Journal of Leukocyte Biology Volume 59, March 1996 363

Regulated expression of a 16-kd galectin-like protein in

activated rat macrophagesGabriel Rabinovich, Leonardo Castagna, Carlos Landa, Clelia M. Riera, and Claudia Sotomayor

Departamentos de Bioquimica Clinica y Quimica Biol#{243}gica, Facultad de Ciencias Quimicas, Universidad Nacional

de C#{243}rdoba, C#{243}rdoba, Argentina

Abstract: We investigated the presence of a

galectin-like protein in rat mononuclear cells using

a polyclonal antibody raised against a soluble lac-tose-binding lectin purified from adult chicken liver

that immunoreacted strongly with a broad protein

band of about 16 kd in Western blot assays. Immu-

nochemical studies revealed a constitutive expres-

sion of this protein in mononuclear cells mainly in

the macrophage (M4) population. Subcellular local-

ization was assessed by Western blot assays of the

cytosolic and membrane fractions of different cell

populations studied: ( 1 ) spleen mononuclear cells,

(2) T cell-enriched, (3) B cell- and M�-enriched

populations, and (4) peritoneal cells, processed in

the presence of lactose. In broad agreement withimmunocytochemical studies of nonpermeabilized

and permeabilized cells, Western blot assays suggest

that this protein is localized mainly in the cytoplasmic

compartment but also associated with the cell sur-

face. By flow cytometric analyses we detected about

a 14% of ED! double-positive cells corresponding

to M4s that constitutively express this galectin-like

protein associated with their cell surface. The cy-

tosolic fraction obtained from the M4-enriched cell

population showed hemagglutinating activity spe-

cilhcally inhibited by �-galactoside-related sugars.

Moreover, this galectin-like protein was retained in

a lactosyl-Sepharose matrix and specifically eluted

with lactose. In this work, evidence is also provided

to show that different stimuli are able to modulate

the expression of the galectin-like protein. Expres-

sion was upregulated in inflammatory and activated

M4s, revealing a significant increase in phorbol es-

ter- and formylmethionine oligopeptide-treated

cells. Both stimuli involving protein kinase C acti-

vation pathway have been able not only to up-regu-

late the total expression of this protein but also to

modulate its subcellular localization. J. Leukoc. Biol.59: 363-370; 1996.

Key Words: galectin . macrophages � activated macrophages

phorbol esters � N-formylmethionine oligopeptides . lipopolysac-

charides

INTRODUCTION

Soluble lactose-binding lectins constitute a family of closely

related carbohydrate-binding proteins distributed among

vertebrates [1, 2] and invertebrates [3]. They show highly

conserved cDNA nucleotide and primary amino acid se-

quences [2, 4] in additionto carbohydrate binding specificity

[4, 5]. According to their molecular architecture this animal

lectin family was subdivided into three groups: prototype,

chimera type, and tandem-repeat type [3], orL-14, L-30, and

L-36 S-type, respectively [6], and more recently a new

systematic name “galectins” [7, 8] has been proposed.

Galectins have been localized intracellularly in the cyto-

plasm [9] and nucleus [10] of several cell types. Moreover,

galectins were also detected in the extracellular milieu or

associated with the cell surface [2]. Their expression is

strongly regulated during development [1, 1 1] and they have

been involved in different biological processes such as cell

proliferation [12, 13], cell adhesion [14], cell recognition

[15], immunomodulation [16, 17], and metastasis [18]. Mac-

2 antigen [19], now called galectin-3, was first identified as a

cell surface component on thioglycollate-elicited murine

peritoneal macrophages (M�s), but not on resident peritoneal

M4s. Cell surface Mac-2 antigen expression has been used to

assess the linear differentiation sequence of M4 cell lines in a

model of normal M4 development [20] and recent studies

report the differential regulation ofMac-2 surface expression

on different murine M4 cell lines [2!].

Abbreviations: AC, adherent cell; BCG, bacillus Calmette-Guerin;

BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; fMLP,

N-formyl-Met-Leu-Phe; HBSS, Hanks’ balanced salt solution; IFN-y,

interferon-y; LPS, lipopolysaccharide; mAb, monoclonal antibody; M$,

macrophage; NAC, nonadherent cell; PC, peritoneal cell; PE, phyco-

erythrin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate;

Pp, proteose peptone; SDS-PAGE, sodium dodecyl sulfate-polyacry-

lamide gel electrophoresis; SpM, spleen mononuclear cell; SS, saline

solution; TNF, tumor necrosis factor; TRITC, tetramethylrhodamine

isothiocyanate.

