Dissociation between systemic and mucosal humoral immune responses in coeliac disease.

Gu , 1991,32,29-35 Dissocia ion be ween sys emic and mucosal humo al immune esponses in coeliac disease S O'Mahony, E A anz, J R Ba on, A Fe guson Abs ac We examined humo al immuni y in coeliac disease as exp essed in se um (sys emic immuni y), and in sali a, jejunal aspi a e, and whole gu la age luid (mucosal immuni y). The aims we e o de ine ea u es o he sec e- o y immune esponse (IgA and IgM con- cen a ions and an ibody alues o gliadin and o he ood p o eins measu ed by enzyme linked immunoso ben assay (ELISA)) in ac i e disease and emission, and o es ablish whe he sec e ions ob ained by ela i ely non- in asi e echniques (sali a and gu la age luid) can be used o indi ec measu emen s o e en s in he jejunum. Se um, sali a, and jejunal aspi a e om 26 adul s wi h un ea ed coeliac disease, 22 ea ed pa ien s, and 28 immunologically no mal con ol subjec s we e s udied, oge he wi h in es inal sec e ions ob ained by gu la age om 15 un ea ed and 19 ea ed pa ien s wi h coeliac disease and 25 con ol subjec s. Jejunal aspi a e IgA and IgM and gu la age luid IgM concen a ions we e signi ican ly aised in pa ien s wi h un ea ed coeliac disease; he la age luid IgM concen- a ion emained highe in pa ien s wi h ea ed coeliac disease han in con ols. Se um and sali a y immunoglobulin concen a ions we e simila in he h ee g oups. Pa ien s wi h un ea ed coeliac disease had highe alues o an ibodies o gliadin compa ed wi h ea ed pa ien s and con ol subjec s in all body luids es ed; hese we e p edominan ly o IgA and IgG classes in se um, and o IgA and IgM classes in jejunal aspi a e and gu la age luid. Values o sali a y IgA an ibodies o gliadin we e signi ican ly highe in un ea ed coeliacs, hough an ibody alues we e gene ally low, wi h a la ge o e lap be ween coeliac disease pa ien s and con ol subjec s. In ea ed pa ien s, wi h p o ed his ological eco e y on glu en ee die , se um IgA an igliadin an i- body alues ell o con ol alues, hough se um IgG an igliadin an ibody alues emained mode a ely aised. In con as , he e was pe sis ence o sec e o y an igliadin an i- bodies in ea ed pa ien s (pa icula ly IgM an ibody) in bo h jejunal aspi a e and gu la age luid. An ibody esponses o be alac o- globulin and o albumin we e simila o hose o gliadin, including pe sis ence o high in es- inal an ibody alues in pa ien s wi h ea ed coeliac disease. The e was a posi i e co ela- ion be ween an ibody alues in jejunal aspi a e and gu la age luid, bu no be ween sali a and jejunal aspi a e; hus sali a y an i- bodies do no e lec in es inal humo al immuni y. Sys emic humo al immuni y in coeliac disease has been he subjec o in ensi e in es iga ion. Nume ous s udies ha e es ablished. ha pa ien s wi h un ea ed coeliac disease ha e high alues o ci cula ing an ibodies o whea de i ed p o eins such as gliadin, and ha an ibody alues all a e a pe iod o ea men wi h a glu en ee die .''4 Es ima ion o se um IgA an igliadin an ibody is now ou inely used bo h as a sc eening es o coeliac disease and as a means o assessing die a y compliance. In con as , in o ma ion on mucosal immuni y in coeliac disease is pa chy. The e ha e been many s udies o mucosal lymphoid cells56 and he e is ci cums an ial e idence o a local cell media ed immune esponse o glu en.78 Se e al s udies ha e ca e ully mapped he numbe s o Ig p oducing plasma cells in he jejunal mucosa o pa ien s wi h un ea ed and ea ed coeliac disease,9 10 showing ha un ea ed pa ien s ha e inc eased numbe s o IgA and IgM (and o a lesse ex en , IgG) jejunal plasma cells. In he 1970s, he p esence o in es inal an ibodies o ood an igens was ecognised by a ela i ely insensi i e p ecipi in echnique," bu he e a e only wo s udies published,'2 1 bo h in child en, on he iso ype o an ibodies o die a y an igens in in es inal sec e ions, and hese gi e con lic ing esul s. The gene al objec i es o his s udy we e wo old. In ela ion o coeliac disease, ou aim was o cha ac e ise, in i o, in es inal humo al immuni y. To