Svetlana A. Didichenko

Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33

In mice, interleukin-18 (IL-18) regulates Th1- or Th2-type immune responses depending on the cytokine environment and effector cells involved, and the ST2-ligand, IL-33, primarily promotes an allergic phenotype. Human basophils, major players in allergic inflammation, constitutively express IL-18 receptors, while ST2 surface expression is inducible by IL-3. Unexpectedly, freshly isolated basophils are strongly activated by IL-33, but, in contrast to mouse basophils, do not respond to IL-18. IL-33 promotes IL-4, IL-13 and IL-8 secretion in synergy with IL-3 and/or FcepsilonRI-activation, and enhances FcepsilonRI-induced mediator release. These effects are similar to that of IL-3, but the signaling pathways engaged are distinct because IL-33 strongly activates NF-kappaB and shows a preference for p38 MAP-kinase, while IL-3 acts through Jak/Stat and preferentially activates ERK. Eosinophils are the only other leukocyte-type directly activated by IL-33, as evidenced by screening of p38-activation in peripheral blood cells. Only upon CD3/CD28-ligation, IL-33 weakly enhances Th2 cytokine expression by in vivo polarized Th2 cells. This study on primary human cells demonstrates that basophils and eosinophils are the only direct target leukocytes for IL-33, suggesting that IL-33 promotes allergic inflammation and Th2 polarization mainly by the selective activation of these specialized cells of the innate immune system.

G-Protein-coupled receptors and Fcgamma-receptors mediate activation of Akt/protein kinase B in human phagocytes.

Activation of the serine/threonine kinase Akt, also called protein kinase B (PKB), was investigated in human neutrophils. Stimulation of the cells with the chemoattractant fMet-Leu-Phe or the chemokines IL-8 and GROα leads to the rapid and transient activation of PKB. Maximum PKB activation correlates with the well documented kinetics of respiratory burst and exocytosis. Wortmannin, a selective inhibitor of phosphoinositide 3-kinases (PI 3-kinases) in neutrophils, abrogates PKB activation. Similarly homo and heterotypic cross-linking of FcγIIA and FcγIIIB causes a transient activation of PKB that is sensitive to wortmannin treatment. Kinase activity measurements in immunoprecipitates from lysates of the myelocytic GM-1 cells or GM-1/CXCR1 cells, which are transfected with the IL-8 receptor 1, confirmed the transient activation of PKB observed in neutrophils. Stimulation of human monocytes with the CC chemokine RANTES (regulated on activation normal T cell expressed and secreted) also results in the activation of PKB. Preincubation of monocytes and neutrophils with Bordetella pertussis toxin inhibits fMet-Leu-Phe and RANTES-stimulated PKB activation, demonstrating that coupling of the receptors to heterotrimeric Gi-protein is required. The data show, that activation of PKB by Gi-protein-coupled receptors is mediated by PI 3-kinase and suggest that PKB is a constituent of neutrophil activating pathways.

Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase.

BACKGROUND Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretory responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling. RESULTS The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effectors, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing non-membrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47phox is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47phox. CONCLUSIONS Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways.

Phosphatidylinositol 3-Kinase C2α Contains a Nuclear Localization Sequence and Associates with Nuclear Speckles

Phosphoinositide 3-kinase C2α (PI3K-C2α) belongs to the class II phosphatidylinositol 3-kinases, which are defined by their in vitro usage of phosphatidylinositol and phosphatidylinositol 4-phosphate as substrates. All type II phosphatidylinositol 3-kinases contain at their C terminus a C2-like domain. Here we demonstrate that Homo sapiensphosphoinositide 3-kinase C2α (HsPI3K-C2α) has dual cellular localization present in the cytoplasm and in the nucleus. A distinct nuclear localization signal sequence was identified by expressing HsPI3K-C2α-green fluorescent protein fusion proteins in HeLa cells. The nuclear localization signal was mapped to a stretch of 11 amino acids (KRKTKISRKTR) located within C2-like domain of the kinase. In the cytoplasm and the nucleus HsPI3K-C2α associates with macromolecular complexes that are resistant to detergent extraction. Indirect immunofluorescence reveals that in the nucleus HsPI3K-C2α is enriched at distinct subnuclear domains known as nuclear speckles, which contain pre-mRNA processing factors and are functionally connected to RNA metabolism. Phosphorylation of HsPI3K-C2α is induced by inhibition of RNA polymerase II-dependent transcription and coincides with enlargement and rounding up of the nuclear speckles. The results suggest that phosphorylation of HsPI3K-C2α is inversely linked to mRNA transcription and supports the importance of phosphoinositides for nuclear activity.

IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils

The contribution of basophils in allergic disease and other Th2-type immune responses depends on their persistence at sites of inflammation, but the ligands and molecular pathways supporting basophil survival are largely unknown. The comparison of rates of apoptosis and of the expression of antiapoptotic proteins in different human granulocyte types revealed that basophils have a considerably longer spontaneous life span than neutrophils and eosinophils consistent with high levels of constitutive Bcl-2 expression. Interleukin-3 (IL-3) is the only ligand that efficiently protects basophils from apoptosis as evidenced by screening a large number of stimuli. IL-3 up-regulates the expression of the antiapoptotic proteins cIAP2, Mcl-1, and Bcl-X(L) and induces a rapid and sustained de novo expression of the serine/threonine kinase Pim1 that closely correlates with cytokine-enhanced survival. Inhibitor studies and protein transduction of primary basophils using wild-type and kinase-dead Pim1-Tat fusion-proteins demonstrate the functional importance of Pim1 induction in the IL-3-enhanced survival. Our data further indicate that the antiapoptotic Pim1-mediated pathway operates independently of PI3-kinase but involves the activation of p38 MAPK. The induction of Pim1 leading to PI3-kinase-independent survival as described here for basophils may also be a relevant antiapoptotic mechanism in other terminally differentiated leukocyte types.

Human basophils activated by mast cell–derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid

The vitamin A metabolite retinoic acid (RA) plays a fundamental role in cellular functions by activating nuclear receptors. Retinaldehyde dehydrogenase-II (RALDH2) creates localized RA gradients needed for proper embryonic development, but very little is known regarding its regulated expression in adults. Using a human ex vivo model of allergic inflammation by coincubating IgE receptor-activated mast cells (MCs) with blood basophils, we observed prominent induction of a protein that was identified as RALDH2 by mass spectroscopy. RALDH2 was selectively induced in basophils by MC-derived interleukin-3 (IL-3) involving PI3-kinase and NF-kappaB pathways. Importantly, neither constitutive nor inducible RALDH2 expression was detectable in any other human myeloid or lymphoid leukocyte, including dendritic cells. RA generated by RALDH2 in basophils modulates IL-3-induced gene expression in an autocrine manner, providing positive (CD25) as well as negative (granzyme B) regulation. It also acts in a paracrine fashion on T-helper cells promoting the expression of CD38 and alpha4/beta7 integrins. Furthermore, RA derived from IL-3-activated basophils provides a novel mechanism of Th2 polarization. Thus, RA must be viewed as a tightly controlled basophil-derived mediator with a high potential for regulating diverse functions of immune and resident cells in allergic diseases and other Th2-type immune responses.

Regulation of the urokinase-type plasminogen activator gene by the oncogene Tpr-Met involves GRB2.

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.

