Dig Dis Sci DOI 10.1007/s10620-014-3203-6
ORIGINAL ARTICLE
Specific Expression of Apolipoprotein A-IV in the Follicle-Associated Epithelium of the Small Intestine Daisuke Tokuhara • Tomonori Nochi • Akiko Matsumura Mio Mejima • Yuko Takahashi • Shiho Kurokawa • Hiroshi Kiyono • Yoshikazu Yuki
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Received: 25 April 2013 / Accepted: 2 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Peyer’s patches (PPs), which are covered by specialized follicle-associated epithelium (FAE) including M cells, play a central role in immune induction in the gastrointestinal tract. This study is to investigate a new molecule to characterize PPs. Methods We generated a monoclonal antibody (mAb 10-15-3-3) that specifically reacts to the epithelium of PPs and isolated lymphoid follicles. Target antigen was analyzed by immunoprecipitation and mass spectrometry. Localization and expression of target antigen were evaluated by immunofluorescence, in situ hybridization and realtime PCR. Results Immunoprecipitation and mass spectrometry revealed that mAb 10-15-3-3 recognized apolipoprotein A-IV (ApoA-IV), a well-known lipid transporter; this finding was confirmed by the specific reactivity of mAb 10-15-3-3 to cells transfected with the murine ApoA-IV gene. Immunofluorescence using mAb 10-15-3-3 showed intestinal localization of ApoA-IV, in which strong expression of the ApoA-IV protein occurred throughout the
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3203-6) contains supplementary material, which is available to authorized users. D. Tokuhara T. Nochi A. Matsumura M. Mejima Y. Takahashi S. Kurokawa H. Kiyono Y. Yuki (&) Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan e-mail: [email protected] D. Tokuhara Department of Pediatrics, Osaka City University Graduate School of Medicine, Osaka, Japan
entire intestinal epithelium during developing period before weaning but was restricted to the FAE in adult mice. In support of these findings, in situ hybridization showed strong expression of the ApoA-IV gene throughout the entire intestinal epithelium during developing period before weaning, but this expression was restricted to the FAE predominantly and the tips of villi to a lesser extent in adult mice. Deficiency of ApoA-IV had no effect on the organogenesis of PP in mice. Conclusions Our current results reveal ApoA-IV as a novel FAE-specific marker especially in the upper small intestine of adult mice. Keywords Apolipoprotein A-IV Mucosal immunology Epithelial cell Peyer’s patch
Introduction The follicle-associated epithelium (FAE) that overlies mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses [1–3]. However, the mechanisms involved in this response and in antigen transport within the FAE remain to be elucidated. To gain insight into the roles of FAE and M cells in these mechanisms, we previously generated the monoclonal antibody (mAb) NKM 16-2-4, which binds specifically to M cells located in the FAE of Peyer’s patches (PPs) and which lacks reactivity to UEA-1-positive goblet cells in intestinal villi, and demonstrated that vaccination with antigen-conjugated NKM 16-2-4 effectively induced protective immunity in both the systemic and mucosal compartments by targeting antigen to M cells [1]. The identification of new FAE- and M-cell-specific molecules likely will contribute toward clarifying the molecular mechanisms
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involved in the mucosal immune response and in the transport of antigens through FAE and by M cells. To acquire additional information regarding the organogenesis of FAE, we here generated a mAb that reacts specifically to this tissue. Using mass spectrometry and transfectant analysis, we determined that the antigen recognized by this mAb is apolipoprotein A-IV (ApoA-IV) and revealed the localization of ApoA-IV expression at the protein and gene levels. ApoA-IV is a component of chylomicron and high-density lipoprotein and thus functions as a lipid transporter molecule [4]; in addition, ApoA-IV is a satiety factor [5] and anti-atherogenic factor [6] in rodents. The expression of the ApoA-IV gene has been studied extensively and reportedly occurs in the villous epithelium (VE) [7–9] and liver of rodents [7, 8]; however, little information regarding ApoA-IV protein expression is available currently [10, 11]. Our immunofluorescence analysis using the anti-ApoA-IV mAb demonstrated the age-dependent expression of ApoA-IV in murine small intestine. Our results reveal ApoA-IV as a novel intestinal FAE-specific marker in adult mice and suggest ApoA-IV as a physiologic signaling or immunomodulatory molecule in the primary immune inductive site of the small intestine.
Materials and Methods Animals Female C57BL/6J and BALB/c mice (pregnant and 1–7 weeks old) were purchased from Japan SLC. Male and female ApoA-IV-/- mice on a C57BL/6J background were purchased from the Jackson Laboratory. All of the mice used in this study were housed in the experimental animal facility at the Institute of Medical Science (University of Tokyo) on a standard 12:12-h light/dark cycle and had ad libitum access to water and food containing 4 % fat. All experiments were approved by the Ethics Committee of the University of Tokyo. Generation of Monoclonal Antibody The FAE-enriched fraction was prepared from murine PPs as previously described [1], with slight modification, by using UEA-1(Biogenesis) and NKM 16-2-4. In brief, cells isolated from murine PPs were fixed in 4 % paraformaldehyde (Wako) and stained with PE-conjugated UEA-1 and FITCconjugated NKM 16-2-4; each antibody was used at 500 ng/ mL. UEA-1- and NKM 16-2-4-positive cells were sorted by using flow cytometry (FACSAria, Becton–Dickinson) and injected into the footpads of Sprague–Dawley rats (106 cells/ rat) 4 times at 2-week intervals, with TiterMax Gold (TiterMax) as an adjuvant. Four days after the final immunization, lymphocytes isolated from the spleen and inguinal
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lymph nodes of the immunized rats were fused with P3X63AG8.653 myeloma cells (CRL-1580; American Type Culture Collection) in the presence of 50 % (wt/vol) polyethylene glycol 1500 (Roche). Established hybridomas were injected into Crlj:CD1-Foxn1nu mice, and mAbs were purified from ascitic fluids by using protein G-Sepharose (GE Healthcare) and labeled with EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific), fluorescein isothiocyanate (FITC, Sigma-Aldrich), or Alexa Fluor 647 (Invitrogen). Immunofluorescence Analysis A mAb (10-15-3-3; rat IgG2a) was selected on the basis of the initial screening and its specificity to FAE of PPs as determined by immunofluorescence analyses, as previously described [1, 12]. In brief, murine small intestinal tissues were perfused intracardially with 4 % paraformaldehyde (Wako) in phosphate-buffered saline (PBS). Perfused small intestines were dissected and further fixed in 4 % paraformaldehyde at 4 °C overnight. Fixed tissues were washed with PBS and incubated in increasing concentrations of cold sucrose (10, 15, and 20 %; total, 24 h). The tissues then were embedded in Tissue-Tek OCT compound (Sakura Finetechnical). Frozen sections (5 lm) were cut and incubated with 1 % BSA for 20 min at room temperature. After a blocking step, each fixed frozen section or fixed tissue sample containing PPs from C57BL/6J, BALB/c, or ApoA-IV-/- mice was stained with 5 lg/mL FITC-conjugated 10-15-3-3 or FITC-conjugated isotype control antibody (FITC-conjugated rat IgG). Some sections were co-stained with 5 lg/mL Alexa-conjugated NKM 16-2-4 to visualize M cells [1]. To examine the immunoreactivities of 10-15-3-3 to FAE at various developmental stages, tissue sections from mice at embryonic day 18 and postnatal day 2 were stained by using 10-15-3-3 followed by antiCD3 and anti-CD4 antibodies (eBioscience) to characterize PP with CD3- CD4? cells as PP inducer cells [13]. These sections were counterstained with 200 ng/mL 40 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 30 min at room temperature and analyzed by using a confocal laser scanning microscope (TCS SP2, Leica, Wetzlar, Germany). To characterize the immunoreactivities of 10-15-3-3 in various organ tissues, samples of brain, lung, liver, spleen, kidney, and colon were immunostained with 10-15-3-3 and counterstained with DAPI. Analysis of Antigen Recognized by 10-15-3-3 To identify the antigen recognized by 10-15-3-3, we performed an immunoprecipitation assay with 10-15-3-3 followed by matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF)/TOF analysis as previously described [12]. In brief, FAE of murine PPs was lysed in
