(Journal of Leukocyte Biology. 2001;70:439-446.)
© 2001
by Society for Leukocyte Biology
Interleukin-12 increases interleukin 8 production and release by human polymorphonuclear neutrophils
Frédéric Ethuin*,
,
Charlotte Delarche*,
Sylvie Benslama*,
Marie-Anne Gougerot-Pocidalo*,
Laurent Jacob and
Sylvie Chollet-Martin*
* Laboratoire d’Immunologie and Unité INSERM 479, Hôpital Bichat, and
Département d’Anesthésie-Réanimation, Hôpital Saint-Louis, Paris, France
Correspondence: Dr. Sylvie Chollet-Martin, Laboratoire d’Immunologie et Unité INSERM 479, Hôpital Bichat, 46 rue Henri Huchard, 75018 Paris, France. E-mail: sylvie.martin{at}bch.ap-hop-paris.fr
 |
ABSTRACT
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Interleukin (IL) 12 is a heterodimeric cytokine mainly produced by
phagocytes—important target cells for IL-12 in particular with a
chemotactic effect—and antigen-presenting cells in response to various
microorganisms. Because IL-8 is a strong chemokine for
polymorphonuclear neutrophils (PMNs), we investigated the effect of
IL-12 on PMN IL-8 production. IL-12 alone had no significant effect,
but with lipopolysaccharide (LPS) it was additive at both protein and
mRNA levels. Actinomycin D at the beginning of culture inhibited IL-8
mRNA induction, whereas late addition affected IL-8 transcript
stability, suggesting gene transcription involvement. Results with
parthenolide and tyrphostin AG490 suggest that nuclear factor-
B and
signal transducer and activator of transcription 4 play a role. The
IL-12 additive effect was restricted to IL-8 release, with no action on
cell-associated IL-8. IL-12 additive effects occurred after 18 h
of culture, with no marked up-regulation of IL-12 receptor expression,
and were blocked by actinomycin D added after 16 h of culture.
Tumor necrosis factor (TNF) 
and interferon (IFN) 
had
intermediate roles; their specific inhibition reduced IL-12’s effect.
IL-12’s chemotactic mechanism seemed mediated by overproduction and
release of IL-8 by human PMNs in the presence of LPS, an effect
involving TNF- and IFN- secretion. These results point to a new
role for IL-12 in inflammation, through an autocrine amplification
loop.
Key Words: phagocyte • chemokine • mRNA • inflammation
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INTRODUCTION
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Interleukin (IL) 12 is a 70-kDa, heterodimeric cytokine composed
of two disulfide-bound subunits named p35 and p40. It is mainly
produced by phagocytes such as monocytes, macrophages,
polymorphonuclear neutrophils (PMNs), and antigen-presenting cells,
particularly in response to bacteria and intracellular parasites. The
main target cells of IL-12 are natural killer (NK) cells and T
lymphocytes, with resulting interferon (IFN) production,
proliferation, and cytotoxicity. IL-12 also enhances phagocyte
functions and is thus considered a bridge between innate and adaptive
immunity against infection [1
]. Recent in vitro studies
have shown that both forms of the heterodimer (IL-12 p40 and IL-12 p70)
can attract macrophage [2
]. Allavena et al. found that
IL-12 was directly chemotactic for human PMNs in vitro
[3
], whereas others have reported an indirect effect
mediated by platelet-activating-factor synthesis [4
].
These effects are mediated by IL-12 binding to specific receptors at
the PMN surface, as shown by flow cytometry, immunoprecipitation, and
reverse-transcriptase (RT)-PCR analysis [3
4
5
]. In vivo,
IL-12 injection promotes chemokine production and subsequent PMN
accumulation in tissues [6
, 7
].
PMNs play an important role in host defense against microorganisms. In
addition to their phagocytic and killing properties, PMNs can
synthesize numerous cytokines, including IL-8, which has a key role in
recruiting circulating PMNs to inflammatory sites [8
].
