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Annals of the Rheumatic Diseases, 1983, 42, 558-562

Leucocyte superoxide dismutase in rheumatoid

arthritis
AMIR A-R. YOUSSEF* AND DENIS N. BARON
From the Departments of Chemical Pathology and Rheumatology, Royal Free Hospital and School of
Medicine, London

SUMMARY Superoxide dismutase (SOD) activity was measured in polymorphonuclear leucocytes

(PMNLs) and mononuclear cells (MNCs) from 60 patients with rheumatoid arthritis (RA) and in
15 controls. In all patients and controls SOD activity (U/mg protein) in MNCs was twice that in
PMNLs. SOD activity in PMNLs and in MNCs from patients with RA was significantly higher than
that in controls. SOD activity in PMNLs (but not in MNCs) from patients treated with cortico-
steroids was significantly higher than that from patients treated with nonsteroidal anti-
inflammatory drugs. There was no relation between SOD activity in both PMNLs and MNCs and
either the patients' age, sex, duration of disease, serum immunoglobulin concentration, IgM
rheumatoid factor, and copper level, or the degree of disease activity.

The superoxide dismutases (SOD; EC 1.15.1.1)
are a group of metalloenzymes whose function
appears to be the protection of cells from the toxic
effects of the endogenously generated superoxide
radical (0O-).1 These enzymes catalyse the reaction
SOD
O2- +O2- +2H+ - H202 + 02
Considerable evidence has accumulated in recent
years suggesting that O- might be generated by
phagocytosing leucocytes during inflammation,2-4
for example, in the joints of patients with rheumatoid
arthritis (RA). This radical may be involved in the
pathological changes of RA which occur in the syno-
vial fluid,56 articular cartilage,7 and synovial tissue.8
Since polymorphonuclear leucocytes (PMNLs)
and mononuclear cells (MNCs) differ both in func-
tion and in morphology, we studied the total SOD
activity separately in each cell type. Peripheral
PMNLs and MNCs were obtained from patients with
RA and from healthy controls.
Patients and methods
PATIENTS
Sixty patients (40 females and 20 males) with classi- by
cal or definite RA' were studied. Their ages ranged means
Accepted for publication 6 August 1982.
Correspondence to Profesor D. N. Baron, Department of Chemical 2-fold diluted blood were layered on 3 parts of
Pathology, Royal Free Hospital, Pond Street, London NW3 2QG.
*Present address: Department of Medicine, Mansoura University blood cells were separated into 2 fractions: an MNC
Hospital, Mansoura, Egypt.
from 37 to 84 years (median 63) and the duration of
disease from one to 27 years (median 9). Twenty-
four patients had extra-articular manifestations
including subcutaneous nodules, peripheral neuritis,
and vasculitic skin ulcers. Immunoglobulin M
rheumatoid factor (IgMRF) was positive in 42
patients. All patients were treated with nonsteroidal
anti-inflammatory drugs (NSAID), and 20 of them
were receiving additional treatment with cortico-
steroids in the form of prednislone 2-5-10 mg/day.
The overall disease activity of the RA patients was
assessed by means of a composite activity index
(CAI) which included both clincal and laboratory
markers combining to form a single score.'° 11 With
the CAI score RA patients were classified into
slightly, moderately, and highly active groups.
A group of 15 apparently healthy subjects (10
females and 5 males) were included as controls. Their
ages ranged from 33 to 76 years (median 58).
METHODS
PMNLs and MNCs were obtained from 8 ml fresh
heparinised peripheral blood. MNCs were isolated
a modification of the method of Boyum,"2 by
of a Ficoll-paque gradient (Pharmacia Fine
Chemicals A B, Uppsala, Sweden). Four parts of
Ficoll-paque solution and then centrifuged. The
layer at the interface between plasma and platelets
558
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Leucocyte superoxide dismutase in rheumatoid arthritis 559

above and Ficoll-paque below, and a bottom layer Other laboratory investigations
containing erythrocytes and PMNLs. The MNC layer The following were measured according to standard
was washed twice with saline (154 mmol/l). PMNLs laboratory methods10 IgMRF, immunoglobulins
1:

