Analysis of Neutrophil Viability and Activation State in LPS Treated Cultured Blood Clots

Abstract

Whole blood clots (BC) cultured for 4 hours at 37 degrees C are a tissue-engineered model for studying innate immune cell responses. Neutrophils, immune cells expressing the CD15 antigen with key roles in wound repair, have a relatively short half-life of 6-12 hours in circulation. It is currently unknown whether neutrophils survive up to 4 hours in cultured BC. Previous work showed that lipopolysaccharide (LPS), an endotoxin, enhances the survival of purified neutrophils and increases chemokine release from cultured BCs. This experiment sought to determine whether the treatment of BC with LPS enhances the survival of neutrophils throughout the cultured BC. We hypothesized that LPS would enhance CD15+ neutrophil counts in cultured BCs and that more neutrophils would be found near the top of the clot. Cryosections from cultured BC +/- LPS (n = 3 healthy human donors, 1 male, and 2 female) were immunostained for CD15, counterstained with Hoechst dye to identify cell nuclei, and then imaged with a fluorescent microscope at 10x magnification. High-density 1.2mm-square fields were taken off the top, middle, and bottom sections of each clot. CD15+ cells and nuclei were counted by hand on an iPad. BC showed an average of 245 CD15+ cells in the top, 135 in middle, and 166 in the bottom fields. BC with LPS had an average of 362 CD15+ in the top, 219 in middle, and 118 in the bottom fields. Analysis of the variance of the mean (ANOVA) determined that LPS stimulated a two-fold increase in % CD15+ cells at the top versus the bottom of the clot (p less than 0.05) without influencing overall neutrophil counts compared to cultured BC without LPS (p = 0.54). The current experiment suggests the BC environment is sufficient to maintain neutrophil viability without LPS and that LPS promotes an asymmetrical cell distribution. This could be due to increased chemokine concentrations in the clot + LPS serum. This work supports using cultured BC to study the neutrophil’s role in inflammation.