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Effects of gold sodium thiomalate on interferon stimulation of C2 synthesis and HLADR expression by human monocytes.

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1032
EFFECTS OF GOLD SODIUM THIOMALATE ON
INTERFERON STIMULATION OF C2 SYNTHESIS AND
HLA-DR EXPRESSION BY HUMAN MONOCYTES
KAREN M. SANDERS, PATRICIA L. CARLSON, and BRUCE H. LITTMAN
Gamma-interferon (y-IFN) is a T cell-derived
lymphokine that has potent macrophage-activating
properties. It increases Fc receptor density, increases
the formation and release of reactive oxygen intermediates, increases the synthesis and release of complement
cascade proteins, especially C2 and factor B, and increases class I1 (HLA-DR) antigen expression. These
effects may play a role in the potentiation of inflammation in rheumatoid arthritis. We examined the possibility that gold sodium thiomalate (GST), an effective
treatment for rheumatoid arthritis, would inhibit yIFN-mediated stimulation of monocyte/macrophages.
GST in concentrations attainable in vivo was shown to
inhibit both spontaneous and yIFN-stimulated C2 production up to 50%. GST inhibition could be only
partially overcome with increasing concentrations of
y-IFN. In addition, GST inhibited y-IFN-stimulated
Thomas Fund publication no. 242.
From the Division of Immunology and Connective Tissue
Diseases, Department of Medicine, Medical College of Virginia,
Virginia Commonwealth University, and the Medical Laboratory
Services, McGuire Veterans Administration Medical Center, Richmond, Virginia.
Supported in part by NIH Postdoctoral Fellowship grant
5-732-CA 09210-08 and a Veterans Administration Merit Review
grant.
Karen M. Sanders, MD: Staff Rheumatologist and Instructor in Medicine, Medical College of Virginia and Virginia Commonwealth University; Patricia L. Carlson, PhD: Laboratory Technologist, Laboratory and Research Services, Veterans Administration
Medical Center; Bruce H. Littman, MD: Chief, Rheumatology
Service, McGuire Veterans Administration Medical Center, and
Associate Professor in Medicine and Microbiology, Medical College
of Virginia and Virginia Commonwealth University.
Address reprint requests to Karen M. Sanders, MD, Department of Medicine (1 l l M), Veterans Administration Medical
Center, 1201 Broad Rock Road, Richmond, VA 23249.
Submitted for publication July 31, 1986; accepted in revised
form March 5 , 1987.
Arthritis and Rheumatism, Vol. 30, No. 9 (September 1987)
HLA-DR expression at the highest concentrations tested
(20-50 pg/ml). GST alone in low concentrations ( 0 . 1 4
yglml) was found to increase HLA-DR antigen expression as quantitated by several methods, including flow
cytometry, cell surface enzyme-linked immunosorbent
assay, and Western blotting. This GST-stimulated increase in HLA-DR antigen expression paralleled an
increased ability of monocytes to present antigen. The
mechanism by which low concentrations of GST stimulate HLA-DR antigen expression is unclear, but was
shown by 35S-methionine cell labeliwg not to involve
increased HLA-DR protein synthesis.
The pathogenesis of rheumatoid arthritis (RA)
is not known, although many etiologies have been
proposed. An initiating stimulus, whether endogenous
or exogenous, apparently triggers a self-sustaining
immune response (1). The continued proliferation and
activation of synovial cells may be due to the secretion
of soluble mediators of inflammation, also known as
cytokines. These factors, synthesized by activated
lymphocytes or monocytes, amplify the inflammatory
cascade by stimulating other effector cells. Some of
these cytokines, including interleukin- 1 (IL-1) and
y-interferon (yIFN) , have been demonstrated within
synovial fluid and tissue samples from RA patients (2).
