Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes

Abstract

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Keywords: CD14; Rac1; high-density lipoprotein; immunology; inflammation; lipid rafts; polymorphonuclear leukocytes; serum amyloid A.

Figure 1

Effect of high-density lipoprotein (HDL) from healthy subjects (HS), n = 7, chronic kidney disease (CKD) patients stage 3 and 4 (CKD), n = 18, hemodialysis (HD) patients (HD), n = 8, on polymorphonuclear leukocytes (PMNLs) from healthy subjects at 0, 10 and 100 µg/mL final concentration. HD(HD): Effect of HDL from HD patients on PMNLs from HD patients, n = 9; the percentage of CD14 positive cells in the absence of HDL was set as 100%. There was no statistically significant difference between the absolute values. (A) Basal CD14 surface expression. (B) CD14 surface expression stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP). * p < 0.05 and ** p < 0.01 versus 0 µg/mL HDL; $ p < 0.05 versus HS; §§ p < 0.01 versus HD; data shown are mean values ± standard error of the mean (SEM).

Figure 2

Effect of serum amyloid A protein (SAA) at a final concentration of 10 µg/mL ((A); n = 3) and of HDL isolated from healthy subjects (HS-HDL) and HS-HDL spiked with SAA (+) at final concentrations of 10 µg/mL and 100 µg/mL ((B); n = 8) on the basal (black bars) and fMLP-stimulated (grey bars) CD14 surface expression. The percentage of CD14 positive cells in the absence of SAA and fMLP (Co: buffer as control, 0 µg/mL HDL) was set as 100%. * p < 0.05 and ** p < 0.01 versus unstimulated values; $ p < 0.05, $$ p < 0.01 versus control (Co: 0.01% bovine serum albumin) for A, versus 0 µg/mL HDL for B; data shown are mean values ± standard error of the mean (SEM).

Figure 3

Effect of methyl-β-cyclodextrin (MβCD) on basal (black bars) and fMLP-stimulated (grey bars) CD14 surface expression. The unstimulated CD11b expression measured as mean fluorescence intensity (MFI) in the absence of MβCD was set as 100%. n = 5 for 0 and 3 mg/mL MβCD; n = 3 for 6 and 9 mg/mL MβCD. * p < 0.05 versus the unstimulated values; $ p < 0.05 and $$ p < 0.01 versus the absence of MβCD; data shown are mean values ± SEM.

Figure 4

Effect of MβCD (M; 6 mg/mL) on basal (black bars) and fMLP-stimulated (grey bars) CD14 surface expression in the absence and presence of 100 µg/mL HDL (H) from healthy subjects ((A); n = 4) and from HD patients ((B); n = 5). The unstimulated CD11b expression measured as mean fluorescence intensity (MFI) in the absence of MβCD was set as 100%. * p < 0.05 versus the unstimulated values; § p < 0.05 and §§ p < 0.01 versus the absence of MβCD; $ p < 0.05 versus the absence of HDL; data shown are mean values ± SEM.

Figure 5

Effect of HDL (10 µg/mL) from healthy subjects (HS), n = 9 and HD patients (HD), n = 7, on the basal Rac1 activity of PMNLs from healthy subjects. The activity in the absence of HDL was set as 100%. ** p < 0.01 versus control; data shown are mean values ± SEM.