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Am J Physiol Lung Cell Mol Physiol 287: L584-L591, 2004. First published May 21, 2004; doi:10.1152/ajplung.00453.2003
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The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells

Steven G. Kelsen,1 Mark O. Aksoy,1 Yi Yang,1 Syed Shahabuddin,1 Judith Litvin,2 Fayez Safadi,2 and Thomas J. Rogers3

Division of Pulmonary Disease and Critical Care Medicine, Departments of 1Medicine, 3Microbiology and Immunology, and 2Anatomy, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Submitted 24 December 2003 ; accepted in final form 16 May 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were ~35% of {beta}-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% {beta}-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was ~78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

G protein-coupled receptors; chemotaxis; inflammation; lung; chronic obstructive pulmonary disease; CXC receptor 3

ACTIVATION OF THE CHEMOKINE RECEPTOR CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells (1, 3, 6, 8, 11). Specifically, in endothelial, mesangial, and microglial cells, activation of CXCR3 by the IFN-{gamma}-inducible CXC chemokines [monokine induced by IFN-{gamma} (Mig/CXCL9); IFN-{gamma}-inducible protein of 10 kDa (IP-10/CXCL10); and IFN-{gamma}-inducible T cell {alpha}-chemoattractant (I-TAC/CXCL11)] regulates cell proliferation, apoptosis, adhesion molecule expression, and chemotaxis (1, 3, 6, 8, 11).

In the respiratory tract, chemokine release by human airway epithelial cells (HAEC) shapes the intensity and nature of the inflammatory cell infiltrate in the respiratory tract (2, 4, 14, 16). For example, HAEC release of IP-10/CXCL10 is increased in the setting of respiratory disease [e.g., chronic obstructive pulmonary disease (COPD)] and appears responsible for recruitment of T lymphocytes, which highly express CXCR3 [i.e., T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells] into the airway (13, 19).

Of interest, precedent exists for HAEC to simultaneously express a chemokine receptor as well as its ligands, at least in the case of the CC chemokine receptor CCR3 and its ligands regulated on activation, normal T cell expressed, and secreted (RANTES)/CCL5, monocyte chemoattractant protein (MCP)-3/CCL7, MCP-4/CCL13, and eotaxin/CCL11 (14, 16, 20). Accordingly, we reasoned that HAEC may express CXCR3 as well as its ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11.

To test this hypothesis, we examined CXCR3 mRNA and protein expression by HAEC in vitro and in vivo and compared CXCR3 mRNA with that of several CC and CXC chemokines and chemokine receptors by expression array. Furthermore, we assessed the functionality of CXCR3 by examining the chemotactic response of epithelial cells to ligands of CXCR3.

Our data indicate high constitutive expression of CXCR3 mRNA and protein by epithelial cells in vivo and in vitro at levels greatly exceeding that of other CC and CXC chemokines and chemokine receptors. Moreover, HAEC express both the classic receptor (i.e., CXCR3-A) and a splice variant (i.e., CXCR3-B) (11). Finally, the receptor is functional as indicated by ligand-induced chemotaxis and reorganization of the actin cytoskeleton in HAEC. Our data suggest the possibility that autocrine or juxtacellular activation of HAEC CXCR3 by Mig/CXCL9, IP-10/CXCL10, or I-TAC/CXCL11 may contribute to airway inflammation/remodeling in obstructive lung disease by affecting HAEC migration and/or proliferation (2, 4).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation and culture. Tracheobronchial epithelial cells were isolated from six living human donors bronchoscopically as previously described (10). Informed consent was obtained from each subject. The protocol was approved by the Institutional Review Board for the Protection of Human Subjects at Temple University. Briefly, a sleeved catheter with 3-mm nylon bristles (Mill-Rose Labs, Mentor, OH) was introduced through the sampling channel of a fiberoptic bronchoscope (Olympus, Lake Success, NY), and the airway mucosa in the vicinity of the right middle lobe and left lingular orifices was brushed under direct vision. Up to five brushings were performed at each location. After removal, the brush was shaken in ice-cold MEM containing penicillin (1,000 U/ml), streptomycin (50 µg/ml), and amphotericin (1 µg/ml). The harvested cell suspension was then filtered through a 100-µM Nitex filter (Tetko, Elmsford, NY). Recovered cells were washed twice in ice-cold PBS and then treated with 50 µg/ml of DNase and 0.5g/l of trypsin in 0.6 mM EDTA at 37°C. Fetal bovine serum was added to quench the reaction. Cells were then washed in PBS and resuspended in 2–5 ml of PBS for counting and determination of cell viability by trypan blue (0.4%) exclusion. Cell yield averaged 7 ± 2 million epithelial cells. Cells were cultured in six-well plates coated with a fibronectin/collagen/BSA mixture in LHC-9 complete medium (Biofluids, Bethesda, MD). Experiments were performed on cells at 80–100% confluence from the first and second passages.

