Rowena A.D. Bunning

Studies on type II collagen and aggrecan production in human articular chondrocytes in vitro and effects of transforming growth factor-β and interleukin-1β*

Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.

Tumor necrosis factor α- and interleukin-1β-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1.

OBJECTIVE To investigate tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. METHODS Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. RESULTS An increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPβ on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. CONCLUSION Our findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.

Astrocyte and endothelial cell expression of ADAM 17 (TACE) in adult human CNS

ADAM 17, also known as TACE, is an important sheddase for a number of proteins, including tumor necrosis factor‐α (TNF‐α), transforming growth factor‐α (TGF‐α), L‐selectin, p75, and p55 TNF receptors, and interleukin‐1 receptor II (IL‐1RII). The presence of ADAM 17 mRNA in adult mouse and rat CNS was recently reported (Karkkainen et al. Mol Cell Neurosci 15:547–560, 2000 ). However, the cellular origin of ADAM 17 remains unknown. In this study, we have used an anti‐ADAM 17 antibody in an immunohistochemical study of its distribution in human adult CNS tissue. Cells with astrocytic and endothelial morphology were ADAM 17‐positive. This finding was further confirmed using double immunofluorescence with antibodies against GFAP and von Willebrand factor, which label astrocytes and endothelial cells, respectively. This study demonstrates that ADAM 17 is expressed by astrocytes and endothelial cells in normal brain tissue and may have a role in normal brain function. GLIA 34:267–271, 2001.

ADAMTS-1 and -4 are up-regulated following transient middle cerebral artery occlusion in the rat and their expression is modulated by TNF in cultured astrocytes.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzymes are a recently described group of metalloproteinases. The substrates degraded by ADAMTS-1, -4 and -5 suggest that they play a role in turnover of extracellular matrix in the central nervous system (CNS). ADAMTS-1 is also known to exhibit anti-angiogenic activity. Their main endogenous inhibitor is tissue inhibitor of metalloproteinases (TIMP)-3. The present study was designed to investigate ADAMTS-1, -4 and -5 and TIMP-3 expression after experimental cerebral ischaemia and to examine whether cytokines known to be up-regulated in stroke could alter their expression by astrocytes in vitro. Focal cerebral ischaemia was induced by transient middle cerebral artery occlusion in the rat using the filament method. Our results demonstrate a significant increase in expression of ADAMTS-1 and -4 in the occluded hemisphere but no significant change in TIMP-3. This was accompanied by an increase in mRNA levels for interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra) and tumour necrosis factor (TNF). ADAMTS-4 mRNA and protein were up-regulated by TNF in primary human astrocyte cultures. The increased ADAMTS-1 and -4 in experimental stroke, together with no change in TIMP-3, may promote ECM breakdown after stroke, enabling infiltration of inflammatory cells and contributing to brain injury. In vitro studies suggest that the in vivo modulation of ADAMTS-1 and -4 may be controlled in part by TNF.

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc.

IntroductionThe aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.MethodsReal-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.ResultsLDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.ConclusionsOur data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.

Arthritis and cannabinoids: HU-210 and Win-55,212–2 prevent IL-1 α-induced matrix degradation in bovine articular chondrocytes in-vitro

