This information is current as
of July 12, 2017.
Citrullination of CXCL12 Differentially
Reduces CXCR4 and CXCR7 Binding with
Loss of Inflammatory and Anti-HIV-1
Activity via CXCR4
Sofie Struyf, Samuel Noppen, Tamara Loos, Anneleen
Mortier, Mieke Gouwy, Hannelien Verbeke, Dana Huskens,
Souphalone Luangsay, Marc Parmentier, Karel Geboes,
Dominique Schols, Jo Van Damme and Paul Proost
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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 © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 182:666-674; ;
doi: 10.4049/jimmunol.182.1.666
http://www.jimmunol.org/content/182/1/666
The Journal of Immunology
Citrullination of CXCL12 Differentially Reduces CXCR4 and
CXCR7 Binding with Loss of Inflammatory and Anti-HIV-1
Activity via CXCR41
Sofie Struyf,2* Samuel Noppen,2* Tamara Loos,* Anneleen Mortier,* Mieke Gouwy,*
Hannelien Verbeke,* Dana Huskens,† Souphalone Luangsay,‡ Marc Parmentier,‡
Karel Geboes,§ Dominique Schols,† Jo Van Damme,3* and Paul Proost3*
C
hemokines are small cytokines, structurally characterized
by conserved cysteine residues and first described for
their ability to control leukocyte migration under basal
and inflammatory conditions (1– 4). Unlike most chemokines
whose function and expression are specific and centered around
their role in leukocyte trafficking, both stromal cell-derived factor
1/CXCL12 and its first identified receptor CXCR4 were found to
be expressed in a wide variety of cell types and tissues (3). Furthermore, this ubiquitous chemokine activates a broad spectrum of
leukocytes (including monocytes, lymphocytes, neutrophils, and
hematopoietic progenitor cells) and hence is an important mediator
*Rega Institute for Medical Research, Laboratory of Molecular Immunology and
†
Laboratory of Virology and Chemotherapy, University of Leuven, Leuven, Belgium;
‡
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels, Belgium; and §Department of Pathology, University Hospital Leuven, Leuven, Belgium
Received for publication June 30, 2008. Accepted for publication October 27, 2008.
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.
1
This work was supported by the European Union 6FP EC Contract INNOCHEM,
the Interuniversity Attraction Poles Programme-Belgian Science Policy, the Fund for
Scientific Research of Flanders (Belgium), the Concerted Research Actions of the
Regional Government of Flanders and the Center of Excellence of the K. U. Leuven
(Credit no. EF/05/15; Rega Institute). A.M. is research assistant and S.S. and M.G. are
senior research assistants from the Fund for Scientific Research of Flanders.
2
S.S. and S.N. equally contributed to this manuscript.
3
Address correspondence and reprint requests to Dr. Paul Proost or Dr. Jo Van
Damme, Rega Institute for Medical Research, Laboratory of Molecular Immunology,
K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail address:
[email protected] or [email protected]
www.jimmunol.org
during infection, inflammation, hematopoiesis, angiogenesis, and
tumor metastasis (5– 8). Indeed, CXCL12 has been discovered
rather as a cytokine that promotes pre-B cell growth (9), before its
chemotactic effect was elucidated (10). Importantly, mutant mice
with a targeted deletion of the CXCL12 gene die perinatally, because of marked defects in cardiac septal formation and vascularization of the gastrointestinal tract (11). Furthermore, it was found
that T-tropic (X4) HIV infection requires binding to a coreceptor,
i.e., CXCR4 which is the major functional receptor for CXCL12
(12). Recently, however, the orphan receptor RDC1, now renamed
CXCR7, has been identified as a second receptor for CXCL12,
breaking up the monogamous relationship between CXCL12 and
CXCR4 (13–15). Although CXCL12 does not belong to the cytokine-inducible proinflammatory chemokines, but is rather classified as a constitutive chemokine, several studies have revealed
increased expression of CXCL12 in different models of inflammation (16). In particular, recent reports suggest that this chemokine
may constitute a major determinant in the development of rheumatoid arthritis (RA),4 inducing the recruitment of T cells, neovascularization of rheumatoid synovium, and the release of matrix
metalloproteinase 3 by chondrocytes (17, 18).
4
Abbreviations used in this paper: RA, rheumatoid arthritis; AF647, Alexa Fluor 647;
[Ca2⫹]i, intracellular calcium concentration; CI, chemotactic index; Cit, citrulline;
PAD, peptidylarginine deiminase; PKB, protein kinase B; CHO, Chinese hamster
ovary; PTH, phenyl thiohydantoin.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
Downloaded from http://www.jimmunol.org/ by guest on July 12, 2017
Posttranslational proteolytic processing of chemokines is a natural mechanism to regulate inflammation. In this study, we describe
modification of the CXC chemokine stromal cell-derived factor 1␣/CXCL12 by peptidylarginine deiminase (PAD) that converts
arginine residues into citrulline (Cit), thereby reducing the number of positive charges. The three NH2-terminal arginines of
CXCL12, Arg8, Arg12, and Arg20, were citrullinated upon incubation with PAD. The physiologic relevance of citrullination was
demonstrated by showing coexpression of CXCL12 and PAD in Crohn’s disease. Three CXCL12 isoforms were synthesized for
biologic characterization: CXCL12-1Cit, CXCL12-3Cit, and CXCL12-5Cit, in which Arg8, Arg8/Arg12/Arg20, or all five arginines
were citrullinated, respectively. Replacement of only Arg8 caused already impaired (30-fold reduction) CXCR4 binding and
signaling (calcium mobilization, phosphorylation of ERK and protein kinase B) properties. Interaction with CXCR4 was completely abolished for CXCL12-3Cit and CXCL12-5Cit. However, the CXCR7-binding capacities of CXCL12-1Cit and CXCL123Cit were, respectively, intact and reduced, whereas CXCL12-5Cit failed to bind CXCR7. In chemotaxis assays with lymphocytes
and monocytes, CXCL12-3Cit and CXCL12-5Cit were completely devoid of activity, whereas CXCL12-1Cit, albeit at higher
concentrations than CXCL12, induced migration. The antiviral potency of CXCL12-1Cit was reduced compared with CXCL12
and CXCL12-3Cit and CXCL12-5Cit (maximal dose 200 nM) could not inhibit infection of lymphocytic MT-4 cells with the HIV-1
strains NL4.3 and HE. In conclusion, modification of CXCL12 by one Cit severely impaired the CXCR4-mediated biologic effects
of this chemokine and maximally citrullinated CXCL12 was inactive. Therefore, PAD is a potent physiologic down-regulator of
CXCL12 function. The Journal of Immunology, 2009, 182: 666 – 674.
