Volume 126, Issue 3, Pages 849-858 (March 2004) Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome  Susanne Strand, Dennis Strand, Rudolf Seufert, Amrit Mann, Johannes Lotz, Manfred Blessing, Michael Lahn, Andreas Wunsch, Dieter C. Broering, Uwe Hahn, Eva-Maria Grischke, Xavier Rogiers, Gerd Otto, Gregory J. Gores, Peter R. Galle  Gastroenterology  Volume 126, Issue 3, Pages 849-858 (March 2004) DOI: 10.1053/j.gastro.2003.11.054

Figure 1 Apoptosis in HELLP livers. Similar pattern of TUNEL+ apoptotic cells (green) in a (A) HELLP liver and (B) a mouse liver injected with anti-CD95 antibody. In addition, nuclei are stained in blue. Lower panel shows H&E staining of identical field shown in the upper panel. A selected subset of apoptotic hepatocytes are indicated with arrows. Bar represents 20 μm. (C) Activation of caspase 3, 9, and 8 in hepatocyte extracts from HELLP patients and normal liver tissues (∗∗∗P < 0.001, ∗∗P < 0.01). Caspase activity was measured by release of the fluorochrome AFC from peptide substrates selective for caspase 8 (Ac-IETD-AFC), caspase 9 (Ac-LEHD-AFC), and caspase 3 (Ac-DEVD-AFC). Results are expressed as means ± standard error. Gastroenterology 2004 126, 849-858DOI: (10.1053/j.gastro.2003.11.054)

Figure 2 Cytotoxicity of HELLP sera using (A) Jurkat cells or (B) primary human hepatocytes. Cells were incubated for 48 hours with 20% serum from healthy nonpregnant and pregnant women or 20% serum from HELLP patients and cytotoxicity was determined by using MTS-cytotoxicity assay. Cytotoxicity is increased in HELLP sera (∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05). (C) FACS analysis for apoptosis in Jurkat cells after 48 hours incubation with serum taken from sources indicated above panel. (D) Jurkat cells inactivated for CD95 signaling (Jurkat−Flice, Jurkat−Fadd) have reduced sensitivity to HELLP serum cytotoxicity (∗∗∗P < 0.001, ∗∗P < 0.01). Gastroenterology 2004 126, 849-858DOI: (10.1053/j.gastro.2003.11.054)

Figure 3 Toxicity in HELLP sera can be reduced by a blocking antibody against (A) CD95L or (B) LY498919 (∗∗P < 0.01). Serum was preincubated for 1 hour with 4 μg/mL with the anti-CD95L antibody NOK-1 (A) or with 1 μg/mL LY498919 (B) before application to primary human hepatocytes. Cytotoxicity was determined by MTT assay. Gastroenterology 2004 126, 849-858DOI: (10.1053/j.gastro.2003.11.054)

Figure 4 (A) Patient that developed postpartum HELLP. (B) Serum taken at the indicated times showed increased cytotoxicity for human hepatocytes. Cytotoxicity was determined by MTS assay after incubation of human hepatocytes for 48 hours with 20% serum from the patient. Gastroenterology 2004 126, 849-858DOI: (10.1053/j.gastro.2003.11.054)

Figure 5 (A) CD95L detection by enzyme-linked immunosorbent assay in fractions after gel-sieving chromatography of a patient’s serum before and after development of HELLP. Two microliters of serum was loaded on a superose 12 column, and 0.5-mL fractions were collected. CD95L concentration in the fractions was determined by enzyme-linked immunosorbent assay. The results are derived from a typical experiment of 3 performed. The elution positions of standard proteins are shown left to right: thyroglobulin (667 kD), yeast alcohol dehydrogenase (150 kD), and bovine serum albumin (66 kD). (B) Cytotoxic activity of HELLP serum fractions for Jurkat cells. Fractions containing CD95L (Fr. 14) or before and after elution of CD95L (Fr. 10 and 20). The highest cytotoxicity is in the fractions with CD95L. ∗∗∗P < 0.001. Gastroenterology 2004 126, 849-858DOI: (10.1053/j.gastro.2003.11.054)

Figure 6 (A) Reverse-transcriptase PCR of placenta from normal and HELLP patients showing similar CD95L messenger RNA expression. (B) Confocal laser scanning image of placenta villous immunostained for CD95L. CD95L (red) is mainly localized in membranes of syncytiotrophoblast cells bordering maternal blood spaces. Nuclei were stained in blue. Bar represents 50 μm. (C) Placenta extract is toxic for Jurkat cells but not toxic for Jurkat−Flice and Jurkat−Fadd cells with inactivated CD95 signaling (∗∗P < 0.01, ∗P < 0.05). Twenty percent placental extract was incubated for 24 hours with Jurkat cells. Cytotoxicity was determined by MTS assay. (D) The molecularly engineered DcR3 decoy receptor, LY498919, protects mice against liver damage induced by exposure to placenta extract. Intraperitoneal injection of mice with mouse placental extract increases serum ALT levels (left panel) and liver caspase-3 activity (right panel) after 6 hours. LY498919 prevented placenta extract-induced ALT release and caspase 3 activation (∗P < 0.05). (E) Apoptotic hepatocytes after placenta injection were visualized by TUNEL assay (green). Arrows indicate apoptotic hepatocytes. Bar represents 20 μm. Gastroenterology 2004 126, 849-858DOI: (10.1053/j.gastro.2003.11.054)