Implications in T-Cell Physiopathology"

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"Galectin-1

Induces Central and

Peripheral

Cell Death:

Implications

in

T-Cell

Physiopathology"

C.E.

SOTOMAYOR*

andG.A. RABINOVICH

Inmunologfa.Dpto.Bioqufmica Clfnica Facultad deCienciasQufmicas.Universidad Nacionalde C6rdoba. C6rdoba. Argentina The immune systemhas a remarkablecapacityto maintain astateofequilibriumeven as it

respondsto adiversearrayofforeign proteins and despiteitscontactexposureto

self-anti-gens. Apoptosisis oneof the mechanisms aimedatpreservingthe homeostasis after the

com-pletion of an immune response, thus returning the immune system to a basal state and

warrantingthe elimination of autoagressive cells in both central and peripheral lymphoid organs. Targeted deletions in critical genes involved in the apoptotic death machinery togetherwith naturalspontaneousmutations haveclearlyshown theimportanceofapoptosis

in theregulationof the immuneresponse. Thiscomplex scenario ofstimulatoryand

inhibi-torygeneshas been enriched with thefindingthatgalectin-1,a14.5kDa

[3-galactoside-bind-ing protein,isable to induceapoptosisof immature corticalthymocytesandmatureTcellsby cross-linking cell surface glycoconjugates. Galectin-1 is present not only in central and

peripheral lymphoid organs,but also at sites of immuneprivilege. Inthepresentarticle we will discuss theimplicationsofgalectin-1-induced apoptosisin T-cellphysiopathologyin an

attempttovalidate itstherapeutic potentialin autoimmuneandinflammatorydiseases.

Keywords: galectin-1, apoptosis,immunomodulation,macrophages,autoimmunity

INTRODUCTION

Death,alongwithgrowthand differentiation,is a

crit-icalpart of thelifecycleof the cell. Homeostasis

con-trol of cell number is thought to be the result of the

dynamic balance between cell proliferation and cell

death.Itisonlyinthe pasttenyears,that theattention

has been focusedonthe physiologicaloccurrence of

cell death and its role in the homeostasis.

Apoptosisorprogrammedcelldeath,is a

phenome-nonthatplaysacrucialrole inamyriad of

physiolog-ical and pathological processes. This review will

briefly cover some relevant aspects ofprogrammed

cell deathinthe immune systemin anattemptto

pro-vide valuable information about new molecules

responsiblefortriggeringdeath signals, suchas

galec-tin-1. The implications of this protein will be

dis-cussed in the context of T cell physiology and the

regulationof central andperipheraltolerance.Finally,

novel and intriguing findings will also be discussed

implicatingtheuseof this carbohydrate-binding

pro-tein in the treatment of autoimmune and inflamma-torydiseases.

* Addressedcorrespondence: Dra.ClaudiaE Sotomayor,Laboratoriode Inmunologia.Departamentode BioquimicaClinica.Facultad de

CienciasQuimicas.UniversidadNacionalde C6rdoba.Ciudad Universitaria.(5000)C6rdoba. Argentina. Phone#54-0351-4334164.Fax#

54-0351-4334187.E-mail:csotomay @bioclin.fcq.unc.edu.ar

?Both authorscontributedequallytothis article.

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APOPTOSIS:

A PHYSIOLOGICAL

MECHANISM

OF

HOMEOSTASIS

AND SELF-TOLERANCE IN THE IMMUNE

SYSTEM

Physiologicalcell deathbyactivationofintrinsiccell

suicide program, provides an efficient and critical

control point for eliminating unwanted cells. This

typeof regulation allows theeliminationof cells that

have been produced in excess, have developed

improperly, or have sustained genetic damage

(Schwartzman and Cidlowski, 1993, Thompson,

1995). Asto the initiationof the death program, the

decision ofa cellto undergo apoptosis canbe

influ-encedbyawidevariety ofextrinsicandintrinsic

reg-ulatorystimuli(Thompson, 1995). Onthe otherhand,

the effector stage takes place after triggering a

number of evolutionarilly conserved genes that regu-late a final common cell death pathway, thatis

pre-served from invertebrates to humans (Vaux el al.,

1994; Raft, 1992).

Programmedcell deathis animportant

physiologi-cal process acting both during development and

homeostasis. Aberrations in this process are

impli-catedin abroad range ofdiseases. Whileloss of

apop-totic response can lead to cancer or autoimmune

diseases, an increased apoptoticrateis implicatedin

neurodegenerative diseases, brain ischaemia and

myocardial infarction.

In

this context, the immune

system offersanexcellentscenariofor thediscussion

ofthe relevance of apoptosisinthedevelopmentand

maintenance of homeostasis. Immunologists have

focussed largely on defining the stimuli that induce

growth, differentiation andeffector functions of

lym-phocytes andoverthe lasttwo decades, theessential

feature of lymphocyte activation and immune

responses have been defined in considerable detail.