In contrast, transformed HAEC (the 16-HBE cell line) were cultured in DMEM plus 4 mM glutamine and 10% FBS until 80–100% confluent.

RNA isolation. Total cellular RNA was isolated in guanidinium isothiocyanate phenol-chloroform (Tri Reagent; Sigma Chemical, St. Louis, MO) as described previously (9). Formaldehyde agarose gel electrophoresis was used to check RNA integrity. All RNA samples yielded two distinct bands corresponding to 18S and 28S ribosomal RNA. RNA was stored at –80°C until it was used for expression array and RT-PCR.

RT-PCR. In initial experiments (n = 4) performed in cultured primary HAEC and 16-HBE cells, CXCR3 mRNA expression was assessed by RT-PCR. Primers were synthesized to detect a 542-bp sequence in the 1107-bp coding region of CXCR3 downstream of the intron splice site (i.e., bp 504–1045). This region codes for amino acids 168–348 in the putative fourth to seventh transmembrane domains of the receptor. The 20-mer primers used were sense, 5'-AGCTTTGACCGCTACCTGAA-3', and antisense, 5'-CGGAACTTGACCCCTACAAA-3'.

Recent studies indicate that in addition to the classic receptor, which has been termed CXCR3-A, a functionally active, alternatively spliced variant termed CXCR3-B may be expressed in some cell types (11). The CXCR3 gene contains a single 978-bp intron. The originally described 1627-bp CXCR3-A transcript is formed by removal of the entire 978-bp intron. The 1860 nucleotide CXCR3-B transcript utilizes an alternative splice acceptor site that is 233 nucleotides upstream (5') of the splice acceptor site used to form CXCR3-A. The sequence downstream of the CXCR3-A splice acceptor site is identical in the two splice variants.

Accordingly, in subsequent experiments (n = 3), we used primers specific for each form of the receptor. To detect CXCR3-A, the following primers were used: sense 5'-AACCACAAGCACCAAAGCAG-3' and antisense 5'-TGATGTTGAAGAGGGCACCT-3'. These primers were used to amplify a 466-bp sequence in the region spanning the splice acceptor site for CXCR3-A. To detect CXCR3-B, we used the following: sense 5'-GCTGCTCAGAGTAAATCACAGACTA-3' and antisense 5'-TGATGTTGAAGAGGGCACCT-3'. These primers amplified a 487-bp sequence containing regions unique to CXCR3-B.

RT-PCR was optimized to detect difficult-to-amplify transcripts by using: 1) a mix of oligo(dT)s and random hexamers for the RT reaction; 2) a low annealing temperature for the PCR reaction; and 3) a proprietary buffer for GC-rich sequences in PCR. For RT, a 10-µl annealing mixture of total RNA (3 µg), random hexamers (0.25 µg), and oligo(dT) primers (0.25 µg) was heated for 3 min at 70°C and added to the RT reaction mixture. This final mixture contained RT buffer, dNTPs (0.5 mM), RNase inhibitor (RNasin, 40 units), and Moloney murine leukemia virus (MMLV) reverse transcriptase (200 units) in addition to RNA and primers. It was incubated at 37°C for 60 min and then heated at 95° for 5 min and kept on ice until PCR.

For PCR, the reaction (50 µl) contained RT reaction mix (3 µl) plus PCR buffer, MgSO4 (2mM), primers (0.4 µM), dNTPs (0.2 mM each), GC-rich mixture (to facilitate PCR of GC-rich transcripts; 10 µl; Roche Diagnostics, Mannheim, Germany), and FastStart Taq DNA polymerase (2 units; Roche Diagnostics). The mixture was heated at 95°C for 4 min in a thermal cycler (model 480; Applied Biosystems, Foster City, CA), and PCR was performed using a 35-cycle program of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min. Reaction products were analyzed by electrophoresis (1.5% agarose, 0.5x Tris-buffered saline). The identity of amplicons was confirmed by direct sequencing of both strands (SeqWright, Houston, TX).

cDNA expression array. In subsequent experiments in cultured HAEC (n = 5), the relative expression of both forms of CXCR3 was compared with other chemokines and chemokine receptors by cDNA expression array analysis. A commercially available, nylon-based, low-density cDNA expression array (GEArray; SuperArray, Bethesda, MD) composed of 25 specific cDNAs for genes of interest spotted in duplicate was used. Of note, the sequence of the CXCR3 cDNA spotted on this array was complementary to a region common to both forms of the receptor and, hence, able to detect both CXCR3-A and -B.

Labeled cDNAs were generated from 15 µg of total RNA in the manufacturer's labeling buffer containing dNTPs, [{alpha}-32P]dCTP (Perkin-Elmer Life Sciences, Downers Grove, IL), RNasin (Promega), and MMLV reverse transcriptase (Promega). The mixture was then denatured and neutralized. Membranes were prehybridized and then incubated overnight in fresh hybridization solution plus labeled cDNA probes (50 µCi). The membrane was then washed repeatedly at increasing stringency in SSC/SDS. Radioactivity was visualized by phosphorimager (Fuji Film, Tokyo, Japan), and gene activity was quantitated relative to {beta}-actin. In all experiments, the nonspecific DNA derived from plasmid UC18 (pUC18) spots used to detect nonspecific binding showed no activity.

