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Production of Interleukin-1 Receptor Antagonist and Interleukin-1p by
Peripheral Blood Mononuclear Cells Is Differentially Regulated
By Debra D. Poutsiaka, Burton D. Clark, Edouard Vannier, and Charles A. Dinarello
We studied the relationship between the production of the
23-Kd interleukin-1 receptor antagonist (IL-lra) and IL-lp in
cultures of human peripheral blood mononuclear cells (PBMC)
using a specific radioimmunoassay for IL-lra that had a
sensitivity of 166 f 11pg/mL. PBMC cultured without human
serum made little IL-lra or IL-lp. In the presence of 1% AB
serum, there was no increase in IL-1p(0.25 f 0.13 ng/mL) but
IL-lra production increased sevenfoldt o 3.4 e 0.5 ng/mL. IgG
(2.5 to 100 pg/mL IgG) or granulocyte-macrophagecolonystimulating factor (GM-CSF) (1 to 100 ng/mL) had no significant effect on IL-lp production but increased IL-lra production up to 18-fold (18.2 e 3.9 ng/mL). Using endotoxin as a
stimulant, 82% f 2% of IL-lra was secreted in comparison
with 52% f 9% of IL-1p. Culture conditions of PBMC
influenced the production of IL-lra but not IL-1s. Rocking
endotoxin-stimulated PBMC produced 75% less IL-lra but
the same amount of IL-1p when compared with PBMC
cultured in stationary plastictubes. Rocking IgG-or GM-CSFstimulated PBMC also produced 75% to 80% less IL-lra.
GM-CSF or IL-lp a t concentrationsthat elicited submaximal
production of IL-lra potentiated IgG-induced IL-lra production. The production of IL-lra and IL-1p are under differential
regulation because serum, IgG, and GM-CSF were potent
stimuli for the production of IL-Ira but not IL-lp, and the
prevention of cell-cell contact of PBMC reduced IL-lra but
not IL-1p production.
0 1991by The American Society of Hematology.
I
(Rockford, IL). RPMI 1640 culture medium was obtained from
Whittaker M.A. Bioproducts (Walkersville, MD). Ultrafiltration
filters were obtained from Fresenius AG (Bad Homburg, Germany). Polypropylene tubes, 12 x 75 mm, were obtained from
Becton Dickinson Laboratories (Lincoln Park, IL).
in the
di-iodinated form of the Bolton-Hunter reagent was obtained from
Dupont-New England Nuclear (Boston, MA). Econo-Pac lODG
desalting columns and Tween-20 were obtained from Bio-Rad
Laboratories (Richmond, CA). MaxiSorp F96 microtiter plates
were obtained from Nunc-Immunoplate (Roskilde, Denmark).
Polyacrylamidegels (10%) were obtained from Amersham (Arlington Heights, IL). XAR-5 film was obtained from Kodak (Rochester, NY).An intensifymg screen, Cronex Lightening Plus, was
obtained from Dupont (Wilmington, DE).
Human IgG, clinical grade suitable for intravenous injection, was
a gift of Hyland Laboratories (Duarte, CA), and human recombinant IL-lawas a gift of Dr Peter Lomedico (Hoffman-LaRoche,
Nutley, NJ). Human recombinant IL-lp was a gift of Dr Alan Shaw
(Glaxo Institute for Molecular Biology, Geneva, Switzerland).
Human recombinant GM-CSF was a gift of Dr Steven Clark
(Genetics Institute, Cambridge, MA). Human recombinant IL-lra”
was a gift of Dr Robert Thompson (Synergen, Boulder, CO). Sheep
antirabbit IgG was obtained from Ventrex Laboratories (Portland,
ME). Monoclonal antihuman IL-1p and polyclonal rabbit antihuman IL-1p were the gift of Richard Dondero (Cistron, Pine Brook,
NJ). ImmunoPure alkaline phosphatase-conjugated goat antirabbit IgG and p-nitrophenol phosphate were obtained from Pierce
(Rockford, IL).
