Is the In Vitro Observed NETosis the Favored Physiological Death of Neutrophils or Mainly Induced by an Isolation Bias?

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs—when gently isolated—can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible)

Obesity Correlates with Chronic Inflammation of the Innate Immune System in Preeclampsia and HELLP Syndrome during Pregnancy

HELLP syndrome is characterized by hemolysis, elevated liver enzymes, and a low platelet count and poses an increased risk to the pregnant woman and the unborn child. Individual risk factors such as obesity may alter immunocompetence and influence the course of preeclampsia (PE) or HELLP syndrome. Blood samples were collected from 21 pregnant women (7 healthy, 6 with PE, and 8 with HELLP syndrome) and polymorphonuclear neutrophils (PMNs) were subsequently isolated. Production of radical oxygen species (ROS), cell movement, and NETosis were assessed by live-cell imaging. Surface protein expression and oxidative burst were analyzed by flow cytometry. PE and HELLP patients had significantly higher BMI compared to the healthy control group. Depending on the expression of CD11b, CD62L, and CD66b on PMNs, a surface protein activation sum scale (SPASS) was calculated. PMNs from patients with high SPASS values showed prolonged and more targeted migration with delayed ROS production and NETosis. Obesity is associated with a chronic inflammatory state, which in combination with immunological triggers during pregnancy could modulate PMN functions. Pregnant women with higher BMI tend to have higher SPASS values, indicating activation of the innate immune system that could co-trigger PE or HELLP syndrome

Is the In Vitro Observed NETosis the Favored Physiological Death of Neutrophils or Mainly Induced by an Isolation Bias?

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs—when gently isolated—can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible)