Ligation of CD40 in Human Müller Cells Induces P2X(7) Receptor–Dependent Death of Retinal Endothelial Cells

PURPOSE: Cluster 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. METHODS: Human 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 (P2X(7) 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 P2X(7) was assessed by FACS, quantitative PCR, or immunohistochemistry. RESULTS: Ligation 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 P2X(7) (A-438079) impaired PCD of RECs; CD40 upregulated P2X(7) in RECs, making them susceptible to ATP/P2X(7)–mediated PCD. Diabetic mice upregulated P2X(7) in the retina and RECs in a CD40-dependent manner. CONCLUSIONS: Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X(7) upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X(7) in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.