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S100A8 and S100A9 are important proteins in the pathogenesis of allergy. Asthma is an allergic lung disease, characterized by bronchial inflammation due to leukocytes, bronchoconstriction, and allergen-specific IgE. In this study, we examined the role of S100A8 and S100A9 in the interaction of cytokine release from bronchial epithelial cells, with constitutive apoptosis of neutrophils. S100A8 and S100A9 induce increased secretion of neutrophil survival cytokines such as MCP-1, IL-6 and IL-8. This secretion is suppressed by TLR4 inhibitor), LY294002, AKT inhibitor, PD98059, SB202190, SP600125, and BAY-11-7085. S100A8 and S100A9 also induce the phosphorylation of AKT, ERK, p38 MAPK and JNK, and activation of NF-κB, which were blocked after exposure to TLR4i, LY294002, AKTi, PD98059, SB202190 or SP600125. Furthermore, supernatants collected from bronchial epithelial cells after S100A8 and S100A9 stimulation suppressed the apoptosis of normal and asthmatic neutrophils. These inhibitory mechanisms are involved in suppression of caspase 9 and caspase 3 activation, and BAX expression. The degradation of MCL-1 and BCL-2 was also blocked by S100A8 and S100A9 stimulation. Essentially, neutrophil apoptosis was blocked by co-culture of normal and asthmatic neutrophils with BEAS-2B cells in the presence of S100A8 and S100A9. These findings will enable elucidation of asthma pathogenesis.

S100A8 and S100A9 are associated with myeloid cell differentiation, chemotactic activities, adhesion of neutrophils, and apoptosis. In this study, we investigated the contribution of S100A8 and S100A9 to differentiation of the human eosinophilic leukemia cell line, EoL-1. S100A8 and S100A9 increased the number of vacuole per one cell and the protein expression of EPO and MBP. Rottlerin, an inhibitor of protein kinase C delta (PKCδ), inhibited the EoL-1 cell differentiation induced by S100A8 and S100A9. These results suggest that S100A8 and S100A9 may regulate the differentiation of eosinophilic progenitors. Moreover, these findings may shed light on elucidation of eosinophil differentiation due to S100 proteins.

Although extract of Dermatophagoides farinae (DF) alone had no effect on neutrophil apoptosis, it inhibited neutrophil apoptosis in neutrophils cocultured with lymphocytes in both normal and allergic subjects. DF showed a stronger inhibition on the apoptosis of allergic neutrophils cocultured with allergic lymphocytes than that of normal neutrophils cocultured with normal lymphocytes. DF induced the secretion of IL-6, IL-8, MCP-1, and GM-CSF of the normal and allergic lymphocytes. The cytokine secretion due to DF in allergic lymphocytes is higher than in normal lymphocytes. The cytokine secretion of normal and allergic lymphocytes induced by DF was suppressed by PAR2i, a PAR2 inhibitor, LY294002, a PI3K inhibitor, AKTi, an Akt inhibitor, PD98059, an ER K inhibitor, and BAY-11-7085, an NF-κB inhibitor. Phosphorylation of ERK induced by DF was suppressed by PAR2i, LY294002 and AKTi, and NF-κB activity due to DF was inhibited by PAR2i, LY294002, AKTi, and PD98059.

