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.

Download full-text


Available from: Matthew B Hale, Jan 25, 2014
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes - predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.
    PLoS ONE 08/2014; 9(8):e105197. DOI:10.1371/journal.pone.0105197 · 3.23 Impact Factor
  • Source
    • "To enumerate CD34+ cells, we used an established multiparameter gating strategy as previously described [10]. The expression of phosphorylated ERK 1/2 or p38 MAPK was calculated using an expression index of the specific median fluorescence intensity (sMFI): sMFIstimulated cells/sMFIunstimulated cells [23]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development. We have previously shown that fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies grow from cord blood (CB) of high-atopic risk infants, compared to low-atopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13) could represent an underlying mechanism to explain our previous findings. CB CD34+ cells from healthy donors were cultured with IL-4 or IL-13 (in combination with LPS) and assessed for Eo/B differentiation using methylcellulose cultures and flow cytometry for related intracellular signalling pathways. Pharmacological inhibitors were added to the methylcellulose cultures to determine the effect of blocking intracellular signalling in CB CD34+ cells in relation to Eo/B colony forming unit (CFU) formation. Stimulation of CD34+ cells with IL-4, but not IL-13, reduced Eo/B CFU formation in the presence of LPS; this was found to be dependent on IL-4Rα and not IL-13Rα1. Additionally, IL-4 reduced the expression of ERK 1/2 after LPS stimulation, which was recovered by inhibition of IL-4Rα. While IL-13 did not have an inhibitory effect on ERK 1/2 expression, inhibition of ERK 1/2 significantly reduced Eo/B CFU formation. Thus, the responsiveness of CB CD34+ progenitor cells to LPS is differentially regulated by the TH2 cytokines, IL-4 and IL-13. This may have implications for in utero interactions between placental-derived pro-allergic cytokines and neonatal progenitor cells influencing Eo/B-mediated inflammatory responses in early life.
    PLoS ONE 02/2014; 9(6):e100734. DOI:10.1371/journal.pone.0100734 · 3.23 Impact Factor
  • Source
    • "Myeloid differentiation is regulated by a complex interaction of multiple cytokines. It has been reported that following the engagement of a cytokine to its cognate receptor, a number of intracellular signal transduction proteins become activated, including the Ca 2þ ‐dependent kinases [Cullen and Lockyer, 2002; Irish et al., 2004; Krutzik et al., 2005; Paredes‐Gamero et al., 2012]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Myeloid differentiation is a complex process whereby mature granulocytes or monocytes/macrophages are derived from a common myeloid progenitor through the coordinated action of hematopoietic cytokines. In this study, we explored the role of the Ca(2+) i signaling transduction pathway in the commitment of hematopoietic stem/progenitor cells to either the monocytic or granulocytic lineage in response to macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). M-CSF and G-CSF induce cell expansion and monocyte or granulocyte differentiation, respectively, without affecting the percentage of hematopoietic progenitor cells. Colony-forming units (CFUs) and flow cytometry demonstrated the involvement of phospholipase Cγ (PLCγ) and protein kinase C (PKC) in monocyte/granulocyte commitment. In addition, using flow cytometry and RNA interference, we identified PLCγ2 as the PLCγ isoform that participates in this cell expansion and differentiation. Differences in signaling elicited by M-CSF and G-CSF were observed. The M-CSF-related effects were associated with the activation of ERK1/2 and nuclear factor of activated T-cells (NFAT); the inhibition of both molecules reduced the number of colonies in a CFU assay. In contrast, using flow cytometry and confocal evaluation, we demonstrated that G-CSF activated Jak-1 and STAT-3. Additionally, the effects induced by G-CSF were also related with the participation of Ca(2+) calmodulin kinase II and the transcription factor PU.1. STAT-3 activation and the increase of PU.1 expression were sensitive to PLC inhibition by U73122. These data show that PLCγ2 and PKC are important upstream signals that regulate myelopoiesis through cytokines, and differences in M-CSF and G-CSF downstream signaling were identified. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 08/2013; 115(1). DOI:10.1002/jcb.24653 · 3.26 Impact Factor
Show more