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Leukotriene B4 Enhances Interleukin-6 (IL-6) Production and IL-6 Messenger
RNA Accumulation in Human Monocytes In Vitro: Transcriptional and
Posttranscriptional Mechanisms
By Marek Rola-Pleszczynski and Jana Stahkova
Leukotriene B4(LTB4)is a potent lipid mediator of inflammation, and some of its bioactivities may involve inflammatory
cytokines. Moreover, it may participate in myelopoiesis,
either directly or via the induction of cytokines and growth
factors. When human monocytes were cultured in the presence of graded concentrations of LTB& significant stimulation of production of bioactive and immunoreactive interleukin-6 (IL-6)was observed. Nanomolar concentrations of LTB4
were optimal and the LTB, receptor antagonist LY 255283
could block its activity. The voxidation products of LTBG
20-OH-LTB4 and 20-COOH-LTBc were only 22% and 2%
effective, respectively. LT& was also effective in stimulating
IL-6 production, but only at micromolar concentrations,
whereas 5-HETE and 12-epi-LTB4were ineffective. The signaling induced by LTB4 did not seem to involve protein kinase C
or A, but rather a tyrosine kinase, as suggested by its
inhibition with genistein. LTB4 induced an accumulation of
IL-6 messenger RNA (mRNA) in treated monocytes with a
dose-response similar to that of IL-6 protein production.
Whereas IL-6 mRNA half-life in untreated cells was approximately 1 hour, it was extended t o 3 hours in LTB4-treated
monocytes. Moreover, nuclear transcription of IL-6 mRNA
was augmented at 30 minutes by a factor of 5 in LTB4-treated
cells. Pretreatment of cells with cyclohexamidebefore exposure to LTB4 superinduced IL-6 message expression, but
partially inhibited the effect of LTB4 on IL-6 mRhlA accumulation, suggesting that newly synthesized proteins may be
involved in the transcriptional activation of the IL-6 gene by
LTB4. These findings constitute a first demonstration that
LTB4 stimulates IL-6 production and that the underlying
mechanisms involve both increased IL-6 gene transcription
and message stabilization. This may constitute an important
mechanism through which rapidly produced mediators may
modulatethe subsequent production of regulatory or growthpromoting cytokines.
o 1992by The American Society of Hematology.
I
phages stimulated by platelet-activating factor.% LTB4, and
possibly other lipoxygenase products, have also been suggested to play a role in modulating myelopoiesis and
leukemic cell p r o l i f e r a t i ~ n . ~ ~ , ~
In the present report, we show that LTB4 enhances IL-6
production and messenger RNA (mRNA) accumulation in
human monocytes. Furthermore, we show that this effect
may be mediated, at least in part, by alteration in IL-6
mRNA stability and transcriptional regulation of IL-6 gene
expression.
NTERLEUKIN-6 (IL-6) is a multifunctional cytokine
produced by monocytestmacrophages, endothelial cells,
fibroblasts, keratinocytes, T cells, and some tumor
Its numerous synonyms reflect its various biologic activities
as B-cell stimulatory factor 2, interferon p2, hybridomaplasmacytoma growth factor, and hepatocyte-stimulating
IL-6 is an important regulator of T- and B-cell
functions, hematopoiesis, and acute-phase response^.^-*^
Infectious agents, endotoxin, and the inflammatory cytokines tumor necrosis factor c1 (TNFa) and IL-1 can induce
IL-6 production, while dysregulation of IL-6 expression is
associated with certain chronic inflammatory, autoimmune,
and hematopoietic di~orders.l-~.’~-’~
The findings that IL-6 production is associated with
inflammatory states suggest that its production may be
modulated by inflammatory lipid mediators. The S-lipoxygenase metabolite of arachidonic acid, leukotriene B4 (LTB4),
has been shown to exert numerous immunoregulatory and
proinflammatory a c t i v i t i e ~ . l ~In- ~monocytes,
~
LTB4 was
shown to stimulate cytotoxicity, TNFa production, and
generation of H202?4,25More recently, it was found to
mediate the enhanced TNF production by alveolar macro-
From the Immunology Division, Faculty of Medicine, University of
Sherbrooke, Sherbrooke, Quibec, Canada.
