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Regular Articles |
From the Sir William Dunn School of Pathology,*
University of Oxford, Oxford; the Nuffield Department of
Surgery
and the Department of Cellular
Pathology,
John Radcliffe Hospital,
Headington, Oxford; and the Gastroenterology
Unit,§
Radcliffe Infirmary, Oxford,
United Kingdom
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Abstract |
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Introduction |
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Fractalkine is the only known chemokine shown to mediate strong cell adhesion mediated via in vitro binding to its receptor, a member of the TM7 family of receptors.1,3,4 Monocytes, natural killer cells, T cells,3 and microglia5 express the CX3CR1 receptor, migrate in response to fractalkine, and adhere to immobilized fractalkine in vitro.1,4 Cell adhesion to immobilized forms of fractalkine or transfected cells expressing full-length fractalkine can occur under flow conditions in vitro.6,7 Binding to immobilized forms of fractalkine does not require G-protein signaling, does not require the mucin stalk, and can occur via an integrin-independent pathway.4,7-9 Fractalkine mRNA and protein expression have been shown to be up-regulated in human umbilical cord endothelial cells (HUVECs) treated with a number of inflammatory cytokines1 in vitro and it has been proposed that binding to fractalkine offers an alternative pathway for leukocyte adhesion under conditions of physiological flow.4 Immunocytochemical studies using reagents reactive to peptide sequences taken from the chemokine domain of fractalkine, have shown labeling of neurons in the brain,10 of endothelium, and dendritic cells (DCs) within the tonsil and skin.11 Reagents reactive to a different set of peptides were reported to detect endothelium and epithelial cells in the human gut.12
To identify the distribution of full-length transmembrane fractalkine in vivo, we have developed antibody reagents that specifically detect the intracellular tail of human fractalkine. Using novel polyclonal and monoclonal reagents that specifically recognize the chemokine domain of fractalkine in human tissue samples, we show that epithelial cells are the predominant cell type that expresses transmembrane forms of fractalkine in normal and inflamed tissues. We propose that constitutive epithelial cell production of fractalkine within the periphery may play a role in the trafficking of CX3CR1 receptor-positive DC precursors to these sites.
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Materials and Methods |
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Rabbit and chicken polyclonal antiserum were raised against the synthetic peptide MAEGLRYIPRSCGSNSYVL, which represents amino acids 352 to 370 located in the intracellular tail region of fractalkine, coupled at the C terminus to KLH. Rabbit antisera was raised against the synthetic peptide CADPKEQWVKDA MQHLDRQAAAL that represents amino acids 74 to 96 located within the chemokine domain of fractalkine and previously used to generate an anti-fractalkine N-terminus antisera.1 Peptide-specific IgG was affinity-purified using NH2-activated Sepharose (Pharmacia, Uppsala, Sweden). Peptide was coupled overnight on a wheel at 4°C by using 10 mg immunizing peptide/ml of beads, in coupling buffer containing 0.1 mol/L NaHCO3 and 0.5 mol/L NaCl, pH 8.5, as per the manufacturer’s recommendations.
Human Tissue Samples
Human tonsils were obtained after routine tonsillectomies conducted at the Nuffield Infirmary, Oxford, UK. Noninvolved human skin samples were obtained after excision of skin tumors at the Department of Dermatology, Churchill Hospital, Oxford, UK. Human gut tissue was obtained either during routine colonoscopy or after surgical procedures. Tissue was embedded in OCT mounting medium (Miles Inc., Elkhart, IL), and stored at -80°C. Ethical approval for this study was obtained from the Central Oxford Research Ethics Committee (CORREC No. C98.127). For immunohistochemical and immunofluorescence analysis, 6- to 10-µm frozen tissue sections were mounted onto slides, allowed to dry at room temperature, and either used immediately or transferred to -20°C.
