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Stimulation of b cell differentiation by adherent mononuclear cells in systemic lupus erythematosus.

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76 1
STIMULATION O F B CELL DIFFERENTIATION
BY ADHERENT MONONUCLEAR CELLS IN
SYSTEMIC LUPUS ERYTHEMATOSUS
ROBERT C. JANDL. TERRY A. ADIRIM. and PETER H. SCHUR
We studied B cell proliferation and differentiation in response to factors released by adherent
monocytes in patients with systemic lupus erythematosus (SLE). Adherent cell supernatants (ACS) were
added to peripheral blood mononuclear cells, and the
effects on IgG synthesis, the number of Ig-secreting cells
(ISC), and proliferation were determined. Exposure of
SLE mononuclear cells to autologous ACS caused an
increase (approximately twofold) in IgG production and
ISC numbers. In contrast, exposure of normal mononuclear cells to autologous ACS did not significantly increase IgG production or ISC numbers. Addition of
SLE ACS to cultures of normal mononuclear cells did
not stimulate ISC production. There was no significant
level of ’H-thymidine uptake by cultures of SLE or
normal mononuclear cells in response to either SLE or
normal ACS. In the presence of an excess number of
autologous T cells, ACS stimulation of IgG synthesis was
further enhanced. These findings indicate that adherent
monocytes contribute to B cell hyperactivity in SLE by
stimulating B cell differentiation. SLE mononuclear
cells appear to be more responsive to ACS stimulation
than are normal mononuclear cells.
From the Department of Rheumatology/Imrnunology,
Brigham and Women’s Hospital, Harvard Medical School, Boston,
Massachusetts.
Supported by U S Public Health Service grants AM-37005
and AM-35907, and by the Lupus Foundation of America.
Robert C. Jandl, MD: Instructor in Medicine; Terry A.
Adirim, BA: Research Associate; Peter H. Schur, MD: Professor of
Medicine.
Address reprint requests to Peter H. Schur, MD, Tower
16B, Brigham and Women’s Hospital, 75 Francis Street, Boston,
MA 02115.
Submitted for publication June 11, 1986; accepted in revised form January 23, 1987.
Arthritis and Rheumatism, Vol. 30, No. 7 (July 1987)
‘The response of normal resting B cells to foreign antigens is a complex sequence of events, which
involves activation, proliferation, differentiation, and
immunoglobulin secretion. The importance of an orderly and controlled progression through this sequence is emphasized by the extraordinary array of
regulatory cells and lymphokines that are capable of
acting on various stages of this pathway. Systemic
lupus erythematosus (SLE) is an autoimmune disease
that is associated with excessive B cell activity. Specifically, large amounts of immunoglobulin and numerous autoantibodies are secreted by SLE B cells, and
the proportion of circulating B cells that spontaneously secrete Ig is markedly increased in S L E patients
(1,2). Other abnormalities of B-lineage cells that have
been reported in SLE patients include increased numbers of circulating immunoblasts (3), cells containing
intracyloplasmic Ig (4), low numbers of circulating
IgD-bearing cells (3,and increased surface Ig (sIg)
negative, Ig-secreting cells (ISC) (6).
Together these findings suggest that there are
substantial abnormalities of the circulating B cell population in patients with SLE. Circulating immune
complexes result from B cell hyperactivity and appear
to cause certain of the pathologic manifestations of the
disease (7). In an effort to understand the basis of SLE
B cell hyperactivity, numerous studies have measured
lymphokine levels and regulatory T cell and monocyte
functions in vitro and in vivo. While many abnormalities have been found, it remains unclear at what stage
in the maturation sequence these immunoregulatory
abnormalities might contribute to SLE B cell hyperactivity.
Recent studies at our laboratory have provided
a unique opportunity to study SLE B cell proliferation
762
JANDL ET AL
and differentiation. In previous work, we observed
that factors produced by adherent mononuclear cells
(MNC) stimulate polyclonal Ig and anti-DNA synthesis (8). Adherent MNC, composed of approximately
95% monocytes, produce the supernatant factor or
factors. The activity occurs in molecules with a relative molecular weight of approximately 14,000 and is
blocked by an antiserum against interleukin-1 (IL-I).
In this report, we describe our studies of the effect of
adherent cell supernatants (ACS) on B cell maturation.
Our findings indicate that autologous ACS stimulate B
cell differentiation in SLE mononuclear cell cultures,
and that SLE MNC are more responsive to ACS than
are normal MNC. Stimulation of B cell differentiation
by ACS may contribute to increased numbers of ISC
and excessive Ig production in SLE.
PATIENTS AND METHODS
Patients and controls. We studied patients who were
seen at the Lupus Clinic, Brigham and Women's Hospital,
who met 4 or more of the revised criteria for the classification of SLE (9). All 1 1 SLE patients were women; their
average age was 41 years (range 17-60). Patients with
clinically active and clinically inactive disease were included. In patients who were taking prednisone, blood was
drawn at least 24 hours after a dose.
Control blood samples were obtained from healthy
laboratory and office workers. Three were male and 9 were
female. Their average age was 28 years (range 22-36).
Cell preparations. Peripheral blood was collected in
heparinized Vacutainer tubes (Becton Dickinson, Rutherford, NJ) and layered over Ficoll-Hypaque (Pharmacia,
Piscataway, NJ). The MNC interface was aspirated and
washed 3 times in RPMI 1640 (Gibco, Grand Island, NY).
Cultures were grown in flat-bottomed Linbro wells (Flow
Laboratories, McLean, VA) at 1 x lo6 cells/ml, in 2.0 ml of
RPMI 1640 supplemented with 10% fetal calf serum (FCS;
Microbiological Associates, Walkersville, MD), 2 mM Lglutamine, 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids, 25 mM HEPES buffer, 100 u n i t s h l of penicillin
G , and 100 pghl of streptomycin (hereinafter referred to as
complete, or fresh, medium). Cultures were kept at 37°C in
a 5% C 0 2 humidified atmosphere for 7 days.
Monocyte-enriched plastic adherent cells were obtained by incubating 30 x lo6 MNC in 10 ml of RPMI with
10% FCS at 37°C for 1 hour. Nonadherent cells were washed
off, and the remaining adherent cells were cultured at 37°C in
5 ml of complete medium. Adherent cells consisted of an
average of 95% monocytes, as assessed by Mo-2 monoclonal
antibody (Coulter Immunology, Hialeah, FL) and anaphthyl esterase staining (10). After 20 hours of culture, the
ACS were harvested and used immediately, or were kept
frozen at -70°C.
An adherent and nonadherent lymphocyte preparation was obtained from MNC from which monocytes had
been depleted (see above). Cells (20-70 x i06)were placed in
a 5-ml syringe that had been packed with sterile nylon-wool,
and preincubated for 1 hour with RPMI-5% FCS (1 1). After
incubating cells in the column for 1 hour at 37"C, nonadherent cells were eluted and passed over a second nylon-wool
column, yielding a final nonadherent cell population (group
B) that contained an average of 80% Leu-1 + cells (a CD-5 T
cell-associated antigen [ 12]), < 1% nonspecific esterasepositive cells, and an average of 1.4% sIg+ cells. Adherent
cells (group A) were mechanically dislodged from the first
column; these were composed of an average of 70% sIg+
cells, 6% Leu-I+ cells, and 21% Mo-2+ cells.
IgG stimulation assay. ACS were tested for their
ability to stimulate IgG production as previously described
(8). Two million mononuclear cells were cultured in duplicate 2-ml wells for 20 hours in complete medium at 37°C.
Following this incubation, the cells were vigorously
resuspended and washed once in warm (37°C) RPMI. The
culture wells, which contained small numbers of adherent
cells, were rinsed twice with warm RPMI. After washing,
the MNC were recultured in their original wells with either
fresh complete medium or ACS. At the end of 7 days, the
level of supernatant IgG was determined. Fresh media
cultures have been shown to produce less IgG than do
control, unwashed cultures. IgG synthesis may be restored
in ACS-treated cultures; this indicates the presence of IgG
stimulating activity in ACS. This activity is maximal when
cultured cells are washed and recultured in ACS after the
20-hour preincubation period (8). ACS usually contained
small amounts of IgC. This amount was subtracted from the
final amount, determined at 7 days, to allow a direct comparison of ACS and fresh media-treated cultures.
