Unconventional strategies for the suppression of allergic asthma.
ABSTRACT Allergic asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. And this observation constitutes the base of the hygiene hypothesis. Here we discuss the hygiene hypothesis with emphasis on regulatory cells. We review the evidence for the emergence of regulatory cells, such as CD4(+)CD25(+) T cells during infection or during induction of tolerance by mucosal antigen administration. The review focuses also on the emergence of activated CD8(+) T cells and macrophages, induced by infections or microbial products, which also can result in the suppression of asthma. The underlying mechanisms by which regulatory immune cells suppress asthma may represent novel unconventional strategies controlling asthma.
[show abstract] [hide abstract]
ABSTRACT: Mucosal tolerance can be induced by oral or nasal administration of soluble proteins and results in the suppression of cellular and/or humoral immune responses to the specific antigen. To compare the effect of oral or nasal ovalbumin administration before, during or after immunization on the development of cellular and humoral immune responses by using a murine asthma model. To induce lung allergic inflammation, animals were immunized twice with ovalbumin/aluminum hydroxide gel and challenged twice with ovalbumin. To induce tolerance, BALB/c mice received ovalbumin by the oral or nasal routes for 3 consecutive days. The ovalbumin administration was initiated before (day -7), during (day 0), or after immunization (day 7). Airway eosinophilia, airway hyperreactivity, mucus hypersecretion, and cytokine production were suppressed when oral or nasal ovalbumin administration was initiated before immunization. Oral but not nasal ovalbumin exposure suppressed ovalbumin-specific nonanaphylactic IgG(1) antibodies, whereas both routes suppressed the production of anaphylactic IgG(1) and IgE antibodies. Mucosal ovalbumin administration at day 0 inhibited all T(H)2-mediated allergic parameters but not nonanaphylactic IgG(1) antibodies. Finally, ovalbumin exposure 7 days after immunization was still effective in suppressing lung allergy but not ovalbumin-specific anaphylactic IgG(1) and IgE antibodies. We show that the effectiveness of mucosal tolerance depends on route and time and presents a hierarchical pattern of suppression in the following order: lung allergic responses > anaphylactic antibodies > ovalbumin-specific IgG(1).Journal of Allergy and Clinical Immunology 02/2006; 117(2):283-90. · 11.00 Impact Factor
Article: Regulatory T cells accumulate in the lung allergic inflammation and efficiently suppress T-cell proliferation but not Th2 cytokine production.[show abstract] [hide abstract]
ABSTRACT: Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.Clinical and Developmental Immunology 01/2012; 2012:721817. · 1.84 Impact Factor
Current Drug Targets - Inflammation & Allergy, 2003, 2, 187-195187
1568-010X/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd.
Unconventional Strategies for the Suppression of Allergic Asthma
Daniel de Sousa Mucida, Alexandre de Castro Keller, Eva Christina Fernvik and
Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo- 05508-
900, SP, Brazil
Abstract: Allergic asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by
intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association
between allergic asthma and microbial infections has been observed. And this observation constitutes the base
of the hygiene hypothesis. Here we discuss the hygiene hypothesis with emphasis on regulatory cells. We
review the evidence for the emergence of regulatory cells, such as CD4+CD25+ T cells during infection or
during induction of tolerance by mucosal antigen administration. The review focuses also on the emergence of
activated CD8+ T cells and macrophages, induced by infections or microbial products, which also can result in
the suppression of asthma. The underlying mechanisms by which regulatory immune cells suppress asthma may
represent novel unconventional strategies controlling asthma.
Key Words: asthma, hygiene hypothesis, regulatory T cells, tolerance, immune deviation, macrophages, TLR4 and NO.
Over the last 2 to 3 decades, the prevalence of asthma
and allergic diseases has increased extensively worldwide.
Environmental factors, such as air pollution, allergen
exposure and infections, have been associated with the
development of atopy, mainly in genetically predisposed
Asthma is a chronic disease characterized by intermittent
and reversible airway obstruction, mucous hypersecretion
and airway hyperreactivity (AHR), airway eosinophilia and
IgE production . In atopy asthmatic individuals, the
interaction between allergens and the immune system leads
to activation of CD4+ T lymphocytes that secret type 2
cytokines, e.g., IL-4, IL-5, and IL-13, which in turn recruit
inflammatory cells to the airway tissue [3,4]. As a result of
the chronic inflammatory process, the airways may suffer
profound structural changes referred to as airway remodeling
The understanding of the pathogenesis of asthma is
improved substantially with murine models that have
demonstrated a pivotal role for the Th2 lymphocyte-secreted
cytokines in the development of the disease (reviewed in this
issue and ref.  ). T lymphocytes appear to be a condition
sine qua non for asthma development. Actually, T-cell
deficient mice (TCR-β knock-out (KO)) do not develop
asthma-like responses, after immunization and challenge .