Less is known, however, about the mechanisms that

terminate immune responses. Mechanisms such as

apoptosis are important after a productive immune

responseto aforeignantigen, when theimmune

sys-tem is returned to a state of rest (van Parijs and

Abbas, 1998). This process allows the immune

sys-tem to respond effectively to a new antigenic

chal-lenge.

Moreover,

apoptosis is crucial for achieving

self-tolerance avoiding the developmentof receptors

capableof recognizing self-antigens. Elucidating the

nature of these homeostatic mechanisms may leadto

better strategies for suppressing harmful immune

responses and foraugmenting and sustaining

benefi-cialresponsestomicrobial vaccinesandtumors.

Death Signals inT-lymphocytes Development

ThedevelopmentofTcellsisgoverned bythe

micro-environment of the thymus.

A

large number of

pre-cursorcellsmigrateintothethymusdaily, wherethey

are subjected to selection in a critical process of

thymic education. Distinctstagesof theTcell

differ-entiationand maturation withinthethymushave been

identified and associated with the cells surface

expression of molecules such as CD4, CD8 and the

TCR/CD3 complex. In the subset of CD4+ CD8+

double-positive(DP) lymphocytes,morethan95% of

cells are destined to die by positive and negative

selection.ThemajorityofDP thymocytesfail to

gen-erate afunctionalTCRthatsuccessfullyinteracts with

major histocompatibility complex proteins

(MCH)

and consequently die by a process called death by

neglect. Those cells bearing TCR that recognize

MHC with intermediated affinity are positively

selected.

Moreover,

a subsetofDPcells recognizing

MHC molecules with high affinity are subjected to

negativeselectionandconsequentlydeletedby

apop-tosis (Surh and

Sprent,

1994).

A

high percentage of

thethymocytes generateddailydie withinthethymus.

Hence,

massive cell death is a crucial part of T-cell

development,asreflectedbythe fact that eachnewT

cellundergoesfor self-MHCrestrictionand

self-toler-ance.Thymocytesthatsuccessfully pass through

pos-itive and negative selections down-regulated the

expression of either CD4 or CD8 and differentiate

into functional single-positive cells and go out as

matureTcellstothe periphery.

Apoptosis in

DP

thymocytes is also triggered by

glucocorticoids (Surh and

Sprent,

1994). The

induc-tion of cell death in thymocytes by glucocorticoids

was oneof the firstsystemsstudiedby Wyllie(1980)

and Cohenetal. (1992). Thisworkprovides anearly

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and wasfundamental tothe developmentof the

con-cept of active cell deathas acellularresponserather

thanapassiveactofoverwhelmingcelldamage.

Thy-mocytes, inaddition toT-cells lines, arehighly

sensi-tiveto apoptosis inducedby exposuretoendogenous

glucocorticoids(Sprentetal., 1988)inanactive

proc-ess, requiring de novo gene expression (Wyllie,

1980). In this sense, Ashwell and colleagues

per-formed in vivo experiments Vacchio et al., 1994,

King et al., 1995) demonstrating that a subset of

thymic epithelial cellsaresteroidogenic and that

inhi-bition of steroid synthesis modified the profile of

lymphoid thymic populations. Furthermore, data

obtainedby the creation of transgenic lines of mice

carrying aglucocorticoid receptorantisense (King et

al., 1995), indicatedthatthymocytes that donotbind

toMHCaredeletedby endogenoussteroids.

Elimination of autorreactive lymphocytes may

occurvia activation-inducedcell death: the same

sig-nals that triggeractivationof peripheralmatureTcells

induceapoptosis ofthymocytes (Takayamaand

Sitko-vsky, 1989).The difference between positive selection

ofnormally functional immatureT cells and

elimina-tionofautorreactivecellsmaylie in theaffinityof their

TCRsfor self antigens andincreasingevidencesuggest

thatco-stimulatorysignals couldplayanimportantrole

in thisprocess TakayamaandSitkovsky, 1989,Iwatw

etal., 1992,Miglioratietal., 1992).

In the network ofintrathymic signals, thepositive

selection might also result from antagonism between

glucocorticoid and activation-induced death. This

modelclearlydemonstrates thatthymocytesunableto

bind to the MHC are eliminated via glucocorticoid

signals, those cells that engage T cell receptor

com-plexwithmoderate avidityareprotectedviathe

ster-oid/ TCR antagonism, whereas thymocytes able to

transduceTCR signalsof sufficientstrength,are

elim-inatedviaactivation-inducedcell death.

The

Fas

Death

Factor:

"Turning

Mature

LymphocytesOff"

Among

the most important molecules involved in

triggering apoptosis appearsthe cell surfacereceptor

Fas,

also called APO-1 or CD95, which induced

apoptosis upon binding to its natural ligand

FasL,

APO-1L, or CD95L, or specific agonist antibodies

(Nagata, 1997). This death receptor is a member of

the tumor necrosis factor (TNF) and nerve growth

factor(NGF) receptor superfamily,whichamong

oth-ers, also includes TNF-R1, TNF-R2, low-affinity

NGFR, CD40 and CD30. Whilefamily membersare

defined by the presence of cysteine-rich repeats in theirextracellular domains, CD95 and TNF-R1 also

shareanintracellular region ofhomology, designated

the "death domain",which isrequiredtosignal

apop-tosis.