Reprint requests: Bioquimico Gabriel Rabinovich, Inmunologia,

Facultad de Ciencias Quimicas, Universidad Nacional de C#{243}rdoba, Ci-

udad Universitaria, Suc 16-C.C. 61,5016, C#{243}rdoba, Argentina.

Received August 30, 1995; revised November 2, 1995; accepted

November 3, 1995.

364 Journal of Leukocyte Biology Volume 59, March 1996

Macrophages exhibit a great variety of functions at dif-

ferent levels of the immune response. Identification of the

wide variety of functions has placed M4s as the central

cells in initiating immune responsiveness, as antigen-pre-

senting cells, and in regulating the activities of other cells

of the immune system [22]. These highly adaptative cells

modify their behavior in response to different environ-

mental signals. The rat peritoneal cavity has been a useful

system for studying phenotypic differences between the

resident M4, population, exudate M4s recruited in response

to sterile inflammatory stimuli, and M4s activated by

pathogens, chemoattractants, or chemical agents. Resi-

dent, inflammatory, and activated M�s show different pro-

files of enzymes, antigens, and receptors which are closely

related to functional competence. These functions can be

up- or down-regulated independently by cytokines,

chemokines, and several stimulating agents [22].

In this work we provide immunochemical and immuno-

cytochemical evidence indicating the presence of a 16-kd

galectin-like protein in rat mononuclear cells, its subcellu-

lar localization, and the modulation of its expression by

different stimuli in the rat M4 population.

MATERIALS AND METHODS

Animals

Studies were conducted on 8- to 12-week-old female Wistar rats (aver-

age weight 250 g). Animals were housed and cared for at the Animal

Resource Facilities, Departamento de Bioqulmica Clinics, Universidad

Nacional de C#{243}rdoba, in accordance with institutional guidelines.

Anti-lectin serum preparation

A rabbit antiserum was raised against an endogenous soluble lactose-

binding lectin purified from adult chicken liver as previously described

1231. Briefly, the soluble lactose-binding lectin was purified from adultchicken liver by affinity chromatography on a lactosyl-Sepharose matrix.

Then rabbits were immunized with 50 �tg of the antigen in Freund’s

complete adjuvant by intradermal injection, followed by a 50-�.tg intrad-

ermal booster injection of the antigen in Freund’s incomplete adjuvant

2 weeks later. After 3 weeks the antiserum was obtained and stored in

small aliquots at -20#{176}Cuntil use. The immunoglobulin C (IgG) fraction

was purified by affinity chromatography on protein A-Sepharose and

labeled with fluorescein isothiocyanate (FITC) [24]. This antiserum was

previously used in immunochemical [23J and immunocytochemical

studies [25].

Antibodies

Monoclonal antibodies (mAbs) ED! (monocyte-macrophage lineage)

and FITC-labeled ED! were from Serotec Ltd., England. DM1A mAb(a-tubulin), goat anti-mouse TRITC-labeled IgG, goat anti-rabbit phy-

coerythrin (PE)-conjugated (Fab)2 IgG, anti-mouse FITC-labeled IgG,

and isotype control (mouse FITC-laheled IgG� ic ) were from Sigma, St.

Luis, MO.

Cells

Spleen mononuclear cells (SpMs) were obtained from normal rats.

Spleens were removed and collected in sterile conditions using Hanks’

balanced salt solution (HBSS), pH 7.2. Spleen cells were prepared by

gentle teasing in HBSS, debris was separated, and the single-cell sus-

pension was collected. SpMs were obtained by Ficoll-Hypaque gradient

centrifugation and were collected and resuspended in RPM! 1640 me-

dium. The I cell-enriched population was obtained by collecting nonad-

herent cells (NACs) from SpMs through a nylon wool column [26]. The

B cell and M4-enriched population was recovered from the adherent cellfraction (ACs). Viability of the cells was assessed by means ofthe trypanblue exclusion test.

Peritoneal cells (PCs) were harvested from rats using HBSS contain-

ing 5 U of heparin and 20 ji,JmL gentamicin. The M� population waspurified from PCs (1 x 106 cells) by adherent culture in 100-mm-diame-

ter tissue culture dishes in RPM! 1640 with 20 jig/mL gentamicin and

10% heat-inactivated fetal calf serum. Nonadherent cells were removed

after 2 h at 37#{176}Cand monolayers of adherent cells were incubated

overnight in the same medium and used as described later under stimu-

lation procedures. The resultant M� monolayer was 98% pure according

to morphologic and phagocytic criteria.

