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The Calcineurin B Subunit (CnB) Is a New
Ligand of Integrin αM That Mediates
CnB-Induced Apo2L/TRAIL Expression in
Macrophages
Lixin Liu, Zhenyi Su, Shuai Xin, Jinbo Cheng, Jing Li, Lan
Xu and Qun Wei
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:238-247; Prepublished online 23
November 2011;
doi: 10.4049/jimmunol.1102029
http://www.jimmunol.org/content/188/1/238
The Journal of Immunology
The Calcineurin B Subunit (CnB) Is a New Ligand of Integrin
aM That Mediates CnB-Induced Apo2L/TRAIL Expression
in Macrophages
Lixin Liu,1 Zhenyi Su,1 Shuai Xin, Jinbo Cheng, Jing Li, Lan Xu, and Qun Wei
alcineurin is the only Ca2+/calmodulin-dependent serine/
threonine protein phosphatase; it is a heterodimer composed of a 61-kDa catalytic subunit (calcineurin A subunit [CnA]) and a 19-kDa regulatory subunit (calcineurin B
subunit [CnB]) (1, 2). The enzyme is expressed in many different
tissues and cells and is recognized as a modulator of T cell activation (3, 4), neuronal excitability (5), apoptosis (6, 7), and cardiac hypertrophy (8, 9). Calcineurin is one of the most important
functional proteins in immune system, which activates NFATc by
dephosphorylating it. Activated NFATc is translocated into nucleus and then upregulates the expression of IL-2, which, in turn,
stimulates the growth and differentiation of T cell and B cells (10,
11). The CnB2/2 thymocytes lose all calcineurin activity and are
defective in positive but not negative selection (12).
The role of CnB was traditionally thought to be to regulate the
phosphatase activity of CnA. However, in recent years, it has been
shown that the CnB is not just a simple protein as the regulatory
subunit of CnA. Cytosolic CnB can interact with tubulin, heat
C
Department of Biochemistry and Molecular Biology, Beijing Key Laboratory, Beijing Normal University, Beijing 10087, People’s Republic of China
1
L.L. and Z.S. contributed equally to this work and should be considered co-first
authors.
Received for publication July 14, 2011. Accepted for publication October 25, 2011.
This work was supported by grants from the National Natural Science Foundation of
China, the National Important Novel Medicine Research Project, and the International Cooperation Project.
Address correspondence and reprint requests to Dr. Qun Wei, Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 10087, People’s Republic of China. E-mail address: [email protected]
Abbreviations used in this article: Bmax, maximum binding; CnA, calcineurin A
subunit; CnB, calcineurin B subunit; aMb2, integrin aM b2; MS, mass spectrometry;
PI, propidium iodide; PK, proteinase K; qPCR, quantitative PCR; siRNA, small
interfering RNA.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102029
shock protein 60 (13), procaspase-3 (14), and PSMA7 (15) and
therefore plays very important role in apoptosis and the proteasome pathway, which are not dependent on the phosphatase activity of CnA. In previous work, we found that i.p. injection of
CnB promoted peritoneal macrophage proliferation (data not
shown), enhanced phagocytic activity (16), increased the secretion
of cytokines, and prolonged the survival of mice bearing H22
ascites tumors (17). We therefore suspected that there was a specific surface receptor on peritoneal macrophages responsible for
the activation of macrophages by CnB and for the tumoricidal
activity of CnB. It is noteworthy that there is a high concentration
of calcineurin in blood and amniotic fluid as well as in the cytosol
of cells (18), which would permit CnB to interact with monocytemacrophages in vivo.
Integrin aM (CD11b/CD18, Mac-1) is a subunit of the heterodimeric integrin aMb2. This integrin, a member of the integrin
superfamily, is expressed primarily on the surfaces of monocytes,
macrophages, granulocytes, and NK cells, which perform some
of the central functions of myeloid cells, including adhesion,
migration, chemotaxis, phagocytosis, and respiratory burst activity
(19–21). Integrin aMb2 is the most promiscuous member of the
integrin family. More than 30 protein and nonprotein molecules
have been reported to bind this receptor (22). These ligands include ICAMs 1, 2, and 3 (23), fibrinogen (24), factor X (25),
complement C3 fragment (26), urokinase plasminogen activator
receptor (27), E-selectin (28), catalase, transferrin (29), and some
nonprotein ligands such as LPS (30) and heparin (31), etc.
