Adenovirus type 7 induces interleukin-8 in a lung slice model and requires activation of Erk.

Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.

How do inhibitory phosphatases work?

We present a hypothesis regarding the mode of induction of the inhibitory phosphatases SHP-1 and SHIP in hematopoietic cells. One mode is a general one in which the phosphatase regulates but does not abort signal transduction and biology. Regulator phosphatases are induced by directly or indirectly engaging the amino acid motifs present in the activating receptor, and act to control the biochemical and biological output. The other mode of induction is a specific one, which critically involves paired co-clustering of activating and inhibitory receptors. Phosphatases working in this way act only under conditions of paired co-clustering of activating and inhibitory receptors, and directly bind amino acid motifs present in the inhibitory receptor. However, this mode of induction is apparently more efficient, as cellular activation is completely aborted. This review presents several examples of each mode of inhibition and speculates on their mechanisms.

Visualization of negative signaling in B cells by quantitative confocal microscopy.

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.

Signal transduction by human-restricted Fc gamma RIIa involves three distinct cytoplasmic kinase families leading to phagocytosis.

Recent experiments indicate an important role for Src family and Syk protein tyrosine kinases and phosphatidylinositol 3-kinase in the signal transduction process initiated by mouse receptors for IgG and leading to phagocytosis. Considerably less is known regarding signal transduction by the human-restricted IgG receptor, FcgammaRIIa. Furthermore, the relationship among the Src family, Syk, and phosphatidylinositol 3-kinase in phagocytosis is not understood. Here, we show that FcgammaRIIa is phosphorylated by an Src family member, which results in recruitment and concomitant activation of the distal enzymes Syk and phosphatidylinositol 3-kinase. Using a FcgammaRI-p85 receptor chimera cotransfected with kinase-inactive mutants of Syk or application of a pharmacological inhibitor of Syk, we show that Syk acts in parallel with phosphatidylinositol 3-kinase. Our results indicate that FcgammaRIIa-initiated monocyte or neutrophil phagocytosis proceeds from the clustered IgG receptor to Src to phosphatidylinositol 3-kinase and Syk.

Adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2.

Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

Positive regulation of interleukin-4-mediated proliferation by the SH2-containing inositol-5'-phosphatase.

The SH2-containing inositol 5'-phosphatase (SHIP) is tyrosine-phosphorylated in response to cytokines such as interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor. SHIP has been shown to modulate negatively these cytokine signalings; however, a potential role in IL-4 signaling remains uncharacterized. It has been recently shown that IL-4 induces tyrosine phosphorylation of SHIP, implicating the phosphatase in IL-4 processes. Tyrosine kinases, Jak1 and Jak3, involved in IL-4 signaling can associate with SHIP, yet only Jak1 can tyrosine-phosphorylate SHIP when co-expressed. In functional studies, cells overexpressing wild type SHIP are found to be hyperproliferative in response to IL-4 in comparison to parental cells. In contrast, cells expressing catalytically inactive form, SHIP(D672A), show reduced proliferation in response to IL-4. These changes in IL-4-induced proliferation correlate with alterations in phosphatidylinositol 3,4,5-triphosphate levels. However, no differential activation of STAT6, Akt, IRS-2, or p70(S6k), in response to IL-4, was observed in these cells. These data suggest that the catalytic activity of SHIP acts in a novel manner to influence IL-4 signaling. In addition, these data support recent findings that suggest there are uncharacterized signaling pathways downstream of phosphatidylinositol 3,4,5-triphosphate.

The adapter protein LAT enhances fcgamma receptor-mediated signal transduction in myeloid cells.

FcgammaR clustering in monocytes initiates a cascade of signaling events that culminate in biological responses such as phagocytosis, production of inflammatory cytokines, and generation of reactive oxygen species. We have identified and determined the function of the adapter protein linker of activation of T cell (LAT) in FcgammaR-mediated signaling and function. Clustering of FcgammaRs on the human monocytic cell line, THP-1, induces phosphorylation of a major 36-kDa protein which immunoreacts with anti-LAT antisera. Our data indicate that although both the 36-kDa and 38-kDa isoforms of LAT are expressed in THP-1 and U937 human monocytic cells, FcgammaR clustering induces phosphorylation of the 36-kDa isoform only. Co-immunoprecipitation experiments revealed a constitutive association of p36 LAT with both FcgammaRI and FcgammaRIIa immunoprecipitates, and an activation-induced association of LAT with PLCgamma1, Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Transient transfection experiments in COS-7 cells indicated that overexpression of a wild type but not a dominant-negative LAT, that is incapable of binding to p85, enhances phagocytosis by FcgammaRI. Furthermore, bone marrow-derived macrophages from LAT-deficient mice displayed reduced phagocytic efficiency in comparison to the macrophages from wild-type mice. Thus, we conclude that p36 LAT serves to enhance FcgammaR-induced signal transduction in myeloid cells.

