Vol. 169, No. 2, 1990 June 15, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 422-429
L.RlRWTRIRNRS DO NOTRRGULATBINTERLEUKIN 1 PRODUCTION BY ACIWJATED MCROPRAGES Bashir ICI
A. Parkar,
Margaret
Pharmaceuticals,
Received
April
6,
E. McCormick,
Bioscience I, Mereside, Chesire, SK10 4TG,
and Stephen Alderly UK
J.
Park,
Foster Macclesfield,
1990
The objective of this work was to investigate the role of leukotrienes in the production of IL-l by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-l production. LTC4 was detected and preceded IL-l production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20uH), nordihydroguaiaretic acid (0.22uM), phenidone (4.60uM), REV5901 (O.ZOuH), and the Merck 5-lipoxygenase “translocation inhibitor” RK886 (0.02uM) with IC values as shown in parenthesis. However, none of these inhibitors re58 uced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages. 01990 Academic Press, Inc. IL-l
is
Its
a hormone-like
diversity
(2)
of actions
and stimulation
of
that
IL-l
such
as rheumatoid
synovial the
plays fluid
release can also
regulation inhibitors shown
to enhance
the results leukotrienes
macrophages.
IL-l these in
by activated production studies
the
are
(i)
possible
of IL-l selective
resorption in
that whilst
monocytes from
types.
the
Studies
to propose
and
(6).
involved
(7,8),
5-lipoxygenase
rheumatoid to stimulate
of cell
suggest
production
diseases
as collagenase
macrophages.
macrophages
is not
that
ability
a variety
by LPS-stimulated it
regulation
For example
such
and bone
and cycle-oxygenase
production of
(5)
leukotrienes
of fever speculated
inflammatory
and its
PGE2, from
by activated
has been
on observations (4)
enzymes
degradation
of 5-lipoxygenase IL-l
of IL-l
degrading
mediation
It
of chronic
was based
cells.
(l), (3).
pathology
This
that
by phagocytic
stimulation
levels
reported
production
primarily
response
including
cartilage
of IL-l
the
tissue
eicosanoids, has been
T cell phase
in
high
connective
regulate
for
role
arthritis.
induce it
Recently,
include
contained of
produced
the acute
a major
pro-inflammatory It
polypeptide
using
dual
leukotrienes
may
LTB4 has been (9).
Based
a definitive
on role
activated inhibitors
were
Abbreviations: IL-l, interleukin-1; LPS, lipopolysaccharide; LT, leukotriene; PG, prostaglandin; NDGA, nordihydroguaiaretic acid; DMSO, dimethylsulphoxide; PHA, phytohaemagglutinin; TNF, tumour necrosis factor alpha. ooo6-291x/90 $1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any formreserved.
422
not
Vol.
169,
No.
in
these
used
lipoxygenase of IL-l
2, 1990
studies
production
lipoxygenase exerting
and results could it
inhibitory
mechanisms
of
are due
(ii)
inhibitor its
be
products;
also
demonstrated
in
established
conditions
in which
both
inhibitors
macrophages of arachidonic
and evaluated acid
a decrease
reduction
in 5-lipoxygenase used
by interfering inhibition
the same cell.
and IL-l
the effects
of a number these
or 5may
with of IL-l
We have,
leukotrienes under
since
and therefore
production
MATERIALS
of 5-
a frequently
properties
metabolism
COMMUNICATIONS
inhibitors
to interpret
simultaneous
was not
RESEARCH
dual
NDGA is
on IL-l (iii)
leukotrienes activated
with
to either has redox
cell;
BIOPHYSICAL
difficult
although
effect the
AND
obtained
and cycle-oxygenase
cyclooxygenase
redox
BIOCHEMICAL
be other
and
therefore,
are
produced
by
of defined
conditions.
AND METHODS
Materials: RPM1 1640 medium and Ficoll-Hypaque were from Flow laboratories, Herts, U.K. L-glutamine, penicillin, streptomycin, Hepes, foetal calf serum, 96 U-well microtitre plates and 12 well flat bottom plates were from Gibco, Paisley, U.K. Polymyxin-B sulphate, zymosan A, lipopolysaccharide (E.coli Olll:B4), phytohaemagglutinin, human AB serum, phenidone (1-phenyl-3pyrazolidone) and NDGA were from Sigma, Poole, U.K. Dimethylsulphoxide (DMSO) was from BDH, Poole, U.K. Revlon 5901 (2-[3’-(l”-hydrohyhexyl)-phenoxymethylquinone) (10) was obtained from Revlon ICI207968 (2-(3-pyridylmethyl)-indazolinone) (11) and the Health Care Group. Merck 5-lipoxygenase translocation inhibitor MK886 (formally code named L663536) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-y1]-2,2dimethylprop-anoic acid) (12) were synthesi3ed in the chemistry laboralories at ICI Pharmaceuticals. Radiochemicals, [ HI-LTD4 (320Ci/mmol) and [ HI-PGE2 (184Ci/mmol) were from Amersham International, UK. ] A]-thymidine (6.7Ci/mmol) was from NEN (Du Pont), Southampton, UK. Preparation of mononuclear phagocytic cells: Resident peritoneal macrophages were obtained from Alderley Park strain mice by peritoneal lavage with RPM1 1640 culture medium containing 2g/l sodium bicarbonate, 2mM L-glutamine, lOOunits/ml penicillin, lOOug/ml streptomycin, lOpg/ml polymyxin-B sulphate and 10% heat-inactivated foetal calf serum (PCS). The peritoneal cells were washed twice in RPM1 1640 medium, and resuspended at 1x10 /ml in culture medium. One ml of the cell suspension was layered into each well of a 12 well flat bottomed plate and incubated for 2hr at 37’C in 5% C02/95% air, after which time the nonadherent cells were removed by washing 3 times with RPM1 1640 medium. Human monocytes: Heparinized peripheral blood was obtained from healthy Mononuclear cells gere isolated by centrifugation over ficollvolunteers. hypaque and resuspended at 2x10 /ml in culture medium containing 10% heat Five ml of this cell suspension was dispensed inactivated human AB serum. into each well of a 12 well flat bottomed plate and incubated for 2hr as above. Nonadherent cells were removed by washing 3 times with RPM1 1640 medium. In some instances, the adherent cells were further incubated for 18hr with lml of culture medium containing either 10% PCS or AB serum. Experimental Procedure: The medium was removed and replaced with 0.5ml of serum-free RPM1 1640 containing antibiotics and the appropriate concentration of inhibitors of arachidonic acid metabolism, which were dissolved in DMSO After lOmins, 0.5ml of medium containing either (final concentration 0.05%). E.coli LPS (lOug/ml) or Zymosan A (100 or 200ug/ml) was added to give the desired concentration of both inhibitors and stimuli. Control wells received 0.5ml of serum-free medium containing 0.1% DMSO. At subsequent times the cell centrifuged to remove any cell debris, and free supernatants were collected, assayed for LTC4 and IL-l.