Dig Dis Sci
lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.5 % Triton X-100, and protease inhibitor cocktail [Roche]). After 1 h of incubation on ice followed by centrifugation, the lysate (5 mg/mL, 1 mL) was precleared with 40 lL protein G-Sepharose (1:1 in PBS, Amersham Pharmacia Biotech) at 4 °C for 1 h. After centrifugation, the lysate was incubated with mAb 10-15-3-3 (10 lg/mL) or an isotype-matched control antibody (rat IgG, BD PharMingen) for 1 h at 4 °C before the addition of 40 lL protein G-Sepharose (1:1 in PBS) and incubation for 1 h at 4 °C. Immune complexes were washed five times with cold PBS containing protease inhibitor cocktail, eluted in sample buffer (2 % SDS, 5 % b-mercaptoethanol, 50 mM Tris–HCl [pH 6.8], and 20 % glycerol), and separated by SDS–PAGE on a 12 % NuPAGE Bis–Tris Gel (Invitrogen). Fractionated proteins then were electrotransferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked in 1 % BSA, 0.2 % Tween-20 in PBS at 4 °C for 2 h and incubated with biotinylated mAb 10-153-3 (10 lg/mL) at room temperature for 1 h and then with ABC-AP complex (Vector Laboratories) at room temperature for 1 h. Finally, bound proteins were visualized by using an alkaline phosphatase (AP)-conjugated substrate kit (Bio-Rad). To identify the precipitated antigen, MALDI–TOF/TOF analysis was performed after digestion of protein band with 50 nM Trypsin Gold (Promega) as previously described [12]. In brief, 1 lL from each digest was spotted onto a stainless steel MALDI target plate and allowed to dry; 1 lL of matrix then was added to each digested sample and allowed to co-crystallize. Positive ion mass spectroscopic (MS) spectra for each digest were acquired (model 4700, Applied Biosystems) in reflectron mode. Major peaks were sequenced by tandem MS analysis. All spectra were analyzed (MASCOT Server), and MASCOT scores higher than the significant score at a confidence interval of 95 % or greater were assumed to be correct calls. Transfection and Expression of ApoA-IV cDNA in Chinese Hamster Ovary (CHO) Cells ApoA-IV cDNA was transfected into and expressed in CHO cells as previously described with some modifications [12]. FAEs of PPs were dissociated from small intestine samples. Total RNA was isolated from the dissociated epithelial cells by using a High Pure RNA Tissue Kit (Roche Diagnostics) and reverse-transcribed by using SuperScript III (Invitrogen) and oligo (dT)20 primers (Invitrogen). To generate ApoA-IV expression vectors, the full-length murine ApoA-IV gene was reverse-transcribed and amplified by polymerase chain reaction (RT-PCR) from murine cDNA by using the primers 50 -ATGTTCCTGAAGGCTGCGGTGCTG-30 (sense) and 50 - TCAGCTCTCCAGAGGTTTGGGC-30 (antisense) and
inserted into pcDNA3.1 vector (Invitrogen). CHO cells were cultured in MEM medium supplemented with 10 % fetal bovine serum and then transfected with the ApoA-IV– pcDNA3.1 construct (pAPO4-cDNA3.1) by using electroporation (Gene Pulser Xcell, Bio-Rad Laboratories). Mock transfectants were generated by electroporating empty pcDNA 3.1 vector into CHO cells. Four days after transfection, cell lysates and supernatants underwent Western blot analysis by using 10-15-3-3 followed by AP-conjugated anti-rat IgG. Immunoreactive bands were visualized by using the AP Color Development Kit (Bio-Rad Laboratories). In Situ Hybridization Murine ApoA-IV cDNA corresponding to nucleotides 838–1,365 (GenBank accession no., BC010769) was subcloned into pGEM-T vector (Promega) and used to generate sense and antisense RNA probes. Digoxigenin (DIG)labeled RNA probes were prepared by using the DIG RNA Labeling Mix (Roche Diagnostics). Intestinal tissue from 7-week-old C57BL6/J mice was embedded in paraffin and sectioned at 6 lm. Tissue sections were de-waxed with xylene and rehydrated through an ethanol series and PBS. The sections were fixed with 4 % paraformaldehyde in PBS for 15 min, treated with 10 lg/mL proteinase K, refixed with 4 % paraformaldehyde in PBS, and placed in 0.2 M HCl for 10 min. Samples then were acetylated through incubation with 0.1 M triethanolamine–HCl (pH 8.0) and 0.25 % acetic anhydride for 10 min and dehydrated through a graded ethanol series. Probes (100 ng/mL) were hybridized by incubating treated samples in Probe Diluent (Genostaff) at 60 °C for 16 h. After hybridization, sections were washed in 59 HybriWash (Genostaff) and then in 50 % formamide in 29 HybriWash and treated with RNase A (50 lg/mL). Sections were washed, treated with 0.5 % blocking reagent (Roche), and incubated for 2 h with AP–conjugated antiDIG antibody (1:1,000 dilution; Roche). Sections were washed and treated with BM Purple AP Substrate (Roche) overnight for visualization of bound probe. Sections were counterstained with eosin, dehydrated, and mounted. Real-Time RT–PCR Analysis ApoA-IV mRNA was quantified by using the Light-Cycler system (Roche Applied Science) according to the manufacturer’s procedures. PCRs were performed by using a 1:50 final dilution of the RT samples. PCR conditions included initial denaturation 95 °C for 8 min, followed by 40 cycles of 15 s at 95 °C, 10 s at 58 °C, and 15 s at 72 °C. The mRNA levels of GAPDH were quantified by using SYBR Green under PCR conditions of initial denaturation
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Fig. 1 Immunofluorescence reactivity of monoclonal antibody 10-15-3-3 in the small intestine of the adult (7 week old) C57BL/6 mice. Immunoreactivity of mAb 10-15-3-3 is shown in green; nuclei and M cells were visualized by using DAPI (blue) and NKM 16-2-4 (red), respectively. a PP stained by DAPI alone. b PP stained by NKM 16-2-4 alone. Immunoreactive M cells stained red (white
arrows). c PP stained by mAb 10-15-3-3, which reacted to the epithelium of PP (white arrows) but not to villous epithelium. d PP stained by DAPI, NKM 16-2-4, mAb 10-15-3-3. e Villous epithelium stained by DAPI alone. f Villous epithelium stained by mAb 10-15-33, which did not react. Scale bar 50 lm
for 8 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 58–62 °C (depending on the cDNA amplified), and 10 s at 72 °C. The sequences of the primers used for ApoA-IV analysis were as follows: 50 -TGTGGTGTGGG ATTACTTTAC-30 (forward primer); 50 -CCATCAGCATA CGTACTAGCAT-30 (reverse primer); 50 -GCCAAGGAG GCTGTAGAACAGTTTCAGAAGAC-FITC-30 (anchor probe); and 50 -LCred640-GATGTCACTCAGCAGCTCA GTACCCTCTTCC-30 -phospate (detection probe). For normalization of our results, we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous control, the primers for which were as follows: 50 -TG AACGGGAAGCTCACTGG-30 (forward primer); 50 -TCC ACCACCCTGTTGCTGTA-30 (reverse primer); 50 -CTGA GGACCAGGTTGTCTCCTGCGA-FITC-30 (anchor probe); and 50 -LCred640-TTCAACAGCAACTCCCACTCTTCC 0 ACC-3 -phospate (detection probe). Results were expressed as the ratio between the levels of ApoA-IV mRNA and GAPDH mRNA.
Results mAb 10-15-3-3 Reacted with Murine FAE Murine M cells were injected into Sprague–Dawley rats to generate mAbs capable of recognizing cell-surface molecules unique to FAE. The reactivities of the generated mAbs were tested through immunofluorescence analysis using frozen sections prepared from small intestine of the adult (7 week old) C57BL/6J mice. One mAb (designated 10-15-3-3) of the rat IgG2a subclass reacted strongly with FAE but not M cells or VE (Fig. 1c, d, f), whereas an isotype-matched control antibody (mouse IgG) did not react with the epithelium of PPs. These results indicated that mAb 10-15-3-3 is a gastrointestinal FAE-specific marker. On the basis of this initial screening, we selected mAb 10-15-3-3 (rat IgG2a) for the present study.
Statistical Analysis
mAb 10-15-3-3 Reacted with a *46-kDa Protein of Murine FAE
Results are expressed as mean ± 1 SD. The statistical significance of differences was determined by performing an unpaired Student’s t test (Excel, Microsoft). A P value \0.05 or \0.01 was considered significant.
