Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency

Jan Stindt, Stefanie Kluge, Carola Dröge, Verena Keitel, Claudia Stross, Ulrich Baumann, Florian Brinkert, Anil Dhawan, Guido Engelmann, Rainer Ganschow, Patrick Gerner, Enke Grabhorn, A. S. Knisely, Khalid A. Noli, Ieva Pukite, Ross W. Shepherd, Takehisa Ueno, Lutz Schmitt, Constanze Wiek, Helmut HanenbergDieter Häussinger, Ralf Kubitz

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Abstract

Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.

Original languageEnglish (US)
Pages (from-to)524-537
Number of pages14
JournalHepatology
Volume63
Issue number2
DOIs
StatePublished - Feb 1 2016
Externally publishedYes

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Bile Acids and Salts
Antibody Formation
Antibodies
Liver Transplantation
Immunoglobulin G
Perfusion
Recurrence
Taurocholic Acid
Mutation
Immunoglobulin Isotypes
Liver
Cholestasis
Serum
Fluorescent Antibody Technique
Western Blotting
Staining and Labeling
Biopsy
Progressive familial intrahepatic 2 Cholestasis

ASJC Scopus subject areas

  • Hepatology

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Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency. / Stindt, Jan; Kluge, Stefanie; Dröge, Carola; Keitel, Verena; Stross, Claudia; Baumann, Ulrich; Brinkert, Florian; Dhawan, Anil; Engelmann, Guido; Ganschow, Rainer; Gerner, Patrick; Grabhorn, Enke; Knisely, A. S.; Noli, Khalid A.; Pukite, Ieva; Shepherd, Ross W.; Ueno, Takehisa; Schmitt, Lutz; Wiek, Constanze; Hanenberg, Helmut; Häussinger, Dieter; Kubitz, Ralf.

In: Hepatology, Vol. 63, No. 2, 01.02.2016, p. 524-537.

Research output: Contribution to journalArticle

Stindt, J, Kluge, S, Dröge, C, Keitel, V, Stross, C, Baumann, U, Brinkert, F, Dhawan, A, Engelmann, G, Ganschow, R, Gerner, P, Grabhorn, E, Knisely, AS, Noli, KA, Pukite, I, Shepherd, RW, Ueno, T, Schmitt, L, Wiek, C, Hanenberg, H, Häussinger, D & Kubitz, R 2016, 'Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency', Hepatology, vol. 63, no. 2, pp. 524-537. https://doi.org/10.1002/hep.28311
Stindt, Jan ; Kluge, Stefanie ; Dröge, Carola ; Keitel, Verena ; Stross, Claudia ; Baumann, Ulrich ; Brinkert, Florian ; Dhawan, Anil ; Engelmann, Guido ; Ganschow, Rainer ; Gerner, Patrick ; Grabhorn, Enke ; Knisely, A. S. ; Noli, Khalid A. ; Pukite, Ieva ; Shepherd, Ross W. ; Ueno, Takehisa ; Schmitt, Lutz ; Wiek, Constanze ; Hanenberg, Helmut ; Häussinger, Dieter ; Kubitz, Ralf. / Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency. In: Hepatology. 2016 ; Vol. 63, No. 2. pp. 524-537.
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T1 - Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency

AU - Stindt, Jan

AU - Kluge, Stefanie

AU - Dröge, Carola

AU - Keitel, Verena

AU - Stross, Claudia

AU - Baumann, Ulrich

AU - Brinkert, Florian

AU - Dhawan, Anil

AU - Engelmann, Guido

AU - Ganschow, Rainer

AU - Gerner, Patrick

AU - Grabhorn, Enke

AU - Knisely, A. S.

AU - Noli, Khalid A.

AU - Pukite, Ieva

AU - Shepherd, Ross W.

AU - Ueno, Takehisa

AU - Schmitt, Lutz

AU - Wiek, Constanze

AU - Hanenberg, Helmut

AU - Häussinger, Dieter

AU - Kubitz, Ralf

PY - 2016/2/1

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N2 - Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.

AB - Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.

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