Amencan Journal of Pathology, Vol. 147, No.

4, October 1995
Copyright ) American Society for Investigative Pathology

Expression and Function of Galectin-3, a

f-Galactoside-Binding Lectin, in Human
Monocytes and Macrophages

Fu-Tong Liu,* Daniel K. Hsu,* Riaz 1. Zuberi,*

Ichiro Kuwabara,* Emil Y. Chi,t
and William R. Henderson, Jr.t
From the Department ofMolecular and Experimental
Medicine,* The Scripps Research Institute, La Jolla,
California, and the Departments ofPathologyt and
Medicine,* University of Washington, Seattle, Washington

A family of flgalactoside-binding animal lectins

has recently been designated as galectins. One
member of this family, galectin-3, has been
known as cBPfor its IgE-binding activity and as
Mac-2, a macrophage surface antigen, CBP35,
CBP30, L-29, and L-34. Although much information has accumulated on the expression of this
lectin in murine macrophages and human monocytic cell lines, little is known about the expression and function of this protein in normal human monocytes/macrophages. We now report
that galectin-3 is expressed in normal human
peripheral blood monocytes and its level increases dramaticaly as human monocytes differentiate into macrophages upon culturing in
vitro Immunoblot analysis showed that there
was a 5-fold increase in the level of galectin-3
after I day of culture and greater than a 12-fold
increase after 5 days. Immunocytochemical analysis confirmed this progressive increase of
galectin-3 expression in cultured monocytes.
Immunogold cytochemistry/electron microscopy
analysis revealed that galectin-3 was expressed
on the surface of human monocytes and that the
level of ceU surface galectin-3 increasedprogressively as these ceUs differentiated into macrophages. The level of galectin-3 in human monocytes/macrophages was modulated by stimuli
such as lipopolysaccharide and interferon-y,
and galectin-3 was secreted when monocytes
were stimulated by calcium ionophore A23187.
Soluble galectin-3 caused superoxide release
from human monocytes; this activity was depen-

1016

dent on the lectin property of galectin-3, as it

was inhibitable by lactose. Thus, galectin-3 may
modulate the function of this ceU type in an autocrine or paracrine fashion through binding to
ceU surface glycoconjugates. (AmJPathol 1995,

147:1016-1028)

Animal lectins are gaining increased attention with

the prominent examples being the cell adhesion molecules, selectins. A family of soluble ,B-galactosidebinding animal lectins has recently been designated
as galectins.1'2 One member of this family,
galectin-3, has been known as sBP, for its IgEbinding activity,3-5 Mac-2, an antigen first identified
on murine macrophages,67 CBP35,5 CBP30,9
L-34,10 and L-29.11 It has an apparent molecular
weight of approximately 30,000 and is composed of
two domains; the amino-terminal domain is made
primarily of tandem repeats and the carboxylterminal portion has characteristics of an S-type
carbohydrate recognition domain.12
Various functions have been suggested or demonstrated for galectin-3 and it appears that this lectin
is a versatile multifunctional protein (reviewed in Ref.
13). Of particular relevance to this study is the role of
this protein in inflammation. Galectin-3 is known to
be expressed abundantly by various inflammatory
cells, including mast cells,14 eosinophils,15 neutrophils,16 and macrophages.7 While being predominantly a cytosolic protein, galectin-3 is expressed on
The General Clinical Research Center at Scripps Clinic is supported
by National Institutes of Health grant MO1 RR00833. The work was
supported by National Institutes of Health grants Al-20958 (FTL),
Al-32834 (FTL), Al-17758 (WRH), Al-23713 (WRH), and HL-30542
(WRH). DKH and RIZ were supported by National Institutes of
Health training grant T32 AR07144. This is publication number
7831-MEM of the Department of Molecular and Experimental Medicine of The Scripps Research Institute.
Accepted for publication June 23, 1995.
Address reprint requests to Dr. Fu-Tong Liu, Department of Molecular and Experimental Medicine, SBR-4, The Scripps Research
Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037.

