| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
,,,
* Department of Clinical and Experimental Medicine, Section of Rheumatology;
Department of Pathology;
Department of Clinical and Experimental Medicine, Clinical Immunology Branch;
Venetian Institute for Molecular Medicine, Centro di Eccellenza per la Ricerca Biomedica; and
¶
Department of Medical and Surgical Sciences, Section of Otorhinolaryngology, University of Padova, Padova, Italy;
||
Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy; and
#
Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
and produced during Th1-type immune responses that exert their action via an interaction with the CXCR3 (1, 2, 3). Although these three ligands are all induced by IFN-, they appear to mediate distinct in vivo biological phenomena, probably via a differential activation of CXCR3 (4, 5, 6, 7). The expression of CXCR3-targeting chemokines has been demonstrated in several diseases or pathological states, including sarcoidosis, ulcerative colitis, multiple sclerosis, viral infections, allograft rejection, atherosclerosis, delayed-type hypersensitivity skin reactions, chronic skin inflammation, rheumatoid arthritis, diabetes, and glomerulonephritis (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Collectively, these studies suggest a critical role for CXCR3 in the recruitment of activated T cells to sites of immune-mediated inflammation. Although chemokines were first identified as chemoattractants, their functions extend well beyond their role in leukocyte migration. For instance, IFN-inducible CXC chemokines have been shown to be able to inhibit angiogenesis and endothelial cell chemotaxis; however, they induce migration and proliferation of vascular pericytes. Recent evidence indicates that an alternative splicing variant of the CXCR3 gene, i.e., CXCR3-B, mediates the angiostatic activity of CXCR3-binding chemokines, providing a convincing explanation for the proliferation induced by CXCR3 agonists on endothelial cells that express CXCR3-A and a parallel inhibitory activity on endothelial cells expressing CXCR3-B (18).
Sjögren’s syndrome (SS)3 is an autoimmune disease characterized by mononuclear T cell-rich infiltrate around the ducts in the salivary gland. T cell infiltrates lead to salivary gland destruction and, ultimately, salivary hypofunction. Our preliminary data have shown that CXCR3-positive lymphocytes infiltrate salivary glands in SS, and that, most notably, CXCR3 is also expressed in both SS and normal ductal epithelial cells (19). In this study, we evaluated whether CXCR3 expression by epithelial cells has a role in a physiologic setting and in the pathogenesis of SS. We provide definitive evidence that CXCR3, in particular the B form, is constitutively expressed by salivary gland epithelial cells, where it regulates the bioavailability of chemokines, a function that was found to be impaired in SS.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
We studied minor salivary gland (MSG) biopsy specimens from 20 healthy subjects (all female; mean ± SD age, 51.0 ± 20.5 years; range, 20–75) and 18 patients (all female; mean ± SD age, 49.0 ± 15.5 years; range, 29–76) with primary SS fulfilling the new American-European Consensus Group diagnostic criteria for SS (20). MSG biopsy was performed as a routine part of the diagnostic evaluation of SS, and informed consent was obtained from the patients and controls. Control subjects reported sicca symptoms, but were completely negative for all immunologic abnormalities, such as autoantibodies, including anti-SSA, anti-SSB, antinuclear Abs, and rheumatoid factor, and hypergammaglobulinemia. To rule out the possibility of an early, preclinical SS or subclinical infection, we performed oral, ocular, and serologic diagnostic examinations. Test results were normal in all control subjects. With regard to the possibility of a subclinical bacterial or viral infection, periodontitis was excluded in all subjects by a comprehensive oral evaluation. We excluded from the study four subjects in whom CMV genome and mRNA transcript (early and late genes) were detected.
