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Impaired natural killer cell function in systemic lupus erythematosus.

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1316
IMPAIRED NATURAL KILLER CELL FUNCTION IN
SYSTEMIC LUPUS ERYTHEMATOSUS
Relationship to Interleukin-2 Production
WILMER L. SIBBITT, JR., LAURA LIKAR, CRAIG W. SPELLMAN,
and ARTHUR D. BANKHURST
Immune regulation requires clonal expansion of
regulatory T cells which is dependent on the presence of
interleukin-2 (IL-2). Previously, both natural killer
(NK) cell function and IL-2 production were found to be
depressed in systemic lupus erythematosus (SLE). This
study was designed to determine the relationship between IL-2 production and NK cell activity in SLE. NK
activity as determined by a standard 4-hour "Cr-release
assay was impaired in SLE (11.0 & 5.1 lytic units/107
cells) compared with controls (25.1 f 7.1 lytic units/107
cells) (P < 0.05). IL-2 production was induced with
concanavalin A and phorbol ester and quantitated using
the IL-2 dependent cell line HT-2. IL-2 production was
impaired in only 1 SLE patient, despite concomitant
abnormalities in NK function in the SLE group as a
whole. Moreover, in patients with impaired NK activity,
incubation of lymphocytes with exogenous IL-2 did not
restore NK activity to normal levels. These findings
demonstrate that impaired NK activity in SLE is independent of IL-2 production and that defects in IL-2
production in SLE may not be as common as previously
reported.
From the Departments of Medicine and Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico.
Supported by grants ROI CA-24873-04 and 2 T32 AM
07173-07 from the Nakonal Institutes of Health and TSH grant CA30561 from the National Cancer Institute, DHHS.
Wilmer L. Sibbitt, Jr., MD; Laura Likar; Craig W. Spellman.' PhD: Arthur D. Bankhurst. MD.
Address reprint requests to Dr. Wilmer L. Sibbitt, Jr..
Department of Medicine, Division of Rheumatology, University of
New Mexico School of Medicine, Albuquerque, NM 87131.
Submitted for Dublication Februarv, 22.. 1983: acceDted in
revised form July 12, 1983.
Arthritis and Rheumatism, Vol. 26, No. 11 (November 1983)
Immune regulation in both health and disease
requires clonal expansion of immunoregulatory T cell
subsets, a process which requires the presence of a
soluble mediator known as interleukin-2 (IL-2) (1). IL2 appears to be necessary for the support of mitogen- or
antigen-induced T cell proliferation (2), the regulation
of cells responsible for natural cytotoxicity (1,3), the
promotion of helper cell function (4), and the modulation of suppressor cell activity ( 5 ) . Many of these
functions have been reported to be abnormal in systemic lupus erythematosus (SLE) (6-9).
Recently, IL-2 production has been reported to
be impaired in SLE in both active and inactive disease
(10). Such a defect in IL-2 production could contribute
to the multiple abnormalities noted in cell-mediated
immunity in SLE. In particular, natural killer (NK)
cell cytotoxicity is often impaired in SLE (8), and the
NK cell itself appears to be regulated by IL-2 (3).
Thus, a relative defect in IL-2 production could result
in impaired N K function. Indeed, Kaufman (11) has
recently reported that impaired NK activity in SLE
can be restored by preincubation of effector cells with
mitogen-derived supernatants from normal peripheral
blood mononuclear cells. Such supernatants are
known from other studies to contain IL-2 (12).
This study was designed to determine if impaired NK function in SLE was secondary to decreased IL-2 production. The results reported here
demonstrate that impaired NK function in SLE is
independent of IL-2 production. In addition, contrary
to other reports, IL-2 production in SLE was found to
~normal levels~ of NK
be largely normal. ~
activity in SLE cannot be restored by preincubation of
effector cells in supernatants which contain IL-2.
h
NK CELLS IN SLE
PATIENTS AND METHODS
Patient population. Fifteen SLE patients and 15 normal controls were studied. All patients included in the study
fulfilled the criteria for SLE as defined by the American
Rheumatism Association (13). Clinical activity was scored
according to the method of Barada et al (14), where 0 =
complete remission, 1 = mild activity, 2 = moderate activity, and 3 = severe systemic disease. No patients were being
treated with cytotoxic agents and only 3 were receiving
corticosteroids at the time of study.
