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. 2000 Apr;46(4):553–561. doi: 10.1136/gut.46.4.553

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

L Muratori 1, M Parola 1, A Ripalti 1, G Robino 1, P Muratori 1, G Bellomo 1, R Carini 1, M Lenzi 1, M Landini 1, E Albano 1, F Bianchi 1
PMCID: PMC1727874  PMID: 10716687

Abstract

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy

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Figure 1  .

Figure 1  

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Immunoblotting reactivity of a representative liver/kidney microsomal antibody type 1 (LKM1) positive serum with recombinant human CYP2D6 (rCYP2D6) and human liver microsomes (HLM). Serum reacts with a 91 kDa polypeptide, corresponding to rCYP2D6 expressed in Escherichia coli and with a 50 kDa polypeptide corresponding to native human CYP2D6 (A). After preadsorption with rCYP2D6, the reactivity with rCYP2D6 is completely lost, whereas a faint 50 kDa band is still visible with native CYP2D6 (B).

Figure 2  .

Figure 2  

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Confocal microscope images of plasma membrane staining by liver/kidney microsomal antibody type 1 (LKM1) positive sera in freshly isolated rat hepatocytes. The hepatocytes were probed with LKM1 positive sera (diluted 1:50) and fluorescein labelled goat anti-human IgG (diluted 1:200). The images correspond to: (A) non-permeabilised hepatocytes exposed to the serum of a healthy control; (B) permeabilised hepatocytes probed with the serum of a LKM1 positive subject; (C-F) non-permeabilised hepatocytes probed with LKM1 positive sera from a patient with AIH (C,D) or a patient with chronic HCV infection (E,F). Images D and F were obtained by merging nine focal frames taken along the z axis at 1 µm intervals. Images B, C, and E show the focal frame corresponding to the median optical section of the hepatocytes. Fluorescence is shown in pseudocolours increasing from blue to red and white. Original magnifications are 30 × for images A, B, E, and F and 60 × for images C and D. 

Figure 3  .

Figure 3  

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Frequency distribution of the pixels referring to cellular fluorescence in optical sections from hepatocytes stained with liver/kidney microsomal antibody type 1 (LKM1) positive serum (diluted 1:40) and fluorescein labelled goat anti-human IgG (diluted 1:200). (A) Typical distribution in non-permeabilised hepatocytes; (B) distribution in hepatocytes that were permeabilised before fixation as reported in the Methods section.

Figure 4  .

Figure 4  

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Effect of the preadsorption of liver/kidney microsomal antibody type 1 (LKM1) positive sera with recombinant human CYP2D6 on the surface staining of isolated rat hepatocytes. LKM1 sera were preadsorbed as described in the Methods section with human recombinant CYP2D6. Non-permeabilised rat hepatocytes were stained with native (A) and preadsorbed (B) sera at the same dilution (1:200). Original magnification × 10. 

Figure 5  .

Figure 5  

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Confocal microscope images of the same focal section of adjacent rat hepatocytes allowed to react with rabbit anti-CYP2D6 serum and liver/kidney microsomal antibody type 1 (LKM1) positive human serum. The cell preparation was double stained with goat anti-human IgG labelled with fluorescein and with goat anti-rabbit IgG complexed with Texas Red. The same median cell optical section shows the green fluorescence due to fluorescein in (A) and the red fluorescence of Texas Red in (B). (C) Result of the electronic merging of images (A) and (B). Original magnification × 30. 

Figure 6  .

Figure 6  

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Immunoprecipitation of antigens recognised by human liver/kidney microsomal antibody type 1 (LKM1) sera on hepatocyte plasma membranes. Plasma membrane antigens of isolated hepatocytes were immunoprecipitated by using human LKM1 positive sera from either autoimmune or HCV related hepatitis patients complexed with Protein A coated Sepharose beads. The precipitated proteins were submitted to SDS/PAGE and visualised using a rabbit polyclonal anti-human CYP2D6 serum. Lane 1, control beads without attached LKM1 serum; lanes 2 and 3, two different LKM1 positive sera from patients with autoimmune hepatitis; lanes 4 and 5, two different LKM1 positive sera from patients with hepatitis C virus (HCV) hepatitis. G6Pase, glucose-6-phosphatase.

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