To elucidate the antigen recognized by mAb 10-15-3-3, we used it (and its isotype-match control, mouse IgG) to immunoprecipitate FAE. As a result, mAb 10-15-3-3 bound to a protein with a molecular mass of approximately
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C MFLKAAVLTLALVAITGTRAEVTSDQVANVVWDYF TQLSNNAKEAVEQFQKTDVTQQLSTLFQDKLGDAS TYADGVHNKLVPFVVQLSGHLAKETERVKEEIKKEL EDLRDRMMPHANKVTQTFGENMQKLQEHLKPYAV DLQDQINTQTQEMKLQLTPYIQRMQTTIKENVDNLH TSMMPLATNLKDKFNRNMEELKGHLTPRANELKA TIDQNLEDLRRSLAPLTVGVQEKLNHQMEGLAFQM KKNAEELQTKVSAKIDQLQKNLAPLVEDVQSKVKG NTEGLQKSLEDLNRQLEQQVEEFRRTVEPMGEMFN KALVQQLEQFRQQLGPNSGEVESHLSFLEKSLREKV NSFMSTLEKKGSPDQPQALPLPEQAQEQAQEQVQP KPLES
D kDa 100 75 50 ApoA-IV (45kDa)
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Fig. 2 Identification of the antigen by MALDI–TOF/TOF mass spectrometry (MS). a Immunoprecipitation of the lysate with monoclonal antibody 10-15-3-3 showed one major (*46 kDa) band on SDS–PAGE (black arrow). b Peptide mass fingerprinting of trypsin-digested protein. The single band (45 kDa) detected was excised and underwent tryptic digestion, followed by peptide sequencing by using MS. Six selected peptides were fragmented and further analyzed by tandem MS. The tandem MS spectrum of one
(arrow) of these six peaks was identified as NMEELKGHLTPR (methionine oxidation motif), as was another (arrowhead). c Amino acid sequence of ApoA-IV. Peptides (NMEELKGHLTPR) detected by MS are shown in bold and underlined in red. d mAb 10-15-3-3 reacted to supernatants and cell lysates of CHO cells transfected with the cDNA of murine apoA-IV but not those transfected with empty vector (control)
46 kDa (Fig. 2a). This band was not detected by the isotype-matched control antibody or when PBS was used instead of the lysate from murine FAE of PPs (Fig. 2a).
were selected, fragmented, and further analyzed by tandem MS. The tandem MS spectrum of one (1,424.7278) of the six peaks was sequenced and identified as NMEELKGHLTPR (methionine oxidation motif; arrow), as was that of another peak (1,440.7274; arrowhead), which showed complete alignment to amino acids 193–204 of the ApoA-IV sequence (Fig. 2c). Another two peaks (1,041.6031 and 1,064.6116) were identified as partial amino acid sequences of murine keratin; the candidate proteins for the remaining two peaks (940.4632 and 1,240.6924) could not be identified. A search of the MASCOT database verified that the antigen bound by mAb 10-15-3-3 is murine ApoA-IV.
MALDI–TOF–MS and Tandem MS Analysis of 10-153-3-Reactive *46-kDa Protein Resulted in the Identification of Murine ApoA-IV As the next step to identifying the *46-kDa protein recognized by mAb 10-15-3-3, we analyzed those antigens by MS using MALDI–TOF/TOF after tryptic digestion. Several major peaks were identified through this process (Fig. 2b). Six peptides (indicated by asterisks in Fig. 2b)
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Dig Dis Sci b Fig. 3 Immunofluorescence characterization of ApoA-IV expression.
A ApoA-IV expression in the epithelium of the adult murine Peyer’s patch (PP). a–f Adult (7 week old) BALB/c mice. a PP stained by DAPI alone. b PP stained by NKM 16-2-4 alone. Immunoreactive M cells were visualized in red (white arrows). c PP stained by mAb 10-15-3-3. In BALB/c mice, ApoA-IV (green) is expressed in the epithelial cells of PP throughout the small intestine (white arrows). d Merged image of PP stained by DAPI, NKM 16-2-4, and mAb 10-15-3-3. e Magnified image of Fig. A-c. f Magnified image of Ad. ApoA-IV (green) is expressed not in the NKM 16-2-4 positive (red) but NKM 16-2-4-negative epithelial cells. g, h Adult ApoAIV-/- mice. g PP stained by DAPI alone are normal in structure. h ApoA-IV immunoreactivity due to mAb 10-15-3-3 is absent from PP in the epithelium of adult ApoA-IV-/- mice. Scale bar 50 lm. B Localization of ApoA-IV immunoreactivity in the small intestine of adult (7 week old) BALB/c mice. Villi at the upper jejunum stained by a DAPI and b mAb 10-15-3-3, which did not show ApoA-IV immunoreactivity (green). Isolated lymphoid follicles (ILF) at the upper jejunum stained by c DAPI and d mAb 10-15-3-3, which demonstrated ApoA-IV immunoreactivity (green) in the ILF-associated epithelium (d, white arrows). PPs in the lower jejunum and lower ileum stained by e DAPI and f mAb 10-15-3-3. ApoA-IV immunoreactivity (green) was most abundant in upper jejunal PP (A-d) but was decreased in the lower jejunum (B-f, white arrows) and was faint in the lower ileum (B-h, white arrows). Scale bar 50 lm. C Developmental alteration of ApoA-IV expression in the small intestinal epithelium of BALB/c mice. a, b Embryonic day 18. ApoA-IV immunoreactivity (green) was faint in the epithelium of the embryonic small intestine (a, white arrows) and was determined to be associated with PP. c, d Postnatal day 2. ApoA-IV immunoreactivity (green) was prominent in the villous epithelium (c, white arrows) and follicle-associated epithelium (d, white arrows) (d). e, f One-weekold mice. Strong ApoA-IV immunoreactivity (green) was observed in the villous epithelium (e, f) and follicle-associated epithelium (e, f; white arrows). Nuclei (blue) are stained by DAPI (f). g, h Threeweek-old mice. ApoA-IV immunoreactivity (green) was present in the follicle-associated epithelium (g, white arrows) but not the villous epithelium. h Merged image of nuclei (blue), M cells (red), and ApoA-IV (green). Scale bar 50 lm
mAb 10-15-3-3 Specifically Bound to Murine ApoA-IV To confirm that the antigen of mAb 10-15-3-3 is murine ApoA-IV, we transfecting the cDNA of murine ApoA-IV (ApoA-IV–pcDNA3.1 construct) into CHO cells. mAb (1015-3-3) recognized murine ApoA-IV in cell lysates and cell culture supernatants from CHO cells transfected with ApoA-IV–pcDNA3.1 (Fig. 2d) but not those transfected with empty vector (Fig. 2d). In addition, mAb (10-15-3-3) recognized intravenously injected E. coli-derived murine ApoA-IV in ApoA-IV gene knockout mice (Supplemental figure 1). These findings demonstrate that mAb 10-15-3-3 is specific for murine ApoA-IV.
