Role of STK in mouse liver macrophage and
endothelial cell responsiveness during
Debra L. Laskin,*,1Li Chen,* Pamela A. Hankey,†and Jeffrey D. Laskin‡
*Rutgers University, Piscataway, New Jersey, USA;†Pennsylvania State University, University Park, Pennsylvania, USA; and
‡University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA
RECEIVED FEBRUARY 28, 2010; REVISED APRIL 7, 2010; ACCEPTED APRIL 8, 2010. DOI: 10.1189/jlb.0210113
Acute endotoxemia is associated with excessive pro-
duction of proinflammatory mediators by hepatic mac-
rophages and endothelial cells, which have been impli-
cated in liver injury and sepsis. In these studies, we an-
alyzed the role of MSP and its receptor STK in
regulating the activity of these cells. Acute endotox-
emia, induced by administration of LPS (3 mg/kg) to
mice, resulted in increased expression of STK mRNA
and protein in liver macrophages and endothelial cells,
an effect that was dependent on TLR-4. This was cor-
related with decreased MSP and increased pro-MSP in
serum. In Kupffer cells, but not endothelial cells, MSP
suppressed LPS-induced NOS-2 expression, with no
effect on COX-2. LPS treatment of mice caused a rapid
(within 3 h) increase in the proinflammatory proteins
NOS-2, IL-1?, and TNF-?, as well as TREM-1 and
TREM-3 and the anti-inflammatory cytokine IL-10 in liver
macropahges and endothelial cells. Whereas LPS-in-
duced expression of proinflammatory proteins was un-
changed in STK?/?mice, IL-10 expression was reduced
significantly. Enzymes mediating eicosanoid biosynthe-
sis including COX-2 and mPGES-1 also increased in
macrophages and endothelial cells after LPS adminis-
tration. In STK?/?mice treated with LPS, mPGES-1 ex-
pression increased, although COX-2 expression was
reduced. LPS-induced up-regulation of SOD was also
reduced in STK?/?mice in liver macrophages and endo-
thelial cells. These data suggest that MSP/STK signal-
ing plays a role in up-regulating macrophage and endo-
thelial cell anti-inflammatory activity during hepatic in-
flammatory responses. This may be important in
protecting the liver from tissue injury. J. Leukoc. Biol.
88: 373–382; 2010.
MSP, also known as hepatocyte growth factor-like protein, is
an 80-kDa serum protein belonging to the plasminogen-re-
lated growth factor family. The liver is the major source of
MSP, which is released as the single-chain inactive precursor,
pro-MSP [1, 2]. During local inflammatory reactions, pro-MSP
is converted to its biologically active form by proteases present
in serum and on the plasma membrane of macrophages at
sites of injury or infection [3–7]. The receptor for murine
MSP is STK (RON in humans), a disulfide-linked heterodimer
possessing intrinsic tyrosine kinase activity [3, 8, 9]. STK/RON
has been reported to be expressed in mature peritoneal, alveo-
lar, hepatic, and dermal macrophages [10–14]. MSP was iden-
tified originally by its ability to promote murine peritoneal
macrophage chemotaxis and phagocytosis [15, 16]. However,
subsequent studies demonstrated that MSP also exerts inhibi-
tory activity on macrophages, including suppression of LPS-
induced NO and PG production [17–20]. This is thought to
be a result of inhibition of NF-?B activation and expression of
inducible NOS (NOS-2) and COX-2 and up-regulation of
SOCS1 and SOCS3 [17, 19, 21, 22]. These findings are consis-
tent with reports that STK?/?mice are hypersensitive to endo-
toxin-induced inflammation and septic shock and that this is
associated with excessive NO production and increased re-
sponsiveness to IFN-? [22–25].
Hepatic macrophages and endothelial cells play an essential
role in clearance of endotoxin from the body [26–28]. How-
ever, in the presence of excessive amounts of endotoxin, this
clearance mechanism is readily overwhelmed, leading to hepa-
totoxicity, septic shock, and multiple system organ failure [27,
29, 30]. Endotoxin is a potent activator of liver macrophages
and endothelial cells, stimulating these cells to produce proin-
flammatory and cytotoxic mediators, including TNF-? and IL-
1?, as well as NO and eicosanoids, each of which has been
implicated in endotoxin-induced hepatotoxicity [29, 31]. In
the present studies, we investigated the role of MSP and STK
in endotoxin-induced inflammatory responses of liver macro-
1. Correspondence: Department of Pharmacology and Toxicology, Rutgers
University, Ernest Mario School of Pharmacy, 160 Frelinghuysen Rd., Pis-
cataway, NJ 08854, USA. E-mail: email@example.com
Abbreviations: COX?cyclooxygenase, CuZnSOD?copper/zinc superoxide
dismutase, LOX?lipoxygenase, MnSOD?manganese superoxide dis-
mutase, mPGES?microsomal PGE synthase, MSP?macrophage-stimulat-
ing protein, NO?nitric oxide, NOS?NO synthase, PG?prostaglandin,
SOCS?suppressor of cytokine signaling, SOD?superoxide dismutase,
STK?stem cell kinase, TREM?triggering receptors expressed by myeloid
0741-5400/10/0088-373 © Society for Leukocyte Biology
Volume 88, August 2010
Journal of Leukocyte Biology 373
phages and endothelial cells. The contribution of TLR-4 to
these activities was also analyzed.
MATERIALS AND METHODS
Collagenase type IV and Escherichia coli LPS (serotype 0128:B12) were pur-
chased from Sigma Chemical Co. (St. Louis, MO, USA). Polyclonal goat
anti-human MSP? antibody and recombinant human MSP were obtained
from R&D Systems (Minneapolis, MN, USA). Rabbit anti-mouse MnSOD
and CuZnSOD antibodies were from Stressgen Bioreagents (Ann Arbor,
MI, USA) and anti-mouse COX-2 antibody from Cayman Chemical (Ann
Arbor, MI, USA). Goat anti-mouse and donkey anti-goat HRP-conjugated
secondary antibodies, rabbit anti-mouse STK, and NOS-2 antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Male TLR-4 mutant C3H/HeJ mice, control C3H/HeOuJ (C3H/OuJ)
mice, and wild-type C57BL/6 mice (8–12 weeks) were obtained from Jack-
son Laboratories (Bar Harbor, ME, USA). C57BL/6 mice with a targeted
disruption of the p55 TNFR1 gene were kindly provided by Immunex (Se-
attle, WA, USA) and bred in the Rutgers University (Piscataway, NJ, USA)
animal facility. The generation of C57BL/6 mice, with a targeted disrup-
tion of the STK gene, has been described previously . All animals were
housed under specific pathogen-free conditions and allowed free access to
sterile water and food. The animals received humane care in compliance
with the institution’s guidelines, as outlined in the Guide for the Care and Use
of Laboratory Animals prepared by the National Academy of Sciences. To
induce acute endotoxemia, mice were administered a single i.p. injection
of LPS (3 mg/kg), repurified as described previously to exclude contami-
nants with costimulatory activity [32, 33]. Blood was collected by cardiac
puncture of the right ventricle using a 21-gauge needle and allowed to co-
agulate overnight at 4°C. Serum was collected after centrifugation of the
blood at 960 g for 30 min at 4°C.
Hepatic macrophage and endothelial cell isolation
Macrophages and endothelial cells were isolated from the livers of mice
killed with Nembutal (200 mg/kg), as described previously with some mod-
ifications [32, 33]. Briefly, the liver was perfused through the portal vein
with Ca2?/Mg2?-free HBSS (pH 7.3) containing 0.5 mM EGTA and 25 mM
HEPES, followed by Leibovitz L-15 medium containing 100 U/ml collage-
nase type IV for 2 min. All buffers were maintained at 37°C during the per-
fusion. The liver was then extracted and disaggregated, and the resulting
cell suspension was filtered through 220 ?m nylon mesh. Hepatocytes were
separated from nonparenchymal cells by three successive washes (50 g) for
5 min. Macrophages and endothelial cells were recovered by centrifugation
of the supernatant at 300 g for 7 min and then purified according to their
size and density on a Beckman J-6 elutriator (Beckman Instruments, Fuller-
ton, CA, USA), equipped with a centrifugal elutriation rotor set at 2500
rpm. The pump speed was set at 12 ml/min to load the cells. Endothelial cells
were collected at 17 ml/min and macrophages at 33 ml/min. The purity for
macrophages and endothelial cells was ?85%, as determined by differential
staining with Giemsa and transmission electron microscopy .
cDNA synthesis and PCR
Cells were stored in RNA LATER solution (Ambion Inc., Austin, TX, USA)
at –20°C until RNA isolation. DNase I-treated total RNA was extracted us-
ing an RNeasy Miniprep kit (Qiagen Inc., Valencia, CA, USA) following the
manufacturer’s instructions. RNA was quantified spectrophotometrically
using a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE,
USA). First-strand synthesis was performed as described previously [32, 33].
RNA (0.2 ?g) in 9 ?l RNase-free water was denatured at 65°C for 4 min,
cooled rapidly on ice, and then resuspended in a 20-?l final volume con-
taining 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1
mM each dNTP, 20 mM random hexamers, and 200 U Superscript II
RNase H–RT (Invitrogen, Carlsbad, CA, USA). After a 1-h incubation at
37°C, RNase H–(2 U) was added, and the samples were incubated for an
additional 20 min; samples were then denatured at 95°C for 5 min and
chilled on ice.
Relative gene expression was determined by real-time quantitative PCR
using the ABI Prism 7000 sequence detection system. Each reaction con-
tained 0.01 ?g cDNA template, 1? primer set consisting of two unlabeled
PCR primers, and a FAM dye-labeled TaqMan probe and TaqMan Univer-
sal PCR master mix in a 20-?l final volume. All reagents, including primer
sets, were obtained from Applied Biosystems (Foster City, CA, USA). The
thermal cycling parameters were set for the following conditions: one
2-min cycle at 50°C, one 10-min cycle at 95°C (for AmpliTaq gold enzyme
activation), and 40 cycles of 95°C for 15 s and 60°C for 1 min. Normaliza-
tion for the relative quantity of mRNA was accomplished by comparison
with 18S rRNA. mRNA expression data are presented as fold-change rela-
tive to untreated control, which were assigned a value of one arbitrarily.
Primer sequences used were: STK, AACTTGGCAAGGATGGTGTC; NOS-2,
GGCAGCCTGTGAGACCTTTG; IL-1?, CCAAAAGATGAAGGGCTGCT;
TNF-?, AAATTCGAGTGACAAGCCGTA; IL-10, GGTTGCCAAGCCT-
TATCGGA; TREM-1, AAGTCCACATGGGGAAGTTC; TREM-3, CCAAGCT-
GGAGATGAGGAAG; COX-2, CATTCTTTGCCCAGCACTTCAC; mPGES-1,
GGCCTTTCTGCTCTGCAGC; mPGES-2, AGCCCCTGGAAGAGGTCATC;
5-LOX, CAGGGAGAAGCTGTCCGAGT; 12-LOX, AAGTTCCTTGGCA-
GACGCC; 15-LOX, TCGGAGGCAGAATTCAAGGT; MnSOD, CACATTA-
ACGCGCAGATCATG; CuZnSOD, ACCAGTGCAGGACCTCATTTTAA.
For preparation of lysates, cells were suspended in buffer containing 1%
IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 1% protease inhibitor, and
1% phosphatase inhibitors in PBS. Lysates were clarified by centrifugation
at 16,000 g for 15 min at 4°C. Protein concentrations were measured using
a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL,
USA). Samples were fractioned on 10% SDS-polyacrylamide gels and trans-
ferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ,
USA). Nonspecific binding was blocked using 5% milk in TBS. Membranes
were incubated overnight (4°C) with primary antibody in 1% milk-TBS,
washed for 1 h using TBS, and then incubated for 1 h with secondary anti-
body (1:5000) in 2.5% milk-TBS (with 0.1% Tween 20). Antibody binding
was visualized using ECL detection reagents (Amersham Life Science, Ar-
lington Heights, IL, USA). For analysis of pro-MSP and MSP, proteins were
immunoprecipitated from serum using Catch and Release v2.0, according
to the manufacturer’s instructions (Upstate Cell Signaling, Charlottesville,
VA, USA). Briefly, serum (100 ?l) was incubated with polyclonal goat anti-
human MSP antibody (1 ?g). Bound MSP proteins were eluted with 35 ?l
Laemmli buffer (with 2-ME) and boiled for 1 min before fractionation on
SDS-polyacrylamide gels as described above. Antibodies to MSP, NOS-2,
and COX-2 were used at a dilution of 1:1000. Antibodies to MnSOD and
CuZnSOD were used at a dilution of 1:5000. For each analysis, 10 ?g pro-
tein was analyzed per lane on the gels.
Western blot and PCR experiments were repeated three to five times
(n?8–16 mice). Data from real-time PCR assays were analyzed by one-way
ANOVA using SigmaStat 3.5. A P value of ?0.05 was considered statistically
Effects of MSP on liver macrophages and endothelial
MSP has been reported to suppress the expression of proin-
flammatory proteins, including NOS-2, by murine peritoneal
Journal of Leukocyte Biology
Volume 88, August 2010
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