Galectin-3 in Human Monocytes/Macrophages

1017

AJP October 1995, Vol. 147, No. 4

cell surfaces and is secreted by various cells (reviewed in Ref. 13). Galectin-3 may have a role in
IgE-mediated cytotoxicity toward parasite targets by
eosinophils15 and IgE-dependent activation of neutrophils.16 In addition to IgE, various other glycoproteins have been shown to be recognized by
galectin-3, including laminin,17 IgE receptor FcsRI,18
and a protein containing a macrophage scavenger
receptor cysteine-rich domain.19 Galectin-3 has
been shown to be capable of activating mast cells18
and neutrophils,20 probably through recognition
(and cross-linkage) of appropriately glycosylated
cell surface glycoproteins. Therefore, a picture is
emerging that this lectin may be a modulator of
inflammatory responses.21
Since the original description of galectin-3 as a
murine macrophage cell surface antigen recognized
by a monoclonal antibody, information regarding this
protein in macrophages has accumulated. The
expression of this protein has been found to
be markedly increased in macrophages from
thioglycolate-stimulated mice as compared with resting macrophages.6 Other investigators found that
the cell surface galectin-3 level appears to increase
as monocytes differentiate into macrophages, on the
basis of studies with murine macrophage cell lines22
as well as murine blood monocytes and tissue macrophages.23,24 The correlation between cell differentiation and the total cellular level of this protein is not
known. More recently, the expression of galectin-3
was found to be upregulated as human monocytic
cell lines were stimulated to differentiate.25 26 However, little is known about expression and function of
this lectin in normal human monocytes/macrophages.
In this study, we found that galectin-3 was
expressed in normal human peripheral blood
monocytes, and its level increased dramatically as
human monocytes differentiated into macrophages.
Galectin-3 may play a role in mediating inflammatory
reactions as we found that it is secreted by human
monocytes, its level in these cells is modulated by
lipopolysaccharide (LPS) and interferon (IFN)-,y, and
it stimulates human monocyte superoxide production.

Materials and Methods

Reagents
Histopaque 1077 and LPS from Escherichia coli serotype 055:B5 were obtained from Sigma Chemical
Co. (St. Louis, MO). RPMI 1640 with HEPES buffer
was obtained from Whittaker M.A. Products (Walkersville, MD). Recombinant human IFN-y was pro-

vided by P. Trown, Hoffman-La Roche (Nutley, NJ).

IFN-'y activity was determined by the supplier in a
WISHNSV cytopathic effect microtiter assay standardized with NIH human IFN-y reference standard
Gg 23-901-530. Recombinant human galectin-3 was
prepared as reported previously.27 Endotoxins were
not detectable in the concentrated stock of the recombinant protein, by using a commercial kit (Limulus amebocyte lysate, pyrogent, BioWhittaker, Walkersville, MD). On the basis of the results obtained
with the positive control, it was determined that the
endotoxin level was less than 16.8 pg/mI in the solution containing 60 ,tg/ml galectin-3, which was the
highest concentration of galectin-3 used in stimulation of monocytes in this study. Goat anti-rat
galectin-3 antisera and rabbit anti-human galectin-3
antisera were generated by immunizing goat and
New Zealand albino rabbits with recombinant rat
galectin-328 and human galectin-3,27 respectively.
The 1251-labeled galectin-3 was prepared by reacting 10 ,tg of the protein with 0.5 mCi of Na1251 in the
presence of chloramine-T.29 Lactosyl-Sepharose 4B
was prepared as described.30

Toxoplasma gondii
The RH strain of T. gondii was maintained by intraperitoneal passage in BALB/c mice and harvested
as described previously.31 The T. gondii tachyzoites
were suspended in phosphate-buffered saline (PBS)
containing calcium and magnesium and were >95%
viable by trypan blue dye exclusion.

Monocyte Cultures
Human monocytes were prepared from venous
blood of normal volunteers as described previously.32 In brief, after Histopaque separation, human
mononuclear cells (2 x 105 to 3 x 105 monocytes/
ml; >92% viability by trypan blue exclusion) were
added to sterile 60- x 15-mm tissue culture plates
(Costar, Cambridge, MA) in 3 to 4 ml of RPMI 1640
with autologous serum and allowed to adhere for 2
hours in humidified 5% C02/95% air at 370C. The
plates were washed six to eight times with warm
sterile PBS to remove nonadherent cells, and fresh
RPMI 1640 with 15% autologous serum was added.
Greater than 98% of the cells of these adherent
monolayers had the characteristic appearance of
monocytes when examined by light microscopy with
Diffquick (Difco Laboratories, Detroit, Ml) staining.
The monolayers were maintained in 5% C02/95% air
with humidity at 37C, with fresh media added the
day after isolation and every other day thereafter.

1018

Liu et al

AJP October 1995, Vol. 147, No. 4

At specified intervals after adherence, human

monocyte monolayers were washed six to eight
times with warm sterile Tyrode's buffer. Reaction
mixtures, containing the components described in
the figures and tables, were added in a final volume
of 3 ml of Tyrode's buffer, and the tissue culture
plates were incubated at 370C in 5% C02/95% air for

dilution), followed by alkaline phosphatase-labeled

goat anti-rabbit IgG. A chemiluminescent detection
system with a Tropix kit (Bedford, MA) was employed
for visualization. Quantitation of galectin-3 from immunoblots was performed by densitometry of exposed film with an LKB Ultroscan XL.

