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T Lymphocyte Adhesion to Human Synovial Fibroblasts. Role of Cytokines and the Interaction Between Intercellular Adhesion Molecule 1 and CD11aCD18

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1245
T LYMPHOCYTE ADHESION TO
HUMAN SYNOVIAL FIBROBLASTS
Role of Cytokines and the Interaction Between
Intercellular Adhesion Molecule 1 and CD 1la/CD18
RAYMOND F . KRZESICKI, WILLIAM E. FLEMING, GREG E. WINTERROWD,
CHERYL A. HATFIELD, MARTIN E . SANDERS, and JIA E N CHIN
We studied the adhesion of human peripheral
blood T lymphocytes to human synovial fibroblasts
stimulated with interferon- y (IFNy), tumor necrosis
factor a (TNFcu), interleukin-lp (IL-lp), or combinations of these cytokines. T lymphocytes bound poorly to
untreated human synovial fibroblasts. IFN y treatment
resulted id the largest increase in adhesion, followed by
TNFa and IL-1p. Combinations of IFNy TNFa and
IFNy I L - l p had a synergistic effect on intercellular
adhesion molecule 1(ICAM-1) expression and adhesion.
The increase in cellular adhesion induced by cytokines
correlated with the up-regulation of the number of cells
expressing ICAM-1 and the density of antigedcell.
There was no synergistic effect on leukocyte functionassociated antigen 3 (LFA-3) or on HLA class I or class
I1 antigen expression. Adhesion was only partially inhibited by anti-ICAM-1, anti-LFA-1, or anti-CD18.
These findings suggest the existence of ICAM-1independent and CDll/CDlS-independent adhesion
mechanisms. Anti-LFA-3 was completely ineffective as
an inhibitor of adhesion. There was no additive or
synergistic advantage of using combinations of antibod-
+
+
~
From the Department of Hypersensitivity Diseases Research and the Department of Clinical Pharmacology, The Upjohn
Company, Kalamazoo, Michigan.
Raymond F. Krzesicki, MS: Department of Hypersensitivity Diseases Research; William E. Fleming. MS: Department of
Hypersensitivity Diseases Research: Greg E. Winterrowd. MS:
Department of Hypersensitivity Diseases Research: Cheryl A . Hatfield, MS: Department of Hypersensitivity Diseases Research; Martin E. Sanders, MD: Department of Clinical Pharmacology; Jia En
Chin, PhD: Department of Hypersensitivity Diseases Research.
Address reprint requests to Jia En Chin, PhD, Hypersensitivity Diseases Research, The Upjohn Company, 301 Henrietta
Street, Kalamazoo, MI 49001.
Submitted for publication July 6 , 1990: accepted in revised
form May 30, 1991.
Arthritis and Rheumatism, Vol. 34, No. 10 (October 1991)
ies to increase the level of inhibition, i.e., anti-ICAM-1
anti-LFA-3, anti-ICAM-1
anti-CD18, or antiICAM-1 anti-LFA-1 (CDlla). Our data indicate that
proinflammatory cytokines may play a prominent role
in the formation and exacerbation of synovial hyperplasia, by regulating the recruitment and retention of T
lymphocytes via the up-regulation of adhesion molecules
on synovial fibroblasts.
+
+
+
It is clear that T lymphocyte autoreactivity can
lead to inflammatory synovitis in rheumatoid arthritis
(RA), although the initiating event remains elusive
( I , 2 ) . This phenomenon is also evident in other inflammatory diseases, such as multiple sclerosis and psoriasis (3,4). The mechanisms through which the T
lymphocytes are continually recruited into the synovium in RA, to set up the inflammatory lesion which
leads to the eventual degradation of cartilage and
bone, are being deciphered. This is due, in part, to
growing understanding of the specific, and enhanced,
expression of adhesion molecules on leukocytes (5-8)
and nonimmune cells (9-1 2) by proinflammatory mediators, such as cytokines. The involvement of adhesion molecules in the pathogenesis of RA has received
increased attention as a crucial component in promoting and exacerbating this chronic autoimmune disorder (13-15).
