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Fractalkine Is an Epithelial and Endothelial
Cell-Derived Chemoattractant for
Intraepithelial Lymphocytes in the Small
Intestinal Mucosa
Andreas Muehlhoefer, Lawrence J. Saubermann, Xuibin Gu,
Kerstin Luedtke-Heckenkamp, Ramnik Xavier, Richard S.
Blumberg, Daniel K. Podolsky, Richard P. MacDermott and
Hans-Christian Reinecker
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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 © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 164:3368-3376; ;
doi: 10.4049/jimmunol.164.6.3368
http://www.jimmunol.org/content/164/6/3368
Fractalkine Is an Epithelial and Endothelial Cell-Derived
Chemoattractant for Intraepithelial Lymphocytes in the Small
Intestinal Mucosa1
Andreas Muehlhoefer,2* Lawrence J. Saubermann,2† Xuibin Gu,* Kerstin LuedtkeHeckenkamp,* Ramnik Xavier,* Richard S. Blumberg,† Daniel K. Podolsky,*
Richard P. MacDermott,*‡ and Hans-Christian Reinecker3*
T
he epithelium layer forms the interface between the external and the internal environments of the gastrointestinal tract. The intestine-associated lymphoid tissue serves
as an immunological barrier against a wide range of infectious
agents. It has become clear that intestinal mucosal barrier function
is regulated by interaction of intestinal epithelial cells with intestinal lymphocytes.
Fractalkine (Neurotactin/NKAF) is a novel chemokine that is
characterized by a CX3C spacing of the cysteine motif and has a
unique membrane-bound structure (1–3). The domain organization
includes a 37-residue intracellular tail, a short membrane-spanning
region, and an extended, mucin-like stalk, which presents the Nterminal chemokine domain at the cell surface. This unique architecture represents a novel mechanism of cell adhesion that differs
from soluble CXC, CC, and C chemokines, which have heparin-
*Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114; †Division of Gastroenterology, Brigham and Women’s Hospital
and Harvard Medical School, Boston, MA 02116; and ‡Division of Gastroenterology,
Albany Medical College, Albany, NY 12208
Received for publication July 23, 1999. Accepted for publication January 10, 2000.
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 National Institutes of Health Grants DK 51003 and
54427 (H.-C.R.), DK 21474 (R.P.M.), DK 41557 (D.K.P.), DK 43351 (H.-C.R.,
D.K.P., R.P.M., R.X.), DK 02532 (L.J.S.), DK44319, DK51362, and AI33911
(R.S.B). A.M. was supported by a grant from the Deutsche Forschungsgemeinschaft.
2
binding domains that may promote immobilization by cell surface
proteoglycans or extracellular matrix components (4). Also, the
juxtamembraneous part of fractalkine contains a dibasic motif
(Thr-Arg-Arg-Gln) that can be cleaved, to yield chemoattractant
soluble fractalkine. Thus, fractalkine represents a new class of chemokines that exhibits properties of both traditional chemokines
and adhesion molecules (3). Fractalkine binds specifically to the
chemokine receptor V28, now termed CX3CR1 (5, 6). CX3CR1
appears to be a highly specific receptor for fractalkine, since no
additional chemokines that bind or compete for the receptor have
been identified, with the exception of the HHV-8-derived virus
chemokine vMIP-II (6 – 8). Surface expression of the CX3CR1 has
been demonstrated in NK cells, monocytes, CD8⫹ T cells, and to
a lesser extent in CD4⫹ T cells (5).
Intestinal intraepithelial lymphocytes (iIEL)4 are thought to represent a first-line defense against pathogens and may be critical for
intestinal integrity. Most iIEL express the CD8⫹ phenotype with
either CD8 heterodimeric ␣/␤-chains or homodimeric ␣/␣-chains.
iIEL exhibit a number of important immunological functions, including cytotoxic activity (9, 10); secretion of cytokines, including
IL-2, IL-3, IL-5, TNF-␣, TGF-␤, and IFN-␥ (11); and may regulate epithelial cell proliferation and regeneration (12). In addition,
intestinal epithelial cells are capable of expressing MHC class II
molecules and can act as effective APCs that can induce and activate T cells in vitro (13–15). Furthermore, intestinal epithelial
cells may selectively activate T cells with suppressor function via
CD1d molecule, which subsequently down-regulate immune responses (16 –20). Intestinal epithelial cells also express IL-15 (21),
A.M. and L.J.S. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Hans-Christian Reinecker, Gastrointestinal Unit, Jackson Building, R711, Massachusetts General Hospital, 32 Fruit
Street, Boston, MA 02114. E-mail address: [email protected]
Copyright © 2000 by The American Association of Immunologists
4
Abbreviations used in this paper: iIEL, intestinal intraepithelial lymphocyte; LPL,
lamina propria lymphocyte.