IgG assay. Supernatant IgG levels were determined
by enzyme-linked immunosorbent assay. Culture supernatants were harvested and dialyzed against phosphate buffered saline (PBS), pH 7.0. Costar 3590 polystyrene microtiter wells (Costar, Cambridge, MA) were coated with
unconjugated anti-IgC in 0.lM carbonate buffer, pH 9.6, at
4°C overnight. After washing, plates were incubated in PBS
with 5% FCS for 1 hour at room temperature. Supernatant
samples diluted in PBS-5% FCS and 0.05% Tween 20 were
added to the washed microtiter wells and incubated for 90
minutes at room temperature. After washing again, alkaline
phosphatase-conjugated goat anti-human IgG (Sigma, St.
Louis, MO) was added to the wells, and incubation was
continued for 90 minutes at room temperature. Freshly made
p-nitrophenyl phosphate disodium salt substrate (Sigma) was
added for 60 minutes at room temperature. The reaction was
stopped with 1N NaOH and the optical density was determined at 405 nm with a Titertek Multiscan MC (Flow
Laboratories). All samples were assayed in triplicate.
Hemolytic plaque assay. Ig-secreting cells were determined using a Staphylococcus protein A reverse hemolytic
plaque assay (13). Briefly, sheep red blood cells (SRBC)
were treated with 2 m g h l of Staphylococcus protein A
(Pharmacia) for 60 minutes at 30°C, in saline that contained
0.133 mg/ml of CrCI3. Test MNC from duplicate wells were
pooled. and mixed with a 30% suspension of Staphylococcus
protein A-treated SRBC, a 1:200 dilution of rabbit antihuman Ig (Cappel, West Chester, PA), an optimum concentration (1 :40) of guinea pig complement (Gibco), and 0.8%
agarose (Seaplaque; FMC Corporation, Rockland, ME).
B CELL DIFFERENTIATION IN SLE
This mixture was poured into 60 X 15-mm plastic petri
dishes that had been precoated with 1% agarose. After
incubation for 2 hours at 37"C, plaques were counted using a
dissecting microscope. The arithmetic mean number of
plaque-forming cells from triplicate plates was determined.
'H-thymidine incorporation. Two hundred thousand
mononuclear cells per 0.2 ml were cultured in complete
medium in 96-well culture plates (Costar 3596). After incubation intervals ranging from 2 days to 7 days, each well
received 1.0 pCi of 3H-thymidine (specific activity 6.7 Ci/
mmole; New England Nuclear, Boston, MA), and incubation continued overnight. Cells were harvested onto glassfiber filters using a semiautomated cell harvester, and counts
per minute were determined in a liquid scintillation counter
(Packard Instrument Company, Downers Grove, IL).
Statistical analysis. Data concerning ISC are expressed as the antilogs of geometric means, times or divided
by the standard error, since the numbers of ISC in different
experiments were not normally distributed (14).
A 2-sample test of the t statistic, which did not
assume equal variances in the 2 populations, was used to
determine the significance of differences between means.
Data were considered to be significant at P < 0.05.
RESULTS
The effect of ACS on IgG synthesis was studied
in 9 SLE patients and 9 normal individuals. Autologous ACS o r fresh media control supernatants were
used to reculture MNC that had been washed after 20
hours of culture. After 7 days, levels of IgG in the
supernatants were determined. In patients with SLE,
levels of IgG in fresh media-treated cultures averaged
1,182 ? 717 ng/ml ( k S E ) , while levels in the ACStreated cultures averaged 2,144 1,516 ng/ml ( P <
0.01). Mean IgG levels in cultures of normal MNC
were 559 + 226 ng/ml in fresh media cultures and 738
-t 273 ng/ml in ACS-treated cultures. This difference
was not statistically significant.
