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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. Segal for supplying antibodies. This
work was funded by the Arthritis & Rheumatism
Council for Research (ARC) grant no. H0508.
R
1. Eyre DR. The collagens of articular cartilage. Semin
Arthritis Rheum 1991;21:2–11.
2. Greenwald RA. Oxygen radicals, inflammation, and
arthritis: pathophysiological considerations and implications for treatment. Semin Arthritis Rheum
1991;20:219–40.
3. Bates EJ, Harper GS, Lowther DA, Preston BN. Effect
of oxygen-derived reactive species on cartilage proteoglycan-hyaluronate aggregates. Biochem Int 1984;8:629–37.
4. Dean RT, Roberts CR, Forni LG. Oxygen-centred free
radicals can efficiently degrade the polypeptide of
proteoglycans in whole cartilage. Biosci Rep
1984;4:1017–26.
5. Panasyuk A, Frati E, Ribault D, Mitrovic D. Effect of
reactive oxygen species on the biosynthesis and structure
of newly synthesized proteoglycans. Free Radical Biol
Med 1994;16:157–67.
6. Bates EJ, Lowther DA, Handley CJ. Oxygen freeradicals mediate an inhibition of proteoglycan synthesis
in cultured articular cartilage. Ann Rheum Dis
1984;43:462–9.
7. Bates EJ, Johnson CC, Lowther DA. Inhibition of
proteoglycan synthesis by hydrogen peroxide in cultured
bovine articular cartilage. Biochim Biophys Acta
1985;838:221–8.
8. Bates EJ, Lowther DA, Johnson CC. Hyaluronic acid
synthesis in articular cartilage: an inhibition by hydrogen
peroxide. Biochem Biophys Res Commun 1985;132:714–
20.
9. Baker MS, Feigan J, Lowther DA. Chondrocyte
antioxidant defences: the roles of catalase and glutathione peroxidase in protection against H2O2 dependent
inhibition of proteoglycan biosynthesis. J Rheumatol
1988;15:670–7.
10. Baker MS, Feigan J, Lowther DA. The mechanism of
chondrocyte hydrogen peroxide damage. Depletion of
intracellular ATP due to suppression of glycolysis caused
by oxidation of glyceraldehyde-3-phosphate dehydrogenase. J Rheumatol 1989;16:7–14.
11. Stevens CR, Benboutra M, Harrison T, Smith EC, Blake
DR. Localization of xanthine-oxidase to synovial
endothelium. Ann Rheum Dis 1991;50:760–2.
12. Tiku ML, Liesch JB, Robertson FM. Production of
hydrogen peroxide by rabbit articular chondrocytes. J
Immunol 1990;145:690–6.
13. Rathakrishnan C, Tiku K, Raghavan A, Tiku ML.
Release of oxygen radicals by articular chondrocytes: A
study of luminol-dependent chemiluminescence and
hydrogen peroxide secretion. J Bone Miner Res
1992;7:1139–48.
14. Henrotin Y, Deby-Dupont G, Deby C, De Bruyn M,
Lamy M, Franchimont P. Production of active oxygen
species by isolated human chondrocytes. Br J Rheumatol
1993;32:562–7.
15. Rathakrishan C, Tiku ML. Lucigenin-dependent chemiluminescence in articular chondrocytes. Free Radical
Biol Med 1993;15:143–9.
16. Shingu M, Takahashi S, Ito M et al. Anti-inflammatory
effects of recombinant human manganese superoxide
528
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 5
dismutase on adjuvant arthritis in rats. Rheumatol Int
1994;14:77–81.
Wooley PH, Whalen JD. The influence of superoxide
scavenging compound CTC 23 on type II collageninduced arthritis in mice. Agents Actions 1992;35:273–9.
Dayer JM, Beutler B, Cerami A. Cachectin/tumor
necrosis factor stimulates collagenase and prostaglandin
E2 production by human synovial cells and dermal
fibroblasts. J Exp Med 1985;162:2163–8.
Benton HP, Tyler JA. Inhibition of cartilage proteoglycan synthesis by interleukin-1. Biochem Biophys Res
Commun 1988;154:421–8.
Buchan G, Barrett K, Turner M, Chantry D, Maini RN,
Feldmann M. Interleukin-1 and tumor necrosis factor
mRNA expression in rheumatoid arthritis: prolonged
production of IL-1a. Clin Exp Immunol 1988;73:449–55.
