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Increased spontaneous activity of antibody-forming cells in the peripheral blood of patients with active sle.

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829
INCREASED SPONTANEOUS ACTIVITY
OF ANTIBODY-FORMING CELLS IN
THE PERIPHERAL BLOOD OF PATIENTS
WITH ACTIVE SLE
DANIEL R. BUDMAN, E. BRUCE MERCHANT, ALFRED D. STEINBERG, BERNARD DOFT.
M. ERIC GERSHWIN, ELAINE LIZZIO. and J. PATTON REEVES
Thirty-seven patients with criteria for systemic
lupus erythematosus (SLE) and 18 normal controls were
studied for their spontaneous background IgM antibody
plaque-forming cell number to specific chemical haptens.
Active SLE patients had significantly more plaque-forming cells in their peripheral blood to a total of five chemical
determinants than did patients with inactive disease or
controls. This increased number of plaque forming-cells
correlated with depressed serum C3 levels by Spearman
rank-order analysis. The finding of elevated numbers of
spontaneous IgM plaque-forming cells to defined chemical
haptens supports the concept that active SLE demonstrates a generalized increase in 9-cell activity toward a
variety of antigens.
Systemic lupus erythematosus (SLE) remains a
disease of unknown etiology characterized by major
From the Arthritis and Rheumatism Branch, National Institute o f Arthritis, Metabolism and Digestive Diseases, National Institutes of Health. and the Immunohematology Branch, Division of
Blood and Blood Products, Bureau of Biologics. Bethesda, Maryland.
Daniel R. Budman, M.D.: Clinical Associate, ARB, NIAMDD:
E. Bruce Merchant, M.D.: Chief, IHB, DBBP. BB; Alfred D. Steinberg,
M.D.: Senior Investigator, ARB, NIAMDD; Bernard Doft, M.D.:
Research Associate, IHB. DBBP. PP: M. Eric Gershwin, M.D.: Clinical Associate, ARB. NIAMDD: Elaine Lizzio, B.S.: Biologist, IHB.
DBBP. BB: J . Patton Reeves. M.S.: Chemist, ARB. NIAMDD.
Address reprint requests to Alfred D. Steinberg, M.D., Building 10. Room 8D-19, National Institutes of Health, Bethesda. Maryland 20014.
Submitted for publication July 9, 1976: accepted August 12,
1976.
Arthritis and Rheumatism, Vol. 20,
No. 3 (April 1977)
alterations of the immune system (1-4). Several investigators have suggested that chronic viral infection
may be the underlying cause of the immune aberrations
(5-7), perhaps operating through a loss of regulatory
(suppressor) cells which normally control antibody responses (8). Indeed, in the New Zealand mouse model of
human SLE, both chronic viral infection and the absence of suppressor cells have been demonstrated
(9-1 I ). In addition, autoimmune phenomena such as
Coombs’ positive hemolytic anemia can be alleviated by
replacement therapy with suppressor thymus cells ( 12).
If these hypotheses are correct, it could be predicted that, i n the absence of normal immune regulation, there would be generalized B-cell hyperactivity in
patients with active SLE. This increased B-cell activity
should be reflected in excessive antibody production to a
variety of antigens, rather than solely toward autoantigens. Patients with SLE are known to have peripheral
blood cells that resemble activated lymphocytes (13). as
well as circulating antibody-producing plaque-forming
cells (PFCs) with specificity for DNA (l4,15). The present study was designed to determine whether or not this
PFC activity was limited to autoantigens.
MATERIALS AND METHODS
Patient Selection
Patients who satisfied American Rheumatism Association criteria for SLE and who were followed in the outpatient
or inpatient service of the Arthritis Branch of the National
BUDMAN ET A L
830
Institutes of Health were studied for their spontaneous PFC
activity as two groups: Group 1 consisted of a carefully selected subpopulation of SLE patients on low-dose corticosteroid therapy whose renal function was at least 60% of normal
(Table I ) , and Group 2 was composed of patients selected a t
random without regard to clinical status, laboratory parameters, or modalities of treatment. In both groups only females
were studied. All patients were graded as having active or
inactive SLE disease by their clinic physician. Patients with
questionable degrees of activity were not included in the study.
