Although nickel hypersensitivity is known as a delayed-type hypersensitivity mediated by nickel-specific T cells, it is greatly influenced by other immune cells. IFN–secreting cells. The involvement of NK cells in the innate response to NiSO4 was confirmed since we could observe a significant reduction of the frequency of nickel-reactive cells in NK cell-depleted mice. Furthermore, the number of IFN- secreting cells was significantly reduced in the ELISPOT assays when NKG2Deb was blocked by anti-NKG2Deb antibody. These results suggest that there is usually an early and quick innate immune response to nickel, which is usually mediated by NK cells and the NKG2Deb receptor. The significance of the innate response to nickel is usually that it may contribute to development of the late T cell-mediated delayed type hypersensitivity against nickel. nickel activation and ELISPOT assay Wells of MultiScreen-IP dishes (Millipore, Billerica, MA) were coated with 50 l either one of capture rat antibodies dissolved in PBS that were specific for mouse IFN- (100 g/ml), IL-2 (100 g/ml) or IL-4 (100 g/ml). After incubation overnight at 4, unbound antibody was removed by three occasions of washing with PBS. The coated wells were blocked with 1% BSA portion V (Sigma-Aldrich, St. Louis, MO). After 2 h at room heat, the blocking medium was discarded and wells were washed three occasions with PBS. Then 1 106 mouse splenic cells were plated buy 485-49-4 in buy 485-49-4 total RPMI 1640 medium (94% RPMI 1640 + 5% FBS + 1% L-glutamine) within each well and treated with LPS, anti-CD3 antibody, or numerous concentrations of NiSO4. RPMI 1640 was from BioWhittaker (Walkersville, MD); FBS from Gibco-Invitrogen (Carlsbad, CA). After 24 h of incubation at 37 on 5% CO2, wells were washed three occasions with PBS and three occasions with PBS/0.05% Tween-20 to remove cells. To detect secreted cytokines, 50 l of 50 g/ml biotinylated detection antibody against mouse IFN-, IL-2, or IL-4 were added per well. After incubating overnight at 4, the dishes were washed three occasions with PBS/0.05% Tween-20 and then incubated with streptavidin-HRP in PBS/BSA/Tween for 2 h at room temp. The spots were designed by using AEC (Pierce Pharmaceuticals, Denmark) development answer and the reaction was halted by washing dishes with tap water. Spots were counted by using Immunospot S4 Pro Analyzer (Cellular Technology Ltd., Cleveland, Oh yea). All antibodies for ELISPOT were purchased from BD Biosciences (San Jose, CA). Nickel sensitization, NK cell depletion, and circulation cytometric analysis To sensitize mice to nickel, mice were intraperitoneally shot with 300 l of 10 M NiSO4 mixed with 300 l alum (Inject Alum, Pierce). 2 or 4 weeks later after injection, mouse splenocytes were used for the ELISPOT analyses. For depletion of NK cells in other experiments, mice were shot intraperitoneally with 25 g anti-NK1.1 (BioLegend, San Diego, CA) in 300 Rabbit Polyclonal to NDUFB10 t PBS on days 0, 3 and 6. On day 8, mice were sacrificed and spleens were gathered. Depletion of NK cells was confirmed by circulation cytometric analysis. The conditions for the ELISPOT analysis were same as explained above. Anti-NK1.1-biotin, anti-CD49b-biotin, streptavidin-PE and streptavidin-FITC (BD Biosciences) were used for circulation cytometric analyses. To analyze splenocytes, reddish blood cells were lysed by incubation in lysis buffer made up of 17 mM Tris and 140 mM NH4Cl for 5 min at room heat. Cells were washed with PBS, counted and incubated for 30 min at 4 with antibodies and washed three occasions with PBS made up buy 485-49-4 of 2% FBS and 0.05% sodium azide. Data purchase and analysis was carried out on FACSCalibur (BD Biosciences) using CellQuest software. Statistical analysis For statistical analysis, Microsoft Excel 2003 buy 485-49-4 (Microsoft Corporation, Redmond, WA) and SPSS version 14 (SPSS Inc., Chicago, IL) were used. < 0.05 was considered statistically significant for all assessments. Additional post-tests for ANOVA were performed only when ANOVA showed significant difference. Acknowledgements This work was supported by a grant from (01-PJ3-PG6-01GN12-0001) from the 2001 Good Health R & Deb Project, Ministry of Health and Welfare, Republic of Korea. K.H. gratefully acknowledges a financial support from the BK 21 Project of the Korean Ministry of Education. Abbreviations CDRcomplementary determining regionELISPOTenzyme-linked immunosorbent spotNK cellnatural monster cellRAGrecombination activating geneSPFspecific pathogen-freeTh1T helper 1.