Injected luminol glows blue revealing immune cells' misdeeds

Mar 22 2009

Detectives on television shows often spray crime scenes with a compound called luminol to make blood glow.

Researchers at Washington University School of Medicine in St. Louis have applied the same compound to much smaller crime scenes: sites where the immune system attacks the body's own tissues.

The authors report in Nature Medicine that injected luminol glows blue at sites of active immune inflammation in living mice, and that they can detect this glow from outside the mice with scientific cameras.

Immune inflammation is thought to be a critical component of arthritis and other autoimmune diseases, atherosclerosis, some forms of cancer and neurodegenerative diseases. Imaging such inflammation non-invasively should help scientists better understand and control it, according to the researchers.

"It's quite striking how specific and sensitive this approach is," says senior author David Piwnica-Worms, M.D., Ph.D. "For example, we have evidence that this technique can highlight inflamed tissue that is on the way to becoming cancerous but not yet discernible via visual or tactile inspection."

Piwnica-Worms, professor of radiology and of developmental biology, notes that cardiologists now believe immune inflammation is a key component that makes an arterial plaque dangerous. Such inflammation causes platelets to bind to plaques, leading the plaques to rupture or break away and putting the patient at risk of heart attack, stroke or lung clots.

For now, blood vessels of the chest and torso are too deep within the body to image with this approach. But vessels of the leg and neck are close enough to the skin that the technique may be "directly translatable" to use in human patients, according to Piwnica-Worms.

Lead author Shimon Gross, Ph.D., a postdoctoral fellow, proposed that luminol might be used to image inflammation when he found earlier studies linking luminol bioluminescence with myloperoxidase (MPO), a protein some types of immune cells use to make bleach during the inflammatory process. When activated, cells known as phagocytes use MPO to make the bleach in pockets. They seek out and swallow invaders, and then push the invaders into these bleach-filled pockets to kill them.

In television dramas like CSI, detectives spray a mixture of hydrogen peroxide and luminol onto crime scenes. The mixture reacts with iron from blood, which in that context acts a catalyst, causing the luminol to glow. In the living body, though, iron isn't as accessible. The iron in hemoglobin, for example, is still inside red blood cells and is often bound to oxygen, blocking the reaction with luminol.

Gross and Piwnica-Worms realized this only after their initial experiments. They injected luminol into mice anticipating that they would need a way to distinguish immune inflammation from other processes that might also cause the luminol to luminesce. Instead, they found the compound only glowed at sites of immune inflammation involving MPO.

"Everything's kept compartmentalized when it's still in the body," says Piwnica-Worms. "When it comes to making luminol glow, the only places where all the necessary ingredients come together in concentrated form in the living body are in active phagocytes containing MPO."

When scientists dabbed an irritant onto the ears of normal mice and injected luminol, immune cells that migrated to the irritation site glowed. But in mice lacking the MPO gene supplied by Jay Heinecke, M.D., Ph.D., of the University of Seattle, no glow could be detected.

To further test the new technique, Lee Ratner, M.D., Ph.D., of Washington University School of Medicine, provided a line of mice that models a type of tumor known to be rife with active immune cells. Injected luminol not only lit up established tumors, it also highlighted areas of inflammation that weeks later would become tumors.

Scientists also used the technique to show inflammation in a mouse model of acute arthritis. Piwnica-Worms speculates that applying luminol in this context could improve arthritis patient management and enable rapid assessment of the effectiveness of new treatments.

Piwnica-Worms and his colleagues are currently working to modify luminol chemically to improve its clinical potential.

Gross S, Gammon ST, Moss BL, Rauch D, Harding J, Heinecke JW, Ratner L, Piwnica-Worms D. Bioluminescence imaging of myeloperoxidase activity in vivo. Nature Medicine, online March 22.

Funding from the National Institutes of Health supported this research.

Washington University School of Medicine's 2,100 employed and volunteer faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Children's hospitals. The School of Medicine is one of the leading medical research, teaching and patient care institutions in the nation, currently ranked third in the nation by U.S. News & World Report. Through its affiliations with Barnes-Jewish and St. Louis Children's hospitals, the School of Medicine is linked to BJC HealthCare.