In Group 1 the estimate of disease activity had t o be confirmed
by two additional physicians.
Active SLE disease was defined as the presence of
clinically identifiable active disease (e.g. arthritis, serositis,
nephritis) in at least one organ system, not including the skin.
Anemia (<12 g of hemoglobulin/deciliter), leukopenia
( <4000/mm2), depressed complement (C3 <84mg/deciliter),
and/or elevated serum DNA binding were not sufficient criteria by themselves to classify a given patient as'active. Three
patients in Group 1 were excluded because of rapidly changing
clinical status within the week of observation. N o patient had
an infection at the time of the study. Alternate day corticosteroid dosage was treated as one-half that dosage on a daily
basis. Normal control subjects were selected from personnel at
the National Institutes of Health.
Plaque Assay for Determining the Numbers of
IgM Antibody-Producing Cells
All study blood was collected in heparin (30 U/ml
blood) without preservative (Fellows Medical Manufacturing
Co, Anaheim, CA). The peripheral blood lymphocytes were
isolated by isopycnic centrifugation through a Ficoll-Hypaque
gradient (16). The isolated mononuclear cells were then suspended in 16 X 125 mm plastic tubes (Falcon Plastics, Oxnard,
CA) and washed twice in Hank's balanced salt solution without calcium or magnesium (Grand Island Biological Co,
Grand Island, NY) to remove adherent mononuclear cells.
A modification of the Jerne plaque assay was used to
quantitate the number of spontaneous Ig M-producing peripheral blood white cells (17). Hapten conjugated SRBC (5%)
and 2 X lo8 human mononuclear cells were combined in 0.9%
agarose and plated in petrie dishes. After 60 minutes at 37°C.
guinea pig complement was added. and the dishes were incubated for an additional 60 minutes at 37°C. Then the plates
were washed with phosphate-buffered saline and the plaques
counted. In one experiment these direct plaques were inhibited
by antihuman IgM serum, a finding confirming that the
plaques were produced by antibodies of the IgM class.
To detect these spontaneous background plaque-forming cells in the circulation, five different chemical haptens were
selected as ligands. The haptens studied were dansyl, nitroiodophenyl (NIP), trinitrophenyl (TNP), 2,4 dinitrophenyl
(DNP), and sulfonate. The chemical haptens were each linked
to a tripeptide spacer, then coupled to sheep red blood cells.
The active azide coupling procedure affords comparable sensitivity levels for each hapten-specific PFC assay system (18).
All results were expressed as geometric mean values
with the standard error of the mean. None of the subjects
tested had known prior immunization with these haptens.
Serologic Tests
The presence and magnitude of antibodies to both
single-stranded DNA and double-stranded DNA were determined by a previously described Farr technique (19). Quantitative serum immunoglobulins (Behring Diagnostics, Somerville. NJ) and C3 (Hyland Laboratories, Costa Mesa, CA)
were measured by radial immunodiffusion.
Statistical Analysis
The student's t test was used to compare active SLE,
inactive SLE, and control groups. All PFC data were compared only after log,, transformation, because PFC are lognormally distributed. Geometric mean values of PFC for the
activity against each individual hapten were reconverted to
arithmetic values for presentation in the tables. Correlations
between PFC and clinical data were performed by Spearman
rank-order analysis.
RESULTS
Group 1 consisted of 9 active SLE patients, 13
inactive SLE patients, and 10 control patients. Active
and inactive SLE patients were similar with regard to
age, creatinine clearance, immunoglobulin levels, total
leukocyte count, and absolute lymphocyte count (Table
I). As expected, patients with active disease had higher
levels of DNA binding (Table I).
