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Deficiency of T cell mediated regulation of anti-dna production in systemic lupus erythematosus.

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24
DEFICIENCY OF T CELL MEDIATED REGULATION
OF ANTI-DNA PRODUCTION IN
SYSTEMIC LUPUS ERYTHEMATIOSUS
JOHN D. CLOUGH, SHEILA A. FRANK, and LEONARD H. CALABRESE
Isolated B cells from normal subjects and patients with systemic lupus erythematosus (SLE) could
be stimulated to produce IgM anti-DNA with pokeweed
mitogen. Normal but not SLE allogeneic T cells abrogated this response. Normal but not SLE autologous T
cells promoted a switch from IgM to IgG anti-DNA production. SLE is characterized by at least two types of
immunoregulatory abnormalities: a defect in T suppressor function and a defect in the IgM to IgG switchover.
Several investigators have documented diminished immunoregulation mediated by T lymphocytes in
systemic lupus erythematosus (SLE) (1-6). Phenomena
subject to regulation that have been examined in SLE
include DNA and protein synthesis by cultured mononuclear cells (l), mixed lymphocyte responses (2,3), immunoglobulin production (4-6), and most recently,
anti-DNA production (5).
SLE is characterized by presence of antibodies
against native DNA. These antibodies have been implicated in some aspects of tissue damage that occurs in
this disease. Such antibodies are found in significant
quantity only in patients with SLE, although very small
From the Department of Rheumatic and Immunologic Disease and Department of Immunopathology, The Cleveland Clinic
Foundation, Cleveland, Ohio.
Supported by grants from the Northeast Ohio Arthritis
Foundation and the Ohio Lupus Foundation.
Address reprint requests to John D. Clough, MD, Cleveland
Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44 106.
Submitted for publication July 9, 1979; accepted in revised
form September 1 I , 1979.
Arthritis and Rheumatism, Vol. 23, No. 1 (January 1980)
amounts of IgM anti-native DNA can be found in most
normal sera (7). We have recently reported evidence
suggesting relatively greater impairment of the IgM to
IgG switchover in the anti-DNA response in severe SLE
as compared to mild SLE (8).
We now extend these findings to show defective
allogeneically stimulated suppression of IgM anti-DNA
production in the presence of SLE T cells. Furthermore
SLE T cells appear to be relatively deficient in the ability to promote the switch from IgM to IgG anti-DNA
production by autologous B cells that characterizes autologous normal T and B cell combinations.
MATERIALS AND METHODS
Patients. Heparinized blood was obtained from 8
American Rheumatism Association criteria for SLE (9). None
was taking corticosteroids or immunosuppressive drugs. All
patients had circulating antibody against native DNA, reduced total hemolytic complement, and active urinary sediments at the time of sampling.
Cultures. Mononuclear cells were isolated on FicollHypaque, washed, incubated for one hour at 37”C, then applied to 12 ml columns of Sephadex G-200 conjugated with
immunoabsorbent-purified rabbit anti-human F(ab)’, as described by Chess et al (10). Cells passing through the column
(T cell enriched fraction, henceforth referred to as “T cells”)
and cells retained on the column, then eluted with Cohn fraction I1 (B cell enriched fraction, henceforth referred to as “B
cells”), were washed 5 times through 3 ml heat-inactivated
fetal calf serum.
Cultures were then established containing 2.5 x lo5 B
cells from each individual. To some of these cultures 5 x lo5
T cells from the same or other donors were added. All cultures
were brought to 2 ml with RPMI 1640 supplemented with
ANTI-DNA IN SLE
25
Table 1. Cell survival after 7 days in culture with PWM
Initial
Type of culture
Autologous coculture
Allogeneic coculture
T cells only
B cells only
Cells x lo-'
7.5
7.5
5.0
2.5
Viable cells/culture
Dead cells/culture
Cells x lo-'
%initial
7.7 f O . I *
9.4 f 0.2
3.0 f 0.2
2.1 fO.l
I03
Cells x lo-'
1.2 f 0.3*
125
60
84
1.6 f 0.8
0.8 f 0.2
0.1 rt 0.1
%initial
16
21
17
4
* Arithmetic mean f standard error.
glutamine, penicillin-streptomycin, and 10% heat-inactivated
fetal calf serum to which was added 0.1 ml 1 : 50 pokeweed
mitogen (PWM). Cultures were maintained for 7 days at 37°C
in a humidified atmosphere of 95% ai.r and 5% CO,. Supernates were then harvested and stored at -20°C until assayed.
Additional cultures containing 10" unseparated mononuclear cells in 2 ml of the same culture medium were cultured under the same conditions. Supernates from these cultures were harvested and stored in the same way until assay.
