Galphai2 is required for chemokine-induced neutrophil arrest

Abstract

Chemokines, including CXCL1, participate in neutrophil recruitment by triggering the activation of integrins, which leads to arrest from rolling. The downstream signaling pathways which lead to integrin activation and neutophil arrest following G-protein-coupled receptor engagement are incompletely understood. To test whether Galpha(i2) is involved, mouse neutrophils in their native whole blood were investigated in mouse cremaster postcapillary venules and in flow chambers coated with P-selectin, ICAM-1, and CXCL1. Gnai2(-/-) neutrophils showed significantly reduced CXCL1-induced arrest in vitro and in vivo. Similar results were obtained with leukotriene B(4) (LTB(4)). Lethally irradiated mice reconstituted with Gnai2(-/-) bone marrow showed a similar defect in chemoattractant-induced arrest as that of Gnai2(-/-) mice. In thioglycollate-induced peritonitis and lipopolysaccaride (LPS)-induced lung inflammation, chimeric mice lacking Galpha(i2) in hematopoietic cells showed about 50% reduced neutrophil recruitment similar to that seen in Gnai2(-/-) mice. These data show that neutrophil Galpha(i2) is necessary for chemokine-induced arrest, which is relevant for neutrophil recruitment to sites of acute inflammation.

Figure 1

Gα_i2 and PLC in neutrophils are required for neutrophil chemokine-induced arrest on P-selectin/ICAM-1. (A) Carotid cannulas were placed in Gnai2^−/− mice (□) and Gnai2^+/+ mice (■) and connected to autoperfused flow chambers coated with P-selectin and ICAM-1 alone or in combination with CXCL1 (10 μg/mL). The wall shear stress in all flow chamber experiments was 5.94 dyn/cm². There were at least 3 mice and 4 flow chambers per group. To account for the 2.5-fold increase of neutrophils in Gnai2-deficient mice compared with littermate controls (Table 1, the number of adherent neutrophils was normalized to systemic neutrophil counts. Number of normalized adherent cells per field of view is presented as means plus or minus SEM. (B) Number of adherent neutrophils on P-selectin and ICAM-1 in combination with CXCL1 of U73122-pretreated whole blood after 6 minutes. Data presented as means plus or minus SEM from 3 mice. ^#P < .05.

Figure 2

Reduced chemokine-induced arrest in cremaster venules of Gnai2-deficient mice in vivo. (A) Number of adherent cells (normalized to blood neutrophil counts) in cremaster muscle postcapillary venules of Gnai2^−/− mice and littermate control mice after intravenous injection of 600 ng CXCL1. Data were recorded and analyzed for 1 minute starting 15 seconds after CXCL1 injection. Data presented are the means plus or minus SEM from 4 mice. (B) Representative pictures of cremaster muscle postcapillary venules of Gnai2^+/+ and Gnai2^−/− mice before and 1 minute after CXCL1 injection (leukocytes are circuited: rolling leukocytes, · · ·; arrested leukocytes, —). Scale bar equals 10 μm. (C-D) Adherent leukocytes in postcapillary venules of Gnai2^−/− mice (D) or littermate control mice (C) after injection of CXCL1. Each line represents the number of adherent leukocytes per square millimeter in 1 venule of 1 mouse after CXCL1 injection. Data presented as means plus or minus SEM from 4 mice. ^#P < .05.

Figure 3

Gnai2-deficient mice cannot induce leukocyte arrest in postcapillary venules of the cremaster muscle in response to LTB₄. Number of adherent leukocytes in cremaster muscle postcapillary venules of Gnai2^−/− mice and Gnai2^+/+ mice after intravenous injection of 5 μg LTB₄. Data were recorded and analyzed for 1 minute starting 15 seconds after CXCL1 injection. Data presented as means plus or minus SEM from 4 mice. ^#P < .05.

Figure 4

Gα_i2 in neutrophils is responsible for chemokine-induced neutrophil arrest. Number of adherent leukocytes in cremaster muscle postcapillary venules of Gnai2^−/− → Gnai2^+/+ chimeric mice and Gnai2^+/+ mice after intravenous injection of 600 ng CXCL1 (normalized for blood neutrophil counts). Data were recorded and analyzed for 1 minute starting 15 seconds after CXCL1 injection. Data presented as means plus or minus SEM from at least 4 mice.

Figure 5

Neutrophil recruitment in a model of LPS-induced lung inflammation and thioglycollate-induced peritonitis is partially dependent on Gα_i2 in neutrophils. (A) Neutrophil recruitment into the alveolar compartments of the lung with or without LPS inhalation (24 hours) was determined by flow cytometry. Gnai2^−/− mice, Gnai2^+/+ mice, and Gnai2^−/− → Gnai2^+/+ chimeric mice showed different recruitment patterns of neutrophils in the alveolar compartment (n = 4). Data are means plus or minus SEM. (B) Peritoneal neutrophil influx 4 hours after injection of 4% thioglycollate into Gnai2^−/− mice (4 mice), Gnai2^−/− mice (3 mice), Gnai2^−/− → Gnai2^+/+ chimeric mice (5 mice), and Gnai2^+/+ → Gnai2^+/+ mice (5 mice). Total number of neutrophils (× 10⁶) in the peritoneal cavity counted using Kimura-stained samples. Horizontal bars are means of 5 replicates. *P < .05 versus other groups; ^#P < .05.