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* Cardiff School of Biosciences, Cardiff University,
Institute of Nephrology, University of Wales College of Medicine, Cardiff, United Kingdom; and
Department of Cell Biology, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression was
restricted to human peritoneal mesothelial cells. Stimulation of human
peritoneal mesothelial cells with OSM induced phosphorylation of gp130
and OSM receptor , which was accompanied by activation of
STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant
protein-1 and IL-6. Although OSM itself did not modulate CXC
chemokine ligand 8/IL-8 release, it effectively suppressed
IL-1-mediated expression of this neutrophil-activating CXC
chemokine. Moreover, OSM synergistically blocked IL-1-induced CXC
chemokine ligand 8 secretion in combination with the IL-6/sIL-6R
complex. Thus suggesting that OSM and sIL-6R release from infiltrating
neutrophils may contribute to the temporal switch between neutrophil
influx and mononuclear cell recruitment seen during acute
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with the inflammatory properties of IL-6, OSM exhibits both pro- and anti-inflammatory characteristics (8, 9, 10, 11, 12). Several reports have documented clear distinctions in the cellular origin and profile of OSM expression in various disease states (6, 8, 9, 13). For example, in acute lung injury OSM was transiently secreted by infiltrating neutrophils (13), whereas OSM levels in synovial fluid from rheumatoid arthritis patients were more sustained (9) and originated from activated macrophages and T lymphocytes (6, 8). Consequently, OSM may elicit distinct events during acute and chronic inflammation, which may relate to the pro- and anti-inflammatory characteristics of this cytokine.
Recurrent bacterial peritonitis is a principle cause of treatment cessation in end-stage renal failure patients undergoing continuous ambulatory peritonitis dialysis (CAPD). This condition provides a unique opportunity to study the cytokine network in a human inflammatory process (14, 15, 16, 17). As with other inflammatory diseases, the profile of leukocyte recruitment encountered during peritoneal infection is critical to the outcome of the condition and is characterized by an initial influx of neutrophils that are rapidly replaced by a more sustained population of mononuclear leukocytes (14, 15). This temporal switch in leukocyte phenotype is central to the resolution of an inflammatory episode, and regulation of this event ultimately depends upon communication between invading leukocytes and the mesothelium (14, 18). Recently, we identified that soluble IL-6R (sIL-6R) shed from infiltrating neutrophils combines with mesothelial-derived IL-6 to differentially control chemokine expression and regulate the pattern of leukocyte recruitment (17). This scenario has subsequently been supported by an in vitro study based on the activation of endothelial cells by IL-6 and its soluble receptor (19).
In this current study, we show that raised i.p. levels of OSM during bacterial infections originate from infiltrating neutrophils and regulates mesothelial expression of IL-6 and inflammatory chemokines. Ultimately, the regulation of these factors by OSM may directly effect leukocyte recruitment during inflammatory episodes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant OSM was purchased from Autogen Bioclear
(Calne, U.K.) and R&D Systems (Abingdon, U.K.). Human recombinant
IL-1, IL-6, CC chemokine ligand (CCL)2, CXC chemokine ligand
(CXCL)8, CXCL10, CCL5, CCL3, and the anti-gp130 mAb (MAB-230) were
obtained from R&D Systems. All cell culture supplements and media were
from Life Technologies (Paisley, U.K.). Primary Abs against OSM
receptor (OSMR) (AN-A2), leukemia inhibitory factor (LIF)
receptor (AN-E1), gp130 (H-255), STAT1 (M-22), and STAT3 (C-20) were
from Santa Cruz Biotechnology (Santa Cruz, CA). The phosphotyrosine Abs
4G10 and PY-20 were from Upstate Biotechnology (Lake Placid, NY) and
Santa Cruz Biotechnology, respectively. Control and conjugated
secondary Abs were from DAKO (Ely, U.K.). The m67 SIE consensus
oligonucleotide was synthesized by Life Technologies and the
[
32P]dTTP obtained from Amersham Pharmacia
Biotech (Amersham, U.K.). All other chemicals were purchased from
Sigma-Aldrich (Poole, U.K.). Prof. S. Rose-John (Institüt
für Biochemie, Christian-Albrecht-Universität zu Kiel,
Kiel, Germany), kindly provided HYPER-IL-6.
Collection of peritoneal dialysis effluent
Dialysis effluent was collected from end-stage renal failure patients currently being treated at the University Hospital of Wales (Cardiff, U.K.) in accordance with ethical approval from Bro Taf Health Authority. A uniform protocol for patient management was adopted and details are described elsewhere (17). The collected effluent was centrifuged and supernatants frozen at -80°C until required. Neutrophils or macrophages were directly isolated from the cellular fraction and used immediately.
Isolation and culture of neutrophils and macrophages
Peritoneal neutrophils were isolated from dialysis fluid collected on day 1 of infection using established methods (17). Differential cell counts confirmed that the neutrophil population represented 84 ± 3% of the total cell count. Peritoneal macrophages were isolated from dialysis fluid collected from noninfected (overnight dwell) patients (20). Macrophages constituted 52 ± 5% of the cell population, whereas neutrophils represented <5% of the total cell count. Neutrophils (105 cells/ml) and macrophages (105 cells/ml) were incubated in RPMI medium containing penicillin and streptomycin for 24 h at 37°C, 5% CO2. Neutrophils were incubated in the presence or absence of 1–10 µM hydrocortisone, whereas macrophages were stimulated with a defined dose of cell-free supernatant derived from Staphylococcus epidermidis, which is a major causative organism in dialysis-related episodes of peritonitis (17, 20). Culture supernatants were rendered cell-free by centrifugation and stored at -80°C.
Isolation and culture of human peritoneal mesothelial cells (HPMCs)
HPMCs were isolated as previously described (18). Briefly, omental tissue from consenting patients undergoing abdominal surgery was digested with 0.1% (w/v) trypsin/0.02% (w/v) EDTA diluted in PBS. Cells were cultured in Earle’s buffered-199 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml transferrin, 5 µg/ml insulin, and 0.4 µg/ml hydrocortisone at 37°C in a humidified atmosphere containing 5% CO2. HPMC monolayers were growth arrested for 48 h in the absence of FCS before stimulation. Under these conditions, HPMCs remain in a viable and quiescent state for up to 96 h (18). All experiments were performed in the absence of FCS on isolates no older than the second passage.
Flow cytometry
Cellular expression of OSMR, LIFR, and gp130 was analyzed
using flow cytometry (FACSCalibur; BD Biosciences, Cowley, U.K.)
and primary Abs specific for the extracellular portion of OSMR,
LIFR, and gp130 (MAB-230). Ab labeling was detected with either
PE-tagged anti-mouse IgG or nonspecific rabbit IgG and compared
with cells labeled with secondary PE-conjugated Abs alone. Data were
acquired from 10,000 gated events per sample.
Immunoprecipitation and Western blotting
HPMC monolayers (75-mm2) were stimulated
with OSM (10 ng/ml), HYPER-IL-6 (50 ng/ml), or medium alone
for 30 min at 37°C in a humidified 5% CO2
incubator. Cells were washed with ice-cold saline and lysed with RIPA
buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 3 mM sodium
orthovanadate, and a mixture of protease inhibitors) containing 1%
(v/v) Nonidet P-40. Lysates containing 
2 mg of protein were
precleared with 100 µl (50% slurry in RIPA buffer) protein
A-conjugated Sepharose (Amersham Pharmacia Biotech) for 2 h at
4°C. The supernatant was incubated overnight at 4°C with 2 µg/ml
polyclonal anti-gp130 Ab (C-20; Santa Cruz Biotechnology) and
immune complexes recovered by addition of 50 µl protein A-conjugated
Sepharose. Samples were washed in RIPA buffer and finally solubilized
in 62.5 mM Tris-HCl (pH 6.8) containing 2% (v/v) SDS, 0.05% (w/v)
bromophenol blue for analysis by Western blotting. Immunoprecipitated
proteins were separated under reducing conditions in a 7.5%
SDS-polyacrylamide gel and electroblotted onto Hybond-P membrane
(Amersham Pharmacia Biotech). For analysis of gp130, proteins were
separated under nonreducing conditions. Membranes were blocked with 5%
(w/v) BSA/PBS containing 3 mM sodium orthovanadate for 2 h at room
temperature, and probed with either a mixture of two monoclonal
phosphotyrosine Abs (2 µg/ml PY-20 and 1 µg/ml 4G10) or 2 µg/ml
rabbit polyclonal anti-gp130 (H-255). Membranes were washed and
incubated for 1 h at room temperature with HRP-conjugated
anti-mouse or anti-rabbit IgG (DAKO). Immunolabeled proteins
were visualized by ECL (SuperSignal; Pierce, Rockford, IL). To detect
OSMR, the membrane was stripped with 62.5 mM Tris-HCl (pH 6.8), 2%
(w/v) SDS, and 100 nM 2-ME for 30 min, blocked overnight with 5% (w/v)
BSA. Blots were then probed with 2 µg/ml anti-OSMR
(AN-A2).
Preparation of nuclear extracts and EMSA
HPMC monolayers (75 mm2) were stimulated
with 10 ng/ml OSM as indicated in Fig. 3
, B and
C. Cells were washed with ice-cold PBS and collected by
gentle scrapping. Nuclear extracts were prepared using a rapid
technique for the extraction of DNA-binding proteins. Briefly, isolated
cells were resuspended in cold buffer A (10 mM HEPES-KOH (pH 7.9), 1.5
mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) and
allowed to swell on ice for 10 min. The cells were vortexed and
centrifuged. The pellet was resuspended in 50 µl of buffer B (20 mM
HEPES-KOH (pH 7.9), 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.3 mM DTT, 0.2 mM PMSF) and
incubated on ice for a further 20 min. The supernatant was separated
from the cellular debris by centrifugation and stored at -80°C until
required. EMSA were performed as described previously
(21). Oligonucleotides containing a STAT-consensus binding
motif (SIE-m67, 5'-cgaCATTTCCCGTAAATCG-3' and
5'-cgaCGATTTACGGGAAATG-3') were annealed for use in EMSA. These
double-stranded fragments were radiolabeled with
[-32P]-dTTP using the Klenow fragment of DNA
polymerase I. Specific STAT/DNA complex was identified by supershift
using anti-STAT1 (M-22) or anti-STAT3 (C-20) polyclonal
Abs.
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Cytokine levels were quantified using sandwich ELISA techniques. Human OSM, LIF, IL-6, sIL-6R, CXCL8, CXCL10, CCL5, and CCL3 levels were determined using matched Ab pairs from R&D Systems. Human CCL2 was monitored using a matched Ab pair OptEIA kit from BD Biosciences.
Statistical analysis
Results were analyzed using unpaired Student t test
or one-way analysis of variance. Analysis of regression fit presented
(see Figs. 1B and 7A) were determined using
MINITAB statistical analysis package.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peritoneal effluent was obtained from dialysis patients with
defined episodes of peritonitis over the first 5 days of infection and
from the same individuals 3–6 mo postinfection. Consistent with the
profile of IL-6 expression (17), OSM levels were
significantly elevated within the first 24 h of diagnosis and
rapidly decreased to the limit of detection by days 2–3 (Fig. 1
A). In contrast, i.p. LIF
concentrations were barely detectable throughout the 5-day period.
Intraperitoneal OSM concentrations were found to correlate
(r = 0.83, p < 0.01) with the degree
of neutrophil infiltration (Fig. 1B), emphasizing that
neutrophils are either the primary source of OSM, or that OSM
influences their recruitment. To determine the source of OSM,
neutrophils were isolated from dialysis fluid collected on day 1 of
infection and cultured overnight in the absence of serum. As shown in
Fig. 1C, neutrophils actively secrete OSM, and this release
could be blocked by inclusion of hydrocortisone, suggesting the
involvement of an inflammatory signal. In contrast, isolated peritoneal
macrophages and mesothelial cells were found not to release OSM, while
OSM secretion by these cells was not induced by a cell-free supernatant
derived from S. epidermidis (a bacterial peritonitis
pathogen) or proinflammatory cytokines (IL-1/TNF-; data not
shown).
OSM activates HPMCs via OSMR/gp130 signaling
The elevation of OSM in dialysis fluid during peritonitis suggests
that this cytokine may perform a regulatory role during the
inflammatory response. OSM elicits its biological effects through
binding either a cognate LIFR or specific OSM receptor OSMR, which
heterodimerize with gp130 to initiate a signaling event (22, 23). To characterize expression of these receptors, FACS
analysis was performed on HPMCs using specific Abs. As illustrated in
Fig. 2, HPMCs express gp130 and the
cognate OSMR, but no LIFR. Although neutrophils and mononuclear
leukocytes showed no expression of LIFR or OSMR, low levels of LIFR
were detected on lymphocytes (data not shown). These data suggest that
within the context of peritoneal infection, mesothelial cells are the
likely responders to OSM.
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, HPMCs were stimulated with 10 ng/ml
OSM for 30 min. Cell lysates were prepared and coimmunoprecipitation
techniques used to examine the phosphorylation of gp130 and OSMR
(Fig. 3A). Immunoprecipitation
of gp130, followed by Western blot analysis with
anti-phosphotyrosine Abs (PY-20 and 4G10 mixture) identified two
bands of 130 and 160 kDa, suggesting heterodimerization of gp130
with the OSMR. To identify each component, blots were stripped and
reprobed with either an anti-gp130 Ab or a monoclonal
anti-OSMR Ab. Although gp130 was detected in all cellular
extracts, OSMR was only observed in OSM-stimulated lysates. Control
stimulations using the IL-6-sIL-6R fusion protein HYPER-IL-6 resulted
in the detection of a single 130-kDa band (Fig. 3A). EMSAs
confirmed activation of STAT by OSM (Fig. 3B), and showed
that OSM preferentially induced STAT3, but not STAT1, activation in
nuclear extracts derived from HPMCs (Fig. 3C). OSM stimulates cytokine expression in HPMCs
OSM stimulated a dose- and time-dependent release of CCL2
and IL-6 from HPMCs (Fig. 4).
Although optimal induction of CCL2 and IL-6 was observed with 10 ng/ml
OSM, the time course of their secretion was distinct (Fig. 4, A and C). Maximal expression of CCL2 was observed
8 h after stimulation, whereas IL-6 levels gradually increased
over the 24-h assay period (Fig. 4, B and D). In
contrast, no expression of CXCL8 (IL-8), CXCL10 (IFN-
-inducible
protein of 10 kDa), CCL3 (macrophage-inflammatory protein-1),
or CCL5 (RANTES) was observed following stimulation with OSM (Table I).
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or anti-gp130 Abs
resulted in a concentration-dependent blockade of OSM-induced CCL2 and
IL-6 (Fig. 5, A and
B) expression (anti-OSMR: 80% for CCL2 and 65% for
IL-6; anti-gp130: 80% for CCL2 and 95% for IL-6). Inclusion of a
neutralizing LIFR Ab had no effect on IL-6 (Fig. 5C) or CCL2
(data not shown) expression. None of the Abs used influenced the basal
mesothelial release of CCL2 and IL-6 (data not shown). Consistent with
the low affinity of OSM for soluble gp130 (sgp130; Ref.
24), addition of this naturally occurring antagonist had
no affect on OSM-induced IL-6 (Fig. 5C) or CCL2 (data not
shown) secretion by HPMCs.
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-induced cytokine expression in HPMCs
The inflammatory response initiated during acute bacterial
peritonitis is primarily driven by the proinflammatory cytokines IL-1
and TNF- (14, 25). Interestingly, increases in i.p.
IL-1 (25) coincide with increases in OSM. Consequently, we
examined whether OSM affects IL-1-induced gene expression. Using a
suboptimal dose of IL-1 (1 pg/ml) for activation of HPMCs (18, 26), experiments examined the effect of OSM on
IL-1-stimulated release of CCL2 and IL-6 (Fig. 6A). Costimulation of HPMCs
with IL-1 and OSM significantly elevated CCL2 and IL-6 secretion in
a marginally synergistic manner. In contrast, IL-1-induced
expression of CXCL8 was blocked in a dose-dependent manner by OSM, with
10 ng/ml OSM showing an 80% inhibition of CXCL8 release (Fig. 6B). Blockade of CXCL8 by OSM was specific for this
neutrophil-activating chemokine because OSM had no affect on
IL-1-induced secretion of CCL5 and CXCL10 (data not shown).
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Through analysis of sIL-6R and OSM levels in peritoneal dialysis
fluid from infected individuals, it is evident that on day 1 of
bacterial peritonitis, OSM concentrations show a strong correlation
(r = 0.8, p < 0.01) with those of
sIL-6R (Fig. 7A). Indeed, the
effect of OSM on IL-1-mediated release of CCL2 and, in particular,
CXCL8 expression is similar to the action of the IL-6/sIL-6R complex
(17). Consequently, we investigated the potential
interaction between OSM and the sIL-6R/IL-6 complex in coregulating
CCL2 and CXCL8 expression. The sIL-6R occurs as two distinct isoforms,
which are released via proteolytic cleavage of the cognate IL-6R
(PC-sIL-6R) and differential mRNA splicing (DS-sIL-6R)
(27). However, neutrophils do not release DS-sIL-6R
(28). Fig. 7, B and C shows the
effect of OSM on PC-sIL-6R-mediated signaling. Stimulation of HPMCs
with a suboptimal dose of PC-sIL-6R (10 ng/ml) and IL-6 (10 ng/ml)
induced a small but significant increase in CCL2 expression, which when
added in combination with OSM caused an accumulative production of CCL2
(Fig. 7B). In contrast, OSM acted synergistically with the
sIL-6R to enhance inhibition of IL-1-induced CXCL8 expression (Fig. 7C). Indeed, when OSM was added in combination with
PC-sIL-6R/IL-6, inhibition of CXCL8 secretion was significantly
(p < 0.01) greater than the effect observed
upon individual stimulation. Similar regulation of CCL2 and CXCL8 was
observed if recombinant DS-sIL-6R was used (data not shown). Thus,
release of OSM and sIL-6R from infiltrating neutrophils may suppress
neutrophil-activating chemokine expression and ultimately block
neutrophil recruitment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of peritoneal effluent from dialysis patients with clinical peritonitis showed that concentrations of OSM were elevated within the first 24 h of infection and directly correlated with the degree of neutrophil infiltration. Neutrophils have previously been shown to produce OSM in response to inflammatory mediators (13, 29, 30), and consistent with these reports, the infiltrating neutrophil population was identified as the primary source of OSM. Although others have characterized mononuclear leukocytes as being major OSM producers (5, 6, 31), resident peritoneal macrophages were found not to secrete OSM, while detection of OSM in bacterial peritonitis did not coincide with the infiltration of mononuclear leukocytes (days 3–4; Ref. 17). These contradictory findings may reflect a differential action for OSM in acute and chronic inflammatory conditions, since OSM release by mononuclear leukocytes has been observed in more progressive clinical disorders (6, 31).
The extremely rapid and transient profile of OSM expression during
episodes of bacterial infection is striking, and emphasizes that its
biological activities must be tightly regulated. Expression of the
cognate OSMR was confined to mesothelial cells, and was not detected
on neutrophils or mononuclear leukocytes. Consequently, mesothelial
cells are likely to be the primary responders to OSM within the
peritoneal cavity, and stimulation of HPMCs by OSM resulted in the
phosphorylation of a gp130/OSMR heterodimer, and activation of
STAT3. However, it remains unclear what in vivo regulatory processes
control the action of OSM. Previous studies have emphasized that the
naturally occurring antagonist sgp130 can inhibit OSM signaling. sgp130
is present at 250 ng/ml in human plasma and its activity has
been shown to influence neutrophil recruitment (17) and to
suppress development of colitis (32). However, sgp130
concentrations known to inhibit sIL-6R-mediated signaling did not
affect OSM-induced cytokine expression. This may be related to the
apparent low affinity of OSM for sgp130 (24). Thus,
blockade of OSM signaling in vivo may require more specialized
mechanisms, such as down-regulation of the OSMR (33) or
the generation of a soluble antagonistic form of the OSM receptor. To
date, no such soluble receptor has been identified.
OSM stimulated the secretion of CCL2 and IL-6 by HPMCs; however,
time-course analyses showed a burst of CCL2 release that was maximal
following a 6-h stimulation with OSM. In contrast, IL-6 secretion
increased gradually over time. These differences were specific for OSM,
since Abs directed against gp130 and OSMR selectively blocked the
induction of both mediators. A similar pattern of IL-6 secretion has
also been reported in U373 astrocytes, where the authors speculated
that IL-6 transcription might require two regulatory processes
(12).