There is also a cell-associated form of IL-8 in PMNs, which can be
rapidly released. IL-8 production by PMNs can be triggered by various
stimuli, such as lipopolysaccharide (LPS) alone or LPS in combination
with proinflammatory cytokines [tumor necrosis factor (TNF) ,
IFN-, granulocyte macrophage colony stimulating factor (GM-CSF),
IL-1ß, etc.] [8
, 9
]. However, the effect
of IL-12 on IL-8 production by PMNs is unknown.
We postulated that IL-12 might play a role in IL-8 production and
release by PMNs. Our results confirmed a synergistic effect of IL-12 on
LPS-induced IL-8 production, at both the mRNA and protein levels.
Moreover, TNF- and IFN- seemed to play an intermediate role in
this effect, demonstrating a new autocrine amplification loop for
cytokine production and release in human PMNs.
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MATERIALS AND METHODS
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Purification of human PMNs
Venous blood was obtained from healthy volunteers, and PMNs were
purified as previously described [10
]. Briefly,
leukocytes were isolated in endotoxin-free conditions by sedimentation
on a separating medium containing 9% Dextran T-500® (Pharmacia,
Uppsala, Sweden) and 38% Radioselectan® (Schering, Lys-lez-Lannoy,
France). After red blood cell sedimentation, the leukocyte-rich
suspension was centrifuged on a Ficoll-Paque® density gradient
(Sigma, St. Louis, MO). Contaminating erythrocytes were removed by
hypotonic lysis. To further purify PMNs, contaminating monocytes and
lymphocytes were removed by 30 min of incubation with pan-antihuman
human leukocyte antigen class II-coated magnetic beads (Dynabeads
M-450; Dynal A.S, Oslo, Norway). Pure PMNs were then resuspended in
RPMI 1640 culture medium (BioWhittaker, Verviers, Belgium) supplemented
with 10% heat-inactivated fetal calf serum (BioWhittacker, Gagny,
France), L-Glutamine (2 mmol/mL), penicillin (100 IU/mL),
and streptomycin (100 µg/mL).
Cell culture
Purified PMNs (2 x106/mL) were cultured for up
to 24 h at 37°C with 5% CO2 in the presence of
various concentrations of recombinant human (rh) IL-12 (R&D Systems,
Abingdon-Oxon, United Kingdom) alone or combined with 100 ng/mL of LPS
derived from Escherichia coli (055:B5; Sigma). The effect of
IL-12 was compared with that of other stimulating agents, including
rhTNF- at 10 ng/mL (R&D Systems), rhIFN- at 250 IU/mL (R&D
Systems), and rhGM-CSF at 5 ng/mL (kindly provided by Schering Plough)
in the presence or absence of LPS (100 ng/mL).
The inhibitory effects of 1 to 30 ng/mL of rhIL-10 (R&D Systems) and
10-10 to 10-4 M dexamethasone (DEX; Sigma)
were studied by adding them for 30 min at 37°C before stimulation
with LPS (100 ng/mL), with or without IL-12 (10 ng/mL). To elucidate
the mechanism of IL-8 gene transcription in the presence of IL-12, we
examined the activation of two transcription factors, nuclear factor
(NF)-B and signal transducer and activator of transcription 4
(STAT4). Freshly purified PMNs were preincubated for 1 h with a
specific NF-B inhibitor, parthenolide, at 5, 10, and 20 µM
(Sigma); tyrphostin AG490 (Sigma) was used at 50 µM to block Janus
kinase (JAK)-2 stimulation and, thus, the STAT4 pathway. Cells were
then cultured with LPS alone (100 ng/mL) or in combination with IL-12
(10 ng/mL).
In some experiments, PMNs were preincubated with 10 ng/mL of
cycloheximide (CHX; Sigma) for 30 min at 37°C and then further
incubated with LPS (100 ng/mL) in the presence or absence of IL-12 (10
ng/mL) for up to 24 h at 37°C.
