Na Rae Lee

House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4.

House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.

Regulation of Constitutive Neutrophil Apoptosis Due to House Dust Mite Allergen in Normal and Allergic Rhinitis Subjects

House dust mite (HDM) is a primary allergen in allergic rhinitis (AR) and asthma. Neutrophil apoptosis is associated with allergic diseases and innate immunity to infection. The present study examined how HDM affects constitutive neutrophil apoptosis in normal and AR subjects. Total IgE increased in AR subjects when compared to normal subjects, and patients with AR were HDM-specific IgE positive (+), which is specific IgE to Dermatophagoides pteronissinus and Dermatophagoides farinae. In normal and AR subjects, neutrophil apoptosis was inhibited by extract of Dermatophagoides pteronissinus (DP), but not by extract of Dermatophagoides farina (DF). Aprotinin (serine protease inhibitor) and E64 (cysteine protease inhibitor) have no effect on neutrophil apoptosis due to DP. The anti-apoptotic effect of DP was blocked by TLR4i, an inhibitor of TLR4, rottlerin, an inhibitor of PKCδ, PD98059, an inhibitor of ERK, and BAY-11-7085, an inhibitor of NF-κB. DP induced PKCδ, ERK, and NF-κB activation in a time-dependent manner. DP inhibited the cleavage of procaspase 3 and procaspase 9. The expression of IL-6, IL-8, TNF-α, G-CSF, GM-CSF, and CCL2 increased in the supernatant collected from the normal and AR neutrophils after DP treatment and the supernatant inhibited the apoptosis of normal and AR neutrophils. In summary, DP has anti-apoptotic effects on neutrophils of normal and AR subjects through the TLR4/PKCδ/ERK/NF-κB pathway, and this finding may contribute to solution of the pathogenic mechanism of allergic diseases triggered by DP.

House dust mite allergen suppresses neutrophil apoptosis by cytokine release via PAR2 in normal and allergic lymphocytes

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.

Induction of the Neutrophil Migration in Normal Subjects due to Asthmatic Bronchoalveolar Lavage Fluid (BALF)

Human neutrophils play an essential role in the innate immune response and are involved in the pathogenesis of the severe and corticosteroid-resistant asthma. Asthma is characterized by an infiltration of inflammatory cells into the lung and by a cytokine release. The aim of this study is to investigate the effects of a bronchoalveolar lavage fluid (BALF) on the chemotaxis and apoptosis of neutrophils which were isolated from healthy subjects. The BALF of subjects with asthma induces the blood neutrophil chemotaxis in the opposite of that in normal subjects. The IL-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1) levels in BALF were higher in subjects with asthma than in normal subjects. The BALF of normal and asthmatic subjects has no effect on neutrophil apoptosis of BALF. MCP-1 delays the constitutive apoptosis of normal blood neutrophils, but has no effect in normal BALF neutrophils. These results may indicate that inflammatory factors secreted by the lung tissue of patients with asthma trigger the neutrophil chemotaxis and also induce the neutrophil dysregulation.

Cytokine secreted by S100A9 via TLR4 in monocytes delays neutrophil apoptosis by inhibition of caspase 9/3 pathway

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.

Different anti-apoptotic effects of house dust mite allergen on eosinophil apoptosis between atopic and non-atopic asthmatic subjects

House dust mite is a major allergen in allergic diseases such as asthma. In this study, we investigated the effects of house dust mite on constitutive eosinophil apoptosis in normal and asthmatic subjects. We classified asthmatic subjects into those with atopic and non-atopic asthma depending on the presence of DP-specific IgE or/and DF-specific IgE in serum. Both blood and bronchoalveolar lavage fluid (BALF) eosinophils in atopic asthma were elevated when compared with normal and non-atopic eosinophils. Dermatophagoides pteronissinus extract (DP) inhibited constitutive eosinophil apoptosis of atopic asthmatic subjects, but not that of normal and non-atopic subjects. DF had no effect on eosinophil apoptosis in normal and asthmatic subjects. The anti-apoptotic effects of DP were not altered by E64, a cysteine protease inhibitor, or aprotinin, a serine protease inhibitor, and Der p1 and Der p 2 had no effect on eosinphil apoptosis of normal and asthmatic patients, including atopic and not-atopic patients. Anti-apoptotic signaling mediated by DP in atopic asthma was associated with the TLR4/PI3K/Akt/ERK/NF-κB pathway. Activation of procaspase 3 and procaspase 9 was delayed by DP stimulation. Our results indicate that DP induces eosinophilic inflammation by inhibiting eosinophil apoptosis, and that the inhibitory effect is associated with exposure of asthma subjects to DP. A better understanding of the difference between atopic and non-atopic asthma will help elucidate the pathogenesis and develop methods for treatment of asthma.

