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RECEPTORS AND SIGNAL TRANSDUCTION

CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways

Departments of Medicine and Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania

Submitted 31 August 2005 ; accepted in final form 30 January 2006

	ABSTRACT

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Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 ± 88% (mean ± SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca²⁺] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5–10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

G protein-coupled receptor; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; cytoskeleton

In this study, we examined the potency and efficacy of several CXCR3 ligands as chemoattractants for human airway epithelial cells. First, we compared the dose-response relationships of I-TAC, Mig, and IP-10 as chemoattractants in this cell type. Second, we examined the signaling pathways mediating the resultant chemotactic response. Specifically, we assessed the role of a pertussis toxin (PTX)-blockable G protein, intracellular calcium, the MAPKs (ERK, p38, and JNK), and the phosphatidylinositol 3-kinase (PI3K) as signaling pathway components in the chemotactic responses to CXCR3 activation.

Our data indicate that I-TAC, Mig, and IP-10 induce chemotaxis in dose-dependent manner with similar potency but with a rank order of efficacy of I-TAC = IP-10 > Mig. The efficacy of the CXCR3 ligands is 50% that of epidermal growth factor (EGF), a highly potent chemoattractant for airway epithelial cells (9, 27, 37). Finally, CXCR3-induced chemotaxis is G protein dependent and requires signaling by the p38 and PI3K pathways but is not dependent on those activated by changes in intracellular calcium (Ca²⁺) or ERK.

	MATERIALS AND METHODS

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Reagents and antibodies. Anti-human CXCR3 mouse IgG₁ monoclonal antibody (clone 49801.11) was purchased from R&D Systems (Minneapolis, MN). Phospho- and total MAPK (anti-p38, anti-ERK1/2, and anti-JNK) and PI3K (anti-Akt) antibodies were obtained from Cell Signaling (Beverly, MA). The MEK1/2 inhibitor U0126, p38 inhibitor SB 20358, and PI3K inhibitor wortmannin were purchased from Calbiochem (San Diego, CA). Fura-2 AM was obtained from Molecular Probes (Eugene, OR). Fibronectin, collagen VI, fatty acid-free BSA, bradykinin (BK), EGF, and PTX were obtained from Sigma-Aldrich (St. Louis, MO). Human recombinant I-TAC, IP-10, and Mig were purchased from R&D Systems.

Cell culture. Normal human bronchial epithelial cells (NHBEC), obtained from Clonetics-BioWhittaker (Walkersville, MD), were grown on collagen type VI-coated plates (25 µg/ml) in serum-free defined growth medium (BEGM; Clonetics) in 5% CO₂ at 37°C. Medium was changed every 2–3 days until cells were 80–100% confluent. Cells from passage 2 or 3 were used as previously described (20). Transformed human airway epithelial cells (16-HBE cell line) were cultured in DMEM plus 4 mM glutamine and 10% FBS (20).

Chemotaxis assay. Chemotaxis in NHBEC and 16-HBE cells was assessed using a commercially available 96-well modified Boyden chamber chemotaxis system (ChemoTx; Neuroprobe, Gaithersburg, MD) as previously described (9, 27). In this system, the upper surface of each well was separated from a lower chamber containing the chemoattractant ligands by a polycarbonate membrane. A circular area of the membrane in the region that covers each lower well is enclosed by a hydrophobic mask to retain the cell suspension within this area. To facilitate epithelial cell movement, we coated the membrane (pore size 8 µm for 16-HBE and 12 µm for NHBEC) with human fibronectin (10 µg/ml) as previously described (9, 27). The system was prepared by loading the bottom wells with 30-µl aliquots of chemotaxis medium (serum-free RPMI, 0.1% BSA), with or without Mig, IP-10, or I-TAC in a range of concentrations. The membrane was then placed over the lower chamber, and a suspension of epithelial cells (0.5–1 x 10⁵ cells in 50 µl) was delivered onto each of the hydrophobically limited regions of the upper surface of the membrane. Chemotaxis was then allowed to proceed at 37°C in 5% CO₂ for 6 h. After this period, the membrane was removed and its topside was carefully wiped to eliminate nonmigrated cells. The membrane was then fixed with methanol and stained with Hema 3 (Fisher Scientific). Chemotaxis was assessed by counting the number of cells that entered a pore or passed through to the underside of the membrane. Migrated cells in the entire cross-sectional area of each well were counted under a microscope (x40 magnification). The number of migrated cells was compared in CXCR3 ligand-containing wells and in wells containing buffer only (i.e., serum-free RPMI medium, 0.1% BSA), which served as a negative control. EGF (1 ng/ml), a potent chemoattractant for epithelial cells, was used as a positive control (9, 27, 37). Triplicate wells were used for each condition, and results were averaged.

