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Induction of peripheral blood lymphocyte transformation by autologous synovial fluid lymphocytes and synovial fluid.

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523
INDUCTION OF PERIPHERAL BLOOD
LYMPHOCYTE TRANSFORMATION BY
AUTOLOGOUS SYNOVIAL FLUID
LYMPHOCYTES AND SYNOVIAL
FLUID
JAMES E. CROUT, FREDERIC C. McDUFFIE, and ROY E. RITTS, Jr.
Synovial fluid lymphocytes and synovial fluid from
most patients with rheumatoid arthritis induced blastogenesis of autologous peripheral blood lymphocytes. In vitro
transfer of the mitogenic activity of the synovial fluid to
peripheral blood lymphocytes was not accomplished, but
diminished response of the peripheral blood lymphocytes
to phytohemagglutinin stimulation after incubation in
synovial fluid was demonstrated. These findings suggest
that a similar blastogenic response in vivo may induce the
lymphoid hyperplasia regularly observed in rheumatoid
arthritis.
Lymphocytes are the predominant infiltrating
cells found in rheumatoid synovium and as such probably play a significant pathogenic role in rheumatoid
arthritis (RA). A considerable amount .of evidence has
accumulated to implicate immune responses to IgG
(rheumatoid factors) and to other urlknown antigens in
From the Mayo Graduate School of Medicine, the Department of Microbiology, and the Division of Rheumatology,
Department of Medicine, Mayo Clinic and Mayo Medical School,
Rochester, Minnesota.
James E. Crout, M.D.: formerly Fellow in Rheumatology,
Mayo Clinic and Mayo Medical School (present address: Naval
Regional Medical Center, Oakland, California 94627); Frederic C.
McDuffie, M.D.: Professor, Departments of Medicine, Microbiology, and Immunology; Roy E. Ritts, Jr., M.D.: Professor and
Chairman, Department of Microbiology.
Address reprint requests to Roy E. Ritts, Jr., M.D., Department of Microbiology, Mayo Clinic, Rochester, Minnesota 55901.
Submitted for publication August 19, 1975; accepted
October 24, 1975.
Arthritis and Rheumatism, Vol. 19, No. 3 (May-June 1976)
the production of this disease (1). B lymphocytes in
synovium may be present as precursors of plasma cells
producing these antibodies. T lymphocytes may contribute t o the production of antibody as helper cells or may
respond directly to antigens and thus produce a local
form of cell-mediated injury in the synovium (2). Further evidence for a crucial role for lymphocytes is found
in reports of clinical improvement in patients with severe RA after depletion of lymphocytes through thoracic duct fistula drainage (3,4).
Bloch-Shtacher et a1 ( 5 ) and Moller (6) have
reported induction of blastogenic transformation of unsensitized lymphocyte in vitro by immune complexes,
and Kinsella (7,8) has found that rheumatoid synovial
fluid containing intracellular inclusions of immunoglobulin and complement is often mitogenic for
autologous and homologous peripheral lymphocytes.
Thus the present authors reasoned that synovial fluid
lymphocytes, either altered by antigen or affected by
immune complexes, might stimulate newly arrived lymphocytes from the peripheral circulation to undergo
blastogenesis. Synovial fluid granulocytes known to
contain complement-immunoglobulin complexes (9,lO)
might produce a similar effect. Therefore the present
study was designed to assess the in vitro blastogenic
response of peripheral blood lymphocytes from patients
with RA and other inflammatory joint diseases to autologous synovial fluid lymphocytes, synovial fluid granulocytes, and cell-free synovial fluid. The results indicate
that autologous synovial fluid lymphocytes do induce
5 24
CROUT ET AL
DNA synthesis in peripheral b l o o d lymphocytes. T h e
nature of t h e stimulating material h a s n o t yet been identified.
MATERIALS AND METHODS
Selection of Participants. Patients* selected by physicians of the Division of Rheumatology were those who had
knee effusions containing enough lymphocytes for study. All
patients with rheumatoid arthritis had classic o r definite disease by the ARA criteria ( I I). Salicylates were discontinued
for 18 hours before blood and synovial fluid samples were
collected for study. N o patient was receiving more than 4 mg
of prednisone or its equivalent daily. None had received gold,
antimalarial drugs, or cytotoxic agents. None had received
intraarticular injections of steroids in the 3 months prior to
study.
