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Evidence of a local intestinal immunomodulatory effect of sulfasalazine in rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM Volume 37
Number 8, August 1994, pp 1138-1145
0 1994, American College of Rheumatology
1138
EVIDENCE OF A LOCAL INTESTINAL IMMUNOMODULATORY
EFFECT OF SULFASALAZINE IN
RHEUMATOID ARTHRITIS
LARS KANERUD, ANNIKA SCHEYNIUS, and INGIALD HAFSTROM
Objective. To analyze whether the intestinal mucosa in rheumatoid arthritis (RA) is immunologically
abnormal and whether sulfasalazine (SSZ) possesses any
local intestinal immunoregulatory effect.
Methods. Lymphocyte subpopulations and
HLA-DR expression were evaluated in biopsy specimens from the duodenal-jejunal mucosa and in peripheral blood samples obtained from 17 patients with
RA, both before and after 16 weeks of SSZ treatment.
The same mucosal assays were also performed in 7
controls.
Results. The mucosa of the small intestine in RA
patients showed no differences in morphology, HLA-DR
expression, or the amounts and distribution of CD3+,
CD4+, CD8+, and y/S+ lymphocytes compared with
the control group. However, there was a reduction in
mucosal CD3+ and y/S+ lymphocyte numbers after
SSZ therapy, which did not correspond to a change in
peripheral blood CD3+ lymphocyte number. SSZ treatment also tended to diminish the peripheral blood
CD4+:CD8+ cell ratio (P = 0.05).
Conclusion. No signs of inflammation or immunologic abnormalities were seen in RA duodenal-jejunal
mucosa. In this part of the intestine, however, SSZ
exerted immunoregulatory effects that were not encountered in the peripheral blood.
Supported by grants from the Swedish Medical Research
Council, the Swedish Association Against Rheumatism, Professor
Nanna Svartz' Foundation, King Gustaf V's 80-Year Fund, the Alex
and Eva Wallstrom Foundation, the Ulla and Gustaf af Ugglas
Fund, Bode Dahlin's Fund, and the 'Forenade Liv' Mutual Group
Life Insurance Company, Stockholm, Sweden.
Lars Kanerud, MD, PhD: Stockholm Soder Hospital,
Karolinska Institute, Stockholm, Sweden; Annika Scheynius, MD,
PhD: Stockholm Soder Hospital, Karolinska Institute; Ingidd Hafstrom, MD, PhD: Stockholm S d e r Hospital, Karolinska Institute.
Address reprint requests to Lars Kanerud, MD, Department of Rheumatology, Stockholm Soder Hospital, s-118 83 Stockholm, Sweden.
Submitted for publication May 28, 1993; accepted in revised
form February 27, 1994.
Rheumatoid arthritis (RA) is a disease of unknown etiology and only partially elucidated pathogenesis. However, the association of joint disease with
a variety of bowel disorders (l), together with reports
of abnormalities in the gastrointestinal tract in RA
patients (2-9), have led to speculation about a possible
etiopathogenic relationship between the gut and RA
(10-13). A common hypothesis is that phlogistic luminal macromolecules (e.g., bacterial cell wall fragments
and dietary proteins) might gain access to the intestinal
lamina propria and the systemic immune system via a
disrupted or otherwise altered intestinal mucosal barrier (14) and elicit an autoimmune response, possibly
by molecular mimicry (15).
There is evidence of altered bowel permeability
in RA (2,16,17) and of subclinical intestinal inflammation, as revealed by "'indium-labeled leukocyte scanning (3,4,16). Furthermore, vasculitic and inflammatory changes have been found in various parts of the
gastrointestinal tract in RA patients (5-7), including
the stomach, colon, and rectum. In the duodenum and
jejunum, various findings have been described, including normal histology (I@, increased numbers of inflammatory cells (€9, and a high frequency of partial
villous atrophy (9).
The immunohistology of the gut mucosa in RA,
however, is largely unknown, despite speculation concerning the role of the gut in the chronic immune
activation characteristic of RA. The use of sulfasalazine (SSZ)-a
drug commonly used to treat colitis-as
an antirheumatic drug has further focused on the
possibility of an alteration of the immune function in
the gut in RA patients. The mode of action of SSZ,
however, is obscure. Different immunomodulatory
effects on peripheral blood leukocytes in vitro have
been reported (19-22), but most require drug concentrations higher than those normally attained therapeutically in the circulation. On the other hand, high drug
LOCAL GUT IMMUNOMODULATORY EFFECT OF SSZ
levels have been reported in the intestinal mucosa (23)
and in ileostomy effluent (24), which makes the notion
of a local gut-mediated immune effect plausible (25).
