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.

Figure 1

Luminol bioluminescence is dependent on MPO in vitro and ex vivo. (a) A schematic representation of the biochemical basis of luminol bioluminescence. HOCl produced by MPO can directly or indirectly oxidize luminol to produce light. Alternatively, MPO can also use the superoxide anion (dashed line) or other ROS (for example, NO^•) as substrates for peroxidase-catalyzed oxidation of luminol. (b) Dynamic luminol-BLI of PMA-stimulated whole blood. At t = 0, PMA (5 μM; bottom) or vehicle (top) were added, and bioluminescence was monitored for 90 min. Images show sequential color-coded maps of photon flux superimposed on black and white photographs of the assay plates. (c) 4-ABAH inhibits luminol bioluminescence in vitro. Peak bioluminescence was determined for solutions containing luminol, purified MPO, H₂O₂ and increasing concentrations of 4-ABAH (0-500 μM). (d,e) 4-ABAH inhibits luminol bioluminescence in PMA-stimulated whole blood ex vivo. Fresh heparinized whole blood was incubated with increasing concentrations of 4-ABAH and luminol in MEBSS. At t = 0, PMA (5 μM) or vehicle were added, and bioluminescence was monitored for 90 min. Luminol bioluminescence (fold increase over initial) was plotted as a function of time (d) and concentration of 4-ABAH (e), as derived from the 22.5-min time point. (f) Fresh heparinized whole blood from Mpo^+/+ or Mpo^−/− mice was incubated with luminol in MEBSS. BLI (0–55 min) was initiated instantly upon stimulation with PMA and plotted as a function of time after PMA treatment. (g) Contribution of NO^• radicals and Phox-generated O₂^•− to luminol bioluminescence in cellulo. Fresh blood from an Mpo^+/+ mouse was incubated with the NOS inhibitor l-NMMA (1 mM) or the Phox inhibitor diphenyleneiodonium (DPI, 10 μM), respectively, and imaged 20 min after stimulation with PMA (5 μM). Error bars represent ± s.e.m.

Figure 2

Luminol-BLI of MPO and glucose oxidase (GOX) implants in vivo. (a) Vehicle (V; PBS), GOX, MPO or MPO plus GOX were embedded in Matrigel, and subcutaneous implants were established in nu/nu mice (n = 3). Shown is a bioluminescence image taken at t = 0 (before luminol administration). Scale bar, 1 cm. (b) BLI (5–60 min at 5-min intervals) was initiated after i.p. injection of luminol (200 mg per kg body weight). Scale bar, 1 cm. (c) Representative time-course of photon fluxes from the various implants in one mouse.

Figure 3

Lack of luminol bioluminescence in vivo in Mpo^−/− mice during acute inflammatory insults. (a) Luminol bioluminescence was dependent on MPO in an acute dermatitis model. Dermatitis was generated by topical application of PMA on the left ear lobe of Mpo^+/+ or Mpo^−/− mice (shown are three representative mice for each genetic background, n = 5 for each group). The right ears served as vehicle (ethanol) controls. Twenty-four hours after application of PMA, luminol was administered (i.p.) and mice were imaged. Scale bar, 1 cm. (b) LPS was injected into the ankle joints of the left lower limbs of wild-type Mpo^+/+ (n = 5) or Mpo^−/− mice (n = 5). Vehicle (PBS) was injected into the joints of the right lower limbs. At t = 0 h (before LPS injection) and 24 h, 48 h, 72 h, 96 h and 120 h after LPS injection, luminol was administered (i.p.), and mice were imaged. Representative images taken at t = 48 h are shown. Scale bars, 1 cm. (c) Background-subtracted LPS-induced luminol bioluminescence (± s.e.m.) quantified as fold increase over vehicle-treated foot for each Mpo genotype at the indicated time points. (d) Histological analysis of the joint region showing massive infiltration of neutrophils 48 h after LPS injection in both Mpo^+/+ and Mpo^−/− mice. Inset, high-resolution images. Scale bars, 10 μm. (e) Mean number of neutrophils per high power field (± s.e.m.).

Figure 4

Luminol-BLI of allergic contact hypersensitivity. (a,b) Acute allergic dermatitis was induced by passive immunization with dinitrophenol-specific IgE (anti-DNP IgE) and topical challenge with dinitrofluorobenzene on the left ear lobes of Mpo^+/+ and Mpo^−/− mice. Right ears served as vehicle controls and mice challenged with dinitrofluorobenzene (DNFB), but not anti-DNP IgE, served as negative controls. Twenty-four hours after application of DNFB, luminol was administered, and mice were imaged. (a) Shown are one Mpo^+/+ and one Mpo^−/− mouse. Scale bar, 1 cm. (b) Background (vehicle on right ear)-subtracted DNFB-induced luminol bioluminescence (± s.e.m.) for each Mpo genotype. n = number of mice. (c) Histological analysis of proximal ear lobes, showing massive edema and eosinophilia within a mixed cell infiltrate 24 h after DNFB treatment of both Mpo^+/+ and Mpo^−/− mice, but not vehicle-treated mice; lower right panel shows eosinophils in a treated Mpo^−/− mouse. Insets show immunostaining with monoclonal antibody to dibromotyrosine of the same specimens as the main panel, documenting the presence of bromotyrosine adducts in treated ears of both Mpo^+/+ and Mpo^−/− mice (brown staining), but not vehicle-treated controls. Scale bars, 50 μm. (d) Mean number of eosinophils or neutrophils per HPF ± s.e.m.

Figure 5

Luminol-BLI of spontaneous LGL tumors in Gzmb:Tax mice. (a) H&E staining of a large (∼1-cm) tumor from the tail of a Gzmb:Tax mouse, showing that the majority of the cell mass is made of neutrophils (polymorphic nuclei; magnification ×630). (b) MPO immunostaining of the same specimen as in a showing localization of MPO within neutrophils (brown staining). (c) H&E staining of a very small (∼200-μm) tumor from the earlobe of a Gzmb:Tax mouse (arrow). Scale bars in a–c, 50 μm. (d–g) Luminol-BLI of tumor-bearing Gzmb:Tax mice showing colocalization of luminol bioluminescence with small tumor foci (yellow arrows). Scale bars in d and e, 1 cm; scale bars in f and g, 1 mm.