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Reduction of leukocyte and interleukin-1 concentrations in the synovial fluid of rheumatoid arthritis patients treated with methotrexate.

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ARTHRITIS & RHEUMATISM Volume 36
Number 9, September 1993, pp 1244-1252
8 1993, American College of Rheumatology
1244
REDUCTION OF LEUKOCYTE AND
INTERLEUKIN-lp CONCENTRATIONS IN
THE SYNOVIAL FLUID OF
RHEUMATOID ARTHRITIS PATIENTS
TREATED WITH METHOTREXATE
RANJENY THOMAS and GRAEME J. CARROLL
Objective. To examine the effect of methotrexate
(MTX) on the numbers of leukocytes in the peripheral
blood (PB) and synovial fluid (SF) of patients with active
rheumatoid arthritis (RA).
Methods. Twelve patients were treated with
MTX; 5 patients not taking MTX served as controls.
Samples of PB and SF were collected at 0, 1, 4, and 8
weeks of the study. Disease activity was scored, and total
leukocytes, neutrophils, lymphocytes, and CD4+,
CDS+, DR+, and CD25+ lymphocyte subsets were
analyzed in PB and SF. Interleukin-lp (IL-1P) concentrations in SF were determined.
Results. Patients treated with MTX showed significant clinical improvement. No change in PB leukocytes or lymphocyte subsets was observed in either
patient group over the 8-week study period. In contrast,
the number of leukocytes, the number and proportion of
neutrophils, and the concentration of IL-1P in the SF of
patients treated with MTX were reduced. In addition, in
MTX-treated patients, there was an appreciable decrease in SF CDS+ lymphocytes, but not CD4+, DR+,
or CD25+ lymphocytes.
Conclusion. These findings suggest that in RA,
MTX acts, at least in part, by reducing the migration of
From the Department of Rheumatic Diseases, Royal Perth
Rehabilitation Hospital, Shenton Park,Western Australia, Australia.
Supported by a grant from the Medical Research Foundation of Royal Perth Hospital.
Ranjeny Thomas, MBBS; Graeme J. Carroll, MBBS.
Address reprint requests to Graeme J. Carroll, MBBS,
Department of Rheumatic Diseases, Royal Perth Rehabilitation
Hospital, Selby Street, Shenton Park, Western Australia 6008,
Australia.
Submitted for publication September 25, 1992; accepted in
revised form March 10, 1993.
leukocytes into the inflamed synovium. Local reduction
of IL-1P secretion may contribute to this effect.
Pulse therapy with low-dose methotrexate
(MTX) has been used in rheumatoid arthritis (RA)
since the early 1970s (1) and has been shown to be
effective in double-blind studies in both the short and
long term ( 2 4 ) . The mechanism of action of MTX is
still poorly understood, but it is known to inhibit the
enzyme dihydrofolate reductase and other folatedependent enzymes, thereby inhibiting the synthesis
of DNA precursors and limiting cell replication (5).
Although it is possible that low-dose MTX may exert
its effects in RA by an antiproliferative action on bone
marrow leukocyte precursors, studies of peripheral
blood (PB) lymphocyte subpopulations have shown no
consistent effect of MTX administration (2,4,6,7).
Clinical experience has demonstrated a relatively
rapid onset of action compared with most diseasemodifying antirheumatic drugs (DMARDs) (3), and it
has been proposed that the mechanism of action may
be partly antiinflammatory (5,8). MTX could therefore
exert a more specific reduction of the inflammatory
response in the joint (9). This could occur by reduced
lymphocyte proliferation within the synovium or reduced migration of inflammatory cells to the joint.
The migration of leukocytes into tissues results
from receptor-mediated interactions between leukocytes and the endothelial cells of postcapillary
venules. Functional and morphologic changes in inflamed endothelial cells, as in RA, enhance the migration of lymphocytes and neutrophils. Thus, the cytokines interferon-y, tumor necrosis factor a, and
interleukin-1 (IL-1) stimulate the binding of neutrophils and lymphocytes to endothelial cells, partly
RA SYNOVIAL FLUID AND MTX TREATMENT
through inducing the expression of intercellular adhesion molecule 1 and E-selectin, which are involved in
binding and migration (10). It is therefore possible that
MTX could exert its action at the level of the endothelial cell itself (1 1) or at the level of the binding or
migration of leukocytes. Furthermore, MTX could
afFect the production or activity of the cytokines
involved in migration. IL-1 is detectable in large
amounts in rheumatoid synovium and is produced by
synovium-derived mononuclear cells, both spontaneously and after lipopolysaccharide stimulation (10).
Increased synovial concentrations of IL-1 correlate
with disease activity, and increased IL-I production in
vitro is associated with the onset or exacerbation of
disease (12,13). Thus, the production of IL-1 is closely
related to the degree of inflammatory activity in RA.
To date, there have been no studies of serial
changes in inflammatory cells in the PB or synovial
fluid (SF) after initiation of MTX treatment. Therefore, we examined serial cellular changes in response
to MTX in the PB and SF of patients with active RA,
and investigated whether changes in leukocyte concentrations were associated with a decline in the level
of synovial IL-1p. The results show a reduction in
neutrophil and lymphocyte numbers in the SF, but not
the PB, of patients with RA treated with low-dose oral
MTX for 8 weeks. This indicates that MTX indeed
exerts a specific effect on the inflammatory response in
the joints in RA.
PATIENTS AND METHODS
Patient population. Patients were recruited for the
study if they satisfied the American College of Rheumatology (formerly, the American Rheumatism Association) 1987
criteria for RA (14), had clinically active disease, with at
least a small effusion in 1 knee, and gave informed consent.
Patients with cancer, leukopenia, thrombocytopenia, significantly altered liver enzymes, or renal disease were excluded. Seventeen patients entered the study.
Twelve patients began taking MTX (7.5 mg orally
each week). The remaining 5 patients continued with existing therapy (1 taking prednisolone 5 mg on alternate days
and sulindac 100 mg twice a day, 1 taking sulfasalazine 2
gm/day and intramuscular gold 50 &month, 1 taking sulindac 200 mg twice a day and indomethacin suppositories 100
mg/day, 1 taking piroxicam 20 mg/day, and 1 taking aspirin
as needed); these 5 patients served as controls. The control
patient who continued DMARD therapy had been taking
stable doses of gold for 10 years and sulfasalazine for 18
months. All others had ceased taking DMARD therapy at
least 2 months prior to entering the study. None of the 12
who received MTX were taking other DMARDs, nor had
they received MTX previously. The median number of
1245
DMARDs previously taken by the MTX-treated group and
the control group was 2. All patients were taking nonsteroidal antiinflammatory drugs. All medications were continued
at the same dosage throughout the study, but patients were
allowed to take paracetamol.
The median age of the MTX-treated patients was 65
and that of the control patients was 75. Eight MTX-treated
patients and 4 controls were female. The median disease
duration was 13 years for the patients treated with MTX, and
16 years for the controls. Rheumatoid factor was present in
92% of the MTX-treated patients, and 100% of the controls.
There were no statistically significant differences between
MTX-treated patients and control patients in any of the
demographic features.
Clinical measurements. The following indices of disease activity were measured by the same clinical metrologist
on each occasion: patient’s assessment of pain, function,
and overall well-being (by visual analog scale ranging from 0
to 99), mean grip strength in each hand (average of 3
measurements), the number of tender joints, the number of
swollen joints, the Ritchie index (15), and the Lee index (16).
Study design. Patients were seen on 5 occasions over
an 8-week period: at 30 minutes prior to receiving MTX,
then at 1, 7, 28, and 56 days after the first dose of MTX.
Controls were seen at the same intervals but did not receive
MTX. PB was drawn and S F was aspirated from a single
knee joint with active RA at each time point. PB, but not SF,
was collected from 1 patient in each of the groups; both PB
and SF were collected from all other patients in the study.
Intraarticular injections of steroids had been avoided in the 3
months prior to the study and were not used during the
study.
