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Real-time imaging reveals that P2Y2 and P2Y12 receptor agonists are not chemoattractants and macrophage chemotaxis to complement C5a is phosphatidylinositol 3-kinase (PI3K)- and p38 mitogen-activated protein kinase (MAPK)-independent.

ABSTRACT: 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'-(β-thio)-diphosphate (ADPβS)). HPLC revealed that these synthetic P2Y(2) (ATPγS) and P2Y(12) (ADPβS) receptor ligands were in fact slowly degraded. We also found that ATPγS, but not ADPβS, 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.

SUBMITTER: Isfort K

PROVIDER: S-EPMC3247992 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Radiation biodosimetry based on transcriptomic analysis of peripheral blood is a valuable tool to detect radiation exposure after a radiological/nuclear event and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including inflammation, can potentially obscure the predictive power of the method. The mitogen-activated protein kinase (MAPK) p38 is normally regulated by the MAP3K-MAP2K pathway in mammalian cells. Members of the p38 MAPK family respond to pro-inflammatory signals and environmental stresses. However, in T cells there is an alternative pathway for p38MAPK activation. This pathway has been shown to play an important role in antigen-receptor-activated T cells and participate in immune and inflammatory responses. Radiation is known to induce a pro-inflammatory response. To examine the role of p38MAPK in response to radiation, we used two mouse models expressing either a p38αMAPK dominant negative (DN) mutation that would be expected to globally suppress p38MAPK signaling or a double knock-in (DKI) mutant, which would be expected to inhibit specifically T cell receptor activation. We exposed wild-type and p38MAPK mutant mice to 7 Gy x-rays and 24 h later we isolated whole blood and performed genome microarray and gene ontology analysis. Comparison between the two mutants (DN, DKI) suggest that the alternative p38MAPK pathway may have wider functions in the response to radiation exposure beyond its well established role as a downstream effector of activated T-cell receptor. Among the differentially enriched pathways, irradiation leads to a significant overrepresentation of pathways associated with morbidity and mortality as well as organismal cell death. In contrast, these pathways were significantly underrepresented in p38MAPKDN and p38MAPKDKI mutant mice, implying that p38MAPK suppression protects mice from the deleterious effects of radiation. Furthermore, radiation exposure resulted in an enrichment of a small number of phagocytosis-related pathways; however, considerably more phagocytosis-related pathways were overrepresented in the p38MAPK mutant mice, implying that p38MAPK signaling, normally, restricts phagocytosis of apoptotic cells after radiation and may, thus, contribute to persistent inflammation. Finally, despite the significant changes in gene expression, we were still able to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of p38MAPK status.

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Real-time imaging reveals that P2Y2 and P2Y12 receptor agonists are not chemoattractants and macrophage chemotaxis to complement C5a is phosphatidylinositol 3-kinase (PI3K)- and p38 mitogen-activated protein kinase (MAPK)-independent.

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