Immunohistochemical analysis
Tissue samples from the labial salivary glands of SS patients were fixed in buffered-formalin, embedded in paraffin, and stained with H&E. For immunostaining, sections were stained using the standard avidin-biotin complex method (Vectastain ABC Kit; Vector Laboratories) as we previously described (12). Briefly, after microwave Ag retrieval procedure, slides were placed in a 2-l glass beaker containing 0.01 M citrate buffer (pH 5.9) and microwaved at full power (800 W for 5 min, three times) before cooling and equilibration in PBS. Anti-CXCR3 mAbs (clone 49801.111 (R&D Systems) or clone 1C6 (BD Biosciences) both used at 1/100) were incubated for 1 h in a humified chamber at 37°C and detected using biotinylated secondary Ab, followed by 30-min incubation with avidin-peroxidase (1/200) and 3,3-diaminobenzidene tetrahydrochloride as chromogen. The intensity of Ab staining was classified in four groups: strong, weak, moderate, and negative. Parallel control slides were prepared either lacking primary Ab or lacking primary and secondary Abs or were stained with normal sera to control for background reactivity. Furthermore, to rule out nonspecific staining, a matching immunohistological assay was performed in which an isotype-negative control was used in place of the antigenically specific primary Ab.
Indirect immunofluorescence analysis
Epithelial cells grown in chamber slides (Nalge; Nunc) were fixed in 2% paraformaldehyde for 10 min and permeabilized in 0.1% Triton X-100 for 4 min. After soaking in PBS containing 1% BSA for 30 min, slides were incubated with the primary Ab at room temperature for 45 min in PBS containing 0.5% BSA. After washing with PBS, slides were stained with FITC-conjugated goat anti-mouse IgG and incubated for 3 min in PBS containing 0.5 µg/ml propidium iodide for nuclear staining before fluorescence microscopy.
Primary cultures of epithelial cells from salivary gland tissues
Primary ductal epithelial cells cultures were established from minor salivary gland biopsies by the explant outgrowth technique (21). Briefly, tissues were minced into fragments of 1 mm3 and cultured in a mixture (1/3) of DMEM (Invitrogen Life Technologies) and Ham’s F-12 (Invitrogen Life Technologies) containing 2.5% FCS, 2 mM L-glutamine (Invitrogen Life Technologies), 0.5 mg/ml insulin (Wako Biochemicals), 0.4 mg/ml hydrocortisone (Sigma-Aldrich), and 10 ng/ml epidermal growth factor (Sigma-Aldrich). When cultures were 
70% confluent, cells were detached using trypsin/EDTA and subcultured in 75-cm2 tissue culture flask (Nunc). The epithelial cell origin of the primary cultures was confirmed by staining with a cytokeratin-specific Ab (ICN Pharmaceuticals). Experiments were performed after subculture when cells were 80% confluent. Cells were used from passages 4–6.
Flow cytometric analysis of CXCR3
The expression of CXCR3 on epithelial cells was assessed by flow cytometric analysis using direct immunofluorescence assay. After incubation at 37°C with FITC-conjugated anti-CXCR3 mAbs (R&D Systems) or the matched control mAb, cells were washed and analyzed. For FACS analysis, 3 x 104 cells were scored using a FACSCalibur analyzer (BD Biosciences), and data were processed using CellQuest (BD Biosciences). The threshold of positivity was set at the nonspecific binding observed in the presence of irrelevant control Ab. Mean log fluorescence intensity (MFI) values were obtained by subtracting the MFI of the isotype control sample from the MFI of the positively stained sample. To evaluate whether the differences between the peaks of cells were statistically significant with respect to control, the Kolmogorov-Smirnov test for analysis of histograms was used according to the CellQuest software guide (BD Biosciences), with D/s values >10 considered significant.
Confocal microscopy
Epithelial cells, grown in chamber slides, were incubated with CXCL10 (200 ng/ml) for 0–30 min. Cells were washed three times in HBSS (37°C), fixed with 0.25% glutaraldehyde in PBS for 10 min, blocked with 0.05 M Tris-HCl (pH 7.5) for 20 min, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were then stained with anti-CXCL10 Abs (clone 49801.111; R&D Systems; 1/150) and Alexa 488 (Sigma-Aldrich)-conjugated goat anti-mouse Ig (1/200) secondary Abs. Samples were analyzed by confocal microscope (2100 Multiphoton; Bio-Rad) with a x60 objective lens (Nikon), using laser excitation at 488 nm. Images were analyzed using the Adobe Photoshop 7.0 program.