Subcellular fractionation

SpMs, NACs, ACs, PCs, and M4s (1 x 10� cells) were washed with

phosphate-buffered saline (PBS) and resuspended in PBS supplemented

with 4 mM mercaptoethanol, 2 mM EDTA, and 300 mM lactose (lysis

buffer). Cells were disrupted by five cycles of sonication at 12 kHz for5 S at 4#{176}Cand total cell lysate was centrifuged at 600g for 10 mm in

order to remove cell debris. Then the total cell lysate was centrifuged at

!00,000g for 60 mm and the resultant supematant and pellet were used

as cytosolic and membrane fractions, respectively. Before SDS-PAGE,

membrane fractions were washed with PBS in order to eliminate soluble

contaminants and expression of a-tubulin, as a predominant cytoplas-

mic protein, was used as a control.

Affinity chromatography and hemagglutination assay

The affinity chromatography was performed on a lactosyl-Sepharose

matrix as previously described [23]. Briefly, cytosolic fraction from the

M4-enriched cell population was dialyzed against PBS to remove lactose

and applied to the lactosyl-Sepharose column. The excluded material

was collected and after several washes the adsorbed material was spe-

cifically eluted with PBS supplemented with lactose.

The hemagglutination assay was done following the procedure de-

scribed by Nowak et al. [27], using serial twofold dilutions of sample in

microtiter U plates and trypsin-treated glutaraldehyde-fixed rabbiterythrocytes.

Stimulation procedures

PCs collected from untreated animals were used as resident M�s. Aninflammatory M� population was obtained from PCs of animals injected

intraperitoneally with 3 mL of 10% (w/v) proteose peptone (PP) (inflam-

matory agent) 3 days before cell collection. PCs from animaLs injected

with 3 mL of pyrogen-free saline solution (55) were used as controls of

in vivo stimulation.

Activated M� populations were obtained as follows: (1) in vitro treat-

ment of cells with 1 j.tgfmL phorbol 12-myristate 13-acetate (PMA) for60 mm at 37#{176}Cand 5% CO2; (2) in vitro treatment with 200 nM

N-formyl-Met-Leu-Phe (fMLP) for 60 mm at 37#{176}Cand 5% C02; (3) M4s

obtained from animals injected intraperitoneally with bacillus Calmette-

Gu#{233}rin(BCG) (5 x 106 live microorganisms) 7 days before cell collec-tion; (4) in vitro treatment of inflammatory M� populations with 1�ig/mL lipopolysaccharide from E. coli (LPS serotype 01 1 1:B4, Sigma)

for 18 h at 37#{176}Cand 5% C02 [28-30]. Ms were also incubated in

medium alone to use it as a negative control of in vitro stimulation.

SDS-PAGE and Western blot

SDS-PAGE was done in a Miniprotean II electrophoresis apparatus (Bio

Rad, Richmond, CA) as described by Laemmli [31]. Proportions of eachfraction were normalized as amounts of proteins or number of cells.

Equal proportions of each fraction were resolved in a 12.5% separating

polyacrilamide slab gel. Samples were diluted in 62 mM Tris, pH 6.8,

containing 2% (w/v) sodium dodecyl sulfate, 5% (w/v) �-mereap-

toethanol, and 10% (v/v) glycerol and then heated for 5 mm at 90#{176}C.Protein bands were detected using Coomassie brilliant blue staining.

A B

kD�)M Pc

IS

Ag SpMNAC AC PC Ag PC

Rabinovich et al. Galectin-like protein increased in activated macrophages 365

After electrophoresis, the separated proteins were transferred onto0.45-jim nitrocellulose membranes and incubated with the anti-lectin

serum as previously described [23]. Briefly, blots were blocked for 30mm with PBS containing 0.05 % Tween 20 (PBS-Tween) and 5 % (w/v)

nonfat dry milk, and incubated for 3 h with a 1:150 dilution of the

anti-lectin serum in PBS-Tween. This dilution was selected as the opti-

mal dilution when the antiserum was titered in a range between 1:75

and 1:300. After several washes with PBS-Tween, blots were further

incubated for 2 h with 1 �igfmL peroxidase-labeled protein A in PBS-Tween. Finally, the peroxidase reaction product was developed in 50

mM Tris (pH 7.4) containing 0.05% (w/v) 4-chloro-1-naphthol and0.03% (v/v) hydrogen peroxide. Controls were performed by incubation

of blots with rabbit preimmune serum to evaluate nonspecific reactions.

All procedures were carried out at room temperature. The relative

amounts of immunoreactive protein bands were quantified by densi-

tometry scanning comparing the total integrated areas under the respec-

Live peaks using a Shimadzu Dual Wavelength Cromato Scanner.