Submitted December 4,1991; accepted April 27, 1992.
Supported by grants from the Medical Research Council of Canada.
J.S. is a Research Scholar of the Fonds de la Recherche en Santi du
Quibec.
Address reprint requests to Marek Rola-Pleszczynski, MD, Head,
Immunology Division, Faculty of Medicine, University of Sherbrmke,
3001 N 12th Ave, Sherbrooke, Qdbec, JIH 5N4 Canada.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fdct.
0 1992 by The American Society of Hematologv.
0006-4971I92 /8004-0005$3.00/0
1004
MATERIALS AND METHODS
Reagents. LTB4, &, 5-HETE, and 12-epi-LTB4were a generous gift from Dr A. Ford-Hutchinson (Merck-Frosst, PointeClaire, Canada). 20-OH-LTB4 and 20-COOH-LTB4 were a gracious gift from Dr P. Poubelle (Ste-Foy, QuBbec, Canada). LY255283 was a generous gift from Dr D.K. Herron (Lilly, Indianapolis, IN). Polyvalent rabbit antihuman IL-6 antibody (Cedarlane,
Hornby, Ontario, Canada) had a specific activity of 10,000 IL-6
neutralizing units per milligram of IgG. It had no crossreactivity
with IL-la, IL-lp, TNFa, or granulocyte-macrophage colonystimulating factor (GM-CSF). Human cDNA probe for IL-6 was a
gracious gift from Dr Lucien Aarden (Amsterdam, The Netherlands) and the cDNA probe for glyceraldehyde-3-phosphatedehydrogenase (GAF’DH) was from American Type Culture Collection
(Bethesda, MD). Muramyl dipeptide (MDP) was obtained from
Behring Diagnostics (La Jolla, CA). Cyclohexamide (CHX), H7,
Staurosporin, catalase, superoxide dismutase (SOD), a-tocopherol, and thiourea were from Sigma (St Louis, MO); HA1004 was
from Seikagaku (Tokyo, Japan); and Genistein was from Biomol
Research Lab (Plymouth Meeting, PA).
Preparation of monocytes. Human venous blood from healthy
medication-free volunteers was collected on citrate/dextrose/
adenine. Cell preparation was performed at room temperature.
The blood was diluted 111with 0.9% NaCl solution, layered over a
Ficoll-Paque (Pharmacia, Uppsala, Sweden) cushion, and centrifuged at 7OOg for 20 minutes according to the method of B o y ~ m . ~ ~
The peripheral blood mononuclear leukocytes (PBML) were
collected at the interface and were washed twice in Ca2+/Mg2+free Dulbecco’s phosphate-buffered saline (PBS) and then susBlood, VOI 80,NO4 (August 15), 1992: pp 1004-1011
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1005
L T B ~STIMULATES IL-6 GENE EXPRESSION
pended in RPMI 1640 medium with 10% heat-inactivated fetal
bovine serum (FBS). Monocytes were then purified by adherence
to the surface of plastic petri dishes previously coated with baby
hamster kidney cell microexsudate. This was effective in enriching
the adherent population to greater than 90% monocytes as
assessed by nonspecific esterase staining. This population was
removed with EDTA (0.01 mol/L) in RPMI 1640 medium and
resuspended in RPMI 1640 medium, 5% FBS,at a final concentration of 1 x 106 cells/mL. This concentration proved to be optimal
in our assays. All reagents were endotoxin-free as assessed by the
Limulus amebocyte lysate test (M.A. Bioproducts). Because LTB4
was dissolved in ethanol before dilution in culture medium, control
cultures contained ethanol in equivalent maximal concentration,
which never exceeded 0.08% vol/vol and had no effect in the assays
used in this study.