Alexa-conjugated M-cell-specific mAb (NKM 16-2-4) with DAPI counterstaining in adult (7 week old) BALB/c mice (Fig. 3A-a, b, c, d). This analysis revealed that ApoA-IV was expressed by FAE (Fig. 3A-c, e). In addition, ApoAIV was expressed in NKM 16-2-4-negative FAE, that is, FAE without M cells (Fig. 3A-f). Together with the finding that mAb 10-15-3-3 reacted to the FAE of C57BL/6 mice, these results revealed that ApoA-IV is expressed in both BALB/c and C57BL/6 mice. We then examined the immunoreactivity of mAb 10-153-3 in ApoA-IV-/- adult (7 week old) mice on a C57BL/6J background. mAb 10-15-3-3 showed no immunoreactivity on FAE and VE (Fig. 3A-h), thus confirming the FAEspecific expression of ApoA-IV in both non-transgenic BALB/c and C57BL/6 adult mice (Figs. 1c, d, 3A-c-f). Although we hypothesized that ApoA-IV might be a physiologic factor in PP organogenesis, the PPs of ApoAIV-/- mice were normal in structure (Fig. 3A-g). In further analysis of ApoA-IV expression in the FAE throughout the small intestinal epithelium of adult (7 week old) BALB/c and C57BL6 mice, mAb 10-15-3-3 revealed that ApoA-IV wasn’t strongly expressed on VE (Fig. 3B-b) but rather was expressed predominantly on the epithelium of PPs and (isolated lymphoid follicles) ILFs (Fig. 3A-c, B-d). Furthermore, the expression of ApoA-IV on FAE was decreased in the lower small intestine (Fig. 3B-f, h). In addition, mAb 10-15-3-3 showed that ApoA-IV was weakly expressed throughout the epithelium of the murine embryonic small intestine (Fig. 3C-a, b). After birth, ApoA-IV expression increased in parallel with pup development, reaching a maximum at 1 week of age (Fig. 3C-c, d, e, f) in both the FAE and VE. After weaning, however, ApoA-IV expression levels became weaker and restricted to FAE (Fig. 3C-g, h). These results demonstrate that ApoA-IV expression has a unique developmental pattern in the murine small intestine, with prominent expression in both the FAE and VE before weaning but FAE-restricted expression after weaning. We then used mAb 10-15-3-3 to investigate the tissue distribution of ApoA-IV. Neither brain, lung, liver, kidney, spleen, nor colon showed ApoA-IV-specific immunoreactivity (data not shown). In comparison, the kidneys of neonatal mice (Fig. 4e, h, k), but not those of embryonic or weaned mice (Fig. 4b, n, q), showed ApoA-IV immunoreactivity. Localization of ApoA-IV mRNA Expression in the Small Intestine
Immunofluorescence Characterization of ApoA-IV To prove that ApoA-IV is specifically expressed by FAE, we performed an immunofluorescence analysis of intestinal epithelium by using FITC-conjugated 10-15-3-3 and
Using in situ hybridization with a DIG-labeled antisense RNA probe, we investigated the localization of ApoA-IV gene expression in small intestinal tissues. Expression of the ApoA-IV gene was limited mainly to the epithelial cells
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Fig. 4 Immunofluorescence characterization of ApoA-IV expression in kidney. Developmentally associated variation of ApoA-IV expression in kidney from BALB/c mice. At each developmental stage, nuclei were stained by DAPI (blue), and ApoA-IV was stained by mAb 10-15-3-3 (green). High ApoA-IV immunoreactivity was
present in kidney from 2-day-old (white arrows) and 7-day-old (white arrows) mice but not from embryonic day 18, 3-week-old, or 6-week-old mice. k, l Higher magnification images of ApoA-IV expression (white arrows) in the kidney of 7-day-old mice (from h and i, respectively). Scale bar 50 lm
of PPs and the villous tips of the jejunum in adult mice (Fig. 5a, b, d, e). The ileum showed a weak, sparse positive signal in the epithelial cells of PPs and villous tips in adult mice (Fig. 5g, h, j, k). However, signals corresponding to ApoA-IV mRNA were abundant in the FAE and VE in 1and 2-week-old mice. ApoA-IV gene expression was not observed in lamina propria (Fig. 5B-a, b, c, d, e, f). Hybridization signals were not obtained by using the sense probe (Fig. 5A-c, f, i, l).
Finally, ApoA-IV gene expression was significantly higher in PP than in PP-denuded jejunum and ileum (Fig. 6c).
Real-Time PCR Analysis of ApoA-IV Gene Expression Quantitative PCR analysis demonstrated that gene expression of ApoA-IV was significantly higher in the jejunum than the ileum at all developmental stages evaluated (Fig. 6a). Confirming the results of our immunohistochemical analysis, ApoA-IV gene expression in mice was detectable at embryonic day 18, increased after birth, and reached the maximum at 1 and 2 weeks of age (Fig. 6a). After weaning, ApoA-IV gene and protein levels decreased. When compared among various organ tissues at each developmental stage, the ApoA-IV gene was predominantly expressed in small intestine, especially jejunal tissue, at all developmental stages (Fig. 6b). In addition, ApoA-IV gene expression was expressed in liver albeit to a lower degree than that in the jejunum, which is compatible with previous findings in rat [5]. Unlike our immunohistochemical results, ApoA-IV mRNA transcripts were not detected in kidney at any developmental stage (Fig. 6b).
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Discussion In the present study, our newly generated mAb, which was selected in light of its specific reactivity to intestinal FAE, revealed intestinal FAE-specific expression of ApoA-IV in mice. ApoA-IV is known to be expressed in humans [6, 9, 14], rats [5, 7, 15], piglet [16], and mice [8]. Whereas its synthesis is restricted to the enterocytes in humans [9], ApoA-IV is produced by both the liver and small intestine in rodents [4, 7, 15]. At the gene level, ApoA-IV is expressed predominantly in small intestinal epithelium and to a lesser extent in liver in rodents [7, 15]; however, because of the limited information regarding ApoA-IV protein expression [11], the posttranscriptional features of this factor are not fully understood. In this regard, we here obtained detailed information regarding ApoA-IV gene expression and protein localization. That the FAE-specific expression of ApoA-IV was unknown until currently may have arisen from the previous general acceptance of ApoA-IV gene expression in VE cells and lack of interest regarding FAE. In addition, our current study focused on the creation of FAE-specific antibodies for the identification of FAE-specific proteins, thus enabling us to demonstrate the novel expression of ApoA-IV in the intestinal FAE rather than VE. On the other hand, ApoA-IV showed strong expression in the FAE of not lower but upper small
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Fig. 5 In situ hybridization of ApoA-IV. A Small intestinal tissue from 7-week-old mice was hybridized with antisense or sense probe to the murine ApoA-IV sequence. a, b, d, e ApoA-IV mRNA was expressed strongly in the epithelium of PP and to a lesser extent in the villi of the upper jejunum. d, e In the villous epithelium, ApoA-IV gene was expressed in the tips rather than the crypts. g, h, j, k ApoAIV gene expression became weaker in the lower part of small intestine compared with the upper part (a, b, d, e). Scattered expression was present in the epithelium of PP (A-g, h), and faint expression occurred
in the epithelium of villous tips (j, k). c, f, i, l The sense probe failed to stain the murine ApoA-IV sequence in the murine small intestine. B In 1-week-old mice, ApoA-IV was predominantly expressed in the a, b PP and a, c villi throughout the epithelium of the entire small intestine. In the villous epithelium, ApoA-IV gene was strongly expressed in both the tips and crypts. In 3-week-old mice, strong expression of ApoA-IV was present in the d, e PP-associated epithelium and d, e epithelial cells of villous tips
intestine; thus, it is necessary to create additional specific markers to detect ileal FAE. One of important aspect in this study is the specificity of mAb (10-15-3-3) to murine ApoAIV. In this regard, mAb-recognized murine ApoA-IV in cell lysates and cell culture supernatants from CHO cells transfected with ApoA-IV pcDNA but not those transfected with empty vector, in addition, mAb recognized intravenously injected E.coli-derived ApoA-IV in ApoA-IV gene knockout mice. These evidence clearly demonstrated the specificity of mAb (10-15-3-3) to murine ApoA-IV. Based on the above specificity of mAb to ApoA-IV, the present study indicated a novel role of ApoA-IV as an upper intestinal FAE-specific marker in the mature murine small intestine. Because our result presented the different feature of ApoA-IV protein compared to the previous studies, there is a potential that FAEspecific ApoA-IV has different structure, coding sequence, or truncation; thus, further study is necessary to elucidate the difference between the FAE-specific ApoA-IV and the conventional ApoA-IV in the VE. Our developmental analysis of ApoA-IV at the protein level showed that FAE-specific expression of ApoA-IV is characteristic of the small intestine of weanling and adult