Cell Lines Used
The human colorectal adenocarcinoma cell line, DLD-1, the murine fibroblast line NIH/3T3, and the hamster epithelial line CHO-K1 were obtained from ATCC (Manassas, VA). The CD84-transfected cell line 300.19 CD-8413 was kindly provided by Dr. P. Engel, Department of Cell Biology and Pathology, University of Barcelona, Spain. NIH/3T3 cells were grown in Dulbecco’s modified Eagle’s medium, DLD-1 and 300.19 CD-84 were grown in RPMI 1640 media, and CHO-K1 was grown in Ham’s F-12K medium, with each medium supplemented with 10% fetal calf serum, glutamine (2 mmol/L), and penicillin/streptomycin (5 IU/ml, 50 µg/ml, respectively; Lifetech, Paisley, UK) and maintained under standard culture conditions. In addition the 300.19 CD-84 cells were maintained in the presence of 1 mg/ml G418. In some experiments, DLD-1 cells were grown to confluence on 15-mm diameter glass coverslips (BDH, Poole, UK) placed within 6-well dishes (Corning Costar Corp., Cambridge, MA) containing culture media, as described above.
Cell Transfections
CHO-K1 or NIH/3T3 cells were plated into 9-cm Petri dishes and grown to 80% confluence then transfected using a ratio of 2 µg DNA/60 µg of lipofectamine (Lifetech) with either full-length fractalkine cloned in the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA), or ß-galactosidase cloned in the same expression vector. Cells were washed three times in ice-cold phosphate-buffered saline (PBS) and incubated at room temperature for 5 to 10 minutes with PBS containing 2 mg/ml lidocaine (Sigma-Aldrich, St. Louis, MO) and 0.05 mol/L ethylenediaminetetraacetic acid (Sigma-Aldrich). Cells were harvested by vigorously pipetting the solution over the surface of the dishes. Collected cells were washed and either resuspended in PBS containing 10% fetal calf serum and used for either cytospin or fluorescence-activated cell sorting (FACS) studies, or placed in lysis buffer containing protease inhibitors (Complete mini; Roche, Lewes, UK) on ice for 20 minutes before centrifugation at 10,000 x g for 20 minutes, and stored at -20°C before use in Western blotting analysis.
Cytospin Studies
Transfected NIH/3T3 cells were suspended at a concentration of 1 x 106 cells/ml and then 200 µl was applied to 1% gelatin-coated glass laboratory slides (BDH) using a Cytospin 3 centrifuge (600 rpm, 6 minutes; Shandon, Pittsburgh, PA). Slides were air-dried and stored at -20°C until used.
FACS Studies
DLD-1 cells were washed and fixed in 2% paraformaldehyde in PBS for 30 minutes at 4°C. Cells were then washed and permeabilized in 0.5% saponin/0.5% bovine serum albumin/PBS (Sigma-Aldrich) containing 5% normal human serum (National Blood Service, Bristol, UK), a solution used for all subsequent staining steps. Primary antibodies were applied for 20 minutes at 4°C, cells were washed, and fluorescein isothiocyanate-conjugated secondary antibodies applied for 20 minutes at 4°C in the dark. Cells were subsequently washed, fixed in 2% paraformaldehyde in PBS, and analyzed by FACS, using a FACScan and CellQuest software (Becton Dickinson, Franklin Lakes, NJ).
Isolation of Total RNA and Semi-Quantitative Reverse Transcriptase-Polymerase Chain Reaction (PCR)
DLD-1 cell pellets were resuspended in total RNAzol B isolation reagent (Biogenesis, Poole, UK) and total RNA isolated according to the manufacturer’s instructions. Dried RNA pellets were resuspended in nuclease-free water and stored at -80°C before analysis. HUVEC cDNA, was a kind gift from Dr. Dicken Koo, Nuffield Department of Surgery, University of Oxford, Oxford, UK. Total RNA was reverse-transcribed using oligo dT12-18 and Superscript reverse transcriptase (Lifetech). Reactions were incubated at 42°C for 40 minutes and enzyme-inactivated at 95°C for 5 minutes. Triplicate PCR reactions were assembled containing cDNA from 25 ng of total RNA and Taq DNA polymerase (Bioline, London, UK). PCR for the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) was performed using the primers 5'-AATTATGGACAG GACTGAACGTC-3' (forward) and 5'-CGTGGGGTCCTTTTCACCAGCAAG-3' (reverse), generating a 386-bp PCR product. PCR for fractalkine was performed using the primers 5'-CACGTGCAGCAAGATGACATC-3' (forward) and 5'-CACTCGGAAAA GCTCCGTGC-3'(reverse), generating a 462-bp PCR product. Reactions were subjected to touchdown PCR using a PTC-200 thermal cycler (MJ Research, Watertown, MA) with the following parameters: after an initial denaturing step of 96°C for 1 minute, five cycles of 96°C for 25 seconds, 70°C for 45 seconds, and 72°C for 45 seconds; followed by 31 cycles of 96°C for 25 seconds, 60°C for 50 seconds, and 72°C for 45 seconds; and finally four cycles of 96°C for 25 seconds, 55°C for 1 minute, and 72°C for 2 minutes. After agarose gel electrophoresis PCR products were analyzed under a UV lamp and product intensities measured by AlphaEase image analysis software (Alpha Innotech Corporation, San Leonardo, CA). Fractalkine PCR product intensities were divided by those of the HPRT PCR product intensities to give a fractalkine:HPRT ratio to generate comparative fractalkine mRNA data. The specificity of fractalkine PCR products was confirmed by digestion with BsmA1 restriction endonuclease (New England Biolabs, Hitchin, UK; data not shown).
