Copyright ©The Histochemical Society, Inc.

Immunohistochemical Characterization of the Distribution of Galectin-4 in Porcine Small Intestine

Melissa A. Wooters1, Michael B. Hildreth, Eric A. Nelson and Alan K. Erickson

Veterinary Science Department (MAW,MBH,EAN,AKE), Biology/Microbiology Department (MBH), South Dakota State University, Brookings, South Dakota

Correspondence to: Alan K. Erickson, Veterinary Science Department, N. Campus Drive, PO Box 2175, South Dakota State University, Brookings, SD 57007. E-mail: alan.erickson{at}sdstate.edu


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Galectins are an evolutionarily conserved family of 15 different lectins found in various combinations in virtually every type of animal cell. One of the primary galectins expressed in intestinal epithelium is galectin-4, a tandem-repeat galectin with two carbohydrate-recognition domains in a single polypeptide chain. In the current study, we produced an anti-galectin-4 monoclonal antibody (MAb) for determining the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its functions in the small intestine. In immunohistochemistry studies, this MAb detected galectin-4 primarily in the cytoplasm of absorptive epithelial cells lining intestinal villi. Mature epithelial cells at the villous tips stained the most intensely with this MAb, with progressively less intense staining observed along the sides of villi and into the crypts. In addition to its cytoplasmic localization, galectin-4 was also associated with nuclei in villous tip cells, indicating that some galectin-4 may migrate to the nucleus during terminal maturation of these cells. In intestinal crypts, a specific subset of cells, which may be enteroendocrine cells, expressed galectin-4 at a relatively high level. Galectin-4 distribution patterns were similar in all three regions (duodenum, jejunum, and ileum) of porcine small intestine. (J Histochem Cytochem 53:197–205, 2005)

Key Words: galectin-4 • porcine intestine • monoclonal antibodies • epithelial cells


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
GALECTINS ARE AN EVOLUTIONARILY conserved family of carbohydrate-binding proteins (lectins) found in a wide variety of animal cells and tissues. Members of the galectin family share binding affinity for ß-galactosides and N-acetyllactosamine-enriched glycoconjugates, and conserved amino acid sequence elements in their carbohydrate-recognition domains (CRDs) (Mandrell et al. 1994Go). The 15 members of the galectin family that have been identified can be divided into three groups based on their domain structure (Hirabayashi and Kasai 1993Go): (a) proto-type galectins (galectins-1, -2, -5, -7, -10, -11, -13, and -14) contain one CRD and exist as either monomers or non-covalently bound homodimers, (b) chimera-type galectins (galectins-3 and -15) contain a non-lectin domain and a CRD in a single polypeptide chain, and (c) tandem-repeat-type galectins (galectins-4, -6, -8, -9, and -12) have two distinct CRDs in a single polypeptide chain. Generally, every animal expresses multiple members of the galectin family with virtually every cell expressing at least one galectin or, more commonly, a combination of various galectins (Liu et al. 2002Go). It appears that individual galectins are expressed in a cell- and tissue-specific manner (Colnot et al. 1998Go). Some galectins are widely distributed among different types of cells and are found in high concentrations in multiple organs. For example, galectin-1 is expressed in epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, and neurons found in lung, liver, lymph nodes, and colon (Rabinovich 1999Go). Other galectins are predominantly expressed in a single-cell type and are found only in a limited number of organs. For example, galectin-4 is found only in the epithelium of the organs along the alimentary canal (Rabinovich 1999Go; Huflejt and Leffler 2004Go).

The early occurrence of galectins in animal evolution, along with the expansion of the family during evolution, suggests that galectins perform critical functions within cells (Yang and Liu 2003Go). Numerous important biological functions have been assigned to various galectins including regulation of pre-mRNA splicing, apoptosis, and cell-cycle progression; activation of inflammatory cells; and adhesion of cells to each other and to the extracellular matrix (reviewed in Rabinovich 1999Go). Galectins have also been shown to be involved in cancer cell invasion and metastasis (Van Den Brule and Castronovo 2000Go; Huflejt and Leffler 2004Go). Just as there is variation in the distribution of galectins in cells and tissues, there also seems to be variation in the biological functions of each galectin in specific cells at different times during development and growth (reviewed in Rabinovich 1999Go). Consequently, to gain an overall understanding of the diversity of functions performed by galectins, individual galectins need to be evaluated in specific cells at particular times during the development and growth of an animal, organ, or cell.

