A Polyglutamic Acid Motif Confers IL-27
Hydroxyapatite and Bone-Binding Properties
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J Immunol 2013; 190:2931-2937; Prepublished online 6
February 2013;
doi: 10.4049/jimmunol.1201460
http://www.jimmunol.org/content/190/6/2931
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References
Aurélie Jeanne Tormo, Linda Ann Beaupré, Greg Elson,
Sandrine Crabé and Jean-François Gauchat
The Journal of Immunology
A Polyglutamic Acid Motif Confers IL-27 Hydroxyapatite
and Bone-Binding Properties
Aurélie Jeanne Tormo,* Linda Ann Beaupré,* Greg Elson,† Sandrine Crabé,*,1 and
Jean-François Gauchat*
I
nterleukin-27 is an IL-12–like type I cytokine known to
regulate the function and differentiation of multiple immune
and nonimmune cell lineages such as T cells, B cells, NK
cells, mast cells, osteoblasts, and stem cells (1–4). IL-27 plays an
important role in the resolution of inflammation in different
models of infection and has marked beneficial effects in the experimental autoimmune encephalitis model of multiple sclerosis
(5–10). IL-27 has also been shown to have potent beneficial antitumor effects in leukemia and solid cancer models in mice (11–
19). Moreover, IL-27 has been described to inhibit osteoclastogenesis and to protect against bone destructive disorders (20).
The therapeutic potential of IL-27 might, however, be compromised
by the large range of cells it regulates, as previously observed for
other type I cytokines with limited target specificity (1–4).
The IL-27 complex comprises p28, a four a helix-bundle cytokine
subunit, and EBV-induced gene 3 (EBI3), a subunit structurally
similar to soluble type I cytokine receptors (1). The polypeptide
loop connecting the p28 C and D a helices contains a stretch of
polyglutamic acids (poly-E) unique among helical cytokines and
highly conserved between species (1). This poly-E stretch is highly
similar to the domains mediating bone sialoprotein-hydroxyapatite (HA) binding (21–24) and HA chromatography was used
previously to purify recombinant p28 (25). HA is the main bone
*Département de Pharmacologie, Université de Montréal, Montreal, Quebec H3T1J4,
Canada; and †NovImmune SA, CH-1228 Plan-les-Ouates, Switzerland
1
Current address: Wittycell, Evry, France.
Received for publication May 25, 2012. Accepted for publication January 8, 2013.
This work was supported by Canadian Institutes of Health Research Grant MOP57832.
Address correspondence and reprint requests to Dr. Jean-François Gauchat, Université de Montréal, Pavillon Roger Gaudry, 2900 Edouard-Montpetit, Montreal, Quebec
H3T 1J4, Canada. E-mail address: [email protected]
Abbreviations used in this article: CLC, cardiotrophin-like cytokine; CLF, cytokinelike factor 1; CNTF, ciliary neurotrophic factor; EBI3, EBV-induced gene 3; HA,
hydroxyapatite; IMAC, immobilized metal affinity chromatography; IRIC, Institute
for Research in Immunology and Cancer; MFI, mean fluorescence intensity; MSC,
multipotent stromal cell; WT, wild-type.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201460
mineral constituent, and the bone sialoprotein poly-E stretch has
been shown to target the protein to growing bone in vivo (26).
More generally, acidic amino acid tags are used to direct recombinant proteins to bones (27, 28). We therefore compared the
properties of wild-type (WT) p28 and IL-27 with those of mutants
lacking the poly-E acidic motif. We observed that the poly-E motif
confers HA- and bone-binding properties to IL-27 and that IL-27
immobilized on HA retains its biological activity. The C-D loop
acidic motif could therefore confer IL-27–specific pharmacological properties beneficial for therapeutic applications targeting
osteoclasts, stem or immune cells residing at the endosteal surface
of bone marrow. Similarly, it may serve to enhance the antitumor
activity of IL-27 on leukemic or cancer metastatic cells with bone
tropisms.
Materials and Methods
Experimental animals
All procedures conformed to the Canadian Council on Animal Care
guidelines and were approved by the Animal Ethics Committee of the
Université de Montréal. Six- to 8-wk-old female C57BL/6 mice were
purchased from The Jackson Laboratory (Bar Harbor, ME).