Immunofluorescence microscopy. Immunocytochemistry for CXCR3 was performed on primary cultured HAEC (passage 2) and 16-HBE cells (passages 22–29) seeded onto chamber slides (Lab-tek; Nalge/Nunc, Naperville, IL) and serum-starved for 24 h before study. Freshly isolated tracheocytes were pelleted by cytospin (Cytofuge2; IRIS, Norwood, MA). All cells were fixed in 100% methanol, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% goat serum in PBS. A mouse anti-CXCR3 MAb, IgG1 clone 1C6, which recognizes both CXCR3-A and -B, was used as the primary antibody. Two additional anti-CXCR3 antibodies that, like 1C6, detect both CXCR3-A and -B, were used to confirm results: a mouse MAb IgG1 (clone 49801.111; R&D Systems, Minneapolis, MN) or a goat polyclonal IgG (N-15; Santa Cruz Biotechnology, Santa Cruz, CA) (n = 3). In addition (n = 2), to specifically detect expression of CXCR3-B, an antibody against the first 51 NH2-terminal amino acids unique to CXCR3-B (mouse MAb IgG1 clone PL1), developed by Lasagni et al. (11) (a generous gift of Dr. Paola Romagnani, Univ. Florence, Italy), was used at a final concentration of 5 µg/ml. (Of note, no CXCR3-A-specific antibody is available.) A fluorescent cyanine dye (Cy3)-conjugated donkey anti-mouse (Jackson ImmunoResearch, West Grove, PA) and a Cy3-labeled donkey anti-goat antibody were used as the secondary antibodies. To rule out nonspecific staining, a matching isotype negative control was used in place of the antigenically specific primary antibody.

For cytokeratin, an epithelial cell marker, an FITC-conjugated mouse MAb (IgG1 clone J1B3; Beckman-Coulter) was used.

Nuclei were stained with 4',6'-diamidino-2-phenylindole dilactate (DAPI). Cells were imaged using a fluorescence microscope (Eclipse E800; Nikon, Tokyo, Japan) with digital video interface (DEI-750 CE Digital Output; Optronics). Images were processed using Adobe Photoshop 7 (Adobe Systems, San Jose, CA).

Flow cytometry. The possibility that CXCR3 was expressed by HAEC in vivo was assessed using freshly isolated tracheocytes obtained by bronchoscopy as described above (n = 4 experiments). Cells were washed and resuspended in 50 µl of PBS plus 2% FBS, pH 7.4. The Fc{gamma}III/II receptors were blocked with anti-human CD16/CD32 (0.5 µg/µl; BD Biosciences, San Diego, CA). Samples were labeled with fluorochrome-conjugated mouse IgG1 MAb antibodies against human CXCR3 (clone 1C6, BD Biosciences, or clone 49801.111, R&D Systems). An FITC-conjugated mouse MAb IgG1 (clone J1B3; Beckman-Coulter) against cytokeratin, an epithelial cell marker, was used to confirm cell phenotype. Appropriately labeled naïve mouse IgG1 was used as isotype control. Fluorescence was analyzed using a FACScan flow cytometer (Becton Dickinson).

Similar procedures were used in experiments with 16-HBE cells (n = 7 experiments).

Radioligand binding. CXCR3 receptor density and equilibrium binding affinity (Kd) were determined in cultured 16-HBE cells by competitive displacement of I125-labeled IP-10/CXCL10 with unlabeled IP-10/CXCL10. Cells were incubated with 80 pM 125I-IP-10/CXCL10 (2,200 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) plus unlabeled IP-10/CXCL10 (80 pM-200 nM) in 50 mM HEPES, 1 mM CaCl2 2H2O, 5 mM MgCl2, and 0.5% BSA at pH 7.2. The cells were shaken at 4°C for 120 min and then filtered through GF/B glass-fiber filters using a Brandel model M-48R harvester (Gaithersburg, MD) and cold NaCl (500 mM), HEPES (50 mM), CaCl2 2H2O (1 mM), and MgCl2 (5 mM), pH 7.2. Filters were counted in a gamma counter (model 1282; LKB Wallac, Perkin-Elmer Life Sciences).

For each binding curve, the counts per minute (cpm) values for bound ligand fell to a nadir (i.e., minimum) at high concentrations of cold ligand, providing a value for nonspecific binding (see Fig. 6). Specific bound cpm was taken, therefore, as the difference between total cpm and the nadir cpm value. Specific binding (fmol/sample) was then calculated as specific cpm/total added cpm x 16 fmol (i.e., the amount of 125I-IP-10 added). Kd was calculated from the specific binding data as follows: Kd = IC50 – [R], where IC50 was calculated by regression analysis and R was the 125I-IP-10 concentration (i.e., 80 pM). Bmax was determined by the formula: Bmax = specific binding/fractional receptor occupancy, where fractional receptor occupancy = [R]/Kd + [R]. Receptor number per cell was calculated as: receptors/cell = specific binding x 6.08 x 1023/cell number per sample.