the specified time periods. At the end of the incubation, the monolayers were washed twice with fresh
RPMI without serum and then lysed with 1 ml of the
lysing buffer (10 mmol/L Tris-HCI, pH 7.4, 5 mmol/L
EDTA, 0.15 mol/L NaCI, 1% Triton X-100) containing
protease inhibitors (0.24 lU/ml aprotinin, 1 ,tg/ml
leupeptin, 0.5 ,ug/ml pepstatin, and 0.5 mmol/L phenylmethylsulfonyl fluoride). The lysates were centrifuged at 40C in an Eppendorf centrifuge (15,600 x g)
for 30 minutes and the clear fluids were stored at
-700C until use. The protein content of each lysate
was determined by the Bradford method with reagents from Pierce (Rockford, IL). The protein concentration was typically approximately 1.5 mg/ml.
Some monolayers were fixed in paraformaldehyde
for transmission electron microscopy studies described below.
In other experiments, adherent monolayers were
detached after 2 hours with PBS containing 5
mmol/L EDTA and 2% trypsin (GIBCO, Grand Island, NY). The detached cells were then washed
twice with RPMI 1640 containing 5% autologous
serum, resuspended in the medium containing
15% serum, and cultured in Teflon beakers (ColeParmer, Chicago, IL) at 370C. After 2 and 5 days of
culture, aliquots of the cell suspension were lysed
as described above for immunoblotting. Other
cells were prepared for immunoperoxidase staining as described below.

Immunocytochemical/Histochemical
Detection of Galectin-3

Immunoblot Analysis of Cell Lysates for

Galectin-3
The cell lysates (containing 110 ,ug of protein) were
mixed with 25 ,ul of lactosyl-Sepharose 4B for 2 hours
at 40C. The beads were then washed with the lysing
buffer and bound proteins were eluted with sodium
dodecyl sulfate polyacrylamide gel electrophoresis
sample buffer. For detection of galectin-3 by immunoblotting, the eluted proteins were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12.5% polyacrylamide gels. The separated proteins were transferred to polyvinylidene
difluoride membranes (Immobilon P, Millipore, Bedford, MA). The galectin-3 was then probed with
rabbit anti-human galectin-3 antiserum (1:10,000

Monocytes/macrophages, either freshly isolated or

cultured in Teflon beakers for 2 and 5 days were
placed in Lab-tek chambers (Nunc, Naperville, IL)
and adhered for 2 hours at 370C. The adherent cells
were washed twice with PBS and fixed with 3%
paraformaldehyde in PBS. The cells were subsequently treated for 1 hour at room temperature with
mouse monoclonal anti-galectin-3 antibody AlD6
(F-T Liu, DK Hsu, RI Zuber, A Shenhar, PN Hill, and
Kuwabara, manuscript in preparation) or an
isotype-matched control monoclonal antibody that
does not bind galectin-3. The cells were then
washed five times and stained by using an immunoperoxidase kit from BioGenex (San Ramon, CA)
according to the manufacturer's directions.

Immunogold/Transmission Electron
Microscopy
Detection of Cell Surface Galectin-3
After incubation for either 0 or 5 days in tissue culture, human monocyte monolayers were fixed in 1%
paraformaldehyde for 5 minutes at 200C, rinsed with
0.1 mol/L sodium cacodylate buffer, pH 7.4, twice
and PBS twice and then incubated in the absence or
presence of rabbit anti-human galectin-3 (1:50 dilution in PBS) for 40 minutes at 200C. After rinsing with
PBS twice, the preparations were incubated in the
absence or presence of goat anti-rabbit IgG labeled
with 15-nm gold particles (1:10 dilution in PBS; AuroProbe EM GAR G15, Janssen Life Science Products, Olen, Belgium) for 16 hours at 40C, followed by
fixation in 2% glutaraldehyde for 20 minutes at 200C.
The preparations were postfixed with 1% osmium
tetroxide (Ted Pella, Inc., Redding, CA), stained with
0.5% uranyl acetate (Polysciences, Inc., Warrington,
PA), dehydrated with ethanol, and embedded in
Epon (Ted Pella, Inc.). Some monolayers were incubated with 20 mmol/L lactose for 10 minutes at 200C
before the initial paraformaldehyde fixation. The
samples were examined with a JEOL 100B electron
microscope (Japan Electron Optics Laboratory,
Tokyo, Japan) at 60 kV as described previously.33

Galectin-3 in Human Monocytes/Macrophages

1019

AJP October 1995, Vol. 147, No. 4

Detection of Intracellular Galectin-3

Human macrophages were fixed in 2% glutaraldehyde for 1 hour and then centrifuged to remove the
supernatant. To the cells were added PBS containing 0.01% Triton X-100 followed by mouse antigalectin-3 monoclonal antibody A1D6 (5 ,g/ml) or
control monoclonal antibody, and the mixtures were
incubated at room temperature for 5 minutes. After
being washed with PBS, the cells were again incubated with the monoclonal antibodies in PBS for 30
minutes at room temperature. The cells were subsequently fixed in 2% glutaraldehyde, washed in PBS,
and then treated with AuroProbe BLplus goat antimouse IgG (Janssen Life Science; diluted 1:10) for 3
hours at room temperature. Afterward, the cells were
fixed in glutaraldehyde and processed for electron
microscopy as described above.