TRAIL is a member of the TNF family of cytokines, which
induces apoptosis of a variety of tumor cells by engaging the death
receptors DR4 and DR5, despite displaying no cytotoxicity against
most normal cells; this makes it a promising new agent for cancer
therapy (32–34). Although TRAIL is constitutively expressed in
a wide variety of normal tissues, its main role appears to be in
the immune system. It is expressed on activated T cells, NK cells,
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We showed previously that the calcineurin B subunit (CnB) plays an important role in activation of peritoneal macrophage, but the
underlying mechanism remained unknown. To examine whether there is a CnB receptor on peritoneal macrophages, we performed
the radioligand binding assay of receptors. The receptor saturation binding curve demonstrated high-affinity and specific binding;
the maximum binding was 1090 fmol/105 cells, and the Kd was 70.59 pM. Then, we used a CnB affinity resin to trap potential
receptors from highly purified peritoneal macrophage membranes. Mass spectrometry analysis showed that the binding protein
was mouse integrin aM. We next performed a competition binding experiment to confirm the binding of CnB to integrin aM. This
showed that FITC-CnB bound specifically to peritoneal macrophages and that binding was blocked by the addition of integrin aM
Ab. We observed that CnB could induce TRAIL gene expression in peritoneal macrophages in vitro and in vivo. Integrin aM Ab
blocking, RNA interference, and ligand competition experiments demonstrated that CnB-induced TRAIL expression is dependent
on integrin aM. Furthermore, the tumoricidal activity of CnB-activated peritoneal macrophages is partially dependent on
TRAIL. In addition, CnB treatment significantly prolongs the survival of mice bearing H22 ascites tumors, which has a positive
correlation with the induction level of TRAIL. These results reveal a novel function of the CnB in innate immunity and cancer
surveillance. They also point to a new signaling pathway leading to induction of TRAIL and suggest a possible application of CnB
in cancer therapy. The Journal of Immunology, 2012, 188: 238–247.
The Journal of Immunology
Materials and Methods
Materials
Recombinant human CnB protein was prepared in our laboratory (the amino
acid sequences of human, mouse, and rat CnB protein are identical). Endotoxin was removed with Cellufine ETclean S endotoxin-removing beads
(Chisso). The purity of CnB was $98%, and LPS contamination was ,4
EU/mg. CnA was purified in our laboratory. Albumin egg (OVA), avidin,
fibrinogen, LPS, unfractionated heparin, and polymyxin B sulfate were
purchased from Sigma-Aldrich. Proteinase K (PK) was from Roche
Diagnostics and IFN-g from Canspec Scientific Instruments (Shanghai,
China). Anti-mouse integrin aM and the rat IgG2b k isotype control
(functional grade purified) were purchased from eBioscience (San Diego,
CA). TRAIL Ab for Western blotting was from Proteintech Group (Chicago, IL), and neutralizing TRAIL Ab was a gift from Dr. Dexian Zheng
(Chinese Academy of Medical Sciences, Beijing, China). WGA-Sepharose
and cyanogen bromide-activated Sepharose 4B were from Pharmacia.
Small interfering RNA (siRNA) was purchased from Ribobio (Guangzhou,
China), and the Annexin V/propidium iodide (PI) apoptosis detection kit
was from Bender MedSystems.
Animals
Specific pathogen-free male Kunming mice and BALB/c mice, 6–8 wk of
age, were purchased from the Department of Laboratory Animal Science
of Peking University Health Science Center and Vital River Laboratories
(Beijing, China), respectively. All animals were housed in microisolator
cages with autoclaved food and bedding to minimize exposure to viral and
microbial pathogens, and all procedures were approved by the Institutional
Animal Care and Use Committee.
fiber filters (Whatman) using a Brandel cell harvester (Brandel) and
washed four times with cold PBS. The radioactivity retaining on the filters
was measured using a Beckman 5500 g-counter (Beckman Coulter). To
calculate specific binding, nonspecific binding (in the presence of an excess of unlabeled CnB) was subtracted from total binding (in the absence
of unlabeled CnB). The maximum binding (Bmax) and Kd were determined by Scatchard analysis.
Separation and purification of peritoneal macrophage
membranes
Mouse peritoneal macrophages (5 g) were mixed with 5 volumes homogenate buffer (50 mmol/l HEPES [pH 7.4], 1 mmol/l CaCl2, 1 mmol/l
PMSF, and 13protease inhibitor mixture), then homogenized with ∼30
strokes of a glass Dounce homogenizer on ice. The suspension was
centrifuged at 4˚C and 600 3 g for 10 min to remove unbroken cells and
nuclei. The pellet was discarded, and the supernatant was centrifuged at
8,000 3 g for another 10 min to remove mitochondria. The membranes
were pelleted by ultracentrifugation (100,000 3 g, 20 min) and further
purified by sucrose density gradient centrifugation. Thereafter, they were
solubilized in 50 mmol/l HEPES (pH 7.4), 3% Triton X-100, 0.1 mmol/l
PMSF, and 13 protease inhibitor mixture and incubated for 30 min at 4˚C
with stirring. The suspension was clarified by centrifugation at 4˚C and
200,000 3 g for 30 min. The supernatant (7 ml) was mixed with 2 ml
WGA-sepharose and incubated at 4˚C with stirring for 20 h. The sepharose
was then transferred to a column and washed thoroughly with washing
buffer (50 mmol/l HEPES [pH 7.4], 0.1% Triton X-100, 150 mmol/l NaCl,
10 mmol/l MgCl2, 0.1 mmol/l PMSF, and 13 protease inhibitor mixture),
and the column was eluted with 15 ml elution buffer (50 mmol/l HEPES,
0.1% Triton X-100, 0.3 mol/l N-acetylglucosamine, 0.1 mmol/l PMSF, and
13 protease inhibitor mixture). One-milliliter fractions were collected, and
protein concentrations were determined with a BCA Protein Assay Kit
(Pierce).
