Crime Scene Compound Shines Light on Immune Inflammation
 David Piwnica-Worms, M.D., Ph.D. Washington University School of Medicine |
 John W. Chen, M.D., Ph.D. Massachusetts General Hospital and Harvard Medical School |
You've seen it on TV shows like "CSI" — a compound known as luminol, sprayed at crime scenes to make blood glow. Today researchers are examining potential uses for luminol in detecting immune inflammation in living animals and say the compound could eventually make its way to clinical application.
The lead author of a new study on bioluminescence imaging (BLI) published in the April 2009 issue of Nature Medicine, David Piwnica-Worms, M.D., Ph.D., a professor of radiology and director of the Molecular Imaging Center at Washington University School of Medicine in St. Louis, and colleagues injected luminol into living mice and found that the compound glowed blue at sites of active immune inflammation. Most surprisingly, it glowed only when the enzyme myeloperoxidase (MPO) was present.
In blood samples ex vivo, researchers noted that luminol is known to be a responsive indicator of a process known as oxidative burst. "This involves the production of hypochlorous acid, a strong oxidizing agent commonly known as bleach," said Dr. Piwnica-Worms.
"Part of the function of activated macrophages and neutrophils—two special types of white cells—is to engulf invading bacteria into a special vesicle inside the cells called the phagosome," he continued. "In the phagosome, when activated, the cells will secrete myeloperoxidase—which will, in that environment, catalyze the reaction of hydrogen peroxide and chloride to make hypochlorous acid. And then it kills the bacteria with that 'bleach' inside these phagosomes."
In Vivo Sensitivity, Specificity Achieved
To determine whether luminol, in vivo, might be specific and sensitive for MPO activity, researchers injected luminol into the peritoneal cavity of living normal and MPO gene-deleted mice. From there the compound circulated into the bloodstream.
"You systemically inject luminol like any compound or drug and it distributes diffusely throughout the body, but you will only see the light where you see the activated MPO—that is where there are areas of inflammation indicating activated neutrophils and macrophages," said Dr. Piwnica-Worms.
"In some ways it might have been expected that you would get some signal from the luminol, based on prior literature," he continued. "What did surprise us were the sensitivity and the MPO specificity in vivo. There was no signal emitted from the MPO gene-deleted mice."
In researching imaging MPO activity for MR and nuclear imaging, John W. Chen, M.D., Ph.D., an assistant professor of radiology in the Division of Neuroradiology and the Center for Molecular Imaging Research at Massachusetts General Hospital and Harvard Medical School, both in Boston, has developed activatable imaging agents that can be modified by MPO, resulting in a substantially higher signal. Dr. Chen recently demonstrated that these agents could be used to sensitively and specifically detect MPO activity in vivo in mouse models of heart attack, multiple sclerosis and stroke.
Dr. Chen noted that luminol has not been known to be specific to MPO in vitro, but the study by Dr. Piwnica-Worms and colleagues demonstrated that in vivo luminol is specific to MPO. "What causes this difference in MPO selectivity in vitro versus in vivo is very interesting and needs further investigation," he said.
Researchers who injected luminol into living mice found that the compound glowed blue at sites of active immune inflammation. Lipopolysaccharide (LPS) was injected into the left ankle joint of pairs of wild type (Mpo+/+) mice (left panel) or MPO gene-deleted (Mpo-/-) mice (right panel). Vehicle (saline) was injected into the right ankle joints. After 48 hours, luminol was administered by intraperitoneal injection (200 mg/kg body weight) and mice were imaged 10 minutes later with a cooled charge-coupled device (CCD) bioluminescence imaging (BLI) system. Note: The bioluminescence signal emitted from the LPS-induced arthritic joints in Mpo+/+ mice, but not Mpo-/- mice. Bar = 1 cm. Image courtesy of David Piwnica-Worms, M.D., Ph.D. |
While luminol has been used extensively in forensic investigations, its toxicity needs to be assessed when administered to humans in doses required for imaging, Dr. Chen added.
Bright Future Envisioned for Clinical Application
Although clinical application of luminol in detecting immune inflammation is a long way off, it does offer some intriguing possibilities, such as earlier detection of cardiovascular disease.
"There has been some very provocative data where MPO seems to have an important role in being a marker of active plaque," said Dr. Piwnica-Worms. "Serum analysis of MPO is currently under active investigation for being a marker of active plaque. If there's an active plaque, compared to chronic plaque, that's a sign that there may be pending stroke or heart attack."
Other possible applications include inflammatory diseases of the brain as well as inflammation of the eyes and lesions of the skin.
Instruments such as endoscopes and bronchoscopes could be optimized to perform local interrogation in conjunction with systemic injection of luminol, said Dr. Piwnica-Worms. New types of instruments or transducers need to be developed for some potential clinical applications.
Dr. Chen agreed. "Bioluminescence currently has little clinical application because light has limited depth of penetration," he said. "Therefore, before luminol and bioluminescence can be used in a clinical context, more sensitive and/or specialized equipment needs to be developed that allows human body parts such as the breast to be imaged.
"Despite the lack of depth penetration of BLI, one can imagine that in the future, luminol/BLI could be used to assess active arthritis," Dr. Chen continued. "Another example could be endovascular or endoscopic detection of MPO in vascular or gastrointestinal diseases."
Dr. Piwnica-Worms suggested that for pre-clinical studies and for academic radiologists, there is bound to be some immediate impact in near-term utility for studying diseases in mouse models with a new BLI tool.
"Luminol provides surprisingly sensitive and highly specific bioluminescence readout of MPO and MPO-mediated inflammation in vivo," he concluded. "In terms of actual translation to the clinic, there are many barriers, but it does open up the possibility in the future of a new clinical optical imaging application. Stay tuned."