The Journal of Immunology
Materials and Methods
Reagents and cells
Recombinant CXCL12 and synthetic CXCL12, which was COOH-terminally labeled with Alexa Fluor 647 (CXCL12AF647) were obtained from
R&D Systems and Almac Sciences, respectively. PAD purified from rabbit
skeletal muscle was purchased from Sigma-Aldrich.
Synthetic CXCL12 isoforms were prepared by fluorenylmethoxycarbonyl solid-phase peptide synthesis with appropriate side-chain protection
groups on a 433A peptide synthesizer (Applied Biosystems) as previously
described (30). The synthetic CXCL12 forms were deprotected and cleaved
from the resin for 90 min at room temperature in 10 ml of trifluoroacetic
acid containing 0.75 g of phenol, 0.5 ml of thioanisole, 0.25 ml of ethanedithiol, and 0.5 ml of water. Subsequently, peptides were precipitated and
washed in diethyl ether, dissolved in water, and loaded on a Source 5RPC
column (4.6 ⫻ 150 mm; GE Healthcare Bio-Sciences). The proteins were
eluted from the RP-HPLC column with an acetonitrile gradient in 0.1%
trifluoroacetic acid and the UV absorption of peptides was monitored at
220 nm. Part of the column effluent (0.7 v/v %) was split to an Esquire LC
ion trap mass spectrometer (Bruker) and averaged profile spectra were
deconvoluted to determine the Mr. Proteins with the correct Mr were folded
for 2 h in 150 mM Tris (pH 8.6) containing 1 M guanidinium chloride, 3
mM EDTA, 0.3 mM reduced glutathione, and 3 mM oxidized glutathione
and repurified by RP-HPLC on a C8 Aquapore RP-300 column (2.1 ⫻ 220
mm; PerkinElmer). Ion trap mass spectrometry was used to select the fractions that contained the folded peptides.
Fresh PBMC were obtained from healthy donors and isolated by hydroxyethyl starch sedimentation and Ficoll-sodium metrizoate centrifugation (31, 32). CD14⫹ monocytes (98% pure CD14⫹ cells as determined by
flow cytometry) were obtained by positive selection with magnetic beads
as outlined by the manufacturer (MACS; Miltenyi Biotec). By culturing
PBMC in PHA (2 ␮g/ml) for 4 days and subsequently 2 wk in IL-2 (50
U/ml), activated lymphocytes were generated (33). The MT-4 lymphocytic
cell line and monocytic THP-1 cells were cultured in RPMI 1640 (Cambrex) supplemented with 10% FBS. Human CXCR4 or CXCR7 were stably expressed in Chinese hamster ovary K1 (CHO-K1) cells (American
Type Culture Collection) and the cells were cultured in Ham’s F-12 medium (Cambrex) supplemented with 10% FBS and 1 mM sodium pyruvate.
Selection agents added to this medium were 400 ␮g/ml geneticin and 250
␮g/ml zeocin for the CHO-CXCR4-G␣16-transfected cells and 400 ␮g/ml
geneticin for the CHO-CXCR7 transfectants.
Detection of CXCL12 and PAD by immunohistochemistry
Staining with Abs directed against CXCL12 (mouse monoclonal antihuman CXCL12, clone 79018; R&D Systems) and against human PAD
(rabbit polyclonal anti-PAD, catalog no. NB100-1853; Novus Biologicals)
was performed on formalin-fixed, paraffin-embedded tissue samples from
the normal small intestine and colon, obtained from patients operated for
diverticular disease and on biopsies from the small intestine and colon
obtained from patients operated for Crohn’s disease. Disease activity in
patients with Crohn’s disease was scored according to the D’Haens score
(34). First, endogenous peroxidase activity was blocked with 2% hydrogen
peroxide in methanol. Subsequently, sections were incubated in normal
swine serum to reduce nonspecific binding. Ag retrieval was performed by
boiling the sections in 10 mM citrate buffer (pH 6), followed by incubation
with the primary Abs. For the demonstration of coexpression, slides were
first stained for CXCL12 immunoreactivity using Envision reagents (DakoCytomation) and 3-amino-9-ethylcarbazol, resulting in bright red immunoreactive sites. After thorough washing with distilled water, anti-PAD
was added and visualized by Envision⫹ secondary reagents. As a chromogen for HRP 3,3⬘-diaminobenzidine tetrahydrochloride was used, resulting
in dark brown immunoreactive sites. To confirm specific immunoreactivity, primary Abs were omitted and staining was performed with the secondary Abs only (Fig. 1D and data not shown). The slides were counterstained with Harris hematoxylin.