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  		Fig. 6. IP-10 radioligand binding in 16-HBE cells. Displacement of bound [125I]IP-10 from 16-HBE cells by cold IP-10 or Mig. Y-axis, specific [125I]IP-10 binding. X-axis, concentration of total ligand. Shown is 1 experiment of 4. CPM, counts per minute.

 
Western blotting. 16-HBE cell lysates (25–50 µg) were electrophoresed and immunoblotted as previously described (9) using Jurkat cell lysate (100 µg) as a positive control. Membranes were washed, blocked overnight, and incubated with the goat polyclonal anti-CXCR3 antibody (N-15) used for immunocytochemistry (Santa Cruz Biotechnology). Membranes were washed, incubated with horseradish-conjugated rabbit anti-goat IgG and then visualized by chemiluminescence (SuperSignal; Pierce, Rockford, IL) on X-ray film.

Chemotaxis of HAEC. The effects of the CXCR3 ligand I-TAC/CXCL11 on chemotaxis of 16-HBE cells were assessed using a 96-well system (ChemoTx; Neuroprobe, Gaithersburg, MD) (n = 6). Briefly, 30-µl aliquots of serum-free RPMI medium containing 0.1% BSA, with or without ligand, were dispensed into the bottom wells of the system. After placement of a polycarbonate membrane (8-µm pore size) coated with fibronectin (10 µg/ml overnight) over the lower wells, 16-HBE cells (1–1.5 x 105 cells in 50-µl aliquots) were seeded onto the surface of the membrane on specially treated circular areas enclosed by a hydrophobic mask to restrict the cell suspension to the application site. Triplicate wells were used for each condition, and epidermal growth factor (10 ng/ml) was used as a positive control. Chemotaxis was allowed to proceed at 37°C in 5% CO2 for 4–5 h. The top side of the membrane was then wiped, fixed with methanol, and stained with Hema 3 (Fisher Scientific). Migrated cells in the entire area of the membrane were counted under a microscope (x40 magnification) and the results were averaged.

The role of chemokinesis vs. chemotaxis in cell migration was assessed by standard checkerboard analysis (n = 4). Concentrations of I-TAC ranging from 0.1 to 100 nM in serum-free RPMI medium containing 0.1% BSA were placed in either the lower well, the upper well, or both the upper and lower wells of the apparatus, and the reaction was allowed to proceed as usual. In addition, the role of CXCR3 in the chemotactic response to I-TAC at a single concentration (1 nM) was assessed by pretreating (30 min) epithelial cells with an anti-CXCR3 blocking antibody (10 µg/ml; clone 49801.111; R&D Systems) (n = 3).

Actin cytoskeleton. CXCR3-induced reorganization of F-actin filaments was assessed in cultured 16-HBE cells treated with IP-10/CXCL10 or vehicle (PBS, 0.1% BSA) for 2, 10, and 30 min at 37°C. Cells were then washed, fixed with 3.7% formaldehyde, permeabilized using 0.1% Triton X-100, and stained with Alexa Fluor 488-conjugated phalloidin (5 U/ml; Molecular Probes, Eugene, OR). Slides were subsequently examined by fluorescence microscopy.

Statistical analysis. Group data were expressed as means ± 1 SE. Differences in sample means were assessed by paired t-test with a P value <0.05 considered statistically significant. For chemotaxis experiments, a one-way ANOVA was used to examine the relationship of cell migration to ligand concentration.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In all RT-PCR experiments performed, total RNA from 16-HBE and cultured tracheocytes yielded a single 542-bp cDNA product for a region common to both forms of the receptor. The sequence of this amplicon exactly matched the known sequence of CXCR3 cDNA (GenBank accession no. NM_001504.1; Fig. 1A). Furthermore, experiments using primers specific for CXCR3-A and -B yielded products of the expected size (466 and 487 bp, respectively) with sequences that also exactly matched the published sequences (GenBank accession no. NM_001504.1 for CXCR3-A and no. AF469635 for CXCR3-B) (11) (Fig. 1, B and C). Reactions lacking RT yielded no PCR products, ruling out contamination with genomic DNA. These results indicated the presence of CXCR3 transcripts of both splice variants CXCR3-A and -B in both sources of airway epithelial cells.