Peripheral blood mononuclear cells (PBMC). PBMC
were isolated from heparinized whole blood by centrifugation on Hypaque-Ficoll gradients (15). Cells from the interface were washed 3 times with phosphate buffered saline
(PBS) and resuspended in medium RPMI 1640 (Grand Island
Biological Co., Grand Island, NY) which contained penicillin (100 IU/ml), streptomycin (100 pg/ml), and 10% heat
inactivated human AB serum. Cell viability was greater than
99%, as determined by trypan blue exclusion, and contained
from 2-20% monocytes by benzidine peroxidase staining
(16). Glass wool adherent cells were removed as previously
described (17). Final suspensions contained 0-1% monocytes as determined by peroxidase staining. In some experiments, effector cells were incubated with concanavalin A
(Con A) (C-2010, Sigma Chemical Co., St. Louis, MO) at 5
&ml for 16 hours at 37°C in 5% CO? prior to being placed in
cytotoxic assays. Cell viability was greater than 96%.
NK assay. K562 (18) is a standard target for the
human NK assay and was used exclusively in this study.
Target cells at 5 x lo6 per 200 pl of fetal calf serum were
incubated with 0.1 ml Naz "Cr O4 (New England Nuclear,
Boston, MA, 1.0 mCi/ml) for 45 minutes at 37°C. The cells
were washed 3 times with RPMI 1640 containing AB serum
and then placed into the wells of round bottom microtiter
plates (Linbro, Hamstead, CT). Various numbers of effector
cells were added to 1 x lo4 labeled target cells, such that
effector:target (E:T) ratios were 100: 1,50: 1, and 25: 1. The
final volume in each well was 200 p1. The plates were
incubated for 4 hours at 37°C and then centrifuged at 400g for
10 minutes. One hundred microliters of supernatant was
removed, placed in glass tubes, and counted in a gamma
counter. Spontaneous release was assessed by a series of
wells containing no effector cells. Maximum release was
obtained by treating a series of wells with Lyzerglobin (J.T.
Baker Chemical Co., Bethlehem, PA). The percentage of
NK activity was calculated as follows:
% N K activity
100 x
=
experimental "Cr release - spontaneous "Cr release
maximum "Cr release - spontaneous "Cr release
A lytic unit (LU) was defined as the number of effector cells
necessary to result in 30% lysis of I x lo4 targets. These
values were determined from individual effector titration
curves by linear regression analysis. The relative NK activity was expressed as lytic units per lo7 effector cells (LU/107
cells).
Proliferation assays. PBMC were cultured in RPMI
1640 supplemented with L-glutamine, penicillin-streptomy-
1317
cin, and 10% fetal calf serum. Con A was titrated to
determine suboptimal and optimal mitogen concentrations.
Each microtiter well contained 2 3 x lo5 cells. Samples were
analyzed in triplicate and were incubated at 37°C in 5% COz
for 72 hours. Cell proliferation was assessed by the addition
of 0.2 pCi of tritiated thymidine (New England Nuclear)
during the last 8 hours of culture. Cultures were harvested
on a Mash I1 Harvester and were counted in a liquid
scintillation counter.
The production of IL-2 from human PBMC. PBMC
were resuspended at 1 x lo6 cellslml in 5-ml culture volumes
using 60 x 20-mm tissue culture Petri dishes. These cells
were induced with 5 pglml Con A and 5 nglml phorbol
myristic acetate (PMA, P8139, Sigma). After 48 hours of
incubation at 37°C and 7% COz, supernatants were collected
by centrifugation.
IL-2 assay. The cell line used to detect the presence
of IL-2 was HT-2, an IL-2 dependent continuous cell line of
BALBlc origin (19). It was maintained in RPMI 1640 supplemented with IL-2 derived from mitogen-stimulated rat
spleen cells. This cell line is sensitive to human as well as
rat-derived IL-2. IL-2 in supernatant was quantitated as
previously described (20). Briefly, samples were evaluated
in triplicate in microtiter plates. Supernatants to be tested
were diluted 1 :2, 1 :4, 1 :8. and 1 : 16, and 100 pl was added to
each well. These wells also received 100 p1 containing HT-2
cells at 2.5 x lo5 cells/ml. These HT-2 cells had been washed
twice in medium to remove all IL-2 before use in the assay.
Positive controls were wells containing dilutions of known
IL-2 containing medium; negative controls were wells containing medium without IL-2. Cells were incubated for 24
hours at 37°C in 7% COz and each well was pulsed with 25 p1
of medium containing 0.6 pCi 'H-thymidine and incubated
for 6 hours at 37°C in 7% CO?. Samples were harvested on an
automated Brandell cell harvester and counted on automatic
liquid scintillation counter. Because IL-2 containing supernatants were quantitated immediately after induction, different experiments were normalized using the following equation:
% relative IL-2 =
cpm sample
-
cpm medium
cpm IL-2 standard - cpm medium
x 100
The IL-2 standard was a preparation of rat IL-2 known to
contain IL-2 with an optimal growth potentiating dilution of
1 :16 which was aliquoted and stored at -70°C. IL-2 titer was
defined as the maximal dilution of an IL-2 containing sample
which could maintain maximal HT-2 proliferation.
