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Depressed in Vitro B В ЭLymphocyte Differentiation in Systemic Lupus Erythematosus.

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Peripheral blood lymphocytes from 20 patients
with systemic lupus erythematosus (SLE) and 21 normal
donors were incubated with pokeweed mitogen in order to
assess in vitro terminal-differentiation of B lymphocytes
into cells synthesizing intracytoplasmic immunoglobulin
(Ig). Although the percentage (mean k SEM) of B lymphocytes bearing surface Ig in the initial cell suspensions
was not statistically different in SLE than in normal subjects (15 f 2.2% versus 16 f 1.9%, respectively), the
frequency of cells containing intracytoplasmic Ig per lo3
mononuclear cells was significantly lower in mitogen-stimulated cultures derived from the patients than from the
normal controls (10 f 2.3 versus 56 f 13.0 for IgM, P <
0.01; 21 f 3.6 versus 63 f 10.4 for IgG, P < 0.01; 13 f
3.1 versus 24 f 3.8 for IgA, P < 0.05 respectively).
Coculturing active SLE lymphocytes with cells from norFrom the Division of Rheumatic Diseases, Department of
Medicine, University of Connecticut School of Medicine, Farmington,
Connecticut 06032. and The Veterans Administration Hospital, Newington, Connecticut 061 I I ,
Presented in part at the 40th Annual Scientific Session of the
American Rheumatism Association, Chicago, Illinois, June 10- I I , 1976.
Supported in part by an Arthritis Foundation Center grant
and in part by Grant MRIS #I006 from the United Stares Veterans
Arthur M. Bobrove, M.D.: Formerly Assistant Professor
Medicine, University of Connecticut. Veterans Administration Hospital, Newington, Connecticut 061 11: Patricia Miller: Research Technician, Veterans Administration Hospital, Newington. Connecticut
Address reprint requests to Arthur M. Bobrove. M.D.. Palo
Alto Medical Clinic, 300 Homer Avenue, Palo Alto, California 94301.
Submitted for publication April 28, 1977; accepted May 19.
Arthritis and Rheumatism, Vol. 20.
No. 7 (September-October 1977)
ma1 subjects resulted in a significant (P < 0.05) increase
in the frequency of cells containing intracytoplasmic IgG
when stimulated with pokeweed mitogen. Moreover, culturing SLE lymphocytes in cell-free media derived from
activated normal lymphocytes also resulted in a significant
increase in the frequency of IgG-containing cells. These
results suggest that B-lymphocyte differentiation in vitro
is depressed in SLE and may, at least partially, be reversed by products derived from normal lymphocytes.
A state of disordered immunity appears to exist
in systemic lupus erythematosus (SLE). Cell-mediated
immunity as judged by delayed cutaneous hypersensitivity is frequently depressed, especially when the disease is
clinically active (1-4). Thymus-derived (T) lymphocytes
are commonly decreased in the peripheral blood during
flares in disease activity (5,6) and lymphocytotoxic antibodies with relative specificity for T cells (7) can be
identified in a high percentage of patients (8,9). In vitro
tests of T-cell function, such as blast transformation
with nonspecific mitogens ( 3 , l O ) and sheep erythrocyte
rosette formation (5,6), are commonly diminished when
compared to normal controls. The humoral immune or
bone marrow-derived (B)-lymphocyte system, in contrast, exhibits signs of increased activity. In addition to
synthesizing numerous autoantibodies, patients with
SLE commonly have hypergammaglobulinemia ( 1 I),
elevated viral antibody titers (12,13), and increased
turnover rates of immunoglobulin (Ig) (14). Cultured
peripheral blood leukocytes from patients with active
SLE synthesize increased a m o u n t s of Ig in vitro (15) a n d
cells resembling p l a s m a cells can occasionally be identified o n smears o f peripheral blood (16).
These observations suggest a generalized hyperreactivity of t h e B-cell system in SLE and p r o m p t e d a
study t o assess B-lymphocyte reactivity to in vitro stimulation with t h e polyclonal activator, pokeweed mitogen
PWM provides a nonspecific signal for terminal
differentiation in vitro of normal B lymphocytes i n t o Igproducing plasma cells (17) and h a s been used as a
probe t o elucidate t h e cellular defects in several immunodeficiency states (l7,18). Our results indicate t h a t in
vitro differentiation of peripheral blood B-lymphocytes
into cells containing intracytoplasmic Ig is diminished in
SLE a n d c a n b e a t least partially restored by supernatants derived from activated normal lymphocytes.
