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Immunologic studies of rheumatoid arthritis patients treated with methotrexate.

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Arthritis & Rheumatism
Official Journal of t h e American Rheumatism Association
IMMUNOLOGIC STUDIES OF
RHEUMATOID ARTHRITIS PATIENTS
TREATED WITH METHOTREXATE
NANCY J. OLSEN, LEIGH F. CALLAHAN, and THEODORE PINCUS
Immunologic functions of peripheral blood
mononuclear cells were studied in rheumatoid arthritis
(RA) patients treated with methotrexate (MTX). Spontaneous IgM rheumatoid factor (IgM-RF) synthesis by
unstimulated cultured blood mononuclear cells was seen
in only 3 of 18 MTX-treated patients, compared with 31
of 54 RA patieDts who were not receiving long-acting
drugs. Total IgM production by unstimulated cultured
mononuclear cells, pokeweed mitogen-induced antibody
synthesis, and plasma levels of IgM-RF were also lower
in MTX-treated patients than in other RA patients. The
numbers of circulating B cells, T4 and TS cells, the
T4:TS cell ratio, and mitogen-induced proliferation
indices were similar in WTX-treated and non-MTXtreated patients. Eleven additional patients were studied
prospectively after initiation of MTX therapy. All
showed significant decreases in spontaneous IgM-RF
synthesis, with declining IgM-RF:IgM ratios, including
all of the 9 who were studied during the first 24 hours of
treatment. The results indicate that MTX has rapid
effects on IgM-RF synthesis, and this action might be
agsociated with its therapeutic efficacy in RA.
Recent reports indicate that the antimetabolite
methotrexate (MTX) provides effective therapy for
rheumatoid arthritis (RA) (1-4). Despite extensive
knowledge of its effects on cancer cells (5), the mode
of action of MTX in RA remains largely unknown.
The development of sensitive techniques to
measure production of IgM rheumatoid factor (IgMRF) in vitro (6,”) has facilitated analyses of drug
effects on the production of this autoantibody by
peripheral blood mononuclear cells of RA patients. In
previous studies, a profound reduction in IgM-RF
synthesis in vitro was seen in patients treated with
gold salts and penicillamine, long-acting agents traditionally used in RA (8-10). This reduction was correlated with changes in clinical parameters (8) and
therapeutic responses (9) to these long-acting drugs.
These observations led us to study in vjtro
IgM-RF synthesis as well as other immunologic functions in blood mononuclear cells of patients treated
with MTX. We report marked differences in IgM-RF
synthesis in MTX-treated patients compared with RA
patients not treated with long-acting drugs. Significant
changes were noted within 1 day of the first dose of
methotrexate.
From the Department of Medicine, Division of Rheumatology and Immunology, Vanderbilt University, School of Medicine,
Nashville, Tennessee.
Supported in part by the Arthritis Foundation, the Maury
County Lupus Fund, NIH General Clinical Research Centers grant
5MOlRR-00095, and NIH Biomedical Research Support grant
RR-05424.
Nancy J. Olsen, MD: Assistant Professor of Medicine;
Leigh F. Callahan, BS: Senior Research Associate; Theodore
Pincus, MD: Professor of Medicine and Microbiology.
Address reprint requests to Nancy J. Olsen, MD, B-3219
Medical Center North, Vanderbilt University, Nashville, TN 37232.
Submitted for publication April 4, 1986; accepted in revised
form November 18. 1986.
PATIENTS AND METHODS
Patients. The initial study population consisted of 97
patients with definite or classic RA, as defined by the
American Rheumatism Association criteria ( 1 1 ) . These pa-
Arthritis and Rheumatism, Vol. 30, No. 5 (May 1987)
481
OLSEN ET AL
tients were derived from 3 sources: 71 from the Vanderbilt
University Medical Center Clinic, 18 from the Nashville
Veterans Administration Hospital, and 8 from the private
practice of Dr. Joseph Huston (Nashville, TN). The patients
were categorized into 3 groups according to treatment,
which continued for at least 2 months. Group 1 received
methotrexate (18 patients), group 2 received parenteral gold
salts (25 patients), and group 3 did not receive MTX,
parenteral gold, penicillamine, azathioprine, hydroxychloroquine, or cyclophosphamide (54 patients). Most patients
were also treated with nonsteroidal antiinflammatory agents
or salicylates, and 46% of the patients were treated with oral
corticosteroids. Seven RA patients were excluded from this
study because they were receiving long-acting agents other
than MTX or parenteral gold. Eleven additional RA patients
who were beginning treatment with MTX were studied
prospectively.
