Protective Role of Eosinophils and TNFa after Ozone Inhalation.
Introduction: Exposure to ozone induces deleterious responses
in the airways that include shortness of breath,
inflammation, and bronchoconstriction. People
with asthma have increased airway sensitivity to
ozone and other irritants. Dr. Allison Fryer and
colleagues addressed how exposure to ozone affects
the immune and physiological responses in
guinea pigs. Guinea pigs are considered a useful
animal model for studies of respiratory and physiological
responses in humans; their response to
airborne allergens is similar to that in humans and
shares some features of allergic asthma.
Fryer and colleagues had previously observed
that within 24 hours of exposure, ozone not only
induced bronchoconstriction but also stimulated
the production of new cells in the bone marrow,
where all white blood cells develop. As a result
of ozone exposure, increased numbers of newly
synthesized white blood cells, particularly eosinophils,
moved into the blood and lungs.
The central hypothesis of the current study was
that newly synthesized eosinophils recruited to
the lungs 3 days after ozone exposure were beneficial
to the animals because they reduced ozoneinduced
bronchoconstriction. The investigators
also hypothesized that the beneficial effect seen
in normal (nonsensitized) animals was lost in animals
that had been injected with an allergen, ovalbumin
(sensitized). They also planned to explore
the effects of inhibitors of certain cytokines (cellsignaling
molecules).
Immune responses in sensitized animals are
dominated by a Th2 pattern, which is characterized
by the synthesis of cytokines (interleukin
[IL]-4, IL-5, and IL-13) and the Th2 subset of CD4+
T lymphocytes and the cells they activate (predominantly
eosinophils, and B lymphocytes that
switch to making immunoglobulin E [IgE]). Thus,
sensitized animals were used as a model of allergic
humans, whose immune responses tend to be
dominated by IgE.
Approach: Fryer and colleagues exposed normal and sensitized
(allergic) guinea pigs to 2 ppm ozone or filtered
air for 4 hours and measured changes in cell
numbers and airway responses 1 or 3 days later.
They counted the numbers of eosinophils and other
white blood cells (macrophages, neutrophils, and
lymphocytes) in bone marrow, blood, and bronchoalveolar
lung lavage fluid. The investigators
also measured important physiological responses,
including bronchoconstriction. Some animals were
pretreated with etanercept and monoclonal anti-IL-5,
which block tumor necrosis factor-a (TNFa) and IL-5,
respectively. TNFa and IL-5 blockers have been used
to treat patients with asthma.
A key feature of the study was a technique to distinguish
which white blood cells were synthesized after
exposure from those that already existed, by injecting
animals with bromodeoxyuridine (BrdU). BrdU
is a thymidine analogue that is incorporated into the
DNA of dividing cells, serving as a marker of newly
produced cells. Therefore, a snapshot can be obtained
of the proportion of newly synthesized (BrdU-positive)
versus pre-existing (BrdU-negative) cell types.
Key results: 1. Allergic and normal animals differed in the time
course of bronchoconstriction and changes in cell
types after ozone exposure. In normal animals,
bronchoconstriction increased substantially at
day 1 but decreased by day 3 after ozone exposure.
In contrast, in allergic animals bronchoconstriction
remained high at day 3. Ozone also increased
the percentage of newly formed, BrdU2
positive eosinophils in the bone marrow and
lungs of normal but not allergic animals.
2. Pretreatment with the TNFa blocker etanercept
had complex effects, which differed between
normal and allergic animals. In normal animals,
etanercept decreased ozone-induced new synthesis
of eosinophils in the bone marrow and
blocked eosinophil migration to the lung; it also
increased bronchoconstriction at day 3 (relative
to day 1 without etanercept). In allergic animals,
etanercept had no effect on any cell type in the
bone marrow or lung after exposure to ozone and
did not change bronchoconstriction compared
with allergic animals not treated with etanercept.
Etanercept tended to increase the numbers of
blood monocytes and lymphocytes in air- and
ozone-exposed normal and allergic animals at
day 3, but had no effect on eosinophils in blood
at this time point. This was one of the few statistically
significant findings in the blood of exposed
animals in the study.
3. Anti-IL-5 reduced bronchoconstriction at day 3 after
exposure of allergic animals to ozone. In contrast,
bronchoconstriction was greatly increased
in normal animals treated with anti-IL-5.
Conclusions: Fryer and colleagues explored the airway and cellular
responses in guinea pigs exposed to ozone. The
HEI Review Committee, which conducted an independent
review of the study, agreed that the findings
supported the authors’ hypothesis (1) that exposure
to ozone stimulates production of eosinophils in bone
marrow, (2) that these newly formed eosinophils migrate
to the lungs, and (3) that those eosinophils play
a delayed but potentially beneficial role in reducing
ozone-induced inflammation in the airways of healthy
normal animals, but not in allergen-sensitized animals.
The Committee also agreed that guinea pigs
were a good model for studying responses to an allergen,
because a major subtype of asthma (the high
Th2 or allergic type) is associated with high levels of
eosinophils in the blood.
A novel finding was that the TNFa blocker etanercept
decreased ozone-induced formation of eosinophils
in the bone marrow and blocked eosinophil
migration to the lung in normal animals. However,
because injecting etanercept had little effect on eosinophils
and did not decrease bronchoconstriction in
allergic guinea pigs, the potential for treating patients
with allergic asthma with TNFa blockers is uncertain.
This is consistent with the poor performance of TNFa
blockers in clinical studies of asthma treatment.
Blocking the cytokine IL-5 with an anti-IL-5 antibody
substantially decreased bronchoconstriction in
sensitized animals. This suggests that therapies targeting
IL-5 and eosinophils would be promising in at
least some types of asthma. The Committee expressed
caution toward experiments with cytokine blockers,
both in animal models and humans, because such
blockers are often not specific to a particular cell type
and may differ at different sites in the body. Without
further detailed confirmation of the effects of the
blockers, interpreting these experiments can be challenging.
The Committee concluded that the study by Fryer
and colleagues raises several intriguing directions for
future research, including exploring ways in which
newly formed eosinophils differ from pre-existing
ones, and how such findings apply to humans with
allergy or asthma.