Chemokine treatment with PAD
CXCL12 was citrullinated with PAD in 40 mM Tris-HCl (pH 7.6) and 2
mM CaCl2 at 37°C. The reaction was stopped by adding 0.1% trifluoroacetic acid. CXCL12 treated with PAD was spotted on polyvinylidene
difluoride membranes (ProSorb; Applied Biosystems) and the NH2-terminal sequence of the protein was determined by Edman degradation. The
phenyl thiohydantoin (PTH) derivatives of the individual amino acids were
separated by RP-HPLC on a 0.8 ⫻ 250-mm PTH Column (Applied Biosystems) and the height of the peaks was used to calculate the relative
amount of arginine converted into Cit. The PTH derivative of Cit eluted
between the derivates of threonine and glycine (24). To obtain CXCL12
with five citrullinated arginines for use in bioassays, synthetic CXCL12
was treated with PAD for 5 h, folded, and purified by C8 RP-HPLC on an
Aquapore RP-300 column (2.1 ⫻ 220 mm). Approximately 2% of the
column effluent was converted to the on-line ion trap mass spectrometer.
Detection of chemotactic activity
The chemotactic activity of CXCL12 for monocytes was determined in
Boyden chemotaxis chambers as previously described (35). Cell suspensions and sample dilutions were prepared in HBSS without calcium or
magnesium, supplemented with 1 mg/ml HSA. All samples were tested in
triplicate. CD14⫹ monocytes purified from PBMC using magnetic beads
(2 ⫻ 106 cells/ml) were allowed to migrate at 37°C for 2 h through 5-␮m
pore size polycarbonate filters (GE Osmonics). Filters were removed, cells
were fixed, stained, and counted microscopically. Chemotactic activity for
monocytic THP-1 cells or activated lymphocytes was tested in 96-well Multiscreen-MIC polycarbonate plates with a pore size of 5 ␮m (Millipore). Cells
(3.5 ⫻ 106 cells/ml for THP-1 cells or 2 ⫻ 106 cells/ml for activated lymphocytes) and samples were suspended in RPMI 1640 medium without phenol
red supplemented with 0.1% (w/v) BSA (Sigma-Aldrich). After 3 h (THP-1
cells) or 2 h (activated lymphocytes) of incubation at 37°C and 5% CO2, the
bottom compartment of the Multiscreen-MIC plate was centrifuged at
220 ⫻ g and cell migration was quantified by measuring the amount of
ATP present in the cells in the bottom compartment with a luminescence
detection assay (ATPlite; PerkinElmer). Results are expressed as chemotactic index (CI) corresponding to the number/luminescence value of cells
migrated to the sample divided by the number/luminescence value of cells
that migrated to control medium.
Binding studies
Competition for CXCL12AF647 binding was measured on CXCR4- or
CXCR7-transfected CHO cells as described previously (36). Briefly, 1.5 ⫻
106 cells/ml (300,000 cells in 200 ␮l) were incubated for 1 h at 4°C with
20 ng/ml CXCL12AF647 and varying concentrations of unlabeled chemokine in RPMI 1640 plus 2% FBS. Cells were washed three times with PBS
plus 2% FBS and finally suspended in PBS plus 2% FCS and 2% formaldehyde. The fluorescence present on the cells was measured by flow
cytometry (FACSCalibur flow cytometer; BD Biosciences). Aspecific
binding was determined by adding a 100-fold excess of unlabeled ligand
over the concentration of labeled ligand. Specific binding was obtained by
subtraction of aspecific binding from total binding. Results are expressed as
the percentage of remaining specifically bound CXCL12AF647.
Intracellular signaling assays
The intracellular Ca2⫹ concentrations ([Ca2⫹]i) were determined spectrofluorometrically using the fluorescent dye fura 2 and were calculated
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In view of the broad spectrum of producer cells, it is largely
unknown whether CXCL12 remains fully active or is rather processed or degraded in different body compartments during pathologic conditions. Indeed, upon secretion, chemokines can be rapidly processed by proteases coexpressed during an immune or
inflammatory response (1). For example, CXCL12-producing cells
may express membrane-bound or soluble CD26, which rapidly
cleaves off the two NH2-terminal residues due to its dipeptidyl
peptidase activity (19, 20). In serum, CXCL12 is rapidly converted
by CD26 and carboxypeptidase N (21–23). Such minor posttranslational modification has a major impact on the biologic effects,
since CD26-truncated CXCL12 has impaired CXCR4 signaling,
lacks chemotactic activity, and its protective effect against HIV-1
is abrogated (19). Recently, a novel posttranslational modification,
i.e., deimination of arginine by peptidylarginine deiminase (PAD),
has been identified on natural CXCL8 and CXCL10 (24, 25). This
modification resulted in reduced activity for both chemokines. In
view of the potential role of CXCL12 and PAD in autoimmune
diseases (26 –29), the interaction of PAD with CXCL12 was studied. PAD promotes the conversion of arginine into citrulline (Cit);
it was therefore investigated whether citrullination of CXCL12 has
consequences for its receptor interactions and biologic activities. It
was found that depending on the degree of citrullination, CXCL12
became gradually weaker as leukocyte chemoattractant and HIV
inhibitor.
667
668
CITRULLINATION DOWN-MODULATES CXCL12 ACTIVITY
from the Grynkiewicz equation as previously described (35). For desensitization experiments, CHO-CXCR4 cells were stimulated first with native
or modified CXCL12 and 100 s later with native CXCL12 at a concentration (10 ng/ml) that induced a significant increase in [Ca2⫹]i after prestimulation with buffer.
CXCL12-dependent phosphorylation of ERK1 plus ERK2 and Akt/protein kinase B (PKB) was evaluated in receptor-transfected CHO cells as
previously described (33). Briefly, cells were grown in 6-well plates in
Ham’s F-12 medium with 10% FBS for 24 h. Subsequently, the cells were
cultured overnight in serum-free starvation medium followed by a preincubation of the cells for 15 min at 37°C in medium with 0.5% BSA before
stimulation with test sample for 5 min at 37°C. Signal transduction was
stopped by chilling on ice and adding ice-cold PBS. Afterward, cells were
washed twice with ice-cold PBS and cell lysis was performed in PBS
containing 1 mM EDTA, 0.5% Triton X-100, 5 mM NaF, 6 M urea, protease inhibitor mixture for mammalian tissues, and phosphatase inhibitor
mixtures 1 and 2 (100 ␮l/well; Sigma-Aldrich). After 10 min, cells were
scraped off and the lysates were collected and incubated for 45 min on ice
and clarified (10 min, 1200 ⫻ g). The protein concentration in the supernatants was determined by the bicinchoninic acid protein assay (Pierce).
