194 research outputs found
RIP Links TLR4 to Akt and Is Essential for Cell Survival in Response to LPS Stimulation
Receptor-interacting protein (RIP) has been reported to associate with tumor necrosisâassociated factor (TRAF)2 and TRAF6. Since TRAF2 and TRAF6 play important roles in CD40 signaling and TRAF6 plays an important role in TLR4 signaling, we examined the role of RIP in signaling via CD40 and TLR4. Splenocytes from RIPâ/â mice proliferated and underwent isotype switching normally in response to anti-CD40âIL-4 but completely failed to do so in response to LPSâIL-4. However, they normally up-regulated TNF-α and IL-6 gene expression and CD54 and CD86 surface expression after LPS stimulation. RIPâ/â splenocytes exhibited increased apoptosis and impaired Akt phosphorylation after LPS stimulation. These results suggest that RIP is essential for cell survival after TLR4 signaling and links TLR4 to the phosphatidylinositol 3 kinaseâAkt pathway
WIP and WASP play complementary roles in T cell homing and chemotaxis to SDF-1a
ProducciĂłn CientĂficaHoming of lymphocytes to tissues is a biologically important multistep process that involves selectindependent
rolling, integrin-dependent adhesion and chemokine-directed chemotaxis. The actin
cytoskeleton plays a central role in lymphocyte adhesion and motility. WiskottâAldrich syndrome
protein (WASP), the product of the gene mutated in WiskottâAldrich syndrome, and its partner,
the WiskottâAldrich syndrome protein-interacting protein (WIP), play important roles in actin
re-organization in T lymphocytes. We used mice with disruption of the WASP and WIP genes to
examine the role of WASP and WIP in T cell homing. T cell homing to spleen and lymph nodes in vivo
was deficient in WASP / and WIP / mice and severely impaired in WASP / WIP / double knockout
(DKO) mice. Deficiency of WASP, WIP or both did not interfere with selectin-dependent rolling or
integrin-dependent adhesion of T cells in vitro. Chemotaxis to stromal cell-derived factor-1a (SDF-1a)
in vitro was mildly reduced in T cells from WASP / mice. In contrast, it was significantly impaired in
T cells from WIP / mice and severely reduced in T cells from DKO mice. Cellular F-actin increase
following SDF-1a stimulation was normal in WASP / and WIP / T cells, but severely reduced in
T cells from DKO mice. Actin re-organization and polarization in response to SDF-1a was abnormal in
T cells from all knockout mice. Early biochemical events following SDF-1a stimulation that are
important for chemotaxis and that included phosphorylation of Lck, cofilin, PAK1 and extracellular
regulated kinase (Erk) and GTP loading of Rac-1 were examined in T cells from DKO mice and found to
be normal. These results suggest that WASP and WIP are not essential for T lymphocyte rolling and
adhesion, but play important and partially redundant roles in T cell chemotaxis in vitro and homing
in vivo and function downstream of small GTPases
Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome
ProducciĂłn CientĂficaBackground: Proliferation and IL-2 production in response to
T-cell receptor ligation are impaired in patients with Wiskott-
Aldrich syndrome (WAS). The transcription factors nuclear
factor-kB (NF-kB), nuclear factor of activated T cells (NF-AT),
and activating protein-1 (AP-1) play a critical role in IL-2 gene
expression.
Objective: To investigate the mechanisms of impaired IL-2
production after T-cell receptor ligation in T cells deficient in
WAS protein (WASP).
Methods: T cells from WASP2/2 mice were stimulated with
anti-CD3 and anti-CD28. Nuclear NF-kB, NF-AT, and AP-1
DNA-binding activity was examined by electroshift mobility
assay. NF-ATp dephosphorylation and nuclear localization
were examined by Western blot and indirect immunofluorescence.
Phosphorylation of the mitogen-activated protein
kinases Erk and Jnk, and of their nuclear substrates Elk-1 and
c-Jun, was examined by Western blot. Expression of mRNA for
IL-2 and the NF-kBâdependent gene A20 and of the AP-1
components c-fos and c-Jun was examined by quantitative
RT-PCR.
Results: Nuclear translocation and activity of NF-kB were
normal in T cells from WASP2/2 mice. In contrast, NF-ATp
dephosphorylation and nuclear localization, nuclear AP-1
binding activity, and expression of c-fos, but not c-Jun, were all
impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal.
