Discussion
Humulus pollen is an important allergen that causes asthma in
East Asia. In this study, we showed that, compared with IP
sensitization, SC sensitization provides a murine model that better
recapitulates the typical clinical characteristics of allergic asthma.
Using this model, we successfully developed a rapid SCIT model for
HSE-induced asthma. This SCIT model elucidated the mechanism of
desensitization
treatment, and provided a rationale for the use of HSE in clinical SCIT.
Compared with animals in the IP.HSE group, animals in the SC.HSE group
showed significantly higher levels of allergen-specific IgE and
significantly higher quantity and proportion of eosinophils in BALF.
These findings are consistent with
the typical characteristics of allergic asthma
patients[16]. In our study, mice
sensitized via the IP route showed an elevated number of eosinophils,
but no change in their proportion. Following airway stimulation by
atomized pollen, the number of inflammatory cells is globally elevated;
thus, enhancement of absolute eosinophil number does not necessarily
reflect the occurrence of a typical type I hypersensitivity reaction.
Allergic asthma is characterized by elevated expression of Th2 cytokines
(i.e., IL-4 and IL-13) and reduced expression of Th1 cytokines (i.e.,
IFN-γ)[20].
IL-4 is a representative Th2 cytokine that can stimulate the
proliferation of B and T cells; it enhances IgE
synthesis[21] and induces eosinophil
infiltration into the airway[22]. In
this study, IL-4 levels in the BALF and SHS were significantly higher in
mice in the SC.HSE group, compared with mice in the SC.PBS and IP.HSE
groups. This may explain why mice in the SC.HSE group showed higher
HSE-specific IgE levels and higher proportions of BALF eosinophils,
while mice in the IP.HSE group did not. IL-13 has been proposed to
induce inflammatory cell infiltration into the airway, as well as mucus
secretion and AHR[23]. Compared with
the respective control groups, BALF IL-13 levels were significantly
higher among mice in both SC.HSE and IP.HSE groups. Thus, IL-13 may have
promoted inflammatory cell infiltration, mucus secretion, and AHR in
these animals, although IL-4 levels were not elevated in the IP.HSE
group. However, IFN-γ levels in BALF and SHS of mice in the SC.HSE and
IP.HSE groups only showed a slight downward trend, but no significant
reduction.
The SC and IP sensitization routes may produce different immune
responses because of the presence of different local antigen-presenting
cell populations at the site of antigen contact. In the skin, dendritic
cells, which vary in quantity and phenotype, are reportedly found in
atopic individuals in a manner that favors type 2 immune
responses[24]. In addition, mast
cells , which can produce Th2 cytokines and amplify allergic response,
are widely distributed in the dermis and act as sentinels at sites of
initial antigen exposure[25]. In
contrast, in the peritoneal cavity, macrophages are found in large
numbers and tend to favor the induction of Th1/Th0 immune
responses[26].
Our group previously found that the approximately 10-kDa component is a
major allergen in H. scandens pollen among Chinese individuals;
we also identified the complete amino acid sequence of this protein
(GenBank: ADB97919.1). Except for this 10-kDa component ,Park et
al.[27] reported that three protein
components (13 kDa,74 kDa, and 80 kDa) from Humulus pollen
extract were bound to IgE, among sera from more than 50% of the
patients. Sun et al.[28] reported
that 30.8% of patient serum samples exhibited binding interactions with
100.5-kDa proteins in Humulus pollen. The immunoblotting
experiments in the present study showed that the IgE antibodies of
sensitized mice were mainly bound to relatively large fragments (48–100
kDa), and rarely bound to the 10-kDa band. This finding is consistent
with the study work of U.S. Eitzer et
al.[29] who found that mice
sensitized with whole protein of timothy-grass pollen did not produce
IgE antibodies against PHLP1, the major allergen of timothy in humans.
These findings suggested that mice and humans may be sensitive by
somewhat different proteins.
Clinically, SCIT typically requires SC injection twice per week for 3–5
years. Considering differences in the life cycles of mice and humans, we
used a rapid desensitization treatment method (eight treatments every
other day) and effectively alleviated pulmonary inflammation and AHR in
asthmatic mice. Our results were similar to those of a study in which
mice received weekly treatments for a continuous 8-week
period[30]. This short-term and rapid
desensitization method saves time and reduces the cost of mouse
maintenance.
Despite the short treatment duration, allergen-specific IgE was
significantly lower in the treatment group than in the vehicle group;
anti-IgE therapy has been effective for some allergic
diseases[31]. In addition, SCIT has
been shown to significantly enhance allergen-specific IgG2a levels.
Elevated levels of allergen-specific IgG4 in humans (similar to IgG2a in
mice) are accompanied by improvement of clinical
symptoms[32]. Therefore, the
therapeutic effect of SCIT in our model may have been achieved by
inhibiting production of allergen-specific IgE and enhancing production
of allergen-specific IgG2a in serum, as in previous
studies[33].
SCIT corrected the imbalance of Th1/Th2 immune responses in asthmatic
mice and converted the immune response to a Th1 type. This may be the
underlying mechanism of desensitization treatment. IL-10 is a
pleiotropic cytokine that has both pro- and anti-inflammatory effects
during immune responses[34]. In our
study, IL-10 levels were significantly increased in asthmatic mice and
significantly decreased after SCIT, indicating that this cytokine may
play a role in exerting pro-inflammatory effects.
There were some limitations in this study. Changes in the systemic
immune response were examined by analysis of SHS because of limited
laboratory conditions; the sensitivity of this method is relatively low,
we will try to co-culture spleen cells with specific allergens in vitro
to observe changes in the systemic immune response in the further
studies.
In conclusion, we successfully developed a better murine asthma model
for Humulus pollen allergy through SC sensitization and
atomization challenge. Using this model, short-term and rapid SCIT
improved symptoms and pathophysiology in asthmatic mice. The murine
models of asthma and SCIT for Humulus pollen allergy developed in
this study may be valuable tools for studying the mechanisms of asthma
and SCIT for airborne allergens, as well as for designing more
efficacious therapies for allergic
asthma.