Fas,

a 36 kDa type I membrane protein, is

expressed on a wide variety of cell types including

hematopoietic andepithelial cells. Expression ofFas

on T and B-lymphocytes increases after antigen

receptor-mediated

activation(Nishimuraetal.,

1995).

This moleculeis also expressed in cells transformed

withhuman T-cellleukaemia virus(HTLV-1),human

immunodeficiency virus (HIV) orEpstein-Barr virus

(EBV).

Many

nonlymphoid tissues, suchasliver, also

expressFas (Nagata, 1997; Galleetal., 1995).

Fas-L is a 40 kDatype II transmembrane protein

thatmediatesapoptosisby crosslinkingofFasin sen-sitive target cells and in contrast to the widespread

distribution of

Fas,

its ligand exhibits a highly

restricted pattern of expression. FasL is induced on

mature CD4+ and CD8+ T lymphocytes following

activation, butitisnotexpressed byother

hematopoi-eticcells(Tanakaetal., 1995;Sudaetal., 1995).This

molecule has been showntoplayaroleinthe

mainte-nanceof peripheralT- and B-cellhomeostasis.

Note-worthy, in some circumstances FasL can be

proteolitically cleaved from the membranebya

met-alloproteinase,occuringin asoluble form thatcan act

as a cytotoxic effector molecule or an inhibitor of

apoptosis(Strandand Galle,

1998).

The Fas-mediated apoptotic cascade is initiatedby

the direct association of the death receptorFas with

the adapter molecule

FADD

(Fas-associated protein

with death domain) and the effectorprotease FLICE

(FADD homologous ICE/ CED-3-1ike prtease,

FADD-like ICE), a member of the

ICE/Ced-3/cas-pase family (Henkart,

1996).

The assembly of this

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Proto

:

14,5

:

14

ip: 4,5

Sltes of

T-cell

apoptosls

Sites

of

Immune

Mlege

Thymus

Cornea

Lymph

node8

Braln

Spleen

Testes

Llver

Prostate

Placenta

MM:Motecutar

mass,

A.A:

Amino acidresidues,tp:tsoetectric point

FIGURE Galectin-1, characteristicand particular expression

activated caspase members can cleave various

sub-strates resulting in characteristic apoptotic

morphol-ogy ofcytoplasmandnuclei.

Activated T-cells express Fas as well as FasL.

Although theyuseFasLto kill theirtargets, theycan

alsousethis molecularweapon againsteach otherto

limit their own numberprotectingactivatedmatureT

cells fromcontinued secretion ofpotentially harmful

levels of cytokines. These cells are eliminated from

the circulationbyactivation ofthe cell deathprogram

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Roleof Fas-mediated cell death in thesuppression

of immuneresponse,self-tolerance

and autoimmunity

Activation-inducedapoptosisofmatureTcells occurs

via

Fas

andFasligand

(FasL)

interactions.

A

setof in

vitro experiences using cell lines or T-cells hybrids

provided successful information about the relevance

ofFasdeathsignals(Bruneretal., 1995;Dhein etal.,

1995;Juetal.,

1995).

Intherecentyears,muchwork

has focusedon the molecular mechanisms by which

thesemoleculesregulate apoptosis speciallythe

rela-tionship ofFas-FasLinteractionwithmembers of the

Bcl-2 family in thecontextofperipheralimmune

tol-erance(vanParijsetal., 1998).

vanParijsandcolleagues clearlydemonstrated that

Bcl-2 protects T cells from apoptosis caused by the

absence of growth factors and activation stimuli, a

process called passive cell death and prolongs the

response ofT cells to amodel offoreign antigen in

vivo. In contrast,Fas induces apoptosis in

autorreac-tiveTcellsorinactivatedTcellsthatarerestimulated

with high concentrations of antigen by a process

called activation-induced cell death. In vivo,

Fas-mediatedapoptosisisresponsibleforeliminating

T cells responding to a model systemic self-antigen

and for preventing autorreactivehelper

T

cells from

activating self-reactive

B

cells.

Genetic defects that predispose to autoimmunity

areprovidingvaluable information about the

mecha-nisms responsiblefor terminating T-cellresponses to

self-antigens. In this sense, the importance of Fas/

FasL interaction in peripheral tolerance, has been

highlighted bythe MRL-lpr/lpror C3H-gld/gldmice

strains whichcarry spontaneousmutationsinFasand

FasL

genesrespectively. These mice exhibitmultiple

autoimmune systemic disorders characterized by the

presence of autoantibodies,

hypergammaglobuline-miaand immune complex nephritis, all features ofa

lupus-like syndrome

(Russell

etal., 1993;Russell and

Wang,

1993).

Nagata

and colleagues (Adachi et al.,

1995)

createdaFas-/-miceby targeteddeletion of the

Fas gene.These micedisplayedenhanced and

accel-erated lymphoproliferation in comparison to lpr/lpr

mice(Adachietal.,

1995).

