Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity.
ABSTRACT The chemokine IFN-gamma-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines, forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was unable to induce recruitment of activated CD8+ T cells into the airways of mice after intratracheal instillation. Use of a different IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding. Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instillation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in vivo.
- [Show abstract] [Hide abstract]
ABSTRACT: Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.Pharmacological reviews 01/2014; 66(1):1-79. · 18.55 Impact Factor
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ABSTRACT: Chemokines are small proteins best known for their role in controlling the migration of diverse cells, particularly leukocytes. Upon binding to their G-protein-coupled receptors on the leukocytes, chemokines stimulate the signaling events that cause cytoskeletal rearrangements involved in cell movement, and migration of the cells along chemokine gradients. Depending on the cell type, chemokines also induce many other types of cellular responses including those related to defense mechanisms, cell proliferation, survival, and development. Historically, most research efforts have focused on the interaction of chemokines with their receptors, where monomeric forms of the ligands are the functionally relevant state. More recently, however, the importance of chemokine interactions with cell surface glycosaminoglycans has come to light, and in most cases appears to involve oligomeric chemokine structures. This review summarizes existing knowledge relating to the structure and function of chemokine oligomers, and emerging methodology for determining structures of complex chemokine assemblies in the future.Progress in molecular biology and translational science 01/2013; 117:531-78. · 3.11 Impact Factor
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ABSTRACT: Structural analyses of protein-protein interactions are required to reveal their functional mechanisms, and accurate protein-protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein-protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s-1). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein-protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.Quarterly Reviews of Biophysics 02/2009; 54(2):123-140. · 11.88 Impact Factor
Oligomerization of CXCL10 Is Necessary for Endothelial Cell
Presentation and In Vivo Activity1
Gabriele S. V. Campanella,* Jan Grimm,†Lindsay A. Manice,* Richard A. Colvin,*
Benjamin D. Medoff,* Gregory R. Wojtkiewicz,†Ralph Weissleder,†and Andrew D. Luster2*
The chemokine IFN-?-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T
lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines,
forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the
functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was
able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was
unable to induce recruitment of activated CD8?T cells into the airways of mice after intratracheal instillation. Use of a different
IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding.
Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instil-
lation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and
could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for
presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in
vivo. The Journal of Immunology, 2006, 177: 6991–6998.
NKT cells. IP-10 exerts its effect on these cells by binding to the
seven-transmembrane, G protein-coupled receptor CXCR3, which
it shares with two other ligands, IFN-inducible T cell-?-chemoat-
tractant (I-TAC/CXCL11) and monokine-induced by IFN-? (Mig/
CXCL9). IP-10 is up-regulated in a wide range of human inflam-
matory diseases, including skin diseases (1–3), atherosclerosis (4),
multiple sclerosis (5, 6), allograft rejection (7, 8), viral hepatitis
(9), and others. In murine models of human diseases, IP-10 has
been shown to play a role in T cell recruitment and disease pa-
In addition to binding to its high affinity receptor CXCR3, IP-10,
like many other chemokines, also binds to glycosaminoglycans
(GAGs) (14, 15). It is generally accepted that GAGs help sequester
and retain chemokines on the endothelium and extracellular matrix
(15–18). For example, it has recently been demonstrated that en-
dothelial heparan sulfate is required both for in vitro chemokine
presentation on endothelial cells and for in vivo chemokine-in-
duced recruitment of leukocytes (19). In addition, we have shown
he chemokine IFN-inducible protein of 10 kDa (IP-103;
CXCL10) regulates the in vivo migration of effector T
cells and other effector lymphocytes, such as NK and
that chemokines can be secreted bound to GAGs as high molecular
mass complexes (20). Similarly, it has been demonstrated that che-
mokines can oligomerize on GAGs (18, 21) at physiological, low
nanomolar concentrations, where they would normally be present
The physiological role of chemokine dimerization and oli-
gomerization has not yet been fully established and remains an
important question that needs to be answered to fully understand
chemokine function. Earlier in vitro studies done with monomeric
chemokine mutants suggested that monomers can bind and acti-
vate their corresponding chemokine receptors similar to the wild-
type protein (22–25). This led to the conclusion that chemokines
are biologically active as monomers. Consistent with this view,
mutants of IL-8 (CXCL8) with reduced potential to form dimers
were found to be fully active in vivo (26), although it is not known
whether these mutants could form oligomers on GAGs. However,
a more recent study demonstrated that monomeric IL-8 was
cleared more rapidly from the lung, suggesting that the ability to
dimerize plays a role in the retention of this chemokine in tissue
(27). In support of a physiological role for chemokine oligomer-
ization, studies with three CC chemokines, RANTES (CCL5),
MCP-1 (CCL1), and MIP-1? (CCL4), demonstrated that mono-
meric mutants had markedly reduced potential to recruit cells in
vivo, although they were active in in vitro chemotaxis (28). How-
ever, no mechanism for this effect was established. Oligomeriza-
tion was also important for RANTES-induced CCR1-mediated
monocyte arrest in vitro (29). Furthermore, although the heparin
binding affinity of some monomeric chemokine variants is only
slightly lower than the wild-type proteins, oligomerization was
found to increase the avidity for GAGs by positive cooperativity
(21), suggesting that oligomerization plays a role in chemokine-
In prior studies, we demonstrated that IP-10, a CXC chemokine,
forms high molecular mass complexes in solution as well as on the
plasma membrane (15). However, no data are available on the
physiological role of oligomerization for IP-10 or any other
CXCR3 ligand. We therefore studied a monomeric variant of IP-10
*Division of Rheumatology, Allergy, and Immunology, Center for Immunology and
Inflammatory Diseases, and†Center for Molecular Imaging Research, Massachusetts
General Hospital, Harvard Medical School, Charlestown, MA 02129
Received for publication June 1, 2006. Accepted for publication August 30, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant RO1-CA69212 (to
A.D.L.) and R24-CA92782 (to R.W.).