al immunoglobulins and speci ic an ibodies o gliadin and o wo an igens which a e no oxic in coeliacs we e measu ed in h ee di e en mucosal sec e ions. Un ea ed and ea ed pa ien s wi h coeliac disease we e s udied o de e mine whe he abno mali ies o sec e o y immuni y a e pe manen and in insic o he coeliac dia hesis o a e only p esen in ac i e disease. Sepa a ely, and o ele ance o he clinical in es iga ion o mucosal immuni y, we s udied he ela ions be ween sys emic and in es- inal an ibodies, and we examined ou da a o es ablish whe he pa e ns o immunoglobulins and an ibodies in jejunal luid a e mi o ed in o he sec e ions which can be ob ained wi hou in uba ion. The sali a y glands a e conside ed pa o he common mucosal immune sys em,'4 and we he e o e s udied pu e pa o id sali a. We also used a whole gu la age echnique o he non-in asi e collec ion o in es inal sec e ions. 5 Me hods PATIENTS STUDIED AT THE TIME OF JEJUNAL BIOPSY Specimens o sali a, jejunal aspi a e, and se um we e collec ed a he same ime as jejunal biopsy on 76 occasions in 69 pa ien s. The e we e 41 pa ien s wi h coeliac disease (se en s udied wice), (23 women and 18 men; median age 42 yea s, ange 15-78) and 28 con ol pa ien s (14 Uni e si y o Edinbu gh Gas oin es inal Uni , Wes e n Gene al Hospi al, Edinbu gh S O'Mahony E A anz J R Ba on A Fe guson Co espondence o: D S O'Mahony, Gas oin es inal Labo a o y, Wes e n Gene al Hospi al, C ewe Road Sou h, Edinbu gh EH4 2XU. Accep ed o publica ion 5 Ma ch 1990 29 on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om O'Mahony, A anz, Ba on, Fe guson women, 14 men, median age 35 yea s, ange 14-75). Con ol subjec s had jejunal biopsy o exclude coeliac disease - jejunal his ology in hese pa ien s was no mal, no o he signi ican pa hology was ound, and a inal diagnosis o unc ional bowel disease was made. Twen y six o he pa ien s wi h coeliac disease we e un- ea ed and his ological examina ion o he jejunal biopsy specimens showed sub o al o se e e pa ial illous a ophy. Se en o hese and a u he 15 pa ien s wi h ea ed coeliac disease (all wi h p e ious diagnos ic biopsy specimens) unde wen biopsy again while on a glu en ee die . The median pe iod on glu en ee die was h ee yea s ( ange 3 mon hs - 17 yea s). Ele en had en i ely no mal jejunal his ology (all o hese had been aking a glu en ee die o a leas wo yea s), and 11 had mino his ological changes - o example inc eased in aepi helial lympho- cy es (mos pa ien s in his g oup had been aking a glu en ee die o less han one yea ). PATIENTS STUDIED BY WHOLE GUT LAVAGE Gu la age was ca ied ou in 15 un ea ed coeliac disease pa ien s, 19 wi h ea ed disease, and 25 con ol pa ien s. These included 10, eigh , and wo pa ien s espec i ely om each g oup who had also had collec ion o jejunal aspi a e. The median pe iod on a glu en ee die in pa ien s wi h ea ed coeliac disease unde going gu la age was eigh yea s ( ange 3 mon hs - 19 yea s). Ele en o he pa ien s on a glu en ee die had in he pas shown a clinical and his ological esponse o he die , bu did no unde go biopsy again a he ime o his s udy. Gu la age was ca ied ou in 25 con ol pa ien s (16 women and nine men, median age 52, ange 21 - 92 yea s). These subjec s we e ei he heal hy olun ee s o pa ien s wi h unc ional bowel diso de . SPECIMEN COLLECTION AND PROCESSING Sali a: pa o id sali a y low was s imula ed wi h 5% ci ic acid sublingually in ou 0 5 ml aliquo s o e i e minu es, and collec ed ia a Ca lsson- C i enden cup placed o e he pa o id duc , wi h gen le aspi a ion o main ain posi ion and suc ion. We collec ed s imula ed sali a only. Jejunal aspi a e: samples we e collec ed om a poin jus dis al o he duodenal-jejunal junc ion, h ough he ubing o he C osby capsule, be o e aking he biopsy specimen. The p o ease inhibi o phenylme hyl sulphonyl luo ide (PMSF, Sigma) 100 mM in 95% alcohol (20 ,ul pe ml o aspi a e) was added be o e aliquo ing.16 Se um was ob ained om all pa ien s. Gu la age: he la age luid used was a poly- e hylene glycol (PEG) based elec oly e la age solu ion (Goly ely). A e an o e nigh as , pa ien s d ank his solu ion a a a e o 250 ml e e y 15 minu es o a pe iod o ou hou s, making he o al olume consumed ou li es. Specimen collec ion began once he ma e ial passed pe ec um became liquid, clea , and ee o aecal ma e ial. App oxima ely 200 ml was collec ed and il e ed in o 50 ml polyp opylene ubes; specimens we e cen i uged and ea ed wi h p o ease inhibi o s as desc ibed by Gaspa i e al.'5 All he abo e specimens we e aliquo ed and s o ed a -70°C. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Re e ence ma e ials and epo ing o esul s Fo assays o immunoglobulins in he a ious sec e ions, se ial wo old dilu ions o a s anda d p epa a ion we e used o p oduce a s anda d cu e. Fo example, o IgA assays dilu ions anging om 1250-19-35 ng/ml o a human colos al IgA s anda d (Sigma) we e used in each es un. Se ial dilu ions o es samples ( a ying in ini ial dilu ion depending on he ype o specimen) we e also assayed. Only when he op ical densi y esul s o a leas wo o hese sample dilu ions ell wi hin he ange o he s anda d cu e was he assay conside ed echni- cally sa is ac o y. The IgA con en o he sample was hen de e mined by aking he mean IgA con en o hese wo sample dilu ions. Fo o al IgM and IgG in sec e ions human e e ence se um (P o ein Re e ence Uni , She ield) was used as a s anda d. In he assays o speci ic an ibodies, expe i- men s we e ca ied ou o de ine op imal es condi ions o each an igen, iso ype, and sec e- ion. Se um om a pa ien wi h un ea ed coeliac disease, p e iously ecognised as ha ing high i es o an ibodies o all iso ypes o a wide a ie y o die a y an igens, was used as a e e ence s anda d. The e e ence specimen and es specimens we e s udied a sui able dilu ions, a ying o he di e en assays, and he pla es we e ead when he op ical densi y o he s anda d eached 1-0. Resul s o es specimens a e exp essed as op ical densi y eadings, % o his s anda d. Resul s a e hus exp essed as non- pa ame ic da a; an ibody alues a e no di ec ly p opo ional o he an igen binding capaci y o he sample. This is a ea u e o all such assays. Immunoglobulins (jejunal aspi a e, gu la age luid, and sali a) Assays we e pe o med in 96 well mic o i e ELISA pla es (Dyna ech). All eac an s we e added in olumes o 0-125 ml pe well and all washes we e done h ee imes using saline wi h 0 05% Tween-80 added. Fo he assay o o al IgA, wells we e coa ed wi h 100 ng/ml a ini y pu i ied goa an ihuman IgA (No heas Labs) in 0-1 M ca bona e bu e , pH 9 6, and incuba ed o e nigh a 4°C and washed. A e washing, se ial wo old dilu ions o s anda d and samples (ini ial sample dilu ion 1/100) we e added o he coa ed wells. Pla es we e incuba ed o e nigh a 4°C and washed. Goa an ihuman IgA con- juga ed wi h alkaline phospha ase (No heas Labs) dilu ed (in saline wi h 1% e al cal se um and 0 05% Tween-80) o a p ede e mined op imal alue was added and pla es we e in- cuba ed o h ee hou s a 20°C. A e washing, pa ani ophenylphospha e (PNPP, Sigma) 1 mg/ml in 10% die hanolamine (DEA) bu e , pH 9-8, was added. Pla es we e ead a op ical densi y 405 in an MR580 mic oELISA eade (Dyna ech). The IgA con en o any gi en sample was de e mined as desc ibed abo e. 30 on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om Dissocia ion be ween sys emic and mucosal humo al immune esponses in coeliac disease The me hod used o de e mine o al IgM and IgG in sec e ions was simila ; ini ial sample dilu ion was 1/25. Se um immunoglobulins we e measu ed by au oanalyse using an immuno u bidime ic me hod. Food an ibodies The assay was simila o ha desc ibed abo e. Immulon 2 (129B) ELISA pla es (Dyna ech) we e used. Wells we e coa ed wi h an igen (gliadin, be alac oglobulin and o albumin) a a concen a ion o 5 , g/ml. Be alac oglobulin and o albumin we e supplied by Sigma; gliadin was supplied by D S e an S obel. Re e ence s an- da d and samples we e added in duplica e dilu- ions o he coa ed wells. The ollowing sample dilu ions we e used: se um: 1/100 (IgA and IgM) and 1/200 (IgG); jejunal aspi a e: 1/10; gu la age