Moreover, De Sanctis et al. have shown that in non-
immunized animals, AHR is strictly αβ-T cell dependent
. In contrast to the conventional αβ-T cells, γδ-T cells
appear to protect against the development of AHR. Indeed, it
has been demonstrated that γδ-T cell-deficient animals
present increased AHR when compared to wild-type mice
*Address correspondence to this author at the Departamento de
Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo,
São Paulo- 05508-900, SP, Brazil; Phone-Fax 55 11 3091 7377; E-mail
[7,9]. On the other hand, depending on the experimental
models used, γδ-T lymphocytes have also been
demonstrated to be fundamental for optimal IgE production
and airway eosinophilia .
It has been shown that IL-4 and IL-13 are both involved
in the production of IgE and mucous [4,11-15].
Furthermore, IL-5 has been demonstrated to be responsible
for the growth of eosinophils and their differentiation,
survival, and activation . It is believed that migration
and activation of eosinophils in the lung induce cell damage
and are thereby directly associated with the development of
AHR . However, murine models of hyperreactivity have
demonstrated that depending on mouse strain, the AHR
might be more or less dependent on one or another cytokine
related to asthma, and thereby associated with different
mechanisms. Foster et al. have shown that IL-5 deficiency
abolished antigen-induced eosinophilia and the development
of AHR . In contrast, Corry et al. reported that IL-4, but
not IL-5, is required to activate mast cells by an IgE-
dependent pathway and thereby generate AHR .
Recently, it has also been demonstrated that animals lacking
IL-13, even in the presence of high levels of IL-4 and IL-5,
do not develop AHR . Moreover, it seems that both IL-4
and IL-13 can exercise a direct effect on the airway cells
inducing AHR . Finally, it has been demonstrated that
the genetic background of the mouse strain can determine the
resistance, susceptibility or even the magnitude of the
development of AHR [22,23].
Current pharmacotherapies in asthma use corticosteroids
and leukotriene antagonists as anti-inflammatory agents and
β2-agonists as bronchodilators to relieve bronchospasms.
Other approaches to treat asthma are humanized monoclonal
antibodies directed against T cells, co-stimulatory
molecules, type 2 cytokines, or IgE antibodies (reviewed in
). Although pharmacotherapies have been proven to be
effective in controlling asthma symptoms, they are not
curative. Moreover, persistent treatments might have side
188 Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 Russo et al.
effects, such as immunosuppression, or exacerbation of Th1
responses leading to autoimmunity.
THE HYGIENE HYPOTHESIS AND REGULATORY
The hygiene hypothesis attempts at rendering the
question on the incidence of allergic diseases worldwide
increasing. The classical view of the hygiene hypothesis uses
the balance between Th1 and Th2 cells to explain the inverse
relation between infections and allergy [25-27]. The Th1
profile is associated with viral and bacterial infections and
autoimmune diseases (AID), and production of IFN-γ, TNF-
α, and IL-2. The Th2 profile is associated with helminth
infections and atopic allergic diseases, and production of IL-
4, IL-5, IL-9 and IL-13. According to the hygiene
hypothesis, a decreased occurrence of bacterial infections in
early childhood results in an insufficient stimulation of Th1
cells, which cannot counterbalance the expansion of Th2
cells, leading to a predisposition to allergy (commented by
Yazdanbakhsh et al. ). Increasing evidence is, however,
accumulating, showing that the first interpretations of the
hygiene hypothesis might not be totally correct. Firstly, an
increase in Th1 mediated AID, e.g., type 1 diabetes has been
found to be associated with the increased prevalence of
allergy in the developed countries . Furthermore, there is
no correlation between the occurrence of helminth infections
and allergic disease, despite both conditions being associated
with a Th2 profile. Actually, some helminth diseases, such
as chronic schistosomiasis, can suppress atopy in children,
probably by their induction of interleukin-10 . Finally,
conflicting results have been reported. For instance, Bager et
al. have shown that subjects with a higher number of
infections during the first two years of life have higher atopy
rates. The authors also found an increased risk of atopy in
individuals who had measles during the first year of life
. These contradictory findings do not fit into the frames
of the classical hygiene hypothesis. It, therefore, seems that
the tide is moving out on the hygiene hypothesis.
