K. Mark Coggeshall

Inhibitory signaling by B cell FcγRIIb

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+.

Negative signaling in B cells: SHIP Grbs Shc

Negative signaling in B cells is initiated by co-crosslinking of the antigen receptor and the Fcy receptor, resulting in cessation of B-cell signaling events and, in turn, inhibiting B-cell proliferation and antibody secretion. Here, a competitive role is proposed for SHIP in blocking the interaction of Shc with the Grb2-Sos complex of proteins that lead to Ras activation in B cells.

Activation of Nuclear Factor of Activated T Cells by Human T-Lymphotropic Virus Type 1 Accessory Protein p12I

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is the agent of an aggressive malignancy of CD4+ T lymphocytes, called adult T-cell lymphoma/leukemia, and is associated with numerous immune-mediated diseases. To establish infection, HTLV-1 must activate targeted T cells during early stages of infection. We recently demonstrated that the HTLV-1 accessory protein p12I is critical for persistent infection in vivo and for viral infectivity in quiescent primary lymphocytes, suggesting a role for p12I in lymphocyte activation. To test whether p12I modulates signaling pathways required for T-lymphocyte activation, we examined AP-1-, NF-κB-, and nuclear factor of activated T cells (NFAT)-driven reporter gene activity in p12I-expressing Jurkat T cells compared to vector-transfected control cells. HTLV-1 p12I specifically induced NFAT-mediated transcription approximately 20-fold in synergy with the Ras/mitogen-activated protein kinase pathway, but did not influence AP-1- or NF-κB-dependent gene expression. Inhibition of calcium-dependent signals by cyclosporin A, BAPTA-AM [glycine, N,N′-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl]-[bis(acetyloxy)methyl ester], and a dominant negative mutant of NFAT2 abolished the p12I-mediated activation of NFAT-dependent transcription. In contrast, inhibition of phospholipase C-γ and LAT (linker for activation of T cells) did not affect p12I-induced NFAT activity. Importantly, p12I functionally substituted for thapsigargin, which selectively depletes intracellular calcium stores. Our data are the first to demonstrate a role for HTLV-1 p12I in calcium-dependent activation of NFAT-mediated transcription in lymphoid cells. We propose a novel mechanism by which HTLV-1, a virus associated with lymphoproliferative disease, dysregulates common T-cell activation pathways critical for the virus to establish persistent infection.

Vav activation and function as a rac guanine nucleotide exchange factor in macrophage colony-stimulating factor-induced macrophage chemotaxis.

ABSTRACT Signal transduction mediated by phosphatidylinositol 3-kinase (PI 3-kinase) is regulated by hydrolysis of its products, a function performed by the 145-kDa SH2 domain-containing inositol phosphatase (SHIP). Here, we show that bone marrow macrophages of SHIP−/− animals have elevated levels of phosphatidylinositol 3,4,5-trisphosphate [PI (3,4,5)P3] and displayed higher and more prolonged chemotactic responses to macrophage colony-stimulating factor (M-CSF) and elevated levels of F-actin relative to wild-type macrophages. We also found that the small GTPase Rac was constitutively active and its upstream activator Vav was constitutively phosphorylated in SHIP−/− macrophages. Furthermore, we show that Vav in wild-type macrophages is recruited to the membrane in a PI 3-kinase-dependent manner through the Vav pleckstrin homology domain upon M-CSF stimulation. Dominant inhibitory mutants of both Rac and Vav blocked chemotaxis. We conclude that Vav acts as a PI 3-kinase-dependent activator for Rac activation in macrophages stimulated with M-CSF and that SHIP regulates macrophage M-CSF-triggered chemotaxis by hydrolysis of PI (3,4,5)P3.

The biochemical basis of transmembrane signalling by B lymphocyte surface immunoglobulin

The specificity of humoral immune responses is determined primarily at the level of antigen interaction with B lymphocytes which express antigen-specific receptor immunoglobulin. When receptor immunoglobulin is crosslinked by antigen or anti-receptor antibodies there isgeneration and transduction of signals which result in new membrane Ia antigen expression and, in some instances, entry of cells into cycle. Until recently, the molecular basis of signal transduction across membrane immunoglobulins has remained enigmatic. Here John Cambier and colleagues discuss studies which indicate that membrane appears similar to thrombin receptors, muscarinic receptors, al adrenergic receptors and many others in transducing signals via initiation of phosphoinositide hydrolysis, yielding diacylglycerol and inositol phosphates which in turn appear to activate protein kinase and calcium mobilization, respectively.

CD95 Rapidly Clusters in Cells of Diverse Origins

We have shown that CD95-mediated cell death requires a clustering of the receptor in distinct sphingolipid-rich domains of the cell membrane (Grassmé et al., 2000, Cremesti et al., 2000). These domains form in response to acid sphingomyelinase (ASM)-induced ceramide generation. However, recent studies challenged the finding of early CD95 clustering (Algeciras-Schimnich et al., 2002). Here, six independent groups tested clustering of CD95 in diverse cell type including primary cells ex vivo and established cell lines. The studies show clustering of CD95 within seconds to minutes in all cell types tested by the different groups. In addition, clustering of CD95 was detected after stimulation of cells using three agonistic anti-CD95 antibodies (CH11, APO-1-3 and JO2), CD95 ligand and stimuli that induce an upregulation and activation of the endogenous CD95/CD95 ligand system. The data confirm our previous studies and suggest rapid, i.e. within seconds to minutes, CD95 clustering as a general phenomenon occurring in many cell types.

Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

ABSTRACT Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcγRII) 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 FcγRII 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 FcγRII−/− 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 corecruited to the BCR, regardless of the presence or absence of FcγRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcγRII 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/FcγRII 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.

B cell antigen receptor endocytosis and antigen presentation to T cells require Vav and dynamin.

Antigen binding to the B cell antigen receptor (BCR) initiates an array of signaling events. These include endocytosis of ligand-receptor complexes via clathrin-coated pits, trafficking of the internalized ligand to lysosomes, degradation of the associated proteins to peptides, and peptide presentation on nascent major histocompatibility complex class II to T cells. The signal transduction events supporting BCR internalization are not well understood. We have identified a pathway supporting BCR internalization that includes the Vav1 and/or Vav3 isoforms and the GTPase dynamin. Vav1 and -3 are not required for B cell development and maturation, nor for a variety of BCR-induced signaling events nor for BCR signaling leading to major histocompatibility complex class II and CD80 expression, but Vav1 and/or -3 are absolutely required for BCR endocytosis and BCR-induced Rac-GTP loading. This is the first demonstration of a link between Vav and Rac in BCR internalization leading to antigen presentation to T cells.

Vav and Rac Activation in B Cell Antigen Receptor Endocytosis Involves Vav Recruitment to the Adapter Protein LAB

The signal transduction events supporting B cell antigen receptor (BCR) endocytosis are not well understood. We have identified a pathway supporting BCR internalization that begins with tyrosine phosphorylation of the adapter protein LAB. Phosphorylated LAB recruits a complex of Grb2-dynamin and the guanine nucleotide exchange factor Vav. Vav is required for activation of the small GTPases Rac1 and Rac2. All these proteins contribute to (and dynamin, Vav, and Rac1/2 are required for) BCR endocytosis and presentation of antigen to T cells. This is the first description of a sequential signal transduction pathway from BCR to internalization and antigen presentation.

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 p70S6k, 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.