Western blot
Epithelial cells (4 x 105 for each assay) were resuspended and lysed in 62 mM Tris-HCl buffer (pH 6.8) containing 5% glycerol, 0.5% SDS, and 0.5% 2-ME. Samples were then subjected to SDS-PAGE (10% gels), transferred to nitrocellulose membranes, and immunostained with anti-phosphotyrosine (PY-20) mAb (ICN Biotechnology) or anti-human CXCR3 mAb (BD Pharmingen) using an ECL detection system (Amersham Biosciences). The blots were then stripped and reprobed with anti-
-actin mAb (Sigma-Aldrich). Cell extracts from chronic lymphocytic leukemia cells were used as a positive control. Before using these cells in any experiment we evaluated CXCR3 expression by means of flow cytometry, confirming that >95% of leukemic cells were positively stained.
RT-PCR analysis
Total cellular RNA was extracted, using the RNA Mini Kit isolation and purification system (Qiagen), from 3 to 4 x 106 cells. On-column DNase digestion was performed according to the manufacturer’s instructions, and RNA was quantified by measuring absorbance at 260 nm. cDNA was synthesized from 1 µg of total RNA at 42°C for 15 min in the presence of AMV reverse transcriptase (2.5 U) using 2.5 mM oligo(dT) primer and reaction conditions described by the manufacturer (Promega).
Real-time quantitative PCR amplification reactions were conducted in an ABI PRISM 7000 sequence detection system (Applied Biosystems) in a 15-µl volume. SYBR Green PCR Master Mix was purchased from Applied Biosystems (P/N 4309155), containing AmpliTaq Gold DNA polymerase and optimized buffer components. A fraction of 5 µM primers and 1.5 µl of cDNA was added to the SYBR Green Master Mix to make a final 15-µl reaction volume. The primers used for CXCR3 and GAPDH amplifications were: CXCR3-A: forward, ACC CAg CAg CCA gAg CAC C-3'; reverse, 5'-TCA TAg gAA gAg CTg AAg TTC TCC A-3'; CXCR3-B: forward, 5'-TgC CAg gCC TTT ACA CAg C-3'; reverse, 5'-TCg gCg TCA TTT AgC ACT Tg-3' and 5'-ATg CCA TgA CCA gCT TTC ACT-3'; reverse, 5'-TTA Agg CAg gCC CTC Agg TA-3'; and GAPDH: forward, 5'-AAT ggA AAT CCC ATC ACC ATC T-3'; reverse, 5'-CgC CCC ACT TgA TTT Tgg-3'. PCRs were performed under the following conditions: 10-min denaturation at 95°C, followed by 95°C for 15 s and 60°C for 1 s, cycled 50 times. Each quantitation target was amplified in triplicate samples. A no-template control for each Master Mix and three standard curves were generated for GAPDH, CXCR3A, and CXCR3B using cDNA in serial dilutions of 1/1, 1/10, 1/100, and 1/1000. The relative amounts of mRNA were determined by comparison with standard curves. The results of each sample were normalized for GAPDH expression. To distinguish specific amplicons from nonspecific amplifications, a dissociation curve was generated.
Cell cycle analysis
For cell cycle analysis, 1 x 106 epithelial cells were washed twice with PBS, fixed with 70% cold ethanol for 30 min at 4°C, and incubated with a solution containing 50 mg/ml propidium iodide and 1 mg/ml RNase (Sigma-Aldrich). The tubes were placed at 4°C in the dark for 30 min before analysis using a FACSCalibur analyzer (BD Biosciences). For each sample, 10,000 cells were analyzed using CellQuest software (BD Immunocytometry Systems).
Cytosolic calcium measurement
Changes in the intracellular calcium concentration were measured in epithelial cells using the fluorescent indicator fura-2-AM. Briefly, 20 x 106 cells were incubated with a solution containing 2.5 µM fura-2-AM, 20% pluronic acid, and 0.25 mM sulfinpyrazone (to prevent fura-2 release into the medium) at 37°C for 30 min. After the loading procedure, aliquots of the cells (2 x 106) were rapidly washed and resuspended in a magnetically stirred thermostatted cuvette. The incubation medium contained 1 mM CaCl2. Excitation and emission wavelengths were 340 and 500 nm, respectively, the excitation slit width was 5 nm, and the emission slit was 10 nm. Control experiments without sulfinpyrazone gave essentially the same results, except for a slowly increasing baseline due to fura-2 leakage. The chemokines were used at 1 µg/ml; anti-Ig was used at 500 ng/ml. Finally, 1 µM ionomycin was added to obtain the maximal effect.