Enzymatic activity of the Src homology 2 domain-containing inositol phosphatase is regulated by a plasma membrane location.

The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with FcgammaRIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore this issue, we investigated the effect of SHIP phosphorylation, receptor tyrosine engagement by its Src homology 2 domain, and membrane recruitment of SHIP on its enzymatic activity. We examined two SHIP phosphorylation kinase candidates, Lyn and Syk, and observed that the Src protein-tyrosine kinase, Lyn is far superior to Syk in its ability to phosphorylate SHIP both in vitro and in vivo. However, we found a minimal effect of phosphorylation or receptor tyrosine engagement of SHIP on its enzymatic activity, whereas membrane localization of SHIP significantly reduced cellular phosphatidylinositol 3,4, 5-triphosphate levels. Based on our results, we propose that a membrane localization of SHIP is the crucial event in the induction of its phosphatase effects.

Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis.

Anti-CD20 antibodies may reduce or eliminate non-Hodgkin's lymphoma B cells in patients, although the mechanism of action is not clear. To explore mechanism(s), we examined the induction of signal transduction events using anti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkin's lymphoma Ramos B cell line. We found that while Rituximab (a human-mouse hybrid mAb) alone induced apoptotic cell death, other murine anti-CD20 mAbs induced apoptosis of Ramos B cells only upon clustering with a secondary antibody. CD20 clustering was accompanied by activation of tyrosine protein kinase activity, PLCgamma2 phosphorylation, influx of Ca(2+), and activation of caspase 3. All signaling events, as well as the subsequent apoptosis, were blocked by PP2, a selective inhibitor of Src-family kinases. Treatment of Ramos with EGTA and BAPTA to block changes in cytoplasmic Ca(2+) likewise prevented CD20-induced apoptosis. Our findings support a model in which CD20 clustering activates members of the Src family of protein tyrosine kinases, leading to phosphorylation of PLCgamma2 and increased cytoplasmic Ca(2+). These early signal transduction events activate caspase 3 to promote apoptotic cell death of NHL B cells.

The SH2-containing 5'-inositol phosphatase (SHIP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes.

Current models of Fc gamma R signal transduction in monocytes describe a molecular cascade that begins upon clustering of Fc gamma R with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of Fc gamma RIIa or the gamma-chain subunit of Fc gamma RI and Fc gamma RIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ultimately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5'-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after Fc gamma R clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either Fc gamma RIIa or Fc gamma RI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of Fc gamma RIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that Fc gamma RIIb and Fc gamma RIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits Fc gamma R-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif-containing cytoplasmic domains of Fc gamma RIIa and Fc gamma RI-associated gamma-chain.

Analysis of tyrosine phosphorylation-dependent interactions between stimulatory effector proteins and the B cell co-receptor CD22.

The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22-derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2.Sos complex was observed to bind exclusively to the fourth phosphotyrosine motif (Y828ENV) from CD22 and does so via a direct interaction based on Far Western and reverse Far Western blotting. Although both PLCgamma and PI3K were observed to bind to multiple phosphopeptides in precipitation experiments, subsequent studies using reverse Far Western blot analysis demonstrated that only the carboxyl-terminal phosphopeptide of CD22 (Y863VTL) binds directly to either one. This finding suggests that PLCgamma and PI3K may be recruited to CD22 either through a direct interaction with Tyr863 or indirectly through an association with one or more intermediate proteins.

Negative signaling in health and disease.

Signal transduction induced by receptors can elicit intracellular biochemical events that either support or inhibit cell activation. Induction of the latter has been termed "negative signaling" and can be triggered by receptors on immune cells that are distinct from activating receptors while other growth-promoting receptors induce both positive and negative signaling events. Here, the biochemistry leading to cell activation or inhibition and induced by receptors on immune cells are reviewed. Furthermore, recent experimental evidence is reviewed that indicates an important contribution of negative signaling to the intracellular survival of infectious pathogens.

