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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 92, Num. s2, 1997, pp. 223-226
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 92, Suppl. II, pp. 223-226
Role of Eosinophilic Airway Inflammation in Models
Jose R Lapa e Silva*/+, C Ruffie, J Lefort, M Pretolani, BB Vargaftig
Unite de Pharmacologie Cellulaire, Unite Associee Institut Pasteur/lNSERM
no. 285, Paris, France
* Laboratorio Multidisciplinar de Pesquisa e Servico de Pneumologia,
Hospital Universitario Clementino Fraga Filho, Universidade Federal do Rio
de Janeiro and Laboratorio de Pesquisa em Anatomia Patologica, Hospital
Evandro Chagas, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de
Janeiro, RJ, Brasil
^+ Corresponding author, FAX: +55-21-270.2193. E-mail:
jrlapa@omega.lncc.br
Received 3 September 1997; Accepted 30 September 1997.
Code Number:OC97192
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Eosinophils play a central role in the establishment and outcome of
bronchial inflammation in asthma. Animal models of allergy are useful to
answer questions related to mechanisms of allergic inflammation. We have
used models of sensitized and boosted guinea pigs to investigate the nature
of bronchial inflammation in allergic conditions. These animals develop
marked bronchial infiltration composed mainly of CD4+ T-lymphocytes and
eosinophils. Further provocation with antigen leads to degranulation of
eosinophils and ulceration of the bronchial mucosa. Eosinophils are the
first cells to increase in numbers in the mucosa after antigen challenge
and depend on the expression of alpha4 integrin to adhere to the vascular
endothelium and transmigrate to the mucosa. Blockage of alpha4 integrin
expression with specific antibody prevents not only the transmigration of
eosinophils but also the development of bronchial hvperresponsiveness (BHR)
to agonists in sensitized and challenged animals, clearly suggesting a role
for this cell type in this altered functional state. Moreover, introduction
of antibody against Major Basic Protein into the airways also prevents the
development of BHR in similar model. BHR can also be suppressed by the use
of FK506, an immunosuppressor that reduces in almost 100% the infiltration
of eosinophils into the bronchi of allergic animals. These data support the
concept that eosinophil is the most important pro-inflammatory factor in
bronchial inflammation associated with allergy.
Key words: eosinophil - alpha4 integrin - Major Basic Protein -
FK506
Asthma as an inflammatory eosinophilic disease
A silent revolution took place in the field of asthma pathogenesis over the
last ten years. Until then, asthma was considered a disease caused mainly
by bronchial smooth muscle disfunction (Kay 1991). Bronchial inflammation
has been known as a feature of asthma for decades but it was always
associated with terminal stages of the disease, and related to death caused
by status asmaticus. Descriptions of severe bronchial infiltration
by eosinophils and mononuclear cells, epithelial shedding, luminal plugs
formed by exsudated proteins and inflammatory cells were tipically seen as
hallmarks of terminal asthma (Dunnill 1960). Introduction of safe
endoscopic procedures allowed physicians to examine the bronchial mucosa of
mild stable asthmatics, and some of the features seen in post-mortem
examination were also present in this more benign form of asthma (Jeffery
et al.1989, Azzawi et al. 1990). The flurry of information that followed
these seminal observations firmly established asthma as an inflammatory
disease. The recent definition proposed by the Global Initiative for Asthma
stated that asthma is a chronic inflammatory disease with many cells
contributing to its pathogenesis (NHLBI/WHO 1995). Eosinophils seem to play
a key role in the establishment of bronchial inflammation in this
condition, together with T lymphocytes that produce a set of cytokines
typical of the Type 2 classification of Mossman and Coffman (Kay et al.
