Gerald Krystal

Monomeric IgE Stimulates Signaling Pathways in Mast Cells that Lead to Cytokine Production and Cell Survival

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.

While previous studies with truncated erythropoietin receptors (EpRs) have suggested that the tyrosine phosphorylation of the EpR does not play a role in Ep‐induced proliferation, we have found, using a more subtle, full length EpR mutant, designated Null, in which all eight of the intracellular tyrosines have been substituted with phenylalanine residues, that Null cells require substantially more Ep than wild‐type cells in order to proliferate as efficiently. A comparison of Ep‐induced proliferation with Ep‐induced tyrosine phosphorylation patterns, using wild‐type and Null EpR‐expressing cells, revealed that Stat5 tyrosine phosphorylation and activation correlated directly with proliferation. Moreover, studies with a Y343F EpR point mutant and various EpR deletion mutants revealed that both Ep‐induced proliferation and Stat5 activation were mediated primarily through Y343, but that other tyrosines within the EpR could activate Stat5 in its absence.

SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts

The hematopoietic-restricted protein Src homology 2–containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP−/− mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Similar to pagetic osteoclasts, SHIP−/− osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP−/− mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP−/− long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis.

Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A : association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases

Subsets of T and natural killer (NK) lymphocytes express the CD94‐NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94‐NKG2A heterodimer inhibits both antigen‐driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma‐specific human T cell clones. Similarly, CD16‐mediated NK cell cytotoxicity is extinguished by cross‐linking of the CD94‐NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine‐based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP‐1 and SHP‐2, but not with the polyinositol phosphatase SHIP. Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho‐ITIM interact directly with the SH2 domains of SHP‐1 and SHP‐2 with a high affinity. Engagement of the CD94‐NKG2A heterodimer therefore appears as a protein‐tyrosine phosphatase‐based strategy that negatively regulates both antigen‐induced T cell response and antibody‐induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.

Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.

We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin-induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR.

Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells

To investigate the role of the src homology 2 (SH2)‐containing inositol 5′ phosphatase (SHIP) in growth factor‐mediated signalling, we compared Steel factor (SF)‐induced events in bone marrow‐derived mast cells (BMMCs) from SHIP−/− and SHIP+/+ littermates. We found SF alone stimulated massive degranulation from SHIP−/− but none from SHIP+/+ BMMCs. This SF‐induced degranulation, which was not due to higher c‐kit levels in SHIP−/− cells, correlated with higher intracellular calcium than that in SHIP+/+ cells and was dependent on the influx of extracellular calcium. Both this influx and subsequent degranulation were completely inhibited by PI‐3‐kinase inhibitors, indicating that SF‐induced activation of PI‐3‐kinase was upstream of extracellular calcium entry. A comparison of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) levels following SF stimulation of SHIP+/+ and SHIP−/− BMMCs suggested that SHIP restricted this entry by hydrolyzing PIP3. Although PI‐3‐kinase inhibitors blocked the release of intracellular calcium, implicating PIP3, and PLCγ‐2 was slightly more tyrosine phosphorylated in SHIP−/− cells, the increase in inositol‐1,4,5‐trisphosphate (IP3) and intracellular calcium levels were identical in SHIP−/− and SHIP+/+ BMMCs. These results suggest that SHIP prevents SF from triggering degranulation of normal BMMCs, and does so by hydrolyzing PIP3, which in turn limits extracellular calcium entry at a step after the release of intracellular calcium.

SHIP Negatively Regulates IgE + Antigen-Induced IL-6 Production in Mast Cells by Inhibiting NF-κB Activity

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5′-phosphatase (SHIP)−/− than in SHIP+/+ bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP+/+ BMMCs requires the enzymatic activity of SHIP, because SHIP−/− BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP+/+ BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP−/− BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP−/− cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-κB and that IκB phosphorylation/degradation and NF-κB translocation, DNA binding and transactivation are much higher in SHIP−/− BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of IκB and NF-κB DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-κB. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-κB activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.

Comprehensive microRNA expression profiling of the hematopoietic hierarchy

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

Dysregulated FcεRI Signaling and Altered Fyn and SHIP Activities in Lyn-Deficient Mast Cells

Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn−/− mice (Lyn−/− BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn−/− BMMCs release more β-hexosaminidase than wild-type BMMCs following FcεRI cross-linking and show that multiple mast cell responses to FcεRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase Cγ isoforms, the mobilization of Ca2+, the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the α4β1 integrin, VLA-4) are slow to initiate in Lyn−/− BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn−/− BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn−/− BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn−/− BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcεRI signaling in Lyn−/− BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn−/− BMMCs may additionally contribute to their altered signaling properties.

Altered responsiveness to chemokines due to targeted disruption of SHIP

SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase- (PI-3K-) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell-derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP(-/-) cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.