Capillarization of hepatic sinusoid by liver endothelial cell-reactive autoantibodies in patients with cirrhosis and chronic hepatitis

Abstract

The special features of liver sinusoidal endothelium (LSE) are crucial for normal liver physiology. Cirrhotic livers, especially in primary biliary cirrhosis (PBC), are characterized by transformation of the LSE into a continuous, vascular type. The transformation is important for disease progression and explains some of the pathological hallmarks of the cirrhotic liver. Here, we investigated the presence of liver sinusoidal endothelial cell (LSEC)-reactive autoantibodies (Abs) in the sera of patients with autoimmune liver diseases, and assessed the ability of these Abs to transform LSE into vascular endothelium. Compared to healthy individuals (9%), significantly higher numbers of patients with PBC (59%; P < 0.001) and autoimmune hepatitis (AIH) (32%; P < 0.05) had Abs against LSECs. Incubation of primary LSEC cultures with F(ab')(2) fragments of anti-LSEC Abs isolated from sera of patients with PBC and AIH, induced 1) cell surface expression of vascular endothelium-associated markers, CD31, and factor VIII-related antigen; 2) significant production of fibronectin, laminin and collagen type IV; 3) loss of fenestrae, formation of tight junctions and Weibel-Palade bodies. Deposition of immunoglobulins on LSECs were found in liver biopsies of AIH and PBC patients. Thus, anti-LSEC autoAbs transform LSE into a vascular type and may therefore play an important role in the development of hepatocellular failure and portal hypertension in PBC and AIH patients.

Figure 1.

a: Scanning electron micrograph showing liver endothelial cells cultured in transwell tissue culture inserts displaying fenestrations (arrowheads) in the cytoplasm. b: TEM micrograph showing an endothelial cell (C) attached to the membrane (M) without any matrix (*). c: No formation of tight junctions are seen. Bars: a, 1 μm; b, 200 nm; c, 1 μm.

Figure 2.

a: Normal LSECs did not express CD31 (dark gray line), FVIIIRAg (light gray line), and VCAM (dashed line) but expressed ICAM (dotted line). b: Treatment of LSECs with cytokines TNF-α and IFN-γ did not induce the cell surface expression of these molecules. c: CD31 (dark gray line) but not FVIIIRAg (light gray line) was expressed intracellularly in normal saponin-treated LSECs. d: Treatment of LSECs with anti-LSEC Abs from PBC and AIH patients induced the expression of CD31 (dark gray line), FVIIIRAg (light gray line), and VCAM (dashed line), and increased expression of ICAM (dotted line). Secondary antibodies served as negative control (black line). A representative histogram from one patient is shown.

Figure 3.

a: Normal liver sinusoidal endothelial cells did not produce the basement membrane components collagen type IV or laminin, nor did treatment with immunoglobulin fractions from normal control sera induce their production. However, some deposition of fibronectin was observed. b and c: Intense staining for the deposition of fibronectin, collagen type IV, and laminin was observed by LSECs after treatment for 72 hours with anti-LSEC Abs from PBC and AIH patients, respectively. A representative result from each patient group is shown.

Figure 4.

Western blot analysis of cell lysates from 1 × 10⁶ untreated LSECs (lane 1), LSECs treated with IgG F(ab)₂ fractions from PBC patients (lane 2), LSECs treated with IgG F(ab)₂ fractions from AIH patients (lane 3), and protein controls (lane 4) stained with anti-fibronectin, anti-collagen type IV, and anti-laminin antibodies. Controls consisted of recombinant human fibronectin, collagen type IV, and laminin (lane 4). Under reducing conditions fibronectin was detected as a Mr 220,000 band, collagen type IV as Mr 180,000 band and laminin as a Mr 200,000 band. Normal LSECs expressed small amounts of fibronectin but not collagen type IV and laminin. However, anti-LSEC Abs induced expression of all three basement membrane proteins. A representative result from each patient group is shown.

Figure 5.

TEM and SEM micrographs of transformed cultured LSECs after treatment with anti-LSECs from PBC and AIH patients. a: TEM micrograph showing adjacent endothelial cells containing endothelial-specific granulae (G) (Weibel-Palade bodies) and pinocytotic vesicles (arrowheads). N, nucleus. b: Enlargement from the area marked as a box showing a tight junction (arrowheads) between two endothelial cells. G, Weibel-Palade bodies. c: SEM micrograph showing loss of fenestrae. Bars: a, 1 μm; b, 100 nm; c, 1 μm.

Figure 6.

Staining of rat kidney tissue for anti-mitochondrial antibodies showed the presence of these antibodies before (a) and even after (b) absorption with LSECs of immunoglobulin fractions from PBC patients. c: A representative histogram of flow cytometric analysis from one PBC patient showing the presence of reactivity to HUVECs and HKMECs before (dashed lines) and after (dotted lines) absorption with LSECs. However, all reactivity to LSECs was completely removed after absorption. Similar results were observed with AIH patients.

Figure 7.

Gene expression of CD31, FVIIIRAg, and ABL in HUVECs, unstimulated LSECs and LSEC treated with the F(ab)₂ fragments of anti-LSEC Abs from PBC/AIH patients (LSEC+Ab). ABL is an endogenous reference gene used for correction of variations in cell number. Abs-treated LSEC had higher gene expression of CD31 and FVIIIRAg as compared to unstimulated LSEC. Quantitative analysis with real-time RT-PCR showed that the expression of CD31 and vWF had increased 3 log and 4.5 log, respectively.

Figure 8.

a: Staining pattern of sinusoidal endothelial cells in normal, PBC, and AIH liver sections. Sections were stained with anti-DC-SIGN2, an antibody specific for sinusoidal endothelial cells of the liver. b: Staining liver sections with anti-human IgG and IgM showed weak or almost no deposition of immunoglobulins in the sinusoidal endothelial cells of normal or PSC specimens. However, intense deposition of IgG and IgM on sinusoidal endothelial cells in the liver sections of PBC and AIH patients was observed. c: Double staining of PBC and AIH liver sections with anti-human IgG and IgM (CY-3, red) and anti-DC-SIGN2 (FITC, green) clearly showed coincident sites of reactivity (yellow) in many areas. Thicker and more intense deposition of immunoglobulins was observed in PBC livers as compared to AIH specimens.