Antibodies Used
Tissue and cytospin samples were labeled using a range of
chemokine-specific and lineage-related reagents (Table 1)
. The immunostaining conditions were
optimized for each antibody reagent used in this study. The following
reagents and fixation conditions were used: murine anti-human
monoclonal IgG1 antibodies reactive to
fractalkine chemokine domain (1 to 10 µg/ml, clone 51636.11; 1
µg/ml, clone 51637.11; R&D Systems, Minneapolis, MN), anti-human CD1a
(1 to 10 µg/ml, clone NA1/34, isotype IgG2a;
Serotec, Kidlington, UK) anti-cytokeratin AE1/AE3 (1 µg/ml, clone
AE1/AE3 isotype IgG1; DAKO AS, Glostrup,
Denmark); goat anti-fractalkine chemokine domain polyclonal (5 µg/ml;
R&D Systems); rabbit anti-fractalkine N-terminal peptide1
(Chemocentryx, San Carlos, CA) used at 0.1 to 2 µg/ml; rabbit
anti-fractalkine C-peptide reagent, chicken anti-fractalkine C-peptide
reagent, used at 1 to 5 µg/ml. Murine anti-human CD84 mAb
(IgG1) was obtained from the Seventh
International Workshop on Human Differentiation Antigens, Harrogate,
UK, 2000. These reagents were used on sections fixed using either 4%
paraformaldehyde in PBS containing 1 mmol/L HEPES at room temperature
or ice-cold acetone for 10 minutes. Specificity controls used for the
polyclonal antibodies were their specific absorption with 10x molar
excess of recombinant human fractalkine chemokine domain (362-CX-025,
R&D Systems) or immunizing peptide for 30 minutes at room temperature
and subsequent application to serial tissue sections. For the
monoclonal reagents, isotype control antibodies of immunoglobulin
classes IgG1 and IgG2a
(Serotec) were applied to tissue sections at the same concentration as
the specific reagents and assessed for reactivity. In addition to the
two anti-fractalkine chemokine domain mAb clones described above, two
further clones (clones 51621.11, 51643.11; R&D Systems) also showed the
same patterns of reactivity as clone 51636.11 on human tonsil tissue.
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All antibodies were diluted in PBS containing 1% bovine serum albumin and 0.2% Triton X-100. Single labeling involved incubation of primary antibodies for 2 hours at 4°C. Concentrations of the secondary and tertiary reagents was typically 0.1 mg/ml. In cases in which a red label was required for visualization, samples were incubated with anti-species-specific IgG conjugated to the fluorochrome Texas Red (Jackson Laboratories, Bar Harbor, ME) for 1 hour at 4°C. In cases in which a green label was required for visualization, samples were incubated with anti-species-specific IgG conjugated to biotin (DAKO), for 1 hour at 4°C. Finally a streptavidin-fluorescein isothiocyanate reagent (Amersham, Amersham, UK) was applied for 30 minutes at 4°C. Double-immunofluorescent labeling involved mixing the mouse and rabbit primary antibodies, during the first incubation period and the biotinylated and Texas Red-conjugated anti-species-specific IgG during the second. Finally a streptavidin-fluorescein-isothiocyanate reagent (Amersham) was applied for 30 minutes at 4°C.