The intent of the present study was to investigate the ability of cytokine “primed” human synovial
fibroblasts to bind nonactivated extravasated peripheral blood T lymphocytes, as a possible mechanism of
cellular retention in the inflamed synovium. Once the
T lymphocytes are sequestered in the synovium, the
probability of activation via antigen-specific or nonantigen-specific pathways by proinflammatory cyto-
KRZESICKI ET AL
1246
kines in t h e synovial milieu, leading to clonal expansion, are greatly enhanced. This correlates well with
t h e fact that human synovial fibroblasts stimulated
with interleukin-lp (IL-lp), tumor necrosis factor a
( T N F a ) , interferon- y (IFNy), or combinations of cytokines demonstrate increased expression of adhesion
molecules, such as intercellular adhesion molecule 1
(ICAM-1) (16). Further support for this hypothesis
comes from our observation that prolonged stimulation with interleukin-2 (IL-2) and/or IL-4 without
a n t i g e n can induce a p h e n o t y p i c change from
CD45RA-positive to CD29-positive T lymphocytes
a n d can enhance adhesion to the endothelium a n d
synovium (Winterrowd GE: manuscript submitted).
Two adhesion events that are of particular
interest are t h e interaction between ICAM-I (CD54)
and C D l l d C D 1 8 complex, a n d between CD2 a n d
leukocyte function-associated antigen 3 (LFA-3)
(CD58), not only because of their demonstrated role in
cell-to-cell adhesion (17-19), but also because of their
ability to facilitate antigen presentation to T lymphocytes a n d cytolysis (20-23). Therefore, w e used a
quantitative in vitro cellular adhesion system t o exam-
ine the relative contributions of ICAM-1-CD1 ldCD18
a n d of CD2-LFA-3 to T lymphocyte adhesion to
cytokine-activated human synovial fibroblasts. We
found that I L - l p , T N F a , a n d IFNy could all increase
cellular adhesion (listed in increasing o r d e r of efficacy). Synergy was observed with the combinations of
IFNy + TNFa a n d IFNy + I L - l p , a n d increased
adhesion could be correlated with a concomitant increase in the expression of ICAM-1, but not of LFA-3.
We further demonstrated that monoclonal antibodies
against ICAM-1, CD18, a n d CD1 la (LFA-1) were able
to partially reduce T lymphocyte adhesion, but antiLFA-3 was ineffective. These findings indicate that t h e
adhesive coupling of ICAM-1 a n d CDI 1dCD18 may
be an important and integral component in inflammatory synovitis in RA, but it cannot account f o r all the
cellular interactions that take place.
MATERIALS AND METHODS
Antibodies and recombinant cytokines. Recombinant
human IL-lp (24) and recombinant human TNFa were
produced at The Upjohn Company (Kalamazoo, MI). Their
specific activities were 2 x lo7 units/mg protein and 3 x lo7
units/mg protein, respectively. Recombinant human IFN y ,
recombinant human IL-4, and recombinant human IL-6 were
obtained from Genzyme (Boston, MA). Recombinant human
granulocyte-macrophage colony-stimulating factor (GMCSF) was from Amgen (Thousand Oaks, CA). Murine mono-
clonal anti-LFA-3 (TS2/9, IgG l), murine monoclonal antiCD1 l a (MHM24, IgGl), murine monoclonal anti-ICAM-1
(84H10, IgGl), and murine monoclonal anti-CD18 (MHM23,
IgGl) were kindly supplied by Dr. Stephen Shaw (National
Cancer Institute, Bethesda, MD). MOPC21 was obtained
from Organon Teknika (West Chester, PA), and murine
myeloma IgGl and monoclonal anti-CD3 (OKT3) were from
Ortho (Raritan, NJ). Anti-CD16 (3G8) was a generous gift
from Dr. David Segal (Bethesda, MD). Murine monoclonal
anti-HLA-DR (2.06, IgG 1), anti-HLA-A,B ,C (W6/32,
IgG2a), and 63D3 were from American Type Culture Collection (Rockville, MD).
Monolayer culture of human synovial fibroblasts. Human synovial fibroblasts in monolayers were derived from
human synovial tissue obtained, as described previously
(16), from RA or osteoarthritis patients undergoing joint
replacement. The cell surface expression of ICAM-1,
LFA-3, and class I and class I1 major histocompatibility
complex antigens in cytokine-stimulated and unstimulated
synovial cells, isolated and cultured in a manner similar to
that reported here, has been previously described (16).