Effect of house dust mite on neutrophil apoptosis by cytokine secretion in lymphocytes

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.

Inhibitory effect of arazyme on the development of atopic dermatitis-like lesions in BALB/c and Nc/Nga mice

Arazyme is a metalloprotease released by Aranicola proteolyticus that was shown to inhibit cytokine release in HaCaT and endothelial cells. However, the regulatory effects of arazyme in atopic dermatitis remain to be fully understood. In the present study, the anti‑inflammatory effects of arazyme in BALB/c and Nc/Nga mice induced with 2,4‑dinitrochlrobenzene (DNCB) were investigated. BALB/c mice were sensitized with DNCB and were subsequently administered arazyme for 4 weeks either orally, dorsally or orally/dorsally. Arazyme administration significantly reduced epidermal thickening and infiltration of inflammatory cells into the dermis compared with the DNCB group. However, serum immunoglobulin E (IgE) levels were not altered by arazyme treatment. Additionally, the level of secretion of interleukins (IL)‑4, ‑5 and ‑13 in the splenocytes of BALB/c mice was elevated following stimulation with concanavalin A, while the increase of IL‑4 and IL‑13 was inhibited by arazyme. Administration of arazyme (25 mg/kg in phosphate‑buffered saline) to Nc/Nga mice that had been sensitized with DNCB for 6 weeks reduced the skin severity score compared with that in the DNCB group and inhibited the histological manifestations of atopic dermatitis‑like skin lesions. In addition, the serum IgE levels were reduced in the arazyme‑treated NC/Nga mice relative to the DNCB group. Collectively, these results indicated that arazyme attenuates the development of atopic dermatitis‑like lesions via lowering the levels of IgE and inflammatory cytokines. The results of the present study will aid in the development of effective therapeutic strategies for the treatment of allergic diseases, including atopic dermatitis.

Protective effect of a novel herbmedicine, Hepad, on apoptosis of SH-SY5Y cells and a rat model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. In this study, we investigated the effects of a novel herb formula, Hepad, on PD. Dose-dependent treatment with 1-methyl-4-phenylpyridinium (MPP+) decreased the viability of SH-SY5Y cells, and Hepad inhibited the toxic effect of MPP+. Hepad blocked the production of reactive oxygen species (ROS) induced by MPP+ in SH-SY5Y cells, and suppressed the activation of caspase 9 and caspase 3 due to MPP+. A rat model of PD was generated by 6-hydroxydopamine (6-OHDA) injection into the left medial forebrain bundle (MFB) of SD rats. In D-amphetamine sulfate-induced rotational behavioral tests, Hepad administration attenuated circling behavior relative to the 6-OHDA-treated disease group. In addition, Hepad treatment significantly increased the tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta (SNpc) that had decreased in response to 6-OHDA treatment (P<0.05). OX-6 expression, which indicates the presence of microglial cells, decreased significantly after treatment of Hepad in contrast to the 6-OHDA-treated disease group (P<0.05). These results indicate that Hepad may be a useful neuroprotective material for the treatment of neurodegenerative disorders such as PD.

Chemotactic effect of S100A8 and S100A9 on human eosinophilic leukemia cells, EoL-1 through TLR4

BackgroundsS100A8 and S100A9 function as key factors in inflammatory responses including cell survival, differentiation and chemotactic activity. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy with eosinophilia.MethodsIn this study, we investigated the contribution of S100A8 and S100A9 to chemotaxis of the human eosinophilic leukemia cell line, EoL-1. A chemotaxis assay, western blot analysis and a NF-κB transcription factor assay were conducted to investigate the chemotactic mechanism.ResultsS100A8 and S100A9 induced the migration of EoL-1 cells via the phosphorylation of PKCδ, AKT, and MAPKs and the translocation of NF-κB; however, they had no effect on eosinophils from normal peripheral blood. PKCδ, AKT, and MAPKs such as ERK, p38 MAPK, and JNK are upstream regulator molecules of NF-κB activation. Iκ-Bα degradation is needed for NF-κB activation.ConclusionThese findings suggest that S100A8 and S100A9 induce the cell movement and contribute to an understanding of S100A8 and S100A9 in eosinophil biology and the pathogenic mechanism of hematological malignancy.