A simple assay to measure phagocytosis of live bacteria.
BACKGROUND
The phagocytosis of pathogens is essential for fighting infections. No assay is available, however, to measure both engulfment and degradation of bacteria under conditions similar to those in vivo. We sought to develop a flow cytometric assay to measure the engulfment and degradation of live bacteria by human blood monocytes and granulocytes.


METHODS
We generated enhanced green fluorescent protein (EGFP)-expressing Eschericha coli by transforming E. coli with the plasmid vector pEGFP. We used these bacteria in a flow cytometric assay to measure both engulfment and degradation of living bacteria by monocytes and granulocytes in human whole blood from fresh, heparinized venous blood samples. To determine whether the test detected differences between healthy individuals and patients with secondary immunodeficiencies, we compared the phagocytosis of monocytes and granulocytes measured in blood samples from immunosuppressed kidney transplantation patients and from patients with postoperative sepsis in immunoparalysis with phagocytosis measured in samples from age-matched healthy individuals.


RESULTS
In samples from healthy individuals, we found that in both monocytes and granulocytes bacterial degradation was negatively correlated with the age of the sample donor. Furthermore, we detected decreased bacterial engulfment in granulocytes from septic patients and decreased bacterial degradation in monocytes from immunosuppressed kidney transplantation patients.


CONCLUSIONS
This flow cytometric assay measures the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. By means of this assay we detected significant differences between healthy controls and patients with secondary immunodeficiencies that may contribute to the increased incidence of infection complications seen in these patients.
Bicker
Heike
H
Institute of Medical Immunology and Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charité, Berlin, Germany.
Höflich
Conny
C
Wolk
Kerstin
K
Vogt
Katrin
K
Volk
Hans-Dieter
HD
Sabat
Robert
R
D004926:Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D006098:Granulocytes / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007108:Immune Tolerance; D016030:Kidney Transplantation; D009000:Monocytes / Q000276:immunology; D010587:Phagocytosis; D011183:Postoperative Complications / Q000097:blood / Q000276:immunology; D018805:Sepsis / Q000097:blood / Q000276:immunology
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins