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Opposite effect of interferon-γ on PGE2 release from interleukin-1-stimulated human monocytes or fibroblasts
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1197-1204
December 30, 1988
OPPOSITE EFFECT OF INTERFERON-y ON PGE 2 RELEASE FROM INTERLEUKIN-I-STIMULATED HUMAN MONOCYTES OR FIBROBLASTS
L a u r e n c e Friteau, Elisa Francesconi, D a n i e l l e Lando, Bernard Dugas and Chantal Damais* Laboratoire d'Immunologie, D 4 p a r t e m e n t de Biotechnologies, * CNRS/Roussel, Roussel-Uclaf, 93230 Romainville, France Received September 27, 1988
S t i m u l a t e d monocytes produce p r o s t a g l a n d i n s (PGE2) in response to lipopolysaccharide (LPS), Muramyl d i p e p t i d e (MDP) or Interleukin-i (IL-I). This response could be m o d u l a t e d in different ways by Interferon-y (IFN-y) . This lymphokine, known to p o t e n t i a t e IL-I p r o d u c t i o n by LPSor M D P - s t i m u l a t e d monocytes, suppressed different Ii-i activities such as PGE 2 release by the same cells. By contrast, an impairement of s u p p r e s s i o n by IFN-y was e v i d e n c e d in r I L - i B - i n d u c e d PGE 2 release from human dermal fibroblasts. Salmon calcitonin (sCT) , another inhibitor of I L - l - i n d u c e d bone resorption, was able to prime monocytes to potentiate. PGE.2 elaboration by LPS , but failed to modulate PGE 2 1 zberatzon from either rIL-iB-stimulated monocytes or fibroblasts. ® 1988 A c a d e m i c Press, I n c .
Interferon-y
(IFN-y),
reported p r e v i o u s l y to IFN-7
act as a
On the other hand,
by microbial
agents
However,
I and II
IFN-y
such IFN-y
(IL-I) as
only
activity
as
a priming
and enhances the
and Tumor Necrosis Factor
lipopolysaccharide which
a n t i p r o l i f e r a t i v e properties have
by increasing
MHC antigen expression
could act
or bactericidal
induction of Interleukin-i
has been
"macrophage a c t i v a t i n g factor".
m e t a b o l i s m and class
factor for tumoricidal
(3,4).
derived lymphokine,
could stimulate directly m o n o c y t e s / m a c r o p h a g e s
oxidative (1,2).
a T-cell
possesses been shown
(LPS) antiviral
(TNF) or MDP and
to inhibit certain
* To w h o m c o r r e s p o n d e n c e should be addressed.
Abbreviations: rIFN-Y: r e c o m b i n a n t human Interferon-Y; rIL-IB: r e c o m b i n a n t h u m a n Interleukin-IB; PGE2: p r o s t a g l a n d i n type E2; LPS: lipopolysaccharide; MDP: Muramyl DiPeptide; sCT: salmon calcitonin; MHC: major histocompatibility complex; TNF: tumor n e c r o s i s factor; HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; SEM: standard error of the mean.
1197
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inflammatory reactions especially synthesis and
al.
(7)
bone resorption
have shown
IL-l-stimulated
that
those
associated
with PGE 2
(5,6). More recently,
IFN-7
suppressed
human monocytes
and
Ghezzi
d e s c r i b e d the specific inhibition by IFN-7 of
PGE 2
Browning et release by
and
Dinarello
(8)
IL-I production by
I L - l - s t i m u l a t e d monocytes. Interleukin-i broad
set of
them,
IL-I
is a p l e i o t r o p i c factor capable
immunologic and inflammatory
stimulates PGE 2 release in vivo
as well as in vitro
Recently, for
(ii), and
(12). Two
(ii). They were termed
IL-I~ and -B and despite their limited amino e x h i b i t e d similarities mechanisms
(9,10). Among
potentiates human fibroblast p r o l i f e r a t i o n
distinct forms of IL-I have been described
(13,14).
of eliciting a
events
acid
homology they
in size, binding and biological activities
several laboratories have suggested different
regulation
numerous n o n - r e l a t e d
of
IL-I
molecules
production
or
factors
their potent IL-l-inhibitory activity In the present study,
we
or
were
action,
and
described for
(9,10).
confirmed that
IFN-7
was
able to
deeply inhibit the PGE 2 release from human m o n o c y t e s stimulated with rIL-IB. triggering
However, agents
opposite results were obtained with other
such
as
inhibited the p r o l i f e r a t i o n b l o c k PGE 2 release
LPS or MDP.
Moreover,
IFN- 7
of normal fibroblasts was
by these cells
which
unable to
stimulated with recombinant
Interleukin-iB.
