(Arazyme)はサイトカイン発現を抑制し、 (Skin barrier …

1 MOLECULAR MEDICINE REPORTS 8: 2013 (Arazyme) (Skin barrier protein) HaCaT THP-1 (human monocytic cell) EoL-1 (human eosinophilic cell) (Dermatophagoides pteronyssinus) (DpE) MCP-1/CCL2, IL-6 IL-8 THP-1 (LPS) MCP-1, IL-6, IL-8 (mast cell) 12- 13- (phorbol 12-myristate13-acetate) (calcium ionophores) IL-6 IL-8 HaCaT tumor necrosis factor- (TNF- ) interferon- (INF- ) thymus and activation-regulated chemokine (TARC)/CCL17, MCP-1, IL-6 IL-8 TNF- INF- (filaggrin) , (involucrin) (loricrin) (filaggrin) , (involucrin) (loricrin) (atopic dermatitis) , Dermatophagoides pteronissinus (DpE) , immunoglobulin E (IgE) (exploits defect) Interleukin(IL)-6, IL-8 monocyte chemotactic protein-1 (MCP-1)/CCL2 (neutrophils)

2 Th2 Thymus and activation-regulated chemokine (TARC)/CCL17 (keratinocytes) (filaggrin), (involucrin) (loricrin) Nephila clavata, aerobic gram-negative symbiotic bacterium ( ) , Serratia proteamaculans Aranicola proteolyticus (metalloprotease) transforming growth factor- (TGF- )/Smad pathway SMP30 (ant-oxidant protein) An in vitro (skin keratinocytes) , Materials and Methods ( )Enzyme purification ( ) m (Pall Life Science, Port Washington, NY, USA) 50mM potassium phosphate ( ) DEAE- 400ml/h 10kD (Pall Life Sciences) 20ml 50mM potassium phosphate ( ) Sephadex G-75 (GE Healthcare Life Sciences, Pittsburgh, PA, USA)

3 10kD -20 C Cell Culture ( ):THP-1 (human monocytic cell) American Type Culture Collection (Manassas, VA, USA) EoL-1 (human eosinophilic cell) Riken Cell Bank (Tsukuba, Japan) 2 RPMI-1640 HMC-1 HaCaT 10% (heat-inactivated fetal bovine serum) , (100 U/ml) (100 g/ml) Iscove Dulbecco s modified Eagle Cell viability assay ( ) (Roche Korea, Seoul, Korea) 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) 100 l THP-1,EoL-1, HMC-1 HaCaT 96-wellplate 1 g 50 g/ml (well) 37 C 24 10 l MTT 4 (100 l) (well)

4 24 ELx808enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek Instruments Inc., Winooski, VT, USA) 550nm ELISA (Enzyme-Linked immunosorbent Assay): 30 THP-1, EoL-1, HMC-1 HaCaT Korea National Arthropods of Medical Impotance Resource Bank (Seoul, Korea) DpE MCP-1, IL-6, IL-8 TARC tumor necrosis factor- (TNF- ) OptEIA Set(BD Biosciences, San Jose, CA, USA) sandwich ELISA Western Blotting ( ) : 5x106 /ml HaCaT 6 (well-plate) TNF- interferon(IFN- ) 50 l lysis buffer ( -20mM HEPES, 400mM Nacl, 1mM EDTA, 1mM EGTA, 25% glycerol, 1mM dithiothreitol, Na3VO4 protease inhibitor) 4 C 15 12,000 x g (50 g/lane) 10% Blots( ) (filaggrin), (involucrin) (loricrin) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Amersham Pharmacia Biotech.)