0022-1767/00/$02.00
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Fractalkine is a unique chemokine that combines properties of both chemoattractants and adhesion molecules. Fractalkine mRNA
expression has been observed in the intestine. However, the role of fractalkine in the healthy intestine and during inflammatory
mucosal responses is not known. Studies were undertaken to determine the expression and function of fractalkine and the
fractalkine receptor CX3CR1 in the human small intestinal mucosa. We identified intestinal epithelial cells as a novel source of
fractalkine. The basal expression of fractalkine mRNA and protein in the intestinal epithelial cell line T-84 was under the control
of the inflammatory mediator IL-1␤. Fractalkine was shed from intestinal epithelial cell surface upon stimulation with IL-1␤.
Fractalkine localized with caveolin-1 in detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells. Cellular
distribution of fractalkine was regulated during polarization of T-84 cells. A subpopulation of isolated human intestinal intraepithelial lymphocytes expressed the fractalkine receptor CX3CR1 and migrated specifically along fractalkine gradients after
activation with IL-2. Immunohistochemistry demonstrated fractalkine expression in intestinal epithelial cells and endothelial cells
in normal small intestine and in active Crohn’s disease mucosa. Furthermore, fractalkine mRNA expression was significantly
up-regulated in the intestine during active Crohn’s disease. This study demonstrates that fractalkine-CX3CR1-mediated mechanism may direct lymphocyte chemoattraction and adhesion within the healthy and diseased human small intestinal mucosa. The
Journal of Immunology, 2000, 164: 3368 –3376.
The Journal of Immunology
which is a strong inducer of iIEL activation and proliferation (22).
Together, these observations suggest that intestinal epithelial cells
can provide regulatory signals for T cells to maintain appropriate
homeostasis in the gastrointestinal tract. However, it is not clear
how iIEL are continually attracted toward intestinal epithelial cells
and then retained within the highly dynamic intestinal epithelium.
To determine the functional role of fractalkine expression in the
intestinal mucosa, we have examined the source and function of
the fractalkine-CX3CR1 ligand-receptor system in the healthy and
diseased small intestinal mucosa.
Materials and Methods
Abs and cytokines
Cell culture
The human intestinal epithelial cell line T-84 was obtained from American
Type Culture Collection (Manassas, VA). Cells were maintained in
DMEM/Ham F12 (50/50 v/v) medium (Cellgro; Mediatech, Herndon, VA)
supplemented with 10% heat-activated FCS (Sigma), 1% penicillin, and
streptomycin (Life Technologies, Gaithersburg, MD). Cells were grown at
37°C in a 5% CO2 atmosphere within a humidified incubator. Cells were
always used for experiments at 70% confluence.
RT-PCR for the detection of CX3CR1 mRNA
The nucleotide sequence of CX3CR1 (GenBank accession number
U28934) was analyzed and two primer generated: 5⬘-TTG AGT ACG ATG
ATT TGG CTG A-3⬘ and 5⬘-GGC TTT GGC TTT CTT GTG G-3⬘. PCR
conditions were 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C for 40 cycles.
Northern blot analysis
Total RNA was extracted from T-84 cells and intestinal biopsy specimens
or resected intestinal tissues using Trizol reagent (Life Technologies), according to the manufacturer’s instructions. Crohn’s disease, ulcerative colitis, or normal intestinal control tissues were obtained from the tissue bank
of the Center for the Study of Inflammatory Bowel Disease at Massachusetts General Hospital, according to the guidelines for the use of discarded
human material at Massachusetts General Hospital. Resected tissue from
three patients with Crohn’s disease and three specimens from control patients were analyzed.
Fractalkine cDNA was generated by RT-PCR using the following primers: 5⬘-ACT CTT GCC CAC CCT CAG C-3⬘, and 5⬘-TGG AGA CGG
GAG GCA CTC-3⬘. The PCR products were subcloned into pCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced. cDNA probes were labeled
with [␣-32P]dCTP by a random hexamer priming method using the Rediprime random primer labeling kit (Amersham Life Science, Arlington
Heights, IL). Membranes were hybridized in Quickhyb solution (Stratagene, La Jolla, CA) at 68°C for 1 h. The membranes were washed and the
blots analyzed by autoradiography. Expression of fractalkine mRNA was
corrected for GAPDH expression and quantitated densitometrically with
the NIH Image 1.6.1 analysis software.
Western blot analysis
Cells were placed in lysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl,
1% Triton X-100, 5 mM EDTA, 5 mM NaF, 10 mg/ml aprotinin, 10 mg/ml
leupeptin, 1 mM PMSF, and 1 mM sodium-orthovanadate (protease/phosphatase-inhibitor mixture)). After 20 min on ice, cell lysates were cleared
by centrifugation at 20,000 ⫻ g for 15 min at 4°C. Protein concentration in
each sample was quantified by the Bradford method, and 20 ␮g protein was
used for Western blot analysis. Proteins were separated by SDS-PAGE,
using 10% Tris-glycine gels, and subsequently transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes
were incubated in blocking solution (1⫻ TBS, 0.025% Tween 20 (TBS-T),
1% BSA) at room temperature for 1 h. After an overnight incubation with
the first Ab (anti-fractalkine C-18 1:1000 or C-20 1:200 in blocking solution) at 4°C, membranes were washed four times in TBST. The HRPlabeled second Ab was dissolved (1:6000) in TBS-T, supplemented with
5% dry milk. After incubation with the second Ab for 1 h at room temperature, the membranes were washed four times in TBS-T and proteins
were detected by the ECL method (Amersham, Piscataway, NJ), according
to the instructions of the manufacturer. Western blots were quantitated
densitometrically with NIH Image 1.6.1 analysis software.
