Granulocyte colony-stimulating factor improves deficient in vitro neutrophil transendothelial migration in patients with advanced liver disease

Abstract

Bacterial infections are frequent complications in patients with liver cirrhosis. Cirrhotic patients present abnormalities in both innate and adaptive immune responses, including a deficient neutrophil recruitment to infected sites. The purpose of this study was to assess neutrophil-endothelium interactions in cirrhotic patients and evaluate the effects of G-CSF on this process. We studied neutrophil adhesion and transendothelial migration in 14 cirrhotic patients and 14 healthy controls. We also analyzed neutrophil expression of the adhesion molecules CD62L and CD11b in whole blood by flow cytometry. Cirrhotic patients expressed higher levels of CD11b than healthy controls, whereas CD62L expression was significantly lower, suggesting exposure of neutrophils to activating agents within the bloodstream. Neutrophils from cirrhotic patients showed increased adhesion to both resting and tumor necrosis factor alpha-stimulated microvascular endothelial cells and decreased transendothelial migration. Granulocyte colony-stimulating factor (G-CSF) (100 ng/ml) significantly enhanced neutrophil adhesion to microvascular endothelial cells in healthy controls but not in cirrhotic patients. G-CSF also significantly improved neutrophil transmigration in cirrhotic patients and healthy controls. In conclusion, cirrhotic patients exhibit increased neutrophil adhesion to microvascular endothelium and deficient transendothelial migration. G-CSF enhances neutrophil transendothelial migration in cirrhotic patients despite having no effect on neutrophil adhesion. Therefore, G-CSF may be able to increase neutrophil recruitment into infected sites in these patients.

FIG. 1.

Flow cytometry analysis of the expression of adhesion molecules CD11b and CD62L in blood neutrophils. Neutrophils from cirrhotic patients expressed significantly higher levels of CD11b receptor than neutrophils from healthy controls while expressing lower levels of CD62L (∗, P < 0.01). Data are means ± SD. ABS, antibody binding sites.

FIG. 2.

Neutrophil adhesion to resting and TNF-α-stimulated HMEC-1 monolayers (100 IU/ml) in healthy controls and cirrhotic patients after 4 h of incubation (∗, P < 0.01). Data are means ± SD.

FIG. 3.

Effect of G-CSF on the number of neutrophils adhering to resting and TNF-α-stimulated HMEC-1 monolayers (100 IU/ml) in healthy controls. G-CSF significantly increased neutrophil adhesion to both resting (P < 0.01) and TNF-stimulated (P < 0.05) HMEC-1 monolayers after 4 h of incubation in healthy controls. Each data point is the mean ± SD for five wells, and P values represent differences between groups by paired t tests.

FIG. 4.

G-CSF failed to increase neutrophil adhesion to resting (P < 0.12) or TNF-α-stimulated (P < 0.48) HMEC-1 monolayers after 4 h of incubation in cirrhotic patients. Each data point is the mean for five wells, and P values represent differences between groups by paired t tests.

FIG. 5.

Quantification of neutrophil transendothelial migration in cirrhotic patients and healthy controls. Neutrophil migration across TNF-stimulated HMEC-1 monolayers following fMLP gradients was significantly reduced in cirrhotic patients compared to healthy controls (∗, P < 0.01 for comparison between groups by the Mann-Whitney test). Data are presented as box plots: the extents of the boxes indicate the 25th and the 75th percentiles, and the lines inside the boxes mark the 50th percentile (median) values. Capped bars indicate the 10th and 90th percentiles, and open circles mark the 5th and 95th percentiles.

FIG. 6.

Effect of G-CSF on neutrophil transendothelial migration. Addition of G-CSF (100 ng/ml) to the upper well of the transmigration system results in significantly higher migration of neutrophils across monolayers of TNF-α-stimulated HMEC-1 cells following a fMLP gradient in both healthy controls and cirrhotic patients after 2 h of incubation. Each data point is the mean of three wells, and P values represent differences between groups by paired t tests.