An alternative explanation for the inverse correlation
between infections and allergy is that infections are actually
needed for the emergence of regulatory T (Treg) cells. The
Treg cells are thought to be able to down-regulate both
allergic diseases (Th2) and AID (Th1). The mechanisms
proposed to the inhibitory function of Treg cells are by
secretion of immunosuppressive cytokines, such as IL-10
and TGF-β, and by cell-cell contact, via the cytotoxic T
lymphocyte-associated antigen 4 (CTLA-4) or membrane-
associated TGF-β [32-34].
The microenvironment plays an important role in the
generation of the Treg cells. For example, the way the
antigens are presented by antigen presenting cells (APCs)
can result in generation/activation of Treg cells. It has been
shown that dendritic cells (DCs) expressing molecules as
Serrate 1 (a notch 1 receptor ligand), and inducible co-
stimulator protein (ICOS) are involved in the generation of
Treg cells. Indeed, interactions between ICOS and ICOS
ligand can induce Treg cells and inhibit the development of
AHR by an IL-10-dependent mechanism [35,36].
A vast literature has described the activity/specificity of
Treg cells [37,38]. In general, Treg cells are considered to be
activated in an antigen specific manner; while their effector
function is non-specific, they can suppress the T cells either
directly or indirectly, via APCs . However, no
conclusive study has defined whether Treg cells really are a
specialized population of T cells or they are generated
depending on the context. Among the various cell surface
molecules identified on CD4+ Treg cells, including CD5,
CD45RB, CD25, CD62L, and CD38, the expression of the
α-chain of the IL-2 receptor (CD25) on Treg cells has been
shown to be highly specific for the protection in AID [40-
42]. Despite that the double-marker CD4+CD25+ has been
used by the majority of researchers in this field, many
studies have demonstrated that cells which do not express
CD25 can also have immunosuppressive activity ([43,44]
and commented by Curotto de Lafaille ). Other cells,
with regulatory properties, such as Tr1, and Th3 have been
found to express some Treg markers, such as CD4 and
CD25; both are, however, suggested to originate from a
CD4+CD25- population [32,46-48]. The Tr1 cells produce
IL-10, TGF-β, IFN-γ, and very low or non-detectable levels
of IL-2 and IL-4, while Th3 cells mainly produce TGF-β,
but also IL-10, and IL-4 [47,49].
The contact with pathogens can generate Treg cells.
Recently, in a Th1-colitis model, Kullberg et al. showed a
potent suppressive capacity of bacterial-triggered
CD4+CD45RBlow (either CD25+ or CD25-) Treg cells .
The suppressive activity was shown to be depend on IL-10
and not on TGF-β. Furthermore, in a model of experimental
asthma, Zuany-Amorin et al. have shown that treatment of
mice with killed Mycobacterium vaccae was sufficient to
generate allergen-specific CD4+CD45RBlow Treg cells,
which suppressed airway inflammation . This activity
was mediated by both IL-10 and TGF-β. The results
highlighted the importance of Treg cells in controlling both
Th1 and Th2 oriented inflammation (see Figure 1).
Here arises a question whether pathogen-associated
infections are essential for triggering the T cell associated
regulatory activity, or in other words, if we need microbial
infections to generate Treg cells.
MUCOSAL TOLERANCE AND REGULATORY T
The mucosa is the largest surface that is exposed to
exogenous antigens, either innocuous or pathogenic. The
total area of the mucosa is hundred-fold larger than that of
the skin. In humans, the area of the small intestine alone is
estimated to be 300 m2 . The gut-associated-lymphoid-
tissue (GALT) consists of 70% constantly activated T cells,
which, at normal conditions are harmless. The majority of
contacts established at the mucosal surfaces are with non-
pathogenic microbial antigens (the microbiota) and the
dietary antigens. The interaction between GALT and the
microbiota is relevant to the maturation of the immune
system, as demonstrated in germ-free models . Absence
of the microbiota can, e.g., reduce the susceptibility to oral
tolerance induction, as observed in neonatal mice, while
LPS can reverse this inability in germ-free mice [53-56]. It
has been suggested that the effect of the microbiota can be
mediated by its interaction with Toll-like receptors (TLRs)
present on GALT cells . As an extension of the classical
hygiene hypothesis, discussed above, it is assumed that even
Unconventional Strategies for the Suppression of Allergic AsthmaCurrent Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 189
Fig. (1). Infections and/or bacterial products induce the appearance of regulatory T cells (Treg) that in turn, suppress Th2 responses
and lung eosinophilic inflammation.