Chemotaxis of ductal salivary gland epithelial cells
The effects of the CXCR3 ligands, CXCL9–11 and CXCL4, on chemotaxis of epithelial cells were assessed using a 48-well modified Boyden chamber (AC48; NeuroProbe). Different chemotactic stimuli were loaded in the bottom chamber, and cells were added to the top chamber. A polyvinylpyrrolidone-free polycarbonate 8-mm membrane with 3- to 12-µm pores was placed between the chambers. The bottom face of the filter was pretreated with 10 µg/ml fibronectin to maximize the attachment of the transmigrated cells to filters. Fibronectin-treated filters did not induce spontaneous chemotaxis in the absence of chemokines. Briefly, 28-µl aliquots of serum-free HEPES-buffered RPMI 1640 supplemented with 0.1% BSA, with or without ligand (CXCL9–11 and CXCL4; 100, 200, and 500 ng/ml) were dispensed into the bottom wells of the chamber. Fifty-microliter aliquots of epithelial cells (2 x 106 cells/ml) resuspended in HEPES-buffered RPMI 1640 were added to the top wells. Chambers were incubated at 37°C with 5% CO2 for 2 h. The membrane was then removed, washed with PBS on the upper side, fixed, and stained with DiffQuik (Baxter). Cells were counted microscopically at x800 magnification in four fields per membrane. All assays were performed in triplicate.
Chemokine assays
Chemokine concentrations in cell-free supernatants harvested from salivary epithelial cells were measured by specific ELISA for CXCL9 (DUOSET DY 392), CXCL10 (DUOSET DY266), and CXCL11 (Quantikine DCX110) purchased from R&D Systems. Detection limits were 60 pg/ml for CXCL9, 30 pg/ml for CXCL10, and 13.9 pg/ml for CXCL11.
Migration of 300-19/hCXCR3 cells toward the supernatants of CXCL10-exposed epithelial cells
The migration of human CXCR3-transfected murine 300-19 B cells (300-19/hCXCR3) was measured in a 48-well modified Boyden chamber (AC48 NeuroProbe). The CXCR+ and CXCR3– cell lines (300-19 B cells; provided by Dr. B. Moser, Theodor Kocher Institute, University of Bern, Bern, Switzerland) were used as positive and negative controls. Directional migration of 300-19/hCXCR3 was quantified toward 200 ng/ml CXCL10 that had been preincubated with salivary duct epithelial cells. Culture medium and nontreated CXCL10 were used as additional negative and positive controls, respectively. Twenty-eight microliters of supernatant, chemokines, or control medium were added to the bottom chambers, and 50 µl of 5 x 106 cells/ml 300-19/hCXCR3 cells resuspended in RPMI 1640 were added to the top chambers. The chamber was incubated at 37°C in 5% CO2 for 2 h. The membranes were removed, washed with PBS on the upper side, fixed, and stained with DiffQuik (Dade). Cells were counted in three fields per membrane at x800 magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 37°C with anti-human CXCR3 mAb at a concentration of 20 µg/ml.
Statistical analysis
Data are expressed as the mean ± SEM, and comparisons between values were made using the ANOVA test. A value of p <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
To determine whether CXCR3 is expressed in vivo by normal salivary gland epithelial cells, we analyzed MSG biopsy specimens from 20 healthy subjects. The expression of CXCR3 was determined by immunohistochemistry of gland tissues. Histologic examination revealed a moderate expression of CXCR3 on ductal epithelial cells and sometimes on acinar epithelial cells in all subjects (Fig. 1, A and C). Histological sections from SS patients revealed a similar staining pattern (Fig. 1, B and D). Additional analyses of MHC class II molecule expression showed that CXCR3 expression did not correlate with the activation state of the epithelial cells. To confirm our findings, we used cultured epithelial cells derived from MSG. Immunocytochemistry clearly demonstrated CXCR3 expression on cultured MSG epithelial cells (Fig. 1, E and F). To exclude the possibility that detachment of cells induces receptor expression, we also cultured several MSG explants on slide chambers. Epifluorescence microscopy on adherent cells revealed a similar staining pattern (Fig. 1G). To exclude the possibility that the addition of FCS to culture medium induces CXCR3 expression, we repeated the analyses before and after 24-h serum starvation. Serum deprivation did not alter CXCR3 expression (data not shown). We then analyzed CXCR3 expression by means of flow cytometry and found that all cells were dimly positive (Fig. 2A). To confirm the epithelial origin of cultured cells, an experiment comprised of double staining for CXCR3 and cytokeratins was performed. To confirm our findings, we assessed cultured MSG cells for CXCR3 expression at the mRNA and protein levels using RT-PCR and Western blotting, respectively. Both CXCR3 protein (Fig. 2B) and mRNA (Fig. 2C) were clearly visible in all cultures. Notably, CXCR3-B, the alternatively spliced variant of CXCR3, was the predominant type of CXCR3 present in the primary cultures of MSG epithelial cells.