mice, and both FAE and VE are high-expression sites for ApoA-IV during developmentally immature periods. A previous study similarly described this elevated gene expression in the small intestine during the neonatal period [7], but our study provided the further feature of the different localization of ApoA-IV protein before and after weaning. The reason for these age-dependent differences in expression warrants elucidation; however, we speculate that enhanced lipid flux during the neonatal period, perhaps from high-fat breastmilk feeding, induced the predominantly elevated villous enterocyte fat transport. A previous study demonstrated neonatal intestinal ApoA-IV mRNA levels that were more than fourfold those of adult intestine, with a subsequent gradual decrease in ApoA-IV mRNA levels over the next 14 days [7]. Accumulated studies revealed that ApoA-IV synthesis was upregulated after intestinal lipid infusion [17–20]. On the other hand, Black DD et al. demonstrated that intraduodenal lipid infusion induced the elevation of jejunal apo A-IV mRNA expression in newborn piglet, but those lipid responsiveness decreased as the piglets were weaned from a high-fat breast milk diet [21]. Taken together with restriction of expression of ApoA-IV in the FAE after weaning, we suspect
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ORIGINAL ARTICLE
Specific Expression of Apolipoprotein A-IV in the Follicle-Associated Epithelium of the Small Intestine Daisuke Tokuhara • Tomonori Nochi • Akiko Matsumura Mio Mejima • Yuko Takahashi • Shiho Kurokawa • Hiroshi Kiyono • Yoshikazu Yuki
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Received: 25 April 2013 / Accepted: 2 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Peyer’s patches (PPs), which are covered by specialized follicle-associated epithelium (FAE) including M cells, play a central role in immune induction in the gastrointestinal tract. This study is to investigate a new molecule to characterize PPs. Methods We generated a monoclonal antibody (mAb 10-15-3-3) that specifically reacts to the epithelium of PPs and isolated lymphoid follicles. Target antigen was analyzed by immunoprecipitation and mass spectrometry. Localization and expression of target antigen were evaluated by immunofluorescence, in situ hybridization and realtime PCR. Results Immunoprecipitation and mass spectrometry revealed that mAb 10-15-3-3 recognized apolipoprotein A-IV (ApoA-IV), a well-known lipid transporter; this finding was confirmed by the specific reactivity of mAb 10-15-3-3 to cells transfected with the murine ApoA-IV gene. Immunofluorescence using mAb 10-15-3-3 showed intestinal localization of ApoA-IV, in which strong expression of the ApoA-IV protein occurred throughout the
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3203-6) contains supplementary material, which is available to authorized users. D. Tokuhara T. Nochi A. Matsumura M. Mejima Y. Takahashi S. Kurokawa H. Kiyono Y. Yuki (&) Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan e-mail: [email protected] D. Tokuhara Department of Pediatrics, Osaka City University Graduate School of Medicine, Osaka, Japan
entire intestinal epithelium during developing period before weaning but was restricted to the FAE in adult mice. In support of these findings, in situ hybridization showed strong expression of the ApoA-IV gene throughout the entire intestinal epithelium during developing period before weaning, but this expression was restricted to the FAE predominantly and the tips of villi to a lesser extent in adult mice. Deficiency of ApoA-IV had no effect on the organogenesis of PP in mice. Conclusions Our current results reveal ApoA-IV as a novel FAE-specific marker especially in the upper small intestine of adult mice. Keywords Apolipoprotein A-IV Mucosal immunology Epithelial cell Peyer’s patch
Introduction The follicle-associated epithelium (FAE) that overlies mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses [1–3]. However, the mechanisms involved in this response and in antigen transport within the FAE remain to be elucidated. To gain insight into the roles of FAE and M cells in these mechanisms, we previously generated the monoclonal antibody (mAb) NKM 16-2-4, which binds specifically to M cells located in the FAE of Peyer’s patches (PPs) and which lacks reactivity to UEA-1-positive goblet cells in intestinal villi, and demonstrated that vaccination with antigen-conjugated NKM 16-2-4 effectively induced protective immunity in both the systemic and mucosal compartments by targeting antigen to M cells [1]. The identification of new FAE- and M-cell-specific molecules likely will contribute toward clarifying the molecular mechanisms
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involved in the mucosal immune response and in the transport of antigens through FAE and by M cells. To acquire additional information regarding the organogenesis of FAE, we here generated a mAb that reacts specifically to this tissue. Using mass spectrometry and transfectant analysis, we determined that the antigen recognized by this mAb is apolipoprotein A-IV (ApoA-IV) and revealed the localization of ApoA-IV expression at the protein and gene levels. ApoA-IV is a component of chylomicron and high-density lipoprotein and thus functions as a lipid transporter molecule [4]; in addition, ApoA-IV is a satiety factor [5] and anti-atherogenic factor [6] in rodents. The expression of the ApoA-IV gene has been studied extensively and reportedly occurs in the villous epithelium (VE) [7–9] and liver of rodents [7, 8]; however, little information regarding ApoA-IV protein expression is available currently [10, 11]. Our immunofluorescence analysis using the anti-ApoA-IV mAb demonstrated the age-dependent expression of ApoA-IV in murine small intestine. Our results reveal ApoA-IV as a novel intestinal FAE-specific marker in adult mice and suggest ApoA-IV as a physiologic signaling or immunomodulatory molecule in the primary immune inductive site of the small intestine.
Materials and Methods Animals Female C57BL/6J and BALB/c mice (pregnant and 1–7 weeks old) were purchased from Japan SLC. Male and female ApoA-IV-/- mice on a C57BL/6J background were purchased from the Jackson Laboratory. All of the mice used in this study were housed in the experimental animal facility at the Institute of Medical Science (University of Tokyo) on a standard 12:12-h light/dark cycle and had ad libitum access to water and food containing 4 % fat. All experiments were approved by the Ethics Committee of the University of Tokyo. Generation of Monoclonal Antibody The FAE-enriched fraction was prepared from murine PPs as previously described [1], with slight modification, by using UEA-1(Biogenesis) and NKM 16-2-4. In brief, cells isolated from murine PPs were fixed in 4 % paraformaldehyde (Wako) and stained with PE-conjugated UEA-1 and FITCconjugated NKM 16-2-4; each antibody was used at 500 ng/ mL. UEA-1- and NKM 16-2-4-positive cells were sorted by using flow cytometry (FACSAria, Becton–Dickinson) and injected into the footpads of Sprague–Dawley rats (106 cells/ rat) 4 times at 2-week intervals, with TiterMax Gold (TiterMax) as an adjuvant. Four days after the final immunization, lymphocytes isolated from the spleen and inguinal
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lymph nodes of the immunized rats were fused with P3X63AG8.653 myeloma cells (CRL-1580; American Type Culture Collection) in the presence of 50 % (wt/vol) polyethylene glycol 1500 (Roche). Established hybridomas were injected into Crlj:CD1-Foxn1nu mice, and mAbs were purified from ascitic fluids by using protein G-Sepharose (GE Healthcare) and labeled with EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific), fluorescein isothiocyanate (FITC, Sigma-Aldrich), or Alexa Fluor 647 (Invitrogen). Immunofluorescence Analysis A mAb (10-15-3-3; rat IgG2a) was selected on the basis of the initial screening and its specificity to FAE of PPs as determined by immunofluorescence analyses, as previously described [1, 12]. In brief, murine small intestinal tissues were perfused intracardially with 4 % paraformaldehyde (Wako) in phosphate-buffered saline (PBS). Perfused small intestines were dissected and further fixed in 4 % paraformaldehyde at 4 °C overnight. Fixed tissues were washed with PBS and incubated in increasing concentrations of cold sucrose (10, 15, and 20 %; total, 24 h). The tissues then were embedded in Tissue-Tek OCT compound (Sakura Finetechnical). Frozen sections (5 lm) were cut and incubated with 1 % BSA for 20 min at room temperature. After a blocking step, each fixed frozen section or fixed tissue sample containing PPs from C57BL/6J, BALB/c, or ApoA-IV-/- mice was stained with 5 lg/mL FITC-conjugated 10-15-3-3 or FITC-conjugated isotype control antibody (FITC-conjugated rat IgG). Some sections were co-stained with 5 lg/mL Alexa-conjugated NKM 16-2-4 to visualize M cells [1]. To examine the immunoreactivities of 10-15-3-3 to FAE at various developmental stages, tissue sections from mice at embryonic day 18 and postnatal day 2 were stained by using 10-15-3-3 followed by antiCD3 and anti-CD4 antibodies (eBioscience) to characterize PP with CD3- CD4? cells as PP inducer cells [13]. These sections were counterstained with 200 ng/mL 40 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 30 min at room temperature and analyzed by using a confocal laser scanning microscope (TCS SP2, Leica, Wetzlar, Germany). To characterize the immunoreactivities of 10-15-3-3 in various organ tissues, samples of brain, lung, liver, spleen, kidney, and colon were immunostained with 10-15-3-3 and counterstained with DAPI. Analysis of Antigen Recognized by 10-15-3-3 To identify the antigen recognized by 10-15-3-3, we performed an immunoprecipitation assay with 10-15-3-3 followed by matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF)/TOF analysis as previously described [12]. In brief, FAE of murine PPs was lysed in