To examine the effect of CXCR3 blocking antibody, PTX, or inhibitors of signaling pathways, we incubated cells with the appropriate agent for 30 min before and during the chemotaxis assay. Neither the anti-CXCR3 antibody (10 µg/ml; clone 49801.11, R&D Systems) nor PTX (1 µg/ml) affected epithelial cell viability over a 6-h incubation period (n = 2 experiments).

The nonspecific effects of the MEK1/2 inhibitor U0126, the p38 inhibitor SB 20358, and the PI3K inhibitor wortmannin on epithelial cell chemotaxis were assessed using vehicle-treated medium (n = 4 experiments). U0126 had no effect on chemotaxis over the concentration range used (0.1 to 10 µM). SB 20358 and wortmannin inhibited chemotaxis slightly (25–35%) at the highest inhibitor concentrations used (3 µM and 100 nM, respectively).

Calcium mobilization. Intracellular Ca²⁺ mobilization ([Ca²⁺]_i) was measured as described previously (6). Briefly, NHBEC were grown to 50–60% confluence on 12-mm coverslips (Fisherbrand, PA) and loaded with the fluorescent Ca²⁺ indicator fura-2 AM (5 µM). Cells were incubated in Hanks' buffered saline solution (HBSS) supplemented with fura-2 AM for 45 min and in HBSS alone for a further 15–60 min to allow deesterification of the dye. Coverslips were placed in a custom-designed bath and transferred to the stage of an inverted epifluorescence microscope equipped with a C&L Instruments fluorimeter system. Fifty microliters of a chemokine, BK, ATP, or HBSS were added to the cells, and fura-2 fluorescence (excitation wavelengths, 340 and 380 nm; emission wavelength, 520 nm) of single cells was acquired at a frequency of 1 Hz. The excitation ratio (340 nm/380 nm) of the fluorescence signals obtained was converted to Ca²⁺ concentration according to the method of Grynkiewicz et al. (17).

Western blot analysis. Cells were grown in six-well plates in full medium until subconfluent and then placed in depleted-medium 24 h before experiments (i.e., BEGM without EGF and bovine pituitary extract for NHBEC, or serum-free DMEM for 16-HBE cells). Cells were then treated with 100 ng/ml I-TAC for 1, 5, 10, or 30 min, washed twice in ice-cold PBS, and then lysed for 10 min in lysis buffer (20 mM Tris, pH 7.5, 120 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na₃VO₄, and 1 mM PMSF). Lysates were stored at –80°C. Protein concentrations were determined using the DC protein assay kit (Bio-Rad).

Cell lysates (25–50 µg) were electrophoresed using SDS-PAGE on a 12% acrylamide gel and electrophoretically transferred to a nitrocellulose membrane as previously described (4). The membrane was blocked with 5% nonfat milk in 1x Tris-buffered saline and 0.1% Tween 20 for 1 h while shaking at room temperature. ERK, SAPK/JNK, p38, and PI3K phosphospecific antibodies were used as directed by the manufacturer. Membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, and then visualized using chemiluminescence (SuperSignal; Pierce, Rockford, IL) on X-ray film. After detection of the phosphoprotein, the blot was stripped and hybridized with antibodies specific for total ERK, SAPK/JNK, p38, or PI3K as appropriate.

Statistical analysis. Results are given as means ± SE. The statistical significance of differences in group mean data was assessed using one-way and two-way ANOVA and Student's t-tests. The level of significance was set at P < 0.05. Curve fitting of chemotactic responses was performed using linear regression with a second-order polynomial equation.