Galectin-4 is expressed in the epithelium lining the entire length of the alimentary canal in mammals (reviewed in Huflejt and Leffler 2004Go). The long-term goal of our research is to determine the biological functions of galectin-4 in the small intestine of postnatal animals. Because galectin-4 is a tandem-repeat galectin with two distinct CRDs in a single polypeptide chain, it seems logical that it accomplishes some of its biological functions by crosslinking glycoconjugates. The molecules that are crosslinked by galectin-4 and the subsequent effects of that crosslinking are not known. In the current study, we determined the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its crosslinking functions in the intestine. Using immunohistochemistry (IHC) with an anti-galectin-4 monoclonal antibody (MAb), we found that galectin-4 is primarily expressed in the cytoplasm of the absorptive epithelial cells covering the villi in the three major regions (duodenum, jejunum, and ileum) of porcine small intestine. The highest level of expression of galectin-4 was observed in mature epithelial cells at villous tips where, in addition to cytoplasmic staining, we also observed nuclear staining, indicating that the localization of galectin-4, and quite possibly its function, change during the terminal maturation of intestinal epithelial cells. We also identified a subset of intestinal crypt cells, which may be enteroendocrine cells that express galectin-4 at a high level.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Production of Anti-galectin-4 MAbs
Galectin-4 was purified from porcine small intestine using the procedure described by Mandrell et al. (1994)Go, which employs affinity chromatography on a lactosyl-Sepharose column (Levi and Teichberg 1981Go) as its key purification step. The identity and purity of porcine galectin-4 was verified by amino acid sequence analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Anti-galectin-4 MAbs were prepared following the procedures described by Harlow and Lane (1988)Go. Highly purified porcine galectin-4 [75 µg in 200 µl of phosphate-buffered saline (PBS), 15 mM KH2PO4, 8 mM Na2HPO4, 137 mM NaCl, 2.6 mM KCl, pH 7.4) containing 2 mM EDTA and 1 mM DL-dithiothreitol (DTT)] was mixed with an equal volume of Freund's complete adjuvant (Sigma; St Louis, MO) and injected intraperitoneally (IP) into BALB/c AnN mice. Booster injections of equal volumes of galectin-4 (75 µg in 200 µl of PBS containing 2 mM EDTA and 1 mM DTT) and Freund's incomplete adjuvant were given at 2-week intervals for a total of four IP injections. Two days before the harvest of splenic lymphocytes, mice were immunized intravenously through the tail vein with galectin-4 (20 µg in 50 µl PBS). Splenic lymphocytes were harvested and fused with NS-1 myeloma cells to produce hybridomas (Harlow and Lane 1988Go). Hybridoma cells were allowed to grow for 2 weeks in selective hybridoma media containing hypoxanthine, aminopterine, and thymidine (Harlow and Lane 1988Go). Culture supernatants from the growing hybridomas were screened for the presence of anti-galectin-4 antibodies using the galectin-4-specific ELISA described below. Hybridomas that produced antibodies specific for galectin-4 were subcloned by limit dilution to get a single clone of hybridoma per well. These hybridomas were allowed to grow for 2 weeks and then screened using the galectin-4-specific ELISA. Ascites containing anti-galectin-4 MAbs was produced by priming mice with pristane and then 2 weeks later injecting the hybridoma cells producing anti-galectin-4 antibody. Ascites fluid was collected 10–12 days after hybridoma injection. A mouse monoclonal antibody isotyping kit (Serotech; Kidington, UK) was used to determine the isotype of the MAb.

Purification of MAbs from Ascites Fluid
To remove lipids, ascites fluid was filtered through glass wool. Filtered ascites fluid was clarified by centrifugation (30 min, 20,000 x g, 4C), diluted 10-fold with 0.1 M sodium acetate, pH 5.0 and loaded onto a HiTrap protein G column (5 ml; Pharmacia, Piscataway, NJ) equilibrated with 0.1 M sodium acetate, pH 5.0. The column was washed with five column volumes of 0.1 M sodium acetate, pH 5.0. Bound MAbs were eluted from the column with 0.1 M glycine–HCl, pH 2.8. To minimize exposure of MAbs to low pH conditions, 50 µl of 2 M Tris base was added per milliliter of eluate as fractions from the column were collected. The MAb-containing fractions were pooled, dialyzed against PBS at 4C with two buffer changes, and stored at –20C.