Dysregulation of neutrophil apoptosis causes pathogenesis and aggravation of allergy. S100A9 exists as one of the proteins in the neutrophils, triggering inflammatory responses by activating the immune cells. In this study, we investigated whether S100A9 affects constitutive neutrophil apoptosis by activating the monocytes in normal and allergic subjects. Supernatant from human monocytic THP-1 cells after treatment with S100A9 suppressed normal neutrophil apoptosis by inhibiting the activations of caspase 9 and caspase 3. S100A9 upregulated the release of MCP-1, IL-6, and IL-8 in THP-1 cells. An increase in cytokine was suppressed by CLI-095, a Toll-like receptor (TLR) 4 inhibitor, PP2, a Src inhibitor, rottlerin, a PKCδ inhibitor, MAP kinase inhibitors, including PD98059, SB202190, and SP600125, and BAY-11-7085, an NF-κB inhibitor. Src, PKCδ, ERK1/2, p38 MAPK, and JNK were phosphorylated by S100A9. The phosphorylation of Src and PKCδ was suppressed by CLI-095, and the activation of ERK1/2, p38 MAPK, and JNK was inhibited by CLI-095, PP2, and rottlerin. S100A9 induced NF-κB activity, and the activation was suppressed by CLI-095, PP2, rottlerin, and MAPK kinase inhibitors. In normal and allergic subjects, supernatant from normal and allergic monocytes after stimulation with S100A9 suppressed normal and allergic neutrophil apoptosis, respectively; MCP-1, IL-6, and IL-8 in the supernatant was increased by S100A9. The cytokine secretion induced by S100A9 is related to TLR4, Src, PKCδ, ERK1/2, p38 MAPK, JNK, and NF-κB. Taken together, S100A9 induces anti-apoptotic effect on normal and allergic neutrophils by increasing cytokine secretion of monocytes. These findings may help us to better understand neutrophil apoptosis regulated by S100A9 and pathogenesis of allergic diseases.

Atopic dermatitis (AD) is an inflammatory skin disorder caused by immunological dysregulation and genetic factors. Whether the expression levels of cytokine and skin barrier protein were altered by S100 calcium binding protein A8 (S100A8) and S100A9 in human keratinocytic HaCaT cells was examined in the present study. Alterations of cytokine expression were examined by ELISA following treatment with S100A8/9 and various signal protein‑specific inhibitors. Activation of the mitogen activated protein kinase (MAPK) pathway and nuclear factor (NF)‑κB was evaluated by using western blotting and an NF‑κB activity test, respectively. The expression levels of interleukin (IL)‑6, IL‑8 and monocyte chemoattractant protein‑1 increased following treatment with S100A8 and S100A9, and the increase was significantly blocked by specific signaling pathway inhibitors, including toll‑like receptor 4 inhibitor (TLR4i), rottlerin, PD98059, SB203580 and BAY‑11‑7085. Extracellular signal‑regulated kinase (ERK) and p38 MAPK pathways were activated in a time‑dependent manner following treatment with S100A8 and S100A9. Phosphorylation of ERK and p38 MAPK were blocked by TLR4i and rottlerin. S100A8 and S100A9 induced translocation of NF‑κB in a time‑dependent manner, and the activation of NF‑κB was inhibited by TLR4i, rottlerin, PD98059 and SB203580. In addition, S100A8 and S100A9 decreased the expression of skin barrier proteins, filaggrin and loricrin. These results may help to elucidate the pathogenic mechanisms of AD and develop clinical strategies for controlling AD.

House dust mite (HDM) is an essential allergen in allergic diseases such as allergic rhinitis and asthma. The pathogenic mechanism of allergy is associated with cytokine release of lymphocytes and constitutive apoptosis of neutrophils. In this study, we examined whether HDM induces cytokine release of lymphocytes and whether the secretion of cytokines is involved in modulation of neutrophil apoptosis. In normal and allergic subjects, extract of Dermatophagoides pteronyssinus (DP) increased IL-6, IL-8, MCP-1, and GM-CSF secretion in a time-dependent manner. This secretion was suppressed by PAR2i, an inhibitor of PAR2, in a dose-dependent manner, as well as by LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, and BAY-11-7085, and an inhibitor of NF-κB. DP induced ERK and NF-κB activation in a time-dependent manner. ERK activation was suppressed by PAR2i, LY294002, and AKTi, and NF-κB activation was blocked by PAR2i, LY294002, AKTi, and PD98059. Supernatants collected from normal and allergic neutrophils after DP treatment inhibited the apoptosis of normal and allergic neutrophils through suppression of caspase 9 and caspase 3 cleavage. DP inhibited neutrophil apoptosis in coculture of normal neutrophils with normal lymphocytes, similar to the anti-apoptotic effects of DP on neutrophils alone. DP more strongly inhibited apoptosis of allergic neutrophils cocultured with allergic lymphocytes than allergic neutrophils without lymphocytes. In summary, DP induces the release of cytokines through the PAR2/PI3K/Akt/ERK/NF-κB pathway, which has anti-apoptotic effects on neutrophils of normal and allergic subjects. These results will facilitate elucidation of the pathogenic mechanism of allergic diseases.