	RESULTS

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CXCR3-induced airway epithelial cell chemotaxis. I-TAC stimulated chemotaxis of NHBEC and 16-HBE cells in a concentration-dependent manner (P < 0.03 for both cell types by ANOVA; n = 8–12 experiments) (Fig. 1, A and B). The maximum response to I-TAC equaled 349 ± 88% of the control response (109 ± 27 cells/well in medium alone) in NHBEC and 210 ± 32% of the control response (173 ± 52 cells/well in medium alone) in 16-HBE cells.

View larger version (10K): [in this window] [in a new window]   Fig. 1. CXC chemokine receptor CXCR3 agonists induce chemotaxis in airway epithelial cells. Effects of three CXCR3 ligands, I-TAC (), Mig (), and IP-10 (), on chemotaxis in normal human bronchial epithelial cells (NHBEC) are shown in A. Effects of I-TAC () and Mig () in the 16-HBE cell line are shown in B. Control values represent migration with buffer alone. Values are means ± SE of 8–12 experiments.

In 16-HBE cells, CXCR3 blocking antibody (10 µg/ml) eliminated I-TAC-induced chemotaxis (185 ± 52 and 91 ± 4% of control for I-TAC alone and I-TAC plus anti-CXCR3 antibody, respectively; n = 3) (Fig. 2A). These data indicate that cell migration was receptor mediated.

View larger version (11K): [in this window] [in a new window]   Fig. 2. A: anti-CXCR3 monoclonal antibody (MAb) eliminates chemotactic responses to I-TAC (10 ng/ml) in 16-HBE cells. IgG-ve control indicates effects of pretreatment with nonfunctional isotype control (10 µg/ml); anti-CXCR3 indicates effects of pretreatment with anti-CXCR3 blocking MAb (10 µg/ml). B: pertussis toxin (PTX) blocks I-TAC induced chemotaxis in NHBEC. Cells were pretreated with PTX (1 µg/ml; +PTX) or vehicle (–PTX) for 30 min before and during exposure to I-TAC (10 ng/ml). Values are means ± SE of 4 experiments for both A and B.

I-TAC-mediated chemotaxis is blocked by pertussis toxin. I-TAC-induced (10 ng/ml) chemotaxis in NHBEC was completely blocked by PTX (1 µg/ml) pretreatment (101 ± 54% of control; n = 4) (Fig. 2B). These data indicate that the CXCR3-mediated chemotaxis involves a PTX-blockable GTP-binding protein.

I-TAC, IP-10, and Mig do not alter intracellular calcium. Neither I-TAC, IP-10, nor Mig induced a change in [Ca²⁺]_i in NHBEC (Fig. 3; n = 3). In contrast, BK or ATP markedly increased [Ca²⁺]_i (Fig. 3). Similar results were obtained in 16-HBE cells (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3. CXCR3 agonists do not induce a Ca2+ flux in NHBEC. NHBEC were loaded with fura-2 AM, and intracellular Ca2+ concentration ([Ca2+]i) was recorded over time. Cells were stimulated with 1 µM I-TAC (A and B), IP-10 (C), or Mig (D). To ensure responsiveness, we stimulated cells several minutes later with bradykinin (BK; 10 µM) (A) or ATP (10 µM) (B–D), which served as positive controls. Arrows indicate the points at which the stimulus was given. Results shown are from 1 experiment representative of 3.

View larger version (13K): [in this window] [in a new window]   Fig. 4. I-TAC induces ERK, p38, and phosphatidylinositol 3-kinase (PI3K) phosphorylation in NHBEC. Western blots are shown for total and phospho-ERK, total and phospho-p38, and total and phospho-Akt. Cells were treated with I-TAC for 1–30 min. Results are from 1 experiment representative of 4.

p38 but not MEK/ERK inhibition affects I-TAC-stimulated chemotaxis. The MEK inhibitor U0126 (0.01–10 µM) (15) had no significant effect on I-TAC (10 ng/ml)-induced chemotaxis in NHBEC (Fig. 5A). In contrast, the p38 inhibitor SB 20358 (36) completely abrogated I-TAC-induced chemotaxis in a concentration-dependent manner (0.03–3 µM) (P <0.05 by ANOVA) (Fig. 5B).