Recently, a human syndrome of autoimmunity

associated lymphadenophathy has been described,

carryingvarious inherited abnormalities in

Fas-medi-ated killing. These abnormalities include the

inherit-ance of two mutant Fas alleles and unknown

signalling defects (Fisher et al., 1995; Rieux-Laucat

etal.,

1995).

Itislikelythat other alteration inFasor

downstream signalling intermediates will be

identi-fiedascause of autoimmunesyndromes. Recently, an

inhereted human caspase 10 mutation has been

described showing defective lymphocyte and

den-driticcell apoptosis in autoimmune

lymphoprolifera-tivesyndrome (type II) (Wangetal., 1999).

The elucidation of the mechanisms involvedin

T

cell survival in vivo will leadto rational approaches

for controlling autorreactivity, while enhancing

immunological memory.

Roleof Fas-mediated cell death inimmunological

privilegestissues

Theimmuneprivilegedtissuesare vulnerable sites in

thebodywhere evenminorcellularimmunereactions

andtheirassociated inflammatoryresponsecancause

irreversible damage. Therefore, protective

mecha-nismsare required to avoid unwanted immune

reac-tions that could result in impaired organ functions.

Interestingly, not only are these "immune privileged

sites" protected against overwhelming inflammatory

responses, but they can also support allogenic or

xenogenictissuegrafts. Some explanation about how

immune privileged is maintained, involve physical

barriersand cytokinesprofiles (Streilin, 1993).

FasL has been also reported to be constitutively

expressed in twoimmunologically privilegedtissues,

such as the eye and the testis. Griffith et al.

(1995)

showed that the constitutive expression of

FasL

on

parenchymalcells within the anterior chamber of the

eyescan maintain theintegrityof this

immune-privi-leged site. It can be reported that Fas+ lymphoma

cells can be induced to undergo apoptosis when

exposedin vitro toexplantsof cornea andiris-ciliary

body from eyes of normal mice, but not when

exposed to eyes ofgld mice, which do not express

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expressed in Sertoli cells of the testis (Bellgrau,

1995).

Taken together, these finding unequivocally

sug-gest that FasL plays a crucial role by avoiding the

damageinflictedbyactivatedTcellstothesetissues.

The expression of high levels of FasL represents a

defensive mechanism to prevent damage caused by

inflammation through an induction of apoptosis of

activatedcells expressing elevated levels ofFas

anti-gen(Osborne, 1996).

Recent data confirm that expression offunctional

FasL might confer the status ofimmune privilegeto

tumor cells, representing an active defense

mecha-nismresulting in the elimination ofimmune

compe-tentanti-tumorlymphocytes (O’Connelletal.,

1999).

In this sense, FasL expression could be associated

with the later tumor recurrence which is commonly

observedinseveraltumorssuchasmelanomas. These

tumorsoftenrecurafter 20yearsormore, andthis can

be explainedbyaloss ofimmunosurveillance.Hence,

FasL expression might contribute to tumor

progres-sion,invasion ormetastasis.

GALECTINS:

A FAMILY OF

CARBOHYDRATE-BINDING PROTEINS

WITH IMMUNOREGULATORY PROPERTIES

DefinitionandBackground

Galectins are agrowing family ofanimal

I-galactos-idebinding proteins,definedbytwo common

charac-teristics: (a) affinity for

poly-N-acetyllactosamine-enriched glycoconjugates and (b) significant

sequencehomology in the carbohydrate binding site

(Barondes et al., 1994a; Barondes et al., 1994b). In

thepastfewyears,therehas beenprogressin

identify-ing new galectins in mammals and other species,

cloningthem and ascertainingthe structural features

thatdeterminecarbohydrate binding. Tenmammalian

galectins have been well characterized and studied

(Leffier, 1997). Structural analyses of various

galectinsindicatethepresenceofhomodimersof

car-bohydrate-binding domains in galectin-1 and

galec-tin-2,a monomerof the carbohydrate-bindingdomain

ingalectin-5 andasinglepolypeptidechain withtwo

carbohydrate-binding domainsjoined by a link

pep-tide in galectins-4,-6,-8 and-9. Galectin-3 has a

carbohydrate-bindingdomain,ashortN-terminal

seg-ment, consisting of PGAYPG

(X)

repeats and an

intervening stretch ofaminoacids,enriched with

pro-line, glycine and tyrosine. Expression analysis have

revealed that certain galectins displayarestricted

dis-tribution, e.g. galectin-2 in hepatoma, galectin-4 in

small intestine, galectin-5 in erythrocytes and

galec-tin-7 in keratinocytes. Galectins with a broad tissue distribution include galectin-1, expressed in cardiac,

smooth and skeletal muscle, macrophages, neurons,

thymus, kidney and placenta, galectin-3 present in

blood cells suchasmonocytes,mastcells, andtumor

Cellsandgalectin-8expressedinliver, kidney,cardiac

muscle, lung and brain (Rabinovich, 1999).

Exten-sively studied among them is galectin-1, an

homodimer with anMrof approximately 14,500 Da.