Stimulation of human synovial fibroblasts with cytokines. Human synovial fibroblasts were seeded in 24-well
tissue culturk plates and fed with Ham’s F12 supplemented
with 10% fetal calf serum (FCS). Cultures that were -80100% confluent were incubated with cytokines for the times
indicated, prior to the adherence of T lymphocytes.
Flow cytometric analysis. Human synovial fibroblasts
were grown in 150-cm2flasks, and the cells were stimulated
with cytokines for the times indicated. At the end of the
incubation period, the cells were washed with phosphate
buffered saline without calcium or magnesium, and the cell
layers were trypsinized as previously indicated (16). The
cells were stained as described (16). Samples were analyzed
on an EPICS 753 flow cytometer using an argon laser
emitting 488 nm light at 400 mW power. Fluorescein isothiocyanate emission was measured through a 525-nm bandpass
filter. Bit maps were set to include viable cells. Fluorescence
intensity was measured on a 3-decade log amplifier. Linearity of the amplifier was confirmed using previously described
methods (25). Mean channel fluorescence values were then
converted to relative linear values, for direct comparison of
intensity differences (26).
Purification of human peripheral blood resting T
lymphocytes. Mononuclear leukocytes were obtained from
normal donors, by leukapheresis. T cells were prepared from
the pheresis product using magnetic immunoselection by
incubation with a cocktail of monoclonal antibodies (63D3,
3G8, and 2.06) at 4°C for 30-60 minutes. After washing,
antibody-coated monocytes, B cells, natural killer cells, and
DR positive T cells were removed by mixing the cells with
anti-IgG-coated iron particles (Advanced Magnetic, Cambridge, MA) for 30 minutes at 4”C, and subsequent application of a strong magnet. T cells obtairled by this negative
selection process were >98% CD3 positive. Monocytes
were undetectable by flow cytometry, and the B cell population was <1% in all experiments.
Chromium labeling of T lymphocytes. T lymphocytes
were labeled with sodium chromate (NEN, Boston, MA) as
previously described (27). Briefly, 50 pCi of sodium chromate was added to 1 X lo7 T lymphocytes in 0.5 ml of
T LYMPHOCYTE ADHESION TO SYNOVIAL FIBROBLASTS
Hanks' balanced salt solution (HBSS; Ca free and Mg free).
The suspension was incubated for 60 minutes at 37°C. The
labeled cells were washed twice with 30 ml of HBSS and
resuspended in binding medium (RPMI 1640 containing 20%
FCS), to a concentration of 2 x lo6 cells/ml.
T lymphocyte-human synovial fibroblast adhesion assay. Labeled T lymphocytes (0.5 ml/well) were added to
human synovial fibroblast monolayers cultured in 24-well
culture plates. The T lymphocytes were allowed to adhere to
the human synovial fibroblasts at 37°C for 1.5 hours. At the
end of the incubation period, nonadherent cells were removed by 3 gentle washings with binding medium maintained at 37°C. The human synovial fibroblasts and adherent
cells were lysed with 0.1 N sodium hydroxide and counted on
a gamma counter. T lymphocyte adhesion was expressed as
the amount of 5'Cr bound to the human synovial fibroblast
SD of triplicate determinations). In
monolayers (mean
experiments where antibodies were used to block adhesion,
T lymphocytes were incubated with anti-LFA-I, anti-CD18,
or MOPC21 for 15 minutes prior to addition to human
synovial fibroblasts. Similarly, human synovial fibroblasts
were pretreated with anti-ICAM-1, anti-LFA-3, or MOPC21
antibodies prior to the addition of T lymphocytes. The
percent inhibition of adhesion was calculated using the
following formula:
*
1-
Adhesion in the presence of antibody (counts per minute)
x 100
Adhesion in the presence of isotype-matched control (cpm)
RESULTS
Effects of IL-lP, TNFa, and IFNy on the ability
of human synovial fibroblasts to bind T lymphocytes.
Figure 1 shows that HSF stimulated with IL-1p (0.1100 unitshl), T N F a (0.5-1,000 unitshl), and I F N y
(1-1,000 unitshl) for 20 hours had an increased ability
to bind T lymphocytes, in a dose-dependent manner,
above the level exhibited by untreated control cells.