Affinity purification of the CnB receptor and MS
CnB protein was coupled to cyanogen bromide-activated Sepharose 4B
(Pharmacia). Fractions eluted from the WGA-sepharose column were incubated with CnB-sepharose for 20 h at 4˚C with stirring. Thereafter, the
sepharose was transferred to a column and washed thoroughly with
washing buffer (50 mmol/l HEPES [pH 7.4], 0.1% Triton X-100, 1 mol/l
NaCl, 0.1 mmol/l PMSF, and 13 protease inhibitor mixture) and then
eluted with 10 ml elution buffer (50 mmol/l HEPES, 0.1% Triton X-100,
0.6% octyl glucopyranoside, 1 mol/l NaCl, 0.1 mmol/l PMSF, and 13
protease inhibitor mixture). Specific binding of [125I]-CnB to the fractions
was measured using a polyethylene glycol assay (47). In brief, fractions
were incubated with [125I]-labeled CnB for 16 h at 4˚C in binding buffer
(50 mM Tris-HCl [pH 7.4], 1% Triton X-100, and 0.1% BSA) in the
presence or absence of excess unlabeled CnB. The receptor–[125I]–CnB
complex was separated from free [125I]-CnB by adding bovine g-globulin
and PEG 6000, followed by centrifugation. The supernatants were aspirated, and the radioactivity of the pellets was counted in a g counter. The
Cell isolation and culture
Resident peritoneal macrophages were harvested from the Kunming mice,
washed, and resuspended in RPMI 1640 serum-free medium containing
50 U/ml penicillin and 50 mg/ml streptomycin. These cells were used for
receptor purification and saturation binding assays or cultured on plates for
TRAIL-related experiments. In most cases, they were plated in 12-well
culture plates (2.5 3 106 cells/well) and incubated for 2 h at 37˚C in a 5%
CO2 atmosphere in a humidified incubator. Nonadherent cells were removed by washing three times with RPMI 1640 medium, and the adherent
macrophages were cultured overnight in serum-free conditions before
being exposed to drugs. RAW264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS. H22 hepatocarcinoma cells
were obtained from H22 tumor-bearing Kunming mice and cultured in
RPMI 1640 medium containing 10% heat-inactivated FBS.
Radiolabeling of CnB and receptor binding assays
CnB was iodinated with [125I]Na by the 1,3,4,6-tetrachloro-3a,6a,diphenylglycoluril method (46). Peritoneal macrophages (2.55 3 105) were
incubated with [125I]-CnB (15–185 pM) in the presence (control) or absence of an excess of unlabeled CnB for 20 h on ice. At the end of the
period, the cell suspensions were filtered through Whatman GF/B glass
FIGURE 1. Scatchard analysis of the binding of [125I]-CnB to peritoneal
macrophages. Peritoneal macrophages (2.55 3 105) were incubated with
different concentrations of [125I]-CnB (15–185 pM) for 20 h at 0˚C. Following incubation, the cells were washed, and radioactivity was counted
with a g-radiation counter. Specific binding was calculated by subtracting
nonspecific binding (in the presence of excess [40 mM] unlabeled CnB).
Bmax and Kd were determined by Scatchard analysis. This figure is one
representative result of three independent experiments.
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macrophages, monocytes, dendritic cells, and neutrophils and
plays important roles in tumor surveillance and defense against
viral infections and many other physiological and pathological
processes (35, 36). In immune cells, TRAIL is induced by several
pathways including the type I or II IFN-mediated JAK/STAT (37–
40) and TLR agonist-mediated TLR pathways (41–43). Histone
deacetylase inhibitors induce TRAIL in human breast tumors via
the transcription factor Sp1 (44), and proline oxidase promotes
TRAIL expression via NFAT (45).
To examine whether there is a CnB receptor on peritoneal
macrophages, we used a CnB affinity resin to trap potential receptors from highly purified peritoneal macrophage membranes.
Mass spectrometry (MS) analysis showed that the binding protein
was mouse integrin aM. Because CnB increased the expression
of TRAIL, we tested whether this expression was mediated by
integrin aM. To our knowledge, there is no report that TRAIL
expression is correlated with integrin aM. If present, they would
add new insights into the mechanisms regulating TRAIL expression and point to a strategy for drug screening based on the receptor.
239
CnB IS A NEW LIGAND OF INTEGRIN aM
240
fraction with maximum [125I]-CnB binding activity was concentrated by
centrifugal ultrafiltration and analyzed by SDS-PAGE (7.5% separating gel
and 4% stacking gel). Protein bands were excised from the SDS-PAGE
gels and transferred to a siliconized Eppendorf tube for trypsin digestion,
and the resulting fragments were analyzed on a Qstar Pulsar I Quadrupole
TOF-MS (Applied Biosystems/MDS Sciex, Toronto, ON, Canada). Proteins were identified either by peptide mass fingerprinting or tandem MS
ion fragmentation using MASCOT software by searching the Swiss-Prot
database.
RNA extraction and real-time PCR
Total RNA was extracted from peritoneal macrophages using an AxyPrep
Multisource Total RNA Miniprep Kit (Axygen). Reverse transcription was
performed with a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio).
