IL-8 into the local femoral environment but not of the other Th1 and Th2 cytokines studied. Reaming, produced significant elevation in local femoral IL-6 and IL-8 concentration, suggesting a local femoral response as a result of this procedure. Possibly, local femoral environment may act as a cell-priming or stimulating zone, for circulating inflammatory cells

Introduction: Metal-on-metal (MoM) articulations for THA are used successfully from CoCr-alloys. Low or high carbon hydride metals contain less or more than 0.2% carbon in the alloy. The systems show encouraging clinical results and lower rates of aseptic loosening in midterm results. Hypersensitivity reactions to high carbide MoM articulations were reported. The immune response is characterized by a perivascular T-/B-lymphocyte infiltration of the capsular tissue around the hip replacement. The present study examines if lymphocytic reactions are present in low carbide MoM THA and if distinct cytokines are released to joint fluids. Retrieval tissues from 28 patients were used. Joint fluids were aspirated at the time of surgery. Materials and Methods: Tissues were collected from 25 patients undergoing 26 aseptic revisions of MoM THA (CoCrMo, Sikomet. ®. , Plus Orthopaedics). The patients had following symptoms: Hip and femoral pain; recurrent dislocation and clicking noises. 8 patients had osteolysis, 12 patients showed a metallosis. The peripros-thetic tissues were examined by standard histology and immunohistochemistry. Joint fluids were frozen at the time of surgery. The control groups were patients with osteoarthritis of the hip (n=10), revisions from Al2O3-UHMWPE articulations (n=6), revisions of MoM with hypersensitivity reaction (n=18), and MoM without hypersensitivity reactions (n=8). The fluids were analyzed for various Interleukins, Il-1 receptor antagonist, G-CSF, GM-CSF, IFN gamma, MIP-1ß, and TNF-α. Results: 18 out of 26 cases showed diffuse and follicular lymphocyte infiltrations in the revision tissues. Perivas-cular T- and B-lymphocytes and few macrophages were also seen. In low and excessive metallosis no lymphocytes were observed. The tissue response in low carbide MoM is similar to high carbide MoM. Analysis of the cytokine profile in the joint fluids showed markers of osteoclast activation (Il-6 and −10) in all MoM articulations. TNF-α increase was increased in all loosening groups but was further increased in MoM. Il-5, IFN gamma, MIP-1ß, and GM-CSF were increased in all fluids from loosening cases but were further increased in MoM with lymphocyte activation. Discussion: Activation of lymphocytes in failed MoM THA’s is not necessarily related to high carbide MoM. Hypersensitivity also occurred in low carbide MoM THA. The cytokine profiles in the joint fluids showed distinct characteristics. Il-6 and Il-10, markers of osteo-clast activation, were elevated in all cases of bone loss and osteolysis. The increase in TNF-α may account for a regulation of the OPG/RANKL system TNF-a which can induce osteolysis. The elevated levels of Il-5, IFN gamma, MIP-1ß, and GM-CSF in MoM failures with hypersensitivity represent markers of chemotaxis and lymphocyte activation may account for index markers of hypersensitivity reaction

Introduction: Metal/metal total hip arthroplasties (THA) with improved qualities of the alloys and encouraging midterm clinical results are widely used. Hyperergic reactions have been observed in revision tissues in a series of failures. This study examined synovial fluids of patients with aseptic loosening of THA from metal/metal and ceramic/polyethylene endoprostheses or arthritis of the hip by analysis of various released cytokines. Materials and Methods: The aspirations of synovial fluids from 11 patients with arthritis of the hip, 6 THA revisions with ceramic polyethylene articulations, and 22 metal/metal articulations were retrieved. 15 of the 22 cases showed lymphocytic infiltration in the histologies. The aspirates were examined with a commercially available assay using a Multiplex Reader. The interleukins Il-1 beta, -2, -5, -6, -10, -12, -13, -15,-17 and IL-1 receptor antagonist (Il-1ra) were measured. Further G-CSF, GM-CSF, IFN gamma, MIP 1 beta, MIP alpha, MCP 1, and TNF alpha were assayed. Results: Samples from patients with aseptic loosenings showed increased Il-10 and MCP compared to osteoarthritis. TNF alpha, MIP alpha, and Il-1β were increased in metal/metal THA. Il-5, Il-12, Il-13 and Il-17 were only increased in patients with lymphocytic reactions, but not in ceramic/polyethylene articulations. GM-CSF, G-CSF, IFN gamma, Il-1ra Il-2, and Il-6 were only elevated in THA with lymphocytic reactions compared to metal/metal cases without. Diskussion: Aspirates from aseptic loosened THA are important diagnostic tools. The data showed a distinctly different cytokine profile joint fluids in aseptic loosenings of metal/metal THA vs. ceramic-UHMWPE articulations and fluids from osteoarthritis patients. The data may contribute to establish a cytokine profile to determine failures due to lymphocytic infiltrations before revision of metal/metal articulations