B9 hybridoma proliferation assay for ZL-6. Bioactivity of IL-6
was assayed using the IL-&sensitive B9 hybridoma cell line (gift
from Dr J. Gauldie, Hamilton, Ontario, Canada) according to
Aarden et
Briefly, cell-free supernatants from monocyte
cultures were added in serial twofold dilution aliquots to cultures
of B9 cells (5 x 104 cells/mL in 100 pL volume) and incubated at
37°C for 72 hours. Proliferation of B9 cells was measured by a
colorimetric assay using M I T (Sigma). Plates were read at 590 nm
on a BioRad Microplate Reader model 3550 (BioRad, Richmond,
CA) and IL-6 contents were determined in comparison to a
standard recombinant human IL-6 (rhIL-6) dilution curve using
probit analysis. Bioactivity of IL-6 could be neutralized by antihuman IL-6 antibody.
ZL-6 enzyme immunoassay. Immunoreactive human IL-6 was
measured using the IL-6 enzyme-linked immunosorbent assay
(ELISA) commerciallyavailable from R & D Systems (Minneapolis, MN).
mRNA studies. After appropriate treatment, cells were pelleted in 15-mL polypropylene tubes (Falcon, Becton Dickinson
Labware, Lincon Park, NJ) and total cellular RNA was isolated by
acid guanidium thiocyanate-phenol-chloroformextraction according to Chomczynski and S a ~ c h iRNA
. ~ ~ was separated by electrophoresis on 1% agarose and transferred onto a Hybond-N (Amersham, Arlington Heights, IL) membrane for Northern analysis.
IL-6 cDNA probe that comprised the Taq-Xba fragment of the IL-6
coding region32was labeled with multiprime DNA labeling system
(Amersham) using ( c ~ P dCTP
~ ~ ) (specific activity, > 3,000 Ci/
mmol/L; Amersham).
Membranes were prehybridized for 4 hours in a mixture containing Tris 120 mmol/L, NaCl600 mmol/L, EDTA 8 mmol/L, NaPP
0.1%, sodium dodecyl sulfate (SDS) 0.2%, and heparin 100
pg/mL; hybridization was performed overnight at 68°C in the same
mixture in which the concentration of heparin was increased to 625
pg/mL and dextran sulfate at 10% was added.33The membranes
were then washed once at room temperature for 20 minutes in 2x
SSC (1X SSC: 0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH
7); once with 0.1 x SSC, 0.1%SDS at 68°C for 60 minutes; and then
rinsed at room temperature with 0.1 x SSC. The membranes were
exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY)
with intensifying screens for 24 hours at -70°C. Signal intensity
was quantitated by densitometry with Pharmacia LKB Ultrascan
XL (Pharmacia, Canada). The membranes were then stripped of
the probe by washing for 60 minutes with 0 . 0 2 ~SSC, 0.2% SDS at
90°C and rehybridized with 1-kbPst I cDNA probe (ATCC) for the
control “housekeeping” gene GAPDH. Densitometric values are
expressed as ratios of IL-6/GAPDH densitometry quantification.
In selected experiments, to assess stability of IL-6 mRNA, cells
were treated with Actinomycin D (10 kg/mL) 1 hour after
stimulation with LTB4 or medium to arrest transcription and the
cultures were stopped at various intervals (0 to 3 hours) thereafter.
Northern analysis was performed as indicated above. In additional
experiments, to assess the involvement of protein synthesis in the
transcriptional or posttranscriptional regulation of IL-6 gene
expression by LTBd, the protein synthesis inhibitor, CHX was used
at 10 kg/mL.
In vitro nuclear transcription. The in vitro nuclear transcription
assays were performed according to Greenberg and
with
several modifications. Briefly, nuclei were prepared from human
monocytesby resuspending cells in a lysis buffer with 0.1% Nonidet
P-40 (NP-40), followed by sedimentation through a 30% sucrose
solution. The nuclei were then either frozen in liquid hydrogen in a
50% glycerol solution or used immediately. In vitro transcription
was performed in the presence of [ ( U - ~ ~UTP
P ] (> 600 Ci/mmol).
