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Article in English | WPRIM | ID: wpr-167181


Autophagy has been investigated for its involvement in inflammatory diseases, but its role in asthma has been little studied. This study aimed to explore the possible role of autophagy and its therapeutic potential in severe allergic asthma. BALB/c mice were sensitized with ovalbumin (OVA) on days 0 and 14, followed by primary OVA challenge on days 28–30. The mice received a secondary 1 or 2% OVA challenge on days 44–46. After the final OVA challenge, the mice were assessed for airway responsiveness (AHR), cell composition and cytokine levels in bronchoalveolar lavage fluid (BALF). LC3 expression in lung tissue was measured by western blot and immunofluorescence staining. Autophagosomes were detected by electron microscopy. 3-Methyladenine (3-MA) treatment and Atg5 knockdown were applied to investigate the potential role of autophagy in allergic asthma mice. AHR, inflammation in BALF and LC3 expression in lung tissue were significantly increased in the 2% OVA-challenged mice compared with the 1% OVA-challenged mice (P<0.05). In addition, eosinophils showed prominent formation of autophagosomes and increased LC3 expression compared with other inflammatory cells in BALF and lung tissue. After autophagy was inhibited by 3-MA and Atg5 shRNA treatment, AHR, eosinophilia, interleukin (IL)-5 levels in BALF and histological inflammatory findings were much improved. Finally, treatment with an anti-IL-5 antibody considerably reduced LC3 II expression in lung homogenates. Our findings suggest that autophagy is closely correlated with the severity of asthma through eosinophilic inflammation, and its modulation may provide novel therapeutic approaches for severe allergic asthma.

Animals , Asthma , Autophagy , Blotting, Western , Bronchoalveolar Lavage Fluid , Eosinophilia , Eosinophils , Fluorescent Antibody Technique , Inflammation , Interleukins , Lung , Mice , Microscopy, Electron , Ovalbumin , Ovum , RNA, Small Interfering
Article in English | WPRIM | ID: wpr-126956


PURPOSE: Radiation-induced autophagy has been shown to play two different roles, in malignant glioma (MG) cells, cytocidal or cytoprotective. However, neither the role of radiation-induced autophagy for cell death nor the existence of autophagy-induced apoptosis, a well-known cell-death pathway after irradiation, has been verified yet. MATERIALS AND METHODS: We observed both temporal and dose-dependent response patterns of autophagy and apoptosis to radiation in MG cell lines. Additionally, we investigated the role of autophagy in apoptosis through knockdown of autophagy-related proteins. RESULTS: Autophagic activity measured by staining of acidic vesicle organelles and Western blotting of LC-3 protein increased in proportion to radiation dose from day 1 to 5 after irradiation. Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose) polymerase demonstrated relatively late appearance 3 days after irradiation that increased for up to 7 days. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosis after irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, which decreased apoptosis significantly. Inhibition of autophagy in Atg5 knockdown cells was shown to be beneficial for cell survival. Stable transfection of GFP-LC3 cells was observed after irradiation. Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicating autophagy in immunofluorescence. Some of these punctuated GFP-LC3 bearing cells formed conglomerated spots and died in final phase. CONCLUSION: These findings suggest that autophagy appears earlier than apoptosis after irradiation and that a portion of the apoptotic population that appears later is autophagy-dependent. Thus, autophagy is a pathway to cell death after irradiation of MG cells.

Apoptosis , Autophagy , Blotting, Western , Cell Death , Cell Line , Cell Survival , Fluorescent Antibody Technique , Glioma , Organelles , Poly(ADP-ribose) Polymerases , Transfection
Article in English | WPRIM | ID: wpr-162251


In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5Kalpha) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kalpha is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kalpha mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kalpha knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1beta and TNFalpha. PIP5Kalpha knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-kappaB and the degradation of IkappaB-alpha, indicating that PIP5Kalpha knockdown interfered with the ganglioside-activated NF-kappaB signaling. Together, these results suggest that PIP5Kalpha is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-kappaB activation in ganglioside-stimulated astrocytes.

Animals , Astrocytes/metabolism , Cells, Cultured , Gangliosides/metabolism , Gene Knockdown Techniques , Inflammation/metabolism , Interleukin-1/metabolism , NF-kappa B/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation
Immune Network ; : 67-75, 2006.
Article in English | WPRIM | ID: wpr-79622


BACKGROUND: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. METHODS: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. RESULTS: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T cell lysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. CONCLUSION: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.

Cell Proliferation , Cytoplasm , Killer Cells, Natural , Phosphotyrosine , Protein Tyrosine Phosphatases , T-Lymphocytes , Transfection
Article in Korean | WPRIM | ID: wpr-86131


In order to develop a method for the detection of minimal residual leukemic disease (MRD) in T cell acute lymphocytic leukemia (T cell ALL), T cell leukemia cell line was used to detect clonal TCR p chain mRNA and to synthesize CDR3 specific oligonucleotide probe. For the Jurkat cell line clonal TCR p chain cDNA was amplified by using RT-PCR with oligonucleotide primer, Vp universal primer. As the result of RT-PCR an approximate 300 bp fragment of the TCR chain was obtained, and the partial identification of the TCR p chain gene and the amino acid sequence of the fragment were done by gene cloning and sequencing. The gene sequence of TCR p obtained was identified as Vp8-Jp1.2-Cp2. Diversity gene segment could not be found. Within the p chain, the CDR3 region was identified as 12 amino acids (SFSTCSANYGYT). It is kown that TCR is expressed in about 40% of the all T cell ALL. However it is not kown what percentage of the TCR p chain mRNA expression translates into the actual TCR molecule. It is not certain how many patients with MRD can be detected by this method used in this study, but this technique might be useful to detect MRD in at least 40% of the patients with T-cell ALL.

Amino Acid Sequence , Amino Acids , Cell Line , Clone Cells , Cloning, Organism , DNA, Complementary , Humans , Jurkat Cells , Leukemia, T-Cell , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Antigen, T-Cell , RNA, Messenger , T-Lymphocytes