NTERLEUKIN-1 (IL-I) exhibits many different effects
in vivo and in vitro.’ Specific inhibitors of the various
biologic activities of IL-1 have been described since the
early 1980s: The “IL-1 inhibitor” was initially described as
a 23- to 25-Kd protein purified from the urine of patients
with monocytic leukemia3J or from human monocytes
cultured on immune complex-coated surfaces.6 Natural
IL-1 inhibitor blocked the activity of IL-1 in several bioa s s a y ~ . ~The
” ~ ~IL-1
. ~ inhibitor blocked the binding of IL-1 in
a competitive fashion to receptors on T cells and fibroblast~.~”
This IL-1 inhibitor was purified from adherent
monocyte^^^'^ and subsequently was cloned.“ As opposed to
IL-lp, the cloned protein lacked agonist activity in inducing
prostaglandin E, in human dermal fibroblasts.’”Therefore,
it was renamed the interleukin-1 receptor antagonist
(IL-lra). An identical protein was cloned from U937 cells
and reported as the IL-1 receptor antagonist protein
(IRAP).12
The measurement of IL-1 inhibitors has been based on a
reduction in the activity of IL-1 in various bioassays. This
approach does not allow for specific and independent
measurements of IL-1 and IL-lra. Without independent
assays, the relationship between IL-1 and IL-lra in health
and disease states cannot be fully explored. Recent studies
used an enzyme-linked immunosorbant assay (ELISA) for
IL-lra to study the differential gene expression of IL-lra
and IL-lp.”
We describe here a specific and sensitive radioimmunoassay (RIA) for the measurement of IL-lra. Using this
assay and a specific ELISA for IL-lp, we have studied the
regulation of the production of IL-lra and IL-1p proteins
from peripheral blood mononuclear cells (PBMC) cultured
in the presence of endotoxin, granulocyte-macrophage
colony-stimulating factor (GM-CSF), IgG, or IL-1. In
addition, we examined the role of cellular contact in the
production of IL-lra and IL-1p.
MATERIALS AND METHODS
Materials. Ficoll type 400 and endotoxin (Escherichia coli,
serotype 055:B5) were obtained from the Sigma Chemical Company (St Louis, MO). Hypaque-M, 90%, was obtained from
Winthrop Pharmaceuticals (New York, NY).Sterile pyrogen-free
water and 0.9% saline were obtained from Abbott Laboratories
Blood, Vol78, No 5 (September I), 1991: pp 1275-1281
From the Department of Medicine, Division of Geographic Medicine and Infectious Diseases, New England Medical Center Hospitals
and Tufts University School of Medicine, Boston, MA.
Submitted February 12,1991; acceptedApril 29,1991.
Supported by National Institutes of Health Grant AI 15614 and
Barter Healthcare Corporation Extramural Program, Deerfield, IL.
Address reprint requests to C A . Dinarello, MD, New England
Medical Centeq Division of Geographic Medicine and Infectious
Diseases, Department of Medicine, 750 Washington St, Boston, MA
02111.
The publication costs of this article were defrayed in part by page
charge payment. l%is article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I 9 9 1 by TheAmerican Society of Hematology.
0006-4971/91/7805-0032$3.0010
1275
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POUTSIAKA ET AL
1276
Study population. The study was approved by the Human
Investigation Review Committee of the New England Medical
Center Hospitals. Informed consent was obtained from each
subject. Healthy male and female volunteers 20 to 50 years of age
taking no medications were recruited from laboratory personnel.
Preparation of PBMC. PBMC were isolated from human blood
as described previ~usly.'~~'~
Some experiments used human AB
serum that had been collected under sterile, pyrogen-free conditions, heated at 56°C for 30 minutes, filtered (0.22-pm pore size),
and then frozen at -70°C. PBMCwere incubated in polypropylene
tubes at 37°C in a humidified atmosphere containing 5% CO,. In
experiments where tubes were rocked, the first hour of incubation
occurred in the incubator with gentle agitation every 20 minutes, to
equilibrate the concentration of CO,. Tubes were tightly sealed
with paraffin film and cultured at 37°C on a rocking table set at 20
cycles/minute. Control experiments showed that tightly sealing the
culture tubes had no adverse effect on IL-lra or IL-1$ production
in stationary tubes (data not shown). Cells were cultured with the
following: human AB serum, endotoxin, human IgG, human
recombinant IL-la, human recombinant IL-l$, and human recombinant GM-CSF. After 20 to 24 hours of incubation, cultures were
frozen at -70°C and subjected to three cycles of freeze-thawing
before assay for IL-lp or IL-lra. In some experiments, cells were
separated from supernatants by centrifugation at 1,500g for 10
minutes at room temperature. The resulting cell pellets were
resuspended in 1 mL of fresh culture medium. The cell and
supernatant fractions were frozen as described previously.