2Address correspondence and reprint requests to Dr. Andrew D. Luster, Massachu-
setts General Hospital, Building 149, 13th Street, Charlestown, MA 02129. E-mail
3Abbreviations used in this paper: IP-10, IFN-?-inducible protein of 10 kDa; GAG,
glycosaminoglycan; HMEC, human microvascular endothelial cells; CHO, Chinese
hamster ovary; BAL, bronchoalveolar lavage; DTPA, diethylenetriaminepentaacetic;
CT, computed tomography; SPECT, single-photon emission CT; Sulfo-EGS, sulfo-
ethylene glycolbis(sulfosuccinimidylsuccinate); BS3, bis(sulfosuccinimidyl)suberate.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
and compared its in vitro and in vivo activities with those of the
wild-type chemokine. We found that oligomerization plays an im-
portant role for the in vivo activity of IP-10 and have elucidated
the mechanism by which oligomerization is required for the func-
tion of IP-10, which may serve as a paradigm for other chemokines
that require oligomerization for their in vivo activity.
Materials and Methods
Materials and mice
Chemically synthesized IP-10 (wild-type or monomeric variant) was ob-
tained from the University of British Columbia (Vancouver, Canada). Re-
combinant IP-10 and mutants of IP-10 were purified as previously de-
scribed (14). C57BL/6 mice were purchased from the National Cancer
Institute. The OT-I TCR mice on the C57BL/6 background were obtained
from Jackson ImmunoResearch Laboratories. All protocols were approved
by the Massachusetts General Hospital Subcommittee on Research and
Cell culture and transfection
300-19 cells were maintained in complete RPMI containing 10% FCS and
transfected with human or murine CXCR3 as described (30). Human mi-
crovascular endothelial cells (HMEC) were maintained in complete Clo-
netics EGM medium, Chinese hamster ovary (CHO) cells and Beas2B
cells, a cell line derived from human bronchial epithelial cells transformed
with a hybrid adeno-SV40 virus, in complete F12 medium.
Heparin Sepharose and S Sepharose chromatography
Aliquots of 20 ?g of IP-10 were loaded on a 1-ml Heparin HiTrap or S
Sepharose FF (cationic exchange) column (both from Pharmacia) equili-
brated in 50 mM Tris, pH 7.5, on an AKTA machine (Pharmacia). The
mutants were eluted with a 20-ml gradient of 0–2 M NaCl in 50 mM Tris,
pH 7.5, and their elution time were measured by OD214.
Superdex 200 gel filtration
Aliquots of 20 ?g of IP-10 were loaded on a Superdex 200 column (Am-
ersham Biosciences) equilibrated in either PBS or 25 mM sodium phos-
phate buffer, pH 7.5, with 0.5 M NaCl on an AKTA machine (Pharmacia).
The proteins were eluted in the same buffer over 1.5 column volumes (30
ml), and protein elution was measured by OD214.
For native gels, 1 ?g of IP-10 was loaded onto a 4–20% Ready PAGE gel
(Bio-Rad), and run with reversed charge at 140 V, without the addition of
SDS or DTT. Cytochrome c (30 ?g; Sigma-Aldrich; 15 kDa; pI 10.5) was
used as a dye front to determine how far the gel had run. For cross-linking
experiments, IP-10 was diluted into PBS and incubated for 30 min before
the addition of a 50-fold molar excess of bis(sulfosuccinimidyl)suberate
(BS3) or sulfo-ethylene glycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS;
Pierce). After 60 min of incubation at 37°C, the reaction was terminated by
the addition of 50 mM Tris. Samples were spun for 10 min to remove any
precipitate and run on a 4–20% SDS-PAGE gel with DTT. Proteins were
visualized with Silver-Stain ProteoPlus (Sigma-Aldrich).
Receptor binding assay
Binding assays were performed as previously described (14).
Chemotaxis assays were performed as described (14). Briefly, chemokine
dilutions were added to the bottom well of a 96-well chemotaxis plate
(NeuroProbe). Activated OT-I cells, days 8–11 in culture with IL-2, were
added on top of the membrane (2.5 ? 104cells) and allowed to migrate at
37°C for 2 h, after which cells in the bottom wells were counted under a
microscope. For transendothelial migration, HMEC cells were grown to
confluency on the bottom side of the chemotaxis filter. HMEC were
washed twice with cold RPMI 1640, and, where indicated, chemokines
were added to the cells in chemotaxis media and incubated for 60 min at
37°C. Nonbound chemokines were removed by washing the cells four
times with cold RPMI 1640 before proceeding with the chemotaxis assay
as described above.