MATERIALS
AND
METHODS
Reagents: R e c o m b i n a n t human I n t e r l e u k i n - I B (i pg to i00 n g / m l ) ( 1 0 7 U / m g of protein) and recombinant human Interferon-7 (i to 1,000 U/ml) (2x107 antiviral units/mg protein) were provided by Roussel-Uclaf. Endotoxin testing was p e r f o r m e d using the Limulus A m e b o c y t e Lysate (LAL) test. All the batches contained <20pg/10ug of the recombinant protein. At lug/ml p r e p a r a t i o n did not inhibit the LAL assay in the presence of known concentration of endotoxin. LPS from Salmonella enteritidis (phenol-water extraction) was obtained from Difco lab. (Detroit, MI), MDP was purchased from Sigma (St Louis, MO) and salmon calcitonin (sCT) was from C a l b i o c h e m (San Diego, CA). All media, buffers, preparations and products used in the experiments were pyrogen-free. Preparation of supernatant fluids from human monocytes: Human peripheral blood m o n o n u c l e a r cells from normal donors (H6pital Avicennes, Bobigny) were enriched in monocytes by lh-plastic adherence (>90% nonspecific esterase positive cells (NSE+)). Cells were then cultured at 5 x 106 NSE+cells/ml for 24 to 48 hours at 37°C in a 5% CO 2 incubator in complete 1198
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
medium: s e r u m - f r e e R P M I 1640 m e d i u m ( M i c r o b i o l o g i c a l A s s o c i a t e s Inc., MD) s u p p l e m e n t e d w i t h a n t i b i o t i c s a n d 0 . 0 1 M H E P E S buffer. C u l t u r e s w e r e i n c u b a t e d in the p r e s e n c e or in t h e a b s e n c e of the s t i m u l a n t s d e s c r i b e d a b o v e at d i f f e r e n t doses. A t the end of the incubation period, cell-free supernatants were c o l l e c t e d and s t o r e d at - 2 0 ° C or - 8 0 ° C u n t i l used. Dermal fibroblast cultures: Human dermal fibroblasts kindly provided by Dr A s s e l i n e a u (CIRD, France), were used before the 15th passage. Trypsinized cells were p l a t e d at 1 0 4 / w e l l in 9 6 - w e l l m i c r o t i t e r p l a t e in 10% foetal c a l f s e r u m (Hyclone, Logan, U T ) - e n r i c h e d M E M medium. Cells were incubated 48 h o u r s w i t h or w i t h o u t r I L - I B b e f o r e an o v e r n i g h t p u l s e (0.04 MBq/well) of [3H]methyl-Thymidine (Sp. act. = 37 G B q / m m o l e , CEA, Saclay, France). D a t a w e r e e x p r e s s e d as the mean counts in experimental cultures over the counts incorporated by unstimulated fibroblasts. Standard deviation u s u a l l y v a r i e d f r o m <10% f r o m t h e mean. In parallel experiments, supernatant fluids were collected a n d s t o r e d at - 8 0 ° C for PGE2 assay. P r o s t a g l a n d i n assay: T h e p r e s e n c e of p r o s t a g l a n d i n t y p e E2 (PGE2) in supernatant f l u i d s was e v a l u a t e d b y a c o n v e n t i o n a l r a d i o - i m m u n o a s s a y u s i n g an antiserum anti-PGE2 (Institut Pasteur). PGE2 content was e x p r e s s e d in ng/ml. I L - I a n d T N F assay: P r e s e n c e of m o n o k i n e s T N F a n d IL-I w a s d e t e c t e d w i t h c l a s s i c a l m e t h o d s of L929~ c y t o t o x i c i t y a n d a c t i v a t i o n of m u r i n e t h y m o c y t e s p r e v i o u s l y d e s c r i b e d in d e t a i l s (15).
RESULTS
Effect
of r I F N - y
In o r d e r of
PGE2,
different or MDP
on P G E 2 r e l e a s e
to a s s e s s human
doses
doses
of r I L - i B
fluids not
(i0,000
dramatic
dose
PGE 2 data
decrease of LPS
was
optimal
but
24
as b o t h 1
to
with
or in t h e a b s e n c e
PGE 2 LPS
release
48 h w i t h LPS
(i0 ng/ml)
of
rIFN~
showed
at
even
differentially
to rEL-IB,
and
of i00 U / m l
concentration
1199
at
of
1
and
which
huge
modulate
to
did
dose PGE 2
agent:
i) a
2) no s i g n i f i c a n t
3) a s y n e r g i s t i c
of r I F N - y
chosen
of s u p e r n a t a n t rIFN-¥
of t h e t r i g g e r i n g
of the r e s p o n s e stimulation,
However,
se
in a d o s e
Results
PGE 2 c o n t e n t
per
shown)
liberation
a n d MDP.
of s t i m u l a n t .
on t h e n a t u r e
The concentration
on m o n o c y t e
U/ml).
release not
depending
modification
1
on f i g u r e
U/ml,
liberation
incubated
stimulated
as w e l l
from o p t i m a l
stimulate
MDP.
rIL-IB
of r I F N - ¥
(I p g to i00 ng/ml),
(i to 1,000
fashion
be presented
were
in the p r e s e n c e
As expected, dependent
from human monocytes.
influence
monocytes
(I ug/ml)
different
the
effect
presented i0 U / m l
with
in f i g u r e were
also
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5°I 4O
30"
20-
iO-
C
rIFN- 7
MOP
PIL-~
LPS +rIFN- 7
Figure 1 :
active. and
The
the
the
inhibition even
synergism
response
Effect
Experiments
release case
LPS w a s
2),
neither
presented
lymphokine
addition
+LPS
+L+IFN
with
fibroblasts
were
effect
rIL-IB
effect whatever
to
1200
instead induced
the d o s e
of
PGE 2 In t h a t
employed,
to rIL-IB.
rIFN-y
effect
suppression
proliferation shown).