In most patients, MTX was given as a weekly oral
dose of 7.5 mg, which was taken in a single dose. Some of
the prospectively studied patients received an initial dose of
12.5 mg intramuscularly. This was followed by the weekly
oral regimen. Blood samples were drawn before the weekly
dose was given.
Clinical assessment. Clinical assessment of these patients included 6 types of measures, as follows. Functional
class (12) was assessed by the physician. Westergren erythrocyte sedimentation rate (ESR) was measured according to
standard methods. Joint counts were performed using a
modified Ritchie index (13) to assess tenderness in 42 joints,
including finger, proximal interphalangeal (I0 joints), and
metacarpophalangeal(10joints), the wrist (2joints), elbow ( 2
joints), shoulder (2 joints), hip (2 joints), knee ( 2 joints),
ankle ( 2 joints), and metatarsophalangeal (I0 joints). Radiographs of the hands were analyzed quantitatively for erosions, using a modification of the method described by Sharp
et a1 (14). Activities of daily living (ADL) were assessed by
self-report, using a modified health assessment questionnaire
(IS) to determine the level of difficulty in performing standardized activities. Grip strength was measured according to
standard methods (16).
Cell cultures. Blood mononuclear cells were obtained
by centrifugation on Ficoll-Paque cushions (Pharmacia,
Piscataway, NJ), washed 7 times, and resuspended in RPMI
1640 tissue culture medium (Gibco, Grand Island, NY)
containing 10% fetal calf serum (Flow Laboratories,
McLean, VA), glutamine, penicillin, and gentamycin. Cells
were cultured in 0.20-ml volumes in round-bottom 96-well
microtiter plates (Costar, Cambridge, MA), at a concentration of lo6 cells/ml. A standard stock solution of pokeweed
mitogen (PWM; Gibco) was added to some cultures, at a
final concentration of 1 : 100. After a 13-day incubation
period, supernatants were collected, pooled, and stored at
-70°C until assayed.
Radioimmunoassays. IgM and IgM-RF concentrations in culture supernatants were determined by solid-phase
radioimmunoassay, as previously described (8). Briefly,
polyvinyl chloride microtiter plates (Falcon, Oxnard, CA)
were coated far 4 hours with a 5 pg/ml solution of goat
anti-human IgM (Tago, Burlingame, CA). The plates were
washed and blocked with 10% newborn calf serum (Gibco)
and 0.050-ml volumes of supernatant, diluted plasma, or
medium alone, added in duplicate. A pool of standardized
normal human serum was used for calibration. After overnight incubation and washing, a dilution of ‘251-labeled
F(ab’Iz anti-IgM (Tago) was added to each well. After a
second overnight incubation, wells were cut, and the bound
radioactivity was counted in a gamma counter (Beckman
Instruments, Irvine, CA).
IgM-RF was measured using the same procedure,
except that purified heat-aggregated human IgG (Pel-Freez,
Rogers, AR) was used as the first layer. Values were
normalized by the use of curves constructed for every
determination with 5 seropositive RA plasmas. Results were
expressed as nanograms of F(ab’)2anti-IgM bound per well.
IgM anti-tetanus antibodies were similarly measured
using dialyzed tetanus toxoid (Connaught, Swiftwater, PA)
as the first layer. Values were normalized using sera of
different titers; the same sera were used in each assay.
Results were expressed as nanograms of F(ab’)z anti-IgM
bound per well.