Generation of stable Ba/F3 cells expressing gp130
The cDNA coding for mouse gp130 (Image ID 6834623; Open Biosystems) was recloned in the expression vector pMG (InvivoGen, Cedarlane,
Burlington, ON, Canada). Linearized plasmids were transfected by electroporation in Ba/F3 cells. Stable transfectants were selected using hygromycin (1 mg/ml). Clones were expanded using in-house–produced mouse
hyper–IL-6 (5 ng/ml) and selected for proliferation in response to mouse
IL-27 (10 ng/ml; R&D Systems, Minneapolis, MN).
Expression and isolation of recombinant proteins
The construct for the expression of mouse ciliary neurotrophic factor
(CNTF) was described previously (29). The p28 cDNA (RIKEN clone
number I830047N17) was obtained from the FANTOM Consortium (The
Institute of Physical and Chemical Research, Saitama, Japan) Genome
Exploration Research Group (provided by K.K. DNAFORM, Ibaraki, Japan). The sequence coding for the mature form of p28 was modified in 39
with a sequence coding for c-Myc (EQKLISEEDL) and 123 His epitopes
and cloned in the expression vector pET24d (30). To generate a vector
coding for mouse p28-poly-A, the codons coding for the aa 163–177 were
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The p28 subunit of the composite cytokine IL-27 comprises a polyglutamic acid domain, which is unique among type I cytokines.
This domain is very similar to the acidic domain known to confer hydroxyapatite (HA)-binding properties and bone tropism to bone
sialoprotein. We observed IL-27 binding to HA, in accordance with previous studies reporting successful p28 HA chromatography.
The IL-27 polyglutamic acid domain is located in a flexible inter-a helix loop, and HA-bound IL-27 retained biological activity.
Using IL-27 alanine mutants, we observed that the p28 polyglutamic acid domain confers HA- and bone-binding properties to IL27 in vitro and bone tropism in vivo. Because IL-27 is a potent regulator of cells residing in endosteal bone marrow niches such as
osteoclasts, T regulatory, memory T, plasma, and stem cells, this specific property could be beneficial for therapeutic applications.
IL-27 has potent antitumoral and antiosteoclastogenic activities. It could therefore also be useful for therapies targeting hematologic cancer or solid tumors metastasis with bone tropism. Furthermore, these observations suggest that polyglutamic motifs
could be grafted onto other type I cytokine inter-a helix loops to modify their pharmacological properties. The Journal of
Immunology, 2013, 190: 2931–2937.
2932
IL-27 BINDS BONE HA
mutated to code for alanine. Recombinant proteins were expressed in
BL21 Star (DE3) bacteria (Life Technologies) under denaturing condition
and purified by immobilized metal affinity chromatography (IMAC) as
described previously (29). For renaturation, WT and mutant p28 were dialyzed against PBS supplemented with 2 mM reduced and 0.2 mM oxidized gluthatione. Denatured and renatured proteins were quantified by
Western blotting using 63 His-tagged mouse CNTF as a standard and antipenta His mAb (Qiagen, Toronto, ON, Canada).
For the production of WT and mutant IL-27, a construct coding for the
sequences of EBI3, a flexible 33 (SGGGG) linker and WT or poly-A p28
were generated using a synthetic EBI3 cDNA (Geneart, Life Technologies
Burlington, ON) and cloned in the insect cell expression vector pIB/V5His (Life Technologies) using standard molecular biology techniques (31).
cDNAs coding for WT and mutant IL-27 were transfected in the High Five
insect cell line (Life Technologies). Supernatants of transfected cells were
collected and concentrated as described previously (32). Recombinant
proteins were purified by IMAC and quantified by Western blotting using
63 His-tagged mouse CNTF as a standard.
Binding to HA beads
Binding to secreted bone matrix in vitro
Multipotent stromal cells (MSC) (a gift from Dr. C. Beauséjour, Université
de Montréal) were isolated from bone marrow using polystyrene adherence
(33). To induce osteocyte differentiation, cells were cultured in 6-well
plates in DMEM containing 100 nM dexamethasone, 50 mM vitamin C,
50 nM vitamin D3, and 10 mM b-glycerophosphate for 10 d. Osteogenic
differentiation was confirmed using alizarin red S (Sigma-Aldrich, St.
Louis, MO) staining. For binding assays, the cell cultures were incubated
with 1 mg WT or mutant IL-27 purified from High Five cell supernatant in
0.5 ml DMEM culture medium for 1 h in ice. After extensive washing with
ice-cold DMEM, the cultures were lysed in 8 M urea and 400 mM
phosphate (pH 7.3) complemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Rockford, IL). Lysate protein contents
were quantified by bicinchoninic acid after dilution to reduce urea concentration (Thermo Fisher Scientific). Aliquots of 50 mg total protein were
analyzed by Western blotting. IL-27 was detected using biotinylated goat
anti-mouse p28 Ab purified by affinity chromatography (R&D Systems)
and HRP-labeled streptavidin (Thermo Fisher Scientific).
Measurement of STAT1 and STAT3 activation by flow
cytometry
The proliferation of Ba/F3-gp130 transfectants was measured using an
Alamar blue fluorometric assay as described previously (32).
Hydrodynamic transfection with IL-27 and IL-27-poly-A
cDNAs
Chimeric cDNA coding for the sequences of EBI3, a flexible 33 (SGGGG)
linker and the WT or poly-A p28 tagged in carboxy with an Avitag (34)
and 83 His were generated using standard molecular biology techniques
(31). These chimeric cDNAs were fused with sequences coding for a T2A
autoclivable peptide (35) and a secreted derivative of the biotin ligase BirA
(36, 37). The WT or mutant IL-27-BirA bicistronic cDNA as well as the
secreted BirA cDNA were recloned in the mammalian expression vector
pcDNA5-His (Life technologies). LPS-free plasmid DNA was purified
using an endotoxin-free maxi-prep plasmid isolation kit (Qiagen). To test
the constructs, the plasmids were transfected in HEK-293 Flp-In cells (Life
Technologies) (32), and the cell culture medium was subjected to Western
blot analysis with HRP-labeled anti-p28 and HRP-labeled streptavidin. For
hydrodynamic transfection experiments, groups of two to three C57BL/6
mice were injected in the tail vein with 50 mg of the pcDNA5-His
derivatives containing WT IL-27-T2A-BirA cDNA, mutant IL-27-T2ABirA cDNA, or BirA cDNA under hydrodynamic transfection conditions
(38). Briefly, the plasmids were diluted in Ringer’s solution supplemented
with 0.5 mM D-biotin to a final volume corresponding to 10% of the weight
of the mice. Injections were performed by a blinded operator in 5 s in mice
subjected to gaseous anesthesia (isoflurane 2–3% in oxygen). Mice were
sacrificed after 48 h, and serum, liver, and bone were isolated. Femur bone
marrows were flushed, and the bones cavities were repetitively washed
for 15 min with 1 ml 8 M urea and 400 mM phosphate. Livers were homogenized in 50 mM Tris (pH 7.4), 100 mM NaCl, 50 mM sodium
phosphate, 5 mM EDTA, 1% Triton X-100, and 10% glycerol. The bone
and liver extracts were subjected to IMAC on Ni-NTA agarose (Qiagen),
according to the manufacturer’s instructions. The proteins isolated by
IMAC were analyzed by Western blotting using “high sensitivity” HRPconjugated streptavidin (Thermo Fisher Scientific).
Measurement of bone marrow cell STAT1, STAT3, and STAT5
activation in mice subjected to hydrodynamic transfection with
IL-27 and IL-27-poly-A cDNAs
Forty-eight hours posthydrodynamic transfection, mice were sacrificed.
Bone marrow mononuclear cells were purified by centrifugation on a
Histopaque-1083 cushion (Sigma-Aldrich). Cells were fixed with 2%
formaldehyde and stained with a combination of PE-labeled anti-CD8 and
either anti–phospho-STAT1, STAT3, or STAT5 mAbs (all from BD Biosciences). Blood samples were collected by cardiac puncture. Sera were
diluted 20-fold in 50 mM Tris (pH 7.4), 100 mM NaCl, and 20 mM imidazole and subjected to IMAC with 15 ml Ni-NTA agarose. The IMAC
eluates were analyzed by Western blotting using “high sensitivity” HRPconjugated streptavidin.