The mechanism whereby ACS increase IgG
levels in MNC cultures was investigated by determining the effects of ACS on B cell differentiation. The
number of Ig-secreting cells in MNC cultures was
determined, by a reverse hemolytic plaque assay, after
7 days of culture in the presence of either undiluted
ACS or fresh medium. The mean number of ISC in
patients with S L E was 126 X 1.6 (the geometric mean
times or divided by the SE) ISC/106 in fresh media
cultures and 253 X 1.3 ISC/106 in ACS-treated cultures
( P < 0.01). In contrast, the mean number of ISC in
cultures of normal MNC showed no response to
autologous ACS; results were 120 ? 1.3 ISC/106 in
fresh media cultures and 106 5 1.4 ISC/106 in ACStreated cultures. This difference was not statistically
significant. In 6 of the cultures of normal MNC,
*
763
autologous ACS actually decreased the number of ISC
to levels that were 72-85% lower than those found in
fresh media controls. Decreases in the number of ISC
in response to autologous ACS were not observed in
any SLE culture tested.
Experiments were next performed to test the
possibility that factors in SLE ACS account for the
increased ISC stimulation seen in S L E cultures. Thus,
SLE or normal ACS were added to 8 normal MNC
cultures that had been washed after overnight incubation, and the effects on the number of ISC were
compared. The mean number of Ig-secreting cells
induced by SLE ACS (67 ? 1.2 ISC/106) was not
significantly different from the mean induced by normal ACS (83 ? 1.4 ISC/I06). Neither S L E ACS nor
normal ACS stimulated ISC to levels that were significantly higher than those in fresh media (65 ? 1.3
ISC/I06).
Since in many experimental systems, proliferation of normal B cells precedes differentiation to Ig
secretion (15),the effect of ACS on cell proliferation
was investigated. Thymidine incorporation in response to ACS was determined in experiments using
MNC from normal subjects and from SLE patients.
The MNC were cultured in fresh medium or ACS from
unrelated S L E patients. Thymidine uptake was measured on the fifth day of culture, since earlier studies
showed that maximum ISC stimulation occurred by
day 5. Increases in the number of ISC served as
positive controls for the effects of ACS. The results of
these experiments are shown in Table 1.
ACS-induced increases in ISC were seen in all 4
SLE cultures tested. In 2 cultures, no significant
increase in proliferation was seen. In the other 2
cultures, 3H-thymidine uptake increased 2.l-fold
and 2.8-fold in response to ACS. In contrast, only 1 of
4 normal cultures showed increased ISC in response to
ACS, and in no instance was ACS-induced proliferation observed. Furthermore, different dilutions of
normal and S L E ACS (range 1 :2 to 1 :64),and different concentrations of cells (range I x 104to 1 x lo6
per 0.2 ml), failed to reveal consistent ACS-induced
proliferation.
Since 3H-thymidine uptake apparently increased in some of the cultures, it was of interest to
determine whether proliferation could be detected
prior to day 5. Time-course experiments were therefore performed, assaying daily for 3H-thymidine uptake and ISC values. Autologous ACS had no effect on
3H-thy~nidineuptake on days 2,3,o r 4,compared with
764
JANDL ET AL
Table 1.
The effect of SLE ACS on the proliferation and differentiation of normal and SLE
MNC*
'H-thymidine uptake
(cpm)
Cell source
S L E patients
Exp. 1
Exp. 2
Exp. 3
Exp. 4
Norma! subjects
Exp. I
Exp. 2
Exp. 3
Exp. 4
Fresh media
241 2
816 ?
3,280 2
1,227 ?
15
570
634
503
6,087 2 2.265
2,101 1,138
5,645 5 1,571
2,532 2 242
*
Ig-secreting cells
(/lo5 starting MNC)
ACS
Fresh media
ACS
288 2
707 2
6,894 2
3,441 2
63
169
1,538
131
460 ?
323 2
164 2
38 2
30
29
33
9
797 -C
706 ?
211 ?
166 2
62
50
12
23
4,814 2
2,861
5,264 2
2,756 2
866
98 5 10
57 2 10
207 2 19
81 2 6
114 ?
68 ?
195 2
231 5
18
7
21
37
* 1,147
1,947
239
* Values are mean t SD of quadruplicate culture wells (3H-thymidine uptake) or of triplicate culture
wells (Ig-secreting cells). SLE = systemic lupus erythematosus; ACS = adherent cell supernatants;
MNC = mononuclear cells.
fresh medium, but it did progressively increase the
number of ISC in cultures of SLE MNC. These results
confirm that in most instances, ACS-induced increases
in 3H-thymidine incorporation do not accompany or
precede increases in ISC numbers.