View larger version (9K): [in this window] [in a new window]   Fig. 5. p38 MAPK and PI3K inhibitors block I-TAC-induced chemotaxis in NHBEC. The inhibitory effects of U0126, an ERK1/2 inhibitor (A), SB 20358, a p38 inhibitor (B), or wortmannin, a PI3K inhibitor (C), on I-TAC-induced (10 ng/ml) chemotaxis are shown. Values are means ± SE of 4–8 experiments.

	DISCUSSION

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Expression of the chemokine receptor CXCR3 has previously been described in a variety of immune (Th1 lymphocytes, natural killer cells, hematopoietic progenitor cells) and structural cells (endothelial cells, microglia, hepatic and renal pericytes, and other) (5, 11, 19, 22, 28, 33). In cells that express CXCR3, activation of this receptor regulates cell movement (5, 8, 10, 22, 33, 34). However, the effects of CXCR3 activation on cell movement are cell type dependent and include both stimulation (e.g., lymphocytes, renal mesangial cells) (5, 33) and inhibition of cell migration (e.g., microvascular endothelial cells) (22). Different chemotactic responses to CXCR3 activation by the same ligands appear to be explained by different signaling pathways. For example, positive chemotactic responses induced by CXCR3 activation are mediated by activation of phospholipase C (PLC) in Th1 lymphocytes and by both PI3K and the ERK MAPK in hepatic stellate cells (5, 33). In contrast, negative chemotactic responses are mediated by G_i-independent, cAMP-dependent pathways in microvascular endothelial cells (22).

Work in our laboratory (20) recently demonstrated that human airway epithelial cells constitutively express the chemokine receptor CXCR3. The present study indicates that both normal airway epithelial cells (NHBEC) and the 16-HBE cell line demonstrate robust chemotactic responses to activators of CXCR3. In fact, in normal airway epithelial cells, the efficacy of the CXCR3 ligand I-TAC was approximately one-half the response to EGF, a highly potent chemotactic agent for airway epithelial cells (9, 27, 37).

In NHBEC, the several CXCR3 ligands demonstrated similar potency but different maximal responses (efficacy). I-TAC and IP-10 were equally efficacious and approximately two times more efficacious than Mig.

Initial experiments designed to elucidate the signaling pathways involved in CXCR3-induced chemotaxis examined possible involvement of a PTX-blockable, GTP-binding protein such as G_i. Pretreatment of human airway endothelial cells with PTX completely eliminated chemotactic responses to I-TAC, supporting the role of G_i in this process (25, 26).

In most but not all cells, activation of chemokine receptors also induces an increase in cytosolic calcium, [Ca²⁺]_i. However, in our study, the several CXCR3 ligands did not change [Ca²⁺]_i. These results differ from studies in T cells and renal mesangial cells in which CXCR3 agonists increased [Ca²⁺]_i (10, 28). Of interest, the absence of a [Ca²⁺]_i response has been reported with other chemokine receptors and their ligands. For example, RANTES/CCL5 induces CCR5-mediated T-lymphocyte chemotaxis without changing [Ca²⁺]_i (35). Furthermore, IL-8/CXCL2 induces CXCR2-mediated neutrophil migration without an increase in [Ca²⁺]_i in cells from PLC-2/3 knockout mice (23). Together, our data and those of others (23, 35) suggest that changes in [Ca²⁺]_i are not always necessary for directional sensing and cell shape change.

Our data indicate that I-TAC induces phosphorylation of p38, ERK1/2, and PI3K, strongly suggesting that these pathways are activated by CXCR3 in HAEC. The role of these several pathways in mediating chemotaxis was assessed using specific inhibitors.

The role of p38 signaling pathways in CXCR3-induced chemotaxis was assessed using the selective p38/ isotype inhibitor SB 20358 (36). SB 20358 dose-dependently and completely blocked I-TAC-induced chemotaxis. These data indicate that the p38/ pathways are necessary for CXCR3-induced airway epithelial cell movement.

