Allergic diseases include asthma, allergic rhinitis, and atopic dermatitis depending on a target organ. House dust mite (HDM) is an essential allergen of allergic diseases (Gaffin and Phipatanakul, 2009; Kemp, 2009; Leung and Guttman-Yassky, 2014). HDM allergens contain Dermatophagoides pteronyssinus (DP) and Dermatophagoides farina (DF), which affect pathogenesis of allergic diseases (Mili?n and Diaz, 2004; Kang et al., 2014). Damage-associated molecular pattern (DAMP) is associated with immune response in the noninfectious inflammation such as allergy and includes S100A8 and S100A9. Both S100A8 and S100A9 belong to the S100 protein family and are involved in allergic disease, cancer and autoimmune disease (Donato et al., 2013; Kim et al., 2013). Neutrophils function as inflammatory cells. Inhibition of constitutive neutrophil apoptosis aggravates pathogenic lesions via continuous accumulation of neutrophils in allergic diseases such as asthma (Scheel-Toellner et al., 2004; Monteseir?n, 2009; Lee et al., 2014).

This study was approved by the Institutional Review Board of Eulji University for normal volunteers. All participants in this study gave their written informed consent. Recombinant S100A8 and S100A9 proteins were produced as follows. Total RNA of human neutrophils was extracted using TRIzol reagent (Life Technologies Inc.) and first strand cDNA was synthesized with AccuPower RT PreMix (Bioneer, Daejeon, Korea). Primers S100A8-1 (5′-ttccatatgatgttgaccgagctggagaa), S100A8-2 (5′-ccgctcgagctactctttgtggctttctt), S100A9-1 (5′-ttccatatgatgacttgcaaaatgtcgca), and S100A9-2 (5′-ccgctcgagactgtggtcttagggggtgc) were used for cDNA synthesis of S100A8 and S100A9. Double-stranded cDNA of human S100A8 and S100A9 was synthesized using polymerase chain reaction (94°C/30 sec, 60°C/30 sec, 72°C/50 sec, 30 cycles) and subsequently cloned into pET28 expression vector (Merck Millipore, Darmstadt, Germany). Recombinant S100A8 and S100A9 expressions were induced with 1 mM isopropyl β-D-thiogalactoside in E. coli BL21 (DE3, Merck Millipore) for 4 h and 16 h at 37°C, respectively. Thereafter, the bacteria were centrifuged at 5,000 g for 10 min and the pellet was lysed in BugBuster Protein Extraction reagent (Merck Millipore). Next, the lysate was centrifuged at 55,000 g for 30 min at 4°C, after which the supernatant was collected. Recombinant His-Tag S100A8 and S100A9 were purified using a nickel column and the purified proteins were verified by SDS-PAGE and western blotting. Human neutrophils were isolated from the heparinized peripheral blood of healthy persons using Ficoll-Hypaque gradient centrifugation and a CD16 microbeads magnetic cell sorting kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were washed after hypotonic lysis to remove erythrocytes and then resuspended at 3 × 10⁶/ml in RPMI 1640 medium with 1% penicillin-streptomycin and 10% FBS. An annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Diego, CA, USA) was used to detect neutrophil apoptosis. Isolated neutrophils were incubated with an FITC-labeled annexin V and propidium iodide (PI) for 15 min at room temperature. Apoptotic neutrophils were analyzed using a FACSCalibur with CellQuest software (BD bioscience) and were determined as the percentage of cells showing annexin V+/PI? and annexin V+/PI+.

Since DP, S100A8, and S100A9 are associated with regulation of neutrophil apoptosis in our previous study (Kim et al., 2015), we examined whether they alter neutrophil apoptosis in new ex vivo environment similar to disease circumstance. Neutrophil apoptosis is not altered by co-culture with eosinophils and lymphocytes, except during incubation with monocytes (Fig. 1). DP, S100A8, and S100A9 had inhibitory effects on neutrophil apoptosis in neutrophil alone without other immune cells. DP and S100A8 suppressed neutrophil apoptosis in combinations of neutrophils, eosinophils, lymphocytes or monocytes. S100A9 significantly inhibited neutrophil apoptosis in some combinations such as neutrophils/lymphocytes and neutrophils/monocytes. S100A8 inhibited similar or stronger neutrophil apoptosis in a mixed culture of immune cells than in a culture of only neutrophils, and the anti-apoptotic effects of S100A9 in the mixture of immune cells is similar to that in neutrophils. Alteration of T helper (Th) cytokines such as Th1, Th2, Th9, Th17, exist in the lung during asthma progress. We investigated whether DP, S100A8, and S100A9 suppress neutrophil apoptosis in the alteration of Th circumstance. DP, S100A8, and S100A9 blocked neutrophil apoptosis, regardless of pretreatment with a T helper (Th) 1 cytokine (IFN-γ), Th2 cytokines (IL-4 and IL-10), a Th9 cytokine (IL-9), a Th17 cytokine (IL-17), a Treg-producing cytokine (TGF-β) (Fig. 2) or combinations of these cytokines. These results may contribute to a better understanding of the regulation of spontaneous neutrophil apoptosis in normal and asthmatic states.