Internalization of CXCR3
Internalization of murine CXCR3 on 300-19 cells was measured as previ-
ously described (31). Internalization experiments with human CXCR3-
transfected cells yielded similar results (data not shown).
In vivo OT-I recruitment.
OT-I cells were prepared as previously de-
scribed (32, 33) and harvested on day 6 with Lympholyte (Cedarlane Lab-
oratories). OT-1 cells (5–7 ? 106) were injected i.p. into male C57BL/6
mice. After 48 h, 0.5–50 ?g of IP-10 in 50 ?l PBS were injected intra-
tracheally. After 18 h, lungs were lavaged with six aliquots of 0.5 ml of
PBS containing 0.6 mM EDTA. RBC were lysed with RBC lysis buffer
after which total cells in the bronchoalveolar lavage (BAL) were counted.
Cells were incubated for 10 min with 2.4G2 anti-Fc?III/II receptor (BD
Pharmingen) and were then stained with FITC-conjugated anti-murine
murine CD8 at 4°C for 20 min. Cells were fixed with 1% paraformalde-
hyde, and cytofluorimetry was performed using a FACSCalibur cytometer
Concentrations of IP-10 in the BAL were measured using a human IP-10
ELISA kit (R&D Systems) according to the manufacturer’s instruction.
Wild-type and monomeric IP-10 were conjugated to diethylenetriamine-
pentaacetic (DTPA) dianhydride (Sigma-Aldrich) at a 1:5 molar ratio in 50
mM sodium phosphate buffer, pH 7.4 for 30 min on ice, and dialyzed
extensively to remove unbound DTPA. For radiolabeling, DTPA-IP-10
(100–150 ?g) was incubated with 1–2 mCi of111InCl3at pH of 6.5 for 30
min. After separation of remaining free111In by size exclusion, the radio-
labeled IP-10 was immediately injected intratracheally.
Single-photon emission computed tomography (SPECT) data were ac-
quired on a combined small animal SPECT-computed tomography (CT)
scanner (XSPECT; Gamma Medica) with a submillimeter resolution. After
the SPECT acquisition (radius of rotation 3 cm, 32 projections, 60 s/pro-
jection) a CT scan was acquired (256 projections, 50 kV, 500 mA) and
coregistered with the SPECT dataset for image fusion and exact three-
dimensional anatomical localization of the tracer signal. Imaging took
place after injection and after 4, 24, 48, and 120 h. After 24 h, some mice
were sacrificed for conventional biodistribution analysis by harvesting the
indicated organs and measuring their activity in a Wallac 1480 Wizard
gamma counter (PerkinElmer). SPECT data analysis was performed to
obtain the number of counts per region in the lung.
Binding of IP-10 to endothelial and epithelial cells
Wild-type or mutant IP-10 was biotinylated with EZ-Link Sulfo-NHS-Bi-
otin (Pierce) at a 1:5 molar ratio in 50 mM sodium phosphate buffer over-
night at pH 6.5 to preferentially label the N terminus free amine group. The
reaction was quenched by the addition of 50 mM Tris, pH 7.0. Coupling of
the wild-type and monomeric IP-10 to biotin was similar as determined by
ELISA. IP-10 was added to cell suspensions (1.5 ? 105) in complete me-
dium for 1 h at 37°C. Cells were washed with cold PBS and stained with
Streptavidin-allophycocyanin. For control experiments, cells were prein-
cubated with anti-CXCR3 Ab (clone 1C6 from BD Biosciences or clone
49801 from R&D Systems) or control IgG Ab for 10 min at 10 ?g/ml
before addition of IP-10. In some experiments, HMEC (5 ? 105) were
incubated with 50 ?g of heparin for 10 min, washed with PBS, and incu-
bated with 10 U of heparinase I, 5 U of heparinase III, and 5 U of chon-
droitinase ABC (all from Sigma-Aldrich) in basal medium supplemented
with 0.5% BSA for 4 h at 37°C before proceeding with the binding assay.
In vitro characterization of monomeric IP-10
To analyze the aggregation state of wild-type or monomeric IP-10,
we loaded the protein on a Superdex 200 gel filtration column.
Under physiological salt conditions, wild-type IP-10 did not elute
from the gel filtration column, suggesting that it forms oligomers
that are unable to elute from the column. A synthetic obligate
monomer of IP-10, with the mutation L27NMe, that was designed
to solve the nuclear magnetic resonance structure of IP-10 (34),
eluted at the expected elution volume (Fig. 1A). In contrast, the
previously described mutant R22E, which has reduced heparin and
CXCR3 binding affinity, behaved like wild-type IP-10. At a higher
6992OLIGOMERIZATION OF IP-10 IS REQUIRED FOR ACTIVITY
salt concentration (0.5 M NaCl), wild-type, monomeric, and R22E
IP-10 all eluted at the same elution volume (Fig. 1B), suggesting
that higher ionic strength interrupts oligomer formation. On a na-
tive gel, monomeric IP-10 ran at a lower molecular mass than the
wild-type or R22E mutant protein (Fig. 1C), demonstrating again
that wild-type and R22E IP-10s have a distinctly higher molecular
mass than the monomeric variant. Because native gels do not allow
size determination, cross-linking experiments were performed, and
cross-linked proteins were run on an SDS-PAGE gel, which allows
estimation of molecular mass. Wild-type and R22E IP-10 dis-
played monomeric, dimeric, and octameric forms on an SDS-
PAGE gel after cross-linking with Sulfo-EGS or BS3(Fig. 1D),
and trimeric and tetrameric forms were also occasionally observed
after cross-linking with BS3(data not shown). These results do not
preclude the existence of even higher molecular mass oligomers
for wild-type IP-10, which might not form stable complexes with
the cross-linkers used. In contrast, monomeric IP-10 displayed
only the monomeric form (Fig. 1D).
