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Ia specific antilymphocyte antibodies in rheumatoid arthritis.

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486
Ia SPECIFIC ANTILYMPHOCYTE ANTIBODIES I N
RHEUMATOID ARTHRITIS
ROBERT P. SEARLES, KUNIO OKUDAIRA, SUSAN M. SAVAGE, and JAMES S. GOODWIN
Antilymphocyte antibodies (ALA) in rheumatoid
arthritis (RA) have increased reactivity with phytohemagglutinin (PHA) activated lymphoblasts which are
known to have increased expression of Ia antigen. The
present experiments suggest that part of this reactivity
represents an Ia specificity of ALA. Fifteen of 18 RA
sera tested were able to inhibit the binding of monoclonal anti-Ia antibodies as measured by a rosette method.
RA sera did not inhibit the binding of other monoclonal
antibodies: anti-OKT3, anti-OKT4, and anti-OKTS.
The ability of RA sera to inhibit the binding of anti-Ia
antibody was eliminated after absorption of the RA sera
with an Ia positive human cell line (B35M) but not by an
Ia negative line (MOLT4). Blocking of anti-Ia binding
was greater in the IgG fraction of the RA sera but also
occurred in the IgM fraction. Experiments including
ultracentrifugation, pepsin digestion of RA sera, and
preincubation of lymphoblasts with aggregated IgG
demonstrated that Fc binding by RA sera was not a
factor. Both monoclonal anti-Ia and anti-Ia heteroantiserum also had increased reactivity with lymphoFrom the Research Division, Lovelace Medical Foundation
and the Department of Medicine, University of New Mexico School
of Medicine, Albuquerque, New Mexico.
Supported in part by Clinical Investigator Award #AM00793-02 from the National Institutes of Health and in part by grants
from the Arthritis Foundation.
Robert P. Searles, MD: Chairman, Department of Clinical
Immunology, Lovelace Medical Foundation; Kunio Okudaira, MD:
Research Associate, University of New Mexico School of Medicine;
Susan M. Savage, BS: Research Technologist, Lovelace Medical
Foundation; James S. Goodwin, MD: Chief, Division of Gerontology, University of New Mexico School of Medicine.
Address reprint requests to Robert P. Searles, MD, Research Division, Lovelace Medical Foundation, 5400 Gibson Blvd.,
SE, Albuquerque, NM 87108.
Submitted for publication March 17, 1982; accepted in
revised form October 8, 1982.
Arthritis and Rheumatism, Vol. 26, No. 4 (April 1983)
blast target cells. Pepsin digested Fab fragments of the
anti-Ia heteroantiserum were able to block the activity
of cytotoxic RA serum. However, ALA cytotoxic to
lymphoblasts did not correlate with anti-Ia by rosette
method. Ia-specific ALA by rosette method was associated with donor variability but did not appear to be
HLA-DR restricted. Ia-specific ALA did correlate with
disease activity. These data suggest that anti-Ia activity
is present in RA sera and may play an immunoregulatory role in this disease.
Antilymphocyte antibodies (ALA) are a heterogeneous group of antibodies found in several diseases
thought to occur from abnormalities in immune function (1). One such disease is rheumatoid arthritis (RA)
characterized by an immune complex-mediated synovitis with synovial membrane Ig synthesis (2,3), local
complement consumption (4), and phagocytic cells (5).
Abnormalities of cell mediated immunity are present
in RA in vivo with synovial infiltrates of predominantly T lymphocytes (6), loss of skin test reactivity (7),
and in vitro with decreased mitogen responsiveness
(8). In addition, RA is characterized by the presence of
autoantibodies including rheumatoid factor, antinuclear antibodies, and ALA (9).
ALA have a broad spectrum of specificities
including cell membrane antigens such as lymphocyte
surface antigens (lo), HLA antigens (11-13), and lymphoid cell lines (14). ALA also cross-react with tissue
antigens including nuclear antigens ( 1 3 , human brain
tissue (16), and trophoblastic antigens (17). The exact
function of ALA has yet to be determined. However,
ALA have been implicated in the loss of suppressor
cells and natural killer cells and are considered immunoregulatory (18,19). Alternatively, ALA may repre-
Ia SPECIFIC ALA
sent an environmental marker, but exhaustive studies
t o implicate a virus have been unrevealing thus far
(20).
