Effects Of Estrogen On Apoptosis Of Polymorphonuclear Neutrophils In Systemic Lupus Erythematosus

Background:Systemic Lupus Erythematosus （SLE）, an autoimmune disease, affectsmany organs and systems and is high morbidity among reproductive women.There are many studies on the pathogenesis of SLE and most of themconcentrate on the role of lymphocytes. However, the pathological role ofanother large group of cells, neutrophils, is poorly undertood. In recent years,studies found that neutrophil apoptosis was involved in some inflammatorydiseases, such as autoimmune hepatitis and ulcerative colitis. There are fewstudies to explore whether the apoptosis of neutrophils is related to thepathological process of SLE. It has been widely accepted that estrogen andestrogen receptors （ER） play key parts in the pathological process of SLE, butthe functions and mechanisms is unclear. Research in the affect of neutrophilsby estrogen is inadequate.Objectives:To analyse the changes of cell apoptosis in SLE by detecting theapoptosis rate of polymorphonuclear neutrophils（PMNs） from the patientswith SLE （experimental group） and healthy women （the control group）. Toexplore the mechanism of affects by estrogen and ER on PMNs apoptosis inSLE by investigating the expression of proapoptosis gene bax, activation ofapoptosis signal passway and the change of proinflammatory cytokines afterPMNs were treated by estrogen.Methods:1. A total of60SLE patients were investigated and the fasting peripheral venous blood were collected. These samples were treated by EDTAanticoagulant and plasmapheresis was conserved at-80℃. At the same time,PMNs were separated aseptically. Clinical characteristics of the patients werecollected.50healthy women were collected as the control group.2. The apoptosis rate of PMNs was measured by flow cytometry （FCM）.The relationship between the apoptosis of PMNs and inflammatory pathologicprocess of SLE was identified by analyzing the difference between theexperimental group and the control group. The correlation was analyzedbetween apoptosis rate and some clinical indices, such as C3and IgG touncover the relationship between apoptosis of PMNs and SLE diseaseactivity.3. The concentrations of E₂, IFN-α, IL-10,TNF-α and IL-17in sera weremeasured by using enzyme-linked immunosorbent assay （ELISA）respectively. The difference was compared between the SLE patients and thecontrol group. The analysis of the correlation between E₂and cytokinesdemonstrated role of indicators in SLE.4. After PMNs were treated with or without estradiol （E₂） for24h, thecells and supernatant were collected respectively. The supernatant wasconserved at-80℃. The concentration of TNF-α was measured by ELISA.The cells were analysed as follows:（1） The apoptosis rate was measured by FCM. The results werecompared with that of the control group.（2） Total RNA was extracted and bax mRNA level was measured byreal-time quantitative polymerase chain reaction （RT-qPCR）. The results werecompared with that of the control group.（3） To explore the mechanism of PMNs activation, Activities of Caspase-3，8，9were measured by colorimetry and the results were comparedwith that of the control group by analyzing the activation variations ofCaspase-3, Caspase-8and Caspase-9.（4） To explore the affect of PMNs apoptosis on the pathologic process ofSLE, the correlation was analysed between TNF-α concentration andapoptosis rate.Results:1. The concentrations of cytokine from the female patients with SLEincreased significantly compared to those from the healthy women:concentration of serum E₂（114.64±36.99pg/ml vs80.86±28.60pg/ml，P=0.000）; concentration of IFN-α（66.61±18.74pg/ml vs31.08±7.68pg/ml，P=0.000）; concentration of IL-10（4.69±2.00pg/ml vs2.85±0.72pg/ml，P=0.000）; concentration of TNF-α（15.21±6.87pg/ml vs8.26±2.47pg/ml，P=0.000）; concentration of IL-17（29.31±10.71pg/ml vs15.41±5.87pg/ml，P=0.000）. A positive correlation was found between the concentration of E₂and IFN-α（P=0.005）or IL-10（P=0.000）or TNF-α（P=0.000）, but nosignificant correlation was found between E₂and IL-17（P=0.298）.2. PMNs apoptosis rate of female patients with SLE was higher than thatof healthy women （3.98±1.95%vs1.85±0.68%，P=0.000）.3. PMNs apoptosis rate of female patients with SLE had noting with theSLEDA（IP=0.0816）. There was significant negative correlation between theconcentration of C3and PMNs apoptosis rate（P=0.000）. However, positivecorrelation between the level of E₂, IgG, TNF-α and PMNs apoptosis rate wasfound（P=0.000）.4. PMNs of female patients with SLE was treated with or without E₂at the concentration of1×10-6mol/L for24hours, then the results werecompared:（1） the experimental group: PMNs apoptosis rate reduced（49.39%±10.53%vs46.15%±10.13%，P=0.002）; level of bax mRNAdecreased（0.0224±0.0155vs0.0269±0.0172，P=0.031）; activities ofCaspase-3diminished（14.93±3.30vs16.59±3.69，P=0.010）; however, dataon activities of Caspase-8（3.91±2.01vs3.90±1.98，P=0.876）, activities ofCaspase-9（14.61±5.98vs14.73±5.94，P=0.374）and the concentration ofTNF-α （21.2610.92pg/ml vs20.26±9.43pg/ml，P=0.629） in cultured serumhad no statistically significant differences.（2）the control group: PMNs apoptosis rate（41.83%±13.25%vs42.24%±13.95%，P=0.679）, level of bax mRNA（0.0140±0.0071vs0.0137±0.0065， P=0.475）, activities of Caspase-3（10.40±3.52vs10.61±3.46，P=0.064）, activities of Caspase-8（1.96±1.28vs1.93±1.23，P=0.621）, activities of Caspase-9（10.78±3.78vs11.21±3.67，P=0.093）andthe concentration of TNF-α （17.297.83vs17.91±8.28，P=0.715） were notsignificantly different.Conclusions:1. The concentrations of plasma E₂, IFN-α, IL-10, TNF-α and IL-17fromfemale patients with SLE were obviously higher than that of healthy women.There was positive correlation between PMNs apoptosis rate and theconcentration of IFN-α, IL-10, TNF-α. The results indicated that the disorderof PMNs apoptosis was relative to SLE.2. PMNs apoptosis rate of female patients with SLE was obviouslyhigher than that of healthy women and was significantly correlative with some SLE pathological indices. It was indicated that apoptosis of PMNs wasinvolved in the SLE.3. E₂at certain concentration can inhabit the activity of Caspase-3,decrease the expression of bax mRNA, and eventually inhibit apoptosis ofPMNs in the SLE pathological processes.