In t h a t and only
at 1 ng/ml.
nor response
dose-related
(data n o t
culture. abolished
fibroblasts
sensitive
specific
fibroblasts.
hereabove,
of rIFN-y
was
i).
from human
a maximal
and rIL-IB-induced
any cytotoxic
(figure
background
a
activity
a d d e d to t h e
spontaneous
induced
IL-I
LPS a n d r I L - l B w a s
reproduced
from the cells with
spontaneous
of t h e
on P G E 2 r e l e a s e were
Nevertheless, this
when
between
As r e s u l t s
(figure
modify
observed
to LPS w a s m a i n t a i n e d
of rIFN-y
monocytes.
+rlFN-~
Effect of rIFN-y (100 U/ml) on PGE 2 release from human monocytes triggered with different stimuli: MDP (I0 ~g/ml) LPS (i0 ng/ml), or Interleukin-iB (i0 ng/ml). Bars represent the mean (± SEM) of four separate experiments.
evidenced
case,
+rIFN- 7
(figure
of
since both
3) w i t h o u t
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
20"
o
t5-
x E
z o
lO-
o o o
I
t
Figure
Effect and
of
2:
100
t
PIL-t~
t0 + PIFN-7
t00
Effect of rIFN-y (from 1 to i00 U/ml) on PGE 2 release from human dermal fibroblasts stimulated with recombinant Interleukin-iB (i0 n g / m l ) . Vertical bars r e p r e s e n t s S E M of a t l e a s t s i x separate experiments.
calcitonin
(sCT)
on
PGE2
release
from
human
monocytes
fibroblasts. Calcitonin
activities at
t0 PIFN- T
high
and
on
rIFN-y
IL-l-induced
dose
(from
1 ng
have bone to
1
been
shown
resorption ug/ml)
to (16).
share
similar
Although,
calcitonin
was
unable
~.0"
8-
r.1 E
6-
oJ
0.
4
i C Figure
3:
iO iO0 PIFN- 7
I
rZL-i~
I0 I00 + PIFN-7
Effect of rIFN~ (from 1 t o i00 U/ml) o n p r o l i f e r a t i o n of rIL-IB-stimulated fibroblasts. Bars represent a typical experiment out of at least six. V e r t i c a l b a r s represent SEM for triplicates.
1201
even to
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I: Salmon calcitonin (sCT) m o d u l a t i o n of rIL-IB- or LPS-induced PGE 2 release from human m o n o c y t e s or fibroblasts
PGE 2 (ng/ml) release from monocytes
fibroblasts
Control
1.5 + 0.3 a
0.6 + 0.2
Calcitonin 0.lug
1.8 + 0.2
0.5 + 0.2
rIL-IB 10ng/ml
7.5 _+ 0.6
7.0 + 1.0
rIL-IB + sCT
8.7 + 0.5
7.8 + 0.9
LPS 100ng/ml
22.0 + 3.0
NT b
LPS + sCT
40.0 + 5.2
NT
* similar results were obtained from Ing/ml to lug/ml. a SEM of at least 6 different experiments. b not tested.
suppress
or to modulate
fibroblasts described from
stimulated with
latex
LPS-stimulated
calcitonin
did
monocytes, TNF
not
2).
an autocrine
(17),
(Table IL-I
enhanced
either
(Table
calcitonin
monocytes
i).
On
of LPS,
sCT
also
(data not
as
PGE 2 release the
other
slightly
with
hand,
or release
by
inhibited
production
evident
or
However,
or TNF production
was
stimulant
monocytes
enhanced i).
intracellular
enhancement
monocyte
from
rIL-iB
in t h e p r e s e n c e
and This
with
stimulate
but,
release
(Table
PGE 2 release
of
rIL-iB
IL-I
used
as
shown).
DISCUSSION
Lymphokines, during
inflammatory
influence monocytes autocrine range as
soluble
factor
(8)
bone tissue network
were
Thus,
non-related
even
produced immune
has
been
though
numerous
molecules
responses,
This
IL-I
various
inhibitors
factors
1202
such
produced
described displayed
target pituitary
pleiotropic
leukocytes
can profoundly
mainly
cytokine
different muscle,
requires
or
activated
recently
this
liver,
(9,10). which
by
Interleukin-l,
on completely
marrow,
connective
and
capacities.
and macrophages
regulatory mechanisms.
