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Measurement of an adhesion molecule as an indicator of inflammatory disease activityUp-regulation of the receptor for hyaluronate CD44 in rheumatoid arthritis.

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1434
MEASUREMENT OF AN ADHESION MOLECULE
AS AN INDICATOR OF
INFLAMMATORY DISEASE ACTIVITY
Up-Regulation of the Receptor for Hyaluronate (CD44)in Rheumatoid Arthritis
BARTON F. HAYNES, LAURA P. HALE, KAREN L. PATTON,
MARGARET E. MARTIN, and REX M. McCALLUM
The hyaluronate receptor (CD44) molecule is a
multifunctional cell surface protein involved in T cell
activation, monocyte cytokine release, fibroblast locomotion, and lymphocyte binding to high endothelial
venules. To study the roles CD44 molecules play in
inflammatory synovitis, we measured expression of
CD44 in inflamed and noninflamed synovial fluid and
tissue, using indirect immunofluorescence assays on
tissue sections and quantitative Western blot analysis.
The ability of purified CD44 protein to modulate T cell
responses was tested in T cell activation assays in which
CD44-containing liposomes were added in vitro. CD44
was widely expressed on many synovial cell types, and
synovial tissue from rheumatoid arthritis (RA) patients
contained 3.5 times more CD44 than tissue from osteoarthritis patients and 10.7 times more than tissue
from patients with joint trauma. The level of soluble
From the Department of Medicine, the Division of Rheumatology and Immunology, the Department of Microbiology and
Immunology, and the Duke University Arthritis Center, Duke
University Medical Center, Durham, North Carolina.
Supported by N I H grants CA-28936, AR-34808, and AR39162.
Barton F. Haynes, MD, Department of Medicine, Division
of Rheumatology and Immunology. Department of Microbiology,
and Duke University Arthritis Center; Laura P. Hale. PhD, Department of Medicine, Division of Rheumatology and Immunology,
Department of Microbiology, and Duke University Arthritis Center;
Karen L. Patton, AB, Department of Medicine, Division of Rheumatology and Immunology, and Duke University Arthritis Center;
Margaret E. Martin, BA, Department of Medicine, Division of
Rheumatology and Immunology, and Duke University Arthritis
Center; Rex M. McCallum. MD, Department of Medicine, Division
of Rheumatology and Immunology, and Duke University Arthritis
Center.
Address reprint requests to Barton F. Haynes, MD, Box
3258, Duke University Medical Center, Durham, NC 27710.
Submitted for publication February 19, 1991; accepted in
revised form June 18, 1991.
Arthritis and Rheumatism, Vol. 34, No. 11 (November 1991)
CD44 in RA synovial fluid was elevated only in fluids
with low cell counts (s7,0001mm3), and not in RA
synovial fluid with higher cell counts. Soluble purified
CD44 protein in liposomes partially suppressed T cell
activation in vitro. These data demonstrate that 0 4 4 is
up-regulated on many synovial cell types in patients with
RA, and that the level of CD44 present in synovial tissue
is related to the degree of synovial inflammation. Determination of ways to inhibit the proinflammatory functions of immune cell membrane CD44 molecules may
provide new therapeutic modalities for RA.
Recent work has defined the importance of cell
adhesion molecules in immune cell function (for review, see refs. 1-3). Cell adhesion molecules that are
receptors for soluble molecules (4).receptors for viruses (for review, see refs. 1.5, and 6). and ligands for
other cell surface molecules (for review, see refs. 1-3)
have been described. On immune cells, cell adhesion
molecules mediate a wide variety of normal cell functions, including cell movement, adherence to other
cells, adherence to extracellular matrix proteins,
mononuclear cell homing, and monocyte cytokine
release (for review, see refs. 14).
The CD44 molecule has been of recent interest
because this protein has multiple proinflammatory
functions, exists in soluble form in serum and plasma,
and regulates the function of other adhesion molecules
(for review, see refs. 2 and 4). On T lymphocytes,
ligand binding to the CD44 molecule promotes T cell
activation, T cell interleukin-2 (IL-2) release, and T
cell adhesion to monocytes (7). On monocytes, ligand
binding to CD44 induces IL-I release (7.8). CD44 on T
and B cells has been implicated as one molecule
involved in lymphocyte homing (9.10). Recent work
CD44 IN RA SYNOVIUM
has demonstrated that CD44 is the primary receptor
for hyaluronate in rodents and humans (1 1-13). Thus,
CD44 is a critical molecule that mediates diverse
functions of immune cells.
To study the role that CD44 plays in the pathogenesis of inflammatory synovitis, we have deter-
mined the distribution of expression of the CD44
molecule in synovial tissue and synovial fluid from
patients with trauma, osteoarthritis (OA), and rheumatoid arthritis (RA). Moreover, because synovitis in RA
is T cell mediated, we investigated the effect of CD44
molecules in liposomes on activation of T lymphocytes. We found that expression of CD44 was markedly up-regulated in RA synovial tissue and fluid, and
that soluble CD44 in liposomes partially inhibited
CD2-mediated T cell activation in vitro.
