Interactive Ca²⁺ signaling in the RPE by anaphylatoxins

Abstract

Purpose : Through the identification of genetic risk factors, it has become apparent that dysregulation of the complement system plays a role in the pathology of age-related macular degeneration (AMD). This would predict that a less efficiently controlled alternative activation pathway would in turn harm the retinal pigment epithelium (RPE). Rather than investigating the potentially harmful effects of the activity of the terminal complement complex (membrane attack complex) on the RPE, we focused here on examining the effects of the soluble anaphylatoxins C3a and C5a, products of C3 and C5 convertases.

Methods : ARPE-19 cells, human RPE cells in primary culture and freshly isolated mouse RPE cells were investigated for their expression of the anaphylatoxin receptors by means of PCR or immunohistochemistry. Second messenger activation by the anaphylatoxins C3a or C5a was studied by Ca²⁺ imaging techniques using fura-2 as a Ca²⁺-sensitive fluorescence probe.

Results : ARPE-19 cells express the anaphylatoxin receptors C3aR and C5aR1 both on mRNA and protein level. This expression pattern was confirmed in human RPE in primary culture and in freshly isolated mouse RPE cells at the mRNA level. Application of C3a or C5a individually both led by to an increase in intracellular free Ca²⁺; although the Ca²⁺ signal in response to C5a was larger in amplitude and duration compared to that elicited by C3a. The simultaneous application of C3a and C5a did not show an additive effect; the resulting response in amplitude and duration was the same as that with C5a alone. Interestingly, when C5a was applied at the moment when the C3a-induced Ca²⁺ response has reached its steady-state level, this led to a decrease in the Ca²⁺ signal. Analysis of the downstream events using blockers against protein kinases known to control anaphylatoxin-mediated signaling in T-cells revealed that the C5a-stimulated Ca²⁺ response depended on the activation of Akt and PI3 kinases, whereas surprisingly the C3a-evoked response was increased by inhibition of Akt kinase.

Conclusions : The RPE is able to respond to complement activation already at the level of the anaphylatoxins. Both anaphylatoxins seem to use different down-stream Ca²⁺ induced signaling pathways. However, combined receptor activation results in an interactive behavior that appears to be dominated by C5aR engagement. Thus the anaphylatoxin-dependent signaling in the RPE might have important impacts on the etiology of AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.