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Phenotypic analysis of synovial tissue and peripheral blood lymphocytes isolated from patients with rheumatoid arthritis.

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1230
PHENOTYPIC ANALYSIS OF SYNOVIAL TISSUE AND
PERIPHERAL BLOOD LYMPHOCYTES ISOLATED
FROM PATIENTS WITH RHEUMATOID ARTHRITIS
JOHN J . CUSH and PETER E . LiPSKY
Cytofluorometric analysis was performed to
characterize the surface phenotype and activation status
of freshly isolated synovial tissue lymphocytes (STL) and
peripheral blood lymphocytes (PBL) from 7 patients
with rheumatoid arthritis (RA). Proliferative synovium
was enzymatically digested to obtain tissue-derived lymphocytes. Indirect immunofluorescent staining of patient PBL and STL with a variety of monoclonal antibodies failed to reveal a consistent alteration in the
number of CD4+ (helperhnducer) PBL or STL. However, there was a significant decrease in the number of
CD8+ (suppressor/cytotoxic) cells in rheumatoid STL
(P < 0.05). A significant reduction in the density of the
T cell differentiation antigens CD3 and CD4 was observed in RA PBL and STL, compared with control
PBL. These differences in antigen density were not seen
when normal PBL were subjected to the same enzymatic
digestion. Both RA PBL and STL manifested increased
expression of HLA-DR antigens, without augmentation
of interleukin-2 receptor expression. Alterations in the
expression of the T cell differentiation antigens and
activation antigens by patient PBL closely paralleled the
abnormalities observed in STL. In contrast, STL of
patients with RA exhibited an increase in the expression of the adhesion-related glycoproteins (leukocyte
function-associated 1 [LFA-I] and very late activation 1
[VLA-11 antigens), not observed with autologous PBL.
These studies demonstrate that lymphocytes isolated
From the Harold C. Simmons Arthritis Research Center,
University of Texas Southwestern Medical Center, Dallas.
Supported by USPHS grants AR-09989 and AR-39169.
John J. Cush, MD: Instructor in Internal Medicine; Peter E.
Lipsky, MD: Professor of Internal Medicine and Microbiology.
Address reprint requests to Peter E. Lipsky, MD, University of Texas Southwestern Medical Center, Department of Internal
Medicine, 5323 Harry Hines Boulevard, Dallas, TX 75235.
Submitted for publication February 22, 1988; accepted in
revised form April 27, 1988.
Arthritis and Rheumatism, Vol. 31, No. 10 (October 1988)
from the synovial tissues of RA patients bear an activated phenotype, exemplified by the modulation of CD3
and CD4 and the expression of HLA-DR. The enhanced
expression of the adhesion molecules LFA-1 and VLA-1
by STL may play a role in the localization of these cells
to the inflamed synovium.
Rheumatoid arthritis (RA) is a chronic inflammatory disease whose articular and systemic manifestations are thought to be governed by immunologic
processes within the synovial tissue (1,2). The synovium of patients with active RA is infiltrated with a
variety of mononuclear cells, of which T lymphocytes
predominate (3). It is thought that the persistent immunologic activity of synovial tissue T lymphocytes
responding to an unknown antigen or polyclonal activator may initiate a cascade of events that accounts for
many of the characteristic features of RA (4). However, considerable uncertainty remains regarding the
activation status of T lymphocytes within the synovium of patients with RA. For example, numerous
reports have documented the intense staining of synovial tissue lymphocytes (STL), synovial fluid lymphocytes (SFL), and peripheral blood lymphocytes (PBL)
(2,5) with monoclonal antibodies to HLA-DR, yet
only minimal expression of interleukin-2 (IL-2) receptors (6,7). in addition, production of certain cytokines
secreted by activated T cells, such as IL-2, is thought
to be diminished in the synovium (8). Finally, observations by electron microscopy have indicated that
STL may have the characteristics of resting lymphocytes (9).