FcgammaRIIb modulation of surface immunoglobulin-induced Akt activation in murine B cells.

We examined activation of the serine/threonine kinase Akt in the murine B cell line A20. Akt is activated in a phosphoinositide 3-kinase (PtdIns 3-kinase)-dependent manner upon stimulation of the antigen receptor, surface immunoglobulin (sIg). In contrast, Akt induction is reduced upon co-clustering of sIg with the B cell IgG receptor, FcgammaRIIb. Co-clustering of sIg-FcgammaRIIb transmits a dominant negative signal and is associated with reduced accumulation of the PtdIns 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3), known to be a potent activator of Akt. PtdIns 3-kinase is activated to the same extent with and without FcgammaRIIb co-ligation, indicating conditions supporting the generation of PtdIns 3,4,5-P3. We hypothesized that the decreased Akt activity arises from the consumption of PtdIns 3,4,5-P3 by the inositol-5-phosphatase Src homology 2-containing inositol 5-phosphatase (SHIP), which has been shown by us to be tyrosine-phosphorylated and associated with FcgammaRIIb when the latter is co-ligated. In direct support of this hypothesis, we report here that Akt induction is greatly reduced in fibroblasts expressing catalytically active but not inactive SHIP. Likewise, the reduction in Akt activity upon sIg-FcgammaRIIb co-clustering is absent from avian B cells lacking expression of SHIP. These findings indicate that SHIP acts as a negative regulator of Akt activation.

Protein interactions of Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP): association with Shc displaces SHIP from FcgammaRIIb in B cells.

Our recent studies revealed that the inositol phosphatase Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP) is phosphorylated and associated with Shc exclusively under negative signaling conditions in B cells, which is due to recruitment of the SHIP SH2 domain to the FcgammaRIIb. In addition, we reported that SHIP-Shc interaction involves both SHIP SH2 and Shc phosphotyrosine binding domains. These findings reveal a paradox in which the single SH2 domain of SHIP is simultaneously engaged to two different proteins: Shc and FcgammaRIIb. To resolve this paradox, we examined the protein interactions of SHIP. Our results demonstrated that isolated FcgammaRIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP but not FcgammaRIIb. In contrast, SHIP isolates contain both proteins, revealing two separate pools of SHIP: one bound to FcgammaRIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with FcgammaRIIb but slower and more transient association with Shc. Affinity measurements using a recombinant SHIP SH2 domain and phosphopeptides derived from FcgammaRIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an approximately equal rate of binding but a 10-fold faster dissociation rate for FcgammaRIIb compared with Shc phosphopeptide and yielding in an affinity of 2.1 microM for FcgammaRIIb and 0.26 microM for Shc. These findings are consistent with a model in which SHIP transiently associates with FcgammaRIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and dissociates from FcgammaRIIb.

GM1 inhibits early signaling events mediated by PDGF receptor in cultured human glioma cells.

Binding of platelet-derived growth factor receptor (PDGF) to its receptor (PDGFR) activates its receptor tyrosine kinase which autophosphorylates tyrosine residues. The p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) binds to specific phosphotyrosines on PDGFR-beta and through the associated p110 catalytic subunit of PI 3-kinase catalyzes the formation of lipids that are involved in intracellular signaling. We examined if GM1 affects interactions between PDGFR-beta and specific proteins involved in PDGFR-mediated signaling. U-1242 MG cells were studied under different growth conditions using immunoprecipitation and Western Blot analysis. PDGF-stimulated the association of PDGFR-beta with p85, ras GTPase-activating protein and PLC gamma. GM1 decreased these associations in parallel with decreased tyrosine phosphorylation of PDGFR. PDGF augmented the activity of PI 3-kinase associated with PDGFR-beta, and this was attenuated by GM1. However, GM1 did not alter SH2 domains of p85. GM1 probably inhibits PDGF-induced signaling proteins with PDGFR-beta by inhibiting phosphorylation of specific tyrosines on the receptor which bind to SH2-domains on signaling proteins.

Chimeric receptors composed of phosphoinositide 3-kinase domains and FCgamma receptor ligand-binding domains mediate phagocytosis in COS fibroblasts.