Aim: The purpose of this study was to evaluate the cytokine molecules present in a rat tendinopathy model and in the torn edge of human rotator cuff tendon in an attempt to understand their role in tendon degeneration. Methods: A rat tendon overuse model was used with custom microarrays consisting of 5760 rat oligonucleotide features in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery.Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery.Specimens were analysed for the presence of interleukins 18, 15, 12, 11, 6, 2, macrophage inhibitory factor (MIF), and tumour necrosis factor ƒÑ by semiquantitative RT-PCR and immunohistochemistry. Tendinopathy was assessed on a basic histological scale. Results: Rat Microarray analysis: Upregulation of IL-6, IL-11 and IL18 receptor was noted in the degenerated rat supraspinatus tendon. Downregulation of IL-2 was noted. No other cytokine signal was expressed. Histological analysis: All torn human supraspinatus tendons changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Cytokine mRNA expression: TNF-£\ mRNA expression was found to be significantly elevated (p< 0.01) in subscapularis tendon compared to torn supraspinatus samples. The expression levels of IL-18, IL-15, IL-6 and MIF was significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and normal control tendon (p< 0.001). Immunohistochemical analysis: Presence of IL-18, IL-15, Il-6, MIF and TNF-£\ was confirmed in all samples of torn supraspinatus tendon. Significantly increased levels of IL-18, IL-15, IL-6 and MIF were found in torn supraspinatus. (p< 0.01) compared to matched and normal subscapularis. Conclusions: Cytokines have been shown to promote the intensive production of reactive O2 metabolites . 1. and are potent agonists of protein kinases . 2. Our finding of significantly increased cytokine levels may suggest that these molecules when expressed during the degenerate and healing phases of tendon injury result in the subsequent production of reactive O2 species and protein kinases. 3. causing tendon damage or failure of the normal reparative process. Our finding of marked tendinopathy in matched subscapularis tendon may also provide a useful human tendinopathy model

Summary. Cytokines produced within the degenerate disc induce expression of neurotrophic factors and pain related peptides which could be important in nerve ingrowth and pain sensitisation leading to low back pain. The intervertebral disc (IVD) is considered the largest aneural and avascular structure within the human body, yet during degeneration vascularisation of the IVD is seen to be accompanied by nociceptive nerves. Low back pain is a highly debilitating condition affecting around 80% of the population, 40% of which are attributed to IVD degeneration. Discogenic pain was largely thought to be a result of irritation and compression of the nerve root, yet recent data suggests that pain may be attributed to the sensitisation of sensory nerves by the synthesis of pain related peptides, calcitonin gene related peptide (CGRP) and substance P. It is known that cytokines and chemokines produced by nucleus pulposus cells elicit various effects including the production of matrix degrading enzymes, and decreased matrix molecules. Here, we investigate the hypothesis that cytokines regulate both neurotrophic factor and pain related peptide synthesis within nucleus pulposus and nerve cells which may elicit algesic effects. Real-Time PCR was performed to investigate gene expression of the neurotrophic factors NGF, BDNF, NT3 and their receptors Trk A, B and C along with Substance P and CGRP on directly extracted RNA from human NP cells and NP cells cultured in alginate for 2 weeks prior to treatment for 48hours with IL-1, IL-6 or TNFα at 0–100ng/mL. Similarly SH-SY5Y neuroblastoma cells were differentiated in retinoic acid for 7 days prior to stimulation with IL-1, IL-6 or TNFα at 0ng/mL and 10ng/mL for 48hours. Immunohistochemistry was used to localise neurotrophic factor receptors Trk A, B and C in both degenerate discs and neuronal cells. NGF expression was present in normal and degenerate disc samples, however only degenerate discs expressed the high affinity receptor TrkA. Similarly Trk B was present in 22% of normal samples increasing to 100% expression within degenerate disc samples. All cytokines increased expression of NGF in NP cells (P≤0.05). TNFα also increased BDNF significantly, whereas no significant affects were seen in NT3 expression in NP cells. Trk B expression was significantly increased by IL-1 and TNFα treatment of NP cells. Conversely Trk C was down regulated by IL-6. Substance P was significantly increased by IL-1 and TNFα treatments whilst IL-6 and TNFα increased CGRP expression in NP cells. In SH-SY5Y cells, IL-1 significantly increased BDNF whilst IL-6 and TNFα failed to induce significant differences in neurotrophic factors. All cytokines increased Trk expression in the nerve cell line; however this failed to reach significance. Immunohistochemistry confirmed the presence of Trk receptors within the neuronal cell line. Here we have demonstrated that a number of cytokines known to be up regulated during disc degeneration and disc prolapse, induce expression of various neurotrophic factors, their receptors and pain related peptides within human NP cells, as well as SH-SY5Y cells. This data suggests that the presence and production of cytokines within the degenerate disc may be responsible for nerve ingrowth and sensitisation of nerves which may result in discogenic pain

