Cell Biology International 31 (2007) 655e662www.elsevier.com/locate/cellbi

Expression and immunohistochemical localization ofgalectin-3 in various mouse tissues

Heechul Kim a,b, Jeeyoung Lee a,b, Jin Won Hyun b,c, Jae Woo Park b,d,Hong-gu Joo a,b,**, Taekyun Shin a,b,*

a Department of Veterinary Medicine, Cheju National University, Jeju 690-756, South Koreab Applied Radiological Science Research Institute, Cheju National University, Jeju 690-756, South Korea

c Department of Biochemistry, College of Medicine, Cheju National University, Jeju 690-756, South Koread Department of Nuclear and Energy Engineering, Cheju National University, Jeju 690-756, South Korea

Received 2 October 2006; revised 22 November 2006; accepted 29 November 2006

Abstract

The expression and immunohistochemical localization of galectin-3, a b-galactoside-binding protein, was studied in several mouse tissues.Galectin-3 expression was low in the cerebrum, heart, and pancreas, and moderate in the liver, ileum, kidney, and adrenal gland. High expressionof galectin-3 was found in the lung, spleen, stomach, colon, uterus, and ovary. The results of Western blot analysis largely matched the immu-nohistochemical findings for galectin-3. These findings suggest that galectin-3 is differentially expressed in a variety of organs in the mouse. Thisstudy provides valuable information for research on galectin-3.� 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Galectin-3; Immunohistochemistry; Mouse

1. Introduction

Galectin-3, a b-galactoside-binding animal lectin, has car-bohydrate-recognition domains and multiple related functions(Barondes et al., 1994). Intracellular galectin-3 plays a pivotalrole in functions as diverse as cell growth, anti-apoptosis, andmRNA splicing (Liu et al., 2002). In addition, extracellulargalectin-3 mediates inflammation and the adhesion of leuko-cytes, and is involved in the recruitment and activation ofneutrophils (Almkvist and Karlsson, 2004).

* Corresponding author. Department of Veterinary Medicine, Cheju National

University, Jeju 690-756, South Korea. Tel.: þ82 64 754 3363; fax: þ82 64

756 3354.

** Corresponding author. Department of Veterinary Medicine, Cheju Na-

tional University, Jeju 690-756, South Korea. Tel.: þ82 64 754 3379; fax:

þ82 64 756 3354.

E-mail addresses: jooh@cheju.ac.kr (H.-g. Joo), shint@cheju.ac.kr

(T. Shin).

1065-6995/$ - see front matter � 2006 International Federation for Cell Biology

doi:10.1016/j.cellbi.2006.11.036

Recently, it was reported that galectin-3 is expressed in avariety of epithelial cells of the urogenital system, including thekidney (Nio et al., 2006) and boar epididymis (Kim et al.,2006), and in the bovine respiratory and digestive tracts duringfetal development (Kaltner et al., 2002). Although it is highlyprobable that galectin-3 is expressed in a variety of mousetissues, little is known about its differential expression in thesetissues.

The aim of this study was to examine the expression andcellular localization of galectin-3 in various murine tissues, in-cluding the cerebrum, heart, lung, liver, spleen, stomach, pan-creas, ileum, colon, adrenal gland, kidney, ovary, and uterus,using Western blot analysis and immunohistochemistry.

2. Materials and methods

2.1. Animals

We used five male and five female BALB/c mice (25 g, 8e10 weeks old).

Tissues from both male and female mice were used for Western blot analysis,

. Published by Elsevier Ltd. All rights reserved.

656 H. Kim et al. / Cell Biology International 31 (2007) 655e662

while four mice were used for histology (three to five samples per gender). All

of the experiments were carried out in accordance with the National Research

Council’s Guide for the Care and Use of Laboratory Animals.

2.2. Tissue preparation

Murine cerebrum, heart, lung, liver, spleen, stomach, pancreas, ileum,

colon, adrenal gland, kidney, ovary, and uterus were dissected and homoge-

nized in lysis buffer for protein analysis. Pieces of tissues were fixed in 4%

paraformaldehyde in phosphate buffer and embedded in paraffin. Paraffin

sections were stained with hematoxylin and eosin, and also used for

immunohistochemistry.

2.3. Antibodies

The rat anti-galectin-3 monoclonal antibody (1 mg/ml) was purified from

the supernatant of hybridoma cells (TIB-166; ATCC, Rockville, MD, USA)

(Lee et al., 2004). Biotinylated isolectin B4 (IB4; derived from Griffoniasimplicifolia) and mouse monoclonal anti-b-actin were obtained from Sigma

(St. Louis, MO, USA).