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  		Fig. 1. CXCR3 mRNA expression by RT-PCR in cultured human airway epithelial cells (HAEC). A: ethidium bromide-stained agarose gel (1.5%) showing 542-bp cDNA amplicons for CXCR3 obtained by RT-PCR using primers for a sequence common to CXCR3-A and -B. Shown from left to right are the amplicons obtained by: 1) PCR of the plasmid Bluescript SK (pBSK), which contains a full-length CXCR3 cDNA used as a positive control (a gift from Dr. Philip Murphy, National Institute of Allergy and Infectious Diseases); 2) RT-PCR of total RNA from a pooled mixture of 10 human cell lines (Universal Reference RNA; BD Biosciences, Palo Alto, CA) also used as a positive control; 3) total RNA from 16-HBE cells; and 4) total RNA from cultured human tracheocytes. Each RNA sample is shown with a paired sample that underwent PCR without prior reverse transcription (–RT) to control for contamination with genomic DNA. B: CXCR3-A. Top: lanes 1–4 show 466-bp cDNA amplicons generated from total RNA using primers specific for CXCR3-A. Lane 1, pooled human RNA; lanes 2 and 3, RNA from cultured tracheocytes from 2 human subjects; lane 4, RNA from 16-HBE cells. Bottom: lanes 5–8 show paired samples that underwent PCR but no RT (–RT) to control for genomic contamination. C: CXCR3-B. Top: lanes 1–4 show 487-bp cDNA amplicons generated from total RNA using primers specific for CXCR3-B. Lane 1, pooled human RNA used as a positive control; lanes 2 and 3, RNA from cultured tracheocytes from 2 human subjects; lane 4, RNA from 16-HBE cells. Bottom: lanes 5–8 show paired samples that underwent PCR but no RT (–RT) to control for genomic contamination.

 
Subsequent experiments using expression array technology with a cDNA probe that detects both splice variants indicated high constitutive expression of CXCR3 in cultured primary HAEC in all six experiments performed (Fig. 2). Of note, expression of CXCR3 was significantly greater than that of CCR3 (35 ± 12% and 3 ± 2% of the {beta}-actin signal, respectively, P = 0.016; Figs. 2 and 3A). In addition to CXCR3 and CCR3, detectable constitutive expression was observed for the following chemokines: IP-10/CXCL10, Mig/CXCL9, I-TAC/CXCL11, fractalkine, macrophage inflammatory protein-1{beta} (MIP-1{beta}), macrophage-derived chemokine, thymus and activation-regulated chemokine, and I-309; and for the following cytokines: IL-1{beta}, TNF-{alpha}, IL-12A and B, IL-15, IL-16, and IL-l8 (Fig. 2). In contrast, little or no detectable expression was observed for the following chemokines: eotaxin, MIP-1{alpha}, RANTES; chemokine receptors: CCR4, CCR5, CCR8, and CX3CR1, the fractalkine receptor; and cytokines: IFN-{gamma} (Fig. 2). Similar results were obtained in all six experiments (Fig. 3). A similar pattern was obtained for 16-HBE cells.



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  		Fig. 2. Representative expression array obtained from a single culture of tracheocytes showing [32P]dCTP activity for 25 genes of interest. In addition, the housekeeping genes {beta}-actin and GAPDH and the plasmid negative control pUC18 are also shown. CXCR3 expression appeared to exceed that of CCR3. Note that the CXCR3 probe contained sequences common to CXCR3-A and -B and, hence, detected both transcripts. RANTES, regulated on activation, normal T cell expressed, and secreted; IP-10, IFN-{gamma}-inducible protein of 10 kDa; I-TAC, IFN-{gamma}-inducible T cell {alpha}-chemoattractant; MIG, monokine induced by IFN-{gamma}; FKN, fractalkine; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; TARC, thymus and activation-regulated protein.

 


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  		Fig. 3. Expression array group data in primary cultured tracheocytes. Constitutive gene activity for all 6 subjects (means ± SE). Expression was quantitated by densitometry, and results are shown as a ratio of the {beta}-actin value. ND, not detected. A: chemokine receptors. Note that the CXCR3 probe contained sequences common to CXCR3-A and -B and, hence, detected both transcripts. B: chemokine ligands. C: cytokines. Note that CXCR3 expression (A) appeared to exceed the activity of CCR3 (~35% vs. 3% {beta}-actin value, respectively).

 
Strong CXCR3 immunoreactivity was present in primary cultured HAEC (n = 5) and 16-HBE cells using antibodies that detected both CXCR3-A and -B (Fig. 4). Similar results were obtained using an antibody (clone PL1) specific for CXCR3-B (Fig. 5). Immunoblots of 16-HBE cells also showed a 40- to 45-kDa band indicative of CXCR3 (data not shown). These results indicate expression of CXCR3 at the protein level in both types of HAEC.



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  		Fig. 4. CXCR3 immunoreactivity in primary cultured tracheocytes. Fluorescence microscopy of cultured human tracheocytes using an antibody that recognized both CXCR-A and -B (clone 49801.111, R&D Systems). Cells were double labeled for CXCR3 (red, Cy3) and cytokeratin (green, FITC). Cell nuclei (blue) were visualized with 4',6'-diamidino-2-phenylindole dilactate (DAPI). Panel 1: cells stained for CXCR3. Panel 2: cells stained for cytokeratin. Panel 3: CXCR3 negative control; only nuclei are visible. Panel 4: cells shown in panel 3 stained for cytokeratin. Magnification, x1,000.