423
Vol.
BIOCHEMICAL
169, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E4 and PGE2 assay: Immunoreactive LTC4 was measured in duplicate by specific raised in rabbits. RIA-using an-zdy The cross reactivity profile of the antibody-was as follows: LTD4(100%), LTC (145%), LTE4(41%), LTB,, racemic 5-HETE, 15-HETE, PGE2, PGD , PGFlal ha, $xB cysteine, glutathione and arachidonic acid were all ? 0.03%. ?mmunoregitive PGE2 was measured by specific RIA as previously described (13). IL-l bioassay: IL-l activity was measured by the enhancement of thymocyte proliferation induced by PHA accordigg to the method of Mizel et a1.(14) with slight modifications. Briefly, 1x10 thymocytes from Balb C m=ere seeded into 96-well round-bottomed plates and triplicate incubations were carried out with 1OOul of various dilutions of culture supernatant ‘n the presence of a suboptimal dose of PHA (5ug/ml). After 48hr, 1uCi of [ 3 HI-thymidine (6.7Ci/mmol) was added for the last 5hr of culture. The results are expressed as the mean (cpm)+ - s.e.m. RESULTS Kinetics
of IL-l
release Both
from
and LTC4 release:
2hr
adherent
LPS and zymosan
attained
maximal
caused
In order
production
it
nor
and IL-1 when
macrophages
show the
time
course
peritoneal zymosan. dependent increase increase
were
of IL-l
macrophages increase with in
in
IL-l
which
macrophages, production. in
human monocytes, production
which
these
IL-l
production
elevated
for
in
these
from
Zymosan cultures,
with
also
LPS and zymosan
caused
to rise
for
be
Figures
2 and 3
(18 hr)
either
mouse
LPS or
and LPS caused
caused
whereas
in IL-l
of both
could
adherent
zymosan
macrophage
release
18 hr.
stimulated both
continued
which
for
which
at least
may have
Such conditions
to adhere
and human monocytes IL-l
under
of IL-1
LPS or zymosan.
leukotrienes
conditions
and LTC4 production
LTC 4 production,
in
LTC4 production
role
allowed
course
with
increase
be demonstrated.
In mouse peritoneal in
Similarly
to find
could
achieved
the
time
and remained
LPS stimulated
to establish
was essential
leukotrienes
1 and 4hr
the
stimulated
a time-dependent
between
zymosan
cultures.
1 shows
mouse macrophages
levels
Neither
18 hr.
Figure
a time-
a time-dependent
LPS was inactive. a time-dependent 18hr.
LTC4 was
Time (hours)
Figure 1. Time course of IL-l release from mouge peritoneal macrophages following adherence for Zhr. Macrophages (1x10 /well) were stimulated with either lOug/ml LPS or lOOpg/ml zymosan. Control shows unstimulated macrophages. Values shown (cpm) are the means of triplicates The -+ s.e.m. Figure shown is typical of two experiments. 424
Vol.
169, No. 2, 1990
1
BIOCHEMICAL
2
3
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
18
1
Time (hours)
2
3
18
4
Time (hours)
0=-z-z---1
03
18 Time (hours)
0-i-J-n--
18 Time (hours)
Figure 2. Time course of IL-l (a) and LTC4 (b) production from mouse peritoneal macrophages following adherence for 18hr. Macrophages (lx106/well) were stimulated with either lOug/ml LPS or lOOug/ml zymosan. Control shows unstimulated macrophages. For the sake of clarity, control values are offset from zero on the ordinate axis. The IL-1 assay values shown (cpm) are the means of triplicates + s.e.m. LTC was measured by RIA and the values are means of duplicates. -The Figure sRown is typical of two experiments. Figure 3. Time course of IL-l (a) and LTC4 (b) production from human peripheral blood monocytes following adherence for 18hr. Monocytes were stimulated with either lOug/ml LPS or 200ug/ml opsonized zymosan. Control shows unstimulated macrophages. For the sake of clarity, control values are offset from zero on the ordinate axis. For the IL-l assay values shown (cpm) are the means of triplicates + s.e.m. LTC4 was measured by RIA and the values are means of duplicates. The-Figure shown is typical of two experiments.
detected
and
its
synthesis
and human monocytes adherent
cells
although
low
effects
of
therefore Effect the
stimulated
effects
inhibitors
inhibitors
out
of several Revlon of
using
inhibitor
production
zymosan. LPS.