Radiolabeling of IL-Ira. Human recombinant IL-lra was radioiodinated with ISI by the method of Bolton and Hunter.I6 Radiolabeled protein was separated from unbound
by chromatography
on a desalting column equilibrated with 0.05 mol/L phosphate
buffer, pH 7.5, containing 0.25% gelatin. The specific activity,
calculated from the disintegrations per minute (dpm) comprising
the protein peak, assuming a 70% recovery, ranged from 15 to 77
pCi/pg.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis. Approximately 10,000 cpm of '251-IL-lra was
solubilized in 15 p,L of loading buffer (62 mmol/L Tris-HC1, p H
6.8, 10% glycerol, 1% SDS, 1% 2-mercaptoethanol, 0.0005%
bromophenol blue) and heated at 100°C for 3 minutes. Prestained
protein molecular weight standards (myosin, M, 30,000; phosphorylase b, M, 92,500, bovine serum albumin [BSA], M, 69,000;
carbonic anhydrase, M, 30,000, trypsin inhibitor, M, 21,500; and
lysozyme, M,14,300) were used on each gel. The gel was dried with
heat in vacuo and autoradiography was performed by exposure of
XAR-5 film for 2 days at -70°C using an intensifying screen.
RIA for IL-Ira. A polyclonal anti-IL-lra antiserum was raised
in New Zealand white rabbits immunized with IL-lra. Dilutions of
anti-IL-lra from 1:100 to 1:6,400 were added to 1251-IL-lra(10,000
cpm). After 24 hours at room temperature, immunoprecipitates
were formed by the addition of sheep antirabbit IgG and polyethylene glycol, at respective final concentrations of 1% and 3%. After 1
hour at room temperature, immunoprecipitates were recovered by
centrifugation at 1,500g for 15 minutes at room temperature.
Dilutions of anti-IL-lra precipitating approximately 20% of the
added "%IL-lra were used in the RIA.
Tween-20 and phenylmethylsulfonyl fluoride at final concentrations of 0.1% and 1 p,mol/L, respectively, were added to PBMC
samples, which were then kept on ice for 1 hour. Undiluted or
diluted PBMC samples were added to RIA buffer (0.01 mol/L
phosphate-buffered saline [PBS], pH 7.4,0.25% BSA, and 0.05%
sodium azide) containing 0.1% Tween-20. Diluted anti-IL-lra was
added to the samples, which were kept at room temperature
overnight. On the second day, "%IL-lra (10,000 cpm) was added
to each sample. On the third day, immunoprecipitates were
formed, recovered, and counted as described above.
The maximum specific binding was calculated by subtracting
nonspecific binding (cpm precipitated in tubes containing 'SI-ILIra but no anti-IL-lra or unlabeled IL-lra) from total binding
(cpm immunoprecipitated from tubes containing lSI-IL-lra and
anti-IL-lra but no unlabeled IL-lra). The percent specific binding
for each sample was calculated by dividing the specific binding of
the sample by the maximal specific binding and multiplying by 100.
All samples and standards were run in duplicate. IL-lra concentration in each sample was read on a logit plot of the percent specific
binding versus the log concentration of a diluted IL-lra standard
from the linear portion of the curve (usually between 35% and 85%
specific binding).
Determination of IL-I$. Human IL-lp was quantified using an
ELISA." Standard curves and IL-lp concentrations in samples
were determined using Microplate Manager software (Bio-Rad
Laboratories) on a Macintosh SE computer (Apple Computer, Inc,
Cupertino, CA). The sensitivity of the assay (limit of detection at
the 95% confidence level) was 40 to 80 pg/mL.