2.4. Western blotting

Tissue samples were homogenized in Pro-prep protein extraction solution

(iNtRON Biotech, Seoul, Korea). The homogenate was centrifuged at

14,000 rpm for 20 min, and the supernatant was harvested. For the immunoblot

assay, the protein concentration of the supernatant was quantified using the Brad-

ford protein assay (Bio-Rad, Hercules, CA, USA). In a preliminary experiment,

it was difficult to compare all the tissues in one panel because of very intense ga-

lectin-3 expression in some organs, including the lung, spleen, stomach, colon,

uterus, and ovary. Therefore, samples were loaded in three separate panels: the

first panel contained kidney, cerebrum, heart, pancreas, and liver; the second

panel contained kidney, lung, spleen, stomach, and ileum; and the third panel

contained kidney, colon, adrenal gland, uterus, and ovary.

Samples containing 20 mg/lane were loaded, electrophoresed, and blotted

onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA).

The membrane was blocked using 5% non-fat milk solution, and subsequently

probed with rat anti-galectin-3 (1:5000 dilution) antibody and horseradish

peroxidase-conjugated anti-rat IgG (Santa Cruz Biotechnology, Santa Cruz,

CA, USA). The membranes were developed using a chemiluminescent

substrate (WEST-one Kit; iNtRON Biotech), according to the manufacturer’s

instructions, and exposed to Agfa medical X-ray film (Agfa Gevaert, Mortsel,

Belgium). After imaging, the membranes were stripped and reprobed using

anti-b-actin antibody (Sigma).

The density (OD/mm2) of each band (n ¼ 3/organ) was measured with

a scanning laser densitometer (GS-700; Bio-Rad). The kidney band in each

panel was used as a standard for normalization, and the relative density of

each band was calculated. The results are presented as the mean � S.E. The

results were analyzed statistically using one-way analysis of variance followed

by the post hoc Student-NewmaneKeuls procedure for multiple comparisons.

In all cases, p < 0.05 was considered significant.

2.5. Immunohistochemistry

Sections of paraffin-embedded tissues (5 mm thick) were deparaffinized

and allowed to react with anti-galectin-3 antibody. Immunoreactivity was

visualized with the avidin-biotin peroxidase reaction (Vector Elite, Vector

Labs, Burlingame, CA, USA). Peroxidase was developed with diaminobenzi-

dine (DAB; Vector Labs). The sections were counterstained with hematoxylin

prior to being mounted.

To visualize the co-localization of galectin-3 and IB4 in the mouse tissues,

the sections were reacted with biotinylated IB4 (Sigma), followed by TRITC-

labeled streptavidin (Zymed, San Francisco, CA, USA). The sections were

then reacted with the anti-galectin-3 antibody, followed by FITC-labeled

goat anti-rat IgG (Zymed).

To reduce or eliminate lipofuscin autofluorescence, the sections were

washed three times for 1 h each in PBS at RT, dipped briefly in distilled

H2O, treated with 10 mM CuSO4 in 50 mM CH3COONH4 buffer [pH 5.0]

for 20 min, dipped briefly again in distilled H2O, and returned to PBS. The

double-immunofluorescence-stained specimens were examined under an

FV500 laser confocal microscope (Olympus, Tokyo, Japan).

3. Results

3.1. Western blot analysis of galectin-3

Western blot analysis was used to examine galectin-3 expres-sion in various murine tissues, and was performed in three sep-arate blots containing mouse tissues with low to high expressionof galectin-3 (Fig. 1AeC). Galectin-3 expression was low in thecerebrum (Fig. 1A), heart (Fig. 1A), pancreas (Fig. 1A), and il-eum (Fig. 1B); moderate in the liver (Fig. 1A), kidney (Fig. 1A),and adrenal gland (Fig. 1C); and high in the lung (Fig. 1B),spleen (Fig. 1B), stomach (Fig. 1B), colon (Fig. 1C), uterus(Fig. 1C), and ovary (Fig. 1C).