The amount of ERK and PKB/Akt phosphorylation in the supernatant (in
pg phospho-ERK or phospho-Akt per mg total protein) was determined
using specific ELISAs (R&D Systems) for phospho-Akt (S473) and phospho-ERK1 (T202/Y204) plus phospho-ERK2 (T185/Y187).
Antiviral assay
MT-4 cells were infected with the CXCR4 using (X4) strain NL4.3 or the
R5/X4 HIV-1 isolate HE. Briefly, 5-fold dilutions of the chemokines were
added to 96-well flat-bottom plates. Then, to each well, 7.5 ⫻ 104 MT-4
cells were added in 50 ␮l of medium, followed by 50 ␮l (500 pg/ml p24
Ag) of diluted HIV-1 stock. The cytopathic effect induced by the virus was
checked microscopically at regular times. When a strong cytopathic effect
was observed (mostly after 4 or 5 days) in untreated HIV-infected cells, the
supernatant of all samples was collected simultaneously and stored at
–20°C. Productive HIV-1 infection was determined by measuring the p24
Ag concentration in culture medium using a p24 Ag ELISA commercial kit
(PerkinElmer). Finally, the IC50 value of the chemokines (i.e., the concen-
tration of the chemokine required for 50% reduction in HIV replication as
measured by the p24 Ag production) was calculated.
Statistical analysis
The nonparametric Mann-Whitney U test was used for statistical analysis
(ⴱ or §, p ⬍ 0.05; ⴱⴱ or §§, p ⬍ 0.01). Asterisks indicate significant
differences compared with dilution buffer; § indicates significant differences between unmodified CXCL12 and a specific modified CXCL12
isoform.
Results
Coexpression of CXCL12 and PAD in Crohn’s disease
Citrullination was reported to play a crucial role in the initiation of
autoimmune diseases (27, 29). Inflammatory mediators, including
chemokines, are involved in the pathogenesis of RA, multiple sclerosis, and inflammatory bowel diseases (17, 18, 37, 38). Therefore,
it was investigated whether PAD and CXCL12, an ubiquitously
expressed chemokine linked with inflammation and angiogenesis,
are coexpressed in patients suffering from Crohn’s disease. Staining characteristics for CXCL12 and PAD were similar in normal
colon and ileum and in diseased colon and ileum. Although a positive immunohistochemical staining for CXCL12 was observed in
the normal small intestine and colon in various cell types (including intestinal epithelial cells, mononuclear cells of the lamina propria and submucosa, occasional endothelial cells, and enteroglia), in
affected areas from patients with Crohn’s disease, CXCL12 expression was markedly up-regulated (Fig. 1). Pronounced staining was
seen in epithelial cells adjacent to mucosal defects, where epithelial
cells covering the ulceration or erosion were strongly positive. Expression levels decreased with increasing distance from the damaged
mucosa. Furthermore, many stromal cells as well as endothelial cells
lining vascular spaces were CXCL12 positive. The expression in the
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FIGURE 1. Immunohistochemical detection of
CXCL12 and PAD expression in the normal gastrointestinal tract and in colon affected by Crohn’s disease. In
the biopsy shown, the disease is severely active (score
10/16). A and B show immunohistochemical detection
of CXCL12 expression in normal colon and colon tissue
affected by Crohn’s disease, respectively. The epithelial
staining for CXCL12 is most prominent in the mucosa
adjacent to ulcerations and erosions and decreases to
normal already at a short distance from the mucosal defect. C illustrates expression of PAD in a biopsy from a
patient with Crohn’s disease. As observed for CXCL12,
PAD expression is most distinct in the lesions. D shows
a negative control staining of colon tissue affected by
Crohn’s disease in which the primary Ab against PAD
was omitted. Immunoreactive sites are stained bright red
in A–D and these microphotographs were taken with a
⫻5 objective. E and F show coexpression of CXCL12
and PAD in autoimmune disease (⫻20 and ⫻40 objective). The colon biopsy of a patient with Crohn’s disease
was stained with Abs against CXCL12 (red) and PAD
(brown).
The Journal of Immunology
669
FIGURE 2. Conversion of CXCL12 by PAD. CXCL12 was treated with
PAD at an enzyme:substrate ratio of 1:20 for 5– 45 min at 37°C and the
percent conversion of arginine to Cit on positions 8 (⽧), 12 (䡺), and 20
(‚) was determined by Edman degradation.
Citrullination of CXCL12 by PAD
Recombinant CXCL12 was treated with purified PAD and citrullination of individual arginine residues was evaluated by Edman
degradation on the first 21 NH2-terminal amino acids of CXCL12.
The conversion of Arg8, Arg12, and Arg20 in the CXCL12 sequence proceeded with equal velocity, indicating that these three
arginines are equally accessible and sensitive for the active site of
PAD (Fig. 2). After 5 min at a 1:20 enzyme:substrate ratio, as
much as 50% of these arginine residues were converted into Cit
and 90% of arginines were citrullinated within 30 min. No Arg8,
Arg12, or Arg20 (at least 97% conversion) was detectable after
incubation for 45 min of CXCL12 with PAD. Sequence analysis of
the incubations was preferred over mass spectrometry, because
partial citrullination of an arginine causes only a shift of ⬍1 mass
unit, which is too close to the accuracy of the ion trap mass spectrometer. Furthermore, using Edman degradation we could monitor each individual arginine in the NH2 terminus of CXCL12.