Enzyme-Linked Immunosorbent Assay

(ELISA) for Detection of Galectin-3
Flat-bottom 96-well microtiter plates were coated
with affinity-purified goat anti-rat galectin-3 antibodies at 1 ,ug/ml overnight at 37C. To each well
was added 200 ,ul of 1% (w/v) bovine serum albumin in PBS, pH 7.4, and the plates were incubated
at 370C for 1 hour. After two washes with PBS,
samples (50 ptl) containing purified recombinant
human galectin-3 from 200 pg/ml to 500 ng/ml or
serial dilutions of culture supernatants or cell lysates were added to the individual wells. The
plates were incubated at room temperature for 2
hours and then washed five times with PBS. Afterward, the bound galectin-3 was detected with
affinity-purified rabbit anti-human galectin-3, followed by horseradish peroxidase-labeled goat
anti-rabbit antibodies, each step involving incubation for 1 hour at 370C and subsequent washing
with PBS five times. One hundred microliters of the
substrate 2,2'-azino-bis(3-ethylbenzthiazolene-6sulfonic acid) were added to each well, the plates
were incubated at 200C, and the color developed
after 30 minutes was read on a Titertek multiscan spectrophotometer. The concentration of
galectin-3 in the test samples was determined by
a standard curve that was linear from 1 ng/ml to
500 ng/ml.

Assay for Superoxide Production

Superoxide production by monocytes was determined by the well established cytochrome c assay,34

on the basis of the reduction by superoxide of ferric

cytochrome c to ferrous cytochrome c, resulting in an
absorbance change at 550 nm. Human monocytes
(2.5 x 10 cells/ml), suspended in 250 [lI of PBS (pH
7.4) containing 0.5 mmol/L MgCO2, 1.1 mmol/L CaC02,
7.5 mmol/L glucose, 75 ,umol/L cytochrome c, and 5
,ug/ml cytochalasin B were placed in wells of 96-well
microtiter plates. After a 5-minute preincubation at
370C, galectin-3 was added and the absorbance
change was monitored with a kinetic microplate
reader (ThermoMax, Molecular Devices, Menlo Park,
CA). The data were analyzed by using enzyme kinetic software (Softmax version 2.01) as described.35 The control wells also contained superoxide dismutase (60 ,ug/ml). Some of the wells received
lactose (25 mmol/L final concentration) in addition to

galectin-3.

Results
Expression of Galectin-3 in Relation to
Monocyte Differentiation
The presence of galectin-3 in human monocytes was
probed by immunoblot analysis. A Mr 30,000 protein
was detected in the lysate of freshly isolated human
monocytes (Figure 1). A minor low molecular weight
band was also seen, which probably represents a
proteolytic cleavage product of galectin-3. The
question of whether galectin-3 expression is dependent on the differentiation of monocytes was addressed by comparing the level of galectin-3 in
freshly isolated human monocytes with those cultured for 1 to 5 days in plastic plates. A progressive
increase in galectin-3 was noted with increasing time
of culturing (Figure 1A). By densitometric analysis of
chemiluminescent exposure on film, we determined
that there was a 5-fold increase in the level of
galectin-3 after 1 day and a 12.5-fold increase after
5 days. Similar results were obtained in two separate
experiments.
To confirm the upregulation of galectin-3 expression concomitant with monocyte differentiation, we
cultured human monocytes in nonadherent conditions. The monocytes were cultured in Teflon beakers and cell aliquots from the cultures were analyzed at different time intervals by either immunoblot
analysis or immunoperoxidase staining. As shown in
Figure 1B, there were 5- and 11-fold increases in
galectin-3 after 2 and 5 days of culture, respectively,
measured by densitometric analysis of chemiluminescent exposure on film. Similar results were obtained in two separate experiments. There was also

1020

Liu et al

AJP October 1995, Vol. 147, No. 4

B
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18.4A
-14.3

18.4

14.3

human monocyte-s (1 Xio7 cells

human monocytesImacrophages. A: Freshly isolated
after adherence (lane 2) or cultured for 1 day (lane 3) or -5 days (lane 4). The cells uwere then
lysed, and galectin- 3 in the lysates was analyzed by immunoblotting as described in Materials and Methods. Lane 1 represents the detection of
galectin-3 in HeLa cells. B: Human monocytes (I X 107 cells per condition) were lysed 2 hours after adherence (lane 1) or after 2 days (lane 2) or
5 diy.s (lane 3) of culture in Teflon beakers. The positions of the mw markers ( X 10- -3) are denoted on the left margin.

Figure

1.