In this study we examined the duodenal-jejunal
mucosa in untreated RA patients, by using immunohistochemical techniques to define the amount and
distribution of CD3 +, CD4+, CD8+, and y/6+ lymphocytes, as well as the expression of HLA-DR antigens.
Further, by comparing the findings in the intestinal
mucosa with those in peripheral blood of the same
patients, we studied whether there is any local effect of
SSZ treatment on the mucosal distribution of these
cells and on the expression of HLA-DR antigens.
PATIENTS AND METHODS
Patients. Twenty-one patients (17 women and 4 men)
with clinically active RA were included in the study. All
fulfilled the 1958 criteria of the American College of Rheumatology for a diagnosis of classic or definite RA (26), and all
were seropositive for rheumatoid factor. Their mean age was
51 years (range 24-71), and the mean duration of disease was
73 months (range 6-204). None of the patients had undergone upper gastrointestinal surgery or complained of gastrointestinal symptoms.
None of the patients had received treatment with
gold, penicillamine, chloroquine, sulfasalazine, corticosteroids, or immunosuppressants for the 3 months prior to
study inclusion. Four patients were taking nonsteroidal
antiinflammatory drugs (NSAIDs); these 4 patients demonstrated no clinical or laboratory differences from the other
patients, either before or after SSZ therapy, nor was there
any difference in their response to therapy.
Clinically active disease was defined as the presence
of at least 2 of the following 3 criteria: duration of morning
stiffness 260 minutes, tenderness and/or swelling of 2 6
joints, and Westergren erythrocyte sedimentation rate (ESR)
230 mdhour.
Controls. Seven ambulatory patients (6 women and 1
man, with a mean age of 30 years [range 21401) who had
discrete abdominal symptoms but no clinical or laboratory
signs of inflammatory bowel disease or malabsorption,
served as controls. Some of these patients were seen at the
rheumatic disease ward because of muscle or joint symptoms, but none had RA or any clinical or laboratory evidence
of inflammatory joint disease. None of the control patients
was receiving continuous antiinflammatory medication.
Study design. The RA patients were started on a
regimen of 1.5-3 gm of SSZ daily. The following investigations were undertaken both before and after 16 weeks of
therapy. Disease activity was assessed using an index based
on the Lansbury joint index, the duration of morning stiffness, and the ESR, as described elsewhere (27). Biopsy of
the small intestine was performed, and tissues were analyzed
by immunohistochemical methods, as well as routine histologic techniques (on most samples). Peripheral blood was
1139
drawn, and the numbers of leukocytes and lymphocytes of
different subpopulations were counted.
The small intestine was also biopsied in the control
patients, and tissues were evaluated in the same manner as
above.
All subjects had given their informed consent prior to
the study. The study was approved by the local Ethics
Committee.
Biopsies. After an overnight fast, small bowel biopsy
specimens were taken from the duodenal-jejunal junction
with either a Watson capsule or a Baumgartner capsule,
under fluoroscopic guidance. The latter instrument allows
for multiple biopsies; the former, only one. Depending upon
the amount of intestinal mucosa obtained, the biopsy samples were either divided into parts or used whole. Samples
were snap-frozen in liquid nitrogen and stored at -70°C for
subsequent immunohistochemical staining. For those with
enough material, a second sample was fixed in formalin and
subjected to routine histologic examination.
Immunohistochemical staining. An indirect peroxidase method (28) was used. Biopsies were sectioned in a
cryostat, and sections (6p) were fixed in acetone for 5
minutes at 4°C. Endogenous peroxidase was blocked by
incubation in 0.3% H202 in phosphate buffered saline (PBS)
for 10 minutes. To reduce nonspecific staining, sections were
allowed to react with normal rabbit serum (diluted 1:lO) for
10 minutes.
The following mouse monoclonal antibodies were
used as primary antibodies: anti-Leu-4 (CD3), anti-Leu-3a
(CD4), anti-Leu-2a (CD8), and anti-HLA-DR, all from
Becton Dickinson (Mountain View, CA), and TCRSl (recognizing $8 T cells) from T Cell Sciences (Cambridge, MA).
