Topography of lectin binding sites in celiac sprue

Lectins are ubiquitious, naturally occurring proteins largely derived from planes that are present in the human diet. They are released from food during mastication and digestion to remain biologically intact during passage through the small intestine. In this study, the topographic distribution of binding sites in the small intestine for six different lectins (derived from wheat germ, castor bean, gorse, horsegram, jackbean and peanut) were explored in 16 patients with celiac sprue a long with controls. For each lectin, the pattern of binding in the small intestinal biopsy specimens was distinct and specifically inhibited by a different carbohydrate residue. Lectin labelling patterns were different for both controls and celiac patients. in addition, the epithelial cell membrane surface and goblet cells exhibited a gradient of fluorescence intensity along the crypt-villus column. This gradient appeared to parallel the degree of enterocytic differentiation consistent with the concept that sequential addition of sugar residues to form completed oligosaccharide side chains occurs with enterocytic differentiation and maturation. These results also suggest that the process of epithelial cellular differentiation, although present in celiac enterocytes, is altered substantially.

Topographie des sites de liaison de la lectine en presence de sprue coeliaque RESUME: Les lcctines sont Jes protc ines naturelles o mnipresences, de rivees princ ipa lement de plantes utilisces clans l'alimentation huma ine.Elles sont libcrces a partir des aliments du rant la mastica tion et la digestio n et dcmeurcnt biologiquement inrnc tes au coL11•s du passage vers le petit intestin.Dan s cette crude, la distributio n topographique des sites de liaison a u niveau du petit intestin pour six lectines diffcrentcs (ti recs de germe de blc, de grnines de ric in, d 'aj oncs, Je pois do li4ues de type aspe rge, de pois sabre et d 'arachides ) a e tc etudiee chez L ECTINS ARE UBIQUITOU~.Nt\ TL:R- a lly occurring protei ns and glycl1protcins largely derived from plants ( l ).Interestingly, lcctim ha,•e been detected in foods t hat arc hecom ing incrcm,ingly imr,ortant in diets of the h ealth cnn,ciou~ cnnsumer including d number of ,wplc~, ~uch as p1.ltatoes, 11eas, henn,, soybeans, va rious nu ts, tomatoes and wheat ge rm (2) .Lecti ns may he released from these d ietary suhstances d uring mast ication and/or d igest il1n and may remain biologically in tact during passage, even in to t he di,wl small intestine (3).Some lcc t im arc poten t b,ological agents that bind tn speci(ic sugar resiJues, agglut inate fetal and neop la~tic cell , inhibit in \'ivo and in v itni tumnur growth and stimulate lymplwcyte proliferat ion ( I ).
Celiac sprue (ccliac d isease, gluten-,cnsit ivc enteroparh y) is a d isorde r characterized hy a typica l, although not specific, smal l in testi nal mucosa!lesion t h.i t is improveJ with restriction of d ietary gluten (4).Impai red ahsorptinn of one or more nutrients may abo occur (4).Although the primary defect m il requires elucidation, gluten may initiate ce ll tox ic ity hy acting as a lecrin and binding to Jefcctive brush border membrane compo nents in genetically predisposed patients (5) .An iden tical lecti n hypothesis (5 ) has been developed fo r soy protein in tolerance, a small intestinal m ucosa!disorJer, cli nically and h isrologically indistingubhablc from ccliac ~prue (6).Je res id us de sucrc pour former des cha, n cs lateralcs d'oligosaccha rides comple tes se produit en presence de differentiation el Je maturation cnccrocyra ircs.Ces rcsultats suggl:rent egalement q uc le processus de d ifferentiation cellulairc epitheliale, bien quc present a u nivcau Jes c nterocytes cocliaqucs est cons iderablcment a ltcre.
Given thei r potentially di ve rse ro le in hoth health a nd disease, chi~ ~tudy explorcJ the cellular kKa li zat ion and topographic distribution of lecrin hind -ing sites in bio p~ie~ from no rmall y differentiating proxi ma l small inte~cina l mucosa and compared these findings with c ha nges seen in poorly differcn-  tiatc<l mucu,,a l hinp,ic,-frn m pat ient, with celiac sprue.The observations ind icate that unique l:1hclling pat terns for individ ual lecti n:, circ prc,cnt in the small inte~tinc and that cellu lar J i(fc rc ncc:, in hoth me m hrnnc a nd mucin glycocnnjugates lik ely refl ect differences in the degree of epithelia l cell d1ffcrentintion.

