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British Journal of Rheumatology 1997;36:522–529 DETECTION OF PROTEIN AND mRNA OF VARIOUS COMPONENTS OF THE NADPH OXIDASE COMPLEX IN AN IMMORTALIZED HUMAN CHONDROCYTE LINE P. J. MOULTON, T. S. HIRAN, M. B. GOLDRING* and J. T. HANCOCK Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY and *Harvard Medical School, Massachusetts General Hospital, East Charlestown, MA, USA SUMMARY The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 × 10−2 nmol/min/106 cells (... = 20.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1b and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox, p47-phox and p67-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox and p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions. K : Cytokines, Human chondrocyte, NADPH oxidase, Superoxide. T onset of rheumatoid arthritis (RA) is characterized by an acute inflammatory response in the affected joint. During this inflammatory response, tissue destruction, particularly of the cartilage matrix, occurs within the joint [1]. This destruction may involve reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and hydroxyl radicals [2]. As well as having a direct role in the destruction of the joint tissue [3–5], ROS have also been shown to inhibit the synthesis of new molecules that comprise the cartilage [6–10], a situation which would exacerbate tissue loss. A number of sources for ROS have been suggested. These include the large number of neutrophils which infiltrate the joint during the early stages of RA [9], the xanthine oxidase enzyme complex in cells of the synovial endothelium [11] or chondrocytes within the cartilage matrix [12–15]. Further evidence for the role that active oxygen species play in the onset and development of arthritis comes from studies of the anti-inflammatory effects of superoxide dismutase (SOD) [16] and the anti-arthritic activity of superoxide scavenging compounds [17]. The breakdown of cartilage, as well as inhibition of the synthesis of the cartilage matrix, has also been shown to be influenced by the presence of several signalling molecules. These include interleukin-1b (IL-1b) and tumour necrosis factor-a (TNF-a) [12, 18–25]. The presence of both these molecules has been shown in inflamed joints and their actions have been implicated in the aetiology of RA. Neutrophils recognize and bind to infecting microorganisms, and subsequently destroy them by the release of ROS, derived from superoxide, into the developing phagosome. This superoxide is produced by a membrane-bound enzyme complex known as NADPH oxidase. The NADPH oxidase enzyme complex consists of a plasma membrane-associated low-potential cytochrome b which consists of two subunits: an a subunit of 22 kDa (p22-phox) and a b subunit of 91 kDa (gp91-phox) [26–28]. The cytochrome b also contains binding sites for FAD and NADPH [29, 30]. Cytosolic components of the NADPH oxidase system include three polypeptides of 40, 47 and 67 kDa known as p40-phox, p47-phox and p67-phox, respectively [31–33]. The exact interaction of these three proteins, along with a GTP-binding protein (p21−rac) and the membrane-bound component of the NADPH oxidase system has not been fully elucidated, but several mechanisms have been proposed [34, 35]. In this paper, we show that the immortalized human chondrocyte (IHC) cell line (C-20/A4) can generate ROS and that the enzyme responsible is related to NADPH oxidase. Previous work using this cell line has shown it to be a good representative model system for the study of chondrocyte function [36, 37]. Although it has been suggested that chondrocytes contain the NADPH oxidase complex [12], we show here for the first time the presence of NADPH oxidase polypeptides and the expression of the respective genes in a cartilage-derived cell type. MATERIALS AND METHODS Materials Human recombinant (hr) interleukin-1b (hrIL-1b) and hr tumour necrosis factor-a (hrTNF-a) were a kind gift from Dr N. Topley, Institute of Renal Medicine, University of Wales College of Medicine, Cardiff Royal Infirmary, Cardiff, or were supplied by, along with hr interleukin-4 (hrIL-4), R&D Systems (Abingdon). Primary antibodies were kindly provided by Prof. A. W. Segal, Department of Medicine, University College London, London, and Prof. O. T. G. Jones, Department of Biochemistry, University of Bristol, who also provided some polymerase chain reaction Submitted 4 June 1996; revised version accepted 1 November 