Previous studies characterizing the cellular constituents of the rheumatoid synovium have largely
been limited to immunohistologic analyses. Although
these methods are generally regarded as effective
means of understanding the organization of cellular
123 1
STL AND PBL ANALYSIS
Table 1. Monoclonal antibodies used in indirect immunofluorescence analyses of peripheral blood
lymphocytes and synovial tissue lymphocytes from patients and controls
Monoclonal
antibodies
Specificity
Distribution
T cell differentiation
antigens
OKT3
OKT4
OKTS
Activation antigens
Anti-Tac
L243
CD3
CD4
CD8
All mature T cells
Helperlinducer T cells
Suppressorkytotoxic T cells
Interleukin-2 receptor
HLA-DR
Activated T cells and B cells
Resting monocytes, B cells, activated
T cells
Adherence molecules
60.3
TS217
p chain of CDI 1 complex
Most bone marrow-derived cells
Persistently proliferating T cells
Controls
P1.17
MOPC-3 IC
Very late activation
antigen
Unknown
Unknown
elements and the nature of their interrelationships,
they are less capable of providing an accurate assessment of the density of various determinants expressed
by resident tissue lymphocytes. Since the density of
various T cell surface antigens has been shown to
change with the activation status of the T cell (lo),
reliance on immunochemical assays may limit the
appreciation of subtle evidence of lymphocyte activation. Moreover, immunochemical analysis of intact
tissue may be subject to sampling errors related to
regional variability of cellular populations within the
synovial tissue and the limitation of cell numbers that
can be analyzed by this method. Analyzing the entire
population of T cells digested from the sample of
synovium for surface antigen expression by flow cytometry obviates these problems.
The advent of flow cytometry has provided a
technologic advance in cellular analysis by virtue of
the capacity to analyze the density of surface antigens
expressed on a large number of cells (1 1). This makes
it possible to detect more subtle phenotypic changes
observed with activation. To date, few studies have
used cytofluorometric analysis for phenotypic characterization of lymphocytes from RA patients, and none
has specifically examined the phenotype of lymphocytes eluted from synovial tissues.
In this study, flow cytometric analysis was
carried out to characterize the lymphocytes infiltrating
the rheumatoid synovium. The phenotypic characteristics of paired PBL and STL samples from RA
patients were compared with each other and with PBL
from control subjects. STL were obtained by enzymatic digestion of proliferative synovium and, along
with paired PBL samples, were stained with a variety
of monoclonal antibodies (MAb). These immunofluorescence studies revealed a significant reduction in the
number of CDS+ (suppressorkytotoxic) cells in the
synovial tissues when compared with peripheral
blood. Normal PBL, RA PBL, and RA STL contained
comparable percentages of CD4+ helperlinducer cells.
The STL and PBL demonstrated an activated phenotype, manifested by modulation of the surface density
of the T cell differentiation antigens CD3 and CD4.
Many of these cells were HLA-DR+, yet did not
express IL-2 receptors. These studies document that,
in RA patients, T cells in the peripheral blood and
synovial tissue, especially CD4+ cells, are activated.
Moreover, synovial tissue-derived cells exhibited an
increased complement of adhesion antigens (very late
activation 1 [VLA-I] and leukocyte function-associated 1 [LFA-11). These findings are consistent with the
conclusion that the rheumatoid synovium is populated
by activated T lymphocytes.
PATIENTS AND METHODS
Patient selection. Seven patients who fulfilled the
American Rheumatism Association revised criteria for definite or classic RA (1 2) and who were undergoing synovectomy or joint replacement surgery were studied. Patient
selection was based only on the availability of synovial
tissue. Synovial samples were obtained from the wrist (2
samples) or the metacarpophalangeal joint (5 samples).
Paired peripheral blood samples were obtained from 6 patients for comparative analysis. As a control, PBL were
obtained from 8 normal, healthy volunteers.