Immunofluorescence assays

PCs (1 x 106 cells) were washed twice with cold PBS containing 0.1%

(w/v) BSA (washing buffer), incubated with PBS containing 3% (w/v)

BSA (blocking-dilution buffer), and processed according to flow cy-tometry or fluorescence microscopy protocols.

In order to determine the presence of the galectin-like protein asso-

ciated with the cell surface, cells were subsequently incubated with a1:50 dilution of the anti-lectin serum, with a 1:40 dilution of the PE-

conjugated goat anti-rabbit (Fab)2 IgG, and finally with an appropriate

dilution of FITC-labeled ED! mAb. Then cells were fixed with 1%paraformaldehyde for 20 mm at room temperature, washed with PBS,and analyzed for relative fluorescence intensity on a FACStart-Plus

instrument (Becton Dickinson, Mountain View, CA). FACS plot program

Lysis II was used for data analysis. Controls included the incubation of

cells with a rabbit preimmune serum and the isotype control (anti-mouse

FITC-labeled IgGi K).

To assess the presence of the galectin-like protein at the cytoplasmic

compartment, the cells were permeabilized with 0.1% Triton X-100-IFA(10 mM HEPES, 150 mM NaC1, and 4% heat-inactivated fetal calf

serum) for 5 mm at 4#{176}Cand were subsequently incubated with theappropriate dilution of ED! mAb, washed, and then incubated with a

1:250 dilution of rat anti-mouse TRITC-labeled IgG. After several

washes, a direct immunofluorescence reaction was performed by incu-

bation of these cells with a 1:30 dilution of the anti-lectin FITC-labeledIgG. To check the access of the antibodies to the cytoplasmic compart-

ment, cells were incubated with a 1:200 dilution of DMJA mAb followedby incubation with a 1:250 dilution of rabbit anti-mouse FITC-laheled

IgG. Finally, cells were fixed as indicated above, mounted in PBS

containing 90% (v/v) glycerol, and observed on a Zeiss epifluorescence

microscope. Controls included the incubation of cells with a rabbit

preimmune serum and the omission of the first antibody step. All the

incubation procedures were carried out for 30 mm at 4#{176}C.

RESULTS

Immunoreactivity of rat mononuclear cell proteinswith the anti-lectin serum

To study the presence of a galectin-like protein in rat

mononuclear cells, different cell-enriched populations

from SpMs and PCs were obtained and Western blot assays

were performed using a rabbit antiserum raised against a

soluble lactose-binding lectin purified from adult chicken

liver.

SDS-PAGE analysis in Fig. !A evidences the protein

profiles of total cell lysates from SpMs, Pcs, and the sol-

uble lactose-binding lectin purified from adult chicken

Fig. 1 . Immunoreactivity of rat mononuclear cell proteins with the

anti-lectin serum. (A) SDS-PAGE. Protein profiles oftotal cell lysates (1

x106 cells) ofspleen mononuclearcells (SpMs) and peritoneal cells (PCs)

visualized by Coomassie brilliant blue staining. (B) AfterSDS-PAGE, the

separated proteins corresponding to SpMs, nonadherent cells (NACs),adherent cells (ACs), and PC total lysates were transferred to nitrocellu-

lose membranes and incubated with a 1 : 150 dilution ofthe immune serum(IS). Soluble lactose-binding lectin from chicken liver (Ag) (10 (ig) was

incubated with the immune serum and used as control of positive im-munoreaction. Total lysates from PCs, incubated with the same dilution

of preimmune serum (PS), were used as a control of specific immunore-

action. Molecular weight standards are shown on the left. The immunore-

active protein band is indicated by the arrow.

liver (Ag). In Fig. !B we show the Western blot assay

performed with the anti-lectin serum that strongly reacted

with a broad protein band of about 16 kd present in total

cell lysates obtained from rat SpMs and PCs. The presence

of this protein band was also detected when two subpopu-

lations were prepared from SpMs: (!) a T cell-enriched

population (NAC) and (2) a B cell- and M�-enriched popu-

lation (AC). Reactivity detected by densitometry quantifi-

cation of the protein bands -was threefold higher in total

cell lysates obtained from PC� than in those obtained from

SpMs, NACs, and ACs. When PC total cell lysates were

incubated with a rabbit preimmune serum, no significant

immunoreactivity was detected (Fig. 1B, PS); and similar

results were obtained by incubation of 5pM, NAC, and AC

lysates with the same preimmune serum (result not shown).

Finally, the soluble lactose-binding lectin purified from

adult chicken liver was included as a control of positive

immunoreaction (Fig lB. Ag).