were isolated by a modification of the method of (IgG, IgM, IgA), and copper in serum.
Baron and Ahmed.13 The bottom layer was diluted
twice with saline (154 mmol/l). A dextran solution Statistical analysis
(mol mass 250 kDa, 6% wt/vol) in isotonic TC 199 Comparison between groups was performed by the
(Glaxo Ltd, London) was added in the proportion of Mann-Whitney U test (2-tailed). Correlations were
1 part to 4 parts of blood vol/vol. The tube was left calculated by means of Pearson's correlation
vertical for 30 min at room temperature to allow coefficient (r).
sedimentation of erythrocytes under gravity. The
supernatant was then centrifuged at 300 g. Con- Results
taminating erythrocytes in both MNCs and PMNLs
were lysed by exposure to deionised water. Isoton- Comparison between SOD activity in PMNLs and
icity was restored by the addition of 4-fold isotonic MNCs (Table 1)
TC 199. Isolated leucocytes were suspended in phos- In patients and controls the median SOD activity in
phate buffered saline (PBS), pH 7.4, and cell viability MNCs was approximately twice that of PMNLs. The
was assessed by the dye impermeability test."4 Viabil- range of SOD activity in MNCs was greater than that
ity of both MNCs and PMNLs was >90%. Microscopi- of PMNLs. In both the patient and the control groups
cal examination of the isolated leucocytes was carried no significant correlation was found between SOD
out to test for purity: >90% of MNCs were lympho- activity in PMNLs and in MNCs.
cytes and <10% monocytes; PMNLs were >99%
pure. The isolated leucocytes were sonicated on ice Comparison between SOD activity in PMNLs and
with an MSE ultrasonic disintegrator Mk2 (MSE MNCs from patients and controls (Table 1)
Scientific Instruments, Sussex, England). The cellu- The median SOD activity in the PMNLs of the RA
lar debris was removed by centrifugation. The result- patients (0-8 U/mg protein) was significantly higher
ing supernatant was used for protein estimation and than that of the control group (0-6 U/mg protein,
for the enzyme assays. The protein concentration in p<0.02). The median SOD activity in the MNCs of
the supernatant was determined by a microadapta- the RA patients was also significantly higher (1.7
tion of the method of Lowry et al.15 with bovine U/mg protein) than that of the control group (1 -2
albumin as a standard. U/mg protein, p<0.005).
Superoxide dismutase assay
Reagents. Xanthine grade 3, xanthine oxidase grade SOD activity in PMNLs and MNCs from patients with
1, nitroblue-tetrazolium (NBT) and SOD standard RA in relation to the factors listed below
(type one from bovine blood) from Sigma Chemical Patients' age (years). There was no correlation
Co. Ltd, Surrey, England. Freshly prepared buffer between the patients' age and SOD activity in
(pH 10.2) containing 50 mmol/l anhydrous sodium PMNLs and MNCs.
carbonate, 100 ,mol/l disodium ethylene diamine
tetra-acetate (EDTA), 25 ,umoVl NBT, and 0-01 units
xanthine oxidase. Xanthine oxidase (150 U/l).
Xanthine 100 ,umol/l. SOD standard (2900 U/mg
protein). Table 1 SOD activity in PMNLs and MNCs ofpatients and
controls
Assay. SOD activity in PMNLs (=20x109) and
MNCs (=10x109) was assayed by the method of SOD activity
Beauchamp and Fridovich.16 SOD is detected by its
ability to inhibit the reduction of NBT mediated PMNLs MNCs
by O- which is generated during the oxidation of U/mg U/mg
protein protein
xanthine by xanthine oxidase. The reduction of NBT
was followed at 560 nm at 25°C with an SP1800 n=15 n=15
spectrophotometer (Pye Unicam Ltd, Cambridge, Normal Median 0-6 1 2
England). The decrease in the rate of NBT reduc- controls (Range) (0-4-1-3) (0.6-2-0)
n=60 n=57
tion/min was plotted against the amount of SOD. The Rheumatoid Median 0-9* 1-7t
activity of SOD in the unknown sample was then arthritis (Range) (0-3-37) (0-37-1)
determined from a standard curve. SOD activity in n =Number of determinations.
each supernatant was related to the amount of pro- Significantly different from controls (p<0.02).
tein and expressed as U/mg protein. tSignificantly different from controls (p<0.005).
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560 Youssef Baron

Table 2 SOD activity in PMNLs and MNCs ofpatients with RA in relation to patients' sex, presence or absence of
extra-articular manifestations, and presence or absence of rheumatoid factor (RF)
SOD activity (U/mg protein)
PMNLs MNCs
Male RA patients vs Female RA patients Male RA patients vs Female RA patients
n 19 41 19 38
Median 1-0 0-9 2-0 1-6
(Range) (0.3-2-0) (04-3 7) (05-5-2) (0.3-7-1)
p NS NS
RA patients with extra- vs RA patients without extra- RA patents with extra- vs RA patients without extra-
articular manifestations articular manifestations articular manifestations articular manifestations
n 24 36 22 34
Median 0-9 0-8 2-2 1-5
(Range) (0.5-1-8) (0-3-3.7) (0.9-7-1) (0.3-5-2)
p NS <0.04
RA patients with vs RA patients with RA patients with vs RA patents with
RF-positive RF-negative RF-positive RF-negative
n 42 18 39 18
Median 0-9 1-0 1-7 1-6
(Range) (0-3-3.7) (0.5-2.5) (0.5-7-1) (0.3-5.2)
p NS NS
n =Number of determinations. NS =Not significant.

