Ask most people what antibodies do, and they’ll respond that these immune agents kill bacteria, viruses, and other harmful microbes that seek to infect the body. An immunologist standing nearby would probably correct that answer by pointing out that antibodies don’t actually do the dirty work.
According to mainstream immunology, these Y-shaped proteins merely bind to bits of microbes. This targets the germs for destruction by other molecules, such as a family of proteins called complement, or by immune cells. In this view, one can think of an antibody as the laser beam that soldiers use to identify the target of a smart bomb.
But what if these immune proteins carry a grenade or two with them? A recent study suggesting just that threatens to upset the traditional notion of what antibodies do.
In the Sept. 26 Proceedings of the National Academy of Sciences, Richard Lerner of the Scripps Research Institute in La Jolla, Calif., and his colleagues report that almost every antibody they’ve tested can generate hydrogen peroxide from molecular oxygen. Since hydrogen peroxide—a widely used antiseptic—readily converts into toxic molecules that damage DNA and proteins, antibodies probably do more than passively label dangerous microbes for destruction, says Lerner. Antibodies themselves are germ killers, he contends.
Lerner holds that his group’s discovery compels a reassessment of the evolution and function of antibodies. Some investigators suggest that his findings complement their work showing various enzymatic properties of antibodies.
Other scientists caution that, so far, there are no data showing that the vertebrate immune system depends on these chemical talents of antibodies or that it ever did in its evolutionary past. Nonetheless, some researchers are intrigued by these possibilities.
Lerner’s finding “is very exciting because it suggests that antibodies have the potential to be microbicidal molecules,” says immunologist Arturo Casadevall of Albert Einstein College of Medicine in New York City.
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Lerner’s new work grew out of a discovery made almost 15 years ago. At that time, he and other scientists developed an unlikely way of creating new enzymes, proteins that facilitate chemical reactions between other molecules. Their novel source of these catalysts was the vertebrate immune system, specifically its antibodies.
The researchers found that immunizing animals with specially designed molecules could generate antibodies that catalyze reactions in which those same molecules participate (SN: 4/22/89, p. 252). In the years since, catalytic antibodies, sometimes called abzymes, have attracted commercial, scientific, and medical interest. Seeking to treat people addicted to cocaine, for example, scientists have generated antibodies that help the body quickly chew up the drug (SN: 10/10/98, p. 239).
A pioneer in the field, Lerner often lectured on catalytic antibodies. Someone would invariably ask him whether enzymelike properties occur among the body’s normal antibodies to invaders. Dismissing the claims of a few scientists, Lerner for years answered that there was no compelling evidence to support the existence of natural abzymes.
Lerner continued to hold that view until some recent experiments with a catalytic antibody that binds to a synthetic organic molecule called trans-stilbene. When irradiated with ultraviolet light, this antibody complex emits intense blue light, an unexpected reaction that Lerner, Kim A. Janda, and their other Scripps colleagues report in the Oct. 13 Science.
While exploring the chemistry of this fluorescent reaction, the researchers noticed that irradiating the antibody alone also produced hydrogen peroxide. Wondering if the catalytic antibody was somehow unique, they gathered natural antibodies from laboratories throughout their institute. All produced hydrogen peroxide when irradiated in a saline solution similar to the liquid component of blood.
The investigators were still skeptical. “We ordered 100 antibodies from around the world, and they all did it,” recalls Lerner.
They even demonstrated that pure, crystallized forms of antibodies can generate hydrogen peroxide when they’re exposed to ultraviolet light. That rules out contaminants as the source of the chemical reaction, contends Lerner.
The investigators don’t completely understand how antibodies generate hydrogen peroxide. Their experiments indicate the key is singlet oxygen, a high-energy oxygen that’s created by the ultraviolet light. The antibody somehow adds an electron to singlet oxygen, converting it to another form of oxygen called superoxide anion. When superoxide anions get together, they spontaneously react to create hydrogen peroxide.
The investigators are reluctant to say for certain that antibodies catalyze the singlet-oxygen reaction. By strict definition, a catalyst doesn’t change in a reaction. Although the investigators have identified particular amino acids that appear to mediate the reaction within antibodies, they’re not sure what happens to the antibody molecules when the singlet oxygen molecules receive their electrons.
Antibodies are “almost surely catalytic, but we tend not to use that word until we know the whole mechanism,” says Lerner.
