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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1413-1421
Oncostatin M Production and Regulation by Human Polymorphonuclear
Neutrophils
By
Alain Grenier,
Monique Dehoux,
Anne Boutten,
Montserrat Arce-Vicioso,
Geneviève Durand,
Marie-Anne Gougerot-Pocidalo, and
Sylvie Chollet-Martin
From the Laboratoire de Biochimie, Hôpital de Montfermeil,
Montfermeil, France; the Laboratoire de Biochimie, INSERM U 408, Laboratoire d'Hématologie et d'Immunologie et INSERM U 479, Hôpital Bichat, Paris, France; and the Laboratoire de Biochimie,
Faculté de Pharmacie, Chatenay-Malabry, France.
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ABSTRACT |
Oncostatin M (OSM) is an interleukin-6 (IL-6) family cytokine known
in particular to induce the synthesis of acute-phase proteins by
hepatocytes. Because human polymorphonuclear neutrophils (PMN) can
secrete numerous cytokines, the potential production of OSM by PMN was
investigated. Highly purified PMN were found to contain an
intracellular stock of preformed OSM that was rapidly mobilized by
degranulating agents such as phorbol myristate acetate and granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, PMN produced OSM after a few hours of stimulation by various agonists. The most potent effect was observed with the combination of
lipopolysaccharide and GM-CSF, which had a concentration- and
time-dependent effect at both the protein and mRNA levels. Actinomycin
D strongly reduced OSM mRNA induction, suggesting the involvement of
gene transcription. Cycloheximide inhibited OSM protein synthesis but
did not affect the release of preformed stores. In addition, OSM
production was downregulated by dexamethasone, whereas IL-10 had no
effect. The OSM produced by PMN was biologically active, as
demonstrated by its ability to induce  1-acid glycoprotein synthesis
by HepG2 cells. OSM secretion thus occurs through a two-step mechanism in PMN, consisting of early release of a preformed stock, followed by
de novo protein synthesis. This would allow rapid and sustained OSM
release to occur at inflammatory sites, and may contribute to the
modulation of local inflammation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
POLYMORPHONUCLEAR neutrophils (PMN) are
circulating phagocytes that participate in the immune and inflammatory
responses to endogenous and exogenous stimuli. In addition to their
phagocytic and killing properties, PMN synthesize numerous cytokines
such as proinflammatory agents (tumor necrosis factor- [TNF-],
interleukin-1 [IL-1], and IL-1 ), the antiviral agent
interferon- (IFN-), chemokines (macrophage inflammatory
protein-1 [MIP-1], MIP-1, IL-8, and IP-10),
growth factors (granulocyte colony-stimulating factor [G-CSF],
macrophage colony-stimulating factor [M-CSF], and
granulocyte-macrophage colony-stimulating factor [GM-CSF]) and
anti-inflammatory mediators (IL-1ra and transforming growth factor-
[TGF-]).1,2 PMN are far more abundant than other inflammatory cells in the early stages of the inflammatory response, suggesting that some of the cytokines released by PMN may be critical for regulating the inflammatory process.
Oncostatin M (OSM) is a cytokine originally isolated from the
supernatants of U937 histiocytic leukemia cells differentiated into
macrophage-like cells by treatment with phorbol myristate acetate. It
was identified by its ability to inhibit the replication of A375
melanoma cells.3 OSM acts on a wide variety of cells and
elicits a multitude of biological responses, including modulation of
cell growth and differentiation, low-density lipoprotein
(LDL) receptor upregulation, and induction of
hematopoietic factors (reviewed in Wallace et al4). OSM
belongs to the IL-6 cytokine family, which includes IL-6, leukemia
inhibitory factor (LIF), IL-11, ciliary neurotrophic factor (CNTF), and
cardiotrophin-1 (CT-1). These cytokines are mainly produced during
inflammatory disorders, in which they induce a systemic acute-phase
response marked by the enhancement of acute-phase protein synthesis by hepatocytes.5 Activated T lymphocytes6 and
monocytes7 are the only human cells known to produce OSM.