the non-pathogenic bacteria (e.g., the microbiota) can
generate Treg cells, which are important in the modulation
of both Th1 and Th2 responses and also in the establishment
of tolerance . The phenomenon of tolerance is constantly
generated on these surfaces and is important for the
maintenance of their “physiological inflammation” .
Menezes et al. have studied the interactions between
GALT and exogenous antigens derived from dietary
proteins. They observed that the immune system of the mice
reared in a diet without proteins, replaced by equivalent
amounts of amino acids, (protein-free mice) resembles that
of germ-free mice or neonates, with low levels of serum- IgA
and -IgG, and secretory IgA, but normal levels of serum
IgM. Moreover, the lymphoid cells of protein-free mice
displayed a Th2 cytokine profile, with high production of
IL-4 but low production of IFN-γ . Taken together,
these results suggest the importance of antigenic
interactions, which might exclude interactions with
pathogens or infectious agents, in the regulation of immune
Indeed, exposure to soluble antigens in the absence of
infection or adjuvant, induce a state of peripheral
immunological tolerance. The two major forms of peripheral
tolerance induced at mucosal surfaces are either oral or
respiratory tolerance. Oral tolerance has been defined as a
suppression of cellular and/or humoral immune responses to
an antigen by prior administration of the antigen via the oral
route . The induction of oral tolerance depends on
different factors, such as dose of antigen, and way of
administration [61,62]. Voluntary ingestion of small doses
of antigen seems to be most effective in tolerance induction,
while intragastric administration (gavage) induces a weaker
state of tolerance [63-66]. In addition, the nature of the
antigen, the genetic background, and immunological status
of the host influence the outcome of oral tolerance [67,68].
Another way to induce mucosal tolerance is by inhalation
of the antigen, known as respiratory tolerance (reviewed in
this issue by Macaúbas et al.). Recently, we have found that
mice fed with a protein-free diet are more refractory to the
induction of oral or respiratory tolerance (Mucida et al.,
manuscript in preparation), a finding that confirms that the
immunological status of the host is important in
establishing mucosal tolerance.
It is well established that previous contact with an
antigen by the respiratory tract can suppress both the cellular
responses and the IgE production [69-71]. However, in
contrast to oral tolerance, respiratory tolerance cannot
suppress neither IgG1 nor IgG2a specific antibody
McMenamin and colleagues suggested that respiratory
tolerance is mediated by immuno-deviation via CD8+γδ T
cells that secrete IFN-γ . Moreover, γδ-T cells might
also be involved in the induction and maintenance of oral
tolerance [75,76]. However, subsequent studies showed that
respiratory or oral tolerance could be fully induced in the
absence of γδ T cells, IFN-γ, or CD8+ T cells [9,77]. Thus,
the later experiments do not support the notion that
immuno-deviation is the major mechanism involved in
tolerance induction [9,77,78]. Actually, reports have shown
that both Th1 and Th2 responses can be suppressed during
mucosal tolerance [9,72,79-82].
Other immunological mechanisms have been ascribed to
mucosal tolerance, such as anergy, deletion (activation-
induced cell death) or ignorance (lack of critical mass of T
cells or antigen). However, a great number of recently
published articles describe an active cellular regulation as the
main mechanism of mucosal tolerance [49,70,83,84]. In fact,
Treg cells have been suggested to play a pivotal role in both
oral and respiratory tolerance [36,70,84,85].
As commented by Weiner  it appears that the
different outcomes in oral and respiratory tolerance might be
related to the different milieus observed in the lung and gut
. The IL-10 producing pulmonary DCs stimulate the
development of CD4+ cells which produce IL-10 and IL-4,
190 Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2Russo et al.
