| Objective:Metal nickel has been widely used for industry manufacturing and daily routine life,it is also a common root sources causing allergic contact dermatitis.It could persist for extended period of time or even life-long once nickel allergy as occurred.The most effective treatment for nickel allergy to date is to relieve the symptoms and prevent it from further deteriorating but not eliminating.As such the investigation of nickel allergy pathogenesis and the indicative biomarker of early allergy symptoms becomes in dispensable important.In this study,we focus on the changes of lymphocyte subsets in peripheral blood and serum cytokines in nickel exposed workers to explore the early effective indicatiors for nickel allergy.In further,we establish nickel-sensitized mouse models to research the proliferation and activation of nickel-specific lymphocytes,which play important roles in the process of nickel induced sensitization.Methods:We conducted a cross-sectional molecular epidemiology study of 70 nickel exposed workers and 126 unexposed controls.The exposed workers were devided into patch-negative and patch-positive subgroups by skin patch test with 2.5%and 5.0%nickel sulfate.Air borne samples in workplace were collected and determined by flame atomic absorption spectrometry,the levels of nickel in skin wiping samples collected from exposed workers were measured by inductively coupled plasma mass spectrometry,urinary nickel was measured using dimethylglyoxime spectrophotometry.Peripheral blood samples were collected from each subject for the complete blood cell count,lymphocyte subsets analysis,and serum separation used for cytokines detection.Eight-week-oldfemale C57BL/6 mice were used to establish the nickel-ACD mouse model to determine the effects of nickel exposure on lymphocytes proliferation,activation,and cytotoxic activity of spleen NK cell in allergic mcie.Mice were sensitized by intradermal injection combined with skin exposure on the upper back and challenged on the both lower back and right ear.The model was evaluated by the difference of the bilateral ears weight,the ear swelling,and localized skin histopathological changesat 24 or48 hours after the challenge.Flow cytometry analysis of lymphocyte proliferation was performed by EdU incorporation,the lymphocyte subsetsin peripheral blood and the lymphocytes activation in spleen and lymph nodes were detected by FACS,cytotoxic activity of spleen NK cells were detected by CCK-8 and CFSE/Annectin-V/7-ADD.The Nickel-specific proliferation and activationof lymphocytes were determined by culture of spleen lymphocytes with NiSO4 in vitro for 24 hours.Unadjusted summary measures are presented for all end points.Linear regression was used to test for differences in the serum concentration of cytokines and Lymphocyte subsets between exposed and control workers,as well as among patch-positive,patch-negative,and control groups.Pearson correlations for lymphocyte subsets and nickel exposure,level of urinary nickel,working years were analyzed in all subjects or exposed workers only.For IL-1β,IL-4,IL-6 and TNF-a,which had a large percentage of non-detectable values in the overall study population(>50%),differences in concentrations between exposed workers and control workers were evaluated using Spearman correlations and the Wilcoxon rank sum test,rather than linear regression.In the animal experimental study,t-testw as used to analyze the differences in all end points between control group and nickel treated group.The statistical analyses were performed using SPSS 22.0Results:1.Molecular epidemiological study:The nickel concentration in the workshop was 0.0054mg/m3～0.3757mg/m3,which was lower than the maximum allowable concentration of soluble nickel compounds(0.5mg/m3)specified by ’ Hygienic Standard for Nickel and Its Inorganic Compounds in the WorkshopAir’(GB16210-1996);The level of skin exposure and the urinary nickel in exposed workers was 0.42(0.23,0.60)μg/cm2 and 13.60(7.35,28.76)μg/L,respectively,and the positive correlation between skin exposure concentration and urinary nickel level was observed(r=0.332,P=0.012).The level of urinary nickel in exposure group was significantly higher than that of control group(6.02(3.93,10.97)μg/L,.P=0.000).There were 14 patch-positive workers in the exposed group,andthe positive rate was 20.00%(14/70),suggesting the current hygienic standards for nickel can’t protect the susceptible population from nickel sensitization.The peripheral blood white blood cells,CD4+/CD8+,and neutrophilsin the exposed group were 7.03±1.67×109/L,1.68±0.72,and 4.54±1.45×109/L,respectively,which were significantly higher than those in the control group(6,23± 1.35×109/L,1.27±0.43,3.71±1.07×109/L)(P<0.05).The CD8+T cells,NK cells and platelets in exposed workers were 0.52±0.26×109/L,0.34×0.18×109/L,200.66±69.01×109/L respectively,which were significantly lower than those of control