Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Interleukin-6 subfamily cytokines and rheumatoid arthritis:role of antagonists

Many cytokines have been implicated in the inflammatory pathways that characterize rheumatoid arthritis (RA) and related inflammatory diseases of the joints. These include members of the interleukin-6 (IL-6) family of cytokines, several of which have been detected in excess in the synovial fluid from RA patients. What makes the IL-6 group of cytokines a family is their common use of the glycoprotein 130 (gp130) receptor subunit, to which they bind with different affinities. Several strategies have been developed to block the pro-inflammatory activities of IL-6 subfamily cytokines. These include the application of monoclonal antibodies, the creation of mutant form(s) of the cytokine with enhanced binding affinity to gp130 receptor and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor-binding site(s). The rationale for the use of anti-cytokine therapy in inflammatory joint diseases is based on evidence from studies in vitro and in vivo, which implicate major cytokines such as interleukin-1 (IL-1), tumour necrosis factor (TNF)-alpha and IL-6 in RA pathogenesis. In particular, IL-6 subfamily antagonists have a wide range of potential therapeutic and research applications. This review focuses on the role of some of the IL-6 subfamily cytokines in the pathogenesis of the inflammatory diseases of the joints (IJDs), such as RA. In addition, an overview of the recently developed antagonists will be discussed.

Blockade of interleukin 6 signaling improves the survival rate of transplanted bone marrow stromal cells and increases locomotor function in mice with spinal cord injury

Bone marrow stromal cells (BMSCs) have the potential to improve functional recovery in patients with spinal cord injury (SCI); however, they are limited by low survival rates after transplantation in the injured tissue. Our objective was to clarify the effects of a temporal blockade of interleukin 6 (IL-6)/IL-6 receptor (IL-6R) engagement using an anti-mouse IL-6R monoclonal antibody (MR16-1) on the survival rate of BMSCs after their transplantation in a mouse model of contusion SCI. MR16-1 cotreatment improved the survival rate of transplanted BMSCs, allowing some BMSCs to differentiate into neurons and astrocytes, and improved locomotor function recovery compared with BMSC transplantation or MR16-1 treatment alone. The death of transplanted BMSCs could be mainly related to apoptosis rather than necrosis. Transplantation of BMSC with cotreatment of MR16-1 was associated with a decrease of some proinflammatory cytokines, an increase of neurotrophic factors, decreased apoptosis rates of transplanted BMSCs, and enhanced expression of survival factors Akt and extracellular signal-regulated protein kinases 1/2. We conclude that MR16-1 treatment combined with BMSC transplants helped rescue neuronal cells and axons after contusion SCI better than BMSCs alone by modulating the inflammatory/immune responses and decreasing apoptosis. © 2013 by the American Association of Neuropathologists, Inc.

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

Blockade of interleukin-6 effects on cytokine profiles and macrophage activation after spinal cord injury in mice

The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. MR16-1 antibodies versus isotype control antibodies or saline alone was administered immediately after thoracic SCI in mice. MR16-1-treated group samples showed increased neuronal regeneration and locomotor recovery compared with controls. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. MR16-1 treatment promoted arginase-1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site and enhanced positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Serum procalcitonin, interleukin-6, soluble intercellular adhesin molecule-1 and IgG to short-chain exocellular lipoteichoic acid as predictors of infection in total joint prosthesis revision

The diagnosis of prosthetic joint infection and its differentiation from aseptic loosening remains problematic. The definitive laboratory diagnostic test is the recovery of identical infectious agents from multiple intraoperative tissue samples; however, interpretation of positive cultures is often complex as infection is frequently associated with low numbers of commensal microorganisms, in particular the coagulase-negative staphylococci (CNS). In this investigation, the value of serum procalcitonin (PCT), interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) as predictors of infection in revision hip replacement surgery is assessed. Furthermore, the diagnostic value of serum IgG to short-chain exocellular lipoteichoic acid (sce-LTA) is assessed in patients with infection due to CNS. Presurgical levels of conventional serum markers of infection including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell count (WBC) is also established. Forty-six patients undergoing revision hip surgery were recruited with a presumptive clinical diagnosis of either septic (16 patients) or aseptic loosening (30 patients). The diagnosis was confirmed microbiologically and levels of serum markers were determined. Serum levels of IL-6 and sICAM-1 were significantly raised in patients with septic loosening (P=0.001 and P=0.0002, respectively). Serum IgG to sce-LTA was elevated in three out of four patients with infection due to CNS. In contrast, PCT was not found to be of value in differentiating septic and aseptic loosening. Furthermore, CRP, ESR and WBC were significantly higher (P=0.0001, P=0.0001 and P=0.003, respectively) in patients with septic loosening. Serum levels of IL-6, sICAM-1 and IgG to sce-LTA may provide additional information to facilitate the diagnosis of prosthetic joint infection.