Proliferation assays. Spontaneous and mitogeninduced proliferative responses by blood mononuclear cells
were measured in 5-day cultures. Cells (at I06/ml) were
incubated in quadruplicate flat-bottom microtiter wells
(Costar) with or without PWM, at 1 : 100 and 1 :250, and
purified phytohemagglutinin (PHA-P; Sigma, St. Louis,
MO), 1 CLg/ml. For ’the terminal 6 hours, 1 pCi of 3Hthymidine (New England Nuclear, Boston, MA) was added
to each culture. Cells were harvested on glass-fiber filter
strips (Bellco, Vineland, NJ), and individual wells were
placed in vials with scintillation fluid and counted. Results
were expressed as counts per minute or, for mitogenstimulated cultures, as a stimulation index (SI), where SI =
(cpm with mitogen)/(cpm without mitogen).
T cell subsets. Phenotypic markers oh peripheral
blood lymphocytes were quantitated by Diaclin Laboratories
(Nashville, TN), using automated flow cytometry. According to that laboratory, normal values (expressed as the mean
percent of total lymphocytes -t standard deviation) are as
follows: total T cells (T3) 67.7 i 9.0; T helper cells (T4) 43.2
t 7.2; T suppressor cells (T8) 22.7 2 5.9; total B cells (B1)
10.8 5.2; human natural killer cells (HNK-I) 8.6 ? 5 . 2 .
Statistical methods. The data were entered into a
computer using the CLINFO software package (Bolt,
Beranek and Newman, Boston, MA) and analyzed on a
VAX 11/780 using the Statistical Package for the Social
Sciences (17). Variables were analyzed in the 3 patient
groups (i.e., those treated with methotrexate, parenteral
gold, or no long-acting agent), by one-way analysis of
variance. Duncan’s test for multiple comparisons was used
to determine which groups differed statistically at the P <
0.05 level. Because the assumption of homogeneity of variances was not met with some variables, the Kruskal-Wallis
nonparametric test was used to test for significant differences between the groups (18). Two-tailed chi-square tests
were used to test for significance of differences among
dichotomous variables.
*
RESULTS
RA patient population. The RA study patients
were analyzed as 3 groups, according to whether they
483
MTX IN RA
were being treated with MTX, parenteral gold salts, or
no long-acting agent for at least 2 months. The patients
had a mean age of 57.3 years, mean disease duration of
11.9 years, and mean formal education level of 11.0
years (Table 1); 59% were female, 85% were white,
46% were taking prednisone, and most were also being
treated with salicylates or nonsteroidal antiinflammatory drugs. There were no significant differences in
any of these measures among patients in groups 1, 2,
and 3.
Assessment of clinical and laboratory measures
indicated more favorable status in methotrexatetreated patients in 4 of the 6 results: number of tender
joints, self-reported ADL difficulty score, grip
strength, and Westergren ESR (Table 1). However,
only the differences in ESR were statistically significant. These data are consistent with trends toward
better clinical status in RA patients who have been
treated with MTX, compared with other RA patients,
but larger numbers and prospective longitudinal studies are needed to establish statistically significant
results.
IgM-RF synthesis in RA patients treated with
MTX, gold, or other therapies. Individual values of
spontaneous IgM-RF synthesis by blood mononuclear
Demographic, clinical, and laboratory features of 97
rheumatoid arthritis patients treated with methotrexate (MTX),
parenteral gold, or other drugs*
Table 1.
All
Group 1 Group 2 Group 3
patients (MTX) (gold) (other)
Demographic data
Age (years)
Duration of disease (years)
Formal education level
(years)
Clinical data
Functional class
No. of tender joints
Radiographic erosion score
Grip strength (mm Hg)
ADL difficulty score (selfreport)
% treated with prednisone
Laboratory data
ESR (Westergren)
(mdhour)
No. of lymphocytes/mm3
% B cells
% T3 cells
T4:TS ratio
51.3
11.9
11.0
57.6
11.0
12.2
61.0
12.5
10.7
55.4
11.9
10.8
2.3
14.8
1.63
93.6
2.05
2.2
12.1
1.79
102.8
1.87
2.3
15.1
1.36
101.0
1.91
2.3
15.5
1.69
87.7
2.17
46
33
48
50
31.4
24.1
28.2
45.9
1,834
11.5
65.0
2.3
1,625
11.8
63.7
2.1
1,989
11.1
67.7
2.6
1,818
11.5
64.2
2.2
~~
~
* Values are means. Groups 1 and 3 and groups 2 and 3 differed
significantly, at P < 0.05, in erythrocyte sedimentation rates (ESR);
no other comparisons were significant at this P level. ADL =
activities of daily living. See Patients and Methods for details of
determinations.