Recently we have demonstrated a new specificity of A L A involving increased reactivity against mitogen activated lymphoblasts (21). Possible explanations
for this phenomenon include an increased sensitivity
against blast cells surviving the mitogen culture or an
increased expression of antigens on activated lymphoblasts. In this regard, Ia antigen has an increased
expression on T lymphoblasts (22,23) and sera from
patients with systemic lupus erythematosus (SLE)
have anti-Ia antibodies (13). In this report we show
evidence that the increased A L A activity in R A sera
against lymphoblasts represents in part an Ia specificity. W e further demonstrate that Ia-specific A L A are
found predominantly in the IgG fraction of RA sera
and can be absorbed with an Ia positive tumor cell
line.
PATIENTS AND METHODS
Patients. Serum samples were selected from 20 patients with rheumatoid arthritis as determined by the American Rheumatism Association criteria (24). All patients had
either stage I or I1 disease (25) and had varying degrees of
clinical activity. Patients were divided into those having
active or inactive disease activity; overall assessment of
clinical activity was based on the judgment of the clinician
caring for the patient at the time of blood drawing. The
following criteria were used: morning stiffness, grip strength,
walking ability, joint count (pain, tenderness, swelling), and
erythrocyte sedimentation rate. Medications included nonsteroidal antiinflammatory drugs, aspirin, gold, penicillamine, hydroxychloroquine, and azathioprine. Serum samples from 11 normal donors and 6 patients with degenerative
joint disease were selected as controls.
Sera. All sera were heat inactivated at 56°C for 30
minutes before use. Some sera were pepsin digested at pH
4.0, 37°C for 16 hours using 2 mg pepsinhl of whole serum.
After pepsin digestion, the precipitate was ultracentrifuged
at 100,OOOg for 20 minutes and the supernatant was dialyzed
overnight against 0.005M phosphate buffer (pH 7.4). Pepsindigested sera showed no residual Fc fragments by immunodiffusion.
In addition, some sera were fractionated into IgM
and IgG using a Sephacryl S-300 column (2.6 X 80 cm).
Immunoglobulins were eluted with 0.05M Tris buffer (pH
7.4). Fractions were pooled, concentrated, and tested for
purity of IgM or IgG by Ouchterlony double immunodiffusion. The purified IgM and IgG were found to be free of IgG
and IgM, respectively. Concentration of IgM was 0.34 mg/ml
and IgG was 10.8 tng/ml as determined by radial immunodiffusion.
Preparation of lymphoblasts. Lymphocytes were separated from heparinized whole blood of normal human
donors by Ficoll-Hypaque gradient centrifugation (26). T
487
cells were usually used and were obtained from sheep
erythrocytes treated with AET and rosetted overnight at 4°C
(27). Rosetted cells were treated with NHdC1-Tris buffer to
lyse sheep cells. Final T cell preparations contained 295%
E-rosette positive cells and 5 1 % sIg positive cells.
The cells were then adjusted to 106/mlin RPMI-1640,
20% fetal calf serum, and 100 unitshl penicillin, 100 pg/ml
streptomycin, and 2 mM L-glutamine were added to 24-well
tissue culture plates. To elicit mitogenic response, 0.2 ml
phytohemagglutinin-P (PHA) at a 1 :200 dilution was then
added to each well and incubated at 37°C in 5% COz
atmosphere for 7 days. After incubation, cells were centrifuged at l50g for 6 minutes.
Preparations of purified lymphoblasts were separated by a discontinuous density gradient centrifugation using
polyvinylpyrolidone (PVP) coated colloidal silica (Percoll
Pharmacia) as previously described (21). Briefly, the heterogeneous population of cells was mixed in isotonic solution,
referred to as 100% Percoll solution. Sequential dilutions of
60%, so%, and 20% solutions made by adding O.15M phosphate buffered saline (PBS) to the 100% solution were
carefully layered over the 100% solution containing the cells.
The density of each layer was determined using Density
Marker Beads (Pharmacia). The 100% layer measured 1.121
buoyant density (gm/ml), 60%-10.75,
509’&1.062, and the
20% layer--1.033. Centrifugation was carried out at 450g for
20 minutes at 4°C.
The cells in the band with a density of 1.062 were
determined as large blasts, cells in the 1.075 density were
intermediate blasts, and the cells floating on 1.121 density
were small lymphocytes. Size and morphology were determined by using cytocentrifuge preparations and Wright’s
stain. Most dead cells floated on top of the 1.033 density
band. After 7 days in culture double-rosetted T cells were
usually 100% large blasts, with few intermediate forms
found. Viability of large lymphoblasts remained at 295%
and these cells were used in all experiments.