Statistical analysis. Statistical analysis was performed using
Stat-View software (Abacus Concepts, Inc, Calabasas, CA) on a
Macintosh SE computer. Two-tailed paired t-tests and analysis of
variance (ANOVA) using Fisher's least significant difference were
used. Data are expressed as the mean 2 standard error of the
mean (SEM). Differences were considered significant for P < .05.
RESULTS
SDS-PAGE ana4si.s of ZL-lra. Radiolabeled IL-lra was
resolved by SDS-PAGE analysis on a 10% polyacrylamide
gel" to observe its integrity and purity. The autoradiogram
in Fig 1 shows a single band of M, 18,000, indicating that
.
-
. . .-
.-..
-
= 92500
= 69000
n
-46000
0"
W
~30000 cn
z
21 500
-
= 14300
Fig 1. Autoradiograph of SDS-PAGE analysis of 'WL-lra. Radiolabeled IL-lra, 10,000 cpm, was resolved by SDS-PAGE on a 10%
polyacrylamide gel.
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1277
REGULATION OF IL-1RA AND IL-lP PRODUCTION
IL-lra had not undergone aggregation or breakdown during radioiodination.
RLA for IL-lra. The sensitivity of the assay was 166 & 11
pglmL, determined from five standard curves. The variation
in six replicates of a PBMC sample was 5.9%. The assay was
specific because the presence of IL-la or IL-1p (10 nglmL)
did not interfere with the measurement of IL-lra (Fig 2).
IL-lra (10 ng/mL) added to each tube in the RIA for IGla
or IL-1p had no influence on either assay (data not shown).
Influence of standard culture conditions on IL-lra production. Fresh, uncultured, and lysed PBMC contained no
IL-lra (data not shown). To study the effect of normal
human serum on IL-lra production, PBMC were cultured
in the presence or absence of 1%AB serum. Total IL-lra
and IL-lp were measured (Fig 3). In serum-free medium,
PBMC produced small amounts of IL-1p or IL-lra. PBMC
cultured in the presence of serum produced sevenfold more
IL-lra compared with PBMC in serum-free medium
(P < .05). Maximal production of IL-lra (13.6 2 0.8 ngl
mL) was observed in the presence of the lowest concentration of AB serum tested (0.25%). There was no change in
the production of IL-lp when serum was added. All
subsequent experiments were performed in the absence of
serum.
Differential induction of IL-lra and IL-1p production by
IgG and GM-CSF. PBMC were cultured in the presence
of increasing concentrations of human IgG, GM-CSF, or
IL-la. After 24 hours, total IL-lra and IL-1p were measured. As shown in Fig 4A, human IgG was a potent
stimulus for IL-lra production. As little as 1 pg/mL IgG
induced significant IL-lra production compared with baseline (P < .01). In contrast, the presence of up to 100 pg/mL
IgG had little effect on IL-1p production in the same
cultures.
GM-CSF was also a potent stimulus of IL-lra production
(Fig 4B). Significant augmentation of IL-lra production
over baseline was observed in cultures containing as little as
1ng/mL (P < .OS). Over the range of concentrations tested,
there was no effect of GM-CSF on the production of IL-lp.
The production of IL-1 and other cytokines is influenced
by IL-1, interferon, and IL-1ra.19-zz
The effect of IL-1 on
IL-lra production is unknown. Therefore, PBMC were
cultured with IL-la or IL-1p (0.1 to 100 ng/mL) for 24
4000 1
=
7
.
E
3000
m
n
Y
.-Yc
0
*
0
-
2000
1000
0
IL-I
p
I L - I ra
Fig 3. The effect of 1% AB serum on the production of IL-lg or
IL-lra by PBMC. PBMC were cultured in stationary polypropylene
tubes in serum-free medium or in the presence of 1% AB serum. After
24 hours, the cultures were harvested and assayed for total IL-lg or
IL-Ira. The data from three experiments are expressed as the mean f
SEM.
hours and total IL-lra was measured. Although there were
increases in IL-lra production over baseline levels, they did
not reach statistical significance (data not shown).
