ABSTRACT
We recently found that macrophages from RhoA/RhoB double knockout mice had increased motility of the cell body, but severely impaired retraction of the tail and membrane extensions, whereas RhoA- or RhoB-deficient cells exhibited mild phenotypes. Here we extended this work and investigated the roles of Rho signaling in primary human blood monocytes migrating in chemotactic gradients and in various settings. Monocyte velocity, but not chemotactic navigation, was modestly dependent on Rho-ROCK-myosin II signaling on a 2D substrate or in a loose collagen type I matrix. Viewed by time-lapse epi-fluorescence microscopy, monocytes appeared to flutter rather than crawl, such that the 3D surface topology of individual cells was difficult to predict. Spinning disk confocal microscopy and 3D reconstruction revealed that cells move on planar surfaces and in a loose collagen matrix using prominent, curved planar protrusions, which are rapidly remodeled and reoriented, as well as resorbed. In a dense collagen type I matrix, there is insufficient space for this mode and cells adopt a highly Rho-dependent, lobular mode of motility. Thus, in addition to its role in tail retraction on 2D surfaces, Rho is critical for movement in confined spaces, but is largely redundant for motility and chemotaxis in loose matrices.
Subject(s)
Cell Movement , Monocytes/physiology , rho GTP-Binding Proteins/metabolism , Cells, Cultured , Humans , Imaging, Three-Dimensional , Microscopy, Fluorescence , Time-Lapse Imaging , rho GTP-Binding Proteins/antagonists & inhibitorsABSTRACT
Adenosine 5'-triphosphate (ATP) has been implicated in the recruitment of professional phagocytes (neutrophils and macrophages) to sites of infection and tissue injury in two distinct ways. First, ATP itself is thought to be a chemotactic "find me" signal released by dying cells, and second, autocrine ATP signaling is implicated as an amplifier mechanism for chemotactic navigation to end-target chemoattractants, such as complement C5a. Here we show using real-time chemotaxis assays that mouse peritoneal macrophages do not directionally migrate to stable analogs of ATP (adenosine-5'-(γ-thio)-triphosphate (ATPγS)) or its hydrolysis product ADP (adenosine-5'-(ß-thio)-diphosphate (ADPßS)). HPLC revealed that these synthetic P2Y(2) (ATPγS) and P2Y(12) (ADPßS) receptor ligands were in fact slowly degraded. We also found that ATPγS, but not ADPßS, promoted chemokinesis (increased random migration). Furthermore, we found that photorelease of ATP or ADP induced lamellipodial membrane extensions. At the cell signaling level, C5a, but not ATPγS, activated Akt, whereas both ligands induced p38 MAPK activation. p38 MAPK and Akt activation are strongly implicated in neutrophil chemotaxis. However, we found that inhibitors of phosphatidylinositol 3-kinase (PI3K; upstream of Akt) and p38 MAPK (or conditional deletion of p38α MAPK) did not impair macrophage chemotactic efficiency or migration velocity. Our results suggest that PI3K and p38 MAPK are redundant for macrophage chemotaxis and that purinergic P2Y(2) and P2Y(12) receptor ligands are not chemotactic. We propose that ATP signaling is strictly autocrine or paracrine and that ATP and ADP may act as short-range "touch me" (rather than long-range find me) signals to promote phagocytic clearance via cell spreading.
Subject(s)
Adenosine Triphosphate/immunology , Chemotaxis/physiology , Complement C5a/immunology , Macrophages, Peritoneal/immunology , Phosphatidylinositol 3-Kinases/immunology , Purinergic P2Y Receptor Agonists/immunology , Receptors, Purinergic P2Y12/immunology , Receptors, Purinergic P2Y2/immunology , p38 Mitogen-Activated Protein Kinases/immunology , Adenosine Diphosphate/genetics , Adenosine Diphosphate/immunology , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Autocrine Communication/drug effects , Autocrine Communication/physiology , Chemotaxis/drug effects , Complement C5a/genetics , Complement C5a/metabolism , Macrophages, Peritoneal/metabolism , Mice , Mice, Knockout , Paracrine Communication/drug effects , Paracrine Communication/physiology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Pseudopodia/genetics , Pseudopodia/immunology , Pseudopodia/metabolism , Purinergic P2Y Receptor Agonists/metabolism , Purinergic P2Y Receptor Agonists/pharmacology , Receptors, Purinergic P2Y12/genetics , Receptors, Purinergic P2Y12/metabolism , Receptors, Purinergic P2Y2/genetics , Receptors, Purinergic P2Y2/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Chemotaxis, the movement of cells along chemical gradients, is critical for the recruitment of immune cells to sites of inflammation; however, how cells navigate in chemotactic gradients is poorly understood. Here, we show that macrophages navigate in a gradient of the chemoattractant C5a through the release of adenosine triphosphate (ATP) and autocrine "purinergic feedback loops" that involve receptors for ATP (P2Y(2)), adenosine diphosphate (ADP) (P2Y(12)), and adenosine (A2a, A2b, and A3). Whereas macrophages from mice deficient in pannexin-1 (which is part of a putative ATP release pathway), P2Y(2), or P2Y(12) exhibited efficient chemotactic navigation, chemotaxis was blocked by apyrase, which degrades ATP and ADP, and by the inhibition of multiple purinergic receptors. Furthermore, apyrase impaired the recruitment of monocytes in a mouse model of C5a-induced peritonitis. In addition, we found that stimulation of P2Y(2), P2Y(12), or adenosine receptors induced the formation of lamellipodial membrane protrusions, causing cell spreading. We propose a model in which autocrine purinergic receptor signaling amplifies and translates chemotactic cues into directional motility.