Arner EC, Pratta MA. Independent effects of interleukin1 on proteoglycan breakdown, proteoglycan synthesis,
and prostaglandin E2 release from cartilage in organ
culture. Arthritis Rheum 1989;32:288–97.
Henderson B, Pettipher ER. Arthritogenic actions of
recombinant IL-1 and tumor necrosis factor a in the
rabbit: evidence for synergistic interactions between
cytokines in vivo. Clin Exp Immunol 1989;75:306–10.
Ismaiel S, Atkins RM, Pearse MF, Dieppe PA, Elson CJ.
Susceptibility of normal and arthritic human articular
cartilage to degradative stimuli. Br J Rheumatol
1992;31:369–73.
Goldring MB, Fukuo K, Birkhead JR, Dudek E, Sandell
LJ. Transcriptional suppression by interleukin-1 and
interferon-g of type II collagen gene expression in human
chondrocytes. J Cell Biochem 1993;54:1–15.
Krane SM, Conca W, Stephenson ML, Amento EP,
Goldring MB. Mechanisms of matrix degradation in
rheumatoid arthritis. Ann NY Acad Sci 1990;580:340–
54.
Segal AW. Absence of both cytochrome b−245 subunits
from neutrophils in X-linked chronic granulomatous
disease. Nature 1987;326:88–91.
Parkos CA, Allen RC, Cochrane CG, Jesaitis AJ.
Purified cytochrome b from human granulocyte plasma
membrane is comprised of 2 polypeptides with relative
molecular weights of 91,000 and 22,000. J Clin Invest
1987;80:732–42.
Teahan C. Rowe P, Parker P, Totty N, Segal AW. The
X-linked CGD gene codes for the b chain of cytochrome
b−245. Nature 1987;327:720–1.
Rotrosen D, Yeung CL, Leto TL, Malech HL, Kwong
CH. Cytochrome b558: The flavin-binding component of
the phagocyte NADPH oxidase. Science 1992;256:1459–
62.
Segal AW, West I, Wientjes F et al. Cytochrome b−245 is
a flavcytochrome containing FAD and the NADPHbinding site of the microbicidal oxidase of phagocytes.
Biochem J 1992;284:781–8.
Wientjes FB, Husan JJ, Totty NF, Segal AW. p40-phox,
a third cytosolic component of the activation complex of
the NADPH oxidase to contain src homology 3 domains.
Biochem J 1993;296:557–61.
Volpp BD, Nauseef WM, Clark RA. Cloning of the
cDNA and functional expression of the 47 kDa cytosolic
component of human neutrophil respiratory burst
oxidase. Proc Natl Acad Sci USA 1989;86:7195–9.
Leto TL, Lomax KJ, Volpp BD et al. Cloning of a
67 kDa neutrophil oxidase factor with similarity to a
noncatalytic region of P60C-SRC. Science 1990;48:727–
30.
34. Cross AR, Yarchover JL, Curnutte JT. The superoxidegenerating system of human neutrophils possesses a
novel diaphorase activity. J Biol Chem 1994;269:21488–
94.
35. de Mendez I, Garret MC, Adams AG, Leto TL. Role of
p67-phox SH3 domains in assembly of the NADPH
oxidase system. J Biol Chem 1994;269:16326–32.
36. Goldring MB, Birkhead JR, Suen LF, Yamin R. Use of
immortalized human chondrocytes in studies of IL-1mediated gene expression. Arthritis Rheum 1993;36:S49.
37. Goldring MB, Birkhead JR, Suen LF, Yamin R, Mizuno
S. Interleukin-1b-modulated gene expression in immortalized human chondrocytes. J Clin Invest 1994;94:2307–
16.
38. Desikan R, Hancock JT, Coffey MJ, Neill SJ.
Generation of active oxygen in elicited cells of
Arabidopsis thaliana is mediated by a NADPH
oxidase-like enzyme. FEBS Lett 1996;382:213–7.
39. Kummer W, Acker H. Immunohistochemical demonstration of 4 subunits of neutrophil NAD(P)H oxidase in
type-I cells of carotid-body. J Appl Physiol
1995;78:1904–9.
40. Topley N, Brown Z, Jorres A et al. Human peritoneal
mesothelial cells synthesise interleukin-8 synergistic
induction by interleukin-1b and tumour necrosis
factor-a. Am J Pathol 1993;142:1876–86.
41. Jones SA, Hancock JT, Jones OTG, Neubauer A, Topley
N. The expression of NADPH oxidase components in
human glomerular mesangial cells: detection of protein
and mRNA for p47-phox, p67-phox, and p22-phox. J
Am Soc Nephrol 1995;5:1–9.