Statistical methods. The two-tailed Student's t-test
was used to determine the significance of differences between the means of 2 groups. Linear regression analysis was
used to compute linear correlation coefficients (r values) and
P values between 2 variables.
RESULTS
NK activity in SLE. As can be seen from Figure
1, NK activity was significantly decreased in SLE
patients (n = 15) relative to normal controls (n = 15) at
SIBBITT ET AL
1318
Table 1. Interleukin-2 (IL-2) activity
erythematosus (SLE) and normal controls
T
lot
Subjects
Natural killer
activity (lytic
units/l~’cells)
Control
I
2
3
4
5
6
Average
27.5
17.7
25.0
29.4
33.3
41.7
28.6 f 8.3
SLE
1
2
3
4
5
6
7
Average
16.6
14.2
20.0
16.6
9.0
8.3
6.5
13.1 ? 5.0
in
systemic
lupus
1L-2 activity, dilutions*
1:2
1:4
1:8
1:16
57.1
42.7
56.25
70.7
59.0
73.4
76.4
87.3
75.0
100.0
74.0
100.0
85.3
91.5
59.3
59.8
100
87.5
96.2
87.3
42.2
26.11
40.2
71.8
66.8
80.6
66.2
81.5
35.2
103.0
95.3
75.2
84.2
64.9
78.9
15.2
75.0
65.6
89.8
85.4
86.6
91.7
4.8
73.0
37.5
63.6
62.4
50.3
66.8
43.6
120.3
93.8
* IL-2 activity is defined as (cpm IL-2 sample/cpm IL-2 standard)
125.1
25:l
5O:l
100:I
x 100 where the IL-2 standard is known to have maximum IL-2
activity at a 1 : 16 dilution.
E:T
Figure 1. Natural killer activity of I5 patients with systemic lupus
erythematosus (SLE) (0)and 15 controls (A)at 4 different effector: target (E:T) ratios, lytic units/lO’ cells (mean f standard error):
SLE = 11.0 ? 5.1; controls = 25.1 +- 7.1 (P< 0.05).
4 different effector:target ratios ( P < 0.05). This
impairment in NK function persisted after 24, 48, and
72-hour culture (data not shown). Of the 15 SLE
patients, 9 had impaired NK function (normal NK
function was defined as greater than 15 LU/107 cells).
IL-2 production. Six controls and 7 S L E patients were tested for IL-2 production. As seen in
Table 1, 4 SLE patients had severely impaired NK
activity (less than 15 LU/107 cells). No differences in
IL-2 production were present in SLE patients with
normal NK, SLE patients with impaired NK, or
normal controls. Of the 7 SLE patients tested, only 1
had decreased IL-2 production (titer < 1:2). IL-2
production did not appear to be related to therapy,
disease activity, or NK activity (Table 2). The response to Con A as measured by 3H-thymidine incorporation was the same for SLE patients (16,083 k
10,679 cpm) and controls (17,927 -t 5,478 cprn) at a
Con A concentration of 5 pg per ml.
Effect of preincubation with exogenous IL-2.
PBMC from both controls and SLE patients were
cultured with a known IL-2 standard (maximum IL-2
activity at 1:4-1:16 dilutions) at a 1:4 dilution or
media containing 200 unitdm1 of a-interferon (Sigma)
for 72 hours. As can be seen in Table 3, both ainterferon and IL-2 increased NK activity in controls
and SLE patients. However, SLE patients with impaired NK function did not have restoration of function to normal levels. In fact, augmentation of NK
activity by interferon (17 ? 11.4%) was greater than
that seen with IL-2 (11.2 2 16.58), although this
difference was not significant. Thus, exogenous IL-2
Table 2. Natural killer (NK) activity and interleukin-2 (IL-2) titer
as related to systemic lupus erythematous (SLE) disease activity
and therapy
Disease
activity
score
1
1
1
2
2
2
2
SLE
patient
NK
activity
(lytic
units/
I 07)
Prednisone
IL-2
titer’
4
6
3
2
7
1
5
16.6
8.3
20.0
14.2
9.0
16.6
6.5
20 mgt
0
0
0
10 mglday
0
10 mg/day
l:l6
1 :4
1:16
1:16
1 :4
1:16
<1:2
* IL-2 titer was the maximum dilution of the IL-2 containing sample
which would maintain maximal HT-2 proliferation, as measured by
tritiated thymidine incorporation.
t Every other day.