Cell preparation. Peripheral blood mononuclear cells
(PBMCs) were obtained from 8 healthy adult volunteers and
from 6 patients at surgery. The PBMCs were prepared from
heparinized venous blood samples by density gradient cen-
1232
CUSH AND LIPSKY
Table 2. Characteristics of 7 patients with rheumatoid arthritis who were selected for study on the basis of availability of synovial tissue*
Patient
Tissue source
Ageisex
Disease duration (years)
Joints affected
Morning stiffness > 1 hour
Wrist
52iF
4
Oligo
Medications
Gold
Pred
Napr
Colch
-
MCP
63iF
24
Poly
+
Hyd
Pred
Feno
MCP
63lF
30
Poly
+
Gold
ASA
Wrist
S7lF
24
Poly
+
ASA
MCP
SSIF
3
Oligo
+
Napr
MCP
38iF
26
Poly
MCP
67iF
-
-
DP
ASA
Ibu
I0
Poly
* MCP = metacarpophalangeal joint; Oligo = oligoarticular: Poly = polyarticular; Gold = intramuscular aurothiomalate; Pred = prednisone;
Napr = naproxen; Colch = colchicine; Hydr = hydroxychloroquine; Feno = fenoprofen; ASA = acetylsalicylic acid: DP = D-penicillarnine;
Ibu = ibuprofen
trifugation with sodium diatrizoateiFicol1 gradients, and
were washed 3 times with Hanks’ balanced salt solution
(HBSS). Patient PBL were prepared by passage of PBMCs
over nylon-wool columns for T cell enrichment, as previously described (1 3).
Synovial tissue digestion and preparation of STL.
Sterile synovial tissue specimens (4-10 gm) were promptly
delivered from the operating suite for tissue digestion and
STL preparation as described in detail elsewhere (14).
Briefly, after vigorous sterile mincing with scissors and
forceps. synovial tissue was added to a 100-ml solution of
HBSS, 1% fetal bovine serum (FBS), and 5% HEPES buffer
containing 0.3 gm of oollagenase, 0.1 gm of hyaluronidase,
and 0.01 gm of deoxyribonuclease. The synovial tissue was
digested for 90 minutes at 37°C with constant mixing. After
washing twice in HBSS containing 10% FBS, the synovial
tissue cell suspensions were passed rapidly over a nylonwool column for depletion of adherent cells and enrichment
of T lymphocytes without the loss of activated, larger
lymphocytes. The number of STL recovered following digestion and purification ranged from 20 million to 120 million
cells per sample.
Monoclonal antibodies. The MAb listed in Table 1
were used for immunofluorescence analysis and included
OKT3, OKT4, OKT8, L243, MOPC-31C, P1.17 (all from
American Type Culture Collection, Rockville, MD), antiTac (a gift from Dr. T. Waldmann), 60.3 (a gift from Dr. P.
Beatty), and TS2/7 (a gift from Dr. M. Hemler).
Indirect immunofluorescent staining, fixation, and
analysis. Lymphocytes (4-10 x 105/sample) were stained
with saturating concentrations of MAbs against T cell differentiation antigens and a variety of activation antigens and
adhesion molecules. Briefly, after washing with 2% normal
human serum in phosphate buffered saline (PBS) and 0.1%
sodium azide, cells were stained with saturating concentrations of the appropriate MAb at 4°C for 30 minutes. The cells
were then washed and counterstained with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin. Serum and excess immunoglobulin were removed by washing,
and the cells were then fixed with 1% paraformaldehyde in
PBS (pH 7.4) for 12 minutes at room temperature. Samples
were analyzed using the Ortho system 50HH flow cytometer
(Ortho Diagnostic Systems, Raritan, NJ) interfaced with a
Data General 2150 computer, as previously described (14).
Lymphocytes were analyzed by selective gating based on
the parameters of forward and orthogonal scatter. The
appropriateness of these criteria was confirmed by staining
with T cell markers. Antigen density was indirectly ascertained by assessing the mean fluorescence intensity (MFI) of
cells analyzed. For each analysis of patient PBL and STL,
and control PBL, fluorescence signals were standardized
using identical photomultiplier tube settings on the flow
cytometer (15).