Rosette inhibition method. Lymphoblasts (5 x 10’)
were incubated with 50 ~1 of media, normal human serum
(NHS), or patient serum for 30 minutes at 4°C and followed
by 10 pl of monoclonal anti-Ia (clone L243, Becton Dickinson, Mountainview, CA) incubated for 45 minutes at 4°C.
This reagent reacts with the common core determinant of
human Ia anti en and precipitates 28,000 and 34,000 dalton
chains from I labeled NP-40 extracts of human B cell lines
(28). After incubation, lymphoblasts were rosetted by addition of 100 pI bovine erythrocytes sensitized with goat antimouse IgG (Tago, Inc, Burlington, CA) by use of the
chromic chloride method (29). The preparations were centrifuged at 200g for 10 minutes and then incubated at 4°C for 30
minutes. Three hundred cells were counted and positive
rosetting cells were determined to be 3 or more erythrocytes
attached to a lymphoblast. When the bovine erythrocytes
coated with goat anti-mouse IgG were incubated with T
lymphoblasts in the presence of RA serum and no monoclonal anti-Ia, the rosettes were always 51%.
An anti-Ia heteroantiserum (a gift from Dr. R.C.
Williams, Jr., University of New Mexico) used as a positive
control was found to have 67.5 2 10.6% inhibition of anti-Ia
binding to the lymphoblasts. This serum was prepared by
injecting rabbits with Ia antigen purified from B35M cells by
B
488
a method modified from that previously described by Welsh
et al (30). Alternative experiments were conducted using
mouse hybridoma antibodies of the OK series (Ortho, Raritan, NJ) in place of the anti-Ia hybridoma. These monoclonal
antibodies included OKT3, reacting with all T cells (31);
OKT4, reacting with helper T cells (31); OKT8, reacting with
suppressor T cells (32);and OKMI, reacting with myelomonocytic cells (33).
Anti-Ia antibody by ELISA. To determine whether
RA sera have a blocking effect on the binding of monoclonal
antibodies to the la positive tumor cell line (B35M), an
enzyme-linked immunosorbent assay (ELISA) was used as
previously described (34). Briefly, B35M cells (5 x lo4
cells/well)were added to flexible microtiter plates (Dynatech
Laboratories Inc, Alexandria, VA) coated with poly G
lysine. After washing with PBS, the unbound sites in the
wells were blocked with 50% fetal calf serum (FCS). Then 25
pl of serum samples were added to the wells in triplicate and
incubated 1 hour at room temperature, and 25 pl of monoclonal anti-Ia antibody (4 pg/ml in PBS + 10% FCS) was then
added to the wells (final concentration of monoclonal anti-Ia
antibody was 2 pg/ml) and incubated for 30 minutes at room
temperature.
After washing, 50 pI of peroxidase-conjugated, affinity-purified goat anti-mouse IgG (Tago, Inc) was added
(1 : 1,000 dilution in PBS with 10% FCS) and incubated at 4°C
for 30 minutes. After washing, 100 pI of the substrate, 0.02%
2.2-azino-di-3-athylbenzthiazolin-sulfonate)
in 2.3% citric
acid buffer (pH 4.0) with 0.17% hydrogen peroxide, was
added to the wells. After 30 minutes incubation at room
temperature, the reaction was stopped by adding citric acid
buffer, pH 2.8. The light absorbance of wells was measured
by an automated ELISA reader (Dynatech Laboratories,
Inc, Dynatech Corp). To construct a standard curve, different final concentrations of monoclonal anti-Ia antibody were
added to the wells in the presence of normal human serum
(NHS) or FCS.
ALA determination. ALA were determined by the 2step microcytotoxicity method of Terasaki (35). Target cells
were resting lymphocytes or mitogen activated lymphoblasts. Serum was incubated 1 hour at 4°C followed by
addition of rabbit complement (Pel Freeze, Rogers, AR),
then incubated for 2 hours at 22°C.Dilutions of RA sera were
made with WMI-1640 and 20% human AB positive serum.