MATERIALS AND METHODS
Patients and biops ies: S ma ll intc:,tinal biopsies from 16 adu lt pmic nt, wi th untreated ccliac , prue and ,ix nllrmal controb were u,cd.T he c lin ica l d<:ta1b from each patient arc summari:ed in Tahlc l.T he c l1 rll c,tl fea ture, llf patient, I to 3 were Jescrihcd in earlier report~ (7)(8)(9), wh ilc biops ies fro m ~ume patient s h ave hee n u,cJ fo r lllher studi es ( J0-12).Except fo r patient, 3 and 6, a ll had d inrrhca and weight lu,s.Pati ent 3 initially presented with a perforated small in testinal ulcer due w lymphoma and was later proven to have ce li ac sprue (9).Patie nt 6 initi al! \ pre~ented with iro n defic iency anemia a lone wi th no diarrhea.A ll contrub were investigated fo r diarrh ea wit h nn cause found ; a ll h ad normal s1rn1 1l 1nte,ti na l hio ps ie~.
To descri be furth er a lcctin labelli ng reactivi ty, a semiquantitati ve scheme of grading fluore •cence intensity was used as reported previous!y ( l 3) to permit expression of the hbrochcmical dam in tabular form.A n absence of J crecrablc fluorescence was expressed as 0, trace fluorescence as l + and inc reasing mtcnsities o ( fluorescence as 2 + to 4 +.

RESULTS
Al l patients a nd controls shnwed positive staining wi th eac h fluoresceinconjugared lectin used, .)ndhinpsies from all paticn h shl1wcd posi ti ve labelli ng of the surface epithelium ,mJ/or goblet cel l muc m~.Adj ace nt control sections wid, inhibitor ~ugar were uniformly negative fnr each kc tin itudied.Re~ulrs arc de~c rihed helow; grading of fl uorescence in tensity b tabulated in T ables 3 and 4.

Triticwn t•ulgare: Incense labelling Df
the lu minal epithelial cell surface a nd goblet cells fro m the base co t he top of the vil lus was obs<.:rvcd in biopsies from normal control s (Figure l ) .Trncc nuorescence or an absence of fl unresccnce was obse rved in the crypt regions.In contrast.eel iac specimens showed labelling of both the lum innl epithelial cell surface and goblet cell muc us from the ba al to apical rcghms of c rypts (Fi gure 2).ln bot h contro l and ccliac hiop-11c ,agrad ic nt ofO uorescence intensity fnr both the lumin,tl epi thelial cell surface and gohlc t cell muci n was ob-,erved.The intensity of Ouoresccnce appeared to increase fro m the hase of the crypts to the luminal aspect of Li1c biopsies for huth cont ro l :rnd ccliac patients.lnc r<.:asingly, the luminal cell 1urface a nd gohkt c<.: 11 of the crypts in ccliac hinpsies labe lled more intensely than the s<1m e region in control hiopsi<.:s (P<0.05).
(1 .5±0.  3).In contrast, ccliac hinpsiesshmved bnt h luminal epithe lial cell surfrtee and gnhlet cell lahe ll ing from the basal tll apical regions of the biop~ies (Figure 4 ).In both comnil and ccli,K hiop~ic~, a ::,imilar grn dient of fluorescence inte nsi ty fo r both the luminal C['ithcl ial ce ll su rfa ce and gnhlct cell muc in ll'as nhsen•e,I.The intensi ty of ll uore~cence appea red to increase from the hase ol the crypt~ LO the mnst luminal aspect of the biopsic::, in both conrwl and cc liac patient~.T h is differenti al gradi ent of fluorescence intensity appeared to be less for ccliac than contrd biopsies.
Ulex euro/1e11s: For contro l hinpsie::,, fluorescence was present in gob let cclb and nlting the luminal epithelial ce ll surface with a differential hinding gr,1d ient from the ha~e of crypts tn the lllp of \' illi (Figure 5).In cuntrast, ccliac

