Research On The Frequency Of Peripheral Blood NKT Cells In Nonalcoholic Fatty Liver Disease

Objective and backgroundNonalcoholic fatty liver disease(NAFLD) is a clinical pathological syndrome characterized by lesion of steatosis and fat storage in hepatic lobules but without alcoholic abuse . It is a common form of liver disease, and the prevalence of the disease increases yearly. Epidemic studies showed that NAFLD occurrence was 17-33% worldwide, while Fan et al reported that it was 15.4% when 3,175 adults were surveyed in Shanghai. NAFLD represents a spectrum of disease ranging from simple steatosis through nonalcoholic steatohepatitis (NASH) to fibrosis and cirrhosis.NAFLD is commonly accompanied by obesity, hyperlipermia and type Ⅱ diabetes and its essence is metabolic syndrome manifested in liver.The pathogenic mechanisms by which NAFLD develops remain unclear but NAFLD might be related to insulin resistance, oxidative stress, cytokines, or genetic factor. The center point of the pathogenic mechanisms of NAFLD is involved in insulin resistance (IR) and hyperlipodieresis. Two basic metabolic deficiencies, hyperinsulinimia and high free fatty acids, which develop at meanwhile, might cause glucose and lipid toxicity, which subsequently damage the liver cells and induce systemic metabolic stress, leading to metabolic disorder.Further studies showed that the innate immune system played a role in the incidence of NAFLD, and the nature killer T (NKT) cells were extensively involved in NAFLD. NKTcells are a subgroup of T cell family. They have been identified as a component of the innate immune defense recently. NKT cells are characterized by an expression of both the nature killer cell marker and the invariant T cell receptor, TCR α/β (Vα 24 / Vβ11 in human beings, Vα 14/Vβ8 in mice), and are activated by recognizing and binding of their TCR α/β to CD1d molecules on antigen-presenting cells, releasing cytokines such as INF-γ (interferon-γ) and IL-4 (interleukin-4). NKT cells play critical roles in anti-infection, anti-tumorigenesis, transplanting immune response, and auto-immune diseases by regulating a balance between Th1 cytokines (proinflammatory factors) and Th2 cytokines (anti-inflammatory factors). It has been shown that the number of NKT cells decreases in the mouse livers in many animal models of NAFLD. Furthermore, an increase in the number of NKT cells by different methods could reduce efficiently the level of lipids in the mouse livers in animal models of NAFLD. However, all these results are only based on animal experiments. How NKT cells change in NAFLD patients is still unknown.This study is to explore the roles of NKT cells in the pathogenic mechanisms ofNAFLD through studing the peripheral blood frequency of NKT cells between normaladult and patients.Materials and methods1. Normal adults and NAFLD patientsAll participants (normal adults and patients) were physical examinees from September 2005 to October 2005 in our hospital. 60 NAFLD patients were selected based on NAFLD diagnostic standards established in 2002 by the Fatty Liver and Alcoholic Liver Diseases Group of Chinese Medical Association Liver Branch, and were excluded from the following circumstances: (1) Microbial infection or physical injury within the most recent three months.(2)History of past illness of hepatitis,malignant tumor and autoimmune disease. (3)Using medication to treat hyperlipemia, hyperglycemia, and hypertension within the most recent three months.(4) Age above 65 or under 18. Of 60NAFLD patients, 50 were male patients and 10 were female patients. Among those patients, their ages ranged from 25 to 65, but their mean age was 43.0±10.15. Accordingly, 60 normal adults were selected with the same exclusive criteria, the same sex ratio, and corresponding ages (difference in ages between two corresponding individuals is less than 3 years). The control group (normal adults) was liver and gall disease-free by ultrasonic examination, and did not take alcohol over 40 grams each week. 2.Clinical and experimental design and data collectionHeight, weight, waist and hip circumferences, and blood pressure of both normal adults and NAFLD patients were measured by standard methods, and their body mass index (BMI) and waist-hip ratio (WHR) were then calculated. 10 ml of fasting blood sample was collected under the calm condition in these two control and experimental groups in the morning, and assessed for a proportion of NKT cells, the levels of metabolic enzymes in the liver (ALT, AST, GGT), blood lipids (TG, TC, HDL-C, LDL-C and VLDL-C) and fasting blood sugar (FBS), and diagnosis of hepatitis. 