The effect of cell-cell contact on IL-lra Production. Culture conditions of monocytes influence their functional
state.lssu-zsTherefore, PBMC in serum-free medium or
stimulated with human IgG or GM-CSF were cultured in
either stationary or rocking polypropylene tubes. After 24
A
I
I
U
IL-1
U
U
p
m
)I
Q
I 4
95
-
--c
noIL-I
*
u
T
1
10
E:
M
.x
tii
80
-
60
40
-
20
-
0
.01
.1
100
GM-CSF (nglml)
I
100
1000
10000
100000
IL-lra (pg/ml)
Fig 2. Effect of IL-la or IL-1g on the standard curve for the IL-lra
RIA. IL-la or IL-lg, 10 ng/mL, was added to each tube of diluted IL-lra
standard. The binding curves for each condition is shown. The data
are expressed as the percent binding (left) or logit binding (right)
versus the log of the concentration of IL-lra in pg/mL.
Fig 4. The induction of IL-lg and IL-lra in PBMC by human IgG or
GM-CSF. PBMC were cultured in serum-free medium in the presence
of increasing concentrations of human IgG (A) or GM-CSF (B). After 24
hours, the cultures were harvested and assayed for total IL-1g and
SEM of data from three
IL-lra. Each point represents the mean
individuals. One asterisk indicates statistically significant differences
compared with baseline for P < .05; three asterisks indicate differences for P < .001.
*
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1278
POUTSIAKA ET AL
A
A
.
20000
-
*
*
stationary
--o-- rocking
*
*
E
-
D
P
P
D
z
stationam
20000
.
E
.-
I+
U
10000
-
2
1
0
B
-E
10
--o-- rocking
.
1
Endotoxin (ng/ml)
10
100
B
+ stationary
20000
.1
0
100
IgG (uglml)
=
20000
---C stationary
0rocking
-- -
.
m
E
Q
P
m
a
:10000
2
U
10000
r
4
O
0
0
.01
.1
1
GM-CSF (ng/ml)
10
100
Fig 5. The effect of rocking on IL-lra production by PBMC. PBMC
were cultured in the presence of serum-free medium, human IgG (A)
or GM-CSF (B). The tubes either remained stationary or were rocked
for 24 hours. The cultures were harvested and assayed for total IL-lra.
Points represent mean f SEM for data from three experiments.
Asterisks indicate statistically significant differences between stationary and rocking cultures at a given concentration of stimulus (one
asterisk, P < .05; two asterisks,P < .01).
hours, total IL-lra was measured. Both IgG (Fig 5A) and
GM-CSF (Fig 5B) were potent stimuli for IL-lra production in stationary cultures (some of the data are displayed in
Fig 4). However, in the presence of IgG, rocking cultures of
PBMC produced significantly lower levels of IL-lra. The
same phenomenon was observed in cultures of PBMC in
the presence of GM-CSF, although the reduction was less.
We also compared the effect of these culture conditions
on the production of IL-1p and IL-lra in PBMC stimulated
by endotoxin. After 24 hours, total IL-16 and IL-lra were
measured. Endotoxin was a strong stimulus for IL-lra
production in stationary cultures (Fig 6A). However, there
was a substantial reduction in IL-lra production in rocking
cultures of endotoxin-stimulated PBMC. In contrast, rocking had no significant effect on endotoxin-induced IL-lp
production (Fig 6B).
Distribution of IL-lra and IL-1p between cellular and
supematant compartments. PBMC in stationary polypropylene tubes were cultured with endotoxin (10 ng/mL). After
24 hours, cells were separated from supernatants by centrifugation. Each fraction was assayed for IL-lp and IL-lra.