DISCUSSION
A novel histochemi cal Hpl'roac h was used in the prese n t invesLigario n m label spec ific suga r residue~ in th e , m,111 intestinal epiLhclium.LcCLins arc n a turally occu rring proteins, deri ved mainly fro m pla nt~ ( l ), that can hind to epithelial ce ll me mbrane surfaces and goblet cells ti rats a nd hum ans ( 13, l 5, 16).Distinc t labelling patterns for e ac h lectin in bn d1 control ,md ccliac biopsies were evide nt, rcf1cc Ling the unique copographi c distrihut in n of spec ifi c car-bohy<lrnte comp\1ne nts in complex cell membrane pro te ins known Lo be precnt in huma n small intestine.
Striking diffe re n ces in fluorescence intensit y a long the c rypt-v illu~ column were a lsn e vide nt in thc~e studies in both con trol and ccliac hiopsk s, reflecting rhe pre;,ence o f a grndient in rhc topographi c distrihuti ()n of spec ific lectin binding sites in s1rn1II intcstin:11 epithelia l cells with m c rcasing c ntcrocyte differentiatio n.This i~ consistent with the con cept th a t epithe lial celb migrate from the c rypt LO v illus region and , as c m c rocytic differcnti;ni on :is well as maturatio n occ urs, sugar residues a rc ~equemi a ll y added tti fo rm comple ted n ligtisuccharidc s ide c h ...i ins (17)(18)(19)(20).This diffcrentiatinn prncess occurs in norma l small imest inal epithelium and (as shnwn in this study) in adult ccliac epiLhclium .
In the adult celiac, howe ver, the wpographic di strihutio n nflectin hindmg sites was unique and differed from control hiops ies, indicating that the Mfcrentiminn pnicess in ccliac enten1cytcs, although present , is :1ltcrcd suhrantia l ly.A lrl1\ 1ugh pre, •inus studies Figure 6) Fl11orescein 1sothioc)•anate-conjugaied-U lex curopeus labelling of expancld cry/H regl(Jn m d1toclenal hiopsy from celiac {)atiem .Cell surface and gohler cell {l1torescence is increased com puree/ w1r/1 controls ( fi!{ure 5) have ex plored la be lling differe nces in lcc tin binding to the mi c rov illus surfa ces in c hildren with ccliac disea~cs (2 1,22 ), these in vestiga rinns, mcluding the prescnL repo rt , have n t1t d e termined if these ohservmiuns arc spec ific (m rhis disnrder.A n on spec ific a lternti o n in the cnt c n lC)'lC diffe rcntiall\ll1 11roccss cnu ld re-,u It fro m a ny form o f injury or tox ic ity that a lte r-, the structure of th e sma ll imestine.Additiona l studies -pnss ibl y empltiying biopsies tro m othe r sma ll intestin,d disord ers th:it result in ,1 ' flat' vi llus les io n (cg, tropical sprue, soy 1 1rote in c nteroparh y, mi c rovillus inclus io n dise ase , e tc )might further define the spec ificity n f th e l:ihelli ng c ha nges recorded in the prese nt in vestigation .
A clear unde r~randing o f the 1 x nhogcnl's is n( celi ac sprue rem a ins l' lusive.Presum a bly, in gen e tically predisposed indi v idu a ls, glute n (or une uf its compone nts) provokes a l,lx ic reactio n , m:1inly in the proximal ~mall intestine, possihly by binding to intestina l surface struc tures.l t has heen suggested char de fi c ienc y of intes tina l peptidase acti v ity results in toxic glu ten degradati o n produc ts that bind to surface struc tures and induce immunoregulatory pa tho mechanisms .Alterna tively , a prinrnry immune de fec t may be responsible.
O the rs ( 2 3} h ave hypo thesized thac glute n acts as a lectin a nd b inds to spec ifi c () ] igos,1cc h,iride struc ture~ l if glycnprnte ins or glycnlipids in the mteslinal hrush hnrde r leading Lo cell tox ic it y.St1me e vidence h as acc umulat ed to suppn rt this glute n -lec tin h ypo thes is 111 thc pnthogenes i, of cel1 ac d bease.G I iadin and glut e n pe ptide~ bl'• have like lcc tins and h ave hce n sh ow n tll hind to high manno,e-rypc glycllproteins in hu1rnm ,crum as well as i111maturl' c rypt cclb in ra t in tcs tii1L' ( 24, 2 5 ).Mo reo ver, gli adin pc pt itb fro m hread whea t may reversihl y agglutina te poorly diffe re ntiated hum,m K562(S) cell s; hinding a nd agglutinating ac t iviL y was appnrcnt ly inhibited by ma nnan, H mannosc po lysacc ha ride a nd N-ace t yl-g lucosamin e (26).Fina lly, 1rn1nna n a nd oligomc rs of N-acctylglucosaminc we re rece ntly repo rted to e xhibi t protective effe cts to gliadin in vit ro using c ulLUrcd small ime~tinal bi opsies from c hi ldren wit h ac tive ccliac Jiseasc ( 27).This intriguing o bse rva ti on sti ll requires in vivo examinatio n.Although the presem study has sh own that the nume rou~ sma ll inte~tina l hinding sites for lec tins arc present in adult c cliacs, a carboh ydra te receptor -~pec ific tn celiac disea,ethat could bind gli adin require, e luc idatitm.