Immunoprecipitation from T-84 cytosol protein and membrane
protein fraction
T-84 cells were washed three times with ice-cold PBS, and 6 ml hypotonic
lysis buffer (10 mM Tris, pH 7.4, 1 mM MgCl2, supplemented with the
protease/phosphatase inhibitor mixture) was added to two 10-cm dishes.
After incubation on ice for 20 min, the cells were disrupted by douncing for
15 times. By adding 5 M NaCl, iso-osmolar conditions were obtained. The
cell lysate was cleared by centrifugation twice for 10 min at 1000 ⫻ g. The
supernatant was then centrifuged at 100,000 ⫻ g for 35 min at 4°C. The
supernatant (cytosolic fraction), was adjusted to a final concentration of
0.1% sodium deoxycholate, 0.1% SDS, and 0.1% Triton X-100. The pellet
was solubilized in 1% sodium deoxycholate, 1% SDS, and 1% Triton
X-100, and after an incubation for 20 min on ice, diluted to obtain identical
conditions as in the cytosolic fraction. The lysate was dounced 10 times
and cleared by a centrifugation at 10,000 ⫻ g for 15 min at 4°C, and
referred to the membrane fraction. Equal (600 ␮g) amounts of protein, as
determined by the Bradford method, were subjected to immunoprecipitation. After preclearing for 2 h with protein A/G beads (Calbiochem, La
Jolla, CA), the lysates were incubated overnight with 2 ␮g/ml C 20 fractalkine Ab. After washing twice with washing buffer (10 mM Tris, pH 7.4,
150 mM NaCl, 1 mM MgCl2, 0.5% Triton X-100), the immunoprecipitate
was denaturated by boiling in Laemmli buffer and subjected to Western
blot analysis.
Isolation of detergent-insoluble glycolipid-enriched membrane
microdomains
Glycolipid-enriched membrane microdomains or detergent-insoluble glycolipid rafts were isolated as described before (23) (24), with minor modifications. Four dishes of T-84 cells (10 cm diameter) were washed three
times with ice-cold PBS, and the cells were solubilized in 1 ml of lysis
buffer (25 mM MES, pH 6.8, 150 mM NaCl, 1% Triton X-100 supplemented with the protease/phosphatase inhibitor mixture). After incubation
for 30 min on ice, the lysate was dounced gently 10 times and cleared by
a centrifugation for 5 min at 1000 ⫻ g. The supernatant was transferred to
the bottom of a centrifugation tube and adjusted to 40% sucrose with an
equal volume of 80% sucrose in MBS (25 mM MES, pH 6.8, 150 mM
NaCl supplemented with the protease/phosphatase inhibitor mixture). A
sucrose step gradient with 30%, 25%, 20%, 15%, and 5% in MBS (2 ml
each) was layered on top. This combination of sucrose concentrations gave
the best resolution between 15% and 25% (data not shown). After a centrifugation in a SW 41 rotor (Beckman Coulter, Fullerton, CA) at 39.000
rpm for 14 –16 h at 4°C, fractions of 1 ml each were taken, starting from
the top (fraction 1), going to the bottom (fraction 12). For the preparation
of cytosol and membranes, two dishes of T-84 cells (10 cm diameter) were
three times washed with ice-cold PBS and taken up in 2 ml buffer A (20
mM Tris, pH 7.8, 1 mM EDTA, 0.25 M sucrose supplemented with the
protease/phosphatase inhibitor mixture). The lysate was sheared 10 times
and cleared by a centrifugation for 5 min at 1000 ⫻ g. One-fourth of the
supernatant was taken for preparation of the cytosol. Four milliliters of
buffer A were added, and a centrifugation for 60 min at 100,000 ⫻ g was
performed. The middle, clear fraction referred to the cytosolic fraction. For
membrane isolation, the remaining three-fourths of the cleared supernatant
were layered on top of a 30% Percoll solution in buffer A, supplemented
with 8.5% sucrose. After centrifugation for 30 min at 100,000 ⫻ g, the
resulting interface, the membrane fraction, was isolated. Protein measurement of the cellular fractions was performed with BCA Protein Assay
Reagent, according to the manufacturer’s instructions (Pierce, Rockford,
IL). Equal amounts of protein were subjected to Western blot analysis, as
described above.