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lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.5 % Triton X-100, and protease inhibitor cocktail [Roche]). After 1 h of incubation on ice followed by centrifugation, the lysate (5 mg/mL, 1 mL) was precleared with 40 lL protein G-Sepharose (1:1 in PBS, Amersham Pharmacia Biotech) at 4 °C for 1 h. After centrifugation, the lysate was incubated with mAb 10-15-3-3 (10 lg/mL) or an isotype-matched control antibody (rat IgG, BD PharMingen) for 1 h at 4 °C before the addition of 40 lL protein G-Sepharose (1:1 in PBS) and incubation for 1 h at 4 °C. Immune complexes were washed five times with cold PBS containing protease inhibitor cocktail, eluted in sample buffer (2 % SDS, 5 % b-mercaptoethanol, 50 mM Tris–HCl [pH 6.8], and 20 % glycerol), and separated by SDS–PAGE on a 12 % NuPAGE Bis–Tris Gel (Invitrogen). Fractionated proteins then were electrotransferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked in 1 % BSA, 0.2 % Tween-20 in PBS at 4 °C for 2 h and incubated with biotinylated mAb 10-153-3 (10 lg/mL) at room temperature for 1 h and then with ABC-AP complex (Vector Laboratories) at room temperature for 1 h. Finally, bound proteins were visualized by using an alkaline phosphatase (AP)-conjugated substrate kit (Bio-Rad). To identify the precipitated antigen, MALDI–TOF/TOF analysis was performed after digestion of protein band with 50 nM Trypsin Gold (Promega) as previously described [12]. In brief, 1 lL from each digest was spotted onto a stainless steel MALDI target plate and allowed to dry; 1 lL of matrix then was added to each digested sample and allowed to co-crystallize. Positive ion mass spectroscopic (MS) spectra for each digest were acquired (model 4700, Applied Biosystems) in reflectron mode. Major peaks were sequenced by tandem MS analysis. All spectra were analyzed (MASCOT Server), and MASCOT scores higher than the significant score at a confidence interval of 95 % or greater were assumed to be correct calls. Transfection and Expression of ApoA-IV cDNA in Chinese Hamster Ovary (CHO) Cells ApoA-IV cDNA was transfected into and expressed in CHO cells as previously described with some modifications [12]. FAEs of PPs were dissociated from small intestine samples. Total RNA was isolated from the dissociated epithelial cells by using a High Pure RNA Tissue Kit (Roche Diagnostics) and reverse-transcribed by using SuperScript III (Invitrogen) and oligo (dT)20 primers (Invitrogen). To generate ApoA-IV expression vectors, the full-length murine ApoA-IV gene was reverse-transcribed and amplified by polymerase chain reaction (RT-PCR) from murine cDNA by using the primers 50 -ATGTTCCTGAAGGCTGCGGTGCTG-30 (sense) and 50 - TCAGCTCTCCAGAGGTTTGGGC-30 (antisense) and
inserted into pcDNA3.1 vector (Invitrogen). CHO cells were cultured in MEM medium supplemented with 10 % fetal bovine serum and then transfected with the ApoA-IV– pcDNA3.1 construct (pAPO4-cDNA3.1) by using electroporation (Gene Pulser Xcell, Bio-Rad Laboratories). Mock transfectants were generated by electroporating empty pcDNA 3.1 vector into CHO cells. Four days after transfection, cell lysates and supernatants underwent Western blot analysis by using 10-15-3-3 followed by AP-conjugated anti-rat IgG. Immunoreactive bands were visualized by using the AP Color Development Kit (Bio-Rad Laboratories). In Situ Hybridization Murine ApoA-IV cDNA corresponding to nucleotides 838–1,365 (GenBank accession no., BC010769) was subcloned into pGEM-T vector (Promega) and used to generate sense and antisense RNA probes. Digoxigenin (DIG)labeled RNA probes were prepared by using the DIG RNA Labeling Mix (Roche Diagnostics). Intestinal tissue from 7-week-old C57BL6/J mice was embedded in paraffin and sectioned at 6 lm. Tissue sections were de-waxed with xylene and rehydrated through an ethanol series and PBS. The sections were fixed with 4 % paraformaldehyde in PBS for 15 min, treated with 10 lg/mL proteinase K, refixed with 4 % paraformaldehyde in PBS, and placed in 0.2 M HCl for 10 min. Samples then were acetylated through incubation with 0.1 M triethanolamine–HCl (pH 8.0) and 0.25 % acetic anhydride for 10 min and dehydrated through a graded ethanol series. Probes (100 ng/mL) were hybridized by incubating treated samples in Probe Diluent (Genostaff) at 60 °C for 16 h. After hybridization, sections were washed in 59 HybriWash (Genostaff) and then in 50 % formamide in 29 HybriWash and treated with RNase A (50 lg/mL). Sections were washed, treated with 0.5 % blocking reagent (Roche), and incubated for 2 h with AP–conjugated antiDIG antibody (1:1,000 dilution; Roche). Sections were washed and treated with BM Purple AP Substrate (Roche) overnight for visualization of bound probe. Sections were counterstained with eosin, dehydrated, and mounted. Real-Time RT–PCR Analysis ApoA-IV mRNA was quantified by using the Light-Cycler system (Roche Applied Science) according to the manufacturer’s procedures. PCRs were performed by using a 1:50 final dilution of the RT samples. PCR conditions included initial denaturation 95 °C for 8 min, followed by 40 cycles of 15 s at 95 °C, 10 s at 58 °C, and 15 s at 72 °C. The mRNA levels of GAPDH were quantified by using SYBR Green under PCR conditions of initial denaturation
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Fig. 1 Immunofluorescence reactivity of monoclonal antibody 10-15-3-3 in the small intestine of the adult (7 week old) C57BL/6 mice. Immunoreactivity of mAb 10-15-3-3 is shown in green; nuclei and M cells were visualized by using DAPI (blue) and NKM 16-2-4 (red), respectively. a PP stained by DAPI alone. b PP stained by NKM 16-2-4 alone. Immunoreactive M cells stained red (white
arrows). c PP stained by mAb 10-15-3-3, which reacted to the epithelium of PP (white arrows) but not to villous epithelium. d PP stained by DAPI, NKM 16-2-4, mAb 10-15-3-3. e Villous epithelium stained by DAPI alone. f Villous epithelium stained by mAb 10-15-33, which did not react. Scale bar 50 lm
for 8 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 58–62 °C (depending on the cDNA amplified), and 10 s at 72 °C. The sequences of the primers used for ApoA-IV analysis were as follows: 50 -TGTGGTGTGGG ATTACTTTAC-30 (forward primer); 50 -CCATCAGCATA CGTACTAGCAT-30 (reverse primer); 50 -GCCAAGGAG GCTGTAGAACAGTTTCAGAAGAC-FITC-30 (anchor probe); and 50 -LCred640-GATGTCACTCAGCAGCTCA GTACCCTCTTCC-30 -phospate (detection probe). For normalization of our results, we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous control, the primers for which were as follows: 50 -TG AACGGGAAGCTCACTGG-30 (forward primer); 50 -TCC ACCACCCTGTTGCTGTA-30 (reverse primer); 50 -CTGA GGACCAGGTTGTCTCCTGCGA-FITC-30 (anchor probe); and 50 -LCred640-TTCAACAGCAACTCCCACTCTTCC 0 ACC-3 -phospate (detection probe). Results were expressed as the ratio between the levels of ApoA-IV mRNA and GAPDH mRNA.
Results mAb 10-15-3-3 Reacted with Murine FAE Murine M cells were injected into Sprague–Dawley rats to generate mAbs capable of recognizing cell-surface molecules unique to FAE. The reactivities of the generated mAbs were tested through immunofluorescence analysis using frozen sections prepared from small intestine of the adult (7 week old) C57BL/6J mice. One mAb (designated 10-15-3-3) of the rat IgG2a subclass reacted strongly with FAE but not M cells or VE (Fig. 1c, d, f), whereas an isotype-matched control antibody (mouse IgG) did not react with the epithelium of PPs. These results indicated that mAb 10-15-3-3 is a gastrointestinal FAE-specific marker. On the basis of this initial screening, we selected mAb 10-15-3-3 (rat IgG2a) for the present study.
Statistical Analysis
mAb 10-15-3-3 Reacted with a *46-kDa Protein of Murine FAE
Results are expressed as mean ± 1 SD. The statistical significance of differences was determined by performing an unpaired Student’s t test (Excel, Microsoft). A P value \0.05 or \0.01 was considered significant.