Immunohistochemistical Labeling
Primary antibodies were incubated from times ranging from 2 to 12 hours at 4°C. Neutralization of endogenous peroxidases was achieved by incubating tissue sections with 3% H2O2 in PBS containing 0.1 g/ml NaN3 (Sigma-Aldrich) for 10 minutes at room temperature. Concentrations of the secondary and tertiary reagents were typically 0.1 g/ml. Double labeling involved mixing the mouse and rabbit primary antibodies during the first incubation period, and the biotinylated and horseradish peroxidase-conjugated anti-species-specific IgG (DAKO) during the second. Finally a streptavidin-alkaline-phosphatase reagent (DAKO) was applied for 30 minutes at 4°C. The sections were then developed using diaminobenzidine tetrahydrochloride (PolySciences, Warrenton, PA), washed in PBS and then incubated with streptavidin-alkaline-phosphatase for 30 minutes at room temperature and developed using BCIP/VBT substrate (Vector Laboratories).
Western Blot Analysis
To test the specificity of the anti-peptide antiserum raised against the carboxyl terminus of human fractalkine, Western blotting was performed on protein lysates of CHO-K1 cells transfected with full-length fractalkine cloned in the mammalian expression vector pcDNA31 (Invitrogen, Carlsbad, CA), or untransfected CHO-K1 cells. Samples, which included supernatant taken from the fractalkine-transfected cells, were run under reducing conditions on 7.5% polyacrylamide gels (Pharmacia) and transferred to nitrocellulose membranes (Hybond C; Amersham) overnight at 4°C. Membranes were probed using goat anti-chemokine domain polyclonal and chicken and rabbit anti-C peptide reagents at 0.5 µg/ml. Specific signal was amplified by addition of horse anti-species-specific IgG conjugated to horseradish peroxidase and visualized using an enhanced chemiluminescence system (ECL, Amersham).
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Results |
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To differentiate between transmembrane-expressed fractalkine and
cleaved forms of the molecule, we developed a number of polyclonal
anti-peptide reagents (rabbit 
-C-pep and chicken -C-pep), using a
peptide sequence from the intracellular tail of the molecule.
We tested the specificity of these anti-C-peptide reagents in the
following ways. First, cell lysates prepared from CHO-K1 WT or CHO-K1
transfected with a construct1
containing the full-length
transcript of fractalkine and samples of supernatant taken from the
transfected cells, were separated via electrophoresis under reducing
conditions and analyzed by Western blotting. Figure 1A
shows a representative experiment
comparing the reactivity of goat anti-fractalkine chemokine domain
polyclonal (R&D Systems) and the chicken anti-C-peptide polyclonal.
Both reagents detected two bands of similar size, 
95 kd, within the
transfected cell lysates (Figure 1
, lanes 2 and 5). These two bands may
represent different glycosylated forms of the molecule. No nonspecific
bands were detected by either reagent within WT CHO-K1 samples (Figure 1
, lanes 1 and 4). The anti-chemokine domain reactive polyclonal
detected a broad band of 85 to 90 kd (Figure 1
, lane 3) within the
transfected cell supernatant whereas the anti-C-peptide reagent showed
no reactivity against proteins within the supernatant (Figure 1
, lane
6). In other experiments neither anti-C-peptide reagent detected
various recombinant forms of fractalkine- or macrophage-derived
chemokine (R&D Systems, data not shown). Thus it is possible to use
these reagents to discriminate between full-length and cleaved forms of
fractalkine. Second, we compared the anti-C-peptide reagents ability to
detect nondenatured fractalkine expressed on transiently transfected
NIH/3T3 cells with other anti-fractalkine reagents. Each of the
anti-fractalkine reagents used: mouse anti-chemokine domain mAb clone
51636.11 (Figure 1C
, R&D Systems), goat anti-chemokine domain reagent
(Figure 1E)
, chicken anti-C-peptide reagent (Figure 1F)
, rabbit
anti-C-peptide (Figure 1H)
, and anti-N-peptide reagent1
(Figure 1I)
strongly stained the surface of 80% of the cells. In
contrast the murine IgG1 isotype control mAb
(Figure 1B)
, no primary antibody control (1D), and rabbit IgG control,
showed no specific staining of the cell surface.