1991, Robinson et al. 1992). The presence of activated eosinophils,
displaying positivity for the cleaved form of the eosinophil cationic
protein (ECP) was correlated with bronchial hyperresponsiveness to
metacholine. Eosinophilia was identified in all types of asthma, such as
atopic, non-atopic, and occupational (Saetta et al. 1992). The possible
role of eosinophils in asthma pathogenesis led to the development of
diagnostic tools, such as the measurement of ECP in serum, bronchoalveolar
lavage fluid or sputum of asthmatic patients, that can also be used as
markers of disease progression or severity (Virchow et al. 1992). However,
the evidences for the actual role of eosinophils in many aspects of asthma
remain circumstantial. A mechanistic approach to answer these standing
questions demands the use of animal models of allergy.
Contribution of animal models to establish causality in allergic
inflammation
The booster injection of antigen in sensitized guinea pigs leads to
bronchial eosinophilia - We have used an animal model to investigate
the establishment of bronchial allergic inflammation. Sensitization of
guinea pigs with ovalbumin, followed by a booster injection of antigen two
weeks later induced a marked bronchial inflammation characterized by the
presence of increased numbers of eosinophils and CD4+ T lymphocytes (Lapa e
Silva et al. 1992, 1993a). These events were temporally associated with
enhanced bronchoconstric-tion to PAF and other agonists, and with release
of secondary mediators in the effluent of the isolated perfused lungs
(Pretolani et al. 1988).
Antigen challenge in boosted animals further increases bronchial
eosinophilia and results in extensive epithelial shedding - Sensitized
and boosted animals were further challenged with ovalbumin after protection
with anti-histaminic and 24 hr after the provocation a further increase in
eosinophil numbers infiltrating the bronchial mucosa was seen, together
with staining of extracellular granular material suggestive of eosinophil
degranulation. Marked mucosal ulceration and epithelial shedding were noted
in most animals of this group (Lapa e Silva et al. 1993b).
Antigen challenge in boosted animals results in infiltration of the
bronchial mucosa by dendritic cells - Animals provoked as above had
their bronchial mucosa infiltrated 24 hr after challenge by cells with
slender processes that stained very positive with a MHC Class II marker but
failed to exhibit positivity for macrophages markers, thus suggesting that
they were true dendritic cells (Lapa e Silva et al. 1994). The presence of
increased numbers of dendritic cells in the bronchial mucosa could indicate
persistence of the immune response by continuing antigen-presentation to T
cells.
Adhesion of VLA-4+ cells to vascular endothelium is a rapid event after
antigen challenge and precedes bronchial inflammation - Guinea pigs
sensitized by repeated ovalbumin inhalation were killed at 3, 6, 24, and 72
hr after the challenge. Sections were stained with the monoclonal antibody
HP1/2 against the alpha4 chain of the integrin VLA-4. Positive cells
adhering to the vascular endothelium of bronchial vessels were maximal at
the first time point and remained high 6 hr after antigen challenge but
failed to show differences at later time points (Lapa e Silva, unpublished
observations).
Eosinophils are the first inflammatory cell type to increase in
bronchial mucosa after challenge and remain high at all time points -
Using the above model, we examined the nature of bronchial inflammation at
the same time points. Eosinophil numbers were significantly higher than
controls 3 hr after challenge and remained higher at all time points
examined, whereas CD4+ cells were only higher than controls 24 hr after
challenge (Lapa e Silva et al. 1995a).
Use of immunologic tools to investigate causality in allergic
inflammation
Antibody against alpha4 integrin prevents the development of
eosinophilic bronchitis without affecting systemic eosinophilia - We
used the monoclonal antibody HP1/2 as a tool to investigate the role of
alpha4 integrins in the development of eosinophilic inflammation. Guinea
pigs were sensitized by repeated inhalations of ovalbumin and provoked by
the same route 14 days later and HP1/2 antibody was injected intravenously
1hr before and 4 hr after antigen challenge. The monoclonal antibody
totally prevented eosinophilia in the bronchoalveolar lavage (BAL) fluid
and markedly inhibited the rise of eosinophil peroxidase levels in the BAL
fluid of treated animals, suggesting that neutralization of alpha4 integrin
prevented not only the homing of eosinophils to the airways but also their
degranulation (Pretolani et al. 1994). Treatment with HP1/2 entirely
prevented mucosal infiltration by eosinophils and CD4+ T cells. Blood
eosinophilia was selectively increased by the alpha4 integrin treatment,
indicating that inhibition of eosinophil recruitment to the alveolar
compartment may partially account for their accumulation in the circulation
(Pretolani et al. 1994).