The relative density of the galectin-3 immunoreaction in eachorgan is summarized in a bar graph (Fig. 1D). Briefly, the expres-sion of galectin-3 in the lung, spleen, stomach, colon, uterus, andovary was significantly greater than in the cerebrum, heart,pancreas, liver, ileum, kidney, and adrenal gland ( p < 0.05).The expression of galectin-3 in the ovary was significantlylower than in the lung, spleen, stomach, colon, and uterus( p < 0.05). Furthermore, the expression of galectin-3 in the co-lon was significantly greater than in the lung, spleen, stomach,and uterus ( p < 0.05) (Fig. 1D). These findings suggest thatthe expression of galectin-3 varied depending on the organ.

3.2. Localization of galectin-3 in various murine tissues

In the cerebrum, galectin-3 was very weakly immuno-stained in some ependymal cells of the cerebral ventricle,but not in neurons and glial cells (Fig. 2A). In the heart, galec-tin-3-positive round cells were occasionally found in theinterstitial tissues of the heart, but cardiomyocytes were notpositive for galectin-3 (Fig. 2C). In the lung, galectin-3 immu-noreactivity was found in the covering epithelia of the bron-chioles, and in some macrophages (Fig. 2D). In the spleen,galectin-3-positive cells were mainly located in the red pulp,with few cells in the white pulp (Fig. 2E, F).

In the gastrointestinal tract (Fig. 3AeC), galectin-3 was de-tected in some round cells in the lamina propria, as well as inthe epithelial cells of the stomach, small intestine (ileum), andlarge intestine (colon). In the liver, galectin-3 immunostainedsome cells along the sinusoids that were identical to Kupffercells based on morphology and localization, but did not stainhepatocytes (Fig. 3D). In the pancreas, a few galectin-3-posi-tive round cells were occasionally found in the interstitium,but not in exocrine and endocrine cells (Fig. 3E).

In the kidney, galectin-3 was immunostained in the medul-lary and cortical collecting tubules, but was scarcely detectedin the glomeruli and proximal and distal tubules in the cortex(Fig. 4A). In the adrenal gland, galectin-3-positive cells were

657H. Kim et al. / Cell Biology International 31 (2007) 655e662

Fig. 1. Western blot analysis of galectin-3 in various mouse tissues. (A) Representative photomicrograph of the Western blot analysis of galectin-3 in the kidney,

cerebrum, heart, pancreas, and liver. (B) Representative photomicrograph of the Western blot analysis of galectin-3 in the kidney, lung, spleen, stomach, and ileum.

(C) Representative photomicrograph of the Western blot analysis of galectin-3 in the kidney, colon, adrenal gland, uterus, and ovary. Arrowheads indicate the

positions of galectin-3 (AeC, approximately 29 kDa). The film exposure time for each panel was adjusted to visualize the moderate density of galectin-3 in

the kidney as a normalization control in the three separate panels (AeC). (D) The quantitative analysis of galectin-3 expressed in a variety of organs based on

densitometric data (n ¼ 3 samples/tissue). The density for the kidney in each panel was used as a standard (1 arbitrary unit) to compare the relative density in

the other organs. The relative density is presented as the mean � S.E. *p < 0.05, compared to the cerebrum, heart, pancreas, liver, kidney, ileum, and adrenal

gland. #p < 0.05, compared to the ovary. $p < 0.05, compared to the lung, spleen, and stomach.

detected mainly in the medulla, but were occasionally found inthe adrenal cortex (Fig. 4B). In the ovary, galectin-3 showedintense immunostaining in some luteal cells, and some cellsin the stroma, but was not found in the growing follicles(Fig. 4C). In the uterus, galectin-3 immunostaining wasdetected in some parts of the covering endometrial epithelium,as well as in some cells in the loose connective tissues of theuterine mucosa (Fig. 4D). The immunoreactivity and cellularlocalization of galectin-3 largely matched the results obtainedwith Western blot analysis.

3.3. Double staining of galectin-3 withisolectin B4 in various murine tissues

In the lung, galectin-3 immunoreactivity was co-localizedin IB4-positive cells, identical to macrophages (Fig. 5AeC,

arrows). In the lamina propria of the ileum, galectin-3-positiveround cells were positive for IB4, which suggests that someIB4-positive macrophages express galectin-3 (Fig. 5DeF,arrows). In the pancreas, little galectin-3 was immunodetectedin IB4-positive macrophages (Fig. 5GeI, arrow), whereas IB4immunoreactivity was also localized in exocrine cells(Fig. 5H) (Laitinen, 1987). In the uterus, galectin-3-positiveround cells were detected in the loose connective tissues ofthe uterine mucosa, and these cells were co-localized withIB4, suggesting that some macrophages in the uterine mucosaexpress galectin-3 (Fig. 5JeL).