NK CELLS IN SLE
1319
Table 3. The effect of exogenous interleukin-2 (IL-2) and ainterferon on natural killef (NK) cell function*
Baseline
Controls (n = 6)
SLE with normal
N K ( n = 3)
SLE with impaired
NK (n = 5 )
44.2
52.6
17.9
?
2
f_
IL-2
a-interferon
18.3
24.2
55.7 ? 29.5
62.2 ? 32.9
66.3
66.9
5.2
29.0
35.7 t 13.5
2
18.2
?
?
31.3
1.0
* Expressed as % "Cr release at a 50: 1 effector:target ratio (mean
* standard deviation). SLE = systemic lupus eiythematosus.
did not selectively restore to normal the depressed
levels of NK function in SLE.
DISCUSSION
This study demonstrates that the defect in NK
cell function in SCE is independent of IL-2 production. Although IL-2 may be important in the regulation
of the NK effector cell, it does not seem to be
implicated in the impairment of NK cell activity seen
in SLE. Only I of the SLE patients tested had impaired IL-2 praduction, despite decreases in NK activity in the SLE patients as a whole. Moreover, exogenous IL-2 derived from normal human PBMC did hot
restore NK function to normal levels. IL-2 is necessary for clonal expansion of cytotoxic cell subsets,
induding those with NK activity (1). However, recent
experimental evidence has demonstrated that there is
not a depletion of potential NK effector cells in SLE,
as one might expect with a relative IL-2 deficiency
(21). Rather, there appears to be a defect in the lytic
mechanism of NK cells, which are present in normal
numbers. In our study, the fact that many SLE patients with normal IL-2 production had impaired NK
function suggests that defects in IL-2 production are
not responsible for defective NK function in SLE.
Alcocer-Varela and Alarcon-Segovia ( 10) found
that IL-2 production was uniforrrily decreased in SLE
patients. The results reported in the present study
show that this conclusion is by no means universal.
Most of our patieuts with active disease had normal
levels of IL-2 production, suggesting that defects in
IL-2 production are not responsible for either disease
activity or the initial pathogenesis of SLE. However, it
must be realized that these same authors also reported
a decreased respQnse to and absorption of IL-2 by
SLE T cells. If such defects in IL-2 response or
absorption of IL-2 existed in the N K effector cell
subpopulation in SLE, then further abnormalities in
NK cell function could be postulated even in the
presence of normal IL-2 production. This is an interesting hypothesis and is not necessarily inconsistent
with results reported in the present study.
The discrepancies in IL-2 production between
our results and those reported earlier are of considerable concern and may well reflect differences in the
experimental techniques used to detect the presence of
IL-2. In the present study, the cell line HT-2 was
selected as the IL-2 responder cell because of its
absolute requirement for IL-2 (20). IL-2 must be
present for HT-2 to grow or remain viable. However,
in the study which reported impaired production of IL2 in SLE, normal human T cells were used as the
responder cells after prolonged culture in mitogenderived supernatants (10). Such supernatants often
contain factors other than IL-2 (22). Prolonged culture
of T cells in the presence of such supernatants does
not necessarily insure that these cells will develop an
absolute growth requirement for only IL-2. Other
factors in these sbpernatants could conceivably be
contributing to the differences in support of T cell
growth noted in mitogen-derived supernatants from
normal controls and SLE patients. Indeed, there have
been reports of intpaired production of other immunoregulatory factors by mitogen-stimulated lymphocytes
from SLE patients (22). Thus, in order to assay 1L-2
activity, we believe that a cell line with a demonstrated
absolute requirement for IL-2 is necessary. Therefore,
it is quite likely that the assay system used in the other
study was not entirely specific for IL-2. This probably
explains the discrepancies between the 2 studies.
Kaufman (1 1) has recently reported that supernatants from normal PBMC stimulated with Con A
could restore defective N K function in SLE to normal
levels. However, in the present study, similar supernatants with known IL-2 activity could not restore
impaired N K activity to normal levels, although some
enhancement was noted. This enhancement was no
greater than that induced by interferon. In the study by
Kaufman, no enhancement by Con A supernatants
was noted in normal controls ( 1 1). However, this latter
finding was unexpected since such supernatants are
known to Contain both IL-2 and interferon and would
be expected to enhance normal NK activity (12,22).
The NK cell may be important in immunoregulation (23), and thus may have some role in the
pathogenesis of SLE. The present study demonstrates
for the first time that impaired NK functioh in SLE
appears to be independent of IL-2 production. Thus,
the proposed role of impaired IL-2 production in the
pathogenesis and activity of systemic lupus erythema-
SIBBITT ET AL
1320
tosus may not be an important factor as previously
suggested.
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