Monocytic C-C chemokine receptor 5 expression increases in in vitro intermittent hypoxia condition and in severe obstructive sleep apnea patients

Abstract

Purpose: Obstructive sleep apnea (OSA) patients have higher risk of cardiovascular disease. C-C chemokine receptor 5 (CCR5), as an important receptor for monocyte recruitment and the initiation of atherosclerosis, was studied under intermittent hypoxia and in OSA patients.

Methods: The expression and function of CCR5 regulated by intermittent hypoxia in monocytic THP-1 cells were investigated in an in vitro intermittent hypoxia culture system. The expression levels of protein and mRNA were analyzed by western blot and RT/real-time PCR analysis. Cell adhesion assay and transwell filter migration assay were carried out to investigate the adhesion and chemotaxis of monocytes. In addition, the mRNA expression of CCR5 in monocytes isolated from peripheral blood of 72 adults was analyzed.

Results: Intermittent hypoxia upregulated the expression of CCR5 in THP-1 cells and enhanced the adhesion and chemotaxis of monocytes to vascular endothelial cells mediated by RANTES. The CCR5 expression induced by intermittent hypoxia was inhibited by inhibitor for p42/44 MAPK. Besides, the expression of CCR5 in monocytes increased along the AHI value especially in severe OSA patients that was statistically significant compared with mild and moderate OSA groups.

Conclusions: This study demonstrated the increased monocytic CCR5 gene expression in patients with severe OSA. Intermittent hypoxia, the characteristic of OSA, induced monocytic CCR5 gene expression and the enhanced RANTES-mediated chemotaxis and adhesion through p42/44 MAPK signal pathways.

Keywords: Chemokine receptor; Chemotaxis; Intermittent hypoxia; Monocyte; Obstructive sleep apnea.

Fig. 1

Intermittent hypoxia enhanced CCR5 gene expression in monocytic THP-1 cells. Monocytic THP-1 cells were treated with normoxia or intermittent hypoxia as described in the Material and methods section. a RNA was isolated for the analysis of CCR5 gene expression by RT/real-time PCR. b Membrane proteins were prepared for western blot analysis. c Human peripheral monocytes were treated with the same conditions as in (a) and total RNA was isolated for the analysis of CCR5 gene expression by RT/real-time PCR. (Data are presented as mean ± SEM, *p < 0.05 vs. Normoxia, ^†p < 0.05 vs. Intermittent hypoxia)

Fig. 2

Intermittent hypoxia enhanced RANTES-induced chemotaxis of monocytic THP-1 cells. Monocytic THP-1 cells were treated with normoxia or intermittent hypoxia as described, and RANTES-mediated chemotaxis were processed. a Photos represented for normoxia-treated or intermittent hypoxia-treated monocytic THP-1 cells that migrated toward lower chamber through the transwell filter. Chemotaxis cells were indicated by black arrow. Scale bar = 100 μm. b Statistical results from three experiments showed significantly enhance the chemotaxis toward RANTES. (Data are presented as mean ± SEM; *p < 0.05 vs. Normoxia)

Fig. 3

Intermittent hypoxia enhanced the RANTES-stimulated adhesion of monocytic THP-1 cells to vascular endothelial cells. Pretreated monocytic THP-1 cells with normoxia or intermittent hypoxia were activated by 30 ng/ml RANTES for another 18 h, and then processed for adhesion assay. a Photos represented for monocytic THP-1 cells after cell adhesion assay. Black arrow indicated the adhered cells. Scale bar = 100 μm. (Normoxia: without any treatment, Normoxia + RANTES: with RANTES stimulation only, Intermittent hypoxia: with intermittent hypoxia pretreatment only, Intermittent hypoxia + RANTES: with intermittent hypoxia pretreatment and RANYES stimulation.) b Statistical results from three independent experiments showed intermittent hypoxia treatment synergistically promoted the adhesive activity of monocytic THP-1 cells. (Data are presented as mean ± SEM; *p < 0.05 vs. Normoxia, ^†p < 0.05 vs. Normoxia + RANTES, ^‡p < 0.05 vs Intermittent hypoxia)

Fig. 4

P44/42 antagonist inhibited the increase of CCR5 expression induced by intermittent hypoxia. Monocytic THP-1 cells were pretreated for 1 h with a 10 μM PD98059 or b 20 μM MSB202190 to inhibit p44/42 or p38 MAPK pathway respectively, followed by treatment with intermittent hypoxia, then cultured in normal incubator for 18 h. RNA was isolated for the analysis of CCR5 mRNA expression by RT/real-time PCR. (Data are presented as mean ± SEM, *p < 0.05 vs. Normoxia, ^†p < 0.05 vs. Intermittent hypoxia + PD98059)

Fig. 5

CCR5 mRNA expression increased in monocytes of OSA patients. a The CCR5 mRNA expression of 72 patients from four groups with different OSA severity was analyzed by RT/real-time PCR. (Data are presented as mean ± SEM, *p < 0.05 vs. AHI ≤ 5.) b Linear regression demonstrated the positive correlation between ODI and CCR5 mRNA expression levels in monocytes (p = 0.013, r = 0.295). c Linear regression demonstrated the positive correlation between CCR5 mRNA expression levels in monocytes and mean SpO₂ (p = 0.038, r = 0.249), but not lowest SpO₂ or time with SpO₂ < 85%