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

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. © 2005 Elsevier B.V. All rights reserved.This work was supported by Spanish Ministries of Health (FIS grants 01/113, PI0408827 and PI0408850 and Redes Temáticas de Investigación Cooperativa C03/01 and C03/14) and Education and Science (grants SAF2002-02160, SAF2004-06856, BIO2002-04091-C03-01, BIO2003-09056-CO2-O2) and by Comunidad Autónoma de Madrid (grant GR/SAL/0854/2004).Peer Reviewe