Next, we analyzed the heparin binding affinity of wild-type and
monomeric IP-10 by elution from a heparin-Sepharose column.
Wild-type IP-10 eluted from the heparin column at 0.95 M NaCl.
Monomeric IP-10 eluted at 0.65 M NaCl, 0.30 M less than the
wild-type (Fig. 2A). To determine the specific binding of the che-
mokines to heparin as compared with electrostatic interactions,
wild-type IP-10 was loaded onto a cationic exchange column and
eluted at 0.73 M NaCl, a 0.22 M reduction compared with the
heparin-Sepharose column. In contrast, monomeric IP-10 eluted at
0.59 M NaCl, only 0.06 M less than from the heparin column.
These data indicate that monomeric IP-10 has lost most of its spe-
cific binding to heparin.
In competitive binding assays using 300-19 B cells expressing
CXCR3, monomeric IP-10 competed for the binding of125I-la-
beled IP-10 with an IC50of 1.3 nM, whereas wild-type IP-10 had
an IC50of 0.16 nM (Fig. 2B). This 10-fold reduction of binding
affinity to the high affinity, seven-transmembrane receptor is sim-
ilar to what has been described for other monomeric or heparin-
binding reduced mutant chemokines (28). In 300-19 cells express-
ing CXCR3, monomeric IP-10 was able to induce comparable
CXCR3 internalization as wild-type IP-10, but only at 10-fold
higher concentrations (Fig. 2C). Monomeric IP-10 was also able to
induce chemotaxis of activated CD8?T cells expressing CXCR3
but again, a 10-fold higher concentration of the monomer was
needed to achieve chemotactic indices similar to those induced by
wild-type IP-10 (Fig. 2D). However, monomeric IP-10 was as ef-
ficacious at its peak concentration as the wild-type protein.
A novel in vivo recruitment assay for IP-10
To test the in vivo activity of IP-10, we developed a new, physi-
ologically relevant in vivo recruitment assay. For this, we purified
CD8?T cells from OT-I mice, which are transgenic for the TCR
specific for the OVA peptide SIINFEKL bound to class I MHC
(32, 33). After 6 days in culture with IL-2 and IL-12, CD8?T cells
were adoptively transferred by i.p. injection into C57BL/6 mice.
Two days after injection of CD8?cells, IP-10 (wild-type or mu-
tant) was injected intratracheally into the mice. The following day,
lungs were lavaged, and CD8?cell recruitment into the airways
was measured by flow cytometry. As seen in Fig. 3A, after intra-
tracheal injection of PBS, a very low number of CD8?or CD4?
T cells was observed in the BAL. However, after injection of 5 ?g
of wild-type IP-10, a large influx of CD8?, but not CD4?, T cells
tography. 10 ?g of IP-10 (wild type (wt), monomeric (mono), or R22E)
was loaded onto a Superdex 200 column equilibrated in either PBS (A) or
50 mM sodium phosphate, pH 7.4, supplemented with 0.5 M NaCl (B) and
eluted with 1.5 column volumes of the respective buffer. C, Native gel; 1
?g of IP-10 (wild type, monomeric, or R22E) was loaded onto a 4–20%
PAGE gel and run with reversed charge at 140V without SDS in loading
or running buffer. Cytochrome c (cytoc.c; 30 ?g) was used as a dye front.
D, Cross-linking gel. IP-10 (1 ?g/lane) was incubated with a 50-fold molar
excess of Sulfo-EGS or BS3for 60 min of incubation at 37°C. Samples
were run on a 4–20% SDS-PAGE gel. Abs (214 nm), OD214.
Oligomerization of IP-10. A and B, Gel filtration chroma-
binding. IP-10 (20 ?g; wild type or monomeric) was loaded onto a 1-ml
Heparin HiTrap column and eluted with increasing concentration of NaCl.
One representative curve from two to three experiments is shown for A–C.
B, Competitive receptor binding assay. The binding of125I-IP-10 to 300-
19/human CXCR3 cells was competed by increasing the concentration of
unlabeled IP-10 (wild type or monomeric). Each data point represents the
mean ? SD of duplicate values of three experiments. C, CXCR3 internal-
ization. Cell surface expression of CXCR3 was measured after incubation
of 300-19/mCXCR3 cells with the indicated concentrations of IP-10 for 20
min at 37°C and stained with anti-murine CXCR3-PE Ab. D, Chemotaxis.
Chemotaxis of activated OT-I CD8?T cells in response to IP-10 (wild type
or mutants) was performed in duplicate using a Neuroprobe chamber. One
representative assay of three experiments is shown for C and D. Abs (214
nm), OD214; MFI, mean fluorescence index; max, maximum.