group(0.60±0.22×109/L,0.45±0.25×109/L,229.85±51.48x109/L)(P<0.05).The detection rate of serum IL-4(10%)and the level of serum IL-8(6.70(5.61,9.61)pg/mL)in exposed group was significantly lower than that of the control group[51.6%and 10.45(4.62,40.90)pg/mL,respectively](P<0.001);Serum MIP-1β of exposed group was 51.72(27.43,79.29)pg/mL,which was significantly higher than that of the control group[33.66(17.32,55.97)](P=0.001).The significant correlation between urinary nickel level and leukocytes,CD4+/CD8+,neutrophils,serum IL-8 was observed in all subjects with pearson correlation coefficients ranging from-0.15 to 0.28.We further analyzed the exposure-response relationships for lymphocyte subsets and cytokines across controls and nickel exposed workers,which was devided into lower and higher exposure subgroups based on the median level of urinary nickel in the exposed group.A significant exposure dependent decline in IL-8 and significant increase in leukocytes,CD4+/CD8+,neutrophils with increasing urinary nickel leves was observed across all subjects.There were no correlations between working yearsand serum cytokines or peripheral blood lymphocyte subsets(P>0.05).The level of urinary nickel of patch-positive group was 13.66(7.71,30.04)μg/L,which was similar to patch-negative group[10.82(2.46,31.05)μg/L](P=0.513);The peripheral blood lymphocytes and T cells of the patch-positive group were 1.69±0.42×09/L and 1.12±0.28×09/L,which were significantly lower than those of patch-negative group(2.08±0.67×109/L,1.45±0.51×109/L)and of control group(2.10±0.51×109/L,1.41±0.38×109/L)(P<0.05).The level of serum MCP-1 of the patch-positive group was 116.49(25.04,215.53)pg/mL,which was significantly higher than thatof patch-negative group[57.03(37.43,85.85)pg/mL](P=0.002)and of control group[58.07(47.21,73.25)pg/mL](P=0.008).While those 3 indicators were similar between patch-negative sgroup and the control group,it indicated that those 3 indicators might be used as the early effect indicators for screening nickel allergy.2.Animal experimental study:The difference of the bilateral ears weight at the 48 hours after the challenge,the ear swelling at 24 hours and 48 hours after the challenge of the nickel-sensitized mice were 5.00±3.81 mg,0.05±0.04 mm and 0.06±0.03 mm,respectively,which were significantly higher than those of the control mice(0.17±1.47mg,0.00±0.01mm,0.01±0.01mm,P<0.05).There was capillaries congestion,reddish skin in challenged ears of nickel-sensitized mice,while no megascopic changes in the back skin were observed.Obvious lymphocytes infiltration presented in ear and dorsal skin of nickel-sensitized mice,indicated that the immune reaction to nickel was presented in treated mice.The peripheral blood leukocytes and B cells of nickel-sensitized mice were 4.62±1.0×109/L and 0.14±0.04×109/L,respectively,which were significantly lower than those of the control mice(6.10±0.93×109/L and 0.19±0.05×109/L)(P<0.05);The proliferation rate of spleen T cells and CD8S^ cells innickel-sensitized mice were(1.51 ±0.26)%and(1.16±0.22)%,respectively,which were significantly higher than thoseof control mice[(1.16±0.22)%,(0.42±0.11)%,P<0.05],suggesting that CD8+T cells might involve in the process of nickel-induced allergic contact dermatitis(ACD).CD69 expression of spleen lymphocytes in nickel-sensitized mice(0.80±0.24)%was significantly lower than that of control mice[(1.58±0.32)%,=0.001],and there was no significant difference in CD44 expression between two groups;However,CD44 expression of lymphocytes in lymph node of the nickel-sensitized mice(70.95±3.31)%was significantly higher than that of the control mice[(60.23±4.96)%,P=0.001],suggesting that the lymphocytes in lymph nodeof nickel-sensitized mice had been activated by nickel.There was no significant differencein cytotoxic activity of spleen NK cellsbetween nickel-sensitized miceand control mice(P>0.05).For control mice,the total numbers of CD8+ T cells and NK cells were significantly declined after co-cultured with 15ug/mL NiSO4 for 24h,suggesting the cytotoxicity of nickel.For nickel-sensitized mice,the total numbers of CD4+ T cells and T cells were significantly increased after co-cultured with 15ug/mL NiSO4 for 24h,suggesting the proliferation of nickel-specific CD4+ T cells and T cells,however,the significant increase of CD8+ T cells was not found,suggesting that the cytotoxicity of nickel to CD8+ T cells was stronger than proliferation of nickel-specific CD8+ T cells.Conclusions:1.Lower level of nickel exposure could alter lymphocyte subsets and cytokine expressions in peripheral blood,and leukocyte,CD4+/CD8+,neutrophilsand the serum IL-8 were more sensitive to nickel exposure.2.Lymphocytes and T cells in peripheral blood and serum MCP-1 might possibly be used as early effective markers for screening of nickel allergy.3.Both CD4+ T cells and CD8+ T cells could participate in the development of nickel-induced ACD,while nickel had obvious toxic effect on CD8+ T cells. |