Given their involvement in numerous inflammatory processes, we
investigated whether purified human PMN could synthesize and secrete
OSM and studied the regulation of this production.
We report for the first time that highly purified PMN contain a
preformed stock of OSM and that bioactive OSM can be synthesized and
released after stimulation with various agonists; these findings suggest that OSM production by PMN constitutes a new element of the
acute-phase response.
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MATERIALS AND METHODS |
Purification of human PMN.
Human PMN were purified as previously described.8,9
Briefly, blood PMN from healthy volunteers were isolated in sterile conditions by sedimentation in medium containing 9% Dextran T-500 (Pharmacia, Uppsala, Sweden) and 38% Radioselectan (Schering, Lys-lez-Lannoy, France); the leukocyte suspension was then centrifuged on Ficoll-Paque medium (Pharmacia). The cell pellet was washed with
phosphate-buffered saline and erythrocytes were removed by hypotonic
lysis. PMN were further purified by 20 minutes of incubation with pan
antihuman HLA class II-coated magnetic beads (Dynal, Oslo, Norway) to
deplete B lymphocytes and activated T lymphocytes and
monocytes, but not resting T cells. PMN were then resuspended in
culture medium (see below). Nonspecific esterase staining always showed
less than 0.5% of monocytes, and flow cytometry showed the absence of
CD14+, CD3+, CD19+ cells,
confirming the recovery of highly purified PMN.
T lymphocyte preparation.
After Ficoll-Paque centrifugation, the mononuclear cell ring was
treated with magnetic beads as described above to deplete B cells,
activated T cells, and monocytes, retaining only purified resting T cells.
Cell culture.
Pure PMN (107/mL) were cultured for up to 48 hours at
37°C with 5% CO2 in 24-well tissue culture plates
(Costar, Cambridge, MA). The culture medium was RPMI 1640 (Sigma, St
Louis, MO) supplemented with 2 mmol/L glutamine, antibiotics, and 10%
heat-inactivated fetal calf serum (Biowittacker, Gagny, France).
Stimulating agents were added to the culture medium at the following
optimal concentrations determined in preliminary concentration-response
experiments: lipopolysaccharide at 100 ng/mL (LPS; from Escherichia
coli 055:B5), phorbol myristate acetate at 100 ng/mL (PMA),
N-formylmethionyl-leucyl-phenylalanine at 10 5 mol/L
(fMLP), TNF- at 100 U/mL (TNF), IFN- at 500 U/mL (IFN), and
GM-CSF at 100 U/mL; all reagents were from Sigma, except for recombinant cytokines, which were from Genzyme (Cambridge, MA). The
inhibitory effects of IL-10 (kindly provided by Schering-Plough Research Institute, Kenilworth, NJ) and dexamethasone (DEX; Sigma) were
studied by adding them for 30 minutes at 37°C before stimulation with LPS plus GM-CSF. In selected experiments, pure resting T lymphocytes (5 × 105/mL) were cultured alone or
cocultured with 107 PMN/mL after stimulation with LPS plus
GM-CSF to test the role of contaminating resting T cells in OSM
production. Cell-free PMN culture supernatants were collected at
various times and stored at  20°C until OSM assay. To
determine cell-associated OSM, 107 PMN/mL were incubated
for 15 minutes at 37°C with or without 100 ng/mL PMA or 100 U/mL
GM-CSF; cell-free supernatants were collected and the cell pellets were
sonicated for 30 seconds to measure cell-associated OSM. Both
supernatants and cell pellets were stored at 20°C until OSM
assay. In some experiments, neutrophils (107/mL) were
preincubated with or without 10 µg/mL of cycloheximide (CHX) for 30 minutes at 37°C and then further incubated with 100 U/mL of GM-CSF
for 15 minutes or 3 or 8 hours.
OSM assay.
OSM was quantified by using a commercial enzyme-linked immunosorbent
assay (ELISA) kit (Quantikine; R&D Systems, Abingdon, UK) following the
manufacturer's instructions; the detection limit was 2.1 pg/mL.
Immunocytochemical staining of intracellular OSM.