The numbers of lymphocytes forming anitodies
to the haptens studied are in Table 2. Because PFC are
log-distributed, the geometric mean is presented in
both arithmetic and log,, notation, with the standard
error of the mean in log,, notation. Both the five individual chemical haptens and sheep red blood cells are
shown. Elevated numbers of PFC were found in the
Table I . C'liiii(ul (itid Lohorurori~PorutiicJiersit1 Group I SL E Purieiirs
Active
No. of patients (all female)
Mean age (years)
Corticosteroid dosage
(mg prednisone/day )
Creatinine Clearance
(ml/minute)
Serum IgA (normal 0.8-2.0 mg/ml)
Serum IgG (normal 7.7-1 I .3)
Serum IgM (normal 0.9-1.7)
Serum complement (C3)
(normal 84-170 mg%)
Percentage of single-stranded
DNA binding (control < 31.1)
Percentage of douhle-stranded
DNA binding (control < 14.2)
Total leukocyte count
Absolute lymphocyte count
* Mean f standard deviation.
t By student's t test. P < 0.05,
Inactive
9
33.7 f 10.3*
13.3 f 13.1
13
30.0 f 10.2
9.7 f 6.8t
88.9 f 27.8
16.6 f 27.4
3.10 f 1.38
16.0 f 6.4
2.43 f 1.49
70.6 f 26.0
2.01 f 1.62
14.4 f 5.5
1.57 f 0.58
113.3 f 39.4t
61.1 f 28.0
34.1 f II.lt
34.8 f 34.8
4.6 f 9.4t
6400 f 2520
1135f 553
6840 f 2508
I o o O f 575
ANTIBODY-FORMING CELLS IN SLE
83
Table 2. Number of PFCs in Peripheral Blood of SLE Patienrs and Normal Controls, with Specijiciry for Five Chemical Haptens and SRBC'
(Spontaneous Backpround Acriviri,)
Dansvl
TNP
NIP
DNP
Sulfonate
SRBC
~~
Active SLE
(9 patients)
Inactive SLE
(13 patients)
Controls
(10 subjects)
P value of active vs control
P value of active vs inactive
32
101
89
19
266*
(2.424 f 0.090)t (2.003 f 0.29 I ) (1.949 f 0.302) (1.274 f 0.405) (1.509 f 0.313)
10
40
28
5
17
(1.605 f 0.203) (1.452 k 0.187) (1.230 f 0.235) ( 1.006 f 0.244) (0.718 f 0.185)
9
7
1
20
28
( I ,291 f 0.290) (1.440 f 0.270) (0.871 f 0.218) (0.956 f 0.243) (0.838 f 0.136)
< 0.005
< 0.10
< 0.005
< 0.25
< 0.03
< 0.005
< 0.10
< 0.005
< 0.30
< 0.05
I24
(2.092 3~0.IXX)
53
(1.726 i 0.140)
35
(1.548 i 0.320)
< 0.01
< 0.05
* Geometric mean antihapten cell number per 10' lymphocytes.
t Geometric mean PFC in log,, notation f standard error o f the mean in parentheses.
peripheral blood of active SLE patients when compared
with inactive patients and controls (Table 2). This increased disease activity was statistically significant for
the haptens dansyl, TNP, and sulfonate, and also for
sheep red blood cells. Inactive patients did not have
significantly increased numbers of PFC when compared
to controls.
Table 3 shows the total number of circulating
PFC to the five chemical haptens for both Group 1 and
Group 2. Both groups of patients with active SLE had
increased numbers of antibody-producing lymphocytes
with specificity for defined chemical haptens. Group 2
patients had more of these cells in their peripheral blood
when the SLE was active than did Group 1 patients.
However, active SLE patients in both Groups 1 and 2
demonstrated significant elevations in PFC when compared to inactive SLE patients or normals.
The total antihapten PFC per lo7 lymphocytes
was compared by Spearman rank-order analysis to that
patient's peripheral leukocyte count, absolute lymphocyte count, single-stranded DNA binding, doublestranded DNA binding, and serum C3. Elevated numbers of PFC in the circulating blood were found to
correlate with depressed C3 ( r = 0.47, P < 0.025), but
not with other hematologic values. Rank-order values of
no statistical significance included total leukocyte count
( r = 0.024, P > 0.5), absolute lymphocyte count ( r =
-0.153, P > 0.5), single-stranded DNA binding ( r =
0.150, P > OS), and double-stranded DNA binding ( r =
0.348, P > 0.1).
DISCUSSION
The factors controlling the release of antibodyforming cells into the peripheral blood in humans remain unknown. However a breakdown of the normal
regulatory control of B-cell function might be expected
Table 3 . Total A ntihanten A cticitv in Perinheral Blood
~~
Group 2
Group I
Active SLE
No.
