Bioluminescence imaging of myeloperoxidase activity in vivo

Abstract

The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo −/− mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions. The heme-containing enzyme MPO is a key component of the cytotoxic armamentarium of phagocytic white blood cells 1,2 . MPO is by far the most abundant protein product in azurophilic granules of neutrophils (5%), constitutes approximately 1% of monocyte protein and is found in the lysosomes of other polymorphonuclear leukocytes and macrophages. The phagosomal oxidative burst is initiated by a stimulus-dependent assembly of the phagocytic NADPH oxidase (Phox), a multimeric protein complex located on the phagosomal membrane. Phox then reduces molecular oxygen to produce superoxide anion (O 2•− ), which further dismutates to yield the relatively unreactive hydrogen peroxide (H 2 O 2 ) 1 . Upon phagocytic activation, large quantities of active MPO are secreted into phagosomes, catalyzing the production of highly bactericidal hypochlorous acid (HOCl) with H 2 O 2 and chloride ions (Cl − ) as substrates (Fig. 1a) 1 .

Myeloperoxidase-induced Genomic DNA-centered Radicals

“…produced from acetaldehyde/xanthine or peroxynitrite produced from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) decomposition. These observations are in agreement with a recent study which showed that luminol is a specific probe for imaging HOCl produced by MPO in vivo (20). Fifty l of 10 M luminol were, therefore, mixed in each well of a 96-well black microplate with an equal volume of resveratrol dissolved in 100 mM Chelex-NaP buffer, pH 7.4.…”

“…To test whether resveratrol inhibits HOCl-triggered DNA oxidation, we measured the ability of resveratrol to block DNA-DMPO nitrone adduct formation and luminol oxidation by HOCl. Although luminol has been recently used to image HOCl production by MPO in vivo (20), it is known that luminol reacts with peroxynitrite and O 2 . , which are also produced during inflammation (32,43).…”

Cutting Edge: The Murine High-Affinity IgG Receptor FcγRIV Is Sufficient for Autoantibody-Induced Arthritis

“…Activated neutrophils and inflammatory monocytes/macrophages can release myeloperoxidase. Myeloperoxidase can oxidize luminol, leading to photon emission that can be detected by bioluminescence imaging (22). Following luminol injection, luminescence was detected in the joint areas, as early as 1 d after K/BxN serum injection in both wt and 5KO mice (Fig.…”

Enhanced detection of myeloperoxidase activity in deep tissues through luminescent excitation of near-infrared nanoparticles

“…Noninvasive imaging of MPO enzymatic activity with luminol was previously reported 8 . These researchers found that luminol specifically interacts with hypochlorous acid to produce an emission of luminescent light that can be detected through optical imaging.…”