ELISA for Detection of Anti-galectin-4 MAbs
Purified galectin-4 (2 µg/well; diluted in 50 mM sodium carbonate, pH 9.6) was immobilized to 96-well Immulon I polystyrene plates (Dynatech; Alexandria, VA) at 37C for 2 hr. The wells were washed three times with PBS containing 0.05% Tween 20 (PBS-Tween). The wells were blocked with 2% bovine serum albumin (BSA) in PBS for 1 hr at 37C and then washed three times with PBS-Tween. Culture supernatants from hybridomas or dilutions of ascites fluid were added to the wells and incubated for 1 hr at 37C. The plate was washed three times with PBS-Tween. Goat-anti-mouse peroxidase conjugate (100 µl of 0.047 mg/ml conjugate in PBS containing 0.01% BSA; ICN, Costa Mesa, CA) was added to the plate and incubated for 1 hr at 37C. The wells were then washed three times with PBS-Tween and once with PBS. Peroxidase activity was detected using 2,2'-azino-di-3-ethyl-benzthiazoline sulfonic acid (ABTS) as previously described (Erickson et al. 1992Go).

Immunoblotting
Lactose-binding proteins were prepared from the jejunum of a 5-week-old pig using the procedure of Mandrell et al. (1994)Go as modified by Wooters et al. (unpublished data). The resulting lactose-binding proteins were dialyzed against Milli-Q water (Millipore; Bedford, MA) and dried in a Speed Vac (Thermo Savant; Holbrook, NY). Lactose-binding proteins were dissolved in XT SDS-PAGE sample buffer containing XT reducing agent (Bio-Rad; Hercules, CA) and heated to 95C for 5 min prior to electrophoresis. Lactose-binding proteins (5 µg/lane) were separated on Criterion X Precast Bis-Tris SDS-PAGE gels (12% resolving gel; Bio-Rad) using XT MES running buffer (Bio-Rad) according to the manufacturer's suggested protocol. Separated proteins were electrotransferred to polyvinylidene fluoride (PVDF) sheets (0.45 µm pore size; Millipore) as previously described by Towbin et al. (1979)Go. The proteins on the PVDF membrane were stained with Ponceau S (0.1% in 1% acetic acid) for 5 min. Unbound stain was removed from the membrane by washing the membrane twice with 1% acetic acid for 5 min. A digital image of the stained membrane was acquired using a Hewlett-Packard Scanjet 4400C (Palo Alto, CA) scanner. The Ponceau S stain was removed from the proteins by three 10-min washes in PBS-Tween. The membranes were blocked for 1 hr with PBS-Tween containing 2% BSA and washed three times for 5 min each with PBS-Tween containing 0.1% BSA. Purified MAb A3-166 (0.1 µg/ml in PBS-Tween containing 0.1% BSA) was incubated with the membrane for 1 hr at room temperature (RT). The membrane was washed three times for 5 min with PBS-Tween containing 0.1% BSA and incubated with peroxidase-conjugated rabbit anti-mouse IgG H+L (0.18 µg/ml in PBS-Tween containing 0.1% BSA; Jackson ImmunoResearch, West Grove, PA) for 1 hr at RT. The membrane was washed three times for 5 min each with PBS-Tween containing 0.1% BSA and once for 5 min with Milli-Q water (Millipore). Bound peroxidase activity was detected by incubating the membrane in a stabilized solution of 3,3',5,5'tetramethylbenzidine (TMB) for horseradish peroxidase (Promega; Madison, WI) for 10 min. Color development was stopped by rinsing the membrane three times with Milli-Q water.