events
monocytic
of a c t i v i t i e s
brain,
factors
by
as an a broad
tissues
such
glands
and
feature
implies
inhibitory
feedback
were
recognized
which
as g l u c o c o r t i c o i d s ,
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 2: Salmon calcitonin (sCT) modulation of TNF and IL-I production by human monocytes incubated with LPS and/or rIFN-y
TNF activity -
IL-I activity
(U/ml)
+
sCT
(Acpm x 10 -3 ) + sCT i00 ng/ml a
-
i00 ng/ml a control
0b
LPS i00 ng/ml
41
rIFN-y i00 U/ml
0
rIFN-y + LPS
0
1 / 2c
30
49 / 20
NT d
1623
NT
1 / 2 50 / 34
1 / 2
NT
67 / 26
NT
a similar results were obtained from ing/ml to lug/ml. b U/ml defined as inverted dilution that results in lysis of 50% of the L929~ cells. c Acpm x 10-3:3H-TdR incorporated by C3H/HeJ thymocytes stimulated with lug/ml PHA-P in the presence of 1/8 diluted supernatants and lysates of monocytes (supernates / lysates). SEM usually varied <10% from the mean. d not tested.
neuropeptides addition, lymphokine depicted such
(~-MSH),
IFN-y with MAF able
to
report
used.
Thus,
monocytes:
rIFN-y
As well
(16)
~hown u n a b l e and
diverse
shown and
TNF
to
to
IFN-y
activity IL-I
In
was
activities
release
could
and bone
act differently the
These
results
et al.
PGE 2
release
cell
from
of LPS e f f e c t , were
in
(5) w i t h m u r i n e
P M A o r zymosan.
evidenced
target
PGE 2 r e l e a s e
no m o d u l a t i o n
activity.
of
and
differently
w i t h MDP,
done by Boraschi
rIFN-y
IFN-y,
been
that
with A23187,
rIL-IB was
particle-stimulated
[L-I
factor)
the stimulant
rIL-IB
of these
as
has
activity
by
with
proliferation
(20),
of
stimulated
inhibition
stimulated
(18,19).
antiproliferative
PGE 2 i n d u c i n g
could modulate
with the work
macrophages no
specifically
of
synergism
suppression
agreement
proteins
antiviral,
activating
(8),
indicates
the nature
human and
inhibit fonction
or u r i n a r y as
(16).
The present according
known
(macrophage
to
as a u t o c r i n e
resorption
TGF-B,
well
By c o n t r a s t ,
from fibroblasts
even at doses which
affected
cells. calcitonin to
(sCT),
interfere
affect
activities.
a calciotropic IL-I
prostaglandin
human monocytes
stimulate
with
directly However,
1203
(17).
bone pathway
sCT
in l a t e x
I n o u r study,
human monocytes slightly
hormone
resorption
sCT was
to produce reduced
TNF
Vol, 157, No. 3, 1988
production
but potentiated
production did
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not modulate
monocytes
PGE 2
and
monocytes.
release
intracellular
On the
either
other
IL-I
hand,
sCT
from rIL-lS-stimulated
or f i b r o b l a s t s .
In c o n c l u s i o n , specificity
of
the results
rIFN-7
and c a l c i t o n i n , As o b s e r v e d
physiopathologic depressive
or
reported
the r e g u l a t o r y
chosen.
different
PGE 2 release
by L P S - s t i m u l a t e d
capacity
according
of
to stimulant,
in vitro w i t h d i f f e r e n t
situations enhancing
compartments
here
might
effect
of
sets
forth
molecules cells stimuli,
preferentially IFN-¥
or
the high such
as
and a c t i v i t y the d i v e r s e determine
calcitonin
in t h e
or t i s s u e s .
ACKNOWLEDGEMENTS
The a u t h o r s are m o r e grateful to Pr. G.Milhaud discussions, to Mrs. Colette Leridant and Denis expert technical assistance.