Receptors for the Fc portion of IgG (FcgammaR) initiate phagocytosis of IgG-opsonized particles by a process involving the assembly of a multi-molecular signaling complex. Several members of this complex have been identified, including Src family kinases, Syk/ZAP 70 family kinases, and phosphoinositide 3-kinase (PI3-K). To test directly the role of PI3-K in mediating phagocytosis, we assessed the phagocytic ability of chimeric receptors composed of FcgammaR extracellular and transmembrane domains fused to regions of the p85 subunit of PI3-K. We found that chimeric receptors with cytoplasmic tails composed of the entire p85 subunit of PI3-K or the inter-Src homology 2 portion of p85 triggered phagocytosis in transfected COS fibroblasts. These two chimeras also showed phosphoinositide kinase activity in vitro when immunoadsorbed. In contrast, a chimera containing only the carboxyl-terminal Src homology 2 domain of p85 that does not interact with the catalytic p110 subunit of PI3-K did not trigger phagocytosis, nor did it show kinase activity in vitro. These data suggest that localization and direct activation of PI3-K at the site of particle attachment is sufficient to trigger the process of phagocytosis.

Inhibitory signaling by B cell Fc gamma RIIb.

The fact that B cells undergo feedback suppression, or negative signaling, through the interaction of secreted antibody with specific antigen has been extensively documented but the mechanisms involved in the process have been elusive. Experiments over the past year using B cell deletion mutants and dominant-negative enzymes have firmly established an important role for SH2-domain-containing inositol 5-phosphatase (SHIP) in negative signaling. Negative signaling through SHIP appears to inhibit the Ras pathway through SH2 domain competition with Grb2 and Shc and may involve consumption of intracellular lipid mediators that act as allosteric enzyme activators or that promote entry of extracellular Ca2+.

Role of SHIP in FcgammaRIIb-mediated inhibition of Ras activation in B cells.

Previous studies by our lab and others established that co-crosslinking sIg and IgG receptor FcgammaRIIb in B cells in a feedback suppression model (negative signaling) promoted tyrosine phosphorylation of the inositol 5-phosphatase SHIP and its interaction with Shc and that these events were associated with inhibition of the Ras pathway. We therefore hypothesized a competition model in which the SH2 domain of SHIP competes with that of Grb2 for binding to phospho-Shc to inhibit the Ras pathway. Here, we provide evidence consistent with this hypothesis. First, FcgammaRIIb-deficient B cells, which do not undergo SHIP tyrosine phosphorylation nor interaction with Shc, displayed an active Ras pathway under negative signaling conditions; reconstitution of FcgammaRIIb expression restored the block in Ras. Second, under conditions of negative signaling leading to SHIP-Shc interaction in wild-type B cells, we observed a profound reduction in the activation-induced association of Grb2 to Sos. Experiments reported here and elsewhere revealed the Grb2-Sos interaction required the engagement of the Grb2 SH2 domain by phospho-Shc. Third, we demonstrated that phospho-Shc cannot concomitantly bind Grb2 and SHIP, indicating that the two proteins competed for the same phospho-tyrosine residue on Shc. These data are consistent with the proposed competition model, and further indicate that the activation induced Grb2-Sos association is rate limiting for Ras activation.

Activation-induced bi-dentate interaction of SHIP and Shc in B lymphocytes.

SHIP is a SH2 domain-containing inositol polyphosphatase that is selectively tyrosine phosphorylated and associated with the adapter protein Shc in B lymphocytes upon co-crosslinking surface immunoglobulin and Fc gamma RIIB1. We previously observed that this stimulation condition is associated with a reduction in the interaction of Grb2 with phosphorylated Shc, an enhanced interaction of Shc with SHIP, and a block in the Ras signaling pathway. We proposed that the SH2 domain of SHIP competes with Grb2 in binding to phospho-Shc, resulting in a block in Ras signaling. To test this model, we examined the mode of SHIP-Shc interaction. Using recombinant Shc and SHIP interaction domains and purified Shc and SHIP phosphopeptides, we show that the interaction is bi-dentate such that the SH2 domain of SHIP recognizes phosphorylated Y317 and doubly-phosphorylated Y239/Y240 of Shc and the Shc PTB domain recognizes phosphorylated NPxpY motifs within SHIP. We observed no role for the Shc SH2 domain in the interaction. These findings are consistent with our earlier model that SHIP and Grb2 compete for binding to phospho-Shc and support the notion that, in addition to the hydrolysis of inositol phosphates and phospholipids, SHIP contributes to anti-proliferative biochemistry by blocking protein-protein interactions.