In particular, y I F N has multiple proinflammatory effects that may be important in potentiating
inflammation in rheumatic diseases (3). For example,
it increases Fc receptor density on macrophages (4),
increases formation of reactive oxygen intermediates
(9,and increases multinucleate giant cell formation
(6). In addition, y I F N increases class I1 (HLA-DR)
expression on many types of cells (7,8), and increases
the bactericidal and tumoricidal ability of monocytes
GST EFFECTS ON MONOCYTES
(9,lO). It is the major macrophage-activating factor
present in activated lymphocyte supernatants (11).
IFN has been shown to increase the synthesis of C2
and factor B on a pretranslational basis (12). Additionally, as noted above, y I F N has been demonstrated in
synovial fluid, serum samples, and joint tissues from
RA patients (13-15).
Gold sodium thiomalate (GST) has been a popular and effective treatment for RA for more than 50
years (16). However, its mechanism of action remains
unknown. Histopathologic studies have shown that
gold accumulates within lysosomes of mononuclear phagocytes (17-20). After prolonged chrysotherapy, more
than 50% of total body elemental gold may be found in
the liver, lymph nodes, spleen, and bone marrow. This
concentration within cells of the reticuloendothelial
system may be responsible for its multiple effects on
cells of the immune system (21,22). Lymphocytes and
monocytes are predominantly affected. Gold salts decrease monocyte secretory and phagocytic capacity
(23-25), impair monocyte chemotaxis and migration
(26,27), and inhibit lymphocyte proliferation to antigens and mitogens (28,29). C1 and several lysosomal
enzymes are inactivated by gold salts (30-32). These
effects serve to down-regulate the exuberant inflammatory response characteristic of active RA.
Previously, our findings have demonstrated that
GST inhibits the production of C2 by monocytes when
they are stimulated by unfractionated lymphokine
preparations (33). Concentrations of GST between 5
and 50 pdml are inhibitory without being cytotoxic,
and do not impair synthesis of DNA or protein. These
effects were shown to be dependent on the presence of
elemental gold, and were not duplicated by thiomalic
acid alone. We have now directly examined whether
GST can inhibit y-IFN-mediated stimulation of
monocytes and macrophages. Inhibition of cytokine
activity would explain the clinical efficacy of gold in
reducing inflammation in RA. We studied C2 synthesis
and HLA-DR expression as 2 markers of the effect of
IFN on human monocytes. We found that GST inhibits y I F N stimulation of C2 synthesis in a dose-related
manner. GST also inhibits the y-IFN-stimulated
HLA-DR expression, but only at higher concentrations. Low concentrations of GST actually stimulate
HLA-DR expression and increase the antigenpresenting ability of monocytes.
MATERIALS AND MBTHODS
Reagents. Monoclonal antibody to HLA-DR framework antigen was obtained from the supernatant of mouse
1033
hybridoma cell line L243 (American Type Culture Collection, Rockville, MD). The antibody was precipitated with
ammonium sulfate, resuspended in phosphate buffered saline (PBS), and dialyzed to remove residual ammonium
sulfate. This stock murine IgG2a antibody was stored at 1.16
mg/ml at -70°C. Another monoclonal antibody against DR
framework antigen (Cappel Worthington, Malvern, PA) was
used on rare occasions. Both antibodies gave identical
results. Control IgG2a murine immunoglobulin was purchased from Meloy Laboratories (Springfield, VA). Recombinant human y I F N was kindly provided by Genentech
(South San Francisco, CA).
Isolation of cell populations. Heparinized blood was
diluted with Hanks’ balanced salt solution. Monofiuclear
cells were obtained by density gradient centrifugation using
lymphocyte separation medium (Litton Bionetics, Kensington, MD), and monocytes were purified by adherence as
previously reported (24). These cells were 98% phagocytic
by neutral red staining and 95% viable by trypan blue
exclusion. They were either added directly to 24-well plates
(Costar, Cambridge, MA) or were cultured for 24-72 hours in
20-ml Teflon bottles (Nalgene; Fisher Scientific, Fairlawn, NJ)
at 1 x 106 celldm1 in medium containing various combinations
of GST (0.1-50 pglml) or y-IFN (0.05-10 ng/ml).