To elucidate the antigen recognized by mAb 10-15-3-3, we used it (and its isotype-match control, mouse IgG) to immunoprecipitate FAE. As a result, mAb 10-15-3-3 bound to a protein with a molecular mass of approximately
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C MFLKAAVLTLALVAITGTRAEVTSDQVANVVWDYF TQLSNNAKEAVEQFQKTDVTQQLSTLFQDKLGDAS TYADGVHNKLVPFVVQLSGHLAKETERVKEEIKKEL EDLRDRMMPHANKVTQTFGENMQKLQEHLKPYAV DLQDQINTQTQEMKLQLTPYIQRMQTTIKENVDNLH TSMMPLATNLKDKFNRNMEELKGHLTPRANELKA TIDQNLEDLRRSLAPLTVGVQEKLNHQMEGLAFQM KKNAEELQTKVSAKIDQLQKNLAPLVEDVQSKVKG NTEGLQKSLEDLNRQLEQQVEEFRRTVEPMGEMFN KALVQQLEQFRQQLGPNSGEVESHLSFLEKSLREKV NSFMSTLEKKGSPDQPQALPLPEQAQEQAQEQVQP KPLES
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Fig. 2 Identification of the antigen by MALDI–TOF/TOF mass spectrometry (MS). a Immunoprecipitation of the lysate with monoclonal antibody 10-15-3-3 showed one major (*46 kDa) band on SDS–PAGE (black arrow). b Peptide mass fingerprinting of trypsin-digested protein. The single band (45 kDa) detected was excised and underwent tryptic digestion, followed by peptide sequencing by using MS. Six selected peptides were fragmented and further analyzed by tandem MS. The tandem MS spectrum of one
(arrow) of these six peaks was identified as NMEELKGHLTPR (methionine oxidation motif), as was another (arrowhead). c Amino acid sequence of ApoA-IV. Peptides (NMEELKGHLTPR) detected by MS are shown in bold and underlined in red. d mAb 10-15-3-3 reacted to supernatants and cell lysates of CHO cells transfected with the cDNA of murine apoA-IV but not those transfected with empty vector (control)
46 kDa (Fig. 2a). This band was not detected by the isotype-matched control antibody or when PBS was used instead of the lysate from murine FAE of PPs (Fig. 2a).
were selected, fragmented, and further analyzed by tandem MS. The tandem MS spectrum of one (1,424.7278) of the six peaks was sequenced and identified as NMEELKGHLTPR (methionine oxidation motif; arrow), as was that of another peak (1,440.7274; arrowhead), which showed complete alignment to amino acids 193–204 of the ApoA-IV sequence (Fig. 2c). Another two peaks (1,041.6031 and 1,064.6116) were identified as partial amino acid sequences of murine keratin; the candidate proteins for the remaining two peaks (940.4632 and 1,240.6924) could not be identified. A search of the MASCOT database verified that the antigen bound by mAb 10-15-3-3 is murine ApoA-IV.
MALDI–TOF–MS and Tandem MS Analysis of 10-153-3-Reactive *46-kDa Protein Resulted in the Identification of Murine ApoA-IV As the next step to identifying the *46-kDa protein recognized by mAb 10-15-3-3, we analyzed those antigens by MS using MALDI–TOF/TOF after tryptic digestion. Several major peaks were identified through this process (Fig. 2b). Six peptides (indicated by asterisks in Fig. 2b)
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Dig Dis Sci b Fig. 3 Immunofluorescence characterization of ApoA-IV expression.
A ApoA-IV expression in the epithelium of the adult murine Peyer’s patch (PP). a–f Adult (7 week old) BALB/c mice. a PP stained by DAPI alone. b PP stained by NKM 16-2-4 alone. Immunoreactive M cells were visualized in red (white arrows). c PP stained by mAb 10-15-3-3. In BALB/c mice, ApoA-IV (green) is expressed in the epithelial cells of PP throughout the small intestine (white arrows). d Merged image of PP stained by DAPI, NKM 16-2-4, and mAb 10-15-3-3. e Magnified image of Fig. A-c. f Magnified image of Ad. ApoA-IV (green) is expressed not in the NKM 16-2-4 positive (red) but NKM 16-2-4-negative epithelial cells. g, h Adult ApoAIV-/- mice. g PP stained by DAPI alone are normal in structure. h ApoA-IV immunoreactivity due to mAb 10-15-3-3 is absent from PP in the epithelium of adult ApoA-IV-/- mice. Scale bar 50 lm. B Localization of ApoA-IV immunoreactivity in the small intestine of adult (7 week old) BALB/c mice. Villi at the upper jejunum stained by a DAPI and b mAb 10-15-3-3, which did not show ApoA-IV immunoreactivity (green). Isolated lymphoid follicles (ILF) at the upper jejunum stained by c DAPI and d mAb 10-15-3-3, which demonstrated ApoA-IV immunoreactivity (green) in the ILF-associated epithelium (d, white arrows). PPs in the lower jejunum and lower ileum stained by e DAPI and f mAb 10-15-3-3. ApoA-IV immunoreactivity (green) was most abundant in upper jejunal PP (A-d) but was decreased in the lower jejunum (B-f, white arrows) and was faint in the lower ileum (B-h, white arrows). Scale bar 50 lm. C Developmental alteration of ApoA-IV expression in the small intestinal epithelium of BALB/c mice. a, b Embryonic day 18. ApoA-IV immunoreactivity (green) was faint in the epithelium of the embryonic small intestine (a, white arrows) and was determined to be associated with PP. c, d Postnatal day 2. ApoA-IV immunoreactivity (green) was prominent in the villous epithelium (c, white arrows) and follicle-associated epithelium (d, white arrows) (d). e, f One-weekold mice. Strong ApoA-IV immunoreactivity (green) was observed in the villous epithelium (e, f) and follicle-associated epithelium (e, f; white arrows). Nuclei (blue) are stained by DAPI (f). g, h Threeweek-old mice. ApoA-IV immunoreactivity (green) was present in the follicle-associated epithelium (g, white arrows) but not the villous epithelium. h Merged image of nuclei (blue), M cells (red), and ApoA-IV (green). Scale bar 50 lm
mAb 10-15-3-3 Specifically Bound to Murine ApoA-IV To confirm that the antigen of mAb 10-15-3-3 is murine ApoA-IV, we transfecting the cDNA of murine ApoA-IV (ApoA-IV–pcDNA3.1 construct) into CHO cells. mAb (1015-3-3) recognized murine ApoA-IV in cell lysates and cell culture supernatants from CHO cells transfected with ApoA-IV–pcDNA3.1 (Fig. 2d) but not those transfected with empty vector (Fig. 2d). In addition, mAb (10-15-3-3) recognized intravenously injected E. coli-derived murine ApoA-IV in ApoA-IV gene knockout mice (Supplemental figure 1). These findings demonstrate that mAb 10-15-3-3 is specific for murine ApoA-IV.
