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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Am J Transplant. 2012 Dec 27;13(2):299–311. doi: 10.1111/ajt.12016

Figure 1.

Figure 1

Figure 1

Figure 1

Figure 1

HLA I antibodies trigger Weibel-Palade body exocytosis via intracellular calcium.

(a) Human aortic endothelial cells (HAEC) were treated with isotype control mIgG at 5μg/mL, HLA I antibody (clone W6/32, murine IgG2a) at 5μg/mL, thrombin at 10U/mL, PMA at 200nM, or histamine at 10mM for 1 hr. Supernatant was collected and vWF was measured by ELISA. An experiment showing the average optical density from duplicate measurements +/− SEM (upper panel). Bar graph in lower panel shows mean fold increase in optical density (OD) from 2 (PMA and histamine) or 3 (mIgG, HLA I antibody and thrombin) independent experiments for each condition. * p<0.05, ** p<0.01 versus mIgG.

(b) HAEC were treated with mIgG, anti-CD105 antibody, or HLA I antibody at 5μg/mL for 30min, thrombin at 1U/mL for 15min, PMA at 200nM for 20min, or histamine at 10mM for 10min and detached using Accutase. Cell surface P-selectin was stained with conjugated anti-P-selectin-PE and analyzed by flow cytometry. Bar graph shows mean fold increase in P-selectin positive cells from multiple independent measurements for each condition: mIgG (n=5), anti-CD105 antibody (n=2), HLA I antibody (n=10), thrombin (n=3), PMA (n=6), histamine (n=23). * p<0.05, ** p<0.001 versus untreated.

(c) HAEC were pretreated with BAPTA-AM at 20μM for 30min, then stimulated with HLA I antibody at 5ug/mL for 1 hour (h) and P-selectin was measured as in (b). Bar graph shows summary of data from 6 independent experiments. Black bars show the fold increase in P-selectin expression without inhibitor, and white bars show P-selectin induction with inhibitor.

(d) In order to monitor intracellular Ca2+ concentration, HAEC were loaded with Fura 2-AM and then treated with HLA I antibody at 1μg/mL, thrombin at 0.01U/mL and 1U/mL, or isotype control antibody (mIgG) at 1μg/mL. Intracellular calcium was monitored in real-time using a fluorimeter. Data are presented as the ratio of emission at 340nm/380nm, and are representative of 2 (mIgG and thrombin 0.01U/mL) or 3 (HLA I antibody and thrombin 1U/mL) independent measurements. The time of introduction of reagents is marked with an arrow.