for h e l p f u l H e r b e r t for
REFERENCES
1. Nathan, C.F., Murray, H.W., Wiebe, M.E., and Rubin, B.J. (1983) J. Exp. Med., 158, 670-689. 2. Rosa, F., and Fellous, M. (1984) Immunol. Today, 5, 261-262. 3. Arenzana-Seisdedos, F., and Virelizier, J.-L. (1983) Eur. J. Immunol., 13, 437-440. 4. Damais, C., Parant, M., and Chedid, L. (1985) Int. J. Immunopharmac., 7, 361. 5. Boraschi, D., Censini, S., Bartalini, M., and Tagliabue, A. (1985) J. Immunol., 135, 502-505. 6. Gowen, M., and Mundy, G.R. (1986) J. Immunol., 136, 2478-2484. 7. Browning,J.L.,and Ribolini,A. (1987) J. Immunol., 138, 2857-2863. 8. Ghezzi, P., and Dinarello, C.A. (1988) J. Immunol., 4238-4244. 9. Dinarello, C.A. (1984) Rev. Inf. Dis. 6, 51-95. i0. Durum, S.K., Schmidt, J.A., and Oppenheim, J.J. (1985) Ann. Rev. Immunol. 3, 263-287. ii Dukovitch, M., Severin, J.M., White, S.J., Yamazaki, S., and Mizel, S.B. (1986) Clin. Immunol. Inumunopath., 38, 381-389. 12 Okusawa, S., Gelfand, J.A., Ikejima, T., Connolly, R.J., and Dinarello, C.A. (1988) J. Clin. Invest., 81, 1162-1172. 13 Oppenheim, J.J., Kovacs, E.J., Matsushima, K., and Durum, S. (1986) Immunol. Today, 7, 45-56. 14 Rupp, E.A., Cameron, P.M., Ranawat, C.S., Schmidt, J.A., and Bayne, E.K. (1986) J. Clin. Invest. 78, 836-839. 15 Jupin, C., Anderson, S., Damais, C., Alouf, J.E., and Parant, M. (1988) J. Exp. Med., 167, 752-761. 16 Gowen, M., Wood, D.D., Mundy, G.R., and Russell, R.G.G. (1985) In the physiologic, metabolic, and immunological actions of Interleukin-i (M.J. Kluger, J.J. Oppenheim, and M.C. Powanda, Eds.), pp. 85-93. Alan R. Liss Inc., New York. 17. Stock, J.L., and Coderre, J.A. (1984) Prostaglandins, 27, 771-779. 18. Robertson, B., Dostal, K., and Daynes, R.A. (1988) J. Immunol., 140, 4300-4307. 19. Fontana, A., Bodmer, S., and Frei, K. (1988) Lymphokines, 14, 91-121. 20. Milhaud, G. (1985) Rec. Res. Cancer Res., 99, 67-70.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1197-1204
December 30, 1988
OPPOSITE EFFECT OF INTERFERON-y ON PGE 2 RELEASE FROM INTERLEUKIN-I-STIMULATED HUMAN MONOCYTES OR FIBROBLASTS
L a u r e n c e Friteau, Elisa Francesconi, D a n i e l l e Lando, Bernard Dugas and Chantal Damais* Laboratoire d'Immunologie, D 4 p a r t e m e n t de Biotechnologies, * CNRS/Roussel, Roussel-Uclaf, 93230 Romainville, France Received September 27, 1988
S t i m u l a t e d monocytes produce p r o s t a g l a n d i n s (PGE2) in response to lipopolysaccharide (LPS), Muramyl d i p e p t i d e (MDP) or Interleukin-i (IL-I). This response could be m o d u l a t e d in different ways by Interferon-y (IFN-y) . This lymphokine, known to p o t e n t i a t e IL-I p r o d u c t i o n by LPSor M D P - s t i m u l a t e d monocytes, suppressed different Ii-i activities such as PGE 2 release by the same cells. By contrast, an impairement of s u p p r e s s i o n by IFN-y was e v i d e n c e d in r I L - i B - i n d u c e d PGE 2 release from human dermal fibroblasts. Salmon calcitonin (sCT) , another inhibitor of I L - l - i n d u c e d bone resorption, was able to prime monocytes to potentiate. PGE.2 elaboration by LPS , but failed to modulate PGE 2 1 zberatzon from either rIL-iB-stimulated monocytes or fibroblasts. ® 1988 A c a d e m i c Press, I n c .
Interferon-y
(IFN-y),
reported p r e v i o u s l y to IFN-7
act as a
On the other hand,
by microbial
agents
However,
I and II
IFN-y
such IFN-y
(IL-I) as
only
activity
as
a priming
and enhances the
and Tumor Necrosis Factor
lipopolysaccharide which
a n t i p r o l i f e r a t i v e properties have
by increasing
MHC antigen expression
could act
or bactericidal
induction of Interleukin-i
has been
"macrophage a c t i v a t i n g factor".
m e t a b o l i s m and class
factor for tumoricidal
(3,4).
derived lymphokine,
could stimulate directly m o n o c y t e s / m a c r o p h a g e s
oxidative (1,2).
a T-cell
possesses been shown
(LPS) antiviral
(TNF) or MDP and
to inhibit certain
* To w h o m c o r r e s p o n d e n c e should be addressed.
Abbreviations: rIFN-Y: r e c o m b i n a n t human Interferon-Y; rIL-IB: r e c o m b i n a n t h u m a n Interleukin-IB; PGE2: p r o s t a g l a n d i n type E2; LPS: lipopolysaccharide; MDP: Muramyl DiPeptide; sCT: salmon calcitonin; MHC: major histocompatibility complex; TNF: tumor n e c r o s i s factor; HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; SEM: standard error of the mean.
1197
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inflammatory reactions especially synthesis and
al.