Patients Studied. Mononuclear cells from 20 patients
with SLE, 19 female and 1 male were studied and the results
compared with those from 21 normal subjects matched for age
(within 5 years) and sex with the patients. All of the patients
had well-documented SLE and fulfilled the preliminary classification criteria of the American Rheumatism Association
(19). Six of the patients with active SLE, as judged clinically by
the presence of fever, arthritis, serositis, mucosal ulceration,
skin rash or central nervous system involvement, were studied
prior t o corticosteroid administration. The remainder were
taking prednisone ranging in dose from 2.5 to 60 mg per day,
with a median dose of 15 mg per day. None had received
cytotoxic drugs. None exhibited renal failure.
Mononuclear cells from 4 individuals without SLE but
treated with corticosteroids for other diseases (see Table I )
were also studied.
Mononuclear Cell Separation. Mononuclear cells were
separated from whole blood on Ficoll-Hypaque (20) and
washed twice with Hanks balanced salt solution (HBSS)
(Microbiological Associates, Bethesda, Maryland) at 37" C.
The cells were resuspended at a concentration of 1 X lo7
Table 1. Pokeweed Mitogen-Induced Development of Cells Containing
Intracytoplastiiic Immunoglobulin Arising in Viiro from Lymphocytes
Obtained jrorn Individuals Treated with Corticosteroids for Disorders
Other than SLE
Allergic rhinitis
Bronchial asthma
Bronchial asthma
Cells with Cytoplasmic Ig per IDS
(IgM IgG t IgA)*
20 mg/day
I0 mg/day
107 (86)t
60 mg/day
80 mg/day
* Mean + SEM = 175 i= 44.9 N = 144 f 17.9.
t Count prior to institution of steroids.
mononuclear cells/ml in HBSS containing 10% heat-inactivated (56" for 30 minutes) fetal calf serum (FCS) (Microbiological Associates), incubated at 37" for 30 minutes and
washed once more at 37" C. The washing and incubation at
37" C were done t o try t o remove cold-reactive antilymphocyte
antibodies (21) and much of the cytophilic Ig (22) from the
mononuclear cell surface.
Identification of B Lymphocytes. B lymphocytes were
identified by the presence of surface Ig, using direct immunofluorescence. One tenth milliliter of 1 X lo7 mononuclear
cells/ml suspended in HBSS with 5% FCS was added to 0. I ml
of fluorescein-conjugated rabbit antiserum to the heavy chains
of human IgM, IgG, and IgA (Cappel Laboratories, Downingtown, Pennsylvania) diluted 1 :8 with HBSS with 5% FCS.
The cell suspensions were incubated for 30 minutes a t 4" C.
Unbound antiserum was removed by passing the cells through
1.5 ml of FCS and then washing once in hBSS with 5% FCS.
The cells were smeared, air-dried, fixed in 95% ethanol, and
mounted in buffered-glycerol (23).
Culture Conditions. Mononuclear cells for culture were
resuspended at a concentration of 5 X IV cells/ml in RPMl
1640 (Microbiological Associates) supplemented with L-glutamine (2 mM/ml), penicillin (100 p/ml), streptomycin (100
pg/mI), and 10% FCS. Cells were cultured in 12 X 75 mm
tissue culture tubes (Falcon Plastics, Oxnard, California) in a
volume of 0.5 ml. When mononuclear cells from 2 unrelated
donors were cultured together, 0.25 ml of the cell suspensions
from each of the donors was combined. Pokeweed mitogen
(Grand Island Biological Company, Grand Island, NY) was
added to the appropriate cultures at the start of incubation at a
concentration of 10 pl/ml (determined in preliminary experiments to be optimal). The tubes were incubated in a humidified atmopshere of 5% C02, 95% air at 37" C for 72 to 168
hours. T o assess the amount of D N A synthesis, I rCi/ml *Hthymidine, specific activity 2 Ci/mM (New England Nuclear,
Boston, Massachusetts) was added t o triplicate cell cultures 16
hours prior to harvesting. Viability was determined by trypan
blue exclusion.
Activated Lymphocyte Supernatants. Mononuclear
cells (4 X 10') from normal individuals were cultured in 1 ml
of RPMl 1640 with Hepes Buffer supplemented with L-glutamine (2 MM/ml), penicillin (100 p/ml), streptomycin (100
pg/mI), and 10% FCS and incubated at 37" for 2 or 3 days.
PWM (10 pl/ml) was added either at the start or at the end of
the culture period. The culture supernatants were collected
and 0.25 ml was promptly added t o an equal volume of fresh
culture media containing 2.5 X lo6mononuclear cells. Freshly
reconstituted PWM was then added and the cultures were
incubated for 7 days.