|
|
Human microvascular endothelial cells selectively express CXCR3-B, although this expression is limited to the S/G2-M phase of their cell cycle (22). To test whether CXCR3 on MSG epithelial cells is also associated with certain cell cycle phases, we compared the number of CXCR3-positive cells both from cultures that were far from confluent with a high proliferation rate and from confluent, growth-arrested cultures. However, the proportions of CXCR3-expressing cells were found to be similar, making it unlikely that this chemokine receptor is strictly associated with a certain cell cycle phase. Consistent with these findings, double staining with FITC-conjugated anti-CXCR3 mAbs and propidium iodide analyzed by flow cytometry demonstrated that the presence of CXCR3 was independent of the DNA content.
CXCR3 agonists do not mediate changes in cytosolic Ca2+
Activation of chemokine receptors often leads to elevation of cytosolic Ca2+; for example, in T cells CXCR3 agonists lead to Ca2+ mobilization (23). To determine whether epithelial cells express functional CXCR3, we first investigated the ability of CXCL4, -9, -10, and -11 to induce changes in intracellular Ca2+. CXCR3-positive epithelial cells did not respond with calcium flux to any of these four agonists, whereas addition of CXCL12, used as a positive control, resulted in an increase in intracellular calcium (Fig. 3, A–D). This is consistent with previous observations that CXCL9, -10, and -11 do not elicit Ca2+ mobilization in HMEC-1 CXCR3-B transfectants (18).
|
In T cells, CXCR3 binding activates several protein kinases. The internalization of many GPCRs, including at least two chemokine receptors, CXCR4 and CCR5, is mediated by the phosphorylation of C-terminal residues by the G protein-related kinases (24). We therefore examined the effects of CXCR3 agonists, CXCL10, CXCL9, and CXCL4, on tyrosine phosphorylation in CXCR3-positive epithelial cells. The addition of 200–500 ng/ml CXCL9, CXCL10, or CXCL4 increased intracellular tyrosine phosphorylation, with a peak response at 30 min (Fig. 3, E and C). Although suggesting that CXCR3 receptors on MSG epithelial cells are able to signal, measurement of the increased tyrosine phosphorylation does not indicate what the functional outcome may be in epithelial cell.
Reduced surface expression of CXCR3 upon ligand binding
Upon ligand binding, in most GPCRs the association of phosphorylated receptors with the cytoplasmic adaptor, -arrestin, ultimately leads to receptor internalization (25). Therefore, we addressed the possibility that CXCL9, -10, and -11 induce CXCR3 internalization in epithelial cells. Results showed that during a 5- to 60-min period of incubation with the agonists, cell surface expression of CXCR3 decreased by 80% (Fig. 3F). This indicates that on epithelial cells CXCR3 is connected to an intracellular machinery mediating internalization in a receptor-dependent manner.
CXCR3 agonists do not induce chemotaxis of epithelial cells
To investigate an eventual functional role for the epithelial CXCR3, chemotactic responses were tested. CXCL9–11 and CXCL4 did not induce CXCR3-positive epithelial cell migration (Fig. 3G). Taken together, these data show that CXCR3 on epithelial cells is capable of recognizing ligands, but is functionally incapable of activating the two known signaling pathways of chemokine receptors, calcium mobilization and chemotaxis, although they undergo internalization. It was reasoned that CXCR3 on epithelial cells may buffer agonists, behaving as a scavenger receptor.