Mediation of the effect of IL-12 by the synthesis of other cytokines
was tested by measuring TNF-, IFN-, and GM-CSF and by adding the
following specific inhibitors to the culture medium: human soluble
TNF- receptor II at 0.1 µg/mL, anti-human IFN- antibody (0.125
µg/mL), and anti-human GM-CSF antibody (0.25 µg/mL) (R&D Systems).
Moreover, a time-course study was done in the presence of actinomycin D
(5 µg/mL) added after 16 h of culture (the additive effect of
IL-12 was observed after 18 h of culture).
At the end of the culture periods, cell-free supernatants were
harvested, and the cell pellets were sonicated for 30 s on ice to
measure cell-associated IL-8. Both supernatants and cell pellets were
stored at -70°C until IL-8 assay.
Cytokine assays
IL-8, TNF-, IFN-, and GM-CSF were quantified by using
enzyme-linked immunosorbent assays (R&D Systems and Immunotech Beckman
Coulter) after the manufacturer’s instructions; the detection limit of
the assays was 10 pg/mL.
Quantification of IL-8 mRNA
For RNA analysis, 7 x 107 highly purified PMNs
were incubated for 1 h in standard culture medium with or without
LPS (100 ng/mL) and/or IL-12 (10 ng/mL). In some experiments, PMNs were
preincubated for 15 min with 5 µg/mL of actinomycin D (Sigma) to
block transcription. In other selected experiments, actinomycin D was
added after the 1-h stimulation period with LPS and/or IL-12, for a
further 30, 60, or 90 min to study the stability of IL-8 transcripts.
Total cellular RNA was isolated from PMNs with RNA-B® (Bioprobe
systems, Montreuil-sous-Bois, France) according to the manufacturer’s
instructions; briefly, cells were lysed in guanidium thiocyanate, and
RNA was extracted with chloroform and then precipitated with
isopropanol and washed with 75% ethanol. The final preparation was
redissolved in water, and the RNA concentration was determined
spectrophotometrically at 260 nm. Twenty micrograms of total RNA were
analyzed by electrophoresis on 1% agarose-formaldehyde gel to check
RNA purity and integrity. mRNA species specific for IL-8 and
glyceraldehyde phosphate dehydrogenase (GAPDH) were then quantified in
each sample using the Quantikine® mRNA kit (R&D Systems, Minneapolis,
MN), according to manufacturer’s instructions. Briefly, 0.1 µg of
each RNA sample was hybridized with gene-specific biotin-labeled
capture oligonucleotide probes (IL-8 or GAPDH) and digoxigenin-labeled
detection probes in a microplate, in a 65°C water bath for 60 min.
The hybridization solutions were then transferred to a
streptavidin-coated microplate, and the RNA/probe hybrids were captured
at room temperature for 60 min. After a wash to remove unbound
material, an antidigoxigenin alkaline phosphatase conjugate was added
for 60 min. After washing steps, a substrate solution and then an
amplifier solution were added, and color was developed in proportion to
the amount of IL-8 or GAPDH mRNA. Color development was stopped, and
the intensity was measured at 490 nm with wavelength correction at 650
nm. Results were expressed in picograms per milliliter of IL-8 or GAPDH
mRNA per microgram of total RNA. The detection limit was 2.6 pg/mL of
IL-8 and 1.9 pg/mL of GAPDH mRNA.
Time course study of IL-12 receptor expression at the PMN surface
Highly purified PMNs (2 x106/mL) were cultured
for up to 24 h in the presence of LPS (100 ng/mL) alone or
combined with IL-12 (10 ng/mL). At time zero (before culture
initiation), and after 4, 18, and 24 h of culture, PMNs were
washed in phosphate-buffered saline (PBS) supplemented with 0.5% human
serum albumin (HSA) (LFB, Courtaboeuf, France), then resuspended in
PBS/HSA and Fc-blocked by treatment with purified human immunoglobulin
(Ig) G (1 µg/105 PMNs, Tegeline®; LFB) for 15 min at
room temperature. Samples were then incubated on ice with
fluorescein-conjugated antihuman IL-12Rß1 monoclonal antibody (clone
69310.111; R&D) for 45 min. After one wash with ice-cold PBS/HSA, PMNs
were resuspended in 1% paraformaldehyde-PBS and kept on ice until flow
cytometry. Nonspecific binding was determined on cells incubated with
the same concentration of an irrelevant fluorescein-labeled IgG1
antibody (R&D). Flow cytometry was done using a Becton Dickinson
FACScan (Immunocytometry Systems, San Jose, CA) with a 15-mV, 488-nm
argon laser. Ten thousand events were counted per sample, and the
fluorescence pulses were amplified by 4-decade logarithmic amplifiers.