Immunoprecipitation with MAb A3-166
MAb A3-166 was immobilized to Protein G-Sepharose beads using a Seize X Protein G Immunoprecipitation kit (Pierce Biotechnology, Inc.; Rockford, IL). Briefly, MAb A3-166 (0.4 ml of 0.24 mg/ml in PBS) was incubated with 0.1 ml of packed Immunopure Immobilized Protein G Plus beads at RT for 1 hr with end-over-end rotation. This solution was centrifuged at 3000 x g for 1 min in a Handee Spin Cup Column (Pierce). The beads were washed three times with PBS by suspension in 0.5 ml of PBS followed by centrifugation at 3000 x g for 1 min in a Handee Spin Cup Column. The Protein G beads were suspended in 0.4 ml of PBS. To immobilize the MAb to the Protein G beads, 25 µl of a solution containing disuccinimidyl suberate (DSS; 25 mg/ml DSS in dimethyl formamide) was added to the beads and incubated with mixing for 1 hr at RT. To remove unbound antibody and quench the DSS reaction, the beads were washed five times with Immunopure IgG elution buffer and three times with IP buffer (50 mM sodium acetate buffer, pH 5.0, 500 mM NaCl, 0.1% SDS, 1% NP-40) by suspending the beads in 0.5 ml of the appropriate buffer followed by centrifugation at 3000 x g for 1 min in a Handee Spin Cup Column. The washed beads were incubated for 16 hr at 4C with lactose-binding proteins (400 µg in IP buffer) from porcine intestine. Unbound antigen was removed by washing the beads five times with 0.4 ml of IP buffer and once with 0.4 ml Milli-Q water (Millipore). Bound antigen was eluted from the beads by incubating the beads for 5 min at 95C with 200 µl of the XT SDS-PAGE sample buffer (Bio-Rad) with no reducing agent. The eluted antigen was collected by centrifugation at 3000 x g for 1 min in a Handee Spin Cup Column and 10 µl of XT reducing agent (Bio-Rad) was added to each sample. The resulting samples were then analyzed using the immunoblotting technique described above with 10 µl of immunoprecipitation sample run in each lane.

Immunohistochemistry
Small intestine was harvested from a 4-week-old pig immediately after euthanasia. The small intestine was rinsed with PBS containing fresh 2 mM PMSF. Tissue segments (75 mm) were cut from the duodenum, jejunum, and ileum. The tissues were then fixed in 10% neutral buffered formalin for 48 hr. The tissues were trimmed, dehydrated through a graded ethanol and propanol series, cleared through three changes of propar, and infiltrated and embedded with paraffin. Five-µm-thick sections were cut using a Leica Microtome (Nussloch, Germany) and placed on Surgipath Snowcoat Xtra slides (Winnipeg, Canada). The slides were air dried overnight and then placed at 60C for 1 hr. The tissues were deparaffinized and treated with 30% hydrogen peroxide. The tissues were washed three times with deionized water and soaked in TBS-Tween (0.05 M Tris-HCl buffer containing 0.3 NaCl and 0.1% Tween 20, pH 7.6) for 5 min. Slides were loaded into a DAKO Autostainer (DakoCytomation; Carpinteria, CA) where they were incubated with primary antibody, MAb A3-166 (0.0048 mg/ml) for 30 min at RT and rinsed with TBS–Tween. Antibody binding was detected using the DAKO Envision+ System–HRP (DakoCytomation) according to the manufacturer's protocol. The tissues were counterstained with Meyer's hematoxylin, coverslipped, and examined under the microscope. Negative controls were performed using the same steps as above with the following changes. For the negative control without antibody, no MAb A3-166 was incubated with the tissue. For the control using an antibody for a non-intestinal target, a MAb for NS-1, a mouse tumor cell line, was incubated on the tissue in place of MAb A3-166. For the blocking control, a 10-fold molar excess of purified galectin-4 was incubated with MAb A3-166 for 30 min at RT prior to the MAb being placed on the tissue. A Fujix Digital Camera HC-300Z (Fuji Photo Film Co. Ltd.; Tokyo, Japan) was used for IHC photographs. Exposure time for each picture was selected by the photomicroscopy system.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Specificity of Anti-galectin-4 MAb A3-166
A clone of hybridoma cells that produces an IgG1 MAb (designated MAb A3-166) that specifically recognizes porcine galectin-4 was identified using a galectin-4-specific ELISA screening procedure. The affinity and binding specificity of MAb A3-166 was then further evaluated using ELISA, immunoblotting, and immunoprecipitation techniques. In ELISA tests, highly purified galectin-4 was recognized by MAb A3-166 at levels as low as 0.5 µg/well, indicating that this MAb can detect the presence of low amounts of galectin-4 (data not shown). For use in immunoblotting and immunoprecipitation experiments, lactose-binding proteins were purified from porcine intestinal mucosa extracts using affinity chromatography on a lactosyl-Sepharose column (Levi and Teichberg 1981Go; Mandrell et al. 1994Go). This preparation contained five bands that stained with Ponceau S (Figure 1A , Lane 2), including single bands at 14, 17, and 34 kD, and a doublet between 18 kD and 22 kD. Amino acid sequencing studies (Wooters MA, et al., unpublished data) have shown that the 34-kD lactose-binding protein is porcine galectin-4 (Chiu et al. 1994Go). The proteins comprising the 18- to 22-kD doublet are likely proteolytic degradation products of the 34-kD galectin-4 (Wooters MA, et al., unpublished data), similar to the galectin-4 degradation products identified in rat intestine by Leffler et al. (1989)Go and Tardy et al. (1995)Go. The 14-kD protein may be galectin-1, which is not found in intestinal epithelial cells but is found in the subepithelial connective tissue of intestine (Wasano and Hirakawa 1997Go), whereas the 17-kD protein is either a degradation product of a higher molecular mass galectin like galectin-3 or galectin-4, or a previously uncharacterized lactose-binding protein. Also present in the lactose-binding protein preparation was a 29-kD protein, which is likely galectin-3, that is not detected by Ponceau S staining (Figure 1A, Lane 2) but can be detected with more sensitive protein-staining techniques like silver staining (data not shown). In immunoblot experiments with lactose-binding proteins from porcine intestine, MAb A3-166 recognized the 18- to 22-kD doublet and the 34-kD protein but not the 14-, 17-, or 29-kD proteins (Figure 1A, Lane 3). When the primary antibody (MAb A3-166) was left out of the immunoblotting procedures, no nonspecific binding of the secondary antibody to lactose-binding proteins was observed (Figure 1A, Lane 4). To further evaluate the specificity of MAb A3-166, immunoprecipitation studies were performed. Similar to the immunoblotting results, MAb A3-166 immunoprecipitated the 18- to 22-kD and 34-kD proteins but not the 14-, 17-, and 29-kD proteins (Figure 1B, Lane 2). Together these experiments clearly show that MAb A3-166 specifically recognizes intact 34-kD galectin-4 and 18- to 22-kD degradation products of galectin-4 but not other lactose-binding proteins.