Unstimulated blood smears from healthy controls were air-dried for 24 hours and fixed in cold methanol/acetone. Smears were then incubated
with a polyclonal rabbit antihuman-OSM antibody (50 µg/mL; Genzyme),
followed by incubation with a biotinylated antibody and then alkaline
phosphatase-labeled streptavidin, as recommended by the manufacturer
(LSAB kit-AP; Dako, Carpinteria, CA). Staining was completed by
incubation with a chromogenic substrate solution; smears were
counterstained with hematoxylin and ammonia water. Positive staining
developed as a fuschia-colored reaction product. Smears incubated with
a control Ig (Sigma) served as negative controls and smears incubated
with an anti-CD11b monoclonal antibody (Immunotech, Marseille, France)
served as positive controls.
Northern blot analysis.
For RNA analysis, 5 × 107 PMN were incubated for up
to 18 hours in 2 mL of standard culture medium containing the
appropriate stimuli. In selected experiments, actinomycin D (5 µg/mL;
Sigma) was added to the medium to block transcription. Total cellular RNA was isolated from PMN with RNA-B (Bioprobe, Cergy-Pontoise, France)
according to the manufacturer's instructions, and the RNA
concentration was determined at 260 nm. Twenty micrograms of total RNA
was analyzed by electrophoresis on 1% agarose-formaldehyde gel and
transferred to nylon filters (Amersham, Les Ulis, France). The filters
were prehybridized in 5× SSC, 25 mmol/L sodium phosphate (pH
6.4), 5× Denhardt's reagent, 0.1% sodium dodecyl sulfate (SDS), and 0.2 mg/mL denatured salmon sperm DNA for 12 hours at 42°C. Human OSM mRNA was detected by hybridization with
32P-labeled oligonucleotide probes (R&D Systems) for 16 hours at 42°C in 5× SSC, 25 mmol/L sodium phosphate (pH 6.4),
5× Denhardt's reagent, 0.1% SDS, 50% formamide, and 0.2 mg/mL
denatured salmon sperm DNA. The membranes were then washed in 2×
SSC, 0.1% SDS for 5 minutes at room temperature and then for 30 minutes at 42°C. The oligonucleotide probes were labeled with
[32P]ATP (specific activity, 3,000 Ci/mmol; Amersham)
by using the Ready To Go T4-Polynucleotide Kinase (Pharmacia) and
further purified on Micro Bio-Spin columns (Biorad, Ivry, France)
following the manufacturers' instructions. The glyceraldehyde
phosphate dehydrogenase (GAPDH) and actin cDNA probes (Clontech, Palo
Alto, CA) were labeled with [32P]dCTP by random
priming using the Rediprime DNA labeling sytem (Amersham). The blots
were then exposed for autoradiography.
Biological activity of PMN OSM.
The HepG2 hepatoma cell line was cultured in minimum essential medium
Eagle (MEM) containing Glutamax, 25 mmol/L HEPES, and Earle's salts (Life Technologies, Cergy Pontoise, France) plus 10%
heat-inactivated fetal calf serum. Cells were plated in 24-well culture
plates (Costar) and allowed to grow to confluence at 37°C in
humidified air containing 5% CO2. Monolayers were then
incubated for 24 hours with 1 mL of RPMI 1640 medium supplemented with
10% fetal calf serum, antibiotics, 2 mmol/L glutamine, pyruvate (Life Technologies), and 106 mol/L dexamethasone and were
stimulated with either 10 ng/mL recombinant human OSM (rhOSM;
Genzyme) or the culture supernatant of PMN stimulated with LPS + GM-CSF
(PMN conditioned medium [CMP]). In some cases, before HepG2
stimulation, rhOSM and CMP were preincubated at 37°C for 30 minutes
with polyclonal rabbit antibodies against human OSM (20 µg/mL; Genzyme).
The cell-free supernatants of HepG2 cultures were then harvested and
stored at 20°C until ELISA measurement of 1-acid
glycoprotein.10 Cell total DNA content was
quantified11 to express the amount of secreted 1-acid
glycoprotein in nanograms per microgram of DNA. All samples were tested
in duplicate.