In addition, to strengthen these data and to better mimic physiologic conditions, we incubated CXCL12 (700 nM) with PAD in the
presence of other proteins, i.e., HSA (2.25 ␮M) and/or a competing
substrate for PAD, keratin (350 nM). Also, in these experimental settings (enzyme:substrate ratio of 1:20 for CXCL12), Arg8, Arg12, and
Arg20 in CXCL12 were readily converted into Cit, as evidenced by
Edman degradation. We can conclude that in addition to the chemokines CXCL8, CXCL10, and CXCL11, CXCL12 is a substrate for
PAD (24, 25). In view of the changes in charge accompanying the
FIGURE 3. Chemotactic activity of CXCL12 isoforms. CXCL12 (f),
CXCL12-1Cit (⽧), CXCL12-3Cit (Œ), and CXCL12-5Cit (E) were tested for
their ability to induce chemotaxis of THP-1 cells (upper panel), CD14⫹ monocytes isolated from peripheral blood (middle panel), or activated lymphocytes
(lower panel). Results represent the mean (⫾SEM) CI of three to six independent experiments. Statistically significant migration compared with spontaneous migration induced by dilution buffer is indicated (ⴱ, p ⬍ 0.05).
conversion of arginine residues, located in the NH2-terminal region,
into Cit, we hypothesized that receptor interactions and biologic activities of CXCL12 might be altered after citrullination.
Chemical synthesis of CXCL12 isoforms
To be able to investigate the influence of specific arginine modifications in the CXCL12 protein structure, authentic CXCL12,
CXCL12-1Cit (with Cit only on position 8), and CXCL12-3Cit
(with Cit on positions 8, 12, and 20) were chemically synthesized,
purified by RP-HPLC, and folded. The experimentally determined
average Mr of CXCL12 (7959.3), CXCL12-1Cit (7960.5), and
CXCL12-3Cit (7962.6) were comparable to the theoretical average
Mr of the three proteins, i.e., 7959.5, 7960.5, and 7962.5, respectively (data not shown). The presence in the synthetic proteins of
Cit in position 8 (CXCL12-1Cit) and in positions 8, 12, and 20
(CXCL12-3Cit) was confirmed by Edman degradation (data not
shown). In addition, a CXCL12 form with all five arginines (also
including Arg41 and Arg47) modified to Cit was prepared by incubation of synthetic CXCL12 with PAD.
Effect of citrullination on the chemotactic activity of CXCL12
The activity of authentic CXCL12 and CXCL12 with one, three, or
five Cit residues instead of arginine was compared in in vitro chemotaxis experiments on monocytic THP-1 cells, on freshly isolated blood
monocytes, and on activated lymphocytes, which show a rather Th1
type expression pattern of chemokine receptors (Fig. 3). On THP-1
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epithelial cells appeared as a basolateral membranous staining with
some granular cytoplasmic staining.
In contrast, PAD staining was mostly cytoplasmic and present in
mononuclear stromal cells of the lamina propria (lymphocytes and
monocytes) in control, normal tissue. In samples from areas affected by Crohn’s disease, a clear positive PAD staining was demonstrated in additional cell types: neutrophils and fibroblast-like
cells. These cell types were morphologically identified by the presence of a segmented nucleus and absence of staining granules for
neutrophils and a specific irregular and elongated cell shape and
nucleus for fibroblasts, respectively. The strong PAD expression in
neutrophils coincided with the pronounced CXCL12 immunoreactivity in epithelial cells adjacent to ulcers, where neutrophils permeate in between the surface epithelial cells (Fig. 1). This implicates that both normal and diseased tissue express PAD and
CXCL12 in various cell types, but that in diseased areas cells
expressing PAD and CXCL12 are adjacent (Fig. 1, E and F). To
address whether PAD could posttranslationally modify CXCL12
in vitro, the chemokine was incubated with this enzyme.
670
cells, a maximal chemotactic response was obtained with CXCL12
at 3 nM and the minimal effective concentration was 0.3 nM ( p ⬍
0.05). For CXCL12-1Cit, a 10-fold higher dose was required to
obtain a maximal response and the minimal concentration that induced significant ( p ⬍ 0.05) chemotaxis was 3 nM. In addition,
CXCL12-1Cit reached a maximal CI of 5.2 at 30 nM compared
with a CI of 11.6 for unmodified CXCL12 at 3 nM. Furthermore,
this maximal CI of CXCL12-1Cit could already be reached by
stimulating the cells with 1 nM CXCL12 (CI, 7.8), indicating that
both the potency and efficacy of CXCL12 are reduced by citrullination of its Arg8. CXCL12-3Cit and CXCL12-5Cit were completely devoid of any chemotactic activity for THP-1 cells at
concentrations up to 100 nM. On freshly isolated monocytes, a
maximal chemotactic response was obtained with 100 nM intact
CXCL12. Comparable to the results on THP-1 cells, CXCL121Cit was significantly less chemotactic for monocytes and
CXCL12-5Cit was inactive. Finally, we tested the chemotactic
activity of three different CXCL12 isoforms (CXCL12,
CXCL12-1Cit, and CXCL12-5Cit) for activated lymphocytes,
which are representative for lymphocytes that infiltrate autoimmune inflammatory lesions where citrullinated CXCL12 might
be generated. The minimal effective concentration of CXCL12
and CXCL12-1Cit for activated lymphocytes was 1 and 30 nM,
respectively, whereas CXCL12-5Cit was inactive up to 100 nM.
Thus, also on this cell type, the chemotactic activity of CXCL12
is affected by citrullination.
