| Natural kiler cell is critical in the defence against viral infections and tumors, the most important role of NK cell is the cytotoxicity. They exert their cytotoxicity through forming'immunological synapse'with their target cells, where they can recognize target cells and release lytic molecules (eg, Granzymes, Perforin etc). Granzyme B enter the target to induce apotosis by the micropore made by perforin. Granzyme B induced cell apotosis is closely related to caspase that cleaves after aspartic acid residues of pro-caspase. The perforin and granzyme in the target cells will be hydrolyzed by protease after exerting their cytotoxic fuction, on the other hand, if NK cell will be destroyed by perforin and granzymes in immunological synapse? The turnover of released lytic molecules are still largely unknown. There are mechanism of cytotoxic lymphocytes to deal with misdirected graznyme B and perforin. For example, serine proteinase inhibitor-9, the specific inhibitor of human granzyme B, could inactivate granzyme B leaked from vesiciles. Moreover, it is well known that NK cell could kill target cells serially, so we can speculate that realeased lytic molecules could be recovered and used again.Human malignant non-Hodgkin's lymphoma NK92 cell was used as a model to study the fate of released lytic molecule granzyme B under target cell stimulation.1. The study of secondary endocytosis of NK92 cell under target cell stimulation.1.1 Observation of secondary endocytosis of NK92 cell by FM1-43FM1-43 dyes reversibly partition into cell membranes. They have almost no fluorescent properties in aqueous solution, but they fluorescence intensely upon membrane binding. FM1-43 has the added advantage of being easily removed with PBS from the surface but not from the insides of cell membrane. FM1-43 prestained NK92 cell were stimulated by PEC for 1h, then endocytosis of NK92 cell were observed under confocal microscope. NK cell labeled with FM1-43 but neither stimulated by PEC nor washed showed fluroscence in cell membrane; NK cell stimulated by PEC and washed with PBS showed flurescence inside the cell, suggested that NK cell exerted membrane internalization after endocytosis.1.2 Membrane internalization under target cell stimulation was clathrin dependentEndocytic pathways are known to at least include both a clathrin-dependent and a caveolin-dependent pathway. NK92 cell were pretreated with CPZ (clathrin specific inhibitor) and labeled with FM1-43 dye, then cell were subjected to confocal microscope to observe fluroscence inside the cell. Our results showed that fluroscence inside NK92 cell was significantly decreased compared with control. It suggests that NK92 cell membrane internalization is calthrin-dependent. At the same time, fluorescence pretreated with CPZ observed by flow cytometry was 48% of control. NK92 cell were labeled with FM1-43 and separated to two groups: cells were stimulated by PEC cell at 4℃or 37℃, data showed that cells at 4℃cannot exert membrane internalization. It suggests that endocytosis of NK92 cell were dependent temperature.2. Endocytosis stimulated by target cell was associated with the recovery of granzyme B2.1 Recovered granzyme B is sorted to early endosomeNK92 cells were pretreated with CPZ and stimulated with target cells, then cells were harvested at 15min,30min and viewed under confocal microscope to observe colocalizaton of EEA-1 and GzmB. Our results showed that without PEC stimulation, an initial endosomal marker EEA-1 and granzyme B were localized in different compartments of NK cells. After 15 min of stimulation with PEC, most of the EEA-1 had colocalized with some of the granzyme B in NK cells. At 30 min, colocalization of EEA-1 and granzyme B became less prominent than before at 15 min.Further, the clathrin heavy chain targeting siRNA was transfected to NK92 using nucleofection. Transfected cell were viewed under confocal microscope to observe The clathrin heavy chain targeting siRNA, CHC siRNA treatment obtained similar results with CPZ treatment.2.2 Recovered granzyme B was sorted to LAMP-1-positive lysosomeClassical clathrin-dependent endocytosis pathway includes form early endosome, late endosome and lysosome. NK92 cell stimulated by target cell for 60min, cells were harvested, viewed under confocal microscope to observe colocalization of GzmB