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Natural killer cell activity of mononuclear cells from rheumatoid patients measured by a conjugate-binding cytotoxicity assay.

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1440
NATURAL KILLER CELL ACTIVITY OF
MONONUCLEAR CELLS FROM RHEUMATOID
PATIENTS MEASURED BY A CONJUGATE-BINDING
CYTOTOXICITY ASSAY
ELIZABETH REINITZ, P. ANDREW NEIGHBOUR, and ARTHUR I. GRAYZEL
The natural killer cell activity of synovial fluid
mononuclear cells and synovial membrane mononuclear
cells from patients with rheumatoid arthritis was compared with that of paired peripheral blood mononuclear
cells from many of these patients. Natural killer cell
activity was measured by the use of a conjugate-binding
cytotoxicity assay with an erythroleukemic cell line,
K562, as target. There was no significant difference
when 15-minute target binding by peripheral blood
mononuclear cells was compared with synovial fluid
mononuclear cells. Target binding of synovial membrane mononuclear cells was significantly greater (P <
0.05) than that of peripheral blood cells. Three-hour
target killing, however, was significantly greater when
synovial fluid mononuclear cells were compared with
the peripheral blood cells, P < 0.01, and when synovial
membrane cells were compared with the peripheral
blood cells (P < 0.05). The products of binding and 3hour killing, a reflection of the total number of mononuclear cells participating in cytotoxicity, were significantly greater when either synovial fluid or synovial
membrane mononuclear cells were compared with peFrom the Departments of Medicine, Pathology, and Microbiology & Immunology, Montefiore Hospital & Medical Center, and
the Albert Einstein College of Medicine, Bronx, NY.
Supported in part by a Clinical Research Center Grant from
the Arthritis Foundaton and research grants NS-15541, NS-I 1920,
AI-09807 and AM-07273 from the National Institutes of Health.
Elizabeth Reinitz, MD: Assistant Professor of Medicine,
formerly Fellow in Rheumatology; P. Andrew Neighbour, PhD:
Assistant Professor of Pathology and Microbiology & Immunology;
Arthur I. Grayzel, MD: Professor of Medicine, the Albert Einstein
College of Medicine.
Address reprint requests to Arthur I. Grayzel, MD, Montefiore Hospital & Medical Center, 111 E. 210 Street, Bronx, NY
10467.
Submitted for publication March IS, 1982; accepted in
revised form July 19, 1982.
Arthritis and Rheumatism, Vol. 25, No. 12 (December 1982)
ripheral blood mononuclear cells (both P < 0.01).
Electron microscopy confirmed the presence of large
granular lymphocytes, representing 20% of the peripheral blood cells and 37% of the synovial fluid mononuclear cells. Interferons were detected in 10/12 rheumatoid and 3/12 nonrheumatoid synovial fluid samples
studied. These findings indicate that functional natural
killer cells are selectively increased in the rheumatoid
joint and may contribute to the overall increase in
immunologic activity found in the joints of these patients.
Some mononuclear cells bearing Fc receptors
with no surface immunoglobulins have been recognized to have spontaneous cytotoxicity for a variety of
target cells in the absence of prior sensitization. These
target cells have included tumor, virus-infected, and
hematopoietic cells. This cytotoxicity is antibody independent and does not require major histocompatibility complex identity. The effector mononuclear cells
have become known as natural killer (NK) cells. In the
human, they are thought to play an important role in
tumor surveillance and in resistance to acute and
chronic viral infections (1). They have been morphologically described as large granular lymphocytes.
These are mononuclear cells that are larger than most
lymphocytes and characterized by crescent-shaped
nuclei and abundant cytoplasm with azurophilic granules (2).
Many patients with certain chronic diseases,
such as systemic lupus erythematosus, multiple sclerosis, and Crohn’s disease, have low or absent NK cell
activity in their peripheral blood mononuclear cells
(PBMC) as compared with normal donors, and many
of these cells fail to respond in vitro to interferon with
the increase of NK cell activity seen with normal
NK CELL ACTIVITY IN RA
1441
donors (3-5). Patients with rheumatoid arthritis, however, have normal NK cell activity in their PBMC, a n d
this activity can be boosted with interferon as in
normal donors (4).