Monoclonal antibodies (MAb). Mouse MAb OKT4
(defines the CD4 T cell subset) and OKT8 (defines the CD8
T cell subset) were obtained from Ortho Diagnostics (Raritan, NJ). Anti-human IL-2 receptor (CD25) was obtained
from Becton Dickinson (Sunnyvale, CA). Unclustered
FMC4 (Ia/DR) was a gift from Flinders Medical Centre
(Adelaide, South Australia). HLA-DR is distributed on
resting monocytes and B cells, and activated T cells. CD25 is
distributed on activated T cells, B cells, and monocytes.
Cell preparation and flow cytometry. The PB leukocyte count and differential white blood cell (WBC) count
were measured using a Technicon H6000 automated hematology analyzer (Technicon, Tarrytown, NY). The erythrocyte sedimentation rate (ESR) was measured by the Westergren technique. Leukocytes in SF were counted at a 1:20
dilution in the improved Neubauer hemocytometer (Weber
Scientific Instruments, Middlesex, UK). Heparinized PB
was diluted with phosphate buffered saline (PBS), pH 7.2,
and then layered on Ficoll-Hypaque gradients (Sigma, St.
Louis, MO). S F cells were suspended in PBS, pH 7.2, then
centrifuged. Mononuclear cells from both PB and S F were
washed and resuspended in PBS at a concentration of 1 x
lo6 ceIls/ml.
Cells were incubated with saturating concentrations
of the appropriate MAb at room temperature for 5 minutes,
washed twice in PBS, and then incubated with fluorescein
isothiocyanate (FITCtlabeled goat anti-mouse immunoglobulin (GAMIg) for 5 minutes. After 2 further washes in
PBS, the cells were fixed in 1% formalin. Cells for the
1246
THOMAS AND CARROLL
Table 1. Effect of MTX treatment on clinical indices in RA patients after 8 weeks*
MTX-treated patients
(n
=
Control patients
12)
(n = 5 )
RA activity index
Week 0
Week 8
Week 0
Pain (VAS)
Functional index
Grip strength (mm Hg)
Right hand
Left hand
No. of tender joints
No. of swollen joints
Global index
Ritchie index
Lee index
ESR (mdhour)
72 t 5
69 t 5
27 t 5
35 t 7
53 9
43 t 11
14 t 8
64 t 14
76 t 6
77 t 7
122 1
6 2 1
76 + 5
17 f 2
15 f 1
67 t 9
86 f 8
91 f 10
7 2 1
3 2 1
36 f 7
8+1
10 f 2
43 + 8
97 f 12
85 t 12
9 t 3
4 2 1
44t3
10 + 4
12 f 1
48 f 7
91 t 14
83 + 12
I1 5 2
I t 1
64 + 14
13 t 2
12 t 1
53 5
*
Week 8
~
*
~~
* Twelve patients with rheumatoid arthritis (RA) were treated with methotrexate (MTX) for 8 weeks;
5
RA patients who did not take MTX served as controls. Disease activity was assessed before (week
0) and 8 weeks after MTX was started. Peripheral blood was drawn for determination of the
erythrocyte sedimentation rate (ESR) at the same time points. Values are the mean t SEM for each
group. VAS = visual analog scale. See Patients and Methods for additional details.
negative controls were stained with FITC-labeled GAMIg
alone. Samples were analyzed using the Ortho Spectrum
Flow Cytometer. Lymphocytes were selectively gated based
on the parameters of forward and orthogonal scatter.
IL-1p assay. IL-1p was measured in S F samples by
enzyme-linked immunosorbent assay (ELISA). To eliminate
interference by rheumatoid factor (17), SF samples were
mixed with an equal volume of Sepharose 4B-human IgG
conjugate (Pharmacia, Uppsala, Sweden) slurry, and incubated for 1 hour at room temperature with gentle agitation.
All samples were then centrifuged at 1,000g for 1 minute to
sediment the solid phase. Supernatants were then assayed.