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Initial analysis of human tonsil sections with both the anti-C
peptide reagents revealed a very different pattern of staining to
published patterns, which were obtained using an anti-fractalkine
N-terminal peptide reagent.12
The predominant cell type
detected with our anti-C-terminal peptide reagents was epithelial
cells. To confirm that epithelial cells express full-length
transmembrane fractalkine we used the adenocarcinoma cell line DLD-1.
We stained confluent monolayers of unstimulated DLD-1 cells for the
presence of transmembrane fractalkine by immunofluorescent double
labeling (Figure 2A)
using mouse
anti-chemokine domain (clone 51636.11; green) mAb and the rabbit
anti-C-peptide reagents (red). This revealed a subset of DLD-1 cells
strongly positive for both epitopes, whereas most of the rest of the
cells were positive for the chemokine domain of fractalkine. The
specificity of the anti-fractalkine chemokine domain mAb was verified
(Figure 2B)
by double labeling using a mouse IgG control mAb (green)
and rabbit anti-C-peptide reagents (red). Furthermore, the reactivity
of the mAb could be competed away by the addition of a 10x molar
excess of recombinant human fractalkine (Figure 2C)
.
Interestingly, when these cells were double-labeled for the
presence of cytokeratin AE1/AE3 (green) and the intracellular epitope
of fractalkine (red), the transmembrane fractalkine-positive cells
correspond to those cells with the highest levels of cytokeratin
expression (Figure 2D)
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for 24 hours. A
representative experiment is presented in Figure 2E
where DLD-1 samples
show abundant expression of fractalkine message transcripts with and
without TNF- treatment. In contrast HUVECs show a weak level of
constitutive expression that is slightly increased with TNF-
treatment.
Finally we compared the anti-N-peptide reagents reactivity to
fractalkine expressed by permeabilized DLD-1 cells with a range of
anti-fractalkine chemokine domain and anti-C-peptide reagents via FACS
(Figure 2F)
. The bold traces show cells labeled with either 1) mouse
anti-fractalkine chemokine domain (clone 51636.11) mAb, 2) goat
anti-fractalkine chemokine domain polyclonal reagent, 3) rabbit
anti-C-peptide polyclonal reagent, or 4) rabbit anti-N-peptide
polyclonal reagent. The thin traces show labeling of the cells with 1)
mouse IgG1 isotype control, 2) 10% goat serum in PBS control, 3) and
4) rabbit IgG control. Both anti-fractalkine chemokine domain reagents
(1 and 2) and the anti-C-peptide reagent (3) detect clear shifts in
fluorescence relative to their controls, indicating the detection of
fractalkine on these cells, however the anti-N-peptide reagent shows no
shift in fluorescence relative to the rabbit IgG control antibodies.
This failure of the anti-N-peptide reagent to detect fractalkine on the
DLD-1 line was also observed when staining confluent DLD-1 via
immunofluorescence (data not shown).
Polyclonal Antibodies Derived from a Fractalkine N-Terminal Peptide Cross-React with Human CD84
During a screening of human tonsil with a panel of
antibodies from the seventh Human Leukocyte Differentiation Antigens
workshop we noticed that the staining pattern of the anti-fractalkine
N-terminal peptide reagent (-N-pep) (Figure 3A)
was very similar to that for human
CD84 (admixture of -CD84 mAb from HLDA human CD84 panel) (Figure 3B)
. CD84 is a cell surface antigen of the Ig super family, with no
ascribed function.13
To test whether -N-Fkn
cross-reacted to CD84 protein we used 20 ng of recombinant human
fractalkine (Rh fractalkine) containing the N-terminus and mucin stalk
(R&D Systems) and 100 ng of cell lysate from the human CD84-transfected
cell line 300.19 CD-84.13
Duplicate Western blot membranes
were incubated with -N-pep, -CD84 (clone 2G7), goat -Fkn, and
chicken -C-pep antisera (Figure 3C)
. The -N-pep reagent clearly
detects Rh fractalkine (lane 1) at the predicted size of 90 kd and also
a degradation product (70 kd), it also strongly detects a band 70
kd in size in the lysate from the CD84-transfected cell line (asterisk,
lane 2). The -CD84 reagent does not detect Rh fractalkine (lane 3)
but clearly detects an 70-kd band (asterisk, lane 4) in
CD84-transfected cells, consistent with the reported size of human
CD84.13
The goat -Fkn reagent also detects the major
90-kd band and degradation band within the Rh fractalkine sample (lane
5) but does not detect any proteins within the CD84-transfected cell
line sample (lane 6). In contrast the -C-pep reagent does not detect
Rh fractalkine (lane 7) or any proteins within the CD84-transfected
cell line sample (lane 8).