5 , Piscataway, NJ, USA) ERK2 Statistical analysis ( ) +/- (SD) one-way ANOVA SPSS (version ; SPSS Inc., Chicago, IL, USA) P< Results ( )Arazyme inhibits the secretion of MCP-1 and IL-8 in THP-1 and EoL-1 cells ( THP-1 EoL-1 MCP-1 IL-8 ) THP-1, EoL-1, HMC-1 HaCaT MTT HMC-1 1 g 50 g/ml THP-1 EoL-1 1 g 50 g/ml HaCaT 5 g/ml 10 50 g/ml DpE THP-1 LPS MCP-1, IL-6 IL-8 ( A B) DpE MCP-1 IL-8 (p< ) MCP-1,IL-6 IL-8 LPS ( 2B) EoL DpE MCP-1,IL-6 IL-8 MCP-1 IL-8 ( 3)

6 IL-6 EoL-1 IL-6 THP-1 Figure1: THP-1, EoL-1, HMC-1 HaCaT THP-1, EoL-1, HMC-1 ( A) HaCaT ( B) ( ) 24 MTT-based viability assay ( 100% ) 3 , MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-disphenyltezolium bromide +/- (SD) Figure2: THP-1 DpE( A) or LPS( B) MCP-1, IL-6, IL-8 THP-1 24-well plate (well) RPMI-1640 37 C 24 10 g/ml 24 ELISA 3 +/-SD P< P< vs DpE , DpE vs MCP: monocyte chemotactic protein-1 IL: interleukin DpE: Dermatophagoides ptronissinus Figure3.

7 EoL-1 DpE MCP-1, IL-6 IL-8 EoL-1 24-well RPMI-1640 37 C 24 10 g/ml DpE 24 ELISA 3 +/-SD P< P< vs DpE , DpE vs MCP: monocyte chemotactic-1 IL: interleukin DpE: Dermatophagoides ptronissinus Arazyme inhibits the secretion of IL-6 and IL-8 in HMC-1 cells and the production of TARC, MCP-1, IL-6 and IL-8 in HaCaT cells ( HMC-1 IL-6 IL-8 HaCaT TARC, MCP-1, IL-6 IL-8 )HMC-1 phorbol 12-myristate 13-acetate (PMA) calcium inophore (CaI) IL-6, IL-8 TNF- PMA CaI IL-6 IL-8 (p<.)

8 4) HaCaT TNF- IFN- TARC, MCP-1, IL-6 IL-8 IL-8 TARC, MCP-1 IL-6 1-5 monocyte, eosinophils, (mast cell) keratinocyte Figure4: HMC-1 PMA CaI IL-6 IL-8 HMC-1 24-well RPMI-1640 37 C 24 50ng/ml PMA 1 MCaI 24 ELISA 3 +/-SD P< P< vs PMA+CaI , PMA+CaI vs IL: interleukin DpE: Dermatophagoides ptronissinus PMA: phorbol 12-myristate 13-accetate, CaI: calcium ionophore Figure5: HaCaT TNF- INF- TARC, MCP-1 IL-6 HaCaT 24-well RPMI-1640 37 C 24 10ng/ml TNF- IFN- 24 ELISA 3 +/-SD P< P< vs TNF- +IFN- , TNF- +IFN- vs TNF- : tumor necrosis factor- IFN- : interferon.

9 TARC: thymus and activation-regulated chemokine. MCP-1: monocyte chemotactic protein-1. IL: interleukin. Arazyme increases the expression of filaggrin, involucrin and loricrin in HaCaT cells ( HaCaT filaggrin, involucrin loricrin ) filaggrin, involucrin loricrin , keratinocyte HaCaT TNF- IFN- HaCaT filaggrin, involucrin loricrin ( 6A) HaCaT filaggrin, involucrin loricrin ( 6B) Figure6: TNF- IFN- ilaggrin, involucrin loricrin Serum-starved HaCaT ( A) 10ng/ml TNF- IFN- Serum-starved HaCaT ( B) 5 g/ml 1 10ng/ml TNF- IFN- 24 48 filaggrin, involucrin loricrin western blot analysis ERK TNF- : tumor necrosis factor- IFN- : interferon.

10 Discussion THP-1 EoL-1 MCP-1 IL-6 IL-8 HMC-1 IL-6 IL-8 HaCaT TARC, MCP-1, IL-6 IL-8 HaCaT filaggrin, involucrin loricrin (metalloprotease) CC14 (keratinocyte) (effector cells) THP-1, EoL-1, HMC-1 HaCaT IL-8 THP-1, EoL-1 HaCaT MCP-1 MCP-1 IL-8 , (neutrophil) (neutrophil) HMC-1 HaCaT IL-6 THP-1 EoL-1 IL-6 DpE IL-6 IL-8 (protease)