Intracytoplasmic Ig. Mononuclear cells synthesizing
cytoplasmic Ig in vitro were assessed at the end of the culture
period. Cultured cells were washed once in HBSS with 5%
FCS, smeared, air dried, and immediately fixed in 95% ethanol
for 20 minutes (24). The slides were rehydrated in PBS, then
covered with a drop of FITC-conjugated rabbit antihuman
serum to the heavy chains of either IgM, IgG, or IgA. A cover
slip was applied to spread the drop and the slides were incubated in a wet chamber at room temperature. The cover
slips were removed and the slides rinsed in PBS two times for
10 minutes each, fixed in 95% ethanol for 20 minutes, and
mounted in buffered glycerol.
Cultures established to assess DNA synthesis were
harvested as previously described ( 2 5 ) . Culture tubes previously incubated at 37" C were placed in an ice bath and the
cells were washed three times with cold PBS. The nucleoprotein was precipitated with cold 5% trichloracetic acid,
washed once in 5% trichloracetic acid, and dehydrated with
100% methanol. The precipitates were dissolved with 0.5 ml
hyamine hydroxide by incubation for 3 hours at 56" C. The
contents of the tubes were then transferred to glass scintillation vials with 12 rnl of LSC (New England Nuclear) and
counted on an lsocap 300 Liquid Scintillation Counter (Nuclear Chicago, Chicago, Illinois). The results were expressed as
cpm per culture.
Slides for immunofluorescence staining were examined
using a Zeiss Photomicroscope I1 equipped with an HBO 200
mercury arc (Osram), BG38 and BGl2 excitation filters, and
510 barrier filter. The frequency of cells with surface or cytoplasmic Ig was determined by counting the total number of
cells in each field under tungsten illumination and the number
of fluorescent cells in the same field under ultraviolet illumination. A minimum of 200 cells was counted for quantitative Igbearing lymphocytes and a minimum of los cells for quantitative cytoplasmic-staining cells.
Significance of statistical differences between mean
values were examined by Student's 1 test for unpaired values.
Frequency of Cells Containing Intracytoplasmic
Ig. T h e capacity of peripheral blood B lymphocytes
from 20 patients with S L E and 21 normal controls t o
differentiate into cells containing intracytoplasmic IgM,
IgG, or IgA in response to P W M stimulation, is shown
in Figure 1. T h e mean number of cells containing intracytoplasmic Ig per 1 Oa mononuclear cells developing
after 7 days of incubation was significantly lower for the
patients with S L E than for the normal controls for each
of the 3 immunoglobulin classes studied (IgM, P < 0.01;
IgG, P < 0.01; a n d IgA, P < 0.05). T h e range for IgM-,
IgG-, and IgA-containing cells per 103 cells was 1-241,
15-172, and 6-41 for the normal controls a n d 0-34,
2-56. and 0-40 for the S L E patients, respectively.
Percentage of B Lymphocytes. To assess whether
there were any differences in the concentration of B
lymphocytes being added t o the cultures of either the
patients or the normal controls, t h e percentage of Igbearing lymphocytes present in the initial mononuclear
cell suspensions was determined. There was n o significant difference between the percentage (mean & S E M )
of Ig-bearing lymphocytes stained for IgM, IgG, or IgA
present in the suspensions from the S L E patients (14.5 f
2.2%) and the percentage in the normal controls (15.8 f
Effect of Corticosteroid Administration. Many of
the patients with S L E in this study were already receiv-
Fig. 1. Pokeweed mitogen-induceddevelopment of cells containing intracytoplasmic immunoglobulin arising from culturedlymphocytes obtained
from 20 patients with SLF
and 21 normal controls II,incubatedfor 7
days. The means f SEA1 are shown for each immunoglobulin class
* P < 0.01. t P < 0.05
ing corticosteroids t o control the clinical manifestations
of the disease. To determine whether the disease or the
administration of corticosteroids was playing a role in
t h e reduced in vitro response exhibited by the patients' B
cells, the findings derived from 6 clinically active patients who had not receiyed steroids were compared with
those of the normal controls and the total patient group.
Lymphocytes from these untreated S L E patients were
also impaired significantly ( P < 0.001) with fewer cells
containing intracytoplasmic IgM, IgG, or IgA than normal controls (Figure 2) and there was no significant
difference between treated a n d untreated patients with
SLE. T h e effect of corticosteroid administration was
also investigated in individuals with diagnoses other
than SLE. Corticosteroids did not appear to significantly affect the number of Ig-containing cells after
PWM stimulation (Table 1).