All the results were obtained with a constant photomultiplier gain. The
data were analyzed with LYSIS II software, and the mean fluorescence
intensity was used to quantify the responses.
Statistical analysis
Results are presented as means plus or minus SE. The
significance of differences was determined by using Wilcoxon’s paired
test. P values of <0.05 were considered significant.
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RESULTS
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IL-12 increases LPS-induced IL-8 production and release by PMNs
The ability of IL-12, alone or combined with LPS, to stimulate
IL-8 production by PMNs was tested over the concentration range of 0.5
to 100 ng/mL. As shown in Figure 1
, IL-12 alone had no significant effect on IL-8 release by PMNs
cultured for 24 h, whatever the concentration used. By contrast,
IL-12 significantly enhanced the capacity of LPS (100 ng/mL) to
stimulate PMN production of IL-8, in a concentration-dependent fashion,
reaching a plateau after 50 ng/mL. At this concentration of IL-12, the
percentage increase was 175 ± 71 relative to LPS alone. Because
no further significant stimulation was observed with 50 or 100 ng/mL of
IL-12 compared with 10 ng/mL, the latter concentration was used in
subsequent experiments.
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Figure 1. Concentration-response effect of IL-12 on IL-8 production by PMNs. PMNs
(2 x106/mL) were incubated in medium alone.
Cell stimulation was performed with LPS alone (100 ng/mL) and also with
increasing concentrations of IL-12, either alone or in the presence of
LPS. After 24 h, IL-8 was determined in the cell-free supernatants
by enzyme-linked immunosorbent assay. Results are expressed as the
means ± SE of five independent experiments. *,
P < 0.05 as compared with cells incubated with LPS
alone.
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IL-8 up-regulation by LPS plus IL-12 was observed at both the protein
and mRNA levels. As shown in Table 1A
, IL-8 mRNA was detectable after 1 h in both control PMNs and
PMNs incubated with IL-12 alone. IL-8 mRNA accumulation induced by LPS
was further enhanced by IL-12. Addition of the transcription inhibitor
actinomycin D at the beginning of the culture period strongly reduced
IL-8 mRNA accumulation induced by LPS plus IL-12. GAPDH mRNA levels
were not modified in any of the culture conditions (Table 1A)
. After
stimulation with LPS plus IL-12 for 1 h (corresponding to the
steady-state mRNA peak in Table 1A
), actinomycin D was added for 30,
60, or 90 min to assess IL-8 transcript stability. In these conditions,
IL-8 mRNA levels decreased rapidly (Table 1B
). Taken together, these data suggest that the induction of
this gene by LPS plus IL-12 might take place, at least in part, at the
transcriptional level.
Comparative stimulation of IL-8 production and release by IL-12 and
other proinflammatory cytokines
The capacity of IL-12 to enhance LPS-induced IL-8 production was
compared with that of other cytokines by incubating PMNs with 100 ng/mL
of LPS plus IFN- (250 IU/mL), TNF- (10 ng/mL), or GM-CSF (5
ng/mL) at optimal concentrations previously determined in our
laboratory [9
]. As shown in Figure 2
, LPS-induced IL-8 production was significantly enhanced by IFN
(7,113 ±1,556 pg/mL), TNF- (10,693 ±1,233 pg/mL) and
GM-CSF (13,048 ±1,885 pg/mL), as compared with IL-12
(2,845 ±456 pg/mL) (P <0.05). Concomitant addition
of IL-12 with IFN-, TNF-, or GM-CSF did not further enhance IL-8
production (Fig. 2) .