NHS and diluent controls had 510% dead cells in each
experiment. Positive cytotoxicity was determined as 230%
dead cells. Blocking experiments were conducted using 2
rabbit heteroantisera directed at lymphocyte surface antigens-anti-&-microglobulin (a gift from Dr. S. Ferone,
Scripps, CA) and anti-Ia (see above).
Fluorescence-activated cell sorter (FACS) analysis.
The effect of RA serum on binding of analyzed monoclonal
antibodies was also studied by an immunofluorescencetechnique. T cells (lo6)were incubated for 30 minutes at 4°C with
50 pl mouse hybridoma antibody or mouse IgGz purified on
Sepharose protein A. After washing, the cells were incubated with 50 liters of FITC labeled goat anti-mouse Ig (Tago,
Inc) for 30 minutes at 4°C. The cells were washed with
10,OOO cells counted on the fluorescence-activated cell sorter. The percentage of positive cells was calculated after
SEARLES ET AL
correction for background staining with the control sample
(mouse IgG2).
RESULTS
The number of lymphoblasts that bound monoclonal anti-Ia antibody by the rosette technique was
31.0 ? 8.54 (mean t standard deviation) per 100 cells
counted compared with 10% when using resting cells.
When the lymphoblasts were preincubated with sera
from patients with RA, a blocking effect of 15-85%
was found; mean inhibition was 50.4 t 16.1% (Table
1). The mean inhibition of RA sera was significantly
different from the inhibition noted with NHS, 10.4 t
7.4% ( P < 0.001).
In addition, some donor variability was present
when the rosette inhibition method was used; that is, a
given RA serum had some variation in blocking effect
on the binding of monoclonal anti-Ia antibody to
lymphoblasts from different donors. In general, less
blocking effect was noted against donor A compared
with the other 4 donors. HLA-DR typing was done in
4 of the 5 donors and donor A was HLA-DR1,4.
However, this decreased reactivity with donor A was
not complete since RA12, RA14, RA17, and RA18 sera
had a high blocking effect on the binding of monoclonal anti-Ia. HLA-DR typing of the RA sera donors
was not done. Finally, sera from 6 patients with
nonimmunologic arthritis (degenerative joint disease)
were tested and found to have 12.8 f 6.7% inhibition,
which is not different from normal human sera.
To determine whether RA serum has a similar
blocking effect on other monoclonal antibodies, lymphoblasts were assessed for reactivity with anti-Ia,
OKT3, OKT4, OKT8, and OKMI, with and without
preincubation with 4 RA sera (Table 2). The data show
that the largest inhibition by the RA serum occurred
with anti-Ia antibodies (31.5 f 8.1) whereas lower
inhibition similar to NHS (10.4 7.4) was found with
other monoclonal antibodies ( P < 0.01).
The specificity of this blocking effect of RA
serum for anti-Ia antibodies was assessed in 3 RA sera
by absorption experiments with 2 tissue culture lines,
one Ia-positive (B35M) and one Ia-negative (MOLT4)
(Figure I). In this representative experiment, 22% of
cells were Ia positive before preincubation with RA
serum 14. After blocking with the RA serum, 10% of
cells were positive. However, if the RA serum was
absorbed with the Ia-positive cell line (B35M), the
inhibitory effect of the RA serum was almost entirely
removed. Absorption with an Ia-negative cell line
*
Ia SPECIFIC ALA
489
la positive lymphoblasts from 5 normal lymphocyte donors (A through E) whose T cells
were activated and then reacted with monoclonal anti-Ia antibody. Blocking effect was observed by
preincubation of lymphoblasts with sera of rheumatoid arthritis (RA) patients
Table 1.
% Ia positive cells from lymphocyte donors*
Sera
Media
NHS 8
RA 01
RA 02
RA 03
RAo4
RA 05
RA 06
RA 07
RA 08
RA 09
RA 10
RA I I
RA 12
RA 13
RA 14
RA 15
RA 16
RA 17
RA I8
A(DRI, 4)
B(ND)t
C(DR6, 6)
D(DR7, 8)
E(DR3. 7)
36
33
26
27
42
32
20
18
31
28
26
30
7
5
15
13
13
17
14
I1
13
9
I1
18
12
I1
12
13
5
30
29
20
8
4
3
4
8
16
33
22
5
26
21
16
70mean
inhibition$
10.4 t 7.4
28
61
41
53
52
52
58
63
58
46
40
62
85
68
34
15
52
40
50.4 t 16.1
Total RA
* Percent Ia positive cells when lymphoblasts were preincubated serially with either media alone, RA
sera, or normal human sera (NHS) followed by monoclonal anti-Ia antibody. Binding by anti-Ia
determined by rosetting with ox erythrocytes conjugated with goat anti-mouse IgG.
t ND = not done.