Because t h e site of disease activity in patients
with rheumatoid arthritis is in the synovium, we used a
conjugate-binding cytotoxicity assay t o study the NK
cell activity of synovial fluid mononuclear cells
(SFMC) and synovial membrane mononuclear cells
(SMMC). We also measured interferon levels in synovial fluids and quantitated the large granular lymphocytes in several mononuclear cell samples.
MATERIALS AND METHODS
Patients. Effector cells were isolated from the synovial fluid and simultaneous peripheral blood samples of 10
patients with definite or classic rheumatoid arthritis. SFMC
without paired blood samples were obtained from another 3
patients. SFMC were isolated from 6 patients; from 3 of
these patients, PBMC were also obtained. Three patients
were receiving gold injections, 3 were receiving penicillamine, 2 were on low doses of corticosteroids, and all were on
nonsteroidal antiinflammatory drugs. SFMC were obtained
from 1 patient with rheumatoid arthritis before, and 2 and 4
weeks after, intraarticular injection with cortocosteroids.
Synovial fluid samples were obtained from patients with
rheumatoid arthritis, osteoarthritis, traumatic arthritis, and
crystal-induced arthritis for measurement of interferon levels.
Efector cells. PBMC were isolated by Ficoll-Hypaque (Pharmacia Fine Chemicals) density gradient centrifugation. These cells were then put through a nylon wool column
to remove adherent monocytes and B cells (6), a procedure
known not to deplete NK cells. The nonadherent cells were
then used as effector cells. Synovial fluids were incubated
with DNase (50 pg/ml) (Sigma #D-4763) and bovine testicular hyaluronidase (20 pg/ml) (Sigma #H-2376) for 30 minutes
on a rocking tray at 37°C. They were filtered through gauze
and then treated in the same way as blood samples. Synovial
membranes were dissected free of fat tissue and minced. In
addition to DNase and testicular hyaluronidase (added in the
same concentrations used in synovial fluids), collagenase (50
p g h l ) (Sigma #C-0130) was added. After incubation for 5
hours at 37"C, the cells were filtered through sterile gauze.
The effector cells were then isolated by the above method for
peripheral blood. To control for the effect of these enzymes
on NK cell function, 2 blood samples were divided in half.
One-half was treated with the enzymes used for synovial
fluids and membranes at the same concentrations; the other
half was not. The medium used throughout was RPMI
(Gibco) with 10% fetal calf serum.
Target cells. K562 cells, a human erythroleukemic
cell line, were maintained in continuous suspension culture.
These cells are known to be susceptible to NK cell-mediated
lysis.
Conjugate formation and cytotoxicity assay. We determined the ability of single effector cells to bind and lyse K
562 target cells by using a modification of the method of
Grimm and Bonavida (7) described in detail elsewhere (8).
Equal volumes of effector and target cells were mixed at a
concentration of 4 x lo5 cells and centrifuged at 1,000
revolutions per minute (rpm) for 5 minutes. After the pellets
were incubated for 10 minutes at 3 7 T , we gently resuspended them by pipetting up and down 6 times with a
Pasteur pipette. The percent of viable mononuclear cells
binding to target cells (percent TBC) was determined. The
effector-target mixtures were resuspended in 1% agarose in
medium, spread on glass slides, and allowed to cool. Slides
were then incubated at 37°C in medium. At 3 hours, the
slides were stained with 1%trypan blue at room temperature
for 5 minutes and then washed thoroughly in phosphate
buffered saline. The percent of conjugates containing dead
cells was determined microscopically. The minimum number
of conjugates scored was 200. The percent NK cells was the
product of percent TBC and percent conjugates with dead
targets. Each sample was prepared and scored in duplicate.
Measurement of interferon levels in synovial fluid
samples. Twelve synovial fluid samples from rheumatoid
patients and 12 synovial fluid specimens from patients with
other diseases were assayed for interferon levels. In 6
patients the same synovial fluid was analyzed for both
interferon and NK cell activity. Interferon was titrated by
the inhibition of vesicular stomatitis virus plaques on human
amniotic cells (WISH-2; American type culture collection)
(9). Titers were expressed as the reciprocal of the dilution
that produced 50% reduction of plaques after standardization to the NIH human reference interferon (G023-901-527).
Statistical methods. The group mean values of percent TBC, dead conjugates, and percent N K cells for
synovial fluid and membrane samples were compared with
PBMC effectors by the Mann-Whitney test. Mean interferon
titers were compared by Student's t-test on log,,, data
transference. Significance was accepted throughout at the P
< 0.05 level.