Distinct dose-related increases in adhesion were observed after stimulation with IFNy and T N F a ; although treatment with IL-l p also increased adhesion,
the maximal response was less than that observed with
either I F N y or T N F a , and the relationship between
the adhesive response and the dosage was not as
clearly defined. Maximal cell adhesion was attained
after 6 hours of human synovial fibroblast stimulation
with each of the cytokines, and peak adhesion was
maintained for at least 48 hours (data not shown). All
subsequent assays were performed with cells that had
been stimulated for 20 hours, unless otherwise indicated.
Synergy between cytokines in the induction of
ICAM-1 expression by human synovial fibroblasts. The
surface expression of 2 adhesion molecules, ICAM-1
and LFA-3, by cytokine-stimulated human synovial
fibroblasts was measured by flow cytometric analysis.
1247
I
Cytoklne (units/ml)
Figure 1. T lymphocyte adhesion to human synovial fibroblasts
treated with interleukin-lp (IL-lp), tumor necrosis factor a (TNFa),
or interferon y (IFNy). Human synovial fibroblasts in 24-well
culture plates were treated with various concentrations of IL-lp,
T N F a , or I F N y for 20 hours, and the adhesion of "Cr-labeled T
lymphocytes was determined. Adhesion experiments were performed in triplicate, and the results from 3 separate experiments are
expressed as the mean and S E M increase in T lymphocyte adhesion
in cultures treated with cytokines, compared with untreated cultures
(control level = 1).
The results are shown in Table 1 and Figure 2. The
cells were treated with maximally effective concentrations of IL-lp (50 unitdml), T N F a (500 unitshl), or
Table 1. Synergy between cytokines in their induction of intercellular adhesion molecule 1 (ICAM-I) expression in human synovial
fibroblasts"
Marker, treatment
ICAM- I
Control
IL-IP
TNFa
IFN y
IL-Ip + T N F a
IL-Ip + I F N y
TNFa + IFNy
HLA class I 1 antigens
Control
IL-IP
TNFa
IFNy
IL-Ip + T N F a
IL-Ip + l F N y
TNFa + IFNy
* Human
%
positive
MCF
Increase in
MCF
(-fold)
27.7
72.4
83 .O
90.7
85.2
97.9
99.3
99
373
560
720
465
1,340
1,760
3.8
5.7
7.3
4.7
13.5
17.8
-
-
1 .o
5.0
1.o
26.9
5.0
27.4
27.7
93
-
71
88
-
0.76
0.95
synovial fibroblasts were treated 'with interleukin-lp (1L-
Ip; 50 unitsiml), tumor necrosis factor a ( T N F a ; 500 unitsiml),
interferon y (IFNy; 1,000 u n i t s h l ) , or combinations of the cytokines at the same concentrations. Cells were prepared for flow
cytometric analysis 40 hours after the start of cytokine treatments.
The mean channel fluorescence (MCF) valu,es have been converted
from logarithmic units to linear values.
KRZESICKI ET AL
1248
A
TNF
0.1
1.0
10
100
B
0.1
1.0
10
1
c l
0.1
1.0
10
100
Figure 2. Flow cytometric analysis of intercellular adhesion molecule 1 expression in human synovial fibroblasts stimulated with
various cytokines. Cells were maximally stimulated for 40 hours
with A, interleukin-lp ( I L l ; 50 units/ml) and tumor necrosis factor a
(TNF; 500 unitdml), B, ILl (50 units/ml) and interferon-y (IFN;
1.000 units/ml), or C, T N F (500 units/ml) and IFN (1,000 unitdml).
Background staining was determined using isotype-matched irrelevant antibodies. Fluorescence intensity (logarithmic scale, 3-decade) is
shown on the abscissa, and the frequency is shown on the ordinate.
IFNy (1,000 units/ml) for 40 hours prior to analysis.
Combinations of IL-1p and TNFa, IL-lp and IFNy,
and T N F a and IFNy at the same concentrations were
also tested, and histograms of the results are shown in
Figure 2. Human synovial fibroblast ICAM-1 expression coinduced by IL-1p and IFNy and by T N F a and
IFNy showed increases of 27% and 40%, respectively,
above the additive levels of each cytokine alone. The
combination of IL-1p and T N F a produced a less-thanadditive effect, although the net effect was 5-fold
greater than with untreated cultures. The increases in
cell surface ICAM-1 expression in human synovial
fibroblasts treated with each of the 3 cytokines, alone
or in combination, were reflected in the increases in T
lymphocyte adhesion. There was no synergy between
cytokines in their induction of LFA-3 or HLA class I
antigen expression (data not shown), and there was
antagonism between I L - l p and IFNy on the induction
of HLA class I1 antigens, as has been reported previously (28).