Fig. (2). Mucosal Ag administration generates different regulatory T cells that secrete anti-inflammatory cytokines that inhibit Th2
but not IFN-γ, characteristics associated with Tr1 cells. On
the other hand, DCs present in mesenteric LNs (MLNs)
produce increased amounts of TGF-β and induced TGF-β
producing CD4+ cells, characteristics associated with Th3
cells. Alpan et al. have demonstrated that in mice lacking
Peyer´s patches, B cells and M cells can still develop oral
tolerance . In these mice, activated DCs present in MLN
are thought to play an important role in TGF-β production
and T cell activation, and thereby in the induction of
tolerance to “harmless” environmental antigens.
It has been suggested that APCs can endocyte
tolerosomes, a vesicular structure present in the serum that
carries MHC II bound to antigenic peptides, and that they
under normal conditions can produce IL-10 and present low
co-stimulatory molecules, promoting the development of
regulatory T cells after antigen stimulation. Moreover, it has
been demonstrated that this serum factor is able to inhibit an
established OVA-mediated response possibly via CD25+
regulatory T cells [88,89].
Despite the differences between oral and respiratory
tolerance, the state of peripheral tolerance induced by both
the oral and airway route suppresses the majority of
deleterious immune responses to innocuous antigens, e.g.,
anaphylactic antibodies (IgE), pulmonary inflammation,
airway eosinophilia, AHR, and mucus formation [90,91]
(see Figure 2).
The involvement of Th3 cells after induction of oral
tolerance has been described in autoimmune murine models.
However, literature is particularly scarce regarding the effect
of oral tolerance on Th2-mediated diseases, such as asthma.
We have previously shown that oral tolerance can prevent the
development of lung and bone marrow eosinophilia in B6
mice . Other reports, using similar approaches, have
shown that high dose oral tolerance was effective in
preventing antigen-induced eosinophil infiltration in the
It is known that human asthma varies between different
populations and even between individuals. As discussed
above, different mouse strains mimic this heterogeneity
since they present different outcomes of allergic asthma. We
selected four different mouse strains that present key features
of asthma to study the influence of this heterogeneity during
the induction of oral tolerance. The strains were BALB/c
mice that develop an IL-4-dependent asthma ; BP2 mice
that present high AHR, largely dependent on IL-5
production ; IL-5 transgenic mice that exhibit
hypereosinophilia ; and γδ-T cell-deficient mice that are
hyper-responsive to metacholine and refractory to oral
tolerance induction [7,75,76]. We used a five-day continuous
oral antigen administration instead of a single gavage. We
found that this protocol of oral OVA administration was
very effective in preventing asthma-like responses in all the
mouse strains described above . However, contrary to
what was expected, we noted a decreased IL-10 production
and no TGF-β production in tolerized mice. Moreover,
treatment with anti-TGF-β, at the time of antigen challenge
did not reverse oral tolerance. These results indicate that
suppressive cytokines are not generated in the airways after
OVA challenge. However, these suppressive cytokines may
operate at different sites, such as in regional bronchial lymph
nodes, or in the spleen, or peak at a time point that could
not be measured.
We also found that oral OVA treatment starting at the
same day, or up to 7 days after OVA immunization, was
still effective in suppressing key phenotypes of asthma, such
as anti-OVA IgE synthesis, IL-4 and IL-5 production, airway
eosinophilia, AHR and mucus production. However, OVA
feeding 14 days after immunization exacerbated the airway
inflammation and IgE production. These findings indicate
that oral tolerance may represent a two-edge sword. It
appears that the inductive phase of mucosal tolerance has an
optimal timing-window, and that it depends on the context
of antigen priming and T cell activation. Actually, once the
immune response is established (tolerance or immunity) a
state of “immunological inertia” is developed, that is hard to
break. However, according to Cohn, in contrast to what is
found in murine models, human allergic patients tolerate
Unconventional Strategies for the Suppression of Allergic AsthmaCurrent Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 191
Fig. (3). Bacterial products induce the secretion of IL-12 by APCs that in turn expand allergen specific CD8+ T cells that inhibit CD4+
well mucosal antigen administration without flares in
disease, and some have shown symptomatic improvement
. More importantly, in atopic patients, oral, sublingual
or inhaled administration of mite or pollen antigens for one
or two years led to a reduction in symptoms and decreased
local inflammation. The different effects of mucosal antigen
administration in murine and human asthma might be
ascribed to different states of T cell activation. Whatever the
mechanisms are, it is clear that infections, mucosal antigen
exposure, and microbiota can influence the immune system
in the development of regulatory pathways that can result in
the suppression of asthma-like responses.