Statistics. Paired patient PBL and STL samples were
analyzed using Student’s t-test for paired samples. Comparisons of population means were carried out using the 2sample t-test.
RESULTS
Patient population. Seven women with RA were
selected on the basis of the availability of synovial
tissue. Patient data are shown in Table 2. Disease
duration ranged from 3 t o 30 years (median 24). All
patients had active synovitis and radiographic erosions
at the time of surgery and were taking antiinflammatory drugs. Two patients were receiving low-dose
prednisone and 4 were taking disease-modifying drugs
(2 gold, 1 penicillamine, 1 hydroxychloroquine) at the
time of surgery. There was no obvious relationship
between disease duration or medication and the number of STL obtained.
Flow cytometric analysis of normal and patient
samples. In general, there was little variation in the
immunofluorescent staining profiles of the 8 normal
control subjects studied. Representative histograms of
staining patterns in control subjects and RA patients
are shown in the figures. When PBL from 2 normal
controls were treated with t h e same solution used for
tissue digestion, no alteration of surface antigen
expression was noted.
Lymphocytes from the peripheral blood and
STL AND PBL ANALYSIS
1233
Table 3. Expression of T cell differentiation antigens by peripheral blood lymphocytes (PBL) and synovial tissue lymphocytes (STL)
obtained from normal control subjects and rheumatoid arthritis (RA) patients*
CD3
CD4
CD8
Cells (n)
% positive
MFI
% positive
MFI
Control PBL (8)
RA PBL (5)
RA STL (7)
70.5 2 3.0
69.7 ? 4.5
66.7 2 3.9
104.5 2 8.St
58.5 2 8.8
54.6 7.5
45.8 2 1.7
47.7 ir 7.7
48.7 2 4.7
53.7 2 3.1$
40.7 2 6.4
32.4 2 5.0
*
% positive
31.9
35.0
22.3
?
2
2
1.8
3.8
4.00
MFI
*
129.2 5.9
94.9 2 15.8
102.7 t 12.1
* Values are the mean ? SEM. MFI = mean fluorescence intensity (arbitrary units).
t P < 0.001 versus RA PBL and RA STL.
t P = 0.01 versus RA PBL and RA STL.
Ei P = 0.03 versus RA PBL and control PBL.
synovial tissues of RA patients exhibited different
staining patterns from those manifested by the control
population.
T cell differentiation antigens. PBL from the 8
controls showed little variation in percentage positivity and MFI when stained for the T cell differentiation
antigens CD3, CD4, and CD8. Similarly, when PBL
were assessed for the percentages of CD3 + , CD4+,
and CD8-t T cells, the 7 RA patients did not differ
significantly from controls (Table 3 and Figure 1).
Moreover, the percentage of CD4+ cells did not differ
X (+I: 69
MFI: 444
%l+):48
MFl:55
AL
x (+I:
85
MFI: 4 7
CDB
CD4
CD3
X L+1:73
MFI:34
x (+I
:35
MFI:117
mludd-4
*A I+]:
26
MFI:92
L L.
X:73
MFI:26
%(+):24
%(+):63
MF1:47
MFI:i00
c.1_^___...j
40
80 i20 i80
40
80
(20 160 2
40
80 420 i B 0 2 0
FLUORESCENCE INTENSITY-
Figure 1. Expression of the T cell differentiation antigens CD3,
CD4, and CD8 by normal peripheral blood lymphocytes (PBL) and
by PBL and synovial tissue lymphocytes (STL) from rheumatoid
arthritis (RA) patients. Cells from a representative experiment were
analyzed at the same fluorescence signal intensity, and the mean
fluorescence intensity (MFI) is given in arbitrary units.
among normal PBL, RA PBL, or RA STL. In contrast,
significantly fewer CD8+ cells were found in the STL
compared with autologous and normal PBL ( P <
0.03).