To our knowledge, the role of p38 MAPK in CXCR3-induced chemotaxis has not been studied previously in any cell type. However, a role for p38 has been demonstrated in chemotaxis induced by EGF in human bronchial epithelial cells (the BEAS-2B line) (9) and by hepatocyte growth factor in corneal epithelial cells (32). These latter studies, along with our own, support a role for p38 in epithelial cell migration induced by both G protein-coupled receptors (chemokine receptors) and tyrosine kinase receptors (EGF receptor). In fact, p38 may act by regulating the activity of proteins (heat shock protein-27) that control F-actin polymerization, an essential step in generating lamellipodia, which are required for cell movement (16).

In this study, the role of PI3K was assessed using the selective PI3K inhibitor wortmannin (1). Wortmannin dose-dependently but incompletely inhibited I-TAC-induced chemotaxis with maximum inhibition (75% inhibition) at 10 nM. At this concentration, wortmannin selectively inhibits PI3K without affecting PI3K, , or . Of interest, our results on PI3K inhibition are in agreement with results in hepatic stellate cells in which IP-10-induced chemotaxis was only partially inhibited (60%) by wortmannin at 100 nM (5). In contrast, I-TAC-induced chemotaxis in T cells was not blocked by wortmannin at concentrations specific for PI3K (<100 nM), strongly suggesting that this kinase does not play a role in CXCR3-induced T-cell movement (33).

Complete inhibition of I-TAC-induced chemotaxis by SB 20358 coupled with the wortmannin results indicating only partial inhibition of chemotaxis suggests that PI3K is one of several mechanisms that regulate p38 (30) and that p38 can be activated in PI3K-independent fashion (2, 12, 21, 39).

The ERK1/2 pathway has been shown to mediate CXCR3-induced chemotaxis in some cell types, e.g., hepatic stellate cells (5). However, inhibition of ERK1/2 in the present study with the use of U0126 had no effect on I-TAC-induced chemotaxis, suggesting that this pathway is not involved in CXCR3-induced chemotaxis in HAEC. ERK1/2 activation may nonetheless be important in other CXCR3-mediated responses of HAEC, e.g., cell proliferation.

To our knowledge, no prior studies have examined the effects of CXCR3 activation on JNK. I-TAC treatment did not phosphorylate JNK MAPK, suggesting that this pathway does not mediate CXCR3-induced chemotaxis in HAEC. However, JNK activation in HAEC by other CXC chemokine receptors, i.e., CXCR4, has been described (13).

In summary, our data indicate that in HAEC, CXCR3-induced chemotaxis is G protein mediated and is critically dependent on signaling by the p38 and PI3K/Akt pathways. CXCR3 agonists do not alter [Ca²⁺]_i, indicating that [Ca²⁺]_i elevation is not necessary for chemotaxis in this cell type. In contrast, the complete elimination of chemotaxis by inhibition of p38 suggests that the p38 pathway is necessary for chemotaxis induced by CXCR3. Partial inhibition of chemotaxis by inhibiting PI3K suggests that the PI3K pathway is not essential for chemotaxis but, rather, acts as one of several mechanisms by which p38 is regulated. A model depicting our thinking is shown in Fig. 6.

View larger version (9K): [in this window] [in a new window]   Fig. 6. A model of CXCR3 signaling in human airway epithelial cells. ERK1/2 and p38 MAPK and PI3K/Akt are all activated by I-TAC. However, only p38 and PI3K/Akt pathways appear to regulate chemotaxis. The p38 pathway is viewed as being essential to chemotaxis in this cell type and can be activated by both PI3K-dependent and -independent pathways. Although the PI3K-independent pathway components are unclear, the small GTPases, Cdc42, and Rho (not shown for clarity) appear to be prime candidates (2, 12, 21, 39).

	GRANTS

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This work was supported by Temple University and the Philip Morris Foundation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. O. Aksoy, 762 Parkinson Pavilion, Temple Univ. Hospital, 3401 N. Broad St., Philadelphia, PA 19140 (e-mail: mark.aksoy{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.

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