What does any of this complicated test-tube chemistry have to do with the immune system? When immune cells engulf microbes trapped by antibodies, they hold them inside special pouches called phagosomes. One way that the phagosomes then destroy microbes is to create a supply of superoxide anions, which starts a cascade of reactions that produces hydrogen peroxide and then more toxic molecules, such as hydroxyl radical and hypohalous acid.
At two separate points, singlet oxygen is a by-product of this cascade. Lerner speculates that antibodies produce an extra dollop of hydrogen peroxide within the phagosome by converting this singlet oxygen into superoxide anions, just as antibodies do in the experiments where the singlet oxygen is generated by ultraviolet light.
Such an antibody-based reaction in the phagosome would focus the toxic molecules on the cell’s intended target, Lerner points out. “The antibody is holding onto the [microbe], so the reaction is happening in the right place,” he says.
The Scripps researchers speculate that antibodies both target and kill invaders—or at least
they held both roles early in the evolution of vertebrates. “First, there were antibodies that could kill on their own. Later on, some of the killing potential was passed off to cells. But the antibody retains the killing capacity of these cells, at least in the test-tube,” says Lerner.
Some scientists argue that such speculation extends too far beyond the published data. Hydrogen peroxide production “is a new feature of [antibodies]. It remains to be determined how general it is and if it is actually effective in killing,” says John J. Marchalonis of the University of Arizona in Tucson, who studies the evolution of the immune system.
Lerner responds that work his team has already completed but not yet published addresses that issue. “We know that antibodies, under the right circumstance, can kill bacteria via this method. There’s no question about that,” he asserts.
Showing that antibodies can destroy microbes in test tubes is one thing, but determining that the proteins do so in animals is a more difficult challenge. The vertebrate immune system has many different weapons with which it can destroy a bacterium, so it’s difficult to tease out the importance of each piece of the arsenal.
“In something as important as killing, nature has built in a lot of redundancy,” says Lerner. Antibodies could backfire on occasion. In a variety of situations, the body creates singlet oxygen. The Scripps researchers speculate that in autoimmune diseases and atherosclerosis, for example, cell or tissue damage may result when antibodies and singlet oxygen come together inappropriately.
“Part of the price [of an effective immune system] may be that antibodies can make bad stuff in the wrong place,” says Lerner.
Sudhir Paul shares that sentiment, although he and Lerner agree on little else. For about a decade, Paul, who is director of the chemical immunology center at the University of Texas–Houston Medical School, and a few other scientists have championed the idea that some of a person’s antibodies may cleave DNA or proteins.
In the past, however, Lerner and other scientists working with catalytic antibodies dismissed such findings, attributing them to contamination with traditional enzymes. Or, Paul says, they argued that the enzymatic nature of antibodies in the body is so weak that the phenomenon is unimportant.
Yet Paul has linked enzymatic antibody activity to a variety of medical conditions, particularly autoimmune disorders. His team, for example, has found that people with an autoimmune thyroid disorder harbor antibodies that cleave thyroglobulin, a precursor for two thyroid hormones.
In some of their earliest work, Paul and his colleagues reported that people with asthma have antibodies that cleave an antiinflammatory protein called vasoactive intestinal polypeptide. Other researchers have identified DNA-destroying antibodies in people with lupus.
Whether or not such antibodies contribute to disease, Paul would like to use some of them to fight a virus. In one recent project funded by the National Institutes of Health, Paul and his group isolated several antibodies that destroy gp120, a protein used by the AIDS virus.
Curiously, Paul’s team discovered this antiviral activity in antibodies obtained from people with lupus, not from those infected with HIV.
The scientists now plan to investigate whether injections of gp120-cleaving antibodies can help animals fight HIV. They’re also looking into ways to prompt an immune system to make such antibodies on its own. “There’s a lot of hope that catalytic antibodies will permit us to knock out specific targets like HIV,” contends Paul.
Lerner remains unpersuaded by Paul’s data, but the scientists concur that the chemistry of the antibody is going to be much more complicated than people have traditionally thought. Hinting at unpublished results, Lerner predicts that hydrogen peroxide production will be just one of the antimicrobial reactions in which antibodies participate.
That’s a welcome thought to some immunologists. The study of antibodies “has been in the doldrums for about 30 years,” says Casadevall. “Everybody feels that everything that needs to be known about antibodies has been discovered. [Lerner’s] paper tells you that’s not the case.”