Moeed Akbar

Effect of chromium and cobalt ions on primary human lymphocytes in vitro

Cobalt-chromium (Co-Cr) alloy metal-on-metal hip resurfacing is increasingly common among younger more active patients suffering from osteoarthritis. Recent reports have increased awareness of metal ions leaching from metallic articulations; this ion exposure may have adverse effects on the immune system. As previous studies reported alterations in lymphocyte number and function in patients with Co-Cr implants, we investigated effects of clinically relevant concentrations of Cr6+ and Co2+ on primary human lymphocytes in vitro. Here, both resting and activated (anti-CD3 ± anti-CD28 antibodies) primary human lymphocytes were exposed to Cr6+ or Co2+ (0.1–100 µM). Following 24 or 48 h of exposure, cell viability, proliferation, cytokine [interferon-γ (IFNγ and interleukin-2 (IL-2)] release, and apoptosis (with and without pre-treatment of cells with a caspase-3 inhibitor) were assessed. Exposure to 10 and 100 µM Cr6+ significantly decreased cell viability and increased apoptosis in both resting and activated lymphocytes. Cell proliferation and cytokine release were also significantly reduced in activated lymphocytes following exposure. The exposure of resting lymphocytes to 100 µM Co2+ resulted in significant decreases in cell viability accompanied by a significant increase in apoptosis. Activated lymphocytes also showed this response after exposure to 100 µM Co2+; in fact, activated cells were significantly more sensitive to Co2+ toxicity. Exposure to 10 µM Co2+ led to significant decreases in cell proliferation and cytokine release, but no significant increase in apoptosis, in activated cells. The results indicate that exposure to high concentrations of metal ions initiate apoptosis that results in decreased lymphocyte proliferation. IL-2 release is inhibited by both metal ions at concentrations that are not overtly toxic. However, metal ion concentrations not directly cytotoxic to lymphocytes may affect events at a molecular level, thereby impeding lymphocyte proliferation. Hence, this may contribute to altered immune system function in patients with Co-Cr implants.

MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease.

MicroRNA (miRNA) has the potential for cross-regulation and functional integration of discrete biological processes during complex physiological events. Utilizing the common human condition tendinopathy as a model system to explore the cross-regulation of immediate inflammation and matrix synthesis by miRNA we observed that elevated IL-33 expression is a characteristic of early tendinopathy. Using in vitro tenocyte cultures and in vivo models of tendon damage, we demonstrate that such IL-33 expression plays a pivotal role in the transition from type 1 to type 3 collagen (Col3) synthesis and thus early tendon remodelling. Both IL-33 effector function, via its decoy receptor sST2, and Col3 synthesis are regulated by miRNA29a. Downregulation of miRNA29a in human tenocytes is sufficient to induce an increase in Col3 expression. These data provide a molecular mechanism of miRNA-mediated integration of the early pathophysiologic events that facilitate tissue remodelling in human tendon after injury.

Acute inflammatory response to cobalt chromium orthopaedic wear debris in a rodent air-pouch model

This study used a rodent air-pouch model to assess the acute inflammatory response to cobalt–chromium (CoCr) alloy wear debris from a metal-on-metal hip resurfacing implant that may contribute to joint failure. Air-pouches were injected with either sterile phosphate-buffered saline, 1 μg lipopolysaccharide (LPS) or 2.5 mg CoCr wear debris. The in situ inflammatory response was monitored 4, 24, 48 and 72 h and 7 days later. A flow cytometric analysis of the inflammatory exudates showed that CoCr wear debris induced a different inflammatory pattern compared with LPS. LPS induced a strong early (4 h) neutrophil influx, with monocyte/macrophage influx peaking at 24 h, whereas CoCr wear debris initiated almost equal numbers of early monocyte/macrophage and neutrophil recruitment. Histological analyses also showed CoCr debris accumulated in the pouch wall and this was accompanied by vast cellular infiltration and fibrosis around the debris throughout the duration of the experiment. Assessment of inflammatory gene transcripts from air-pouch tissue showed that CoCr wear debris increased the expression of cytokines involved in promoting inflammation and fibrosis (IL-1β, TGF-β) and chemokines that promote the recruitment of neutrophils and monocytes/macrophages (CXCL2 and CCL2). The data suggest that inflammatory responses to CoCr debris induce a specific acute process in which the recruitment of monocytes/macrophages is key.

Distribution of metal released from cobalt–chromium alloy orthopaedic wear particles implanted into air pouches in mice

Metal-on-metal hip replacement implants generate wear debris and release ions both locally and systemically in patients. To investigate dissemination of metal, we determined blood and organ levels of cobalt (Co), chromium (Cr), and molybdenum (Mo) following the implantation of Co-Cr alloy wear debris in mice using skin pouches as a model system. We observed increased metal levels in blood for up to 72 h; the levels of Co were highest and remained elevated for 7 days. Co levels were elevated in all organs studied (liver, kidney, spleen, lung, heart, brain, and testes), with the peak at 48 h; highest levels were measured in liver and kidney (838.9 ± 223.7 ng/g in liver, and 938.8 ± 131.6 ng/g in kidney). Organ Cr levels were considerably lower than Co levels, for example, Cr in kidney was 117.2 ± 12.6 ng/g tissue at 48 h. Co is more mobile than Cr, reaching higher levels at earlier time points. This could be due to local tissue binding of Cr. Exposure to Co-Cr particles in vivo altered antioxidant enzyme expression and activities. We observed induction of catalase protein in the liver and glutathione reductase (GR) and peroxidase (GPx) proteins in the spleen. Activities of catalase and GPx in the liver were significantly increased while that of GR was decreased in the kidney. Organs of mice with Co-Cr particle implantation were exposed to increased metal levels capable of inducing reactive oxygen species scavenging enzymes, suggesting the tissue may be subjected to oxidative stress; however, the overall antioxidant defence system was not markedly disturbed.

