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The Journal of Immunology, 2005, 175: 5396-5402.
Copyright © 2005 by The American Association of Immunologists

Altered CXCR2 Signaling in {beta}-Arrestin-2-Deficient Mouse Models1

Yingjun Su2,{dagger}, Sandeep K. Raghuwanshi2,§,¶, Yingchun Yu{dagger}, Lillian B. Nanney{ddagger}, Ricardo M. Richardson§,¶ and Ann Richmond3,*,{dagger}

* Department of Veterans Affairs, Nashville, TN 37212; Departments of {dagger} Cancer Biology and {ddagger} Plastic Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232; § Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707; and Department of Biochemistry, Meharry Medical College, Nashville, TN 37208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1–3 and CXCL5–8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein {beta}-arrestin-2 ({beta}arr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of {beta}arr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of {beta}arr2-deficient ({beta}arr2–/–) and their wild-type ({beta}arr2+/+) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca2+ mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of {beta}arr2 resulted in increased Ca2+ mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of {beta}arr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of {beta}arr2–/– mice. Wound re-epithelialization was also significantly faster in {beta}arr2–/– mice than in {beta}arr2+/+ mice. Taken together, the data indicate that {beta}arr2 is a negative regulator for CXCR2 in vivo signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Appropriate recruitment of neutrophils in tissues is essential for host defense in infection. This is exemplified by the fact that bacterial endotoxin-induced depression of neutrophil recruitment severely compromises host defense and exacerbates the clinical outcome of patients with septic shock (1, 2, 3, 4). Persistent accumulation of neutrophils in tissues is also an important provoking factor for many diseases including cardiac vascular disease, inflammatory bowel disease, chronic wounds, and lung injury (5, 6, 7, 8, 9, 10, 11, 12). The trafficking of neutrophils after extravasation from the lumen of microvessels into peripheral tissues is based on the ability of cells to move toward the gradient of certain chemoattractants; this process is termed chemotaxis. A wealth of substances have been identified as chemotactic factors capable of initiating the chemotaxis of various cell types. Among these molecules, the bacterial-derived N-formylated peptides (fMLP), C5a, and the family of chemotactic cytokines or chemokines, which includes CXCL1–3 and CXCL5–8, have been identified as potent chemoattractants for neutrophils both in vitro and in vivo (13, 14).

Chemokines are small proteins that bind seven transmembrane G protein-coupled receptors (GPCRs),4 regulate leukocyte trafficking, and are highly important in the pathogenesis of inflammatory diseases. Based on the arrangement of the first two cysteine residues in the N terminus, chemokines can be classified into CC, CXC, CX3C, and C subfamilies (15). Chemokine receptors couple predominantly to pertussis toxin-sensitive G{alpha}i proteins to activate phospholipase C, resulting in the generation of the intracellular messengers diacylglycerol and inositol 1,4,5-triphosphate. Whereas inositol 1,4,5-triphosphate mobilizes Ca2+, diacylglycerol in combination with elevated Ca2+ activates protein kinase C (16, 17). A series of responses in GPCR internalization and recycling are crucial for receptor signaling, and various groups of GPCRs are regulated differently during these processes.

Despite being extensively studied, the mechanisms underlying the signaling and trafficking of chemokine receptors are still not fully understood. The complexity of this issue is evident as more and more adaptor/scaffold proteins are being found to associate with GPCR endocytosis, recycling, signaling, and cell chemotaxis. Arrestin, a versatile family of adaptor proteins, forms complexes with most GPCRs after agonist binding and phosphorylation of receptors by G protein-coupled receptor kinases. These complexes are important for receptor desensitization as well as downstream signaling (18). Two members of the arrestin family, {beta}-arrestin-1 ({beta}arr1) and {beta}-arrestin-2 ({beta}arr2), which share a high degree of homology, bind to the CXC chemokine receptors CXCR1 and CXCR2 and regulate cellular responses to these receptors (19, 20). We have previously shown that {beta}arr2 plays a crucial role in transducing signals of the CXCL8 receptors CXCR1 and CXCR2 (20). Clathrin-mediated CXCR2 endocytosis, followed by interaction with the Rab11-family interacting protein-2 and myosin Vb were also shown to regulate receptor recycling (21, 22). We have also shown that different domains of the C terminus of CXCR2 interact with protein phosphatase 2A, adaptin 2 (AP-2), and the heat shock cognate/heat shock protein 70-interacting protein to promote receptor sequestration, internalization, and trafficking (23, 24, 25). These processes likely play important roles in CXCR2-mediated downstream signaling.