Statistical analysis.
Data are expressed as the means ± SEM; differences were considered
statistically significant if P < .05 in Wilcoxon's paired test.
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RESULTS |
OSM synthesis can be induced by various stimuli.
Because PMN have been described to synthesize several cytokines de
novo, the ability of PMN to synthesize OSM in various conditions of
stimulation for 20 hours of culture was tested. As shown in Fig 1, fMLP, IFN-, and LPS alone
moderately induced OSM release as compared with unstimulated PMN
(respectively, 218 ± 50, 251 ± 88, and 455 ± 93 pg/107 PMN v 126 ± 39 pg/107 PMN;
P < .05; n = 6). TNF- and GM-CSF were potent inducers
(respectively, 508 ± 163 and 828 ± 173 pg/107 PMN;
P < .05). The strongest OSM production was obtained using PMA
alone or LPS plus GM-CSF, giving values of, respectively, 1,300 ± 400 and 1,294 ± 133 pg/107 PMN (P < .05). OSM upregulation by LPS, GM-CSF, LPS plus GM-CSF, and PMA was
observed at both the protein and mRNA levels. As shown in
Fig 2, OSM mRNA was undetectable after 1 hour in control PMN, whereas stimulated PMN accumulated OSM mRNA. As
shown in Fig 3, PMN incubation at the
outset of culture with the transcriptional inhibitor actinomycin D
strongly reduced the OSM mRNA accumulation induced by LPS plus GM-CSF.
After stimulation with these agonists for 1 hour (steady-state mRNA
peak in Fig 2), actinomycin D was added to assess OSM transcript
stability. In these conditions, OSM mRNA levels decreased rapidly
(Fig 4), with a calculated half-life of 45 ± 8 minutes (n = 3, regression analysis). Taken together, our data
suggest that the induction of this gene by LPS plus GM-CSF might take
place at the transcriptional level.
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| Fig 1.
Stimulation of PMN OSM production. PMN
(107/mL) were incubated for 20 hours with various
stimulating agents at the concentrations indicated in Materials and
Methods. OSM was assayed in the cell-free supernatants. Control cells
(CTRL) were incubated with medium alone. Results are expressed as the
means ± SEM of six independent experiments. *P < .05 compared with control cells.
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| Fig 2.
OSM mRNA expression in human PMN. PMN (5 × 107) were cultured for up to 18 hours in the presence of
LPS (100 ng/mL) plus GM-CSF (100 U/mL) (right panel). In other
experiments, PMN were cultured for 1 hour in the presence of LPS
and/or GM-CSF, PMA (100 ng/mL), or in medium alone (control)
(left panel). Total RNA was extracted and Northern blots of OSM and
GAPDH were run as specified in Materials and Methods.
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| Fig 3.
Effect of actinomycin D on expression of OSM transcripts
induced by LPS plus GM-CSF. PMN (5 × 107) were incubated
for 15 minutes in the presence or absence of 5 µg/mL actinomycin D
(ACT D) and then stimulated for 1 hour with LPS (100 ng/mL) plus GM-CSF
(100 U/mL). Controls cells (CTRL) were incubated in the medium alone.
Total RNA was extracted and processed for Northern blot analysis of OSM
and actin mRNAs as described in Materials and Methods. Results are from
one experiment representative of two.
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| Fig 4.
Stability of OSM transcripts in LPS plus GM-CSF-treated
PMN. PMN (5 × 107) were incubated with LPS (100 ng/mL)
plus GM-CSF (100 U/mL) for 1 hour and then actinomycin D (5 µg/mL)
was added for the indicated times. Total RNA was extracted and Northern
blot analysis of OSM and actin mRNAs was performed as indicated in
Materials and Methods.
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OSM production increased in a concentration-dependent manner and
reached a steady level with 100 U/mL GM-CSF and 100 ng/mL LPS
(Fig 5). A fixed concentration of GM-CSF
(100 U/mL) combined with increasing concentrations of LPS (1 to 1,000 ng/mL) had an additive effect (not shown). Other combinations,
including LPS plus IFN- or TNF-, had the same additive effects
(not shown).