Immunoblotting analysis of galectin-3

per condition)

were

either

analyzed directly

in cultured

hours

progressively more intense immunoperoxidase

staining for galectin-3 as the cells were cultured for 2
and 5 days, as compared with freshly isolated
monocytes (Figure 2). It is interesting that there was
a prominent nuclear staining in many of the cells.
This result is consistent with previous reports of nuclear localization of this protein in proliferating
cells.36
To exclude the possibility that the difference in the

level of galectin-3 was a result of varying degrees of

enzymatic degradation of galectin-3 during cell lysis,
1251-labeled galectin-3 was mixed with the cells at
the end of the culture period and before lysis. The
total lysates were then analyzed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis followed
by autoradiography. There was no appreciable variation in the intensity of the 1251-labeled galectin-3
band in samples from freshly isolated monocytes or
from monocytes cultured for 1 or 5 days (data not
shown).

Immunogold/Electron Microscopic Analysis

of Galectin-3 on the Surface of Monocytes!
Macrophages
Previously, we demonstrated that galectin-3 is expressed on the surface of rodent mast cells and
macrophages and that galectin-3 can be removed
from the cell surface by lactose.14 In this study, we
examined whether surface expression of galectin-3
also occurs on human monocytes and macrophages. Furthermore, we also determined whether,
as monocytes are cultured in vitro, there would be
increased cell surface expression of galectin-3, paralleling the increased total cellular galectin-3 content. Freshly isolated human monocytes and day 5
monocyte-derived macrophages underwent immunogold cytochemistry, and the labeled cells were
examined by transmission electron microscopy. As
shown in Figure 3, freshly isolated monocytes exhibited the typical monocyte morphology (Figure 3A),

Galectin-3 in Human Monocytes/Macrophages

1021

AJP October 1995, Vol. 147, No. 4

Figure 2. Immunoperoxidase staining forgalectin-3 in human monocytes cultured in suspension. Freshly isolated human monocytes and monocytes
cultured in Teflon beakersfor 2 and 5 days were stained with murine monoclonal anti-galectin-3 antibody or an isotype-matched control monoclonal
antibody by an immunoperoxidase method ( brown reaction product) and with hematoxylin (blue) as a counterstain. A, B, and C: Days 0, 2, and
5 monocyteslmacrophages stained with the anti-galectin-3 antibody. D, E, and F: DaYs 0, 2, and 5 monocytes/macrophages stained with the control
antibody.

whereas the cells cultured for 5 days acquired the

characteristics of monocyte-derived macrophages
(Figure 3B). Gold particles specific for galectin-3
were observed on the surface of the freshly isolated
monocytes (Figure 3C). Increased cell surface gold
particle deposition was noted in day 5 monocytederived macrophages (Figure 3D). The number of
immunogold particles on the surface of monocytes
cultured for various periods was determined by morphometric analysis, with cells incubated in the absence or presence of 20 mmol/L lactose for 10 minutes at 200C before initial paraformaldehyde fixation.
As shown in Figure 4, the number of galectin-3-

specific immunogold particles increased progressively when the cells were cultured for longer periods
of time. As expected, the number of immunogold
particles decreased substantially (by 65 to 82%)
when cells were treated with lactose before the immunogold cytochemical analysis.
Immunogold/electron microscopic analysis was
also performed with permeabilized cells for detection of intracellular galectin-3. As shown in Figure 3,
E and F, galectin-3-specific gold particles were
found throughout the cytoplasm and in close association with the nuclear envelope and cytoplasmic
vesicles of the rough endoplasmic reticulum.

1022

Liu et al

AJP October 1995, Vol. 147, No. 4

A~

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Figure 3. Immunogold cytochemical localization ofgalectin-3 expression in human monocyteslmacrophages. Huiman monocytes werefixed in 10%
paraformaldehyde as described in Materials and Mcethods either 2 hours after adherence (day 0) (A; bar, 2 pm) or after tissue culture for 5 days (B;
bar, 2 Asm). The cell seen in A exhibits the typical monocyte morphology with a central nucleus (N) and small rim ofperipheral cytoplasm. Golgi
apparatus (G) and other intracellular structures are of normal appearance. Short microvilli (MV; arrowheads) are present on the cell surface. The
cell in B has the characteristics of a monocyte-derived macrophage. The nucleuis (N) is lobulated and there is an increase in the cytoplasmic volume.
Abundant mitochondria (Al) and Golgi complexes (G) are present. Long microvillus projections (arrowheads) are noted on the cell surface. Day 0
monocytes (C; bar, 1 ,uLm) and day 5 monocyte-derived macrophages (D; bar, 1 ,um) underwent immunogold cytochemistry to localize galectin-3
on cell surface as described in Materials and Methods. Gold particles (arrows) specific for galectin-3 are observed on the surface of the day 0
monocytes (C). Increased cell surfacegold particle deposition (arrows) is noted in day5 monocyte-derived macrophages (D). Day 5 monocyte-derived
macrophages also underwent immunogold cytochemistry after being permc-abilized with Triton X-100 as described in Materials and Methods for
dctection of intracellulargalectin-3 (E and F; bar, 1 ,u m). Galectin-3 is located throughout the cytoplasm (gold particles circled) and also in close
association with the nuclear membrane (arrowheads; E). Many galectin-3-specific gold particles are associatced with cytoplasmic vesicles (arrows)
of the rouigb endoplasmic reticulum (F).