T cells were identified by the standard sheep erythrocyte rosette assay (1 1). B cells were identified by direct immunofluorescence using FITC-labeled F(ab)'2 fragments of antiimmunoglobulin antibodies (12).
Assays. DNA antibody levels in the IgM and IgG
classes were assayed in the supernates by a recently developed
solid-phase radioimmunoassay which has been described in
detail (7). Briefly, formalinized human group 0 Rh negative
erythrocytes were coated with double-stranded calf thymus
DNA in the presence of 0.1% CrCl, such that 10' cells bound
about 20 p g of Millipore-filtered DNA. Aliquots of these
DNA-coated cells containing 4.8 X 10" cells (with about 0.96
pg DNA) suspended in veronal-buffered saline with 0.1% gelatin (VBS-G) were exposed to 0.2 ml aliquots of culture supernate in duplicate and incubated for 15 minutes at 37°C.
After the cells were washed 3 times in VBS-G, duplicates of
each combination were incubated with monospecific "'I-labeled anti-IgM and anti-IgG for 15 minutes at 37°C. The cells
were again washed three times with VBS-G and were transferred to counting vials and counted in a Beckman Biogamma
counter.
Controls included a duplicate run of each supernate
against erythrocytes exposed to CrCl, but not coated with
DNA, and blanks containing uncoated or coated cells with
each of the labeled anti-Ig sera, but without supernates. From
the radioactivity bound in each instance, the quantity of
DNA-binding immunoglobulin in each class could be calculated using standardization factors determined for each of the
radioiodinated anti-Ig; these were derived by testing each of
the radioiodinated anti-Ig against known amounts of corresponding purified Ig bound to the formalinized erythrocytes
as previously described (7).
Polyclonal IgM and IgG were measured by doubleantibody radioimmunoassay.
Expression of results. Except where otherwise indicated, results are expressed as geometric means of nanograms of immunoglobulins measured in the culture supernate
+- standard error (log transformed). The Mann-Whitney U
test for comparing samples not known to have normal distribution was employed for determination of P values.
RESULTS
Nature of cells in separated populations. T cell
fractions contained about 85% sheep erythrocyte rosette
forming cells and t0.5% surface immunoglobulin positive cells. None of the cells were phagocytic for latex
particles. The B cell fractions contained less than 0.5%
sheep erythrocyte rosette forming cells. About 56% had
surface immunoglobin and about 40% phagocytosed latex particles.
Incubation of separated cells with iron carbonyl
removed 30-50% of the cells and eliminated the latex
particle phagocytosing population, presumably macrophages, from the B cell fraction. A similar proportion of
the T cell population was also removed by this procedure. No studies were done to characterize the cells removed from the T cell fraction. In the studies reported
here iron carbonyl was not used to remove macrophages, since it appeared to remove unknown lymphocyte populations as well.
Cell viability. Viable cell counts after 7 days of
culture of normal cells in various autologous and allogeneic combinations are shown in Table 1. An approximate 25% increase in cell number occurred during the
culture period in the allogeneic T and B cell combinations. This increase was not seen in the autologous combinations. Some losses in total cell numbers were noted
with cultures of T cells or B cells alone. A mean of 15%
nonviable cells was noted at the end of the culture period by trypan blue exclusion.
Production of polyclonal immunoglobulins by
cultured lymphocytes. The results of studies of polyclonal immunoglobulin production are shown in Figure
1. B cells freed of T cells by anti-Fab immunoabsorbent
chromatography produced relatively small amounts of
immunoglobulin when cultured for 7 days with PWM.
IgM production was about twice the amount of IgG
production by both normal and SLE B cells. Mean total
immunoglobulin production was somewhat greater by
normal B cells than by SLE B cells, although this difference was not statistically significant.
CLOUGH ET AL
26
when T cells were not irradiated, and 2370 ng/culture
of autologous T and B cells.
In contrast to allogeneic combinations of normal
T and B cells, the allogeneic combinations of SLE T
cells with normal B cells did not produce significantly
less IgM than did autologous normal T and B cell recombinations. Moreover this allogeneic combination
actually produced slightly more IgG than did the normal autologous combinations.
Unfractionated normal or SLE lymphocytes produced significant quantities of IgM and IgG when cultured for 7 days with PWM (data not shown). As noted
5000 r
ji0I
ln
W
I
c-
z
>
ln
z
5
100-
3
m
0
50-
J
W
0
z
=I
z
E
m
;[
V
T
N
A U I
N
ALLO.
N
SLE
ALLO.
N
SLE
AUTO.
SLE
N
ALLO.