The IL-1p ELISA was performed according to the
method recommended by the manufacturer (Cistron Biotechnology, Pine Brook, NJ). Briefly, assay wells, precoated
with anti IL-lp MAb, were incubated with PBS containing
1% bovine serum albumin for 1 hour at 37°C to block
nonspecific binding. One hundred microliters of either assay
buffer, IL-1p standard, or test sample was then added to
each well and incubated for 2 hours at 37°C. The wells were
washed 3 times with wash buffer supplied with the kit, after
which 100 pl of rabbit anti-IL-lp antiserum was added, and
incubation was continued for 2 hours at 37°C. After washing,
100 pl of goat anti-rabbit IgG conjugated to horseradish
peroxidase was added to the wells and incubated for 30
minutes at room temperature. After washing, 100 pl of
substrate solution (containing o-phenylenediamine and hydrogen peroxide) was added to each well for 15 minutes at
room temperature. The reaction was stopped by the addition
of 50 pl of 4M sulfuric acid, and the absorbance was read at
490 nm. Results were calculated by interpolation from a
linear standard curve.
Statistical analysis. Because variables could not be
assumed to be normally or log-normally distributed, differences between MTX-treated patients and control patients
were assessed using nonparametric methods. The MannWhitney or Wilcoxon test was used for comparison between
groups or matched pairs as appropriate. Matched pairs
included (a) clinical score or cell count in MTX-treated (or
control) patients at week 0 compared with week 8; (b)
MTX-treated group versus control group clinical score or
cell count at week 0 or week 8; (c) the difference between
scores or counts obtained at week 0 and those obtained at
week 8, for the MTX-treated versus the control group.
Differences were considered to be significant at a level of P
< 0.05. Bonferroni corrections were made for multiple
statistical tests, but uncorrected P values are also shown to
illustrate the trends observed. Pearson correlation coefficients were used to examine relationships between variables.
RESULTS
Clinical response to MTX. Patients in the MTX
treatment group had more active disease at the beginning of the study (Table 1). This is reflected in the
higher global index ( P = 0.002) and higher functional
index (P = 0.038). No improvement was observed in
the control group over the 8-week period of observation; in fact, a clear trend toward deterioration was
evident for most of the indices, although no statistically significant differences were observed.
Comparisons of the changes in clinical indices
between 0 and 8 weeks between the 2 groups showed
improvement in the MTX group in indices of pain (P=
O . O l l ) , function ( P = 0.015), grip strength (right P =
0.036; left P = 0.011), and Ritchie index ( P = 0.012).
The Bonferroni correction for multiple tests on clinical
variables gave a critical value for a ( P ) of 0.0014.
Thus, with this correction, none of the observed
differences were statistically significant. There was no
statistically significant improvement before 8 weeks in
any of the indices. The change in the ESR at 8 weeks
RA SYNOVIAL FLUID AND MTX TREATMENT
1247
T
T
D
100
2o
L
z
E
E
50
K
u)
w
0
I
1
I
J
0
7
28
56
0
I
!
I
I
0
7
28
56
Time (days)
Time (days)
Figure 1. Effects of treatment with methotrexate (MTX) on clinical indices and leukocyte concentrations in synovial fluid (SF) and peripheral blood (PB). MTX-treated patients (0)received MTX for 8
weeks, and control patients (0)did not take MTX. Measurements of A, pain (visual analog scale
[VAS]), B, erythrocyte sedimentation rate (ESR), C, PB leukocyte count (WBC), and D, SF leukocyte
count (WBC) were made before (0) and at 7,28, and 56 days after MTX was started. Leukocytes were
counted by hemocytometer. Values are the mean and SEM for each patient group.
Table 2. Peripheral blood (PB) leukocytes and lymphocyte subsets in MTX-treated RA patients and in controls*
MTX-treated patients (n
Cells in PB
Total leukocytes
Total neutrophils
% neutrophils
Total lymphocytes
% lymphocytes
Lymphocyte subsets
No. CD4+
% CD4+
No. CD8+
% CD8+
CD4:CD8 ratio
No. DR+
% DR+
No. CD25+
% CD25+
Week 0
7,497
5,644
15
1,311
15
f 814
f 630
f3
f 212
5 3
687 2 135
56 t 3
342 f 72
24 ? 3
2.90 t 0.5
160 t 27
142 I
63 f 15
5 2 1
=
12)
Control patients (n
=
5)
Week 8
Week 0
Week 8
7,148 t 1,030
5,530 k 824
76 t 3
1,917 t 575
21 t 6
6,432 t 1,205
4,344 t 823
67 t 1
1,873 t 138
25 t 1
6,165 5 1,293
4,538 t 1,301
70 t 6
1,698 2 221
25 t 4
1,049 t 363
49 5 3
422 t 94
24 t 3
2.62 t 0.5
264 t 112
11 t 2
79 t 27
3 t 1
1,037 f 144
55 h 4
468 f 102
25 t 4
2.61 t 0.64
236 f 53
13 t 3
5 9 " 13
3 t 1
933 t 204
53 t 5
366 f 48
21 t 2
2.49 h 0.23
214 t 34
13 2 2
29 t 7
2 t 1
* Leukocytes were either counted on a hemocytometer or stained with monoclonal antibody as described in Patients and Methods and analyzed
by fluorescence-activated cell sorter. Lymphocytes were selectively gated based on light scatter properties. The number of cells was
determined by multiplying the percent positive by the total number of lymphocytes. Values are the mean 2 SEM ( X IO-'/liter) for each group.
See Table 1 for definitions.
THOMAS AND CARROLL
Table 3. Synovial fluid (SF) leukocytes and lymphocyte subsets in MTX-treated RA patients and in controls*
MTX-treated patients (n = 11)
Control patients (n = 4)
Cells in SF
Week 0
Week 8
Week 0
Week 8
Total leukocytes
Total neutrophils
% neutrophils
Total lymphocytes
% lymphocytes
Lymphocyte subsets
No. CD4+
% CD4+
No. CD8+
% CD8+
CD4:CD8 ratio
No. DR+
% DR+
No. CD25+
% CD25+
11,691 f 3,011
8,001 2 2,239
66 f 4
2,601 f 661
27 t 4
5,693 2 1,774
4,412 2 1,294
48 f 9
1,747 f 341
33 ? 8
6,000 2 2,581
3,332 t 2,026
47 f 10
2,504 2 234
37 2 6
7,650 t 2,989
4,849 t 3,101
43 f 15
2,428 f 239
28 t 6
1,127 t 307
39 f 4
941 f 286
28 f 4
1.92 k 0.46
1,158 f 450
34 f 6
249 t 117
7 f 1
746 t 97
43 f I
394 t 73
22 f 2
2.19 2 0.2
535 f 86
31 f 2
83 20
4 f 1
1,345 f 157
53 t 1
627 2 76
25 2 4
2.26 f 0.39
568 f 75
27 f 5
204 f 66
8 f 2
843 f 323
38 t 17
637 t 158
24 t 4
1.82 t 0.69
669 t 52
28 f 5
98 3 48
5 2 2
* Values are the mean
2
*
SEM ( X lO-%ter) for each group. See Tables 1 and 2 for further details and definitions.
compared with week 0 was greater in the MTX group
than the controls ( P = 0.0036) (Figure 1B and Table 1).
The Bonferroni correction for multiple tests on laboratory values gave a critical value for a (P)of 0.0031.
Therefore, with correction, this difference was not
statistically significant.
PB leukocyte response to MTX. The leukocyte
subpopulations in the MTX and control groups are
shown in Table 2. At the start of the study, the total
Table 4.
leukocyte count tended to be higher ( P = 0.051) and
the percentage of lymphocytes lower ( P = 0.045) in the
MTX group compared with the controls. No correlation was observed between the WBC count and any of
the clinical indices at week 0. In the PB, there was no
significant change in the WBC count or the CD4+,
CD8+, or DR+ lymphocyte subsets in either the MTX
or the control group during the 8-week period of
observation.
Synovial fluid (SF) leukocytes and neutrophils in MTX-treated RA patients and in controls*
SF levels at week 0
Patient
MTX-treated
MI
M2
M3
M4
MS
M6
M7
M8
M9
M10
M11
Control
c1
c2
c3
c4
Leukocytes
( X 10-61
liter)
Neutrophils
(x 10-9
liter)
31,200
12,000
2,000
30,000
5,700
9,300
7,000
12,000
7,500
7,000
10,900
4,700
13,000
5,700
600
SF levels at week 8
neutrophils
Leukocytes
( X 10-9
liter)
Neutrophils
( X 10-9
liter)
neutrophils
21,528
8,400
960
25,800
3,249
960
3,850
7,320
4,575
5,810
5,559
69
70
48
86
57
36
55
61
61
83
51
200
6,200
600
15,000
4,400
13,300
NA t
13,100$
3,300
100
1,025
160
3,782
456
11,100
2,464
6,517
NA
8,515
198
0
123
80
61
76
74
56
49
NA
65
6
0
12
2,726
8,580
3,021
72
58
66
53
12
9,000
15,000
5,900
700
5,130
11,850
2,655
42
57
79
45
6
%
%
* Eleven patients with RA were treated with MTX, and 4 patients did not take MTX (controls). See Tables 1 and 2 for further details and
definitions.
t NA = no SF could be aspirated at 8 weeks. At 4 weeks, the SF leukocyte count was 1,000 X 1O6/Iiter, neutrophils were 20 x IO'/liter (2%).
t SF leukocytes at 16 weeks were 450 x 106/liter, with 0 neutrophils.
RA SYNOVIAL FLUID AND MTX TREATMENT
A
*Oo0
r
T
*
0
0
0
2ooo
1
T
0
2ooo
U
R
500
0
i
1
1
T
I
1
I
I
0
7
28
56
Figure 2. Effect of MTX on SF lymphocyte subsets. MTX-treated
patients (0)received MTX for 8 weeks, and controls (0)
did not
take MTX. SF was collected before (0) and at 7, 28, and 56 days
after MTX was started. SF leukocytes were stained with either A,
anti-CD4,B, anti-CD8, or C, anti-DR and analyzed by fluorescenceactivated cell sorter. The percentage of positive cells was determined after gating lymphocytes. The number of cells in each subset
was determined by multiplying the percent positive by the total
number of SF lymphocytes. Values are the mean and SEM for each
patient group. See Figure 1 for definitions.
1249
SF leukocyte response to MTX. In contrast to
the PB leukocyte counts, the number of SF leukocytes
declined in patients receiving MTX, while in control
patients there was a trend toward an increase (Tables
3 and 4 and Figure 1D). The change in the number of
leukocytes between weeks 0 and 8 differed appreciably
between the 2 groups (P = 0.045), although with
correction, the difference was not statistically significant. In the MTX group, the reduction in SF leukocytes correlated weakly with 3 of the indices of clinical
response: patient's assessment of function (r = 0.53, P
< 0.03), overall score (r = 0.59, P < 0.02), and grip
strength (r = 0.47, P < 0.05).
SF neutrophil response to MTX. In both the
MTX and control groups the changes observed in total
leukocyte numbers were closely reflected by those
observed for neutrophils. A reduction in the number of
neutrophils in the SF was observed in the MTX group,
whereas an increase was seen in the control group
(Tables 3 and 4). The difference was not statistically
significant (P= 0.23). The proportion of neutrophils in
the synovial fluid fell in the MTX group but not the
controls (P = 0.019), although with correction the
difference was not statistically significant. There was
no change in the number or proportion of SF monocytes in the MTX group or the controls (data not
shown).
SF lymphocyte subset responses to MTX. A
reduction in the number, although not the proportion,
of lymphocytes was seen in the MTX group compared
with the controls, but this was not statistically significant. Reductions in the numbers of CD4+ lymphocytes were observed in both the MTX and control
groups (Figure 2A). These changes were not statistically significant in either group. Furthermore, the
proportion of CD4+ lymphocytes did not change in
either group. No change was observed in the number
of CD8+ lymphocytes in the control group, but in the
MTX group reduced numbers of CD8+ lymphocytes
were observed (P = 0.0833) (Table 3 and Figure 2B).
In the MTX group the CD4:CD8 ratio increased from
a mean k SEM of 1.92 k 0.46 to 2.19 ? 0.2, whereas
in the control group, it decreased from 2.26 0.39 to
1.82 ? 0.69. The difference was not statistically significant. An early but not statistically significant reduction in the number of DR+ lymphocytes was observed
only in the MTX group (Figure 2C). Reduced numbers
of c ~ 2 5 +lymphocytes were observed in both the
MTX and control groups, but these differences also
were not statistically significant- No statistically significant changes in leukocytes, neutrophils, or lym-
*
THOMAS AND CARROLL
1250
phocytes were observed in SF prior to the week-8
assessment.
Response of IL-lP concentration to MTX. A
reduction in the SF IL- 1p concentration was observed
at 8 weeks in the MTX group but not in controls (P =
0.0376) (Figure 3). With correction, the difference was
not statistically significant.
1000
500
DISCUSSION
In this study of serial changes in clinical parameters and in PB and SF leukocytes following the
administration of MTX, our results confirm the effectiveness of low-dose oral MTX in RA. The rapidity of
clinical improvement was notable, and consistent with
previous reports (3). Although the control group patients had less active RA than did those in the MTX
treatment group, there was nevertheless a clear clinical response to MTX treatment compared with the
treatment taken by the controls. The control group
tended to have less active RA because they were
considered to have stable disease and did not require
additional treatment.
In this study, no significant changes in PB
leukocytes were observed in patients receiving MTX.
The results of previous studies of the effect of MTX on
PB inflammatory cell populations have been inconsistent. Weinblatt et a1 (2), for example, found a significant increase in the percentage of PB CD3+ and
CD4+ lymphocytes in patients treated with MTX for 2
years (compared with baseline). Furst and Kremer (5)
found a decrease in the total PB lymphocyte count and
in the CD8+ lymphocytes after a mean of 28 months of
treatment with MTX. Andersen et a1 (4) found an
increase in the percentage of PB CD3+ cells and a
decrease in monocytes after 14 weeks of MTX therapy. And, Olsen et a1 (6), who compared patients
treated with MTX for varying periods of time with
non-MTX-treated RA patients, found no difference in
the numbers of PI3 B cells, CD4+ and CD8+ lymphocytes, or the CD4:CD8 ratio. Our study provides
further evidence that the effect of MTX on the numbers or proportions of major circulating PB leukocyte
populations is relatively minor. However, differences
in the patient populations studied or the timing of the
measurements may be responsible for some of the
inconsistencies noted. One patient in our control
group continued taking DMARD treatment throughout
the study (patient C3, Table 4). Although she had been
on a stable regimen for at least 18 months and no
change in PB or SF leukocytes would have been
0
L
I
I
I
0
7
28
56
Time ( d a y s )
Figure 3. Effect of MTX on SF interleukin-lp (IL-lp) concentration. MTX-treated patients ( 0 )received MTX for 8 weeks, and
controls (0)did not take MTX. SF was collected before (0) and at 7,
28, and 56 days after MTX was started. SF IL-1p was measured by
enzyme-linked immunosorbent assay. Values are the mean and
SEM for each group. See Figure 1 for other definitions.
expected over the 8-week study period, such a change
in response to DMARD therapy cannot be excluded.
In this study, appreciable reductions were observed in the total number of leukocytes and in the
number and proportion of neutrophils in the SF of
patients treated with MTX for 8 weeks. Decreases in
the total number of SF lymphocytes and in the CD8+
and DR+ lymphocyte subsets were also apparent in
patients treated with MTX. Although a decline in SF
CD4+ and CD25+ lymphocytes was seen in the MTX
group, a similar downward trend was observed in the
control group. Our study is limited by the small
number of patients examined and the high degree of
variability between patients in both the treatment and
control groups, consistent with the findings of others
(7). However, the trends in this study suggest that in
the first 8 weeks of treatment, MTX reduces the
number and proportion of neutrophils and the number
of lymphocytes in the SF, the latter of which could not
be ascribed to any particular lymphocyte subset examined in this study.
The reduction in leukocyte numbers correlated
with clinical improvement in response to MTX. This
suggests that the reduction in SF leukocytes is important for the clinical response to MTX. Several explanations for the reduction in inflammatory cells in the
RA SYNOVIAL FLUID AND MTX TREATMENT
SF, based on the known actions of MTX, are possible.