were
in both
However,
Unstimulated
detected. acid
zymosan
macrophages
synthesis
inhibitors
of arachidonic
5901
(10)
production whilst
produced
to evaluate on IL-l
from no LTC4
the
production
were
as stimulus. on LTCA and IL-l
PGE2 and IL-l
mouse macrophages
no LTC4 was detected
Experiments
inhibitors
LTC4 generation.
leukotriene
lipoxygenase
with
IL-l
of arachidonic
of LTC4,
macrophages.
with
of IL-l
of lipoxygenase
production
inhibit
treated
levels
carried
preceded
by adherent These
were
compounds
MK886 acts 425
acid
metabolism
(18 hr)
and MK886 (12) by different
production:
are
I shows
on the
mouse peritoneal
both not
mechanisms. by a novel
Table
potent redox Revlon
mechanism
and selective agents 5901 namely
and is
a 5-
Vol.
169,
No.
Table
2, 1990
I.
BIOCHEMICAL
Effect of inhibitors and IL-1 release
AND
BIOPHYSICAL
COMMUNICATIONS
of arachidonic acid metabolism on LTC4, PGE2 from mouse peritoneal macrophages
values (uM) 1c2Pc4 PGE2
COMPOUNDS
RESEARCH
SELECTIVITY RATIO
% IL-l INHIBITION
HK886
0.02
>3
>150
0
REVLON 5901
0.20
>lO
>50
2
ICI 207968
3.20
>lOOO
>300
5
NDGA
0.22
2.4
11
3
PHENIDONE
4.60
11.4
2.5
7
Macrophage cultures (1~10~) were stimulated for 18hr with zygosan (leOug/ml) in the presence or absence of a range of concentrations (10 M - lo- H) of the test agents. Supernatants were collected and assayed for LTC PGE and IL-l. LTC4 and PGE2 were measured by specific radioimmunoassays. 16’ va 1 ues were determined from plots of the percent inhibition of LTC4 or PGEz’production versus test agent concentration at the point where 50% inhibition of the respective eicosanoiod was seen, and are means of duplicate values. The selectivity ratio was calculated by dividing IC50(PGE2) by IC O(LTC4). IL-1 was measured by the thymocyte proliferation assay. Supernatan i s from control macrophage cultures stimulated with zymosan, in the absence of inhibitor, typically gave 7254 + 463 cpm. Percent inhibition of IL-l production was calculated-by dividing the mean cpm in the presence of test agent by the mean control cpm x 100. Values shown are those calculated at concentrations 100 times their respective ICsp value the foliofor inhibition of LTC4. Total inhibition of LTC4 production occurred a wing concentrations as shown in parenthesis, HK886 (2uM); REV5901 (10nM); ICI207968 (1OOuM); NDGA (1OuM) and Phenidone (5OuM).
preventing
translocation
of 5-lipoxygenase
membrane(l2),
a process
which
enzyme
ICI207968
caused
(15).
zymosan-stimulated selective also
are
have marked
production
from
concentrations in Table
thought
inhibitor
dual
inhibitors
redox
properties. ranged
inhibition
ICI207968 with moderate
for
cell
activation
of
to be a (11). NDGA
inhibited
macrophages
to 100 times
their
ICso
the
from
and cycle-oxygenase agents
mouse peritoneal lo-‘M
to the
has been shown redox potential
None of these
from
cytosol
of LTC4 production
of 5-lipoxygenase
zymosan-stimulated which
the
to be required
selective
mouse macrophages.
5-lipoxygenase
and phenidone
is
from
which IL-l
at values
as shown
I. DISCUSSION
IL-l,
a 16kD polypeptide,
inflammation
(16).
has implicated rheumatoid IL-l,
then
effects is
in
IL-l
mediates
Evidence
arthritis
(17).
compounds
which
the
based
as the major
treatment
If inhibit of
several
aspects
on tissue mediator
explant of
leukotrienes
synthesis arthritis
implicated. 426
and chronic
and cell
tissue are
their
rheumatoid
of acute damage
involved could and other
culture
studies
in diseases in
such
the generation
have
as of
beneficial
diseases
where
IL-l
Vol. 169, No. 2, 1990 Our studies
have
macrophage
culture
Zymosan
BIOCHEMICAL
demonstrated conditions
stimulated
IL-l.
both
Interestingly,
deep-rough
significant
LPS in activating
which
l-4hr.
kinetics (22)
differ who showed
kinetics
of
(lhr) It
is
peak
with
et al
(23)
mRNA.
18 hrs
restimulation Arenzana-Seisdedos periods
incubation
their
overnight
IL-l
when
stimulated also
It
is
transiently of 18 hr Under the
et al
(24)
show
perturbs restores
inhibitors
who also with
pool, induces
24 hr.
IL-l
been
for
a
upon
the the
Haq et al
loose
of
of membrane
their
by
macrophages
to LPS.
during
However,
light,
reported
resting
response
upon
Restimulation
macrophages
have
which
a transient
re-expression
resting
that
the adherence
In this
itself
gamma interferon to LPS.
be due to
peak activity
IL-l
rapid
their
human monocytes
et al
The rapid
and
to produce
showed
diminishes
not
These
might
kinetics. in
results
resting (25)
for
Moreover, period
have
ability
shown
to secrete
LPS.
that
freshly
that
the macrophage
isolated
adherent isolation since
leukotriene
generation,
ability
to produce
their whereby
leukotriene
of leukotriene inhibition
that
did
under
hrs.
isolation
after
ability
similar
to respond with
shows
LPS
synthesis.
diminishes
their
culturing,
possible
conditions
significant
Indeed,
ability
after
LTC4.