42. O’Bryan JP, Frye RA, Cogswell PC et al. AXL. A
transforming gene isolated from primary human myeloid
leukemia cells, encodes a novel receptor tyrosine kinase.
Mol Cell Biol 1991;11:5016–31.
43. Parkos CA, Dinauer MC, Walker LE, Allen RA, Jesaitis
AJ, Orkin SH. Primary structure and unique expression
of the 22 kDa light chain of human neutrophil
cytochrome b. Proc Natl Acad Sci USA 1988;85:3319–
23.
44. Wolthuis A, Boes A, Grond J. Cell-density modulates
growth, extracellular-matrix, and protein synthesis of
cultured rat mesangial cells. Am J Pathol 1993;143:1209–
19.
45. Jones SA. PhD Thesis. University of Bristol, 1995.
46. Rosen GM, Freeman BA. Detection of superoxide
generated by endothelial cells. Proc Natl Acad Sci USA
1984;81:7269–73.
47. Garrett IR, Boyce BF, Oreffo ROC, Bonewald L, Poser
J, Mundy GR. Oxygen-derived free-radicals stimulate
osteoclastic bone-resorption in rodent bone in vitro and
in vivo. J Clin Invest 1990;85:632–9.
48. Meier B, Radeke HH, Selle S et al. Human fibroblasts
release reactive oxygen species in response to interleukin1 or tumour necrosis factor-a. Biochem J 1989;263:539–
45.
49. Hiran T, Moulton P, Hancock JT. Superoxide production in articular chondrocytes by a phagocyte-like
NADPH oxidase. Eur J Clin Invest 1995;25(S2):A26.
50. McPhail LC, Shirley PS, Clayton CC, Snyderman R.
Activation of the respiratory burst enzyme from human
neutrophils in a cell-free system. Evidence for a soluble
cofactor. J Clin Invest 1985;75:1735–9.
51. Guerne PA, Sublet A, Lotz M. Growth-factor responsiveness of human articular chondrocytes: Distinct
profiles in primary chondrocytes, subcultured chondrocytes and fibroblasts. J Cell Physiol 1994;158:476–84.
MOULTON ET AL.: NADPH OXIDASE IN HUMAN CHONDROCYTES
52. Zhou Y, Lin G, Murtaugh MP. Interleukin-4 suppresses
the expression of macrophage NADPH oxidase heavy
chain subunit (gp91-phox). Biochim Biophys Acta
1995;1265:40–8.
53. Abramson SL, Gallin JI. Interleukin-4 (IL-4) inhibits
superoxide production by human mononuclear phagocytes. Clin Res 1989;37:825A.
54. Meier B, Jesaitis AJ, Emmendorffer A, Roesler J, Quinn
MT. The cytochrome b558 molecules involved in the
fibroblast and polymorphonuclear leucocyte superoxidegenerating NADPH oxidase systems are structurally and
genetically distinct. Biochem J 1993;289:481–6.
55. Tsunawaki S, Mizunari H, Nagata M, Tatsuzawa O,
Kuratsuji T. A novel cytosolic component, p40-phox, of
respiratory burst oxidase associates with p67-phox and is
absent in patients with chronic granulomatous disease
who lack p67-phox. Biochem Biophys Res Commun
1994;199:1378–87.
56. Cross AR, Jones OTG. Enzymatic mechanisms of
57.
58.
59.
60.
529
superoxide production. Biochim Biophys Acta
1991;1057:281–98.
Schreck R, Rieber P, Baeuerle PA. Reactive oxygen
intermediates as apparently widely used messengers in
the activation of the NFkb transcription factor and
HIV-1. EMBO J 1991;10:2247–58.
Fujisawa K, Aono H, Hasunuma T, Yamamoto K, Mita
S, Nishioka K. Activation of transcription factor NFkb
in human synovial cells in response to Tumor-NecrosisFactor-a. Arthritis Rheum 1996;39:197–203.
Stevenson MA, Pollock SS, Coleman CN, Calderwood
SK. X-irradiation, phorbol esters and H2O2 stimulate
mitogen-activated protein-kinase activity in NIH-3T3
cells through the formation of reactive oxygen intermediates. Cancer Res 1994;54:12–5.
Burke-Wolin T, Abate CJ, Wolin MS, Gurtner GH.
Hydrogen peroxide induced pulmonary vasodilation—
role of guanosine 3',5' cyclic monophosphate. Am J
Physiol 1991;261:L393–8.