It has been postulated that this protein recognizes a

wide variety of extracellular receptors such as

fibronectin (Ozeki et al.,

1995)

and laminin (Zhou

and Cummings, 1993) and cell surfaceglycoproteins

suchasCD45 and CD43 (Baumetal., 1995a, Perillo

etal.,

1995)

through deciphering specific glycocodes

(KasaiandHirabayashi,

1996).

By

virtue of this specific recognition, this

evolu-tionarily conserved family of animal lectins have

been implicatedin avariety of functions thatinclude

cell growthregulation (Sandford and Harris-Hooker,

1990; Wells and Mallucci, 1991), cell adhesion

(Cooper et al., 1991; Zhou and Cummings, 1993;

Rabinovich et al., 1999a), neoplastic transformation

(Akahani etal., 1997a), immuneresponses (Ofneret

al., 1990;

Levy

etal., 1983)and T-cellapoptosis

(Per-illo etal., 1995; Rabinovich et al., 1998; Iglesias et

al.,

1998a). However,

the widespread expression of

multiple members of the galectin family and

pre-sumed overlaps in carbohydrate-binding specificities

have madeit difficult toestablish thein vivo function

of individual members ofthisclass ofproteins

(Poir-rierand Robertson, 1993).

All known members of this family lack a signal

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cytosolandareisolated assoluble proteins.

However,

thereis evidencethatsomemembersareexternalized

byanatypical secretorymechanism(Cooperand

Bar-ondes, 1990).

The expression pattern of different galectins

changes during development (Colnotetal.,

1997)

and

thispatternisalso alteredatsitesof inflammation and

in breast, colon, prostate and thyroid carcinomas

(Akahani, et al. 1997b). The level of expression of

some galectins by tumor cells has been show to be

correlated with metastatic potential. Although

galectins exert their effects throughrecognition ofa

spectrum of appropriately glycosylated proteins on

the surface ofa variety of cells, the precise

mecha-nism and signal transduction pathways involved in

these functionsremainlargelyunknown.

GALECTIN-I: EXPRESSION WITHIN

THE

IMMUNE SYSTEM AND IMPLICATIONS

IN T-CELL PHYSIOLOGY

Participationof Galectin-I as a

Gear

of the

Central and Peripheral Cell Death Machinery

Galectin-1 has been shown to be expressed in sites

where T-cellapoptosistakesplaces includingthe

thy-mus (Baumet al., 1995a), spleen (Rabinovich etal.,

1996)

andlymphnodes (Baum, etal., 1995b). Ithas

been particularly found in thymic epithelial cells

(Baum et al., 1995a), activated macrophages

(Rab-inovich, et al., 1998) and effectorT cells (Blaser, et

al., 1998)(Figure 1).

The firstevidence suggestingthat galectin-1 could

be involved in central immune tolerance was first

suggested by Goldstone and Lavin (1991), who

reportedanincrease in thelevels of galectin-1

mRNA

during apoptosis induced by glucocorticoids. As

clearly stated, the interplay between thymic steroids

and TCR signals modulate cell death within the

thy-mus (Wyllie,

1980).

It is well known thatthymocyte

maturationalso requires the participation thymic

epi-thelial cells and extracellular matrix components

(Andersonetal., 1994; Andersonetal.,

1996).

Inthis

sense, Baum etal.

(1995a)

demonstrated that human

thymic epithelial (TE) cells produced high levels of

galectin-1 which boundspecificallyto the surface of

corticalthymocytes.Thisendogenouslectinmediated

the adhesion ofthymocytestoTEcells.Sensitivityof

T

cellstogalectin-1 was foundtobe modulatedbythe

expression of glycosiltransferaseenzymes that might

modifytheavailability ofoligosaccharideligands for

galectin-1. Perilloetal(1997)provided then

conclud-ing evidence that galectin-1 inducedapoptosis oftwo

distinct subpopulations of non-selected and

nega-tively-selected CD41ow, CD81ow immature cortical

thymocytes (Perilloetal., 1997). Nullmutantmice in

galectin-1 gene will be useful to confirm whether

galectin-1 playsacriticalrole in the central cell death

machinery for postive and negative selection of

developing thymocytes.

Activation-induced cell death ofmatureT cells is

one of the mechanisms aimed at turning off the

immune response and preventing the expansion of

autoagresive clones. In addition to its role in central

tolerance, Perillo et al. (1995) clearly showed that

galectin-1 inducedapoptosis also inactivated mature

T cells. Recently, Blaser et al. (1998) found that

galectin-1 expression was strongly up-regulated in

effectorT cells and inhibited antigen-induced

prolif-eration of naive and memory CD8+ T cells. This mechanism was mediated by an arrest in cell cycle

progression at the level of S and G2/M stages

(Allioneetal., 1998).

Moreover,

wehaverecently shown thepresenceof

agalectin-l-like protein,which wasdifferentially

reg-ulatedinresident,inflammatoryandactivated

macro-phages (Rabinovich et al.,

1996).