LPS.
the macrophages
that
Our studies
alter
of up to 7 days
of
restores
not
12-24 to attain
to rapid
in
2hr
and Lonnemann
conditions
protein
LPS resulted
Our experience
does
with
with
(21)
between these
used
observed
the same cell.
et al
adherence
which
only
macrophages
in
required
rise
for were
an intracellular
that
we have
with
LTC4 formation.
activity
of macrophage
to form
shown
of IL-l
macrophages
and IL-2
longer
trauma
LPS gives
have
release
rested of
the
time
by de novo
the macrophages
time-dependent
period
for
the
In addition synergizes
study
to adhere from
occur
we see under
since
both
stimulation
these
levels
that
be accounted
stimulate
further
IL-l
LPS from
macrophages. ionophore
of IL-l
by Hazuda
that
only
(Re to Rc chemotypes)
In our
produced
production.
shown
induce
can be released reported
IL-l
that
levels not
have
from
do not
and the
LPS stimulated
calcium
LTC4.
which
are
release
possible
Fuhlbrigge
IL-l
that
preformed
cannot
step
those
of IL-l
release
IL-l
leukotrienes from
that
had been allowed
and peak
Therefore
where
LTC4 and IL-l whereas
and E.coli
to release
of E.coli
between conditions
for
of LTC4 release
strains
any leukotrienes,
of stimulus
(18 and 19)
demonstrated
macrophages
release
type
release
minnesota
amounts have
the
important
et al
macrophages
from wild-type Furthermore,
are
of Salmonella
and Cohn (20)
both
LTC4 and IL-l Luderitz
mutants
stimulated Aderem
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of IL-l
at doses
preceded
tested
in our
which
completely
427
do not
produce
procedure in some way resting them for a period
leukotrienes
production
synthesis
cells
when
stimulated.
IL-l
release,
study
caused
abrogated
none
any leukotriene
of
Vol.
169,
No.
production. production simultaneous Moreover, inhibitory production
but only
adhered for demonstrate
interesting
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
inhibition of LTC4 and IL-l was not demonstrated in their study. NDGA has redox properties, and therefore, may be exerting its effect
on IL-l
production
by other
of 5-lipoxygenase.
cellular
In our hands,
in LPS- or zymosan-stimulated
redox systems NDGA inhibits
macrophages
inhibition macrophages
rather IL-l
that
2hr (data not shown), conditions under which we failed leukotriene production. It is an enigma that we could
have to not
of IL-l production by NDGA in LPS- or zymosanthat had been allowed to adhere for 18 hr. It
to note that
inhibitor
AND
Kunkel and Chensue have reported that NDGA inhibited the of IL-l from zymosan-stimulated macrophages (8). However,
than by inhibition
demonstrate stimulated
BIOCHEMICAL
2, 1990
of lipoxygenase
in a previous
report
and cyclooxygenase,
(26),
SK&F 86002,
which
also
is
a dual
has redox properties,
suppressed the formation of IL-l, though the authors do not allude to the mechanism of action. More recently, it has been reported that leukotrienes may be involved in the regulation of TNF (27,28). Although our data do not support
a role
for leukotrienes in the regulation of IL-l this may not be the case for other cytokines
macrophages, respect, Kovacs et al (29) and TNF are differentially conclusion, regulation
our data suggest of IL-l
production
by activated such as TNF. In this
and Scales et al (30) have demonstrated regulated at the messenger RNA level. that
leukotrienes
are not involved
from LPS- or zymosan-activated
that In
IL-l
in the
macrophages.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13.
Mizel, S.B. (1982). Immunol. Rev., 63, 51-72. Dinarello, C.A. and Wolff, S.M. (1982). Am. J. Med., 72, 799-819. Kampschmidt, R.F., Upchurch, H.F., Eddington, C.L., and Pulliam, L.A. (1973). Am. J. Physiol., 224. 530-533. Fontana, A., Hengartner, H., Weber, E., Fehr, K., Grob, P.J. and Cohen, G. (1982). Rheumatol. Int., 2, 49-53. Saklatavala, J., Sarsfield, S.J. and Townsend, Y. (1985). J. Exp. Med., 162, 1208-1222. Gowen, M., Wood, D.D., Ihrie, E.J., McGuire, M.K.B. and Russel, R.G. (1983). Nature, 306, 378-380. Dinarello, C.A., Bishai, I., Rosenwasser, L.J. and Coceani, F. (1984). Int. J. Immunopharmac., 6, 43-50. Kunkel, S.L. and Chensue, S.W. (1985). Biochem. Biophys. Res. Commun, 128, 892-897. Rola-Pleszczynski, M. and Lemaire, I. (1985). J. Immunol.,l35, 3958-3961. Coutts, S.M., Khandwala, A., van Ingwegan, R., Chakraborty, U., Musser, J ., Bruens, J., Jariwala, N., Dally-Meade, V., Ingram, R., Pruss, T., Jones, H., Neiss, E. and Weinryb, I (1985). In Prostaglandins, Leukotrienes and Lipoxins. ed. Bailey, M.J. ~~627-637, New York: Plenum Press. Foster, S.J., Bruneau, P., Walker, E.R.H. and McMillan, R.M. (1990) Br. J. Pharmacol. (In press). Gillard, A. and Ford-Hutchinson, A.W. eta1 (1989). Can. J. Physiol. Pharmacol., 67, 456-464. Forder, R.A. and Carey, F. (1984) Prostaglandins. 28, P666. 428