PATIENTS AND METHODS
Synovial tissue and synovial fluid. Synovial tissue
from patients undergoing joint surgery was obtained as
discarded tissue from the Duke University Department of
Pathology. Synovial fluid from patients undergoing arthrocentesis was obtained as discarded fluid from the Duke
University Clinical Immunology Laboratory. Human tissue
and fluid were obtained according to a protocol approved by
the Duke Institutional Review Board, for the use of discarded tissue.
Histopathologic techniques. Synovial tissues were
processed, cut, and studied in indirect immunofluorescence
(IF) assays as previously described (14). Using light and IF
microscopy, an inflammation score was assigned to each
synovial sample based on the degree of T cell, B cell, and
monocyte infiltration, vessel proliferation, and fibroblast and
synovial lining cell proliferation, as described (1516). Synovial sections from each patient were also evaluated for
degree of inflammation by a modification of the method of
Rooney et a1 (15).
Cell types were identified by indirect IF assay or light
microscopy, and the histologic features were scored using
the following criteria. Macrophages were identified as LeuM3+ cells and were recorded as follows: &3/high power
field (HPF) = O +; 3-1OIHPF = l + ; 1&30/HPF = 2+;
3&5O/HPF = 3+; >SO/HPF = 4+. T and B lymphocytes
were identified by immunofluorescence for CD2 or B 1,
respectively, and were recorded as follows: rare scattered
cells = O+; <1 aggregate110 HPF = l + ; 1 aggregate/2-3
HPF = 2+; 1-2 aggregates/HPF = 3+. Synovial lining cell
depth was determined by examination of hematoxylin and
eosin-stained sections and recorded as follows: 1-2 cells
thick = O+; 2-4 cells thick = l + ; 5-8 cells thick = 2+; 9-12
cells thick = 3+; >12 cells thick = 4+.
Vessel proliferation was determined using antibody
V2 and recorded as follows: rare vessels = O + ; l-S/HPF =
1+; 6-1OIHPF = 2+; 11-201HPF = 3+; >2O/HPF = 4+.
Perivascular lymphocytes were identified and sections
stained with hematoxylin and eosin, and the numbers were
1435
recorded as follows: rare = O+; 1-2Aow power field (LPF) =
l + ; 3 4 L P F = 2+; 5-8/LPF = 3+; >8/LPF = 4+. The size
of lymphoid aggregates on stained sections was recorded as
follows: rare cells/HPF = O+; 1-10 cells/HPF = l + ; 11-20
cells/HPF = 2+; 21-50 cells/HPF = 3+; >50 cellsIHPF =
4+. Collagen-containing fibroblasts were assessed using the
anti-procollagen monoclonal antibody (MAb) M38, as follows: none = 0; <IO/HPF = I + ; 10-30/HPF = 2+; 30-501
HPF = 3+ ; >5O/HPF = 4+,
The scores for the individual items were added, and
the sum was used as an estimate of the overall intensity of
inflammation by histologic criteria. The degree of reactivity
of CD44 and antifibronectin antibodies was graded 1+ to 4+,
with 1+ signifying 525% of synovial tissue area reactive, 2+
signifying >25% but 550% of synovial tissue area reactive,
3+ signifying >50% but 575% of synovial tissue area
reactive, and 4+ signifying >75% of synovial tissue area
reactive.
Monoclonal antibodies. The following monoclonal
antibodies were used: CD44 (AlG3 and A3D8) (17,181,
antifibronectin (FN-15; Sigma, St. Louis, MO) 187.1 rat
anti-mouse K chain (American Type Culture Collection,
Rockville, MD), 35.1 (CD2) (19), 9-1 (CD2) (201, and P3x63/
Ag8 ascites fluid as a negative control.
Flow cytometry. Flow cytometric analysis was performed on synovial fluid cells, using a FACS Star Plus flow
cytometer (Becton Dickinson, Mountain View, CA) in IF
assays, as described (14,21).
Characterization of CD44 from synovial tissue. Synovial tissue was thawed, homogenized with a Dounce homogenizer in 0.6-1 .O ml extraction buffer (10 mM Tris, pH 8.0,
150 mM NaCl, 1% Triton X-100,20 pdml soybean trypsin
inhibitor, 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl
fluoride [PMSF]), and centrifuged (15,ooO revolutions per
minute for 1 minute). The protein content of supernatants
(tissue extracts) was determined using a copperhicinchoninic acid assay (16) (BCA Protein Assay; Pierce, Rockford,
IL). Tissue extracts were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on
7% or 10% minigels (Mini-Protean 11; Bio-Rad, Richmond,
CA), followed by Western blot analysis using alkaline phosphatase-conjugated goat anti-mouse immunoglobulin, along
with the color development substrates BCIP (S-bromo-4chloro-3 indolyl phosphate) and NBT (nitroblue tetrazolium)
as developing reagents.