This study was designed to further investigate the in vivo role of {beta}arr2 in CXCR2 signaling and CXCR2-mediated neutrophil recruitment into inflamed sites. We compared neutrophil activation and chemotaxis in two mouse models of inflammation on {beta}arr2+/+ and {beta}arr2–/– mice: a dorsal air pouch and an excisional punch wound. The data show that {beta}arr2 deficiency leads to enhanced CXCR2 signaling which is exemplified by a significant increase in neutrophil activation and superoxide production as well as neutrophil mobility within two differing in vivo models. The data also suggest that {beta}arr2 deficiency promotes re-epithelization in the mouse excisional wound healing model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

All experiments were approved by and confirmed to the guidelines of the Animal Care Committee of Meharry Medical College, Nashville, TN. Animals were housed five per cage in a room at 22 ± 5°C with an alternate 12-h light-dark cycle. {beta}arr2–/– mice (C57BL/6 background) were kindly provided by Dr. Robert J. Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC). Male and female mice were evaluated, and controls were age- and sex-matched littermates.

Genotyping

All mice were genotyped at the age of 3 wk; DNA samples were prepared from the tail tips with DNeasy tissue kit from Qiagen and subjected to triplex PCR as described (26, 27, 28).

Murine skin air pouch model of inflammation

Mice (6–8 wk) were anesthetized with isofluorane, and dorsal air pouches were raised by injecting 3 ml of sterile air s.c. on days 0 and 3 as described previously (29, 30, 31). On day 6 and while the mice were anesthetized with isofluorane, inflammation in the air pouch was induced by local injection of recombinant murine CXCL1 (100 pmol) dissolved in 0.5 ml of sterile PBS. Mice were sacrificed 4 h after CXCL1 injection by CO2 asphyxiation, and air pouches were lavaged three times with 3 ml of PBS. Cells were collected by centrifugation at 2000 rpm for 5 min at room temperature. The supernatants were removed, and the cells were resuspended in 10 ml of PBS and counted. Aliquots of the cell suspension were stained with Wright-Giemsa and enumerated by light microscopy (~90% of the exudates are neutrophils).

Peritoneal recruitment of leukocytes

Zymosan was prepared in PBS to a final concentration of 1 mg/ml, and 1.0 ml was injected into the peritoneum of control and {beta}arr2–/– mice. Mice were euthanized by CO2 asphyxiation, and the peritoneal cavity was lavaged at 4 h postinjection with 8 ml of ice-cold RPMI containing 2% FBS and 2 mM EDTA. Cells were collected by centrifugation, counted, and stained with Diff-Quick to assess the percentage of neutrophils.

Intracellular Ca2+ mobilization, receptor internalization, and GTPase activity

Zymosan-elicited peritoneal neutrophils (3 x 106 cells) were washed with HEPES-buffered saline and loaded with 1 µM Indo-1-acetoxymethyl ester for 30 min at room temperature. The cells were washed and resuspended in 1.5 ml of HEPES-buffered saline, and intracellular Ca2+ mobilization was measured as described (32). For receptor internalization, 5 x 105 neutrophils were resuspended in 250 µl of HBSS containing 25 mM HEPES and 0.1% BSA. Cells were treated with recombinant murine CXCL1 (100 nM) at 37°C for different time periods. Reactions were stopped by adding 1 ml of ice-cold HBSS followed by centrifugation at 1000 rpm for 2 min. Then cells were washed three times and assayed for 125I-labeled CXCL1 binding as previously described (33). For GTPase activity, cells were isolated, pooled, and frozen at –80°C until use. Membranes were prepared and assayed for GTPase activity (10 µg of membrane preparations per assay) as described (32, 34) in the presence and absence of 1 µM recombinant murine CXCL1.

Superoxide anion production

The production of superoxide anion (O2-) was measured as described previously (35). Briefly, murine peritoneal neutrophils were resuspended in physiological buffer containing 130 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 1 mM CaCl2, 5 mM glucose, and 20 mM HEPES (pH 7.4) at 1 x 107 cells/ml. Cells were seeded in 96-well plates, and then 25 µl of cytochrome c solution (type VI, 10 mg/ml) were added to the cells in the presence or absence of 20 µl of superoxide dismutase (1 mg/ml). After incubation at 37°C for 5 min, cells were stimulated with 100 nM CXCL1, RANTES, or PMA. After incubation for 60 min, the optical density of reduced cytochrome c in the supernatant was measured in a microplate reader (Bio-Rad) at 550 nm. Superoxide anion production was calculated as nanomoles of cytochrome c reduced per 106 cells with an extinction coefficient of E550 = 21 nM–1 cm–1.