Concentration-dependent effects of antiICAM-1, anti-CD18, and anti-LFA-1 on T lymphocyte
human synovial fibroblast adhesion. Since stimulation
of human synovial fibroblasts with the combination of
T N F a (500 unitdml) and IFNy (1,000 unitslrnl) resulted in the highest level of ICAM-1 expression
(Table I ) and T lymphocyte adhesion (data not
shown), this protocol was used to assess the concentration of neutralizing antibodies against ICAM- 1,
LFA-3, CD18, and LFA-1 (CDlla) required to inhibit
T lymphocyte-human synovial fibroblast adhesion. As
shown in Figure 3, the lowest concentration of antiICAM-1 antibody that was needed to inhibit cellular
adhesion induced by T N F a and IFNy was 0.1 pglwell
( P < 0.0001 versus T N F a + IFNyinduced adhesion).
Increasing the antibody concentration to 0.6 pg/well or
1 pg/well (data not shown) did not further reduce the
adhesive interaction obtained with 0.3 pg/well. T
lymphocyte-human synovial fibroblast adhesion was
blocked by only -39% compared with the adhesion
obtained using an isotype-matched irrelevant antibody
control, MOPC21 (1 pg/well).
The ability of antLCD18 and anti-LFA-1 antibodies to block the adhesion between cytokinestimulated human synovial fibroblasts and T lymphocytes was concentration dependent (Figure 3).
Significant reduction in adhesion was attained using
anti-CD18 at a concentration of 0.03 pglwell and
anti-LFA-1 (CD1la) at a concentration of 0.01 pg/well
( P 0.01 and P < 0.001, respectively, compared with
the adhesion using antibody control). Maximal inhibition of T lymphocyte binding achieved with anti-CD18
T LYMPHOCYTE ADHESION TO SYNOVIAL FIBROBLASTS
and anti-LFA-1 was -30% and 39%, respectively,
using a 0.3-pg/well concentration of each antibody.
Inhibition of cytokine-induced T lymphocytehuman synovial fibroblast adhesion using anti-CD18
and anti-LFA-1. Figure 4 shows the adhesion between
T lymphocytes and human synovial fibroblasts treated
with each of the 3 cytokines, IL-lp (50 unitshl),
TNFa (500 unitshl), and IFNy (1,000 unitshl), and
with 3 combinations of the cytokines. The figure also
shows the effect of adding an isotype-matched antibody, MOPC21. Addition of this antibody had no
inhibitory effect on binding when compared with cells
in the absence of any antibody. The addition of either
anti-CD18 or anti-LFA-1 at 0.3 pg/well was equally
effective in reducing adhesion between the 2 cell types
under all 6 different experimental conditions. However, there were still significant differences (all P <
0.01) in the adherence of T lymphocytes to human
synovial fibroblasts treated with TNFa, IFNy, IFN y
+ TNFa, and IFNy + IL-Ip, when compared with
untreated HSF, in the presence of either of the 2
antibodies. This indicates that anti-CD18 and antiLFA-1 were only partially effective in interfering with
T lymphocyte adhesion. The inhibition of T lymphocyte adhesion showed some variability among some of
the experiments. Table 2 shows the mean percent
inhibition of T lymphocyte adhesion using anti-LFA- 1
-5
1249
a100
No antlbody
0MOPC21
9000
I
antl-CD18
rZa antl-LFA-1
I
:
E
2600
P)
c
0
6 2000
0
c)
::
h
c
1100
P
-E,
1000
I
k
4
100
r
'",
0
Control IL-18
TNFa
IFNy
IL-18
IFNy
IFNy
TNFy
TNFa
IL-la
+
+
+
Figure 4. Effect of anti-LFA-I and anti-CD18 antibodies on T
lymphocyte adhesion to human synovial fibroblasts treated with
cytokines. Human synovial fibroblasts were pretreated with maximally effective concentrations of interleukin-lp (IL-lp; 50 units/ml),
T N F a (500 unitdml), I F N y (1,000 units/ml), or combinations of the
cytokines for 20 hours prior to measurement of T lymphocyte
adhesion. Anti-LFA-l or anti-CD18 antibodies were added at a
concentration of 0.3 pg/well. MOPC21 (0.3 pg/well) was used as the
isotype-matched control antibody. Experiments were performed in
triplicate, and the results are expressed as the mean and S D
adhesion of T lymphocytes to human synovial fibroblasts. See
Figure 3 for additional definitions.
or anti-CD18, in 2-4 separate experiments for each of
the 6 different conditions of human synovial fibroblast
pretreatment. The inhibition of adhesion ranged from
40% to -70%, irrespective of the cytokine used.
