Objectives: 

The objective of this study was to investigate the intracellular pathways involved in the protection provided by melatonin against apoptosis induced by tumour necrosis factor-alpha (TNF-alpha) in human neutrophils and lymphocytes.

Materials: 

Peripheral blood leucocytes were purified from healthy individuals by Ficoll centrifugation. Cells were treated for 2 hours with TNF-alpha (100 ng/ml) in the presence of cycloheximide (CHX; 10 micrograms/ml), which promotes caspase-8 activation by eliminating endogenous caspase-8 inhibitor, c-FLIP. Protein expression levels of caspase-3, caspase-9, caspase-8, cFLIP, Bid, ERK1/2, and phospho-ERK1/2 were evaluated by Western blotting. Also, apoptotic cell death was analysed by redistribution of phosphatidylserine (PS) in the presence of propidium iodide (PI).

Results: 

Treatment with TNF-alpha plus CHX led to apoptotic cell death, as ascertained by Annexin V/PI staining. Likewise, in the presence of CHX, TNF-alpha treatment induced cFLIP down-regulation and subsequent caspase-8 activation, thus directly triggering caspase-3, on the one hand, and causing Bid truncation and subsequent caspase-9 activation, on the other hand. Conversely, pre-treatment with 1 mM melatonin for 1 hour inhibited TNF-alpha plus CHX-evoked leukocyte apoptosis. Similarly, pre-treatment with melatonin restored cFLIP protein levels, thereby preventing TNF-alpha plus CHX-mediated caspase activation. Blockade of melatonin membrane receptors MT1/2 and extracellular signal-regulated kinase (ERK) pathway with luzindole and PD98059, respectively, abolished the protective effects of melatonin on leukocyte apoptosis evoked by TNF-alpha plus CHX.

Conclusions: 

Our results suggest that melatonin seemingly requires membrane receptor MT1/2 interaction and ERK activation to counteract TNF-alpha-induced leukocyte apoptosis.

Supported by MICINN-FEDER grant (BFU2010-15049). J. Espino is beneficiary of grant from MEC (AP2009-0753).