 


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  		Fig. 5. CXCR3-B immunoreactivity in primary cultured tracheocytes. Fluorescence microscopy of cultured tracheocytes using an antibody specific for CXCR3-B (a gift from Dr. Paola Romagnani, Univ. of Florence). Cells were double labeled for CXCR3 (red, Cy3) and cytokeratin (green, FITC). Cell nuclei (blue) were visualized with DAPI. Panel 1: cells stained for CXCR3. Panel 2: cells stained for cytokeratin. Panel 3: CXCR3 control; only nuclei are visible. Panel 4: cells shown in panel 3 stained for cytokeratin. Magnification, x1,000.

 
Radioligand binding of IP-10/CXCL10 in airway epithelial cells (16-HBE cells) demonstrated ~80,000 receptor sites/cell (Bmax = 78,153 ± 24,706 sites/cell; n = 4) having high affinity for this ligand (Kd = 2.2 ± 0.7 nM; Fig. 6). Mig/CXCL9 bound with considerably lower affinity (Kd of ~200 nM; Fig. 6) as previously described (5, 12, 21).

Flow cytometry of freshly isolated tracheocytes obtained bronchoscopically using antibodies that recognize both CXCR3-A and -B indicated strong expression in vivo. A majority (68 ± 12%) of cytokeratin-positive cells (i.e., the epithelial cell population) stained positively for CXCR3 (n = 4; Fig. 7, A and B). The mean fluorescence intensity of the CXCR3-positive population was 29-fold greater than isotype controls (P < 0.03). In addition, all three major epithelial cell phenotypes, ciliated, basal cells, and secretory cells, stained positively for CXCR3 by immunocytochemistry (Fig. 7C).



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  		Fig. 7. CXCR3 immunoreactivity in bronchoscopically isolated tracheocytes. A: flow cytometry. Histogram showing cychrome fluorescence of anti-CXCR3 (black) and isotype control (gray). B: dual parameter plot of CXCR3 (cychrome) vs. cytokeratin (FITC) fluorescence in double-labeled cells. Note that the majority of keratin-positive cells are also CXCR3 positive. Shown is 1 subject representative of 4. C: fluorescence microscopy. Cells were double labeled for CXCR3 (panel 1, red) and cytokeratin (panel 2, green). Panel 3 shows cells in Nomarski optics. C, ciliated; B, basal cells. Panels 4–6 show second cell field. Panel 4, CXCR3 negative control. Magnification x1,000.

 
Similar flow cytometry results were obtained for 16-HBE cells that stained positively in all seven experiments performed.

Finally, CXCR3 was functional. I-TAC/CXCL11 induced migration of 16-HBE cells in a characteristic concentration-dependent manner (Fig. 8; n = 6). Cell migration was maximum at ~0.1 nM and equaled 232 ± 70% SE of the medium control (n = 6; P = 0.008).



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  		Fig. 8. Chemotaxis induced by I-TAC in 16-HBE cells. Migration of 16-HBE cells over 5 h in response to I-TAC (0–10 nM). Note the concentration-dependent increase in cell migration in response to I-TAC with maximum at ~0.1 nM. Means ± SE of 6 experiments (P = 0.008 by ANOVA).

 
Checkerboard analysis indicated that cell movement occurred primarily in response to a concentration gradient (data not shown).

Pretreatment of epithelial cells with a CXCR3 blocking antibody (clone 49801.111, R&D Systems) eliminated the response to I-TAC/CXCL11. For example, cell migration in response to I-TAC (1 nM) was 141 ± 5% of the medium control value (n = 3 experiments). Pretreatment of cells with the anti-CXCR3 antibody (10 µg/ml) decreased the I-TAC response to 94 ± 6% of the medium control.

Furthermore, CXCR3 ligands induced actin reorganization in 16-HBE cells. In response to IP-10/CXCL10, stress fibers were more obvious and redistributed to the cell periphery. Apparent cell diameter also increased, suggesting cell flattening (Fig. 9). Changes were most obvious at 10–30 min of treatment.



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  		Fig. 9. Actin reorganization with IP-10 in cultured 16-HBE cells. Cells stained for F-actin with Alexa Fluor 488-conjugated phalloidin. Note IP-10 treatment (10 nM for 10 min) increased stress fibers and cell diameter compared with vehicle. Shown is 1 experiment representative of 4.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous studies indicate that the CXCR3 receptor is expressed by activated Th1 lymphocytes, natural killer cells, some endothelial cells, kidney mesangial cells, microglial cells, and eosinophils (3, 6, 8, 13, 15, 17). The present study extends these observations and demonstrates for the first time that HAEC express CXCR3, the cognate receptor for the interferon-{gamma}-inducible, non-ELR (lacking the glutamine-leucine-arginine motif at the NH2 terminus) CXC chemokines, Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Moreover, the receptor is highly expressed. The multigene array indicates that the CXCR3 message level approximates 30% of the {beta}-actin value, and the radioligand binding data indicate ~80,000 ligand binding sites/cell. [For comparison, the {beta}2-adrenergic receptor, another G protein-coupled receptor, is expressed at the level of ~5,000 sites/cell in cultured HAEC (9).] Of interest, the present study confirms constitutive expression of the CC chemokine receptor CCR3 on airway epithelial cells (20) but indicates that the CXCR3 mRNA level appears to exceed that of CCR3. CXCR3 mRNA also appeared to equal or exceed that of its ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 and that of the CC chemokines eotaxin/CCL11 and RANTES/CCL5. Our study also indicates that both CXCR3-A and -B are expressed in HAEC.