0
0
0
0
0
0
0
_____
-L
P
0
O’
M
F
GOLD
OTHER
Figure 1. Spontaneous IgM rheumatoid factor (IgM-RF) synthesis
by blood mononuclear cells obtained from 97 rheumatoid arthritis
patients in 3 treatment groups. MTX = methotrexate (see Patients
and Methods for details). Dashed line indicates the upper limit of
normal values.
cells (Figure 1) indicated that 3 of the 18 patients (17%)
receiving MTX produced spontaneous RF, compared
with 9 of 25 patients (36%) receiving parenteral gold
and 31 of 54 patients (57%) not receiving any longacting agent. The differences in these distributions
between the MTX-treated group and patients not
taking long-acting agents was statistically significant
( P < 0.05).
Mean spontaneous IgM-RF synthesis for MTXtreated patients was 0.5 ng of anti-IgM, compared with
0.9 ng of anti-IgM for parenteral gold-treated patients
and 1.7 ng of anti-IgM for patients not receiving any
long-acting drug (Table 2). The difference between the
MTX-treated group and the group receiving no longacting drugs was statistically significant ( P = 0.0004),
although the difference between the MTX and
parenteral gold groups was not significant. PWM-
484
OLSEN ET AL
Table 2. Rheumatoid factor (RF) and IgM values in 97 rheumatoid arthritis patients treated with
methotrexate (MTX), parenteral gold, or other drugs*
Plasma levels
IgM-RF (ng of anti-IgM)
IgM (pdml)
In vitro cultures
Spontaneous
IgM-RF (ng of anti-IgM bound)
IgM (ndml)
PWM-induced
IgM-RF (ng of anti-IgM bound)
IgM (ndml)
All
patients
Group 1
(MTX)
Group 2
(gold)
Group 3
5,806
2,110
3,939
2,001
5,021
1,608
6,750
3,031
0.061
0.03$
1.2
1,737
0.5
768
0.9
1,365
1.7
2,252
0.0004t
0.082
1.7
3,597
1.5
1,955
0.9
2,470
2.1
4,693
O.Ol$
0.112
(other)
P
* Values are means. The P values refer to three-way comparisons of groups 1, 2, and 3, and were
derived from one-way analysis of variance. PWM = pokeweed mitogen.
t Groups 1 and 3 differed at P < 0.05, by post hoc analysis.
$ Groups 2 and 3 differed at P < 0.05, by post hoc analysis.
induced IgM-RF synthesis was also lower in the
methotrexate- and gold-treated patients, but differences were less marked than were those of spontaneous IgM-RF synthesis. No significant correlations
could be demonstrated between in vitro R F synthesis
and the MTX dosage (5-12.5 mg/week), the interval
since the last dose (1-7 days), or the duration of
treatment (2-27 months) (data not shown).
Studies of total IgM synthesis, both spontaneous and PWM-induced, indicated that the lowest levels were seen in the MTX-treated group (Table 2).
\
z 4-1
L
I
4
24
HOURS
I
48
Figure 2. Spontaneous IgM rheumatoid factor (IgM-RF) synthesis
in 9 rheumatoid arthritis patients, measured within 48 hours of
beginning methotrexate treatment.
Only 2 of the 17 MTX-treated patients tested had
spontaneous IgM synthesis >2,000 ng/ml of IgM,
while 12 of 50 patients not taking long-acting drugs
produced IgM in this range. Similarly, PWM-induced
IgM was lower in the group receiving MTX. No
patients treated with MTX produced more than 2,000
ng/ml of PWM-induced IgM; however, 8 of the 52
patients who were not taking long-acting drugs produced >2,000 ng/ml of PWM-induced IgM. When
individual ratios of spontaneous IgM-RF:IgM were
averaged for each group, we found that the value for
was almost twice that of group 1
group 3 (2.68 X
(1.47 x
and that of group 2 (1.44 X lop3).
