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. 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 (O2•-) which further dismutates to yield hydrogen peroxide (H2O2)1. Upon phagocytic activation, large quantities of active MPO are secreted into phagosomes, catalyzing the production of highly bactericidal hypochlorous acid (HOCl) using H2O2 and chloride ions (Cl-) as substrates.
Luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione) is a redox-sensitive compound that emits blue luminescence (lambdamax = 425 nm) when exposed to an appropriate oxidizing agent. High stability and low cost has rendered luminol useful in a variety of fields ranging from metallurgy, analytical chemistry, biochemistry, clinical diagnostics and forensic sciences for detecting reactive intermediates. Luminol-enhanced luminescence can detect extraordinarily low concentrations of oxidizing species in complex biological systems and indeed, luminescence of isolated phagocytes and whole blood was introduced 25-30 years ago2,3. Luminol-enhanced luminescence enables analyses ex vivo of the phagocytic oxidative burst upon stimulation with a myriad of soluble activators, opsonized particles, or intact microorganisms4,5. Luminol-enhanced luminescence also is used clinically to screen neutrophils ex vivo for defects in oxidative metabolism such as chronic granulomatous disease (CGD)4 and MPO deficiency6. Luminol is relatively nontoxic, well absorbed and rapidly excreted upon systemic administration7, and was used to treat humans with alopecia areata in the 1960’s8. We have therefore developed a method to image MPO activity at sites of inflammation in small laboratory animals9. We have also demonstrated the unique specificity of luminol bioluminescence to MPO activity, but not to other oxidizing species (e.g., H2O2, superoxide anion or nitric oxide) in whole blood samples ex vivo or other peroxidases (e.g., eosonophil peroxidase) in vivo. Thus, this powerful technique provides the means to continuously monitor MPO activity in real-time by bioluminescence imaging (BLI) upon systemic administration of luminol. Herein we present a protocol to image MPO activity in a simple mouse model of acute dermatitis, induced upon topical application of phorbol 12-myristate 13-acetate (PMA) on the ear lobe. To demonstrate the specificity of this imaging technique, we use both wild type and MPO-/- animals.