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169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Mizel, S.B., Oppenheim, J.J. and Rosenstreich, D.L. (1978). J. Immunol., 120, 1497-1503. Rouzer, C.A. and Kargman, S. (1988). J.Biol.Chem., 263, 10,980-10,988. Durum, S.K., Schmidt, J.A. and Oppenheim, J.J. (1985). Ann. Rev. Immunol. 3, 263-287. Henderson, B., Pettipher, E.R. and Higgs, G.A. (1987). Brit. Med. Bull. 43, 41.5-428. Luderitz, T., Schade, U. and Rietschel, E.T. (1986). Eur. J. Biochem. 155, 377-382. Luderitz, T., Brandenburg, K., Seydel, U., Roth, A., Galanos, C. and Rietschel, E.T. (1989). Eur. J. Biochem., 179, 11-16. Aderem, A.A. and Cohn, Z.A. (1988). J. Exp. Med., 167, 623-631. Hazuda, D.J., Lee, J. and Young, P.R. (1988). J. Biol. Chem., 263, 84738479. Lonnemann, G., Endres, S., Van de Meer, J.W.M., Cannon, J.G., Koch, K.M. and Dinarello, C.A. (1989). Eur. J. Immunol., 19, 1531-1536. Fuhlbrigge, R.C., Chaplin, D.D., Kiely, J-M. and Unanue, E.R. (1987). J. Immunol., 138, 3799-3802. Arenzana-Seisdedos, F., Virelizier, J.L. and Fiers, W. (1985). J. Immunol., 134, 2444-2448. Haq, A.U., Rinehart, J.J. and Maca, R.D. (1985). J. Leuk. Biol., 38, 735-746. Lee, J.C., Griswold, D.E., Votta, B. and Hanna, N. (1988). Int. J. Immunopharmac., 10, 835-843. Dubois, C.M., Bissonnette, E. and Rola-Pleszczynski, M. (1989). Am. Rev. Respir. Dis., 139, 1257-1264. Schade, U.F., Burmeister, I., Engel, R., Reinke, M. and Wolter, D.T. (1989). Lymphokine Research., 8, 245-250. Kovacs, E.J., Radzioch, D., Young, H.A. and Varesio, L. (1988). J. Immunol., 141, 3101-3105. Scales, W.E., Chensue, S.W., Otterness, I. and Kunkel, S.L. (1989). J. Leuk. Biol., 45, 416-421.
429
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 422-429
L.RlRWTRIRNRS DO NOTRRGULATBINTERLEUKIN 1 PRODUCTION BY ACIWJATED MCROPRAGES Bashir ICI
A. Parkar,
Margaret
Pharmaceuticals,
Received
April
6,
E. McCormick,
Bioscience I, Mereside, Chesire, SK10 4TG,
and Stephen Alderly UK
J.
Park,
Foster Macclesfield,
1990
The objective of this work was to investigate the role of leukotrienes in the production of IL-l by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-l production. LTC4 was detected and preceded IL-l production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20uH), nordihydroguaiaretic acid (0.22uM), phenidone (4.60uM), REV5901 (O.ZOuH), and the Merck 5-lipoxygenase “translocation inhibitor” RK886 (0.02uM) with IC values as shown in parenthesis. However, none of these inhibitors re58 uced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages. 01990 Academic Press, Inc. IL-l
is
Its
a hormone-like
diversity
(2)
of actions
and stimulation
of
that
IL-l
such
as rheumatoid
synovial the
plays fluid
release can also
regulation inhibitors shown
to enhance
the results leukotrienes
macrophages.
IL-l these in
by activated production studies
the
are
(i)
possible
of IL-l selective
resorption in
that whilst
monocytes from
types.
the
Studies
to propose
and
(6).
involved
(7,8),
5-lipoxygenase
rheumatoid to stimulate
of cell
suggest
production
diseases
as collagenase
macrophages.
macrophages
is not
that
ability
a variety
by LPS-stimulated it
regulation
For example
such
and bone
and cycle-oxygenase
production of
(5)
leukotrienes
of fever speculated
inflammatory
and its
PGE2, from
by activated
has been
on observations (4)
enzymes
degradation
of 5-lipoxygenase IL-l
of IL-l
degrading
mediation
It
of chronic
was based
cells.
(l), (3).
pathology
This
that
by phagocytic
stimulation
levels
reported
production
primarily
response
including
cartilage
of IL-l
the
tissue
eicosanoids, has been
T cell phase
in
high
connective
regulate
for
role
arthritis.
induce it
Recently,
include
contained of
produced
the acute
a major
pro-inflammatory It
polypeptide
using
dual
leukotrienes
may
LTB4 has been (9).
Based
a definitive
on role
activated inhibitors
were
Abbreviations: IL-l, interleukin-1; LPS, lipopolysaccharide; LT, leukotriene; PG, prostaglandin; NDGA, nordihydroguaiaretic acid; DMSO, dimethylsulphoxide; PHA, phytohaemagglutinin; TNF, tumour necrosis factor alpha. ooo6-291x/90 $1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any formreserved.
422
not
Vol.
169,
No.
in
these
used
lipoxygenase of IL-l
2, 1990
studies
production
lipoxygenase exerting
and results could it
inhibitory
mechanisms
of
are due
(ii)
inhibitor its
be
products;
also
demonstrated
in
established
conditions
in which
both
inhibitors
macrophages of arachidonic
and evaluated acid
a decrease
reduction
in 5-lipoxygenase used
by interfering inhibition
the same cell.
and IL-l
the effects
of a number these
or 5may
with of IL-l
We have,
leukotrienes under
since
and therefore
production
MATERIALS
of 5-
a frequently
properties
metabolism
COMMUNICATIONS
inhibitors
to interpret
simultaneous
was not
RESEARCH
dual
NDGA is
on IL-l (iii)
leukotrienes activated
with
to either has redox
cell;
BIOPHYSICAL
difficult
although
effect the
AND
obtained
and cycle-oxygenase
cyclooxygenase
redox
BIOCHEMICAL
be other
and
therefore,
are
produced
by
of defined
conditions.
