Lymphocyte response to virus antigens in systemic lupus erythematosus.

1271
LYMPHOCYTE RESPONSE TO VIRUS
ANTIGENS IN SYSTEMIC LUPUS
ERYTHEMATOSUS
ROBERT E. WOLF and MORRIS ZIFF
The cell-mediated immune response of lymphocytes to rubella, measles, parainfluenza types 1,2, and
3, varicella-zoster, and herpes virus type 1 virus antigens
was evaluated in 15 SLE patients and 15 matched controls
by incorporating 3H-thymidine in whole blood cultures as
a measure of blastic transformation. SLE patients were
less responsive than normal individuals to six of eight
virus antigens tested. Culture of washed SLE cells in AB
plasma did not reverse the hyporesponsiveness.The results
indicated that a functional impairment of the circulating
lymphocytes appeared to be responsible for the in vitro
hyporesponsiveness of SLE patients to virus antigens.
From the Rheumatic Diseases Unit, Department of Internal
Medicine, University of Texas Southwestern Medical School, and
Veterans Administration Hospital. Dallas, Texas.
Supported by Postdoctoral Fellowship and Clinical Study
Center Grants from The Arthritis Foundation, USPHS Grants
AM09989 and AM051 54, a Veterans Administration Research Associateship, and a grant from the Oklahoma Lupus Foundation.
Robert E. Wolf, M.D., Ph.D.: Arthritis Foundation Postdoctoral Fellow. University of Texas Southwestern Medical School
(presently Assistant Professor of Medicine, University of Texas Southwestern Medical School, and Research Associate, Dallas VA Hospital); Morris Ziff. Ph.D., M.D.: Professor o f Medicine, Chief, Rheumatic Diseases Unit, University of Texas Southwestern Medical
School. and recipient, Research Career Award, National Institutes of
Health.
Address reprint requests to Robert E. Wolf, M.D., Rheumatic Diseases Unit, Department of Internal Medicine, University of
Texas Southwestern Medical School, 5323 Harry Hines Boulevard,
Dallas. Texas 75235.
Submitted for publication January 6, 1976; accepted April 22.
1976.
Arthritis and Rheumatism, Vol. 19, No. 6 (November-December 1976)
Consideration of viruses in the etiology and pathogenesis of systemic lupus erythematosus (SLE) is based
on a number of observations. Tubuloreticular structures
superficially resembling paramyxoviruses have been detected in various tissues from patients with SLE (1-4),
but it has recently been concluded (5) that these structures are not specifically related to myxo- or paramyxoviruses and probably represent a nonspecific response to injury. Elevated antibody titers have been
found in the serum (6,7); however they have not implicated a single virus and may represent nonspecific responses (8). More recently, evidence for enhanced type
C RNA virus expression has been reported in SLE
(9-1 1 ) as well as in the New Zealand mouse (12-14) and
canine (1 5) animal models.
A generalized, nonspecific diminution of cellmediated immunity in SLE has been indicated by decreased responses to plant mitogens and common bacterial and fungal antigens (16-29). However conflicting
results have been reported (23-26,30,3 1 ). Low levels of
circulating thymus-dependent (T) lymphocytes (23,
32-34) have also been observed. Although some investigations have demonstrated inhibitory effects of sera
from SLE patients on normal T cells in vitro (26,35-37),
serum factors d o not appear to be responsible for the
decreased cell-mediated immunity (CMI) in SLE patients (26-28). Diminution of CMI appears to correlate best with disease activity (1 6,18,29,38).
CMI to virus antigens has been little studied in
SLE. In the only previous investigation, Utermohlen et
WOLF AND ZIFF
1272
Table 1. Virus Antigens Used for Lymphocyte Stimulation
a1 (39), using the leukocyte migration inhibition technique, observed specific hyporesponsiveness t o measles
antigen in SLE patients. They observed t h a t this antigen
failed t o inhibit migration, whereas inhibition of migration w a s normal in t h e presence of rubella and parainfluenza type 1 antigens.
In t h e present studies t h e ability of lymphocytes
from SLE patients t o u n d e r g o blastic transformation in
t h e presence of rubella, measles, parainfluenza types I ,
2, a n d 3, varicella-zoster, and herpes virus type 1 antigens
w a s measured b y t h e incorporation of tritiated thymidine (3H-thymidine) during d e novo DNA synthesis.