Effect of citrullination on CXCL12-mediated signal transduction
In CHO-CXCR4 cells, CXCL12 induced a significant increase in
[Ca2⫹]i from 0.3 nM onward (Fig. 4). To obtain a similar response
with CXCL12-1Cit, ⬃30-fold higher amounts were required. As
observed in the chemotaxis experiments, CXCL12-3Cit and
CXCL12-5Cit were completely inactive at concentrations up to
100 nM. Compared with authentic CXCL12, CXCL12-1Cit was
also 30-fold less efficient at desensitizing the calcium responses to
FIGURE 5. Phosphorylation of ERK or Akt/PKB induced by CXCL12
isoforms. The influence of CXCL12 (f), CXCL12-1Cit (⽧), CXCL123Cit (Œ), and CXCL12-5Cit (E) on the phosphorylation of ERK1 and
ERK2 (upper panel) or Akt/PKB (lower panel) was evaluated in CHOCXCR4 cells. CHO-CXCR4 cells were treated with different concentrations of the CXCL12 isoforms for 5 min. Levels of phosphorylated ERK
and Akt/PKB in the cell lysates were determined using specific ELISAs
and the relative increase compared with medium-treated cells was calculated. Results represent the mean rise in phosphorylated ERK or Akt/PKB
levels (⫾SEM) of three independent experiments. Statistically significant
differences compared with control stimulation with medium alone are indicated (ⴱ, p ⬍ 0.05).
1 nM CXCL12, whereas CXCL12-3Cit and CXCL12-5Cit failed
to desensitize CXCR4.
CXCL12 has been reported to induce phosphorylation of ERK1,
ERK2, and Akt/PKB (39). After 5 min, significant ERK phosphorylation was detected when CHO-CXCR4 cells were treated with
0.1 nM CXCL12 and optimal phosphorylation of ERK was obtained upon stimulation with 1- 10 nM CXCL12 (Fig. 5). Incubation of CXCR4-transfected cells with 3 nM CXCL12-1Cit resulted
in significant phosphorylation of ERK but optimal stimulation was
only obtained with 30 –100 nM CXCL12-1Cit. In addition to ERK,
also Akt/PKB phosphorylation was detected in CXCL12-treated
CHO-CXCR4 cells. Akt/PKB phosphorylation was detected with
0.1 nM CXCL12 and an ⬃10-fold higher dose of CXCL12-1Cit
was required to obtain a comparable Akt/PKB phosphorylation.
Neither CXCL12-3Cit nor CXCL12-5Cit induced CXCR4-dependent ERK or Akt/PKB phosphorylation at concentrations as high
as 100 nM. In CHO-CXCR7 cells, up to 300 nM CXCL12 was
unable to induce ERK or Akt/PKB phosphorylation (data not
shown).
Effect of citrullination on CXCL12-mediated receptor binding
Competition for CXCL12 binding to the receptors CXCR4 and
CXCR7 was investigated with CXCL12AF647 since two commercial preparations of 125I-CXCL12 only weakly bound to CHOCXCR4 cells, although they efficiently and specifically bound to
CHO-CXCR7 cells (data not shown). CXCL12 efficiently competed for CXCL12AF647 binding to CXCR4 and CXCR7 (Fig. 6).
One-half of the labeled CXCL12 was displaced on the CHOCXCR4 or CHO-CXCR7 cells after addition of 4.3 nM or 7.7 nM
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FIGURE 4. CXCR4-dependent calcium signaling of citrullinated
CXCL12. CXCL12 (f), CXCL12-1Cit (⽧), CXCL12-3Cit (Œ), and
CXCL12-5Cit (E) were compared for their ability to induce a rise in the
[Ca2⫹]i in CHO-CXCR4 cells (upper panel). Results represent the mean
increase in [Ca2⫹]i ⫾ SEM of four or more experiments. To test for the
desensitizing capacity of CXCL12 (lower panel), cells were first stimulated
with different concentrations of various CXCL12 forms, followed by stimulation with 1 nM native CXCL12 (n ⱖ 4). The percentage inhibition of the
response to the second stimulus is shown. Statistically significant differences compared with dilution buffer (applied as first stimulus) are indicated
(ⴱ, p ⬍ 0.05).
CITRULLINATION DOWN-MODULATES CXCL12 ACTIVITY
The Journal of Immunology
671
Table I. Anti-HIV-1 activity of CXCL12 isoforms in lymphocytic MT-4
cellsa
IC50 (nM)
NL4.3
NL4.3
HE
HE
Chemokine
Expt. 1
Expt. 2
Expt. 1
Expt. 2
CXCL12
CXCL12-1Cit
CXCL12-3Cit
CXCL12-5Cit
18.8
⬎500
⬎500
⬎500
3.5
⬎500
⬎500
⬎500
11.1
⬎500
⬎500
⬎500
8.6
⬎500
⬎500
⬎500
a
MT-4 cells were infected with HIV-1 (the NL4.3 strain or HE isolate) in the
presence of CXCL12 isoforms as described in Materials and Methods. Results from
two independent experiments are shown.
Effect of citrullination on CXCL12-mediated anti-HIV-1 activity
of unlabeled chemokine, respectively. In accordance with the signaling data, CXCL12-1Cit weakly competed (50% inhibition of
binding at 50 nM CXCL12-1Cit) and CXCL12-3Cit and CXCL125Cit failed to compete for binding to CXCR4. Surprisingly,
CXCL12-1Cit was equally efficient compared with CXCL12 to
compete for binding of CXCL12AF647 to CXCR7. Moreover,
CXCL12-3Cit still moderately competed for CXCL12AF647 binding, but CXCL12-5Cit failed to bind to CXCR7.
FIGURE 7. Effect of citrullination on the anti-HIV-1 activity of
CXCL12. Lymphocytic MT-4 cells were incubated with varying concentrations of CXCL12 (f), CXCL12-1Cit (⽧), CXCL12-3Cit (Œ), or
CXCL12-5Cit (E) at the time of infection with the X4 NL4.3 HIV-1 strain.
Results from triplicate cultures are expressed as the mean percentage inhibition of HIV-1 replication as described in Materials and Methods.
