gms | German Medical Science

Objectives: The function of phagocytic cells is of crucial importance to i) ingest and degrade the infectious agent, and ii) to sensitize the host against secondary infections. Measuring phagocytosis and oxygen radical formation has long been accepted to monitor granulocyte function. Thus chemiluminescence measures were applied to study host defence against pathogens and to evaluate damage of endothelial and epithelial lining cells in various diseases including sepsis. We aimed at a new method to study phagocytosis and concomitant digestion using whole blood

Methods: Based on the pH-sensitive properties of a green-fluorescent protein- (GFP) like dye (Eos-FP), cloned from a coral Lobophyllia hemprichii that switches its fluorescence emission from green (516 nm) to red (581 nm) upon irradiation with approximately 400-nm light (1), we developed a whole blood assay using fixed Eos-FP transfected E. coli. Following incubation of 106 bacteria in 1ml of 1+4 diluted whole blood, we followed the dual fluorescence emission spectrum in granulocytes, monocytes and dendritic cells by conventional two colour flow cytometry.

Results and Conclusion: Patients with local infections, and sepsis were compared with patients suffering from septic shock. Remarkably, the uptake of bacterial numbers per cell as well as the digestive capacity of the PMN fraction varied in patients, but was fairly constant in healthy individuals. The fluorescent light emission ratio of green (fluorescence 1) and red (fluorescence 2) after 1h of incubation was a suitable measure to evaluate the digestive capacity of the PMN fraction of an individual patient. In healthy individuals, whole blood incubation with Eos-FP E. coli resulted in a mean channel ratio of 2.5 to 3.5 after 60min. In addition, we found that the assay is also suitable to study the uptake and digestion of Eos-FP bacteria by dendritic cells. Pre-loading with other infectious and particulate agents completely inhibited Eos-FP E. coli uptake. Results contribute to our understanding of impaired antigen presentation following major tissue damage.