Real-time PCR primers were designed with Primer 5.0 software, and the
sequences were as follows: mouse TRAIL forward, 59-GCCACAGACACTTTCGGTGTT-39 and reverse, 59-TGATCTCATTTTGCGGAAAGAA-39;
integrin aM forward, 59-AAACCACAGTCCCGCAGAGA-39 and reverse,
59-CGTGTTCACCAGCTGGCTTA-39; and b-actin forward, 59-AGAGGGAAATCGTGCGTGAC-39 and reverse, 59-CAATAGTGATGACCTGGCCGT-39.
PCR was performed on an Applied Biosystems 7500 Real Time PCR
system using SYBR Green Master Mix reagent (Applied Biosystems).
Reactions were performed in triplicate.
Western blot analysis was performed on whole cell lysates of the peritoneal
macrophages. Cell lysates were resolved by SDS-PAGE under reducing
RNA interference
RAW264.7 macrophages were transfected with siRNA targeting mouse
integrin aM or negative control siRNA using HiPerFect Transfection
Reagent (Qiagen) according to the manufacturer’s instructions. In brief,
cells were seeded at 3 3 105 cells/well in 12-well plates and incubated for
6 h in the presence of 750 ng siRNA complexed with 18 ml HiPerFect.
Knockdown efficiency was assessed by quantitative PCR (qPCR) 48 h after
transfection. The sequences of the siRNAs targeting integrin aM were as
follows: siRNA1 sense, 59-GCACUGAGAUCCUGUUUAA dTdT-39 and antisense, 39-dTdT CGUGACUCUAGGACAAAUU-59; and siRNA2 sense, 59GGAGAAUACUUAUGUGAAU dTdT-39 and antisense, 39-dTdT CCUCUUAUGAAUACACUUA-59.
Flow cytometry
Competitive binding and apoptosis assays were performed by flow cytometry. In brief, peritoneal macrophages isolated as above were incubated
with 10 mg/ml FITC-labeled avidin or 10 mg/ml FITC-labeled CnB for 1 h
at 4˚C in the presence or absence of 10 mg/ml integrin aM mAb. The
cells were washed twice and analyzed with an FACSVantage SE (BD
Biosciences). H22 cells were cocultured with CnB-activated peritoneal
FIGURE 2. Affinity purification of the CnB receptor from peritoneal macrophage membranes and SDS-PAGE analysis. A, CnA but not OVA binds to
CnB affinity resin. CnA or OVA protein was incubated with CnB affinity resin or empty resin for 16 h at 4˚C. The fractions eluted from resins were
concentrated and subjected to SDS-PAGE. Lanes 1 and 4, CnA and OVA protein input; lanes 2 and 5, fractions eluted from CnB affinity resin; and lanes 3
and 6, fractions eluted from empty resin. Proteins were stained with Coomassie brilliant blue. B, Binding activity profile of [125I]-CnB to fractions of CnB
affinity chromatography. The fractions eluted from CnB affinity resin were incubated with [125I]-labeled CnB (1.5 3 105 cpm) at 4˚C for 16 h in the
presence or absence of excess unlabeled CnB. The receptor–[125I]–CnB complexes were separated from free [125I]-CnB by the polyethylene glycol assay as
mentioned above, and the radioactivity of pellets was counted in a g counter. To calculate the specific binding, the nonspecific binding (in the presence of
excess unlabeled CnB) was subtracted from the total binding (absence of unlabeled CnB). C, SDS-PAGE analysis of fractions eluted from the CnB affinity
resin. The fraction with maximum [125I]-CnB binding activity was concentrated and subjected to SDS-PAGE followed by Coomassie brilliant blue staining.
Lane 1, fraction eluted from empty resin; and lane 2, fraction eluted from CnB affinity resin. This figure is one representative result of three independent
experiments.
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Western blot analysis
conditions and then electrotransferred to a nitrocellulose membrane
(Millipore). The membrane was blocked with 5% nonfat milk and incubated
with primary Ab for 2 h at room temperature, followed by biotin-conjugated
species-specific secondary Ab for 1 h, then with avidin-HRP for 30 min.
Protein bands were detected with SuperSignal ECL reagents (Pierce) and
visualized by autoradiography.
The Journal of Immunology
Table I.
No.a
1
2
3
241
Identification of protein bands by MS
Protein Name
Database Accession No.b
Mass (kDa)c
Species
Integrin aM precursor
Calcineurin B subunit type 1
Trypsin inhibitor A precursor
(Kunitz-type trypsin)
P05555
CANB1_MOUSE
P01070
128.6
19.3
24.3
Mouse
Mouse
Soybean
a
The protein bands can be seen on the SDS-PAGE gel in Fig. 2C.
Accession numbers were obtained from the Swiss-Prot database.
The predicted molecular mass values were derived from the Swiss-Prot database.
b
c
macrophages for 30 h, and then apoptosis of the H22 cells was examined
by Annexin V–FITC/PI staining. The H22 cells were washed twice with
ice-cold PBS and stained with 10 ml Annexin V and 5 ml PI in 200 ml
binding buffer for 15 min at room temperature. After staining, 300 ml
binding buffer was added to each tube, and samples were analyzed on the
flow cytometer. Unstained cell sample and cells stained with Annexin V or
PI only were prepared for fluorescence compensation.
mixture and so present in the washing buffer and elution buffer.