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| Fig 5.
Concentration-response of GM-CSF- and LPS-induced OSM
production by PMN. PMN (107/mL) were stimulated with
increasing concentrations of LPS or GM-CSF for 20 hours. OSM was
assayed in the cell-free supernatants. These data are representative of
one typical experiment of three.
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Because resting T cells are the only cells potentially not removed by
HLA class II-coated magnetic beads, their participation in OSM
production was evaluated. T lymphocytes alone (5 × 105) did not produce detectable OSM (<2.1 pg/mL) when
stimulated by LPS plus GM-CSF for 20 hours; moreover, PMN alone
(107) and combined with 5 × 105
autologous T lymphocytes produced similar levels of OSM (data not shown).
Time course study of OSM release by PMN.
The small amounts of OSM released by control cells were detectable
after 4 hours of culture. By contrast, in optimal conditions of
stimulation (LPS + GM-CSF), OSM was detectable after 1 hour, reached a
plateau by 24 hours, and gradually accumulated for up to 48 hours of
culture (n = 3; Fig 6). It is noteworthy
that 70% of the total amount of OSM was synthesized within 8 hours.
Maximal expression of OSM mRNA by PMN stimulated by LPS plus GM-CSF was observed as early as 1 hour and disappeared after 6 hours (Fig 2).
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| Fig 6.
Time course of OSM release by PMN. PMN
(107/mL) were stimulated with GM-CSF (100 U/mL) plus LPS
(100 ng/mL). Control cells were incubated with the medium alone.
Supernatants were collected at the times indicated and OSM was assayed
in an ELISA method. Results are expressed as the mean ± SEM of three
independent experiments.
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Human PMN contain an intracellular pool of OSM.
Because time-course studies showed that OSM was released as early as 1 hour after PMN stimulation, we investigated whether PMN contained a
preformed stock of OSM in two different ways. First,
immunocytochemistry showed the presence of intracellular OSM in
unstimulated PMN in whole-blood smears (Fig
7). Second, degranulating experiments were conducted with pure isolated
PMN incubated with or without PMA or GM-CSF (both potent inducers of
neutrophil degranulation8,12) for 15 minutes at 37°C.
Released and cell-associated OSM were then measured separately. As
shown in Fig 8, the amount of
cell-associated OSM was 70 ± 8 pg per 107 PMN, whereas
the OSM concentration was below the detection limit in the supernatant
of resting PMN maintained for 15 minutes at 37°C in the absence of
degranulating agents. PMA or GM-CSF stimulation led to a reduction in
cell-associated OSM, matched by a parallel increase in the
extracellular OSM level (Fig 8); the total amount of extracellular plus
cell-associated OSM was similar to the total amount of cell-associated
OSM in resting PMN. These results suggested that a preexisting pool of
OSM was released. Similar experiments with incubation times of 5 and 30 minutes gave similar results, in keeping with a maximal degranulating
effect of PMA after as little as 5 minutes (data not shown).
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| Fig 7.
Immunocytochemical staining of PMN in whole-blood smears.
(A) Negative control; no staining was seen with a control Ig. (B)
Intracellular fuschia staining was observed in PMN with specific
polyclonal anti-OSM antibodies; smears were examined by light
microscopy at ×1,200.
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| Fig 8.
Effect of PMA and GM-CSF on OSM secretion by human PMN.
PMN (107/mL) were incubated for 15 minutes without
(resting) or with 100 ng/mL PMA or 100 U/mL GM-CSF. Secreted and
cell-associated OSM were assayed with a specific ELISA. Results are
expressed as the mean ± SEM of four independent experiments.
#P < .05 compared with OSM secreted without degranulating
agent. *P < .05 compared with cell-associated OSM without
degranulating agent. ND, not detected (<2.1 pg/mL).
