Alteration in the gene expression pattern of primary monocytes after adhesion to endothelial cells

Abstract

Monocytes originate from precursors made in the bone and remain in the circulation for nearly 24 h. Much effort has been done to identify the molecules regulating transendothelial migration of monocytes during inflammatory conditions. In contrast, considerably less is known about the process of constitutive monocyte emigration although nearly 340 million monocytes leave the circulation each day in healthy individuals. Previous studies indicated that chemokines were up-regulated in monocytes cocultured with endothelial cells that induce the retraction of the latter cell type, thereby increasing vascular permeability. Thus, we hypothesized that the utilities required for efficient constitutive monocyte extravasation are generated by monocytes themselves because of adhesion to naïve endothelial cells. To test this hypothesis, cDNA microarray analysis was performed to determine the changes in the gene expression pattern of primary monocytes that have been attached to endothelial cells compared with monocytes that were held in suspension, and we were able to identify three major groups of genes. The first group includes genes such as matrix metalloproteinase 1, monocyte chemoattractant protein 1, and tissue transglutaminase 2, which are likely required for monocyte extravasation. The second group consists of genes that are expressed in phagocytes such as caveolin-1 and CD74. Finally, the third group comprises genes that are expressed in cells of endothelial tissue and cartilage including E-selectin, fibronectin-1, matrix Gla protein, and aggrecanase-2. In summary, we conclude that adhesion of peripheral blood monocytes to naïve endothelial cells has two effects: mandatory extravasation-specific genes are regulated, and the differentiation program of monocytes is initiated.

Fig. 1.

E-selectin is up-regulated in a small subset of HUVEC-attached monocytes. (A) ICC (a) and combined ICC plus RISH (c) show that E-selectin expression is not detectable in suspension monocytes. In contrast, E-selectin protein (b) and E-selectin mRNA (d) are detectable in HUVEC-attached monocytes. CD14 expression of monocytes appears in red, and E-selectin expression (on both a protein level and an mRNA level) is shown in green. Arrows indicate the intracellular presence of E-selectin expression in HUVEC-attached monocytes. (Scale bars: 20 μm.) (B) Verification of E-selectin up-regulation in HUVEC-attached monocytes by flow cytometry. The expression of the molecule of interest is shown as a shaded histogram. Open histograms represent staining with isotype-matched control monoclonal antibodies. On average 2.19 ± 1.25% of HUVEC-attached monocytes were positive for E-selectin expression. The results shown are representative of at least three independent experiments.

Fig. 2.

CTGF and fibronectin-1 expression is up-regulated in a subset of HUVEC-attached monocytes. (A) CTGF expression is detectable on both a protein level (a and b) and an mRNA level (c and d) in suspension and HUVEC-attached monocytes. However, the CTGF protein expression level is higher in HUVEC-attached monocytes (b, arrows) as compared with control cells (a, arrowheads) indicating CTGF up-regulation. (B) Fibronectin-1 expression is detectable on both a protein (a and b) and mRNA (c and d) in suspension and HUVEC-attached monocytes. However, the fibronectin-1 protein expression level is higher in HUVEC-attached monocytes (b, arrows) as compared with control cells (a, arrowheads) indicating fibroenctin-1 up-regulation. CD14 expression of monocytes appears in red, and CTGF and fibronectin-1 expression (on both a protein level and an mRNA level) is shown in green. (Scale bars: 20 μm.) The results shown are representative of at least three independent experiments.

Fig. 3.

tTG-2, but likely not MMP-1, is up-regulated in HUVEC-attached monocytes. (A) tTG-2 expression is detectable on both a protein level (a and b) and an mRNA level (c and d) in suspension and HUVEC-attached monocytes, and tTG-2 is more highly expressed in HUVEC-attached monocytes. tTG-2 is localized in the plasma membrane of both control (a, arrowhead) and HUVEC-attached (b, arrows) monocytes. (B) The expression levels of MMP-1 appear to be similar in suspension (a and b) and HUVEC-attached (c and d) monocytes. Like tTG-2, MMP-1 is found in the plasma membrane of monocytes (a, arrowheads; b, arrows). CD14 expression of monocytes appears in red, and tTG-2 and MMP-1 expression (on both a protein level and an mRNA level) is shown in green. (Scale bars: 20 μm.) The results shown are representative of at least three independent experiments.

Fig. 4.

Schematic overview of the changes in the gene expression profile of HUVEC-attached monocytes. During constitutive adhesion to endothelial cells, the gene expression pattern of primary monocytes is altered and genes are regulated that are required for subsequent transendothelial migration. Additionally, the differentiation program of monocytes into phagocytes and other lineages is initiated.