and lamp-1. Our results showed that colocalization was significant decreased treated with CPZ or CHC siRNA.Proteins sorted for degeneration will be cleaved by lysosomal enzymes and cannot be detected by lysosome surface maker co-localization assay, so the observed colocalization of granzyme B and LAMP-1 means the granzyme B was not recovered for degeneration. The colocalization was not increased treated by lysosomal inhibitor leupepetin, it would appear reasonable to suggest that most of, if not all, the recovered granzyme B was sorted to LAMP-1+ lysosomes, re-cycled, and used again.3. Inhibition of clathrin-dependent endocytosis attenuated the cytotoxicity of NK92 cells.The cytotoxicity of NK92 cell against K562 at different E/T ratio (2.5:1,5:1,10:1) was analysed by FCM. Results showed that cytotoxicity of NK92 cell pretreated with CPZ decreased compared with control (16.93% vs 1.45% ,20.44% vs 1.69%,30.22% vs 2.79%).Similar results obtained from NK92 cell transfected with CHC siRNA , Results showed that cytotoxicity of NK92 cell pretreated with CHC siRNA decreased compared with control(23.18% vs 10.63% ,25.31% vs 13.02%,38.69% vs 15.51%)。4. Quantity of granzyme B in the supernatants and NK92 cellsTo examine the effect of CPZ on exocytosis of NK92 cell, at the indicated time(1h, 3h, 5h) of NK92 cell incubated with K562, supernatants were collected for granzyme B measurement. At the same time, NK92 cell were collected and lysed for western-blot. Granzyme B in the supernatant of NK92 and incubated PEC cells increased in the CPZ pre-treatment group as time went on. The quantity of granzyme B had risen slightly at the 1h and 5h junctions compared with control (1.8 ng/ml vs 2.0 ng/ml,2.45 ng/ml vs 2.8 ng/ml), but decreased compared at 3h with control (2.7 ng/ml vs 2.25 ng/ml). In contrast, CPZ pre-treatment caused a decrease of intracellular granzyme B.5. Conclusion1. NK92 cell could undergo clathrin-dependent endocytosis under target cell stimulation.2. Endocytosis stimulated by target cell was associated with the recovery of granzyme B.3. Granzyme B is recovered into early endosome,late endosome and lamp1+ lysosome to be used again.4. Inhibition of clathrin-dependent endocytosis attenuated the cytotoxicity of NK92 cell. Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that is both clinically and pathologically heterogeneous. Mechanistic studies of the complex pathogenesis of MS have relied extensively on animal models of CNS demyelination. The most commonly used animal model for MS is EAE. EAE is induced in multiple species either by immunization of animals with myelin antigens in adjuvant or by adoptive transfer of activated myelin-specific T cells into animals.Interleukin (IL)-33 is a recently described member of the IL-1 family that also includes IL-1βand IL-18. Like IL-1 and IL-18, IL-33 was found to have strong immunomodulatory functions. However, unlike IL-1 and IL-18, which mainly promote T helper (Th) type 1 responses, IL-33 has been shown to induce the production of Th2 cytokines (IL-5 and IL-13) . IL-33 was identified as the ligand for the orphan receptor ST2 (also known as IL-1RL1). The ST2 gene encodes two isoforms of ST2 protein: ST2L, a membrane-bound form; and soluble ST2 (sST2). IL-33 mRNA is expressed by multiple organs and cells types in human and mice. IL-33 is expressed by fibroblasts, epithelial cells, endothelials, activated macrophages, particularly in high endothelial venules. IL-33 appears to play an important role in several inflammatory disorders, including asthma, rheumatoid arthritis and anaphylactic shock.IL-33 is highly expressed in CNS, so in this study, we examine the expression and sourse of IL-33 in normal mice and mice with EAE, to further investigate the contribution of IL-33 to the induction and augmentation of EAE.1. Expression of IL-33 in the CNS of mice1.1 Expression of IL-33 in the CNS of normal miceWe investigated the expression of IL-33 in various tissues (including spinal cord, brain, skin, lung, kidney, heart, spleen, liver) of normal C57BL/6 mice by PCR and western-blot. IL-33 mRNA transcripts of the central nerve system tissues (including spinal cord and brain) were more abundant compared with liver and spleen (12.5-fold increase, P<0.01). Immunofluroscence demonstrates that IL-33 is also highly expressed in gray matter of spinal