Rabbit anti-mouse immunoglobulin (diluted 1:40; Dakopatts,
Copenhagen, Denmark) was used as secondary antibody,
and sections were incubated for 3 @ 4 0 minutes. Preformed
complexes of horseradish peroxidase and mouse monoclonal
anti-horseradish peroxidase (Dakopatts) were used, and the
reaction was developed with 3-amino-9-ethylcarbazole before counterstaining with Mayer’s hematoxylin. Optimal
dilutions of antibodies were determined using sections from
normal lymph nodes. Specificity tests included omission of the
primary antibodies; staining was not observed in these tests.
All sections were examined under light microscopy
and read under coded conditions by the same observer (LK).
At least 2 sections per antibody and per biopsy were
examined, and in each section at least 5, but often many
more, villi and the corresponding area above the muscularis
mucosa were evaluated. Staining was scored on a scale of
0-3+, where 0 = none, 1+ = few, 2+ = moderate, and
3+ = many positive cells. Only those lymphocytes with the
entire circumference stained were considered positive. Epithelial expression of HLA-DR antigens was also estimated
semiquantitatively on a scale of 0-3+, according to the
extent and intensity of staining. The scale was adjusted for
each antibody; thus, a grade of 3+ represents the maximal
number of positive cells or the maximal extent and intensity
of staining within all specimens.
Peripheral blood leukocytes. Total leukocyte counts
and differential cell counts were assessed using a Technicon
H 6000 system (Tarrytown, NY). Lymphocyte subpopulation percentages were determined by flow cytometry. Mono-
KANERUD ET AL
1140
nuclear cells were isolated from heparinized samples of
venous blood using Lymphoprep (Pharmacia,Uppsala, Sweden) density gradient centrifugation and washed twice in
PBS containing 1% bovine serum albumin. Cells were incubated using combinations of the following fluorescein
isothiocyanate-conjugated or phycoerythridconjugatedmonoclonal antibodies: anti-Leu-M3 (CD14), anti-Leu-4 (CD3),
anti-Leu-3a (CD4), anti-Leu-2a (CD8), and anti-HLA-DR
(Becton Dickinson), and polyvalent anti-human Ig (recognizing
B cells; Kallestad, Chaska, MD) plus isotype-matched negative controls.
Incubations were performed at 4°C for 30 minutes
using 1 x lo6 cells per tube and saturating amounts of
antibody. After washing once, the cells were resuspended,
and flow cytornetry was performed using a fluorescenceactivated cell sorter (FACStar; Becton Dickinson) equipped
with an argon-ion laser tuned at 488 nm. The analysis gate
was set for live lymphocytes, using forward-angle scatter
and 90” side scatter to gate out debris and anti-CD14 antibody to ensure that the gate contained not more than 2%
monocytes. Data were stored in ‘‘list’’ mode and analyzed
using the Consort 30 data program (Becton Dickinson).
Statistical analysis. Only nonparametric 2-tailed tests
were used. For comparisons between RA patients before
and after therapy, the Wilcoxon matched pairs test was
applied, and for comparisons between RA patients and
controls, the Mann-Whitney U test was used. Spearman’s
rank correlation test was used for correlation purposes.
P values less than 0.05 were considered significant.
RESULTS
Three patients had to stop SSZ therapy because
of potentially serious side effects. Except for the
patient in whom the immunohistochemical analyses
could be performed, the data from these 3 patients are
not included. Two patients did not want to participate
in the intubation procedure, one because of nausea
experienced previously, and the other because of
pregnancy. These patients are excluded from the
immunohistologic studies. The flow cytometry tests
were incomplete because of technical problems or
sampling errors in a few patients.
Disease activity. The 18 patients who completed
the full 16-week protocol of SSZ treatment responded
with significantly diminishing inflammatory activity
(27) (Figure 1). The disease activity index declined
from 77.6 k 32.1 to 27.4 -t 18.4 units (mean -t- SD)
(P < 0.001). No patient’s condition deteriorated during
therapy.
Findings of routine histologic examination of
biopsy tissues. No gross pathologic features were seen
on formalin-fixed, hematoxylin and eosin-stained sections from the 17 RA patients and the 7 control
I
150
Morning stiffness
I
1
2 120
1
\
E
E 90
ffl
+
.-
5
60
rn
e,
c
3
.c
_
30
E
0
untreated
a f t e r 1 6 w SASP
Figure 1. Mean disease activity in 18 rheumatoid arthritis patients,
according to the duration of morning stiffness, an articular index of
tender andlor swollenjoints, and the erythrocyte sedimentation rate
(ESR; Westergren) before and after 16 weeks of treatment with
sulfasalazine (SASP). Mean -C SD values before sulfasalazine therapy were 127 f 125 minutes, 100 5 39 units, and 47 f 21 mmlhour,
respectively, and were 8 f 11 minutes, 53 2 37 units, and 21 f 13
mmlhour, respectively, after 16 weeks. * = P < 0.001, by Wilcoxon
matched pairs test.
patients studied. In 2 RA patients, only biopsy tissues
obtained after therapy were examined.
Findings of immunohistological staining. Biopsy
specimens from 17 RA patients before and after SSZ
therapy and from 7 controls were examined. In all
samples, CD3+ lymphocytes were frequent, both in
intraepithelial areas and in the lamina propria. There
was a dominance of CD4+ over CD8+ cells in the
lamina propria and the opposite configuration in the
epithelium. There was a significant correlation between the number of CD3+ and CD8+ cells in the RA
patients, both before and after therapy (r, = 0.64, P <
0.01 and r, = 0.59, P < 0.05, respectively), as well as
in the control patients (r, = 0.75, P < 0.001).
Gammaldelta-positive T cells were preferentially located intraepithelially but were not infrequent
in the lamina propria. Seemingly, the higher the number of ylS T cells in the section, the higher the
proportion that were located within or adjacent to the
epithelium. There was considerable variation in ylS T
cell numbers among patients and controls. A correlation between the numbers of y’S T cells and CD8+
cells was found in the RA patients after therapy (r, =
0.53, P < 0.05) and a tendency was seen before
therapy (r, = 0.47, P < 0.1). No significant difference
in the number (Table 1) or location of CD3+ , CD4+,
1141
LOCAL GUT IMMUNOMODULATORY EFFECT OF SSZ
Table 1. Semiquantitative evaluation of lymphocyte subpopulations and HLA-DR antigen expression in the duodenal-jejunal mucosa of untreated patients with rheumatoid arthritis (RA) and controls
~~
CD3+
CD4+
Score
RA
Controls
1+
2+
3+
7
10
CD8+
RA
Controls
RA
-
-
-
4
3
15
5
2
2
11
CD8+, or y/S T cells was found between the untreated
RA patients and the controls.
After SSZ treatment, there was a significant
decrease in the numbers of CD3+ cells and ylS T cells
(for both cell types in 5 of 17 RA patients; P < 0.09,
whereas the numbers of CD4+ and CD8+ cells were
unchanged (Table 2). There was no difference in
clinical improvement or other clinical or laboratory
variables in the patients showing a decrease in CD3+
or y/S T cells compared with the other patients.
Moreover, there was no correlation between the
amounts of the different lymphocyte subpopulations
and the age of the RA patients.
HLA-DR expression. Positive epithelial staining
was noted in all patients and controls, but there was a
clear variation in the distribution and intensity of the
staining among different biopsy specimens as well as
within the same specimen. Staining was most promiTable 2. Semiquantitative evaluation of lymphocyte subpopulations and HLA-DR antigen expression in the duodenal-jejunal
mucosa of patients with rheumatoid arthritis, before and after
sulfasalazine (SSZ) therapy
Cell subset,
score before SSZ
CD3+
1+
2+
3+
CD4+
1+
2+
3+
CD8+
1+
2+
3+
yl6+
1+
2+
3+
HLA-DR+
1+
2+
3+
Score after SSZ
1+
2+
6
4
14
1
1
5
-
3
1
1
3
1
3+
HLA-DR+
yl6+
Controls
RA
Controls
RA
Controls
1
-
7
3
4
6
4
1
6
-
3
5
7
7
1
3
3
nent along the luminal end of the enterocytes, covering
the tips and the lateral borders of the villi, but basolateral staining was also seen. Epithelial expression
diminished toward the crypts, where staining was
mostly weak or absent. Granular staining in the cytoplasm of the enterocytes was noted, and was always
most dense near the lumen. In the lamina propria,
positive-stained cells of different types were abundant.
