Effects of hyperglycemia, hyperinsulinemia, and hyperosmolarity on neutrophil apoptosis.
BACKGROUND
Hyperglycemia is an independent risk factor for increased mortality of critically ill surgical patients, but despite the recognized clinical benefits of early insulin treatment, there is a lack of understanding of the cellular and molecular mechanisms behind this phenomenon. We hypothesized that polymorphonuclear neutrophils, the first line of the innate immune defense system, suffer from altered apoptotic turnover when exposed to hyperglycemic conditions, ultimately decreasing the number of viable cells active at a site of infection.


METHODS
Venous blood samples were drawn from 10 volunteers and incubated for 0.5 or 24 h in a 1:10 dilution with RPMI 1640 medium at various glucose and insulin concentrations. Mannitol was used to control for increased osmolarity. In addition, all samples were incubated either with low-dose lipopolysaccharide (LPS) (1 ng/mL) or without LPS. Neutrophils were extracted using Ficoll-Hypaque density centrifugation and stained with annexin V and propidium iodide. Fluorescence was detected by flow cytometry and analyzed using CellQuest software.


RESULTS
The mean percentage of apoptotic neutrophils after 24 h of incubation at physiologic glucose concentrations (100 mg/dL) was 42.2 +/- 4.1%; exposure to low-dose LPS decreased this number to 18.4 +/- 2.5% (p < 0.01). Neither the exposure to low (10 mg/dL) nor increasingly high (200 or 400 mg/dL) glucose concentrations altered these percentages significantly. Exposing whole blood to increasing osmolarity (addition of 5.5 mM and 16.5 mM mannitol to simulate 200 and 400 mg/dL glucose) led to a mean absolute reduction of the percentage of apoptotic neutrophils to 34.6 +/- 3.6% (+5.5 mOsm; p < 0.05) and 32.3 +/- 4.5% (16.5 mOsm; p < 0.01), respectively.


CONCLUSIONS
The ability of neutrophils to enter their apoptotic program in cultured whole blood withstands short-term changes in glucose and insulin concentrations. Neither hyperglycemia nor hypoglycemia led to a significant alteration of the apoptotic turnover of these cells, suggesting that the increased rate of infectious complications in short-term hyperglycemic critically ill patients may not be traced to increased apoptosis of neutrophils. However, isolated hyperosmolarity reduces neutrophil apoptosis, an observation that may warrant future investigation.
Turina
Matthias
M
Department of Surgery, Price Institute of Surgical Research, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA. matthias.turina@louisville.edu
Miller
Frederick N
FN
Tucker
Colleen
C
Polk
Hiram C
HC
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002469:Cell Separation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006943:Hyperglycemia / Q000276:immunology*; D006946:Hyperinsulinism / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D009994:Osmolar Concentration; D011183:Postoperative Complications / Q000276:immunology; D011419:Propidium / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D017304:Annexin A5; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D011419:Propidium