Control Of G Protein Signaling: Role Of The RGSs

中性粒细胞RGS/Galphai2的相互作用限制了CXCR4介导的骨髓保留信号，这是正常的跨内皮和间质迁移所必需的。炎症性损伤调动BM中性粒细胞进入循环系统。为了离开血液进入炎症部位，中性粒细胞与毛细血管后内皮细胞（ECs）形成一系列粘附相互作用，最终通过跨内皮迁移（TEM）破坏内皮。先前的研究表明，EC结合的化学引诱剂与中性粒细胞g蛋白偶联受体（gpcr）结合，激活异三聚体Gi蛋白，从而触发中性粒细胞迁移所需的细胞内信号级联反应。g蛋白信号（RGS）蛋白的调节因子通过加速Gi的内在GTPase活性来影响GPCR信号的开始和持续时间。为了了解它们在中性粒细胞炎症反应中的重要性，我们使用了Gi2蛋白不再结合RGS蛋白的小鼠（G184S KI小鼠）以及四种不同的成像平台。首先，我们使用双光子活体显微镜对骨髓中性粒细胞的动员进行成像。骨髓显像研究显示G184S小鼠骨髓生态位扩大，G184S KI细胞的整体运动性增强。静脉注射趋化因子KC可调动对照中性粒细胞，但调动不佳的G184S KI中性粒细胞。CXCR4拮抗剂AMD3100也不能正确调动G184S KI中性粒细胞。然而，AMD3100和KC联合使用能够调动G184S KI中性粒细胞。因此，过度保留和动员不良都是中性粒细胞从骨髓中释放不良的原因。这证明了RGS蛋白在骨髓中性粒细胞的释放和保留中的功能作用。接下来，我们用野生型或G184S KI骨髓重建小鼠的肺成像。我们惊讶地发现血管内的中性粒细胞池扩大了。同样，当我们对重组小鼠的肝脏成像时，我们发现了一个扩大的G184S KI中性粒细胞池。我们用流式细胞术证实了外周中性粒细胞群体的扩大。因此，尽管中性粒细胞减少，在G184S KI小鼠中，中性粒细胞边缘池扩大。接下来，我们建立了一个四维活体成像系统来评估肌细静脉和周围组织中中性粒细胞的分布和动态。我们通过局部注射il -1 β诱导急性炎症，并用荧光标记的PECAM-1和Gr-1抗体分别标记cremaster血管和内源性中性粒细胞。我们观察到WT中性粒细胞迅速粘附在发炎的小静脉上，并在相邻的ECs之间进行了透射电镜检查。相比之下，不仅G184S KI中性粒细胞较少；但那些确实出现的，在它们挣扎着迁移时，在发炎的小静脉中积累起来。在间质迁移过程中，G184S KI没有适当地极化和保持前缘。最后，我们用刀豆蛋白A诱导小鼠炎症，在G184S KI骨髓重建小鼠中诱导多系统血管炎，而野生型小鼠则很少或没有血管炎。这些结果表明，中性粒细胞转运的各个方面都需要中性粒细胞RGS蛋白，而中性粒细胞中RGS蛋白的丢失会导致血管损伤，这是由于衰老的中性粒细胞清除不当造成的。

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 four 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. The CXCR4 antagonist AMD3100 also did not properly mobilize the G184S KI neutrophils. However, the combination of AMD3100 and KC was able to mobilize the G184S KI neutrophils. Thus, both excessive retention and poor mobilization accounts for the poor neutrophil release from the bone marrow. This argues for functional roles for RGS proteins both in the release and retention of bone marrow neutrophils. Next, we imaged the lung of mice reconstituted with either wild type or G184S KI bone marrow. We were surprised to find an expanded intravascular pool of neutrophils. Similarly, when we imaged the liver of the reconstituted mice, we found an expanded pool of G184S KI neutrophils. We confirmed the expanded populations of peripheral neutrophils by using flow cytometry. Thus, despite neutropenia, the marginated pool of neutrophils is expanded in the G184S KI mice. Next, 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. Those G184S KI that did transmigrate failed to properly polarize and maintain a leading edge during interstitial migration. Finally, we induced inflammation by treating the mice with Concanavalin A, which induced a multisystem vasculitis in the G184S KI bone marrow reconstituted mice, whereas the wild type mice had minimal or no vasculitis. These results indicate that neutrophil RGS proteins are needed for all aspects of neutrophil trafficking and that the loss of RGS proteins in neutrophil results in vascular damage due to the improper clearance of aging neutrophils. Transendothelial and interstitial lymphocyte 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. Establishment of methodology to assess F-actin and myosin IIA dynamics during B cell and neutrophil migration. Using a flow-based assay, we examined the chemoattractant mediated signaling pathway that induced F-actin formation in resting B cells. F-actin levels increased with 5 seconds and peaked at 20 seconds after CXCL13 exposure. F-actin formation depended upon increases in both branched and linear F-actin, Galpha i protein nucleotide exchange, Dock2, and Rac activation. Inhibiting Erk or Src activation, or Myosin IIA reduced, but did not eliminate CXCL13 induced F-actin. Inhibiting Gbeta/gamma signaling slightly reduced the induction of F-actin, while inhibition of BTK or RhoA had no effect. Activated Galpha i recruited Elmo1 to the plasma membrane. These studies support a prominent role for a Galpha i/Elmo1/Dock2/Rac/Wave regulatory complex signaling pathway in the initial F-actin response of B cells to CXCL13. Using ICAM-1 and chemoattractant coated imaging chambers along with an agarose overlay, we imaged the motility of LifeAct GFP and Myosin IIA-GFP expressing B-lymphocytes and neutrophils. In the under agarose assays the neutrophils migrated much more vigorously than did the B cells, however, a 6 h exposure to LPS significantly improved B cell migration, while a 24-48 h exposure dramatically altered B cell migration, not only did the 48 hour activated B cells migrate more quickly, but they developed multiple, transient lamellipodia. In both B cells and neutrophils F-actin accumulated at the leading cell edge and in the pseudopods of the migrating cells. Inactivating Galpha i nucleotide exchange markedly impaired the motility of both the nave and 6 h LPS activated B cells, however, it had only modestly impacted the 48 h LPS activated B cells. Myosin IIA weakly accumulated at the leading edge and strongly in the pseudopods. Myosin IIA also accumulated on the lateral edge of turning cell on the side opposite the direction of the turn. Intravital imaging of myosin IIA-GFP B cells confirmed the dynamic behavior of Myosin-IIA during transendothelial and interstitial migration of B cells in vivo. These studies are extending of the signaling pathways and molecules that control B cell movement 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 developin