Gold effects on y-IFN stimulation of C2 synthesis.
Peripheral blood monocytes (PBMC; 4 x 10’) in 200 pl of
supplemented medium 199 were added to each well of a
24-well plate. Recombinant y-IFN and GST were added at
appropriate dilutions to triplicate cultures. Plates were incubated in humidified air, 5% COz at 37°C for 5 days. Duplicate
100-pl samples were taken from each well and stored frozen
at -20°C until assayed for C2 hemolytic activity, as previously described (34). The presence of GST has been shown
to have no effect on the C2 assay (33). Cumulative production of C2 per culture was expressed as effective molecules
x 10-*/105 cells.
Gold effects on yIFN stimulation of HLA-DR expression. Cellular enzyme-linked immunosorbent assay (CELISA).
CELISA was performed by the method of Morris (39, with
modification. Briefly, Teflon-cultured monocytes were suspended at lo7 cells/ml in veronal buffered saline with 2%
bovine serum albumin (VBS-BSA). Sodium azide (AZ)
(0.02%) was added to prevent capping and internalization of
the membrane complex. Twenty microliters of this suspension was added to each well of a 96-well V-bottom polyvinyl
chloride plate (Thomas Scientific, Swedesboro, NJ). Ten
microliters of L243 antibody diluted to 15 pg/ml in VBS-BSA
was added to the cells. Murine IgG2a was used in equivalent
amounts in control wells. The plate was incubated at 4°C on
a rocker for 60 minutes and then washed 3 times by centrifugation at 1,500g with VBS-1% BSA. Alkaline phosphatase
conjugated to anti-mouse IgG (Sigma, St. Louis, MO) was
added to the resuspended cell pellet at a 1 :100 dilution. The
plate was again incubated (90minutes at 4°C) and washed as
above. The cells were resuspended in 75 p1 of 2 mM MgC12
in distilled water. The flexible microtiter plate (Dynatech,
Alexandria, VA) was snap-fitted over a rigid 96-well polystyrene plate (Immulon 11; Fisher Scientific), and holes were
punched through each V well. Cells were then transferred by
brief centrifugation, and 75 pl of 2 x diethanolamine buffer
with 2 x concentrated substrate (p-nitrophenyl phosphate;
1034
Sigma) was added. After a 30-minute incubation, optical
density was read at 405 nm.
Flow cytometric analysis of fluorescence. Tefloncultured monocytes were washed 3 times in PBS with 10 mM
AZ and 2% BSA (PBS-AZ-BSA) and were resuspended in 50
pl. Antibody L243 or control murine IgG2a was added at a
concentration of 23 pdml to the cell pellet in 50 pl. The cells
and antibodies were incubated on ice for 30 minutes, and
then the cells were washed again with PBS-AZ-BSA, and
were then resuspended in 100 pl. An equal volume of sheep
fluorescein-conjugated F(ab');! fragment of anti-mouse IgG
(Cappel Worthington) at a dilution of 1 :12 from stock was
added to the cell suspension. Following another 30-minute
incubation at 4"C, the cells were washed free of antibody as
described above, fixed in 2% formaldehyde, washed again,
and resuspended in filtered PBS for flow cytometric analysis
with an Ortho Cytofluorograph 50H. Fluorescent photomultiplier tube gains were selected so that more than 95% of the
cells treated with control mouse IgG2a were in the first 100
channels of a 1,000-channel histogram. Cells staining with
L243 antibody were in the following 900 channels and are
expressed as percent positive. Ten thousand cells in each
treatment group were analyzed.