Time-course of IL-12 enhancement of LPS-induced IL-8 production and
release
In the presence of LPS, with or without IL-12, small amounts of
IL-8 were detected in cell-free supernatants after 4 h and
gradually increased for up to 24 h (Fig. 3
). It is noteworthy that the additive effect of IL-12 was not
observed before 18 h of culture and was significant at 24 h.
In the presence of CHX, an inhibitor of protein synthesis, very low
levels of IL-8 were rapidly detected in cell-free supernatants of
LPS ± IL-12-stimulated PMNs, remaining stable over time. This
suggested that a small pre-existing pool of IL-8 was rapidly released
upon LPS stimulation and was not affected by exogenous IL-12.
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Figure 3. Time course of LPS ± IL-12-induced IL-8 release by PMNs and
effect of CHX. PMNs (2 x106/mL) were stimulated with
LPS ± IL-12 in the presence or absence of CHX (10 ng/mL).
Supernatants were collected at the times indicated, and IL-8 was
assayed. Results are representative of one typical experiment out of
three.
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Time course of IL-12Rß1 expression on the PMN surface
Because the effect of IL-12 was not observed before 18 h of
culture, IL-12 receptor expression was quantified both before and at
various times during culture. Freshly purified PMNs positively stained
with the fluorescein isothiocyanate-labeled monoclonal antibody
specific for the human low-affinity IL-12Rß1 chain (Table 2
). The mean fluorescence intensity of the cells was moderately but
significantly increased relative to samples incubated with the IgG1
isotype control (4 ±2, n =4,
P <0.05). IL-12Rß1 expression was not modified after
4 h of culture with LPS alone or combined with IL-12; in contrast,
IL-12Rß1 expression increased slightly but significantly after
18 h of culture with LPS and remained stable until 24 h of
culture. The addition of IL-12 to LPS in the culture medium reduced
IL-12Rß1 expression insignificantly. Taken together, these results
suggest that IL-12Rß1 is constitutively expressed on the PMN surface
in basal conditions and can be slightly up-regulated by LPS
stimulation.
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Table 2. Time Course of IL-12 Rß1 Expression on the PMN Surface before Cell
Culture (0) and after Stimulation with LPS Alone (100 ng/mL) or
LPS + IL-12 (10 ng/mL)
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Effect of IL-12 on cell-associated IL-8
Because IL-8 exists as a membrane-bound and intracellularly stored
cytokine, we measured IL-8 in PMN lysates after treatment with LPS with
and without IL-12. As shown in Table 3
, cell-associated IL-8 was detected in the lysates of PMNs cultured
for 24 h without exogenous stimuli. This cell-associated pool of
IL-8 increased significantly with LPS and LPS plus IL-12 but not with
IL-12 alone. IL-12 significantly enhanced LPS-induced IL-8 release in
cell-free supernatants but had no effect on levels in cell lysates
(Table 3)
.
The time-course of PMN-associated IL-8 was also studied.
Cell-associated IL-8 increased in LPS-stimulated PMNs, throughout the
24-h culture period. IL-12 did not increase LPS effects, even towards
the end of the culture period. In the presence of CHX, the amount of
cell-associated IL-8 remained similar to that of the constitutive pool
(data not shown).
Indirect effect of IL-12 on IL-8 release and role of intermediate
TNF-, IFN-, and GM-CSF potential production
Because the additive effect of IL-12 was observed only after
18 h of culture, actinomycin D was added to the culture after
16 h to block the transcription of IL-8 mRNA (Fig. 4
). This late addition of actinomycin D reduced LPS plus
IL-12-induced IL-8 production at 24 h of culture relative to cells
incubated without actinomycin D; IL-8 levels were similar to those
obtained after LPS treatment alone. These results suggested that late
addition of actinomycin D might block a second wave of IL-8 production
potentially induced by other mediators in response to IL-12.