(7)
bone resorption
have shown
IL-l-stimulated
that
those
associated
with PGE 2
(5,6). More recently,
IFN-7
suppressed
human monocytes
and
Ghezzi
d e s c r i b e d the specific inhibition by IFN-7 of
PGE 2
Browning et release by
and
Dinarello
(8)
IL-I production by
I L - l - s t i m u l a t e d monocytes. Interleukin-i broad
set of
them,
IL-I
is a p l e i o t r o p i c factor capable
immunologic and inflammatory
stimulates PGE 2 release in vivo
as well as in vitro
Recently, for
(ii), and
(12). Two
(ii). They were termed
IL-I~ and -B and despite their limited amino e x h i b i t e d similarities mechanisms
(9,10). Among
potentiates human fibroblast p r o l i f e r a t i o n
distinct forms of IL-I have been described
(13,14).
of eliciting a
events
acid
homology they
in size, binding and biological activities
several laboratories have suggested different
regulation
numerous n o n - r e l a t e d
of
IL-I
molecules
production
or
factors
their potent IL-l-inhibitory activity In the present study,
we
or
were
action,
and
described for
(9,10).
confirmed that
IFN-7
was
able to
deeply inhibit the PGE 2 release from human m o n o c y t e s stimulated with rIL-IB. triggering
However, agents
opposite results were obtained with other
such
as
inhibited the p r o l i f e r a t i o n b l o c k PGE 2 release
LPS or MDP.
Moreover,
IFN- 7
of normal fibroblasts was
by these cells
which
unable to
stimulated with recombinant
Interleukin-iB.
MATERIALS
AND
METHODS
Reagents: R e c o m b i n a n t human I n t e r l e u k i n - I B (i pg to i00 n g / m l ) ( 1 0 7 U / m g of protein) and recombinant human Interferon-7 (i to 1,000 U/ml) (2x107 antiviral units/mg protein) were provided by Roussel-Uclaf. Endotoxin testing was p e r f o r m e d using the Limulus A m e b o c y t e Lysate (LAL) test. All the batches contained <20pg/10ug of the recombinant protein. At lug/ml p r e p a r a t i o n did not inhibit the LAL assay in the presence of known concentration of endotoxin. LPS from Salmonella enteritidis (phenol-water extraction) was obtained from Difco lab. (Detroit, MI), MDP was purchased from Sigma (St Louis, MO) and salmon calcitonin (sCT) was from C a l b i o c h e m (San Diego, CA). All media, buffers, preparations and products used in the experiments were pyrogen-free. Preparation of supernatant fluids from human monocytes: Human peripheral blood m o n o n u c l e a r cells from normal donors (H6pital Avicennes, Bobigny) were enriched in monocytes by lh-plastic adherence (>90% nonspecific esterase positive cells (NSE+)). Cells were then cultured at 5 x 106 NSE+cells/ml for 24 to 48 hours at 37°C in a 5% CO 2 incubator in complete 1198
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
medium: s e r u m - f r e e R P M I 1640 m e d i u m ( M i c r o b i o l o g i c a l A s s o c i a t e s Inc., MD) s u p p l e m e n t e d w i t h a n t i b i o t i c s a n d 0 . 0 1 M H E P E S buffer. C u l t u r e s w e r e i n c u b a t e d in the p r e s e n c e or in t h e a b s e n c e of the s t i m u l a n t s d e s c r i b e d a b o v e at d i f f e r e n t doses. A t the end of the incubation period, cell-free supernatants were c o l l e c t e d and s t o r e d at - 2 0 ° C or - 8 0 ° C u n t i l used. Dermal fibroblast cultures: Human dermal fibroblasts kindly provided by Dr A s s e l i n e a u (CIRD, France), were used before the 15th passage. Trypsinized cells were p l a t e d at 1 0 4 / w e l l in 9 6 - w e l l m i c r o t i t e r p l a t e in 10% foetal c a l f s e r u m (Hyclone, Logan, U T ) - e n r i c h e d M E M medium. Cells were incubated 48 h o u r s w i t h or w i t h o u t r I L - I B b e f o r e an o v e r n i g h t p u l s e (0.04 MBq/well) of [3H]methyl-Thymidine (Sp. act. = 37 G B q / m m o l e , CEA, Saclay, France). D a t a w e r e e x p r e s s e d as the mean counts in experimental cultures over the counts incorporated by unstimulated fibroblasts. Standard deviation u s u a l l y v a r i e d f r o m <10% f r o m t h e mean. In parallel experiments, supernatant fluids were collected a n d s t o r e d at - 8 0 ° C for PGE2 assay. P r o s t a g l a n d i n assay: T h e p r e s e n c e of p r o s t a g l a n d i n t y p e E2 (PGE2) in supernatant f l u i d s was e v a l u a t e d b y a c o n v e n t i o n a l r a d i o - i m m u n o a s s a y u s i n g an antiserum anti-PGE2 (Institut Pasteur). PGE2 content was e x p r e s s e d in ng/ml. I L - I a n d T N F assay: P r e s e n c e of m o n o k i n e s T N F a n d IL-I w a s d e t e c t e d w i t h c l a s s i c a l m e t h o d s of L929~ c y t o t o x i c i t y a n d a c t i v a t i o n of m u r i n e t h y m o c y t e s p r e v i o u s l y d e s c r i b e d in d e t a i l s (15).