Preparation of Cell Cultures. Blood and synovial fluid
samples were collected in heparinized syringes. Lymphocytes
from both samples were obtained by Ficoll-Hypaque gradient
centrifugation (12). HyalHronidase was found to diminish
phytohemagglutinin respowie of SFL in preliminary studies
and was not used in the separation process of SFL. Cells at
the interface were removed and washed twice in Medium
199 (GIBCO, Grand Isla'nd, New York) with HEPES
buffer (Calbiochem, Los Angeles, California) (12). The cells
were counted and diluted in this buffer, which contained 15%
heat-activated fetal calf serum (Reheis Chemical Co, Phoenix,
Arizona), 100 U/ml of penicillin, and 100 pg/ml of streptomycin. Lymphocyte preparations were examined using
myeloperoxidase and Wright's stained smears. Differential
counting of peripheral blood lymphocyte preparations (PBL)
averaged 95% medium o r small lymphocytes and 5% monocytes and neutrophils. In synovial fluid lymphocyte preparations (SFL), small and medium sized lymphocytes averaged
85%, with neutrophils and monocytes comprising the remaining 15%. Final cell concentration was then adjusted to 1.0 X
IV lymphocytes/ml. Additional portions of lymphocytes were
adjusted to 2.0 X los lymphocytes/ml and 4.0 X lo6
lymphocytes/ml.
Lymphocytes to be treated with mitomycin (designated as mSFL o r mPBL) were suspended in the above medium and incubated at 37°C for 30 minutes with Mitomycin-C
(Nutritional Biochemical Corp, Cleveland, Ohio) at 10 pg per
1 X lo@lymphocytes. The cells were washed three times and
resuspended in medium a t appropriate concentrations.
One-way mixed lymphocyte cultures in quintuplicate
were prepared by adding 0.1 ml of each lymphocyte
suspension at I X los lymphocytes/ml(O.2 ml of medium and
2 X lo6 lymphocytes in each culture). To test the effect of
increasing the concentration of possible antigens, additional
cultures were prepared by adding 0.1 ml of mSFL or mPBL at
concentrations of 2 X lo8 and 4 X lo8 lymphocytes/ml to 0.1
ml of PBL or SFL at a concentration of 1.0 X lo6
lymphocytes/ml. Final total cell densities were 1.5 X lo8 lymphocytes/ml and 2.5 X 10' lymphocytes/ml respectively.
Synovial fluid neutrophils (SFN) were obtained by
* The protocol was approved by the Mayo Clinic Human Studies
Committee. Informed consent was obtained from each participant.
collecting the cell button from the bottom of the FicollHypaque gradient tube and treating it with approximately
15 ml of 0.87% ammonium chloride to lyse the red blood
cells. Cells were centrifuged and washed three times in Medium
199 with HEPES buffer. Differential counts averaged 96%
polymorphonuclear neutrophils and 4% lymphocytes and
monocytes. After adjustment to 1.0 X IOB neutrophils/ml,
0.1-ml aliquots were added to 0.1 ml of peripheral blood
lymphocytes containing 1 .O X lo6 lymphocytes/ml.
The cell-free fraction of synovial fluid (SF) was prepared by centrifuging an aliquot of S F at 6700g. An aliquot of
each supernatant was examined under light microscopy for the
absence of cells. Then 0.1-ml aliquots of dilutions of cell-free
S F were added to 0.1 ml of PBL and SFL containing 1 X 108
lymphocytes/ml.
Lymphocyte response to mitogen was assessed by adding 25 pg of Phytohemagglutinin-M (Difco, petroit, Michigan) to 0.2-1111 aliquots of PBL and SFL concentrated at 1 X
lo6 lymphocytes/ml.