Subcellular distribution of the 1 6-kd galectin-likeprotein in different rat cell-enriched populations

In order to analyze the relative contribution of soluble and

membrane-bound forms of the immunoreactive protein

bands, SpMs, NACs, ACs, and PCs were disrupted in lysis

buffer, and the cytosolic and membrane fractions were oh-

tamed after centrifugation procedures.

Equal proportions of both fractions were subjected to

SDS-PAGE. Figure 2A shows the protein profiles of cy-

tosolic and membrane fractions obtained from SpMs,

NACs, ACs, and PCs. When these proteins were trans-

ferred onto nitrocellulose membranes and incubated with

the anti-lectin serum, they evidenced the Western blot

pattern shown in Figure 2B. The immunoreactive protein

A B

.7, IS’ 1�’ 15’

FLI-�.F1uor..c.’�. �*

366 Journal of Leukocyte Biology Volume 59, March 1996

bands were quantified by densitometry and expressed as a

subcellular distribution index (SD!) (Fig. 2C). In every cell

population under study the reaction in the cytosolic frac-

tion was stronger than in the membrane fraction.

Parallel experiments evidenced similar amounts of the

immunoreactive protein in both membrane and cytosolic

fractions when lactose was omitted in the lysis buffer (re-

sults not shown). The significant increase of the immunore-

active protein detected in the cytosolic fractions when

lactose was added to the lysis buffer suggests that the

presence of this sugar was able to transform the galectin-

like protein to a soluble form. This result, however, does

not rule out the possibility that a minor amount of this

protein can be tightly bound to the membrane fraction.

Expression of �-tubulin as a predominant cytoplasmic pro-

tein was used to check contamination between membrane

and cytoplasmic fractions (data not shown).

A SpM NAC Ac PCkDa� mf cf mf Cf mf cf mf

B

I

C

� 120

0 so

�0II Ii2 I.IiI.I.

Fig. 2. Subcellular distribution of the galectin-like protein in cytosolic

and membrane fractions. Different mononuclear cell populations (5pM,

NAC, AC, and PC) were disrupted in lysis buffer, centrifuged, and the

cytosolic and membrane fractions (cfand mf respectively) were obtained.

(A) Equal amounts of protein (75 �tg) were subjected to SDS-PAGE and

the protein bands were visualized by Coomassie brilliant blue staining.

(B) After SDS-PAGE, the separated proteins were transferred to nitrocel-lulose membranes and incubated with a 1 : 150 dilution of the anti-lectin

serum. Molecular weight standards are shown on the left. The immunore-

active protein band is indicated by the arrow. (C) The immunoreactive

protein bands were quantified by densitometry and expressed as subeel-

lular distribution index (SD!): cf or mf I [cf + mf] x 100. Controls were

performed by incubation of the cytosolic and membrane fractions with a1:1000 dilution ofanti-a-tubulin -(DM1A) followed by a 1:500 dilution

of peroxidase labeled goat anti-mouse IgG.

A�:

l�

�11�,

i�.

- pLt.�UiCJE

2.44

�:

Fig. 3. Surface expression of galectin-like protein in resident M$s.

Dual-parameter counter plot. (A) Increasing green fluorescence intensity

(ED!) is plotted on the x axis versus increasing red fluorescence intensity

(anti-lectin serum) on the y axis. Cells were subsequently incubated with

a 1:50 dilution of the anti-lectin serum, with a 1:40 dilution of the

PE-conjugated goat anti-rabbit (Fab)2 IgG, and finally with appropriate

dilution of FITC-labeled ED! mAb. (B) Cutoffwas determined according

to fluorescence background represented by the reaction of the cells with

the isotype control antibody.

Immunocytochemical localization of thegalectin-Iike protein

In order to analyze the presence of the galectin-like protein

associated with the cell surface or in the intracellular corn-

partment, immunofluorescence assays were performed with

nonpermeabilized and permeabilized PCs.

Flow cytometric analyses of nonpermeabilized PCs were

performed using an FITC-labeled ED! mAb to detect the

M4 population. The fluorescence distribution of resident

M4s stained with the anti-lectin serum and the isotype

control are shown as counter plot graphics in Figure 3A

and B. About !4% double-positive cells were detected,

corresponding to M4s that constitutively express the

galectin-like protein associated with their cell surface.

On the other hand, permeabilized cells (Fig. 4A) were

immunostained with the anti-lectin FITC-labeled IgG (Fig.