Since it has been reported that T cells regulate
autoantibody synthesis by SLE B cells (16), we investigated the effect of T cells on ACS-induced IgG
synthesis. Our previous studies showed that ACS had
no effect on IgG production by enriched B cell populations. It was also observed, however, that when T
cells were added to the B cells at a ratio of 3 : 1, the IgG
stimulatory response to ACS was reconstituted (8).
These results indicate that in order to stimulate IgG
synthesis, ACS require T cells. Experiments were
therefore performed to determine whether the effects
of ACS on IgG synthesis could be enhanced by using
larger numbers of T cells. To perform these experiments, SLE and normal T cells and B cells were
separated according to their adherence to nylon-wool.
Nylon-wool-adherent cells (group A) are predominantly B cells, while nylon-wool-nonadherent cells
(group B) are predominantly T cells (see Patients and
Methods). Group A (adherent) and group B (nonadherent) cells were washed and recultured in autologous
ACS after overnight incubation. The effect on IgG
synthesis is shown in Table 2 .
As expected, MNC from 1 normal subject
showed no response to ACS. However, MNC from a
second normal subject and from both SLE patients
Table 2. ACS-induced IgG synthesis by fractionated MNC*
Unseparated
MNC
Normal subject 1
Fresh media
ACS
Normal subject 2
Fresh media
ACS
S L E patient 1
Fresh media
ACS
S L E patient 2
Fresh media
ACS
Group A
Group B
Recombined
MNC
15
16
8
5
4
2
14
16
1,483
2,248
128
108
110
1,595
405
1,063
573
720
54
20
330
970
680
790
2,180
3,890
520
220
150
1,680
1,420
2,930
* Values are average ng/ml of IgG in duplicate culture wells. Group A = nylon-wool-adherent cells
(average of 70% B cells and 6% T cells); group B = nylon-wool-nonadherent cells (average of 1.4%
B cells and 80% T cells). ACS = adherent cell supernatants; MNC = mononuclear cells; S L E =
systemic lupus erythematosus. See Patients and Methods for details.
B CELL DIFFERENTIATION IN SLE
tested produced 1.3-1.8 times more IgG in ACStreated cultures than in fresh media cultures. IgG
stimulation increased in group B lymphocyte cultures
to levels that were 3-, 11-, and 15-fold higher in
ACS-treated cultures than in fresh media cultures.
This degree of stimulation has not been observed in
previous cultures using unfractionated normal or SLE
MNC. In all experiments involving group A lymphocyte cultures, IgG synthesis was lower in ACS-treated
cultures than in fresh media, indicating that ACS also
has suppressive effects on a subpopulation of MNC.
These observations support the hypothesis that
T cells play an important role in the stimulation of IgG
synthesis by ACS. Further studies are required to
determine how specific monocyte factors interact individually with T cells and B cells, and to determine
the relative importance of T cells and ACS on IgG and
autoantibody synthesis in SLE patients versus normal
subjects.
DISCUSSION
The complex immunoregulatory mechanisms
governing maturation of normal resting B cells into
committed Ig-secreting cells have been the subject of
intense interest. Most evidence indicates that resting,
mature B cells become capable of Ig secretion after a
series of maturation steps (17). The first of these steps
involves activation and entry into cell cycle. After
repeated cell divisions and clonal expansion, the cells
then evolve into mature, terminally differentiated
plasma cells. In addition, factors that directly stimulate resting B cells to differentiate have been described
(15). Numerous cells and lymphokines regulate B cell
maturation, many of which function abnormally in
SLE patients. To date, however, the events that
culminate in B cell hyperactivity in SLE remain poorly
understood.
Early reports indicated that the proportion of
circulating lymphocytes identified as B cells, by EACrosettes or surface Ig, is low or normal in patients with
SLE (18,19). However, the proportion and absolute
number of cells secreting lg are markedly elevated in
SLE patients; most studies have shown IgG-secreting
and IgA-secreting cells predominating (2,14). Moreover, increased numbers of circulating plasma cells
have been reported (4). These data are consistent with
the hypothesis that an abnormally large proportion of
circulating B cells have matured to the point of Ig
secretion in SLE.