Appropriate control of leukocyte recruitment from an initial neutrophil
influx to a more sustained population of mononuclear leukocytes is an
underlining feature in the resolution of inflammatory conditions such
as peritonitis (16, 34). Using a series in vitro and in
vivo approaches, we have recently defined roles for IFN-
(26), IL-6, and sIL-6R (17) in the control of
this critical step. The data presented in this study suggest that OSM
may directly or indirectly contribute to the regulation of this
event. Through direct activation of the mesothelium, OSM has the
capacity to induce CCL2 expression and to inhibit proinflammatory
cytokine activation of CXCL8. Thus, OSM may contribute to the
cessation of neutrophil recruitment and concurrent attraction of
mononuclear leukocytes. This is also emphasized by the synergistic
inhibition of IL-1-induced CXCL8 expression by OSM and the
sIL-6R/IL-6 complex, while the secretion of IL-6 in response to OSM
might further contribute to the control of leukocyte recruitment by
sIL-6R. Because modulation of chemokine secretion in response to OSM
has been reported in other cell types (8, 9, 10, 11, 12), it is
conceivable that this model of events may be applicable to other
inflammatory conditions.
This study demonstrates that OSM performs an important regulatory role during acute inflammation. OSM may be included in an increasing list of mediators that are released from the infiltrating neutrophils so as to suppress neutrophil attraction (e.g., L-selectin and sIL-6R), and as is the case for the IL-6/sIL-6R complex, promote recruitment of mononuclear leukocytes. Ultimately, regulation of these neutrophil-derived mediators might be central to orchestrating the transition of innate to acquired immunity seen in various inflammatory settings.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Suzanne M. Hurst at the current address: Center for Research in Biomedicine, Faculty of Applied Sciences, University of West of England, Bristol, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, U.K. E-mail address: Suzanne.Hurst{at}uwe.ac.uk
3 Abbreviations used in this paper: OSM, oncostatin M; CAPD, continuous ambulatory peritonitis dialysis; sIL-6R, soluble IL-6R; sgp130, soluble gp130; LIF, leukemia inhibitory factor; CXCL, CXC chemokine ligand; CCL, CC chemokine ligand; OSMR, OSM receptor ; HPMC, human peritoneal mesothelial cell.
Received for publication May 24, 2002. Accepted for publication August 19, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and tumor necrosis factor . Am. J. Pathol. 142:1876.[Abstract]
autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin. J. Immunol. 167:3435., interleukin-1- and interleukin-1 receptor antagonist in dialysate and serum from patients on continuous peritoneal dialysis. Am. J. Kidney Dis. 27:402.[Medline]
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  E. Larrea, R. Aldabe, I. Gonzalez, V. Segura, P. Sarobe, I. Echeverria, and J. Prieto Oncostatin M Enhances the Antiviral Effects of Type I Interferon and Activates Immunostimulatory Functions in Liver Epithelial Cells J. Virol., April 1, 2009; 83(7): 3298 - 3311. [Abstract] [Full Text] [PDF]
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 P. G. Tiffen, N. Omidvar, N. Marquez-Almuina, D. Croston, C. J. Watson, and R. W. E. Clarkson A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo Mol. Endocrinol., December 1, 2008; 22(12): 2677 - 2688. [Abstract] [Full Text] [PDF]
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) or 1 ng/ml (B) or 3 ng/ml
(D) OSM (•) for specified times. ELISA measured CCL2
(A and B) or IL-6 (C and
D) concentrations in the collected supernatant. Results
are the mean ± SEM of five separate experiments.
) or 1–10
µg/ml neutralizing gp130 Ab (
) were incubated with growth-arrested
HPMCs for 24 h in the presence of either 1 ng/ml
(A) or 3 ng/ml (B) of OSM. ELISA measured
CCL2 (A) or IL-6 (B) in the supernatants.
Results are the mean ± SEM of five separate experiments, where
*, p < 0.01 represents a significant inhibition
in both OSM-induced CCL2 or IL-6 expression. C, A total
of 1–10 µg/ml LIFR Ab (
) or 1–5 µg/ml sgp130 (
) were
incubated with HPMCs in the presence of 3 ng/ml OSM and IL-6 was
measured in the supernatant after 24 h. Results are the mean
± SEM of four separate experiments.
) or in the
presence 10 ng/ml IL-6 (•), or 10 ng/ml PC-sIL-6R plus 10 ng/ml IL-6
(