Effect Of Four Types Chemical Allergens On Cytokine Expression

Objectives To investigate change of multiple cytokines of THP-1derived macrophagesfollowing four types chemical allergens exposure in order to establish the cytokinesperum of sensitization reaction. Meanwhile, specific cytokine expression will bevalidated in animal experiments to identify the sensitive markers of the skin sensitization.Our data will provide base and reference for the establishing in vitro methods for skinsensitization.Methods1The human monocytic cell line THP-1was induced by phorbol ester (PMA,0.1μg/ml) for72hours and make it transform into THP-1macrophages with adherentcapabilities. Four kinds of chemical sensitizers including nickel sulfate (NiSO4),potassium dichromate (K2Cr2O7),2,4-dinitrochlorobenzene (DNCB), p-phenylenediamine (PPDA), isocyanate (TDI) and two kinds of non-chemical allergens sodiumdodecyl sulfate (SDS), isopropyl alcohol (IPA) were selected as our the test substance,ELISA assay was used to detect IL-6, TNF-α expression level of cell culture supernatant.And the liquid flow multiple protein quantification (CBA) was applied to detectd the IL-1β, IL-8, IL-4, IL-10, INF-γ expression levels of the cell culture supernatant.230BALB/C mice was randomly divided into the control group,0.2%NiSO4and0.5%NiSO4group. Mice in0.2%NiSO4,0.5%NiSO4were treated with NiSO4DMSO solution for3days. Mice in control group were treated with the same amount DMSO. Multi-proteinliquid flow technology (CBA) was applied to expression of IL-1β, IL-6, TNF-α. EDUmethod was used to detect T cell proliferation.Results1When THP-1cells were treated with NiSO4, DNCB, K2Cr2O7for6h andPPDA and TDI for12h, IL-6, TNF-α, IL-1β, IL-8content in cell supernatants werereached to highest levels.2At the6h or12h treatment, IL-6level secreted by THP-1cellexposed to NiSO4, DNCB, K2Cr2O7PPDA and TDI were40,25,20,50,50folds of thatin the control group. Meanwhile, TNF-α levels were20,12,20,8,5folds, IL-1β levelswere30,60,25,30,45fold and15,12,15,12,7folds in terms of IL-8levels.3To ourinteresting, no significant changes were seen in terms of TNF-α, IL-1β, IL-8expressionwhen THP-1drived macrophage cells were treated with SDS or IPA. However IL-6levels were increased.4IL-4, IL-10, INF-γ cytokines were not detected changes.5Mice were exposed to NiSO4for3days, The serum TNF-α levels in0.5%NiSO4group and2%NiSO4groups were7.88pg/ml,18.66pg/ml, higher than that of control group (2.98pg/ml),and the difference was statistically significant (P<0.05). Meanwhile the serum IL-6levelsin were10.94pg/ml and22.55pg/ml, higher than that of control group (4.79pg/ml), andthe difference was markedly significant (P<0.05). However, There was no significantdifference in term of serum IL-1β levels among two NiSO4groups and the control group.The IL-6, TNF-α, IL-1β levels in spleen cell culture supernatant of NiSO4exposuregroups were significantly higher than those that in control group (P<0.05).6Mice wereexposed to NiSO4for3days, T cell proliferation in2%NiSO4group was significantlyhigher than that in control and there was a significant difference between the two groups(P<0.05). No significant different was seen between0.5%NiSO4group and controlgroup.Conclusions Four types of chemical allergens can cause TNF-α, IL-1β, IL-8and othercytokines increase in THP-1cell. Animal test of metal allergens also proved the cellexperiment study. Our data suggested that cytokines expression including TNF-α, IL-1β,IL-8of THP-1macrophages following allergens exposure can serve as biomarker. Thetreatment point might selected6h as cytokine secretion to detect metal allergenicity.