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Figure 4. Effect of actinomycin D (Act D) on the time course of LPS ±
IL-12-induced IL-8 release by PMNs. PMNs (2 x106/mL)
were stimulated with LPS ± IL-12, and actinomycin D was added at
16 h of culture. Supernatants were collected at 16, 18, and
24 h, and IL-8 was assayed by ELISA. Results are representative of
one typical experiment out of three.
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Because TNF-, IFN-, and GM-CSF are potent inducers of IL-8
production by PMNs, we postulated that they might play an indirect role
in the observed effect of IL-12 on IL-8 production. Indeed, TNF- and
IFN- but not GM-CSF were detected in cell-free supernatants of LPS
plus IL-12-stimulated PMNs (Table 4
). Specific inhibitors were thus added during culture. As shown in
Figure 5
, the additive effect of IL-12 on IL-8 production by PMNs was
significantly reduced in the presence of soluble TNF-RII or
anti-IFN- antibodies, whereas anti-GM-CSF antibodies had no effect.
Inhibitory effects of IL-10 and DEX
As shown in Figure 6
, 30 min of PMN pretreatment with increasing concentrations of DEX
resulted in a significant concentration-dependent inhibition of IL-8
release by LPS-stimulated PMNs. When IL-12 was combined with LPS to
stimulate the cells, the inhibitory effect of DEX was unaffected (Fig. 6)
. When IL-10 was used as the inhibitory agent, LPS-induced IL-8
production by PMNs was also inhibited in a concentration-dependent
manner (Fig. 7
) but to a lesser extent than with DEX. Addition of IL-12 did not
modify this inhibitory effect.
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Figure 6. Effect of DEX on LPS ± IL-12-induced IL-8 production by PMNs.
PMNs (2 x106/mL) were incubated for 30 min with
increasing concentrations of DEX and then stimulated by LPS (100
ng/mL), with or without IL-12 (10 ng/mL), for 24 h. IL-8 was
assayed in the cell-free supernatants by ELISA. Results are expressed
as the means ± SE of three independent experiments.
*, P < 0.05 as compared with LPS + IL-12.
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Figure 7. Effect of IL-10 on LPS ± IL-12-induced IL-8 production by PMNs.
PMNs (2 x106/mL) were stimulated by LPS (100 ng/mL)
with or without IL-12 (10 ng/mL) in the presence of increasing
concentrations of IL-10. After 24 h of culture, IL-8 was assayed
in the cell-free supernatants by ELISA. Results are expressed as the
means ± SE of three independent experiments. *,
P < 0.05 as compared with LPS + IL-12.
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Involvement of NF-B and STAT4 transcription factors
Both the NF-B and STAT4 transcription factors seemed to be
involved in the effect of IL-12 on IL-8 release. Indeed, as expected
(Table 5 ), the NF-B inhibitor parthenolide strongly decreased
LPS-induced IL-8 production in a concentration-dependent manner; in the
presence of IL-12, there was a insignificant trend towards more
pronounced inhibition in the presence of 5 µM parthenolide. Another
NF-B inhibitor, caffeic acid phenyl ester, used at 10 µg/mL, gave
similar results (data not shown). Tyrphostin AG490, a JAK-2 inhibitor,
did not modify LPS-induced IL-8 production but prevented the additive
effect of IL-12 (Table 5)
. Because JAK-2 is involved in the tyrosine
phosphorylation of STAT4, we infer that both the NF-B and JAK-2
pathways are involved in the increased IL-8 production in response to
LPS plus IL-12.
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Table 5. Effect of Parthenolide and Tyrphostine AG490 on IL-8 Release by PMN
Cultured for 24 h with/without LPS (100 ng/mL), in the Presence or
Absence of IL-12 (10 ng/mL)
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DISCUSSION
|
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This study identified a new mechanism underlying the chemotactic
effect of IL-12 on PMNs, because IL-12 increased LPS-induced IL-8
production and secretion by human PMNs at both the mRNA and protein
levels. Kinetic and inhibition studies suggested that this effect is
mediated by IFN- and TNF- but not by GM-CSF. Given the key role
of IL-8 in PMN recruitment to inflammatory sites, these findings point
to a new role of IL-12 in inflammation, via an autocrine amplification
loop.