luid: 1/2; sali a: 1/2. An app op ia e dilu ion o s anda d was included in each assay; hese dilu ions ga e op imal op ical densi y eadings. We es ablished ha hese an ibodies we e speci ic by incuba ing samples wi h he ele an TABLE I Immunoglobulin concen a ions (median ( ange)) in se um (mglml), jejunal aspi a e, gu la age luid, and sali a (Isg/ml) Ig Un ea ed T ea ed Con ol class coeliac pa ien s coeliac pa ien s subjec s Se um IgA 2-4(1-3-3-8) 2-0(0 7-3 3) 1-8(0-9-4-1) IgM 1 1(03-34) 09(0-3-3-1) 1-3(03-4-5) IgG 10-5 (5-6-15-7) 10-7 (5-4-20-1) 9-5 (7-3-15-9) Jejunal aspi a e IgA 290* (25-2282) 140-7 (20-1558) 102-5 (22 9-540 6) IgM 29.8* (1 9-357 9) 9-6 (0-174-4) 4.5 (0-39 5) IgG 23-9* (3-261-8) 11-8 (0-3-271-4) 7-3 (1-2-29-3) Gu la age luid IgA 146-2 (10-7-487-5) 90-8 (8-8-621-8) 139-7 (7 9-403) IgM 17-5* (2-1-100-1) 25.7* (0-192-2) 5 3 (0-37) IgG 2-7 (0-1-34-9) 0-9 (0-1-11-4) 1-0 (0-12-2) Sali a IgA 123-8(48 6-454) 118-1 (32-2-838-2) 165-8 (66-5-606 9) IgM 1-2(0-1-138-9) 1-4(0-1-12-4) 1 1(0-1-3-6) IgG 1-3 (0-25-1) 0-9 (0-1-10-6) 0.5 (0-34-3) *Immunoglobulin concen a ion signi ican ly highe (p<005) han in con ol subjec s. Immunoglobulin concen a ion signi ican ly highe (p<005) han in ea ed coeliac disease pa ien s. TABLE II Se um ood an ibody alues (median ( ange)) Un ea ed T ea ed Con ol Ig coeliac pa ien s coeliac pa ien s subjec s An igen class (n=26) (n=22) (n=28) Gliadin IgA 43-7* (4-6-150) 5-8(0-3-41-9) 5 3(0-44-5) IgM 43 0(19-3-95-4) 38-6 (10-5-87-6) 47-9(16-1-108-8) IgG 82-4* (9-8-137-4) 41-6* (6-8-107-4) 21 (0-105-2) BLG IgA 14.7* (1I 4-150) 6-6*(2-1-87-5) 3-3(0 2-36 4) IgM 33-6(9-125-5) 38-7(11-3-94-6) 27-4 (3 5-82 2) IgG 94.7* (5-150) 70-2 (8-6-150) 30 9 (0-131-8) OVA IgA 18-8* (2-6-150) 14-8 (4 8-606) 11-4 (0 9-35-9) IgM 31-1(4-87) 37-8 (9-5-123-4) 30-0 (5-7-102-6) IgG 64-4 (2-6-150) 63-4* (8-4-150) 27-6 (3-6-150) *An ibody alues signi ican ly highe (p<005) han in con ol subjec s. An ibody alues signi ican ly highe (p<005) han in ea ed coeliac disease pa ien s. BLG=be alac oglobulin; OVA=o albumin. TABLE III Jejunal aspi a e an ibody Un ea ed T ea ed Con ol Ig coeliac pa ien s coeliac pa ien s subjec s An igen class (n=26) (n=22) (n=28) Gliadin IgA 265* (0-7-134-5) 4.0*(03-342) 1-1(0-13-3) IgM 80 5* (0-1-150) 19-6* (2-1-114-4) 1-9(0-11-7) IgG 1-8* (0-23 4) 1-0* (0-4 8) 0-2 (0-2 2) BLG IgA 25.3* (0-6-150) 9-2(1 1-110 9) 50 (0-1-60-8) IgM 16-4* (0-150) 9-2* (1 -1-78- 3) 1-6(0-11-7) IgG 4.5*(0-104) 1-6(0-35-1) 1-0(0-7-7) OVA IgA 15-8* (4-150) 19-6* (1-5-6-5) 4-2 (0-100-5) IgM 18-7* (0-97) 8-4* (0- 1-52.5) 1*1(0-17-7) IgG 1-3* (0-26.2) 1 1(0-11-7) 0 5 (0-26 5) *An ibody alues signi ican ly highe (p<005) han in con ol subjec s. An ibody alues signi ican ly highe (p<0-05) han in ea ed coeliac disease pa ien s. BLG=be alac oglobulin; OVA=o albumin. an igen and showing ha he an ibody was speci ically abso bed ou . In a s udy o 20 samples, he wi hin pla e op ical densi y coe i- cien o a ia ion was 7-3%, and he be ween pla e op ical densi y a ia ion was 11-1%. I he op ical densi ies o he duplica e sample dilu- ions a ied by > 15%, he assay was epea ed. STATISTICAL METHODS Di e ences in an ibody alues and immuno- globulin con en we e analysed using he Mann- Whi ney U es . Fo co ela ions, Spea man's es was used. Resul s IMMUNOGLOBULIN CONCENTRATIONS (TABLE I) Se um No signi ican di e ences we e obse ed. Jejunal aspi a e Un ea ed coeliac disease pa ien s had signi i- can ly highe jejunal aspi a e concen a ions o IgA, IgM, and IgG compa ed wi h con ols, and highe IgM compa ed wi h pa ien s wi h ea ed coeliac disease. Values o ea ed coeliac disease pa ien s we e no signi ican ly di e en om hose o con ol subjec s. Gu la age luid IgM con en was signi ican ly highe in bo h un ea ed and ea ed coeliac disease pa ien s han in con ol subjec s . Sali a No signi ican di e ences we e obse ed. ANTIBODIES TO FOOD PROTEINS Se um (Table II) Un ea ed coeliac disease pa ien s had high alues o se um IgA an igliadin an ibody, wi h alues o ea ed coeliac disease pa ien s simila o con ol