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Role of the pro-inflammatory cytokines, LIGHT and CD40L, in platelet-mediated inflammation in sickle cell anemia

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Vanessa Tonin Garrido
Total Authors: 1
Document type: Doctoral Thesis
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
Defense date:
Examining board members:
Nicola Amanda Conran Zorzetto; Licio Augusto Velloso; Erich Vinicius De Paula; Claudia Regina Bonini Domingos; Antonio Fabron Junior
Advisor: Nicola Amanda Conran Zorzetto; Fernando Ferreira Costa

Sickle cell disease results from a single amino acid substitution in the gene encoding the ?-globin subunit, leading to hemoglobin S production in red blood cells. Polymerization of deoxygenated sickle hemoglobin leads to decreased deformability of red blood cells, resulting in hemolytic anemia and vaso-occlusive events. Platelets appear to play an important role in the vaso-occlusive process, as following their activation they express and secrete mediators that induce an inflammatory response in endothelial cells and leukocytes. The purpose of this study was to investigate the production and expression of LIGHT and CD40L on platelets, the presence of this protein in the plasma of controls (healthy subjects; CON), sickle cell anemia patients (AF) and sickle cell anemia patients on hydroxyurea therapy (AFHU). In addition, this study evaluated the involvement of platelets and their mediators, LIGHT and CD40L, in the activation of leukocytes and endothelial cells. Plasma levels of both cytokines were significantly higher in AF and AFHU individuals than in control individuals and interestingly, HU therapy was not associated with a reduction in these levels. A significant correlation was observed between levels of LIGHT with plasma levels of CD40L, IL-8, ICAM-1, Thrombospondin-1 and TNF-?, whereas the plasma concentration of CD40L correlated with levels of TNF-? and especially with plasma Thrombospondin-1. LIGHT expression was significantly higher on the surface of platelets from AF and AFHU subjects, compared with CON individuals and this expression demonstrated a correlation with markers of platelet activation. LIGHT secretion was determined by ELISA and significant concentrations of this cytokine could be detected in the supernatant of platelets from CON and AF individuals, indicating that platelets may be an important source of LIGHT. Although CD40L expression was not detected on the platelet surface in patients or controls, sickle platelets secreted an increased amount of CD40L, compared to controls. A significant correlation was observed between CD40L and LIGHT release in sickle cell patients, indicating that the production of these two proteins may be tightly coupled. The expression of LIGHT (HVEM and LT?R) and CD40L (CD40) receptors was evaluated by flow cytometry on the surface of platelets, neutrophils, lymphocytes and monocytes. An increased HVEM receptor expression was observed on the platelets and lymphocytes of sickle cell patients, whereas the expression of the CD40 receptor was elevated on platelets, neutrophils, lymphocytes and monocytes from sickle cell patients, compared to control subjects. Evaluating the contribution of platelets to leukocyte activation, we observed that platelets from sickle cell anemia individuals increased the expression of the activation marker, CD69, on lymphocytes and also induced a pro-inflammatory phenotype on monocytes. Co-culture of HUVEC with platelets demonstrated that sickle cell platelets have an increased ability to induce ICAM-1 expression on endothelial cells than platelets from control subjects. Furthermore, in the presence of anti-CD40L antibodies, a drastic reduction was observed in this increase. Although the expression of endothelial ICAM -1 was also reduced in the presence of anti-LIGHT antibodies, these results were not statistically significant. Interestingly, high plasma concentrations of LIGHT were associated with elevated tricuspid regurgitant velocity, indicative of pulmonary hypertension in sickle cell anemia. A significant association was also found between high levels of CD40L and patients with a lifetime history of acute chest syndrome. LIGHT and CD40L appear to contribute to leukocyte and endothelial activation, playing an important role in the pathophysiology of sickle cell anemia and apparently in the clinical manifestations of this disease. These results highlight the important role that platelets and their inflammatory mediators may play in the vascular inflammation that is known to occur in sickle cell anemia (AU)

FAPESP's process: 11/17553-7 - Expression of the inflammatory cytokine light in platelets of Sickle Cell disease patients: evaluation of activation of leukocytes and endothelial cells
Grantee:Vanessa Tonin Garrido
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)