As for the CnB band, we suspect that it was the endogenous protein that had bound to integrin aM in the process of membrane
In vivo tumor models
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In the therapeutic model, BALB/c mice were i.p. inoculated with H22
hepatocarcinoma cells (1 3 106). Two days after injection of tumor cells,
mice were treated with CnB, IFN-g, or normal saline for 10 d (i.p.).
Survival following tumor challenge was recorded. In the prophylactic and
therapeutic model, Kunming mice were treated with CnB, heat-inactivated
CnB, or normal saline for 3 d (i.p.). Mice were then i.p. inoculated with
H22 cells (1 3 106), followed by drug treatment for 7 d. All mice were
sacrificed 60 d after tumor challenge.
Statistical analysis
Data are expressed as means and SEM. In vivo data were analyzed by twotailed t tests and the Gehan-Breslow-Wilcoxon test. A p value ,0.05 was
considered statistically significant.
Results
CnB binds specifically to murine peritoneal macrophages
To determine if CnB binds specifically to murine peritoneal
macrophages, we incubated the macrophages with different concentrations of [125I]-CnB on ice for 20 h, measured cell-bound
radioactivity, and calculated the specific binding of [125I]-CnB.
The receptor saturation binding curve (Fig. 1) demonstrated specific binding; the Bmax was 1090 fmol/105 cells, and Kd was
70.59 pM. These findings suggested that a high-affinity receptor
for CnB was present on peritoneal macrophages.
Integrin aM is a receptor for CnB on murine peritoneal
macrophages
We next investigated whether there was a receptor for CnB on
the peritoneal macrophages. First, we assessed the availability and
specificity of the CnB affinity resin. CnA was used as a positive
control, and OVA was used as a negative control. It was shown that
CnA but not OVA could specifically bind to CnB affinity resin (Fig.
2A), which is consistent with our knowledge of CnB. Next, highly
purified peritoneal macrophage membranes prepared as described
in Materials and Methods were incubated with CnB affinity resin
for 16 h at 4˚C, and bound proteins were eluted with octyl glucopyranoside buffer. Fractions 7 and 8 showed specific binding of
[125I]-labeled CnB (Fig. 2B). The fraction with maximum binding
activity was concentrated and subjected to SDS-PAGE analysis.
We observed three major bands at ∼160–170, 22–24, and 18 to 19
kDa, respectively (Fig. 2C). MS analysis showed that the three
bands were mouse integrin aM, Kunitz-type trypsin inhibitor,
and CnB (Table I). Integrin aM is a subunit of the integrin aM
b2 (aMb2) molecule, also known as Mac-1 or CR3. Kunitz-type
trypsin inhibitor (24 kDa) is a known ligand of integrin aM
(29). It is a widely used component of the protease inhibitor
FIGURE 3. Integrin aM Ab blocks binding of FITC-CnB to peritoneal
macrophages. A, Peritoneal macrophages (1 3 106 /ml) were incubated
with 5 mg/ml integrin aM Ab (M1/70) or 5 mg/ml isotype Ab (rat IgG2b)
for 30 min at room temperature, followed by staining with FITC-conjugated anti-rat IgG for 15 min at room temperature. Cells were washed three
times before being analyzed by flow cytometry. The percentages of
integrin aM-positive cells are indicated by the line M1. B, Integrin aM Ab
blocks binding of FITC-CnB to peritoneal macrophages. Peritoneal macrophages (5 3 106/ml) were incubated with 10 mg/ml FITC-CnB or FITCavidin for 1 h at 4˚C in the presence or absence of 10 mg/ml integrin aM
Ab. Cells were washed twice and subjected to flow cytometry. The percentages of integrin aM-positive cells are indicated by the line M1. This
figure is one representative result of three independent experiments.
242
preparation and had then bound to the CnB affinity resin complexed with integrin aM.
Integrin aM Ab blocks binding of FITC-CnB to peritoneal
macrophages
The above experiment showed that CnB could interact with integrin
aM in the presence of detergent. We next asked whether it could
bind under physiological conditions. First, we examined expression of integrin aM on the surface of peritoneal macrophages. As
expected, .70% of the peritoneal macrophages were integrin aM
positive (Fig. 3A). Next, we performed a competition binding
experiment to confirm the binding of CnB to integrin aM. This
CnB IS A NEW LIGAND OF INTEGRIN aM
showed that FITC-CnB bound specifically to peritoneal macrophages and that binding was blocked by the addition of integrin
aM Ab (Fig. 3B).
CnB induces TRAIL gene expression in peritoneal
macrophages in vitro and in vivo
Because CnB bound specifically to integrin aM on peritoneal
macrophages, we tested whether it could activate the macrophages
and induce the expression of important functional molecules. To
address these questions, we measured the expression of several
proinflammatory factors (TNF-a, IL-6, and IL-1b), chemokines
(MIP-2, keratinocyte chemoattractant, and LPS-induced CXC
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FIGURE 4. CnB upregulates TRAIL expression in peritoneal macrophages in vitro and in vivo. A, CnB upregulates TRAIL expression at the mRNA
level. Peritoneal macrophages were plated in 12-well culture plates (2.5 3 106 cells/well) and incubated with CnB or control proteins (avidin and fibrinogen) for 10 h. Total RNA was extracted, and TRAIL expression was analyzed by qPCR. Results were normalized to b-actin expression and are
presented as fold increases over the medium-only control. B, CnB upregulates TRAIL expression at the protein level. Peritoneal macrophages were plated
in six-well culture plates (6 3 106 cells/well) and incubated with different concentrations of CnB (5, 20, and 100 mg/ml) for 24 h. At the indicated time
points, cells were lysed, and the lysates were immunoblotted with TRAIL Ab. C, Polymyxin B abrogates LPS-induced TRAIL expression but not CnBinduced TRAIL expression. Peritoneal macrophages were plated in 12-well culture plates (2.5 3 106 cells/well) and incubated with CnB (20 mg/ml) or LPS
(30 ng/ml) for 10 h in the presence or absence of 10 mg/ml polymyxin B. TRAIL expression was analyzed by qPCR. D, PK digestion abolishes CnBinduced but not LPS-induced TRAIL expression. Peritoneal macrophages were incubated with CnB (20 mg/ml), PK-digested CnB (20 mg/ml), LPS (100 ng/
ml), or PK-digested LPS (100 ng/ml) for 10 h. TRAIL expression was analyzed by qPCR. E, CnB induces TRAIL expression in peritoneal macrophages
in vivo. Kunming mice were divided into four groups (five mice/each group) and i.p. injected with different doses of CnB (20, 100, or 400 mg/mouse) or
PBS. Mice were sacrificed at 12 h after injection, and peritoneal macrophages were harvested for RNA extraction. TRAIL expression was analyzed by
qPCR. All experiments were performed more than three times, and the results are expressed as means 6 SE. *p , 0.05, **p , 0.01.
The Journal of Immunology
chemokine) and some members of the TNF superfamily (Fas ligand,
4-1BBL, TRAIL, TNF-like weak inducer of apoptosis, LIGHT, and
TL1). As expected, CnB upregulated expression of the proinflammatory factors chemokines and members of the TNF superfamily such as 4-1BBL, TRAIL, and TL1 (data not shown).
However, CnB was no more effective than the control proteins used
(fibrinogen and avidin) in inducing these genes (data not shown)
except in the case of TRAIL. CnB markedly increased the expression of TRAIL in a dose-dependent manner at the mRNA and
protein levels (Fig. 4A, 4B). Fibrinogen is a known ligand of aMb2
and was used as a receptor-related control, whereas avidin was used
as a receptor-unrelated control. Because CnB was expressed and
purified from bacterial cultures, and LPS can also induce TRAIL
expression (30), we sought to distinguish the presumed CnB-induced
TRAIL expression from LPS-induced TRAIL expression. We found
that LPS-induced TRAIL expression was markedly inhibited by
243
polymyxin B sulfate, whereas CnB-induced TRAIL expression was
not (Fig. 4C). Conversely, PK-digested LPS retained the ability to
induce TRAIL expression, whereas PK-digested CnB did not (Fig.
4D). These data demonstrate that the CnB-induced TRAIL expression was not due to contamination by LPS. Next, we further examined whether CnB could induce TRAIL expression in peritoneal
macrophages in vivo. Kunming mice were divided into four groups
and i.p. injected with different doses of CnB (20, 100, or 400 mg/
mouse) or PBS. We observed that CnB treatment groups showed
more extensive production of TRAIL than the PBS treatment group,
and the CnB 100 mg group showed the most extensive production of
TRAIL comparing to the 20 and 400 mg groups (Fig. 4E).
CnB-induced TRAIL expression is dependent on integrin aM
Because CnB increased the expression of TRAIL, we tested
whether this expression was mediated by integrin aM. We found
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FIGURE 5. Integrin aM mediates CnB induced-TRAIL expression in primary macrophages and RAW 264.7 macrophage cells. A, Integrin aM Ab
blocks CnB induced-TRAIL expression in peritoneal macrophages. Peritoneal macrophages were plated in 12-well culture plates (2.5 3 106 cells/well) in
serum-free conditions. They were pretreated with integrin aM Ab (final concentrations, 10, 20, and 30 mg/ml) or isotype Ab (final concentration, 20 mg/ml)
for 30 min, followed by stimulation with 20 mg/ml CnB for 10 h. At the indicated time points, total RNA was extracted, and TRAIL expression was
analyzed by qPCR. Results were normalized to b-actin expression and are presented as fold increases over the medium-only control. B, Unfractionated
heparin inhibits CnB induced-TRAIL expression in peritoneal macrophages. Cells were pretreated with unfractionated heparin (final concentrations, 10, 30,
and 300 U/ml) for 30 min, followed by stimulation with 20 mg/ml CnB for 10 h. TRAIL expression was analyzed by qPCR. C, Fibrinogen inhibits CnBinduced TRAIL expression in peritoneal macrophages. Cells were pretreated with fibrinogen (final concentrations, 100 and 300 mg/ml) for 30 min, followed
by stimulation with 20 mg/ml CnB for 10 h. TRAIL expression was analyzed by qPCR. D, Efficiency of knockdown of integrin aM by siRNA in RAW
264.7 cells. RAW 264.7 cells were transfected with either negative control siRNA or integrin aM siRNA, and knockdown efficiency was assessed 48 h after
transfection by qPCR. Integrin aM knockdown inhibits CnB induced-TRAIL expression (E) but not IFN-g induced-TRAIL expression (F) in RAW264.7
macrophages. Cells were transfected with either negative control siRNA or integrin aM siRNA. Thirty-six hours after transfection, cells were treated with
20 mg/ml CnB or 200 U IFN-g for 12 h, and TRAIL gene expression was analyzed by qPCR. All experiments were performed more than three times, and
the results are expressed as means 6 SE.