The results are shown in Fig 7. Similar to previous res u l t ~ the
~ ~ distribution
~'
of IL-1p between cellular and
supernatant fractions was variable. In contrast, the distribution of IL-lra between cellular and supernatant compartments was consistent. Approximately 80% of the IL-lra was
Fig 6. The effect of rocking on IL-lra and IL-lp production by
PBMC in response to endotoxin. PBMC were cultured in the presence
of serial dilutions of endotoxin in stationary or rockingtubes. After 24
hours, the cultures were harvested and assayed for IL-lra (A) or IL-lp
(B). These data from three donors are expressed as mean -c SEM.
Asterisks indicate a statistically significant difference between stationary and rocking cultures (P< .05).
secreted into the supernatant. The differences in IL-lra
content between the cell-associated and supernatant fractions was statistically significant (P < .001). The difference
between the proportions of IL-lra and IL-lP in the cellassociated fraction was statistically significant as was the
Q)
loo
1
cell-associated
IL-1
p
I L - 1 ra
Fig 7. The distribution of IL-lra and IL-1p in cellular and supernatant compartments. PBMC were cultured in the presence of 10 ng/mL
of endotoxin. After 24 hours, culture supernatants were separated
from cells by centrifugation. Each sample was assayed for IL-1p and
IL-lra. The bars represent the mean f SEM of the percentage of the
total found in the intracellular or extracellular portions. The data are
from four experiments.
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REGULATION OF IL-IRA AND IL-16 PRODUCTION
1279
difference between the proportions in the supernatants
(P < .05).
Potentiationof IgG-inducedIL-lra production by GM-CSF
or ZL-1p. Because low (picogram) levels of cytokines exist
in
we questioned whether potentiation of IgGinduced IL-lra production by GM-CSF or IL-1 existed in
vitro. PBMC were cultured in stationary tubes for 24 hours
with IgG (1 pg/mL) in the presence or absence of GM-CSF
(0.1 ng/mL). These concentrations stimulated submaximal
production of IL-lra when used separately (Fig 4). In
addition, PBMC were cultured with IgG (1 pg/mL) with or
without IL-lp (100 ng/mL). The results are shown in Fig 8.
Potentiation of IgG-induced IL-lra production was observed in the presence of GM-CSF (Fig 8A) or IL-1p (Fig
8B).
DISCUSSION
We have investigated the relationship between IL-lra
and IL-lp production by PBMC using an RIA sensitive and
specific for IL-lra. This RIA enabled the independent
measurement of IL-lra in the same samples containing
IL-1, a complicated task when using bioassays. The primary
finding of these studies was the existence of differential
regulation of the production of IL-lra and IL-1p. This
conclusion is based on (1) human AB serum, human IgG,
and GM-CSF induced IL-lra but not IL-lP; (2) the
prevention of cell-cell contact substantially decreased ILA
20000
-E
4
.
a
a
w
t
10000
r
i
0
--.
20000
E
a
n
w
2 'IO000
%-
=!
0
GMCSF
IgG/GM-CSF
IL-1 p
IgGIIL-1 p
is
i
IgG
Fig 8. Potentiationof IgG-induced IL-Ira production by GM-CSF or
IL-1p. PBMC were cultured with IgG ( I Fg/mL), OM-CSF (0.1 ng/mL),
or IgG in combination with GM-CSF (A), or IL-1p (100 ng/mL) with or
without IgG (B). After 24 hours, IL-Ira was measured. The line within
the IgG/GM-CSF or IgG/IL-lp bar indicates the predicted additive
effect on cytokine production of IgG plus GM-CSF or IgG plus IL-lp.
The data are expressed as the mean SEM of three experiments.
Ira production but had no effect on IGlp production; and
(3) approximately 80% of IL-lra produced by PBMC was
secreted into the medium whereas only 50% of IL-lp was
secreted. We also observed the potentiation of IgG-induced
IL-lra production by GM-CSF or IL-1p. The existence of
differential regulation of IL-lra and IL-1p production was
in agreement with studies on the synthesis of mRNA for
each protein.I3
This and other studies suggest that IL-lra production
would occur in tissue rather than in the circulation. Such a
view is supported by observations that the production of
IL-lra or other IL-1 inhibitors similar or possibly identical
to IL-lra is increased under conditions associated with
monocyte differentiation. For example, IL-lra or IL-1
inhibitor production increased in macrophage-monocyte
cultures maintained for several weeks with or without
GM-CSF3' or in freshly obtained PBMC stimulated with
GM-CSF, as in the present study. In contrast, the prevention of cell-cell contact (as might occur in the circulation)
resulted in a marked decrease in IL-lra production.
