Abstract

Abstract 1015

Introduction:

Platelets are mediators of inflammation as well as thrombosis. Platelets are necessary for normal inflammation and play a primary role in the immune clearance of certain pathogens. Platelets secrete cytokines when they are activated, and can also synthesize proteins de novo when activated, such as IL-1b. Sickle Cell Disease (SCD) is characterized by an activated immune system, and it is now recognized that much of the pathology of SCD results from an inflammatory vasculopathy. Platelets may mediate some of the vascular injury through their inflammatory function, as indicated by: 1) elevated platelet counts in SCD, 2) platelets circulating in an activated state in SCD, as judged by activation dependent surface markers, and 3) elevated blood levels of platelet derived inflammatory proteins, such as soluble CD40 ligand.

Hypothesis:

A direct mechanism through which platelets could impact the immune system in SCD would be to produce and secrete cytokines. We hypothesized that the identity profile and quantity of cytokines produced and secreted by platelets would be altered in SCD compared to controls, and that those alterations would be associated with clinical status. We measured the expression levels of cytokine mRNA in resting and activated platelets in patients with SCD and compared those to controls. We also measured thrombin-induced secretion of cytokines from highly purified platelets in buffer and compared that to controls. Cytokines representing TH1, TH2, and TH17 immune phenotypes were measured.

Methods:

Whole blood was collected from adult patients with SCD undergoing exchange transfusions. Platelets were isolated by sequential centrifugation followed by passage through an affinity column for glycophorin-A and CD45. Platelets were activated with 0.125U/ml thrombin for 18 hours in M199 medium at 37C, and then the supernatant (the platelet releasate) was analyzed for cytokine content. Cytokines were measured using BD Cytometric Bead Array for Th1/Th2/Th17 profile: IL-1β (driver of inflammation, secreted from platelets), IL-2, IL-4, IL-6 (induction/maintenance of autoimmunity), IL-10, IL-17a (maintenance of autoimmunity), TNF-α, IFN-γ, and sCD40L (secreted from platelets). Total RNA was isolated from either fresh platelets or platelets after activation and analyzed by QRT-PCR. Relative mRNA quantity was measured using certified mRNA primer sets and SYBR green detector, using β-actin as an internal control.

Results:

Both IL-6 and TGFβ mRNA levels were significantly increased to more than nine- and six-fold over control levels, respectively. IL-1β and IL-10 mRNA levels were also increased over controls, but this did not reach significance. Platelet secretion of IL-6, IL-1β, and soluble CD40 ligand was increased approximately 300-, 21-, and 5-fold, respectively, in sickle cell platelets compared to controls. The alterations of cytokine mRNA and cytokine secretion were more pronounced in patients with alloantibodies than in those without. These preliminary findings support the idea of platelets as active modulators of SCD inflammation, and indicate a novel mechanism through which antiplatelet agents may be beneficial in SCD.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.