Loss of skeletal muscle in cancer: biochemical mechanisms

Patients with cancer often undergo a specific loss of skeletal muscle mass, while the visceral protein reserves are preserved. This condition known as cachexia reduces the quality of life and eventually results in death through erosion of the respiratory muscles. Nutritional supplementation or appetite stimulants are unable to restore the loss of lean body mass, since protein catabolism is increased mainly as a result of the activation of the ATP-ubiquitin-dependent proteolytic pathway. Several mediators have been proposed. An enhanced protein degradation is seen in skeletal muscle of mice administered tumour necrosis factor (TNF), which appears to be mediated by oxidative stress. There is some evidence that this may be a direct effect and is associated with an increase in total cellular-ubiquitin-conjugated muscle proteins. Another cytokine, interleukin-6 (IL-6), may play a role in muscle wasting in certain animal tumours, possibly through both lysosomal (cathepsin) and non-lysosomal (proteasome) pathways. A tumour product, proteolysis-inducing factor (PIF) is produced by cachexia-inducing murine and human tumours and initiates muscle protein degradation directly through activation of the proteasome pathway. The action of PIF is blocked by eicosapentaenoic acid (EPA), which has been shown to attenuate the development of cachexia in pancreatic cancer patients. When combined with nutritional supplementation EPA leads to accumulation of lean body mass and prolongs survival. Further knowledge on the biochemical mechanisms of muscle protein catabolism will aid the development of effective therapy for cachexia.

Pro-inflammatory cytokines IL6, TNF-α, IL1β, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis

Objective: To evaluate the serum levels and diagnostic value of cytokines and acute phase proteins in patients with infective endocarditis (IE). Patients and methods: Serum samples from 63 patients diagnosed with IE and 71 control patients were analysed for the following markers: interleukin-6 (IL6), tumour necrosis factor-α (TNF-α), interleukin 1-β (IL1β), procalcitonin (PCT), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). Results: Serum levels of IL6, IL1β and CRP were significantly elevated in patients with IE as compared to controls. PCT, TNF-α and LBP were not elevated. Conclusion: Serum CRP and IL6 are elevated in IE. IL 6 may aid in establishing the diagnosis. There was no correlation between IL 6 levels and CRP, causative microorganism, echocardiographic features or outcome. © 2007 The British Infection Society.

Polarized secretion of leukemia inhibitory factor

Background: The direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor LIF) belongs to the interleukin-6 IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1 beta is known to promote IL-6 secretion from the stimulated membrane apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1 beta stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF. Results: When Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1 beta increased LIF production. After treating the apical surface with IL-1 beta, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones. Conclusion: The site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.

Targeting the glycoprotein 130 receptor subunit to control pain and inflammation

The glycoprotein 130 (gp130) is a shared signal-transducing-membrane-associated receptor for several hematopoietic cytokines. Its activation is implicated in pain and in a variety of diseases via signaling of proinflammatory cytokines. These include interleukin-6 (IL-6) subfamily cytokines, many of which play important roles in the pathogenesis of diseases such as rheumatoid arthritis, Castleman's disease, and Kaposi's sarcoma. Several strategies have been developed to block gp130-receptor-mediated signaling. These include the application of monoclonal antibodies, the creation of mutant form(s) of the gp130 with increased binding affinity for such ligands as IL-6/sIL-6R complex, and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor binding site(s). Other strategies include targeting gp130-mediated signaling pathways such as that involving signal transducer and activator of transcription-3. This review provides a summary of the latest research pertaining to the role of gp130 in the pathogenesis of inflammatory and other diseases in which the gp130 receptor is implicated. An overview of antagonists targeting the gp130 receptor is included with particular emphasis on their mechanism of action and their limitations and potential for therapeutic application.

Peptidoglycan derived from Staphylococcus epidermidis induces Connexin43 hemichannel activity with consequences on the innate immune response in endothelial cells