"S-methionine labeling of cells and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Monocytes were cultured in Teflon bottles as above, with
addition of 250 pCi of 3SS-methionine per ml of culture for
the last 18 hours. Cell lysis and immunoprecipitation were
performed as has been previously published (36). The cells
were washed 3 times in PBS and lysed in wash buffer (0.1M
Tris, O.05M NaC1,l mM MgC12,Z mM phenylmethylsulfonyl
fluoride, 0.01M iodoacetamide at pH 7.4) containing 0.5%
NP-40. Lysis took place on ice for 30 minutes. The lysates
were centrifuged at 17,300g for 20 minutes and then were
precleared by incubation with normal mouse IgG attached to
protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden)
in the proportion of 1 ml of 1ysate:lOO pl of packed
Sepharose. This was incubated on a rocker overnight at 4°C
and then centrifuged. Twenty microliters of the supernatant
was precipitated with trichloroacetic acid and counted in a
liquid scintillation counter.
Equal precipitable counts per minute of protein were
added in 1 ml of buffer to 100 pl of dry-packed protein
A-Sepharose CL-4B beads with attached L243 antibody
(approximately 1.O mg antibody/ml beads), and incubated 3
hours with agitation at 4°C. The beads were then washed
serially with the following reagents: twice in 0.01M Tris,
0.5% NP-40, 1 mM EDTA, pH 7.4 (NTE buffer); once in
0.01M Tris, 0.5M NaCl, 0.5% NP-40, I mM EDTA, p B 7.4;
once in NTE buffer; once in 0.01M Tris, 0.15M NaCl with 5
mg/ml sodium deoxycholate; and last, in NTE buffer. The
beads were suspended in 0.01M Tris with I% SDS and 1%
2-mercaptoethanol, and boiled to elute bound protein material. This supernatant was added to an equal volume of
Laemmli sample buffer and boiled again for 5 minutes. The
immunoprecipitates were subjected to SDS-PAGE through
12% discontinuous slab gels, according to the method of
Laemmli (37). The stacking gel was 4 cm in height; the
separating gel was 16 cm in height. The gel was soaked in
Enhance (NEN, Arlington Heights, IL), and then dried and
autoradiographed at -70°C for 1-2 weeks.
SANDERS ET AL
Western blotting. Monocytes were cultured in Teflon
bottles containing media with gold, IFN, and appropriate
controls. The cells were lysed, immunoprecipitated, and
electrophoresed as described above. The gel was electroblotted to nitrocellulose, which was then immunostained
with L243 or Cappel anti-DR antibodies. The paper was
washed in PBS with 0.05% Tween 20, and developed with an
anti-mouse IgG immunoassay utilizing an avidin-biotin
complex linked to horseradish peroxidase (Vectorstain; Vector, Burlingame, CA). The substrate utilized was 4-chloronaphthol.
Antigen presentation. Teflon-cultured monocytes
were washed and added at lo5 cells/well to a round-bottom
96-well polystyrene plate (Nunclon; Nunc, Raskilde, Denmark and Fisher Scientific). Autologous lymphocytes were
magnetically depleted of adherent phagocytic cells after
incubation with carbonyl iron particles for 90 minutes at
37°C. These lymphocytes were then added at lo6 cells/well
to the monocytes. Tetanus toxoid (Wyeth, Philadelphia, PA)
or streptokinase-streptodornase(Lederle, Pearl River, NY)
was used as the antigenic stimulus. Control cultures of
monocytes only and lymphocytes only were included to
confirm that neither population was antigen-responsive. For
the last 18 hours of a 5-day incubation, the wells were pulsed
with 1 pCi of 3H-thymidine. The cells were harvested and
thymidine incorporation was determined. Results are expressed as the mean + SEM cpm of quadruplicate wells.
RESULTS
Gold inhibition of y-IFN-stimulated C2 production. GST was found to decrease spontaneous and
y-IFN-stimulated C2 production by human PBMC
(Figure 1). Spontaneous C2 production was decreased
by 30-50%. Gold concentrations of 25 pg/ml and 50
pg/ml inhibited most effectively. In addition, when
these cells were stimulated by 0.3-3.0 ng/ml of y-IFN,
there was a linear relationship between GST concentration and inhibition of C2 production. At the highest
concentrations of GST (50 pg/ml), there was total
inhibition of y-IFN-stimulated C2 production. This
effect was demonstrated in 3 separate experiments.
