Stable expression of human muscle-specific kinase in HEp-2 M4 cells for automatic immunofluorescence diagnostics of myasthenia gravis

Abstract

Muscle-specific kinase (MuSK) belongs to the nicotinic acetylcholine receptor complex which is targeted by pathogenic autoantibodies causing Myasthenia gravis. While up to 95% of patients with generalized Myasthenia gravis were shown to be positive for acetylcholine receptor-specific autoantibodies, up to 70% of the remaining patients develop autoantibodies against MuSK. Discrimination of the autoantibody specificity is important for therapy of Myasthenia gravis. Recently, the new automatic fluorescence assessment platform AKLIDES has been developed for immunofluorescence-based diagnostics of autoimmune diseases. In order to establish an AKLIDES procedure for the detection of MuSK-specific autoantibodies (anti-MuSK), we developed a recombinant HEp-2 cell clone expressing the human MuSK cDNA. Here we show at the mRNA and protein level that the cell clone HEp-2 M4 stably expresses human MuSK. We provide evidence for a localization of MuSK at the cell membrane. Using cell clone HEp-2 M4 on the AKLIDES system, we investigated 34 patient sera that were previously tested anti-MuSK positive by radioimmunoassay as positive controls. As negative controls, we tested 29 acetylcholine receptor-positive but MuSK-negative patient sera, 30 amytrophic lateral sclerosis (ALS) patient sera and 45 blood donors. HEp-2 M4 cells revealed a high specificity for the detection of MuSK autoantibodies from 25 patient sera assessed by a specific pattern on HEp-2 M4 cells. By using appropriate cell culture additives, the fraction of cells stained positive with anti-MuSK containing sera can be increased from 2-16% to 10-48%, depending on the serum. In conclusion, we provide data showing that the novel recombinant cell line HEp-2 M4 can be used to screen for anti-MuSK with the automatic AKLIDES system.

Figure 1. HEp-2 M4 cells keep morphology and proliferation characteristics of parental HEp-2 cells.

A. Phase contrast images of exponentially growing transfected HEp-2 M4 and non-transfected HEp-2 parental cells. Images were obtained with microscope CKX41 and 10× objective (scale bar 50 µm). B. Cell doubling times of exponentially growing cultures of both cell lines were determined as described in materials and methods.

Figure 2. Detection of MuSK expression in HEp-2 M4 cells.

A. Exponentially growing transfected clone HEp-2 M4 and non-transfected parental HEp-2 cells were used for RNA preparation and RT-PCR analysis of heterologously expressed MuSK, with (+) or without (−) reverse transcriptase (RT), as described in materials and methods. 1, HEp-2 M4+RT; 2, HEp-2 M4−RT; 3, HEp-2+RT; 4, HEp-2−RT. Amplicons were analyzed by agarose electrophoresis with 50 bp DNA size marker as control (5). B. HEp-2 M4 cells growing on glass slides were fixed and processed for indirect immunofluorescence with anti-V5 antibody to detect expression of the MuSK-V5 fusion protein as described in the methods section. Photo was taken by AKLIDES system (10× objective, TRANSFECT mode). C. Living HEp-2 M4 cells grown on glass slides were incubated with commercial anti-MuSK primary antibody followed by fixation and secondary antibody staining as described in the methods section. The image was taken by an Olympus fluorescence microscope IX81 (40× objective). MuSK-specific signals appear as characteristic speckled pattern. D. Living HEp-2 M4 cells grown on glass slides were incubated with anti-MuSK autoantibody positive patient sera (1) and further processed for double immunofluorescence staining as described in materials and methods. A second serum pretested by RIA to be negative for anti-MuSK autoantibody (2) was used as negative control. Detection of the V5 epitope by V5-specific primary antibody and Cy3 labeled secondary antibodies, and simultaneous detection of MuSK with patient autoantibodies and Alexa Fluor 488 conjugated secondary antibodies is illustrated as indicated with V5 (Cy3) and patient serum (FITC channel used), respectively. Double immunofluorescence staining images were obtained by merging both images as indicated (merge). The double immunofluorescence staining analysis shows strong evidence for specific binding of MuSK autoantibodies to only those HEp-2 M4 cells expressing the MuSK-V5 fusion protein. Images were taken by the Olympus fluorescence microscope IX81 (40× objective). Scale bar 50 µm.

Figure 3. ESM increases MuSK expression in HEp-2 M4 cells.

A. Exponentially growing HEp-2 M4 cells were treated (right chart) or not treated (left chart) with the epigenetic supplement mixture (ESM) as indicated. Cell cycle phases were analyzed by FACS. Although there is an increase of cells in G2/M phase with ESM, this treatment apparently does not induce cell cycle arrest. B. Cell extracts from exponentially growing transfected HEp-2 M4 or untransfected parental HEp-2 cells were processed for Western Blotting. Cells had been treated or not with the epigenetic supplement mixture (ESM) as indicated. Detection of the MuSK-V5 fusion protein was performed as described in the materials section. Expected band of expressed MuSK is indicated by arrow, other bands are probably degradation products. C. Clone HEp-2 M4 or parental HEp-2 cells were treated or not with ESM as indicated, and MuSK expression was analyzed by immunofluorescence using MuSK autoantibody-positive patient serum (1). A serum pretested by radioimmunoassay to be negative for anti-MuSK autoantibodies was used as control (2). Immunofluorescence images were evaluated by AKLIDES system (10× objective, TRANSFECT modus).

Figure 4. Anti-MuSK detection on HEp-2 M4 cells.

HEp-2 M4 cells were seeded onto glass slides, and living cells were cultivated in the presence or absence of ESM. Cells were incubated with MG patient sera pretested by RIA to contain anti-MuSK autoantibodies or with control sera. Cells were further processed for indirect immunofluorescence testing by using the protocol described in the materials section along with AKLIDES system (10× objective, TRANSFECT modus). Each experiment was repeated two or three times, respectively. Four photographs were taken automatically by AKLIDES from each well and evaluated manually as described in in the materials section. Anti-MuSK autoantibody binding of HEp-2 M4 treated with ESM (1) was compared to HEp-2 M4 (2) and HEp-2 cells (3), both without ESM treatment. ESM-treated HEp-2 M4 were incubated with MG patient sera positive for anti-AChR (4), sera from ALS patients (5) and sera from healthy blood donors (6). The control groups were also tested on HEp-2 M4−ESM as well as on HEp-2 cells, but only the results for HEp-2 M4+ESM are shown. The median is indicated as line. ESM-treated HEp-2 M4 cells incubated with anti-MuSK antibodies showed significantly more positive cells compared to AChR control group (p value = 2.7E-5).