Immunofluorescence staining for the detection of fractalkine
Intestinal biopsy specimen was obtained during diagnostic endoscopy after
prior patients’ consent. The tissue was fixed with OCT-mounting medium
(Miles, Elkhart, IN), immediately frozen in liquid nitrogen, and stored at
⫺80°C until use. The tissue was cut at ⫺22°C into 5-␮m slices and put on
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The following Abs were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA): anti-fractalkine C-18, directed against the extracellular domain
of fractalkine; anti-fractalkine C-20, directed against the intracellular domain of fractalkine; anti-caveolin-1; and anti-E-cadherin. Anti-goat HRP
was obtained from Amersham (Piscataway, NJ); anti-goat biotin, antimouse/rabbit biotin, and streptavidin-FITC were obtained from Dako (Carpenteria, CA). Streptavidin-PE, streptavidin-rhodamine, anti-rabbit biotin,
anti-CD4 FITC, anti-CD8 biotin, and anti-CD8 PE used for FACS analysis
were obtained from PharMingen (San Diego, CA). CX3CR1 expression
constructs and antisera were a gift from P. M. Murphy (National Institute
of Allergy and Infectious Disease, Bethesda, MD). Fractalkine, IL-1␤, and
IL-2 were obtained from R&D Systems (Minneapolis, MN).
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FRACTALKINE IS A CHEMOATTRACTANT IN THE HUMAN INTESTINE
positively charged glass slides and stored at ⫺80°C until use. The slides
were thawed for 5–10 min, dipped into acetone (⫺20°C) for 30 s, and air
dried. T-84 cells were seeded at a density of 10,000 cells/ml and grown on
coverslips (Lab-Tek, Naperville, IL) for 3 days. Cells were fixed as the
intestinal biopsy specimens. After air drying, the cells or biopsy specimens,
respectively, were blocked for 20 min in 5% (v/v) donkey serum (Santa
Cruz Biotechnology) in PBS (blocking solution). All steps were conducted
at room temperature in a humidified, light-protected chamber. After an
incubation with anti-fractalkine C-20 (2.5 ␮g/ml), anti-cadherin, or anticaveolin-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution for 2 h, slides were washed with PBS (2 ⫻ 2 min) and incubated with anti-goat biotin Ab in blocking solution (1:50) for 30 min. After
an additional washing step, the slides were incubated with streptavidinFITC (1:50) for 1 h. After washing, slides were mounted with Vectashield
(Vector Laboratories, Burlingame, CA). Staining was analyzed with an
IX70 Olympus-fluorescent microscope.
Isolation of iIEL and FACS analysis
Intestinal jejunal tissue was obtained during gastric bypass surgery for
obesity. iIEL were isolated as described earlier (25). iIEL were either used
freshly for FACS analysis or kept in culture, as described before (26).
FACS analysis was performed, as described before (25), using a FACScan
flow cytometer (Becton Dickinson, Mountain View, CA). The antiCX3CR1 Ab containing antisera was used at a dilution of 1/50; negative
control experiments were performed with the preimmune serum also at a
1/50 dilution.
FIGURE 2. Induced expression of membrane-bound fractalkine by
IL-1␤ is accompanied by secretion of soluble fractalkine. Western blot
analysis of fractalkine protein expression in T-84 cell lysates after stimulation with 10 ng/ml IL-1␤. A total of 20 ␮g of protein per lane was
resolved on 4 –12% SDS gel and immunoblotted with Abs recognizing the
intracellular region of fractalkine (A) or the fractalkine chemokine domain
(C). B and D, Represent the densitometrically analysis of membrane-bound
fractalkine in A or soluble fractalkine in C, respectively. Fractalkine was
immunoprecipitated from cytosolic or membrane fractions of T-84 cells
with an Ab directed against the cytoplasmic region of fractalkine and resolved by SDS-PAGE, followed by immunoblotting for the presence of
fractalkine (E). A total of 100 ng/ml of glycosylated recombinant fractalkine was used as a control (E, lane 5). F, Represents densitometric analysis of fractalkine protein concentration in E expressed as mean density/
area. One representative experiment of three conducted is shown.
iIEL migration assay
iIEL were stimulated with IL-2 (10 ng/ml) for 3 days. Using a chemotaxis
chamber (Neuroprobe, Cabin John, MD), the lower well was filled with
medium with or without fractalkine, and, in some instances, antifractalkine Ab at concentrations indicated. A 5-␮m polyvinylpyrrolidonefree polycarbonate membrane separated the lower from the upper well,
which was filled with 50 ␮l medium, containing 100,000 iIEL. Each assay
was performed in triplicate. The cells were allowed to migrate for 8 h at
37°C. Cells in the lower well were counted, and the migration index (migrated cells divided by the number of cells that migrated without fractalkine) was calculated.
Results
IL-1␤ up-regulates basal expression of fractalkine mRNA in T84 cells
Fractalkine mRNA expression has been found in different tissues,
including small intestinal and colonic tissue (3). The latter
prompted us to investigate whether fractalkine mRNA is expressed
in intestinal epithelial cells. Northern blot analysis demonstrated
that fractalkine mRNA is expressed in the intestinal epithelial cell
line T-84 (Fig. 1). Incubation with the NF-␬B-inducing cytokine
IL-1␤ was able to induce a transient 4-fold increase in basal steady
state expression of fractalkine transcripts within 2 h (Fig. 1).