1996. Correspondence to: J. T. Hancock, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY. = 1997 British Society for Rheumatology 522 MOULTON ET AL.: NADPH OXIDASE IN HUMAN CHONDROCYTES (PCR) primers. All other materials used were obtained commercially. Culture conditions The C-20/A4 human chondrocyte cell line was derived from juvenile costal chondrocytes from a specimen of rib cartilage which were immortalized using the origin-defective simian virus 40 containing large T antigen (SV40Tag) [36, 37]. These cells were cultured in cell culture flasks with a growth area of 75 cm2 as a monolayer in a 50/50 mixture of Ham’s F-12/DMEM containing 1 × Nutridoma-SP, 4 m -glutamine, 5 U/ml penicillin, 0.005 mg/ml streptomycin, 24 U/ml nystatin and 7.5 mg/ml amphotericin B at 37°C in a 5% CO2 atmosphere. For routine subculture at subconfluence, the cells were washed with sterile phosphate-buffered saline (PBS) and then harvested using 0.05% trypsin. Cells for use in either protein preparations or RNA preparations were also harvested at this time in the same way. Preparation of chondrocyte membrane and cytosolic proteins Chondrocytes were harvested as described above. Cells (02 × 106 ) were resuspended in 1 ml of lysis buffer [10 m Tris–HCl (pH 7.4), 1% NP-40 (v/v), 150 m NaCl, 1 m Na2-EDTA] in microcentrifuge tubes and maintained on ice for 30 min. The cell lysate was centrifuged (2000 g, 5 min, room temperature) and the resulting supernatant was removed to an ultracentrifuge tube and spun (106 000 g, 45 min, 4°C). The supernatant, containing the cytosolic proteins, was removed to microcentrifuge tubes and the protein was precipitated by adding 5 vols of ice-cold acetone and maintaining at −20°C for 20 min. The protein was collected by centrifugation (13 000 g, 5 min, room temperature), the supernatant was removed and discarded, and the pellet that remained was resuspended in sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) loading dye [75 m Tris–HCl (pH 6.75), 40 m dithiothreitol (DTT), 10% glycerol, 2% SDS, 0.001% bromophenol blue]. The membrane proteins, a pellet formed during ultracentrifugation, were resuspended in SDS–PAGE loading dye. Electrophoresis and immunoblotting Protein fractions were separated by SDS–PAGE using a 12% polyacrylamide gel and were subsequently electroblotted onto Immobilon-P for 30 min at 250 V. Blocking, primary antibody addition, secondary antibody addition, washing and developing of the sample membranes were as described in the manufacturer’s instructions in the ECL Western blotting detection system. Incubation times and dilution rates for the primary antibodies were provided by A. W. Segal (personal communication) and from previous work using the antibodies [38, 39]. 523 Preparation of total RNA Chondrocytes were harvested as described previously. The cells were resuspended in RNAzol B (01 ml per 2 × 106 cells) as per the manufacturer’s instructions. To eliminate DNA contamination, the RNA was harvested by centrifugation (13 000 g, 15 min room temperature), washed in 70% ethanol and resuspended in 20 ml DNase I buffer (100 m sodium acetate, 5 m MgCl2 in deionized H2O). DNase I was added at 1 mg/ml (10 000 U/mg) and incubation was at 25°C for 30 min. The RNA was reprecipitated and was stored at −80°C. Preparation of cDNA and polymerase chain reaction The RNA was harvested by centrifugation (13 000 g, 15 min, room temperature), washed with 70% ethanol and resuspended in deionized H2O. Synthesis of cDNA was as described by Topley et al. [40] using Superscript II reverse transcriptase (RT) (Life Technologies Ltd, Paisley) in the presence of RNase inhibitor. The PCR was performed as described by Topley et al. [40]. PCR sense and antisense primers were as described by Jones et al. [41] or were designed for this study (Table I). Determination of superoxide production Chondrocytes were harvested as described previously. The cells were counted and seeded at a fixed density, 0.5 × 105 cells/well, in 24 well cell culture plates, flat bottom wells each with a growth area of 1.88 cm2, and were incubated for a further 24 h in the normal growth medium or in the normal growth medium with various cytokine pre-assay treatments added. Prior to assay, the cells were washed several times in Krebs–Ringer buffer and 1.5 ml of assay buffer, Krebs–Ringer buffer containing 80 m cytochrome c with or without stimuli, was then added to TABLE I Sequences of the oligonucleotide primers used in PCR amplification [41] except for * (this study) Gene a-Actin [42] p22-phox [27, 43] p40-phox [31] p47-phox [32] p67-phox [33] gp91-phox [28] Primer Primer name direction a+ a− 22+ 22− 40+ *40n+ 40− *40n− 47+ *47n+ 47− *47s− *47n− 67+ 67− 91+ 91− Forward Reverse Forward Reverse Forward Forward Reverse Reverse Forward Forward Reverse Reverse Reverse Forward Reverse Forward Reverse Primer sequence (5'–3') GGAGCAATGATCTTGATCTT TCCTGAGGTACGGGTCCTTCC GTTTGTGTGCCTGCTGGAGT TGGGCGGCTGCTTGATGGT GTCTGGGTGCTGATGGATGA CCTATGACTCAGAGCAGGTG TCTTCGTAGTAGTAGCAACG TCAGCGTCCCGGTAATTCAG ACCCAGCCAGCACTATGTGT GTCGGAGAAGGTGGTCTAC AGTAGCCTGTGACGTCGTCT CATCAAGTATGTCTCTGGCT TTGCTTTCATCTGACAGAACC CGAGGGAACCAGCTGATAGA CATGGGAACACTGAGCTTCA TGGGCTGTGAATGAGGGGCT TGACTCGGGCATTCACACAC 524 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 5 TABLE II Superoxide production by the cell line C-20/A4. Differences in the rate of superoxide production by the chondrocyte cell line C-20/A4 when subjected to different treatments were determined as described in Materials and methods. Control data are from cells that had no treatment. The percentage difference is the difference between the rates of superoxide production of the treated and control cells Treatment Average rate of O2− production (2...) Total (×10−2 nmol/ Number of number of Percentage 6 min/10 cells) experiments replicates difference Control Ionomycin (5 mg/ml) 5.5 2 0.7 6.8 2 0.8 6 6 18 18 +24 Control IL-1b (100 ng/ml) 3.7 2 0.7 3.7 2 0.9 4 4 12 11 0 Control IL-4 (100 ng/ml) 1.3 2 0.3 1.3 2 0.3 1 1 3 2 0 Control TNF-a (100 ng/ml) 3.3 2 0.9 3.1 2 1.0 2 2 6 2 −1 Control fMLP (1 m) 2.6 2 0.6 2.7 2 0.8 2 2 6 4 +1 Control PMA (1 mg/ml) 4.6 2 0.4 6.1 2 0.4 3 3 9 9 +31 each well. Control wells were prepared as above with 10 mg/ml superoxide dismutase added. The cells were then incubated for up to 2 h before the first spectroscopic readings were taken. Cytochrome c reduction was determined spectroscopically by the difference in the absorbance at 550 and 540 nm, the production of superoxide being determined with the extinction coefficient of cytochrome c (E550–540 cytochrome c = 19.1 m/cm). Cell numbers were determined by harvesting, as described previously, and counting the cells. RESULTS Superoxide production Using the discontinuous cytochrome c reduction assay for the measurement of superoxide release by C-20/A4 cells, it was found that in the absence of an added stimulus, the cells constitutively generated superoxide at a rate of 05.4 × 10−2 nmol/min/106 cells (... = 20.5, n = 30). The addition of ionomycin (5 mg/ml) or PMA (phorbol myristate acetate) (1 mg/ ml) at the start of the assay enhanced the rate of superoxide generation by 024 or 31%, respectively (Table II). However, no significant stimulation of the generation of superoxide was seen when IL-1b, IL-4, TNF-a or the bacterial peptide f-Met-Leu-Phe (fMLP) were added at the start of the assay. A 24 h pre-treatment of C-20/A4 cells with IL-1b, with a subsequent addition of a stimulatory dose of ionomycin, resulted in a rate of superoxide production that was 26% higher than in those cells cultured without IL-1b and then stimulated with ionomycin (Table III). However, interestingly, pre-treatment with either IL-4 or TNF-a reduced the final rate of release of superoxide by 26 or 11%, respectively. Detection of mRNA for components of the NADPH oxidase complex PCR products of the expected base pair size were obtained from cDNA prepared from unstimulated C-20/A4 RNA using primers for p22-phox (primers 22+/22−; product size 316 bp) (Fig. 1a), p40-phox (primers 40+/40−; product size 380 bp) (Fig. 1b), p47-phox (primers 47+/47−; product size 767 bp) (Fig. 1c) and p67-phox (primers 67+/67−; product size 746 bp) (Fig. 1d). The PCR products obtained with the p67-phox (67+/67−) and p47-phox (47n+/47n−) primers were cloned into the pCRTMII vector using a TA cloning kit (Invitrogen) and partially sequenced. The partial sequence from the p67-phox derived PCR fragment (0200 bp) showed 090% homology to the published human cDNA sequence, while the partial sequence from the p47-phox derived PCR fragment (0151 bp) showed 089% homology to the published human cDNA sequence. a-Actin PCR primers were used as a control to assess RNA integrity (results not shown). PCR amplification using primers for gp91-phox consistently generated three products of 0200, 420 and 480 bp in size, although a product size of 383 bp was expected (results not shown). The most prominent PCR product, the band of 480 bp, was cloned