Lymphocytes from SLE patients w e r e hyporesponsive
t o six of the eight virus antigens tested. In addition,
there was no recovery of responses when washed SLE
lymphocytes were cultured in plasma from a b l o o d
g r o u p AB, R h positive individual. These results suggest
t h a t a lymphocyte a b n o r m a l i t y is t h e main factor responsible for t h e hyporesponsiveness o f SLE lymphocytes t o virus antigens.
mogenization of infected chorioallantoic membranes, but all
others were cell-free culture supernatants. The rubella and
measles antigens had been treated with Tween 80 and ether.
Samples of uninfected tissue cultures were used as controls
with herpes virus type 1 and varicella-zoster antigens. Although
tissue culture controls were not available for the other antigens, specificity of stimulation by measles HA and CF antigens, and by parainfluenza types I , 2, and 3, was indirectly
indicated by the degree of discordant stimulation between the
two measles antigen preparations and between the three parainfluenza antigen preparations, which were grown in the same
tissue substrates under similar culture conditions.
Stimulation of Cells with PHA and Virus Antigens in
Whole Blood Cultures. Blastic transformation in the presence
of virus antigens was determined for lymphocytes from I5 SLE
patients (14 with active disease) and 15 controls ( 1 1 healthy
individuals and 4 outpatients, 2 with hypertension, 1 with
unilateral ovarian enlargement, and 1 with a healed human
bite). The controls were considered as a single group because
no difference in stimulation by antigen or mitogen was detected between the healthy and patient controls. Pertinent
clinical data are presented in Table 2. Patients and controls
were matched for age within 3 years. Racial and sexual characteristics of the two groups were similar.
A modification of the whole blood culture assay of
Pauly el a1 (41) was used t o measure aH-thymidine incorporation. Each culture contained 0.1 ml of fresh, heparinized
whole blood, glutamine (300 pg/ml) (Eastman Kodak Co,
Rochester, N Y ) and RPMI-1640 (Grand Island Biological Co,
Grand Island, N Y ) in a total volume of 3 ml. Virus antigens at
I : 10 and 1 :50 dilutions were added in 0. I-ml amounts to
triplicate cultures. Control cultures containing 0.1 ml of medium or 0.1 ml of PHA (equivalent to 5 pl) (Difco Laboratories, Detroit, MI) were carried out in parallel.
After incubation for 7 days at 37°C in humidified air
with a 5% CO, atmosphere, the cultures were pulsed for 20
hours with I pCi (6.7 Ci/mM) of SH-thymidine (New England
Nuclear, Boston, MA) under the same conditions. Harvesting
was accomplished with a Millipore 3025 Sampling Manifold
(Millipore Corp, Bedford, MA) and 2.4-cm diameter 934AH
glass fiber filters (Reeve Angel, Clifton, NJ). The samples were
washed twice with 3% acetic acid, 10% trichloroacetic acid
(TCA), and 0.12 M phosphate-buffered saline (PBS) at pH 7.2.
MATERIALS AND METHODS
Table 2. Clinical and Laboratory Data of S L E Patients and Controls
Virus
Strain
Herpes virus I
Varicella-Loster
Rubella
Measles
Measles
Parainfluenza I
Parainfluenza 2
Parainfluenza 3
Maclntyre
VZ-I0
B- 127
Philadelphia 26
Philadelphia 26
C-3s
C-39
C-243
Type of
Antigen*
Culture
Source?
CF
CF
HA
HA
CF
HA
HA
HA
EE
HELF
BHK-21
PHAM
PHAM
PMK
PM K
PM K
* CF: complement fixation; HA: hemagglutination inhibition.
t EE: 8-day-old embryonated eggs: HELF: human embryonic lung
fibroblasts; BHK: baby hamster kidney: PHAM: primary human
amnion: PMK: primary rhesus monkey kidney.
Patients. The SLE patients met the criteria of the
American Rheumatism Association for the diagnosis of SLE
(40) and were either inpatients or outpatients at Parkland
Memorial Hospital. Other characteristics of the two groups
are presented below.
Virus Antigen Preparations. The virus antigens used in
these studies were acquired from the National Center for
Disease Control, Atlanta, Georgia, through the cooperation of
Dr. Helen Casey. The antigens were prepared for either hemagglutination inhibition (HA) or complement fixation ( C F )
antibody assays. Table 1 presents the strain and antigen type
for each of the virus antigens and the tissue culture substrate in
which each was prepared.
The herpes virus type I antigen was prepared by ho-
SLE(n
Age (years)
Race
Sex
ESR* (mm/hr)
No. with
hypocomplemen temia
No. with positive
anti-nDN A test$
* Westergren
=
15)
15-44 (mean: 30)
13 black, 2 white
14 female, I male
17-125 (mean: 73)
Control(n
ND
9
ND
erythrocyte sedimentation rate.