alues. High alues o se um IgG an igliadin an ibody we e ound in bo h un- ea ed and ea ed coeliac disease pa ien s, wi h signi ican ly highe alues in he un ea ed pa ien s. The e we e no signi ican di e ences be ween pa ien g oups in alues o se um IgM an ibodies. Pa e ns o se um an ibodies o OVA and be alac oglobulin we e gene ally simila o hose o an igliadin an ibody, as de ailed, wi h s a is ical in o ma ion in Table II. Un ea ed pa ien s had high alues o IgA and IgG an i- be alac oglobulin an ibody and IgA an i- o albumin an ibody. Se um IgA an i-be alac o- globulin and IgG an io albumin an ibody alues we e highe in ea ed pa ien s han in con ol subjec s. Jejunal aspi a e (Table III) The e was e y li le an ibody de ec ed in jejunal 31 on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om O'Mahony, A anz, Ba on, Fe guson Figu e 1:JIejunal aspi a e IgA and IgM an igliadin an ibody alues. (Median an ibody alues shown as ho izon al ba s.) 160 - 140 120 2 100 - 80 s 60 c} a -3 60- z 0 40 20 - 0 IgA p< 0.00001 I p< 0-0002 ' p< 0-002 * * ^ UCD TCD C IgM p < 0.00001 p< 0-005 ' p< 0-00001 . -4- I BP 4- -WY,- 1 _ _ _~~-1 UCD TCD C T ea ed coeliac pa ien s wi h mino jejunal his ological changes T ea ed coeliac pa ien wi h en i ely no mal jejunal his ology I 160 . 140 - 120 - 2 100- Z ! 80- 60 0 40! 20 IgA p<0-002 p < 0-02 p <005 -I- OL _ IgM p < 0-0002 p< 0.05 p < 0-005 sol - - IS!1 0*~- UCD TCD C UCD TCD C Figu e 2: Gu la age luid IgA and IgM an igliadin an ibody alues. (Median an ibody le els shown as ho izon al ba s.) aspi a es om con ol subjec s, bu as de ailed in Table III, o all h ee iso ypes and all h ee an igens s udied, an ibody alues we e signi i- can ly highe in jejunal aspi a es om un ea ed pa ien s han om con ol subjec s (p alues all <002). Fo an igliadin an ibodies, alues in ea ed coeliac pa ien s we e in e media e be ween un ea ed coeliac disease pa ien s and con ol subjec s and signi ican ly di e en om bo h. When an ibody alues in he 11 ea ed pa ien s wi h en i ely no mal jejunal his ology we e compa ed wi h hose in con ol subjec s, IgA an igliadin an ibody alues we e no signi i- can ly highe . Con e sely, IgM an igliadin an i- body alues emained signi ican ly aised (p<O0OO5) in his g oup. An ibodies o be alac o- globulin and o albumin showed a g ea e o e - TABLE IV Gu la age luid an ibody alues Un ea ed T ea ed Con ol Ig coeliac pa ien s coeliac pa ien s subjec s An igen class (n = 15) (n=19) (n=25) Gliadin IgA 53-6* (2-2-137-8) 9-2* (0 4-72 9) 2-2 (0-57 9) IgM 569* (8-150) 17-9* (0-2-150) 3-1 (0-88-7) IgG 0.4* (0-2-19) 0-6 (0-3) 0-2 (0-6-5) BLG IgA 7 9*(1-8-85-5) 8-4(0&6-76 5) 3-5(0-1-71-2) IgM 5-6*(0-14-5) 5.3*(0-53) 0-1(0-22-6) IgG 07(0-21) 04(0-8-1) 04(0-44) OVA IgA 15-0* (3 7-39 3) 14-7* (0 5-46 8) 4-8 (0-76) IgM 6-6*(0 8-33 3) 3-6*(0-46 3) 0 9(0-20 6) IgG 0 3* (0-1-3) 0-0 (0-61-2) 0 0 (0-3 6) *An ibody alues signi ican ly highe (p<0 05) han in con ol subjec s. An ibody alues signi ican ly highe (p<005) han in ea ed coeliac disease pa ien s. TABLE V Sali a y an ibody alues Un ea ed T ea ed Con ol Ig coeliac pa ien s coeliac pa ien s subjec s An igen class (n=26) (n=22) (n=28) Gliadin IgA 5 1* (0-1-29-6) 4-2(0-12-5) 30(0-11-7) IgM 5-1(0-150) 5 4(0-35-9) 2-4(0-15-5) IgG 0.3* (0-17-9) 0-0 (0-4 6) 0-0 (0-7 6) BLG IgA 8-3* (I-9-53-7) 8-3* (0-46.5) 5-3 (0-29 8) IgM 1-0(0-28 3) 1-2(0-14-4) 0-8(0-11-9) IgG 0-4(0-58) 0 0(0-18-8) 0 0(0-2 9) OVA IgA 15-8 (1-9-92-9) 13-2 (06-34) 14-3 (34-30 2) IgM 1-2(0-34 1) 1-4(0-16-7) 1-3(0-13-3) IgG 00(0-13) 0-0(0-3-1) 0-0(0-1-7) *An ibody alues signi ican ly highe (p<005) han in con ol subjec s. An ibody alues signi ican ly highe (p<005) han in ea ed coeliac disease pa ien s. BLG= be alac oglobulin; OVA=o albumin. lap be ween alues in coeliac disease pa ien s and con ol subjec s, bu again high IgM an ibody alues pe sis ed in he ea ed pa ien s. Jejunal aspi a e IgA and IgM an igliadin an ibody alues a e shown in Figu e 1. Gu la age luid (Table IV) As in jejunal aspi a e, high alues o IgA and IgM an ibodies o gliadin we e ound in bo h