Western blot analysis of tissue extracts. To compare
band densities from a given experiment, blots were photographed, and the resulting positive film densities were measured using a laser densitometer. The level of CD44 in
synovial tissue from trauma patients was assigned the value
of 1 , and the level of CD44 in RA and OA fluids was
expressed as the ratio of CD44 in RA or OA tissue:CD44 in
trauma tissue.
Analysis of synovial fluid for CD44 protein. Synovial
fluid specimens were centrifuged, aliquoted, and stored at
-80°C until processed. CD44 protein was immunoprecipitated from aliquots of synovial fluid which were precleared
by incubation with P3-Sepharose (control) beads, then precipitated with either A3D8-Sepharose or P3-Sepharose. Immunoprecipitates were removed from the beads by boiling in
0.06M Tris, pH 6.8, 10% glycerol, 2% SDS and were
HAYNES ET AL
1436
Table 1. Characteristicsof the patients studied
Patient
Disease
duration
(years)
no.*
Age
RA
154
146
68
8
60
>20
90
61
86
38
7
12
68
62
5
> 10
16
10
127
59
15
58
71
60
10
10
5
60
2
244
78
71
169
81
237
74
8
13
0.6
20
148
72
0.3
212
21 1
229
42
1
0.5
1
19
0.8
OA
20
II
36
198
242
Trauma
161
250
22
35
36
52
Medications at
surgeryt
Fibronectin
index
Tissue
Inflammation
CD44
site$
score
index
21
13
14
18
8
13
4+
4+
4+
4+
4+
4+
4+
Shoulder
5
3+
4+
ASA. IAS
ASA
None
NSAID
NSAID
NSAID
NSAID
Knee
Knee
8
8
5
3
14
2+
2+
I+
2+
3t
2t
I t
4+
NSAID
Knee
NSAID
NSAID
None
ASA
None
None
Shoulder
Prednisone
Prednisone, MTX
NSAID, MTX
NSAID, ASA
NSAID, ASA
ASA, Prednisone,
NSAID, gold
Prednisone, TLI
Knee
Knee
PIP
Knee
Knee
PIP
Hip
Hip
Knee
Knee
4
Shoulder
8
3
Toe
5
25
10
5
PIP
I
Elbow
MTP
MCP
4
3+
3+
It
I+
3+
3+
I+
I+
I+
4+
4+
3+
3+
I+
2+
4+
2+
2+
1+
1+
4+
4+
2+
I+
I+
* RA = rheumatoid arthritis (diagnosed according to the American College of Rheumatology
[formerly,the American Rheumatism Association] criteria [40]); OA = osteoarthritis.
7 MTX = methotrexate; NSAID = nonsteroidal antiinflammatory drug; ASA = aspirin; TLI = total
lymphoid irradiation; IAS = intraarticular steroids.
$ PIP = proximal interphalangeal joint; MTP = metatarsophalangeal joint; MCP = metacarpophalangeal joint.
analyzed by SDS-PAGE and Western blot analysis using
alkaline phosphatase-conjugated 187.1 rat anti-mouse immunoglobulin. The level of CD44 in trauma synovial fluid was
assigned the value of 1, and the level of CD44 in RA and OA
fluids was expressed as the ratio of CD44 in RA or OA
fluid:CD44 in trauma fluid, as for synovial tissue. Band
densities of CD44 in gels were determined as described
above for tissue.
Purification of soluble CD44 protein. CD44 (A3D8)
and control IgG 1 (P3x631Ag8) antibodies were conjugated to
cyanogen bromide-activated Sepharose CL-4B (Pharmacia,
Piscataway, NJ) (3.0 mg IgG/ml gel). H u n 8 T cell (CD44+)
lysate was solubilized from 5 x lo9 cells in 50 ml buffer (300
mM NaCI, 10 mM Na2HP0,, pH 7.4, 0.2% [weight/volumel
NaN,, 0.5% [volume/volume] NP40,0.01% [w/vl Tween 80,
0.2 mM PMSF, and 0.1 mM tosyl-L-lysine chloromethyl
ketone) (30 minutes at 0°C). centrifuged (3,OOOg for I5
minutes at 4"C, 23,420g for 30 minutes at 4°C). filtered,
precleared twice over a PfSepharose column, and allowed
to bind to A3D8-Sepharose overnight at 4°C. The column
was washed with 5 column volumes of equilibration buffer,
followed by 5 column volumes of 50 mM Tris, pH 7.4,0.5%
NP40. NP40 was exchanged for octyl glucoside (OG;Sigma)
by washing with 2 column volumes of 50 mM Tris, pH 7.4,
1.5% (w/v) OG.