244
that the induction of TRAIL was inhibited by pretreatment with
integrin aM Ab (Fig. 5A). The fact that the inhibition of TRAIL
expression was incomplete may be due to partial activation of the
macrophages by the binding of integrin aM Ab itself (48) (data
not shown). We further examined whether known ligands of
integrin aM could compete for binding of CnB to the receptor and
reduce TRAIL expression. Unfractionated heparin is a strong ligand of integrin aMb2 and can inhibit binding of fibrinogen,
factor X, and iC3b to this receptor (31, 49). CnB-induced TRAIL
expression was 80% inhibited by preincubation with unfractionated heparin (Fig. 5B) as well as to some extent by fibrinogen
(Fig. 5C). Furthermore, CnB-induced TRAIL expression was 50–
60% inhibited when we knocked down integrin aM in RAW 264.7
macrophages, whereas IFN-g–induced TRAIL expression was
unaffected (Fig. 5E, 5F). These results suggest that CnB-induced
and IFN-g–induced TRAIL expression is mediated by different
receptors and pathways.
The tumoricidal activity of CnB-activated peritoneal
macrophage is partially dependent on TRAIL
FIGURE 6. Tumoricidal activity
of CnB-stimulated peritoneal macrophages. A, Peritoneal macrophages
were plated in 12-well culture plates
(2.5 3 106 cells/well) and incubated
with 20 mg/ml CnB for 10 h. They
were then cocultured with H22 hepatocarcinoma cells at different E:T
ratios for 30–36 h in serum-free
conditions. H22 cells in the supernatants were harvested at the indicated time points and stained with
FITC-Annexin V/PI, followed by
flow cytometry analysis. B, Tumoricidal activity of CnB-stimulated
macrophages at different drug concentrations. CnB- and LPS-stimulated macrophages were cocultured
with H22 hepatocarcinoma cells for
30–36 h, and the H22 cells were
harvested for apoptosis assay by
Annexin V/PI staining. The lower
right quadrant represents early apoptosis, and the upper right represents
late apoptosis. C, The effect of neutralizing TRAIL in the coculture
system. CnB-stimulated macrophages
were cocultured with H22 cells for
30 h in the presence of 20 mg/ml of
neutralizing TRAIL Ab or 20 mg/ml
isotype Ab, and then the H22 cells
were harvested, and apoptosis was
assayed by Annexin V/PI staining.
All experiments were performed more
than three times, and the results are
expressed as means 6 SE.
LPS served as a positive control. We observed that CnB-activated
peritoneal macrophages induced significant apoptosis in H22 cells
from an E:T ratio of 5:1 to 20:1 (Fig. 6A). Moreover, 5 mg/ml CnB
was sufficient to activate the peritoneal macrophages and cause
apoptosis of the H22 cells (Fig. 6B). Next, we examined the role
of TRAIL in tumor eradication by CnB-stimulated peritoneal
macrophages. As expected, the early apoptosis of H22 cells was
markedly inhibited by neutralizing TRAIL (Fig. 6C). These data
indicate that the tumoricidal activity of CnB-activated peritoneal
macrophages is partially dependent on TRAIL.
CnB treatment significantly prolongs the survival of mice
bearing H22 ascites tumors, which has a positive correlation
with the induction level of TRAIL
We had previously reported that i.p. injection of CnB prolonged the
survival of mice bearing H22 ascites tumors. In this study, we
repeated this experiment and further compared CnB with the classic
immunotherapeutic drug IFN-g. We observed that CnB and IFN-g
had equal effect on prolonging survival of mice bearing H22 ascites tumors (Fig. 7A). It is known that induction of TRAIL expression in immune cells is an important antitumor mechanism
of IFN-g. So we wondered whether CnB’s antitumor effect was
consistent with its TRAIL induction ability in peritoneal macrophages. We observed that CnB could not induce TRAIL expression in peritoneal macrophages if we boiled it for 20 min (inset of
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To determine if CnB-activated peritoneal macrophages play a key
role in eradicating the H22 cells and to assess the role of TRAIL in
this process, we established an in vitro model to mimic the killing
process. In this model, H22 cells were cocultured with CnBstimulated peritoneal macrophages, and apoptosis was assayed.
