To the Editor:
Allergic airway diseases, such as rhinitis and asthma, are one of the
most common chronic inflammatory respiratory diseases in the world (1).
Although the mechanisms underlying the pathology and treatment of
allergic airway inflammation have been widely studied, many aspects
remain unclear, including how allergic eosinophilic inflammation
effectively resolves in the airways (2). Recent studies implicated
natural killer (NK) cells in the regulation of eosinophilic airway
inflammation, notably by inducing apoptosis of autologous eosinophilsin vitro (3). Moreover, lipoxin A4(LXA4), a lead member of a larger family of specialized
pro-resolving lipid mediators, can enhance the ability of NK cells to
cause eosinophil-apoptosis in vitro (3) and decrease allergic
eosinophilic inflammation in animal models (4).
In this study, twenty subjects ( mean age 28.07 ± 7.1, ten men
and ten women) with confirmed grass pollen allergic rhinitis were
included. All the subjects underwent two standardized nasal allergen
challenges with either a single pre-titrated threshold dose of a grass
pollen allergen extract or a diluent in a randomized order and at least
4 weeks apart (for details see Supp. Material and Methods). Nasal lavage
fluid and cells were collected at baseline and at different time points
after challenge. The study was approved by the local ethics committee
and all subjects gave informed written consent.
The nasal allergen challenge induced typical allergic symptoms and a
local inflammatory response in all patients that resolved spontaneously
within 72 hrs (Figure S1). No significant response was observed after
the diluent challenge. Leukocyte sub-populations were further identified
in nasal lavage samples by FACS (Figure 1A). Neutrophils were the most
abundant population recruited after allergen challenge, peaking 1 hr
after. As expected, eosinophils were also rapidly recruited.
Interestingly, NK cells were recruited as early as 1 h after the
challenge, persisted for 6 hrs before falling to their baseline levels.
Monocyte and lymphoid cell recruitment was also observed (Figure 1B).
There was a positive relationship between the total number of nasal
lavage NK cells and that of eosinophils 1, 6 and 24 hrs after allergen
challenge (Figure 1C).
As LXA4 can decrease allergic eosinophilic inflammation
and regulate NK cell-eosinophil interaction (3, 4), we next quantified
LXA4 levels in nasal lavage samples after the allergen
challenge. We observed that LXA4 was produced in the
nasal mucosa at baseline and significantly increased in the nasal lavage
fluid 1 hr after allergen challenge (Figure 2A). LXA4 is
produced by multistep enzymatic processes in different cell types,
including neutrophils (6). Interestingly, increased LXA4levels 1 hr after allergen challenge correlated with the peak of nasal
neutrophil infiltration, suggesting a potential role of neutrophils in
LXA4 biosynthesis during the early phase of the allergic
inflammatory response (Figure 2B). LXA4 levels also
significantly increased at 48 and 72 hrs post-allergen challenge when
compared to baseline levels and correlated at these time points to
monocyte recruitment.
As previously shown, we observed that peripheral blood NK cells isolated
from healthy donors induce apoptosis of autologous blood eosinophilsin vitro (Figure S2). Moreover, to induce eosinophil
apoptosis a direct contact and a combined action of
CD56bright and CD56dim NK cells were
needed (Figure S2). Besides a potential pro-resolving effect of NK cells
on eosinophils, recent data has shown that NK cells can also trigger
superoxide release by eosinophils, that can worsen inflammation (5). In
our study, eosinophils isolated from healthy donors were by far the most
important producers of superoxide anion among different leukocyte
populations (Figure 2C). In the presence of NK cells, superoxide release
from eosinophils was significantly reduced after 1 hr of co-incubation.
In contrast, this inhibitor effect was no longer present after 4 hrs of
co-incubation (Figure 2D). When eosinophils from healthy donors were
exposed to LXA4, they significantly reduced their
superoxide release in a dose-dependent manner (Figure 2E). Superoxide
release by eosinophils co-incubated with NK cells in the presence of
LXA4 was still inhibited in a dose-dependent manner 4
hrs later (Figure 2F) in contrast without LXA4 (Figure
2D).
Our study underlines the complex network between cellular and molecular
actors during resolution of allergic airway inflammation. 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
correlate with eosinophilic inflammation. 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 LXA4 biosynthesis.
Moreover, we identified a combined role for NK cells and
LXA4 in mediating resolution of eosinophilic
inflammation in vitro .
Anh Poirot, BSc1;
Guillaume Wacht, MD, MSc2;
Christine Lehalle, MSc3;
Philippe Saas, PhD4;
Nelly Frossard, PhD3;
Bernard Geny, MD, PhD2;
Fréderic de Blay, MD1;
Cindy Barnig, MD, PhD4,5
1 Department of Chest Disease, University Hospital of
Strasbourg, Strasbourg, France
2 EA 3072, University of Strasbourg, France
3 UMR 7200 CNRS / Université de Strasbourg,
Laboratoire d’Innovation Thérapeutique and LabEx MEDALIS, Faculté de
Pharmacie, Strasbourg, France
4 Univ. Bourgogne Franche-Comté, INSERM, EFS BFC,
UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et
Génique, LabEx LipSTIC, F-25000 Besançon, France
5 Department of Chest Disease, University Hospital of
Besançon, Besançon, France