Figure 4
Figure 4. Human serum enhances adhesion molecule expression and PBMC binding on Neu5Gc-loaded endothelium. (A) A representative example of surface E-selectin expression determined on Neu5Ac and Neu5Gc-loaded HUVECs after 5 hours of activation by low-titer serum (S30, right) or high-titer serum (S34) or heat-inactivated forms thereof (bottom). Control peaks (shaded curve) represent E-selectin expression on endothelial cells not activated by serum. (B) As described in panel A, except HUVECs were exposed to serum for 15 minutes only. Heat-inactivated serum controls are included as a negative control (shaded curve). Short serum incubations show a Neu5Gc-dependent expression of P-selectin that requires anti-Neu5Gc antibodies and is sensitive to heat inactivation of serum. This response was most evident in low passage HUVECs (passage # ≤ 3), consistent with the observed (data not shown) and reported passage-dependent loss of P-selectin expression in vitro.46 (C) HUVECs were cultured on 12-mm glass coverslips (Fisher) in 24-well plates and loaded with either Neu5Ac or Neu5Gc as described previously. Loaded cells were incubated with 50% human sera in EBM-2 for 4 hours at 37°C or with heat-inactivated serum as a control. A negative control was not exposed to human serum (NoS) and a positive control was exposed to TNF-α (10 ng/mL) for stimulation. At 4 hours, 105 PBMCs were added to each well and incubated at 37°C on an orbital shaker at 100 revolutions/minute for 1 hour. Cells were washed thrice in PBS and fixed in PBS containing 3% paraformadehyde. Bound PBMCs were counted in 10 randomly chosen fields at ×400 magnification. Data were averaged and expressed as cells/field. Data are presented as mean with the scatter. This panel is a representative example of 5 similar replicates. ***P < .001. (D) Same as panel C except that the α-methyl-glycosides (1mM) were incubated with the S34 before HUVEC stimulation. All groups were Neu5Gc loaded. We observed inhibition of PBMC binding in S34-stimulated HUVECs with Neu5Gc2Me but not Neu5Ac2Me. ***P < .001. Examples of fluorescent images from the experiment in panel D are shown in supplemental Figure 1. All results presented were performed at least 3 times.

Human serum enhances adhesion molecule expression and PBMC binding on Neu5Gc-loaded endothelium. (A) A representative example of surface E-selectin expression determined on Neu5Ac and Neu5Gc-loaded HUVECs after 5 hours of activation by low-titer serum (S30, right) or high-titer serum (S34) or heat-inactivated forms thereof (bottom). Control peaks (shaded curve) represent E-selectin expression on endothelial cells not activated by serum. (B) As described in panel A, except HUVECs were exposed to serum for 15 minutes only. Heat-inactivated serum controls are included as a negative control (shaded curve). Short serum incubations show a Neu5Gc-dependent expression of P-selectin that requires anti-Neu5Gc antibodies and is sensitive to heat inactivation of serum. This response was most evident in low passage HUVECs (passage # ≤ 3), consistent with the observed (data not shown) and reported passage-dependent loss of P-selectin expression in vitro.46  (C) HUVECs were cultured on 12-mm glass coverslips (Fisher) in 24-well plates and loaded with either Neu5Ac or Neu5Gc as described previously. Loaded cells were incubated with 50% human sera in EBM-2 for 4 hours at 37°C or with heat-inactivated serum as a control. A negative control was not exposed to human serum (NoS) and a positive control was exposed to TNF-α (10 ng/mL) for stimulation. At 4 hours, 105 PBMCs were added to each well and incubated at 37°C on an orbital shaker at 100 revolutions/minute for 1 hour. Cells were washed thrice in PBS and fixed in PBS containing 3% paraformadehyde. Bound PBMCs were counted in 10 randomly chosen fields at ×400 magnification. Data were averaged and expressed as cells/field. Data are presented as mean with the scatter. This panel is a representative example of 5 similar replicates. ***P < .001. (D) Same as panel C except that the α-methyl-glycosides (1mM) were incubated with the S34 before HUVEC stimulation. All groups were Neu5Gc loaded. We observed inhibition of PBMC binding in S34-stimulated HUVECs with Neu5Gc2Me but not Neu5Ac2Me. ***P < .001. Examples of fluorescent images from the experiment in panel D are shown in supplemental Figure 1. All results presented were performed at least 3 times.

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