Ozone is Produced by Antibodies During Bacterial Killing and in Inflammation, Say Scientists at The Scripps Research Institute

La Jolla, CA. November 14, 2002-Professor Richard A. Lerner, M.D.,
Associate Professor Paul Wentworth, Jr., Ph.D., and a team of
investigators at The Scripps Research Institute (TSRI) is reporting
that antibodies can destroy bacteria, playing a hitherto unknown
role in immune protection. Furthermore, the team found that when
antibodies do this, they appear to produce the reactive gas ozone.

"[Ozone] has never been considered a part of biology before," says
Lerner, who is Lita Annenberg Hazen Professor of Immunochemistry and
holds the Cecil H. and Ida M. Green Chair in Chemistry at TSRI. The
report will appear in an upcoming issue of the journal Science.

The ozone may be part of a previously unrecognized killing mechanism
that would enhance the defensive role of antibodies by allowing them
to subject pathogens to hydrogen peroxide and participate directly
in their killing. Previously, antibodies were believed only to
signal an immune response.

This ability of antibodies to generate toxic compounds may also link
them to a number of inflammatory diseases, such as atherosclerosis,
lupus, multiple sclerosis, and rheumatoid arthritis. Furthermore,
this research opens up exciting possibilities for new antibody-
mediated therapies for conditions ranging from bacterial and viral
infection to cancer.

Recognition and Killing in the Same Molecule

Also called immunoglobulins, antibodies are secreted proteins
produced by immune cells that are designed to recognize a wide range
of foreign pathogens. After a bacterium, virus, or other pathogen
enters the bloodstream, antibodies target antigens-proteins, fat
molecules, and other pieces of the pathogen-specific to that foreign
invader. These antibodies then alert the immune system to the
presence of the invaders and attract lethal "effector" immune cells
to the site of infection.

For the last hundred years, immunologists have firmly held that the
role of antibodies was solely to recognize pathogens and signal the
immune system to make an immune response. The conventional wisdom
was that the dirty work of killing the pathogens was to be left to
other parts of the immune system.

Now, Lerner, Wentworth and their colleagues have demonstrated that
antibodies also have the ability to kill bacteria. This suggests
that rather than simply recognizing foreign antigens and then
activating other parts of the immune system to the site of
infection, the antibodies may further enhance the immune response by
directly killing some of the bacteria themselves.

Antibodies do this by producing the chemical oxidant hydrogen
peroxide-best known as the foamy formulation used for first-aid.
Hydrogen peroxide is lethal to bacterial cells because it pokes
holes in their cell walls, bursting the cells and killing them.

In the Science paper, the TSRI team reports the effective killing of
E. coli bacteria through hydrogen peroxide production by antibodies
specific for that bacteria.

The Ozone Hole in Each One of Us

Certainly the most surprising result that Lerner, Wentworth, and
their colleagues found was that antibodies appear to make ozone,
which they detected through its chemical signature. They have not
yet demonstrated conclusively that what they found is ozone, but
they are highly confident that ozone is what the antibodies are
producing because no other known molecule has the same chemical

Ozone is a particularly reactive form of oxygen that exists
naturally as a trace gas in the atmosphere, constituting on the
average fewer than one part per million air molecules. But it is
noted mainly where its presence or absence poses a threat to public

The gas is perhaps better known for its crucial role absorbing
ultraviolet radiation in the upper reaches of Earth's stratosphere-
about 25 km above the surface-where it is concentrated in a so-
called ozone layer, protecting life on earth from damaging solar

Ozone is also a familiar component of air in industrial and urban
settings where the highly reactive gas is a hazardous component of
smog in the summer months. Never before has ozone been detected in

"All our analytical data point to this oxidant possessing the
chemical signature of ozone," says Wentworth, "in which case, this
is a new molecule in biology and therefore may have tremendous
ramifications for signaling and inflammation."

Proof for a Proposed Reaction Pathway

All antibodies have the ability to produce hydrogen peroxide, the
report adds, but they need to first have available a molecule known
as "singlet" oxygen-another highly reactive oxygen species-to use as
a substrate.

Singlet oxygen is an electronically excited form of oxygen that
forms spontaneously during normal metabolic processes or when oxygen
is subjected to visible or ultraviolet light in the presence of a
sensitizer. "Phagocytic" innate immune cells, like neutrophils, also
produce singlet oxygen and are the most likely source of the
substrate for antibodies, since during an immune response,
antibodies will recruit neutrophils and other immune cells to the
site of an infection.

Once there, the neutrophils will engulf and destroy bacteria and
other pathogens by blasting them with singlet oxygen and other
oxidative molecules. The antibodies reduce singlet oxygen by
combining it with water to produce hydrogen peroxide, producing
ozone as a side product.

Interestingly, all antibodies have the ability to do this, which
leads the TSRI team to speculate that the removal of singlet oxygen
may have been the original role of antibodies. In a previous report,
the same team speculated an ancient form of antibodies may have
existed-molecules whose role was to catalyze singlet oxygen
destruction, since singlet oxygen can potentially destroy any cell,
making it dangerous to have around. Prior to the evolution of the
modern antibody-mediated humoral immune response in vertebrates
hundreds of millions of years ago, ancient antibodies may have been
responsible for controlling the release of highly reactive and
potentially dangerous singlet oxygen. Later, when antibodies
developed as part of the adaptive arm of the immune system, they
kept their original function because it provided a bit of extra

Another interesting finding is that the antibodies carry the
reaction through an unusual intermediate. Lerner, Wentworth, and
their colleagues postulate that the antibodies carry the reaction
through an intermediate chemical species of dihydrogen trioxide, a
reduced form of ozone.

Dihydrogen trioxide has also never before been observed in
biological systems, and its presence as an intermediate has been the
source of considerable speculation in the scientific community.

The team's reported detection of ozone is strong support of this
proposed dihydrogen trioxide intermediate, and now the team is
tackling the larger question of what it means.

"This is a novel set of observations and very interesting ones--
there are a million questions [we could ask]," says TSRI Professor
Bernard Babior, M.D., Ph.D. "What does the ozone do to the body's
proteins, nucleic acids? Are there lethal concentrations of ozone?
Does it have anything to do with other reactive species in the

The research article, "Evidence for Antibody-Catalyzed Ozone
Formation in Bacterial Killing and Inflammation" is authored by Paul
Wentworth, Jr., Jonathan E. McDunn, Anita D. Wentworth, Cindy
Takeuchi, Jorge Nieva, Teresa Jones, Cristina Bautista, Julie M.
Ruedi, Abel Gutierrez, Kim D. Janda, Bernard M. Babior, Albert
Eschenmoser, and Richard A. Lerner, and appears in the November 18,
2002 "Science Express," the advanced publication edition of the
journal Science. The article will appear in Science later this year.

The research was funded by the National Institutes of Health, The
Skaggs Institute for Chemical Biology, and an A.R.C.S. fellowship.

For more information contact:
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