Mouse skin excisional wounding procedures

Excisional punch wounds were made as described previously with slight modifications (36). Mice (6–8 wk of age and weighing 22–25 g) were anesthetized with an i.p. injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) (Fort Dodge Animal Health). The dorsal surface of the mouse was cleaned, shaved, and sterilized with betadine solution (Purdue Frederick) and 70% ethanol. Two full-thickness excisional wounds were made in the dorsal paravertebral region with a 4-mm-diameter punch (Acu-Punch). Wounds were covered and kept under occlusion by Spray Bandage (Curad; Beiersdorf). Analgesics (Buprenex, Reckitt & Colman Pharmaceuticals) were administered i.p. with at a dosage of 4.5 µg/kg body weight. All wounds were visually monitored daily for signs of infection.

Immunohistochemical assessment of neutrophil infiltration in wound tissues

Mice were sacrificed, and the wound beds with adjacent margin skin tissues were harvested at postwounding days 1, 2, and 3. The tissues were fixed in 4% paraformaldehyde (Fisher) for 24 h and embedded in paraffin blocks. Tissue sections 6-µm thick were cut through the center of wounds, placed on charged slides, and baked overnight at 50°C. After a deparaffinization procedure, sections were rehydrated in TBS-Tween 20 for 5 min before immunostaining. Endogenous peroxidase was diminished by treatment with 0.03% hydrogen peroxide and samples were treated with diluted rabbit serum before the addition of primary Ab. Granulocytes expressing Ly-6G surface markers were immunostained with anti-mouse Ly-6G mAb (BD Pharmingen) diluted 1/50 for 60 min. Sections without primary Ab served as negative controls. The Vectastain ABC Elite (Vector Laboratories) system and NovaRed chromagen (Vector Laboratories) were used to produce localized, visible staining. Slides were lightly counterstained with Mayer’s hematoxylin, dehydrated, and coverslipped for observation under the microscope. For each type of either wild-type or {beta}arr2–/– mouse, four wound samples from 2 mice were excised and analyzed for each time point after wounding. The extent of neutrophil infiltration into the wound tissues was evaluated as previously described (37, 38) on tissue slide images using Image Pro Plus software.

Myeloperoxidase (MPO) assay

MPO activity was assayed as described previously (36, 39). Briefly, wound tissues from postwounding days 1, 2, and 3 were harvested, snap frozen in liquid nitrogen, and homogenized in 1 ml of potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. Hexadecyltrimethylammonium bromide is a detergent that releases MPO from primary granules of neutrophils. After sonication on ice for 15 s (Sonic Model 300; Fisher Scientific), tissue homogenates were centrifuged at 20,000 x g for 20 min at 4°C. The supernatants were collected, and their protein concentrations were measured using the Bio-Rad protein determination assay. Aliquots of the supernatant containing equivalent amounts of 50 µg of protein were mixed with 500 µl of potassium phosphate buffer (50 mM, pH 6.0) containing 0.2% o-dianisidine hydrochloride (Sigma-Aldrich) and 0.0005% H2O2. The reaction was initiated by the addition of hydrogen peroxide. The change in OD490 during a 2-min reaction period was measured spectrophotometrically, using the Beckman-DU 7000 (Beckman Coulter). The optical densities were compared for each aliquot of wound tissue lysate from both wild-type and {beta}arr2–/– mice. The MPO values of non-wounded skin tissues from each genotype served as controls.

Wound closure analysis

Wounds were collected on postwounding days 3, 5, 7, and 10. Each wound was centrally bisected, embedded in paraffin, and sectioned at 6 µm, followed by staining with Gomori’s trichrome. For either wild-type or {beta}arr2–/– mice, eight wound tissues from four mice were excised and analyzed for each time point. The percentage of wound resurfacing was assessed by morphometric analysis (37, 38) on tissue slide images using Image Pro Plus software.