SLE
-
-
N
SLE
Figure 1. Polyclonal immunoglobulin production (geometric mean f
standard error) by 2.5 x lo5 normal (N)or lupus erythematosus
(SLE) B cells alone or cocultured with 5 X lo5 N or SLE T cells in the
presence of pokeweed mitogen. Autologous (AUTO.) and allogeneic
(ALLO.) combinations are indicated. Allogeneic normal T cells suppressed IgM and IgG production by N or SLE B cells whereas allogeneic SLE T cells did not.
Maximal IgM and IgG production were
achieved by recombining autologous T and B cells at a
2: 1 ratio before culture with PWM. This produced an
approximately sevenfold increase in IgM production as
well as a marked increase in IgG production as compared with B cells alone. As with B cells alone, the normal combinations produced more immunoglobulin than
did the SLE combinations; in this circumstance the difference was significant for IgM (Pc 0.02).
Normal B cells cultured with allogeneic normal
T cells produced 86% less IgM than did normal B cells
cultured with autologous T cells (Pc 0.01). SLE B cells
cultured with normal T cells produced 78% less IgM
than did SLE B cells cultured with autologous T cells (P
< 0.02). Similar differences in IgG production were observed in allogeneic combinations where the T cells
were from normal donors. The suppressive effect of allogeneic T cells was almost completely abolished by
1000 r gamma irradiation of this population prior to coculture; allogeneic combinations of normal B cells with
irradiated normal T cells produced a geometric mean of
1790 ng IgM/culture, as compared with 330 ng/culture
50
\
F
a
z
Y
101
a
5.0.
I
F
z
a
3
a
Figure 2. IgM and IgG anti-DNA production (geometric mean f
standard error) by 2.5 X lo5 normal (N)or lupus erythematosus
(SLE) B cells alone or cocultured with 5 x lo5 autologous T cells in
the presence of pokeweed mitogen. Normal B cells switched from
IgM to IgG antibody production with the addition of normal T cells.
This switchover was not seen in the SLE cultures.
ANTI-DNA IN SLE
loor
27
fl
1
....
....
...
-
...
....
La
CI,
c
Y
a
z
4
5.0
F
z
a
I.o
0
w
0.5
T
wdcB
9
0
2 -
N
N
N
SLE
N
SLE
N
SLE
J
a
Figure 3. IgM and IgG anti-DNA production (geometric mean f
standard error) by 2.5 x lo5 normal (N)or lupus erythematosus
(SLE) B cells alone or cocultured with 5 X lo5 allogeneic normal or
SLE T cells. IgM anti-DNA production was suppressed by allogeneic
N but not SLE T cells; IgG anti-DNA production by B cells was minimal and not greatly affected by the addition of allogeneic T cells.
in the section immediately following, however, these
same cultures produced no detectable anti-DNA.
Production of DNA antibody by cultured lymphocytes. B cells cultured with PWM after separation from
T cells produced significant amounts of anti-DNA (Figure 2). This was true whether the cells were obtained
from normal subjects or SLE patients. The anti-DNA
produced in this circumstance was predominantly IgM.
SLE B cells also produced some IgG anti-DNA, significantly more than did normal B cells (P< 0.05).
The recombination of normal T cells with autologous B cells resulted in a highly significant (P< 0.005)
more than tenfold increase in IgG anti-DNA production over that by B cells alone (Figure 2). This was accompanied by a reduction of mean IgM anti-DNA production which was not statistically significant.
Autologous recombinations of separated T and B
cells from SLE patients also showed slight increases in
IgG anti-DNA production and reductions in IgM antiDNA production as compared to SLE B cells alone, but
these differences were small and not statistically significant (Figure 2).
Allogeneic T and B cell combinations behaved
somewhat differently (Figure 3). Addition of normal T
cells to allogeneic normal B cells resulted in 95% suppression of IgM anti-DNA production as compared to B
cells alone (P < 0.02). IgG anti-DNA production remained minimal. Normal T cells also produced 95%
suppression of IgM anti-DNA production by SLE B
cells (P< 0.02); this was accompanied by a modest reduction of IgG anti-DNA production which was not
statistically significant. On the other hand, SLE T cells
produced a lesser degree of suppression of IgM antiDNA production by normal B cells, which did not differ significantly from IgM anti-DNA production by
normal B cells alone; IgG anti-DNA production remained minimal.
Interestingly, there was no detectable anti-DNA
production by unseparated lymphocyte populations cultured with PWM whether they were from normal subjects or SLE patients; separation followed by recombination was necessary to observe this phenomenon.
Neither B cells alone nor recombined T and B cells produced anti-DNA without PWM. T cells alone, with or
without PWM, did not produce antibody.