The densities of the T cell differentiation antigens CD3 and CD4 on rheumatoid PBL and STL were
significantly diminished ( P < 0.001 and P = 0.01,
respectively) compared with normal PBL (Table 3 and
Figure 1). Moreover, when paired rheumatoid PBL
and STL samples were compared, STL generally
exhibited a lower antigen density for CD3 ( P = 0.05)
and CD4 ( P = 0.016) than did autologous PBL. The
mean percentage decrease in STL antigen density for
CD3 and CD4 in paired patient samples was 20% and
27%, respectively. Therefore, the STL of RA patients
exhibited the lowest densities of the surface antigens
CD3 and CD4. Alterations in the density of CD8 were
more variable. However, 5 of 7 patients exhibited CD8
densities on both PBL and STL that were more than
2 SD below that exhibited by normal controls.
Activation antigens. Normal PBL contained a
small percentage of cells expressing IL-2 receptors
and HLA-DR antigens. RA PBL and RA STL exhibited a marked increase in the percentage of lymphocytes expressing HLA-DR ( P 5 0.03). The percentage
of HLA-DR+ cells was greater among the STL (Table
4), ranging from 19.1% to 51.9% positive (mean 34.7).
PBL from the RA patients showed a somewhat lower,
although still increased, percentage of HLA-DR+
cells (Figure 2), ranging from 11.1% to 37.8% (mean
20.6). In contrast with the increased percentage of
HLA-DR expressing lymphocytes, few T cells from
either RA PBL or RA STL expressed IL-2 receptors
(Table 4).
Adhesion antigens. The percentage of rheumatoid PBL and STL expressing LFA-1 was comparable
with that of normal PBL (Table 5). However, the STL
exhibited a greater antigen density of LFA-1 when
compared with patient and normal PBL (P = 0.01). On
CUSH AND LIPSKY
1234
Table 4.
Expression of activation antigens by rheumatoid arthritis (RA) patients and normal control subjects*
HLA-DR
Interleukin-2 receptor
PBL
%
positive
Patient no.
PB L
STL
%
MFI
STL
%
MFI
positive
o/c
MFI
positive
~
ND
37.8
ND
11.1
11.9
21.5
20.6 i 5.4t
1
2
3
4
5
6
Mean
* SEM
Control subjects,
mean f SEMP
* PBL
done.
tP
=
=
8.6
* 0.9
ND
79.0
ND
19.2
26.5
28.0
38.2 2 11.9
46.9
51.9
32.2
27.7
30.6
19.1
34.7 2 4.6$
38.0 2 5.0
8.6 t 0.9
peripheral blood lymphocytes; MFI
=
MFI
positive
~
~~
42.0
37.8
36.0
45.4
89.0
61 .5
52.0 f 7.6
ND
17.6
0.0
6.0
6.4
6. I
7.2 2.5
ND
31.6
35.9
30.6
34.5
15.0
29.5 2 3.3
0.0
3.4
0.0
2.4
0.0
14.9
3.5 2 2.2
24. I
25.2
26.4
23.4
63.8
10.0
28.8 2 6.8
38.0
2.4
38.0 t 3.0
2.4 i 0.6
38.0
f
5.0
*
2
0.6
mean fluorescence intensity (arbitrary units); STL
=
?
synovial tissue lymphocytes; ND
3.0
=
not
0.03 versus controls.
< 0.001 versus controls.
5 n = 6 for HLA-DR; n = 8 for interleukin-2.
$P
TAC
HLA-DR
r
%
( + I :3
MFI: 18
L
L
,
.
.
% (+) : 6
MFI: 31
PBL, LFA-1 was expressed in a low density pattern,
with a minority of cells expressing a higher density of
LFA-I. In contrast, STL from all RA patients studied
showed a marked increase in the number of cells
expressing a higher density of LFA-I antigens (Figure 3).