RESULTS
Effect
of r I F N - y
In o r d e r of
PGE2,
different or MDP
on P G E 2 r e l e a s e
to a s s e s s human
doses
doses
of r I L - i B
fluids not
(i0,000
dramatic
dose
PGE 2 data
decrease of LPS
was
optimal
but
24
as b o t h 1
to
with
or in t h e a b s e n c e
PGE 2 LPS
release
48 h w i t h LPS
(i0 ng/ml)
of
rIFN~
showed
at
even
differentially
to rEL-IB,
and
of i00 U / m l
concentration
1199
at
of
1
and
which
huge
modulate
to
did
dose PGE 2
agent:
i) a
2) no s i g n i f i c a n t
3) a s y n e r g i s t i c
of r I F N - y
chosen
of s u p e r n a t a n t rIFN-¥
of t h e t r i g g e r i n g
of the r e s p o n s e stimulation,
However,
se
in a d o s e
Results
PGE 2 c o n t e n t
per
shown)
liberation
a n d MDP.
of s t i m u l a n t .
on t h e n a t u r e
The concentration
on m o n o c y t e
U/ml).
release not
depending
modification
1
on f i g u r e
U/ml,
liberation
incubated
stimulated
as w e l l
from o p t i m a l
stimulate
MDP.
rIL-IB
of r I F N - ¥
(I p g to i00 ng/ml),
(i to 1,000
fashion
be presented
were
in the p r e s e n c e
As expected, dependent
from human monocytes.
influence
monocytes
(I ug/ml)
different
the
effect
presented i0 U / m l
with
in f i g u r e were
also
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5°I 4O
30"
20-
iO-
C
rIFN- 7
MOP
PIL-~
LPS +rIFN- 7
Figure 1 :
active. and
The
the
the
inhibition even
synergism
response
Effect
Experiments
release case
LPS w a s
2),
neither
presented
lymphokine
addition
+LPS
+L+IFN
with
fibroblasts
were
effect
rIL-IB
effect whatever
to
1200
instead induced
the d o s e
of
PGE 2 In t h a t
employed,
to rIL-IB.
rIFN-y
effect
suppression
proliferation shown).
In t h a t and only
at 1 ng/ml.
nor response
dose-related
(data n o t
culture. abolished
fibroblasts
sensitive
specific
fibroblasts.
hereabove,
of rIFN-y
was
i).
from human
a maximal
and rIL-IB-induced
any cytotoxic
(figure
background
a
activity
a d d e d to t h e
spontaneous
induced
IL-I
LPS a n d r I L - l B w a s
reproduced
from the cells with
spontaneous
of t h e
on P G E 2 r e l e a s e were
Nevertheless, this
when
between
As r e s u l t s
(figure
modify
observed
to LPS w a s m a i n t a i n e d
of rIFN-y
monocytes.
+rlFN-~
Effect of rIFN-y (100 U/ml) on PGE 2 release from human monocytes triggered with different stimuli: MDP (I0 ~g/ml) LPS (i0 ng/ml), or Interleukin-iB (i0 ng/ml). Bars represent the mean (± SEM) of four separate experiments.
evidenced
case,
+rIFN- 7
(figure
of
since both
3) w i t h o u t
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
20"
o
t5-
x E
z o
lO-
o o o
I
t
Figure
Effect and
of
2:
100
t
PIL-t~
t0 + PIFN-7
t00
Effect of rIFN-y (from 1 to i00 U/ml) on PGE 2 release from human dermal fibroblasts stimulated with recombinant Interleukin-iB (i0 n g / m l ) . Vertical bars r e p r e s e n t s S E M of a t l e a s t s i x separate experiments.
calcitonin
(sCT)
on
PGE2
release
from
human
monocytes
fibroblasts. Calcitonin
activities at
t0 PIFN- T
high
and
on
rIFN-y
IL-l-induced
dose
(from
1 ng
have bone to
1
been
shown
resorption ug/ml)
to (16).
share
similar
Although,
calcitonin
was
unable
~.0"
8-
r.1 E
6-
oJ
0.
4
i C Figure
3:
iO iO0 PIFN- 7
I
rZL-i~
I0 I00 + PIFN-7
Effect of rIFN~ (from 1 t o i00 U/ml) o n p r o l i f e r a t i o n of rIL-IB-stimulated fibroblasts. Bars represent a typical experiment out of at least six. V e r t i c a l b a r s represent SEM for triplicates.
1201
even to
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I: Salmon calcitonin (sCT) m o d u l a t i o n of rIL-IB- or LPS-induced PGE 2 release from human m o n o c y t e s or fibroblasts
PGE 2 (ng/ml) release from monocytes
fibroblasts
Control
1.5 + 0.3 a
0.6 + 0.2
Calcitonin 0.lug
1.8 + 0.2
0.5 + 0.2
rIL-IB 10ng/ml
7.5 _+ 0.6
7.0 + 1.0
rIL-IB + sCT
8.7 + 0.5
7.8 + 0.9
LPS 100ng/ml
22.0 + 3.0
NT b
LPS + sCT
40.0 + 5.2
NT
* similar results were obtained from Ing/ml to lug/ml. a SEM of at least 6 different experiments. b not tested.
suppress
or to modulate
fibroblasts described from
stimulated with
latex
LPS-stimulated
calcitonin
did
monocytes, TNF
not
2).
an autocrine
(17),
(Table IL-I
enhanced
either
(Table
calcitonin
monocytes
i).
