CD40L and Its Receptors in Atherothrombosis-An Update

Abstract

CD40L (CD154), a member of the tumor necrosis factor superfamily, is a co-stimulatory molecule that was first discovered on activated T cells. Beyond its fundamental role in adaptive immunity-ligation of CD40L to its receptor CD40 is a prerequisite for B cell activation and antibody production-evidence from more than two decades has expanded our understanding of CD40L as a powerful modulator of inflammatory pathways. Although inhibition of CD40L with neutralizing antibodies has induced life-threatening side effects in clinical trials, the discovery of cell-specific effects and novel receptors with distinct functional consequences has opened a new path for therapies that specifically target detrimental properties of CD40L. Here, we carefully evaluate the signaling network of CD40L by gene enrichment analysis and its cell-specific expression, and thoroughly discuss its role in cardiovascular pathologies with a specific emphasis on atherosclerotic and thrombotic disease.

Keywords: CD40 signaling; CD40L; Mac-1; atherosclerosis; cardiovascular diseases; inflammation; thrombosis.

Figure 1

Model of CD40L–receptor interactions. Membrane-bound CD40L forms biologically active trimers that can interact with one of its known receptors, the integrins αIIbβ3, α5β1, αMβ2 (Mac-1), or its “classical” receptor CD40. While binding to CD40 occurs statically, binding to integrins can be enhanced by activation-induced conformational changes of both subunits that expose the ligand-binding site (inside-out signaling). Binding to Mac-1 occurs predominantly through the activated integrin, binding to α5β1 requires no previous cell activation and switching into the open, high-affinity conformation. Binding to αIIbβ3 usually occurs through the open conformation, but exact binding properties to CD40L have not been investigated in detail. Besides CD40L, CD40 can also interact with the complement-factor C4b-binding protein (C4BP). Binding of CD40L to one of its receptors induces downstream signaling events, except for Mac-1, for which CD40L serves as biased agonist without induction of outside-in signaling. Binding to α5β1 causes an activation of MAP-kinase signaling pathways, ligation to CD40 causes activation of mitogen-activated protein kinase (MAPK)-, phosphoinositide 3-kinase (PI3K)-, and nuclear factor-κB (NF-κB)-signaling events and subsequent pro-inflammatory gene expression. The consequences of CD40 signaling depend on the target cell type. On the contrary, ligation of CD40L to a receptor induces bidirectional signaling events in CD40L-bearing cells, e.g., T cells, B cells, or platelets, possibly by induction of MAPK signaling cascades. Binding of CD40L to its ligands can occur in a homotrimeric fashion, where a trimer of CD40L binds three monomers of CD40, or in a heterotrimeric fashion, where each monomer (of a trimer of CD40L) can bind to different receptors, which was demonstrated for CD40, αIIbβ3, and α5β1.

Figure 2

Gene expression patterns of CD40L and its receptors in human immune cell types. Baseline gene expression of different human immune cell types quantified by RNAseq was extracted from the Protein Expression Atlas of the European Bioinformatics Institute (EMBL-EBI) (33). Expression values were retrieved as FPKMs, underwent hierarchical clustering and normalization as row scores by Morpheus (Broad Institute). Gene names are encoding for the proteins as follows: Cd40lg: CD40L; Itgb2: integrin subunit α2b (CD41); Cd40: CD40 receptor; Itgam: integrin subunit αM (CD11b); Itga5: integrin subunit α5 (CD49e).

Figure 3

Enrichment of the CD40L/CD40 pathway in unstable human plaques. Gene set enrichment of the core CD40-signaling gene signature (Table 3) was tested on published transcriptomes of human stable and ruptured atherosclerotic plaques from laser-microdissected macrophage-rich regions of carotid endarterectomy specimen. Enrichment was tested between ruptured and stable plaques. The expression of the enrichment-driving genes (lower panel) was blotted on a heatmap and colored by a row-score (blue = lowest, red = highest gene expression per row).

Figure 4

CD40L and its receptors in thrombosis and hemostasis. (1) Monomeric CD40L is stored in α- and dense granules of platelets. Upon platelet activation and degranulation, CD40L is mobilized to the cell surface and forms biologically active trimers. These are shed within minutes to hours by ADAMs and matrix metalloproteinases (MMPs) and released as monomers of soluble CD40L (sCD40L). These have been reported to form dimers and trimers in circulation that a biologically active. (2) Platelet-expressed CD40L binds to its receptors on leukocytes, α5β1 and CD40, but not αMβ2 (Mac-1), to form platelet–leukocyte aggregates. Notably, platelet-expressed CD40 can bind to CD40L-expressing leukocytes, such as activated T cells. (3) Ligation of platelet-expressed receptors, α5β1 and CD40, by CD40L initiates platelet activation cascades that eventually result in the surface expression of platelet receptors, such as P-selectin, degranulation, aggregation, and spreading. CD40 ligation on platelets activates a TRAF-2 dependent, but not TRAF-6 dependent, signaling pathway. It is a controversy whether ligation of α2bβ3 by CD40L participates in platelet activation. However, physical interactions between platelet-expressed α2bβ3 and CD40L, as well as between CD40 and CD40L, help to stabilize platelet aggregates and fuel thrombus growth. Binding of platelet CD40L and of sCD40L to its endothelial receptors CD40 and α5β1 promotes endothelial cell activation, pro-inflammatory gene expression, and secretion of coagulation factors, including tissue factor, which in turn can amplify thrombosis and coagulation.

Figure 5

Participation of CD40L pathways in leukocyte recruitment. Several simultaneous mechanisms that contribute to the recruitment and transmigration of inflammatory leukocytes in inflamed tissue have been identified: (1a) endothelial cells (ECs) express membrane-bound CD40L in inflammation. The exact dynamics and stability of endothelial-expressed CD40L is unknown, but shedding by ADAMs and matrix metalloproteinases (MMPs) has been described. The interaction of αMβ2 (Mac-1) with endothelial CD40L mediates slow rolling and firm adhesion, which ultimately favors myeloid cell accumulation in tissues. (1b) It has been proposed that besides αMβ2, also CD40 and α5β1, expressed on leukocytes may participate in rolling and adhesion. (2) Ligation of α5β1 and CD40 on leukocytes, either by sCD40L or membrane-bound CD40L from leukocytes, platelets, or ECs activate pro-inflammatory signaling in leukocytes and increased expression of adhesion receptors that will in turn enhance cell recruitment. (3) α5β1 and CD40 expressed on ECs is ligated by sCD40L or cell-bound CD40L. As a result, ECs upregulate adhesion molecule expression and secretion of chemokines, besides other pro-inflammatory changes.