$ Percent mean inhibition calculated by sum of inhibitions for each RA serum divided by number of
donors tested x 100.
8 Average percent Ia positive cells for all NHS tested against that individual donor. Eleven NHS were
tested: in no case did the inhibition of monoclonal anti-Ia binding to T lymphoblasts by NHS exceed
23%.
(MOLT4) had no effect on this inhibitory capacity.
These data suggest that an Ia specificity of ALA
against lymphoblasts exists in RA serum.
To assess the possibility of interference by
Inhibition of monoclonal antibodies binding to T
lymphocytes by rheumatoid arthritis sera determined by rosette
inhibition method
Table 2.
Monoclonal
% inhibition*
la
OKT3
OKT4
OKT8
OKMl
31.5 t 8.1
3.5 t 7.0
4.8 t 9.5
6.3 5.8
0
*
* Four sera (RAo4, RA05, RA16, RA20) were tested. Lymphoblast
target cells were obtained from normal donors SS (DRI, 41, TH
(DR3, 7) and CS (DR3, 7). Percent inhibition calculated by number
of Ia positive cells with media alone minus Ia positive cells preincubated with rheumatoid arthritis serum divided by number Ia positive
cells with media alone times 100.
immune complexes, RA sera were subjected to ultracentrifugation at 100,OOOgfor 30 minutes. No effect of
ultracentrifugation was found on the blocking of antiIa antibodies when the rosette method was used. In
addition, when lymphoblasts were preincubated with
IgG aggregates, no blockage of anti-Ia binding was
observed. Finally, the blocking effect was determined
after pepsin digestion of RA sera. Two RA sera were
each tested against 2 different donors and no difference
was found between the digested and undigested sera
(50.0
14.7% inhibition by untreated sera versus
48.8 ? 17.5% inhibition by pepsin digested sera). This
lack of effect by pepsin digestion and aggregated IgG
suggests that binding to Fc receptors was not a factor.
Ia antigens are present on cells other than
lymphoblasts. To determine if RA sera had the ability
to inhibit binding of monoclonal anti-Ia to other Ia
positive cells, an Ia positive human cell line, B35M,
was used as target in an ELISA method. Five RA sera
*
490
Z
SEARLES ET AL
25
1
20
-
IA P O S I T I V E
15 -
CELLS
10 -
Table 4. Comparison of anti-Ia activity against resting lymphocytes and activated lymphoblasts as target cells
Resting
RA
'1
CELLS
PR'EINCUBATED
WITH M E D I A
+
ROLT4
CELLS PREINCUBATED
WITH RA SERUR
Figure 1. Blocking effect of rheumatoid arthritis (RA) serum 14 on
binding of monoclonal anti-Ia antibodies to lymphoblasts as determined by rosette inhibition method. Effect of absorption of RA
serum with Ia positive (B35M) and Ia negative (MOLT4) tissue
culture lines.
were examined and a 30.2 ? 18.4% inhibition was
found with the B35M targets compared with 8.3 ?
6.6% inhibition for NHS.
To confirm the existence of anti-Ia specific
activity in RA sera, similar experiments were conducted using the fluorescence activated cell sorter (Table
3); T lymphoblasts were incubated with either media,
NHS, or 4 RA sera followed by monoclonal anti-Ia and
FITC labeled goat anti-mouse Ig. The results demonstrate that anti-Ia inhibition by RA sera is similar in
both methods.
To further characterize this phenomenon,
monoclonal anti-Ia antibody was compared with
known heteroantisera directed at lymphocyte surface
antigens and tested against resting and activated lymphocyte target cells in a cytotoxicity procedure. Both
anti-Ia reagents demonstrated marked increases in
activity against the lymphoblasts while a n t i - b p heteroantiserum showed no significant change (Table 4).
The rabbit anti-Ia and bovine a n t i - b p were
then pepsin digested to prevent Fc binding necessary
Monoclonal anti-Ia
Heteroantiserum
anti-Ia
Heteroantiserum
anti-&rr.
1 :32
l:l.O24t
1:2.048
* Percent positive cells that bind to monoclonal anti-Ia antibody
using rosette technique.
t Titers of heteroantisera positivity determined as 230% dead cells.
