Evaluation of phagocytosis in human neutrophils using enhanced green fluorescent protein (EGFP) expressing E. coli

Abstract

Introduction: Phagocytosis plays a very important role in innate immunity and helps the body against bacterial infections. Patients who have defect in phagocytosis suffer from recurrent bacterial infections that may be life threatening.  It is important to detect the defect in phagocytosis as early as possible in life. Patients who have received immunosuppressive medication may also have suppressed phagocytosis. There are different laboratory tests for evaluation of phagocytosis such as NBT (Nitroblue Tetrazolium) and DHR (Dihydrorhodamine) which use chemical compounds not real bacteria. Nitroblue Tetrazolium is yellow chemical substance, in NBT test neutrophils are isolated first and then for Stimulation of respiratory burst in neutrophils add PMA (Phorbol Myristate Acetate).  PMA and NBT are exposed to neutrophils, if a respiratory burst occurs in neutrophils, the color of NBT changes from yellow to purple, that purple neutrophils can be seen under microscope. In DHR test Dihydrorhodamine 1,2,3 was exposed to neutrophils that were stimulated with PMA. Normal neutrophils oxidize DHR after ingestion; finally neutrophils will be fluorescent that can be detected by flow cytometry. The behavior of neutrophils when exposed to chemicals compounds such as NBT and DHR may be different and abnormal, so when real bacteria such as E. coli are exposed to neutrophils the behavior of neutrophils phagocytosis will be normal. The aim of this study was to evaluate a simple and quick method for testing phagocytosis using real bacteria instead of chemical compounds.

Materials and Methods: An EGFP (Enhanced green fluorescent protein) sequence was cloned into a pColdI expression vector. E. coli (Bl21 strain) was transformed by EGFP containing vector. EGFP expression in bacteria was detected by a fluorescent microscope and flow cytometry. EGFP expressing bacteria were added to the heparinized blood of healthy volunteers. Phagocytosis and digestion of fluorescent bacteria by neutrophils were detected using flow cytometry at different time points.

Results: Neutrophils that engulfed the fluorescent bacteria showed high fluorescent activity and, were identified by flow cytometry. Bacterial digestion over time led to a decrease in fluorescent of neutrophils.

Conclusion: EGFP expressing bacteria and flow cytometry technique can be used to evaluate phagocytosis. It can be optimized for clinical as well as research uses.