No correlation between epithelial expression of
HLA-DR and either the lymphocyte subset numbers
or the patient’s age was noted. Furthermore, no significant difference in epithelial HLA-DR expression
was seen in RA patients versus controls or in the RA
patients before versus after SSZ therapy (Tables 1
and 2).
Peripheral blood leukocyte numbers. The total
number of peripheral blood leukocytes and the numbers of lymphocytes, monocytes, and granulocytes
diminished significantly during SSZ therapy. The values before therapy and the difference after 16 weeks of
therapy are shown in Table 3.
With regard to lymphocyte subsets, the percentage of CD3+ cells increased (though not statistically significant; P = 0.08), while the percentage of
CD8+ cells increased significantly (P < 0.05) (Table
4). However, as the total number of lymphocytes
decreased, the absolute numbers of these two lymphocyte subpopulations did not change. There was no
Table 3. Peripheral blood leukocyte numbers in 17 rheumatoid
arthritis patients before, and the difference after, 16 weeks of
sulfasalazine therapy
Cell
type
Week 0
(mean -+ SD
x lo’iliter)
Difference
at week 16
(mean L SD
change)
Leukocytes
Lymphocytes
Monocytes
Granulocytes
7.5 2 1.5
2.0 2 0.5
0.5 -c 0.2
5.0 t 1.1
-1.1 2 1.4*
-0.3 t 0.4”
-0.05 ? 0.11*
-0.8
1.3*
* P < 0.05.
t P < 0.01.
*
1142
KANERUD ET AL
Table 4. Percentages and absolute numbers of peripheral blood lymphocyte subsets and HLA-DRexpressing lymphocytes in 17 rheumatoid arthritis patients before, and the difference after, 16 weeks
of sulfasalazine therapy
Percentages
Difference
at week 16
(mean f SD
change)
Monoclonal
antibody
No. of
patients
Week 0
(mean SD)
Leu-4 (CD3)
Leu-3a (CD4)
Leu-2a (CD8)
CD4:CDS ratio
Polyvalent
anti-human Ig
HLA-DR
17
15
16
15
14
60.5 2 11.8
41.6 f 12.2
21.8 2 9.7
2.5 f 1.5
16.0 f 7.7
7.0
2.5
3.9
-0.5
10
19.4 f 4.9
-4.5
*
Absolute numbers
2 12.2
f 11.9
f 7.5*
f 1.3t
0.6 2 10.9
f
8.2
Week 0
(mean f SD
x lo’tliter)
Difference
at week 16
(mean i SD
change)
1.22 f 0.51
0.85 i 0.41
0.44 -t 0.24
2.5 f 1.5
0.31 f 0.17
-0.09
-0.12
0.01
-0.5
-0.02
f 1.3t
f 0.19
0.39
-0.12
2
2
0.15
f 0.33
f 0.28
* 0.14
0.20
* P < 0.05.
7P
=
0.05.
significant change in the percentages or the absolute
numbers of CD4+ cells or of polyvalent anti-human
Ig + cells. The CD4 + :CD8+ cell ratio diminished from
2.5 to 1.9 (P = 0.05). Although not significant (P =
O.ll), a decline in HLA-DR+ cell numbers after
therapy was also noted.
DISCUSSION
The present investigation arose from the
hypothesis that the gut may play a pathogenic role in
RA. If this is true, one could expect to find immunologic abnormalities or immunologic changes in the
intestinal mucosa during therapy. We chose to study
the mucosa at the duodenal-jejunal junction because
this part of the intestine is affected in nontropical sprue
(celiac disease) and in Whipple’s disease, both of
which are associated with arthralgias or arthritis (1).
Abnormal function of this intestinal segment, despite
morphologically normal appearance, is also described
in Crohn’s disease (29).
To our knowledge, there are no previous reports regarding studies of lymphocyte subsets in the
intestinal mucosa in RA and, consequently, no data
concerning the effects of SSZ or other antirheumatic
agents on this compartment. In inflammatory bowel
disease, studies of the local effects of SSZ on lymphocyte subsets in the intestinal mucosa are also scarce,
but SSZ has been shown to normalize the increased
mononuclear cell yield from colonic biopsies in patients with ulcerative colitis, as well as to correct the
reduced T cel1:B cell ratio associated with active
disease (30).