This study was undertaken to assess the contribution of fat embolism (FE) to the development of acute lung injury in the presence of resuscitated hemorrhagic shock. Twenty-seven NZW rabbits were randomly assigned into four groups: resuscitated hemorrhagic shock and FE (HR/FE), resuscitated hemorrhagic shock, FE, and control. FE was induced via intramedullary femoral canal pressurization using a 1–1.5 ml bone cement injection. Only HR/FE animals displayed significant proinflammatory cytokine release as compared to controls. These findings suggest that the combination of resuscitated shock with FE initiates an inflammatory response, which may lead to the development of fat embolism syndrome. The objective of this study was to assess the contribution of fat embolism caused by intramedullary femoral canal pressurization to the development of acute lung injury in the presence of resuscitated hemorrhagic shock. Only the animals that underwent resuscitated shock and fat embolism displayed amplified BALF proinflammatory cytokine expression. These findings suggest that the combination of resuscitated shock with fat embolism initiates an inflammatory response, which may play a role in the development of fat embolism syndrome. Only HR/FE BALF IL-8 and MCP-1 levels were significantly higher than controls (0.72 ng/ml vs. 0.26ng/ ml, p=0.03; 18.3 ng/ml vs. 2.0 ng/ml, p=0.01, respectively). Twenty-seven NZW rabbits were randomly assigned into four groups: resuscitated hemorrhagic shock + fat embolism (HR/FE), resuscitated hemorrhagic shock (HR), fat embolism (FE), and control. Shock was induced via carotid bleeding for one-hour prior to resuscitation. For FE induction, the intramedullary cavity was drilled, reamed and pressurized with a 1–1.5 ml bone cement injection. Four hours later, postmortem bronchoalveolar lavage was performed through the right mainstem bronchus. Analyses of bronchoalveolar lavage fluid (BALF) of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were carried out in triplicate and blinded fashion using the ELISA technique. Our findings suggest that FE by itself does not initiate inflammatory lung injury, as there were no apparent differences between the control and FE cytokine levels. Only the HR/FE animals revealed elevated levels of pro-inflammatory cytokines in BALF. These findings are in agreement with our previous results, which displayed neutrophil activation only in the HR/FE group

Cytokine mediated activation of osteoclasts can lead to peri-implant osteolysis and aseptic loosening. The aim of this study was to determine the IL-1β and TNFα mRNA cytokine expression profile of human macrophages when stimulated with polyethylene particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR). Human peripheral blood monocytes or human monocytes from the cell line THP-1 were used in this study. rqRT-PCR conditions were optimized by stimulating human macrophages with 200ng/ml lipopolysaccharide (LPS). The median CV% value for duplicate measures was 12.6 (range 4.5–54). Stimulation assays were performed using unfractionated endotoxin-free commercial polyethylene particles (median size 7μm); or fractionated particles (size range 0.1–1.2μm). Human macrophages were stimulated with high dose unfractionated polyethylene particles at 0, 3500 or 10500 mm. 3. /cell or with fractionated polyethylene particles at 0 and 100mm. 3. /cell at time points 0 and 3 hours. Low dose unfractionated polyethylene stimulation was performed on THP-1 cells at 0, 50, 100, 1000 and 10000 mm. 3. /cell. In all experiments LPS stimulation was used as a positive control. RNA was extracted and rqRT-PCR was performed using standard techniques. High dose unfractionated polyethylene stimulation did not result in a significant difference in cytokine mRNA levels between groups. Using fractionated polyethylene, a small increase in IL-1β mRNA was identified (21% versus maximal stimulation using LPS). Low dose unfractionated polyethylene stimulation of THP-1 cells demonstrated dose dependent decreases in TNFα and IL-1β mRNA expression that was not due to inhibition of RNA extraction or a decrease of cell viability. Endotoxin-free polyethylene particles do not appear to be a major stimulus for IL-1β and TNFα mRNA production as measured by rqRT-PCR. We did observe a small positive effect on IL-1β mRNA expression using a fractionated polyethylene stimulus. However it remains unclear whether this effect is due to fractionation of particles into the submicron range or is due to introduction of endotoxin during the filtration process

