Loss of Cd40 Endogenous S-Nitrosylation During Inflammatory Response in Endotoxemic Mice and Patients With Sepsis

Abstract

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

Tyrosine nitration limits stretch-induced CD40 expression and disconnects CD40 signaling in human endothelial cells

“…35 Thus far, no oxidative modification of CD40 has been described except an endogenous S-nitrosylation in human monocyte/ macrophages that may affect its spatial distribution on the cell surface, thereby preventing an optimum physical interaction of CD154 with CD40. 36 The present data strongly suggest that nitration of Tyr-82 in stretched human cultured endothelial cells results in its subsequent degradation by the chymotrypsin-like activity of the 20S proteasome. The finding that these cells also synthesize CD40 de novo on exposure to cyclic stretch raised the question of whether it is the newly synthesized protein on its way to the membrane that is trapped by nitration, for example, in the trans-Golgi network, and then degraded by the proteasome or the mature membrane-bound protein whose nitration causes its internalization and subsequent degradation.…”

Effect of Enhanced Glycemic Control with Saxagliptin on Endothelial Nitric Oxide Release and CD40 Levels in Obese Rats

“…CD40 expression on inflammatory cells is essential to plaque initiation, progression and stability 32) . Activation of CD40 is regulated by eNOS through direct nitrosylation and changes in cell membrane distribution 5) . This is the first study to show a reduction in CD40 levels with a DPP4 inhibitor and is consistent with the observed reversal in endothelial dysfunction.…”

“…A pathophysiologic relationship between reduced nitric oxide (NO) bioavailability and loss of insulin synthesis 5) . Saxagliptin is a dipeptidyl peptidase-4 (DPP4) inhibitor that causes significant reductions in postprandial plasma glucose and hemoglobin A1C levels 6,7) .…”

Nitrosothiols in the Immune System: Signaling and Protection

“…S-nitrosylation of the CD40 extracellular domain occurs in quiescent macrophages and monocytes, whereas denitrosylation is produced after activation by CD40L (52). This FIG.…”

“…On the other hand, S-nitrosylation of Src may activate NF-jB (To see this illustration in color, the reader is referred to the web version of this article at www .liebertpub.com/ars). modification blocks CD40 signal transduction, such that CD40 denitrosylation could be a prerequisite for macrophage activation via the CD40 pathway (52). Indeed, CD40 denitrosylation was observed in mice injected with LPS and in monocytes from patients suffering severe sepsis or septic shock (52).…”