un ea ed and ea ed coeliac disease pa ien s compa ed wi h con ol subjec s, wi h signi i- can ly highe an ibody alues in he un ea ed compa ed wi h he ea ed pa ien s. High alues o IgA and IgM an ibodies o be alac oglobulin and o albumin we e ound in un ea ed pa ien s; high alues o IgA and IgM an i-o albumin and IgM an i-be alac oglobulin an ibodies pe sis ed in he ea ed coeliac disease pa ien s. Gu la age luid IgA and IgM an igliadin an ibody alues a e shown in Figu e 2. Sali a (Table V) Sali a y an ibody alues we e gene ally low, wi h la ge o e laps beween pa ien g oups. Un- ea ed coeliac disease pa ien s had highe alues o IgA and IgG an igliadin an ibodies compa ed wi h con ol subjec s; only IgA an igliadin an i- body alues we e highe han in he ea ed pa ien s. Highe alues o IgA an i-be alac o- globin an ibody we e ound in bo h un ea ed and ea ed coeliac disease pa ien s compa ed wi h con ol subjec s. SERIAL STUDIES IN COELIAC DISEASE PATIENTS Se en coeliac disease pa ien s who had a clinical and his ological esponse o a glu en ee die we e s udied be o e and a e ea men . Se ial changes in se um IgA an igliadin an ibody and jejunal aspi a e IgA and IgM an igliadin an i- bodies a e shown in Figu e 3. Whe eas se um an ibody alues ell signi ican ly (p<0 05) wi h ea men , he e was no signi ican change in he alues o in es inal an ibody despi e his ological I I s 32 A o on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om Dissocia ion be ween sys emic and mucosal humo al immune esponses in coeliac disease Figu e 3: Se ial changes in alues o se um an igliadin an ibody and jejunal aspi a e IgA and IgM an igliadin an ibodies in 7 coeliac disease pa ien s be o e and a e ea men wi h glu en ee die . ND=no mal die ; GFD=glu en ee die . Se um IgA an i -gliadin 1-5 -o m -n 10 0 -0 Q 05 0- -o 0 15- 1*0- 0-5- Aspi a e IgA an i -gliadin 1 5- 1 0- 0-5- 0 Aspi a e IgM an i -gliadin ND GFD eco e y on a glu en ee die . The e was, in ac , a end owa ds highe alues o IgM an ibody a e ea men . I should be no ed ha hese se en pa ien s had been aking a glu en ee die o six mon hs o less, which may accoun o he ac ha hey beha ed di e en ly om he ea ed coeliac disease pa ien s as a whole, who had signi ican ly lowe jejunal aspi a e an ibody alues compa ed wi h un ea ed coeliac disease pa ien s. CORRELATIONS BETWEEN ANTIBODY VALUES IN JEJUNAL ASPIRATE, GUT LAVAGE FLUID, SERUM, AND SALIVA (TABLE VI) IgA an igliadin an ibody alues we e compa ed in he a ious body luids es ed. In he un ea ed coeliac disease pa ien s, we ound a posi i e co ela ion be ween an ibody alues in se um and jejunal aspi a e ( =0-68, p<0-0001) bu in he ea ed coeliac disease pa ien s his co ela- ion was no main ained as hey had high alues o jejunal aspi a e an ibody, whe eas se um an ibody alues we e low o absen . In he con ol g oup, he e was a posi i e co ela ion ( =0-59, p<0-001) be ween an ibody alues in se um and sali a. No co ela ion was ound be ween sali a y and jejunal aspi a e an ibody alues in any o he h ee g oups. Bo h jejunal aspi a e and gu la age luid had been collec ed om 20 pa ien s ( om all h ee pa ien g oups). A posi i e co ela ion ( =079, p<00l) was ound be ween an ibody alues in jejunal aspi a e and gu la age luid. Discussion In es inal an igliadin an ibodies in coeliac disease pa ien s we e mainly in he IgA and IgM classes, and signi ican amoun s o IgM an ibody pe sis ed in he sec e ions o ea ed coeliac disease pa ien s wi h en i ely no mal jejunal his ology. Sec e ed IgA an ibody was de ec ed bnly in he subg oup o ea ed coeliac disease pa ien s wi h mino his ological abno mali ies, mos o whom had had less han a yea 's ea - men wi h a glu en ee die . I is possible ha he in es inal IgA an igliadin an ibody and mino his ological changes could bo h be due o con- inued inges ion o small amoun s o glu en. Con e sely, in es inal IgM an ibody alues emained highe han in con ol subjec s, e en in pa ien s wi h comple ely no mal jejunal mucosa who had been aking a glu en ee die o some yea s. I is likely ha minu e amoun s o