Results
6
To assess WT and mutant IL-27 biological activity by phosflow, 10 mouse
splenocytes were activated for 15 min at 37˚C with mouse IL-6 (50 ng/ml),
commercial IL-27 (50 ng/ml) (both from R&D Systems), or in house High
Five insect cell-produced WT IL-27 and IL-27-poly-A (50 ng/ml) in a final
volume of 200 ml RPMI 1640 medium supplemented with 20 mM HEPES
(pH 7.4) as described previously (32). Cells were fixed and stained (32)
with either FITC-labeled anti–phospho-STAT1 (Y701) or FITC-labeled
anti-phospho STAT3 (Y705) mAbs (BD Biosciences, Mississauga, ON,
Canada). Fluorescence was quantified by flow cytometry using a FACSCalibur (BD Biosciences). Data were analyzed using the FlowJo software
(Tree Star, Ashland, OR).
For the phosflow assay with the cytokines immobilized on HA beads,
10 ng mouse IL-6 or IL-27 (both from R&D Systems) were incubated with
1 mg HA beads in RPMI 1640 medium supplemented with 20 mM HEPES
(pH 7.4) for 15 h at 4˚C. After extensive washing with RPMI 1640 medium, the beads were resuspended to 2 mg/ml in RPMI 1640 medium
supplemented with 20 mM HEPES (pH 7.4) and incubated with 106
splenocytes for 15 min at 37˚C. For the assessments of cytokine leakage,
cytokine-loaded beads were washed and incubated in RPMI 1640 medium
supplemented with 20 mM HEPES (pH 7.4) and incubated for 15 min at
37˚C. The incubation medium was separated from the beads using 40-mm
cell strainers (BD Falcon) and incubated with splenocytes for 15 min at
37˚C. STAT activation was measured as described above.
The IL-27 poly-E motif confers HA binding properties to the
IL-27 p28 subunit
Both the human and mouse IL-27 amino acid sequences contain an
acidic poly-E motif (1). The p28 poly-E stretch is highly similar to
the acidic domain, which mediates bone sialoprotein binding to
HA (Fig. 1) (21–23), and HA chromatography was used successfully to purify Escherichia coli–produced p28 (39). To investigate whether the poly-E stretch of p28 contributes to HA
binding, we produced E. coli–derived recombinant WT p28 or
a mutant p28 derivative with the glutamic acids of the poly-E
stretch replaced by neutral alanines, using the approach previously used to map the bone sialoprotein HA-binding domain (24).
Although the WT p28 bound HA beads under both denaturing
(Fig. 2A) and nondenaturing (Fig. 2B) conditions, this property
was lost in the p28 mutant (referred to as p28-poly-A) (Fig. 2).
These results indicate that the stretch of polyglutamic acids of the
polypeptide loop connecting the C and D a helices of p28 confers
HA binding properties to p28. Because this connecting loop is
outside the regions known to be directly involved in receptor
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For binding assays under denaturing conditions, 2 mg HA beads (Bio-Rad,
Mississauga, ON, Canada) were washed, equilibrated with 6 M urea, 0.05 M
phosphate (pH 7.3), and 2 mM 2-ME in 10 ml polypropylene columns (BioRad), and incubated with 10 mg denatured WT or mutant p28 in the same
buffer. The columns were washed with equilibration buffer, and proteins
bound to HA were eluted with increasing concentrations of phosphate (50–
400 mM) in 6 M urea, according to the manufacturer’s instructions. For
binding assays under native conditions, the HA chromatography was
performed with renatured WT or mutant p28 in the absence of urea. The
flow-through and elution fractions were analyzed by Western blot as described above.
Proliferation assays
The Journal of Immunology
2933
FIGURE 1. The sequence of the p28 subunit of IL27 contains a polyglutamic acidic motif. The sequences
of mouse (m) and human (h) bone sialoprotein (BSP)
and IL-27 are indicated. The poly-E motifs are boxed.
