Galphai2-mediated signaling events in the endothelium are involved in controlling leukocyte extravasation

Abstract

The trafficking of leukocytes from the blood to sites of inflammation is the cumulative result of receptor-ligand-mediated signaling events associated with the leukocytes themselves as well as with the underlying vascular endothelium. Our data show that Galpha(i) signaling pathways in the vascular endothelium regulate a critical step required for leukocyte diapedesis. In vivo studies using knockout mice demonstrated that a signaling event in a non-lymphohematopoietic compartment of the lung prevented the recruitment of proinflammatory leukocytes. Intravital microscopy showed that blockade was at the capillary endothelial surface and ex vivo studies of leukocyte trafficking demonstrated that a Galpha(i)-signaling event in endothelial cells was required for transmigration. Collectively, these data suggest that specific Galpha(i2)-mediated signaling between endothelial cells and leukocytes is required for the extravasation of leukocytes and for tissue-specific accumulation.

Fig. 1.

Allergen-induced eosinophil accumulation uniquely relies on Gα_i2 signaling mechanisms that are independent of cell autonomous events in eosinophils. OVA-induced eosinophil accumulation in the airway lumen of Gα_i2^−/− (A), but not Gα_i3^−/− (B), mice was significantly lower (∗, P < 0.05) relative to wild-type animals (n = 8 mice per group). (C) Eosinophil transwell chemotaxis assays demonstrated that in the absence of Gα_i2 signaling events, in vitro eosinophil migration to recombinant mouse eotaxin-1 was the same, if not nominally higher, relative to wild type. (D) PTX pretreatment of eosinophils before the transwell chemotaxis assay showed that the blockade of all Gα_i-signaling events abolished eotaxin-1-induced chemotaxis, thus demonstrating that the CCR3 receptor-mediated eosinophil chemotaxis occurring in the absence of Gα_i2 results exclusively from signaling events using the remaining PTX-sensitive Gα subunits, Gα_i1 and/or Gα_i3.

Fig. 2.

Gα_i2 signaling in a non-lymphohematopoietic compartment(s) in the lung is necessary for eosinophil accumulation after allergen provocation. (A) Adoptive transfer of eosinophils into OVA-treated wild-type recipients (n = 8–10 mice per group) demonstrated that Gα_i2^−/− eosinophils have an increased ability to traffic to both the airway lumen and the peribronchial areas of the lung. In contrast, the reciprocal transfer of wild-type eosinophils into OVA-treated Gα_i2^−/− recipients demonstrated that eosinophil accumulation depended on lung-associated Gα_i2 signaling. Ø, transfer of PBS vehicle alone. ∗, P < 0.05; †, significantly different (P < 0.05) from all other OVA groups examined. (B) Bone marrow engraftment of wild type recipients (Donor Marrow) demonstrated that the lack of eosinophil recruitment to the lung observed in Gα_i2^−/− mice (Non-Irradiated) was a consequence of a Gα_i2 signaling event(s) in a lung structural cell type(s); n = 10–12 mice per group, ∗, P < 0.05. (C) OVA-induced pulmonary Th2 cytokines and IFN-γ levels in Gα_i2^−/− mice are unaffected relative to OVA-treated wild type (Non-Irradiated). Moreover, OVA-induced Th2 cytokine and IFN-γ levels in OVA-treated mice after engraftment of Gα_i2 marrow into wild-type recipients was also unaffected relative to OVA-treated wild-type controls (Donor Marrow). All values presented are means ± SEM (n = 8 mice per group).

Fig. 3.

The loss of Gα_i2 signaling in knockout mice leads to nonspecific increases in all circulating white blood cell types and severely limits LPS-induced airway neutrophil accumulation. (A) Increase in all white blood cell types is observed in both allergen-naïve and OVA-treated Gα_i2^−/− mice. The data presented represent means ± SEM (n = 5 mice per group). ∗, significantly different (P < 0.05) from wild-type saline control mice. †, significantly different (P < 0.05) from OVA-treated wild-type mice. (B) LPS administered to Gα_i2^−/− or Gα_i3^−/− mice (n = 7–10 mice per group; wild-type animals served as negative controls) showed that the induced BAL neutrophil levels 16 h after administration were significantly decreased in Gα_i2^−/− mice but unaffected in Gα_i3^−/− animals. ∗, P < 0.05. (C) Transwell chemotaxis assays demonstrated that in the absence of Gα_i2 signaling events, in vitro neutrophil migration to MIP-2 was not lower but instead nominally higher relative to wild type.

Fig. 4.

The loss of Gα_i2 signaling in the vascular endothelium leads to the accumulation of immobilized cells in postcapillary venules through a blockade of diapedesis. (A) Intravital photomicroscopy of LPS-exposed mesentery postcapillary venules showed that the loss of Gα_i2 resulted in a significant increase of stationary leukocytes adherent to the vascular endothelium. Numbered white arrows indicate individual rolling leukocytes and black arrows identify stationary leukocytes. The Gα_i-dependence of lymphocyte binding and migration through an endothelial cell monolayer was assessed by using an ex vivo parallel plate flow chamber under conditions of laminar flow comparable to pressures observed in postcapillary venules [i.e., 2 dynes (1 dyne = 10 μN)/cm²]. (B) VCAM-1-dependent firm adhesion was blocked by antibodies against either endothelial VCAM-1 or lymphocyte associated α₄-integrin but was unaffected by PTX pretreatment of the endothelial cell monolayer. ∗, significantly different (P < 0.05) from no-antibody control group. (C) Lymphocyte migration through an endothelial cell monolayer is blocked in a concentration-dependent fashion by pretreatment of the endothelial cells with PTX. “No treatment,” endothelial cells treated with PBS. ∗, significantly different (P < 0.05) from PBS-treated control group.