Studied
Geometric
Mean
No.
Studied
Geometric
Mean
9
838
(2.023 f 0. I lo)*
I74
(2.241 f 0.130)
146
(2.163 f 0.129)
8
6140
(3.788 f 0.284)
. 216
(2.334 f 0.386)
I84
(2.265 f 0.326)
< 0.003
< 0.005
Inactive SLE
13
Control
10
P valuet of active vs control
P valuet of active vs inactive
* Geometric mean log,,
t By student's t test.
< 0.001
< 0.003
f standard error of the mean in parentheses.
7
8
BUDMAN ET A L
to be accompanied by an increase in circulating cells, as
well as by increased antibody production to a variety of
antigens. This study demonstrates that patients with
active systemic lupus erythematosus have increased
numbers of circulating IgM antibody-producing cells to
defined chemical haptens and to sheep red blood cells.
This increased number of spontaneously occurring PFC
to chemical haptens occurs during active disease, but is
not observed during disease inactivity. Of all the measures of disease activity, depressed C3 correlated best
with increased PFCs.
A given patient may have a high PFC count to
one hapten and a low number of PFCs to another. This
variability in the number of PFCs to different haptens
was also seen in controls. The magnitude of PFC activity to a given hapten may be genetically controlled.
Therefore the sum of the PFC numbers to each of several chemical haptens was thought to be a representative
measure of spontaneous B-cell activity.
The higher number of total PFCs in Group 2,
compared to Group I , active patients may be a result of
the study design. Because Group 1 patients were selected
for low corticosteroid dosage and good renal function,
these criteria would bias against a patient with more
florid SLE disease. Suggestive support for this supposition appears in Table I . Active patients did not have
markedly depressed leukocyte counts or lymphocyte
counts, which are often associated with increased disease activity (20,21). I n contrast, Group 2 patients were
unselected with regard to therapy. Hence the number of
PFCs in the peripheral blood may be a crude indicator
of magnitude of disease activity. Furthermore, 2
patients studied over a period of I year have had high
antihapten PFC counts when active, and low counts
when inactive, a finding suggesting that disease activity
rather than prior immunization is the sole cause of the
elevated antihapten PFC in active SLE patients.
The finding of elevated PFC in the circulation
agrees with previous observations of increased immunoglobulin synthesis by peripheral blood white cells in
patients with active SLE (22). These observations suggest that there is a generalized B-cell activation in
patients with SLE. Our findings indicate that this loss of
normal B-cell function is accompanied by increased
numbers of antibody-producing cells in the peripheral
blood with specificity for chemical haptens and sheep
erythrocytes. Thus excessive B-cell activity in active SLE
patients is apparently not limited to autoantibody production. There seem to be at least three defects in B-cell
function during active SLE: a ) increased synthesis of
immunoglobulins, b ) increased numbers of immuno-
globulin-synthesizing cells in the blood, and c ) increased
production of autoantibodies. The interrelationships between these defects and their underlying mechanisms
remain to be fully clarified.
The increased numbers of PFC in the blood of
patients with active SLE may occur on the basis of
several mechanisms: a) increased release of PFC, b)
decreased egress of PFC, c) increased activation of lymphocytes in the circulation, and d ) increased survival of
PFCs or their precursors in the peripheral blood. Unfortunately, turnover studies of PFC cannot be performed
at this time.
A number of hypotheses have been advanced to
explain the increased B-cell activity in active SLE. An
adjuvant effect (23) may be caused by nucleic acids or
viruses. Alternatively, suppressor cells may be lost or
defective ( I I , 12) and thus allow B-cell proliferation and
activity. Or a lack of thymic hormone (24) may not
allow the proper expression of immunologic regulation
by altering membrane signals which operate through the
adenyl cyclase cyclic AMP system. The results of the
present study are consistent with a generalized adjuvant
effect or a temporary loss of suppressor function during
active SLE.
It is also possible that the elevated PFCs in active
SLE represent a common pathway resulting from dysfunction of one or several different mechanisms of immunologic control. Further study of this B-cell abnormality may lead to better understanding of its
pathogenesis and, perhaps, to more specific therapy.
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ANTIBODY-FORMING CELLS IN SLE
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