CD44 protein was eluted with 2.SM MgC12, 50 mM
Tris, pH 7.4, 1.5% OG, and the column was regenerated by
washing with O.IMTris, 0.5M NaCl, pH 8.5, then with 0.1M
NaC,H302, 0.5M NaCI, pH 4.5 and finally, with phosphate
buffered saline (PBS). Eluted fractions were dialyzed sequentially against 50 rnM Tris, pH 7.4 + 1.5% OG, PBS +
1.5% OG,PBS + 1.25% OG,and PBS + 1% OG,using a
Centriprep-30 device (Amicon, Danvers, MA), and the affinity purification steps were repeated until SDS-PAGE
silver staining of the resulting CD44 protein preparation
revealed only a single band, at 80-85 kd, that reacted
strongly with A3D8 antibody in Western blot analysis.
CD44 liposomes. Liposomes were prepared by the
method of Mimms et al (22), using 1 p M purified CD44 or
control glycophorin protein, 1 nM L-a-dioleoyl lecthin
(Avanti Polar Lipids, Birmingham, AL), and 240 nM OG.
Liposomes were analyzed for content of the appropriate
protein using a novel labeling technique and flow cytometry.
Liposomes were incubated with 5-(N-octadecanoyl) aminofluorescein (Molecular Probes, Eugene, OR) in PBS for 10
minutes at room temperature. Fluoresceinated liposomes
were then reacted with 4.5-mm magnetic beads (Dynabeads
M-450 goat anti-mouse IgG; Dynal, Great Neck, NY)coated
with CD44 (A3D8) or anti-glycophorin (E3,E4,ES) MAb.
After continuous end-over-end rotation for 45 minutes at
4"C, beads were washed 3 times in PBS, using a magnet to
immobilize the beads during PBS changes. Fluoresceinated
liposome-bead conjugates were then analyzed by flow cytometry.
T cell activation assay. Peripheral blood mononuclear
cells (PBMC) from healthy donors were stimulated with
CD44 IN RA SYNOVIUM
1437
*
Mean
SEM inflammation score, CD44 index, and
fibronectin index in synovial tissue from patients with trauma,
osteoarthritis (OA), and rheumatoid arthritis (RA)
Table 2.
Inflammation score
CD44 index
Fibronectin index
Trauma
patients
(n = 6)
OA
patients
(n = 8)
9.2 f 4.2*
2.0 t 0.4
6.6 2 1.3
1.9 2 0.2
2.3 f 0.6
2.4 2 0.6
RA
patients
(n = 7)
13.1
2
2.0
3.6 2 0.2t
3.8
2
O.lt
~
* There was a wide range in the inflammation scores among the 8
patients (range 1-25). Synovium from patients 229 (inflammation
score 5) was used as control tissue in biochemical studies.
t P < 0.001 versus tissue from patients with trauma or OA, by
Student's r-test.
were added to these cultures 20 minutes prior to addition of
CD2 MAb. In some experiments, PBMC were pretreated
with 0.1% bromelain to remove cell membrane CD44(18.23).
Statistical analysis. Data were compared using Student's 1-test or the Spearman rank order correlation test.
RESULTS
Figure 1. CD44 expression in synovium from patients with trauma,
noninflammatory osteoarthritis (OA), and rheumatoid arthritis (RA).
A,B, and C, Synovium from trauma patient 229 (inflammation score
5). A, Hematoxylin and eosin (H & E) stain. B, Reactivity with
antifibronectin monoclonal antibody (MAb) FN IS, by indirect immunofluorescence (IF) (fibronectin index 2+). C, Reactivity with
anti-CD44 MAb AIG3, by indirect IF (CD44 index I +). D,E,and F,
Synovium from OA patient 36 (inflammation score 5). 0. H & E
stain. E. Fibronectin expression, by indirect IF (fibronectin index
I +). F, CD44 expression, by indirect IF ( C W index I +). C,H, and
I, Synovium from RA patient 86 (inRammation score 18). C. Perivascular lymphocytes are indicated by arrows. H & E stain. H, Fibronectin expression, by indirect IF (fibronectin index 4+). I, CD44 expression, by indirect IF (CD44index 3+). J, K,and L,Synovium from RA
patient 7 (inflammation score 13), with pannus formation. J, Perivascular lymphocytes are indicated by arrows. H & E stain. K,Fibronectin expression, by indirect IF (fibronectin index 4+). L,CD44 expression, by indirect IF (CD44 index 4+). As demonstrated, CD44
expression is upregulated in synovium from patients with RA, but not
in synovium from patients with tmuma or OA. (S = synovial lining
cells; V = vessel. Original magnification x 400.)
optimal mitogenic concentrations of CD2 MAb 35.1 and 9-1
as described (7). Where indicated, CD44 or control glycophorin liposomes (final protein range used was 28-140 nM)
Histologic analysis of expression of CD44 and
fibronectin in synovium from patients with trauma, OA,
and RA. We studied CD44 expression in synovial
tissues from 7 RA patients and 8 OA patients who had
surgery for joint replacement and 6 patients who had
joint surgery due to trauma (Table 1). As a control for
CD44, we also studied synovial tissue expression of
the extracellular matrix protein, fibronectin. Previous
studies have shown fibronectin deposition to be increased in synovial tissue of patients with RA (24).