Two of the 5 RA PBL samples examined contained an
increased number of cells expressing high density LFAI . The adhesion molecule VLA-1 was rarely expressed
by normal or RA PBL. However, there was a significant
increase (P < 0.001) in the number of VLA-1+ cells in
the STL (ranging from 36% to 60.3% positive, mean
48.1%), which sharply contrasts with the usual lack of
this marker in normal or rheumatoid PBL (Table 5 and
Figure 3).
DISCUSSION
L
.
% (+I :52
70 ( + ) : 3
MFI:23
MFI:79
CL
W
rn
a-’
fz
&
I
L
6
40 80
420 I60 200
-1
-1
w
0
40 80 120 160 200
FLUORESCENCE INTENSITYFigure 2. Representative histogram of the expression of activation
antigens by normal peripheral blood lymphocytes (PBL) and by
PBL and synovial tissue lymphocytes (STL) from rheumatoid
arthritis (RA) patients. See Figure I for explanation.
In the past decade, several investigators have
reported functional and phenotypic evidence of T
lymphocyte activation in RA patients (1618). There
have been reports of spontaneously proliferating PBL
(19), increased RNA and DNA content of PBL (20),
abnormalities of PBL and SFL proliferative responses
to both rnitogens and autologous stimulator cells (2123), and increased numbers of STL, SFL, and PBL
bearing HLA-DR and other activation antigens (24).
Despite the indication of intense T lymphocyte activation, studies of rheumatoid lymphocytes from all tissue compartments have often demonstrated less than
normal expression of IL-2 receptors, and in vitro
studies have shown PBL, SFL, and tissue-derived
lymphocytes to be deficient in their production of IL-2
(25,26) and poorly responsive t o exogenous IL-2
STL AND PBL ANALYSIS
1235
Table 5. Expression of adhesion antigens (leukocyte function-associated I [LFA-I] and very late activation 1 [VLA-11) by rheumatoid
arthritis patients and normal control sub.iects*
LFA- I
VLA- 1
~
PBL
PBL
STL
STL
Patient no.
positive
MFI
%
positive
MFI
%
positive
MFI
%
positive
MFI
SEM
ND
80.1
93.4
88.2
70.7
79.5
82.4 ? 3.5
ND
17.8
40.0
17.5
45.2
21 .o
28.3 2 5.3
90.8
72.9
84.8
79.4
65.4
60.0
75.6 t 4.4
55.6
38.9
40.7
17.0
64.4
39.0
42.6 2 6.lt
ND
9.7
ND
0.0
45.6
9.9
16.3 8.7$
ND
31.2
ND
26.2
27.5
9.0
23.5 ? 4.3
36.0
60.3
ND
57.2
40.6
46.6
48.1 2 4.25
28.3
21.8
ND
18.9
46.4
11.0
25.3 ? 5.3
78.9 -+ 3.9
21.3
* 1.5
78.9 ? 3.9
21.3 t 1.5
%
1
2
3
4
5
6
Mean
~
?
Control subjects, mean
? SEMT
*
1.9
2
0.5
* PBL = peripheral blood lymphocytes; MFI = mean fluorescence intensity (arbitrary units); STL
done.
t P = 0.01 versus controls.
$ P = 0.1 versus controls.
8 P < 0.001 versus controls.
f n = 7 for LFA-I; n = 5 for VLA-I.
(27,28). Additionally, it has been claimed that STL
have the morphologic characteristics of resting lymphocytes (9). These studies question the activation
status of STL and, by implication, the role of STL in
the pathogenesis of RA. Therefore, cells eluted directly from the synovial tissue were analyzed to provide information about the activation status of lymphocytes from the sites of inflammation in RA.