Cannabinoids have analgesic, immunomodulatory and anti‐inflammatory properties and attenuate joint damage in animal models of arthritis. In this study the mechanisms of action of the synthetic cannabinoid agonists, HU‐210 and Win‐55,212–2, were studied to determine if they affected interleukin‐1 alpha (IL‐1α)‐induced proteoglycan and collagen degradation in bovine nasal cartilage explant cultures and prostaglandin E2 (PGE2) production in primary cultures of bovine articular chondrocytes. The effects of the inactive enantiomer, Win‐55,212–3, were compared with those of the active enantiomer, Win‐55,212–2, to determine if the effects were cannabinoid (CB)‐receptor mediated. The chondrocytes and explants were stimulated by IL‐1α (100 U mL−1 ≡ 0.06 nm and 500 U mL−1 ≡ 0.3 nm, respectively). Proteoglycan breakdown was determined as sulfated glycosaminoglycan (sGAG) release using the dimethylmethylene blue assay. Collagen degradation was determined as hydroxyproline in the conditioned culture media and cartilage digests. PGE2 was determined by ELISA. Expression of cannabinoid receptors, CB1 and CB2; cyclooxygenase‐1 and −2 (COX‐1 and COX‐2); inducible nitric oxide synthase (iNOS); as well as activation of nuclear factor‐kappa B (NF‐κB) in chondrocytes were studied using immunoblotting techniques and immunofluorescence. The results showed that HU‐210 and Win‐55,212–2 (5–15 μm) significantly inhibited IL‐1α‐stimulated proteoglycan (P < 0.001) and collagen degradation (P < 0.001). Win‐55,212–2 (5–10 μm) also significantly inhibited PGE2 production (P < 0.01). At 5 μm, Win‐55,212–2 inhibited the expression of iNOS and COX‐2 and activation of NF‐κB. Chondrocytes appeared to constitutively express cannabinoid receptors CB1 and CB2. It is concluded that biologically stable synthetic cannabinoids protect cartilage matrix from degradation induced by cytokines and this effect is possibly CB‐receptor mediated and involves effects on prostaglandin and nitric oxide metabolism. Cannabinoids could also be producing these effects via inhibition of NF‐κB activation.

Expression of ADAMTS-1, -4, -5 and TIMP-3 in normal and multiple sclerosis CNS white matter

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) -1, -4 and -5 proteases have been identified in the CNS at the mRNA level. These glutamyl endopeptidases, inhibited by tissue inhibitor of metalloproteinases (TIMP)-3, are key enzymes in the degradation of the aggregating chondroitin sulphate proteoglycans (CSPGs), and may therefore play a role in CNS extracellular matrix (ECM) changes in multiple sclerosis (MS). We have investigated ADAMTS and TIMP-3 expression in normal and MS CNS white matter by real-time RT-PCR, western blotting and immunohistochemistry. We report for the first time the presence of ADAMTS-1, -4 and -5 in normal and MS white matter. Levels of ADAMTS-1 and -5 mRNA were decreased in MS compared to normal tissue, with no significant change in ADAMTS-4 mRNA levels. Protein levels of ADAMTS-4 were significantly higher in MS tissue compared to normal tissue. Immunohistochemical studies demonstrated that ADAMTS-4 was associated predominantly with astrocytes with increased expression within MS lesions. TIMP-3 mRNA was significantly decreased in MS compared to controls. These studies suggest a role for ADAMTS-4 in the pathogenesis of MS. Further studies on the activity of ADAMTS-4 will enable a better understanding of its role in the turnover of the ECM of white matter in MS.

Cannabinoids in pain and inflammation.

Cannabinoids exhibit medicinal properties including analgesic, anti-inflammatory and immunosuppressive properties. This paper reviews some of the recent findings in the study of cannabinoids in pain and inflammation. Some of the effects of cannabinoids are receptor mediated and others are receptor independent. Endocannabinoids naturally reduce pain and are cerebroprotective. Natural and synthetic cannabinoids have the potential to reduce nociception, reverse the development of allodynia and hyperalgesia, reduce inflammation and inflammatory pain and protect from secondary tissue damage in traumatic head injury.

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes

OBJECTIVE Interleukin-1β (IL-1β) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. METHODS Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. RESULTS Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. CONCLUSION Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

Expression of ADAM-17, TIMP-3 and fractalkine in the human adult brain endothelial cell line, hCMEC/D3, following pro-inflammatory cytokine treatment

ADAM-17 expression is localised to endothelial cells in the human central nervous system (CNS) and is increased in multiple sclerosis (MS) white matter, suggesting a role in MS pathogenesis. Expression of ADAM-17, TIMP-3, and fractalkine were investigated in a human brain endothelial cell line (hCMEC/D3) after pro-inflammatory cytokine treatment. Tumour necrosis factor (TNF) significantly increased fractalkine mRNA (>100 fold) and protein expression, which was associated with increased shedding of fractalkine from the cell. Fractalkine shedding may regulate immune cell trafficking into the CNS, however, this does not appear to be directly controlled by ADAM-17 activity.