We found that expression of both CD44 and
fibronectin was dramatically upregulated in RA synovial tissue, compared with their expression in OA
tissue or noninflamed tissue from patients with trauma
(Figure 1). In trauma and OA synovium, CD44 and
Table 3. Mean -c- SEM inflammation score, CD44 index, and
relative level of CD44 protein (by Western blot analysis) in synovial
tissue from patients with osteoarthritis (OA) and rheumatoid arthritis (RA)'
OA
RA
patients
patients
(n = 3)
(n
Inflammation score
CD44 index
CD44 protein level
~~~
* The CD44 index
= 5)
f 2.1
2.0 2 0.3
3.5 2 0.7
6.4
~
17.3 2 2.3
4.0 2 O.Ot
2 1.7$
10.7
-
was determined by histologic immunofluores-
cence analysis. CD44 protein levels are arbitrary units relative to the
level in synovium from trauma patient 229 (CD44 protein level set at
1. inflammation score 5).
t P < 0.01 versus tissue from patients with OA. by Student's I-test.
$ P < 0.005 versus tissue from patients with OA, by Student's 1-test.
HAYNES ET AL
1438
antifibronectin MAb reacted with synovial lining cells,
vessels, and fibroblasts (Figures IA-F). In RA synovium, infiltrating lymphocytes and macrophages, as
well as synovial lining cells, vessels, and fibroblasts,
stained brightly for CD44 (Figures 1G-I). In RA synovium with pannus formation, both CD44 and fibronectin were widely expressed throughout synovial
tissues (Figures 1J-L).
The RA tissues studied had a mean ? SEM
inflammation score of 13.1 2 2.0, versus 6.6 2 1.3 in
OA tissues. The degree of CD44 MAb reactivity in the
47-
Table 4. Quantitation of CD44 levels in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA)
Patient no.
RA
18
31
25
6
26
22
30
I5
23
33
13
17
12
35
34
Mean
OA
5
16
24
10
3324
-
29
Mean
~~
A
1
0.8
0.6
0.4
0.2
0.0
198
2
SEM
CD44 protein
1.634
4.047
4,776
NA
5,737
6,080
6,820
8,565
9,472
9,772
11,061
I .55
2.87
3.45
4.81
3.74
2.55
2.87
0.54
1.05
0.70
1.69
level*
19,840
0.00
20,214
23,867
26,024
1 1.279 2 2, I07
0.47
1.31
I .04
1.91 2 0.36
98
780
83 I
1,072
3,469
1,250 2 577
1.38
I .24
0.57
0.73
0.77
0.94 2 0.16
-
* Data are arbitrary units relative to the level in synovial fluid from
198229 7 154 86
1.0
* SEM
Synovial fluid
WBC count
(mm?
229
7
SYNOVIUM
154
86
NUMBER
B
Figure 2. Quantitative Western blot analysis of CD44 protein in
synovial tissue. Equal amounts of tissue were extracted from each
synovium and subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, followed by Western blot analysis with antiCD44 monoclonal antibody A3D8. A, Western blots of synovium
from osteoarthritis patient 198 (inflammation score 3). trauma patient 229 (inflammation score 5 ) , and rheumatoid arthritis patients 7,
154, and 86 (inflammation scores 13, 21, and 18, respectively). B,
Area under the densitometry curve (in arbitrary units) of the CD44
bands shown in A.
trauma patient I I (white blood cell [WBC] count 450/mm3, CD44
protein level set at I). The types of medications taken by RA
patients with WBC counts >8,500/mm3 did not differ from those
medications taken by RA patients with WBC counts <7,000/mm3.
RA patient 17 also had calcium pyrophosphate crystals present in
the joint fluid.
indirect IF assay was graded on a I + to 4+ scale, with
1 representing the least CD44 present and 4 the most
(CD44 index; see Patients and Methods). The mean 2
SEM CD44 index in RA patients was 3.6 & 0.2, versus
1.9 2 0.2 in OA patients ( P C 0.001) (Table 2). Thus,
CD44 up-regulation in RA synovial tissue was caused
by 2 separate mechanisms: 1) an increase in the
expression of CD44 on synovial tissue cell types
(synovial lining cells, vessels, fibroblasts), and 2) an
influx of CD44+ infiltrating immune cells (CD44+T
and B lymphocytes, macrophages).
Direct quantitative assay of tbe relative amount
of CD44 protein in synovial tissue. The relative amount
of CD44 in synovial tissue was determined in synovial
tissue from 5 patients with OA (patients 244, 169,237,
242, and 198), and 3 patients with RA (patients 154,86,
and 7), using quantitative Western blot analysis (Table
3). Analysis of the 5 OA synovial tissues demonstrated
a mean 2 SEM relative amount of CD44 of 3.5 f 0.7
(i.e., an average of 3.5 times more CD44 than in
synovial tissue from trauma patient 229). In contrast,
RA synovial tissue contained a mean 2 SEM relative
amount of CD44 of 10.7 +- 1.7. Representative West-
CD44 IN RA SYNOVIUM
1439
E F Mr x 10 -3
A B C
A B C D E F Mr x 10-'
-110
84
- 47
- 33
-110
84
47
-
-
- 33
- 24
- 24
A
B
Figure 3. Western blot analysis of CD44 protein in synovial fluid from patients with trauma, osteoarthritis (OA), and rheumatoid arthritis (RA).