glu en (comple e compliance o a glu en ee die is di icul o achie e in adul s) main ains a local immune esponse a he han a sys emic one. Ou inding o a pe sis en IgM an ibody es- ponse pa allels he inding o a ela i ely high ac ion o IgM plasma cells in ea ed coeliac disease pa ien s.9 We ound high alues o se um IgA and IgG an igliadin an ibody in un ea ed coeliac disease pa ien s. In he ea ed pa ien s, se um IgA an igliadin an ibody alues we e simila o hose in con ols bu se um IgG an igliadin an ibody alues, hough signi ican ly lowe han in he un ea ed coeliac disease pa ien s, emained signi ican ly highe han in con ol subjec s. These indings ag ee wi h hose o o he epo s.'7 18 Concen a ions o IgA, IgM, and IgG we e all high in jejunal aspi a es om un ea ed coeliac disease pa ien s. Coun s o jejunal plasma cells and in i o immunoglobulin p oduc ion in coeliac disease a e highe han in con ol subjec s o all iso ypes,'0 suppo ing he iew ha he immunoglobulins (a leas IgA and IgM) in coeliac in es inal sec e ions a e p oduced locally. TABLE VI Co ela ion be ween IgA an igliadin an ibody alues in se um, jejunal aspi a e, and sali a Se um and Se um and Sali a and G oup sali a aspi a e aspi a e Un ea ed coeliac disease =0 021 =0-68 =0-293 (n=26) (NS) (p<0-0001) (NS) T ea ed coeliac disease =0-392 =0-04 =0-323 (n=22) (NS) (NS) (NS) Con olsubjec s -0 593 = -0 193 =0-071 (n=28) (p<0 001) (NS) (NS) NS =no signi ican . 33 on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om 34 O'Mahony, A anz, Ba on, Fe guson I is likely ha mos o he jejunal IgG is plasma de i ed as he numbe s o IgG sec e ing plasma cells a e low e en in un ea ed coeliac disease.' We should poin ou ha immunoglobulin measu ed in jejunal aspi a e was o al immuno- globulin and no speci ic sec e o y IgA and IgM. I is possible ha a leas some o he jejunal IgA and IgM is se um de i ed (coeliac disease is a p o ein losing diso de ). We a e cu en ly cha ac e ising jejunal immunoglobulins and an ibody in e ms o molecula weigh and pe cen age o o al Ig which con ains a sec e o y componen . An inc ease in he in es inal luid immunoglobulin con en was no accompanied by equi alen changes in se um immuno- globulins; in ac , he se um IgM con en was no iceably low in some un ea ed coeliac disease pa ien s. We ha e no ye assessed he con ibu ion o speci ic an igliadin an ibody o he inc ease in in es inal immunoglobulin con en in coeliac disease. Falchuk and S obe ,'9 using an a ini y ch oma og aphy echnique, epo ed ha app oxima ely hal o he ne inc ease in IgA and IgM syn hesis (in an in i o model o glu en challenge) was due o syn hesis o an igliadin an ibody. Con e sely, in a mo e ecen epo o in i o sec e ion o an igliadin an ibody by coeliac jejunal mucosal biopsy specimens,20 Cicli e a e al calcula ed ha an igliadin an ibody accoun ed o 2 1%, 12- 1%, and 4 1% o he o al concen a ions o IgA, IgM, and IgG espec i ely. In any e en , enhanced in es inal an ibody p oduc ion in coeliac disease is no limi ed o glu en de i ed p o eins: we ound high alues o in es inal an ibody o be alac oglobulin and o al- bumin in un ea ed coeliac disease pa ien s, wi h pe sis ence o IgM an ibody o hese p o eins in ea ed pa ien s. An ibodies o hese ood p o- eins we e less speci ic o coeliac disease han an igliadin an ibodies. I has been sugges ed ha high alues o se um an ibody o hese p o eins in un ea ed coeliac disease is simply he esul o inc eased in es inal pe meabili y o an igens. Al hough ELISA is now accep ed as he s anda d assay echnique o measu ing an i- bodies o ood p o eins in coeliac disease, se e al di e en ELISA me hods ha e been desc ibed, and di e en e e ence p epa a ions a e used by each g oup o in es iga o s. The me hod we used is essen ially simila o ha o Sa ilah i e al3 and we used c ude gliadin as an igen o he ELISA ( a he han say, alphagliadin), as Ske i e al2' ha e shown ha se a and in es inal aspi a es om coeliac pa ien s con ain an ibodies which bind o se e al di e en gliadin subuni s. We ha e no asc ibed le els o 'posi i i y' o 'nega- i