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Figure 1

Immunoblotting and immunoprecipitation with MAb A3-166. (A) Lactose-binding proteins from porcine intestinal mucosa (5 µg; Lanes 2–4) were separated by SDS-PAGE and transferred to PVDF membranes as described in Materials and Methods. The membranes were stained with Ponceau S (Lane 2) and then probed with MAb A3-166 (Lane 3) and no primary antibody (Lane 4). The migration positions of the major lactose-binding proteins are indicated with arrows at the left of Lane 2. The migration positions of 34-kD galectin-4, and the 18- to 22-kD degradation products of galectin-4 are indicated with arrowheads at the right of Lane 3. (B) Immunoprecipitation reactions using MAb A3-166 were performed on lactose-binding proteins from porcine intestinal mucosa as described in Materials and Methods. The immunoprecipitated proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were probed with MAb A3-166 (Lane 2) and no primary antibody (Lane 3). The migration positions of 34-kD galectin-4, and the 18- to 22-kD degradation products of galectin-4 are indicated with arrowheads at the right of Lane 2. The masses of pre-stained Rainbow molecular weight markers (Lanes 1A and 1B; Amersham, Piscataway, NJ) are indicated in kilodaltons at the left of each panel.

 
Galectin-4 Distribution in Porcine Small Intestinal Tissue Sections
MAb A3-166 was used to detect the distribution of galectin-4 in tissue sections from the three major divisions (duodenum, jejunum, and ileum) of porcine small intestine (Figure 2) . Staining by MAb A3-166 was observed in the absorptive epithelial cells lining the villi of the small intestine (Figure 2), but not in goblet cells (Figure 3B) or intraepithelial lymphocytes. Also, no staining of galectin-4 was observed in the other layers of the intestinal wall including the lamina propria, submucosa, muscularis, or serosa (Figure 2A). Similar staining patterns by MAb A3-166 were observed for all three regions of the small intestine [duodenum (Figure 2A and 2C), jejunum (Figure 2E), and ileum (Figure 2F)]. When the primary antibody was left off the tissues (Figure 2A inset) or when MAb A3-166 was replaced with similar subclass (IgG1) antibody specific for a non-intestinal target protein (NS-1 mouse tumor cell line; Figure 2B), no staining was observed in the intestinal sections, indicating that the staining of intestinal epithelial cells (Figure 2A) is due to binding of MAb A3-166 to intestinal proteins. To demonstrate that MAb A3-166 was specifically recognizing galectin-4, an excess of purified galectin-4 was used to block MAb A3-166 binding to intestinal cells. When a 10-fold molar excess of purified galectin was incubated with the MAb A3-166 prior to it being applied to the tissue section, staining of the intestinal epithelial cells by the antibody was blocked (Figure 2D). Interestingly, staining of other regions of the tissue, like the apical glycocalyx of epithelial cells and the submucosa, increased (Figure 2D). This staining pattern likely results from the binding of complexes of galectin-4 and MAb A3-166, through the lectin-binding activity of galectin-4, to extracellular N-acetyllactosamine-containing glycans that galectin-4 may not normally have access to in native tissues. An anti-galectin-4 IgM subclass MAb (developed in our laboratory) produced similar results as MAb A3-166 in IHC studies with consistent staining observed in the absorptive villous epithelial cells throughout the small intestine (data not shown).



