Background/Purpose: Numerous alterations in signal transduction in SLE T cells have been previously reported.  Diminished expression of the ζ chain of  the TCR/CD3 complex has been shown to be accompanied by the substitutive assembly of  FcεRIγ chain and coupling to Syk PTK (Protein Tyrosine Kinase), rewiring downstream signaling to produce alternative functional outcomes in lupus T cells. Syk  is  100-fold   times enzymatically more potent than ZAP-70.  In this study we examined   the localization of ZAP-70 and Syk in lipid rafts of  lupus T cells; lipid raft is the site in membranes where signaling molecules preferentially associate to form signalosomes.       

Methods: SLE patients were diagnosed according to the American College of Rheumatology 1987 criteria.  Healthy donors were recruited from the blood bank of Hospital Universitario de Caracas.  All individuals signed an informed consent previously approved by the Hospital Bioethics Committee.  Highly enriched T cells, obtained from peripheral blood samples and subjected to RossetteSep^TM  isolation, were adhered to poly-L-lysine coated slides and activated for 5 and 15 minutes at 37°C, with 4.5 μm superparamagnetic polystyrene beads coated with antibodies against CD3ε and CD28. The cells were fixed, permeabilized and stained with antibodies recognizing ZAP-70, Syk and Cholera Toxin Subunit B as a lipid raft marker. The cell-bead complexes were evaluated by confocal microscopy and densitometries were obtained using ImageJ, 1.49t (National Institutes of Health, USA).  

Results: We observed similar localization of Syk  in lipid rafts of unstimulated lupus  T cells as compared to healthy controls (0.216 ± 0.045 vs. 0.287 ± 0.056; Pearson’s  r  ± SME, n= 6).  In contrast, there was a diminished localization  of ZAP-70 in unstimulated lupus T cells  as compared to healthy controls (0.383 ± 0.035 vs. 0.488 ± 0.037; Pearson’s  r  ± SME, n= 6; Student’s t-test, p= 0.046,). After CD3-CD28 costimulation during 15 minutes, we observed an increased  localization of Syk in lipid rafts of lupus T cells  as compared  to healthy controls (0.352 ± 0.033 vs. 0.235 ± 0.042; Pearson’s  r    ± SME, n= 6; Student’s t-test, p= 0.034), whereas  ZAP-70 localization in lipid rafts after costimulation was similar in both groups (0.433 ± 0.040 vs. 0.402 ± 0.031; Pearson’s  r  ± SME, n= 6).     

Conclusion: Our findings suggest  an abnormal pattern in the localization of key tyrosine kinases in lipid rafts after activation of SLE T cells, with a predominant translocation of Syk over the typical T-cell kinase, ZAP-70. This abnormal response  may contribute to known signaling abnormalities such as displacement of transmembrane protein Linker for Activation of T cells (LAT)  from  lipid rafts and downstream deficient activation of Extracellular Signaling Regulated Kinases (ERK),   as previously shown in our laboratory.

Supported by Proyecto COLCIENCIAS contract No. 461-2012, code 1115-569-33414, and Proyecto FONACIT grant No. 2013000348.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-localization-of-spleen-tyrosine-kinase-syk-in-lipid-rafts-of-in-vitro-tcrcd3-cd28-activated-lupus-t-cells/