The mechanism for the stimulation of IL-lra production
is unclear. Stimulation by IgG presumably occurs via the Fc
receptor. Therefore, it is likely that conditions which
increase Fc receptor expression andfor function, such as
adherenceu324
or exposure to cytokines such as interferon or
GM-CSF,33augment IgG-induced IL-lra production. The
observed potentiation of IgG-induced IL-lra production by
GM-CSF may be explained by such a phenomenon. It is
unlikely that augmentation of Fc receptor function is the
mechanism whereby IL-1p potentiated IgG-induced IL-lra
production because IL-1 does not change Fc receptor
function or expression on m0nocytes.3~
It is also unclear why the production of IL-lra by rocking
PBMC regardless of the stimulus was less than that by
stationary PBMC. However, it was apparently not through
an injurious effect of rocking because IL-1p production by
the same cells was not affected. It is possible that when
cell-cell contact or adherence to culture vessels was prevented, there was reduced synthesis of a necessary factor
for IL-lra production. Because induction of IL-1p by
endotoxin was unaffected by rocking, a reduction of endotoxin receptors is an unlikely explanation. Alternatively, the
prevention of cell-cell contact or adherence to culture
vessels might have stimulated the production of a factor
that inhibited IL-lra production.
There is 26% homology between the sequences of IL-lra
and IL-1p." However, there appears to be a signal sequence encoded by the IL-lra gene which is lacking in the
sequence encoded by the IL-lp gene. This implies different
mechanisms of secretion for the two proteins." Disparity
between the amount of IL-lra secreted and the amount of
IL-lp secreted may be explained by this structural difference.
The relevance of the differential regulation of the production of I G l P and IL-lra observed in vitro to the pathogenesis of disease is unknown. Certainly, IL-1p is implicated in
several disease states.' These include septic or endotoxin
inflammatorybowel disease,35and acute myelogenous le~kemia.'~
It is unknown if conditions thought to
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1280
POUTSIAKA ET AL
involve increased IL-1 activity have an accompanying deficiency in IL-lra production. The antagonism of IL-1 by
exogenous IL-lra may afford a therapeutic option in these
diseases. In view of this, exogenous IL-lra (1) blunted
septic shock induced by Escherichia coli in rabbits” and
baboons (Dr Lyle Moldawer, personal communication,
November 1990); (2) reduced mortality induced by endotoxin in rabbits38; (3) blunted fever induced by IL-1 in
rabbit^'^; (4) decreased the inflammation seen in immune
complex colitis4’; and (5) reduced the spontaneous production of GM-CSF by and proliferation of acute myelogenous
leukemia cells.41
Intravenous Ig is effective in disorders characterized by
increased activity of the immune system. These include
graft-versus-host disease:’ Kawasaki
and immune thrombocytopenic
One plausible mechanism of action of Ig is the in vivo induction of IL-lra
production. Given the therapeutic potential of IL-lra,
further in vitro and in vivo work is warranted.
ACKNOWLEDGMENT
We thank Dr Joseph G. Cannon, Dr Eric V. Granowitz, Dr
Laurie C. Miller, Scott F. Orencole, and Sana Isa for their valuable
assistance.
REFERENCES
1. Dinarello CA. Interleukin-1 and interleukin-1 antagonism.
Blood 77:1627,1991
2. Larrick JW: Native interleukin 1 inhibitors. Immunol Today
10:61,1989
3. Seckinger P, Lowenthal JW, Williamson K, Dayer J-M,
MacDonald HR: A urine inhibitor of interleukin 1 activity that
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1991 78: 1275-1281
Production of interleukin-1 receptor antagonist and interleukin-1 beta
by peripheral blood mononuclear cells is differentially regulated
DD Poutsiaka, BD Clark, E Vannier and CA Dinarello
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