Scavenging of CXCL10 by salivary gland ductal epithelial cells
We therefore directly assessed the ability of epithelial cells to bind and sequester relevant agonists. The amount of CXCL9, -10, and -11 produced by epithelial cells was first measured. Quiescent cells showed low constitutive levels of CXCL11 (16 pg/ml), whereas CXCL9 and CXCL10 could not be detected in the culture supernatant. Next, exogenous CXCL10 (200 ng/ml) was added to cell cultures, and its disappearance was monitored by ELISA and confocal microscopy. In preliminary experiments supernatants were harvested at different time points (from 0 to 60 min). Because of its very rapid disappearance, it was not possible to obtain accurate clearance curves for CXCL10. However, we observed decreased CXCL10 concentrations to 5% in 5 min (Fig. 4A). This high fraction of scavenging (95%) was substantially maintained after 2 h. The anti-CXCR3 mAb 49801.111 was used to assess the contribution of receptor-mediated internalization compared with other scavenging mechanisms. The addition of the blocking Ab inhibited chemokine uptake by 20%. These observations are consistent with the hypothesis that epithelial cells may scavenge CXCR3 agonists and that this function is in part mediated by CXCR3-B. To visualize the uptake of chemokine by MSG epithelial cells, we used immunofluorescence labeling with anti-chemokine Abs. Confocal microscopy demonstrated that most cells internalized the chemokine after 5 min (Fig. 4, B and C).
|
Microparticles released from the surface of various cell types contain cell surface protein and cytoplasmic components of the original cell (26). It has been demonstrated that at least the chemokine receptor CCR5 can be released through microparticles (27). To address the possibility that CXCL10 scavenging depends on CXCR3-containing microparticles, we assessed by Western blot analysis the MSG epithelial cell supernatant after centrifugation for 10 min at 500 x g. There was no CXCR3 in the epithelial cell supernatant. This refutes the possibility that CXCL10 scavenge is due to CXCR3-containing microparticles.
Salivary gland ductal epithelial cells inhibit 300-19/hCXCR3 cell migration
In an effort to confirm our results and assess the biological relevance of these findings, we used a chemotaxis assay to quantify directional migration of 300-19 cells transfected with human CXCR3 (300-19/hCXCR3) toward CXCL10. Epithelial cells strikingly down-regulated 300-19/hCXCR3 cell migration. This inhibitory effect was reversed by the addition of anti-CXCR3 mAb 49801.111 (Fig. 4F).
SS salivary gland ductal epithelial cells display defective scavenging of CXCL10
Using cultured MSG epithelial cells from SS patients, we investigated whether these cells have the capability of healthy control cells to scavenge CXCL10. In the supernatants from unstimulated SS epithelial cells, low levels of CXCL11 comparable to those observed in controls were detected, whereas CXCR3 protein expression was slightly reduced. In contrast the fraction of scavenging over a period of 2 h was reduced and ranged from 21 to 70% (Fig. 4A). Immunofluorescence confocal microscopy confirmed this observation, showing reduced CXCL10 internalization 5 min after loading cultured cells with the chemokine (Fig. 4, D and E). Moreover, as expected on the basis of these results, chemotaxis assay showed that 300-19/hCXCR3 cells migrate vigorously toward the supernatants of CXCL10 even in the presence of SS epithelial cells (Fig. 4F). This indicates that SS ductal epithelial cells have lost their ability to scavenge CXCR3 agonists.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
A number of conditions have been described in which cells expressing chemokine receptor fail to respond to appropriate agonists. There are three chemokine-binding molecules, with homology to chemokine receptors, but defective in signaling function, i.e., D6, DARC, and CCX-CKR, which are classified as silent chemokine receptors (30, 31, 32). Despite exhibiting high affinity interactions with chemokines, these receptors are not coupled to the major signaling pathways that are activated by other chemokine receptors upon ligand stimulation and thus do not mediate cell migration. Recently, Fra et al. (33) demonstrated that D6 binds and scavenges most inflammatory chemokine agonists at CCR1 through CXCR5. These data together with the restricted expression of D6 on endothelial cells lining afferent lymphatics suggest that D6 acts as a gatekeeper to prevent excessive transfer of inflammatory chemokine to lymph nodes. Similarly, DARC and CCX-CKR may act as a chemokine buffering and scavenging system, which can internalize their ligands without inducing any detectable signal and target them for degradation (34).