4B) and ED1 mAb (Fig. 4C) and observed by fluorescence

microscopy. Very strong staining in the cytoplasmic corn-

partment was detected, suggesting an intracellular local-

ization of the galectin-like protein. In both experiments,

when the cells were incubated with the rabbit preimmune

serum or when the first antibody step was omitted, no

significant fluorescent reactivity was detected (result not

shown).

Taken together, the results suggest that this galectin-like

protein is localized in resident M4s, mainly in the cyto-

plasmic compartment of permeabilized cells but also asso-

ciated with the cell surface of intact viable cells.

Partial characterization of the galectin-like proteinfrom M4-enriched cell population

The cytosolic fraction obtained from the M4-enriched cell

population showed hemagglutinating activity, which was

specifically inhibited by lactose and thiodigalactoside (re-

kDa#{176}

58....

37-..

26-..

I12....

Fig. 5. Detection of the eluted M$ galectin-like protein blotted on a

membrane with anti-lectin serum. Cytosolic fraction of M4s was applied

onto a lactosyl-Sepharose matrix and the adsorbed material was eluted

with 300 mM lactose. Western blot assays were performed using a

dilution of anti-lectin serum. Molecular weight standards are shown on

the left. The immunoreactive protein band corresponding to cytosolicfraction (co. affinity chromatography excluded material (excl.), affinity

chromatography eluted material (elut.), and soluble lactose-binding lectin

purified from adult chicken liver (Ag) is indicated by the arrow.

A

-S a� � A.E c -J CD u):; 0 0) �. m � � A.Cl) Z U) 0. A. �

‘I

I

B

0

Rabinovich Ct al. Galectm-like protein increased in activated macrophages 367

Fig. 4. Immunocytochemical localization of the galectin-like protein inresident M4s. (A) Phase microscopy ofpermeabilized PCs and respective

immunofluorescence images obtained after incubation with (B) FITC-la-

beled anti-lectin IgG and (C) ED! mAb followed by TRITC-labeled

anti-mouse IgG. Fixed cells were mounted in PBS-glycerol and observed

by Zeiss epifluorescence microscope. Controls for nonspecific trapping of

the antibodies were performed using the rabbit preimmune serum as

described in the text.

sult not shown). In addition, this galectin-like protein was

retained in a lactosyl-Sepharose matrix. Then, when the

adsorbed material was eluted from the column with lactose

and analyzed by Western blot assays developed with the

anti-lectin serum, the strongest reaction was observed with

the !6-kd protein band (Fig 5). This molecular mass was

estimated by SDS-PAGE analysis.

Differential expression of 16-kd galectin-likeprotein on resident, inflammatory, and activatedmacrophages

To study whether different stimuli are potentially able to

modulate the expression of the the galectin-like protein on

rat M4 cells, we performed immunochemical experiments

using the anti-galectin serum and total cell lysates ob-

tamed from different stimulated M4s.

M4-enriched cell populations were purified by plastic

adherence of rat peritoneal cells. Resident M�s, inflamma-

tory M4s (PP-M4), saline M�s (SS-M�), and activated M�s

were used in this experiment.

Western blot assays of total cell lysates obtained from

the different stimulated M�s were performed using the

anti-lectin serum, and the immunoreactive pattern is

shown in Figure 6A. After densitometry quantification of

N

E

E

I �NDE2‘C

Fig. 6. Differential expression of the 16-kd galectin-like protein in

resident, inflammatory, and activated M4ts. (A) M� populations treated

with different stimuli-none, 55, PP. PMA, fMLP, BCG, and LPS-were

disrupted in lysis buffer and centrifuged. Equal amounts of total cell

lysates (1 x 106) were subjected to SDS-PAGE, transferred onto nitrocel-

lulose membranes, and incubated with 1:150 dilution of the anti-lectin

serum. Molecular weight standards are shown on the left. The immunore-

active protein band is indicated by the arrow. (B) The immunoreactive

protein bands were quantified by densitometry and expressed as increase

index (I I)= stimulated M�/resident M4. Resident M4

A) R- CP B) PP-CP

- 1 � 1� 1� 1

D)fMLP. CP

1�) BCG-CP F) LPS-CP

#{149}1 1#{149}1 1� ISP�1-%,1#{149}��* � , 1U� 1� I� I

368 Journal of Leukocyte Biology Volume 59, March 1996

the protein bands, results were expressed as fold increase

compared with the resident M� population (increase index

= II, Fig. 6B). Resident M�s, exhibiting constitutive ex-

pression of the galectin-like protein, were considered

equivalent to unity (II !). PP-M4 increased the expres-

sion of this protein at least twofold (II 2.2). More im-

pressive, we observed up-regulated expression of this

molecule in activated M4s, especially in those treated in

vitro with the chemical agents PMA and fMLP (II 6 and

4.5, respectively). Finally, activation by intracellular bac-

teria or LPS caused intermediate enhanced expression (II

= 2.5 and 2.7, respectively) of this protein in the M4s.