THE HYGIENE HYPOTHESIS AND REGULATORY
CD8+ T CELLS
Since Th1 and Th2 responses are thought to be mutually
exclusive, it was interpreted by the classical hygiene
hypothesis that the prevention of allergic responses could be
explained by the emergence of CD4+ Th1 cells. However,
data obtained by cell transfers of allergen-specific Th1 cells
showed that, instead of counterbalancing Th2-induced
allergic inflammation, Th1 cells caused more severe airway
inflammation [95,96]. These results, obtained in mice, are in
line with previous findings in human asthmatics showing
that IFN-γ is present in serum and BAL fluid [97-100].
Actually, Th1 cells are thought to be involved in the
mechanism whereby viral infections exacerbate asthma .
Although the therapeutic value of strategies based on a
shift to a Th1 pattern may suffer serious concerns, there is
solid evidence that the induction of Th1 cells or type 1
immune responses can suppress Th2 responses. For instance,
immunization with naked DNA plasmids containing cDNA
for allergens, together with bacterial DNA containing
unmethylated CpG dinucleotides or synthetic
oligodeoxynucleotides that mimics microbial DNA, or
together with heat-killed Listeria monocytogenes with or
without IL-18 as adjuvant result in the suppression of
allergic responses [102-105]. Thus, it appears that type 1
immune responses can have both detrimental and beneficial
effects on allergic airway inflammation.
Th1 cells might adversely affect asthma by increasing the
expression of cell adhesion molecules (e.g. VCAM-1)
involved in the recruitment of Th2 cells to airways .
Conversely, the protective effect of type 1 immune response
may be attributed to the activation of CD8+ T cells. Indeed,
it is well established that immunization with DNA plasmids
containing cDNA for allergens, bacterial DNA, or heat-killed
Listeria monocytogenes elicits a strong cellular CD8+-
dominated immune response, an IL-12-dependent IFN-γ
production and increased IL-18 mRNA expression . In
these models, neutralization of IL-12 or depletion of CD8+
T cells restored airway inflammation, while transfer of CD8+
T cells suppressed it [102,104]. These results confirm
previous findings showing that CD8+ T cells act as
suppressor cells in allergic responses, but the mechanism of
suppression remains elusive [107,108] (see Figure 3).
THE HYGIENE HYPOTHESIS AND REGULATORY
Macrophages and/or DCs are essential for an individual's
resistance to infections with intracellular pathogens. During
infection, macrophages or DCs can be activated directly by a
microbial product, or indirectly by cytokines. Bacterial
lipopolysaccharide (LPS), a prototypic cell wall component
of gram-negative bacteria, activates macrophages and DCs
via the transmembrane TLR4, while IFN-γ, a prototypic
type 1 cytokine, activates macrophage via the IFN-γ receptor
LPS are ubiquitous in the environment and are often
present in polluted air and in organic or household dusts
. It has been shown that exposure to airborne LPS can
either protect against asthma or exacerbate it [113,114]. The
beneficial effects of LPS are thought to be mediated by
enhanced secretion of the type-1 cytokines IL-12 and IFN-γ_
that are known to down modulate allergic responses [113-
115]. Conversely, airborne LPS might adversely affect
asthmatics by enhancing an established airway inflammation
and airway obstruction [113-118].
It is well established that Th1-mediated immunity
induces the synthesis of inducible nitric oxide synthase
(NOS)2, which is an essential event in the antibacterial
function of macrophages. LPS is also a potent stimulator of
nitric oxide (NO) production  and a number of studies
have demonstrated the involvement of NO in lung
physiopathology . In non-asthmatic individuals, with
normal airways, exhaled NO is derived from constitutive
endothelial and neural nitric oxide synthases, NOS3 and
NOS1, whereas the increased levels of NO detected in
asthmatics appear to be derived from inducible NOS (iNOS,
or NOS2), expressed by the inflamed airways [121,122].
Whether NO production has a beneficial or deleterious effect
in asthma is still controversial. Data from experimental
asthma models, using gene inactivation of NOS isoforms,
indicate that the induction of airway eosinophilic
inflammation can be both dependent and independent of
NOS2 activity, whereas NOS1, but not NOS2 expression,
seems to be required for protection against AHR [123,124].
In addition, conflicting results were also obtained in studies
192 Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 Russo et al.