All cultires were incubated at 37°C for 136 hours in a
humidified atmosphere. One microcurie of tritiated thymidine
(New England Nuclear Corp, Boston, Massachusetts) (20
pCi/m mole) was added to each culture for the final 18 hours
of incubation. The washed trichloracetic acid precipitate of
these cells was counted in a liquid scintillation counter. The
fifth culture was used to measure cell viability by trypan blue
exclusion.
Preparation of PBL Cultures Incubated with Synovial
Fluid. To determine whether the stimulatory material in the
cell-free SF could be transferred to autologous peripheral
blood lymphocytes, aliquots of peripheral blood lymphocytes
at 1 X 10' lymphocytes/ml were incubated for 1 hour at 37°C
with an equal amount of autologous cell-free SF. The cells
(tPBL) were then washed twice in medium and resuspended at
a concentration of 1 X los lymphocytes/ml. After further
treatment with Mitomycin-C, 0.1-ml aliquots of tPBL and
mtPBL were added to equal volumes of autologous mPBL and
PBL respectively. Cultures were incubated, harvested, and
counted as previously described.
Measurement of Immune Complexes. Precipitin reactions against monoclonal rheumatoid factor and Clq were
performed o n the serum and synovial fluid in most patients by
the methods described by McDuffie et a1 (13).
RESULTS
Significant stimulation ( a mitogenic index of 3 or
greater t h a n the nonstimulated, resting cultures) of
a u t o l o g o u s peripheral blood lymphocytes mixed with
synovial fluid lymphocytes (Table 1, c o l u m n s 5 vs 3 or 4)
occurred in 5 of 6 patients with R A and in the single
patient with Reiter's syndrome. T r e a t m e n t of cells f r o m
each source with mitomycin revealed that the increased
thymidine incorporation represented a response of PBL
t o stimulation b y SFL for each patient studied (Table 1,
c o l u m n s 6 and 7). The use of different concentrations of
s t i m u l a t i n g cells m a d e i t p o s s i b l e to s h o w that
stimulation w a s a dose-dependent phenomenon in 5 of
t h e 6 r e s p o n d i n g pairs. I n patient 8 maximum
PERIPHERAL BLOOD LYMPHOCYTE TRANSFORMATION
stimulation was produced by 2 X lo5 cells, with a lesser
response to 4 X 105 cells. N o stimulatory effect of PBL
on SFL was observed at any of the three concentrations
employed (Table I , column 7).
The responsiveness of PBL to synovial fluid cells
was found to be specific for lymphocytes in that no
stimulation was produced by SFN used in equivalent
concentrations (Table 1, column 8). Lack of stimulation
of peripheral blood lymphocytes by autologous SFN
was also noted in 1 patient with pseudogout and 1
patient with gout. (Data for these findings not listed in
Table 1 because enough SFL could not be obtained to
perform complete studies.) Because the percentage of
monocytes in the neutrophil preparations was about the
same as in lymphocyte preparations (*2%), it is unlikely
that the stimulation produced by lymphocytes was the
result of monocyte contamination.
Addition of autologous cell-free synovial fluid,
however, resulted in significantly increased incorporation of tritiated thymidine by both PBL and SFL
cultures (Table 1, columns 9 and 10). Thus, for 6 of
the cultures of PBL, the dpm (column 9) was greater
than three times the resting values (column 3), and for 5
of 7 SFL cultures thymidine incorporation (column 10)
exceeded three times the control resting value (column
4). Four cultures responded best to undiluted synovial
fluid, but PBL from patients 1,4, and 7 responded better
to diluted material. The maximum responses of PBL to
synovial fluid were greater than those of SFL in all
patients but patient 4. Similarly, inspection of the data
on responses of PBL and SFL to phytohemagglutinin
(PHA) (columns 11 and 12) revealed that PBL
incorporated from 1.5 to 10 times more thymidine than
SFL in response to this mitogen. In 7 of 8 patients the
responses of PBL to PHA stimulation were within the
range of normal (20,OOO-60,000 dpm per culture, TC
199:HEPES being the standard in this laboratory).
