Control Of G Protein Signaling: Role Of The RGSs

National Institute Of Allergy And Infectious Diseases

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Abstract

Neutrophil RGS/Galphai2 interactions limit CXCR4 mediated bone marrow retention signals and are needed for normal transendothelial and interstitial migration. Inflammatory insults mobilize BM neutrophils into the circulatory system. To leave the blood to enter inflammatory sites, neutrophils form a series of adhesive interactions with postcapillary endothelial cells (ECs), ultimately breaching the endothelium via transendothelial migration (TEM). Previous studies have shown that EC bound chemoattractants engage neutrophil G-protein-coupled receptors (GPCRs) activating heterotrimeric Gi proteins, which trigger intracellular signaling cascades necessary for neutrophil migration. Regulators of G-protein Signaling (RGS) proteins affect the onset and duration of GPCR signaling by accelerating the intrinsic GTPase activity of Gi. To understand their importance during neutrophil inflammatory responses, we used mice whose Gi2 proteins no longer bind RGS proteins (G184S KI mice) along with two different imaging platforms. First, we imaged the mobilization of neutrophils from the bone marrow using two photon intravital microscopy. The bone marrow imaging studies revealed an expanded bone marrow niche in the G184S mice and an overall enhanced motility of G184S KI cells. Intravenous injection of the chemokine KC mobilized control neutrophils, but poorly mobilized G184S KI neutrophils. However, the CXCR4 antagonist AMD3100 did mobilize the G184S KI neutrophils arguing that an excessive CXCR4 retention signal limits their mobilization. Second, we established a four-dimensional intravital imaging system to assess the profile and dynamics of neutrophils in the cremaster muscle venules and surrounding tissues. We induced acute inflammation by local injection of IL-1beta and labeled the cremaster vasculature and endogenous neutrophils with fluorescently-labeled PECAM-1 and Gr-1 antibodies, respectively. We observed that WT neutrophils rapidly adhered to the inflamed venules and underwent TEM between adjacent ECs. In contrast, not only did fewer G184S KI neutrophil arrive; but those that did appear, accumulated within the inflamed venules as they struggled to transmigrate. Finally, those G184S KI that did transmigrate failed to properly polarize and maintain a leading edge during interstitial migration. These results indicate that neutrophil RGS proteins are needed for all aspects of neutrophil trafficking from the bone Galphai signaling promotes marginal zone B cell development by enabling transitional B cell ADAM10 expression. The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. In this study, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development. Chronic excessive S1PR1 signaling in B cells disrupts chemokine receptor signaling and the organization of lymphoid organs. Ligand engaged chemoattractant receptors trigger Gaphai subunit nucleotide exchange stimulating downstream effector molecules. Activated receptors also dock G-protein-coupled receptor kinases (GRKs) that mediate receptor desensitization. Adequate B lymphocyte homing into lymph nodes (LNs) depends upon GRK2 tempering Sphingosine-1 phosphate receptor 1 (S1PR1) signaling. We have shown that B cell loss of GRK2 also affects chemokine receptor signaling, which can be rescued by treating with an S1PR1 antagonist. The disrupted S1PR1 and chemokine receptor signaling leads to splenomegaly, yet a shrunken white pulp; small LNs underpopulated with B cells, and nearly absent Peyers patches. Poor homing and accelerated egress accounts for the lack of mature B cells in LNs and bone marrows. Intravital imaging revealed GRK2 deficient B cells adherent to high endothelial venules, yet unable to complete transmigration. Treating Grk2-/-mb1-cre mice with a S1PR1 antagonist partially reversed the severe defects in B cell trafficking. The Grk2-/-mb1-cre mice also have an abnormal distribution of B1 cells, which exhibit altered responses to chemokines and S1P. These findings confirm and extend GRK2 roles in regulating B cell chemoattractant receptor signaling and trafficking. In vivo actin dynamics during lymphocyte transendothelial and interstitial migration revealed by intravital microscopy. Actin is essential for many cellular processes including cell motility. Yet F-actin dynamics during lymphocyte transendothelial migration (TEM) and interstitial migration have not been visualized. We used high-resolution confocal intravital imaging with LifeAct-GFP bone-marrow reconstituted mice, which allowed visualization of lymphocyte F-actin dynamics in vivo. We found that nave lymphocytes preferentially cross high endothelial venules (HEVs) using the paracellular rather than the transcellular route. During both modes of transmigration F-actin levels rise at the lymphocyte leading edge as the cell engages the TEM site. Once the lymphocytes breach the endothelium, they briefly reside in HEV pockets prior to crossing into the parenchyma. During interstitial migration dynamic actin-based protrusions rapidly form and collapse to help drive motility. Using a panel of inhibitors, we established roles for actin regulators and myosin II in lymphocyte TEM. This study provided unprecedented views of lymphocyte TEM and interstitial migration in vivo. CXCR4 anchors pre-selection thymocytes to the thymic cortex. Pre-selection thymocytes are normally retained in the thymic cortex, but the mechanisms responsible for their retention remain incompletely understood. Deletion of genes encoding the E-protein transcription factors E2A and HEB disorders chemokine receptor expression on developing thymocytes and results in escape of pre-selection TCRbeta-CD8+ thymocytes into the periphery. This study shows that CXCR4 expression anchors pre-selection thymocytes to the thymic cortex via interaction with its ligand CXCL12 on cortical thymic epithelial cells, and that disruption of CXCR4-CXCL12 engagements releases pre-selection thymocytes from the thymic cortex. CXCR4 expression must be extinguished during positive selection to allow free migration of TCR-signaled thymocytes out of the thymic cortex into the thymic medulla. Thus, E-protein transcription factors regulate the ordered expression pattern of chemokine receptors on developing thymocytes and it is the interaction of the chemokine receptor CXCR4 with its ligand that retains pre-selection thymocytes in the thymic cortex. This was collaborative study with Dr. Alfred Singers laboratory (NCI).