Volume 62, Issue 3, Pages 846-856 (September 2002) Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-κB dependent up-regulation of IL-8 and MCP-11  Carla Zoja, Stefania Angioletti, Roberta Donadelli, Cristina Zanchi, Susanna Tomasoni, Elena Binda, Barbara Imberti, Maroeska Te Loo, Leo Monnens, Giuseppe Remuzzi, Marina Morigi  Kidney International  Volume 62, Issue 3, Pages 846-856 (September 2002) DOI: 10.1046/j.1523-1755.2002.00503.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Leukocyte adhesion (A) and transmigration (B) in human umbilical endothelial vein cells (HUVEC) treated with Shiga toxin-2 (Stx-2) under flow condition (1.5 dynes/cm2). Endothelial cells were exposed to control medium, Stx-2 (25 pmol/L, 24 hours) or tumor necrosis factor-α (TNF-α; 100 U/mL, 24 hours) as positive control. At the end of perfusion, the number of adherent and transmigrated leukocytes were quantified by digital analysis of videotapes of each experiment (N = 9 experiments). Data are expressed as mean ± SE. *P < 0.01 vs. control. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Scanning electron micrograph of adherent and transmigrated leukocytes in HUVEC challenged with Stx-2. Micrographs show adherent (A) and transmigrated (B) leukocytes at different stages of activation in HUVEC treated with Stx-2 (25 pmol/L, 24 h). Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Expression of interleukin-8 (IL-8;A) and monocyte chemoattractant protein-1 (MCP-1;B) mRNA in HUVEC exposed to Stx-2. (Top) Northern blot experiments were performed using total RNA isolated after a 6-hour period of culture with medium alone (control) or Stx-2 (25 pmol/L, 50 pmol/L and 1 nmol/L). Data shown are representative of N = 5 experiments. (Bottom) Densitometric analysis of autoradiographic signals for IL-8 and MCP-1. The optical density of the autoradiographic signals was quantified and calculated as the ratio of IL-8 or MCP-1 to GAPDH mRNA. Results (mean ± SE) are expressed as the fold increase over control (considered as 1) in densitometric arbitrary units. *P < 0.01 vs. control. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Effect of anti-IL-8 or anti-MCP-1 antibodies on Stx-2 induced leukocyte adhesion and transmigration under flow. Before the adhesion assay, HUVEC treated for 24 hours with Stx-2 (25 pmol/L) were exposed for 20 minutes to anti-IL-8 or anti-MCP-1 antibodies. At the end of perfusion, the number of adherent and transmigrated leukocytes were quantified. Data are mean ± SE (N = 7 experiments). *P < 0.01 vs. control, °P < 0.01 vs. Stx-2, #P < 0.05 vs. Stx-2 +anti IL-8 antibody. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 Stx-2 induces leukocyte adhesion on glomerular endothelial cells (GEC) under flow. Effect of anti-IL-8 or anti-MCP-1 antibodies. GEC were treated for 24 hours with control medium or Stx-2 (25 pmol/L), and then cells were exposed or not to anti-IL-8 or anti-MCP-1 antibodies for 20 minutes. At the end of perfusion, the number of adherent leukocytes were quantified. Data are mean ± SE (N = 3 experiments). *P < 0.01 vs. control, °P < 0.01 vs. Stx-2. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 6 Stx-2 activates the transcription factor NF-κB. (A) EMSA for NF-κB activity was performed in nuclear extracts from HUVEC exposed for 1 hour to Stx-2 (25 pmol/L, 50 pmol/L and 1 nmol/L). Complexes I and II denote the inducible κB specific DNA-protein complexes. A 100-fold molar excess unlabeled (cold) or an unlabeled nonspecific oligonucleotide (irrelevant probe) was added to nuclear extracts from HUVEC treated with Stx-2 (50 pmol/L). The results shown are representative of three independent experiments employing different nuclear extracts. (B) Subunit composition of NF-κB activated by Stx-2. Nuclear extracts from HUVEC treated with Stx-2 (50 pmol/L, 1 hour) were incubated with antibodies against p65, p50, cRel, RelB and p52 subunits. Antibody supershifts produced by binding of the antibody to the DNA-protein complex are indicated. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 7 Expression of IL-8 and MCP-1 mRNA induced by Stx-2 is inhibited by adenovirus-mediated gene transfer of IκBα. Endothelial cells were left untreated or infected with rAd.IkBα for 3 hours, then cells were exposed to medium alone or to Stx-2 (50 pmol/L, 6 hours). Results shown are representative of N = 3 Northern blot experiments. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 8 Leukocyte adhesion and transmigration induced by Stx-2 are regulated by endothelial activation of NF-κB dependent genes. Endothelial cells were left untreated or infected with rAd.IκBα for 3 hours, then cells were exposed to medium alone (control; □) or to Stx-2 (25 pmol/L, 24 h; Stx-2; ). At the end of incubation, HUVEC were perfused with total leukocyte suspension and the number of leukocytes that adhered and transmigrated were quantified. Data are expressed as mean ± SE (N = 3 experiments). °P < 0.05 vs. control; *P < 0.05 vs. non infected Stx-2. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 8 Leukocyte adhesion and transmigration induced by Stx-2 are regulated by endothelial activation of NF-κB dependent genes. Endothelial cells were left untreated or infected with rAd.IκBα for 3 hours, then cells were exposed to medium alone (control; □) or to Stx-2 (25 pmol/L, 24 h; Stx-2; ). At the end of incubation, HUVEC were perfused with total leukocyte suspension and the number of leukocytes that adhered and transmigrated were quantified. Data are expressed as mean ± SE (N = 3 experiments). °P < 0.05 vs. control; *P < 0.05 vs. non infected Stx-2. Kidney International 2002 62, 846-856DOI: (10.1046/j.1523-1755.2002.00503.x) Copyright © 2002 International Society of Nephrology Terms and Conditions