Negative controls of diluent (RPMI + 20% AB positive serum) and
normal human serum were 510% dead cells.
for complement mediated lysis and these Fab fragments were then preincubated with lymphocyte targets. Two RA sera were tested against 3 different
normal donors. In a representative experiment,
marked inhibition of the cytotoxic ALA reactivity in
RA serum 19 occurred after preincubation with either
anti-Ia or anti-&p heteroantisera (Table 5 ) . The complete inhibition of cytotoxicity with either heteroantisera was unexpected. Our studies do not define the
precise nature of this inhibition. Two possible explanations are that the inhibition of cytotoxicity by anti-&p
antisera is due to its steric hindrance of anti-Ia antibody or that both Ia and &p-specific ALA in RA sera
are necessary for cytotoxicity to occur.
Next, the cytotoxicity of RA sera against lymphoblasts was correlated to anti-Ia activity using the
rosette inhibition method. The results indicate a slight
negative correlation between the 2 methods, which
was nonsignificant (n = 16, r = -0.30). As previously
shown, ALA cytotoxic to lymphoblasts is a coldTable 5. Effect of preincubation of target lymphocytes with
heteroantisera on rheumatoid arthritis antilymphocyte antibody (RA
ALA) activity. Anti-p2p or anti-Ia were first pepsin digested at 37°C
for 14 hours. RA serum 19 was tested for microcytotoxicity against
resting or activated lymphocyte targets or lymphocytes
preincubated with Fab fragments of anti-p2p or anti-Ia
FACS
Rosette method
RA 14
RA 15
RA 22
RA 18
50
80
60
47
42
* Percent inhibition
42
33
22
Activated
Sample
Serum
calculated by number of Ia positive cells with
media alone minus Ia positive cells preincubated with RA serum
divided by number Ia positive cells with media alone times 100.
31
Negt
Table 3. Comparison of anti-Ia blocking activity in 4 rheumatoid
arthritis (RA) sera using fluorescence activated cell sorter (FACS)
or the rosette method
% inhibition*
Lymphoblasts
9+
Resting*
Pokeweed
mitogen
Coticanavalin A
* Results are titers of ALA with positivity determined as 30% or
greater dead cells per 100 counted. Dilutions of sera made with
RPMI + 20% AB positive serum. Negative controls of diluent and
normal human serum were 10% or less.
t Preincubation with pepsin digested anti-p2p or pepsin digested
anti-Ia.
Ia SPECIFIC ALA
49 1
Table 6. Comparison of blocking effect of IgM and IgG fractions
of 2 rheumatoid arthritis (RA)sera on the binding of monoclonal
anti-Ia antibodies to lymphoblasts
% inhibition* after blocking with
Serum
RA 14
RA 20
Unfractionated
49
39
IgM
12
17
IgG
38
45
* Calculated as number of Ia positive cells with media alone minus
number of positive cells preincubated with RA serum (unfractionated or fractionated) divided by number of positive with media alone
times 100.
reactive IgM antibody (2 1). Therefore, to determine
which antibody class was involved in the la inhibition
using the rosette method, 2 RA sera were separated
into IgM and IgG fractions using a Sephacryl S-300
column. The results show the blocking effect was
found to a greater extent in the IgG fraction which is
generally considered to be warm reactive, noncytotoxic ALA (Table 6). Some Ia-specific ALA were also
present in the IgM fraction.
The effect of RA disease activity on the inhibition of anti-Ia binding was also assessed (Figure 2).
Results of NHS, inactive RA, and active RA patients
were compared, demonstrating that the most inhibition occurred with active RA sera. Sera from both
inactive and active RA were significantly different
from NHS (P < 0.001). In addition, sera from RA
patients with active disease were found to have significantly greater anti-la activity than sera from patients
with inactive disease (P < 0.05).
target cells. Pepsin digested Fab fragments of an antiIa heteroantiserum were able to block the activity of
cytotoxic RA serum. Ia specific ALA did correlate
with disease activity. Finally, Ia specific ALA did not
appear to be HLA-DR restricted, but some donor
variability was found by using the rosette inhibition
method.