However all of the responses of SFL cultures to PHA
were below the lower limit of normal. The decreased
responsiveness of SFL to synovial fluid and to PHA
could not be explained by death of SFL in culture. At a
cell density of 1.0 X 106/ml, viability of PBL as
estimated by trypan blue exclusion was 58-82% at 120
hours, whereas that of SFL was 41-71%. At higher
densities viability of cells from both sources was slightly
less: 42-67% for PBL and 35-58% for SFL at 2.5 X 108
cells/ml at 120 hours.
The upper half of Table 2 shows results of tests
for rheumatoid factor, complement, and immune complex-like material in the synovial fluid of patients 1
through 8. Rheumatoid factors were present in 3 of the 7
525
patients with RA, immune complex-like material was
found in 4 of the 4 rheumatoid patients tested, and low
levels of complement were demonstrated in 4 of the 7
patients with RA. Thus the ability of SF or SFL to
stimulate autologous PBL apparently did not depend on
the presence of rheumatoid factor or depressed synovial
fluid complement levels. The 1 patient (number 3) whose
mSFL did not stimulate PBL had a normal level of
synovial fluid complement and lacked rheumatoid factor but did have immune complex-like material detectable by both monoclonal rheumatoid factor and Clq in
synovial fluid.
In order to determine whether synovial fluid
might contain a substance, such as an antigen, that
could bind to lymphocytes and thus be responsible for
the stimulation of PBL by SFL, PBL were incubated
with autologous, cell-free synovial fluid and then examined for their ability to stimulate PBL that had not been
similarly treated. The results, shown in Table 3, indicated a modest increase in thymidine uptake of resting
PBL after exposure to synovial fluid in 2 of 6 RA
patients and in 1 patient with osteoarthritis (patients 10,
12, and 14; column 4 compared with column 3). No
significant increase in thymidine incorporation into
DNA occurred, however, when treated PBL were incubated with untreated PBL (column 5 ) . Results were
the same with mitomycin-treated PBL, except for an
increased uptake by cells of patients 10 and 12 which
failed to reach a significant level (column 7).
In the 6 patients with RA response of tPBL to
PHA was consistently decreased in comparison to corresponding cultures of untreated PBL treated with PHA
(columns 8 and 9). Responses of tPBL and PBL from
patients with gout and osteoarthritis were not significantly different upon addition of PHA. Because
lymphocytes treated with synovial fluid from 2 RA
patients had shown increased resting incorporation of
thymidine, it was considered possible that the treated
cells might already be nearly maximally stimulated. The
use of smaller amounts of PHA, however, did not result
in any increase in thymidine uptake (column 9, x and y).
As in the results reported in Table 1, addition of
autologous synovial fluid to PBL from these 6 patients
with RA produced a significant increase in thymidine
uptake (column 10). Synovial fluid from 2 additional
control patients (with gout and osteoarthritis) did not
affect the mitogenic index.
DISCUSSION
Because of the critical role lymphocytes play in
cell-mediated and humoral immunity, several groups of
CROUT ET AL
526
Table 1. Mitogenic Responses of Lymphocytes from Peripheral Blood and Synouial
Column
2
3
4
Patient
Diagnosis
PBL*
SFLt
I
RA
45 1 ( f I 10)
5
PBL
353 ( f 7 2 )
a
b
C
* mPBL
6
+ SFL
2,279 ( f 3 2 5 )
2,359 ( f 2 0 7 )
6, I06 ( f 5 6 5 )
PBL
d
e
f
+ mSFL
2,287 (f 128)
2,224(*314
5,776 ( f 6 0 2 )
2
RA
397 ( f 9 4 )
390(f86)
2,622 ( f 4 1 0 )
3,677(&382)
4,031 ( f 6 0 7 )
2,085 ( f 3 0 7 )
3,007(f412)
7,584(&692)
3
RA
5,054 ( f 9 4 I )
1.926 ( f 3 17)
3,263(+317)
3,522 ( f 2 0 9 )
3,278 ( f 4 0 7 )
5,831 ( f 8 0 2 )
5,054 ( f 7 1 2 )
4,689(&419)
4
RA
2,856 ( f 4 9 8 )
l,5 12 ( f 2 7 4 )
2,647 ( f 2 3 8 )
5,308 ( f 6 0 2 )
9,695 (f1,014)
2,729 ( f 6 0 2 )
9,137 (fI .072)
12,540(+2,694)
5
RA
404 ( f 9 4 )
307 ( f 5 8 )
2,495(*302)
2,680 ( f 4 0 7 )
7,240 ( f 8 9 4 )
1,964 ( f 4 6 7 )
2,499 ( f 8 4 2 )
6,020 (f I ,2 14)
6
Monoartic.