Table 1. Target binding and killing activity of synovial fluid, synovial membrane, and peripheral blood mononuclear cells from patients with
rheumatoid arthritis
Source*
(n)
PBMC (13)
SFMC (13)
SMMC (6)
Binding
(15 minutes)
20.8 t_ 4.2
21.2 ? 2.0
25.7 ? 4.6
Pt
0.8175
0.0485
Killing
(3 hours)
19.0 2 2.5
24.9 t 5.0
23.7 2 3.6
-
0.0029
0.0141
* PBMC = peripheral blood mononuclear cells; SFMC = synovial fluid mononuclear cells; SMMC
1- P value as compared with results for PBMC (Mann-Whitney test).
% natural killer
(binding x killing)
3.9 f 0.9
5.3 2 1.1
6.0 _t 1.2
Pi
=
Pi
-
0.0041
0.0029
synovial membrane mononuclear cells.
REINITZ ET AL
1442
RESULTS
The recovery of cells after nylon wool passage
was the Same for blood (63 7.6%), SYnOvial fluid (66
+ 7.5%), or synovial membrane (65 9.3%).
The percentage Of mononuclear
bound at
15 minutes, the percentage Of bound target
and the percentage of N K cells are shown in Figure I
*
*
g1
5l
7
3
12
I
361
2
7
&
28 -
a
20
am
-
for each blood, synovial fluid, and synovial membrane
examined; the mean values are summarized in Table 1.
The percentage of mononuclear cells bound at
15 minutes was not significantly different when PBMC
were compared with SMFC but was significantly
greater when the SMMC were compared with PBMC
results. Three-hour target cell killing was significantly
greater when SFMC were
with pBMC and
when SMMC were compared with PBMC.
The products of binding and 3-hour cytotoxicity, a measure of the isolated PBMC with NK cell
activity, showed significantly greater results when the
SFMC values or the SMMC values were compared
with PBMC values.
To test whether enzyme treatment of synovial
fluids and membranes affected N K cell activity,
PBMC from 2 patients were examined with and without enzyme treatment at the same concentrations as
used in synovial samples. These experiments showed
that the enzymes had no significant effect on NK cell
activity, (3.42% and 4.32% without enzymes; 3.60%
and 4.00% with enzymes).
SFMC from 1 patient were studied before and 2
and 4 weeks after intraarticular steroid injection. The
initial percentage of N K cells (5.7%) showed a decline
2 weeks after injection (3.5%) and partial recovery at 4
weeks (5.5%).
Since some large granular lymphocytes are
thought to mediate NK cell activity, we used electron
microscopy to look for such lymphocytes. Characteristic large granular lymphocytes accounted for 20% of
the peripheral blood nonadherent cells and 37% of the
synovial fluid nonadherent cells (Figure 2).
Figure 3 shows the interferon levels detected in
the synovial fluid samples. Twelve rheumatoid samples had a mean of 1.73 2 .83 log,, units of interferon;
12 samples from patients with osteoarthritis or crystalinduced arthritis had a mean of 0.80 .55 log,, units of
interferon. These values were found to be significantly
different ( P = 0.0037). In the 6 synovial fluids analyzed
for both interferon and N K cell activity, there was no
significant correlation between the titer of interferon
and cytotoxicity.
*
DISCUSSION
I2PBMC
SFMC
SMMC
Figure 1. The percent target cell binding (bottom panel), 3-hour
target cell killing (middle panel), and percent natural killer (NK)
(upper panel) of mononuclear cells isolated from the peripheral
blood (PBMC), synovial fluid (SFMC), and synovial membrane
(SMMC) of patients with rheumatoid arthritis. The shaded area
indicates the mean ? I S D .
