Interleukin-8 Receptors CXCR1 and CXCR2 Are Not Expressed by Endothelial Colony-forming Cells

Abstract

Endothelial colony-forming cells (ECFCs) are human vasculogenic cells described as potential cell therapy product and good candidates for being a vascular liquid biopsy. Since interleukin-8 (IL-8) is a main actor in senescence, its ability to interact with ECFCs has been explored. However, expression of CXCR1 and CXCR2, the two cellular receptors for IL-8, by ECFCs remain controversial as several teams published contradictory reports. Using complementary technical approaches, we have investigated the presence of these receptors on ECFCs isolated from cord blood. First, CXCR1 and CXCR2 were not detected on several clones of cord blood- endothelial colony-forming cell using different antibodies available, in contrast to well-known positive cells. We then compared the RT-PCR primers used in different papers to search for the presence of CXCR1 and CXCR2 mRNA and found that several primer pairs used could lead to non-specific DNA amplification. Last, we confirmed those results by RNA sequencing. CXCR1 and CXCR2 were not detected in ECFCs in contrary to human-induced pluripotent stem cell-derived endothelial cells (h-iECs). In conclusion, using three different approaches, we confirmed that CXCR1 and CXCR2 were not expressed at mRNA or protein level by ECFCs. Thus, IL-8 secretion by ECFCs, its effects in angiogenesis and their involvement in senescent process need to be reanalyzed according to this absence of CXCR-1 and - 2 in ECFCs.Graphical Abstract.

Keywords: CXCR1; CXCR2; ECFCs; RNAseq; chemokines; interleukin-8.

Fig. 1

Expression of CXCR1 and CXCR2 on whole blood samples by flow cytometry with increasing concentration of antibodies purchased from R&D (a) or Santa-Cruz (b)

Fig. 2

Expression of CXCR1 and CXCR2 on peripheral blood mononuclear cell by flow cytometry with increasing concentration of antibodies purchased from R&D (a) or Santa-Cruz (b)

Fig. 3

Expression of CXCR1 and CXCR2 on CB-ECFCs by flow cytometry with increasing concentration of antibodies purchased from R&D (a) or Santa-Cruz (b)

Fig. 4

Chemotactic migration assay induced by IL-8 at increasing concentrations. a Migration of neutrophils (PMNs) towards Il-8 (n = 9), % of positive control. b Migration of CB-ECFCs towards Il-8 (n = 9), % of positive control. c Migration of CB-ECFCs grown in 10 ng/mL IL-8 for 24 h towards Il-8 (n = 9), % of positive control

Fig. 5

Schematic representation of the transcripts of CXCR1 and CXCR2 with the position of the primers used in the different papers (Kimura et al. [6], Yoon et al. [7], Kwon et al. [16], Audigier et al. [17]/Smadja et al. [3].)

Fig. 6

a Relative level of expression of HGF, IL-6, CDKN2A, NOS3, VWF, SERPINE-1, CDKN1A, CDH5, VEGFA, CCR8, CXCR2, CCR5, CCL3, CCR3, CCL2, IL-1B, CXCL-1, CCL5 by CB-ECFCs, h-iECs and h-iPSCs evaluated by RNA sequencing. b Expression of CXCR1 by CB-ECFCs, h-iECs and h-iPSCs evaluated by RNA sequencing. c Expression of CXCR2 by CB-ECFCs, h-iECs and h-iPSCs evaluated by RNA sequencing