Total and surface

expression of this carbohydrate-binding protein,

called RMGal (for rat macrophage galectin-1) were

foundto beup-regulated when these cellswere

acti-vated with protein kinaseCactivatorssuchasphorbol

esters (PMA) and chemotactic peptides (fMLP).

Whenthisproteinwaspurifiedby affinity

chromatog-raphy andits biochemical properties andamino acid

sequence were determined, a definitive conclusion

was reached concerning its pertenence to the

galec-tin-1 subfamily Rabinovich etal., 1998).

Macrophages play an important role in several

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theyhave greatphagocytic ability and alarge

reper-toire of lytic enzymes and secretory products, they

alsoexpressawidearrayofcytokines, surface

recep-torsabletorecognize specificantigenepitopes.

Acti-vatedmacrophagesare more efficient in theirability

toprocessand present antigensinthe initiation ofan

immune response by virtue of the higher levels of

majorhistocompatibility complexmolecules(Adams

and Hamilton, 1984; Adams etal.

1996). Moreover,

theyarealsokey immunoregulatorycells ableto turn

offanestablished immuneresponse(Aliprantisetal.,

1996). We determined that by using current

tech-niquestoevaluate apoptosis, suchasDNA

fragmenta-tion, TUNEL assay and transmission electron

microscopy that galectin-1 produced by activated

macrophagesis ableto induce apoptosis ofmatureT

cells in acarbohydrate-dependent manner

(Rabinov-ich et al., 1998). The results were comparatively

stronger to those found in an heterologous system

using CLL-I,the 16 kDa chickenisolectin

(Rabinov-ich etal., 1997).

RMGal proteinwasfoundtobe secretedonlywhen

macrophageswereactivated withpotentbiochemical

agents and pro-inflammatory cytokines (Rabinovich

etal., 1999c).

Galectin-1 Expression in

Immune

PrivilegedSites"

aNovel Mechanism of Protection?

Galectin-1 is also present in sites of immune

privi-lege, such asplacenta (Hirabayashiand Kasai, 1988;

Iglesiasetal., 1998a),cornea(Ogdenetal., 1998)and

prostate (Allen et al., 1991; Hirabayashi and Kasai,

1993). The presenceofthis protein in these

vulnera-ble sitesmight contribute to mantain a stateof

toler-ance by inducing apoptosis of inflammatory and

activated T cells that could provoke injury,

autoim-mune damage or infection. Accordingly, galectin-1

could also be proposed as an alternative regulatory

signal to regulate immune privilege. Expression of

thisprotein in firsttermgestationplacentawould

pre-vent inflammatory T cells from harming the fetus

(Iglesiasetal., 1998a). Inagreement,aprotein related

to the galectin family called GRIFIN

(galected-relatedinterfiber protein) has beenrecently

identified inlens,cellularstructuresof theoptical

sys-tem (Ogden etal.

1998).

Furthermore, recent results

reported by Maldonado et al.

(1999)

have clearly

shown by using immunogold techniques, that

galec-tin-1isexpressedin Mller cells inpost-natalchicken

retinaand in mitochondria localizedin theinner

seg-ments of cone cells. Expression of this protein in

theseglialcells suggestsapotential role inmetabolic

and immunomodulatory processes between Mtller

and otherretinalcells.

Thispro-apoptotic proteinwasfoundtobe

up-reg-ulated by metastatic in comparison to non-invasive

tumors.Incertainway,tumorsmightbeconsidered as immune privileged sitesand several mechanisms for

tumorevasionof immune recognition have been

pro-posed, suchas decreasedexpression ofMHCclass I

0rB7.1 co-stimulatory signal,

_TGF-

secretion,

endo-cytosis of tumor antigens and FasL expression

(O’Connelletal., 1999).

In

thissense,oneshould

sus-pect that galectinsin tumorcellscantrigger apoptosis

oftumor-infiltrating lymphocytes (TILs),thus

allow-ing thetumor toescapeimmuneattack.

DeathSignalsin the Periphery

Despite strikingsimilarities intheirlocalization,

criti-caldifferences should bedistinguishedbetweenFasL

and galectin-1. FasL induces apoptosis by a

interac-tionwith itscounterpartFas/APO-1/CD95 within the

same cell (suicide) or a neighbour cell (fraticide),

while galectin-1 is secreted and binds to cell surface

glycoconjugates (Perillo etal., 1997) oncortical

thy-mocytes and T cells. Besides, galectin-1 and FasL

apparentlyuse different signal transduction pathways

to engagethe apoptotic programof the cell. Recently,

Su etal.

(1996)

and Perillo etal.

(1995)

showed that

theT lymphoblastoidcelllineMOLT-4 thatwas

unsen-sitive to FasL-induced apoptosis, was susceptible to

galectin-1.

In

contrast, the T lymphoblastoid cell line

CEM,

which was sensitive to FasL was resistant to

galectin-l-induced apoptosis.These datastrongly

sug-gestthat galectin-l-induced apoptosis are clearly

dis-tinctfrom thosetriggered by Fasengagement.