Gram-positive bacterial cell wall components including PGN (peptidoglycan) elicit a potent pro-inflammatory response in diverse cell types, including endothelial cells, by activating TLR2 (Toll-like receptor 2) signalling. The functional integrity of the endothelium is under the influence of a network of gap junction intercellular communication channels composed of Cxs (connexins) that also form hemichannels, signalling conduits that are implicated in ATP release and purinergic signalling. PGN modulates Cx expression in a variety of cell types, yet effects in endothelial cells remain unresolved. Using the endothelial cell line b.End5, a 6 h challenge with PGN induced IL-6 (interleukin 6), TLR2 and Cx43 mRNA expression that was associated with enhanced Cx43 protein expression and gap junction coupling. Cx43 hemichannel activity, measured by ATP release from the cells, was induced following 15 min of exposure to PGN. Inhibition of hemichannel activity with carbenoxolone or apyrase prevented induction of IL-6 and TLR2 mRNA expression by PGN, but had no effect on Cx43 mRNA expression levels. In contrast, knockdown of TLR2 expression had no effect on PGN-induced hemichannel activity, but reduced the level of TLR2 and Cx43 mRNA expression following 6 h of PGN challenge. PGN also acutely induced hemichannel activity in HeLa cells transfected to express Cx43, but had no effect in Cx43-deficient HeLa OHIO cells. All ATP responses were blocked with Cx-specific channel blockers. We conclude that acute Cx43 hemichannel signalling plays a role in the initiation of early innate immune responses in the endothelium.

The elevation in circulating anti-angiogenic factors is independent of markers of neutrophil activation in preeclampsia

Background - Severe preeclampsia is associated with increased neutrophil activation and elevated serum soluble endoglin (sEng) and soluble Flt-1 (sFlt-1) in the maternal circulation. To dissect the contribution of systemic inflammation and anti-angiogenic factors in preeclampsia, we investigated the relationships between the circulating markers of neutrophil activation and anti-angiogenic factors in severe preeclampsia or systemic inflammatory state during pregnancy. Methods and results - Serum sEng, sFlt-1, placenta growth factor, interleukin-6 (IL-6), calprotectin, and plasma a-defensins concentrations were measured by ELISA in 88 women of similar gestational age stratified as: severe preeclampsia (sPE, n = 45), maternal systemic inflammatory response (SIR, n = 16) secondary to chorioamnionitis, pyelonephritis or appendicitis; and normotensive controls (CRL, n = 27). Neutrophil activation occurred in sPE and SIR, as a-defensins and calprotectin concentrations were two-fold higher in both groups compared to CRL (P < 0.05 for each). IL-6 concentrations were highest in SIR (P < 0.001), but were higher in sPE than in CRL (P < 0.01). sFlt-1 (P < 0.001) and sEng (P < 0.001) were ˜20-fold higher in sPE compared to CRL, but were not elevated in SIR. In women with sPE, anti-angiogenic factors were not correlated with markers of neutrophil activation (a-defensins, calprotectin) or inflammation (IL-6). Conclusions - Increased systemic inflammation in sPE and SIR does not correlate with increased anti-angiogenic factors, which were specifically elevated in sPE indicating that excessive systemic inflammation is unlikely to be the main contributor to severe preeclampsia.

Further preliminary assessment of three human glioma cell lines as models of human astrocytic toxicity in vitro

Three human astroglioma lines U251-MG, U373-MG and CCF-STTG1 have been evaluated further as possible models for astrocytotoxicity (GFAP and IL-6 release). The effects of bacterial lipopolysaccharide, chloroquine diphosphate and acrylamide were studied on GFAP expression and LPS, chloroquine diphosphate, ethanol, trimethyltin chloride (TMTC) and acrylamide were examined on interleukin-6 (IL-6) release in the U373-MG line only. At 4-h LIPS elevated GFAP (17.0±5.0% P < 0.05) above control in the U251-MG cell line only. Chloroquine diphosphate over 4 h in the U251-MG line resulted in an increase in GFAP-IR to 20.3 ±4.2% and 21.1 ± 4.1 % above control levels 0.1 µM (P< 0.05) and 1 µM (P< 0.05) respectively. CQD was associated with decreases in MTT turnover, particularly after 24 h incubation. With the U373-MG line, LPS (0.5 µg/ml) increased IL-6 expression 640% above control (P < 0.001), whilst chloroquine diphosphate (100 µM), ethanol (10mM) and TMTC chloride (1 µM) also increased IL-6. It is possible that batteries of astrocytic human glioma cell lines may be applicable to the sensitive evaluation of toxicants on astrogliotic expression markers such as GFAP and IL-6.

Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development

Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the full value of this emerging field can be realized. The characterization of input material for cell-based therapy bioprocesses from multiple donors is necessary to identify and understand the potential implications of input variation on process development. In this work, we have characterized bone marrow derived human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of the measurable input variation on the development of autologous and allogeneic cell-based therapy manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has been demonstrated that this variation results in a range in the process time between these donor hMSC lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent of donor variability throughout the expansion process. Measuring informative product attributes during process development will facilitate progress towards consistent manufacturing processes, a critical step in the translation cell-based therapies.

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment

Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C-γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (p-ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor-α, interleukin-6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte-macrophage colony-stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages.