Chemiluminescence Quantitative Immunohistochemical Determination of MRP2 in Liver Biopsies
Department of Pharmaceutical Sciences (MG,AR), Pathology Unit of the "F. Addarii" Institute of Oncology (MM,SC,WFG), and Department of Internal Medicine and Gastroenterology (ER), University of Bologna, Bologna, Italy
Correspondence to: Prof. Aldo Roda, Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy. E-mail: aldo.roda{at}unibo.it
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Summary |
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Key Words: biopsy chemiluminescence MRP2 imaging immunohistochemistry liver protein expression
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Introduction |
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An alternative, indirect approach consists in the quantification of the mRNA encoding the target protein through real-time PCR (Bustin 2000). This technique is very sensitive, but working with sterile material is needed to avoid the contamination or degradation of mRNA. In addition, low mRNA contents cannot be easily quantified by real-time PCR because of the formation of nonspecific amplification products that can mask weak specific signals. Also, possible posttranscriptional effects on protein expression cannot be detected by real-time PCR.
A common feature of all these techniques is the presence of preanalytical protein or nucleic acid extraction steps, which lengthen the overall analytical process and may reduce the assay reproducibility. In addition, a minimum amount of sample is required, which could represent a drawback when protein expression has to be evaluated in tiny samples, such as tissue samples from biopsies. It should also be considered that the amount of sample available for analysis could be further reduced if other assays have to be performed or sample archiving is required. A direct, simple, and rapid method for the quantitative evaluation of protein expression in small tissue samples would thus be helpful.
Immunohistochemistry (IHC) techniques with colorimetric and fluorescent detection are routinely used in research and diagnostics for the localization of proteins or other antigens in tissue sections. These techniques are simple, fast, and, if provided a suitable detection antibody, adequately sensitive for most applications. However, they only permit qualitative or semiquantitative analysis of protein expression. Evaluation of samples could also be subjective; indeed, for IHC diagnostics two or more independent assessments from trained personnel are usually required. Thus, quantitative IHC analysis of protein expression requires a sensitive detection principle that permits an objective and quantitative evaluation of the bound antibody.
Chemiluminescence (CL) detection of enzyme labels using chemiluminescent substrates fulfils these requirements: it is much more sensitive than colorimetry, highly specific, and with low background (differently, for example, from fluorescence, which is affected by sample autofluorescence). In addition, the intensity of the CL signal is proportional to the amount of the enzyme label over a wide range of concentration (Kricka 1995; Roda et al. 2000
). Low-light imaging devices, such as thermoelectrically or cryogenically cooled charge-coupled device (CCD) cameras (Lerner 2001
; Van Dyke and Woodfork 2002
), allow the quantitative measurement of the CL signal, even at the single-photon level, and can be connected with an optical microscope to evaluate the spatial distribution of the CL emission from a given tissue sample at the microscopic level (Christenson 2002
). In optimized experimental conditions, the spatial resolution achievable with CL imaging microscopy can be as low as 1 µm (Roda et al. 1996
), thus comparable to that of conventional or fluorescence optical microscopy. Chemiluminescence imaging microscopy thus represents an ultrasensitive analytical tool to localize and quantify biomolecules in single cells and tissue sections. It has been used for the detection of antigens, gene sequences, enzymes, and metabolites in cells and tissue sections by means of immunohistochemical, in situ hybridization, and enzyme or chemical reactions (Roda et al. 2000
,2002
; Creton and Jaffe 2001
).
Despite these characteristics, applications of CL imaging microscopy mainly regarded the sensitive localization of analytes, rather than their quantitative evaluation. The aim of the present study is to demonstrate the potential of CL IHC imaging microscopy for the direct assessment of protein expression levels in tissues. For this purpose, we selected the MRP2 transport protein, a member of the human multidrug resistanceassociated protein family, which represents the most important hepatocellular transporter involved in the excretion of organic anions into bile (Taniguchi et al. 1996). This protein is localized at the apical (canalicular) hepatocyte membrane and mediates the hepatobiliary excretion of sulfated or glucuronidated bile salts, bilirubin glucuronides, glutathione conjugates, and drug metabolites (Madon et al. 1997
). Alteration of MRP2 expression or function may play an important role in the pathogenesis of cholestasis (Trauner et al. 1997
; Kullak-Ublick and Meier 2000
). Indeed, a mutation in the MRP2 gene can cause hereditary cholestasis (Dubin-Johnson syndrome) in humans (Kartenbeck et al. 1996
), and experimental studies in various animal models have shown that a reduced MRP2 expression may contribute to an impaired hepatic excretory function resulting in cholestasis (Paulusma and Oude Elferink 1997
). Expression of MRP2 and other transport proteins in liver tissues has been extensively studied by means of Western blot analysis and real-time PCR techniques (Zollner et al. 2003
), and a reduced MRP2 expression has been also observed in patients affected by primary biliary cirrhosis (PBC), a chronic human liver disease characterized by inflammation and progressive destruction of the small intrahepatic bile ducts resulting in chronic cholestasis (Lee and Boyer 2000
; Kullak-Ublick et al. 2002
).
The aim of the present study is the development of a CL IHC procedure for the direct quantitative evaluation of MRP2 expression in tissue sections from formalin-fixed, paraffin-embedded liver biopsies. Immunohistochemical detection of MRP2 was accomplished by means of a monoclonal anti-MRP2 mouse antibody, followed by a biotinylated anti-mouse secondary antibody and a streptavidin-horseradish peroxidase (HRP) conjugate revealed using a sensitive enzyme substrate based on the luminol/H2O2/enhancer CL system. The assay has been optimized and its main analytical performance in terms of reproducibility and accuracy were carefully evaluated. The CL IHC method was thus applied to study MRP2 expression levels in patients with PBC treated with ursodeoxycholic acid (UDCA). UDCA, an hydrophilic poorly detergent, nontoxic bile acid that represents a minor component of the human bile acids pool, demonstrated its ability to slow the progression of PBC toward its terminal phase (Paumgartner and Beuers 2002). Clinical studies (Zollner et al. 2001
; Kojima et al. 2003
) showed that MRP2 levels increase during treatment with UDCA, and it was proposed that the increased expression of MRP2 ameliorates the impairment of biliary organic anion secretion, thus limiting hepatocellular accumulation of potentially toxic biliary constituents (Shoda et al. 2001
). The obtained results were in agreement with those previously reported in the literature, thus proving the suitability of the CL IHC assay for the fast and direct evaluation of protein expression in tissues.
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Materials and Methods |
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Reagents
The anti-MRP2 monoclonal mouse antibody (M2III-6) was obtained from ALEXIS Biochemicals (San Diego, CA), whereas the nonspecific binding blocking solution (DAKO Protein block serum-free) and the HRP-based mouse antibody detection system (DAKO LSAB Kit) were from DAKO (Hamburg, Germany). The latter is a two-step detection system for primary mouse and rabbit antibodies, which employs biotinylated species-specific secondary antibodies and a streptavidin-HRP conjugate. Chemiluminescent and colorimetric detection were performed using ECL, a commercial CL substrate for HRP based on the luminol/hydrogen peroxide/enhancer CL system (Amersham Bioscience; Little Chalfont, England) and the chromogenic HRP substrate 3,3'-diaminobenzidine (DAKO Liquid DAB + Substrate-Chromogen System). Hematoxylin solution for slides counterstaining was also from DAKO. All other chemicals were of analytical reagent grade.
Immunohistochemistry
For the IHC detection of MRP2, 3-µm tissue sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase activity was blocked using 0.3% H2O2 in methanol for 30 min. Antigen retrieval was performed by treatment with 1 mM EDTA buffer (pH 8.0) in a microwave oven for 5 min (350 W) + 3 min (750 W). Paraffin slides were incubated at 25C with the blocking solution for 10 min, followed by 1:30 (v/v) anti-MRP2 antibody (60 min), biotinylated secondary antibody (30 min), and HRP-streptavidin conjugate (30 min). Negative controls were performed by omitting one of the components of the immunodetection system (anti-MRP2 antibody, biotinylated secondary antibody or HRP-streptavidin conjugate). After each step, slides were washed (3 x 5 min) with TBS buffer (10 mM Tris-HCl, pH = 7.4, containing 150 mM NaCl).
Chemiluminescence Detection
Chemiluminescence detection was performed using an epifluorescence microscope (BX 60; Olympus Optical, Tokyo, Japan) connected to a slow-scan, ultrasensitive CCD camera (LN/CCD Princeton Instruments; Roper Scientific, Trenton, NJ). The camera was equipped with a 512 x 512 pixel detector cooled to 100C to reduce background noise. Resolution of the acquired images varied from 2.5 (10x objective) to 0.6 µm/image pixel (40x objective). An OptiScan ES103 motorized microscope stage (Prior Scientific Instruments Ltd.; Fulbourn, England) allowed reproducible positioning of the slides after processing. The microscope was also enclosed in a dark box to avoid interference from ambient light. For the CL measurement the substrate solution (usually 2550 µl) was added to the slides until the tissue sections were covered, then the CL image was acquired. Image processing and quantitative analysis for the assessment of the localization of MRP2 were performed using the image analysis software Metamorph v. 4.5 (Universal Imaging Corporation; Downington, PA).
Colorimetric Detection
Colorimetric detection of bound HRP was performed using the HRP chromogen 3,3'-diaminobenzidine; slides were then counterstained with hematoxylin. Sections previously used for CL measurement could also be stained by providing a brief washing in TBS buffer to remove the CL substrate. Transmitted light images of stained slides were obtained with the same microscope used for CL measurements, employing a RGB filter (CRI Inc.; Woburn, MA) that allowed acquisition of separate grayscale images corresponding to the RGB colors. The three gray-scale images were then combined into a single high-quality color image by means of the image analysis software.
Analysis of Clinical Samples and Data Processing
Two tissue sections from each biopsy were processed as described previously. For each section, images of the CL signal were acquired from three randomly selected fields using a 5-min exposure time and a 10x objective magnification. Image acquisition started after 12 min on addition of the CL substrate and was completed within 20 min. The area corresponding to the tissue section was defined in each image and the CL signal from this area (expressed in relative light units, which are proportional to the number of emitted photons) was evaluated. Visually damaged tissue areas were, if necessary, manually excluded from the integration area. The real CL signal was then determined by subtracting the background CL signal, measured in a tissue-free area of the image. Finally, the results of the six fields were averaged to obtain the CL signal of the biopsy. Statistical comparison of the CL signals of biopsies was performed using Student's t-test.
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Results |
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Figure 1 shows representative images of MRP2 localization in liver tissue sections performed by means of CL and colorimetric detection. The CL image (Figure 1A) presents localized CL signals over a weak diffuse background, which is consistent with the expected localization (i.e., the canalicular membranes of the hepatocytes) of MRP2. The specificity of the CL detection has been assessed by performing control experiments in the absence of one of the components of the immunodetection system (primary anti-MRP2 monoclonal antibody, biotinylated secondary antibody, or HRP-streptavidin conjugate). No detectable CL signal has been observed in all cases, thus demonstrating the absence of nonspecific binding of the immunoreagents to the tissue, as well as of interference by endogenous peroxidase activity and background emission from the CL substrate. The localization of the CL signal in the canalicular membranes of the hepatocytes has also been confirmed by comparison with the conventional colorimetric detection of MRP2 (Figure 1B).
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The quantitative evaluation of the MRP2 expression level requires a standardized CL measurement procedure.
To define this procedure, we first studied the kinetics of the CL signal. In fact, a steady-state CL emission is preferable because a rigid control of the measurement time is not required to obtain a good reproducibility, and repeated measurements of the same sample are possible. Because the kinetics of the CL signal may depend on its intensity (for example, the fast consumption of the substrate in the presence of an intense emission could determine the decay of the CL signal), we measured the time behavior of the emission in samples with different signals (Figure 2). The data showed that, regardless of the intensity of the emission, the CL signal reached a steady-state value after a few minutes of the addition of the substrate; this value was maintained for at least 20 min. Chemiluminescence measurements performed within this time after the addition of the CL substrate thus allowed the reliable evaluation of the CL emission.
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To perform a reliable comparison of the MRP2 expression levels in different samples, we also studied the reproducibility of the CL measurement.
First, we demonstrated that the measurement of a limited number of areas in a given tissue section allowed for the reliable evaluation of the CL signal of the section. We selected a representative set of sections and measured the CL signal in three randomly selected areas for each section. The measured CL signals (Figure 3A) showed little variability within each tissue section (SEM <10%), thus indicating that such a measurement provided a reliable estimation of the mean CL signal of the whole section. It is worthwhile to note that, using a 5-min integration time to acquire the CL images, the overall measurement time for each tissue section was maintained below 20 min, as required by the kinetics of the CL emission.
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Analysis of clinical samples from patients with PBC was performed by measuring the CL signal of at least two sections for each biopsy, then the changes in the MRP2 expression level in biopsies obtained in different years were evaluated by comparison of the CL signal intensities (Figure 4). Among the examined patients, six (46%) showed a statistically significant increase (p<0.05) of the mean CL intensity. For two patients (15%), the CL signal intensity decreases from the first to the second biopsy, whereas for the others the changes in the mean CL intensities (if any) were not statistically relevant.
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Discussion |
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We applied this technique for the measurement of MRP2 levels in patients affected by PBC and treated with UDCA. The results obtained were in agreement with the data previously reported in the literature that demonstrated an increase in the MRP2 levels following UDCA therapy. We also compared the observed changes in the MRP2 levels and the progression of fibrosis, as deduced from histological examination of biopsies. Indeed, regression of fibrosis stage has been observed mainly for patients with increased MRP2 levels. In fact, out of five patients who showed a regression of the fibrosis, four also gave a statistically significant increase in the mean CL signal. However, this does not necessarily imply a cause and effect relationship between an increase of the MRP2 levels and remission of fibrosis associated to PBC (the main histological parameter used for assessing PBC stage), because the levels of MRP2 may simply reflect the improvement of the overall liver function. More work is required to clarify the relationship between the remission of histological dates of fibrosis and the increased levels of MRP2 in primary biliary cirrhosis and what the role is of UDCA to modulate the expression of transporter in liver.
In conclusion, the developed CL IHC method allowed the direct quantitative evaluation of MRP2 expression in tissue samples from paraffin-embedded liver biopsies by combining the specificity of the IHC reaction with the easy quantitative evaluation of the signal peculiar to CL detection, thus offering significant advantages in comparison to other detection techniques commonly used in IHC (i.e., colorimetry and fluorescence) that permit only qualitative or semiquantitative analysis. The high detectability and the low intrinsic background of CL detection make this method also suitable for the analysis of proteins less abundantly expressed than MRP2, even if as the concentration of the target protein decreases, the interference from the possible aspecific binding of the labeled immunoreagent could become important, and a careful optimization of the experimental procedure would be required to obtain reliable results.
Chemiluminescent IHC could thus represent an alternative technique to Western blotting and real-time PCR for the quantitative evaluation of protein expression in tissue sections. Moreover, the applicability of this technique to formalin-fixed, paraffin-embedded tissue samples makes IHC CL suitable to perform retrospective or epidemiological studies on libraries of archived samples.
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Footnotes |
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Literature Cited |
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