There has been little agreement regarding the
phenotypic abnormalities seen with the PBL, SFL,
and STL subsets in RA. Differences in tissue sampling
and the methods used to process and analyze these
cells have led to a lack of consensus regarding the
cellular abnormalities typical of RA patients. However, over the past few years, an increasing number of
investigators have used the flow cytometer to analyze
the phenotypes of PBL and SFL, and a clearer picture
has begun to evolve. For example, a number of
authors have noted a discordance between the phenotypes of cells present in PBL and SFL (2,29-32). Fox
et a1 have claimed that lymphocytes from the site of
inflammation (i.e., SFL) differ significantly in phenotype compared with those found in the peripheral
blood (32). Thus, SFL were found to contain fewer
CD4+ cells and a greater number of CD8+, HLADR+, and OKTlO+ cells (32). Others have confirmed
that SFL are enriched in CD8+ lymphocytes and are
relatively depleted of CD4+ cells. Such findings contrast with an enrichment of the CD4+ and a decrease
in CD8+ populations of lymphocytes in the synovial
tissues reported previously (9,18) and in this study.
The use of more limited fluorescence micros-
32.4
=
32.4
?
3.6
synovial tissue lymphocytes; ND
=
not
?
3.6
1.9 f 0.5
copy has led many investigators to conclude that the
percentages and ratios of CD4+ and CD8+ PBL are
normal in RA patients. In contrast, flow cytometric
analysis of PBL from these patients has suggested that
those with the most active disease may be deficient in
circulating CD8+ PBL (33-39, although these results
have been disputed (36). In several immunohistologic
studies of STL, investigators have focused on the
tissue architecture and the relative predominance of T
lymphocytes in synovial tissues. These studies have
emphasized the variable nature of the cells infiltrating
the synovium, therefore suggesting that inherent sampling errors may limit their value. To date, there have
been no comparative studies on the phenotypic status
of PBL and STL from RA patients by flow cytometric
analysis of these populations.
The use of in vitro models of T cell activation
has demonstrated that the expression of cell surface
molecules on T cells is not static, but rather, exhibits
predictable changes upon activation (10,37). Typically, activation is characterized by reductions in CD3
and CD4 density, with a parallel increase in the
number of cells expressing IL-2 receptors and HLADR antigens. These changes served as the basis for
analyzing the activation status of PBL and STL in RA
patients.
Obvious diminutions were noted in the antigen
densities of CD3 and CD4 from STL and PBL of
patients with RA. These changes were most marked
when RA STL were compared with normal PBL.
Modulation of the density of these T cell antigens was
comparable with that seen with early activation in
CUSH AND LIPSKY
1236
VLA-I
LFA-1
MFI: 23
ox,(+I: 40
O h (+) :80
MFI:48
MFI :31
%(+):73
% (+I :60
MF1:39
MFI:22
0 40 80 i20 160 200
40 80 120 1 6 0 200
FI I InRFSCENCE INTENSITY-
Figure 3. Representative histogram of the expression of adhesion
antigens (leukocyte function-associated 1 [LFA-I] and very late
activation 1 [VLA-I]) by normal peripheral blood lymphocytes
(PBL) and by PBL and synovial tissue lymphocytes (STL) from
rheumatoid arthritis (RA) patients. See Figure 1 for explanation.
vitro, suggesting that the STL and PBL of RA patients, especially the CD4+ cells, are activated and
may participate in the ongoing immune response that
propagates RA.
Further evidence of an “activated phenotype”
for T cells in RA patients is the finding of a greater
percentage of HLA-DR+ STL compared with patient
PBL and normal PBL. Despite the above activationrelated alterations, an increased number of lymphocytes staining with the anti-Tac MAb was not observed. Other investigators have noted that both PBL
and SFL are enriched in HLA-DR+ T cells, yet
deficient or normal in their expression of IL-2 receptors (23). Similar phenotypic changes, with the appearance of HLA-DR+ and Tac- T lymphocytes, have
been shown to occur in long-term tissue cultures (ref.
38 and unpublished observation), suggesting that the
acquisition of IL-2 receptors may be a limited and
short-lived event in the proliferation and differentiation of T lymphocytes stimulated by antigens or mitogens. There is also evidence that lymphocytes from
RA patients are unable to produce normal amounts of
IL-2 when appropriately challenged (25,26,39). Since
endogenous IL-2 has previously been shown to upregulate 1L-2 receptor expression (38), impaired IL-2
production may contribute to the diminished expression of IL-2 receptors by STL in RA.
