Alpha-Melanocyte Stimulating Hormone (α-MSH) Is a Post-Caspase Suppressor of Apoptosis in RAW 264.7 Macrophages

The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.