(1000
-h
-c
0
4000
Table 2. Inhibition of T lymphocyte adlhesion to human synovial
fibroblasts activated with cytokines, in the presence of antiICAM-1, anti-LFA-I, or anti-CD18
w
r
Q
c
0
2.
=,n
-E
k
% inhibition of T lymphocyte adhesion
2000
2.
Treatment
.-b
'",
1.'
' .
anti-ICAM-1
. .
. .
anti-CDl8
anti-LFA-1
Antibody ( p g l m l )
Figure 3. Dose-dependent effect of anti-intercellular adhesion molecule l (anti-[CAM-l), anti-CD18, and anti-leukocyte functionassociated antigen 1 (anti-LFA-I) antibodies on T lymphocyte
adhesion to human synovial fibroblasts treated with tumor necrosis
factor a (TNFa) and interferon y (IFNy). Human synovial fibroblasts were treated with maximally effective concentrations of
T N F a (500 units/ml) and I F N y ( 1 ,000 unitsiml) for 20 hours prior to
measurement of T lymphocyte adhesion in the presence of antiICAM-1, anti-CD18, anti-LFA-I, or MOPC21 as the control antibody. Experiments were performed in triplicate, and the results are
expressed as the mean and S D adhesion of T lymphocytes to human
synovial fibroblasts.
IFN y
( I ,000 unitsiml)
TNFa
(500 unitsiml)
IL-Ip
(50 unitsiml)
IFNy + T N F a
IFNy + IL-1p
T N F a + IL-1p
~~
Anti-ICAM-1
Anti-LFA-1
AntLCD18
45.1
C
8.7 (6)
58.6
?
6.4 (4)
57.2 2 7.8 (3)
44.9
?
26.1 (6) 40.6
?
3.7 (4)
42.8 2 5.2 (3)
39.7
?
30.6 ( 5 ) 41.7
?
13.2 (3) 49.6
2
9.5 (2)
37.8 ? 10.5 (7) 61.0 t 6.7 (4) 59.6 2 5.6 (3)
42.6 2 8.7 (4) 66.9 ? 3.4 (3) 67.1 2 3.9 (2)
23.0 ( I )
42.9 ? 11.0 (3) 55.2 2 8.9 (2)
~
* Human synovial fibroblasts were treated with the cytokines alone
or in combination as indicated, and the adhesion assays were
performed in the presence of anti-ICAM-1, anti-leukocyte function-associated antigen 1 (anti-LFA-I), anti-CD18, or MOPCZl, all
at 0.3 pg/well, as described in Materials and Methods. The percent
inhibition of T lymphocyte adhesion was calculated using the
formula shown in Materials and Methods. Results shown are the
mean 2 SD from the number of separate experiments performed (in
parentheses). See Table 1 for additional definitions.
KRZESICKI ET AL
1250
0000
-5
6000
C
0
-
RS MOPC21
0antl-ICAM-1
I
antl-LFA-3
rZa antl-ICAM-1 +
antl-LFA-3
4000
r
U
0
so00
0
S
n
-E
L
p
-
2000
2,
1000
VL
0
Control IL-1,9
TNFa
IFNy
I L- l p
t
TNFa
lFNy
IFNy
t
t
TNFa
IL-10
Figure 5. Effect of anti-ICAM-1 and anti-LFA-3 antibodies on
T-lymphocyte adhesion to human synovial fibroblasts treated with
cytokines. Human synovial fibroblasts were pretreated with maximally effective concentrations of interleukin-lp (IL-Ip; 50 units/ml),
TNFa (500 units/ml), IFNy ( I ,000 units/ml), or combinations of the
cytokines for 20 hours prior to measurement of T lymphocyte
adhesion. Anti-ICAM-l or anti-LFA-3 antibodies were added at a
concentration of 0.3 Fg/well (when both anti-ICAM-1 and antiLFA-3 were added concomitantly, the total concentration of antibodies was 0.6 &well). MOPC21 (0.3 p,g/well) was used as the
isotype-matched control antibody. Adhesion of T lymphocytes to
human synovial fibroblasts in the absence of any antibody was
identical to the levels obtained when cultures were incubated with
MOPC21 (data not shown). Experiments were performed in triplicate, and the results are expressed as the mean and SD adhesion of
T lymphocytes to human synovial fibroblasts. See Figure 3 for
additional definitions.