Expression of Galectin-3 in Monocyte

Monolayers Activated by LPS, T. gondii,
and IFN-y
To address the question of whether the expression of
galectin-3 can be modulated by activation of monocytes, we studied the effect of soluble (ie, LPS) and

particulate (ie, T. gondii) stimuli on the level of

galectin-3 in monocytes. Human monocytes cultured
as monolayers for 1 day were incubated with 50
ng/ml LPS for 2 hours, with T. gondii for 4 hours, or
with T. gondii for 4 hours and then with 50 ng/ml LPS
for 2 hours. The cells were then lysed, and the
lysates were analyzed by immunoblotting for levels

Galectin-3 in Human Monocytes/Macrophages

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that there were lower levels of galectin-3 in the lysates of monocytes treated with either LPS or T.
gondii or both, as compared with the untreated control (Figure 5A).
As IFN-y activates human monocytes,7 we examined its effect on galectin-3 expression. Freshly isolated human monocytes were cultured as monolayers in the presence or absence of 100 U/ml IFN- for
5 days. The cells were lysed and the lysates
were subjected to immunoblot analysis. Again, less
galectin-3 was detected in the lysates of the IFN-ytreated monocytes as compared with the control
(Figure 5B). By densitometric analysis of the chemiluminescent exposure on film, we determined that
the IFN-y-treated monocytes contained 62% of
galectin-3 found in the untreated cells (data not
shown).
To quantitate the effect of the above stimuli on
galectin-3 expression, the amount of galectin-3 in
each lysate was determined by ELISA. As shown in

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i/i/i c ultiluri'd ii t/ii' atusC/iC (lane 1 )
orpcIsenc C,i(/ 100 t 1/il INb ylane 2) t/o
'c/ hi' i//i11) uh/ul//llui4). I/ iui/iluls i//i/u' ))iu/cC i//I ir/ (c,,'/it uark rs X 1()

1024 Liu et al
A/P October 1995, Vol. 147, No. 4

Table 1. Quantitation of Galectin-3 in the Lysates of

v-

6
1-r

Human Monocytes

,tg galectin-3/g total

a.

Experiment

Treatment

protein + SD

11

None
LPS
T. gondii
T. gondii + LPS
None

1.58 0.23
0.75 + 0.012
<0.5*
<0.5*
10.2 0.41; 6.01 + 0.44
5.06 + 0.36; 2.58 + 0.47

IFN-y

8-

In experiment 1, human monocytes (1 x 107) cultured for 1

day were incubated with LPS (50 ng/ml) for 2 hours; T. gondii (5
x 107) for 4 hours, or T. gondii (5 x 107) for 4 hours followed by
LPS (50 ng/ml) for 2 hours. In experiment 11, human monocytes (1
X 107) were cultured for 5 days in the presence or absence of
IFN-y (100 U/ml). n = 2 experiments. Determinations were done
by ELISA.
* Below the sensitivity of ELISA.

6-

C)

4-

*,0
0
0
0
ci)

EC.

Table 1, the results support the decrease in the level

of galectin-3 in monocytes treated with either LPS, T.
gondii, or IFN-y.

Induction of Secretion of Galectin-3 from

Monocytes/Macrophages by Calcium
lonophore

2-

0-i_

Lactose

Figure 6. Superoxide produtctioni bh hunmani monocytes iniducedl b

galectin-3. Recombinant human galectin-3 (.30 ug/mli) uas adcled,

We next addressed the question of whether

galectin-3 is secreted by human monocytes/macrophages. Human monocytes were cultured for 3 days
and then exposed to a calcium ionophore, A23187
(10 ,ug/ml) for 30 minutes. The supernatants were
collected, the cell lysates were prepared, and the
galectin-3 content in both were determined by
ELISA. As shown in Table 2, there was a significantly
higher level of galectin-3 in the supernatant of calcium ionophore-treated cells, which is coupled with
a concomitant decrease in the galectin-3 content in
the lysate of these cells as compared with the control. In contrast, there was no change in the level of
lactic dehydrogenase in the lysates of treated cells
as compared with the untreated cells (data not
shown). Similar results were obtained in two separate experiments, suggesting that galectin-3 is secreted from stimulated monocytes.

either alone or togetber unith lactose (25 mnmol L), to human mionocytes
(2.5 X IffInit) in microtiterplate.s and the suiperoxidce generated was
onoizitored as described in Materials and Methods. 7he data represent
mean + SID of'triplicate measurements. Statistical sigtnificatnce was
determinied by, unpcpaired t-test, P < 0.0001.