Using a simple assay system that measures B
765
cell responses to factors produced by adherent
monocytes, we have now shown that SLE monocytes
contribute to B cell hyperactivity by stimulating B cell
differentiation. These studies evolved from earlier
work which demonstrated that if cultured MNC are
deprived of supernatant factors produced early in
culture (by removing the supernatant and washing the
cells), subsequent polyclonal Ig and anti-DNA synthesis will be reduced by about 50% (8). Reculturing the
washed MNC in autologous ACS restores polyclonal
Ig synthesis. The effect can be seen as early as 2-4
hours and as late as 3-4 days into culture. Indeed,
removal of the supernatant factors after 3-4 days of
culture will cause Ig synthesis to virtually cease; this
indicates that the presence of Ig-stimulating factors
over prolonged periods of time is required in this
system.
Earlier studies have also disclosed that Igstimulating activity in ACS occurs in molecules with a
relative molecular weight of approximately 14,000 (by
size-fractionation high performance liquid chromatography) and can be neutralized by an antiserum against
IL-1 (8). Thus, IL-1, and possibly additional factors in
ACS, appears to mediate stimulation of IgG production. The present studies were aimed at determining
how ACS affects B cell maturation in SLE patients
compared with normal subjects. The first experiment
compared the effects of autologous ACS on 1gG synthesis by SLE and normal MNC cultures. Significant
increases in levels of IgG in response to ACS were
observed only in the SLE cultures. A few normal
MNC cultures also showed increased IgG levels in
response to ACS. These findings are consistent with
data from our previous studies, and indicate that both
SLE and normal MNC can increase IgG secretion in
response to autologous ACS, but that the response is
greater in the SLE population.
We next addressed the question of whether
ACS enhanced B cell differentiation. We enumerated
lg-secreting cells in MNC cultures exposed to ACS. A
reverse hemolytic plaque assay was used to identify
ISC, and the results for SLE and normal MNC were
compared. After ACS treatment, the mean number of
ISC approximately doubled in the SLE cultures,
whereas no response to ACS was observed among
MNC from normal individuals. From these data, we
conclude that SLE B cell differentiation is stimulated
by ACS. This stimulation is modest, but highly consistent, in the SLE population and indicates that
approximately half of the spontaneous Ig-secreting
766
cells seen in vitro depend on factors derived from
adherent cells.
Experiments were performed to determine
whether factors present in SLE supernatants could
account for different ISC responses in SLE cultures
compared with normal cultures. SLE ACS were cultured on normal MNC, and the effect on the number of
IS(: was determined. The results showed that neither
SLE nor normal ACS significantly stimulated the
number of ISC in normal MNC cultures. This finding
suggests that ACS alone cannot account for the different autologous ISC responses observed between
the 2 groups. Thus, SLE MNC appear to be more
responsive to ACS than are normal MNC. It could be
argued that normal MNC contain cells that dampen the
response to abnormal factors in S L E ACS and prevent
the ISC response that is seen in SLE. In addition, it is
conceivable that differences in major histocompatibilit y complex antigens between SLE patients and
normal subjects account for differences in the response to ACS. Additional experiments must be performed to investigate these possibilities.
The possibility that ACS-induced B cell differentiation could be accompanied by cellular proliferation was also addressed. 3H-thymidine uptake values
in cultures of normal and S L E MNC were determined
after the addition of SLE ACS or control fresh media.
The results were compared with the effects of SLE
ACS on the number of Ig-secreting cells. In 3 of 6
experiments, it was clear that increased numbers of
ISC occurred in the absence of any increase in 3Hthymidine uptake; this finding indicates that MNC
proliferation is not required for an increase in ISC.
Increases in 3H-thymidine uptake were observed in 2
of 4 S L E cultures tested, however. Thus, in some SLE
cultures, there is a tendency to proliferate in response
to ACS. The significance of this proliferation is not
clear. These data are therefore consistent with the
proposal that ACS primarily stimulates B cell differentiation in SLE. The possibility that proliferation of a
small population of B cells was not detected, or that
there is a subgroup of S L E patients in whom some
proliferation reproducibly occurs in response to ACS,
will require additional studies.
Soluble factors that enhance B cell differentiation are important to our understanding of SLE.