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Because GM-CSF is a physiologic inducer of both neutrophil
degranulation and OSM synthesis, a kinetic study was performed in the
presence and absence of cycloheximide, an inhibitor of protein
synthesis. The amounts of OSM detected after 15 minutes of stimulation
with GM-CSF were not affected by cycloheximide (Fig 9). In contrast, after 3 and 8 hours
of GM-CSF stimulation, a significant reduction in OSM synthesis was
observed after CHX treatment (62% and 68% inhibition, respectively)
as compared with untreated cells (P < .05, n = 4).
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| Fig 9.
Effect of CHX on GM-CSF-induced OSM secretion. PMN
(107/mL) were preincubated for 30 minutes with or without
10 µg/mL CHX and then stimulated with 100 U/mL GM-CSF for the times
indicated; control cells were incubated with medium alone. The OSM
secreted was assayed in the cell-free supernatants with an ELISA
method. Results are expressed as the mean ± SEM of four independent
experiments. #P < .05 compared with the control cells.
*P < .05 compared with the CHX untreated cells. ND, not
detected (<2.1 pg/mL).
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Taken together, these data strengthen the hypothesis that the observed
OSM secretion by PMN is first due to the release of a preexisting
intracellular pool, followed by de novo synthesis.
DEX but not IL-10 inhibits OSM production induced by LPS plus GM-CSF.
Because IL-10 and DEX are potent modulators of cytokine production by
PMN,13,14 we tested their influence on inducible OSM
production. As shown in Fig 10,
pretreatment of PMN with DEX resulted in significant
concentration-dependent inhibition of OSM production induced by LPS
plus GM-CSF (inhibition of 0%, 42%, 61%, and 65% with DEX
concentrations of 1010, 108,
106, and 104 mol/L, respectively;
n = 3). By contrast, IL-10 (1 to 1,000 ng/mL) failed to modulate OSM
production by PMN in optimal conditions of stimulation (not shown). The
same pattern was observed at the OSM mRNA level, as DEX reduced the
level of OSM transcripts in stimulated PMN, whereas IL-10 failed to
affect it (not shown).
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| Fig 10.
Effect of DEX on OSM production by stimulated PMN. PMN
(107/mL) were incubated for 30 minutes with DEX (indicated
concentrations) and then stimulated by GM-CSF (100 U/mL) plus LPS (100 ng/mL) for 20 hours; OSM was assayed in the cell-free supernatants by
using an ELISA method. The data are expressed as the mean ± SEM of
three independent experiments. *P < .05 by comparison with
LPS- plus GM-CSF-stimulated PMN without DEX.
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OSM bioactivity.
OSM is an IL-6-related cytokine that stimulates hepatocytes and
induces the expression of acute-phase proteins such as -1 acid
glycoprotein (AGP).5 We investigated the ability of CMP (culture supernatant of PMN stimulated with LPS plus GM-CSF) to stimulate 1-acid glycoprotein synthesis by HepG2 cells. In a preliminary experiment, we found that AGP synthesis was unaffected by
direct stimulation with LPS plus GM-CSF. Conditioned medium (500 µL) containing 4 ng of OSM (measured by ELISA) was added to HepG2
cell culture medium for 24 hours. As shown in
Fig 11, CMP and rhOSM (10 ng/mL)
stimulated 1-acid glycoprotein synthesis as compared with
unstimulated HepG2 cells (n = 5; P < .05), and antibodies raised against OSM led to a 35% inhibition of CMP-induced AGP secretion (from 29.3 to 18.9 ng AGP/µg DNA; P < .05 as
compared with CMP alone), whereas it completely abolished rhOSM-induced AGP secretion (control).
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| Fig 11.
Biological activity of OSM produced by PMN. HepG2 cells
were incubated with the supernatant from LPS- plus GM-CSF-stimulated
PMN (PMN conditioned medium [CMP]) or rhOSM (10 ng/mL); control cells
(CTRL) were cultured in the medium alone. Anti-OSM antibodies were used
as indicated. Secreted 1-acid glycoprotein (AGP) was determined in
cell-free supernatants after 24 hours (nanograms per microgram of DNA).
Results are expressed as the mean ± SEM of five independent
experiments. *P < .05 compared with control cells. #P < .05 compared with AGP synthesis without anti-OSM.