The reaction was stopped by the addition of tRNA, DNase, and
proteinase K. The radiolabeled RNA was isolated by chloroform/
phenol extraction and trichlorocetic acidhodium pyrophosphate
precipitation. The labeled nuclear RNA was then hybridized to
linearized cDNA probes that were immobilized on Genescreen
plus membranes with a slot blot apparatus. The membranes were
prehybridized in the presence of formamide and salmon sperm
DNA for 2 days. The membranes were then hybridized for 4 days,
washed, and exposed to X-ray film for 2 weeks.
Statistical analysis. Data were analyzed for statistical significance using the Student’s t-test for paired values (for single
comparisons) or analysis of variance with post-hoc testing (for
multiple comparisons). Differences were considered significant at
P < .05.
RESULTS
Enhanced production of IL-6 by LTBcstimulated monocytes. When human monocytes were cultured in the presence of graded concentrations of LTB4, a significant enhancement of IL-6 production was observed (Fig 1). In the
absence of any other stimulus, such as lipopolysaccharide or
MDP, IL-6 production was augmented by 3- to 10-fold,with
- 25
5000
E
4000
-20
\
(D
Y
z
.->
Q)
-15
2000
-10 0
c,
0
2!
‘
C
0
0
0
=!
.-Q)>
3000
(D
d
a
Y
u)
c
C
c.
E
c
1000
0
c
-5
i”
-E
0
C
14
12
10
8
6
-loglO[LTB4], M
Fig 1. Effect of LTB, on IL-6 production by human monocytes.
Monocytes were cultured in the absence (vehicle control:C) or presence of graded concentrations of LTB, for 24 hours. Cell-free supernatants were then analyzed for IL-6 bioactivity ( 0 )using the B9 cell
proliferation assay and immunoreactivity (W) using the ELISA assay
(see Materials and Methods). Data represent mean f SE of six
separate experiments. Statistically significant effects were noted for
LTB, concentrations of 10-lo, lo-*,and 10-6 mol/L (P e .05).
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1006
ROLA-PLESZCZYNSKI AND STANKOVA
a peak effect at
mol/L LTB4. Both bioactive and
immunoreactive IL-6, measured by the B9 cell assay and
ELISA, respectively, were affected in parallel fashion. Prior
addition of the LTB4 receptor antagonist LY-255283blocked
the effect of LTB4 on IL-6 production, suggesting a membrane receptor-mediated event (Fig 2). Kinetics studies
showed enhanced IL-6 production by 6 hours, with optimal
response at 24 hours (Fig 3).
To assess the selectivity of the LTB4-induced IL-6 response, we studied the dose-response effects of several
other related eicosanoids (Fig 4). Compared with LTB4, its
w-oxidation metabolite 20-OH-LTB4was 5 times less effective at
mol/L, whereas 20-COOH-LTB4 was essentially ineffective. In contrast, LTA, induced a significant
IL-6 response, but only at the highest
mol/L) concentration. 5-HETE or 12-epi-LTB4,a nonenzymatic hydrolysis product of LT&, failed to elicit any IL-6 response (data
not shown).
Signal transductionpathways ofL TB4-inducedIL-6production. To assess the potential involvement of protein phosphorylation activities during LTB4-induced IL-6 production, we pretreated monocytes with the protein kinase C
and A inhibitors staurosporine, H7, and HA 1004, as well as
the tyrosine kinase inhibitor genistein, before stimulation
with LTB4. Only genistein partially inhibited the LTB4induced IL-6 response (Fig S), suggesting the involvement
of tyrosine phosphorylation events during this process.
On the other hand, LTB4 is known to induce reactive
oxygen intermediates, such as H202.24To assess their
1 o4
**
3
103
102
1
P
101
I
c
12
10
8
6
-loglO[LTB4], M
Fig 3. Kinetics of IL-6 production by monocytes stimulated with
LTB,. Monocytes were cultured in the absence (C) or presence of
graded concentrations of LTB, for indicated periods. Cell-free supernatants were then collected and bioactive IL-6 contents measured in the
B9 proliferation assay. Data represent mean f SE of three experiments. **P e .01; *P e .05. (0)One hour; (e)6 hours; (m) 24 hours.
potential role in activation of IL-6 production, we pretreated monocytes with the antioxidants a-tocopherol, thiourea, catalase, and superoxide dismutase. None of these
agents had any effect on LTB4-induced IL-6 response (Fig
T
.