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Figure 2

Immunohistochemical detection of galectin-4 expression in porcine small intestine. Tissue sections from porcine small intestine [duodenum (A–D), jejunum (E), ileum (F)] were stained with MAb A3-166 at a dilution of 1:100 (A,E,F) or 1:1000 (C,D). The absorptive epithelial cells along the villi stained intensely (A,C,E,F) with the most intense staining observed in the mature epithelial cells at the tips of villi (arrowheads in C,E,F). Negative controls without antibody (inset on A) and with an antibody for a non-intestinal target protein (B) showed no staining. Blocking studies, performed in the presence of a 10-fold molar excess of purified galectin-4 (D), decreased staining of epithelial cells by MAb A3-166 but increased staining of the apical glycocalyx, submucosa, and serosa. Original magnifications were x40 (A) and x100 (B–F).

 


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Figure 3

Immunohistochemical detection of galectin-4 expression along the crypt–villus axis. Tissue sections from porcine small intestine were stained with MAb A3-166 at a dilution of 1:100. The cytoplasm of absorptive epithelial cells, but not goblet cells (arrowhead in B), along the villi stained intensely (A,B). The most intense staining was observed in the villous tip cells (A) in which high-intensity staining was also observed in epithelial cell nuclei (arrowheads in A). Epithelial cell staining decreased in the crypts (C), except for a subset of heavily stained crypt cells, which may be enteroendocrine cells (arrowheads in C). Original magnifications were x400 (B–C) and x1000 (A).

 
There are some consistent differences in the intensity of MAb A3-166 staining along the crypt–villus axis. The highest intensity staining was observed in epithelial cells at the tip of the villi (Figures 2C, 2E, 2F, and 3A), with progressively less intense detection of galectin-4 along the sides of the villi (Figure 3B) and into the crypts (Figure 3C). This is especially evident when the concentration of MAb A3-166 was decreased 10-fold, revealing that the highest concentration of galectin-4 is present in the epithelial cells at the villous tips (Figure 2C). In most of the epithelial cells lining the villi, the distribution of galectin-4 within intestinal epithelial cells was relatively continuous throughout the cytoplasm with slightly higher intensity binding on the apical side of the cells (Figure 3B). An exception to the cytoplasmic localization of the galectin-4 was observed in the epithelial cells at the tips of the villi where, in addition to cytoplasmic staining, dark staining nuclei were also observed (Figure 3A). Most of the epithelial cells within the crypt stained lightly or not at all with MAb A3-166, whereas a small population of crypt cells contained regions (usually on the basal side or vascular pole of the cells) that stained intensely with this antibody (Figure 3C). These same characteristics of binding intensity along the crypt–villus axis were observed in all three small intestinal regions [duodenum (Figure 2A and 2C), jejunum (Figure 2E), and ileum (Figure 2F)]. Preliminary IHC studies using tissues sections from other animals indicate that MAb A3-166 stains intestinal epithelial cells from rats, but not frogs or sheep (data not shown).