IL-17A mediates inflammatory and tissue remodelling events in early human tendinopathy

Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy. We sought evidence of interleukin 17A (IL-17A) expression in early human tendinopathy and thereafter, explored mechanisms whereby IL-17A mediated inflammation and tissue remodeling in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) along with control biopsies were collected from patients undergoing shoulder surgery. Markers of inflammation and IL-17A were quantified by RT-PCR and immunohistochemistry. Human tendon cells were derived from hamstring tendon obtained during ACL reconstruction. In vitro effects of IL-17A upon tenocytes were measured using RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of IL-17A was detected in ‘early tendinopathy’ compared to both matched samples and non-matched control samples (p < 0.01) by RT-PCR and immunostaining. Double immunofluoresence staining revealed IL-17A expression in leukocyte subsets including mast cells, macrophages and T cells. IL-17A treated tenocytes exhibited increased production of proinflammatory cytokines (p < 0.001), altered matrix regulation (p < 0.01) with increased Collagen type III and increased expression of several apoptosis related factors. We propose IL-17A as an inflammatory mediator within the early tendinopathy processes thus providing novel therapeutic approaches in the management of tendon disorders.

Targeting danger molecules in tendinopathy: the HMGB1/TLR4 axis

Objectives To seek evidence of the danger molecule, high-mobility group protein B1 (HMGB1) expression in human tendinopathy and thereafter, to explore mechanisms where HMGB1 may regulate inflammatory mediators and matrix regulation in human tendinopathy. Methods Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. Markers of inflammation and HMGB1 were quantified by reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction and used through passage 3. In vitro effects of recombinant HMGB1 on tenocyte matrix and inflammatory potential were measured using quantitative RT-PCR, ELISA and immunohistochemistry staining. Results Tendinopathic tissues demonstrated significantly increased levels of the danger molecule HMGB1 compared with control tissues with early tendinopathy tissue showing the greatest expression. The addition of recombinant human HMGB1 to tenocytes led to significant increase in expression of a number of inflammatory mediators, including interleukin 1 beta (IL-1β), IL-6, IL-33, CCL2 and CXCL12, in vitro. Further analysis demonstrated rhHMGB1 treatment resulted in increased expression of genes involved in matrix remodelling. Significant increases were observed in Col3, Tenascin-C and Decorin. Moreover, blocking HMGB1 signalling via toll-like receptor 4 (TLR4) silencing reversed these key inflammatory and matrix changes. Conclusion HMGB1 is present in human tendinopathy and can regulate inflammatory cytokines and matrix changes. We propose HMGB1 as a mediator driving the inflammatory/matrix crosstalk and manipulation of the HMGB1/TLR4 axis may offer novel therapeutic approaches targeting inflammatory mechanisms in the management of human tendon disorders.

Stromal fibroblast activation and inflammation in frozen shoulder

Introduction Frozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the stromal compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder, Methods Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Stromal activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR. Results Immunohistochemistry demonstrated increased expression of stromal activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM). Conclusions These results show that stromal fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated stromal fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

Alarmins in frozen shoulder: a molecular association between inflammation and pain

Background: The pathophysiological mechanisms behind proliferation of fibroblasts and deposition of dense collagen matrix in idiopathic frozen shoulder remain unclear. Alarmins (also known as danger signals) are endogenous molecules that are released into the extracellular milieu after infection or tissue injury and that signal cell and tissue damage. Purpose: To investigate whether the presence of alarmins is higher in patients with idiopathic frozen shoulder than in control subjects. Study Design: Controlled laboratory study. Methods: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients with unstable shoulders (control). The samples were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry using antibodies against alarmin molecules including high-mobility group protein B1 (HMGB1), interleukin 33, S100A8, S100A9, and the peripheral nerve marker PGP9.5. Immunoreactivities were rated in a blinded fashion from “none” to “strong.” Immunohistochemical distribution within the capsule was noted. Before surgery, patient-ranked pain frequency, severity, stiffness, and the range of passive shoulder motion were recorded and statistically analyzed. Results: Compared with control patients, patients with frozen shoulder had greater frequency and severity of self-reported pain (P = .02) and more restricted range of motion in all planes (P < .05). H&E-stained capsular tissue from frozen shoulder showed fibroblastic hypercellularity and increased subsynovial vascularity. Immunoreactivity of alarmins was significantly stronger in frozen shoulder capsules compared with control capsules (P < .05). Furthermore, the expression of the alarmin molecule HMGB1 significantly correlated (r > 0.9, P < .05) with the severity of patient-reported pain. Conclusion: This study demonstrates a potential role for key molecular danger signals in frozen shoulder and suggests an association between the expression of danger molecules and the pain experienced by patients.