About theapoptotic signal trigger by cross-linking

(9)

whit galectin-1 and T lymphoblastoid cell line that

notexpress CD3, demonstrate that thelectin is

capa-ble to activated the death cell program (Pace and

Baum, 1997).

These results suggest that the

mecha-nism by which galectin-1 can induced apoptosis

appears to be distinct from T cell receptor trigger

apoptosis.

Death vs proliferation: Galectin-1 vs Galectin-3

While galectin-1 has been shown to trigger T cell

apoptosis (Perilloeta1.,1995;Rabinovich etal., 1998;

Iglesias etal., 1998a), galectin-3 has been shown to

stimulate proliferation (Yang etal., 1996; Iglesias et

al., 1998b;Inoharaetal.,

1998).

Similarlytomembers

ofthe Bcl-2 family, galectins-1 and -3 belong to an

additional family of proteins with high sequence

homologies but opposite effectsoncell survival. The

balance between the competing activities of

pro-apoptotic proteins suchas

Bax,

Bad and Bak and

on the other hand anti-apoptotic proteins such as

Bcl-2 and Bcl-xL, determines cell fate (Adams and

Cory, 1998).

Proteins most similarto Bcl-2 promote

cell survivalby

_inhibiti,ng

adaptersneeded for

activa-tionof the proteases(caspases)that dismantle the cell,

while moredistant relatives instead promote

apopto-sis apparently through mechanisms that include

dis-placingtheadaptersfrom the pro-survival proteins.

In

this sense, thefamilyof Bcl-2 related proteins

consti-tute one of the most relevant apoptotic regulatory

geneproducts actingatthe effectorstageof apoptosis

(Kr6emmer, 1997).

Hence,

it seems meaningful that

the interplaybetween galectins-1 and-3 could also

representanalternativepathwayinthe normal control

of cell homeostasis.To supportthishypothesisa

strik-inghomology has been found between galectin-3 and

Bcl-2 particularly localized in the NWGR domain

(Yang, 1996; Akahanietal., 1997).

Galectin-1 in

T

cell Adhesion to Extracellular

Matrix

Despite the lack ofa secretionsignalsequence,

galec-tin-1 issecretedintothe extracellular millieu, where it

recognizes poly-N-acetyl-lactosamine chains on

major ECM components, such as laminin (Zho and

Cummings, 1993) and fibronectin (Ozeki et al.,

1995).

By

virtueofthisrecognition,thiscarbohydrate

binding proteinhasbeen suggestedtoactas a

modu-lator of cell-cell and cell-ECM interactions.In

collab-oration with the laboratory ofDr. Ofer Lider in the

Weizmann Instituteof Science, Israel, Rabinovich et

al.

(1999a)

hasrecentlyshown thatgalectin-1 (at

con-centrations below its apoptotic threshold) inhibited the adhesionof humanTcellstoECM glycoproteins

in a dose and carbohydrate-dependent manner. The

inhibition of T-cell adhesioncorrelated with the

abil-ityofthisproteintoblock there-organizationof cell’s

actin cytoskeleton. Finally, the production of

pro-inflammatory cytokines in the context of the

ECM was markedly reduced in the presence ofthis

carbohydrate-binding protein. This is the first report

as tothe role of galectin-1 in Tcell adhesion.

How-ever, this protein has been shown to promote cell

attachmentordettachmentonother cell systemssuch

asmyoblasts (Cooperetal., 1991), melanocytes (van

den Brulleetal., 1995), olfactoryneurons

(Mahanta-happaetal 1994), rhabdomyosarcomacells(Ozekiet

al,

1995)

and fibroblasts (Zhou and Cummings,

1993).

GALECTIN-1

IN T-CELL

IMMUNOPATHOLOGY

Galectin-l, ProgrammedCell Death and

Autoimmunity:an Attractive Association

Autoimmune disease challenges clinical

immunol-ogytosetthesystemright.

An

autoimmune diseaseis

caused,accordingtothe clonalselectionparadigmby

aberrant activation of the immuneresponse and loss

of central-and/or _peripheral immune tolerance to

self-antigens

(Cohen, 1995).

The rational answer for

harmful activation is to find away to deactivate the

pathogenic lymphocytes. As aforementioned,

obser-vations in murine models of systemic autoimmunity

(10)

regula-tionoflymphocyteapoptosisis crucial tothe mainte-nance of peripheral tolerance (Singer et al., 1994;

Fisher etal.,

1995).

Inthis context,oneshould expect

that knock out mice for galectin-1 would evidence

autoimmune manifestations, suchaslupus-like

disor-ders or arthritis, as observed for spontaneous

muta-tions in Fas and FasL in lpr/lpr or gld/gld mice

respectively.

However,

no important phenotypic

changes could be detected in null-mutant mice as

regards galectin-1 gene (Poirrier and Robertson,

1993).

An

exhaustive examinationof the

immunolog-ical system is imperative in these genetically

modi-fied mice notonly atthe central level but also atthe

periphery to search forpotentially harmfull

autoag-gressive clones and signs ofdisregulated apoptosis.

