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Screening of Surface Markers on Rat Intestinal Mucosa Microfold Cells by Using Laser Capture Microdissection Combined with Protein Chip Technology

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Screening of Surface Markers on Rat Intestinal Mucosa Microfold Cells by Using Laser Capture Microdissection Combined with Protein Chip Technology Int J Clin Exp Med 2014;7(4):932-939 www.ijcem.com /ISSN:1940-5901/IJCEM1401061 Original Article Screening of surface markers on rat intestinal mucosa microfold cells by using laser capture microdissection combined with protein chip technology Junyong Zhao*, Xiaoyu Li*, Qifeng Luo, Lei Xu, Lei Chen, Li Chai, Yixiang Huang, Lin Fang Department of Breast and Thyroid Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai, 200072, China. *Equal contributors. Received January 21, 2014; Accepted April 10, 2014; Epub April 15, 2014; Published April 30, 2014 Abstract: Objective: The objective of this research was to investigate the possibility of screening surface markers on rat intestinal mucosa microfold cells (M cells) by using laser capture microdissection (LCM) combined with protein chip technology. Methods: We labeled rat intestinal mucosa microfold cells with Ulex europaeus agglutinin (UEA)-1 antibody and visualized these by immunofluorescence staining. Using the Proteome Profiler rat protein chip, we analyzed the protein expression profiles of LCM M-cells compared to lymph follicle-associated epithelial (FAE) cells, and we identified potential differences to screen for marker proteins. Results: M cells can be clearly distinguished from lymphoid FAE cells under the fluorescence microscope. We successfully cut, isolated, and obtained microfold and lymph FAE cells with more than 95% homogeneity. Six differentially expressed proteins were identified through comparison of the protein chip profiles of these 2 cell types. Among these, VEGF, LIX, CNTF, and IL-1α/IL-1F1 were found to be at significantly lower levels in M cells, IL-1ra/IL-1F3 and MIG/CXCL9 appeared in significantly higher levels in M cells (P < 0.05). Conclusion: The results presented here clearly demonstrate that the combined use of LCM and protein chip technology is effective in the screening of M cell surface markers with high sensitivity and specificity. This will facilitate isolation, identification, and establishment of M cell lines, allowing further characteriza- tion of their functional properties. Keywords: Laser capture microdissection (LCM), proteomics, protein chip, M cells, marker Introduction help to elucidate their functional mechanisms with potential clinical applications. Microfold cells (M cells) are specialized cells found mainly on the follicle-associated epithe- The cellular morphology and structure of M lium (FAE), and they are scattered among epi- cells varies greatly among species and also thelial cells overlying the dome structures of within species, where they can differ morpho- lymphoid follicles in the small and large intes- logically even within anatomic sites. The micro- tines. M cells are especially concentrated in fold structure is present only in human M cells, lymphoid aggregates such as Peyer’s patches which lack microvilli, whereas there are abun- (PPs). They are also abundantly scattered in the dant microvilli on the M cells of rabbits and distal colon and rectal mucosa [1]. M cells play mice. Moreover, the percentage and anatomi- crucial roles in antigen presentation and the cal locations of M cells in FAE vary substantially initiation of immune responses. The infectious among species, ranging from 5% to 10% in mechanisms of pathogens are dependent on human and mouse PPs, yet they are present at interactions with mucosal epithelial cells. In levels of approximately 50% in rabbit and recent years, there has been increasing inter- human caecum [4]. The surface markers of M est in the feasibility of designing orally adminis- cells also vary according to anatomical location. tered vaccines based on the functions of intes- In previous studies using exogenous large mol- tinal mucosa M cells [2, 3]. Therefore, the study ecule lectins to stain FAE in mouse PP, it was of the specific surface proteins of M cells may observed that intestinal mucosa M cells mainly Screening of surface markers on microfold cells express α (1, 2)-linked fucose and can be we screened and marked rat intestinal M cells. detected with Ulex europaeus agglutinin (UEA)- M cells and lymph follicle epithelial cells were 1, which can identify an epitope of M cells that purified using LCM combined with protein chip distinguishes them from intestinal epithelial technology, and we investigated novel M cell cells; however, lectin UEA-1 cannot distinguish membrane proteins as potential specific mark- M cells in the mouse cecal colon. ers, in order to advance future research into the biological characteristics of these cells. Very few M cell-specific ligands and receptors have been identified, and the vast majority of Material and methods those that have been found are not unique to M cells. Some important pathogen recognition Animals and treatment receptors (PRRs) such as