Ligation of CD40 in Human Müller Cells Induces P2X₇ Receptor–Dependent Death of Retinal Endothelial Cells

Abstract

PurposeCluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox.MethodsHuman RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry.ResultsLigation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7–mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner.ConclusionsCluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.

P2X7 as a scavenger receptor for innate phagocytosis in the brain

“…Both nucleoside reverse transcriptase inhibitors and P2X7 antagonists suppressed the clinical symptoms of GA and CNV in animals (Kerur et al ., ; Mizutani et al ., ). P2X7‐mediated retinal cell apoptosis can also be stimulated by ligation of CD40 (Portillo et al ., ) and amyloid β peptides (Wakx et al ., ), for which P2X7 antagonists have shown beneficial effects. It is worth noting that although complete loss of P2X7 receptors in P2X7 −/− mice protected the animals from developing GA (Carver et al ., ; Kerur et al ., ), it failed to protect the animals from developing laser‐induced CNV, a more severe form of late AMD (Mizutani et al ., ).…”

The CD40-ATP-P2X7 Receptor Pathway: Cell to Cell Cross-Talk to Promote Inflammation and Programmed Cell Death of Endothelial Cells

“…Activated by CD40 ligation in Müller glia and triggers release of extracellular ATP 7ATP Secreted by CD40-activated Müller glia and mediates Müller glia-microglia/macrophages and Müller glia-endothelial cell communication (7,8) P2X 7 receptor ATP receptor upregulated in microglia and retinal endothelial cells in a CD40-dependent manner. Induces pro-inflammatory cytokine secretion by macrophages/microglia and programmed cell death in endothelial cells (7,8) TNF-α/IL-1β Upregulated in diabetic and I/R-induced retinopathies, and linked to development of these retinopathies (33-38, 48-50, 53). CD40 promotes TNF-α/IL-1β upregulation (11) ICAM-1 Upregulated in endothelial cells in diabetic and I/R-induced retinopathies and linked to development of these retinopathies (3,29,30,49,54).…”

“…Whereas direct CD40 ligation in retinal endothelial cells does not cause PCD, CD40 stimulation enhances PCD of endothelial cells when they are incubated with CD40 + Müller cells (8). This effect is not driven by NOS2, oxidative stress, TNF-α, IL-1β, or Fas ligand (8).…”

“…As described above, CD40 ligation in Müller glia increases release of ATP. CD40 ligation in retinal endothelial cells upregulates P2X 7 receptor expression making these cells susceptible to ATP-induced PCD (8). Indeed, pharmacologic inhibition of P2X 7 receptor prevents PCD of the endothelial cells (8).…”

“…The CD40 pathway is activated in these retinopathies and CD40 has emerged as a central mediator of inflammatory responses and pathology in these disorders (5)(6)(7). Herein I will review our work that identified CD40 expressed in retinal Müller glia as a trigger for secretion of ATP that in turn engages the P2X 7 receptor leading to pro-inflammatory cytokine production by monocyte/macrophages/microglia and programmed cell death of retinal endothelial cells (7,8). Through this process, CD40 present in a non-hematopoietic cell amplifies inflammation and causes tissue pathology.…”