IL-27 immobilized on HA activates STAT signaling in immune
cells
To investigate whether IL-27 bound to HA retains biological activity or represents a latent form of the cytokine, we tested the effect
of HA beads loaded with single-chain commercial IL-27 on the
activation of STAT1 and STAT3 using unfractionated mouse
splenocytes as targets. This biological activity assay was chosen
because it could be performed under cell culture conditions and in
a timeframe (15 min) unlikely to result in a leakage of the cytokine
from the beads. When splenocytes were incubated with HA beads
preincubated with IL-27, a strong upregulation of both STAT1
and STAT3 phosphorylation could be detected by flow cytometry
(Fig. 3A). No induction was observed when splenocytes were
stimulated with beads preincubated with culture medium only,
indicating that the effect observed was not due to a nonspecific
activation of the splenocytes by HA (Fig. 3A). To exclude effects
mediated by free IL-27 released from HA during the test, we incubated IL-27–loaded HA beads with cell culture medium for
15 min and analyzed the capacity of the conditioned media to stimulate splenocytes once the beads had been removed. No induction
of STAT activation could be detected (Fig. 3B). This indicates that
the activation of STAT1 and STAT3 observed is mediated by
immobilized IL-27 rather than IL-27 leaking from the HA beads
during the phosflow assays.
The IL-27 poly-E motif confers bone-binding properties to the
IL-27 p28 subunit
FIGURE 2. The p28 poly-E motif mediates HA binding. WT (WT p28)
or mutant p28 (p28-poly-A) produced in bacteria were subjected to HA
chromatography under denaturing (A) or native (B) conditions. Input
samples, flow-through, and elution fractions were analyzed by Western
blot using an anti-His tag Ab. Six His-tagged CNTF was used as control.
To investigate whether the HA-binding properties of the IL-27
p28 subunit resulted in bone tropism in vitro, we expressed singlechain WT or mutant IL-27 in which the poly-E motif was replaced
by alanine in the High Five insect cell line (IL-27 poly-A). The two
recombinant proteins, which were tagged in carboxy with a 12His motif, were concentrated and purified by IMAC. The two
recombinant cytokines were biologically active when tested for
their capacity to induce the proliferation of mouse Ba/F3 cells
transfected with gp130 cDNA or to induce STAT phosphorylation of murine splenocytes (Fig. 4). To compare the bone-binding
properties of the WT IL-27 and the poly-alanine IL-27 mutant, we
induced the differentiation of MSC (33) in osteocytes in vitro.
Osteocyte differentiation was paralleled by bone synthesis, detected by staining with a calcium-specific alizarin red S (Fig. 5A).
MSC-derived osteocyte cell cultures were incubated with WT IL27 or the poly-alanine IL-27 mutant produced in insect cells and
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recognition (1, 40, 41), we investigated whether IL-27 immobilized on HA remained biologically active.
2934
IL-27 BINDS BONE HA
FIGURE 3. HA-immobilized IL-27 induces STAT phosphorylation in
splenocytes. (A) Splenocytes were stimulated for 15 min with the cytokines indicated (panels IL-6 and IL-27) or HA beads preincubated with IL6 or IL-27 (panels IL-6+HA and IL-27+HA). All cytokines were from
R&D Systems. Cells were then fixed, permeabilized, and stained with antipSTAT1 or anti-pSTAT3. Fluorescence was measured by flow cytometry.
Filled gray histograms, unstimulated cells (panels IL-6 and IL-27) or cells
stimulated with control HA beads (panels IL-6+HA and IL-27+HA). Mean
fluorescence intensity values of the controls (left) and stimulated cells
(right) are indicated in the histograms. Data are representative of three
independent experiments. (B) HA beads preincubated with IL-6 or IL-27
(panels IL-6+HA and IL-27+HA) were incubated in cell culture medium
for 15 min, and the conditioned medium was used to stimulate splenocytes
for 15 min. Panels IL-6 and IL-27, cells stimulated for 15 min with IL-6 or
IL-27. Filled gray histograms, unstimulated cells. STAT phosphorylation
was assessed as described in (A). Data are representative of three independent experiments.
the protein bound to the cultures was analyzed by Western blot
with an Ab specific for p28 (Fig. 5B). The amount of mutated
IL-27 cytokine recovered was much lower than that of WT IL27, indicating that the polyglutamic acids contributed to the
binding of this cytokine to the osteocytes cell cultures in vitro
(Fig. 5B).
The poly-E motif modifies IL-27 pharmacology in vivo
To analyze the effect of replacing polyglutamic acids by alanine
in vivo, we generated derivatives of the mammalian expression
vector pcDNA5-His coding for WT and poly-A IL-27 tagged in
carboxy with an Avitag biotin ligase peptide substrate (34) and
8 His. The modified cDNA inserted in the mammalian expression
vector were multicistronic, comprising the sequences coding for
a T2A autocleavable peptide (35), followed by a secreted derivative of biotin ligase to catalyze IL-27 biotinylation (34, 36, 37).
The constructs containing the cDNA coding for WT and poly-A
IL-27 were compared for expression and biotinylation by transfection in HEK-293 Flp-In cells. Similar signals were observed
in the cell culture medium of HEK-293 transfected with vectors
coding for either WT or mutant IL-27 using anti-p28 or streptavidin, indicating that the expression, secretion and biotinylation of
WT and IL-27 were identical (data not shown).
To compare the properties of the two IL-27 derivatives in vivo,
we subjected mice to hydrodynamic transfection (38) with the
cDNA coding for WT IL-27 or IL-27 poly-A. This transfection
procedure is known to result in transient liver production and
systemic release of the recombinant proteins (38). It has been used
extensively to analyze the effect of IL-27 in vivo (16, 19, 25).
When livers from mice subjected to hydrodynamic transfection
were analyzed for IL-27 expression by IMAC, followed by
Western blot analysis for biotinylated protein, both WT and polyA mutants IL-27 could be detected (Fig. 5C). As expected, no
biotinylated IL-27 could be detected in mice transfected with the
control plasmid coding only for the secreted biotin ligase (Fig.
5C). To evaluate whether IL-27 binding to bone could be detected
in vivo, we flushed the mice femurs from the bone marrow and
then extensively washed the bone lumens with a urea–phosphate
buffer solution to recover bound IL-27. The extracts were subjected to IMAC and Western blot analysis. Interestingly, we could
only detect IL-27 in the eluates of bones isolated from the mice
transfected with WT IL-27 cDNA and not poly-A IL-27 cDNA
(Fig. 5D).
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FIGURE 4. Recombinant WT and poly-A IL-27 produced in the High
Five insect cell line are biologically active. (A) Ba/F3 cells expressing
endogenous WSX-1 in combination with transfected gp130 were incubated
with the indicated cytokines for 72 h, and proliferation was measured by
fluorescence using the Alamar blue test. Error bars indicate the SEM of
triplicate cultures. (B) Splenocytes were stimulated with 50 ng/ml of the
indicated cytokines for 15 min, fixed, and permeabilized. STAT phosphorylation was assessed as described in Fig. 3. Filled gray histograms,
unstimulated cells. Data are representative of three independent experiments.
The Journal of Immunology
The poly-E motif is required for STAT activation in bone
marrow CD8 cells
We analyzed whether the IL-27 polyglutamic acid motif affects its
capacity to activate immune cells in the bone. Bone marrow cells
from groups of three mice subjected to hydrodynamic transfection
with control plasmid, IL-6, WT IL-27 cDNA, or IL-27-poly-A
cDNA were analyzed for STAT phosphorylation by flow cytometry. Activation of STAT1, 3, and 5 could be detected in bone
marrow CD8 cells isolated from mice transfected with WT IL-27
cDNA (Fig. 6A). The level of activation was low, an observation
that could reflect reduced IL-27 sensitivity of memory CD8 cells
because of the downregulation of gp130 (42). No activation was
observed in the corresponding cells isolated from mice transfected
with IL-27-poly-A cDNA. The lack of STAT stimulation was not
due to the absence of IL-27 poly-A, because the cytokine could be
detected in the sera of the mice transfected with either the WT or
mutant IL-27 cDNA by IMAC and Western blot analysis (Fig.
6B). These observations suggest that the polyglutamic acid motif
influences the biological activity of IL-27 toward resident bone
cells in vivo.
Discussion
Bone sialoprotein is a major constituent of the bone. It comprises
polyglutamic acid domains with HA-nucleating properties (23, 24).
A highly homologous motif is present in the p28 C-D loop (1), and
p28 purification by HA chromatography was reported previously
(39). We confirmed that p28 binds HA under native conditions.
FIGURE 6. The poly E motif is required for the activation of bone
marrow CD8 cells by IL-27. Mice were tail vein-injected with LPS-free
mammalian expression vector DNA coding for biotin ligase (control) IL-6,
WT IL-27, or IL-27-poly-A. Mice were sacrificed after 48 h. (A) Bone
marrow CD8 T cell STAT1, STAT3, and STAT5 activation was analyzed by
flow cytometry. The quantification of STAT phosphorylation in CD8positive–gated cells was performed as described in Fig. 3. Filled gray
histograms, cells from control mice injected with biotin ligase cDNA.
Mean fluorescence intensity values of the controls (left) and stimulated
cells (right) are indicated in the histograms. Data are representative of
three independent experiments. (B) Sera from the mice transfected with
WT IL-27, IL-27-poly-A, or biotin ligase cDNA were analyzed for the
presence of IL-27 by IMAC and Western blot.
Such binding could also be observed under more stringent denaturing conditions. Using mutants in which the acidic amino acids
were replaced by alanines, we could show that the polyglutamic
acid domain confers the HA-binding properties to the cytokine.
IL-27 bound to HA beads retained the capacity to induce STAT1
and STAT3 phosphorylation in splenocytes. This biological activity assay was chosen because it can be performed under experimental conditions preventing leakage of HA-immobilized
IL-27. These results indicate that HA-bound IL-27 is not a latent,
inactive form of the cytokine and concurs with the current structural model of IL-27 according to which the C-D loop is not directly involved in receptor recruitment (1, 40, 41). We cannot,
however, exclude that HA binding modifies the specific activity of
the cytokine. Recently, mutations that reduce the flexibility of the
IL-2 B-C loop have been shown to strongly increase the affinity of
this cytokine for IL-2Rb, indicating loops connecting the a helix
can contribute to the biochemical properties of type I cytokines
(43).
The IL-27 acidic motif resembles the poly-E stretch known to
target the bone sialoprotein to growing bone in vivo (26). It is also
similar to the acidic polypeptide tags used to confer bone targeting
properties to therapeutic proteins (26–28). We therefore analyzed
the bone-binding properties of IL-27 in vitro and in vivo. To assess
this, we induced osteogenic differentiation of mouse multipotent
stromal cell cultures. Binding to the mix of osteocytes and osteocyte-secreted bone matrix could be detected by Western blot
analysis. No binding was observed using mutant IL-27 poly-A,
indicating that the observed binding involved a polyglutamic
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FIGURE 5. IL-27 binds osteocyte cell cultures and can be recovered
from the bones of mice transfected with IL-27 cDNA. (A) Multipotent
stromal cells were differentiated into osteocytes for 10 d. Osteocyte differentiation was detected by Alizarin red S staining. (B) Osteocyte cell
cultures were incubated with medium (lane control), WT IL-27, or poly-A
mutant IL-27 produced in High Five insect cells for 1 h on ice. Lane
commercial IL-27, 100 ng IL-27 from R&D Systems. After extensive
washing, cell cultures were lysed and analyzed for p28 binding by Western
blot using a biotinylated anti-p28 mAb. Data are representative of three
independent experiments. (C and D) Mice (two for each conditions) were
tail vein-injected with LPS-free mammalian expression vector DNA coding for biotin ligase (lanes control), WT IL-27, or IL-27-poly-A. Mice
were sacrificed at 48 h, and proteins from liver homogenates (C) or eluted
from bone lumen using urea and phosphate (D) were analyzed by IMAC
and Western blot with HRP-labeled, high-sensitivity streptavidin. Data are
representative of two independent experiments.
