ARTICLE |
Correspondence to: Rafael Bragado, Dept. of Immunology, Fundación Jiménez Díaz, Avda. Reyes Católicos, 2, 28040 Madrid, Spain. E-mail: rbragado@fjd.es
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Summary |
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The scavenger receptors CLA-1/SR-BI and CD36 interact with native and modified lipoproteins and with some anionic phospholipids. In addition, CD36 binds/transports long-chain free fatty acids. Recent biochemical evidences indicates that the rabbit CLA-1/SR-BI receptor can be detected in enterocytes, and previous studies showed the presence of mRNA for both CLA-1/SR-BI and CD36 in some segments of the intestinal tract. These findings prompted us to study their respective localization and distribution from the human stomach to the colorectal segments, using immunohistochemical methods. Their expression in the colorectal carcinoma-derived cell line Caco-2 was analyzed by Northern blotting. In the human intestinal tract, CLA-1/SR-BI was found in the brush-border membrane of enterocytes from the duodenum to the rectum. However, CD36 was found only in the duodenal and jejunal epithelium, whereas enterocytes from other intestinal segments were not stained. In the duodenum and jejunum, CD36 co-localized with CLA-1/SR-BI in the apical membrane of enterocytes. The gastric epithelium was immunonegative for both glycoproteins. We also found that CLA-1/SR-BI mRNA was expressed in Caco-2 cells and that its expression levels increased concomitantly with their differentiation. In contrast, the CD36 transcript was not found in this colon cell line, in agreement with the absence of this protein in colon epithelium. The specific localization of CLA-1/SR-BI and CD36 along the human gastrointestinal tract and their ability to interact with a large variety of lipids strongly support a physiological role for them in absorption of dietary lipids.
(J Histochem Cytochem 49:12531260, 2001)
Key Words: intestinal lipid absorption, intestinal lipid receptors, CD36, CLA-1/SR-BI
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Introduction |
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FAT DIGESTION and intestinal lipid absorption initially involve the luminal lipolysis of triglycerides to monoglycerides and fatty acids and of cholesterol esters from the bile and diet to free cholesterol and fatty acids. The products of lipid hydrolysis interact with bile salts to form mixed micelles, which pass through an unstirred water layer covering the enterocyte surface, raising the concentration of their components (fatty acids, monoglycerides, and free cholesterol) near the brush-border membrane of enterocytes. Finally, the lipid molecules are released from micelles and enter the enterocyte (
The lipoprotein receptor CLA-1, also known as scavenger receptor BI (SR-BI) (
CD36 is a plasma membrane glycoprotein structurally related to CLA-1/SR-BI, which is expressed in platelets (
The regiospecificity of SR-BI and CD36 has been characterized along the gastrocolic axis of the murine species. In mouse, SR-BI is expressed in the proximal intestine where cholesterol absorption occurs (
Although Northern blotting experiments have revealed the expression of both CD36 and CLA-1/SR-BI in human intestine (
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Materials and Methods |
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Primary Antibodies
The monoclonal antibody (MAb) FA6.152 (
Tissue Samples
Tissue samples were obtained from patients who had undergone surgical resection of different areas of the digestive tract. Normal tissue found adjacent to the resected pathological tissues was used in all cases, as ascertained by routine hematoxylin/eosin staining, with the ongoing approval of the internal review board. Immediately after resection, samples were frozen in isopentane or fixed with 4% paraformaldehyde in PBS for 624 hr at 4C. These fixed samples were embedded in paraffin according to conventional methods (
StreptavidinPeroxidase Methods
Frozen and paraffin sections (5 mm thick) were mounted in silanized slides and allowed to dry overnight before immunohistochemical staining. Paraffin was removed with xylene. Sections were hydrated and endogenous peroxidase activity was inhibited by incubation with 3% H2O2 for 10 min and 0.3% H2O2 in methanol for an additional 20 min. Sections were then washed in Tris-buffered saline (TBS) and incubated in 3% normal serum, goat serum for CLA-1/SR-B1 staining, or horse serum for CD36, with 0.05% Triton X-100 in TBS, pH 7.5, at room temperature (RT) for 30 min. Then the sections were incubated for 12 hr at 4C with the primary antibodies, anti-CLA-1 (1:200) and anti-CD36 (4 µg/ml) diluted in TBS. After washing twice in TBS, sections were incubated with the secondary antibodies for 1 hr at RT. The biotinylated secondary antibodies used were goat anti-rabbit IgG (1:200) for CLA-1/SR-B1 and horse anti-mouse IgG (1:200) for CD36 (both from Vector Labs; Burlingame, CA). Sections were washed in TBS and incubated with the streptavidinperoxidase complex (Zymed Labs; San Francisco, CA) for 30 min and washed in TBS followed by Tris-HCl buffer, pH 7.6. The peroxidase activity was revealed using 3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen (Sigma; St Louis, MO). The reaction product of DAB was intensified with nickel nitrate (10 µl of an 8% solution of nickel nitrate in 1 ml DABH2O2 solution) to obtain a dark black color of immunostained antigens (