Discussion
Chemokines play a relevant role in a number of pathologic conditions, mainly based on their overexpression in diseases associated with leukocyte infiltration (16, 40). CXCL12 is a pluripotent
chemokine, affecting many cell types by interaction with CXCR4
and CXCR7. Unlike most proteins of the chemokine family,
CXCL12, CXCR4, and CXCR7 are well conserved in different
animal species (4, 41). CXCL12 and CXCR4 were originally
found to be essential for B lymphopoiesis and bone marrow myelopoiesis (42). Consistently, mice lacking either CXCL12 or its
receptors exhibit defects that extend beyond the immune system,
revealing roles for this chemokine in nonimmunologic processes,
such as the genesis of the cardiovascular system or CNS (11,
42– 44).
The chemokine system is a target for viral mimicry and is exploited by viruses to gain access into immune cells (45). To infect
human leukocytes, HIV-1 has to bind to CD4 and a coreceptor that
belongs to the family of the G protein-coupled receptors. The majority of HIV-1 strains use either CXCR4 (X4 or T-tropic strains),
CCR5 (R5 or M-tropic strains) or both CCR5 and CXCR4 (dualtropic strains). Since they compete for binding of viral gp120 to the
coreceptors, the natural ligands of CXCR4 and CCR5 block viral
entry in CD4⫹ cells. Also, CXCR7 has been described to act as a
coreceptor for several HIV-2 and SIV strains to infect primary
brain-derived CD4⫹ cells (46). CXCR7 is a peculiar chemokine
receptor, because its ligands do not induce calcium signaling in
MCF-7 cells, which express CXCR7 endogenously (15). It was
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FIGURE 6. Receptor binding properties of CXCL12 isoforms. Competition for binding of CXCL12AF647 to CXCR4- or CXCR7-transfected
CHO cells was evaluated for CXCL12 (f), CXCL12-1Cit (⽧), CXCL123Cit (Œ), and CXCL12-5Cit (E). Results are expressed as the percentage
of remaining specific binding of CXCL12AF647 to transfected CHO cells
(mean ⫾ SEM of four or more independent experiments). Statistically
significant binding compared with medium treatment is indicated (ⴱ, p ⬍
0.05). In the upper panel (CXCR4 transfectants), significant differences
between CXCL12 and CXCL12-1Cit are indicated (§, p ⬍ 0.05). In this
panel, CXCL12 binding was also significantly different from CXCL123Cit or CXCL12-5Cit, but this is not indicated (p ⬍ 0.05). On CXCR7
transfectants (lower panel), the affinity of CXCL12 and CXCL12-1Cit was
not different, whereas CXCL12 binding differed significantly from that of
CXCL12-3Cit or CXCL12-5Cit (§, p ⬍ 0.05).
Since CXCR4 is one of the main coreceptors for HIV infection of
human leukocytes, the effect of citrullination of CXCL12 on its
antiviral activity against the CXCR4-tropic NL4.3 HIV-1 strain
was investigated on lymphocytic MT-4 cells. CXCL12 inhibited
infection of the NL4.3 strain with an IC50 of 31 nM (Fig. 7). In
contrast, CXCL12-1Cit had moderate anti-HIV-1 activity, i.e.,
23% inhibition of infection at 200 nM. However, CXCL12-3Cit
and CXCL12-5Cit failed to inhibit infection of lymphocytes with
the NL4.3 HIV-1 strain at concentrations up to 200 nM. These data
were extended with a second set of experiments in which MT-4
cells were infected with the NL4.3 strain in parallel with the dualtropic HE isolate (Table I). The HE virus was as sensitive to
CXCL12 inhibition as the NL4.3 strain, since the IC50 values of
both strains were comparable for authentic CXCL12. These additional experiments confirmed that citrullination of CXCL12 severely reduced the capacity to block retroviral infection of the
lymphocytic cell line. Even at the highest dose tested, CXCL123Cit and CXCL12-5Cit were unable to protect the cells from infection with either NL4.3 or HE virus.
672
significantly higher in patients with active ulcerative colitis compared with normal controls (60). CXCR4 expression levels of ulcerative colitis patients also correlated with disease activity, suggesting that the CXCL12-CXCR4 axis is involved in the
inflammatory process in these patients. Indeed, blockade of the
CXCL12-CXCR4 axis ameliorated murine experimental colitis
(60). We hypothesized that, in view of the coexpression of
CXCL12 and PAD in Crohn’s disease and the evidence that chemokines are substrates for PAD (24, 25), also crucial NH2-terminal arginine residues in CXCL12 might be converted by this enzyme. Indeed, treatment of CXCL12 with PAD resulted in rapid
deimination (citrullination) of several arginine residues in the primary structure of CXCL12 and in a reduced number of positively
charged amino acids.
Chemically synthesized citrullinated CXCL12 isoforms with
a variable degree of citrullination (one, three, or five arginine
residues converted) were used to study the effect of arginine
deimination on the receptor binding and signaling properties
and on the biologic activities of CXCL12. Even deimination of
only the first arginine (Arg8) in the CXCL12 sequence strongly
reduced the CXCR4 binding potency of this chemokine. In addition, CXCL12-1Cit showed significantly reduced calcium signaling, ERK and Akt/PKB phosphorylation, as well as monocyte and lymphocyte chemotactic activity. Further citrullination
of CXCL12 on Arg8/Arg12/Arg20 (CXCL12-3Cit) or all five
arginine residues (CXCL12-5Cit) completely abolished CXCR4
binding and signaling properties and led to complete loss of leukocyte chemotactic activity. In accordance with the binding and
signaling data on CXCR4, citrullination of CXCL12 reduced its
anti-HIV-1 potency in lymphocytic cell cultures, CXCL12-1Cit
being partially active, whereas CXCL12-3Cit and CXCL12-5Cit
were completely inactive. In contrast to the profound effect of the
deimination of Arg8 on the interaction of CXCL12 with CXCR4,
CXCL12-1Cit fully retained its binding properties on
CXCR7. CXCL12-3Cit still weakly interacted with this newly
identified CXCL12 receptor, but maximal citrullination abrogated
CXCR7 binding. This indicates that different residues in the
CXCL12 sequence are crucial for either CXCR4 or CXCR7 binding. In pathophysiologic conditions, PAD may play an antiinflammatory role, attenuating the recruitment of CXCR4⫹ leukocytes to sites of inflammation. Whether PAD expression levels are
high enough to inactivate all produced CXCL12 remains to be
determined. In contrast to some NH2-terminally truncated chemokines, citrullinated CXCL12, unfortunately, does not block
CXCR4 for binding of active, intact CXCL12 (1).