Statistical analyses

Results are expressed as mean ± SEM. Statistical differences between groups were determined by Student’s two-tailed paired t test, and the statistical difference was claimed for p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Role of {beta}arr2 in CXCR2-mediated signal transduction

Zymosan-elicited peritoneal neutrophils from {beta}arr2–/– and {beta}arr2+/+ animals were analyzed for CXCR2-mediated intracellular Ca2+ mobilization. As shown in Fig. 1A, CXCL1 induced Ca2+ mobilization in neutrophils from both {beta}arr2–/– and {beta}arr2+/+ mice. However, neutrophils from {beta}arr2–/– animals showed a more sustained response to CXCL1 than cells from {beta}arr2+/+ mice. CXCL1-induced G protein activation was also greater in membranes from {beta}arr2–/– neutrophils than those from {beta}arr2+/+ neutrophils as assessed by GTPase activity (Fig. 1B). {beta}arr2 deletion also caused a marked decrease in CXCL1-mediated CXCR2 internalization in {beta}arr2–/– mice relative to that of {beta}arr2+/+ mice (Fig. 1C).



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  		FIGURE 1. Characterization of CXCR2 in {beta}arr2–/– mice. A, For intracellular Ca2+ mobilization, zymosan-elicited peritoneal neutrophils (3 x 106 cells) were Indo-1-acetoxymethyl ester loaded and stimulated with 10 nM murine CXCL1 (MIP-2). Each tracing represents an analysis from a single mouse with the indicated {beta}arr2 genotype, and the data shown are representative of at least five each of {beta}arr2+/+ and {beta}arr2–/– animals. B, For GTPase activity, membranes were prepared from neutrophils and assayed for time-dependent CXCL1-stimulated inorganic phosphate (Pi) released. Results shown are representative of one of two experiments performed in triplicate. C, For internalization, neutrophils (0.5 x 106 cells/assay) were treated with murine CXCL1 (100 nM) at different times, washed, and assayed for 125I-labeled CXCL1 binding. The values are presented as percentage of total which is defined as the total amount of 125I-labeled CXCL1 bound to control (untreated) cells. The experiment was repeated three times with similar results.

 
To further assess the role of {beta}arr2 in CXCR2 signaling, we determined the ability of CXCL1 to mediate superoxide production in neutrophils from {beta}arr2–/– and {beta}arr2+/+ mice by measuring the optical density of superoxide dismutase-inhibitable reduction of cytochrome c (35). As shown in Fig. 2, CXCL1 induced a 68.5-fold increase in H2O2 production in neutrophils from {beta}arr2–/– mice compared with neutrophils from {beta}arr2+/+ mice (1.78 ± 0.22 vs 0.026 ± 0.021-fold over basal for {beta}arr2–/– and {beta}arr2+/+, respectively). Superoxide production in response to RANTES (CCL5 chemokine which activates CCR5) and PMA (which is independent of receptor activation) were also included as controls. CCL5 induced a greater response in {beta}arr2–/– mice (4.17 ± 0.2-fold over basal) relative to {beta}arr2+/+ (0.9 ± 0.05-fold over basal). In contrast, PMA-mediated superoxide production was similar in both {beta}arr2–/– (29.10 ± 1.42-fold over basal value) and {beta}arr2+/+ (26.4 ± 0.57-fold over basal value) neutrophils.



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  		FIGURE 2. CXCL1-induced superoxide production in neutrophils from {beta}arr2 deficient and wild-type mice. Zymosan-elicited peritoneal neutrophils (106 cells) from {beta}arr2 deficient ({beta}arr2–/–) and wild-type ({beta}arr2+/+) mice were incubated in cytochrome c solution with or without superoxide dismutase and stimulated with 100 nM murine CXCL1, CCL5, or PMA for 60 min. Superoxide production was determined by monitoring the optical density of the reduced cytochrome c in the supernatant at 550 nm as described in Materials and Methods. Data are the averaged analysis from two animals (four wounds) with the indicated {beta}arr2 genotype.

 
Role of {beta}arr2 in CXCR2-mediated neutrophil migration in air pouch model

To determine the in vivo role of {beta}arr2 in CXCR2-mediated neutrophil migration, air pouches were created in {beta}arr2–/– mice and their littermates ({beta}arr2+/+). PBS-induced neutrophil recruitment (~1.5 x 106 cells) was similar for mice of either genotype (Fig. 3). By contrast, injection of CXCL1 (100 pM) into the air pouch 6 days after it was raised induced greater neutrophil accumulation (8.26 ± 2.72 x 106 cells) in {beta}arr2–/– mice than in control {beta}arr2+/+ animals (4.04 ± 0.86 x 106 cells; Fig. 3).