The most frequent pathogenic organism in arthroplasty infections is Staphylococcus. The immune response impairment is a frequent finding in elderly people. Objective: to investigate the response of some cytokines and the effect of age in an experimental model of osteomyelitis. Materials and methods. 40 adult male Wistar rats received a stainless steel needle, intramedullarily in the left tibia. Young rats (3 months old) and Old rats (22 months old) were alloted in: Group A: Sterile implant. Group B: Sterile implant + slime producing S. aureus. 9 weeks after surgery, rats were sacrified. Determinations: Cytokines (IL-1b, IL-2, L-4, IL-6, IL-10 and IL-12)(ELISA) in blood (previous to surgery and to sacrifice) and in tibia extract (after sacrifice); the number of bacteria in tibia and implant. The Wilcoxon, Mann-Whitney U test were used (p≤ 0.05 significant). Results. Infection was detected in all the operated tibias in old rats receiving S.aureus, and in 7/10 of young rats. IL-2 levels increased in blood in the S.aureus group after surgery in old and young rats. Pre and postoperative IL-2 levels in blood were higher in old rats in both groups than in the corresponding groups of young rats. There was a decrease with age in blood of IL-4 (previous and after surgery), and a decrease of IL-1. S.aureus groups increased IL-1 levels in the operated tibia independently of age; increased IL-2 and IL-10 levels in young rats in the operated tibia; increased IL-4, IL-6 and IL-12 in old rats in blood, decreased IL-4 and increased IL-2 and IL-10 in blood in young rats. Conclusions. Significant differences in tibia infection were found with age. Old rats presented differences with young rats in cytokine response in an experimental model of osteomyelitis, showing an immune response impairment associated with old age

Phagocytosis of wear particles by perimplant macrophages results in cytokine release and osteoclast activation and osteolysis. Some investigators have proposed that this response may be mediated by adherent endotoxin. The aim of this study was to determine the role of endotoxin in modulating pro-inflammatory cytokine mRNA expression of macrophages when stimulated with titanium particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR). Human peripheral blood mononuclear cells were isolated from healthy subjects and plated in chamber slides. Three types of titanium particles were prepared; commercially pure titanium particles (cpTi), endotoxin stripped particles and endotoxin stripped particles with endotoxin (LPS) added back. Endotoxin levels of 450, 0 and 140 Eu/ml respectively were confirmed by high sensitivity Limulus Amebocyte Lysate assay. Macrophages were stimulated with particle concentrations of 0, 8.3, 83 and 830 particles per cell at time points 0 and 3 hours. LPS (200ng/ml) was used as a positive control. rqRT-PCR was performed using standard techniques. Stimulation of human macrophages with cpTi demonstrated a significant dose dependent increase in TNFα, IL-1A, IL-1B and, IL-6. (Kruskal-Wallis p=0.01, p=0.017, p=0.001 and p=0.013 respectively). IL-18 mRNA levels were not increased (P> 0.05). The expression of mRNA following stimulation with the highest dose of titanium particles was similar to that following LPS stimulation. Endotoxin-free cpTi particles did not elicit any increase in mRNA expression above base line levels (P > 0.05, all cytokines). This lack of response was rescued in endotoxin-stripped particles with LPS added back. Particle dose dependent increases in cytokine mRNA levels were observed for TNFα, IL-1A, IL-1B and, IL-6 mRNA but not IL-18 (p=0.01, p=0.01, p=0.01, p=0.05 and p=0.> 0.05 respectively). Our results show that adherent endotoxin plays a role in modulating particle induced pro-inflammatory cytokine mRNA expression in-vitro. Further study is required in evaluating the role of adherent endotoxin in vivo

