Señalización coestimuladora mediada por el regulador de complemento Crry/p65 en linfocitos Th2

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 21-06-200

Complement Regulatory Protein Crry/p65-mediated Signaling in T Lymphocytes: Role of Its Cytoplasmic Domain and Partitioning into Lipid Rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity

Effect of Roux-en-Y Bariatric Bypass Surgery on Subclinical Atherosclerosis and Oxidative Stress Markers in Leukocytes of Obese Patients: A One-Year Follow-Up Study

Little is known about the mechanisms underlying the cardioprotective effect of Roux en-Y gastric bypass (RYGB) surgery. Therefore, the aim of the present study was to investigate whether weight loss associated with RYGB improves the oxidative status of leukocytes and ameliorates subclinical atherosclerotic markers. This is an interventional study of 57 obese subjects who underwent RYGB surgery. We determined biochemical parameters and qualitative analysis of cholesterol, leukocyte and systemic oxidative stress markers —superoxide production, glutathione peroxidase 1 (GPX1), superoxide dismutase (SOD) activity and protein carbonylation—, soluble cellular adhesion molecules —sICAM-1 and sP-selectin—, myeloperoxidase (MPO) and leukocyte-endothelium cell interactions—rolling flux, velocity and adhesion. RYGB induced an improvement in metabolic parameters, including hsCRP and leukocyte count (p < 0.001, for both). This was associated with an amelioration in oxidative stress, since superoxide production and protein carbonylation were reduced (p < 0.05 and p < 0.01, respectively) and antioxidant systems were enhanced (GPX1; p < 0.05 and SOD; p < 0.01). In addition, a significant reduction of the following parameters was observed one year after RYGB: MPO and sICAM (p < 0.05, for both), sPselectin and pattern B of LDL particles (p < 0.001, for both), and rolling flux and adhesion of leukocytes (p < 0.05 and p < 0.01, respectively). Our results suggest that patients undergoing RYGB benefit from an amelioration of the prooxidant status of leukocytes, metabolic outcomes, and subclinical markers of atherosclerosis

Dissociation of actin polymerization and lipid raft accumulation by ligation of the Inducible Costimulator (ICOS, CD278)

9 páginas, 4 figuras -- PAGS nros. 4-12T lymphocyte antigen activation is facilitated by clustering of membrane glycosphingolipid-enriched microdomains (GEMs, lipid “rafts”) at the T cell/APC contact that is linked to changes in actin cytoskeleton and is one major mechanism of CD28 costimulation. Ligation of CD28 alone, or ligation of the CD28-like molecules CTLA-4 (CD152) and ICOS (CD278) induces actin polymerization with cell elongation and generation of lamellipodia and filopodia in T cells. These changes are dependent on Src, PI3-kinase, Vav, and Rho family GTPases. Whereas CD28 and CTLA-4 have been shown to be functional and physically associated with lipid rafts, the presence of ICOS in lipid rafts or its effect in raft clustering is not known. In this work, we have activated the T cell line D10 with anti-ICOS antibodies, alone or combined with anti-CD3 antibodies, bound or unbound to polystyrene microbeads or glass coverslips. The possible relationship of ICOS-induced changes in actin cytoskeleton to the ICOS localization in membrane rafts was then analyzed by fluorescence microscopy, or by immunoblot of detergent insoluble (“raft”) or soluble (“non-raft”) fractions of cell lysates. Our data show that ICOS promotes TCR/CD3 induction of raft clustering at the site of activation. However, ICOS, which, on its own, can induce accumulations of polymerized actin, is undetectable in membrane rafts, even when using CD3 or ICOS, ligands capable of inducing clear changes in the actin cytoskeletonThis work was supported by Grants from Fondo de Investigación Sanitaria (Ministerio de Ciencia e Innovación, Spain) numbers PI070620 and PI10/00650 (to J.M.R.); PI070484, PI10/00648 (to P.P.) and by AIRC (Milan) (to U.D.). Y.Y.A. is the recipient of a Predoctoral Fellowship of the “Junta de Ampliación de Estudios” (JAE) Program (C.S.I.C., Ministerio de Ciencia e Innovación, Spain). P.P. is a Tenured Scientist of C.S.I.C. at the Centro Nacional de Microbiología, I.S. Carlos IIIPeer reviewe