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High-affinity interleukin-2 receptors on blood lymphocytes are decreased during active rheumatoid arthritis.

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1176
BRIEF REPORT
HIGH-AFFINITY INTERLEUKIN-2 RECEPTORS ON BLOOD
LYMPHOCYTES ARE DECREASED DURING ACTIVE
RHEUMATOID ARTHRITIS
PAUL EMERY, NIGEL WOOD, KAY GENTRY, ALEX STOCKMAN, IAN R. MACKAY, and ORA BERNARD
The expression of receptors for interleukin-2
(IL-2R) was examined in patients with active or inactive
rheumatoid arthritis (RA) and in control subjects. Unstimulated blood lymphocytes from patients with active
RA had increased levels of total IL-2R, as measured by
Tac-positive cells, compared with the levels found in the
other 2 groups. Mitogen-stimulated cells from patients
with active RA expressed less IL-2R per cell, but the
most striking feature was the failure to express highaffinity IL-2R. These changes could reflect persistent
antigenic stimulation and explain the defective cellmediated immunity in RA patients.
The lymphocyte growth factor, interleukin-2
(IL-2), is produced by T cells after stimulation by
mitogen or antigen in the presence of interleukin-I
(1,2). Binding studies have shown that there are 2
From the Walter and Eliza Hall Institute pf Medical Research, and the Department of Rheumatology, The Royal Melbourne Hospital, Parkville, Victoria, Australia.
Supported by grants from the National Health and Medical
Council of Australia and an anonymous grant to the Reid Rheumatism Laboratory of the Walter and Eliza Hall Institute.
Paul Emery, MA, MD, MRCP: Assistant Physician, Clinical Research Unit, Walter and Elira Hall Institute, and The Royal
Melbourne Hospital (current address: Rheumatism Research Wing,
University of Birmingham, Birmingham, UK); Nigel Wood, MB,
BS: Registrar, The Royal Melbourne Hospital; Kay Gentry, BSc:
Technical Scientist, Walter and Elizd Hall Institute; Alex Stockman,
MB, FRACP: Visiting Rheumatologist, The Royal Melbourne Hospital: Ian R. Mackay, MD, FRCP, FRACP, FRCPA: Unit Head,
Clinical Research Unit, Walter and Eliza Hall Institute, and the
Royal Melbourne Hospital; Ora Bernard, PhD: Senior Research
Fellow, Walter and Eliza Hall Institute.
Address reprint requests to Ora Bernard, PhD, Walter and
Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050. Australia.
Submitted for publication July 8, 1987; accepted in revised
form March 1, 1988.
Arthritis and Rheumatism, Vol. 31, No. 9 (September 1988)
classes of 1L-2 receptor (1L-2R): high affinity and low
affinity. The former class binds IL-2 with a thousand
times greater avidity than does the latter (3). The
expression of IL-2R is revealed by a monoclonal
antibody, anti-Tac. IL-2 stimulates T cell proliferation
only when it binds to high-affinity IL-2R and is internalized; low-affinity IL-2R does not internalize after
IL-2 binding (4).Another 1L-Zbinding molecule that
does not bind anti-Tac has recently been identified.
This protein, a 75-kd a chain, together with the 55-kd
p chain, forms the high-affinity IL-2R ( 5 ) . Anti-Tac
recognizes only the p chain, and binding by this
antibody does not result in internalization of 1L-2R.
High-affinity IL-2R comprises only about 3% of all
IL-2R molecules.
In rheumatoid arthritis (RA) and several other
autoimmune disorders affecting humans and in mouse
strains predisposed to autoimmune disease, there are
characteristic, but still unexplained, abnormalities of
1L-2 regulation. In RA, both the histologic appearance
of lymphocytic infiltration of synovial membranes and
the B cell activation suggest that there is excessive
production of IL-2, but measurements of IL-2 have
produced conflicting results (for review, see ref. 6).
Although circulating lymphocytes in RA show phenotypic evidence of activation (7), a consistent abnormality in RA is the reduced ability of blood lymphocytes
in vitro to proliferate in response to mitogens and,
more particularly, to antigens (8). This deficient response is at least partly reversible with exogenous
IL-2 (9).
Since lymphocyte proliferation in the presence
of IL-2 depends on ligand-receptor interaction, we
investigated the expression of high-affinity and lowaffinity IL-2R on blood lymphocytes and its correlation
BRIEF REPORTS
with the total IL-2R in RA. The influence of disease
activity on receptor expression was assessed by specifically selecting patients from the extremes of low levels
of disease activity and high levels of disease activity.