In another experiment (Table I), the effect of
increasing y I F N concentrations while maintaining a
constant gold concentration (25 pg/ml) was studied
further. GST at this dose impaired the response to all
concentrations of y I F N ranging from 0.05-5 .O ng/ml.
Inhibition ranged from 43-80%. Thus, the inhibitory
effect of GST on C2 production could be partially
overcome by higher concentrations of yIFN, unless
>25 pglml of GST was used.
GST effects on HLA-DR expression. GammaIFN has been shown to increase monocyte and
macrophage DR expression within 24-48 hours of cell
culture (8). A marked increase of DR expression on
1035
GST EFFECTS ON MONOCYTES
40
f 301
T
Table 2. Gamma-interferon (yIFN) stimulation of HLA-DR
expression by human monocytes, as determined by cellular
enzyme-linked immunosorbent assay (CELISA)*
,f
Anti-HLA-DR
antibodies
Control media
y I F N , 0.5
unitdm1
yIFN, 8
units/ml
y I F N , 32
unit s/ml
y I F N , 128
units/ml
y I F N , 512
unitdm1
Interferon y
(nslml)
Figure 1. Gold inhibition of yinterferon-stimulated C2 production.
Each point represents the mean C2 hemolytic activity of supernatants from 4 X 1 6 normal human monocytes, incubated for 5 days
in triplicate cultures, with or without added yinterferon. Results are
expressed as effective molecules (e.m.) of C2 ? SEM x lo-'. 0 =
no gold sodium thiomalate (GST) added; A = 12.5 pg/ml GST; 0 =
25 pg/ml GST; 0 = 50 pg/ml GST.
cells from yIFN-stimulated cultures was demonstrated by CELISA (Table 2). Simultaneous gold treatment of yIFN-stimulated cells inhibited the increase
of DR at the highest concentration of GST tested (25
pg/ml) (Table 3). Gold was also tested without added
IFN to assess its effect on cellular DR expression. A
control IgG2a was included to control for Fc receptor
increases with IFN or gold stimulation. Surprisingly,
as shown in Table 4, GST alone was found to increase
HLA-DR expression. Maximum stimulation occurred
Table 1. Effects of increasing yinterferon (yIFN) concentrations on C2 production by gold sodium thiomalate (GST)-treated
monocytes*
C2 production?
yIFN
(ng/ml)
With GST
(25 &ml)
No GST
0.00
0.05
0.10
0.50
0.75 f 0.12
0.74 2 0.03
0.65 f 0.05
1.76 ? 0.25
1.68 f 0.23
3.21 t 0.26
1.24 ? 0.04
1.97 t 0.11
3.17 2 0.16
4.06 f 0.19
6.03 t 0.54
5.67 f 0.39
1 .OO
5.00
* Normal human monocytes were incubated for 5 days in vitro with
y I F N in various concentrations and GST (25 pg/ml).
t Results are expressed as the mean effective molecules
10-8/10s cells.
?
SEM x
%
increase
Control
IgG2a
Difference
35 ? 1.0
39 t 3.5
12 +- 1.0
14 2 1.5
23
25
4.3
53 t 2.4
20 t 2.5
33
43.5
69 f 6.1
22
0.5
47
100.0
1.5
26 f 0.5
59
156.5
80 f 2.5
29 f 4.0
51
121.7
85
f
?
* Normal human monocytes were incubated for 48 hours with y I F N
as shown, washed, and assayed for DR expression by CELISA.
Results are the mean optical density at 405 nm f SEM of quadruplicate wells X 100.
with low doses of gold. Gold, however, also increased
the binding of control antibody, although antiHLA-DR binding was always greater. This effect may
have been mediated through Fc receptor binding.