A large number of in vitro and in vivo studies have demonstrated that
PMNs can release significant amounts of biologically active IL-8 after
contact with various stimuli [8
]. Among the most potent
stimuli were LPS associated with chemotactic factors such as C5a,
N-formyl-methionyl-leucyl-phenytalanine, platelet-activating
factor, leukotriene B4, and proinflammatory cytokines
[8
, 11
]. We now extend these findings to
IL-12. No direct effect on IL-8 secretion in PMN culture supernatants
was observed with IL-12 alone. However, when PMNs were exposed to IL-12
in the presence of LPS, IL-8 release was increased, in an
IL-12-concentration-dependent manner after 24 h of culture. IL-8
production stimulated by LPS plus IL-12 at optimal concentrations was
lower than that obtained with LPS plus IFN-, TNF-, or GM-CSF.
However, it is noteworthy that an IL-12 concentration of up to 10 ng/mL
could be achieved locally in synovial fluid of patients with rheumatoid
arthritis [12
] and during experimental ischemia
reperfusion [13
], for instance.
There is increasing evidence that PMNs contain a preformed stock of
various cytokines, permitting rapid and sustained secretion at
inflammatory sites. Here, we confirmed that large amounts of
cell-associated IL-8 exist in PMNs, largely exceeding those found in
cell-free supernatants. It has been suggested that the IL-8 in PMN
lysates mainly corresponds to receptor-bound IL-8 [14
,
15
]. After stimulation with LPS alone, the amount of
cell-associated IL-8 was maximal and was not further enhanced by IL-12.
Saturation of this system would probably prevent any effect of IL-12 on
cell-associated IL-8, as previously described with IL-4, IL-10, and
IL-13 [16
].
Kinetic studies of cell-free supernatants during 24 h of culture
showed a rapid release of a preformed pool of IL-8, followed by
sustained release induced by LPS, as previously described
[8
]. CHX addition at the beginning of the culture period
significantly reduced IL-8 production after as little as 4 h,
without affecting the release of the preexisting pool, thereby
confirming the de novo protein synthesis. Using a newly described mRNA
quantification method [17
], we confirmed previous
reports from this and other laboratories of the presence of a
constitutive IL-8 mRNA pool in unstimulated PMNs that facilitated the
rapid appearance of the mature protein on stimulation [9
,
18
, 19
]. IL-12 slightly increased IL-8 mRNA
levels after 1 h of culture relative to LPS alone, at least in
part via a transcription-dependent mechanism, as shown both by the
inhibitory effect of actinomycin D added at the beginning of culture
and by the transient maximal expression of IL-8 mRNA.
A striking feature of the additive effect of IL-12 on IL-8 release is
its late appearance, after 18 h of culture, pointing to the
involvement of other mediators. This led us to perform a second set of
experiments, in which actinomycin D was added after 16 h of
culture. The late addition of this inhibitor abolished the effect of
IL-12, restoring IL-8 levels similar to those found in the presence of
LPS alone. Actinomycin D thus probably blocked the transcription of a
second wave of IL-8 mRNA potentially induced by other mediators.