$ 2 2 . 0 pg nDNA bound per I ml of serum.
15)
15-47 (mean: 30)
10 black, 5 white
I5 female
NDt
9
t ND: not done.
=
1273
LYMPHOCYTE RESPONSE IN SLE
The filters were placed in counting vials and dried before
addition of 10 ml of Aquasol (New England Nuclear). The
samples were counted for 5 minutes in a Beckman liquid
scintillation counter. The cpm were adjusted to cpm/2 X lo6
mononuclear cells by using a conversion factor based on the
number of mononuclear cells per culture at the time of preparation. The data were then expressed as a stimulation index
(SI), where SI = cpm stimulated culture/cpm medium control
culture. SI 2 2.0 was considered positive.
Stimulation of Cells with Virus Antigens in Whole Blood
Cultures Containing Autologous or Normal A B Plasma. Whole
blood from 8 active, untreated SLE patients was centrifuged at
I200 rpm for 10 minutes. The cells were washed three times
with warm (37OC) RPMI-1640 and were centrifuged at room
temperature, 1200 rpm for 10 minutes, after each wash. Either
fresh autologous plasma or fresh AB plasma from a single
donor was then added to the original blood volume. These
reconstituted mixtures were used to prepare whole blood cultures as described above. Rubella, measles-CF, parainfluenza
type 1, and parainfluenza type 3 antigens at dilutions of 1 : 10
and 1 : 50 were added in 0. I-ml volumes. The cellular responses
were analyzed as above.
Statistical Analysis. The z test (42) was used to compare rates of PHA stimulation (S1 2 10) in the SLE and
control groups. Cpm incorporated in unstimulated cultures
were analyzed by the student's t test (42). The responses to
individual virus antigens and the number of antigens to which
there was a positive response were compared by x 2 analysis
(42). Nonparametric multiple group comparison by the Kruskal-Wallis test (42) was used to assess the response of SLE
lymphocytes in autologous and A B plasma.
RESULTS
Response of Whole Blood Cultures to PHA. I n
order t o evaluate nonspecific lymphocyte responses,
whole blood samples from 15 SLE patients a n d 15 controls were cultured with PHA, a n d incorporation of 3Hthymidine was determined. A stimulation index (SI)
equal to or greater than 10 was observed in 93% of the
controls but in only 46% o f t h e SLE patients. This
difference w a s highly significant ( P < 0.001). These and
other differences were not d u e t o increased incorporation of 'H-thymidine in unstimulated SLE cultures,
because n o significant difference was found between
the mean c p m incorporated in unstimulated cultures
of the control (510 f 146) a n d SLE (433 f 81) groups.
Response of Whole Blood Cultures to Virus Antigens. Responses to virus antigens were determined in
parallel with the PHA stimulation studies. T h e number
a n d percentage of SLE patients with positive responses
(SI 2 2.0) when whole blood cultures were incubated
with eight virus antigens are presented in Table 3. T h e
SLE patients were significantly less responsive than control individuals to rubella, measles-CF, varicella-zoster,
a n d parainfluenza types 1, 2, a n d 3 antigens. No significant differences were observed with measles-HA and
herpes virus type 1 viruses. In the case of the measlesHA antigen, t h e failure t o observe a significantly decreased incidence of response in SLE patients appeared
to be due t o the controls' decreased frequency of response to this antigen. In the case of the herpes antigen,
lymphocytes from most individuals in both t h e SLE and
control groups were stimulated. Specificity of stimulation by t h e virus antigen preparations was indicated by
the absence of differences in incidence of positive responders to varicella-zoster a n d herpes virus, when medium controls or tissue culture controls were used to
calculate t h e SI (Table 3). In addition, specificity was
suggested by discordant responses to the two measles
antigens in 6 of 13 responsive individuals a n d to the
three parainfluenza antigens in 12 of 21 responders.
In addition t o t h e occurrence of hyporesponsiveness t o most virus antigens individually, SLE patients
also responded t o fewer antigens than did the controls
Table 3. Stimulation of Lymphocytes of S L E Patients and Controls by Virus Antigens
Group
SLE
No. positive$ per
no. tested
Control
No. positive per
no. tested
p9
Rubella
HA
Measles
HA
Measles
CF
Varicella
CF*
Herpes
CFt
Paraflu-l
HA
Paraflu-2
HA
Paraflu-3
HA
3/15
2/15
2/15
3/15
9/14
4/15
3/15
5/15
12/15
7/15
9/15
12/14
12/13
11/14
9/14
12/14
0.003
0.1 I
0.02
0.001
0.19
0.015
0.04
0.01
* Two of 15 SLE patients and 13 of 15 control individuals were positive when SI = cpm stimulated cultures/cpm tissue culture control cultures.