and partially sequenced, but bore no similarity to the published gene sequence for gp91-phox. Further various combinations of forward and reverse primers were also used. The primer combinations 47n+/47n− (496 bp), 47n+/47s− (318 bp) TABLE III Superoxide production by the cell line C-20/A4 after a 24 h pre-incubation with cytokines and subsequent stimulation with ionomycin. Differences in the rate of superoxide production by the chondrocyte cell line C-20/A4 when subjected to different treatments were determined as described in Materials and methods. Control data are from cells that had no pre-treatment but were stimulated with ionomycin. The percentage difference was the difference between the rates of superoxide production of the treated and control cells Treatment Average rate of O2− production (2...) Total (×10−2 nmol/ Number of number of Percentage min/106 cells) experiments replicates difference Control IL-1b (100 ng/ml) 5.3 2 1.2 6.7 2 1.9 3 3 9 9 +26 Control IL-4 (100 ng/ml) 5.3 2 1.2 3.9 2 0.9 3 3 9 9 −26 Control TNF-a (100 ng/ml) 3.2 2 0.7 2.9 2 0.6 2 2 6 6 −11 MOULTON ET AL.: NADPH OXIDASE IN HUMAN CHONDROCYTES 525 F. 1.—Agarose gel electrophoresis of PCR products obtained using primers designed against: (a) p22-phox; (b) p40-phox; (c) p47-phox; (d) p67-phox. PCR amplification was carried out on cDNA derived from unstimulated C-20/A4 cells (+RT) and on the cDNA control reaction (no reverse transcriptase added to the RNA) (−RT) as described in Materials and methods. and 40n+/40− (330 bp) all generated products of the predicted size (results not shown). As a control, cDNA was prepared from human neutrophils and PCR amplified using the 22+/22−, 40+/40−, 47+/47−, 67+/67− and 91+/91− primer sets. PCR products of the expected base pair size were generated for all primer sets tested in this way (results not shown). Immunodetection of components of the NADPH oxidase complex Cytosol and membrane protein fractions from unstimulated C-20/A4 cells cultured in the normal growth media were prepared as described in Materials and methods, and analysed by Western blotting. Polyclonal antisera to p47-phox detected a band at 47 kDa along with a lower band of 25 kDa in the cytosolic fraction of these cells (Fig. 2, Well 2). Polyclonal antisera to p67-phox detected a protein of 67 kDa in the cytosolic fraction along with a larger protein of 082 kDa (Fig. 2, Well 3). Control lanes with proteins obtained from human neutrophils also showed a similar banding pattern, although a smaller protein, 033 kDa, was also seen with the p67-phox antisera (Fig. 2, Well 4). To establish further the presence of NADPH oxidase in these cells, we probed the cell fractions with antisera raised against the other proposed components. Antisera to p40-phox detected a protein with a molecular mass less than that which was expected, while antisera to gp91-phox gave a faint band of the expected size (results not shown). Antisera to both p22-phox and p21−rac, a G protein that is reported to be involved in the activation of NADPH oxidase in neutrophils, did not hybridize to proteins of the expected size. DISCUSSION We have found that the generation of superoxide by the chondrocyte-derived immortalized C-20/A4 cells occurs constitutively at a low level. Constitutive release of ROS by other cell types [41] and of hydrogen peroxide by rabbit chondrocytes [12] has been reported F. 2.—Western blot analysis of protein from the C-20/A4 cell line. Proteins were prepared from unstimulated C-20/A4 cells as described in Materials and methods, separated on 12% polyacrylamide gels and Western blotted. The resulting membranes were hybridized to primary antibodies against components of the NADPH oxidase system which were then detected with secondary antibody and the ECL detection kit (Amersham). Well 1, neutrophil protein, p47-phox antibody; Well 2, C-20/A4 protein, p47-phox antibody; Well 3, C-20/A4 protein, p67-phox antibody; Well 4, neutrophil protein, p67-phox antibody. 