However, nuclear translocation of phosphorylated
Erk and phosphorylation of its nuclear substrate Elk1,
which activates the c-fos promoter, were impaired.
Conclusion: These results suggest that WASP is essential for
NF-ATp activation, and for nuclear translocation of p-Erk,
Elk1 phosphorylation, and c-fos gene expression in T cells.
These defects underlie defective IL-2 expression and T-cell
proliferation in WAS
Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation
ProducciĂłn CientĂficaF-actin polymerization following engagement of the T cell receptor (TCR) is dependent on WASP and is critical for T cell activation. The link between TCR and WASP is not fully understood. In resting cells, WASP exists in a complex with WIP, which inhibits its activation by Cdc42. We show that the adaptor protein CrkL binds directly to WIP. Further, TCR ligation results in the formation of a ZAP-70-CrkL-WIP-WASP complex, which is recruited to lipid rafts and the immunological synapse. TCR engagement also causes PKCtheta-dependent phosphorylation of WIP, causing the disengagement of WASP from the WIP-WASP complex, thereby releasing it from WIP inhibition. These results suggest that the ZAP-70-CrkL-WIP pathway and PKCtheta link TCR to WASP activation
Targeted Inactivation of the IL-4 Receptor α Chain I4R Motif Promotes Allergic Airway Inflammation
The insulin/interleukin-4 (IL-4) receptor (I4R) motif mediates the association of insulin receptor substrate (IRS)-2 with the interleukin-4 (IL-4)Rα chain and transduces mitogenic signals in response to IL-4. Its physiological functions were analyzed in mice with a germline point mutation that changed the motif's effector tyrosine residue into phenylalanine (Y500F). The Y500F mutation abrogated IRS-2 phosphorylation and impaired IL-4âinduced CD4+ T lymphocyte proliferation but left unperturbed Stat6 activation, up-regulation of IL-4-responsive gene products, and Th cell differentiation under Th2 polarizing conditions. However, in vivo the Y500F mutation was associated with increased allergen-induced IgE production, airway responsiveness, tissue eosinophilia, and mucus production. These results define an important role for the I4R motif in regulating allergic inflammation
The Binding Site for TRAF2 and TRAF3 but Not for TRAF6 Is Essential for CD40-Mediated Immunoglobulin Class Switching
AbstractTo define the role of TRAF proteins in CD40-dependent isotype switching in B cells, we introduced wild-type (WT) and mutant CD40 transgenes that lacked the binding motifs for TRAF6 (CD40ÎTRAF6), TRAF2 and TRAF3 (CD40ÎTRAF2/3), or both (CD40ÎTRAFs) into B cells of CD40â/â mice. The in vivo isotype switch defect in CD40â/â mice was fully corrected by WT and CD40ÎTRAF6, partially by CD40ÎTRAF2/3, and not at all by CD40ÎTRAFs transgenes. CD40-mediated isotype switching, proliferation, and activation of p38, JNK, and NFÎșB in B cells were normal in WT and CD40ÎTRAF6 mice, severely impaired in CD40ÎTRAF2/3, and absent in CD40ÎTRAFs mice. These results suggest that binding to TRAF2 and/or TRAF3 but not TRAF6 is essential for CD40 isotype switching and activation in B cells
WIP Regulates Signaling via the High Affinity Receptor for Immunoglobulin E in Mast Cells
Wiskott-Aldrich syndrome proteinâinteracting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcΔRI) in mast cells. WIP-deficient bone marrowâderived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcΔRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcΔRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcΔRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcΔRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement
TACI and BAFF-R mediate isotype switching in B cells
The tumor necrosis factor family members BAFF and APRIL induce Ig isotype switching in human B cells. We analyzed the ability of BAFF and APRIL to induce isotype switching in murine B cells to IgG1, IgA, and IgE. APRIL and BAFF each engage two receptors, transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), on B cells. In addition, BAFF engages a third receptor on B cells, BAFF-R. To determine the role of these receptors in isotype switching, we examined B cells from mice deficient in TACI, BCMA, and BAFF-R. The results obtained indicate that both TACI and BAFF-R are able to transduce signals that result in isotype switching
- âŠ