Krutzik, P.O., Hale, M.B. & Nolan, G.P. Characterization of the murine immunological signaling network with phosphospecific flow cytometry. J. Immunol. 175, 2366-2373

Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, Stanford, CA 94305, USA.
The Journal of Immunology (Impact Factor: 4.92). 09/2005; 175(4):2366-73. DOI: 10.4049/jimmunol.175.4.2366
Source: PubMed


The immune system is a multitiered network that at the first level uses changes to intracellular signaling proteins to commit cells to determined fates. At the second tier, cells interact with one another via specifically expressed surface receptors and their cognate signaling molecules. At the third level, the local environments of immune cells change the outcomes of intracellular signaling pathways and thereby the role of cells during immune challenge. The interplay among these three tiers allows the distinct cell types of the immune system to respond cohesively to eliminate foreign Ags. In this study, using phosphospecific flow cytometry, we analyze elements of these network tiers by generating profiles of single-cell phosphoprotein responses in B cells, T cells, and myeloid cells to a number of mechanistically and clinically relevant cytokines (IFN-gamma, GM-CSF, IL-2, and IL-10) as well as LPS at key regulatory interfaces (Jak-Stat and MAPK pathways). The stimuli typically induced phosphorylation of specific signaling pathways and exerted their effects on distinct subsets of immune cells. However, upon comparison of stimulation in vitro and in vivo, we noted that signaling pathway specificity and cell type specificity were influenced strongly by the external environment. When taken from the in vivo environment, certain cell subsets became hypo- or hyper-responsive, showed profound differences in sensitivity to cytokine levels, or displayed altered phosphorylation kinetics. Thus, simultaneous analysis of the three tiers of the immune system network illustrates the principles by which immune regulation is context dependent and how in vitro culture systems compare with the in vivo environment.

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Available from: Matthew B Hale, Jan 25, 2014
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    • "In contrast, positive controls in each experiment, namely, IFNγ and GM-CSF induced pSTAT1 (Figure 2C and 2E) and pSTAT5 (Figure 2D and 2F), respectively, had higher levels of pSTAT1 or pSTAT5 compared to unstimulated samples. It is to be noted that pSTAT5 is predominantly activated in T cells in response to IL-2 [26] and in myeloid cells in response to GM-CSF [27]. Therefore, we observed only a modest increase in pSTAT5 levels upon GM-CSF treatment compared to unstimulated B cells. "
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