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Langerhans cells, melanocytes, endothelial cells, and dermal
dendrocytes have been reported to express fractalkine on the basis of
staining using the -N-pep polyclonal antisera.11,14
We
were interested in comparing the reactivity of our -C-pep reagent
and goat -Fkn reagents, within similar skin samples. Serial frozen
sections taken from noninflamed human skin were stained with goat
-Fkn (Figure 4A)
or -C-pep (Figure 4B)
. In both cases staining was restricted to the basal keratinocytes
of the epidermis, with no significant staining of structures or cells
within the dermis. In contrast, the -N-pep reagent (Figure 4C)
reproduced the staining reported previously,11,14
with
clear labeling of intraepidermal Langerhans cells and melanocytes along
the basement membrane of the epidermis, along with blood vessels and
cells with a dendritic morphology, within the dermis (Figure 4C)
. Our
independently produced -N-pep antisera obtained an identical
staining pattern to Figure 4C
.
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-N-pep reagent that labels specifically
NIH/3T3-transfected cells (Figure 1I)
fails to detect transmembrane
fractalkine expressed on epithelial cells in the skin but detects other
cell types not labeled by anti-fractalkine chemokine domain-specific or
anti-intracellular epitope-specific reagents. Expression of Transmembrane Fractalkine within the Tonsil and Human Colon
Using our validated antibody reagents we critically examined the
expression of transmembrane fractalkine expression within the human
tonsil and noninvolved and chronically inflamed gut tissue. We
re-examined fractalkine expression within the human tonsil using the
mouse anti-fractalkine chemokine domain mAb (mouse -Fkn, clone
51636.11) and rabbit -C-pep reagents, looking at expression
within the outer pharyngeal epithelium (Figure 5; A, B, and C
) and the
cortex of the tonsil (Figure 5, D, E, and F)
. Fractalkine expression within the pharyngeal epithelium was
reminiscent of the pattern of fractalkine expression seen in the
epidermis, with mouse -Fkn mAb (Figure 5A
, red) strongly labeling
the epithelial cells but not underlying connective or lymphoid tissue.
The staining was not uniform, with the basal layer of the epithelium
more strongly stained than the next higher cell layers and the
strongest expression on the outer layers, no obvious DC labeling was
detected. Negative control antibodies showed no background staining or
autofluorescence (data not shown). This staining pattern was also
detected using the goat -Fkn polyclonal reagent (data not shown).
Again, similar to the epidermis, staining for the intracellular domain
of fractalkine using -C-pep reagents (Figure 5B
; red) was restricted
to the basal layer of the epithelium. Double labeling using mouse
-Fkn mAb (green) and -C-pep reagent (red) clearly showed
expression of both epitopes (orange) restricted to the basal layer of
the epithelium (Figure 5C)
.
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-Fkn mAb
(Figure 5D
, green) showed that the predominant site of fractalkine
expression was cells within the crypt epithelia. This pattern was also
detected using the goat -Fkn polyclonal reagent (data not shown).
There was no staining of blood vessels, or cells within T or B cell
areas, relative to control antibodies. The -C-pep reagent also
showed a staining pattern restricted to the crypt epithelia (Figure 5E
,
red). Double labeling (Figure 5F)
using the mouse -Fkn mAb (green)
and -C-pep reagent (red) clearly showed double labeling of a subset
of cells within the crypt epithelia (orange). Strong green labeling of
the external surface of the crypts is a staining artifact, also
restricted to this surface in the negative controls (data not shown).
There is however also a minor population of chemokine-domain single
positive cells, located closer to the lymphoid edge of the crypts.