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  		FIGURE 3. CXCL1-induced leukocyte migration into murine air pouch in {beta}arr2–/– and wild-type control mice. Six-day air pouches were raised in the dorsum of 6- to 8-wk-old {beta}arr2–/– mice and their littermates ({beta}arr2+/+) as described in Materials and Methods. Mice were injected with 0.5 ml of PBS or PBS containing murine CXCL1 (100 pmol). Exudates were collected after 4 h, and the total number of leukocytes (~90% neutrophils (PMN)) was enumerated. Data represent means ± SD (n = 8). ***, p < 0.0001.

 
Determination of neutrophil infiltration of wound tissues

In addition to the demonstration of differential leukocyte recruitment into chemokine concentrated sites in the air pouch model, a second in vivo mouse model was included. Neutrophil chemotaxis was evaluated in the mouse skin excisional wound healing model. In both wild-type and {beta}arr2–/– mice, the infiltration of Ly-6G-positive neutrophils was prominent within the wound bed as early as postwounding day 1 and during the early phase after wounding (Fig. 4, A and B), then decreased steadily through day 10 (Fig. 4, C and D). With higher magnification, a dense accumulation of infiltrating neutrophils in {beta}arr2–/– wound tissue was evident at the interface between the newly formed wound scab and its underlying s.c. tissue, whereas Ly-6G-positive cells scattered in wound granulation tissue (Fig. 4, E and F). By postwounding days 2 and 3, increased numbers of neutrophils had accumulated at the scab-granulation tissue interface (Fig. 4E, inset b), and Ly-6G-positive cells showed a sporadic distribution in the areas immediately adjacent to the wound bed (Fig. 4E, inset a). The leading epidermal tips advanced from wound edges toward the center of the wound through the Ly-6G-positive neutrophil-dominant zone at the scab-granulation tissue interface (Fig. 4E, inset b). This immunohistological observation indicated that the neutrophil chemotaxis in {beta}arr2–/– mice followed the identical spatial and temporal pattern observed in the excisional wound bed of the wild-type mice (Fig. 4F, insets a and b).



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  		FIGURE 4. Histological observation of excisional wounds in {beta}arr2–/– (A, C, and E) and {beta}arr2+/+ mice (B, D, and F). A and B, Day 3 excisional injuries show a wound bed filled with a provisional matrix and epithelial tips that are advancing down the sides of the crater while massive inflammatory infiltrates containing polymorphonuclear neutrophils have streamed into the defect (trichrome staining; size bars, 1100 µm). C and D, Day 10 wounds are 100% resurfaced, but the {beta}arr2–/– mouse reveals an epidermis that is greatly thickened by comparison. {beta}arr2–/– neodermis is also thickened and shows diminished cellularity that typically accompanies the resolution of the granulation tissue (trichrome staining; size bars, 50 µm). E and F, Day 2 wound edges are immunostained for neutrophils using Ly-6G antisera. Epithelial cells have begun to migrate down the side of the deficit. (size bars, 250 µm). Inset Box a shows the density of Ly-6G immunopositive cells at the dermal-s.c. interface within the adjacent tissue (size bars, 80 µm). Inset Box b shows the extreme density of Ly-6G-immunoreactive cells that have amassed at the interface between the scab and the provisional matrix of the granulation tissue nearer the central region of the excisional wound (size bar, 15 µm). *, Wound edges; arrowheads, advancing epithelial tips.

 
Neutrophil infiltration was further quantitatively determined by MPO assays performed on excisional wound tissue extracts. The data showed that the MPO activity of wild-type mouse wounds increased sharply by 24 h postwounding and was sustained over postwounding day 3 (Fig. 5). The MPO activity at 24 h after wounding (postwounding day 1) in {beta}arr2–/– mice was equivalent to that of the wild-type mice. However, by postwounding day 2, the MPO activity in wounds from the {beta}arr2–/– mice was ~1-fold higher than that of wounds from wild-type mice (p < 0.01). This was followed by a marked decrease in MPO activity to the level exhibited in wounds from wild-type mice at postwounding day 3.



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  		FIGURE 5. MPO activity assay from wound extracts. MPO activity within each wound bed was determined spectrophotometrically as described in Materials and Methods. Mean ± SEM of four wounds for each time point of each mouse genotype is shown. Statistical difference is evaluated by Student’s t test. **, p < 0.01.

