Hale, M.B. & Nolan, G.P. Phospho-specific flow cytometry: intersection of immunology and biochemistry at the single-cell level. Curr. Opin. Mol. Ther. 8, 215-224

Stanford University, Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, 269 Campus Drive, Stanford, CA 94305, USA.
Current opinion in molecular therapeutics (Impact Factor: 3.42). 07/2006; 8(3):215-24.
Source: PubMed


Striving to achieve greater clinical relevance, researchers in basic science and in drug discovery are transitioning from biochemical investigations using cell lines to technologies that garner mechanistic information from primary patient material. Such studies can be broad in scope, despite limited sample material and cell-type heterogeneity. The development of flow cytometry for following intracellular signaling has met some of these demands and opened new avenues for mechanistic exploration. This review covers some of the most recent research to leverage this new technology and follows two new developments: increasing interest in JAK/STAT signaling, and experimental strategies that reveal disease-induced modulation of signaling networks.

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    • "To measure responses in coculture, we used quantitative flow cytometric analysis of protein phosphorylation at the level of individual cells. We considered this approach essential for measuring kinase activation within mixed cell populations, where the effect of interactions between different cell populations is being determined and where isolation of primary cell subpopulations might by itself alter their phenotype (26, 35). Increased levels of phospho-p38 in Gr-1high monocytes were evident at 15 min post-LPS (100 ng/ml) treatment and remained higher at 30 min (Fig. 5A, P < 0.001). "
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    ABSTRACT: Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.
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    ABSTRACT: An understanding of hematological cancer cell signaling processes poses one of the most complex and intractable problems in modern biomedical inquiry. While we understand some of the fundamental players that contribute to oncogenic processes, significant effort is focused upon determining how these individual players relay information to each other to create the composite functions of a cancer cell. Efforts designed to understand these processes at the single cell level will undoubtedly allow for understanding of the heterogeneity of hematological tumors as well as, simultaneously, the function of the 'responding' immune system. I will relate some of the insights our laboratory has developed over the last several years applying single-cell phospho-flow cytometry to the study of signaling in primary patient material and murine models. While it is clear that this analysis now allows us to accomplish phospho-signaling biochemistry at the single cell level with primary cell material, we are only beginning to develop some of the bioinformatics tools to appropriately display the vast amount of information collected by such approaches. These approaches, however, have already allowed us to develop approaches that prognosticate patient outcomes based on signaling status, prior to any treatment, as well as subgroup patient subtypes according to signaling states. The modest efforts to date presage a time where it should be possible to provide far more tailored therapies specific to the varied diseases represented by the hematological malignancies.
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