We provide evidence that PAD functionally affects receptor
signaling immune mediators, i.e., CXCL8 (25), CXCL10 (24),
and CXCL12 (this manuscript), identifying citrullination as a
new regulatory mechanism of chemokine activity. Until now,
proteolytic processing has been described as the major posttranslational modification regulating chemokine activity (1). In
the process of identifying alternatively posttranslationally modified chemokines, we succeeded to isolate naturally citrullinated
CXCL8 and CXCL10 from conditioned medium of stimulated
PBMC (24, 25). In contrast to in vitro citrullination of
CXCL12, only the most NH2-terminal arginine of CXCL5,
CXCL8, and CXCL10 was converted into Cit by PAD (24, 25).
Treatment of the CC chemokine CCL17 with PAD resulted in
citrullination on at least two positions (25). No biologic data are
yet available on the effect of CXCL5 and CCL17 citrullination
(25). The consequences of CXCL8 citrullination on its receptorbinding capacity and in vitro chemotactic activity were limited:
the in vitro chemotactic activity of citrullinated CXCL8 was
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suggested that CXCR7 may be constitutively active in tumor cell
lines to provide a growth/survival advantage and increased adhesiveness of cells (15). In tumor biopsies, both CXCR7 and CXCR4
are expressed on the vasculature (47, 48). Alternatively, CXCR7
has been suggested to act as a silent receptor for CXCL12 that
helps in the establishment of the CXCL12 gradient that guides
progenitor cells toward their targets in the embryo (49). Thus, at
present, the biologic role of CXCR7 is still controversial and therefore it is difficult to speculate on the physiologic consequences of
the altered interactions of citrullinated CXCL12 with CXCR7.
Furthermore, CXCL12 is critically involved in the development
of various other pathophysiologic conditions, including RA, atherosclerosis, cancer growth, and metastasis (6 – 8, 28, 50). In particular, CXCL12 protein is overexpressed in rheumatoid synovia
compared with osteoarthritic synovial membranes (51). In RA,
CXCR4 and CXCL12 were reported to be responsible for influx of
lymphocytes (17) and addition of a CXCR4-specific antagonist
prevented joint inflammation in collagen-induced arthritis (28).
Like CXCL12, PAD is known to play a role in various autoimmune diseases and PAD citrullinates autoantigens in RA patients
that lead to the generation of anti-Cit Abs (52, 53). These Abs are
more potent diagnostic tools than rheumatoid factor and can be
detected in RA patients before the appearance of clinical symptoms (29). Moreover, both human PAD2 and PAD4 have been
detected in synovial fluid from RA patients (54). In multiple sclerosis, citrullination of myelin basic protein is enhanced (27).
In this study, we describe that PAD expression in diseased tissue
from Crohn’s disease was enhanced in fibroblast-like cells and
neutrophils. PAD expression in neutrophils has been reported before. Actually, PAD4 was discovered in HL-60 cells treated with
retinoic acid and methyl sulfoxide to induce differentiation of the
cells to granulocytes (55). Since high levels of proinflammatory
cytokines such as TNF-␣ were reported to correlate with enhanced
concentrations of anti-Cit Abs in RA patients (56) and since treatment of neutrophils with inflammatory mediators leads to enhanced concentrations of deiminated histones in extracellular
chromatin traps (57), the possibility that PAD activity is also upregulated in neutrophils of Crohn’s disease patients at sites of active inflammation cannot be excluded. Furthermore, in patients
with Crohn’s disease, CXCL12 and PAD are coexpressed. At
present, we can only speculate on the function of these molecules
in Crohn’s disease, because reports on the expression of CXCL12
and PAD in this inflammatory bowel disease are scarce. Intestinal
tissue from macroscopically affected and nonaffected areas from
10 patients with inflammatory bowel disease were analyzed by
immunohistochemistry using Abs targeting Cit residues in proteins
as a detection method for PAD expression (58). However, because
this study comprised a mixed group of patients and because also in
nonaffected tissue some inflammation was observed, no strong
conclusions could be made. Citrullinated proteins were present in
a higher number of biopsies from affected tissue and therefore it
was confirmed that citrullination is an inflammation-dependent
process. However, overall, statistically significant differences in
the extent or pattern of citrullination between normal and diseased
tissue could not be observed (58). CXCL12 mRNA expression was
investigated in homogenized colonic biopsies from patients with
inflammatory bowel diseases and CXCL12 mRNA levels were
higher in patients suffering from ulcerative colitis than from
Crohn’s disease (59). We studied protein expression and observed
a regional expression pattern of CXCL12 protein in Crohn’s disease showing marked up-regulation in areas of ulceration and erosion compared with normal colon. Very recently, CXCR4 expression was evaluated on peripheral T cells in patients with
inflammatory bowel disease; CXCR4 immunofluorescence was
CITRULLINATION DOWN-MODULATES CXCL12 ACTIVITY
The Journal of Immunology
Acknowledgments
We thank René Conings, Jean-Pierre Lenaerts, Sandra Claes, Willy Put,
Isabelle Ronsse, and Christel Van Den Broeck for technical assistance.
Disclosures
The authors have no financial conflict of interest.
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CITRULLINATION DOWN-MODULATES CXCL12 ACTIVITY