PATIENTS AND METHODS
Patients. All patients had classic or definite RA,
as defined by the criteria of the American Rheumatism
Association (10). To be classified as inactive disease,
at least 5 of the following 6 manifestations were
necessary: morning stiffness of fewer than 15 minutes
duration, no fatigue, no joint pain, no joint tenderness,
no joint or tendon sheath swelling, and no elevation of
the erythrocyte sedimentation rate (ESR). To be classified as active disease, all of the following manifestations were necessary: morning stiffness of more than
45 minutes duration, an ESR of more than 30
mm/hour, and a Ritchie articular index ( 1 1 ) of more
than 10. All patients with inactive RA and all but I of
the patients with active RA were taking a diseasemodifying antirheumatic drug; no patient was taking a
corticosteroid preparation. The control group (n = 15)
comprised healthy laboratory staff, patients with degenerative joint disease, and patients who were in the
hospital and had no evidence of any inflammatory
disease. The mean ages of the study subjects were as
follows: patients with inactive RA 62 years (range 4477), patients with active RA 58 years (range 28-83),
and control subjects 48 years (range 20-78).
Preparation of cells. Blood was collected into
tubes containing heparin and centrifuged on FicollPaque (Pharmacia, Uppsala, Sweden) to separate
mononuclear cells. Some of these cells were assayed
for IL-2 binding, others were prepared for labeling
with anti-Tac, and the remaining cells were incubated
at a concentration of 5 x lO5/rnl in RPMI (Gibco,
Grand Island, NY), containing an optimal concentration (10 pg/ml) of phytohemagglutinin (PHA), 10%
fetal calf serum (FCS), and 1% mercaptoethanol, in
flasks and microtiter wells. After 40 hours of incubation, the cells being cultured in flasks were washed 3
times, counted, and readjusted to the correct concentration for the IL-2 binding and anti-Tac assays. The
samples were assayed under code. In other experiments, the expression of high-affinity and low-affinity
IL-2R on blood lymphocytes, at different times, was
examined.
IL-2 production and lymphocyte proliferation.
Cells were cultured at 5 x 10S/mlin microtiter wells.
After 48 hours of stimulation with PHA, the cells were
1177
centrifuged, and supernatants were tested in an IL-2
assay, as described by Gillis et a1 (12), using a cytotoxic T cell line and 3H-thymidineincorporation. Calibration was accomplished with the use of preparations of
IL-2 derived from a supernatant of the EL-4 cell line,
and recombinant IL-2 (kindly donated by Cetus Corporation, Emeryville, CA): I unit of IL-2 was defined
as the inverse of the dilution giving 50% of the maximum response. Measurement of lymphocyte proliferation was performed after 48 hours of mitogen stimulation of lymphocytes, by measuring the incorporation
of a 6-hour pulse of ,H-thymidine into lymphocytes. In
other experiments, the response of lymphocytes to
IL-2 alone was examined.
Assay for total IL-2R. The binding of anti-Tac
was estimated as follows. A 50-pl volume of cells, at
107/ml,was incubated at 0°C with 15 pl of fluoresceinconjugated anti-Tac (Becton Dickinson, Orangeburg,
NY) for 45 minutes. Mouse IgG (Becton Dickinson)
was used as the nonspecific antibody control. The cells
were washed 3 times in a balanced salt solution and
fixed with 1% formaldehyde, 5 mM NaN,, and 2%
glucose in phosphate buffered saline prior to analysis in
a fluorescence-activated cell sorter (FACS model IV;
Becton Dickinson). From these data, the percentage of
cells reactive with anti-Tac was determined, and the
mean fluorescence, in arbitrary units, was calculated.
'251-labeledIL-2 binding assay. '2sI-labeled IL-2
(specific activity 20-50 pCilpg) was purchased from
Du Pont (New York, NY), New England Nuclear
(Boston, MA), or Amersham (Buckinghamshire, UK).
The cells were washed 3 times in RPMI containing 1%
bovine serum albumin and 25 mM HEPES, pH 7.5,
prior to incubation in the same medium for 2 hours.
The cells were then centrifuged and resuspended at 5
X lo6 cells/ml, and 200-4 samples were incubated
with various amounts of I2'I-labeled IL-2, as previously described (13), with continuous shaking, for 30
minutes at 0°C or at 37°C. The cells were then layered
over 200 pl of FCS and centrifuged for 5 minutes at
1,500 revolutions per minute. The tips of the tubes
containing the cell pellets were cut off, and tip and
supernatant were counted in a gamma counter. Nonspecific binding was determined by performing the
assay in the presence of a 200-fold excess of unlabeled
IL-2 (Amersham). To remove previously bound IL-2,
cells were incubated with 0.5M acetic acid, pH 2.7,
and 0.17M NaCl for 5 minutes at O"C, and then washed
twice with RPMI prior to performing the 12'I-labeled
IL-2-binding assay.
