Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems

Abstract

Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to target reactive oxygen and nitrogen species during polymorphonuclear neutrophil inflammatory burst following injury. Here, we review the following 1) the broad effect of myeloperoxidase on normal physiology, 2) the difference between myeloperoxidase and other peroxidases, 3) the current optical probes available for use as surrogates for direct measures of myeloperoxidase-derived oxidants, and 4) the range of preclinical options for imaging myeloperoxidase accumulation at sites of inflammation in mice. We also stress the advantages and drawbacks of each of these methods, the pharmacokinetic considerations that may limit probe use to strictly cell cultures for some reactive oxygen and nitrogen species, rather than in vivo utility as indicators of myeloperoxidase function. Taken together, our review should shed light on the fundamental rational behind these techniques for measuring myeloperoxidase activity and polymorphonuclear neutrophil response after injury toward developing safe myeloperoxidase inhibitors as potential therapy for chronic obstructive pulmonary disease and rheumatoid arthritis.

Keywords: bioluminescence; neutrophil chemotaxis; noninvasive imaging; reactive nitrogen species; reactive oxygen species.

Figure 1

Biology of PMNs in response to injury and inflammation. Localized overexpression of E‐selectin and P‐selectin on activated endothelial cells slow the PMN roll upon the endothelium via leukocyte‐derived L‐selectin. Responding PMNs transmigrate through the endothelial cells after LFA‐1 hooks intercellular adhesion molecule 1 (ICAM‐1) and arrive at the site of damage just before diapedesis caused by the increased vascular permeability from histamine released from the mast cells. PMNs undergo phagocytosis of the invasive microbes once they arrive at the infection site. In addition, MPO is secreted from the patrolled PMNs to produce the potent antimicrobial reagent HOCl in response to infection.

Figure 2

Surface representation of the active site of the MPO monomer. PDB numbers: 3F9P, the light chain is shown in pink, and the heavy chain is shown in blue. There are 3 linkages between the heme and protein through Asp⁹⁴ on the light chain and Glu²⁴² and Met²⁴³ on the heavy chain (highlighted as yellow). The catalytically essential residues Gln⁹¹, His⁹⁵, and Arg²³⁹ (highlighted as red) compose the distal heme pocket; in the proximal site, the heme iron is coordinated through the His³³⁶ imidazole residue (highlighted as turquoise).

Figure 3

Fluorescence reaction for measuring MPO activity. (A) General mechanism of ADHP oxidation in a proposed 2‐step reaction, whereby the MPO–H₂O₂ complex generates 2 ADHP radicals that undergo a subsequent enzyme‐independent dismutation reaction to complete formation of 1 resorufin and 1 ADHP molecule. (B) Classic biochemical assays are possible using absorbance changes caused by the MPO–H₂O₂ system and the use of new fluorogenic probes, such as ADHP. Pictures are cuvettes containing increasing concentrations of the MPO–H₂O₂ system, as indicated, at a static ADHP level (40 μM). (C) Stopped‐flow progress curves of resorufin generation by MPO (23 nM) initiated by the addition of H₂O₂ (22 μM) for a series of given ADHP concentrations (adapted from Huang et al. [41]).

Figure 4

In vivo monitoring of MPO activity in mice. (A) Experimental design of the imaging study is shown. (B) A representative image of a mouse 6 d after s.c. injection of Streptococcus pyogenes (3 × 10¹⁰ CFU in phosphate‐buffered saline) in the upper back of the animal shown in white light (right image) and bioluminescence imaging (left image). A yellow dotted circle indicates the region of interest. (C) An example of the hydrogen peroxide (H₂O₂)‐dependence of MPO in vivo activity is shown. Albino C57BL/6 mice injected with 180 nM of MPO with either 400 μM of H₂O₂ (site 1), 2 mM of H₂O₂ (site 2), 4 mM of H₂O₂ (site 3), and 40 mM of H₂O₂ (site 4) were added to the matrigel (100 μl) before s.c. implant. (B–C) To visualize the MPO activity, an i.p. injection of luminol (Sigma‐Aldrich, 3 mg/kg) was given 10 min before luminescence imaging using the IVIS Lumina XRMS system (PerkinElmer, Waltham, MA, USA). The Institutional Animal Care and Use Committee of Auburn University approved all animal procedures.