Alexa-conjugated M-cell-specific mAb (NKM 16-2-4) with DAPI counterstaining in adult (7 week old) BALB/c mice (Fig. 3A-a, b, c, d). This analysis revealed that ApoA-IV was expressed by FAE (Fig. 3A-c, e). In addition, ApoAIV was expressed in NKM 16-2-4-negative FAE, that is, FAE without M cells (Fig. 3A-f). Together with the finding that mAb 10-15-3-3 reacted to the FAE of C57BL/6 mice, these results revealed that ApoA-IV is expressed in both BALB/c and C57BL/6 mice. We then examined the immunoreactivity of mAb 10-153-3 in ApoA-IV-/- adult (7 week old) mice on a C57BL/6J background. mAb 10-15-3-3 showed no immunoreactivity on FAE and VE (Fig. 3A-h), thus confirming the FAEspecific expression of ApoA-IV in both non-transgenic BALB/c and C57BL/6 adult mice (Figs. 1c, d, 3A-c-f). Although we hypothesized that ApoA-IV might be a physiologic factor in PP organogenesis, the PPs of ApoAIV-/- mice were normal in structure (Fig. 3A-g). In further analysis of ApoA-IV expression in the FAE throughout the small intestinal epithelium of adult (7 week old) BALB/c and C57BL6 mice, mAb 10-15-3-3 revealed that ApoA-IV wasn’t strongly expressed on VE (Fig. 3B-b) but rather was expressed predominantly on the epithelium of PPs and (isolated lymphoid follicles) ILFs (Fig. 3A-c, B-d). Furthermore, the expression of ApoA-IV on FAE was decreased in the lower small intestine (Fig. 3B-f, h). In addition, mAb 10-15-3-3 showed that ApoA-IV was weakly expressed throughout the epithelium of the murine embryonic small intestine (Fig. 3C-a, b). After birth, ApoA-IV expression increased in parallel with pup development, reaching a maximum at 1 week of age (Fig. 3C-c, d, e, f) in both the FAE and VE. After weaning, however, ApoA-IV expression levels became weaker and restricted to FAE (Fig. 3C-g, h). These results demonstrate that ApoA-IV expression has a unique developmental pattern in the murine small intestine, with prominent expression in both the FAE and VE before weaning but FAE-restricted expression after weaning. We then used mAb 10-15-3-3 to investigate the tissue distribution of ApoA-IV. Neither brain, lung, liver, kidney, spleen, nor colon showed ApoA-IV-specific immunoreactivity (data not shown). In comparison, the kidneys of neonatal mice (Fig. 4e, h, k), but not those of embryonic or weaned mice (Fig. 4b, n, q), showed ApoA-IV immunoreactivity. Localization of ApoA-IV mRNA Expression in the Small Intestine
Immunofluorescence Characterization of ApoA-IV To prove that ApoA-IV is specifically expressed by FAE, we performed an immunofluorescence analysis of intestinal epithelium by using FITC-conjugated 10-15-3-3 and
Using in situ hybridization with a DIG-labeled antisense RNA probe, we investigated the localization of ApoA-IV gene expression in small intestinal tissues. Expression of the ApoA-IV gene was limited mainly to the epithelial cells
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Fig. 4 Immunofluorescence characterization of ApoA-IV expression in kidney. Developmentally associated variation of ApoA-IV expression in kidney from BALB/c mice. At each developmental stage, nuclei were stained by DAPI (blue), and ApoA-IV was stained by mAb 10-15-3-3 (green). High ApoA-IV immunoreactivity was
present in kidney from 2-day-old (white arrows) and 7-day-old (white arrows) mice but not from embryonic day 18, 3-week-old, or 6-week-old mice. k, l Higher magnification images of ApoA-IV expression (white arrows) in the kidney of 7-day-old mice (from h and i, respectively). Scale bar 50 lm
of PPs and the villous tips of the jejunum in adult mice (Fig. 5a, b, d, e). The ileum showed a weak, sparse positive signal in the epithelial cells of PPs and villous tips in adult mice (Fig. 5g, h, j, k). However, signals corresponding to ApoA-IV mRNA were abundant in the FAE and VE in 1and 2-week-old mice. ApoA-IV gene expression was not observed in lamina propria (Fig. 5B-a, b, c, d, e, f). Hybridization signals were not obtained by using the sense probe (Fig. 5A-c, f, i, l).
Finally, ApoA-IV gene expression was significantly higher in PP than in PP-denuded jejunum and ileum (Fig. 6c).
Real-Time PCR Analysis of ApoA-IV Gene Expression Quantitative PCR analysis demonstrated that gene expression of ApoA-IV was significantly higher in the jejunum than the ileum at all developmental stages evaluated (Fig. 6a). Confirming the results of our immunohistochemical analysis, ApoA-IV gene expression in mice was detectable at embryonic day 18, increased after birth, and reached the maximum at 1 and 2 weeks of age (Fig. 6a). After weaning, ApoA-IV gene and protein levels decreased. When compared among various organ tissues at each developmental stage, the ApoA-IV gene was predominantly expressed in small intestine, especially jejunal tissue, at all developmental stages (Fig. 6b). In addition, ApoA-IV gene expression was expressed in liver albeit to a lower degree than that in the jejunum, which is compatible with previous findings in rat [5]. Unlike our immunohistochemical results, ApoA-IV mRNA transcripts were not detected in kidney at any developmental stage (Fig. 6b).
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Discussion In the present study, our newly generated mAb, which was selected in light of its specific reactivity to intestinal FAE, revealed intestinal FAE-specific expression of ApoA-IV in mice. ApoA-IV is known to be expressed in humans [6, 9, 14], rats [5, 7, 15], piglet [16], and mice [8]. Whereas its synthesis is restricted to the enterocytes in humans [9], ApoA-IV is produced by both the liver and small intestine in rodents [4, 7, 15]. At the gene level, ApoA-IV is expressed predominantly in small intestinal epithelium and to a lesser extent in liver in rodents [7, 15]; however, because of the limited information regarding ApoA-IV protein expression [11], the posttranscriptional features of this factor are not fully understood. In this regard, we here obtained detailed information regarding ApoA-IV gene expression and protein localization. That the FAE-specific expression of ApoA-IV was unknown until currently may have arisen from the previous general acceptance of ApoA-IV gene expression in VE cells and lack of interest regarding FAE. In addition, our current study focused on the creation of FAE-specific antibodies for the identification of FAE-specific proteins, thus enabling us to demonstrate the novel expression of ApoA-IV in the intestinal FAE rather than VE. On the other hand, ApoA-IV showed strong expression in the FAE of not lower but upper small
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Fig. 5 In situ hybridization of ApoA-IV. A Small intestinal tissue from 7-week-old mice was hybridized with antisense or sense probe to the murine ApoA-IV sequence. a, b, d, e ApoA-IV mRNA was expressed strongly in the epithelium of PP and to a lesser extent in the villi of the upper jejunum. d, e In the villous epithelium, ApoA-IV gene was expressed in the tips rather than the crypts. g, h, j, k ApoAIV gene expression became weaker in the lower part of small intestine compared with the upper part (a, b, d, e). Scattered expression was present in the epithelium of PP (A-g, h), and faint expression occurred
in the epithelium of villous tips (j, k). c, f, i, l The sense probe failed to stain the murine ApoA-IV sequence in the murine small intestine. B In 1-week-old mice, ApoA-IV was predominantly expressed in the a, b PP and a, c villi throughout the epithelium of the entire small intestine. In the villous epithelium, ApoA-IV gene was strongly expressed in both the tips and crypts. In 3-week-old mice, strong expression of ApoA-IV was present in the d, e PP-associated epithelium and d, e epithelial cells of villous tips
intestine; thus, it is necessary to create additional specific markers to detect ileal FAE. One of important aspect in this study is the specificity of mAb (10-15-3-3) to murine ApoAIV. In this regard, mAb-recognized murine ApoA-IV in cell lysates and cell culture supernatants from CHO cells transfected with ApoA-IV pcDNA but not those transfected with empty vector, in addition, mAb recognized intravenously injected E.coli-derived ApoA-IV in ApoA-IV gene knockout mice. These evidence clearly demonstrated the specificity of mAb (10-15-3-3) to murine ApoA-IV. Based on the above specificity of mAb to ApoA-IV, the present study indicated a novel role of ApoA-IV as an upper intestinal FAE-specific marker in the mature murine small intestine. Because our result presented the different feature of ApoA-IV protein compared to the previous studies, there is a potential that FAEspecific ApoA-IV has different structure, coding sequence, or truncation; thus, further study is necessary to elucidate the difference between the FAE-specific ApoA-IV and the conventional ApoA-IV in the VE. Our developmental analysis of ApoA-IV at the protein level showed that FAE-specific expression of ApoA-IV is characteristic of the small intestine of weanling and adult
mice, and both FAE and VE are high-expression sites for ApoA-IV during developmentally immature periods. A previous study similarly described this elevated gene expression in the small intestine during the neonatal period [7], but our study provided the further feature of the different localization of ApoA-IV protein before and after weaning. The reason for these age-dependent differences in expression warrants elucidation; however, we speculate that enhanced lipid flux during the neonatal period, perhaps from high-fat breastmilk feeding, induced the predominantly elevated villous enterocyte fat transport. A previous study demonstrated neonatal intestinal ApoA-IV mRNA levels that were more than fourfold those of adult intestine, with a subsequent gradual decrease in ApoA-IV mRNA levels over the next 14 days [7]. Accumulated studies revealed that ApoA-IV synthesis was upregulated after intestinal lipid infusion [17–20]. On the other hand, Black DD et al. demonstrated that intraduodenal lipid infusion induced the elevation of jejunal apo A-IV mRNA expression in newborn piglet, but those lipid responsiveness decreased as the piglets were weaned from a high-fat breast milk diet [21]. Taken together with restriction of expression of ApoA-IV in the FAE after weaning, we suspect
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