Lanes A and B, synovial fluid from trauma patient 11 (cell count 450/mm3,
relative CD44 level I .O); lanes C and D, synovial fluid from OA
patient 29 (cell count 3,469/mm3, CD44 level 0.77); lanes E and F, synovial fluid from RA patient 13 (cell count 1 1,061/mm3,CD44 level 1.69).
A, Lanes A, C,and E are control lanes in which CD44 protein was immunoprecipitated with CD44 monoclonal antibody (MAb) and then run
in Western blot analysis and blotted with control P3x63/Ag8 IgGl paraprotein. Lanes 9, D, and F contain CD44 protein immunoprecipitated
with CD44 MAb and then blotted with CD44 MAb. B, Lanes A, C,and E are from P3-Sepharose-immunoprecipitated material blotted with P3
control antibody. Lanes B, D,and F are from P3-Sepharose-immunoprecipitated material blotted with CD44 MAb A3D8. The 40-kd band in
all gels is shown to be nonspecific since it it present in P3-Sepharose-immunoprecipitated protein blots.
ern blots demonstrating CD44 levels in trauma synovium (patient 229), RA synovium (patients 7, 154, and
86) and OA synovium (patient 198) are shown in
Figure 2A. Figure 2B shows the relative amount of
CD44 in each synovial tissue, as determined by the
actual value obtained by laser densitometry of the
same Western blot gel. Quantitative Western blot
analysis demonstrated that RA tissue contained 3.5fold more CD44 per gram of wet tissue than did OA
tissue and 11-fold more than did trauma tissue, and
that the amount of synovial tissue CD44 correlated
with the degree of inflammation present (Table 3).
Comparison of the relative levels of soluble CD44
in RA versus OA synovial fluid. Because CD44 protein
has been shown to circulate in a soluble form in plasma
and serum (18), we investigated for the presence of
4.0
O.O0'
I
a
'
'
I
'
I
'
I
"
I
20000
Synovial Fluid Cellslmm3
10000
I
'
'
30b00
Figure 4. Rheumatoid arthritis synovial fluid cell counts versus
relative levels of synovial fluid CD44 protein.
soluble CD44 in synovial fluid from the patients with
trauma, RA, and OA. For this analysis, we used
immunoprecipitation of CD44 antigen from 200 pl of
synovial fluid, followed by quantitative Western blot
analysis. In the 5 OA synovial fluids studied, the mean
SEM white blood cell count was 1,250 2 577/mm3
and the mean level of CD44 was 0.94 ? 0.2 (i-e., 0.94
times the level of CD44 found in synovial fluid from
trauma patient 11). In RA synovial fluid, the mean 2
SEM cell count was elevated (1 1,279 k 2,107/mm3; P
< 0.025 compared with OA) and the CD44 level was
near double that in trauma and OA synovial fluid (1.91
2 0.4; P < 0.001) (Table 4). Figure 3 shows examples
of CD44 in RA versus OA and trauma synovial fluids.
RA synovial fluid contained an average of 2-fold
more soluble CD44 than did OA or trauma synovial
fluid, and the mean RA synovial cell count was higher
than for OA (Table 4). However, when individual RA
synovial fluid cell counts were plotted against the
relative level of RA svnovial fluid CD44. we observed
a significant trend in- which higher CD44 levels occurred in synovial fluid samples with lower cell counts
(r = -0.68, P < 0.01) (Figure 4). When RA synovial
fluids were grouped according t o cell count, those with
low cell counts (<7,000/mm3) had 3.3 times more
CD44 than did RA fluids with higher cell counts
(>8,500/mm3) (mean 2 SEM relative CD44 level 2.84
2 0.3 versus 0.85 2 0.35). Thus, higher levels of
soluble CD44 were present in RA synovial fluids with
lower cell counts, and synovial fluid CD44 decreased
to ~ b n o r m a llevels in the fluids from Patients with
more inflammation.
HAYNES ET AL
1440
A
B
C
D
Mr x 10 -3
4 10
m
A B C D Mrxl
-110
84
84
.
)
- 47
I
- 33
A
- 24
47
33
I
24
B
Figure 5. Western blot analysis of CD44 protein in synovial fluid from patients with non-rheumatoid arthritis types of inflammatory synovitis.