In the present study, histologic examination of
the RA mucosal biopsy samples stained with hematoxylin and eosin revealed no abnormalities compared with
controls. This is consistent with findings of an earlier
investigation of the mucosa from the duodenal-jejunal
junction (18), but differs from the results of 2 other
reports, in which there was an increase in inflammatory
cells in the lamina propria in 4 of 18 patients with RA (8),
and a high incidence of partial villous atrophy in 4 of 15
seropositive RA patients (9). In these latter 2 studies,
many patients had symptoms of gastrointestinal disturbances and were receiving various kinds of medical
treatment that might have affected the mucosa.
Using immunohistochemical techniques, we
found no difference in the amounts of CD3 + , CD4+,
and CD8+ cells in patients with untreated RA compared with controls. The distribution pattern, with a
predominance of CD4+ over CD8+ cells in the lamina
propria and a predominance of CD8+ to CD4 + cells in
the epithelium in all patients and controls, is consistent
with earlier studies of normal small intestine (31,32).
After SSZ therapy, we observed a reduction of CD3+
cells in the mucosa of the small intestine of the RA
patients, but no change in the CD4+ and CD8+ cell
profile.
T lymphocytes bear a T cell receptor (TCR) that
is composed of either an cdp or a y/S heterodimer
associated with the CD3 complex. The TCRy/G+ cells
are mostly of the CD3+, CD4-, CD8- phenotype
(double negative), but a minority express either
CD4+, CD8- or CD4-, CD8+ (33,34). Our results
show that there is considerable variation in the num-
LOCAL GUT IMMUNOMODULATORY EFFECT OF SSZ
bers of ylS T cells in the small intestine mucosa, both
in RA patients and in controls, a finding that is
consistent with other studies of normal mucosa
(34,35). The TCR y/S+ cells were preferentially located
in, or adjacent to, the epithelium, but small amounts
were also found in the lamina propria. There was no
difference in the amount and localization of this T cell
subset in untreated RA patients and controls; however, the numbers of TCR y/S+ cells in RA gut mucosa
decreased during SSZ treatment, as was noted with
CD3+ cells. We also found a weak correlation between the amounts of y/S T cells and CD8+ cells in the
RA patients, which might suggest that a significant
proportion of the y/S T cells in RA have the CD3+,
CD4-, CD8-t phenotype. Intraepithelial T lymphocytes of this phenotype in the intestinal mucosa have
been described in up to 50% (36) or more (37) of
normal subjects.
The function of TCRy/S+ cells is unclear. A
possible role in immune surveillance at mucosal sites
has been suggested (38), because early reports showed
that gut mucosal intraepithelial lymphocytes in mice
predominantly express TCR y/S (39). Subsequent immunohistochemical studies in humans, however, have
shown that not more than 10% of the CD3+ intraepithelial lymphocytes are TCR y/S+, which is approximately the same proportion as in peripheral blood
(40,41). There are, however, a few exceptions. In
celiac disease, the proportion of TCR y/S+ intraepithelial lymphocytes is increased also after dietary treatment (35). High proportions of TCRy/G+ intraepithelial T lymphocytes have also been found in dermatitis
herpetiformis (42) and in patients with cow’s milksensitive enteropathy and postenteritis syndrome (43),
and increased numbers have been demonstrated in
patients with Helicubacter pyluri-associated chronic
gastritis of the antrum (44). However, since increased
amounts of y/S T cells do not seem to be part of a
generalized response in intestinal inflammation (36),
our finding of reduced numbers of $8 T cells in RA
patients during SSZ treatment may indicate a local,
specific regulatory effect of SSZ.
In studies of the peripheral blood, we observed
reduced numbers of lymphocytes, monocytes, and
polymorphonuclear granulocytes after SSZ therapy.