IL-1␤-induced expression of membrane-bound fractalkine in
T-84 cells is accompanied by secretion of soluble fractalkine
To determine whether the induction of fractalkine mRNA expression in T-84 cells resulted in increased production of fractalkine
protein, Western blot and immunoprecipitation analysis were conducted with Abs specifically directed against the chemokine domain and the intracellular region of fractalkine. As demonstrated in
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FIGURE 1. Basal fractalkine mRNA expression in T-84 cells is upregulated by IL-1␤. Northern blot analysis of fractalkine mRNA expression
in RNA from T-84 cells (20 ␮g of total RNA/lane). Cells were incubated
for the time indicated with 10 ng/ml IL-1␤. Fractalkine mRNA expression
was quantified with a densitometer (Molecular Dynamics, Sunnyvale, CA)
and NIH Image 1.61 (National Institutes of Health, Bethesda, MD). C,
Represents the densitometrical analysis of fractalkine mRNA expression in
A, normalized to GAPDH mRNA expression in B, expressed in median
density/area. One representative experiment of four conducted is shown.
The Journal of Immunology
FIGURE 3. Membrane-bound fractalkine is localized in caveolin-1 containing detergent-insoluble glycolipid-enriched membrane microdomains.
A, Demonstrates the immunoblot analysis of fractalkine expression in T-84
cell membrane fractions separated on a sucrose gradient layered on top of
the T-84 cell lysate, containing 1% Triton X-100. The sucrose concentrations of relevant fractions are indicated. Equal amounts of protein were
subjected to Western blot analysis, as described in Materials and Methods.
The same blot was stripped and rehybridized with Abs directed against
caveolin-1 (B).
FIGURE 4. Membrane-bound fractalkine expression is regulated during T-84 cell polarization. T-84
were grown on coverslips, and immunofluorescence
was performed, as described in Materials and Methods, for the detection of fractalkine in A, caveolin-1
in C, and E-cadherin in E. The corresponding bright
field exposures are presented in B, D, and F, respectively (magnification ⫻40).
FIGURE 5. CX3CR1 mRNA is expressed in iIEL and lamina propria
mononuclear cells. RT-PCR was conducted with human RNA isolated
from the CD8⫹ iIEL cell line EEI10 (lane 1), three independently freshly
isolated iIEL cell preparations from the small intestine (lanes 2– 4); freshly
isolated LPL (lanes 5–7), and whole small intestinal mucosa (lane 8). RNA
from COS-7 cells nontransfected (lane 9) and after transfection with a
CX3CR1 expression construct (lane 10) was used as positive and negative
controls. RT-PCR conducted with the CX3CR1 primer yielded the expected 653-bp product.
recombinant fractalkine (Fig. 2E, lane 5). More than 82% of the
fractalkine expressed in nonstimulated T-84 cells localized in the
T-84 cell membrane fraction. Stimulation with IL-1␤ for 12 h increased the fractalkine content in the cytosol fraction 2.5-fold and
by 1.3-fold in the membrane fraction (Fig. 2, E and F).
Fractalkine is localized in caveolin-1 containing detergentinsoluble glycolipid-enriched membrane microdomains in T-84
cells
To further characterize the membrane compartment containing
fractalkine, we separated postnuclear membrane protein fractions
by sucrose density-gradient centrifugation. This method separates
detergent-insoluble functional distinct membrane fractions characterized by their specific lipid composition (24, 27, 28). As demonstrated in Fig. 3A, fractalkine was present in low density membrane fractions from T-84 cells. Fractalkine was present in
membrane fractions corresponding to sucrose concentrations of
15% to 24%, with the highest concentration in the fraction corresponding to 22% (Fig. 3A, lane 5). These low density membrane
fractions have been associated with detergent-insoluble glycolipidenriched membrane fractions or rafts (29). In epithelial cells, these
membrane fractions are characterized by the presence of a distinct
scaffolding protein caveolin (30). As demonstrated in Fig. 3B,
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Fig. 2, fractalkine was detected in T-84 cell lysates running with a
molecular size of ⬃95 kDa, which is consistent with the previously
described size of glycosylated fractalkine (3). The basal expression
of fractalkine in T-84 cells was increased 2-fold within 12 h after
incubation with 10 ng/ml IL-1␤ (Fig. 2, A and B). Fractalkine has
been demonstrated to be released in soluble form from its transmembrane region, and we therefore determined whether fractalkine was released from T-84 cells upon stimulation by IL-1␤. As
demonstrated in Fig. 2, C and D, the increase of fractalkine protein
expression in T-84 cell lysates was followed by an elevation of
soluble fractalkine in the T-84 cell media supernatants. The basal
expression of fractalkine in T-84 cell media supernatants increased
5-fold over 24 h after induction by IL-1␤ (Fig. 2, C and D).