We used flow cytometric techniques to better
control for Fc receptor binding and to explore the
effect of GST alone on cellular expression of DR
antigens. Cells were incubated for 48 hours in varying
concentrations of GST and then were analyzed. As
shown in Table 5, all concentrations of GST tested
(0.1-25 pg/ml) caused an increase in the percentage of
DR positive cells, as well as a moderate increase in
mean channel fluorescence. Control mouse immunoglobulin binding was not increased with GST
treatment, ruling out an effect through Fc receptor
binding. In further experiments (data not shown),
GST-mediated increases in DR antigen expression
predominated at the lower concentrations.
Table 3. Effects of gold sodium thiomalate (GST) on yinterferon
(yIFN) stimulation of HLA-DR expression by normal human
monocytes, as determined by cellular enzyme-linked immunosorbent assay*
Anti-HLA-DR
antibodies
Control media
y I F N , 10 units/ml
y I F N + 1.0 pg/ml GST
y I F N + 5.0 pg/ml GST
y I F N + 25 pg/ml GST
61.0 ? 1.9
73.0 f 3.4
73.0 t 3.0
80.0 ? 2.9
62.0 f 2.1
Control
antibodies Difference
31.0 f 1.2
31.0 f 1.2
28.0 2 1.9
31.0 t 0.8
27.0 t 1.3
30
42
45
49
35
~~~~
* Normal human monocytes were incubated for 48 hours in media
containing y I F N with or without the addition of GST. Results are the
mean optical density f SEM of quadruplicate wells X 100.
1036
SANDERS ET AL
Table 4. Effect of gold sodium thiomalate (GST) on HLA-DR
expression by human monocytes, as determined by cellular
enzyme-linked immunosorbent assay*
-
Control media
GST. 5 pg/ml
GST. 10 pg/ml
GST. 25 pg/ml
GST. SO pg/ml
Anti-HLA-DR
antibodies
Control
antibodies
Difference
36.0 +- 9.1
80.2 I 1 1 . 4
91.0 I21.3
74.0 k 12.6
70.2 k 5.2
19.8 2 16.1
30.2 2 11.2
46.8 +- 9.6
53.8 2 6.6
55.8 2 13.2
16
50
44
20
14
~~
* Normal human monocytes were incubated for 48 hours in vitro in
medh containing GST. Results are the mean optical density at 405 nm
-+ SD of quadruplicate samples x 100.
DR antigen content was then studied by Western blot analysis of immunoprecipitated cell lysates.
As shown in Figure 2, moderate concentrations of
GST (10 and 20 pg/ml) did not increase the amount of
precipitable DR protein as compared with control
media (lanes 1 and 2 compared with lane 10). Low
concentrations of GST (0.1 and 1 .O pdml) did result in
increased HLA-DR antigen content (lanes 3 and 4).
The y-IFN control also resulted in a clear-cut increase
in precipitable DR protein (lane 9). There appears to
be minimum inhibition of precipitable DR protein
when 20 pglml of GST is added to y-IFN treatment
(lane 3, but not at the lower gold concentrations.
Although y-IFN is known to increase DR expression by increasing actual synthesis of the protein
via increases in messenger RNA (7), it was unclear
whether gold salts increased DR expression by increasing synthesis or by decreasing the degradation of
already synthesized DR membrane protein. We therefore incubated monocytes with y-IFN or with gold at
various concentrations in the presence of 35S-methionine. Biosynthetically labeled HLA-DR protein was
then immunoprecipitated from cell lysates, subjected
Table 5. Gold sodium thiomalate (GST) stimulation of HLA-DR
antigen expression
Treatment
% DR
positive
Mean
fluorescence
Control media
GST, 0.1 pgiml
GST, 1.0 pg/ml
GST, 5.0 pg/ml
GST, 10.0 pg/ml
GST, 25 Fg/ml
y-interferon, 5 ng/ml
Control antibodies (media)
Control antibodies (GST, 25 pg/ml)
Control antibodies (y-interferon)
76.5
89.5
81.4
87.3
91.1
88.5
75.7
6.0
6.0
8.6
410.0
455.9
446.9
456.1
471.0
443.8
549.1
288.2
23 1.2
343.4
Figure 2. Western blot of immunoprecipitate HLA-DR protein.