Superoxide Production by Human

Monocytes Induced by Galectin-3
To determine the effect of galectin-3 on the function
of monocytes, we tested the ability of galectin-3 to
stimulate superoxide production by this cell type.
Human peripheral blood monocytes were treated
with recombinant galectin-3 in the presence of cytochalasin B and the superoxide generated, as
gauged by the absorbance change at 550 nm resulting from the reduction of ferric cytochrome c, was
monitored. As shown in Figure 6, galectin-3 at 1
,tmol/L (30 ,ug/ml) induced production of superoxide

Table 2. Measurement of Release of Galectin-3 from Human Monocytes Treated with a Calcium Ionophore

Treatment

Supernatant

None
A23187

16.65 - 1.90
63.34 5.78

Amount of galectin-3 (ng)

Lysate
Total
229.60 16.56
184.40 + 14.22

246.25 - 36.92
247.74 40.00

% release

6.8_ 1.6
25.6 4.7*

Human monocytes (1 x 107) cultured for 3 days in autologous serum were treated with either buffer alone or A23187 (10 ng/ml) for 30
minutes. The supernatants were harvested and the cells were lysed with a lysing buffer (see Materials and Methods). Total amount of
galectin-3 from 107 cells was determined by ELISA. Results are shown as mean _ SD.
* Statistical significance was determined by unpaired t-test; P < 0.001.

Galectin-3 in Human Monocytes/Macrophages

1025

AJP October 1995, Vol. 147, No. 4

significantly increased above control. A higher concentration of galectin-3 (2 ,mol/L) induced a comparative level of superoxide production (data not
shown). At both concentrations, the observed reduction of cytochrome c was inhibitable by superoxide
dismutase, indicating that the absorbance changes
were indeed related to superoxide production. Furthermore, the effect of galectin-3 is dependent on its
lectin function, as it could be inhibited by lactose, a
known saccharide ligand of galectin-3 (Figure 6).
Similar results were obtained in three separate experiments. Phorbol myristate acetate, a well known
activator of monocytes, was included for comparison, and the maximal level of superoxide induced by
galectin-3 was approximately 16% of that induced
by phorbol myristate acetate (20 ng/ml). The recombinant protein does not contain a detectable amount
of endotoxin (see Materials and Methods). In addition, the fact that the observed activity of galectin-3
could be inhibited by its specific saccharide ligand
suggests that it was unlikely a result of any possible
contaminant present in the recombinant protein
preparation.

Discussion
The major findings of this work are fourfold. First,
normal human monocytes express galectin-3 and
the total cellular level of this lectin increases dramatically as the monocytes differentiate into macrophages in vitro. Second, the amount of galectin-3
expressed in human monocytes is downregulated by
LPS and IFN--y. Third, galectin-3 is secreted by human monocytes when stimulated by the calcium
ionophore A23187. Fourth, human monocytes can
be activated by galectin-3 as evidenced by superoxide production. It has been well established that
upon culturing in vitro monocytes acquire biochemical and morphological features characteristic of
macrophages.32 Therefore, the marked increase in
the galectin-3 level as human monocytes are cultured in vitro suggests that the expression of this
protein correlates with monocyte/macrophage differentiation. This conclusion is consistent with a previous report that the level of cell surface galectin-3
increases with differentiation of murine monocytes.2223 It is to be noted, however, that the cell
surface expression of galectin-3 does not necessarily correlate with the total cellular levels of this lectin.14'38 In the case of human monocytes/macrophages, the galectin-3 mRNA and protein levels
have been found to be significantly increased in
promonocytic HL-60 cells after treatment with stimuli