2935
2936
Acknowledgments
We thank Dr. J.D. Kulman, (University of Washington, Seattle, WA) and
Dr. J. Krosl and Dr. G. Sauvageau (Institute for Research in Immunology
and Cancer [IRIC], Université de Montréal) for the gift of the secreted biotin
ligase cDNA and B9 cells, respectively. We are indebted to Dr. C. Beau-
séjour (Université de Montréal) for the gift of multipotent stromal cells as
well as help and advice regarding osteogenic differentiation in vitro. We
thank S. Sénechal (Université de Montréal) for help with flow cytometry.
We are grateful to the IRIC genomic platform (IRIC, Université de Montréal) for DNA sequencing.
Disclosures
The authors have no financial conflicts of interest.
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acid–HA interaction rather than binding to cell surface IL-27R
expressed by osteocytes.
To analyze whether the pharmacological properties of IL-27 are
influenced by the poly-acidic motif, we transfected mice with
mammalian expression vector derivatives coding for WT and
mutant IL-27. Both recombinant proteins were tagged in carboxy
with 8-His and a biotin ligase epitope substrate (34). The expression vectors used coded for biotin ligase to catalyze the biotinylation of IL-27 (34, 36, 37). Hydrodynamic transfection results
predominantly in liver expression of the recombinant proteins
(38). We could detect biotinylated WT and mutant IL-27 in
transfected mouse liver. Interestingly, we could also isolate WT
but not mutant IL-27 poly-A from the femur lumen of transfected
mice. This suggests that the poly-E motif confers bone-binding
properties to IL-27. The approach used was selected for its sensitivity and simplicity. It did not, however, allow the quantitation
of the proportion of IL-27 immobilized in the bones, which
remains to be determined. Analysis of STAT1, 3, and 5 phosphorylation in bone marrow CD8 cells in mice subjected to hydrodynamic transfection suggests that the poly-E motif can play
a role in the targeting of bone marrow–resident cells.
It will be interesting to examine whether the poly-E motif
influences other pharmacological properties of IL-27, such as its
capacity to bind serum proteins, endothelial cells, or extracellular
matrix.
The observation that IL-27 binds HA and bone suggests that
the acidic motif confers to this cytokine a tropism for the bone
endosteal surface. This location has been identified as a niche for
hematopoietic stem cells (44–47) whose differentiation is known
to be inhibited by IL-27 (48). This surface is close to niches in
which numerous immune cells targeted by IL-27 can reside. The
bone marrow endosteal surface is a favored location for T regulatory cells (49, 50) whose differentiation is inhibited by IL-27
(51‑53). The bone marrow is also a preferred site for memory CD4
and CD8 cells (54–56), for B cell differentiation (57), and for
long-lived plasma cells (58, 59). Our phosflow analysis results
from mice transfected with WT or mutant IL-27 cDNA indicate
that the IL-27 acidic motif can contribute to the activation of bone
marrow CD8–resident cells. This motif is therefore likely to be
a factor in the potent antitumor activities of IL-27, which are
known to involve CD8 T cells (11–19, 60). As bone marrow
niches are also preferred sites for the metastasis of several solid
tumors and for blood cell malignancies (61, 62), the conjunction
of potent antitumoral activities and of bone binding properties of
IL-27 could be beneficial therapeutically (11–19). This could be
particularly relevant for multiple myeloma treatment as the IL-27
therapeutic activity on this neoplasia is accompanied by a capacity
to inhibit osteoclastogenesis (17, 20, 63) and IL-17 production,
which is known to promote myeloma growth (64). This property
of IL-27 could also be promising for the treatment of bone metastatic prostate cancer, because IL-27 modifies the cross-talk between bone and immune cells (65).
The location of the IL-27 HA-binding motif in a flexible loop
connecting the C-D a-helices suggests that this motif could be
used to engineer type I cytokine derivatives with bone-binding
properties. For example, this could be an attractive modification
for G-CSF, which is used therapeutically for the mobilization of
hematopoietic stem cells.
IL-27 BINDS BONE HA
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