Neutrophil Expression of HNA-2 and mPR3 As Biomarkers for the Development of Preeclampsia

Introduction: The human neutrophil antigen-2 (HNA-2) is clinically important for being involved in immune neutropenias, febrile transfusion reactions and transfusion related acute lung injury (TRALI). HNA-2 is located on the glycoprotein CD177 (NB1) and is expressed on subpopulations of neutrophils, ranging from 0 to 100%. It has been suggested that the HNA-2 is the receptor to the membrane fraction of Proteinase 3 (mPR3), as they are co-expressed in the same subpopulation of neutrophils. Furthermore, HNA-2 binds to PECAM-1 on endothelial cells, facilitating the transendothelial migration of neutrophils. The preeclampsia (PE) is characterized by a neutrophil activation and production of pro-inflammatory cytokines, migration of neutrophils to human umbilical vein, and metabolic and phenotypic changes of neutrophils leading to endothelial dysfunction. The aim of this study was to determine the expression of HNA-2 and mPR3 into three different groups of individuals: nonpregnant women, healthy pregnant women and women with PE in order to investigate the role of these molecules in the pathogenesis of PE.

Methods: A cross-sectional study included samples from 81 nonpregnant women, 75 healthy pregnant women, and 45 women with PE. Blood samples from nonpregnant women were collected at the Blood Center, while blood samples from pregnant women during the 2^nd trimester of gestation were collected at 2 obstetric hospitals. The HNA-2 and mPR3 expression was determined by flow cytometry (FACSCalibur, Becton Dickinson) using the monoclonal antibodies MEM166 conjugated to PE and WGM2 conjugated to FITC, respectively (Abcam, Cambridge, UK). Mouse IgG1 was used as isotype control (Abcam, Cambridge, UK). The data acquisition and analysis was performed by CellQuest® software (Becton Dickinson). The fluorescence intensity was classified as low (≤ 60%), medium (61-80%) and high (> 80%). Subjects were considered HNA-2 or PR3-negative if less than 5% of their neutrophils reacted with the monoclonal antibodies.

Results: The median percentage of neutrophils expressing HNA-2 and mPR3 was significantly higher in women with PE (88%), compared with the expression in healthy pregnant (70%) and nonpregnant women (72%) (p<0.0001; ANOVA). Analyzing the results according to the degree of HNA-2 and mPR3 expression, a higher expression (> 80%) was observed in 64% (29/45) of woman with PE compared to 33% (25/75) of healthy pregnant and 26% (21/81) of nonpregnant women (p=0.0009 and p<0.0001; respectively). Interestingly, we observed that two healthy pregnant women who initially presented increased antigens coexpression (median of 96%), developed PE at the 35^th week of gestation. In addition, historical obstetric analysis of the healthy pregnant women group revealed three other women who had a previous clinical and laboratory diagnosis of PE showed in this study a high expression of antigens HNA-2 and mPR3 (median of 98%).

Conclusion: The present study indicates that the expression of HNA-2 and mPR3 in neutrophils is significantly increased in women with PE. Such antigens overexpression may be associated to the increased migration of activated neutrophils to human umbilical vein endothelium in addition to the local inflammation and tissue destruction, factors already described to be involved in the etiology of PE. In conclusion, the present data suggest that high expressions of HNA-2 and mPR3 measured early in the 2^nd trimester of the pregnancy may be possible biomarkers for the development of PE.