Inhibition of cytokine-induced T lymphocytehuman synovial fibroblast adhesion in the presence of
anti-ICAM-1 and anti-LFA-3. Figure 5 shows the
inhibition of cellular adhesion using treatment protocols similar to those outlined above, in the presence of
anti-ICAM-1 (0.3 pg/well), anti-LFA-3 (0.3 pg/well),
anti-ICAM-1 + anti-LFA-3 (0.3 pg/well each), and
MOPC21 (0.3 pg/well). Anti-LFA-3 was completely
ineffective as an inhibitor of T lymphocyte-human
synovial fibroblast adhesion. Addition of anti-ICAM-1
reduced the amount of adhesion to approximately
basal levels when the human synovial fibroblasts had
been treated with maximally effective concentrations
of IL-Ip, I L - l p TNFa, or IL-1p + IFNy. However,
incomplete inhibition of T lymphocyte binding was
observed when anti-ICAM-1 was added to human
synovial fibroblasts pretreated with TNFa, IFNy, or
the combination of T N F a and IFNy. Even when both
anti-ICAM- 1 and anti-LFA-3 were added concomi-
+
tantly, T lymphocyte adhesion to human synovial
fibroblasts pretreated with TNFa, IFNy, or T N F a +
I F N y remained elevated: 38%, 105%, and 118%
greater than control adherence, respectively.
Table 2 shows the results from up to 7 separate
adhesion experiments with each cytokine or combination of cytokines, using anti-ICAM-1. The range of the
percent inhibition with anti-ICAM- 1 was comparable
with the data obtained with anti-LFA-1 and antiCD18, for human synovial fibroblasts stimulated with
IL-lp, TNFa, or IFNy alone. However, although
anti-LFA- 1 and anti-CD 18 antibodies were equivalent
in their abilities to inhibit adhesion of T lymphocytes
to HSF treated with IFNy + T N F a or with IFNy +
IL- l p , anti-ICAM-1 antibody (84H10) was significantly less effective than either anti-LFA-1 or antiCD 18 antibodies in inhibiting T lymphocyte-human
synovial fibroblast adhesion induced by these cytokine
combinations (P 5 0.005 in each case).
DISCUSSION
There is growing recognition of the complexity
of lymphocyte trafficking through various tissues as a
normal function of homeostasis. Dysregulation of lymphocyte homing to its target tissue appears to be the
hallmark of many autoimmune diseases, such as RA,
asthma, Graves’ disease, Hashimoto’s thyroiditis, and
various cutaneous inflammatory diseases (1,2,29-33).
The regulation of expression of ICAM-1 by
cytokines in human dermal fibroblasts (10,34) and
human synoviocytes/fibroblasts (13-16) has been reported, although the role played by ICAM-1 in the
adhesion between fibroblasts and T lymphocytes is
less well understood. We found that cellular adhesion
was augmented by IL-lp, TNFa, and IFNy. Of these
3 cytokines, IFNy elicited the greatest increase in
ICAM-1 expression and T lymphocyte adhesion, as
has been found with fetal astrocytes (9), thymic epithelial cells ( 3 3 , keratinocytes (36-38), and human
thyroid cells (31). Equally important is the observation
that there was synergy between cytokines in terms of
their effects on ICAM-1 expression and T lymphocyte
adhesion to human synovial fibroblasts. Therefore, in
vivo retention of T lymphocytes in the inflamed synovium may be due to the effects of all 3 of these
cytokines. Expression of either HLA class I or HLA
class I1 antigens by human synovial fibroblasts did not
show similar synergy. This contrasts with the reported
synergism observed with IFNy and TNFa4nduced expression of HLA class 11 antigens in Schwann cells (39).