The majority of these decoy receptors are expressed by endothelial cells, where they act as chemokine sink or transporter, thus contributing to maintain the homeostatic levels of chemokines in the surrounding microenvironment. Our data suggest that CXCR3-B expression by epithelium is also involved in the maintenance of tissue homeostasis. Epithelial tissues are continuously exposed to a wide variety of pathogens. The mammalian immune system has developed intricate mechanisms to provide defense at epithelial surfaces and to control potentially harmful responses to normal symbiotic flora and low virulence pathogens. Epithelial cells are important players in the homeostatic trafficking of lymphocyte subsets through the secretion of tissue-specific chemokines (35). It has been demonstrated that chemokine proteins can be directed to intracellular storage depots. Secretion of these stores enables rapid initiation of chemokine-driven responses without the delay required to initiate transcription (34). Just the opposite, leukocyte recruitment should be tightly regulated by the absorption of chemokines to avoid excessive inflammatory cell infiltration with subsequent damage of the adjacent nondistressed cells.
As the ductal epithelial cells simultaneously express a chemokine receptor and its ligand, we hypothesized that ligand binding may activate an autocrine or juxtacellular loop. Our data demonstrated that CXCR3 on epithelial cells does not mediate cell migration or calcium flux, although it participates in tyrosine phosphorylation. The kinetics of protein tyrosine phosphorylation appear to be slow compared with receptor internalization and CXCL10 scavenging. However, it is known that chemokine receptor internalization and receptor-mediated signaling share only partly common signal transduction steps. In addition, the fate of the receptor after ligand stimulation may affect the length, strength, and type of intracellular signals generated (36). Therefore, the increased intracellular tyrosine phosphorylation, although suggesting that CXCR3 on MSG epithelial cells is able to signal, is not necessarily linked to chemokine trapping and receptor internalization. In this view, because CXCL10, like many others chemokines, binds to cell surface glycosaminoglycans, it is possible that MSG ductal epithelial cells trap CXCL10 on their surface in a receptor-independent manner, thus removing relevant amounts of chemokine. Indeed, it has been recently demonstrated that glycosaminoglycans on CXCR3-expressing cells are not required for ligand binding and signaling, whereas glycosaminoglycans on non-CXCR3-bearing cells may retain and sequestrate CXCL10 close to its site of secretion (37). Finally, because we have shown that the scavenging function is in part reversed by pretreatment of epithelial cells with a CXCR3-blocking Ab (indicating the direct role of epithelial CXCR3 in CXCL10 scavenge), it is also possible that expression of IFN--induced chemokine-scavenging receptors may help the innocent bystander epithelial cells avoid immune attacks by lymphocyte infiltration. The hypothesis that CXCR3-B on epithelial cells acts as a molecular trap for the agonists, down-regulating type 1-dominated inflammatory processes, has been documented in this report by a number of data in both normal subjects and SS patients. We first investigated whether epithelial cells down-regulate the ability of CXCL10 to induce the migration of 300-19 cells transfected with human CXCR3 (300-19/hCXCR3). As expected, normal MSG ductal epithelial cells strikingly down-regulate chemotaxis toward CXCL10. Next, we used cultured MSG ductal epithelial cells from SS patients. The molecular basis for the directed migration of autoreactive T cells leading to epithelitis in SS is presently unknown. It has recently been proposed that IFN- stimulates the production of CXCL9, -10, and -11 in the SS ductal epithelium, and that CXCL9, -10, and -11 are involved in the accumulation of T cell infiltrates in the SS salivary glands (38, 39). Using cultured MSG epithelial cells from SS patients we have shown that the scavenging of CXCL10 is reduced, leading to a striking loss of the ability of these cells to inhibit cell migration. Thus, it is possible that the impairment of this scavenging function might favor chemotaxis, leading to an excessive recruitment of CXCR3-positive T lymphocytes. Therefore, we hypothesize that antagonizing this receptor could have great potential to provide novel therapeutic agents for the treatment of SS.