BRIEF REPORTS
1178
Effect of exogenous IL-2. Blood lymphocytes
were incubated in RPMI and PHA as described above,
except that 10 unitdm1 of IL-2 was added to the
cultures. The cells were washed 3 times with RMPI
and assayed for '251-labeled IL-2 binding, at 400 pM
'251-labeled IL-2, as described above.
Statistical analysis. Nonparametric data were
analyzed by the Mann-Whitney U test and Wilcoxon's
rank sum test, and normally distributed data were
analyzed by Student's t-test, using a Statswork program (Heyden and Son, London, UK). Chi-square test
with Yates' correction was used for analysis of the
data on IL-2 production. Variances are expressed
throughout as the mean SEM.
*
RESULTS
Expression of IL-2R. Total IL-2R expression
was examined before and after PHA stimulation in 27
patients with RA (12 with inactive disease and 15 with
active disease) and in 15 control subjects (Figure 1).
For unstimulated cells, the mean -+ SEM percentage
of Tac-positive cells was greater both in patients with
inactive RA (0.37 t 0.06; P < 0.05) and in patients
with active RA (1.47 f 0.33; P < 0.02) than in control
subjects (0.24
0.02). After PHA stimulation, the
percentage of cells bearing IL-2R increased (mean
>50% for all groups); between-group differences were
not significant. However, the mean ? SEM fluorescence per cell was significantly less in patients with
active RA (63 k 5 ) than in control subjects (132 t 27)
( P < 0.01). The difference in the mean level of fluorescence per cell in patients with inactive RA (77 f 9) versus that in patients with active RA was not significant.
Expression of high-affinity IL-2R. Cells from 1 1
patients with inactive RA, 11 with active RA, and 11
controls were tested for high-affinity IL-2R before and
after PHA stimulation. As expected, no high-affinity
IL-2R were detected on unstimulated cells (data not
shown). After PHA stimulation, there were significant
differences in the expression of high-affinity IL-2R
among the 3 groups (Figure 2A). Lymphocytes from
subjects with inactive RA or from controls bound IL-2
at high affinity, although the binding curve for inactive
RA was saturated at a higher IL-2 concentration.
Lymphocytes from patients with active RA showed
lower binding, which is indicative of a reduced number
*
Unstimulnted
PHA Stimulnted
100
0
ea
a
.-B
c
2u 6C
5
a4
m
+
.
2 4c
am
al
n
0
I'
0
2c
1
Control
Inactive
RA
Active
RA
Control
1
Inactive
RA
I
Active
RA
Figure 1. Percentage of Tac-positive cells in 15 control subjects, 12 patients with inactive rheumatoid
arthritis (RA), and 15 patients with active RA before and after a 48-hour stimulation with phytohemagglutinin (PHA). Bars show the mean f SEM. Cells from 1 patient with active RA (0 8.5) reacted
differently in these assays. Two of the PHA-stimulated active RA cultures were unsuccessful.
BRIEF REPORTS
1179
801
high-affinity IL-2R, as judged by maximum IL-2 binding, but these were probably of somewhat reduced
affinity because the maximum binding was reached at
400 pM rather than at 200 pM as for the controls.
In 3 experiments, preincubation with acid to
strip off any prebound IL-2 did not produce an increase in 1251-labeledIL-2 binding (data not shown).
The binding of IL-2 at 0°C was similar to binding at
3TC, indicating that binding was not due to abnormal
internalization of IL-2.
Effect of exogenous IL-2. The addition of exogenous IL-2 to the cultured lymphocytes from patients
with RA did not result in any increase in binding of
'251-labeledIL-2. In 3 such experiments, the binding of
1251-labeledIL-2 remained the same as that recorded
when exogenous IL-2 was not added to the cultures.
Time course of high-affinity IL-2R expression.
To exclude a premature peak of high-affinity receptor
expression on the RA cells, '251-labeled IL-2 binding
experiments were performed at 24,48, and 72 hours on
cells from patients with active RA and on cells from a
control subject (Figure 2B). There was no evidence of
an early peak in high-affinity IL-2R expression. The
maximum binding to cells from 2 RA patients, for all
timepoints examined at an IL-2 concentration of 400
pM (the concentration of maximum binding), was the
same as that for nonspecific binding in cells from
normal subjects.
IL-2 production and lymphocyte proliferation.