cord and colocolize with nucleus. Immunohistochemistry also demonstrates that IL-33 is expressed in nucleus.1.2 The cell sourse of IL-33 in the spinal cordThe distributions of IL-33 in the spinal cord were examined by double immunostaining of spinal cord frozen sections with specific antibodies to glial fibrillary acidic protein (GFAP), a marker for astrocytes; CD68, a marker for microglia; neuron specific protein, a marker for neurons. Results showed that IL-33 did not colocolize with markers of glial cells and neurons.1.3 The expression of IL-33 in the mice of EAEWe investigated the expression of IL-33 in the spinal cord of mice of EAE by PCR, western-blot and immunohistochemistry. Cytoplasmic and nuclear proteins were prepared from tissues of spinal cords of normal mice and mice with EAE. IL-33 expression is decreased in mice with EAE compared with normal mice. Nuclear fractionation experiment demonstrated that IL-33 was located in the nucleus and cytoplasm, while under EAE, IL-33 was undetectable in the nuclear fraction. This was further confirmed by immunohistochemistry, as shown in staining of parallel sections showed that IL-33 was expressed in cytoplasm and nuclear in normal mice, but under inflammation in EAE, cells were swelling and IL-33 was mainly expressed in cytoplasm.1.4 The expression of ST2 in the spinal cord of normal mice and EAE miceST2 was expressed in in the white matter of normal, but in the mice of EAE, ST2 was expressed in the gray matter.2. Treatment of EAE mice with rIL-33 and IL-33 polyclonal antibody2.1 The effect of IL-33 in EAE miceWe examined the effect of recombinant IL-33 on the development of EAE. C57BL/6 mice were immunized with MOG35-55/CFA and were injected i.p. prior of immunization or daily with IL-33 (1μg per mouse) for 7 days from onset of the disease. Mice treated with IL-33 did not change the clinical score of the disease.2.2 Treatment of IL-33 polyclonal antibody in EAE miceMice received a neutralizing polyclonal antibody against murine IL-33. Anti-IL-33 antibody was given i.p. 150μg per mouse every three days prior of immunization or from onset of disease. As control, mice were given the same amount of rabbit control IgG (purified normal rabbit IgG) by i.p. injection. To our surprise, mice developed significantly more severe disease as assessed by clinical score compared with control. Consistent with these observations, a massive infiltration of CD4+ T cells were observed within the spinal cords of mice treated with anti-IL-33 antibody 21 days after the first immunization.2.3 Development of Th1- and Th17-type responses is enhanced in mice treated with anti-IL-33 antibodyWe harvested spleen cells from WT, IgG and anti-IL-33 antibody treated mice at the peak of the disease. Splenocytes were cultured in the presence of 20μg/ml of MOG35–55 peptide for 72 h. Supernatants of the cultures were harvested to measure T cell cytokine production by ELISA. Wild-type produced none or undetectable amounts of cytokines. Anti-IL-33 antibody treated group showed higher production of and IL-17A, IFN-γand TNF-αcompared with IgG treated group.We next examined the cytokine mRNA expression in the spinal cords of control (IgG treated) mice and anti-IL-33 antibody treated mice with EAE. Spinal cords were harvested at the peak of the disease. Total mRNA was extracted from the tissue in order to measure the cytokine mRNA by real-time RT-PCR analysis. At day 21 after immunization, we found that in the spinal cord of anti-IL-33 antibody treated mice, there was a significant higher expression of IL-17A, IFN-γ, TNF-α, compared with control mice. Anti-IL-33 treatment caused 2.5-fold increase of mRNA for IL-17A, 3.2-fold increase for IFN-γ, 3.8-fold increase for TNF-α.2.4 Anti-IL-33 polyantibody reduced the number of CD11c+CD8α+DC in spleen and draining lymph nodes but did not affect the number of regulatory T cells in spleenWe analyzed the draining lymph nodes and spleen of anti-IL-33 antibody treated mice and found significantly reduced numbers of CD8α+CD11c+ DC by flow cytometry. However, no obvious changes in the frequency of CD4+CD25+Foxp3+ regulatory T cells was observed in spleen treated with anti-IL-33 antibody compared with IgG control. The regulatory T cells of draining lymph nodes were almost undetectable.
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