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. 2005 Oct 15;175(8):5396-402.
doi: 10.4049/jimmunol.175.8.5396.

Altered CXCR2 signaling in beta-arrestin-2-deficient mouse models

Yingjun Su  1 , Sandeep K Raghuwanshi, Yingchun Yu, Lillian B Nanney, Ricardo M Richardson, Ann Richmond
Affiliations

Altered CXCR2 signaling in beta-arrestin-2-deficient mouse models

Yingjun Su et al. J Immunol. .

Abstract

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 (betaarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of betaarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of betaarr2-deficient (betaarr2(-/-)) and their wild-type (betaarr2(+/+)) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca(2+) mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of betaarr2 resulted in increased Ca(2+) 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 betaarr2 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 betaarr2(-/-) mice. Wound re-epithelialization was also significantly faster in betaarr2(-/-) mice than in betaarr2(+/+) mice. Taken together, the data indicate that betaarr2 is a negative regulator for CXCR2 in vivo signaling.

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Figures

FIGURE 1
FIGURE 1
Characterization of CXCR2 in βarr2−/− mice. A, For intracellular Ca2+ mobilization, zymosan-elicited peritoneal neutrophils (3 × 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 βarr2 genotype, and the data shown are representative of at least five each of βarr2+/+ and βarr2−/− 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 × 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.
FIGURE 2
FIGURE 2
CXCL1-induced superoxide production in neutrophils from βarr2 deficient and wild-type mice. Zymosan-elicited peritoneal neutrophils (106 cells) from βarr2 deficient (βarr2−/−) and wild-type (βarr2+/+) 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 βarr2 genotype.
FIGURE 3
FIGURE 3
CXCL1-induced leukocyte migration into murine air pouch in βarr2−/− and wild-type control mice. Six-day air pouches were raised in the dorsum of 6- to 8-wk-old βarr2−/− mice and their littermates (βarr2+/+) 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.
FIGURE 4
FIGURE 4
Histological observation of excisional wounds in βarr2−/− (A, C, and E) and βarr2+/+ 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 βarr2−/− mouse reveals an epidermis that is greatly thickened by comparison. βarr2−/− 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.
FIGURE 5
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.
FIGURE 6
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 βarr2+/+ mice, whereas that in βarr2−/− 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.
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