Fractalkine Is an Epithelial and Endothelial Cell-Derived Chemoattractant for Intraepithelial Lymphocytes in the Small Intestinal Mucosa

Abs and cytokines

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, anti-mouse/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-1β, and IL-2 were obtained from R&D Systems (Minneapolis, MN).

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 [α-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 μg 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 HRP-labeled 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 μg) 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 μg/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-μm slices and put on 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 μg/ml), anti-cadherin, or anti-caveolin-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 streptavidin-FITC (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 anti-CX₃CR1 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.

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, anti-fractalkine Ab at concentrations indicated. A 5-μm polyvinylpyrrolidone-free polycarbonate membrane separated the lower from the upper well, which was filled with 50 μl 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.

IL-1β up-regulates basal expression of fractalkine mRNA in T-84 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-κB-inducing cytokine IL-1β was able to induce a transient 4-fold increase in basal steady state expression of fractalkine transcripts within 2 h (Fig. 1⇓).

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FIGURE 1.

Basal fractalkine mRNA expression in T-84 cells is up-regulated by IL-1β. Northern blot analysis of fractalkine mRNA expression in RNA from T-84 cells (20 μg of total RNA/lane). Cells were incubated for the time indicated with 10 ng/ml IL-1β. 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.

IL-1β-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 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-1β (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-1β. 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-1β (Fig. 2⇓, C and D).

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FIGURE 2.

Induced expression of membrane-bound fractalkine by IL-1β 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-1β. A total of 20 μg 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.

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-1β, 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 anti-fractalkine 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 recombinant fractalkine (Fig. 2⇑E, 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-1β 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 detergent-insoluble 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. 3⇓A, 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. 3⇓A, lane 5). These low density membrane fractions have been associated with detergent-insoluble glycolipid-enriched 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. 3⇓B, 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. 3⇓B), 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).

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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).

Fractalkine expression is regulated during polarization of the intestinal epithelial cells

The human intestinal epithelial cell line T-84 provides a well-established 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. 4⇓A). 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 E-cadherin on the surface of unpolarized as well as at the basolateral surface of polarized T-84 cells (Fig. 4⇓, E and F).

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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).

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.

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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.

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 express CX₃CR1 protein on the cell surface using flow cytometry. As demonstrated in Fig. 6⇓A, the freshly isolated human iIEL consisted of 78% CD8-positive (Fig. 6⇓A) and 7.6% CD4-positive lymphocytes (Fig. 6⇓B). A total of 48.5% of the fresh isolated iIEL expressed the fractalkine receptor CX₃CR1 (Fig. 6⇓C).

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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 anti-CD4 FITC-coupled Abs. Representative experiment from three independently conducted is shown.

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 system, as described (33), and expressed in fold increase of the basal cell migration (Migration Index) (Fig. 7⇓A). 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. 7⇓A, 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. 7⇓B, p < 0.001). Flow cytometry confirmed the presence of CX₃CR1 on iIEL (Fig. 7⇓C) after incubation with IL-2. Furthermore, iIEL retained their CD8-expressing phenotype during IL-2 activation (Fig. 7⇓D). 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 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-μm 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). Ab-blocking 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 (∗, 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).

Fractalkine is expressed by intestinal epithelial cells and endothelial cells within the small intestinal mucosa and up-regulated 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. 8⇓A, 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. 8⇓A, 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 of inflammatory cells (Fig. 8⇓C). 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.

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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 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).

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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 (∗, p < 0.01).