3. Peripheral blood NKT cell assays10 μl FITC-labeled anti-TCRVα24 monoclonal antibody was added to the freshly collected and clbt-resistant EDTA-mediated blood samples (50 μl each in an eppendorf tube), and the blood samples were then incubated at 25 ℃ for 30 min under dark condition. After adding 2 ml red blood cell lysis buffer to those blood samples, mix them very well, and spin down at 1,200 rpm for 5 min. Aspirate supernatant from each sample, wash precipitated cells/pellets with 1 ml of 1X PBS, and then spin down at 1,200 rpm for 5 min once again. Aspirate the supernatant, and repeat washing steps one more time mentioned above. Finally, the cells were resuspended in 1 ml of 1X PBS, and assessed with an instrument. All samples were assessed by using EPICS-XL flow cytometer at a wavelength of 488 nm, and analyzed with X-parameter, FSC (forward scatter), and Y-parameter SSC (side scatter). The lymphocytes in the blood samples were gated on FSC vs. SSC plots, and their FITC intensity was measured. The corresponding IgG1-stained cells were used as negative controls.4. Data analysis with statisticsAll the data collected were analyzed by statistic software, SPSS 13.0. The t-test was used to assess whether the means of two groups (normal adults and NAFLD patients) were statistically different from each other. Sample correlation coefficients were determined by Pearson analysis. Logistic regression analysis was used to assess the potential effects of high risk factors for NAFLD, and their corresponding regression formula was set up. When p value is less than 0.05, change between two groups is significant.Results1 .Comparison of the frequency of peripheral blood NKT cells in normal adults to that in NAFLD patientsWe defined those T cells that are Va24-positive as NKT cells. We found that peripheral blood samples from the normal adults contained a few NKT cells, which constitute 1.62 ±0.55 % of the total lymphocytes while the samples from the NAFLD patients received 1.21 ±0.47 % of the total lymphocytes. Statistical analysis showed that the change of frequency of peripheral blood NKT cells between the normal adults and NAFLD patients was significant (t = 4.275, p< 0.001). 2. Comparison of clinical measurements in normal adults to those in NAFLD patientsIn the NAFLD patients, BMI (body mass index), WC (waist circumference), HC (hip circumference), WHR (waist-hip ratio), and MAP (mean arterial pressure) were 26.9±2.7 kg/m2, 92.0±8.3 cm, 100.3 ±5.4 cm, 0.92±0.05, 96.9±10.6 mmHg, respectively. Moreover, the levels of ALT (alanine aminotransferase), AST (Aspartate aminotransferase), GGT (gamma glutamyl transferase), TG (triglycerides), TC (total cholesterol), and FBS (fasting blood sugar) in those patients were 46.8±28.2 U/L, 36.0 ± 21.2 U/L, 38.9 ± 21.0 U/L, 2.64±1.12 mmol/L, 4.99±0.91 mmol/L, and 5.70±2.43mmol/L, respectively. However, in the normal adults, BMI, WC, HC, WHR and MAP were 22.4 ± 2.2 kg/m2, 80.2 ± 6.0 cm, 93.7±3.9 cm, 0.86±0.04, 88.9±12.6mmHg, respectively, and the levels of ALT, AST, GGT, TG, TC, and FBS were 21.5 ± 13.6 U/L, 24.5 ± 8.3 U/L, 26.3 ± 22.8 U/L, 1.71 ± 1.09 mmol/L, 4.58 ± 0.85 mmol/L, and 4.39 ± 0.48 mmol/L, respectively. Statistic analysis showed that those clinical measurements significantly changed between the normal adults and NAFLD patients (p<0.01).In addition, in the NAFLD patients, the mean age, height, and levels of HDL-C (high-density lipoprotein-cholesterol) and LDL-C (low density lipoprotein-cholesterol) were 43.0±10.15 years, 167.3 ± 7.1 cm, and 1.28 ± 0.25 mmol/L and 2.73 ± 0.76 mmol/L, respectively while they were 43.0 ± 9.7 years, 166.7 ± 6.3 cm, and 1.26 ± 0.21 mmol/L and 2.53 ± 0.74 mmol/L, respectively, in the normal adults. Statistic analysis suggested that those clinical measurements did not significantly change between the normal adults and NAFLD patients (p>0.05).3. Relationship of the frequency of peripheral blood NKT cells with clinical measurements. Pearson analysis indicated that BMI, WC and ALT level in the NAFLD patients werecorrelated negatively with the frequency of peripheral blood NKT cells. Their correlation coefficients were -0.283, -0.251, and -0.258, respectively. The corresponding p values were 0.002, 0.006, and 0.004 , respectively.4. Analysis of NAFLD high risk factorsThe significant clinical variables were selected for regression analysis. Except two clinical variables, weight and waist circumference (WC), all the other 12 clinical variables were defined as active variables, and whether NAFLD occurs or not was accordingly defined as a passive variable (the control group of normal adults was given a value of zero while the group of NAFLD patients was given a value of 1). The four most significant clinical variables and one constant were determined by using Logistic regression analysis. As a result, a regression formula, P = 1 / (1 + e 41.037+2.527NKT-1.372BMI-0.155AST.1.237FBS), was established on the basis of those four clinicalvariables and the constant. All the p values of those four selected clinical variables when tested with t-test were less than 0.05. Therefore, a decrease in the frequency of peripheral blood NKT cells, and an increase in BMI, AST and FBS levels were suggested to be thehigh risk factors for NAFLD.Conclusions1.Lower frequency of peripheral blood NKT cells was observed in NAFLD patients. 2.NKT cells might play an important role in the occurrence and development of NAFLD. 3.Decreased frequency of peripheral blood NKT cells might be one of the high risk factors for the incidence of NAFLD.