In vitro activity of monomeric IP-10. A, Heparin Sepharose
6993The Journal of Immunology
was observed in the BAL. The preferential recruitment of the
adoptively transferred CD8?T cells was further demonstrated by
use of the Thy 1.1 marker (G. S. V. Campanella, A. D. Luster,
manuscript in preparation). IP-10 has been shown to be expressed
in the lung by bronchial epithelial cells (35) as well as other cell
types, leading to recruitment of effector T lymphocytes into the
airways during Th1 and Th2 inflammation. By injecting IP-10 di-
rectly into the murine airways, this assay models the natural en-
vironment in which IP-10 is induced and achieves a robust and
specific in vivo chemotactic response.
Monomeric IP-10 is unable to cause in vivo recruitment
A dose response of wild-type IP-10 showed that 0.5 ?g of IP-10
caused a statistically significant recruitment of CD8?T cells, with
a mean recruitment index of 3.7, whereas 5 and 50 ?g each in-
duced even more recruitment, with mean recruitment indices of
15.7 and 53.6, respectively. However, intratracheal injections of
0.5–50 ?g of monomeric IP-10 induced no recruitment of CD8?T
cells into the airways (Fig. 3B).
Oligomerization rather than heparin binding is important for the
in vivo activity of IP-10
To understand why monomeric IP-10 is unable to induce recruit-
ment of CD8?T cells into the airways, we studied two of our
previously published IP-10 mutants in the in vivo recruitment
model. One of them, mutant R8A, has the same heparin-binding
affinity and oligomerization pattern as wild-type IP-10 but has a
60-fold reduced CXCR3 binding affinity and no chemotactic in
vitro activity (Fig. 2D and Ref. 14). As expected, R8A was unable
to induce recruitment of CD8?cells in vivo (Fig. 3C) and con-
firmed that the recruitment of the CD8?T cells was a CXCR3-
dependent process. Of particular interest was mutant R22E, which
had similarly reduced heparin-binding affinity (elution at 0.61 M
NaCl from a heparin-Sepharose column; Ref. 14) as monomeric
IP-10. The CXCR3-binding affinity of R22E was also similar to
that of monomeric IP-10 (IC503.4 nM; Ref. 14), but R22E was not
as efficacious as wild-type or monomeric IP-10 in chemotaxis (Fig.
2D). However, mutant R22E oligomerizes like wild-type IP-10
(Fig. 1). In the in vivo recruitment assay, mutant R22E induced a
dose-dependent, statistically significant influx of CD8?T cells at
a dose of 5 ?g (mean recruitment index, 4.7) and 50 ?g (mean
recruitment index, 25.5; Fig. 3C). Although this mutant was
clearly less potent at lower doses than wild-type IP-10, higher
concentrations of mutant R22E could overcome its reduced
CXCR3- and heparin-binding affinity. In contrast, monomeric
IP-10 was unable to induce any recruitment of T cells even at a
dose of 50 ?g. This demonstrates that the reduced heparin- and
CXCR3-binding affinity of monomeric IP-10 is not the main rea-
son for its inability to induce T cell recruitment in vivo. Instead, it
suggests that the ability to oligomerize is an essential requirement
for the in vivo activity of IP-10.
Coinjection of monomeric IP-10 does not inhibit wild-type IP-10
It has been reported for the CC chemokines RANTES (36) and
MCP-3 (37), that monomeric or heparin-binding reduced mutants
were able to inhibit the in vivo activity of the wild-type protein. To
test the ability of monomeric IP-10 to inhibit wild-type IP-10, we
coinjected 5 ?g of both wild-type and monomer IP-10 intratrache-
ally into the same mouse. As shown in Fig. 3D, there was no
significant inhibition of wild-type IP-10 by coinjected monomeric
IP-10, suggesting that monomeric IP-10 does not act as a dominant
negative inhibitor of IP-10. In addition, monomeric IP-10 did not
form heterodimers with wild-type IP-10 as determined by gel fil-
tration (data not shown).
Biodistribution of intratracheally injected IP-10
To investigate the biodistribution and retention of wild-type and
monomeric IP-10 after intratracheal instillation, we labeled both
proteins with111In. Labeling IP-10 for biodistribution studies did
not change its IC50values in the competitive CXCR3 binding as-
say, its heparin-binding affinity, its oligomerization state as mea-
sured by gel filtration, or its in vitro chemotaxis dose response
(data not shown).111In-labeled wild-type or monomeric IP-10 was
injected intratracheally into mice and subsequently imaged by
SPECT-CT after 0, 4, 24, 48, and 120 h postinjection. As seen in
Fig. 4, A–D, after instillation, most radioactivity was found in the
lung for both wild-type and monomeric IP-10. Interestingly, even
at later time points, most of the radioactivity in the animal was still
found in the lung for both wild-type and monomeric IP-10, up to
the last time point studied (120 h). Analysis of the imaging matrix
demonstrated that both wild-type and monomeric IP-10 had 70–
90% of the radioactivity in the animal localized in the lungs (Fig.