The pharyngeal epithelium of the tonsil contains a resident population
of immature CD1a-positive DCs. Double-immunofluorescent labeling
(Figure 5G)
using -CD1a mAb detected positive immature DCs (green)
and -C-pep reagent (red) showed that although CD1a-positive DCs are
intimately associated with the transmembrane-expressed fractalkine they
do not express it. Similarly, immature DCs within the crypts were also
shown to be negative for transmembrane fractalkine by
double-immunofluorescent labeling (Figure 5H)
.
Recently Muehlhoefer and colleagues12 reported fractalkine protein expression on the epithelium of the lamina propria of the human gut and on the lamina propria and endothelium of chronically inflamed gut. This study was performed using a mixture of an anti-N-peptide andanti-C-peptide polyclonal reagents (Santa Cruz Biotechnology, Inc.) and thus was not able to demonstrateconclusively that the fractalkine detected was the transmembrane form on these cells. We were not able to duplicate this staining using these reagents.
We therefore stained frozen sections of human colon from Crohn’s and
ulcerative colitis patients with the mouse -Fkn mAb (Figure 5I
,
green) and -C-pep reagent (Figure 5J
, red), confirming the
positivity of the lamina propria for extracellular and intracellular
epitopes of fractalkine. Double labeling showed that the epithelial
cells of the lamina propria express transmembrane fractalkine because
they are clearly positive for both epitopes (Figure 5K
, orange). A
similar expression pattern was detected within adjacent noninvolved
colon tissue (Figure 5L)
. In contrast to results obtained with the
-N-pep reagent (data not shown) no obvious positive endothelial
staining by either the anti-chemokine domain or intracellular
epitope-reactive reagents was detected. The staining pattern revealed
by the -C-peptide reagent was consistent on 16 ulcerative colitis
and 13 Crohn’s disease patient samples (for age and sex breakdown
refer to Table 2
).
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To confirm that fractalkine-positive cells within the epithelial
crypts of the tonsil were epithelial cells, we stained adjacent serial
tonsil frozen sections with -C-pep reagent (Figure 6A
, brown) and -cytokeratin (Figure 6B
, blue). Staining with either antibody was restricted to within the
epithelial crypts, within the sections examined, with a characteristic
labeling of small cuboid cells bordering the lymphoid tissue of the
tonsil and larger cells with dendritic morphology within the epithelial
crypts. Double labeling a section 30 µmol/L further into the block
clearly shows these cells are double-positive (Figure 6C
, purple)
although the single-positive cytokeratin staining is more widespread
(blue). Similar results were obtained by double labeling samples of
human colon (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation.
Anti-fractalkine N-terminal peptide reagent1
fails to
detect fractalkine expression on this cell line. This reagent also
fails to stain epithelial cells within fixed human tissues, showing a
strikingly different staining pattern to the other anti-fractalkine
reagents used in this study. In contrast to other studies, we have been
unable to detect fractalkine expression on endothelium or immature DCs
in either normal or inflamed human tissues. Fractalkine mRNA distribution in man seems to be complex, with many large organs of diverse function, in particular the brain and the heart, showing high levels of expression.1 Cleaved and transmembrane forms of fractalkine may potentially have quite different biological roles. In vitro descriptions of the interaction of fractalkine with its only described receptor CX3CR1 have suggested a role in arrest and extravasation of receptor-positive cells from the bloodstream.4,7 Although the expression of fractalkine mRNA in unactivated HUVECs is low, this is increased significantly when they are stimulated with inflammatory cytokines.1 In a similar manner, cardiac myocyte expression of fractalkine mRNA increases after treatment of rats with either lipopolysaccharide or inflammatory cytokines.13 The reduction in inflammatory influx observed in a model of rat glomerulonephritis when CX3CR1-blocking antibodies were administered has suggested a causal connection between increased fractalkine expression, linked to local inflammation, and the enhanced trafficking of inflammatory cells.16
In man, expression of fractalkine protein has been reported on neurons within the brain,17 and this has been confirmed by in situ hybridization and immunohistochemistry in rodents.5,10 Fractalkine protein expression has also been reported on DCs and endothelium within the human skin and tonsil,11 and recently on the epithelium of the lamina propria in the small intestine.12 Importantly, the same reagent has been used in all these human studies of brain, skin, and tonsil, namely an anti-fractalkine N-terminal peptide reagent,1 whereas a mixture of anti-fractalkine N-terminal and anti-C-terminal antibodies was used to stain the small intestine. Using antibodies raised against the chemokine domain of fractalkine and against the intracellular tail of the molecule, we have confirmed by double labeling that the predominant site of fractalkine expression in the human colon is the lamina propria. In contrast to the previous study12 conducted on samples of human small intestine, we were unable to detect any endothelial staining. This was also true within resection material taken from a large number of Crohn’s and ulcerative colitis resection patients (n = 13 and n = 16, respectively), in which there was a considerable inflammatory infiltrate. This observation is not consistent with a simple model of fractalkine up-regulation on endothelium in response to inflammation. The targeted replacement of CX3CR1 in mice by a GFP reporter construct fails to block monocyte extravasation from blood or migration of DCs from the skin after microbial or contact sensitizer stimulation18 again suggesting that fractalkine’s primary role in vivo is not that of an endothelial adhesion molecule.