Background. Adverse reactions to metal debris are implicated in the failure of metal-on-metal hip arthroplasty. The peri-implant tissues are often infiltrated by leukocytes which may cause observed immunological effects, including soft tissue necrosis and osteolysis. Cobalt ions from orthopaedic implants aberrantly activate the innate immune receptor human toll-like receptor-4 (TLR4), leading to inflammatory cytokine release including interleukin-8 (IL-8). IL-8 has been shown to increase expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These factors are essential for leukocyte adhesion to endothelium, which is required for leukocyte migration into tissues. This study investigates cobalt's effect on gene and protein changes in IL-8, ICAM-1 and VCAM-1 to determine their potential role in immune cell infiltration of peri-implant tissues. Methods. TLR4-expressing human dermal microvascular endothelial cells (HMEC-1) were treated with a range of clinically relevant cobalt ion concentrations. IL-8 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Stimulation with cobalt ions significantly increases IL-8 secretion (n=3) in HMEC-1 cells. This is a TLR4-specific effect as a small molecule TLR4 antagonist inhibited cobalt-induced IL-8 secretion. Following cobalt treatment (0.75mM cobalt chloride) there is a 12-fold increase in ICAM-1 (p-value=0.0004) and a 6-fold increase in VCAM-1 (p-value<0.0001) gene expression. Work will be undertaken to determine the role of TLR4 in these responses. Conclusion. Cobalt increases IL-8 secretion and adhesion molecule gene expression in HMEC-1 cells. This in vitro finding demonstrates the potential for cobalt ions to increase leukocyte adhesion to the endothelial surface. This may contribute to leukocyte infiltration of peri-implant tissues in metal-on-metal hip arthroplasty failure

Objectives. Emerging evidence indicates that tendon disease is an active process with inflammation that is critical to disease onset and progression. However, the key cytokines responsible for driving and sustaining inflammation have not been identified. Methods. We performed a systematic review of the literature using MEDLINE (U.S. National Library of Medicine, Bethesda, Maryland) in March 2017. Studies reporting the expression of interleukins (ILs), tumour necrosis factor alpha (TNF-α) and interferon gamma in diseased human tendon tissues, and animal models of tendon injury or exercise in comparison with healthy control tissues were included. Results. IL-1β, IL-6, IL-10, and TNF-α are the cytokines that have been most frequently investigated. In clinical samples of tendinopathy and tendon tears, the expression of TNF-α tended not to change but IL-6 increased in tears. Healthy human tendons showed increased IL-6 expression after exercise; however, IL-10 remained unchanged. Animal tendon injury models showed that IL-1β, IL-6, and TNF-α tend to increase from the early phase of tendon healing. In animal exercise studies, IL-1β expression showed a tendency to increase at the early stage after exercise, but IL-10 expression remained unchanged with exercise. Conclusions. This review highlights the roles of IL-1β, IL-6, IL-10, and TNF-α in the development of tendon disease, during tendon healing, and in response to exercise. However, there is evidence accumulating that suggests that other cytokines are also contributing to tendon inflammatory processes. Further work with hypothesis-free methods is warranted in order to identify the key cytokines, with subsequent mechanistic and interaction studies to elucidate their roles in tendon disease development. Cite this article: W. Morita, S. G. Dakin, S. J. B. Snelling, A. J. Carr. Cytokines in tendon disease: A Systematic Review. Bone Joint Res 2017;6:656–664. DOI: 10.1302/2046-3758.612.BJR-2017-0112.R1

Background: Non-union of fractures is a common problem faced by orthopaedic surgeons. Although the basic processes of fracture healing have been better elucidated in recent years, in terms of their cellular and molecular biology, the pathogenesis of fracture non-union remains poorly understood. Aims: To examine the pattern of cytokine expression in established non-unions, in particular the inflammatory cytokines interleukin 1 and tumour necrosis factor alpha. Materials and Methods: Tissue was taken from 7 non united fractures at the time of a surgical procedure aimed at effecting union. Part of the tissue was snap-frozen in liquid nitrogen, and a portion of the sample was processed for routine histology. Normal bone tissue was taken from the femoral shaft at the time of arthoplasty, to provide normal control tissue. Total RNA was extracted from the frozen tissue by means of a mortar and pestle and a modified phenol-chloroform extraction protocol. Cytokine expression patterns were examined using the Cytokine Gene Expression plate I (PE Biosystems) and analysed using the Sequence Detection Software and Microsoft Excel. Results: A consistent pattern of cytokine expression was seen in all non-union tissue samples. There was marked suppression of interleukin 1 beta, interleukin 8, interleukin 10 and TNF-alpha when compared to resting bone. This environment is thus one where the stimulus for bone resorption is suppressed, with consequent loss of stimulation of bone formation (theory of “bone coupling”), directly and also possibly through interaction with prostaglandin production. In addition, collagen production is stimulated preferentially. These findings argue against the traditional definitions of fracture non-union, and suggest a possible adjunctive role for the administration of interleukins in the treatment of non-united fractures

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system. Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully. We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response