In conclusion, our results show that salivary gland epithelial cells constitutively express CXCR3-B. We also show that this receptor scavenges CXCL10 from the supernatant without mediating calcium flux or chemotaxis. Finally, we demonstrated that in SS, this scavenging function is impaired so that MSG epithelial cells are not able to inhibit chemotaxis.
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported in part by Grant-in-Aid PRIN 2002 from the Italian Ministry of University, Research, and Education and Grant for Young Researchers Support (Progetto Giovani Ricercatori, 2001) from the University of Padova Medical School, Padova, Italy. 
2 Address correspondence and reprint requests to Dr. Carlo Agostini, Dipartimento di Medicina Clinica e Sperimentale, Università di Padova, Immunologia Clinica, Via Giustiniani 2, 35128 Padova, Italy. E-mail address: carlo.agostini{at}unipd.it
3 Abbreviations used in this paper: SS, Sjögren’s syndrome; MFI, mean log fluorescence intensity; MSG, minor salivary gland.
Received for publication July 6, 2005. Accepted for publication December 8, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187: 2009-2021. -interferon encodes a member of the platelet factor 4 family of cytokines. Proc. Natl. Acad. Sci. USA 87: 5238-5242. interferon-inducible cytokine (IP-10). J. Exp. Med. 166: 1084-1097. -inducible protein-10-dependent mechanism. J. Immunol. 168: 3205-3212. -inducible protein 10, monokine induced by IFN-, and IFN--inducible -chemoattractant for T cell chemotaxis toward epidermis in atopic dermatitis. J. Immunol. 171: 1078-1084. -inducible 10-kd protein and CXC receptor 3 in the salivary gland lesions of patients with Sjögren’s syndrome: comment on the article by Ogawa et al. Arthritis Rheum. 48: 2390-2231. [Medline]-Arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J. Biol. Chem. 277: 49212-49219. -chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors. Am. J. Pathol. 158: 867-877. -induced T cell-attracting chemokines, interferon--inducible 10-kd protein (CXCL10) and monokine induced by interferon- (CXCL9), in the salivary gland lesions of patients with Sjögren’s syndrome. Arthritis Rheum. 46: 2730-2741. [Medline] chemoattractant (CXCL11) in the salivary glands of patients with Sjögren’s syndrome. Clin. Immunol. 112: 235-238. [Medline]This article has been cited by other articles:
|
|
 |
|
  A. E. Cardona, M. E. Sasse, L. Liu, S. M. Cardona, M. Mizutani, C. Savarin, T. Hu, and R. M. Ransohoff Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues Blood, July 15, 2008; 112(2): 256 - 263. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Ji, C. M. Lee, L. W. Gonzales, Y. Yang, M. O. Aksoy, P. Wang, E. Brailoiu, N. Dun, M. T. Hurford, and S. G. Kelsen Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1187 - L1196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Lasagni, R. Grepin, B. Mazzinghi, E. Lazzeri, C. Meini, C. Sagrinati, F. Liotta, F. Frosali, E. Ronconi, N. Alain-Courtois, et al. PF-4/CXCL4 and CXCL4L1 exhibit distinct subcellular localization and a differentially regulated mechanism of secretion Blood, May 15, 2007; 109(10): 4127 - 4134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hansell and R. Nibbs Professional and Part-Time Chemokine Decoys in the Resolution of Inflammation Sci. Signal., May 1, 2007; 2007(384): pe18 - pe18. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Agostini and C. Gurrieri Chemokine/Cytokine cocktail in idiopathic pulmonary fibrosis. Proceedings of the ATS, January 1, 2006; 3(4): 357 - 363. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). Cells were gated against an isotype control Ab (
). Representative results from 10 independent experiments are shown. Unimodal distribution with a statistically significant shift between the sample data and the subclass control data. To role out false positives, we performed preliminary flow cytometric experiments using both R&D Systems 49801.111 and BD Biosciences 1C6 Abs. Both mAbs showed similar staining patterns even if incubated at 4°C. B, Western blotting. Extracts from MSG epithelial cell cultures were run on 10% SDS-PAGE, transferred to nitrocellulose membranes, blotted with the anti-CXCR3 Ab 1C6 (BD Biosciences) developed by chemiluminescence, and detected by autoradiography. The molecular masses of protein standards are indicated on the left. The filter was reprobed with anti-