that promote differentiation of these cells into macrophages.2526 Our study definitively demonstrated
the upregulation of total cellular galectin-3 in normal
human monocytes upon cellular differentiation in
vitro.
The mechanism for the downregulation of galectin-3
expression in human monocytes/macrophages by
LPS, T. gondii, and IFN-,y is unknown. It is possible
that galectin-3 may be modified by oxidants generated by activated phagocytes, similar to the oxidative degradation demonstrated for leukotrienes and
heparin proteoglycan.39'40 Sato and Hughes41 have
shown that activation of thioglycolate-elicited murine
peritoneal macrophages by LPS caused a significant
reduction in cell surface expression of galectin-3.
They postulated that this lectin may be competing
with cytokines for shared surface receptors and that
LPS-induced decrease in cell surface galectin-3 in
macrophages would maximize the cellular response
to activating cytokines. Our results show that the
effect of LPS can be generalized to human monocytes/macrophages and that LPS causes downregulation of the intracellular galectin-3.
Whether galectins are secreted has been the
subject of much discussion, especially as these
lectins do not contain a characteristic signal
sequence.7'42'43 By pulse-chase experiments,
galectin-3 has been shown to be secreted by murine
macrophages from thioglycolate-stimulated mice.7
Recent work confirmed and extended this original
finding, including the demonstration of secretion of
galectin-3 from murine macrophage cell lines.38'41 It
is remarkable that cell lines differ in their capability to
secrete galectin-3 and some cell lines secrete substantial amounts of newly synthesized galectin-3.33
The secretion of this lectin was also demonstrated in
baby hamster kidney cells44 and Madin-Darby canine kidney cells43 and, in the latter case, selective
secretion of the protein from the apical side of the
cells was shown. Our data support that galectin-3 is
also secreted by activated human monocytes. It is
possible that the treatment of monocytes with LPS or
IFN-,y also results in the release of the intracellular
galectin-3 into the media, thus accounting for the
decreases in the intracellular content of this protein.
We, however, did not detect galectin-3 in the
supernatants of these cultures. It is possible that the
galectin-3 released from the freshly isolated monocytes (which contain rather low levels of galectin-3)
after the LPS treatment was below the detection limit
of our ELISA. On the other hand, prolonged culture of
monocytes, as was in the case of IFN-y treatment of
these cells, might have resulted in extensive degradation of the released galectin-3.

1026

Liu et al

AJP October 1995, Vol. 14 7, No. 4

Our results suggest that the function of galectin-3

is most likely associated with more differentiated
macrophages as opposed to monocytes. One such
function is cell-cell or cell-extracellular matrix interaction. The finding that galectin-3 binds laminin in a
carbohydrate-dependent fashion17 suggests that
this lectin may play a role in adhesion of monocytes/
macrophages to basement membranes under inflammatory conditions. Galectin-3 may recognize
glycoproteins and glycolipids with appropriate oligosaccharide ligands present on endothelial cells and
contribute to diapedesis of monocytes/macrophages. Galectin-3 may also play a role in phagocytosis, which is increased in inflammatory as opposed
to resident peritoneal macrophages.5 The role of
lectin in phagocytosis has been previously well demonstrated46; galectin-3 may serve to recognize glycoproteins or glycolipids on the surface of tumor
cells or microorganisms.
Our results also suggest that galectin-3 can activate monocytes/macrophages through its lectin
function. Because galectin-3 is secreted by these as
well as other cell types, the activation of monocytes/
macrophages may occur in an autocrine or paracrine fashion. The concentration of galectin-3 (ie,
approximately 1 ,tmol/L) that induced optimal superoxide release from monocytes is similar to that
shown to cause maximal activation of mast cells18
and neutrophils.20 Because galectin-3 is present
abundantly in the cytoplasm of various cells, it is
possible that when these cells release copious
amounts of their intracellular galectin-3, relatively
high local concentrations of this lectin can be
reached at least transiently in the surrounding milieu.
In fact, we have detected close to 1 ,tg/ml (30
nmol/L) of galectin-3 in bronchoalveolar lavage fluid
from mice with induced airway inflammation (unpublished observation). Considering the dilution factor
one introduces in obtaining the fluid, it is reasonable
to believe that the original concentrations might be
approximately 100-fold higher (ie, in the micromolar
range). Previously, we have proposed that galectin-3
may function as a cytokine and may have an effect
on a variety of cells that express oligosaccharide
ligands recognized by this lectin.21 Indeed,
galectin-3 was found to bind the IgE receptor (FcsRI)
on mast cells and activate IgE-sensitized and unsensitized rat basophilic leukemia cells, possibly
through cross-linking of FcsRI and/or receptorbound IgE.18'47 Furthermore, galectin-3 was found to
stimulate superoxide production by human neutrophils.20 Therefore, the finding that galectin-3 also
activates monocytes provides additional evidence

for a broad modulatory role of galectin-3 in inflammatory response.

The presence of lectins in macrophages and their
role in the various functions of this cell type is a
subject of considerable interest and significance.48
Macrophage mannose receptor, like galectin-3, was
found to be an excellent marker for macrophage
differentiation.48 Experimental data have been
provided to support that this receptor mediates
uptake and killing of microorganisms.49 Galactose/
N-acetyl-galactosamine-specific lectin is also expressed in macrophages and may be involved in
macrophage tumoricidal activity.50'51 Continued investigations of galectin-3 as well as other animal
lectins should provide new insights into the biological functions of mononuclear phagocytes.

Acknowledgments
The authors thank Dr. L. Frigeri for providing purified
goat anti-galectin-3, Gertrude Chiang and Mechthild
Jonas for skilled technical help, and Velda Comstock
for assistance in preparation of the manuscript. Peripheral blood from healthy donors for some of the
experiments was obtained through the service provided by General Clinical Research Center at
Scripps Clinic.

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