T LYMPHOCYTE ADHESION TO SYNOVIAL FIBROBLASTS
In contrast with our findings, a study by Haynes
et a1 (13) indicated that the interaction between
ICAM-1 and LFA-1 was not important for T cell
adhesion to synovial fibroblasts. To further complicate
the issue, Piela and Korn (34) showed that the adhesion of human resting peripheral blood T lymphocytes
to dermal fibroblasts stimulated with I F N y was
ICAM-1 dependent but the increase in ICAM-1
expression induced by IL-1p did not correlate with
adhesion, which implies that the molecule may not be
functional. Our data on cytokine induction of ICAM-1
expression by human synovial fibroblasts correlated
very well with the level of adhesion of T lymphocytes
to activated human synovial fibroblasts, and did not
suggest the existence of nonfunctional ICAM- I molecules. Nevertheless, the data gathered from studies of
fibroblasts are generally consistent with the evidence
obtained regarding ICAM- 1 expression and function in
the endothelium, a tissue that has been much better
characterized (4042).
Even though we have evidence to support the
hypothesis that the interaction between ICAM-1 and
CD1 ldCD18 plays a significant role in the adherence
of T lymphocytes to cytokine-activated human synovial fibroblasts, the data also suggest the importance of
ICAM- 1-independent and CD I l/CD 18-independent
adhesion mechanisms. Table 2 clearly shows that the
reductions in T lymphocyte adhesion were significantly greater when antibodies against LFA-1 or CD18
were used, compared with the inhibition by antiICAM-1 antibody. This may imply that the ICAM-1independent adhesion results from the expression in
human synovial fibroblasts of ICAM-2, which is another ligand for LFA-I (43). Unlike endothelial cells,
human synovial fibroblasts stimulated with IL-4 (1-200
units/ml) did not exhibit any increase in their capacity
to bind T lymphocytes. There was also no synergistic
increase in T lymphocyte adhesion when human synovial fibroblasts were stimulated with IL-4 and IFNy,
TNFa, or IL-lp, as has been reported with endothelial
cells (44). Therefore, it is unlikely that one of the
CD1 UCD18-independent mechanisms, which account
for 40-60% of the binding of T lymphocytes to human
synovial fibroblasts, is due to the interaction of vascular cell adhesion molecule 1 with very late activation
antigen 4 (45,46).
We did not obtain any evidence for the involvement of the CD2/LFA-3 ligand pair in the process of
adhesion between T lymphocytes and human synovial
fibroblasts, which is in direct contrast to a previously
published report (13). This may be due to the differ-
1251
ence between the adhesion assay protocol we used and
that used by Haynes et a1 (13), in which the adhesion
was accomplished at 4°C. Our observation is not
unprecedented in that the adhesion of T lymphocytes
to cultured endothelial cells was not blocked by either
anti-CD2 or anti-LFA-3 (41,47). tt may be speculated
that the function of the CD2/LFA-3-associated interaction between human synovial fibroblasts and T
lymphocytes in RA is not important for adhesion, but
may play a role in T lymphocyte activation and/or
cytolysis (23,48). The exact function of LFA-3 expression on cytokine-activated and unactivated human
synovial fibroblasts remains to be clarified.
The presence of IL-6 and GM-CSF in the
synovial fluid of RA patients has been documented
(49-52), but we failed to demonstrate their ability to
increase T lymphocyte adhesion when used to stimulate human synovial fibroblasts (data not shown). This
may be related to our finding, by radioimmunoassay,
that there was no increase in ICAM-1 expression by
human synovial fibroblasts treated with IL-6 or GMCSF, alone or in combination with other cytokines,
such as IL-lp, TNFa, IFNy, or IL-4 (data not shown).
It is now apparent that the memory population
of T lymphocytes in human peripheral blood can be
induced by 1L-2 and IFNy to express ICAM-1 (53).
This observation emphasizes that T lymphocytederived cytokines have the ability not only to profoundly affect the expression of adhesion ligands on
granulocytes, mononuclear cells, and stromal tissue,
but also to enhance T cell activation and proliferation
via autoregulation. Our data lend further support to
the evolving hypothesis that cytokines have an integrated effect on the activation of synovial fibroblasts
by enhancing the adherence of ‘r lymphocytes, thus
promoting the development of inflammatory synovitis
in RA.
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