Several investigators have demonstrated an increased expression of Ia antigen on T lymphoblasts
(22,23). This phenomenon may represent both synthesis of Ia antigen and uptake of Ia antigen from other
shedding cells (36). The importance of the la positive T
cells is unclear, but their presence appears to represent a state of activation. Increased numbers of Ia
positive T cells have been reported in peripheral
blood, synovial fluid, and synovial tissue of RA patients (37,38). The existence of anti-la antibodies in
RA could represent a response to these cells and may
50
C
1z
C
-
+J
C
L
01
DISCUSSION
Previously we have reported that cytotoxic
ALA in RA sera have increased reactivity against
lymphoblasts (21). The present experiments suggest
that part of this reactivity represents an la specificity
of ALA. Fifteen of 18 RA sera tested were able to
inhibit the binding of monoclonal anti-Ia antibodies as
measured by a sensitive rosette method. There was no
inhibition of the binding of other monoclonal antibodies, and the inhibition was not due to Fc binding or
immune complexes. The ability of RA sera to inhibit
the binding of anti-Ia antibody was eliminated after
absorption of the RA sera with an Ia positive human
cell line (B35M) and not by Ia negative line (MOLT4).
Blocking of anti-Ia binding was greater in the IgG
fraction of the RA sera but also occurred in the IgM
fraction. Both monoclonal anti-Ia and anti-la heteroantiserum had increased reactivity with lymphoblast
KIl
.
80-
a"
a
a
4ol
a8
&
4
I
a
0
I-
20
I:
0
NHS
...
a
a
0
a
Inactive
RA
ACtivQ
RA
Figure 2. Anti-Ia blocking effect of sera from rheumatoid arthritis
(RA) patients in various stages of disease activity was compared
with normal human serum. Sera from patients having both active
and inactive RA were significantly different from normal human
serum (P < 0.001). Sera from patients with inactive RA had
significantly less anti-Ia blocking than those from patients with
active RA (P < 0.05). Percent inhibitions were calculated as
discussed in Patients and Methods section. Vertical bars indicate t I
SD from the mean.
492
SEARLES ET AL
have important pathogenetic significance. Recently we
described anti-Ia antibodies in systemic lupus erythematosus (SLE) which are responsible for a decreased
autologous mixed lymphocyte reaction (13,39). This
implicates an immunoregulatory function in SLE
which might be similar in RA. Sera from patients with
RA have previously been shown to inhibit the allogeneic mixed lymphocyte reaction (MLR) (40). Ia positive stimulator cells appear to be involved in the
induction of this reaction (41). However, it is still
unclear whether anti-Ia antibodies affect the stimulator
or responder populations of the MLR (42). Therefore,
we are presently studying Ia-specific ALA in the MLR
to determine if they account for these abnormalities in
RA.
There was no significant correlation between
anti-Ia activity by rosette method and ALA cytotoxic
to lymphoblasts. Cytotoxic ALA are uncommon
whereas anti-Ia is common, occurring in both cytotoxic ALA positive and negative sera. These differences
could be secondary to the sensitivities of the tests.
Cytotoxic ALA were predominantly in the IgM fraction (21) whereas anti-Ia by rosette inhibition was
predominantly in the IgG fraction, suggesting 2 different antibodies. Nevertheless, similarities were also
found between the 2 antibodies. Donor variability
previously reported with cytotoxic ALA (21) was
present with anti-Ia by rosette inhibition but was less
marked. In addition, cytotoxic ALA were inhibited by
pepsin-digested anti-Ia and anti-&p heteroantisera.
These data suggest that either a portion of cytotoxic
ALA specificity includes Ia antigens or that the inhibition of cytotoxicity by anti-Ia antibody may be by
steric hindrance. However, the fact that anti-p2p can
also block cytotoxic ALA calls into question the
specificity of this cytotoxic response. In the final
analysis, it is probable that both Ia and blast cell
antigens other than Ia are recognized by RA sera with
the cytotoxic methods. This is consistent with the
recent report that activated cells express antigens
other than HLA (43).
Ia-specific ALA did appear to fluctuate with
disease activity in RA patients. This phenomenon is
similar to SLE where ALA and anti-Ia both show
increases with disease activity (1,13). These correlations suggest that ALA may play an immunoregulatory
role in the disease activity of these 2 diseases.
ACKNOWLEDGMENTS
The authors wish to thank Ms Ruth Graham and Ms
Linda Bernhardt for expert secretarial assistance and Ms Isi
Miranda for her assistance with graphics. We would also like
to thank Dr. G. Troup for HLA-DR typing of our donors.
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