The synovium in rheumatoid arthritis is the site
of an intense immune response (10) that is reflected in
the cellular contents of rheumatoid synovial fluid. In
addition to the local synthesis of immunoglobu~insand
rheumatoid factor (1 1, 12), activated dendritic macrolymphocytes are present in synovial
phages and
membranes and fluids (13, 14). Our studies show that
NK CELL ACTIVITY IN RA
1443
Figure 2. Electron micrographs of lymphocytes showing typical large granular lymphocyte-like characteristics from a synovial infiltrate of a
patient with rheumatoid arthritis. Characteristic features include a high cytoplasmic-to-nuclear ratio, crescentic nucleus, intracytoplasmic
granules, and abundant short microvilli. Cells were fixed in suspension with 2.5% gluteraldehyde in Millonings phosphate buffer and postfixed
with 1% osmium tetroxide. Thin sections were stained with uranyl acetate followed by lead citrate and examined in a Siemens 101 electron
microscope at 80 kV.
rheumatoid synovial membranes and synovial fluid
contained more functional NK cells than did blood
samples from patients with rheumatoid arthritis. Recently Fox et al (15), using monoclonal antibodies to T
lymphocyte surface antigens, showed a difference in
the proportion of T cell subsets between synovial fluid
and peripheral blood lymphocytes in patients with
rheumatoid arthritis. These antibodies, however, do
not permit one to enumerate NK cells. We have also
demonstrated the presence of large granular lymphoA crystal induced arthritis
m osteoarthritis
0
rheumatoid arthritis
a
.-
c
3
0
cytes, some of which are responsible for NK cell
activity (2). Finally, interferon, known to increase N K
cell activity, was found in rheumatoid synovial fluids.
The inflamed rheumatoid joint also contains
prostaglandin E (PGE). PGE and interferon may have
antagonistic actions on NK cell activity (16), although
the interaction, even in vitro, is complex (17). One
cannot be certain of the net effect in vivo, but the
evidence from the in vitro assays indicates an increase
in functional NK cells in the synovium as compared
with peripheral blood in these patients. All patients in
our study were treated with nonsteroidal antiinflammatory drugs known to decrease the production of
PGE-a process that might also tip the balance in
favor of increasing NK cell activity.
It is of interest that in 1 patient, SFMC studied
before and after intraarticular steroid injection showed
an initial decline and then recovery of NK cell activity.
This is consistent with our previous report in which
corticosteroids were found to depress NK cell activity
(4).
0
Rheumatoid
Non
Rheumatoid
Figure 3. The interferon levels in log,, transformed units of synovia1 fluid samples from rheumatoid and nonrheumatoid patients. The
shaded areas indicate the geometric mean interferon level 2 I SD.
Values less than 1 unit are considered negative for interferon.
Burmester et a1 (18) investigated the NK cell
activity of PBMC and SFMC from patients with rheumatoid arthritis and found significantly higher activity
using SFMC. In their study, SFMC NK cell activity
was often 3 times greater than the PBMC NK cell
activity, but they used a chromium release cytotoxicity assay with a 12-hour incubation in contrast to the
conjugate binding assay with a 3-hour incubation used
in the present study. The former allows for effector
cell recycling. Each NK cell can bind and lyse more
REINITZ ET AL
1444
than 1 target cell (19). In addition, there may be an
increase in interferon levels during a longer period of
incubation; this may serve to recruit or activate additional NK cells. Because of the immobilization in agar,
recycling does not take place, and only bound effectors can be recruited with the conjugate binding assay.
This assay measures a short-term event involving
lymphocytes, presumably activated in vivo, whereas
the chromium release assay may measure recycling
and more extensive recruitment as well. Taken together, however, the finding of increased NK cell activity
in the "Cr release assay and increased numbers of NK
cells by the conjugate binding assay suggest an absolute increase in the number and function of NK cells in
the rheumatoid joint.
We found a larger percentage of large granular
lymphocytes in SFMC than in PBMC. Since at least
some of these cells are thought to mediate NK activity, it is possible that we are seeing a selective accumulation of these cells in the joint, along with interferon.
Interferon titers are known to rise in response to viral
infections and to be involved with immunologic processes.
It is intriguing to speculate about the reason NK
cell activity is high in the synovium of patients with
rheumatoid arthritis, yet diminished in the blood of
patients with some other diseases, such as systemic
lupus erythematosus. Perhaps in rheumatoid arthritis
we are seeing a reaction to an offending agent or
antigen localized in the joint, whereas in systemic
lupus we are seeing the reflection of a primary immunoregulatory abnormality. At present we can characterize and describe these phenomena but we cannot
define their role in the disease process.
ACKNOWLEDGMENT
We wish to thank Yvonne Kress for the electron
microscopy.
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Hoffman T: Natural killer function in systemic lupus
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