Immunofluorescence Methods
Sections were processed and incubated with the primary antibodies as described above and then incubated with rhodamine (1:150)- or FITC (1:60)-conjugated secondary antibodies (Boehringer Mannheim; Mannhein, Germany) for 60 min in darkness. After washing in TBS, the sections were mounted using Mowiol (SigmaAldrich Quimica; Madrid, Spain) and observed in a Zeiss epifluorescence microscope.
Control Experiments
Although the specificity of the antibodies has been previously described (see above) to further assess their specificity in the immunohistochemical procedures performed in this study the following negative controls were systematically performed for both CLA-1/SR-BI and CD36 staining: (a) omitting the primary specific antibodies, (b) using rabbit preimmune serum or normal mouse serum instead of the primary antibodies, and (c) incubating with an inappropriate secondary antibody after the incubation with the primary antibodies at optimal titers.
Northern Blotting Analysis
Total RNA was extracted from Caco-2 cells, collected at Days 0, 3, 6, 9, and 12 after reaching confluence, using the acid guanidine thiocyanate method (
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Results |
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The distribution of CLA-1/SR-BI and CD36 along the gastrocolic and crypt-to-villus axes of the human gastrointestinal mucosa has been investigated. Both of the immunohistochemical methods we used, streptavidinperoxidase (Fig 1 and Fig 2) and immunofluorescence (Fig 3), led to similar results. The use of the polyclonal antibody anti-CLA-1/SR-BI in formalin-fixed, paraffin-embedded samples and in frozen tissues provided identical results. However, although CD36 was detected in paraformaldehyde-fixed, paraffin-embedded samples, immunolabeling for CD36 on unfixed frozen sections provided the best signal-to-noise ratio. In addition, and as an internal positive control for CD36 immunostaining, this protein was clearly detected in microvascular endothelial cells of the gastrointestinal tract (Fig 3) whereas the endothelium of large vessels was unstained, as has been previously described (
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In stomach, neither CLA-1/SR-BI nor CD36 was detected in epithelial, parietal, mucous and endocrine cells (Fig 1A). In contrast, both CLA-1/SR-BI and CD36 were found in the brush-border membrane of enterocytes at specific segments of the intestinal tract, but not in the nucleus, cytoplasm, or basolateral plasma membrane of enterocytes. Intestinal goblet cells, Paneth cells, and enteroendocrine cells were immunonegative for both glycoproteins. Interestingly, regional differences in the distribution of these glycoproteins along the gastrointestinal tract were noted. Thus, while the duodenal epithelium showed positive staining for both glycoproteins (Fig 1B-1D, Fig 2A2E, and Fig 3A3C), differences in the immunoreactivity along the crypt-to-villus axis were observed. CLA-1/SR-BI was expressed in the brush-border membrane of enterocytes all along the epithelium, from the crypt to the villous tip. Surface enterocytes were the most intensely stained cells for CLA-1/SR-BI. However, CD36 labeling was restricted to the brush border of the enterocytes located in the upper two thirds of the villus and it was absent from crypt cells (Fig 3A3C). On the other hand, CLA-1/SR-BI was also detected on the luminal surface of Brunner's gland cells, where no immunoreactivity for CD36 was observed (Fig 3D and Fig 3E). Moreover, a similar distribution pattern for CLA-1/SR-BI and CD36 in the jejunal mucosa was observed (Fig 1E, Fig 2F2H, and Fig 3F). A negative immunoreaction for CD36 was observed in the epithelium of the ileum, colon, and rectum (Fig 3G and Fig 3H), where the immunoreactivity for CLA-1/SR-BI glycoprotein was intense (Fig 1F1K). In these intestinal segments, CLA-1/SR-BI was found in the apical membrane of enterocytes all along the intestinal epithelium. As observed in small intestine, the immature undifferentiated cells from the base of the crypts showed a positive immunoreaction for CLA-1/SR-BI, whereas those areas of the colon and rectum epithelium mainly composed of goblet cells were unstained (Fig 1K).
