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.