AND METHODS
Materials: RPM1 1640 medium and Ficoll-Hypaque were from Flow laboratories, Herts, U.K. L-glutamine, penicillin, streptomycin, Hepes, foetal calf serum, 96 U-well microtitre plates and 12 well flat bottom plates were from Gibco, Paisley, U.K. Polymyxin-B sulphate, zymosan A, lipopolysaccharide (E.coli Olll:B4), phytohaemagglutinin, human AB serum, phenidone (1-phenyl-3pyrazolidone) and NDGA were from Sigma, Poole, U.K. Dimethylsulphoxide (DMSO) was from BDH, Poole, U.K. Revlon 5901 (2-[3’-(l”-hydrohyhexyl)-phenoxymethylquinone) (10) was obtained from Revlon ICI207968 (2-(3-pyridylmethyl)-indazolinone) (11) and the Health Care Group. Merck 5-lipoxygenase translocation inhibitor MK886 (formally code named L663536) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-y1]-2,2dimethylprop-anoic acid) (12) were synthesi3ed in the chemistry laboralories at ICI Pharmaceuticals. Radiochemicals, [ HI-LTD4 (320Ci/mmol) and [ HI-PGE2 (184Ci/mmol) were from Amersham International, UK. ] A]-thymidine (6.7Ci/mmol) was from NEN (Du Pont), Southampton, UK. Preparation of mononuclear phagocytic cells: Resident peritoneal macrophages were obtained from Alderley Park strain mice by peritoneal lavage with RPM1 1640 culture medium containing 2g/l sodium bicarbonate, 2mM L-glutamine, lOOunits/ml penicillin, lOOug/ml streptomycin, lOpg/ml polymyxin-B sulphate and 10% heat-inactivated foetal calf serum (PCS). The peritoneal cells were washed twice in RPM1 1640 medium, and resuspended at 1x10 /ml in culture medium. One ml of the cell suspension was layered into each well of a 12 well flat bottomed plate and incubated for 2hr at 37’C in 5% C02/95% air, after which time the nonadherent cells were removed by washing 3 times with RPM1 1640 medium. Human monocytes: Heparinized peripheral blood was obtained from healthy Mononuclear cells gere isolated by centrifugation over ficollvolunteers. hypaque and resuspended at 2x10 /ml in culture medium containing 10% heat Five ml of this cell suspension was dispensed inactivated human AB serum. into each well of a 12 well flat bottomed plate and incubated for 2hr as above. Nonadherent cells were removed by washing 3 times with RPM1 1640 medium. In some instances, the adherent cells were further incubated for 18hr with lml of culture medium containing either 10% PCS or AB serum. Experimental Procedure: The medium was removed and replaced with 0.5ml of serum-free RPM1 1640 containing antibiotics and the appropriate concentration of inhibitors of arachidonic acid metabolism, which were dissolved in DMSO After lOmins, 0.5ml of medium containing either (final concentration 0.05%). E.coli LPS (lOug/ml) or Zymosan A (100 or 200ug/ml) was added to give the desired concentration of both inhibitors and stimuli. Control wells received 0.5ml of serum-free medium containing 0.1% DMSO. At subsequent times the cell centrifuged to remove any cell debris, and free supernatants were collected, assayed for LTC4 and IL-l.
423
Vol.
BIOCHEMICAL
169, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E4 and PGE2 assay: Immunoreactive LTC4 was measured in duplicate by specific raised in rabbits. RIA-using an-zdy The cross reactivity profile of the antibody-was as follows: LTD4(100%), LTC (145%), LTE4(41%), LTB,, racemic 5-HETE, 15-HETE, PGE2, PGD , PGFlal ha, $xB cysteine, glutathione and arachidonic acid were all ? 0.03%. ?mmunoregitive PGE2 was measured by specific RIA as previously described (13). IL-l bioassay: IL-l activity was measured by the enhancement of thymocyte proliferation induced by PHA accordigg to the method of Mizel et a1.(14) with slight modifications. Briefly, 1x10 thymocytes from Balb C m=ere seeded into 96-well round-bottomed plates and triplicate incubations were carried out with 1OOul of various dilutions of culture supernatant ‘n the presence of a suboptimal dose of PHA (5ug/ml). After 48hr, 1uCi of [ 3 HI-thymidine (6.7Ci/mmol) was added for the last 5hr of culture. The results are expressed as the mean (cpm)+ - s.e.m. RESULTS Kinetics
of IL-l
release Both
from
and LTC4 release:
2hr
adherent
LPS and zymosan
attained
maximal
caused
In order
production
it
nor
and IL-1 when
macrophages
show the
time
course
peritoneal zymosan. dependent increase increase
were
of IL-l
macrophages increase with in
in
IL-l
which
macrophages, production. in
human monocytes, production
which
these
IL-l
production
elevated
for
in
these
from
Zymosan cultures,
with
also
LPS and zymosan
caused
to rise
for
be
Figures
2 and 3
(18 hr)
either
mouse
LPS or
and LPS caused
caused
whereas
in IL-l
of both
could
adherent
zymosan
macrophage
release
18 hr.
stimulated both
continued
which
for
which
at least
may have
Such conditions
to adhere
and human monocytes IL-l
under
of IL-1
LPS or zymosan.
leukotrienes
conditions
and LTC4 production
LTC 4 production,
in
LTC4 production
role
allowed
course
with
increase
be demonstrated.