i y' o an ibody alues; such a bi a y designa- ions a e o some alue in sc eening es s used in clinical p ac ice bu no in p ospec i e esea ch in es iga ions. Al hough un ea ed coeliac disease pa ien s had s a is ically highe alues o sali a y IgA and IgG an igliadin an ibodies compa ed wi h con- ols, sali a y an ibodies we e gene ally low wi h a la ge o e lap be ween pa ien s and con ols. Fu he mo e, he e was no co ela ion be ween jejunal aspi a e and sali a y an ibody alues. Ou esul s do no sugges ha an ibody es s on sali a ha e any diagnos ic o sc eening po en ial. On he o he hand, gu la age o e s a ela i ely non-in asi e al e na i e o in uba ion o he collec ion o in es inal sec e ions o immunoglobulin and an ibody s udies. An i- body indings in gu la age luid we e b oadly simila o hose in jejunal aspi a e, wi h a posi i e co ela ion o IgA an igliadin an ibody alues in hose pa ien s s udied by bo h echniques. Immunoglobulin concen a ions, howe e , di e ed conside ably in jejunal aspi a e and gu la age luid; all h ee immunoglobulin iso ypes we e aised in jejunal aspi a e in he un ea ed coeliac disease pa ien s, whe eas only IgM was signi ican ly aised in gu la age luid. Gu la age luid is likely o con ain sec e ions om no only he small bowel bu also gas ic juice, bile, panc ea ic sec e ions, and colonic sec e ions. In his espec , i is no a homogeneous luid, unlike jejunal aspi a e, which e lec s e en s in he jejunum only, and is hus mo e likely o ep esen accu a ely local immune phenomena in coeliac disease. Fluid low a e is ano he ac o ha may in luence Ig alues in exoc ine sec e ions; his may pa ly accoun o di - e ences in indings in jejunal aspi a e and gu la age luid. The ele ance o glu en eac i e in es inal B cells and an ibodies o he pa hogenesis o coeliac disease is unce ain; he p esence o an ibodies o glu en de i ed p o eins in pa ien s wi h coeliac disease may be me ely an epiphe- nomenon in he con ex o a T cell media ed en e opa hy wi h expansion o he ele an popula ions o T helpe as well as e ec o cells.22 I is ce ainly possible ha mucosal IgM an i- body is immunopa hogenic, o example by ixing complemen in he immedia e icini y o en e ocy es. This s udy shows he dissocia ion o sys emic and in es inal humo al immune esponses in pa ien s wi h coeliac disease, and is e idence ha o he s udy o immunopa hology o in es inal disease, di ec in es iga ion o he gu is manda o y. M s J Johns one and M N Ande son p o ided echnical assis- ance. 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Rele ance o in es inal mucosal T-cells in he immunology o coeliac disease and ood alle gy. In: Chadwick VS, ed. Mechanisms o gas o- zn es inal in lamma ion. Welwyn: Smi h Kline and F ench, 1984: 85-7. 6 Ma sh MN. Func ional and s uc u al aspec s o he epi helial lymphocy e, wi h implica ions o coeliac disease and opical sp ue. Scand3 Gas oen e ol 1985; 20 (suppl 114): 7 Fe guson A, MacDonald TT, McClu e JP, Holden RJ. Cell- media ed immuni y o gliadin wi hin he small-in es inal mucosa in coeliac disease. Lance 1975; i: 895-7. 8 Bullen AW, Losowsky MS. Cell-media ed immuni y o glu en ac ion III in adul coeliac disease. Gu 1978; 19: 126-31. on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om Dissocia ion be ween sys emic and mucosal humo al immune esponses in coeliac disease 35 9 Baklien K, B and zaeg P, Fausa 0. 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Sec e ion o gliadin an ibody by coeliac jejunal mucosal biopsies cul u ed in i o. Clin Exp Immunol 1986; 64: 119-24. 21 Ske i JH, Johnson RB, He zel PAS, Lab ooy JT, Shea man DJC, Da idson GP. Va ia ion o se um and in es inal glu en an ibody speci ica ions in coeliac disease. Clin Exp Immunol 1987; 68: 189-99. 22 Ma sh MN. Immunocy es, en e ocy es and he lamina p o- p ia: an immunopa hological amewo k o coeliac disease. J R Coll Physicians Lond 1983; 17: 205-12. on Sep embe 1, 2020 a Uni e sidad de Valladolid. P o ec ed by copy igh .h p://gu .bmj.com/Gu : i s published as 10.1136/gu .32.1.29 on 1 Janua y 1991. Downloaded om