DISCUSSION
We have previously demonstrated (6) and reconfirmed here that allogeneically stimulated suppression
of polyclonal IgM synthesis is defective in patients with
SLE. We have extended these observations to include
similar data for polyclonal IgG. The cells that mediate
this suppression are surface immunoglobulin negative
and radiosensitive, and they are presumably T cells.
The possibility that the apparent suppressive effect
demonstrated in the allogeneic T and B cell combinations represents generation of cytotoxic T cells against
allogeneic B cells, eliminating immunoglobulin production by killing the B cells, cannot be entirely ruled out.
However, studies of surviving cells at the end of the culture period show greater numbers present in the allogeneic than autologous cultures. Moreover, our previous
studies showed the presence of a soluble suppressor factor in the allogeneic supernates (6). Finally, the suppressor effect was eliminated from normal T cells by a small
amount of irradiation which would not have been likely
CLOUGH ET AL
28
to eliminate cytotoxic cell function. Furthermore, in animal systems, cell-mediated lympholysis shows genetic
restriction (13); allogeneic suppression does not.
We now extend these observations to show that
allogeneically stimulated suppression of DNA antibody
synthesis mediated by T cells is also defective in patients with SLE. Since DNA antibody is important
pathogenically in some of the clinical features of SLE,
especially glomerulonephritis, this defect in T cell mediated suppression seems relevant to the disease process
itself.
Sagawa and Abdou (5) have also reported defective T cell mediated regulation of anti-DNA production
in SLE. Our findings are unlike theirs, however, in that
we were able to demonstrate significant amounts of
DNA antibody production by normal as well as SLE B
lymphocytes when T cells were absent. The anti-Fab
immunoabsorbant column method of cell separation we
used differs from the sheep erythrocyte sedimentation
method used by Sagawa and Abdou in that the former
technique binds and perhaps additionally stimulates B
cells, whereas the latter does not. That normal B cell
populations contain cells recognizing DNA has been reported previously (14); perhaps the additional stimulus
of column attachment allows such cells to differentiate
to antibody-producing cells in the presence of PWM under these culture conditions.
It is not clear why allogeneic stimulation should
preferentially activate suppressor T cells. Surface markers reported to be present on suppressor cells [y-Fc receptors (1 5 ) , TH-2 antigen (16)] would not be expected
to interact in a special way with allogeneic cells. Special
receptors for allogeneic histocompatibility antigens
have not been reported on suppressor cells. In our allogeneic culture system, B cells appear to provoke T suppression as well as indicate its presence. Sasportes et a1
(17) have reported that the stimulating antigens in this
process are the HLA-DR antigens, which are present in
particularly high concentration on B lymphocytes. They
have pointed out the possible importance of this phenomenon in maintenance of pregnancy and allograft acceptance. The possibility that some “allogeneic-like” interaction between cell populations in the same
individual, e.g. autologous mixed lymphocyte reaction,
plays a role in normal resistance to autoimmunity may
be relevant to this (1 8,19).
In this study only autologous T cells mediated a
switchover from IgM to IgG anti-DNA production in
vitro. In mice the soluble T cell factor responsible for
this type of switch is genetically restricted in its effect
(20). Our data are consistent with the existence of the
same sort of restriction in humans. Alternatively, the
switchover-promoting effect of T cells may simply be
masked in the allogeneic situation by the marked T suppressor effect that is stimulated by the allogeneic cells.
The failure of autologous SLE T and B cell combinations to demonstrate the switchover is consistent
with our earlier finding that among untreated patients
with active SLE nephritis, those with more severe disease had higher IgM :IgG ratios within their serum
anti-native DNA than did those with milder disease (8).
This observation suggested that SLE patients with less
capacity to convert to IgG antibody production have
more severe disease since the immune response can
progress to its conclusion. The present study suggests
that this defect differentiates SLE patients from normal
individuals. It is obvious that this cannot be an acrossthe-board defect, since total serum IgG may be elevated
in SLE. These studies show the defect only for the specific anti-DNA antibody. Nor is it an absolute defect,
since some IgG anti-DNA production is seen in the
SLE cocultures.
The data are consistent with either the interpretation that in SLE there is a T cell defect leading to
failure of stimulation of B cells to switch from IgM to
IgG antibody production or that SLE B cells fail to respond to the switchover signal. The presence of IgG
anti-DNA in supernates of SLE B cells cultured alone
suggests that they are already partially switched over,
but recombination with autologous T cells promotes no
additional IgG antibody production. Studies of identical twins discordant for SLE might help to resolve the
question of whether this defect resides primarily in T
cells, B cells, or both.
ACKNOWLEDGMENT
The authors gratefully acknowledge the skilled secretarial assistance of Ms Judi Wagner.
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