526 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 5 previously. However, it was also noted that the basal levels of superoxide released by the cells varied from experiment to experiment. It has been reported by others that ROS release, along with protein synthesis and extracellular matrix deposition, is influenced by the density at which the cells are grown [44, 45]. In comparison to phagocytic cells, the rates of superoxide production are extremely low (01%), but are comparable to those reported for non-phagocytic cell types such as mesangial cells [41], endothelial cells [46], osteoclasts [47], fibroblasts [48] and pig primary articular chondrocytes [49]. The superoxide generating activity of C-20/A4 cells was enhanced by the addition of the calcium ionophore ionomycin or the protein kinase activator PMA, suggesting that both the intracellular calcium concentration and protein phosphorylation are involved in the activation of the enzyme responsible. Interestingly, superoxide production by the C-20/A4 cell line did not increase in the presence of IL-1b, a cytokine which may be produced as part of an inflammatory response. This is in contradiction to the findings in fibroblasts [48]. Furthermore, neither the cytokine TNF-a nor the bacterial derived peptide f-MLP, two known stimulators of superoxide production in other cell types, caused stimulation of the rate of superoxide production by C-20/A4 cells. Although IL-1b did not stimulate superoxide production in isolation, it was found that pre-assay incubation of the C-20/A4 cells with IL-1b resulted in a priming effect, where an enhanced rate of production was seen on the subsequent addition of ionomycin. Such a phenomenon has been observed in neutrophils [50]. It has been well recognized that neutrophils can be primed by sub-stimulatory doses of stimulant, with an enhanced stimulation seen when the final stimulant is applied. The priming effect of IL-1b was observed after a pre-assay incubation of as little as 1 h (results not shown). This priming effect may be due to the synthesis of new NADPH oxidase components, the recruitment of NADPH oxidase components which have been sequestered in some way, or an enhanced activation of existing NADPH oxidase complex components. We are currently investigating these priming effects further by the use of Northern blot analysis to compare levels of mRNA of various components of the NADPH oxidase complex in cells grown in the presence of various cytokines. It has been reported that IL-1b led to the expression of NADPH oxidase components in growtharrested mesangial cells [41] and a similar effect may be seen here. In contrast to our results, it has been shown that IL-1b can increase the production of hydrogen peroxide by rabbit articular chondrocytes in primary culture [13]. However, IL-1 response profiles have been reported to be different in primary and subcultured chondrocytes [51]. This may be a reason for the lack of immediate response to IL-1b in this study. Clearly, for IL-1b to have a priming effect, the C-20/A4 cells must possess IL-1b receptors and it has been shown previously that IL-1b causes decreased expression of cartilage matrix protein genes and increased expression of genes for non-specific collagens in these cells [24]. Another reason for the lack of immediate generation of superoxide in response to IL-1b may be a partial disruption of the signalling pathways within the cell, possibly caused by the immortalization procedure. On the other hand, pre-treatment with IL-4 caused a reduction in the ionomycin response. This reduction in the rate of superoxide production was probably not due to a loss in cell viability. Using the exclusion of trypan blue as an indicator of cell viability, it was found that the use of IL-4 as a stimulant did not appear to reduce cell viability significantly (results not shown). Furthermore, the rate of cytochrome c reduction in the control cells, cells assayed in the presence of superoxide dismutase, for both the ionomycin-stimulated cells and the IL-4 pre-treated cells that were subsequently stimulated with ionomycin, was comparable. This demonstrated that non-specific reduction of the cytochrome c due to the release of cell contents upon cell death was no different for the two treatments, suggesting that increased cell death did not account for the reduction in the rate of superoxide production in the IL-4 pre-treated cells. Similar effects of IL-4 were also noted in pig macrophages [52], where it was shown that IL-4 caused a reduction in superoxide production via a suppression of the expression of the gp91-phox component of the NADPH oxidase. Similarly, IL-4 was shown to inhibit superoxide production by human mononuclear phagocytes [53]. We are at present studying further the effects of other cytokines/chemokines on superoxide production as well as the effects of activators and inhibitors of signal transduction pathways. One of the major sources of superoxide in phagocytic and non-phagocytic cells is reported to be the NADPH oxidase complex, and we therefore used the NADPH oxidase of human phagocytes as a model. Using RT-PCR to probe for the existence of mRNA encoding the components of NADPH oxidase in these cells, we give evidence for the first time that chondrocytic cells express mRNAs for p40-phox, p47-phox, p67-phox and p22-phox components of the NADPH oxidase. However, we did not find that the gp91-phox component was present in the C-20/A4 cells. Failure to demonstrate the presence of the gp91-phox component has been reported by others in mesangial cells and fibroblasts. It has been suggested that the gp91-phox subunit exists as isoenzyme forms which are different in phagocytic cells and non-phagocytic cells [41, 54]. Our results seem to support this hypothesis. To confirm that the NADPH oxidase was expressed as protein components and not only as mRNA, the membrane and cytosol fractions from C-20/A4 cells were analysed by Western blotting. This demonstrated the presence of the polypeptides p47-phox and p67-phox. The smaller protein detected using the p67-phox antisera has been proposed to be a breakdown product of the p67-phox polypeptide [55]. As these polypeptides have only ever been found associated with the functioning of the NADPH oxidase MOULTON ET AL.: NADPH OXIDASE IN HUMAN CHONDROCYTES complex, it is good evidence, along with the measured release of superoxide, that these cells too have a functional NADPH oxidase. Further work in our laboratory also suggests the presence of the gp91-phox component of the NADPH oxidase enzyme complex (not shown) which supports this view. With the lack of a gp91-phox PCR product, it suggests that even if this polypeptide is immunologically related, it is a different gene product to that characterized in neutrophils [54]. Interestingly, antisera raised to p40-phox showed a band at a smaller molecular weight than expected, possibly due to non-specific binding. Evidence presented here shows that a functioning NADPH oxidase enzyme complex, similar to that found in neutrophils, is present in the chondrocyte-derived immortalized cell line C-20/A4. Superoxide can be generated by a wide variety of cell types [56], although commonality of generation and function has yet to be established, and it seems possible that the presence of cytochrome b−245 isoenzymes may allow differential generation of superoxide by a variety of cell types in response to a number of stimuli. The role of such superoxide release, however, remains unclear. The ROS generation of phagocytes is essentially used in a destructive and killing mode. It has been suggested that the ROS produced by chondrocytes are involved in the homeostatic maintenance of the cartilage matrix [12]. However, mounting evidence suggests that ROS may have a role in cell signalling. It has been reported that hydrogen peroxide promotes proliferation of cells and leads to activation of transcription factors [57]. More recently, it has been reported that TNF-a induced activation of the transcription factor NFkb can be inhibited by the antioxidant agent N-acetyl--cysteine [58]. Hydrogen peroxide has also been shown to activate MAP kinase pathways [59] and guanylyl cyclase [60]. However, the native environment of a chondrocyte is in a relatively anaerobic environment which is poorly supplied by the blood and, therefore, the physiological relevance of chondrocyte superoxide release needs to be established. However, it is possible that if damage occurs to the cartilage, then the environment of a chondrocyte may become more aerobic and, therefore, superoxide release may become relevant to further damage or even repair. Having established the presence of NADPH oxidase in this immortalized cell line, we intend to carry out a full sequence analysis of all the NADPH oxidase components from these cells as well as isolated articular chondrocytes to establish whether there are any differences between the NADPH oxidase complex in neutrophils and chondrocytes. It seems as though this will be particularly important in the case of the gp91-phox component. Further work will also investigate the presence of NADPH oxidase in human chondrocytes isolated from articular tissues. A We acknowledge the following: Prof. O. T. G. Jones, Ashley M. Toye and Marcus J. Coffey (Department of Biochemistry, University of Bristol) for the design and 527 supply of PCR oligonucleotide primers 40+/40−; Professor A. W. 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