Finally, the CXCR3 receptor(s) expressed by HAEC is functional. The receptor binds ligand with high affinity and, when activated, induces chemotaxis and actin cytoskeleton reorganization.

Functional responses to CXCR3-A and -B activation. The CXCR3 receptors coded for by the two splice variants differ in structure, ligand binding properties, cellular expression, and cellular responses (11). The two forms of the receptor also activate different signaling pathways and elicit different cellular responses (3, 11).

Of interest, human microvascular endothelial cells selectively express CXCR3-B that, when activated by IP-10/CXCL10 or platelet factor 4 (PF4)/CXCL4, induces apoptosis and inhibits cell proliferation and migration (11). In contrast, activated T lymphocytes, like airway epithelial cells, express both CXCR3-A and -B, although in T cells, CXCR3-A expression is considerably greater than is expression of CXCR3-B (11). In T lymphocytes, stimulation with IP-10/CXCL10 induces proliferation and chemotaxis, responses presumably mediated by CXCR3-A. Our data demonstrating a chemotactic response of HAEC to I-TAC/CXCL11, therefore, are likely explained by a response mediated by CXCR3-A.

Biological consequence(s) in HAEC of CXCR3-A and -B stimulation. Subjects with COPD, lung allograft rejection, and sarcoidosis manifest a Th1/Tc1 lymphocytic infiltrate in the bronchial mucosa and alveolar wall, with T cells primarily expressing CXCR3 (1, 7, 15, 18). HAEC release of Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11, which selectively recruits Th1/Tc1 lymphocytes, presumably contributes to this process (13, 19). Furthermore, subjects with COPD demonstrate greater than normal expression of IP-10/CXCL10 by bronchial epithelial cells, further implicating HAEC in the development of this pattern of inflammation (18).

We speculate, in turn, that activation of CXCR3 on HAEC by release of Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 could also be important in airway inflammation and remodeling, as occurs in obstructive lung diseases such as COPD and asthma, by inducing effects on the epithelium (2, 4). For example, CXCR3-A-induced airway epithelial cell migration in response to IP-10/CXCL10, Mig/CXCL9, or I-TAC/CXCL11 may help reconstitute a highly differentiated epithelial lining after epithelial denudation. The biological effect(s) of CXCR3 activation in HAEC requires further study. Nonetheless, it is clear that the expression of both CXCR3 splice variants by HAEC adds another level of complexity to the lung cytokine network and suggests the possibility that the CXC ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 produce autocrine or juxtacellular effects on the epithelium.


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 ABSTRACT
 METHODS
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 DISCUSSION
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Grant support was provided by the Philip Morris Foundation (to S. G. Kelsen) and National Institute on Drug Abuse (to T. J. Rogers, Grants DA-14230 and P30-DA-13429).