Implications of galectin-1 in central andperipheral

immunetolerancepromptedustoinvestigateits

ther-apeutic potential incollagen-type II-induced arthritis

(CIA) in DBA/1 mice, an experimental model of

rheumatoid arthritis(Durieetal., 1994). In

collabora-tion withthe laboratoryofDr. Chernajovsky in Lon-don, Rabinovich et al. demonstrated by using gene

and protein therapy strategies that galectin-1

sup-pressed arthritis via T cell apoptosis (Rabinovich et

al., 1999b).

A

single injection of syngenic DBA/1

fibroblast engineeredto secrete galectin-1 at theday

of the disease onset,aswellasdailyadministrationof

recombinant galectin-1, were both able to abrogate

clinical and histopathological manifestations of

arthritis. Bothtreatmentsresultedin the inhibition of

anti-collagentypeII (C-II)antibody levels,inhibition

of thepro-inflammatory responseand ashifttowards

a Th2-mediated immune response, asjudged by the

anti-CII

IgG

isotypes in mice sera atthe end of the

treatment and the cytokine profilein draining lymph

nodecells. Finally, clear-cutevidence wasprovidedto

show that mice engagedin thegenetherapy protocol

with galectin-1 increased their susceptibility to

anti-gen-induced apoptosis, providing thefirstcorrelation

between theapoptoticproperties ofgalectin-1 andits

therapeuticpotentialin vivo.

Rheumatoid arthritis (RA) is a common chronic

autoimmune disease forwhich there is not effective

therapy capable ofpreventing long-term progression

andjoint damage (Feldmann et al., 1996;

Chernajo-vsky et al., 1995). Therefore, effective treatment of

arthritis will require the elimination ofarthritogenic

lymphocytes that initiate and perpetuate joint

inflam-mation, as well as the induction of tissue repair.

Hencegalectin-1-induced apoptosis could provide for

an idealmechanismusing anaturally occurring

pro-tein to terminate the autoimmune T-cell attack,

pre-venting the expansion of dominant autoaggressive

clones (Vaishnaw et al., 1997). It has been clearly

suggestedthat theextentof apoptosisinRAis

inade-quateto counteractongoing proliferation.This

imbal-ancemaybe explainedbythe production of cytokines

such as

IL-I,

which favor synoviocyte and T-cell

proliferation and inhibit susceptibility to apoptosis,

possibly associated with increased expression of the

Bcl-2familyof proteins(Tsuboietal., 1996).

TNF-Which acts as apotent pro-inflammatorymoleculein

RA,

signalspredominantly throughthe nuclear factor

kappa B (NFkB) pathway, promoting theexpression

of adhesion molecules and recruiting additional

cytokines such asGM-CSFandIL-6 in theinflamed

joint. SignalingthroughNF-kB has beensuggestedto

inhibit apoptosis (Fujisawa et al., 1996). Finally, an increase insoluble truncatedFashas been detectedin

RA

synovialfluidthus inhibitingthe functional

inter-actionbetweenFasandFasL

(Hasunuma

etal.,

1997).

Altogether, thesefindings suggestthatadisregulated

activationofprogrammedcell deathis a critical

com-ponent of theethiopathogenyofRA.

Results concerning the role of galectin-1 in

sup-pressing anautoimmuneinflammatoryprocess arein

agreementwiththoseraisedby

Levy

etal.(1983)ina

model experimental autoimmune myasthenia gravis

in rabbits and those raisedby Offneretal.

(1990)

in

experimental autoimmune encephalomyelitis in

Lewisrats.

CONCLUDING REMARKS

Theelucidationof the biochemicalpathwaysand

spe-cific proteins that regulate programmed cell death

provide a remarkable opportunity to manipulate the

life-anddeathdecisionsofthe cells. Thebasic

(11)

pro-grammed cell death will have far-reaching implications for the future health of autoimmune

dis-ease patients.

In

this sense, galectins represent an

attractive target for biomedical research and clinical

intervention.Experimentalevidence is nowemerging

to supportthe use ofgalectin-1 notonly inthe

treat-ment of autoimmune disease, but also in medical

strategies aimed at targeting T-cell physiopathology

such as the inhibition oftransplant rejection, control

ofgraft versushostdisease andinhibitionof chronic

inflammatoryprocesses.

Acknowledgements

We are acknowledged for their contribution to our

work to Drs. Clelia Riera, Yuti Chernajovsky, Ofer

Lider, Gordon Daily, Hanna Dreja, Cristina

Maldo-nado, Amiram Ariel,JunHirabayashi, CarlosLanda,

Leonardo

Castagna,

Mercedes Iglesias, Nidia

Mod-estiandCarlota-WolfensteinTodel.

This workwassupported bygrants from"Consejo

Nacional de Investigaciones Cientfficas y Tdcnicas"

(CONICET),"Consejo de Investigaciones Cientfficas

y Tecnol6gicas de la Provincia de C6rdoba"

(CONI-COR), "SecretarfadeCienciay Tdcnicade laUNC"

(SeCyT-UNC) and

Agehcia

de Promoci6nCientffica

yTecnol6gica

(FONCIT)

PICTN 02189.

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