The expression of the adhesion-related glycoproteins LFA-1 and VLA-1 is unique to rheumatoid
STL. The STL demonstrated an increased percentage
of cells expressing high density LFA-1 antigens when
compared with patient and normal PBL. Resting lymphocytes contain both low and high density populations, with the vast majority of normal PBL expressing
only low density LFA-1. The presence of a greater
number of high density LFA-1+ STL may reflect the
importance of these adhesion molecules in mediating
the emigration of these cells into rheumatoid synovium, since LFA-1 has been shown to play a central
role in mediating cell-cell interactions involving T
lymphocytes, including cytotoxicity (40), antigen recognition by T cells (41), and T cell interactions with
endothelial cells (42). Whether a small number of these
LFA- 1 bright cells preferentially migrate to synovial
tissues or whether the normally low density LFA-1+
cells circulate to the inflamed synovium and then
augment their LFA-1 density as a result of local
activation cannot be determined from these studies.
Nonetheless, documentation of the presence of an
increased percentage of T cells in the rheumatoid
synovium with a high density of LFA-1 suggests a role
for this molecule in perpetuating inflammation.
VLA-1 is not normally expressed on PBL and is
only expressed by chronically activated cells in longterm tissue culture, and has thus been suggested to be
a post-activation antigen (43). VLA-1+ cells have
been found in rheumatoid synovial fluids (43) and in
lymphocytes retrieved from broncho-alveolar lavage
(44). In the RA patients we studied here, the percentage of cells bearing the VLA-1 marker was greatest in
STL. Since few, if any VLA-1+ cells were found in
patient PBL, it is unlikely that a subset of cells
expressing VLA- 1 preferentially migrated to the inflamed synovium. Rather, it is more likely that expression of VLA-1 developed as a result of local activation
and/or proliferation within the synovium.
The adhesion molecules identified by the VLA
and LFA families are part of a larger superfamily of
cell surface receptors called the “integrins,” all of
which are thought to play a role in cellular adhesion
and migration (45). The expression of these surface
STL AND PBL ANALYSIS
proteins may be pivotal to the immunopathogenesis of
RA, either by promoting lymphocyte traffic to inflamed synovial tissues or, once tissue access has been
achieved, by promoting the sequestration of specific T
cell clones within synovial tissues. In RA, the preferential expression of LFA- 1 , and especially VLA-1,
molecules by STL, and not PBL, suggests that these
markers may be acquired locally as a result of activation and proliferation of T cells within the synovium.
The sequestration of such cells may contribute to the
chronicity and the persistent immunologic reactivity
within the joints and may perpetuate the local tissue
destruction and the systemic manifestations of RA.
In summary, this study demonstrates that lymphocytes isolated from the synovial tissues of RA
patients bear an activated phenotype, exemplified by
the modulation of the T cell differentiation antigens,
especially by CD4+ cells, and the expression of HLADR by a large number of these cells. This study is the
first to compare paired PBL and STL from RA patients
by flow cytometry, and it demonstrates that the PBL
exhibit phenotypic alterations comparable with that
observed in freshly eluted STL. STL, and to a lesser
extent PBL, demonstrated an unusual phenotype of
activated T cells manifested by enhanced HLA-DR
positivity without augmentation of IL-2 receptor
expression. In contrast with PBL, the synovial tissues
of these patients were enriched for T lymphocytes
bearing the adhesion molecules LFA-1 and VLA-1. It
is likely that the enhanced expression of VLA-1,
LFA-1, and HLA-DR by STL relates to local sequestration and persistent activation of these cells within
the synovial tissues and that the acquisition of these
adhesion molecules by STL may play a role in the
localization of these cells to the inflamed synovium.
ACKNOWLEDGMENTS
The authors thank Dr. Lee Lankford for providing
the synovial tissue and peripheral blood samples and Ann
Buser for assistance in flow cytometric analysis.
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