To confirm that the fractalkine protein detected in T-84 cell
lysates was membrane bound, T-84 membranes were separated
from cytosol protein fractions before and after stimulation with
IL-1␤, followed by immunoprecipitations with anti-fractalkine
Abs directed against the intracellular region of fractalkine and
Western blotting with Abs directed against the fractalkine chemokine domain. As demonstrated in Fig. 2, E and F, the antifractalkine Abs hybridized to a protein band in the immunoprecipitates from the cytosol as well as the transmembrane protein
fraction, which corresponded to the size of full-length glycosylated
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FRACTALKINE IS A CHEMOATTRACTANT IN THE HUMAN INTESTINE
FIGURE 6. A subpopulation of isolated human iIEL expresses CX3CR1
on the cell surface. FACS analysis of CX3CR1 expression on fresh isolated
human iIEL. In A, isolated iIEL were stained with anti-CD8 PE and FITC
control Abs, and in B, with CD4-FITC and the rhodamine control Abs. C,
Represents the staining of iIEL with specific CX3CR1 antisera and antiCD4 FITC-coupled Abs. Representative experiment from three independently conducted is shown.
Fractalkine expression is regulated during polarization of the
intestinal epithelial cells
The human intestinal epithelial cell line T-84 provides a wellestablished model for the assembly of intercellular junctions and
the development of apical-basolateral polarity (32). Immunofluorescence staining revealed specific staining for fractalkine in T-84
cells (Fig. 4A). Fractalkine was mainly expressed in undifferentiated proliferating T-84 cells that are localized at the edges of
monolayers with developed apical-basolateral polarity (Fig. 4). Polarized T-84 cells still retained some fractalkine expression, which
appeared to be localized to cell-cell contact regions. In contrast,
fractalkine expression in unpolarized T-84 cells demonstrated a
strong granular cytoplasmic and cell surface expression pattern
(Fig. 4, A and B). Because fractalkine demonstrated a strong enrichment in caveolin-1-containing membrane fractions, we determined the expression of caveolin-1 in polarized and unpolarized
T-84 cells. As shown in Fig. 4, C and D, caveolin-1 was strongly
expressed in unpolarized T-84 cells. The specific expression pattern of fractalkine and cavelolin-1 in unpolarized T-84 was not due
to restricted access to the basolateral surface of the polarized epithelial cells because anti-E-cadherin Abs were able to detect Ecadherin on the surface of unpolarized as well as at the basolateral
surface of polarized T-84 cells (Fig. 4, E and F).
FIGURE 7. Fractalkine is a chemoattractant for IL-2-activated iIEL.
Fractalkine was added at the concentrations indicated to the lower well of
a chemotaxis chamber, which was separated from the upper well by a 5-␮m
filter. IL-2-activated iIEL were added to the upper well and allowed to
migrate for 8 h at 37°C, as described in Materials and Methods (A). Abblocking experiments confirm the specificity of fractalkine-induced migration of IL-2-activated iIEL (B). The mean of three independent experiments ⫾ SEM is shown. Statistical analysis was performed using Student’s
t test (ⴱ, p ⬍ 0.01). CX3CR1 expression (C) and CD8-positive phenotype
(D) in IL-2-activated iIEL were confirmed by flow cytometry. In C, the
dotted line represents the fluorescence intensity after incubation of iIEL
with preimmune sera; the solid line after incubation with anti-CX3CR1
antisera. D, Demonstrates the expression of CD8 on iIEL without (dotted
line) or after stimulation for 3 days with IL-2 (solid line).
Human iIEL express functional fractalkine receptor CX3CR1
To determine the potential target cells of fractalkine expression by
intestinal epithelial cell in the intestinal mucosa, we assessed expression of CX3CR1 transcripts in RNA isolated from lymphocyte
population obtained from normal human small intestine. As shown
in Fig. 5, iIEL from three independent cell isolations and two of
three lamina propria lymphocyte isolations demonstrated CX3CR1
mRNA expression. In addition, CX3CR1 mRNA expression was
detected in the iIEL cell line EEI10.
Because iIEL are most likely to interact with intestinal epithelial
cells and both freshly and cultivated iIEL demonstrated a strong
expression of CX3CR1 mRNA, we determined whether iIEL ex-
press CX3CR1 protein on the cell surface using flow cytometry. As
demonstrated in Fig. 6A, the freshly isolated human iIEL consisted
of 78% CD8-positive (Fig. 6A) and 7.6% CD4-positive lymphocytes (Fig. 6B). A total of 48.5% of the fresh isolated iIEL expressed the fractalkine receptor CX3CR1 (Fig. 6C).
Fractalkine mediates chemoattraction of human iIEL
To determine whether the CX3CR1 receptor expressed on iIEL is
able to mediate chemoattraction, the migratory response of isolated
human iIEL was determined in the chemotaxis chamber multiwell
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Western blot analysis of these cellular compartments revealed that
the expression of caveolin-1 was restricted to same sucrose gradients as the fractalkine expression T-84 cells. Caveolin-1 concentration was maximal at apparently 22% sucrose (Fig. 3B), consistent with a previous report (31). This seems to be cell specific for
T-84 cells, as maximal amounts of caveolin are typically detected
at sucrose concentrations of 15–20% in other cell lines (24). Collectively, these data indicate that fractalkine is largely sequestered
in a microdomain of T-84 cell membranes that display the biophysical characteristics of caveolae (29).
The Journal of Immunology
3373
system, as described (33), and expressed in fold increase of the
basal cell migration (Migration Index) (Fig. 7A). Since fresh isolated iIEL do not migrate efficiently toward chemokine gradients,
iIEL were stimulated with IL-2 (10 ng/ml) for 3 days, as previously described (34). As demonstrated in Fig. 7A, soluble fractalkine induced the migration of IL-2-activated iIEL in a dose-dependent fashion. Highest induction was found at a fractalkine
concentration of 1 ng/ml, and activity decreased at higher concentrations. Similar dose-response curves have been reported for
RANTES in the induction of migration of iIEL (33). The fractalkine-induced migration of iIEL could be significantly inhibited in
a dose-dependent fashion by Abs against the chemokine domain
(C-18), but not the carboxy terminus of fractalkine (C-20) (Fig. 7B,
p ⬍ 0.001). Flow cytometry confirmed the presence of CX3CR1
on iIEL (Fig. 7C) after incubation with IL-2. Furthermore, iIEL
retained their CD8-expressing phenotype during IL-2 activation
(Fig. 7D). Together, these experiments demonstrate that iIEL express functional CX3CR1 receptors, and can be chemoattracted by
soluble fractalkine when activated by IL-2.
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FIGURE 8. Fractalkine is expressed in epithelial and
endothelial cells in the human small intestinal mucosa.
Immunohistochemistry of frozen sections was utilized to
localize fractalkine in normal human small intestinal
mucosa (A, B) and in active Crohn’s disease mucosa
(C–F). The immunofluorescence pictures in A and C are
accompanied by the corresponding bright field exposures of the same tissue section on the right (B and D,
magnification ⫻40). E (⫻40) and F (⫻100) show basal
expression of fractalkine in small intestinal epithelial
cells in active Crohn’s disease. Arrows indicate fractalkine expression in endothelial cells (A, C). One representative immunostaining of five conducted in each
group is shown.
Fractalkine is expressed by intestinal epithelial cells and
endothelial cells within the small intestinal mucosa and upregulated in active Crohn’s disease
Immunohistochemistry was utilized to determine whether fractalkine is expressed in human normal or diseased small intestinal
mucosa. Frozen sections of normal and inflamed small intestinal
mucosa (Fig. 8) were stained for the presence of fractalkine with
a combination of Abs against the intracellular (C-18) and extracellular (C-20) regions of fractalkine, followed by incubation with
biotinylated anti-rabbit Abs and FITC-coupled streptavidin. As
demonstrated in Fig. 8A, intestinal epithelial cells in normal small
intestinal tissues stained positive for fractalkine. In addition, a
punctuated positive staining for fractalkine was present in endothelial cells outlining small mucosal blood vessels (Fig. 8A, indicated by white arrows). Fractalkine expression was highly up-regulated in endothelial cells outlining all small blood vessels
observed within the inflamed intestinal mucosa during Crohn’s
disease even in areas of the mucosa that did not show infiltration
3374
FRACTALKINE IS A CHEMOATTRACTANT IN THE HUMAN INTESTINE
of inflammatory cells (Fig. 8C). Furthermore, during intestinal inflammation in Crohn’s disease, intestinal epithelial cells demonstrated strong basolaterally positive staining for fractalkine (Fig. 8,
E and F). Together, these experiments demonstrate that fractalkine
can be expressed by small intestinal epithelial cells and endothelial
cells in the normal and inflamed small intestine.
Fractalkine mRNA expression is up-regulated in the intestine
during active Crohn’s disease
To determine whether fractalkine expression is involved in intestinal inflammation, we assessed fractalkine mRNA expression in
normal small intestine, as well as in intestinal tissues resected from
patients with active Crohn’s disease. As demonstrated in Fig. 9,
fractalkine mRNA was expressed in the normal small intestine
(Fig. 9, lanes 1–3). Fractalkine mRNA was significantly up-regulated in Crohn’s disease (Fig. 9, lanes 4 and 5). The expression of
fractalkine mRNA in small intestine increased from a mean density/area of 32 ⫹ 4.5 in normal intestine up to 85 ⫹ 16.2 in
Crohn’s disease tissues after correction for GAPDH expression
( p ⬍ 0.01).