Although these differences were not significant, the
data suggest that MTX and gold might continue to
preferentially decrease IgM-RF synthesis even though
total IgM synthesis has also decreased.
The 3 treatment groups did not differ in absolute
or relative numbers of circulating B or T lymphocytes
(Table I). In addition, ratios of lymphocytes bearing
the T4 and T8 cell surface markers did not differ in the
3 groups (Table I), nor did absolute numbers of T4,
T8, and HNK cells (data not shown). These findings
suggest that differences in IgM-RF or IgM production
in the 3 groups are not explainable by relative or
quantitative alterations in immunoglobulin-producing
or regulatory cell populations.
Prospective studies of in vitro IgM-RF synthesis
in patients treated with MTX. The above analyses
indicated that methotrexate is associated with lower
levels of IgM-RF synthesis, but their retrospective
nature rendered it impossible to determine whether
decreased IgM-RF synthesis is a direct effect of MTX
MTX IN RA
485
or is associated with a possible selection bias. Previous studies had indicated that changes in IgM-RF
synthesis occur within the first 9 weeks of treatment
with parenteral gold salts or penicillamine (9). Therefore, in vitro IgM-RF synthesis was analyzed prospectively in patients who were beginning therapy with
MTX. Nine patients were studied within 48 hours of
their first dose of MTX (Figure 2 and Table 3). All
showed declines in spontaneous IgM-RF production
during this period, including 3 of 4 patients studied 4
hours after the first dose of MTX. A mean decrease to
36% of baseline spontaneous IgM-RF production was
seen between 24 and 48 hours. In contrast, 3 control
patients who were studied twice within 48 hours,
without change in therapy, showed IgM-RF levels
within 30% of baseline values, with a mean level
similar to that seen at baseline (Table 3).
Eleven patients were studied prospectively at
various intervals after MTX treatment was started.
Mean spontaneous IgM-RF synthesis declined most
markedly during the first 24 hours of treatment, and
remained low during 2 months of evaluation (Figure 3).
Overall, the mean spontaneous IgM-RF synthesis decreased to 51% of the pretreatment value within the
first 8 hours. After 48 hours, this value had decreased
further, to 37% of baseline. The mean level remained
similar in 4 of the 5 patients monitored for 2 months or
longer. In these patients, mean IgM-RF production
decreased to 8% of the corresponding baseline values.
One additional patient showed marked decreases in
IgM-RF synthesis soon after the dose (to 9% of
Table 3. Spontaneous IgM rheumatoid factor synthesis in rheumatoid arthritis patients beginning methotrexate (MTX) treatment,
compared with patients not starting a new drug treatment
% of
baseline
Baseline
<48 hours
10.3
3.1
0.9
4.6
1.2
2.2
3.0
4.7
2.2
3.6
2.2
0.3
0.4
3.0
0.3
0.4
0.6
2.8
1.2
I .2
21
10
44
65
25
18
20
60
55
36
2.4
2.3
1.3
2.0
3.1
2. I
1.6
2.3
129
91
123
114
Patients starting MTX
1
2
3
4
5
6
7
8
9
Mean
Patients not starting MTX
1
2
3
Mean
2
MONTHS
Figure 3. Mean spontaneous IgM rheumatoid factor (IgM-RF) synthesis and IgM-RF/IgM ratios in 11 rheumatoid arthritis patients
beginning treatment with methotrexate.
1
2
7
1
DAYS
baseline at 6 hours and 54% at 24 hours) but, subsequently, had increased IgM-RF production at the 1-,
2-, and 3-month intervals. After 4 months of therapy,
IgM-RF synthesis in this patient again decreased to
36% of baseline.
Serum MTX levels measured in some of these
patients demonstrated a rapid decline within 48 hours
of the dose (Baer A: personal communication). Thus,
decreased IgM-RF synthesis persists after significant
drug levels have been cleared from the serum.