Indeed, low or normal levels of IL-1 (20,21), IL-2
(21,22), interferon (23), and B cell growth and differentiation factors (24) in vitro have all been reported,
suggesting that these specific lymphokines are not
directly involved in B cell hyperactivity. Impaired B
JANDL ET AL
cell responses to mitogen stimulation (25) and preparations containing B cell differentiation factor (26)
have also been described, which implies that the
responsiveness of S L E B cells to these stimuli is
impaired. The ACS-induced differentiation of B cells
described in the present study is therefore a novel
finding which is directly relevant to understanding B
cell hyperactivity.
The ability of T cells to modulate S L E B cell
activity (16) has been reaffirmed in this study, since
very high ratios of T cells to B cells correlated with
augmented ACS stimulation of IgG production. Consistent with our earlier observations, the 2 S L E cultures produced moderate to high concentrations of
IgG, compared with 1 of 2 normal cell cultures (8). In
3 of 4 cultures, B cells contained in nylon-woolnonadherent cells (group B) were exceptionally responsive to ACS, and produced 3, 11, and 15 times
more IgG in ACS-treated cultures than in fresh mediatreated cultures. This degree of stimulation will facilitate further analyses of B cell stimulatory factors in
ACS by providing a more sensitive assay.
In addition, these experiments show that the
mechanism of action of ACS may be complex. Reduced ACS-induced IgG synthesis in B cell-enriched
(group A) cultures could mean that inadequate numbers of T cells were present, that the high numbers of
contaminating monocytes that were present for the
duration of culture suppressed the response (27), or
that the B cells or T cells obtained by this technique
are refractory to the effects of ACS. On the other
hand, the heightened response to ACS seen in T
cell-enriched (group B) cultures may mean that T cells
facilitate ACS stimulation in a concentration- or dosedependent manner, that enough monocytes have been
removed to eliminate suppressive effects, o r that the B
or T cells contained in this fraction are exceptionally
responsive to ACS. Differences in MNC subsets (e.g.,
numbers of preactivated B cells or suppressor T cells)
might explain why SLE and some normal MNC cultures respond to ACS while most normal MNC cultures do not. While further studies will be required to
resolve these questions, the data support the concept
that B cell hyperactivity results from interactions with
T cells as well as rnonocytes, and that ACS may
interact with particular T cell o r B cell subpopulations.
The ability of monocytes to act as a first line of
defense against environmental agents (28) and as producers of an extraordinary array of inflammatory
mediators (29) suggests that they may play an important role in SLE. Preliminary studies indicate that
B CELL DIFFERENTIATION IN SLE
factors in addition to IL-1 are required to mediate ACS
stimulation, since affinity-purified IL- 1 or recombinant
IL-Ip d o not entirely reproduce the effect of ACS on
IgG synthesis (Jandl R, George J , Dinarello C, Schur
P: unpublished observations), and that IL-1 may be
necessary, but not sufficient, for ACS to exert its
effects. This is consistent with the results of recent
studies on normal B cells, which indicate that IL-1 acts
to enhance B cell maturation induced by other factors
(30,31), and that, by itself, IL-1 has little effect on B
cell proliferation or differentiation (3 1,32).
Since it is clear that IL-I is required for the
effects of ACS on B cells, it is possible that activation
of monocytes by stimuli (e.g., immune complexes)
could lead to IL-I production, enhanced B cell differentiation, and increased Ig secretion in SLE. However, this theory must be reconciled with findings of
low levels of mitogen-stimulated IL-1 in SLE (20,21).
One possible explanation consistent with our data is
that the response of SLE B cells to ACS may require
the presence of additional monocyte- or lymphocytederived factors and that only minimum levels of IL-1
are required to facilitate the response. Alternatively,
SLE B cells may hyperrespond to lymphokines, such
as IL-1, in terms of differentiation and Ig secretion.
Moreover, it is clear that stimuli in addition to that of
ACS are involved in disease expression since ACSinduced IgG-stimulating activity can also be found
occasionally in normal individuals. It is likely that
additional genetic abnormalities, B cell abnormalities,
or abnormalities in other MNC subsets are necessary
for the expression of SLE.
These studies raise many questions concerning
the roles of T cells, monocytes, and monocyte factors
in the pathogenesis of lupus. A greater understanding
of why monocytes stimulate B cell differentiation in
SLE will, we hope, provide insights into mechanisms
of normal and abnormal Ig secretion.
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