Patients' sex. SOD activity in PMNLs and in MNCs Discussion

of male and female patients did not differ signifi-
cantly (Table 2).
Duration of disease. There was no correlation
between disease duration (years) and SOD activity in
PMNLs or in MNCs.
Disease activity. SOD activity in both PMNLs and
MNCs did not differ significantly when the values
obtained from the slightly active, the moderately
active and the highly active groups were compared
with each other. No correlation was found between
the CAI score'° 11 and SOD activity in PMNLs or
MNCs.
Extra-articular manifestations. Patients with extra-
articular manifestations had significantly higher SOD
activity in the MNCs but not in the PMNLs than those
without these manifestations (Table 2).
IgMRF. SOD activity in PMNLs and MNCs of
both IgMRF-negative and IgMRF-positive patients
did not differ significantly (Table 2).
Immunoglobulin levels. SOD activity in PMNLs
and MNCs showed no correlation with the level of
any of the immunoglobulins (IgG, IgM, and IgA).
Serum copper level (,umolIl). Serum copper level
showed no correlation with SOD activity either in
PMNLs or MNCs.
Type oftreatment. SOD activity of PMNLs (but not
of MNCs) obtained from RA patients treated with
corticosteroids (1-1 U/mg protein) was significantly
higher than that from those treated with NSAID (0-8
U/mg protein, p<0-02).
The present study supports the findings of other
investigators who showed that leucocytes, particu-
larly PMNLs, contain SOD.23 17 18 Our observation
that SOD activity in MNCs (90% of which were
lymphocytes) is up to twice that of PMNLs is also in
agreement with the work of others,19 I as is the wide
variation in SOD activity in MNCs.19 These findings
may reflect the morphological as well as the func-
tional diversity of these cells. This diversity is exemp-
lified by a study21 comprising SOD activity in T and
non-T lymphocytes (mainly B lymphocytes), where
the former cell type was shown- to possess almost
twice the activity of the latter. It has also been sug-
gested22 that T lymphocytes are more long lived than
B lymphocytes and that the half-life of circulating
PMNLs is only 6-8 h.23 Thus the life span of these
leucocyte populations parallels the amount of SOD
in each cell type.
Although O- is potentially harmful for PMNLs, it
also plays an essential role in the intracellular killing
of phagocytosed bacteria.' Therefore it is not sur-
prising to find relatively low SOD activity in PMNLs.
However, the reason for the presence of higher
SOD activity in MNCs (>90% lymphocytes) is not
clear. It has been suggested21 that high SOD activity
in these cells may relate to such immune functions
as the production of immunoglobulins and lym-
phokines. In the present study the elevated SOD
activity in MNCs from patients with RA was not
related either to immunoglobulin concentration or to
 Downloaded from http://ard.bmj.com/ on March 15, 2015 - Published by group.bmj.com
Leucocyte superoxide dismutase in rheumatoid arthritis 561
rheumatoid factor titres. On the other hand patients
with extra-articular manifestations had significantly
higher SOD activity in MNCs (but not in PMNLs)
than those without such manifestations. Since
immunological processes are involved in the
pathogenesis of RA lesions,25 high SOD activity in
MNCs from such patients, particularly those with
extra-articular manifestations, might suggest the pre-
sence of immunologically hyperactive cells. This con-
cept is supported by the fact that the increase in SOD
activity was not related to either the patients' age,
sex, duration of disease, serum copper level, disease
activity, or type of treatment. The mechanism of
increased SOD activity in MNCs can possibly be
explained by increased O- production by the
hyperactive cells, which in turn may lead to SOD
induction.2628
Our finding of increased SOD activity in PMNLs,
but not MNCs, from RA patients treated with cor-
ticosteroids was not related to any of the factors
studied. This finding is of interest since the increase in
SOD activity might prevent excessive release of OF2
and therefore protect against the effects of this
radical.5`8 This mechanism might explain, at least in
part, the beneficial effect of corticosteroids.
It is of additional interest that cortcosteroids are
potent enzyme inducers29 and cortisol has been
shown to inhibit the production of O- by PMNLs.30
This inhibition could be the result of induced SOD
activity in PMNLs. This finding has also been indi-
rectly confirmed3" by the demonstratic-n of decreased
killing ability by PMNLs incubated with cortisol.
Moreover, the phagocytic activity of PMNLs is defec-
tive in RA patients treated with prednisolone.32
These findings together with our own may account, at
least in part, for the susceptibility to infection of
RA patients,33 particularly those treated with
corticosteroids.
Further work is under way to establish the exact
role of leucocyte SOD and to assess its importance
and possible applications.
We thank Miss Jane Lytle for excellent secretarial help This work
formed part of a thesis (AA-RY) accepted for the degree of PhD
(University of London).
A. A-R. Youssef was supported by a grant from the University of
Mansoura.
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Leucocyte superoxide dismutase

in rheumatoid arthritis.
A A Youssef and D N Baron

Ann Rheum Dis 1983 42: 558-562

doi: 10.1136/ard.42.5.558

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