by Anne-lie Ståhl, Lisa Sartz, and Diana Karpman

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Presentation transcript:

by Anne-lie Ståhl, Lisa Sartz, and Diana Karpman Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli–induced hemolytic uremic syndrome by Anne-lie Ståhl, Lisa Sartz, and Diana Karpman Blood Volume 117(20):5503-5513 May 19, 2011 ©2011 by American Society of Hematology

Circulating platelet-leukocyte complexes and surface-bound C3 in a patient with EHEC-associated HUS. A pediatric HUS patient had increased levels of circulating platelet-monocyte (A) and platelet-neutrophil (B) complexes during the acute phase of disease co... Circulating platelet-leukocyte complexes and surface-bound C3 in a patient with EHEC-associated HUS. A pediatric HUS patient had increased levels of circulating platelet-monocyte (A) and platelet-neutrophil (B) complexes during the acute phase of disease compared with levels obtained after recovery and with pediatric controls (n = 5). Levels of platelet-monocyte (A) and platelet-neutrophil (B) complexes with surface-bound C3 were elevated during the acute phase of disease and decreased at recovery to levels similar to those seen in the pediatric controls. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology

Surface-bound C3 on platelet-leukocyte complexes induced by Stx2, Stx1, Stx1B, O157LPS, and/or O111LPS. Surface-bound C3 on platelet-leukocyte complexes induced by Stx2, Stx1, Stx1B, O157LPS, and/or O111LPS. Incubation of whole blood with Stx2, Stx1, Stx1B, or O157LPS induced C3 deposition on platelet-monocyte (A) and platelet-neutrophil (B) complexes and a lesser increase of C3 on unbound monocytes (C) and unbound neutrophils (D). Data are expressed as box-plots depicting the median and range regarding the percentage of the platelet-monocyte or platelet-neutrophil population or the percentage of unbound monocytes or neutrophils that were positive for C3 (n = 12 experiments). The horizontal line within each box represents the median. The upper and lower limits of the box represent the interquartile range. The lower and upper limits represent the range. C3 deposition on complexes or on unbound monocytes or neutrophils in whole blood incubated with an agonist was compared with PBS-treated whole blood. *P < .05; **P < .01; ***P < .001. NS indicates not significant. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology

Surface-bound C9 on platelet-leukocyte complexes induced by Stx2, Stx1, Stx1B, Ol57LPS, and/or O111LPS. Surface-bound C9 on platelet-leukocyte complexes induced by Stx2, Stx1, Stx1B, Ol57LPS, and/or O111LPS. Incubation of whole blood with Stx2, Stx1, Stx1B, or O157LPS induced C9 deposition on platelet-monocyte (A) and platelet-neutrophil (B) complexes and to a lesser extent on unbound monocytes (C) and unbound neutrophils (D). Data are expressed as box-plots depicting the median and range regarding the percentage of the platelet-monocyte or platelet-neutrophil population or the percentage of unbound monocytes or neutrophils that were positive for C9 (n = 12 experiments). C9 deposition on complexes or unbound monocytes or neutrophils in whole blood incubated with an agonist was compared with that in PBS-treated whole blood. *P < .05; **P < .01; ***P < .001. NS indicates not significant. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology

Inhibition of platelet-monocyte complex formation and complement deposition. Inhibition of platelet-monocyte complex formation and complement deposition. (A) Blocking of Stx2 and/or O157LPS binding by preincubation of whole blood with anti-Gb3 and/or anti-TLR4 before incubation with Stx2 and/or O157LPS reduced platelet-monocyte complex formation significantly. C3 (B) and C9 (C) deposition on the blood-cell complexes was similarly reduced by anti-Gb3 and anti-TLR4 in these experiments. Data are expressed as box-plots depicting median and range (n = 4 experiments). Complex formation and C3 or C9 deposition on complexes in whole blood incubated with an agonist were compared with that in whole blood preincubated with anti-Gb3 and/or anti-TLR4 before the addition of the agonist. *P < .05. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology

Characterization of surface-bound C3 or C9 on platelets and on platelet-derived microparticles. Characterization of surface-bound C3 or C9 on platelets and on platelet-derived microparticles. Incubation of platelet-rich plasma (A) or whole blood (B) with Stx2 or O157LPS induced deposition of C3c, C3b, iC3b, C9neo, or C5b-9 on the surface of platelets (A) and to a lesser degree on platelet-derived microparticles (B). Data are expressed as median ± SD (n = 5 experiments). C3 or C9 deposition on platelets (in platelet-rich plasma) or platelet microparticles (in whole blood) incubated with agonists was compared with that in unstimulated samples. *P < .05; **P < .001. NS indicates not significant. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology

Expression of complement regulators on microparticles. Expression of complement regulators on microparticles. Microparticles from O157LPS-stimulated whole blood were incubated with mouse anti–human CD35:FITC (CR1; A), CD55:APC (DAF; B), or CD46:RPE (MCP; C), showing binding of the antibodies to platelet-, monocyte-, and neutrophil-derived microparticles. Data are presented as the mean fluorescence intensity (MFI) ± SD from 5 independent experiments. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology

Phagocytic uptake of microparticles by neutrophils. Phagocytic uptake of microparticles by neutrophils. Incubation of neutrophils with microparticles isolated from Stx2- or O157LPS-stimulated whole blood induced phagocytic uptake of the microparticles compared with microparticles isolated from unstimulated whole blood. Data are presented as the MFI ± SD from 5 independent experiments. Uptake of microparticles from Stx2- or O157LPS-stimulated whole blood was compared with that from unstimulated whole blood. **P < .01. Anne-lie Ståhl et al. Blood 2011;117:5503-5513 ©2011 by American Society of Hematology