4E). To confirm these results, in some experiments, mice were
sacrificed after 24 h, and the radioactivity in the organs was mea-
sured (Fig. 4F). As seen in the SPECT-CT, most of the labeled
wild-type and monomeric IP-10 was found in the lung, with a
small amount in the kidneys and liver. This small amount of ra-
dioactivity in the kidneys and liver probably reflects a low level of
activated OT-I CD8?T cells in vivo. IP-10 (wild type or mutant) was
injected intratracheally at the indicated concentration after adoptive trans-
fer of activated OT-I CD8?cells 48 h previously. The BAL was harvested
18 h later, and CD8?T cells were analyzed by flow cytometry. A, Flow
analysis of T lymphocytes in the BAL. After intratracheal injection of PBS
or 5 ?g of wild-type IP-10, cells were recovered from the BAL, and the
percentage of CD3?CD8?and CD3?CD4?T cells was determined by
flow cytometry. B, Quantification of CD8?T lymphocytes recruited into
the BAL. Total number of CD8?T lymphocytes recruited into the BAL by
indicated doses of wild-type or monomeric IP-10. C, Dose response of
IP-10 mutants. IP-10 mutants at the indicated doses were injected intra-
tracheally after adoptive transfer of activated CD8?cells 48 h prior. The
BAL was harvested 18 h later, and CD8?T cells were analyzed by flow
cytometry. The recruitment index was calculated in comparison with in-
tratracheal injection of PBS. D, Coinjection of wild-type and monomeric
IP-10. Wild-type or monomeric IP-10 (5 ?g) were either injected intratra-
cheally individually, or premixed for 30 min in PBS before injection. The
rest of the experiment was performed as described in C. Experiments were
performed with four mice per group; one representative experiment out of
at least two is shown.
Monomeric (Mono) IP-10 does not induce recruitment of
6994OLIGOMERIZATION OF IP-10 IS REQUIRED FOR ACTIVITY
IP-10 excretion. It is estimated that after 24 h ?75% of the injected
IP-10 was still in the animal. These imaging experiments clearly
demonstrate that the retention of monomeric IP-10 in the lung is
similar to wild-type IP-10.
To determine whether the IP-10 was in the airway fluid or in the
lung parenchyma, we measured the concentration of wild-type and
monomeric IP-10 in the BAL by ELISA. We found only a very
small fraction of injected wild-type (0.14%) or monomeric IP-10
(0.08%) was recoverable from the airways after 18 h (Fig. 5). In
contrast, in control experiments, most of the IP-10 was recovered
when the lavage was done ten minutes after intratracheal injection
(46.7% for wild-type and 61.8% for monomeric IP-10). Addition-
ally, increasing the salt concentration of the lavage fluid to 1 M
NaCl to release chemokine potentially bound to GAGs on bron-
chial epithelial cells did not release much more IP-10 and yielded
no difference between wild-type and monomeric IP-10. This sug-
gests that within 18 h both wild-type and monomeric IP-10 moved
out of the airways into the lung parenchyma.
Reduced endothelial cell binding of monomeric IP-10
To further explore the mechanisms underlying monomeric IP-10’s
inability to induce in vivo recruitment, we studied the in vitro
binding of wild-type and monomeric IP-10 to endothelial and ep-
ithelial cells. For this purpose, we labeled the chemokines with
biotin, which did not influence their in vitro activity or oligomer-
ization state (data not shown). Analysis of IP-10 binding to HMEC
by flow cytometry demonstrated dose-dependent binding of wild-
type IP-10, whereas monomeric IP-10 failed to show significant
binding even at a concentration of 10,000 ng/ml (Fig. 6, A and B).
There was a low level of background binding of the monomer to
the cells; however, it was not concentration dependent. In contrast,
biotin-labeled mutant R22E bound to HMEC at higher concentra-
tions, although markedly reduced compared with wild-type IP-10.
To exclude that CXCR3, which has been shown by several groups
to be expressed on HMEC under certain conditions (e.g., Refs. 38
and 39), is involved in the binding of IP-10 to HMEC, two Abs
A, Wild-type IP-10 after 30 min; B, wild-type IP-10 after 120 h; C, monomeric IP-10 after 30 min; D, monomeric IP-10 after 120 h. i, axial CT image;
ii, SPECT image; iii, fusion of the corresponding coregistered axial CT and SPECT image (i and ii); iv, coronal reformation of the fused CT/SPECT dataset;
v, three-dimensional volume rendering of the fused CT/SPECT dataset presented in a 0-degree and 90-degree angle. One representative animal of three per
group is shown, the experiment was repeated twice. E, Percent of radioactivity in the lung compared with total radioactivity in the animal. The proportion
of radioactivity in the lung was calculated by SPECT matrix analysis for different time points after injection of111In-labeled IP-10 (n ? 3 animals per
group). F, Organ biodistribution. Animals were sacrificed 24 h post-intratracheal injection of111In-labeled IP-10; the indicated organs were harvested and
counted in a scintillation counter (n ? 3 animals per group). Results are means ? SD.
Molecular imaging of111In-labeled IP-10.111In-labeled IP-10 was injected intratracheally into mice, and mice were imaged by SPECT-CT.
6995The Journal of Immunology
that block CXCR3A and B and detect CXCR3 on HMEC were
used during the binding studies, and did not inhibit the binding of
wild-type or monomeric IP-10 to HMEC (Fig. 6C). To investigate
the mechanism of IP-10 binding to HMECs, the cells were di-
gested with glycosidases before performing the binding assay.
Wild-type IP-10 binding was reduced by 63% after glycosidase
treatment, demonstrating that wild-type IP-10 binding to endothe-
lial cells is mainly dependent on glycosaminoglycans (Fig. 6D). In
contrast, the low level binding observed for monomeric IP-10 was
not affected by glycosidase treatment.
