Discussion
In this study, we used a standardized nasal allergen provocation test
with a single threshold dose that led to an acute self-limited allergic
respiratory inflammatory response. Here we showed for the first time
that NK cells are recruited to the nasal mucosa shortly after the
initiation of the allergic inflammatory response and correlate with
eosinophilic infiltration.
Natural killer (NK) cells are key members of the innate lymphocyte
family with essential roles in host defense against pathogens as well as
in malignancies (20). However, a role for NK cells in the regulation of
type 2 immune responses and allergic diseases has been recently
suggested. Indeed, experimental studies using murine models have shown
that NK cells can participate in the regulation of allergen-induced
eosinophilic airway inflammation (11, 21-23). Moreover, the presence of
activated NK cells has been noted in patients with type 2 diseases,
including asthma (12, 24), allergic chronic rhinosinusitis (25, 26) and
atopic dermatitis (27). Whether NK cells enhance or limit type 2 immune
responses is still debating.
In human in vitro experiments, it has been shown that human NK
cells can promote apoptosis of autologous eosinophils but the molecular
mechanisms underlying these events were only partially addressed (13,
28). In the present study, we confirmed that peripheral blood NK cells
from healthy donors are able to induce the apoptosis of autologous
peripheral blood eosinophils. As eosinophils have the capacity to
release a broad range of toxic mediators into surrounding host tissues
with consequent tissue damage, eosinophil apoptotic cell death followed
by their subsequent phagocytosis by surrounding phagocytes is therefore
critical for the successful resolution of allergic and asthmatic
inflammation (29). Moreover, as suggested earlier (13, 28), we confirmed
that NK cell-induced eosinophil apoptosis needed a direct contact
between eosinophils and NK cells.
Besides potential inflammation pro-resolving effects, activator effects
of NK cells on eosinophils have also been reported. In a previous study,
human NK cells were shown to induce a dose-dependent in vitroactivation of autologous eosinophils after 3 and 12 hours of co-culture
(13). Upon activation, eosinophils are able to release many mediators,
including highly reactive oxygen species that can damage surrounding
tissues and worsen inflammation (30). In the current study, we observed
that among different leukocyte populations involved in type 2 allergic
inflammation, healthy donor eosinophils from were by far the most
important producers of superoxide anion. After 1 hr of co-incubation
with NK cells, the release of superoxide by eosinophils was
significantly reduced. This inhibitor effect was overwhelmed after 4 hrs
of co-incubation, confirming a partial activator effect of NK cells on
eosinophils.
Several classes of natural pro-resolving and anti-inflammatory chemical
mediators are produced at sites of inflammation, including the
respiratory mucosa (4). LXA4 is a lead member of a newly
described class of pro-resolving lipid mediators derived from
arachidonic acid that signals through ALX/FPR2 receptors (5).
LXA4 has been reported to decrease allergic and
asthmatic responses in mouse models (6). In the current study, we showed
that LXA4 was already present in the nasal mucosa at
baseline and significantly increased in the nasal lavage fluid shortly
after the nasal allergen challenge.
LXA4 is produced by multistep enzymatic process
resulting from lipoxygenase (LO) activities in different cell types. One
of the main pathways of LXA4 synthesis involves
lipoxygenation of arachidonic acid by 15-LO in epithelial cells to
generate 15(S)-hydroxyeicosatetranoic acid (15S-HETE) that can then be
utilized as a substrate by the 5-LO expressed in neutrophils to
synthesize LXA4 (31). In our study, neutrophils were the
most abundant population recruited during the nasal allergic response,
peaking 1 hr after the allergen challenge and returning to baseline
levels at 24 hrs. A such transient increase of neutrophils has been
documented earlier after allergen challenge in animal models and human
asthma but their role in allergic disease has been questioned (32-34).
Interestingly, we observed an association between the
LXA4 levels and the peak of nasal neutrophil
infiltration, suggesting the involvement of neutrophils in
LXA4 biosynthesis during the early respiratory allergic
inflammatory response. After falling back to baseline levels,
LXA4 significantly re-increased at 48 and 72 hrs
post-allergen challenge at time points where monocytes were present in
the nasal lavage samples. An increasing number of studies have linked
efferocytosis by macrophages and monocytes with their further production
of pro-resolving mediators, including LXA4 (35).
Both NK cells (12) and eosinophils (36) express the LXA4receptor ALX/FPR2. Since anti-inflammatory effects of
LXA4 on eosinophils were previously reported (19), we
next determined whether LXA4 was able to modulate
eosinophil superoxide release. Activated eosinophils significantly
reduced their superoxide release in a dose-dependent manner when exposed
to LXA4. When eosinophils were co-incubated with NK
cells in the presence of LXA4, superoxide release was
still inhibited in a dose-dependent manner 4 hrs later, in contrast with
the previously observed partial activator effect of NK cells on
eosinophils 4 hrs later.
Our study underlines the complex network between cellular and molecular
actors during resolution of allergic airway inflammation. Our data
suggested that NK cells could represent an endogenous counterregulatory
response to type 2 inflammation, as NK cells are recruited to the nasal
mucosa during an allergic inflammatory response in parallel to
eosinophil accumulation an are able to promote eosinophil apoptosis. The
accumulation of neutrophils along with monocytes during the allergic
inflammatory response may furthermore be an important regulatory
feedback to initiate and promote resolution of allergic inflammation, as
our data suggest involvement of these cells in LXA4biosynthesis, a specific pro-resolving lipid mediator. Finally,
LXA4 appeared to be essential in limiting superoxide
release by eosinophils in the presence of NK cells.
Current treatment strategies in allergic airway diseases have focused on
combating pathologic type 2 inflammation using either broad or selective
immunosuppression. In the next future, the therapeutic paradigm could
shift to the use of pharmacologic agents that enhance endogenous
pro-resolving mechanisms. This presupposes a better understanding of the
roles and interactions between cellular and molecular processes that
drive the resolution of allergic inflammation.
In conclusion, here we report for the first time that NK cells are
recruited to the nasal mucosa of subjects with allergy in response to
nasal allergen challenge and we identify a synergistic role for NK cells
and LXA4 in mediating resolution of allergic eosinophilic inflammation.