I'No difference in the number positive when SI was calculated with medium or tissue culture controls.
$ Stimulation index 2 2.0; SI = cpm stimulated cultures/cpm medium control cultures.
4 xz test.
WOLF AND ZIFF
1274
8r
had received either antiinflammatory or immunosuppressive drugs before the study. The mean SIs in both
autologous plasma and AB plasma were in the nonstimulatory range (<2.0) for all four virus antigens.
Although the mean values tended to be higher for cells
cultured in autologous plasma than in AB plasma, the
differences were not statistically significant for any of
the virus antigens by multiple group comparison based
on Kruskal-Wallis nonparametric analysis.
a " 4-
DISCUSSION
0 1 2 3 4 5 6 7 8
NUMBER OF ANTIGENS RESPONDED TO
Fig 1. Number of viral antigens to which lymphocytes from SLE
patients (solid bars) and control individuals (cross-hatched bars) showed
positive responses. Each bar represenrs the number of subjecrs having an
SI 2 2.0 to each of the eight viral antigens tested.
(Figure I ) . These differences were highly significant (P
< 0.001). Among the SLE patients, 1 1 responded to less
than three virus antigens, and 4 to three or more antigens. Among the controls, on the other hand, only 1
responded to less than three antigens, and 13 responded
to three or more antigens.
Effects of Prednisolone on Responsiveness to Virus
Antigens. Because 8 of the 15 SLE patients had received
some prednisolone, the possible effects of therapy on
responsiveness were also analyzed. The SLE patients
were divided into two treatment groups: a ) 7 patients
who had received no prednisolone, and b ) 8 patients
who were taking prednisolone. Table 4 shows that hyporesponsiveness was observed in both untreated and
treated patients with no significant difference between
them. Thus the decreased responses in the SLE patients
as a group did not appear to result from the effects of
prednisolone on the lymphocytes. Because 14 of the 15
patients had active disease, it was not possible to correlate hyporesponsiveness to virus antigens with disease
activity.
Responses of Washed S L E Cells in Autologous
and Normal AB Plasma. The responses of washed peripheral blood cells from 8 additional patients with active SLE to rubella, measles-CF. parainfluenza type 1 ,
and parainfluenza type 3 virus antigens were simultaneously compared in fresh autologous plasma and fresh
AB plasma to evaluate the influence of serum factors
such as virus antibody or antilymphocyte antibodies
(Table 5 ) . All patients were clinically active, and none
The cellular immune response of patients with
SLE to rubella, measles, parainfluenza types 1, 2, and 3
RNA viruses, and varicella-zoster and herpes virus type
1 DNA viruses was evaluated in a whole blood culture
technique that used incorporation of 3H-thymidine as a
measure of blastic transformation. Decreased responses
were detected with rubella, measles-CF, parainfluenza
types I , 2, and 3, varicella-zoster virus antigens, and
PHA, but not with measles-HA and herpes virus type 1
antigens. SLE patients also responded to significantly
fewer antigens than did the controls.
These results are consistent with a generalized
hyporesponsiveness of SLE lymphocytes to virus antigens and are in accord with the reported decrease of cellmediated immunity (CMI) to plant mitogens and common bacterial and fungal antigens (16-29) in SLE,
rather than implicating any of these viruses as a specific
pathogen. Moreover, it cannot be concluded from the
data presented that the hyporesponsiveness to virus antigens was peculiar to SLE, because other chronic inflammatory diseases were not studied.
Several phenomena that might contribute to the
observed hyporesponsiveness of SLE patients to virus
antigens were evaluated. The administration of corticosteroids may decrease the number of circulating T lymTable 4. Relationship of Prednisolone Dose to Response of SLE
Patients' Lymphocytes to Viral A ntigens
Therapy
No. of Antigens
Responded To
Not Receiving
Prednisolone*
Receiving
Prednisolonet
I 2
23
5
2
6
2
* One patient received
aspirin, 650 mg q 4 hours. None was receiving
other antiinflammatory or immunosuppressive drugs.
t Six patients were taking 10-20 mg/day. Two had received I or 2
doses of 6 0 mg at the time of initial diagnosis. A blood sample was
drawn 16 and 18 hours after last dose.