We also examined the expression of CD36 and CLA-1/SR-BI mRNA in human Caco-2 cells, an enterocytic cell line derived from a colon adenocarcinoma that expresses CLA-1/SR-BI. This cell line differentiates on reaching confluence, forming a polarized monolayer with an apical brush-border membrane, similar to that of the enterocyte. Expression of apoE mRNA was used as a positive control for differentiation (
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Discussion |
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In this study we have demonstrated the localization of CLA-1/SR-BI and CD36 receptors in the brush-border membrane of human enterocytes. Both proteins are co-localized in the enterocytes that cover the surface of the duodenum and jejunum villi. Moreover, and unlike CD36, the expression of CLA-1/SR-BI is not restricted to the duodenal and jejunal villous cells but is also present in the intestinal crypts, Brunner's glands, and in the rest of the intestinal tract, ileum, colon and rectum, where the localization of this glycoprotein is demonstrated for the first time. Accordingly, Caco-2 cells, a colon-derived cell line, expressed mRNA for CLA-1/SR-BI but not CD36.
The broad ligand-binding specificity of both CD36 and CLA-1/SR-BI (
How the intestinal absorption of lipids other than cholesterol takes place is still a matter of debate. It has been shown that the uptake of cholesterol esters, triacylglycerols, fatty acids, and phospholipids by small intestine brush-border membrane vesicles is completely inhibited by proteolytic treatment (
From our experimental observations it is also difficult to ascribe specific and/or differential roles for CD36 and CLA-1/SR-BI. Simultaneous expression of CD36 and CLA-1/SR-BI in the proximal intestine might serve either to guarantee, by acting redundantly, a vital function and/or to allow a fine-tuned modulation of lipid absorption. Interestingly, it has recently been reported that in SR-BI deficient mice, intestinal cholesterol absorption is normal, suggesting the necessity of other molecules able to compensate for the loss of intestinal absorptive activity (
The results of the present study, together with previous evidence, demonstrate the existence of a family of lipid-binding proteins localized in the brush-border membrane of the enterocytes that could mediate the absorption of lipids. Further investigations are necessary to determine the functions of each protein in the different intestinal segments where they are expressed. These studies could provide new therapeutic strategies that, acting on these transporters, would permit regulation of the absorption of lipids, a physiological function directly related to hypercholesterolemia and obesity, two major risk factors for atherosclerosis.
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Acknowledgments |
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Supported by grants from Comisión Interministerial de Ciencia y Tecnología (PM98/0063 to R.B.), Comunidad Autónoma de Madrid (08.2/0004/1998 to R.B.), and Fondo de Investigación Sanitaria (97/0389 to A.M.H. and 99/1046 to M.A.V.). E. Teixeiro is a fellow of the CAM.
Received for publication December 13, 2000; accepted May 2, 2001.
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