 
Increased wound re-epithelialization in {beta}arr2–/– mice

The ability of wild-type and {beta}arr2–/– mice to close the wound was assessed histologically from trichrome-stained sections by comparing the percentage of wound resurfacing at different time points (Fig. 4, A and B). As shown in Fig. 6, the percentages of epidermal resurfacing for wild-type mouse wounds at days 3, 5, and 7 were 32, 38, and 72%, respectively, whereas wound resurfacing percentages from {beta}arr2–/– mice were significantly increased to 40, 61, and 89% at days 3, 5, and 7, respectively (p < 0.05 for comparison between {beta}arr2–/– and {beta}arr2+/+ mouse wounds of days 3 and 7, and p < 0.01 for comparison of wounds of day 5). Wounds from both genotypes were each completely re-epithelialized by postwounding day 10 (Fig. 4, C and D). Wound contraction rates (defined histologically as the distance between wound edges at different time points) showed no significant difference between the two mouse groups (data not shown).



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  		FIGURE 6. Comparison of wound re-epithelialization rates. The percentages of wound resurfacing at postwound days 3, 5, and 7 were 32, 38, and 72% in {beta}arr2+/+ mice, whereas that in {beta}arr2–/– mice were 40, 61, and 89%, respectively. The value of each time point was obtained from eight wounds and is expressed as mean ± SEM. *, p < 0.05; **, p < 0.01 by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
{beta}-Arrestins play a pivotal role in the termination of GPCR-mediated signaling by binding to the phosphorylated form of the receptor, thereby uncoupling the receptor from its trimeric G protein. {beta}-Arrestins also act as scaffold proteins by forming complexes with other proteins, which upon receptor activation and/or internalization activate downstream effectors (40, 41, 42). Previous studies from our laboratories and others using transfected cell lines expressing wild-type and mutant receptors have shown that CXCR2 undergoes internalization via two mechanisms: one is receptor phosphorylation/arrestin-dependent; and the other is receptor phosphorylation/arrestin independent. The later mechanism seems to be mediated by adaptor proteins such as AP-2 and heat shock cognate/heat shock protein 70 interacting protein and requires the C-terminal leucine-rich motifs of the receptors (LLKIL and KILAIH GLI) (23, 24). In this study, we sought to determine the precise role of {beta}arr2 in CXCR2-mediated leukocyte functions using a {beta}arr2–/– mouse model (28). The data herein demonstrated that {beta}arr2 negatively modulates CXCR2-mediated neutrophil activation and motility. First, upon activation by CXCL1, zymosan-elicited peritoneal neutrophils from {beta}arr2–/– displayed greater G protein activation, sustained intracellular Ca2+ mobilization, and robust superoxide production compared with {beta}arr2+/+ animals (Figs. 1 and 2). Second, {beta}arr2–/– mice showed a 2-fold increase in CXCL1-mediated neutrophil accumulation in the dorsal air pouch relative to wild-type animals (Fig. 3). Third, {beta}arr2–/– mice showed greater MPO activity at the site of the wounds (Fig. 5).

An important question addressed in this study deals with the in vivo role of receptor internalization in CXCR2-mediated leukocyte motility. Previous studies from our laboratory have shown that agonist-induced receptor internalization is necessary for CXCR2-mediated cell migration. First, in HEK293 cells expressing CXCR2, overexpression of a dominant negative mutant of dynamin blocked receptor internalization and inhibited CXCL1- and CXCL8-mediated chemotaxis (21). Second, a carboxyl tail-deleted mutant of CXCR2, 331T, resistant to receptor phosphorylation, displayed decreased chemotaxis in RBL-2H3 cell lines, relative to wild-type CXCR2 (20). In RBL-2H3 cells, the decreased chemotaxis was correlated with an increase in phosphoinositide hydrolysis and intracellular Ca2+ mobilization, suggesting that second messenger production rather than internalization plays a feedback regulatory role in chemotaxis (20, 43). The in vivo data, however, indicate that {beta}arr2 is likely involved in CXCR2-mediated neutrophil chemotaxis in a pathway that is independent of its role in receptor internalization. {beta}arr2–/– neutrophils showed a ~50% decrease in CXCL1-mediated CXCR2 internalization but a significant increase in CXCL1-induced neutrophil accumulation in the air pouch relative to wild-type animals (Figs. 1 and 3). Furthermore, in cutaneous excisional wounds, the arrestin status had an impact on two disparate aspects of wound repair. MPO activity in the wound bed as well as re-epithelialization were significantly increased in {beta}arr2–/– mice as compared with {beta}arr2+/+ mice (Figs. 5 and 6).

