Figure 1. Nasal provocation with a single dose of allergen
induces a rapid nasal allergic inflammatory response that resolves
spontaneously over 72 hrs. Subjects (n=20) suffering from confirmed
seasonal grass pollen rhinitis were challenged intranasally with a
single threshold dose of a grass pollen extract or diluent. A,Nasal symptoms were assessed at baseline and at different time points
(15 min, 30 min, 1, 6, 24, 48 and 72 hrs) after nasal challenge. The
total nasal symptom score (TNSS) represents the cumulative score of
sneezing, nasal running (rhinorrhea), itching and nasal blockage
(obstruction) each scored from 0 to 3 using a questionnaire. B,Total inflammatory cells and C, protein levels were measured in
nasal lavage samples collected at the indicated time points after
allergen challenge or diluent. Data are displayed as mean ± SEM;
*P < 0.05, **P < 0.01, ****P< 0.0001; 2-way ANOVA with Sidaks’s multiple comparison test
was used to compare allergen vs diluent at various time points.
Figure 2. NK cells appear early during the allergic
inflammatory response. A, Representative dot plots for flow
cytometric sequential gating of leukocyte populations in nasal lavage.
First, doublets were excluded, then CD45‒ events and
subcellular debris. Leukocyte populations (granulocytes, lymphoid cells
and monocytes) were identified on the basis of CD14 expression and side
scatter. Eosinophil and neutrophils were identified among granulocytes
on the basis of absence or presence of CD16 expression, respectively. NK
cells and T cells were identified among the lymphoid cell population as
CD3-NKp46+ cells and
CD3+ NKp46‒ cells, respectively.B , Time course of neutrophils, eosinophils, NK cells, lymphoid
cells, T lymphocytes and monocytes following allergen nasal challenge or
diluent. Data are displayed as mean ± SEM; *P < 0.05;
**P < 0.01; ***P < 0.001; 2-way ANOVA
with Sidaks’s multiple comparison test was used to compare allergenvs diluent at various time points.
Figure 3. NK cells correlate with eosinophils during the
allergic inflammatory response. A , Correlation between total NK
cells and eosinophils 1, 6 and 24h after the allergen challenge (Pearson
correlation r value and significance are noted).
Figure 4 . NK cells are able to induce apoptosis in autologous
eosinophils and to inhibit superoxide production by eosinophils.A , Peripheral blood eosinophils were incubated (4 hrs, 37°C)
with medium alone, autologous supernatants from past eosinophil
cultures, autologous supernatants from past NK cell-eosinophil
co-cocultures (1:5 ratio) and autologous peripheral blood NK cells (1:5
ratio). Eosinophil apoptosis was assayed by FACS with annexin-V and
7-AAD staining. Results are expressed as means ± SEM, n = 6
individual healthy donors; ****P < 0.0001; one-way ANOVA with
Tukey’s multiple comparison test B , CD56dimand CD56bright NK cells were sorted by flow cytometry
(left panel) and co-incubated (4 hrs, 37°C) alone or together with
autologous eosinophils (1 :5 ratio) (right panel). Eosinophil apoptosis
was assayed by FACS with annexin-V and 7-AAD staining. Results are
expressed as means ± SEM, n = 5 individual healthy donors, ****P
< 0.0001, one-way ANOVA with Tukey’s multiple comparison test.C , Superoxide release was assessed by electron paramagnetic
resonance (EPR) in different leukocyte population involved in type
2-mediated allergic inflammation at rest (vehicle) and when activated by
phorbol myristate acetate (PMA) (10 min, 50 ng/mL). Results are
expressed as means ± SEM, n = 5 individual healthy donors, *P
< 0.05, at rest (vehicle) when cells are compared to each
other; **P < 0.05, after PMA when cells are compared to each
other; £P < 0.05, when cells are compared
between rest (vehicle) and PMA; multiple t -tests with Bonferroni
correction. D , NK cells induce a significant reduction of
superoxide production by FACS isolated eosinophils 1 hr after
co-incubation (37°C, 1:5 ratio) that is overwhelmed after 4 hrs of
co-incubation. Results are expressed as means ± SEM, n = 7
individual healthy donors, *p<0.05, paired Student’s ttest.
Figure 5 . LXA4 is produced in the nasal mucosa
after nasal allergen challenge and correlate to neutrophil and monocyte
recruitment. A , LXA4 concentrations were
quantified by ELISA in nasal lavage samples. Values are expressed as
means ± SEM; *P < 0.05 compared to diluent,$P< 0.05 compared to baseline levels; 2-way
ANOVA with Sidaks’s multiple comparison test was used to compare
allergen vs diluent at various time points and 2-way ANOVA with
Tukey’s multiple comparison test was used to compare levels vsbaseline levels. B , Correlation of LXA4 levels
and neutrophil counts 1 hr after allergen challenge (Pearson correlationr value and significance are noted). C , Correlation of
LXA4 levels and monocyte levels 48 hrs (left panel) and
72 hrs (right panel) after allergen challenge (Pearson correlationr value and significance are noted).
Figure 6 . LXA4 is essential to inhibit NK cells
triggered eosinophil superoxide release in vitro . A ,
Eosinophils were pre-treated (15 min) with increasing doses of
LXA4 (1, 10 or 100 nM or vehicle [0.1% ethanol])
and then stimulated with PMA (10 min, 50 ng/mL), superoxide release was
assessed by EPR. Results are expressed as means ± SEM; n = 6
individual healthy donors; *p<0.05 compared to vehicle;£p<0.05 compared to 1 nM; one-way ANOVA with
Tukey’s multiple comparison test. B , Eosinophils were
pre-tretated (15 min) with LXA4 (10, 100 nM or vehicle)
and co-incubated with NK cells (37°C, 1:5 ratio). Eosinophils showed
still a dose-dependent reduction of superoxide production after 4 hrs of
co-incubation. Results are expressed as means ± SEM, n = 3
individual healthy donors; *P < 0.05; multiple t -tests
with Bonferroni correction.
Figure S1. Detailed clinical scores composing the total nasal symptom
score (TNSS). A, Nasal sneezing, B, nasal itching,C, nasal running (rhinorrhea) and D, nasal blockage
(obstruction). Data are displayed as mean ± SEM; *P <
0.05, ***P < 0.001, ****P < 0.0001;
2-way ANOVA with Sidaks’s multiple comparison test was used to compare
allergen vs diluent at various time points.