Because IFN-, TNF-, and GM-CSF amplify IL-8 release by
LPS-stimulated PMNs in vitro [8
, 9
], we
tested the effect of their specific inhibitors. Both soluble type II
TNF receptor and anti-IFN antibodies reduced LPS plus IL-12-induced
IL-8 production, suggesting that newly synthesized TNF- and IFN
play a role in this IL-8 production. Indeed, TNF- and IFN- were
detected in cell culture supernatants, confirming previous data
[8
, 9
, 20
]. Anti-GM-CSF
antibodies did not modify IL-8 production, and we were unable to detect
GM-CSF using an enzyme-linked immunosorbent assay method; this is in
keeping with previous studies showing that PMNs could produce only
GM-CSF in the presence of monosodium urate crystals [8
,
21
]. The absence of GM-CSF from PMN culture supernatants
provides further indirect evidence for the absence of mononuclear cell
contamination, using the immunomagnetic depletion of human leukocyte
antigen class II-positive cells that we recently described
[10
]. Another mechanism potentially involved in the
delayed effect of IL-12 is IL-12 receptor expression on the PMN surface
late during the 24-h culture period. In keeping with two previous
reports [4
, 5
], we found that PMNs
expressed the low-affinity IL-12Rß1 chain in basal conditions,
suggesting immediate direct stimulation of PMNs by IL-12. Moreover,
slight up-regulation of IL-12Rß1 was observed at 18 and 24 h of
culture, and this could also play a role in the potentiation of the LPS
effect by IL-12. Taken together, our results suggest that IL-12
amplifies release of IL-8 by LPS-treated PMNs in an autocrine/paracrine
manner, chiefly via TNF- and IFN- production; moreover, this loop
may be amplified by IL-12 itself, which is released by PMNs in the
presence of LPS and IFN- [22
]. Slight up-regulation
of constitutive IL-12Rß1 expression on the PMN surface could also
participate in this phenomenon.
Several immunoregulatory mediators are known to down-regulate IL-8
production by PMNs. In our study, the general inhibitors DEX and IL-10
strongly inhibited LPS-induced IL-8 production, in keeping with
previous reports [16
, 23
]. When IL-12 was
combined with LPS, both inhibitors retained their
concentration-dependent inhibitory effects, suggesting that these
mechanisms would be intact in clinical settings. Other inhibitor
experiments were undertaken to determine whether IL-12 increased
LPS-induced IL-8 production via activation of the NF-B and/or STAT4
transcription factors. Two NF-B inhibitors, parthenolide
[24
] and caffeic acid phenethyl ester
[25
], decreased LPS-induced IL-8 production, confirming
the involvement of NF-B in IL-8 production by PMNs
[26
]. The addition of IL-12 did not significantly modify
this pathway. Conversely, IL-12 binding to its receptor was known to
interact with JAK-2 in several cell types [27
] and
subsequently to activate STAT4 [28
]. JAK-2 inhibition by
tyrphostin AG490 modulates some immune functions [29
].
We now show that it is also able to block the effect of IL-12 in our
model, without affecting the action of LPS. Collectively, these
observations suggest that, in human neutrophils, the up-regulating
effect of IL-12 on LPS-induced IL-8 production is dependent on both
NF-B and STAT4 activation.
This study identified a new mechanism underlying the chemotactic effect
of IL-12 on PMNs. Previous studies have suggested a role of
platelet-activating factor [4
], and we now provide
evidence for the involvement of IL-8. Increased IL-8 production at
inflammatory sites where PMNs, IL-12, and endotoxin are found might
play a key role in amplifying PMN recruitment. Our observations are
relevant to several in vivo studies in which local or intravenous IL-12
administration led to PMN influx to the tissues [6
,
7
, 30
]. Moreover, in one of these models,
PMN infiltration was associated with IL-8 and IFN- overproduction
[7
].
In conclusion, our data indicate that IL-12 plays a role in
inflammatory cell recruitment via PMN production of IL-8, the most
potent chemokine for PMNs themselves. We also found that intermediate
synthesis of TNF- and IFN- might participate in this mechanism.
IL-12, produced early during the response to infectious agents, could
thus promote rapid IL-8-induced PMN influx.
|
ACKNOWLEDGEMENTS
|
|---|
This work was supported by grants from Assistance
Publique-Hôpitaux de Paris (CRC 99209 and Fonds d’Etudes et de
Recherche) and grants from Société Françcaise
d’Anesthésie et de Réanimation.
Received October 13, 2000;
revised April 11, 2001;
accepted April 16, 2001.
|
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