arthritis
1,204 ( f 2 8 7 )
647(+114)
9 10 (f 102)
990 (f184)
I,509(f207)
247(f68)
462 ( f 4 7 )
1,503(+247)
7
RA
236(f59)
1,390 ( f 2 10)
4,2 10 ( f 3 0 6 )
20,214 (f3,412)
1,203 (f174)
3,957 ( f 6 2 l )
16.4 I7 (f2.124)
8
Reiter’s
3,991 ( f 4 1 6 )
31,221 (f4,017)
44,947 (f5,624)
12,802 (f1,279)
33,947 (f3.640)
62,619 (f7.821)
27,167 (f1.894)
cultures:
84 (f30).
t mSFL cultures: 3 (&23).
360 (f88)
4,302 ( f 5 9 7 )
All results expressed as dpm f standard deviation.
All cultures contained 2 X 105 lymphocytes ( I X 105 responding cells and I X 105 stimulating cells) except as otherwise indicated.
d = mSFLof I X I@ cells
a = SFL of I X 105cells
b = SFL o f 2 X I05 cells
e = mSFLof 2 X I@ cells
f = m S F L o f 4 X I05 cells
c = S F L o f 4 X 105cells
investigators have examined lymphocyte function in
rheumatoid arthritis. Most of these studies have shown
that lymphocytes from rheumatoid synovial fluid have
a diminished response to PHA stimulation, whereas
the response of peripheral blood lymphocytes is norma1 (15-18). I n one report (19) peripheral blood
lymphocytes had a diminished response to PHA stimulation, but synovial fluid reactivity was not studied.
Menard et a1 (20) noted some increase in the incorporation of tritiated thymidine by unstimulated peripheral blood lymphocyte cultures in patients with RA.
Studies of mixed lymphocyte reactivity of per-
PERIPHERAL BLOOD LYMPHOCYTE TRANSFORMATION
527
Fluid to Autologous Cells and Synouial Flu'd and to Phytohemagglutinin
8
7
~~
~
~
+
j
k
555(f38)
628(f64)
777(f43)
263(f52)
325(f62)
x
Y
2
580 (f74)
741 (f62)
622(f18)
245 (f67)
378 (f96)
1.572(f197)
2,913(f278)
3,134(f402)
4,055(f612)
1,055(f143)
2,738(f468)
2,625(f501)
876 (fI 18)
948(f126)
453 (f201)
555 (584)
631 (f62)
513 (f67)
397(f101)
477(f87)
720(f93)
i2
+ SF
SFL + SF
2,607(41507)
19,584(f3,027)
1,642( f 306)
1,345(f194)
20.445(f3,745)
1,090(f124)
430(f110)
923(f216)
21,083(f3,714)
3,072(f862)
1,282(f127)
1,088 (f3 18)
1,131(f296)
5,256(*746)
1,131(f207)
766(fl10)
9,812(f942)
1,022(f196)
842(f102)
622(f79)
2,666(f564)
1,44I (f296)
388(175)
313
27,408(&4,164)
I1,952(+3,412)
15,997(f2.074)
I1,550(+2.436)
26,522(f4.279)
5,045(f672)
2,738(*375)
3,625(f419)
4,942( f 1,004)
960(f114)
20,781(f3,176)
6,830(f914)
4,050(f795)
1,476(f197)
19,943(f3,176)
2,705(f578)
1,843(*297)
933(f114)
4,942(f627)
1,960(f217)
1,568(+159)
1,228(f174)
3,500(f416)
1,123(f287)
887(f92)
256 (f67)
3,940(f490)
9,690(f1,070)
2,960(f41I )
1.084(fI 19)
56,031(f6,497)
7,382(f874)
31.232(f3.179)
5,03I (f612)
38,846(&5,164)
11,609(f1,475)
PBL
w
II
10
9
PBL SF
Neutrophil
SFL + mPBL
g
h
i
~~~~~~~~
PBL
+ PHA
SFL + PHA
376 (f287)
260( f148)
400(f207)
688(f106)
734(f186)
256 (f61)
375 (f54)
22,253(f4,125)
huff.
Lym phs.
28.188(f3.127)
2.498(f527)
2,309(f365)
7,147(&702)
6,146(f603)
4,304(f597)
4,296(&712)
7,705(f1,074)
3,558(f621)
5,322(f507)
5,669(f812)
9,085(f1.014)
2,785(f612)
589(+117)
390(f62)
56,052(*7,244)
14,752(f1.874)
Abbreuiations: PBL, peripheral blood lymphocytes; SFL, synovial fluid lymphocytes; m, mitomycin-treated; SF, synovial fluid; PHA, phytohemagglutinin.
g
h
i
=
=
=
rnPBL of I X
rnPBL of 2 X
mPBL of 4 X
lo5cells
lo6cells
lo5cells
j
=
k
=
SF neutrophils of I X 10' cells; PBL 2 X 10'
SF neutrophils of 2 X IO'ceIls; PBL 2 X lo6
ipheral blood lymphocytes in RA have revealed diminished responsiveness. Both Astorga and Williams (2 1 )
and Hedberg et a1 (22) found that the D N A synthesis of
PBL from patients with R A , when mixed with
mitomycin-treated lymphocytes from other RA patients, was less than when mitomycin-treated normal
w = undiluted synovial fluid
x = I/lOsynovial fluid
y
=
z
=
1/20 synovid fluid
1/40synovial fluid
lymphocytes were employed for stimulation. In control
mixtures rheumatoid lymphocytes were able to stimulate nonrheumatoid cells normally. In a subsequent
study Williams et a1 (23) noted inhibition of mixed
lymphocyte culture (MLC) reactions of normal individuals by a substance in the IgG fraction of sera from 4 of
CROUT ET AL
528
Table 2. Immunologic Tests on Synouial Fluids
Complement
(units/ml)
Patient
Diagnosis
Rheumatoid Factor
(reciprocal titer)
1
2
3
4
5
6
RA
RA
10,Ooo
7
8
9
10
II
12
13
14
15
16
RA
RA
RA
Monoartic.
arthritis
RA
Reiter’s
RA
RA
RA
RA
RA
RA
Osteo.
Gout
1,280
<20
<20
640
<20
<20
<20
20,480
640
40,960
640
2,560
40,960
<20
<20
Immune Complexes*
Patients
Normalt
MRF
Ch
<5
<5
34
26
<5
13-43
31-60
22-52
N DS
ND
-
1.8-48
+
+
+
+
17
22-51
19-49
+
+
+
ND
ND
24
43
<5
39
<5
<5
<5
<5
20
23
18-48
12-42
21-51
23-53
29-59
30-60
21-51
22-52
0.9-30
I .3-3 I
ND
-
ND
+
ND
ND
-
+
+
-
ND
ND
+
-
* Presence of immune complexes estimated from precipitin reactions with monoclonal rheumatoid factor
(MRF) and Clq.
t Normal ranee for comulement in svnovial fluid determined from total protein concentration (14).
ND = not Zone.
52 patients with RA that correlattd with the presence of
lymphocytotoxic antibody in the serum. A recent report
by Stastny (24) suggests the sharing of an MLC antigenic determinant by certain patients with RA may be
responsible for diminished MLC responses.
T cell-B cell interaction plays a significant role in
the production of antibodies to most antigens, and
Allison and colleagues (25) have suggested that autoimmunity may result either from bypassing the requirement for T-cell helper function or from the presence of a defect in T-cell suppression. Most studies on
the relative numbers of T and B cells in peripheral blood
of patients with rheumatoid arthritis have shown them
to be normal (18,26-28) although a decrease in the
proportion of T cells with active disease has been reported (26). However the percentage of T lymphocytes
in the synovial fluid has been found to be increased
(18,27,29). B-cell percentages in synovial fluid were
found to be less than those in peripheral blood by Winchester et a1 (29) and by Sheldon et a1 (18). The number
of null cells not containing surface membrane receptors,
immunoglobulin, or nonspecific receptors for sheep
erythrocytes in synovial fluid was increased in the study
of Winchester et a1 (29). These findings in synovial fluid
contrast with the results of histologic examination of
rheumatoid synovial membrane in which B cells appear
to predominate (30).