Discussion
In this study, we have determined that the fractalkine-CX3CR1
ligand-receptor system provides a novel mechanism of intestinal
lymphocyte regulation by intestinal epithelial cells. Thus, we have
observed fractalkine expression by intestinal epithelial cells and
endothelial, which can contribute to regulation of the attraction and
migration of intestinal lymphocytes in healthy and diseased intes-
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FIGURE 9. Fractalkine mRNA expression is up-regulated in the intestinal mucosa in active Crohn’s disease. Northern blot analysis of fractalkine mRNA expression (A) in resected mucosal tissues from normal controls (lanes 1–3) and inflamed Crohn’s disease mucosa (C and D, lanes
4 – 6). Fractalkine mRNA expression was densitometrically analyzed after
normalization for GAPDH expression (B) and expressed as mean density/
area (C). Statistical analysis was conducted by Student’s t test (ⴱ, p ⬍
0.01).
tinal mucosa. In the intestine, lymphocytes constantly migrate
through the endothelial cell layer of high endothelial venules and
organize within the lamina propria (35, 36). Some of the lymphocytes continue through the epithelial basal membrane and settle
within the intestinal epithelial cell monolayer forming the iIEL
compartment. During intestinal inflammation, neutrophils are also
able to cross the epithelial barrier. The composition of migrating
intestinal leukocyte populations is dynamically regulated as a reflection of the immunological status of the intestinal mucosa. Chemokine expression by intestinal epithelial cells has been suggested
to contribute to chemotactic gradients necessary to direct leukocyte migration into the intestinal mucosa (37– 40). In the current
models of leukocyte migration, chemokines and their receptors
transduce signals to the rolling leukocyte to induce cell arrest and
firm adhesion by activating the adhesive capacity of integrins (41,
42). However, fractalkine is the first chemokine, which is able to
mediate leukocyte attraction, capture, firm adhesion, and activation
even in the absence of integrins (43, 44).
The detection of fractalkine transcripts in mRNA isolated from
small and large intestine (3) prompted us to determine whether
intestinal epithelial cells are a source of fractalkine mRNA and
protein expression. Our experiments demonstrate that the human
intestinal epithelial cell line T-84 is able to express membranebound and soluble fractalkine under the control of the inflammatory mediator IL-1␤, suggesting that fractalkine may be involved
in directing intestinal epithelial cell-lymphocyte interactions as
well as the attraction of lymphocytes into the intestinal lamina
propria. Induction of fractalkine expression in intestinal epithelial
cells may mediate long lasting effects. Whereas basal expression of
fractalkine mRNA was maximally induced within 4 h after a single
induction by IL-1␤, membrane-bound fractalkine protein levels
increased over 12 h, and the amount of soluble fractalkine increased steadily over 24 h. Furthermore, the expression of fractalkine in intestinal epithelial cells was regulated during polarization
and differentiation of T-84 cells. Immunohistochemistry demonstrated that fractalkine was highly expressed in nonpolarized T-84
cells, which are still able to migrate and to proliferate, whereas
fractalkine expression decreased in T-84 cells after polarization
and differentiation and localized to the basolateral cell surface.
Fractalkine expression in undifferentiated and migrating intestinal
epithelial cells may indicate a role of fractalkine in intestinal
wound healing.
Membrane-bound fractalkine colocalized with caveolin-1 in a
specialized membrane compartment characterized by its specific
lipid composition, which allowed separation on a sucrose gradient
from other membrane protein fractions. This indicates that fractalkine may be specifically expressed in caveolae on the surface of
intestinal epithelial cells. Caveolae are vesicular invaginations of
the plasma membrane characterized by the expression of the
caveolin family of scaffolding proteins (30). Caveolins can assemble signaling molecules into preassembled signaling complexes,
and an increasing number of signaling pathways have been shown
to be associated with caveolae, such as those mediated by G protein-coupled receptors, the ras-raf pathway, and pathways involving the activation of src family tyrosine kinases (30). Signal transduction events important in cell-cell adhesion, such as integrin
signaling, have been shown to be dependent on the presence of
caveolins (45). Consistent with the expression of fractalkine in
caveloae-like structures, the same nonpolarized T-84 cell population stained positive for the expression of caveolin-1. Targeting of
fractalkine to glycolipid-enriched microdomains may be mediated
by the myristylation of cysteines in the membrane or juxtamembrane region of fractalkine (C353, C367, and C386), analogous to
the membrane targeting of LAT (linker for activation of T cells)
The Journal of Immunology
evidence that fractalkine is involved in the attraction of leukocytes
during disease was demonstrated in experiments in which fractalkine-mediated migration of granulocytes isolated after Ab-induced glomerulonephritis could be inhibited by vMIP-2 (7).
The mechanism of lymphocyte migration into healthy and diseased intestinal mucosa is not well understood. Together, our studies support a model in which fractalkine may be involved in the
attraction and retention of iIEL into the intestinal epithelium. Fractalkine expression within the intestinal mucosa may have an important function in intestinal inflammation. Endothelial-expressed
fractalkine may initiate capture of lymphocytes by firm adhesion to
the activated endothelium, and epithelial cell-derived soluble fractalkine may direct lymphocytes to the site of inflammation and
tissue repair. Membrane-bound fractalkine expressed by intestinal
epithelial cells may then help to retain iIEL within the regenerating
intestinal epithelium.
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