The platelet as an immune cell-CD40 ligand and transfusion immunomodulation

Abstract

The discovery that platelets possess cell membrane, cytoplasmic, and secreted forms of the co-stimulatory molecule CD40 ligand (CD40L, also known as CD154) has led to a revolution in the view of this anucleate, differentiated cell fragment, previously thought only to be involved in blood clotting (hemostasis). During the last decade, it has become clear that platelets function in innate and adaptive immunity and possess pro-inflammatory, as well as pro-thrombotic properties. They interact not only with other platelets and endothelial cells, but also with lymphocytes, dendritic cells, and structural cells such as fibroblasts. Soluble forms of CD40L (sCD40L) in the human circulation are almost entirely derived from platelets. Elevated levels of CD40L are associated with clinically important conditions, such as vascular disease, abnormal clotting (thrombosis), lung injury, and autoimmune disease. Each year millions of platelet transfusions are given to patients that contain large amounts of sCD40L. sCD40L in the supernatant of stored platelets can induce cytokines, chemokines, and lipid mediators by activating CD40 bearing cells. Increased levels of sCD40L in transfused blood are associated with transfusion-related acute lung injury, a potentially fatal complication, as well as more common, milder transfusion reactions such as fever and rigors. These effects come under the rubric of transfusion immunomodulation, which postulates that transfusion recipient biology, particularly immune function, is dramatically altered by transfusion of stored allogeneic blood.

Figure 1. Role of Platelet CD40L in Inducing Inflammation

1. Platelets become activated at the site of vessel injury

2. Activated platelets express / secrete CD40L

3. Platelet CD40L serves to trigger the onset of inflammation

Figure 2. Platelets secrete soluble CD40L (sCD40L) during storage prior to transfusion

The concentration of sCD40L in the storage supernatant of washed platelet prepared for transfusion is greatest at 3 or 4 days of storage. Soluble CD40L was measured by standard sandwich ELISA.

Figure 3. Platelet-derived CD40L induces Cox-2 expression in human lung fibroblasts, and induces the production of PGE₂ and IL-6

Panel A: Human lung fibroblasts were stimulated with medium (unstimulated), PCS (platelet concentrate supernatant) or CD40L-depleted PCS. After 24 hours, cells were stained with a Cox-2 specific antibody or an isotype control antibody. Unstimulated cells express low levels of Cox-2, while PCS strongly activated lung fibroblasts to up-regulate Cox-2 protein. The induction of Cox-2 was partly dependent on CD40L, as CD40L-depleted PCS had a significantly reduced capacity for Cox-2 induction. Panels B and C: Cell-free culture medium from similarly treated human lung fibroblast cultures was analyzed for PGE₂ and IL-6 content. Unstimulated cells produced low levels of PGE₂ and IL-6, while PCS caused great amounts of these mediators to be produced by the fibroblasts. These high levels of production were reduced when CD40L was neutralized in the PCS via an anti-CD40L antibody. Mean +/- SEM, n = 9 similar experiments, * p < 0.001 PCS vs. Unstim., ** p < 0.001 PCS + anti-CD40L vs. PCS alone.

Figure 4. CD40L concentration in platelet concentrates is associated with adverse clinical events following transfusion

Panel A shows the mean levels of CD40L in platelet transfusions implicated in cases of transfusion related acute lung injury (TRALI) as opposed to non-implicated control platelet transfusions. CD40L levels were significantly higher in transfusions implicated in TRALI. Panel B displays the rate of transfusion reactions (fever/rigors) versus quintiles of CD40L levels in platelet transfusions. The rate of adverse reactions increases significantly from 0% to 5% as the levels of CD40L increase from the 1st to 5th quintile.

Figure 5. Washed transfusions are associated with improved survival in leukemia patients

A Kaplan-Meier plot of survival of 118 consecutive adult patients (ages 18-80) treated with curative intent for acute leukemia is shown according to whether patients received washed versus unwashed transfusions during their entire course. Patients receiving washed transfusions had significantly better survival, even after adjusting for other prognostic factors by proportional hazards analysis.