Lymphocytes from {beta}arr2–/– mice showed an increase in CXCR4-mediated G protein activation but a decreased chemotaxis in response to CXCL12, relative to {beta}arr2+/+ (28). These data likely indicate that {beta}arr2 uses different cellular pathways to mediate migration of lymphocytes, and these may be different in 1) CXCR4 vs CXCR2-mediated responses, 2) cell types, and 3) various assays for chemotaxis. Supporting this contention is the observation that inhibition of {beta}arr2 expression in HEK293 decreased CXCR4 mediated chemotaxis and p38MAPK activation, but not CXCR2-mediated chemotaxis (42, 44). Furthermore, in mouse embryonic fibroblast (MEF) deficient in both {beta}arr1 and {beta}arr2 ({beta}arr1/2–/–), CXCR4-mediated p38MAPK, JNK, and ERK1/2 phosphorylation were significantly attenuated, whereas CXCR2 activation produced a robust increase in MAPK activation. Interestingly, CXCR4 internalization was totally inhibited in MEF {beta}arr1/2–/– cells, whereas CXCR2 internalization was simply delayed relative to control MEF ({beta}arr1/2+/+). These data further suggest that CXCR2 internalization can occur, at least in part, independent of {beta}arr1/2.

An interesting observation is that the in vivo migration of {beta}arr2–/– neutrophils is increased relative to that of neutrophils from wild-type animals. These results contrasted with our previous in vitro studies using stably transfected RBL-2H3 cells where cells expressing CXCR2 mutant receptors deficient in internalization showed decreased chemotaxis in response to CXCL8 (20). These data likely indicate that the in vivo environment in the air pouch and the cutaneous wound healing models are different from those of the in vitro Transwell assay. Supporting that contention is the observation that zymosan-elicited peritoneal neutrophils from {beta}arr2–/– mice displayed reduced chemotaxis in vitro relative to {beta}arr2+/+ animals (data not shown).

The negative effect of arrestin in CXCR2 chemotaxis could be explained in several ways. First, the two mechanisms of internalization are additive, causing the rapid internalization of the receptor upon agonist activation (~95% in the first 2–5 min) relative to CXCR4 or CXCR1 (~50% after 30–60 min) (20). This rapid internalization diminishes signal length and, as a consequence, MAP kinase activation and chemotaxis. Inhibition of either pathway delays receptor internalization, resulting in increased intracellular signal activation in response to ligand binding and thus cell migration. Second, it is possible that only the AP-2-dependent mechanism for receptor internalization and signal amplification is required for chemotaxis. Inhibition of the arrestin-dependent pathway may promote AP-2-mediated CXCR2 internalization and signal amplification and thus chemotaxis. Third, leukocyte migration in vivo is a complex mechanism, which requires the interaction of the cells with adaptor molecules such as E-selectin for complete arrest, preceding transendothelial migration (45). CXCR2 was recently shown to mediate leukocyte arrest under conditions of vascular flow independent of E-selectin (46). Thus, the absence of {beta}-arrestins, which increases receptor activity, may increase the ability of CXCR2 to mediate neutrophil arrest and chemotaxis in vivo. A fourth explanation could be that the increase in receptor activity results in greater secretion of proteases and tissue permeability thereby facilitating cell migration.

