Expression and function of endothelial selectins during human development

Abstract

Leucocyte trafficking is vital for the immune defence. In adults, early tethering and rolling interactions between leucocytes and endothelial cells are mediated by P-, E- and L-selectins and their ligands. In contrast, the role of selectins in migration of mononuclear cells during fetal development in humans remains unknown. We studied the functions of endothelial E- and P-selectins and their counter-receptors during human ontogeny. Immunohistochemical stainings showed that P-selectin is expressed in megakaryocytes and endothelial cells starting from gestational weeks 7 and 11, respectively. Endothelial E-selectin appeared latest, at week 32. Real-time imaging using in vitro flow chamber assays showed that cord blood mononuclear leucocytes used E-, P- and L-selectin and PSGL-1 to roll on and adhere to endothelium under physiological shear stress. These data show that selectins are synthesized and functional before birth in humans and have the potential to mediate the emigration of mononuclear cells and inflammatory responses.

Keywords: adhesion molecules; cell recruitment/emigration; endothelium; ontogeny; selectins.

Figure 1

Endothelial selectins are expressed during fetal development in humans. (a–b) Expression of P-selectin in (a) megakaryocytes and platelets and (b) vascular endothelium in fetal organs. Gestational age is indicated, and white arrows point to megakaryocytes and platelets and black arrows point to vessels; WP, white pulp. Scale bar, 50 μm. (c, d) Human umbilical vein endothelial cells (HUVEC) isolated from preterm deliveries were (c) left untreated and stained for negative control and P-selectin and analysed by microscopy (blue is DAPI, scale bar, 20 μm) or (d) stimulated with tumour necrosis factor-α for 4 hr and stained for E-selectin using FACS. Representative data from at least three independent experiments with different donors are shown.

Figure 2

Cord blood mononuclear cells (MC) interact with endothelium under flow conditions. (a) Two representative video frames (4 seconds apart) showing two rolling (thick black and open arrows) and two firmly adherent cord blood MC (thin black arrows) on activated human umbilical vein endothelial cells in a flow assay (1 dyn/cm²). (b–d) Comparison of rolling (b, c) and firm adherence (d) of cord blood and adult blood MC under the flow. **P < 0·01.

Figure 3

Endothelial E- and P-selectins support rolling of cord blood mononuclear cells (MC) under flow conditions. The effect of inhibition (mean ± SEM, n = 4 to n = 8 independent experiments using MC and activated human umbilical vein endothelial cells from different individuals) of E-selectin and P-selectin with monoclonal antibodies on (a, b) rolling, (c, d) firm adhesion and (e, f) adhesion efficacy (fraction of adherent cells from all rolling cells) of neonatal and adult MC under the flow. *P < 0·05; **P < 0·01.

Figure 4

Cord blood mononuclear cell (MC) P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin mediate rolling under laminar shear. Cord blood MC and adult peripheral blood mononuclear cells (PBMC) were double stained for CD45 and the indicated adhesion molecules, and analysed using FACS. (a) Representative histograms, (b) percentage of positive cells and (c) mean fluorescence intensities are shown (mean ± SEM; n = 3 to n = 5 different individuals). (d, e) Rolling, (f, g) firm adherence and (h, i) adhesion efficacy of cord and adult blood MC in the flow assay (1 dyn/cm²) was determined in the presence of monoclonal antibodies against PSGL-1, L-selectin, or negative control. The results are (mean ± SEM, n = 4 or 5 independent experiments using MC and activated human umbilical vein endothelial cells from different individuals). (j) Distribution of different leucocyte subpopulations among CD45⁺ cord blood and adult MC was determined using FACS [percentage of positive cells (mean ± SEM; n = 3 different individuals/group)]. *P < 0·05; **P < 0·01.