Wild-type IP-10 also bound strongly to epithelial cells, both to
human bronchial epithelial cell-derived Beas2B cells (Fig. 6E) as
well as CHO cells (Fig. 6F), whereas monomeric IP-10 did not
show significant binding to either cell line. Similarly to what was
seen with the glycosidase-treated endothelial cells, binding of
IP-10 to CHO 745 cells, which are deficient in GAGs, was dras-
tically reduced, demonstrating that epithelial cell binding was also
dependent on GAGs (Fig. 6G). Indeed, it appears as if binding of
wild-type IP-10 to CHO 745 cells was reduced to the level of
monomeric IP-10. Binding to CHO cells was also analyzed by
immunofluorescence and clearly illustrated binding of wild-type
IP-10 to epithelial cell surfaces (Fig. 6H), whereas there was no
significant binding of monomeric IP-10 to CHO cells (Fig. 6I),
implying that IP-10 oligomerization is needed for binding to en-
dothelial and epithelial cells.
Monomeric IP-10 does not immobilize on endothelial cells to
induce transendothelial migration
Leukocytes must traverse the endothelium to enter tissue from the
blood. We therefore studied the ability of monomeric IP-10 to
induce in vitro migration through an endothelial cell layer. HMEC
were grown to confluency on the bottom side of Neuroprobe che-
motaxis filters. In some wells, wild-type or monomeric IP-10 was
added to the endothelial cells for 1 h at 37°C and allowed to im-
mobilize on the endothelial cell layer. Nonbound IP-10 was
washed off the endothelial cells, after which the chemotaxis cham-
ber was assembled without the addition of further IP-10 in the
bottom chamber. In other wells, wild-type or monomeric IP-10
was added in solution to the bottom chamber before adding the
filter with confluent HMEC. Activated CD8?T cells were added
to the top of the chemotaxis filter and allowed to migrate for 2 h
in response to IP-10. Monomeric IP-10 added in solution to the
lower chamber was able to induce chemotaxis of T cells through
the endothelial cell layer similarly as through bare filters (Fig. 7).
In contrast, when IP-10 was bound to the HMEC and washed off
before addition of T cells, monomeric IP-10 was unable to induce
T cells transendothelial migration, whereas wild-type IP-10 was
able to induce chemotaxis of T cells through the HMEC layer. This
for 1 h at 37°C with the indicated concentration of biotinylated wild-type, monomeric, or R22E IP-10. After the cells were washed, binding was measured
by flow cytometry using Streptavidin-allophycocyanin (Strep-APC; A–G) or by immunofluorescence using Streptavidin-FITC (H and I). A, Flow cytometry.
Binding of wild-type, monomeric, and R22E IP-10 to HMEC. Representative flow diagram at 10,000 ng/ml IP-10. B, Quantification of binding to HMEC.
Mean fluorescent intensity (MFI) at indicated concentrations of IP-10 is shown. C, CXCR3 Ab blocking. Mean fluorescent intensity at 5000 ng/ml IP-10
binding to HMEC after pretreatment with Abs. D, Glycosidase-treated HMEC. Mean fluorescent intensity at 5000 ng/ml IP-10 binding to HMEC after
glycosidase treatment. E and F, Flow cytometry. Binding of 5000 ng/ml wild-type or monomeric IP-10 to Beas2B (E), CHO wild-type (E), or CHO 745
cells (F). G, Immunofluorescence of wild-type IP-10 binding to CHO cells. H, Immunofluorescence of monomeric IP-10 binding to CHO cells. Wild-type
IP-10 or monomeric IP-10 (5000 ng/ml) binding to CHO wild-type cells (original magnification, ?600). One representative experiment of at least two is
Binding of wild-type (wt), monomeric, and R22E IP-10 to endothelial and epithelial cells. Cells (HMEC, Beas2B, and CHO) were incubated
injection. Wild-type or monomeric IP-10 (5 ?g) was injected intratrache-
ally into mice. BAL was performed 10 min or 18 h after instillation. After
the 18-h BAL, lungs were lavaged with 1 ml of 1 M NaCl buffer. IP-10
concentration was measured by ELISA. Experiments were performed with
three mice per group and repeated twice. Results are means ? SD.
IP-10 recovery in BAL 10 min or 18 h after intratracheal
6996OLIGOMERIZATION OF IP-10 IS REQUIRED FOR ACTIVITY
suggests that monomeric IP-10 is unable to be retained on the
endothelial cell layer, whereas wild-type IP-10 can be immobilized
on endothelial cells, which is required for transendothelial cell
migration of CXCR3-expressing lymphocytes.
The physiological role of chemokine oligomerization is still un-
clear. Here, we show that oligomerization is essential for the in
vivo activity of IP-10; moreover, we identify a novel mechanism
for this effect, which may be applicable to other chemokines.
