The induction of NOS2 expression by the hybrid cecropin A–melittin antibiotic peptide CA(1–8)M(1–18) in the monocytic line RAW 264.7 is triggered by a temporary and reversible plasma membrane permeation

Abstract: There is an increasing awareness of immune cell modulation by antimicrobial peptides. While this process often requires specific receptors for the peptides involved, several reports point out to a receptor-independent process. The cecropin A–melittin hybrid peptide CA(1–8)M(1–18) (KWKLFKKIGIGAVLKVLTTGLPALIS-amide) modifies gene expression in the macrophage line RAW 264.7 in the absence of any previous macrophage priming, suggesting a membrane permeation process. To further analyze the initial steps of this mechanism, we have studied the interaction of the peptide with these cells. Below 2 μM, CA(1–8)M(1–18) causes a concentration-dependent membrane depolarization partially reversible with time. At 2 μM, the accumulation of the SYTOX green vital dye is one half of that achieved with 0.05% Triton X-100. The binding level, as assessed by fluorescein-labeled CA(1–8)M(1–18), varies from 7.7±1.2 to 37.4±3.9×106 molecules/cell over a 0.5–4.0 μM concentration range. Electrophysiological experiments with 0.5 μM CA(1–8)M(1–18), a concentration that triggers maximal NOS2 expression and minimal toxicity, show a reversible current induction in the RAW 264.7 plasma membrane that is maintained as far as peptide is present. This activation of the macrophage involves the production of nitric oxide, a metabolite lethal for many pathogens that results from unspecific membrane permeation by antimicrobial peptides, and represents a new mode of action that may open new therapeutic possibilities for these compounds against intracellular pathogens. [Copyright &y& Elsevier]