Toll-like receptor-4 (TLR-4), platelet activating factor receptor Sprague-Dawley rats (weighing approximately (PAFR), and α5β1 integrin are expressed on the 100-120 g; male and female) were provided by surface of both human and mouse M cells. By Shanghai Slack Animal Center (Shanghai, interacting with pathogen-associated molecu- China), acclimatized for 2 days, and fasted for lar patterns (PAMPs) such as lipopolysaccha- 24 hours before experimentation. After anes- ride, lipoteichoic acid, peptidoglycan, and bac- thetization via intraperitoneal injection with 3% terial flagellin, these PRRs can activate pentobarbital sodium (40-45 mg/kg), rats were downstream pathways [5]. Although PRRs can sacrificed and whole small intestines collected be found in surrounding enterocytes, the into 0.9% balanced saline solution, scoured expression of these receptors varies with dif- carefully, secretions and excess fecal residue ferent cells. For example, α5β1 integrin is dis- removed, sheared, and the tissue containing persed on the lateral and basolateral surfaces the PPs were located (an average of 8-12 PPs in of enterocytes, whereas in intestinal mucosa M each rat), collected into a tube, and stored at cells, it is distributed only on the apical surface -80°C, after freezing and transport in liquid [3]. nitrogen. When screening mouse intestinal PP with UEA- Experimental materials 1, a large amount of nonspecific binding occurs to tissue, and 40% of UEA-1 reacts strongly All reagents used in this study were domestic, with goblet cells, with only 10% binding to intes- unless otherwise specified. FITC-labeled UEA-1 tinal mucosa M cells. These data illustrate that was purchased from Sigma (Munich, Germany); goblet cells may express the same UEA-1 immu- PEN-coated slides were purchased from Leica, nogenic antigen as M cells; however, work is and the cryotome (Leica CM1850), LCM underway to develop novel M cell surface mark- machine (Leica LMD6500), ECLTM CHEMI ers [6]. Malik et al. [7] developed an alginate- Reagent, SuperRX medical X-ray film, BCA pro- coated UEA-1 lectin ionic gel with an antigen tein quantification kit, and Proteome ProfilerTM desorption function. Research carried out on Arrays Rat Cytokine Array Panel A protein chip Balb/c mice indicated that this type of ionic gel kit were purchased from R & D company. can induce more efficient immune responses compared to a conventional vaccine. Moreover, Preparation and staining of frozen sections Kim et al. [8] found that the complement C5a receptor (C5aR) is expressed on the apical sur- Intestinal tissue was examined to ensure that it face of M cells and that the expression levels of contained intestinal mucosa FAE and selected C5aR mRNA in co-cultured Caco-2 cells was specimens embedded in optimal cutting tem- 6-fold higher than in mono-cultured Caco-2 perature (OCT) Tissue-Tek embedding medium. cells. In addition to C5aR, GP2 protein was also It was then dissected into 10-μm-thick serial detected on the surface of M cells and in mouse sections, subjected to improved H&E and PPs. immunofluorescence staining, and then placed on PEN-coated slides. Improved HE staining As current domestic and overseas research on was performed by staining with hematoxylin for M cells mainly focuses on experimental rat 1 s; ice-water flushing for 10 s; eosin for 10 s; models, SD rats were used in this study. Using dehydration in 85%, 95%, and 100% ethanol UEA-1, an antibody known to recognize M cells, for 10 s each; and clearing in xylene for 2 min. 933 Int J Clin Exp Med 2014;7(4):932-939 Screening of surface markers on microfold cells For immunofluorescence staining, the samples tion of protein concentrations using a BCA pro- were fixed in anhydrous alcohol that was pre- tein quantification kit. Gels were prepared by cooled at -20°C for 5 min, rinsed 3 times with addition of TEMED (10 μl) to 10 ml of 10% sepa- PBS-1% Triton X-100, and blocked with PBST- ration gel, which was mixed, and 8 ml of it was 0.05% BSA in a wet box at 4°C for 50 min. After poured slowly and allowed to set for 20-30 min. discarding the blocking solution, FITC-labeled Next, 10 ml of 10% stacking gel was prepared, lectin UEA-1 (5 μg/ml) was added, and samples TEMED (5 μl) was added to it, mixed, and imme- were incubated in wet boxes overnight at 4°C, diately poured. The comb was then inserted, washed 3 times with PBS, and stored at -80°C. and the gel was allowed to completely polymer- ize (approximately 30 min). The glass plates Laser capture microdissection were then fixed to the electrophoresis equip- ment and placed in the electrophoresis tank. PEN-coated slides were placed face down after Protein standards (10 μl) were measured along staining, and a 0.2-ml Eppendorf tube cap was with 20 µl M cell and lymph follicle epithelial placed on the microdissection machine dedi- cell samples. Sample buffer (3 times the vol- cated tubing rack. Real-time monitored sam- ume) was added to give a final volume of 30 μl. ples were prepared for separation on a com- Before gel loading, samples were denatured in puter screen by using a CCD camera, the target boiling water for 2-5 min.
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