In mouse peritoneal in
Similarly
to find
could
achieved
the
time
and remained
LPS stimulated
to establish
was essential
leukotrienes
1 and 4hr
the
stimulated
a time-dependent
between
zymosan
cultures.
1 shows
mouse macrophages
levels
Neither
18 hr.
Figure
a time-
a time-dependent
LPS was inactive. a time-dependent 18hr.
LTC4 was
Time (hours)
Figure 1. Time course of IL-l release from mouge peritoneal macrophages following adherence for Zhr. Macrophages (1x10 /well) were stimulated with either lOug/ml LPS or lOOpg/ml zymosan. Control shows unstimulated macrophages. Values shown (cpm) are the means of triplicates The -+ s.e.m. Figure shown is typical of two experiments. 424
Vol.
169, No. 2, 1990
1
BIOCHEMICAL
2
3
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
18
1
Time (hours)
2
3
18
4
Time (hours)
0=-z-z---1
03
18 Time (hours)
0-i-J-n--
18 Time (hours)
Figure 2. Time course of IL-l (a) and LTC4 (b) production from mouse peritoneal macrophages following adherence for 18hr. Macrophages (lx106/well) were stimulated with either lOug/ml LPS or lOOug/ml zymosan. Control shows unstimulated macrophages. For the sake of clarity, control values are offset from zero on the ordinate axis. The IL-1 assay values shown (cpm) are the means of triplicates + s.e.m. LTC was measured by RIA and the values are means of duplicates. -The Figure sRown is typical of two experiments. Figure 3. Time course of IL-l (a) and LTC4 (b) production from human peripheral blood monocytes following adherence for 18hr. Monocytes were stimulated with either lOug/ml LPS or 200ug/ml opsonized zymosan. Control shows unstimulated macrophages. For the sake of clarity, control values are offset from zero on the ordinate axis. For the IL-l assay values shown (cpm) are the means of triplicates + s.e.m. LTC4 was measured by RIA and the values are means of duplicates. The-Figure shown is typical of two experiments.
detected
and
its
synthesis
and human monocytes adherent
cells
although
low
effects
of
therefore Effect the
stimulated
effects
inhibitors
inhibitors
out
of several Revlon of
using
inhibitor
production
zymosan. LPS.
were
in both
However,
Unstimulated
detected. acid
zymosan
macrophages
synthesis
inhibitors
of arachidonic
5901
(10)
production whilst
produced
to evaluate on IL-l
from no LTC4
the
production
were
as stimulus. on LTCA and IL-l
PGE2 and IL-l
mouse macrophages
no LTC4 was detected
Experiments
inhibitors
LTC4 generation.
leukotriene
lipoxygenase
with
IL-l
of arachidonic
of LTC4,
macrophages.
with
of IL-l
of lipoxygenase
production
inhibit
treated
levels
carried
preceded
by adherent These
were
compounds
MK886 acts 425
acid
metabolism
(18 hr)
and MK886 (12) by different
production:
are
I shows
on the
mouse peritoneal
both not
mechanisms. by a novel
Table
potent redox Revlon
mechanism
and selective agents 5901 namely
and is
a 5-
Vol.
169,
No.
Table
2, 1990
I.
BIOCHEMICAL
Effect of inhibitors and IL-1 release
AND
BIOPHYSICAL
COMMUNICATIONS
of arachidonic acid metabolism on LTC4, PGE2 from mouse peritoneal macrophages
values (uM) 1c2Pc4 PGE2
COMPOUNDS
RESEARCH
SELECTIVITY RATIO
% IL-l INHIBITION
HK886
0.02
>3
>150
0
REVLON 5901
0.20
>lO
>50
2
ICI 207968
3.20
>lOOO
>300
5
NDGA
0.22
2.4
11
3
PHENIDONE
4.60
11.4
2.5
7
Macrophage cultures (1~10~) were stimulated for 18hr with zygosan (leOug/ml) in the presence or absence of a range of concentrations (10 M - lo- H) of the test agents. Supernatants were collected and assayed for LTC PGE and IL-l. LTC4 and PGE2 were measured by specific radioimmunoassays. 16’ va 1 ues were determined from plots of the percent inhibition of LTC4 or PGEz’production versus test agent concentration at the point where 50% inhibition of the respective eicosanoiod was seen, and are means of duplicate values. The selectivity ratio was calculated by dividing IC50(PGE2) by IC O(LTC4). IL-1 was measured by the thymocyte proliferation assay. Supernatan i s from control macrophage cultures stimulated with zymosan, in the absence of inhibitor, typically gave 7254 + 463 cpm. Percent inhibition of IL-l production was calculated-by dividing the mean cpm in the presence of test agent by the mean control cpm x 100. Values shown are those calculated at concentrations 100 times their respective ICsp value the foliofor inhibition of LTC4. Total inhibition of LTC4 production occurred a wing concentrations as shown in parenthesis, HK886 (2uM); REV5901 (10nM); ICI207968 (1OOuM); NDGA (1OuM) and Phenidone (5OuM).
preventing
translocation
of 5-lipoxygenase
membrane(l2),
a process
which
enzyme
ICI207968
caused
(15).
zymosan-stimulated selective also
are
have marked
production
from
concentrations in Table
thought
inhibitor
dual
inhibitors
redox
properties. ranged
inhibition
ICI207968 with moderate
for
cell
activation
of
to be a (11). NDGA
inhibited
macrophages
to 100 times
their
ICso
the
from
and cycle-oxygenase agents
mouse peritoneal lo-‘M
to the
has been shown redox potential
None of these
from
cytosol
of LTC4 production
of 5-lipoxygenase
zymosan-stimulated which
the
to be required
selective
mouse macrophages.