Kinetics of B-cell Differentiation and Cell Survival. T h e development of cells containing intracytoplasmic IgG in response to PWM was studied on
various days on mononuclear cells from 3 normal subjects a n d 3 patients with SLE. Peak response occurred
on day 7 for cells from normal subjects, whereas n o
increment at all was observed for cells from S L E patients by day 7 (Figure 3). Cultures continued beyond 7
Fig. 3. Pokeweed mitogen-induced development of cells containing intracytoplasmic IgG arising in vitro from lymphocytes obtained from 3
active SLE patients @-a. and 3 normal controls 0 -.- . - . - . 0 , as a
function of lime at 37' C .
Fig. 2. Pokeweed mitogen-induced development of immunoglobulin containing mononuclear cells arising in vitro from lymphocytes obtained
from 6 active-untreated SLE patients. the total (20)SLE patient population, and the 21 normal controls. The means k SEM are those of the
sums of IgM-, IgG- and IgA-containing cells.
* P < 0.01.
days exhibited a markedly reduced cell survival. Cell
survival as determined by trypan blue exclusion and
total cell counts at day 7 was comparable for patients
and normal subjects (64%versus 67%).
Kinetics of DNA Synthesis. To assess, independently, the functional activity of the mononuclear cells
and investigate whether SLE lymphocytes were preferentially damaged by the washing and preincubation at
37" C and showed a significant loss of reactive cells with
increasing duration of incubation, cells from 4 patients
with active SLE and 4 normal controls were incubated
with PWM and the amount of SH-thymidine incorporation was determined. Although the mononuclear cells obtained from the SLE patients initially
responded less well than cells from normal subjects, by
day 7 the response of the two groups was the same
(Figure 4).
Effect of Coculturing SLE with Normal Lymphocytes. To study the possible role of regulatory cells in
this system, lymphocytes from SLE patients were cocultured with lymphocytes from normal subjects in the
presence of PWM. Enhancement or suppression in the
number of cells staining for intracytoplasmic Ig was
determined by comparing the results obtained from the
cocultured lymphocytes stimulated with PWM with the
data from the individual donor lymphocytes cultured
separately with PWM. In general, few Ig-containing
cells developed in cocultures devoid of PWM. The results, expressed as the difference between the number of
Ig-positive cells observed and the number predictid, are
shown in Figure 5. There was a significant enhancement
in the number of IgG-containing cells in cocultures combining lymphocytes from active SLE patients and normal donors, when compared to that observed in cocultures combining cells from pairs of unrelated normal
donors (P < 0.05). No significant enhancement or suppression in IgG-positive cells resulted from cocultures of
lymphocytes obtained from patients with inactive SLE
and normal donors or pairs of unrelated SLE patients.
Unlike that noted for IgG, there was only a minimal change in IgM-positive cells (mean f SD = 1.5 f
4.75) in active SLE-normal cocultures.
SLE patients in the active group exhibited at least
one of the following clinical manifestations: fever ( I )
alopecia (3), active skin lesions (3), objective arthritis
(4), mucous membrane ulcers (2), CNS involvement ( l ) ,
and pleuritis (1). Six of the active patients had signs of
active nephritis. All but one were hypocomplementemic
humoral immunity, possibly due to a defect in immune
regulation, prompted a study in vitro of B-lymphocyte
differentiation in patients with SLE. The finding of a
decrease in the frequency of cells containing intracytoplasmic Ig, with PWM stimulation, in peripheral
blood lymphocytes from SLE patients was unexpected
and was contrary to the signs of heightened humoral
immunity exhibited by the patients in vivo. Indeed, the
patients studied generally had hypergammaglobulinemia as well as the presence of multiple autoantibodies.
Our results have been confirmed independently by others employing PWM to assess the development of
plaque-forming cells in SLE (26) as well as in vitro
synthesis and secretion of Ig (27).
Multiple factors may be acting singly or together
to contribute to the depressed in vitro response.
Lupus B lymphocytes have been shown to have
an increased rate of spontaneous DNA synthesis (28)
and to produce greater amounts of Ig in vitro in the
absence of stimulation than normal cells (15). This suggests B-cell activation in vivo in SLE. Such cells may be
v 20
Fig. 4. Pokeweed mitogen-induced D N A synthesis of peripheral blood
mononuclear cells obtained from 4 SLE patients .---a,
and 4 normal
controls 0-.-.-.-.0,as a f i c t i o n of time at 37’ C.
(mean C’s = 59 mg%) and demonstrated elevated antibody titers to native DNA (mean = 54%) by a modified
Farr assay.