Antibody against alpha4 integrin blocks the development of bronchial
hyperreactivity - The effects described above were also accompanied by
a marked reduction in the bronchoconstrictor response to intravenous
methacholine in animals treated with the anti-a4 integrin antibody,
in comparison with those treated with the corresponding isotype (Pretolani
et al. 1994).
Antibody against eosinophil major basic protein (MBP) prevents the
development of bronchial hyperreactivity without affecting bronchial
inflammation - Guinea pigs were sensitized as above and purified rabbit
anti-guinea pig MBP antibody was injected intranasally 1 hr before and 5 hr
after ovalbumin inhalation. Both BAL eosinophilia and infiltration of the
bronchial mucosa by eosinophils were unaffected by antibody treatment,
remaining similar to those seen in isotype-treated provoked animals.
Bronchial hyperreactivity to acetylcholine, however, was markedly reduced
in animals treated with the anti-MBP antibody, strongly suggesting a
central role for this cationic protein in the development of altered
functional states in allergic inflammation (Lefort et al. 1996).
Use of pharmacologic tools to modulate allergic inflammation
Glucocorticosteroids but not nedocromil sodium decreases eosinophilic
bronchitis - The effects of two standard anti-inflammatory drugs on the
bronchial wall infiltration by eosinophils and other cell types were
investigated using a guinea pig model. Animals were sensitized and boosted
with ovalbumin and treated for five days after the booster injection of
antigen with subcutaneous injection of either dexamethasone or nedocromil
sodium. Bronchial infiltration by eosinophils and CD4+ cells was totally
prevented by dexamethasone. Nedocromil sodium, however, failed to inhibit
the eosinophilic bronchitis that accompanied the booster injection (Lapa e
Silva et al. 1995b).
FK506 markedly reduces bronchial inflammation - The novel
immunosuppressive drug FK506 was used in an animal model of allergic
inflammation to investigate its effects on the bronchial mucosa. Guinea
pigs were sensitized and boosted with ovalbumin and treated for five days
by subcutaneous injections of FK506 from the day of the booster injection.
The drug totally prevented the development of bronchial infiltration by
eosinophils and CD4+ T cells (Lapa e Silva et al. 1995b).
FK506 blocks the development of bronchial hyperreactivity - We
investigated the effects of FK506 on alpha4 integrin expression by
inflammatory cells infiltrating the bronchi and on the emergence of
bronchial hyperresponsiveness to acetylcholine. Guinea pigs received
aerosol of avalbumin at days 0, 2, and were challenged at day 14. FK506 was
administered 1hr before and 5 hr after challenge. Treatment by the
immunosuppressant was accompanied by a marked decrease in numbers of CD4+
cells and eosinophils in the bronchial mucosa. FK506 also reversed the
increase in alpha4 integrin positive cells adhering to the vascular
endothelium and infiltrating the bronchial mucosa. We found preliminary
evidence that the drug also prevented the development of bronchial
hyperesponsiveness to acethylcholine, possibly through its action on cell
recruitment to the bronchial mucosa. One possible mechanism for this action
is down-regulation of alpha4 integrin expression on inflammatory cells
(Lapa e Silva et al. 1996).
This work was supported in part by grants from INSERM, Pasteur Institute,
and by grants to JRLS from the Fundacao Universitaria Jose Bonifacio/FUJB
and Brazilian Research Council/CNPq.
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Copyright 1997 Fundacao Oswaldo Cruz - Fiocruz
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