To investigate the role of IP-10 oligomerization, we utilized a
synthetic mutant variant of IP-10 with an additional N-methyl
group at position L27, which interrupts the main chain hydrogen
bond between residues L27 and I29 on opposing chains. Gel fil-
tration studies as well as sizing gels with or without cross-linkers
clearly demonstrated that the mutant variant L27NMe IP-10 is an
obligate monomer, whereas wild-type IP-10 forms higher order
Although monomeric IP-10 had the ability to induce in vitro
chemotaxis and CXCR3 internalization with efficacy comparable
with that of wild-type IP-10, monomeric IP-10 was unable to in-
duce recruitment of activated CD8?T cells into the airways of
mice even at concentrations 100-fold higher than the effective
wild-type IP-10 concentration. Interestingly, mutant R22E, which
had reduced heparin and CXCR3 binding similar to that of mo-
nomeric IP-10, yet oligomerized like wild-type IP-10, induced ro-
bust recruitment of T cells in vivo at 10-fold higher concentration
compared with wild-type IP-10. We were therefore able to differ-
entiate between heparin binding and oligomerization and demon-
strate clearly that the inability of monomeric IP-10 to induce in
vivo recruitment is due to its inability to oligomerize and not sim-
ply due to its reduced heparin or CXCR3 binding affinity.
The biodistribution of chemokines after release into the body is
not well understood, and we are reporting here one of the first
molecular imaging studies of a chemokine. We found that both
wild-type and monomeric IP-10 were retained for ?5 days in the
lung after intratracheal instillation. Therefore, the difference in in
vivo activity of wild-type and monomeric IP-10 was not due to the
difference in retention at the site of injection. In addition, our bio-
distribution data showed a similar proportion of injected111In-
labeled wild-type and monomeric IP-10 in the kidneys and liver.
Because clearance through the liver and kidney most likely occurs
through the blood stream, these findings suggest that both wild-
type and monomeric IP-10 were similarly transported across the
epithelium and endothelium. Transport of proteins across the al-
veolar epithelial barrier has been well documented for various pro-
teins (reviewed by Hastings et al. in Ref. 40 and Kim and Malik in
Ref. 41) and occurs mainly through paracellular diffusion or trans-
cytosis across the epithelium. The mechanism by which intratra-
cheally instilled IP-10 crosses the epithelial and endothelial barrier
is not known. The results of the BAL ELISA suggest that the vast
majority of intratracheally injected wild-type and monomeric
IP-10 had left the airways after 18 h. However, the imaging studies
demonstrated that most of the IP-10 was retained in the lung,
whereas a small proportion was transported into the blood stream.
This suggests that both wild-type and monomeric IP-10 crossed
the epithelial barrier and were retained in the lung, from where a
small fraction of the proteins moved across the endothelium.
To understand the role of oligomerization for binding to endo-
thelial and epithelial cells, we studied the binding of wild-type and
monomeric IP-10 to both cells types in vitro. Wild-type IP-10
bound strongly to epithelial cells in a GAG-dependent manner. In
contrast, monomeric IP-10 did not bind well to epithelial cells.
Therefore, the process by which IP-10 leaves the airways does not
seem to require strong, GAG-dependent binding, as both wild-type
and monomeric IP-10 had left the airways after 18 h. However,
wild-type IP-10 also bound strongly to HMECs, whereas mono-
meric IP-10 bound very weakly to HMECs, and not in a dose-
dependent manner. Together with the biodistribution data showing
that both wild-type and monomeric IP-10 are transported into the
blood stream equally, this suggests that only wild-type IP-10 is
presented on the pulmonary endothelium under physiological flow
conditions to establish a haptotactic gradient on the endothelium,
which induces T cell recruitment.
To confirm this theory, the ability of wild-type and monomeric
IP-10 to induce transendothelial migration was investigated. The
continued presence of soluble monomeric IP-10 was able to induce
transendothelial migration of T cells similar to that induced by
wild-type IP-10. However, when only endothelial cell bound IP-10
was assayed by washing away unbound IP-10, wild-type IP-10 was
able to induce transendothelial chemotaxis, but monomeric IP-10
was not. This further demonstrates that monomeric IP-10 is unable
to be immobilized on endothelial cells and suggests that presen-
tation of IP-10 on endothelial cells is required for its activity
In summary, we found that after intratracheal injection, wild-
type oligomeric IP-10 crosses the epithelial barrier, is retained in
the lung, and is presented on the endothelium. This binding to
endothelial cells is dependent on oligomerization, and establishes
a haptotactic gradient, which induces recruitment of effector T
cells into the airways (Fig. 8). Monomeric IP-10 also crosses the
epithelial barrier and is retained in the lung after intratracheal in-
stillation. However, in the absence of oligomerization, monomeric
migration of activated CD8?T cells. HMEC were grown to confluency on
the bottom side of the filter membrane, and IP-10 (wild type or monomer)
was added either immobilized on the HMEC or added soluble to the bottom
well of a 96-well Neuroprobe chamber. Experiments were performed in
duplicated and repeated three times. Results are means ? SD.
Monomeric IP-10 does not induce in vitro transendothelial
Schematic view of IP-10 oligomerization in a lung recruit-
6997The Journal of Immunology
IP-10 is not retained on the endothelium and therefore cannot es-
tablish a haptotactic gradient to recruit CXCR3-expressing lym-
phocytes into the airspaces. We propose a novel mechanism of
chemokine regulation that requires oligomerization for presenta-
tion on endothelial cells, which is necessary for chemokine activity
We thank Dr. Andrew Tager for helpful discussions.
The authors have no financial conflict of interest.
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6998OLIGOMERIZATION OF IP-10 IS REQUIRED FOR ACTIVITY