Excisional wounding breaches the skin and represents a formidable challenge to the animal. The result is typically an immediate and intense inflammatory response. We have previously shown that deletion of the gene encoding CXCR2 or inhibition of CXCR2 function significantly delays the recruitment of neutrophils, decreases proliferation of keratinocytes, and delays wound closure in the mouse excisional wound healing model (36, 39). In the present study, deficiency in {beta}arr2 results in enhanced activation of in vivo murine CXCR2 signaling, as illustrated by higher neutrophil infiltration into wounds. This observation is consistent with the increased CXCL1-induced neutrophil chemotaxis in the air pouch model. Altogether the enhanced wound repair in the {beta}arr2–/– mice may result from the combined effects of increased CXCR2-mediated keratinocyte motility, enhanced neutrophil infiltration, as well as effects of other cell types in the wound bed. With regard to the time course of MPO data, MPO activity usually peaks around 24 h after wounding and declines thereafter. We observed that the MPO value was not significantly higher in wounds from {beta}arr2–/– mice as compared with wounds from wild-type mice at day 1. However, in wounds from {beta}arr2–/– mice, MPO activity peaked at a significantly higher level on post wound day 2 than for those from wild-type mice on either day 1 or day 2. We cannot be certain why the MPO activity remains high in {beta}arr2–/– mice on postwounding day 2. There are several possible explanations: 1) neutrophils that migrate into the wound bed in {beta}arr2–/– mice remain viable for a longer period of time, resulting in higher levels of MPO activity in the wound bed on postwound day 2; 2) neutrophils of {beta}arr2–/– mice are slower to degranulate in vivo and therefore retain higher levels of measurable MPO activity in the wound bed on postwound day 2; 3) neutrophils from {beta}arr2–/– mice exhibit higher levels of MPO activity than those from wild-type mice; or 4) neutrophils from {beta}arr2–/– mice continue to respond to the chemokine gradient for a longer period of time, peaking at day 2. We favor possibility 4, based on data from the air pouch experiments described here. Future studies will be directed toward clarifying the mechanism for enhanced/prolonged MPO activity in wounded {beta}arr2–/– mice.

An unanswered question in this study is, "What is the role played by {beta}arr1 in CXCR2-mediated chemotaxis in vivo and in wound healing." In vitro studies using mouse embryonic fibroblasts deficient in {beta}arr1, {beta}arr2, or {beta}arr1/2 have shown that CXCR2 couples to either {beta}arr1 or {beta}arr2 to inhibit CXCL8-mediated respiratory burst (44). Double knockout of {beta}arr1 and {beta}arr2 results in embryonic lethality and thus cannot be tested in in vivo wound healing models (47). Although {beta}arr1–/– mice have been generated, their genotype can be determined only by Southern blotting (51), making it difficult to conduct studies that require a large number of animals. Nonetheless, several studies have shown that there are differences between {beta}arr1 and {beta}arr2 with regard to their affinities for different GPCRs, subcellular distribution, function in GPCR recycling through the endosome, and the activation of receptor mediated downstream signaling (47). CXCR2 undergoes endocytosis in a clathrin/AP2-dependent fashion (22, 28). {beta}arr2 has 6-fold greater affinity for clathrin than {beta}arr1, and AP-2 preferentially binds to {beta}arr2 (48, 49). Therefore, it is likely that CXCR2 interacts preferentially with {beta}arr2 to mediate cellular functions. Supporting this contention is the observation that CXCR2 undergoes rapid ligand-induced endocytosis (within several minutes) with rapid recruitment of {beta}arr2 to cytoplasmic membrane (20). These features are consistent with the conclusion that for class A GPCRs, {beta}arr2 is more efficient than {beta}arr1 in binding and regulating endocytosis (50).

In summary, the data herein indicate that {beta}-arrestin-2 negatively regulates CXCR2-mediated neutrophil functions. Several questions, however, remain to be addressed. Arrestin was shown to bind to both AP-2 and the phosphorylated form of the receptor. It will be important to determine whether the receptor/AP-2/arrestin or receptor/arrestin/AP-2 complex is more important for rapid receptor internalization, polarization of intracellular signals, and optimal chemotaxis. Moreover, further studies are needed to define the functional interaction between {beta}arr2, CXCR2, and other interacting proteins to more fully target CXCR2-mediated chemotaxis in chronic inflammatory disease and in wound healing.


   Acknowledgments
 
We thank Kelly S. Parman of Mouse Pathology and IHC Core Laboratory, Vanderbilt University Medical Center, for technical support with mouse tissue processing and immunostaining. We also thank Dr. Robert J. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, for providing {beta}-arrestin-2–/– mice and for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by a Historically Black Colleges and Universities Grant (to A.R. and R.M.R.); by a Senior Career Scientist Award from the Department of Veterans Affairs (to A.R.); and by the National Institutes of Health through Skin Disease Research Center Grant 2P30 AR41943 (to L.B.N.). Back

2 Y.S. and S.K.R. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Ann Richmond, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232. E-mail address: ann.richmond{at}vanderbilt.edu Back

4 Abbreviations used in this paper: GPCR, G protein-coupled receptor; {beta}arr1, {beta}-arrestin-1; {beta}arr2, {beta}-arrestin-2; AP-2, adaptin 2; MPO, myeloperoxidase; MEF, mouse embryonic fibroblast. Back

Received for publication May 24, 2005. Accepted for publication August 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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