    Discussion
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 Summary
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 Materials and Methods
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 Discussion
 Literature Cited
 
Among normal mammalian tissues, galectin-4 expression is limited to the epithelium lining the alimentary canal from the tongue through the large intestine (Oda et al. 1993Go; Chiu et al. 1994Go; Wasano and Hirakawa 1995Go; Danielsen and van Deurs 1997Go; Huflejt and Leffler 2004Go). Although some studies in normal tissues have suggested that galectin-4 is involved in stabilization of cellular junctions and membranes (Chiu et al. 1994Go; Danielsen and van Deurs 1997Go), the biological functions performed by galectin-4 in the different organs along the alimentary canal have not been clearly defined. The long-term goal of our research is to characterize the biological functions performed by galectin-4 in the small intestine. Structural characteristics of galectin-4 (tandem-repeat galectin with two CRDs with distinct carbohydrate-binding specificities connected by a linker region) suggest that it may accomplish its functions by crosslinking two glycoconjugate ligands in a manner that changes or stabilizes some aspect of cellular activity (Wu et al. 2002Go). Most potential glycoconjugate ligands for galectins, and for lectins in general, are found on the outer surface of cells and in the extracellular matrix, implying that galectin function occurs in an extracellular environment. However, galectins have many characteristics that are similar to intracellular proteins including an acetylated N terminus, no secretion signal peptide, and synthesis on free ribosomes (reviewed in Liu et al. 2002Go). In addition, the carbohydrate-binding and crosslinking activities of galectin-4 are destroyed in the extracellular environment by oxidation of sulfhydryl residues and proteolysis, respectively (Liu et al. 2002Go). The first step in gaining an understanding of the functions of galectin-4 in small intestine is to determine where galectin-4 is normally found in small intestinal tissue. In the current study, we rapidly fixed fresh porcine intestinal tissue with formalin to immobilize the tissue's galectin-4 in its naturally occurring location and then used IHC analysis with a galectin-4-specific MAb to visualize the distribution of galectin-4 in these tissues.

Previously, Danielsen and van Deurs (1997)Go assessed the distribution of galectin-4 in porcine small intestine using IHC and electron microscopy experiments performed with anti-galectin-4 polyclonal antibodies (PAb) made against a partially purified preparation of porcine galectin-4. Many of the results of the current study, which used a highly specific anti-galectin-4 MAb, are consistent with the findings of this previous study (Danielsen and van Deurs 1997Go). Both studies found galectin-4 primarily in the cytoplasm of the absorptive intestinal epithelial cells lining intestinal villi with the most intense staining in the villous tip cells and with no staining in the other layers that comprise the wall of the intestinal tract. Also consistent in both studies, galectin-4 was detected at the highest intensity on the apical side of the absorptive intestinal epithelial cells, suggesting that at least some of the functions of galectin-4 are performed at the microvillar (brush border) surface of the cells. In addition to verifying the findings of the study by Danielsen and van Deurs (1997)Go, the current study provides the first evidence that galectin-4 is associated with nuclei in villous tip cells and identifies a subset of galectin-4 expressing crypt cells that may be enteroendocrine cells. The current study is also the first to demonstrate that galectin-4 distribution is similar in all three major regions of porcine small intestine.

Our results indicate that virtually all galectin-4 is found inside the absorptive intestinal epithelial cells that line the intestinal villi, indicating that galectin-4 performs at least some intracellular functions for these cells. Because it is possible that extracellular galectin-4 may have been washed away during the rinse step before fixation of the tissues, our studies do not eliminate the possibility that some galectin-4 is secreted and has extracellular functions under certain physiological conditions. Within most absorptive intestinal epithelial cells, galectin-4 staining was distributed throughout the cytoplasm with slightly more intense staining observed on the apical side of the cell. This result is consistent with a previous study in which subcellular fractionation techniques were used to demonstrate that ~46% of galectin-4 is bound to microvilli and its associated actin filaments, 29% is bound to tubulovesicular structures, and the other 25% is bound to unrecognizable cytoplasmic components (Danielsen and van Deurs 1997Go). One intracellular function proposed for galectin-4 that is consistent with this distribution pattern is that galectin-4 may be involved in the formation and/or stabilization of lipid rafts which are used for intracellular transport of glycosphingolipids (GSL) and membrane proteins to the apical surface of the plasma membrane of intestinal epithelial cells (Danielsen and van Deurs 1997Go). In support of this hypothesis, galectin-4 has been found to copurify with lipid rafts isolated from villous intestinal epithelial cells (Hansen et al. 2001Go). Lipid rafts form when GSL, cholesterol, glycosylphosphatidylinositol-anchored plasma membrane proteins, and various transmembrane proteins self-associate in the trans-Golgi membrane (Alberts et al. 2002Go). Lectins, like galectin-4, are found associated with these rafts likely functioning to stabilize the raft structure by interacting with the glycans on the GSL and transmembrane proteins. After the lipid rafts form in the trans-Golgi, they pinch off into transport vesicles that are targeted for the apical cell surface. When the lipid raft reaches the apical membrane, it is incorporated into the plasma membrane with the GSL and the associated proteins expressed in the outer leaflet of the plasma membrane. This process would lead to externalization of galectin-4 without the necessity for a secretion sequence on galectin-4. Externalization of galectin-4 into the oxidizing extracellular environment would likely inactivate the galectin leading to its release into the intestinal lumen where its crosslinking ability would be rapidly destroyed by proteases found in the intestinal lumen.