LYMPHOCYTE RESPONSE IN SLE
1275
Table 5 . Responses of Lymphocytes from SLE Patients t o Virus Antigens in Whole Blood Cultures with
Autologous and Normal A B Plasma
Mean SI* f SE
Plasma Source
Rubella
Measles-CF
Parainfluenza
Type I
Parainfluenza
Type 3
Autologous
I .7 f 0.5
0.9 f 0.1
NSt
1.9 f 0.4
I.OfO.l
NS
I .4 f 0.2
0.9 f 0.1
NS
I .6 f 0.3
0.9 f 0.I
NS
AB
P
* SI: stimulation index.
t NS: not significant by multiple group comparison based on Kruskal-Wallis nonparametric analysis.
phocytes (43,44) and alter in vitro correlates of CMI
(45,46). In the present study the administration of prednisolone did not appear to be responsible for the decreased response of SLE lymphocytes to virus antigens,
because equally depressed responsiveness was detected
in both treated and untreated patients. A similar lack of
relationship between corticosteroid therapy and hyporesponsiveness to mitogens or bacterial and fungal antigens in vitro and in vivo has been reported (27-29,33).
In fact, Rosenthal and Franklin (29) have reported recovery of CMI in SLE patients receiving corticosteroid
therapy by in vitro and in vivo parameters.
Both cytotoxic (47-49) and noncytotoxic (50) antibodies which may be T-cell specific (51) have been
detected in the serum of SLE patients. These and other
possible SLE serum factors may nonspecifically decrease
in vitro CMI responses of normal lymphocytes to mitogens (26,33,37) and allogeneic leukocytes (36,37,52),
and they may impair spontaneous rosette formation
with sheep erythrocytes (33). In addition, elevated virus
antibody titers in autologous SLE plasma may have
neutralized the virus antigens.
In order to evaluate the influence of virus antibody or antilymphocyte antibody on the response to
virus antigens, the cells were processed at room temperature and then washed three times with warm (37°C)
medium prior to culture in the presence of AB serum
from a normal donor. The failure to increase the responsiveness of lymphocytes by this treatment argues against
a significant role for serum factors in the observed hyporesponsiveness to virus antigens. Winchester et a1 (50)
have reported a marked reduction of cold-reactive, surface-bound immunoglobulin after washing at 37"C, and
Wernet and Kunkel (52) have demonstrated at least a
partial recovery of response to allogeneic leukocytes by
SLE responder lymphocytes in AB serum, whereas total
suppression of reactivity was observed in autologous
serum. Because overnight incubation before culture has
been reported to alter murine lymphocyte subpopulations (53), the washing procedure used avoided
this potential source of error.
Utermohlen et a1 (39), using a leukocyte migration inhibition assay, observed a decreased response of
CMI to measles-CF antigen but not to rubella and parainfluenza type 1 antigens in SLE patients. These investigators raised the possibility that the decreased reactivity to measles antigen might have been indicative of a
generalized defect of CMI. This interpretation is consistent with the results of the present study. The fact that
negative responses to rubella and parainfluenza type 1
antigens were observed in the present study and not by
Utermohlen and coworkers may be attributable to differences in the method of evaluating responses to virus
antigens. In the present study CMI was measured by 3Hthymidine uptake of lymphocytes in culture, whereas
Utermohlen et a1 measured inhibition of leukocyte migration.
Specificity of the responses to the virus antigens
and not to constituents of the culture media employed
was indicated by two observations. First, herpes virus
type 1 and varicella-zoster antigens showed no significant differences in the number of positive responders
when uninfected tissue culture control material was
compared with medium controls to calculate the stimulation index in the case of these two antigens. Second,
although supernatants containing the measles-CF and
measles-HA antigen were both obtained from infected
primary human amnion cells, stimulation indices of
these two antigens were discordant. Parainfluenza antigens were all obtained from infected primary monkey
kidney cells, but the responses to these three antigens
were also discordant.
The present findings of a diffuse hyporesponsiveness of lymphocytes from SLE patients to virus antigens
appear to be best explained by a basic functional defect
of the lymphocytes of these patients, rather than by the
WOLF A N D ZIFF
1276
effects of serum factors or therapy. Utsinger (27) a n d
Lockshin et af (28) h a v e reached a similar conclusion
regarding t h e response of SLE lymphocytes to mitogens.
16.
ACKNOWLEDGMENT
The authors thank Ms. Olivia White for her very capable technical assistance and Ms. Terrie Harris for
manuscript transcription.
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