Significantly more patients with active RA than control subjects had unmeasurable levels of IL-2 in PHAstimulated supernatants (7 of 11 versus 0 of 11; P <
0.05). Two of 11 patients with inactive RA had unmeasurable levels of IL-2 (Figure 3). There were no
significant differences between the 3 groups in the
results of mitogen-stimulated proliferation studies.
There was no significant correlation between the
expression of IL-2R, either as low-affinity or highaffinity receptors, and lymphocyte proliferation or
production of IL-2.
x
0
IL-2
2 1201 B*
A
U
100
200
Total
300
400
pM
Time (hrs)
Figure 2. Binding of 'ZsI-labeledinterleukin-2 (IL-2) to cells obtained
from patients with active rheumatoid arthritis (RA) (O), patients
with inactive RA (O), and from control subjects (A). A, Binding
activities at various concentrations of IL-2. Bars show the mean
SEM for 11 subjects. B, Time course of binding of IL-2 (at 400 pM)
to cells from 2 patients with active RA and from 1 control subject.
*
of high-affinity receptors. There were significant differences ( P < 0.05) in IL-2 binding levels between
lymphocytes from patients with active RA and those
from controls at all concentrations of IL-2, except for
the lowest concentration tested. The differences in
binding between patients with inactive RA and those
with active RA was significant ( P < 0.05) at IL-2
concentrations of 100 pM and 400 pM. Lymphocytes
from patients with inactive RA had normal numbers of
DISCUSSION
The ability to respond to IL-2, and the specificity of the immune response, are dependent on
expression of high-affinity IL-2R. The normal induction of high-affinity IL-2R in the initial phase of an
immune response may well determine the outcome.
Any defect in the expression of high-affinity IL-2R
could therefore contribute to the occurrence of RA,
particularly if this disease is seen as consequential to a
BRIEF REPORTS
1180
IL-2 Production
1000
Proliferation
a
-
a
f
i
-!
a
a
a
I
3
100
a
-
a
*
-
c
a
t
w
.G
a
3L
1
c
-
10-
I
0
Control
L
Inactive
RA
me*..
Active
RA
1
I
I
I
Control
Inactive
RA
Active
RA
Figure 3. Interleukin-2 (IL-2) production and lymphocyte proliferation in cells obtained from 1 1 control subjects, I 1
patients with inactive rheumatoid arthritis (RA), and I I patients with active RA. Significantly more patients with active RA
(7 of 11) than control subjects (0 of 11) failed to produce 1L-2. Bars show mean levels of proliferation.
failure to eradicate a putative provoking antigen. The
regulation of IL-2 in RA is a subject of controversy
since, although many features of the disease suggest
uncontrolled production of IL-2, there is a consistently
defective cellular immune response, both in vivo and
in vitro, suggestive of reduced IL-2 production (8).
High-affinity IL-2R can be distinguished from
low affinity IL-2R by their binding affinities for IL-2
and by the fact that internalization of ligandheceptor
and cell activation take place only upon occupancy of
high-affinity IL-2R. Total 1L-2R expression has previously been shown to be increased on lymphocytes
obtained from the synovial fluid of patients with RA (7).
We have demonstrated that peripheral blood lymphocytes from patients with RA have an increased expression of total IL-2R, and this is particularly true for
patients with active disease. When blood lymphocytes
from patients with RA were stimulated with PHA, the
number of cells expressing total IL-2R was normal, but
the number of receptors per cell was reduced, especially on cells from patients with active disease.
Our studies of high-affinity IL-2 binding in RA
demonstrated that the expression of functional highaffinity IL-2R was greatly reduced, particularly in
active disease. This defect may reflect preactivation in
vivo and, hence, could occur in nonrheumatic inflammatory diseases. In fact, our observation that the
defect was not evident in patients with inactive disease
indicates that it is not an intrinsic determinant of the
rheumatoid process. Also of relevance here is an
equivalent defect in another immunopathic disorder,
murine lupus (14), in which high amounts of soluble
IL-2R are demonstrable in serum (15).
The actual cause for low expression of highaffinity IL-2R in RA is not clear, but some possibilities
can be excluded. First, the failure to improve IL-2
binding after removing prebound IL-2 with acid, before the binding assay, indicates that low expression
was not due to the prior occupation of receptors by
IL-2. Second, the difference in expression between
patients with inactive RA and those with active RA
implies that genetic factors are not operative. Third,
since there was a reduction in free IL-2 produced in
vitro, this could represent the primary abnormality;
that this would not be the full explanation is suggested
by the between-group differences in IL-2R expression
compared with IL-2 production and the absence of a
significant reduction in cell proliferation, indicating
BRIEF REPORTS
that 1L-2 production was, initially at least, adequate.