Of the 11 patients studied, 1 discontinued treatment after 4 months because of generalized malaise,
although clinical improvement had been noted. He
subsequently showed an increase in IgM-RF synthesis, from undetectable levels to 3.9 ng of anti-IgM
8 months after discontinuing MTX therapy.
The relative specificity of the observed decreases for IgM-RF is indicated by the generally
parallel decrease in the IgM-RF:IgM ratio (Figure 3).
As an additional measure of specificity, IgM antitetanus antibodies were measured in culture supernaTable 4. Relative decreases in spontaneous IgM rheumatoid factor
(IgM-RF), anti-tetanus antibody, and total IgM synthesis in 4
rheumatoid arthritis patients beginning methotrexate treatment
IgM-RF:IgM ( x lo3)
IgM-RFr IgM-tetanus
Patient
Day 0
Day 1
Day 0
Day 1
1
8.9
16.0
45.0
7.9
0.6
5.0
2.4
2.7
0.28
0.39
I .27
1.22
0.10
0. I9
0.27
0.13
2
3*
4
* Studied at baseline
and at 48 hours.
486
tants in 4 patients at baseline and within 48 hours of
the initial dose of MTX. In all 4, the IgM-RF:IgMtetanus ratios decreased by more than 50% (Table 4),
indicating that during the early treatment period, MTX
preferentially affects synthesis of IgM-RF.
Synthesis of PWM-induced IgM-RF decreased
after MTX therapy was begun, but it did so much more
slowly than did spontaneous ZgM-RF. Mean values
were 95% of baseline after 48 hours and 4 weeks of
treatment, although patients monitored for 2 months
or longer showed PWM-induced IgM-RF decreases to
33% of pretreatment levels. These results suggest that
spontaneously activated cells are most susceptible to
the effects of MTX, while other precursors of IgM-RF
production may be affected later.
Additional immunologic changes seen with MTX
therapy. Plasma levels of IgM-RF, measured b y
radioimmunoassay, did not decrease during the first 2
months of evaluation (data not shown). This suggests
that the decreased levels seen in the study group
treated longer than 2 months (Table 2) occurred only
after longer periods of treatment.
Spontaneous and mitogen-induced uptake of
3H-thymidine were measured in parallel with sequential cultures for antibody production, since MTX has
antiproliferative effects (5). Spontaneous 3H-thymidine uptake 24 hours after the methotrexate dose was
80% of the corresponding pretreatment baseline level
and remained at 83% of the baseline level after 4
weeks, indicating that there was no significant effect.
Mean proliferation values in response to PHA actually
increased over the 2 months. These data suggest that
decreased antibody synthesis does not result from
antiproliferative effects. An alternative interpretation
of these results is that uptake of 3H-thymidine is not
the optimal marker for measurement of effects of MTX
on cellular DNA synthesis.
The role of mononuclear cell subsets in regulating the acute decrease in IgM-RF synthesis was studied using measurements of various phenotypic markers at baseline and within 48 hours of the first dose of
MTX. In the 4 patients studied, the mean ratio of
T4:T8 cells decreased to 78% of the pretreatment
value because of an increase in the absolute number of
T8+ cells. The absolute number of B cells increased
1.8-fold. Absolute numbers of HNK cells, as measured by 2 markers, Leu-7 and HNK-l, increased
2.4-fold and 2.8-fold, respectively. These results suggest that decreased R F synthesis during early treatment intervals is accompanied by increases in suppres-
OLSEN ET AL
sor T cells and HNK cells, but not by a decrease in the
number of circulating B lymphocytes.
DISCUSSION
Among 97 RA patients, significantly lower frequencies and levels of spontaneous IgM-RF synthesis
were seen in 18 patients taking methotrexate, compared with those not taking this drug. In addition, total
spontaneous IgM synthesis and PWM-induced IgMR F and IgM synthesis were decreased. Prospective
analysis of patients from the time they began MTX
therapy indicated marked decreases in spontaneous
IgM-RF synthesis within 24 to 48 hours of the first
dose. Therefore, MTX appears to affect those cells
which are most activated, including those producing
RF, early in the course of treatment. With continued
therapy, decreases in mitogen-induced IgM-RF and
total IgM production are seen as part of a more
generalized immunosuppressive effect. These results
may be explained by the changing characteristics of
the circulating cell population. In active RA, large
cells with blastic features (19), which may be metabolically active and more susceptible to MTX, are
present. With treatment and decreased synovitis,
fewer of these cells are observed (20), and the effects
of MTX might then be expected to be less specific.