CnB IS A NEW LIGAND OF INTEGRIN aM
The Journal of Immunology
245
Fig. 7B). This heat-inactivated CnB failed in prolonging the survival of Kunming mice bearing H22 ascites tumors (Fig. 7B),
which was consistent with its weak ability to induce TRAIL expression in peritoneal macrophages. From these data, we speculate
that TRAIL is the main candidate molecule responsible for the
tumoricidal activity of CnB in vivo.
Discussion
We have shown above that CnB is a ligand of integrin aM, a
subunit of the heterodimeric integrin aMb2. Integrin aMb2 is expressed primarily on the surfaces of innate immune cells, which
play important roles in cells adhesion, migration, chemotaxis, and
phagocytosis. However, it remains unclear whether integrin aM
promotes tumor progression or tumor regression. Several groups
have reported that tumor-infiltrating integrin aM-positive cells
support tumor growth by, for example, stimulating angiogenesis
and neovascularization (50–52). In contrast, integrin aMb2 is
crucial for effective FcR-mediated immunity to melanoma (53)
and promotes the survival of mouse NK cells (54). In addition, it
inhibits TLR-triggered inflammatory responses and prevents excessive production of proinflammatory cytokines and type I IFN
(55). Recent research has highlighted a critical role for inflammation in the development of cancers, based on the observation
that innate immune cells drive tumor progression by producing
proinflammatory factors such as TNF-a and IL-6 (56–58). We
have shown above that CnB binds and activates integrin aMb2 on
macrophages and induces high levels of the cytotoxic cytokine
TRAIL but low levels of proinflammatory cytokines (data not
shown) and so converts the inflammatory macrophages into cytotoxic macrophages. Therefore, integrin aMb2 is a double-edged
sword, and whether it promotes or inhibits tumor progression
depends on its ligand.
TRAIL induces apoptosis of a variety of tumor cells while
displaying no cytotoxicity against most normal cells; this makes
it a promising new agent for cancer therapy. It has been reported
that in immune cells, TRAIL is induced mainly by the type I or
II IFN-mediated JAK/STAT (37–40) and TLR agonist-mediated
TLR pathways (41–43). To our knowledge, this is the first report
that TRAIL expression is mediated by integrin aM, and our
findings should add to understanding of the mechanisms regulating
TRAIL expression. They also suggest a strategy for drug screening
based on the integrin aM-TRAIL pathway; this might permit the
identification of a peptide that induces high levels of TRAIL but
low levels of proinflammatory cytokines among mutants of CnB or
other ligands of integrin aM.
In vivo experiments above show that CnB as well as IFN-g can
markedly prolong the survival of mice bearing H22 ascites tumors.
However, IFN-induced immune toxicity has been a limiting factor
that restricts the amount of IFN that can be used for effective
cancer therapy (59). In contrast with IFN-g, CnB has a lower
toxicity in vitro and vivo. Acute toxicity experiment indicates that
mice can endure at least a 50-fold normal dose of CnB (17).
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FIGURE 7. The tumoricidal activity of CnB in vivo.
A, CnB treatment significantly prolongs the survival of
BALB/c mice bearing H22 ascites tumors. Tumor cells
(1 3 106) were injected i.p. Two days after injection
of tumor cells, mice were treated with CnB (filled
squares, 100 mg/mouse/d), IFN-g (gray circles, 4000
U/mouse/d), or normal saline (half-filled circles) for
10 d. Each group contained six mice. B, Heat-inactivated CnB fails in inducing TRAIL as well as in
prolonging mice survival. Heat-inactivated CnB induces low level of TRAIL in mouse peritoneal macrophages (inset). Kunming mice were i.p. injected
with CnB (:, 100 mg/mouse/d), heat-inactivated CnB
(n, 100 mg/mouse/d), or normal saline (d) for 3 d
before tumor challenge. Mice were then i.p. inoculated with 1 3 106 H22 cells, followed by drug
treatment for 7 d. Each group contained 10 mice.
Statistical differences between treatment groups were
calculated by survival curve statistics (Gehan-Breslow-Wilcoxon test) using GraphPad Prism software
(GraphPad). This figure is one representative result of
three independent experiments. *p , 0.05, **p ,
0.01, ***p , 0.001.
CnB IS A NEW LIGAND OF INTEGRIN aM
246
Besides, we observe that the antitumor effect of CnB is consistent
with the TRAIL induction level by CnB. Heat-inactivated CnB
fails in inducing TRAIL expression in peritoneal macrophages as
well as in prolonging the survival of Kunming mice bearing H22
ascites tumors. This indicates that TRAIL is an important candidate molecule responsible for the tumoricidal activity of CnB
in vivo. However, whether TRAIL is the only executing molecule
in CnB-initiated tumoricidal action should be further examined in
future studies.
Research on the functions of calcineurin in the immune system is
mainly focused on aspects of adaptive immunity such as T cell
activation. Our findings add new insights into the roles of CnB in
the regulation of innate immunity. We suspect that the substantial
levels of CnB in sera are important in sustaining innate immune
responses and cancer immunosurveillance by activating monocytes/
macrophages. Our observations also suggest a possible application
of CnB in cancer therapy because of its ability to induce high levels
of TRAIL, low toxicity, and stability.
23.
24.
25.
26.
27.
28.
29.
30.
The authors have no financial conflicts of interest.
31.
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