4. Discussion

Galectin-3 is known to be expressed in several organsincluding the kidney, lung, thymus, breast, prostate, spleen,

658 H. Kim et al. / Cell Biology International 31 (2007) 655e662

Fig. 2. Immunohistochemical localization of galectin-3 in the cerebrum (A), heart (C), lung (D), and spleen (E, F). Galectin-3 immunoreactivity was not seen the

cerebral cortex (A). In the heart, some round cells were positive for galectin-3 (arrow) (C). In the lung, galectin-3 was localized in the covering epithelia of the

bronchioles (arrowheads) and some macrophages (arrow) (D). In the spleen, galectin-3 immunoreactivity was mainly detected in the red pulp (E). The marginal

zones around the white pulp are shown at high magnification (F). No specific reaction product was seen in the lung incubated with non-immune sera (B). Counter-

staining with hematoxylin. Scale bars, 50 mm (A, C, D); 90 mm (B, E); 30 mm (F).

and colon (reviewed by Dumic et al., 2006), suggesting that itis expressed in a variety of cell types in mammals.

In this study, we compared the tissue expression level ofgalectin-3 using immunohistochemistry and Western blot anal-ysis. The present findings suggest that galectin-3 may play animportant role in organs with a high level of expression,including the lung, spleen, stomach, colon, uterus, and ovary.However, we do not exclude the possibility that the role ofgalectin-3 is concealed in those organs with low expression,when the cell phenotype of galectin-3 may be less distinctive.

Galectin-3 has been detected in the epithelial covering ofbovine fetal respiratory and digestive tracts (Kaltner et al.,2002) as well as in the ductal system of the salivary glands,pancreas, kidney, and eye, and in the intrahepatic bile ducts

(reviewed by Dumic et al., 2006). In the digestive tract ofmice, galectin-3 mRNA expression was intense in the large in-testine, including the cecum, colon, and rectum, whereas thestomach and small intestine had weak signals for galectin-3that increased slightly in intensity toward the ileum (Nioet al., 2005). We also found a similar galectin-3 expressionpattern in the mouse digestive tract, which is largely consistentwith the previous report (Nio et al., 2005). The immunoblotanalysis detected high expression of galectin-3 in the stomachand colon, but moderate immunoreactivity in the ileum. Thisdiscrepancy might have been caused by the different detectionsystem and cell types, in situ hybridization for mRNA in epi-thelial cells, but not in the other cells in the lamina propria(Nio et al., 2005). In the case of the kidney, our results match

659H. Kim et al. / Cell Biology International 31 (2007) 655e662

Fig. 3. Immunohistochemical localization of galectin-3 in the stomach (A), ileum (B), colon (C), liver (D), and pancreas (E). In the stomach, galectin-3 immu-

noreactivity was intense in the covering epithelial cells (arrow) and in some cells in the lamina propria (arrowhead). In the ileum (B) and colon (C), some epithelial

cells (arrow) and round cells (arrowhead) were immunopositive for galectin-3. In the liver, galectin-3 was immunostained in some Kupffer cells (D, arrowheads). In

the pancreas, galectin-3 immunoreactivity was detected in some cells (arrowhead) (E). Counterstaining with hematoxylin. No specific reaction product was seen in

the colon incubated with non-immune sera (F). Scale bars, 80 mm (AeC, F); 60 mm (D, E).

those of previous studies showing that galectin-3 expression isfound in the urinary system, including the kidney, ureter, andurinary bladder (Nio et al., 2006). Taking all findings intoconsideration, we postulate that galectin-3 in the coveringepithelia is involved in mucosal defense systems, conferringprotection from exogenous attack. Its function in the ducts

under physiological and pathological conditions remains tobe elucidated.

Galectin-3 has been identified in a variety of cell typesincluding fibroblasts, keratinocytes, granulocytes, dendriticcells, and macrophages (Dumic et al., 2006). In thepresent study, we also found that galectin-3 demonstrates

660 H. Kim et al. / Cell Biology International 31 (2007) 655e662

Fig. 4. Immunohistochemical localization of galectin-3 in the kidney (A), adrenal gland (B), ovary (C), and uterus (D). In the kidney, the immunoreactivity of

galectin-3 in the medulla was more intense than that in the cortex (A). In addition, galectin-3 immunoreactivity in the collecting tubules was more intense

than that in the proximal and distal tubules and renal corpuscles (inset). In the adrenal gland, galectin-3 immunoreactivity was detected in the medulla. In the

ovary, galectin-3 immunoreactivity was detected in some luteal cells (arrow), but not in the unilaminar follicles (arrowhead) (C). In the uterus, the covering

epithelium of the uterine mucosa (arrow) and some cells in the endometrium (arrowhead) were immunopositive for galectin-3 (D). Counterstaining with hema-

toxylin. Scale bars, 400 mm (A, B); 100 mm (C, D); 50 mm (inset).