The absolute number of CD8+ cells did not change;
instead, there was a slight, but not significant, decrease in the absolute numbers of CD4+ cells, which
resulted in a statistically significant increase in the
percentage of CD8+ cells, as well as a close to
significant decrease ( P = 0.05) in the CD4+:CD8+ cell
1143
ratio. These data correspond very well to those reported after successful treatment of RA patients with
penicillamine or chloroquine (45). Thus, the slight
decrease in CD4+ cell numbers does not seem to be
specific for SSZ, but rather, may reflect mutual immunoregulatory effects by these antirheumatic drugs or
simply a decrease in disease activity. In contrast,
Symmons et a1 (19) found no reduction in the median
numbers of total lymphocytes or CD3+, CD4+, or
CD8+ lymphocytes after 12 weeks of SSZ treatment,
despite improvement in most of the clinical variables
except the articular index in patients who were also
treated with NSAIDs. This points to the importance of
detailed recording of medications taken, since NSAID
therapy may have influenced these results (45).
The effect of SSZ therapy on peripheral blood
lymphocyte subpopulations differed from the effect on
the mucosa of the small intestine, where a reduction of
CD3+ lymphocytes was seen. This difference might be
interpreted as a local immunomodulatory effect of SSZ
in the gut mucosa, which is encountered with drug
concentrations considerably higher than those
achieved in peripheral blood and, presumably also,
than those in bone marrow. A similar hypothesis has
been put forward by Sheldon et a1 (25), who proposed
that there is an effect of SSZ on the lymphocyte traffic
pathways of cells emerging from the gut-associated
lymphoid tissue of the small intestine. Those investigators demonstrated suppressive effects of SSZ on T
cell and B cell mitogen-induced lymphocyte transformation of murine spleen cells and human lymphocytes, but only at high concentrations (21,25). Other
functional in vitro studies imply that SSZ in concentrations easily attained in the small intestine has profound effects on both B lymphocytes and T lymphocytes, whereas in the lower concentrations achieved in
peripheral blood, SSZ affects merely B lymphocytes
(19,20,22).
Besides the normal distribution of lymphocyte
subpopulations in the mucosa of the small intestine of
the RA patients, we also identified a distribution of
HLA-DR antigens, which is consistent with earlier
reports of normal proximal small intestine (46,47).
There was no correlation between HLA-DR expression and mucosal lymphocyte subsets within the specimens, and no difference in HLA-DR expression between RA patients and controls or in RA patients
before versus after SSZ treatment.
In studies of peripheral blood, we found that
19% of the lymphocytes were HLA-DR+ in untreated
RA patients (Table 4), which is compatible with find-
KANERUD ET AL
1144
ings of earlier studies (48,49). After SSZ therapy, this
number decreased, although not significantly, to 15%.
In peripheral blood, most HLA-DR+ cells are B cells
and a minority are T cells or monocytes. An elevated
number of HLA-DR+ T cells has been reported in RA
(50-52) and in various other conditions (50), and this
feature is thought to reflect T cell activation. The
reduction (although not statistically significant) of
HLA-DR+ cells in this study cannot be explained by
diminishing numbers of polyvalent anti-human Ig+ cells,
but suggests a small decrease in HLA-DR+ T cells.
The essence of our findings is that the duodenaljejunal mucosa in RA patients does not show any
evidence of abnormalities with regard to morphology,
HLA-DR expression, or the amounts and the distribution of CD3+, CD4+, CD8+, andy/6+ lymphocytes. However, SSZ seems to exert a local immunoregulatory effect in the mucosa, since the amounts of
CD3+ and y/6+ lymphocytes decreased during therapy. We found no corresponding decrease in CD3+
lymphocytes in peripheral blood, which indicates that
this immunoregulatory effect may require higher drug
concentrations than are normally achieved in the
blood. Sulfasalazine therapy does, however, tend to
normalize the imbalance between the CD4+ and
CD8+ lymphocyte subsets in peripheral blood, which
has been described in RA ( 4 9 , as well as the numbers
of HLA-DR+ lymphocytes. These effects may not be
specific for the drug, but instead, may be shared by
other antirheumatic drugs or may merely reflect diminishing disease activity. Whether the effects on mucosal
lymphocyte subsets we describe have any relevance
for the antirheumatic effects of SSZ is still an unresolved question.
ACKNOWLEDGMENTS
We are very grateful to Catharina Johansson, Anne
Svensson, and Katarina Karlstrom for excellent technical
assistance, to Gunilla Kumlien for performing the flow
cytometric analysis, and to Marja Winberg for secretarial help.
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local, effect, intestinal, evidence, sulfasalazine, arthritis, immunomodulator, rheumatoid
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