We could not detect either chemokine domain-reactive or transmembrane
fractalkine-positive DCs within the epithelium of the tonsil and skin.
Instead there was diffuse staining of fractalkine chemokine-domain
single-positive cells throughout the epithelium, with a restricted
expression of the transmembrane double-positive form on the basal
epithelial cells. This is again in contrast to staining reported using
the anti-N-terminal peptide reagent (-N-pep) and repeated in our
hands (Figure 4)
. In this article we demonstrate that that -N-pep
antisera cross-react with human CD84 in CD84-transfected cells (Figure 3C)
and seem to detect CD84-positive cells within the tonsil (Figure 3, A and B)
. Previous reports of strong protein expression on human
epidermal Langerhans cells were surprising given negligible levels of
fractalkine mRNA are detected by reverse transcriptase-PCR from freshly
extracted epidermal human and murine DCs,11,19
rather
message levels increase significantly as they are allowed to mature
in vitro, a situation considered to be analogous to
Langerhans cells that have completed their migratory journey to
secondary lymphoid tissue.
Although our study does not address the role of fractalkine expression within the brain, we have shown an interesting and apparently widespread, constitutive expression of transmembrane fractalkine by epithelial cells in the periphery. If there is active cleavage of fractalkine from these cells, which is suggested by the more widespread detection of cells single labeled for the chemokine domain of fractalkine in the skin and tonsil, a constitutive fractalkine gradient might be produced. Such a constitutive fractalkine gradient might serve to attract CX3CR1-positive cells to peripheral tissues. Indeed this is the model that has been suggested for T cell trafficking into the lamina propria of the human gut.12
A recent report of the transient expression of CCR6 by monocyte-derived DCs produced in the presence of TGF-ß,20 suggested that cells gain or lose responsiveness to chemokines through the regulation of chemokine receptor expression. Chemokines are known to mediate constitutive and inducible leukocyte recruitment and our analysis of fractalkine expression in noninflamed tissues is consistent with fractalkine providing one of these constitutive signals. Cells potentially recruited by fractalkine include monocytes, which respond chemotactically to fractalkine.1,15 The demonstration that monocytes have the capacity to rapidly differentiate into DCs after a series of transmigrations through an endothelial monolayer,21 is consistent with the possibility that constitutive fractalkine expression, within the periphery, may be important in the attraction and differentiation of monocytes into dermal and/or epidermal DCs. Although the targeted replacement of CX3CR1 has not shown obvious defects of DC localization18 the demonstration that cutaneous DCs were strongly positive for the reporter gene, is consistent with the potential importance of precursor/Langerhans cell interactions with epithelial fractalkine. Furthermore, there remains the possibility that fractalkine might act as a ligand for other as yet unidentified chemokine receptors, expressed by responding cells. Further analysis of CX3CR1 regulation on cells of the hemopoietic lineage, which presently is restricted because of a paucity of good reagents, should aid in the clarifying of which cell types are interacting with fractalkine in these distinct anatomical compartments.
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Acknowledgements |
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-CD84 antibodies; and Dr. T. Schall for
providing anti-fractalkine antisera. |
Footnotes |
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Supported by the Arthritis Research Campaign (grant G0553), the National Association of Colitis and Crohn’s, and the Wellcome Trust.
A. D. L. and N. C. contributed equally to this study.
Accepted for publication November 2, 2000.
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