Autocrine control of neutrophil chemotaxis

趋化性允许多形核中性粒细胞（PMN）迅速到达感染和炎症部位。尽管我们对趋化性的理解最近取得了进展，但仍然存在许多悬而未决的问题。趋化剂刺激PMN释放ATP，并通过嘌呤能受体自分泌反馈回路控制趋化。在这个项目中，我们修改了提出的工作范围，以了解趋化信号如何诱导ATP释放，以及由释放的ATP形成的腺苷如何控制负反馈回路，负反馈回路在定义细胞极性和尾足缩回中起关键作用。拟议的项目基于以下修订的工作假设：化学引诱剂受体的刺激诱导快速细胞内事件，导致ATP释放。额外的ATP释放通过不同的机制提供配体，腺苷，抑制A2a腺苷受体。这些受体增加后退边缘的cAMP水平，提供抑制反馈回路，引发全局抑制，促进尾足收缩，确定细胞极性并促进迁移。我们建议通过以下修订后的具体目标来检验这一工作假设：

Chemotaxis allows polymorphonuclear neutrophils (PMN) to rapidly reach infected and inflamed sites. Despite recent progress in our understanding of chemotaxis, many open questions still remain. Chemoattractants stimulate the release of ATP from PMN and autocrine feedback loops via purinergic receptors control chemotaxis. In this project we revised the the scope of the proposed work to understand how the chemotactic signals induce ATP release and how adenosine formed from the released ATP controls negative feedback loops that play a critical role in defining cell polarity and uropod retraction. The proposed project rests on the following revised working hypothesis: Stimulation of chemoattractant receptors induces rapid intracellular events that result in ATP release. Additional ATP release through distinct mechanisms provides the ligand, adenosine, for suppressive A2a adenosine receptors. These receptors increases cAMP levels at the receding edge, providing an inhibitory feedback loop that elicits global inhibition and promotes uropod contraction, defining cell polarity and promoting migration. We propose to test this working hypothesis in by addressing the following Revised Specific Aims: Specific Aim 1. Upstream events leading to ATP release We will determine the upstream signaling pathways that link chemotactic receptor activation and ATP release during gradient sensing. In addition, we will examine different ATP release mechanisms and their contributions to ATP release during gradient sensing, cell polarization, and migration. Emphasis will be placed on the signaling pathway downstream of chemotactic receptors e.g., calcium signaling and MAPK activation that lead to the opening of ATP release channels such as connexins or pannexins and release of ATP via vesicular transport. Specific Aim 2. Global inhibition and purinergic signaling We will study if and how purinergic receptors, e.g., A2a or A2b adenosine receptors induce global inhibition that defines polarity and promotes cell migration. Emphasis will be placed on the suppressive down-stream signaling pathways that elicit global inhibition. We will focus on activation of adenylate cyclases, PKA, and cAMP accumulation. In addition, we will examine how these signaling events block gradient sensing and induce Rho activation and myosin formation that induces uropod contraction at the receding edge. Knowledge of these mechanisms will further define the purinergic mechanisms that control chemotaxis and may allow us to conceive novel therapeutic strategies aimed at treating these purinergic responses to treat inflammatory and infectious diseases.