To examine the possibility that different stimuli could

modulate the expression of the galectin-like protein asso-

ciated with the cell surface, flow cytometry experiments on

different nonpermeabilized M4 cell populations were per-

formed using an FITC-labeled ED! mAb and the anti-

lectin serum followed by PE-conjugated antibody.

Double-positive cells reached low levels of surface lectin

expression in resident and inflammatory M�s (!4 and

!8%, respectively) (Fig. 7A and B). No significant sur-

face reactivity was detected in BCG-treated M4s (Fig. 7E).

LPS-treated cells evidenced intermediate enhanced reac-

tivity (55%) (Fig. 7F); M4s disturbed by strong chemical

agents such as PMA and fMLP expressed the highest

amount of surface reactivity (68%) (Fig. 7C and D).

The total overexpression of this protein and the modula-

Lion of cell surface reactivity suggest that PMA and fMLP

could he powerful modulators of the galectin-like protein

expression.

DISCUSSION

The present report describes the regulated expression of a

!6-kd galectin-like protein in mononuclear cells from rat

spleen and peritoneum. There are two main interesting

findings: (!) the identification of a mammalian galectin-

like protein by using a polyclonal antiserum raised against

a soluble lactose-binding lectin purified from adult

chicken liver; (2) the regulation of expression of this lectin

in M4s by inflammatory and activating agents.

Western blot assays revealed the highest expression of

this protein in PCs; however, it was also present among

5pM, T cell-enriched (NAC), and B cell- and M�-enriched

(AC) populations.

Subeellular distribution assessed by Western blot assays

indicates that the !6-kd galectin-like protein was present

in the different cell-enriched populations predominantly in

the cytosolic fractions. This cytosolic localization was con-

firmed by immunofluorescence microscopy studies per-

formed with permeabilized resident M4s. In addition, low

surface expression of this protein was detected in non-

stimulated resident M�s by means of flow cytometric

analyses. Subcellular localization and surface expression

of galectins have been shown to be under physiological

regulation at the level of protein secretion [32, 33]. Imma-

ture and mature M4s are able to synthesize similar

���1tTht #{149}FIEiL� I4

I

�‘

14.35

,!

i�:A� N.i�#{241}

�.1’#{149}!S

�-

2�74

:�

‘4I

?L.!

�_t�I �18.40

i�I�

:r-

a.i�t �-

Jt

[:S&6

Fig. 7. Surface expression ofgalectin-like protein in resident, inflamma-

tory, and activated M�s. Dual-parameter counter plots. Increasing greenfluorescence intensity (ED!) is plotted on the x axis versus increasing

red fluorescence intensity (anti-lectin serum) on the y axis. (A) Resident

PC (R-PC), (B) proteose peptone-treated PC (PP-PC), (C) PMA-treated

PC (PMA-PC), (D) fMLP-treated PC (fMLP-PC), (E) BCG-treated PC(BCG-PC), and (F) LPS-treated PC (LPS-PC) were processed. Cells were

subsequently incubated with a !:50 dilution ofthe anti-lectin serum, with

a 1:40 dilution of the PE-conjugated goat anti-rabbit (Fab)2 IgG, andfinally with appropriate dilution of FITC-labeled ED! mAb.

amounts of galectin 3 (previously known as Mac-2), but

unlike the latter, immature M4s fail to secret it [32].

Since a large number of galectins have been found in

mammalian tissues in the past few years, the following

criteria were considered to determine whether the !6-kd

immunoreactive protein band belongs to this group of car-

bohydrate-binding proteins: (!) this protein strongly re-

acted with the anti-lectin serum, (2) most of the protein

was detected in a soluble form in the cytosolic fractions

when the lysis buffer was supplemented with lactose, (3)

the hemagglutinating activity detected in the cytosolic

fraction was specifically inhibited by f�-galactoside related

sugars and retained in a lactosyl-Sepharose matrix, (4)

Rabinovich et a!. Galectin-like protein increased in activated macrophages 369

when the adsorbed material was eluted from the affinity

column with lactose it showed a prominent !6-kd protein

band detected in Western blot assays developed with the

anti-lectin serum. Finally, to answer the question whether

this novel galectin-like protein detected with the antiserumraised against the !4-!6-kd chick hepatic lectin is�related

to other mammalian galectins or the antibody reactivity

indicates a new mammalian lectin, more complete charac--

terization of this protein is under progress at present.