,
C
.
,
14
.
,
12
.
,
-
10
.
- .
8
6
01
Fig 2. Effect of LTB, receptor antagonist LY-255283 on LTB,induced IL-6 production by monocytes. Monocytes were pretreated (5
minutes) with 10 or 100 pmol/L LY-255283 and then cultured in the
absence (C) or presenceof graded concentrations of LTB,for 24 hours.
Cell-free supernatants were then collected and bioactive IL-6 contents measured in the B9 proliferation assay. Data represent mean f
SE of three experiments. (m) LTB,; ( 0 ) LTB,
LY-255283 (10
pmol/L); (A)LTB, + LY-255283 (100 pmol/L).
+
,
I
C
14
I
I
12
I
I
10
I
I
8
I
I
6
Fig 4. Comparative effect of (0)
LTA, (A)20-OH-LTBe and (0)
20-COOH-LTB4 on IL-6 production. Monocytes were cultured in the
absence (C) or presence of graded concentrations of indicated eicosanoids for 24 hours. Cell-free supernatants were then analyzed for
immunoreactive IL-6 by ELISA. Data represent mean f SEM of three
experiments. (U)LTB,.
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1007
LTB, STIMULATES IL-6GENE EXPRESSION
A
COtIrrOl
10
8
6
Fig 5. Effect of kinase inhibitors (A) and antioxidants (B) on LTB,-induced IL-6 production. Monocytes were pretreated (30 minutes) with
kinase inhibitors (A) or antioxidants (B) and then cultured in the absence ( C )or presence of graded concentrations of LTB, for 24 hours. Cell-free
supernatants were then assayed for immunoreactive IL-6 by ELISA. Data represent mean f SEM of three experiments.
Enhanced accumulation of IL-6 mRNA in LTBJ-stimuluted monocytes. Adherence-purified but otherwise unstimulated human monocytes that had been “rested” overnight
contained low amounts of IL-6 mRNA (Fig 6). Exposure of
the cells to graded concentrations of LTB4 induced the
appearance of 1.3-kb IL-6 mRNA transcripts. Maximal
effect, which represented a greater than 10-fold increase in
IL-6 mRNA, was induced by
mol/L LTB4 (Fig 6), but
enhanced IL-6 mRNA accumulation was still evident at
lo-“’ mol/L LTB4 in cells exposed to the mediator for 1
hour. Time-course studies showed peak mRNA accumulation at 1 hour, with a gradual decrease thereafter (Fig 7).
Augmented IL-6 mRNA stability in LTBJ-stimulated monocytes. IL-6 mRNA stability was assessed by treating medium- or LTB4-stimulatedmonocyte cultures with actinomycin D (10 pg/mL) at 1 hour, stopping the reaction at
indicated time intervals thereafter (0 to 3 hours), and
performing Northern blot analysis. While the IL-6 mRNA
half-life appeared to be less than 1 hour in medium-treated
monocytes, it was extended to 3 hours in LTB4-treated cells
(Fig S), suggesting that LTB4 markedly reduced the rate of
decay of IL-6 mRNA. In contrast, GAPDH mRNA remained stable in both unstimulated and LTB4-stimulated
monocytes.
Augmented IL-6 nuclear transcription in LTB,stimulated
monocytes. Nuclear run-on analysis was used to determine whether enhanced IL-6 production and mRNA accumulation in LTB4-stimulated monocytes was mediated at
the level of IL-6 gene transcription. Nuclear transcription
studies showed a fivefold increase in IL-6 gene transcription at 30 minutes after stimulation with LTB4, with return
close to baseline levels by 2 hours (Fig 9). Although
LTB4(M)
0
lo4 1 6 ”
-28s
+18s
+28S
Fig 6. Northern analysis of IL-6 mRNA accumulation in monocytes
stimulated with graded concentrations of LTB,. Total RNA (10 pg)
isolated from human monocyte cultures was separated by electrophoresis, transferred, and hybridized with a cDNA probe for human
IL-6 (top panel). The membrane was then stripped and rehybridized
with a cDNA probe for GAPDH (lower panel).