Fig. (4). Activation of macrophages by LPS and IFN-γ result in enhanced expression of NOS2, IDO and HO-1 enzymes that generate
products that suppress T cell responses.
with drug-induced inhibition of NO production, in which
NOS2 inhibition was shown to either exacerbate or attenuate
allergen-induced airway inflammation and AHR [125,126].
A recent report showed that intranasal LPS
administration in immunized and challenged rats drastically
decreased established airway eosinophilia . We
confirmed these results using intranasal or systemic LPS
administration. Systemic LPS administration almost
completely suppressed both the early and the late allergic
reaction and AHR. This suppression was exclusively
dependent on TLR4 and NOS2 activity (manuscript
submitted). The mechanism by which NO suppresses
allergic responses remains to be determined. However, it is
likely that very complex regulatory pathways are operating
in vivo, and these might include inhibition of key cells
involved in allergy, such as mast cells, Th2 lymphocytes,
eosinophils, bronchial epithelial cells, and vascular cells
LPS and/or IFN-γ have been shown to induce the
expression of the tryptophan-catabolizing enzyme
indoleamine 2,3-dioxygenase (IDO) in the lungs [129-131].
Degradation of tryptophan, an essential amino acid required
for cell proliferation, or the generation of tryptophan-derived
catabolites appear to be involved in the mechanisms of IDO-
induced T cell suppression . Interestingly, IDO is
expressed in the placenta, where it prevents the rejection of
the fetus during pregnancy by a mechanism inhibiting
alloreactive T cells . Thus, expression of IDO by APCs
might potentially inhibit T cell proliferation. More recently,
IDO expression was detected on a subset of human DCs
suggesting that these cells may represent a regulatory subset
of APCs in humans . In mice, it has long been known
that engagement of CTLA-4 with B7, a counterligand
present on DCs, delivers inhibitory signals to T cells that
result in peripheral tolerance. However, the mechanism of T
cell suppression induced by CTLA-4 was for a long time
poorly understood. A recent report by Grohmann et al. has,
however, shed light on this issue by showing that long-term
pancreatic islet allograft survival could be achieved with
soluble fusion protein (CTLA-4-immunoglobulin) that was
contingent upon effective tryptophan catabolism in the host.
Thus, CTLA-4 acts as a ligand for B7 molecules and
induces the expression of IDO, that in turn, leads to T cell
Finally, it is well established that interleukin-10 (IL-10)
is a potent anti-inflammatory cytokine that is released by
different cell types, including activated macrophages. In
murine macrophages, IL-10 induces the expression of heme
oxygenase-1 (HO-1) . HO-1 expression can also be
induced by NO or other inflammatory stimulus .
Interestingly, the anti-inflammatory effect of IL-10 is
thought to rely on the ability of HO-1 to degrade heme,
catalyzing the cleavage of the heme ring to form free iron,
carbon monoxide (CO), and biliverdin. Indeed, inhibition of
HO-1 protein synthesis significantly reversed the inhibitory
effect of IL-10 on LPS-induced TNF-production .
Moreover the involvement of CO in the anti-inflammatory
effect of IL-10 in vitro was demonstrated .
Additionally, we have shown that significant levels of IL-10
are found in the BAL fluid of allergic mice, but we were not
able to identify the cell type that is responsible for its
production . It is likely that IL-10 production in the
asthmatic lung is a compensatory mechanism to control
airway inflammation. Others have shown that administration
of rIL-10 inhibited IL-5 production and allergic eosinophilia
Taken together, it is clear that activated macrophages,
through the expression of NOS2, IDO, and HO-1, act as
suppressor cells in asthma (see Figure 4).
Infections, microbial products and tolerance induction
can down modulate the allergen-driven Th2 intrapulmonary
responses observed in asthma. This suppression appears to
result from the emergence of regulatory cells, such as
Unconventional Strategies for the Suppression of Allergic AsthmaCurrent Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 193
CD4+CD25+ T cells, CD8+ T cells, and macrophages. The
mechanism of suppression involves different pathways, such
as cell-to-cell contact, inhibitory cytokines (IL-10 and TGF-
β), and macrophage enzymes (NOS2, IDO, and HO-1) with
immunosuppressive activities. Thus, to better understand
and to be able to control allergy and particularly, asthma, it
would be of interest to further exploit these different
pathways of suppression.
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