Whereas the regulation of glycoconjugate biosynthesis is�

well studied and documented, there is comparativel�r littl1�

information about the regulation of lectin expressi#{246}ni

[34-36], especially in the context of the immune system.

The most striking finding of our study is that expression of

the galectin-like protein was increased mainly when cells

were stimulated in vitro by phorbol esters (PMA) and

chemotactic peptides (fMLP). LPS-activated M4s and cells

exposed to BCG evidenced an intermediate increment of

galectin-like protein expression. On the other hand, in-

flammatory M4s showed twofold higher expression than

resident M4s.

Moreover, flow cytometry experiments showed highly in-

creased expression of the galectin-like protein associated

with the cell surface in M�s stimulated with PMA, fMLP,

and LPS. The highest total expression of this protein in

chemically activated M�s, together with the modulation of

cell surface reactivity, suggests that this molecule might be

involved in immunological processes. Similarly, galectin-3

was found to be expressed at high levels at the cell surface

of thyoglycollate-elicited murine M4s but not on resident

peritoneal cells [2!], supporting the idea that galectins

participate in regulating inflammation and immune func-

Lions [8]. In addition, recent studies indicate that cell sur-

face expression of some lectins may be a marker for

neoplastic transformation and a determinant of metastatic

potential [!8].

Extracellular matrix remodeling would be necessary for

extravasation and diapedesis, and hence lectin binding to

surface glycoproteins may be important in early recruit-

ment of inflammatory M4s. Further activation of these M�s

by interferon-y and LPS appears to down-regulate surface

expression of galectin-3, suggesting that this carbohydrate-

binding protein could have an antagonistic effect on the

activation caused by these stimuli. Loss of galectin-3 from

the M� surface would maximize the cytokine response

[37]. The findings presented herein support the idea that

cells not only regulate the synthesis of proteins such as

galectin-3 but also control their subcellular localization

and activities under different extrinsic signals [38]. In the

case of galectins as well as any animal lectin there could

be multiple physiological partners, either intracellular or

extracellular, that diversify the role played by these mole-

cules [32, 39].

Interestingly, the expression of the 16-kd galectin-like

protein is highly induced by chemical agents such as PMA

and fMLP. The direct effect of phorbol esters on PKC

activation, bypassing the membrane transduction Se-

quence, has been extensively studied and documented

[40]. Chemotactic agonists like fMLP require the involve-

ment of receptor-ligand interaction, G protein coupling,

and second messenger generation in order to activate PKC.

On the other hand, several studies document the role of

protein kinases in the signal transduced by LPS in M4s

[4!, 42]. Once PKC is activated by these stimuli, several

properties of the cell dependent on threonine-senne pro-

tein phosphorylation, such as secretion, synthesis, and in-

tracellular distribution of biological components, are

significantly modified [29]. The fact that treatments lead-

ihg to increased galectin expression could be associated

with the PKC activation pathway makes this system attrac-

Live for exploring the field of signal transduction.

Conservation of galectins during the evolution, their

presence in several tissues of diverse species, and their

developmental regulation strongly imply that they play a

role in some physiological processes requiring protein-car-

bohydrate recognition [8, ! !, 34]. Some lectin molecules

and their glycoconjugates have been shown to have im-

munoregulatory properties, suppressing clinical signs of

autoimmune processes like experimental autoimmune en-

cephalomyelitis (EAE) [17] and experimental autoimmune

myasthenia gravis (EAMG) [!6]. Moreover, they have been

able to induce the release of tumor necrosis factor from

M�s [43] and to reprogram cell migration and T cell-medi-

ated immunity [44].

It should be also kept in mind that the present work has

identified only some stimuli able to modulate galectin-like

protein expression. Characterization of the regulatory

mechanisms is likely to be important in the understanding

of the role of lectins in inflammatory reactions and immu-

nological processes involving activated M4s, such as tumor

cytolysis [45], microbial destruction, and phagocytosis

[46].

ACKNOWLEDGMENTS

This work was supported by “Consejo Nacional de Investi-

gaciones CientIficas y T#{233}cnicas” (CONICET) PID

3357400/92, “Consejo de Investigaciones CientIficas y

Tecnologicas de la Provincia de C#{243}rdoba” (CONICOR)

PID 3!56/94, and “Secretarla de Ciencia y T#{233}cnica de la

UNC,” 1994. Gabriel Rabinovich is a recipient of a schol-

arship from CONICOR.The authors wish to express sincere

thanks to Dr. Gustavo Chiabrando, Silvia Correa, and Pa-

blo Iribarren for their helpful discussions in preparing the

manuscript.

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