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ROLA-PLESZCZYNSKI AND STANKOVA
1008
0
5
K
4
>
a
.-
t-w
0
I
g
L
2
2
W
z
3
c
0
I
E
n
0
4
.-
0
L
(3
-
4
-
0
5
2
0 min
'c)
I
n
P
a
$
B
1
psw2
=!
-
GAPDH-
0
Time, h
1
2
LTB4lOnM
- +
- +
IL-6
- +
augmented in the presence of LTB4, the magnitude of IL-6
gene transcription was approximately half the magnitude of
IL-6 mRNA accumulation or protein synthesis.
Involvement of protein Tnthesis in activation of IL-6 gene
expression. To determine whether de novo protein synthesis was required for LTB4-induccd activation of IL-6, we
treated monocytes with the protein synthesis inhibitor CHX
either 30 minutes before (-30 minutes) or at the time (-3
A
Time (hours)
120 min
c
Smin
30min
120min
-0
Fig 9. Nuclear run-on analysis of IL-6 gene transcription in monocytes stimulated with medium or LTB,. Nuclei were isolated from
monocytes incubated for 0, 5, 30, or 120 minutes with LTE, (lo-'
moi/L). Run-on transcription assays were performed as described in
Materials and Methods. (A) Ratios of the rates of transcription of
IL-6/GAPDH genes are illustrated. Nonspecific binding of PzP]RNAt o
negative control plasmid pSP72 DNA (without a cDNA insert) was
undetectable. Data represent mean of two t o four experiments. (E)
Corresponding autoradiogram.
minutes) of stimulation with LTB4
and lo-'" mol/L).
Both pretreatment and cotreatment with CHX rcsulted in
superinduction of IL-6 mRNA accumulation, suggesting a
negative control of IL-6 mRNA levels by short-lived regulatory proteins (Fig 10). Furthermore, the loss (-30 minutes)
or blunting (-3 minutes) of the LTB4
mol/L effect
suggests that the stimulation of IL-6 mRNA transcription
I M
3
Omin
__t
-
TimeafterActDe)
2
30 min
TIME
3
Fig 7. Kinetics of IL-6 mRNA accumulation. Northern analysis of
11-6 mRNA accumulation in monocytes stimulated for varying periods
mol/L). Northern blot using a cDNA probe for
of time with LTB, (10-n
human IL-6 is shown in the top panel and densitometry ratios of
ILdlGAPDH blots in the bar graph.
0
5 min
control
LTBd
0 1 2 3
0 1 2 3
GAPDHB
Fig 8. Analysis of IL-6 mRNA stability in monocytes treated with ( 0 )medium or (m) LTB, (10-8mol/L). Monocytes were incubated for 1 hour
with medium or LTB, and the levels of 11-6 mRNA were determined before (time 0) and 1,2,and 3 hours after the addition of actinomycin D (10
pglmL). (A) Densitometric analysis of the levels of IL-6 mRNA after actinomycin D treatment expressed as a percentage of the levels in monocytes
at time 0. (E) Corresponding Northern blot autoradiogram.
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LTBd STIMULATES IL-6 GENE EXPRESSION
1009
IL6
CHX
ni I
LTB4
EE %
O
-
0 0
ow
l-
-30min
-3min
EE Ea
O
E PE,
g8
ow-
0
0 0
-
w
Fig 10. Effect of CHX on 1L-8 mRNA accumulation.
Monocytes were either untreated (nil) or pretreated
with CHX (10 pg/mL) for 30 or 3 minutes before
stimulation with LTB, (10-6 and 10-10 mol/L) for 1
hour. Total RNA was then isolated, separated by
electrophoresis, transferred, and hybridized with a
cDNA probe for human IL-6. The membrane was then
stripped and rehybridizedwith a cDNA for GAPDH.
by LTB4 above background levels may be dependent on
newly synthesized proteins.