Lanes A and C are control lanes as in Figure 3. A, Synovial fluid CD44 levels in a patient with gout versus a patient with trauma. Lane B.
synovial fluid from trauma patient I I (CD44 level 1 .O); lane D, synovial fluid from gout patient 32 (CD44 level 3.79). Band at 40 kd in lanes A,
B, and C is a nonspecific band not present in lane D for technical reasons. The nonspecific 40-kd band was present in all P3-Sepharoseimmunoprecipitated protein blots (not shown). 8 , Synovial fluid CD44 levels in a patient with psoriatic arthritis versus a patient with trauma.
Lane B, Synovial fluid from trauma patient I I ; lane D, synovial fluid from psoriatic arthritis patient 100 (CD44 level 7.0).
Effect of elevated synovial fluid cell counts on
soluble CD44 levels in non-RA forms of inflammatory
synovitis. To determine whether elevated synovial
fluid cell counts (>8,500/mm3) were associated with
normal or depressed synovial fluid CD44 levels in
diseases other than RA, 3 non-RA inflammatory synovial fluids were studied (Staphylococcus aureus septic
arthritis, cell count 371,915/mm3[patient 141, gout, cell
count 35,400/mm3[patient 321, and psoriatic arthritis,
cell count, 9,294/mm3 [patient 1001). In all 3 cases,
CD44 levels were elevated above the mean level in the
RA fluids with high cell counts (relative CD44 level
2.45, 3.79, and 7.00 in patients 14, 32, and 100,
respectively) (Figure 5 ) . Thus, the decrease in the
synovial fluid CD44 levels in the presence of high
(>8,500/mm3)cell counts in patients with RA was not
a general phenomenon related solely to the number of
synovial inflammatory cells present.
Effect of soluble CD44 protein incorporated into
liposomes on T cell activation. Because soluble CD44
antigen levels were decreased in RA synovial fluids
with high cell counts but not in fluids from patients
with gout or other non-RA inflammatory synovitis,
and because cell-associated CD44 is involved in T cell
activation (7,25,26), we sought to determine potential
immunologic effects on T cell function caused by the
decreased levels of soluble CD44 in inflammatory RA
synovial fluids. To directly assess the effect of soluble
CD44 protein on T cell activation, CD44 was af€inity
purified from T cell membranes, incorporated into
Table 5. Effect on CD2-mediated T cell activation when soluble
CD44 protein was incorporated into liposomes+
~~
Treatment, addition to culture
Sham-treated PBMC
Media
CD2 MAb
CD2 MAb + CD44 liposomes
CD2 MAb + control liposomes
Bromelain-treated PBMC
Media
CD2 MAb
CD2 MAb + CD44 liposomes
CD2 MAb + control liposomes
c p d l d cells
460
308,230
172,960
255.090
5,380
552,440
646,830
550,000
* Data are from a single experiment representative of 4 experiments
with sham-treated cells and 3 matched experiments with bromelaintreated cells. The protein concentration for both CD44 and glycophorin liposomes added in this experiment was 140 nM. The mean 5
SEM suppression of CD2 proliferation by sham-treated peripheral
blood mononuclear cells (PBMC) in the presence of CD44 liposomes
in 4 separate experiments was 35 f 4% compared with control
liposomes (P < 0.02, paired r-test). Addition of CD44 liposomes to
bromelain-treated PBMC had no significant effect on CD2-mediated
proliferation (mean f SEM increase 5 & 7% compared with control
liposomes in 3 experiments; P not significant). Identical results were
obtained when soluble, free CD44 protein was added to CD2stimulated PBMC cultures (data not shown). MAb = monoclonal
antibody.
CD44 IN RA SYNOVIUM
liposomes, and incubated with PBMC prior to stimulation with CD2 antibodies (a potent stimulus of T cell
activation) (7,27). Initial experiments showed that
CD44 protein-containing liposomes alone had no effect on the proliferation of T cells (data not shown).
However, when CD44 protein-containing liposomes
(CD44 concentration 140 nM) were added to PBMC
prior to the addition of CD2 antibodies, T cell proliferation was significantly decreased, by a mean ? SEM
(n = 4) of 35 k 4% (P< 0.02 compared with addition
of control [glycophorin-containing] liposomes) (Table
5 ) . We have previously shown that the cysteine protease, bromelain, removes surface CD44 from T cells
(18,23). In the present investigation, we observed no
suppression of T cell activation when CD44 proteincontaining liposomes were added to bromelain-treated
PBMC, studied as a control (mean ? SEM suppression -5 ? 7%, n = 3; P not significant)(Table 5 ) .
DISCUSSION
We have demonstrated that the expression of
the hyaluronate receptor (CD44) is up-regulated in
synovial tissue and fluid from patients with rheumatoid
arthritis compared with tissue and fluid from patients
with osteoarthritis or trauma, and that soluble CD44
protein in liposomes partially inhibits CD2-mediated T
cell activation in vitro. The CD44 molecule is an 85-kd
glycosylated molecule with N-terminal sequence homology to cartilage link proteins (28,29). Forms of
CD44 of varying sizes have been described on many
cell types (4,28-30). Variations in the size of CD44
isoforms have been suggested to be due to glycosylation differences, the addition of chondroitin sulfate
molecules to CD44 (30), and in some cases, alternative
splicing of CD44 messenger RNA (31,321. Three forms
of CD44 have been identified on PBMC (Hale LP,
Haynes BF: unpublished observations), and an 85-kd
(presumably secreted) form has been identified in
serum, plasma (18,33), and now, synovial fluid.