G‐Protein Coupled Receptor‐Mediated Activation of PI 3‐Kinase in Neutrophilsa

Stimulation of the respiratory burst of neutrophil leukocytes with chemotactic agonists requires two concomitant signal transduction pathways. One is calcium dependent and leads to activation of phospholipase C, the other is calcium independent but sensitive to the fungal metabolite wortmannin, a specific inhibitor of phosphatidylinositide 3-kinase (PI 3-kinase). Two isoforms of PI 3-kinase have been characterized in neutrophils, the p85/p110 PI 3-kinase alpha and the p101/p120 PI 3-kinase gamma. The relative contribution of the two PI 3-kinases in mediating chemoattractant-stimulated superoxide production and exocytosis in neutrophils in unclear. Here, we report that the protein tyrosine kinase inhibitor genistein markedly attenuates chemoattractant-stimulated phosphatidylinositol (3,4,5)-trisphosphate (PIP3) formation in neutrophils. PI 3-kinase activity in untreated cells is bimodal showing a maximum production after 10-15 sec that protracts with a lower PIP3 formation for approximately 2 min and returns to basal levels after 2-3 min. Genistein at 100 microM strongly inhibits PIP3 elevation and the fMet-Leu-Phe-stimulated respiratory burst. The activity of purified PI 3-kinase, however, is not altered in the presence of genistein, suggesting that the genistein-sensitive intermediate is located between the G-protein-coupled receptor and PI 3-kinase. Expression of a dominant negative form of PI 3-kinase alpha in GM-1/CXCR1 cells, a promyelolocytic cell line transfected with the G-protein-coupled receptors CXCR1, considerably reduces IL-8-stimulated PIP3 formation. The present observations suggest that in phagocytes stimulated with agonists of G-protein-coupled receptors the bulk of PIP3 is generated by PI 3-kinase alpha, which is activated through a genistein-sensitive target, presumably a protein tyrosine kinase.

Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade initiated by IL-5 in eosinophils

BACKGROUND Eosinophil differentiation, activation, and survival are largely regulated by IL-5. IL-5-mediated transmembrane signal transduction involves both Lyn-mitogen-activated protein kinases and Janus kinase 2-signal transducer and activator of transcription pathways. OBJECTIVE We sought to determine whether additional signaling molecules/pathways are critically involved in IL-5-mediated eosinophil survival. METHODS Eosinophil survival and apoptosis were measured in the presence and absence of IL-5 and defined pharmacologic inhibitors in vitro. The specific role of the serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim) 1 was tested by using HIV-transactivator of transcription fusion proteins containing wild-type Pim-1 or a dominant-negative form of Pim-1. The expression of Pim-1 in eosinophils was analyzed by means of immunoblotting and immunofluorescence. RESULTS Although pharmacologic inhibition of phosphatidylinositol-3 kinase (PI3K) by LY294002, wortmannin, or the selective PI3K p110delta isoform inhibitor IC87114 was successful in each case, only LY294002 blocked increased IL-5-mediated eosinophil survival. This suggested that LY294002 inhibited another kinase that is critically involved in this process in addition to PI3K. Indeed, Pim-1 was rapidly and strongly expressed in eosinophils after IL-5 stimulation in vitro and readily detected in eosinophils under inflammatory conditions in vivo. Moreover, by using specific protein transfer, we identified Pim-1 as a critical element in IL-5-mediated antiapoptotic signaling in eosinophils. CONCLUSIONS Pim-1, but not PI3K, plays a major role in IL-5-mediated antiapoptotic signaling in eosinophils.

Evidence for a pool of coronin in mammalian cells that is sensitive to PI 3-kinase

Coronin, a 57 kDa actin binding protein elutes with an apparent molecular mass of 400–600 kDa from gel filtration columns. This fraction is not unrelated to the reported 200 kDa complex where coronin is associated with phox proteins of the NADPH‐oxidase. Phosphatidylinositol 3‐kinase (PI 3‐kinase) solubilizes coronin from the 400–600 kDa complex, thus constitutive active PI 3‐kinase is sufficient to disrupt the complex, whereas wortmannin stabilizes it. Conversely, the phox protein associated pool of coronin is PI 3‐kinase independent. During phagocytosis coronin is recruited together with PI 3‐kinase to membranes of nascent and early phagosomes co‐localizing with the actin cytoskeleton, confirming that coronin contributes to phagocytosis.