First, MTX could reduce chemotaxis and migration of
neutrophils to the inflamed joint by alteration of the
concentration or activity of secreted cytokines or
other chemoattractants. Similarly, the binding and
migration of lymphocytes could be affected either
directly or through an effect on modulating cytokines.
Alternatively, MTX could directly suppress lymphocyte proliferation in the rheumatoid synovium, or less
likely, MTX could suppress the turnover of neutrophil
and lymphocyte precursors in the bone marrow, thus
reducing the number of inflammatory cells which
could potentially reach the inflamed joints.
Utilizing 3H-deoxyuridine uptake as a marker
of DNA synthesis, the antiproliferative effect of MTX
in vitro has been demonstrated in many cell types,
including bone marrow myeloid precursors (1 1,18,19).
Because the changes in lymphocytes and neutrophils
in this study were evident in the SF but not in PB, it is
not likely that MTX is acting via a generalized suppressive effect on PB cells or their precursors. Furthermore, since few T cells appear to proliferate in the
synovium (20-22), it is also unlikely that the preferential accumulation of MTX by actively proliferating
synovial lymphocytes accounts for reduced numbers
of these cells in the SF.
Studies of rheumatoid synovium have shown
that lymphocytes pass through synovial venules to
form perivascular lymphocyte-rich infiltrates in which
CD4+, CD45RO+ T cells with an activated (DR+ and
CD29+) phenotype predominate (23,24). A small proportion of T cells also express CD25 (24). Although not
statistically significant, downward trends in the concentration and proportion of DR+ and CD25+ lymphocytes were observed in the SF of patients treated
with MTX. Thus, it remains possible that MTX could
reduce the activation of lymphocytes within the
synovium.
It is more likely, however, that MTX reduces
the recruitment of cells into the synovium. Our findings indicate that MTX may have a general effect on
leukocyte migration and/or chemotaxis that may be
responsible for the reduced cellularity of the synovial
fluid. MTX has been demonstrated to reduce neutrophi1 chemotaxis and to decrease the in vitro production of the chemoattractant leukotriene B, (LTB,) by
macrophages (25-27). LTB, and complement factor
Sa-induced neutrophil migration was also reduced by
MTX in vivo (26,27). We found reduced levels of
immunoreactive IL-1p in the SF of MTX-treated patients. This was not associated with a concomitant
125 1
decrease in the concentration or proportion of SF
monocytes. Since IL-1 enhances adherence of neutrophils and lymphocytes to endothelial cells and promotes capillary leakiness, it is possible that the
reduced concentrations of IL-1 in SF may have contributed to reduced leukocyte migration (10). It is also
possible, however, that the reduction of both IL-1p
and leukocytes in SF could be the result of another,
as-yet-unknown function of MTX. For example, the
findings could be explained by MTX toxicity to cells
migrating into the joint, including those that secrete
1L-1.
Previous studies using IL- 1-responsive cell
lines have shown either reduced production or reduced activity of IL-1p in monocytes treated with
MTX (28-33), but IL-1p concentrations in inflammatory exudates after treatment with MTX have not
previously been examined. Our findings suggest that
spontaneous monocyte-derived IL-l p production may
be reduced in vivo after MTX treatment. Inasmuch as
IL-1 has pleiotropic effects on articular tissues such as
the activation of chondrocytes, osteoblasts, and osteoclasts, as well as the stimulation of prostaglandin,
metalloproteinase, and collagenase production (34,35),
it is possible that reductions in IL-1 concentrations
may contribute to the decrease in joint inflammation
that accompanies the clinical use of MTX.
The findings in this study are consistent with
the hypothesis that in RA, MTX acts, at least in part,
by reducing the migration and/or proliferation of leukocytes at sites of local inflammation. Although only a
small number of patients were studied, it is evident
that this effect occurs within the first 8 weeks of
therapy, along with the clinical response to MTX.
ACKNOWLEDGMENTS
We thank Beverly Laing for performing the disease
activity measurements, Michael Bell and James Merriman
for technical assistance, Dr. Valerie Burke, Beverly Laing,
and Linda Williams for help with the statistical analysis, and
Dr. Laurie Davis for critical review of the manuscript.
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