Each lane represents the product of lysate from 7 million adherent
monocytes incubated for 48 hours with the indicated amounts of
gold sodium thiomalate (GST) and/or y-interferon (y-IFN) prior to
lysis: Lane I , 20 pglml GST only; lane 2, 10 pg/ml GST only; lane
3, 1.0 pglml GST only; lane 4, 0.1 pg/ml GST only; lane 5 , 10 nglml
y I F N + 20 pg/ml GST; lane 6, 10 ngiml y-IFN + 10 pg/ml GST;
lane 7, 10 ng/ml y I F N + 1.0 pg/ml GST; lane 8, 10 ng/ml y-IFN +
0.1 pg/ml GST; lane 9, 10 ng/ml y I F N only; lane 10, medium.
Lysates were subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and electrotransferred to nitrocellulose, incubated with monoclonal anti-HLA-DR antibody, washed, and reacted with peroxidase-conjugated anti-mouse immunoglobulin prior
to washing again and the addition of substrate. Actin and the
HLA-DR a and p chains are visible.
to SDS-PAGE, and autoradiographed. The autoradiograph of this experiment is shown in Figure 3. Gammainterferon is shown to yield increased incorporation of
methionine into membrane DR protein, as shown by
the dark bands of a and p chains. GST did not result in
any increase in HLA-DR labeled protein over media
control. Therefore, the effects of GST are not related
to increased protein synthesis of new DR membrane
antigens.
Last, because the preceding experiments demonstrated a clear stimulation of DR expression induced
by GST, we determined if this DR increase had
functional implications by studying the ability of GSTtreated monocytes to present antigen to autologous
lymphocytes. When monocytes were pretreated with
GST, washed, and cocultured with autologous lymphocytes, low doses of gold were able to enhance
3H-thymidine incorporation. Higher concentrations of
1037
GST EFFECTS ON MONOCYTES
Figure 3. Autoradiograph of immunoprecipitated HLA-DR protein. Human adherent monocytes were incubated for 48 hours with
the indicated amounts of y-interferon (yIFN) or gold sodium
thiomalate (GST). 35S-methioninewas added for the last 18 hours of
cell culture. Lane 1 , medium; lane 2, 1.0 ng/mlyIFN; lane 3, 0.1
pg/ml GST; lane 4 , 0 . 5 pg/ml GST; lane 5, 1.0 pg/ml GST; lane 6,5.0
pg/ml GST. Cells were lysed and precleared as described in
Materials and Methods. An equal amount of trichloroacetic
acid-precipitable protein (cpm) per culture was immunoprecipitated
with monoclonal anti-HLA-DR antibody on protein A-Sepharose
beads. Immunoprecipitated protein was then subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiographed. The dark spot in the lower left comer is an artifact.
gold, however, suppressed subsequent lymphocyte
proliferation (Table 6).
DISCUSSION
The proliferation of pannus and influx of inflammatory cells into the joints of patients with RA may be
driven by the local release of soluble effector molecules known as cytokines. Activated T cells within
rheumatoid joints (38,39) are the main source of these
factors (40), which interact with synovial lining cells,
endothelial cells, fibroblasts, chondrocytes, polymorphonuclear leukocytes, macrophages, and dendritic
cells. Cellular factors, including cytokines, fragments
of complement components, and immune complexes,
cause macrophage and neutrophil chemotaxis, inhibit
their migration, and cause new blood vessel growth,
fibroblast proliferation, and macrophage activation (1).
One such cytokine, y I F N , is released from activated
T cells and modulates cellular function through a
specific receptor whose kinetics and structure are still
being elucidated (41). The availability of pure recombinant y I F N has made it possible to fully explore its
effects on pure populations of cells.