The findings reported here are consistent with
other evidence that MTX alters immune function.
Methotrexate has been found to be useful in suppressing graft-versus-host disease in transplant recipients
(21), even at a 4-day dosage regimen (22), which
suggests that it has an early onset of action. Short-term
suppression of antibody synthesis has been demonstrated in patients with neoplasms who received intermittent infusions of MTX and cytosine arabinoside
(23). It was hypothesized that the underlying mechanism involved differential killing of activated lymphocytes, with preservation of memory cells.
Immunosuppressive effects of MTX in RA have
been described. In one study, significant decreases in
serum levels of IgG, IgM, and IgA were seen after 14
weeks of MTX treatment (4). Although the serum R F
titer was not significantly decreased over the period of
that study, in another study ( 3 ) , patients had a significant decline in serum R F during 18 weeks of MTX
therapy. Our data indicate that a reduction of plasma
R F titers occurs, but over much longer times than the
early reduction in cellular R F synthesis. In one study,
in vitro immunoglobulin synthesis by blood mononuclear cells from treated patients indicated no signifi-
487
MTX IN RA
cant decreases in IgM production, and actual increases
in total IgG production (4). However, significant decreases in serum levels of immunoglobulins were
reported in that study, and it is unclear how these
results could be accounted for without decreased
immunoglobulin production by the relevant cells.
Consistent with results reported by others (2),
no significant changes in mononuclear cell proliferation were seen in our MTX-treated patients, as assessed using 3H-thymidine incorporation measures.
However, by blocking the enzyme thymidylate
synthetase, MTX may actually make cells more avid
for uptake of thymidine, and it may be optimal to use
different markers to assess effects on DNA synthesis.
Preliminary results using deoxyuridine in place of
thymidine to assess cell division indicate decreased
proliferation of circulating mononuclear cells after
administration of MTX in culture, but not in treated
patients. Antiproliferative effects may thus contribute
to the action of this drug, but it does not appear that
these effects explain the changes observed in RA.
Our results which indicate that there were no
significant differences in T cell subsets in the different
treatment groups are similar to the findings of 2
previous reports (2,4). However, prospective analyses
indicated an increase in absolute numbers of T8+ cells
within 48 hours of the first MTX dose, suggesting a
possible regulatory role for these cells in mediating
decreased antibody synthesis.
We also observed an increase in HNK cells
after the first MTX dose. It has been reported that
MTX can augment HNK cell function in vitro (24).
Since HNK cells have been shown to inhibit B cell
differentiation (25), it is possible that MTX could
suppress antibody synthesis through quantitative or
qualitative increases in natural killer cells.
Regardless of the mechanism of action, our
results indicate that within hours of the initiation of
methotrexate therapy, there are unequivocal effects on
the capacity of mononuclear cells to elaborate IgMRF. Although the role of IgM-RE in RA is not completely understood (10,26), the disease-modifying
potential of MTX appears t o be associated with
the profound effects of this drug on immune activity.
In view of increasing recognition of the long-term
severe morbidity (27,28) and mortality (28-32) of RA,
the rationale for therapy that results in rapid reversal
of the pathogenetic processes may be increasingly
supported.
ACKNOWLEDGMENTS
We thank Drs. Alan Baer, Howard A. Fuchs, and
Joseph W. Huston for help and cooperation in our study of
their patients, and Drs. Paul Nance and Jeremy Kaye for
expert interpretation of the radiographs. Raye Brooks was of
invaluable help in performing the joint counts and functional
evaluations. Dr. Wayne Green and Keith Shults were especially helpful in performing lymphocyte subset phenotyping.
Susan Tewksbury and Lisa Murray provided skilled technical assistance, and Joan Koehler patiently prepared the
manuscript.
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