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DISCUSSION |
This study suggests that human PMN contain an intracellular pool of OSM
that is rapidly released in degranulating conditions. This release is
followed by OSM mRNA accumulation and protein synthesis in PMN
stimulated by various agonists. Given the role of OSM in the
acute-phase response, these findings support the pivotal role of PMN in
the inflammatory process via a newly described two-step mechanism.
Indeed, some cytokines are stored in PMN, such as vascular endothelial
growth factor (VEGF),8 whereas others can be
synthesized de novo, such as TNF-,2 but we concomitantly explored for the first time both secretion processes for the same cytokine.
The PMN isolation method used here yielded highly purified preparations
(>99% PMN). Immunomagnetic depletion of HLA class II-positive cells
(B cells, activated T cells, and monocytes) may have left a very small
proportion of HLA class II-negative cells (resting T lymphocytes), but
these cells were ruled out as a source of the observed OSM release, by
means of PMN and T-lymphocyte coculture assays. Furthermore, we failed
to detect IL-6 (<10 pg/mL/107cells, ELISA) in our PMN
preparations even after LPS stimulation (data not shown), confirming
the absence of contaminating monocytes.2,14
Although the intracellular pool of OSM rapidly released during
degranulation induced by PMA or GM-CSF seemed small (48 ± 6 pg and
37 ± 9 pg/107 PMN, respectively) as compared with the
amount synthesized after 20 hours of culture, this content of
pre-existing OSM is of importance, because it allows a rapid and
significant secretion of OSM at the inflammatory site and could be an
early event in the multistep process of PMN activation. The existence
of this intracellular pool was confirmed by using CHX, which failed to
affect early OSM release. Other cytokines stocked in PMN granules and
rapidly released after exposure to degranulating agents include
VEGF in specific granules8 and TGF- in an
undefined granule type.15 PMA and GM-CSF failed to release
the entire OSM pool, as shown by a measurable residual level of
cell-associated OSM. PMA induces degranulation of specific and
azurophilic granules, whereas the effect of GM-CSF is limited to
specific granules. OSM may thus also be located in another type of
granules or in membrane-bound state.
PMN are relatively short-lived end-stage cells that can synthesize mRNA
encoding various cytokines.2 We found that OSM production
by PMN was upregulated by a wide variety of stimuli, including
chemoattractants and cytokines, corresponding to different mechanisms
of cell activation. GM-CSF, TNF-, and IFN-, all of which
upregulate the expression of several cytokine genes in
PMN,1,16 also induced OSM synthesis; among them, the most
potent effect was observed with GM-CSF. Formyl peptide (fMLP), a
chemotactic agent for PMN,17 showed weak activity. The
protein kinase C activator PMA yielded the most potent induction of OSM
synthesis. LPS alone was a relatively moderate inducer of OSM, and its
combination with GM-CSF (or IFN- or TNF-) was additive, pointing
to the use of different signaling pathways. The low level of OSM
produced by unstimulated cells cultured for 20 hours was usually found with other cytokines and could be due to stimulation by cell
adherence.16 Interindividual variations were also observed,
as commonly described with other cytokines.17,18
Kinetic studies showed that the amount of OSM (70 pg/107
PMN) measured after 1 hour of stimulation by LPS plus GM-CSF (the optimal combination of physiological stimuli) was similar to the intracellular stock of OSM, suggesting that the OSM induced by LPS plus
GM-CSF is initially cell-associated. OSM production then increased and
was strong within 4 hours of stimulation (40% of the total OSM
amount), suggesting direct stimulation rather than induction of a
second messenger. Similar results were obtained with GM-CSF alone.