Functionally, CD44 has been shown to be a
central molecule involved in T lymphocyte adhesion,
T lymphocyte activation, and cytokine release from
monocytes (43-13,28-30). The association of the
CD44 intracellular domain with the cytoskeletal protein ankyrin and with the enzyme protein kinase C
(PKC) (34) has suggested a role for CD44 in signal
transduction of surface events to intracellular molecules. Ligand binding to the CD44 molecule promotes
T cell adherence to monocytes via other adhesion
molecule pathways (intercellular adhesion molecule
lhymphocyte function-associated antigen 1 [LFA- I]
1441
and LFA-3KD2) (7,35), suggesting that CD44 can
serve as a regulator of function of other adhesion
molecules (for review, see refs. 2 and 4).
Recent studies have demonstrated that the
CD44 protein is the primary receptor for hyaluronate
in rodents and humans (1 1-13). Both hyaluronate (36)
and CD44 MAb (7,8) binding to monocytes induce
monocyte IL-1 release. On T cells, hyaluronate and
CD44 MAb ligation of CD44 have disparate effects;
CD44 MAbs augment T cell triggering (7,25,26), while
hyaluronate suppresses T cell triggering (37). Finally,
CD44 MAbs and polyclonal anti-CD44 serum have
been shown to inhibit the binding of lymphocytes to
high endothelial venules at sites of inflammation, such
as synovium (9,10,30), suggesting that lymphocyte
CD44 is one of several molecules involved in organspecific lymphocyte homing. Thus, the hyaluronate
receptor (CD44) molecule, by existing in several isoforms and by virtue of its wide cellular distribution,
functional association with other adhesion molecules,
and physical association with ankyrin and PKC, is a
multifunctional proinflammatory molecule involved in
immune cell activation (for review, see ref. 4).
Hyaluronate, the ligand for CD44, is an important component of synovial fluid and plays a critical
role in maintaining high viscosity of synovial fluid in
normal diarthrodialjoints (for review, see refs. 38 and
39). In RA synovial fluid, the hyaluronate concentration and degree of polymerization are decreased (39).
Reduction in the concentration and degree of polymerization of synovial fluid hyaluronate has been suggested to be an important factor leading to joint
dysfunction and destruction in RA (38,39), and potentially may decrease the immunosuppressive effect of
hyaluronate on T cells (32). Given the striking increase
in CD44 levels in RA synovial tissue, the decrease in
synovial fluid hyaluronate in RA could be due in part
to increased binding of hyaluronate by synovial tissue
CD44.
In RA synovial tissue, up-regulated CD44
expression was due both to increased CD44 expression on synovial lining cells, fibroblasts, and vessels,
and to influx of CD44+ immune cells into synovium.
In contrast to synovial tissue, where CD44 levels
increased in direct proportion to the degree of tissue
inflammation present, in synovial fluid, CD44 levels
were lower in the presence of higher inflammatory cell
counts (Figure 4). Thus, in RA there is differential
regulation of cell-associated CD44 in tissues versus
soluble CD44 in synovial fluid. Synovial fluid from
patients with gout, psoriatic arthritis, and septic arthritis had elevated CD44 levels compared with that from
HAYNES ET AL
1442
patients with trauma, suggesting that the lack of elevated CD44 levels in inflammatory RA synovial fluid
was a result of the pathologic process in RA, and not
a general consequence of elevated synovial cell
counts. The basis for the lack of elevation of soluble
CD44 in RA synovial fluid with elevated cell counts is
not known. Possibilities include adsorption of soluble
CD44 by synovial fluid cells, degradation of soluble
CD44 by synovial fluid proteases, and inhibition of
production of soluble CD44.
Given the multiple proinflammatory immune
functions mediated by the membrane-bound hyaluronate receptor (CD44) molecule, we suspected that
soluble CD44 may have antiinflammatory properties.
In T cell activation assays, soluble CD44 significantly,
though only partially, suppressed T cell activation in
vitro (Table 5 ) . Based on reported functions of CD44
(4,7,8), it is likely that soluble CD44 protein will also
be shown to inhibit CD44-mediated cell adhesion and
CD4Cmonocyte IL- 1 release. Thus, the determination
of modes for interfering with the proinflammatory
functions of the CD44 molecule is a promising area of
investigation for development of new treatment strategies for RA.
Addendum. Since submission of this article, 4 distinct forms of CD44 have been described (31,41431, and a
nomenclature system has been proposed (44).
ACKNOWLEDGMENT
The authors acknowledge the expert secretarial assistance of Kim R. McClammy.
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