DISCUSSION
Leukotrienes are produced by cells of myeloid origin and
possess numerous biologic activities.3s In contrast to the
peptido-leukotrienes LTC4, LTD4, and LTE4, which exert
potent vascular and smooth muscle activities, LTB4 is a
powerful leukotropic, proinflammatory, and immunoregulatory mediator.’RJy Cells that produce LTB4 or become
targets of its actions can also produce other proinflammatory molecules such as free oxygen radicals, prostaglandins,
and various cytokines. To further understand the potential
interactions between lipidic and peptidic mediators of
inflammation and immune regulation, we undertook to
study the regulatory properties of LTB4on IL-6 production
by human monocytes.
Our studies are the first demonstration that LTB4 stimulates monocyte IL-6 production and the first demonstration
that this effect is associated with increased IL-6 mRNA
stability and augmented IL-6 gene transcription. The process is mediated by the LTB4receptor, is stereospecific, and
involves tyrosine kinase activity. Our observations that high
concentrations of LT& can also stimulate IL-6 production
suggest that endogenous metabolites of 5-lipoxygenase may
also be involved.
The relatively short half-life of the mRNA of many
proto-oncogenes, cytokines, and membrane receptors may
be, at least in part, dependent on the presence of AT-rich 3’
untranslated sequences in their gene^.^^.^' The IL-6 mRNA
has a half-life of approximately 1 hour in fibroblasts3xand
monocytes,3ythe latter being confirmed in our studies. As
shown by Hirano et al,12 the 3‘ end of the IL-6 gene has also
two AT-rich motifs.
The cellular processes that regulate mRNA stability are
still poorly understood. While our studies clearly show that
LTB4prolongs IL-6 mRNA half-life, it is unclear whether it
acts by decreasing monocyte ribonuclease production, by
augmenting ribonuclease degradation, by inducing a ribonuclcase inhibitor, or by altering the sensitivity of IL-6 mRNA
to degradation.
The latter mechanism could be associated with transcription of the proximal, rather than the distal, poly(A) addition sequence of the IL-6 gene,” leading to elimination of
the AT-rich segment. Northern blot analysis could not
easily detect such a shift, but stability of the IL-6 mRNA
transcripts would be increased. Our findings that LTB4
affects stability of IL-6 mRNA could be readily explained by
this mechanism, but specific studies will be needed to test
this hypothesis. Elias and
suggested that their
observation of synergistic stimulation of fibroblast IL-6
production by IL-la and TNFa and the associated mRNA
stabilization could be due to such a mechanism involving
different IL-6 mRNA species.
On the other hand, superinduction of IL-6 mRNA
accumulation by treatment with CHX suggest that IL-6
mRNA levels may be under the control of short-lived
regulatory proteins.
Our present report shows that a lipid mediator, LTB4,
can regulate the production of a cytokine, IL-6, by augmenting thc rate of transcription of its gene and by enhancing
the stability of its mRNA. This may constitute an important
mechanism through which rapidly produced mediators may
regulate the subsequent production of inflammatory or
immunoregulatory cytokines. In as much as LTB4 may be an
“endogenous” Ca2+ionophore, as first suggested by k h a n
et a1,4” its endogenous production in mononuclear leukocytes after certain stimuliZhmay allow it to act as an internal
“second messenger” for regulation of cytokine production
at the transcriptional and posttranscriptional level.
ACKNOWLEDGMENT
The authors thank Suzanne BCdard, Diane Bolduc, Louise
Bouvrette, CCline Fouquet, Denis Gingras, and Sylvie Turcotte for
technical assistance, and Carole Jacques for preparation of the
manuscript.
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ROLA-PLESZCZYNSKI AND STANKOVA
1010
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1992 80: 1004-1011
Leukotriene B4 enhances interleukin-6 (IL-6) production and IL-6
messenger RNA accumulation in human monocytes in vitro:
transcriptional and posttranscriptional mechanisms
M Rola-Pleszczynski and J Stankova
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