Many of the effects of y I F N may stimulate the
inflammatory processes within joints. For example,
the increase of HLA-DR antigen expression on
macrophages and other cells increases antigen presentation to lymphocytes. By stimulating oxidative metabolism, IFN increases the production and release of
reactive oxygen intermediates from activated macrophages (5). These reactive molecules have been
thought to be involved in the tissue damage seen in
RA. Likewise, the increased synthesis and release of
complement components coupled with increased Fc
receptor density may feed immune complex-mediated
complement activation, phagocytosis, and release of
degradative enzymes. Gamma-interferon, itself, increases the release of plasminogen activator and increases the proliferation of fibroblasts (42,43). Gamma-interferon has been found in the serum, joint fluid,
and tissues of patients with RA and other autoimmune
diseases. We have previously confirmed that y-IFN is
present within most rheumatoid joints in physiologically active concentrations, and its levels correlate
with total synovial fluid white cell counts (44). It is
reasonable to assume that its effects on immunologically competent cells may be important in the perpetuation of inflammation.
Gold salts suppress many elements of the immune system, although their effects on lymphokinemediated events have not been investigated in detail.
We have previously demonstrated that GST inhibits
the response of normal human monocytes to activated
lymphocyte supernatants as measured by C2 producTable 6. Antigen presentation by gold sodium thiomalate (GSTk
treated normal human monocytes*
Mean cpm
GST
(udml)
No antigen
0.0
0.1
1 .o
5.0
10.0
25.0
514 210
683 -C 122
171 2 21
1,038 f 234
422 f 145
380 f 80
*
-C
SEM
Antigen
*
7,086 435
13,835 f 715
6,466 t 1,218
7,275 ? 767
2,514 -C 531
2,477 r+_ 747
Stimulation
index
13.7
20.2
37.8
7.0
6.0
6.5
* Normal human monocytes (without lymphocytes) stimulated with
antigen incorporated 159 f 22 cpm. Lymphocytes alone stimulated
with antigen incorporated 693 -C 166 cpm.
SANDERS ET AL
tion (33). GST was also shown to inhibit several other
macrophage events that require differentiation, including the spontaneous cytotoxicity for chicken erythrocytes and the loss of myeloperoxidase enzyme activity
(24). However, phorbol myristate acetate-stimulated
superoxide generation from monocytes and lymphokine activation to kill nucleated target cells, events
that did not require differentiation, were not inhibited
by gold treatment. Therefore, gold salts may inhibit
monocyte differentiation into macrophages. We have
demonstrated that GST inhibits the yIFN-stimulated
increase of C2 production from human monocytes in a
dose-response relationship, by an average of 50%.
This effect is seen at concentrations of gold that are
easily achievable in vivo. It is not caused by changes
in cell viability or general synthetic ability, as we (33)
and other investigators (23) have shown previously.
High concentrations of y I F N were able to
overcome gold inhibition of C2 production only partially. In addition, partial inhibition of C2 production
was seen with concentrations of GST as low as 5 pglml
(data not shown). However, GST did not affect HLADR antigen expression in a similar fashion. We found
that concentrations of GST that were able to inhibit C2
production did not inhibit the yIFN-stimulated increase in DR. However, the higher concentrations of
GST (25-50 pg/ml) were inhibitory. The finding that
low concentrations of gold salts are able to increase
DR antigen expression on monocytes is of interest.
This effect of GST was functionally correlated with
better antigen-presenting function. Several other reports have also suggested that gold compounds can be
immunostimulatory (45,46). Clinically, the effects of
low concentrations of GST on HLA-DR expression
could be related to some of the untoward side effects
of gold salts. GST-treated patients may develop autoimmune manifestations such as thrombocytopenia and
nephrotic syndrome. It is speculated that these immunostimulatory effects of GST are related to enhanced HLA-DR expression.
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