5-lipoxygenase
and phenidone
is
from
which IL-l
at values
as shown
I. DISCUSSION
IL-l,
a 16kD polypeptide,
inflammation
(16).
has implicated rheumatoid IL-l,
then
effects is
in
IL-l
mediates
Evidence
arthritis
(17).
compounds
which
the
based
as the major
treatment
If inhibit of
several
aspects
on tissue mediator
explant of
leukotrienes
synthesis arthritis
implicated. 426
and chronic
and cell
tissue are
their
rheumatoid
of acute damage
involved could and other
culture
studies
in diseases in
such
the generation
have
as of
beneficial
diseases
where
IL-l
Vol. 169, No. 2, 1990 Our studies
have
macrophage
culture
Zymosan
BIOCHEMICAL
demonstrated conditions
stimulated
IL-l.
both
Interestingly,
deep-rough
significant
LPS in activating
which
l-4hr.
kinetics (22)
differ who showed
kinetics
of
(lhr) It
is
peak
with
et al
(23)
mRNA.
18 hrs
restimulation Arenzana-Seisdedos periods
incubation
their
overnight
IL-l
when
stimulated also
It
is
transiently of 18 hr Under the
et al
(24)
show
perturbs restores
inhibitors
who also with
pool, induces
24 hr.
IL-l
been
for
a
upon
the the
Haq et al
loose
of
of membrane
their
by
macrophages
to LPS.
during
However,
light,
reported
resting
response
upon
Restimulation
macrophages
have
which
a transient
re-expression
resting
that
the adherence
In this
itself
gamma interferon to LPS.
be due to
peak activity
IL-l
rapid
their
human monocytes
et al
The rapid
and
to produce
showed
diminishes
not
These
might
kinetics. in
results
resting (25)
for
Moreover, period
have
ability
shown
to secrete
LPS.
that
freshly
that
the macrophage
isolated
adherent isolation since
leukotriene
generation,
ability
to produce
their whereby
leukotriene
of leukotriene inhibition
that
did
under
hrs.
isolation
after
ability
similar
to respond with
shows
LPS
synthesis.
diminishes
their
culturing,
possible
conditions
significant
Indeed,
ability
after
LTC4.
LPS.
the macrophages
that
Our studies
alter
of up to 7 days
of
restores
not
12-24 to attain
to rapid
in
2hr
and Lonnemann
conditions
protein
LPS resulted
Our experience
does
with
with
(21)
between these
used
observed
the same cell.
et al
adherence
which
only
macrophages
in
required
rise
for were
an intracellular
that
we have
with
LTC4 formation.
activity
of macrophage
to form
shown
of IL-l
macrophages
and IL-2
longer
trauma
LPS gives
have
release
rested of
the
time
by de novo
the macrophages
time-dependent
period
for
the
In addition synergizes
study
to adhere from
occur
we see under
since
both
stimulation
these
levels
that
be accounted
stimulate
further
IL-l
LPS from
macrophages. ionophore
of IL-l
by Hazuda
that
only
(Re to Rc chemotypes)
In our
produced
production.
shown
induce
can be released reported
IL-l
that
levels not
have
from
do not
and the
LPS stimulated
calcium
LTC4.
which
are
release
possible
Fuhlbrigge
IL-l
that
preformed
cannot
step
those
of IL-l
release
IL-l
leukotrienes from
that
had been allowed
and peak
Therefore
where
LTC4 and IL-l whereas
and E.coli
to release
of E.coli
between conditions
for
of LTC4 release
strains
any leukotrienes,
of stimulus
(18 and 19)
demonstrated
macrophages
release
type
release
minnesota
amounts have
the
important
et al
macrophages
from wild-type Furthermore,
are
of Salmonella
and Cohn (20)
both
LTC4 and IL-l Luderitz
mutants
stimulated Aderem
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of IL-l
at doses
preceded
tested
in our
which
completely
427
do not
produce
procedure in some way resting them for a period
leukotrienes
production
synthesis
cells
when
stimulated.
IL-l
release,
study
caused
abrogated
none
any leukotriene
of
Vol.
169,
No.
production. production simultaneous Moreover, inhibitory production
but only
adhered for demonstrate
interesting
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
inhibition of LTC4 and IL-l was not demonstrated in their study. NDGA has redox properties, and therefore, may be exerting its effect
on IL-l
production
by other
of 5-lipoxygenase.
cellular
In our hands,
in LPS- or zymosan-stimulated
redox systems NDGA inhibits
macrophages
inhibition macrophages
rather IL-l
that
2hr (data not shown), conditions under which we failed leukotriene production. It is an enigma that we could
have to not
of IL-l production by NDGA in LPS- or zymosanthat had been allowed to adhere for 18 hr. It
to note that
inhibitor
AND
Kunkel and Chensue have reported that NDGA inhibited the of IL-l from zymosan-stimulated macrophages (8). However,
than by inhibition
demonstrate stimulated
BIOCHEMICAL
2, 1990
of lipoxygenase
in a previous
report
and cyclooxygenase,
(26),
SK&F 86002,
which
also
is
a dual
has redox properties,
suppressed the formation of IL-l, though the authors do not allude to the mechanism of action. More recently, it has been reported that leukotrienes may be involved in the regulation of TNF (27,28). Although our data do not support
a role
for leukotrienes in the regulation of IL-l this may not be the case for other cytokines
macrophages, respect, Kovacs et al (29) and TNF are differentially conclusion, regulation
our data suggest of IL-l
production
by activated such as TNF. In this
and Scales et al (30) have demonstrated regulated at the messenger RNA level. that
leukotrienes
are not involved
from LPS- or zymosan-activated
that In
IL-l
in the
macrophages.
REFERENCES
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