Moreover, CHX significantly reduced OSM production at 8 hours (by up to
68%), confirming de novo protein synthesis. Northern blotting showed
that OSM mRNA expression by stimulated PMN was rapid (as early as 1 hour). In the same way, Cassatella et al17 and Fujishima
and Aikawa19 demonstrated that IL-8 mRNA expression was
maximal at 1 hour. This maximal expression of OSM mRNA in PMN was
transient, with a short half-life. The combination of LPS plus GM-CSF
acts at least partly at the transcriptional level, as suggested by
Northern blot analysis in the presence of actinomycin D. Taken
together, these findings confirm that PMN make an early and sustained
contribution to the inflammatory process through two different
mechanisms of OSM release: liberation of a preformed stock followed by
de novo protein synthesis. It is noteworthy that neither IL-6 (data not
shown)2,14 nor LIF (data not shown) was found in PMN
culture supernatants. Therefore, in contrast to
monocytes,5,20 among the three major cytokines belonging to the IL-6 family, only OSM was released by PMN whatever the
conditions of stimulation used.
Some immunoregulatory mediators downregulate cytokine production by
PMN. In our study, DEX strongly inhibited OSM production at both the
mRNA and protein levels, in keeping with the inhibitory effects of DEX
on other cytokines produced by PMN, such as IL-8.13 Interestingly, IL-10 preincubation did not inhibit OSM production induced by LPS plus GM-CSF, whereas IL-8 and TNF- release was downregulated in the same experiments (data not shown). The modulation of cytokine production by IL-10 is complex and dependent on the nature
of the stimulus and cell type.20,21
Richards et al22 have shown that OSM has strong
hepatocyte-stimulating activity and that 1-acid glycoprotein, among
other acute-phase proteins, is potently induced by OSM in the
hepatocyte cell line HepG2. In this work, we found that the supernatant
of stimulated PMN upregulated 1-acid glycoprotein production by these cells; moreover, 1-acid glycoprotein secretion was, at least
in part, inhibited by anti-OSM antibodies, suggesting that OSM present
in the conditioned medium was biologically active.
OSM produced by PMN may serve numerous functions, including the
stimulation of acute-phase reactant synthesis22 and the modulation of other regulatory cytokines. OSM produced locally by PMN
in inflamed tissues may act as an anti-inflammatory mediator. First,
OSM inhibits IL-1-induced expression of IL-8 by lung
fibroblasts,23 suggesting that PMN may limit their own
recruitment to sites of local inflammation. Second, OSM is a potent
inducer of several antiproteases in cells of extrahepatic origin. For
example, OSM upregulates the synthesis of 1-antitrypsin and
1-antichymotrypsin by epithelial cells originating from the jejunum,
lung or skin.24-26 Therefore, OSM released by activated PMN
could participate in this local induction of antiproteases, which could
be a mechanism against the detrimental effects of PMN proteases (ie,
elastase). Most acute-phase proteins induced by OSM possess
anti-inflammatory properties in vivo and in vitro.27 For
example, 1-antichymotrypsin and 1-antitrypsin inhibit PMN
superoxide anion production and may have a role in the modulation of
reactive oxygen species-induced tissue damage.28,29 Besides
this anti-inflammatory effect, recent data suggest that OSM could also
be directly proinflammatory.30 Indeed, Modur et
al30 recently showed that OSM induces PMN adhesion and
transmigration, as well as chemokine production by endothelial cells.
OSM produced by PMN may not only participate in the repair discussed
above, but also play a role in initiating the inflammatory response.
In conclusion, our data indicate that PMN at inflammatory sites can
rapidly release an intracellular stock of OSM, followed by de novo OSM
synthesis. This two-step mechanism of cytokine secretion by PMN would
allow rapid and sustained OSM release to occur at inflammatory sites
and may contribute to the modulation of local inflammation.
| |
ACKNOWLEDGMENT |
The authors are grateful to V. Andrieux-Beautru, P. Soler, C. Poüs, and V. Leçon-Malas for expert technical assistance
and to Prof M.A Cassatella (University of Verona, Verona, Italy) and V. Ollivier for helpful discussions and advice.
| |
FOOTNOTES |
Submitted February 24, 1998; accepted October 13, 1998.
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 fact.
Address reprint requests to Sylvie Chollet-Martin, PhD, Laboratoire
d'Hématologie et d'Immunologie, Hôpital Bichat, 16 rue
Henri Huchard, 75018 Paris, France; e-mail:
sylvie.martin{at}bch.ap-hop-paris.fr.
| |
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Abstract
Introduction