Participation in apoptotic pathways is another intracellular function that has been proposed for a number of galectins in a variety of different cells (Perillo et al. 1995Go; Wada and Kanwar 1997Go; Hotta et al. 2001Go; Yang and Liu 2003Go). In the small intestine, epithelial cells are initially produced from stem cells that are found in the intestinal crypts. In the crypts, these cells undergo differentiation followed by a series of mitotic divisions before migrating up the sides of the villi. When these cells reach the villous tips, they undergo apoptosis and are released into the intestinal lumen (Hall et al. 1994Go; Ramachandran et al. 2000Go). In the current study, the highest expression of galectin-4 was observed in the most mature (villous tip) cells. In these villous tip cells, galectin-4 appeared to be associated with the nucleus. Both of these observations are consistent with galectin-4 playing a role in apoptosis in villous tip cells, resulting in death and turnover of these cells. Consistent with our observations, a previous study showed that galectin-4 mRNA was strongly downregulated in colorectal tumor tissue, indicating that lack of expression of galectin-4 might permit cell proliferation by preventing normal apoptosis of intestinal cells (Rechreche et al. 1997Go).

Most cells in the intestinal crypts do not express galectin-4 to any great extent, except for a set of widely dispersed cells that stained with anti-galectin-4 MAb on their basal or vascular side. Based on this distribution pattern, these cells are likely enteroendocrine cells. Enteroendocrine cells have been shown to be scattered among epithelial cells in the crypt and along the villi and to contain small secretory granules, which contain hormones, located on the basal side of the cells (Cheng and Leblond 1974Go). The most intense staining of galectin-4 in this subset of crypt cells was observed on the basal side of the cells, indicating that galectin-4 is likely associated with the components (e.g., hormones) of the cells that are secreted out of the vascular side of the cells. A similar observation of galectin-4 expression in secretory cells of the gastrointestinal tract was recently reported for rat gastric secretory cells including endocrine, parietal, and chief cells (Niepceron et al. 2004Go). More studies in this area need to be performed to establish that these galectin-4-expressing crypt cells are enteroendocrine cells, and then to determine what role galectin-4 performs in these crypt cells.

The distribution pattern of galectin-4 in porcine small intestine established in the current study indicates that galectin-4 likely serves multiple intracellular functions in small intestine. The distribution of galectin-4 in the cytoplasm of the absorptive epithelial cells lining the villi, with slightly higher expression at the apical surface, is consistent with a role for galectin-4 in lipid raft formation and stabilization. The observations that galectin-4 expression is highest and localized to the nucleus in mature epithelial cells at the villous tips are consistent with the hypothesis that galectin-4 promotes apoptosis of mature intestinal epithelial cells. Also, the presence of galectin-4 in enteroendocrine cells indicates galectin-4 may be involved in packaging hormones in secretory granules in these cells. Further studies to test the role of galectin-4 in all of these putative functions are needed. Also, studies designed to gain a better understanding of the mechanisms by which galectin-4 accomplishes these functions are needed. One method to address these mechanisms is to identify the intracellular glycoconjugate ligands that are recognized and crosslinked by galectin-4. The results of the current study suggest that these glycoconjugate ligands will be found inside absorptive intestinal epithelial cells, especially villous tip cells, and also in enteroendocrine cells in intestinal crypts.


    Acknowledgments
 
Supported by NIH Grant DK-056010-01.

We thank Laura Mills, Lori Zobel, Mitzi Trooien, Ying Fang, and Pam Steen for technical advice and assistance.


    Footnotes
 
1 Present address: Merck & Co., Inc., West Point, PA. Back

Received for publication June 10, 2004; accepted November 10, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
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