Effect of Activated Lymphocyte Supernatants. T o
establish that the responding cells in the cocultures
showing enhancement were derived from the SLE cells,
patient lymphocytes were cultured in media containing
cell-free supernatants of PWM-activated normal lymphocytes cultured for 2 to 3 days. Figure 6 illustrates
that in 5 separate experiments the addition to SLE cells
of supernatants harvested from PWM-activated normal
cells resulted in a significant increase in the number of
cells developing intracytoplasmic IgG. When the effects
of the supernatants on normal and SLE lymphocytes
were compared, the degree of enhancement resulting
from the PWM-activated cell supernatants was significantly (P < 0.01) greater with the SLE (214 f 68.9%)
than with the normal (20 f 11.5%) cells (Figure 7).
The apparent immune imbalance, manifested by
a depression in cell-mediated immunity and heightened
- 10
Fig. 5. Eflect of coculturing mononuclear cells from pairs of unrelated
donors on the pokeweed mitogen-induced development of IgG-containing
cells. Each point represents the difference between the number of IgGcontaining cells observed in the cocultures and the number predicted
from the individual donor cells cultured separately with pokeweed mitogen. 1 --I
indicates arithmetic mean of each group.
*P < 0.05.
Fig. 6. Enhancement of the pokeweed mitogen-induced formation of IgG-containing cells, arising from
mononuclear cells obtained from 5 active SLE patients; with supernatants from pokeweed mitogen-activated
normal lymphocytes. Each pair of bars represents a separate experiment. W represents S L E mononuclear
cells cultured with pokeweed mitogen in the presence of supernatant from nonactivated normal lymphocytes.
&j represents SLE cells cultured with pokeweed mitogen in the presence of supernatant from pokeweed
mitogen-activated normal lymphocytes.
unable to differentiate further under in vitro conditions,
be less responsive to an additional stimulus with PWM,
or simply die out in culture.
Antilymphocyte antibodies present in a significant proportion of SLE patients (9,21) could exert an
inhibitory effect on B-cell activity in much the same way
that such antibodies interfere with in vitro T-cell function (29). Efforts to remove antibodies from the cell
surface by incubating the cells a t 37" C for 30 minutes
and washing them at 37" C may be adequate for normal
lymphocytes to shed cytophilic Ig (22), but it may take
up to 24 hours for SLE lymphocytes to shed antibodies
capable of suppressing blast transformation (29). Unfortunately efforts to incubate normal as well as SLE
mononuclear cells at 37' C for 24 hours resulted in a
markedly reduced cell survival in the 7-day cultures.
Circulating immune complexes that can attach to
B-lymphocyte receptors have been identified in the
serum in SLE (30). By blocking C3 receptors, immune
complexes could potentially interfere with B-cell reactivity. In fact, such blockage has been proposed as the
explanation for the decrease in the percentage of B
lymphocytes detectable as complement receptor lymphocytes in the peripheral blood in SLE (5,6). Our results showing that the percentage of B lymphocytes,
identified by the presence of surface Ig, is not significantly lower in SLE than in normal subjects are consistent with the findings of others ( 5 3 1 ) . This supports the
contention that interference with detection rather than a
true decrease may account for the lower percentage of
complement receptor lymphocytes in SLE especially
since these cells and Ig-bearing lymphocytes reportedly
constitute the same population (32). Preliminary experiments designed to study the effect of serum factors in
SLE have not, however, been able to confirm such an
inhibitory effect (33). Short term incubation of normal
lymphocytes in lupus sera prior to PWM stimulation did
not result in significant impairment to proliferation or
B-cell differentiation.
The augmentation of the B-cell response in cultures from some patients with clinically active SLE by
activated-normal lymphocytes or supernatants derived
500 4
conceivable that the evolution of active lupus and antilymphocyte antibodies result in dysfunction of T-helper
cells a s well.
Finally, the depression of in vitro B-cell differentiation in S L E observed in this study may not reflect
events occurring in vivo in sites of major Ig synthesis.
Anatomic compartmentalization of reactive cells as a
result of the disease could conceivably lead t o a depletion of these cells in the peripheral blood. A study of
bone marrow and lymphoid tissue might shed some light
on this point.
We wish to thank Dr. Naomi F. Rothfield for performing the Farr assays and both Drs. Rothfield and Robert
Zurier for their critical review.
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Fig. 7. Effect ?/pokeweed mitogen-activated normal lymphocyte supernatants on the development of IgC-containing cells iti citro from lymphocytes obtained from 5 active SLE patients ;:;:;I,and 5 normal controls
B. Each bar represents a separate experiment and expresses the percentage difference between the eflect of supernatants from pokeweed mitogen-activated and nonactivated normal lymphocytes.
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lupus, differentiation, systemic, erythematosus, эlymphocyte, depressed, vitro
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