Moreover, our finding that exogenous IL-2 did not
induce any increase in high-affinity binding of IL-2 is
consistent with this possiblity. The failure to demonstrate a definite abnormality in proliferation may relate
to the use of a maximal mitogenic stimulus, which
reduces the role of antigen-presenting cells and has
been shown to minimize differences between patients
with RA and control subjects (8). Fourth, since it is
now clear that high-affinity IL-2R is composed of 2
chains, it is possible that the defect in RA is in the
recently described a chain of the IL-2R.
In conclusion, we have shown that an increased
proportion of lymphocytes from patients with RA
circulate in an activated state and, after stimulation,
have a reduced ability to produce IL-2 and, more
particularly, to express high-affinity IL-2R. The degree
of this abnormality of high-affinity 1L-2R expression,
together with the clear association with disease activity, represents a strikingly consistent result for a
disease such as RA, in which there is such a variable
degree of expression. However, the finding that the
defect is maximal in patients with active disease means
that the defect may not be intrinsic to the pathogenesis
of RA. Nonetheless, the defect may be critical, since
the inability to induce high-affinity IL-2R must be
prejudicial to the response of lymphocytes to antigenic
signals, and it could certainly account for the characteristically defective cell-mediated immunity seen in
patients with RA (8).
REFERENCES
Gillis S: Interleukin-2: biology and biochemistry. J Clin
lmmunol 3:l-13, 1983
Robb RJ: Interleukin-2: the molecule and its function.
Immunol Today 5:203-209, 1984
Robb RJ, Greene WC, Rusk CM: Low and high affinity
cellular receptors for interleukin-2: implications for the
level of TAC antigen. J Exp Med 160: 1 1 2 6 1 146, 1984
Greene WC, Depper JM, Kronke M, Leonard WJ: The
human interleukin-2 receptor: analysis of structure and
function. Immunol Rev 92:29-48, 1986
5 . Teshigawara K, Wang H, Kato K, Smith KA: Interleukin-2 high-affinity receptor expression requires two distinct binding proteins. J Exp Med 165:223-238, 1987
6. Emery P, Williamson DJ, Mackay IR: The role of
cytokines in rheumatological inflammation, Concepts in
Immunology, Vol. 4. Autoimmunoregulation and Autoimmune Disease. Edited by JM Cruse, RE Lewis Jr.
Basel, Karger, 1987
7. Burmester GR, Jahn B, Gramatzki M, Zacher J, Kalden
JR: Activated T cells in vivo and in vitro: divergence in
expression of Tac and la antigens in nonblastoid small T
cells of inflammation and normal T cells activated in
vitro. J Immunol 133:123&1234, 1984
8. Emery P, Panayi GS, Cole BC, Welsh KI: Relationship
of HLA DR4 expression to defective cellular immunity
in rheumatoid arthritis using PPD, mycoplasma and
lectin mitogens. J Rheumatol 12:859-864, 1985
9. Emery P, Panayi GS, Nouri AME: Interleukin-2 reverses deficient cell-mediated immune responses in rheumatoid arthritis. Clin Exp Immunol 57: 123-129, 1984
10. Ropes MW. Bennett GA, Cobb S, Jacox R, Jessar RA:
1958 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 9:175-176, 1958
1 1 . Ritchie DM, Boyle JA, McInnes JM, Jasani MK, Dalakos TG, Grieveson P, Buchanan WW: Clinical studies
with an articular index for the assessment of joint
tenderness in patients with rheumatoid arthritis. Q J
Med 147:393406, 1968
12. Gillis S, Ferm MM, Ou W, Smith KA: T cell growth
factor: parameters of production and a quantitative
microassay for activity. J Immunol 120:2027-2032, 1978
13. Bernard 0, Fazekas de St Groth B, Ullrich A , Greene
WC, Schlessinger J: High affinity interleukin 2 binding
by an oncogenic hybrid IL-2-EGF receptor molecule.
Proc Natl Acad Sci USA 84:2125-2129, 1987
14. Santoro TV, Lehmann KR, Batt RA, Kotzin BL: The
role of L3T4+ cells in the pathogenesis of lupus in
Ipr-bearing mice. I. Defects in the production of interleukins 2 and 3. Eur J Immunol 17: 1131-1 136, 1987
15. Balderas RS, Josimovic-Alasevic 0, Diamantstein T,
Dixon FJ, Theofilopoulos AN: Elevated titers of cellfree interleukin 2 receptor in serum of lupus mice. J
Immunol 139: 1496-1500, 1987
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