Altered Lipid raft-associated recruitment of Lyn and Syk in B cells of patients with systemic lupus erythematosus.
-
1
universidad de antioquia, Grupo de inmunología celular e inmunogenética, Facultad de Medicina, Colombia
-
2
Universidad de Antioquia/ Hospital Universitario San Vicente Fundación, Grupo de Reumatología, Facultad de Medicina, Colombia
Introduction: Systemic Lupus Erythematosus (SLE) is the prototype of human systemic autoimmune disease. A central pathogenic participation of T and B cells and numerous immune-regulation abnormalities in SLE have broadened the outlook for new and more specific therapeutic strategies to treat this disease. The activation of B and T cells is regulated inside membrane-associated micro-domains, called lipid rafts (LR) where various post-ligand signaling molecules, such as Src-family kinases, are recruited to their membrane receptor complexes.
Spatio-temporal localization of these specific kinases in LR is a key factor determining signaling activity which is essential to ensure effective and self-limited signage. Lyn is one member of the Src-family of protein tyrosine kinases that has emerged as a key kinase involved in the regulation of the B cell activation. During the B cell activation through its receptor (BCR), Lyn is recruited immediately to LR after ligand binding, and has an inhibitory effect on different downstream signaling molecules. Furthermore, another tyrosine kinase Syk is involved in membrane signals initiated in various immune cells. It is recruited to the LR after the ITAMs got phosphorylated during the BCR-ligand interaction. In inducible knockout mice (KO) for Syk one systemic autoimmune disease similar to human lupus has been described, in addition LYN gene mutations have been shown to be associated with susceptibility to human lupus.
AIM: To evaluate the recruitment of Lyn and Syk to the LR of B cells derived from patients and controls after their activation.
Materials and Methods:Ten patients with Systemic Lupus Erythematosus (SLE) according to the diagnostic criteria of the American College of Rheumatology 1982 and seven healthy controls of similar age and sex were evaluated. The B cells were isolated by rosetting (RossetteSep®, Stem Cell Technologies) and cultured overnight in RPMI. To induce BCR crosslinking, B cells were incubated with 15 ug/mL of affinity pure F(ab´)2 goat anti human IgM + IgG at 37ºC for 10 minutes. Lipid Rafts were isolated at 4ºC from triton X-100 lysates, by their density on sucrose solutions. Briefly, B cells were resuspended in lysis buffer and then mixed in a sucrose solution (45% final concentration, another two sucrose solutions (35 and 5%) were added in order to create a gradient. Samples were centrifugated at 38000 rpm at 4ºC for 20 hours. In total 12 fractions were collected from each sample, 1 to 6 corresponded to lipid rafts. The protein content of these rafts was evalauted by Western blot with specific antibodies.
Results:
a. We observed a Lipid rafts formation in 90% of the SLE patients and all the controls, after the activation with anti-BCR as determined by the presence of flotillin (fractions 1 to 6).
b. Lyn showed and increased signal in all the controls, relative to the baseline levels, while in the SLE patients the presence of Lyn was variable: Lyn signal is similar to resting levels (2/10), in other cases increased (3/10), in some cases there was not observed Lyn recruitment (3/10) and in others a reduction of Lyn was detected (2/10).
c. All controls showed and increased signal of Syk in lipid rafts of B cells stimulated although the presence of this kinase in lipid rafts of SLE patients show more variability and it was different to observed in controls.
Conclusions: Alterations observed in the recruitment of Lyn and Syk to LR could be associated with the state of activation of B cells in SLE patients and their involvement in the pathogenesis of this entity.
Acknowledgements
Colciencias, grant 1115-569-33414
Hospital Universitario San Vicente Fundación
Keywords:
lipid rafts,
B cells,
tyrosine kinases,
Lupus Erythematosus, Systemic,
B Cell Signaling
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Autoimmunity
Citation:
Vasquez
A,
Gonzalez
LA,
Vanegas
A,
Muñoz-Vahos
CH,
Baena
A and
Vasquez
G
(2015). Altered Lipid raft-associated recruitment of Lyn and Syk in B cells of patients with systemic lupus erythematosus..
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00208
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
29 Apr 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
MD, PhD. Gloria Vasquez, universidad de antioquia, Grupo de inmunología celular e inmunogenética, Facultad de Medicina, Medellin, Antioquia, 050016, Colombia, glomavas@gmail.com