A new method to quantify phagocytosis and intracellular degradation using green fluorescent protein‐labeled Escherichia coli: Comparison of cord blood macrophages and peripheral blood macrophages of healthy adults

Abstract

Interactions between innate and adaptive immune functions in neonatal macrophages (MΦ) are still unclear. We therefore established a method to quantify bacterial phagocytosis and intracellular degradation, using green fluorescent protein (GFP)‐labeled Escherichia coli in combination with phenotypic analysis. The kinetics of the proportion of phagocyting MΦ, phagocytosed bacteria per MΦ, and bacterial degradation were comparable for cord blood MΦ of term neonates and MΦ of healthy adults. Phenotyping revealed CD14 and CD16 to be down‐modulated within minutes. GFP‐labeled E. coli may be useful tools to further study MΦ subpopulations and determinants of phagocytosis in cord blood MΦ. © 2006 International Society for Analytical Cytology

Neonatal monocytes express antiapoptotic pattern of Bcl-2 proteins and show diminished apoptosis upon infection with Escherichia coli

“…The phagocytosis assay was carried out as previously described (38). Incubation was performed for 60 min at a multiplicity of infection of 50:1.…”

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants

“…Articles neonates have reported diminished (14), enhanced (12,13), or equivalent capability (11,13). These inconsistent findings are likely to reflect methodological differences, as sample type (e.g., purified cells or whole blood), preparations and species of bacteria, together with the use of humoral factors (e.g., complement) vary considerably between studies (11).…”

“…However, the data, particularly for deficiencies in phagocytosis, are inconsistent (11). There are no definitive comparisons of phagocytosis between adults and neonates; studies have identified positive (12,13), negative (14), and neutral (11,13) trends in functionality between the groups (15). Similarly, the reported correlation between GA and phagocytosis functionality is inconsistent (12,16).…”

The CD95/CD95L pathway is involved in phagocytosis-induced cell death of monocytes and may account for sustained inflammation in neonates

“…PBMCs and CBMCs were isolated by density gradient centrifugation (Biochrom AG, Berlin, Germany) as described (18). Washed cells were resuspended in VLE RPMI-1640 (Biochrom) containing 10% heat-inactivated fetal calf serum (Biochrom) at 2 × 10 6 cells/ml in flat-bottom 24 well cell culture plates (Costar, Bodenheim, Germany).…”

“…Although phagocytic activity and intracellular degradation of bacteria did not differ in comparison with those of monocytes from peripheral blood of adults (PBMOs) (17,18), PICD was greatly diminished in CBMOs (19).…”