The ultimate cause of rheumatoid arthritis is unknown, but evidence is accumulating that the immune
system of RA patients is stimulated by unidentified antigens (31). Immune complex-like material has been
found in the serum and synovial fluid of patients with
RA and may be in part responsible for complementmediated tissue injury (3 1-34). Immune complexes are
capable of stimulating unsensitized lymphocytes in vitro
(5,6) and Kinsella (7,8) has found rheumatoid synovial
fluid to be mitogenic for autologous and homologous
peripheral blood lymphocytes. This stimulatory activity
of synovial fluid was associated with the presence of
phagocytic cells containing immunoglobulin and complement in the synovial fluid but not with the presence of
rheumatoid factor. The present study also revealed that
both PBL and SFL from patients with RA underwent
increased blastogenesis when stimulated by autologous
cell-free synovial fluid. However the ability of synovial
fluid to stimulate PBL or SFL could not be related to the
presence of rheumatoid factor or immune complexes
or to depression of synovial complement levels. Nor
did polymorphonuclear neutrophils, which have been
shown to contain aggregates of immunoglobulins and
470 ( f 7 1 )
130 ( f 2 4 )
356 (f8O)
244(f61)
263 ( f 6 7 )
RA
RA
RA
Osteo.
Gout
12
13
14
15
16
=
=
x
y
w = 25 pg
of PHA
2.5 pg of PHA
1.25 pg of PHA
* mPBL: 46-122.
663 ( f 8 2 )
105 ( f 3 4 )
123(f31)
227 ( f 5 6 )
270 (f110)
604 ( f 7 8 )
256(f79)
X 10'
stimulating cells or 2
2,560 ( f 3 10)
X
Y
X
W
Y
X
W
Y
X
W
Y
X
W
X
W
X
W
X
W
X
W
X
W
X
W
X
W
X
W
3,33O(f329)
204 (f44)
PBL + SF
10
324(f195)
216 ( f 8 2 )
4,640 (f121)
1,230 (f135)
2,120 ( f 143)
1,417 ( f 168)
3,240 (f147)
1.080 (rt2 16)
477(*102)
192 (rt38)
14.721 (f2.742)
895 (f1 17)
21,347 ( f 4 2 1 )
578 ( f 4 8 )
I@ responding cells alone).
46,199 (f4.129)
52,157 (f3,260)
6,022 ( f 6 4 2 )
2, I60 ( f 4 1 6 )
8,230 ( f 9 4 2 )
18,245 (3~3,426)
301 ( f 3 8 )
787 (3Z30)
33,084 (f1.795)
377 ( f 4 1 )
249 ( f 16)
9, I46 ( f 8 4 2 )
12,095 (f1,774)
164 ( f 2 8 )
123 ( f 2 7 )
9,028 ( f 6 2 3 )
tPBL + PHA
9
Abbreviations: PBL, peripheral blood lymphocytes; tPBL, synovial fluid incubated peripheral blood lymphocytes; m. mitomycin-treated; SF,
synovial fluid; PHA, phytohemagglutinin.
All results expressed as dpm (f standard deviation).
All cultures contained 2 X la cells ( I X 106 responding cells and 1
234 ( f 7 9 )
43,156 (f2.197)
178 (524)
204(f37)
407 ( f 8 2 )
352 (f44)
245 ( f 9 4 )
6,397 ( f 2 6 0 )
I25 (f19)
I I1 (f24)
135 ( f 4 l )
122 ( f 6 1 )
6,662 ( f 2 1 5 )
1,125(*174)
143 ( f 2 5 )
1,618 (f157)
35.91 I (f3.517)
181,987 (f26.182)
12,587 ( f 9 6 3 )
1,053 ( f 6 8 )
677 ( f 7 8 )
29,539 (f2,906)
153 ( f 3 8 )
PBL + PHA
8
2,595 ( f 1 9 3 )
653 (f44)
1,391 ( f 1 3 3 )
738 ( f 2 4 )
RA
II
206 ( f 6 8 )
327 ( f 6 1 )
7
mPBL + PBL PBL + mtPBL
1,630 (f152) 2,739(&313)
+ tPBL
6
387 ( f 2 8 )
RA
10
PBL
5
208 (*23)
303 ( f1 10)
RA
9
tPBL
4
392 ( f 4 l )
PBL*
Diagnosis
Patient
3
2
Column
Table 3. Mitogenic Response of Synovial Fluid Incubated Peripheral Blood Lymphocytes to Autofogous Untreated Peripheral Blood Lymphocytes
m
P
r
M
W
ic
CROUT ET AL
5 30
complement (35), stimulate autologous peripheral blood
lymphocytes.
Results of the present study demonstrated that
washed synovial fluid lymphocytes are able to induce
significant transformation of autologous peripheral
blood lymphocytes in most patients with RA. Unfortunately it is impossible t o tell if this finding is
specific for R A because S F L from patients with such
diseases as osteoarthritis, crystal-induced synovitis, etc,
could not be obtained in amounts suficient to carry out
appropriate control studies. T h e nature of the material
present in or on S F L that is responsible for the
stimulation observed is unknown. Because it could not
be transferred from synovial fluid t o lymphocytes by in
vitro incubation, it may be a substance tightly absorbed
to cell membranes and/or released by lymphocytes into
synovial fluid. Presence of lymphokines in cell cultures
may explain the observed mitogenicity of S F L for autologous PBL. Although mitomycin treatment inhibits cell
replication (36), it does not inhibit release of preformed
mediators. Similarly, a population of synovial fluid lymphocytes activated in vivo before treatment with mitomycin could release preformed mitogenic factors that
stimulate PBL. Stastny (2) has found lymphokine-like
substances in effusions and synovial tissue of patients
with RA. It is attractive t o speculate that rheumatoid
S F L may contain some unique antigen t o which the
host’s immune system has been sensitized. Alspaugh et
af (37,38) have found that sera of patients with Sjogren’s
syndiome a n d R A possess antibodies against antigens
present in certain lymphoblastoid cell lines.
The results of the present study a r e in marked
contrast to a prior study by Griffiths and Williams (39),
who found stimulation of S F L by PBL but not the
reverse. The present authors cannot explain the different results of the two studies. However the GriffithsWilliams study did use lower ratios of S F L to PBL
and hyaluronidase in preparing S F L . Of particular
note were the very high levels of thymidine uptake by
resting PBL in the Griffiths and Williams report (in the
order of 30,000 cpm). It is possible that stimulation of a
minority of the PBL population by S F L might have
been missed in the presence of such high background
counts. Menard et a1 have pointed o u t that the relative
amount of stimulation of rheumatoid PBL by P H A is
inversely proportional to the resting thymidine uptake
(20).
Because discordant results have been reported
from two different laboratories, additional studies are
necessary to clarify the exact nature of the interaction
between S F L and PBL in patients with R A . Never-
theless results reported in this study demonstrate
that under the conditions employed S F L and cell-free
synovial fluid from most patients with R A are capable
of inducing significant transformation of autologous
PBL. Though it is not possible to transfer this activity
from SF t o PBL by in vitro incubation, PBL treated
with synovial fluid d o exhibit the same decreased responsiveness t o phytohemagglutinin that freshly isolated S F L show in comparison to untreated PBL. This
inhibitory material is probably distinct from the material in S F L that is responsible for stimulating autologous PBL. The eventual identification of t h e responsible stimulatory and inhibitory substances should
lead t o more complete understanding of lymphocyte
function in rheumatoid arthritis and its possible relationship t o the pathogenesis of the disease.
ACKNOWLEDGMENTS
The authors express their appreciation to Ms. JoAnn
Caron for valuable technical assistance and to their colleagues
in the Division of Rheumatology for patient referral.
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