Gαi2 Is the Essential Gαi Protein in Immune Complex–Induced Lung Disease

Abstract

Heterotrimeric G proteins of the Gαi family have been implicated in signaling pathways regulating cell migration in immune diseases. The Gαi-protein–coupled C5a receptor is a critical regulator of IgG FcR function in experimental models of immune complex (IC)–induced inflammation. By using mice deficient for Gαi2 or Gαi3, we show that Gαi2 is necessary for neutrophil influx in skin and lung Arthus reactions and agonist-induced neutrophilia in the peritoneum, whereas Gαi3 plays a less critical but variable role. Detailed analyses of the pulmonary IC-induced inflammatory response revealed several shared functions of Gαi2 and Gαi3, including mediating C5a anaphylatoxin receptor–induced activation of macrophages, involvement in alveolar production of chemokines, transition of neutrophils from bone marrow into blood, and modulation of CD11b and CD62L expression that account for neutrophil adhesion to endothelial cells. Interestingly, C5a-stimulated endothelial polymorphonuclear neutrophil transmigration, but not chemotaxis, is enhanced versus reduced in the absence of neutrophil Gαi3 or Gαi2, respectively, and knockdown of endothelial Gαi2 caused decreased transmigration of wild-type neutrophils. These data demonstrate that Gαi2 and Gαi3 contribute to inflammation by redundant, overlapping, and Gαi-isoform–specific mechanisms, with Gαi2 exhibiting unique functions in both neutrophils and endothelial cells that appear essential for polymorphonuclear neutrophil recruitment in IC disease.

Insulin secretion stimulated by l-arginine and its metabolite l-ornithine depends on Gαi2

“…Our detailed analysis of G protein levels in the G␣ i2 genetargeted mouse lines corroborates significant upregulation of G␣ i3 (about 45%) in the global G␣ i2 -deficient mice, which has also been described for other tissues and organs (6,17,39,40). In contrast to the global G␣ i2 -deficient islets, the G␣ i3 protein levels were unchanged in G␣ i2 ␤cko islets.…”

“…The generation and characterization of global G␣ i2 -deficient (G␣ i2 Ϫ/Ϫ ) mice has been described (27,29,39). The ␤-cellspecific G␣ i2 -deficient mice, termed G␣ i2 ␤cko (genotype: G␣ i2 fl/fl ;Rip-Cre ϩ/tg ), were generated by crossing Rip-Cre mice (12) with mice carrying floxed G␣ i2 alleles (27).…”

“…The generation of the global G␣i2-deficient mice (G␣i2 Ϫ/Ϫ ) has been described previously (29). To prolong life expectancy of the G␣i2 Ϫ/Ϫ animals, mice were bred and kept in individually ventilated cages (IVC) under specific-pathogen-free conditions (39) and had free access to water and standard chow. ␤-Cellspecific deletion of G␣ i2 (G␣i2 ␤cko ) was achieved by crossing the floxed G␣i2 mouse line (27) and the Rip-Cre ϩ/tg mouse line (12), both on a C57BL/6N background.…”

“…To achieve electrophoretical separation of G␣ i isoforms, separation was performed in gels containing 6 M urea. The proteins were visualized by immunodetection using the following primary antibodies described elsewhere (7,10,21,39): rabbit anti-G␣ i2 (1:8,000 islets, 1:20,000 hypothalamus), rabbit anti-G␣i3 (1:5,000), rabbit anti-G␣o1/o2 (1:2,000), and rabbit anti-G␤common (1:9,000). Antibodies against G␣s(short)/s(long) (1:1,000; SC-383) and G␣q (1:1,000; SC-393) were purchased from Cell signaling.…”

Endothelial LSP1 Modulates Extravascular Neutrophil Chemotaxis by Regulating Nonhematopoietic Vascular PECAM-1 Expression

“…After endothelial cells were treated with or without GATA-2-specific or nontargeting silencing RNA for 24 h, neutrophil transendothelial migration was examined as previously described (39,40) with minor modification. In brief, freshly isolated murine neutrophils were suspended at 5 3 10 6 cells/ml in DMEM medium supplemented with 5% FBS.…”