   ACKNOWLEDGMENTS
 
We thank Dr. Paola Romagnani of the University of Florence, Italy, for graciously providing the antibody against CXCR3-B and for helpful suggestions related to the RT-PCR reaction. We also thank Dr. Xiao Zeng of Superarray for helpful suggestions related to the RT-PCR reaction and for graciously providing primers for this purpose. Finally, we thank Dr. Philip Murphy, National Institute of Allergy and Infectious Diseases, for providing the plasmid Bluescript SK containing the full-length CXCR3 cDNA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. G. Kelsen, 761 Parkinson Pavilion, Temple Univ. Hospital, 3401 N. Broad St., Philadelphia, PA 19140 (E-mail: Kelsen{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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  1. Agostini C, Calabrese F, Rea F, Facco M, Tosoni A, Loy M, Binotto G, Valente M, Trentin L, and Semenzato G. CXCR3 and its ligand CXCL10 are expressed by inflammatory cells infiltrating lung allografts and mediate chemotaxis of T cells at sites of rejection. Am J Pathol 158: 1703–1711, 2001.[Abstract/Free Full Text]
  2. Barnes PJ. Chronic obstructive pulmonary disease. N Engl J Med 343: 269–280, 2000.[Free Full Text]
  3. Bonacchi A, Romagnani P, Romanelli RG, Efsen E, Annunziato F, Lasagni L, Francalanci M, Serio M, Laffi G, Pinzani M, Gentilini P, and Marra F. Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem 276: 9945–9954, 2001.[Abstract/Free Full Text]
  4. Busse WW and Lemanske RF. Asthma. N Engl J Med 344: 350–362, 2001.[Free Full Text]
  5. Cox MA, Jenh CH, Gonsiorek W, Fine J, Narula SK, Zavodny PJ, and Hipkin RW. Human interferon-inducible 10-kDa protein and human interferon-inducible T cell {alpha} chemoattractant are allotropic ligands for human CXCR3: differential binding to receptor states. Mol Pharmacol 59: 707–715, 2001.[Abstract/Free Full Text]
  6. Cross AK and Woodroofe MN. Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro. J Neurosci Res 55: 17–23, 1999.[CrossRef][Web of Science][Medline]
  7. D'Ambrosio D, Mariani M, Panina-Bordignon P, and Sinigaglia F. Chemokines and their receptors guiding T-lymphocyte recruitment in lung inflammation. Am J Respir Crit Care Med 164: 1266–1275, 2001.[Free Full Text]
  8. Jinquan T, Jing C, Jacobi HH, Reimert CM, Millner A, Quan S, Hansen JB, Dissing S, Malling HJ, Skov PS, and Poulsen LK. CXCR3 expression and activation of eosinophils: role of IFN-{gamma}-inducible protein-10 and monokine induced by IFN-{gamma}. J Immunol 165: 1548–1556, 2000.[Abstract/Free Full Text]
  9. Kelsen SG, Anakwe OO, Aksoy MO, Reddy PJ, Dhanesekaran N, Penn R, and Benovic JL. Chronic effects of catecholamines on the {beta}2-adrenoreceptor system in cultured human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 272: L916–L924, 1997.[Abstract/Free Full Text]
  10. Kelsen SG, Mardini IA, Zhou S, Benovic JL, and Higgins NC. A technique to harvest viable tracheobronchial epithelial cells from living human donors. Am J Respir Cell Mol Biol 7: 66–72, 1992.
  11. Lasagni L, Francalanci M, Annunziato F, Lazzeri E, Gianni S, Cosmi L, Sagrinati C, Mazzinghi B, Orlando C, Maggi E, Marra F, Romagnani S, Serio M, and Romagnani P. An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4. J Exp Med 197: 1537–1549, 2003.[Abstract/Free Full Text]
  12. Lu B, Humbles A, Bota D, Gerard C, Moser B, Soler D, Luster AD, and Gerard NP. Structure and function of the murine chemokine receptor CXCR3. Eur J Immunol 29: 3804–3812, 1999.[CrossRef][Web of Science][Medline]
  13. Luster AD. Chemokines–chemotactic cytokines that mediate inflammation. N Engl J Med 338: 436–445, 1998.[Free Full Text]
  14. Nickel R, Beck LA, Stellato C, and Schleimer RP. Chemokines and allergic disease. J Allergy Clin Immunol 104: 723–742, 1999.[CrossRef][Medline]
  15. Panina-Bordignon P, Papi A, Mariani M, Di Lucia P, Casoni G, Bellettato C, Buonsanti C, Miotto D, Mapp C, Villa A, Arrigoni G, Fabbri LM, and Sinigaglia F. The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics. J Clin Invest 107: 1357–1364, 2001.[Web of Science][Medline]
  16. Polito AJ and Proud D. Epithelial cells as regulators of airway inflammation. J Allergy Clin Immunol 102: 714–718, 1998.[CrossRef][Web of Science][Medline]
  17. Romagnani P, Annunziato F, Lasagni L, Lazzeri E, Beltrame C, Francalanci M, Uguccioni M, Galli G, Cosmi L, Maurenzig L, Baggiolini M, Maggi E, Romagnani S, and Serio M. Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J Clin Invest 107: 53–63, 2001.[Web of Science][Medline]
  18. Saetta M, Mariani M, Panina-Bordignon P, Turato G, Buonsanti C, Baraldo S, Bellettato CM, Papi A, Corbetta L, Zuin R, Sinigaglia F, and Fabbri LM. Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 165: 1404–1409, 2002.[Abstract/Free Full Text]
  19. Sauty A, Dziejman M, Taha RA, Iarossi AS, Neote K, Garcia-Zepeda EA, Hamid Q, and Luster AD. The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells. J Immunol 162: 3549–3558, 1999.[Abstract/Free Full Text]
  20. Stellato C, Brummet ME, Plitt JR, Shahabuddin S, Baroody FM, Liu MC, Ponath PD, and Beck LA. Expression of the C-C chemokine receptor CCR3 in human airway epithelial cells. J Immunol 166: 1457–1461, 2001.[Abstract/Free Full Text]
  21. Weng Y, Siciliano SJ, Waldburger KE, Sirotina-Meisher A, Staruch MJ, Daugherty BL, Gould SL, Springer MS, and DeMartino JA. Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors. J Biol Chem 273: 18288–18291, 1998.[Abstract/Free Full Text]



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