On
of LPS,
sCT
also
(data not
as
PGE 2 release the
other
slightly
with
hand,
or release
by
inhibited
production
evident
or
However,
or TNF production
was
stimulant
monocytes
enhanced i).
intracellular
enhancement
monocyte
from
rIL-iB
in t h e p r e s e n c e
and This
with
stimulate
but,
release
(Table
PGE 2 release
of
rIL-iB
IL-I
used
as
shown).
DISCUSSION
Lymphokines, during
inflammatory
influence monocytes autocrine range as
soluble
factor
(8)
bone tissue network
were
Thus,
non-related
even
produced immune
has
been
though
numerous
molecules
responses,
This
IL-I
various
inhibitors
factors
1202
such
produced
described displayed
target pituitary
pleiotropic
leukocytes
can profoundly
mainly
cytokine
different muscle,
requires
or
activated
recently
this
liver,
(9,10). which
by
Interleukin-l,
on completely
marrow,
connective
and
capacities.
and macrophages
regulatory mechanisms.
events
monocytic
of a c t i v i t i e s
brain,
factors
by
as an a broad
tissues
such
glands
and
feature
implies
inhibitory
feedback
were
recognized
which
as g l u c o c o r t i c o i d s ,
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 2: Salmon calcitonin (sCT) modulation of TNF and IL-I production by human monocytes incubated with LPS and/or rIFN-y
TNF activity -
IL-I activity
(U/ml)
+
sCT
(Acpm x 10 -3 ) + sCT i00 ng/ml a
-
i00 ng/ml a control
0b
LPS i00 ng/ml
41
rIFN-y i00 U/ml
0
rIFN-y + LPS
0
1 / 2c
30
49 / 20
NT d
1623
NT
1 / 2 50 / 34
1 / 2
NT
67 / 26
NT
a similar results were obtained from ing/ml to lug/ml. b U/ml defined as inverted dilution that results in lysis of 50% of the L929~ cells. c Acpm x 10-3:3H-TdR incorporated by C3H/HeJ thymocytes stimulated with lug/ml PHA-P in the presence of 1/8 diluted supernatants and lysates of monocytes (supernates / lysates). SEM usually varied <10% from the mean. d not tested.
neuropeptides addition, lymphokine depicted such
(~-MSH),
IFN-y with MAF able
to
report
used.
Thus,
monocytes:
rIFN-y
As well
(16)
~hown u n a b l e and
diverse
shown and
TNF
to
to
IFN-y
activity IL-I
In
was
activities
release
could
and bone
act differently the
These
results
et al.
PGE 2
release
cell
from
of LPS e f f e c t , were
in
(5) w i t h m u r i n e
P M A o r zymosan.
evidenced
target
PGE 2 r e l e a s e
no m o d u l a t i o n
activity.
of
and
differently
w i t h MDP,
done by Boraschi
rIFN-y
IFN-y,
been
that
with A23187,
rIL-IB was
particle-stimulated
[L-I
factor)
the stimulant
rIL-IB
of these
as
has
activity
by
with
proliferation
(20),
of
stimulated
inhibition
stimulated
(18,19).
antiproliferative
PGE 2 i n d u c i n g
could modulate
with the work
macrophages no
specifically
of
synergism
suppression
agreement
proteins
antiviral,
activating
(8),
indicates
the nature
human and
inhibit fonction
or u r i n a r y as
(16).
The present according
known
(macrophage
to
as a u t o c r i n e
resorption
TGF-B,
well
By c o n t r a s t ,
from fibroblasts
even at doses which
affected
cells. calcitonin to
(sCT),
interfere
affect
activities.
a calciotropic IL-I
prostaglandin
human monocytes
stimulate
with
directly However,
1203
(17).
bone pathway
sCT
in l a t e x
I n o u r study,
human monocytes slightly
hormone
resorption
sCT was
to produce reduced
TNF
Vol, 157, No. 3, 1988
production
but potentiated
production did
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not modulate
monocytes
PGE 2
and
monocytes.
release
intracellular
On the
either
other
IL-I
hand,
sCT
from rIL-lS-stimulated
or f i b r o b l a s t s .
In c o n c l u s i o n , specificity
of
the results
rIFN-7
and c a l c i t o n i n , As o b s e r v e d
physiopathologic depressive
or
reported
the r e g u l a t o r y
chosen.
different
PGE 2 release
by L P S - s t i m u l a t e d
capacity
according
of
to stimulant,
in vitro w i t h d i f f e r e n t
situations enhancing
compartments
here
might
effect
of
sets
forth
molecules cells stimuli,
preferentially IFN-¥
or
the high such
as
and a c t i v i t y the d i v e r s e determine
calcitonin
in t h e
or t i s s u e s .
ACKNOWLEDGEMENTS
The a u t h o r s are m o r e grateful to Pr. G.Milhaud discussions, to Mrs. Colette Leridant and Denis expert technical assistance.
for h e l p f u l H e r b e r t for
REFERENCES
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