immunostaining in monocyte-like cells (presumably tissue-resident macrophages) in the connective tissues of all organsstudied. Because the expression of galectin-3 in macrophagesis observable in the loose connective tissues of most organs,we postulate that intracellular galectin-3 in macrophagesmay support cell survival in an autocrine manner because in-tracellular galectin-3 shares an anti-apoptotic motif, NWGR,with bcl-2, which is associated with cell survival (Yanget al., 1996). If galectin-3 is released from connective tissuemacrophages, extracellular galectin-3 may confer cell deathto invading inflammatory cells. Furthermore, it has beenreported that galectin-3 is involved in pro-inflammatory activ-ities, including cell activation and migration, or has immuno-modulatory roles through inducing T cell anergy (Stillmanet al., 2006). Galectin-3 has also been detected in the T cellsand intestinal epithelium in inflammatory bowel disease

(Muller et al., 2006). These results suggest that galectin-3has a dual role in connective tissue, either in cell death and/or survival, depending on it being expressed either intracellu-larly or extracellularly (Dumic et al., 2006).

To our knowledge, this is the first report showing theexpression of galectin-3 in the ovary. Galectin-3 was stronglyexpressed in the luteal cells, as well as in some stromal mac-rophages. Considering the role of galectin-3 in cell survival,we postulate that it may play a role in the maintenance (orsurvival) of luteal cells during pregnancy, and/or that galec-tin-3 is involved in protecting luteal cells from the surroundingenvironment, including immune cell attack.

In the spleen, immunohistochemistry showed that the ex-pression level of galectin-3 in the red pulp was much higherthan that in the white pulp, clearly demonstrating the regionaldistribution of galectin-3 in the spleen; the majority of resident

661H. Kim et al. / Cell Biology International 31 (2007) 655e662

Fig. 5. Immunofluorescent co-localization of galectin-3 with isolectin B4 in the lung (AeC), ileum (DeF), pancreas (GeI), and uterus (JeL). Arrows indicate

galectin-3 immunoreactivity in isolectin B4-positive macrophages (C, F, I, and L). The * (AeC) and # (GeI) indicate bronchioles and pancreatic islets, respec-

tively. C, F, I, and L are merged images. Scale bars ¼ 50 mm.

macrophages and dendritic cells occur in the red pulp, whereasT and B lymphocytes are present in the white pulp. The pres-ent results further support those of Ho and Springer (1982),who showed that galectin-3, previously named Mac-2, isexpressed in the spleen. Furthermore, a recent study by a mem-ber of our research group clearly showed that naive spleniclymphocytes express only trace amounts of galectin-3,whereas activated lymphocytes express substantial amounts(Joo et al., 2001). We postulate that the present immunohisto-chemistry technique was insufficient to detect the expressionof galectin-3 in naive lymphocytes of the white pulp. Conse-quently, we do not exclude the possibility that naive T cells

contain galectin-3 in a normal unstimulated mouse spleen.Dendritic cells and macrophages play a critical role in immuneresponses, either by themselves or with other immune cells, in-cluding lymphocytes; therefore, our findings provide valuableinformation about the potential role of galectin-3 in those celltypes in the spleen, based on its role in adhesion and its anti-apoptotic activity (Stillman et al., 2006; Woo et al., 1990).

In the adrenal gland, we found that galectin-3 was stronglyexpressed in the medullary cells, from which epinephrine andnorepinephrine are secreted. We have little information on theexpression of galectin-3 in endocrine tissues, and thus furtherstudy is needed on this organ.

662 H. Kim et al. / Cell Biology International 31 (2007) 655e662

Taking all our results into consideration, we postulate thatgalectin-3 is expressed in all the organs examined in this study,and that it plays a distinctive role in each one. Further researchis required to elucidate the function of galectin-3 in each organand/or cell type.

Acknowledgments

This work was supported by a program of the Basic AtomicEnergy Research Institute (BAERI), which is a part of theNuclear R&D Programs funded by the Ministry of Science &Technology (MOST) of Korea.

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