A simple method for determining polymeric IgA-containing immune complexes

Journal ol Immunolr,gt( al Methods. 60 ! 1983 ) 305- 317 Elsevier

305

A Simple Method for Determining Polymeric IgA-Containing Immune Complexes Jaime Sancho, Jesfis Egido ~ and Eva Gonz,ftlez Sert'tcto de Nelrologia. Fundacton Jtmenez Diaz ~L'ntt'erstdad Aut6nomaL .4 t'd~t. Reve~ (-atohco~ 2. .'qadrtd 3. Spatn I Recel,.ed 20 Ju[,. 1982. accepted 10 January 1983~

A simplified a~sa} to measure polymeric IgA-immune complexes m biological fluid~ t,,, described. The assay is based upon the specdtc binding of a secretory component for pol),meric Ig~X. In the firq step, m u l h m e n c IgA (monomertc and polymeric) immune complexes are determined by the standard Ral~ cell assa}. Secondly. labeled secretoD component added to the assay is bound to pobmeric lgA-Jmmune complexes pre~iousb fixed to RaJt cells, but not to monomeric IgA immune complexes. To a~o~d false positives due to possible complement-fixing IgM immune comple,,es, prior IgM immunoadsorption ~ performed. Using anti-lgM antiserum coupled to CNBr-acuvated Sepharose 4B thl~ step is not tmle-consuming. Polymeric IgA has a Iov, affinity constant and brads v, eakly to Raji cells, as Scatchard analysis of the data shows. Thus. polymeric IgA immune complexes do not brad to Raji cells directly through Fc receptors, but through complement breakdov, n products, as ",,,ith I~G-mmmne complexes U~mg thts method, we ha',e been successful in detecting specd~c pol}menc-lg~X tmmune complexes m patlent~ ~ith IgA nephropathy (Berger'~ disease) and alcohohc h~er d~sease, as ,xell as m normal ~ubjects after meal., of high protein content. This nev,. simple, rapid and reproducible assa~ might help to stud} the physlopathological role of pol,,menc IgA immune complexe~ m humans and animals. Kes v.ords: IgA polvmert( tmmune ~ o m p l e x e s - Ralt tell assa~--I.g.4 nephropathy--ah'ohohc dt tease

ht'er

Introduction Recentl~ techniques for detection of igA-containing immune complexes (Levinsky and Soothill, 1977; Penner et al., 1978: Hall et al., 1980: Kauffman et al., 1980) have been used to show the presence of IgA immune complexes in a variety of diseases such as dermatitis herpetiformis (Hall et al., 1980), Henoch-Sch0nlein purpura (Levinsky and Barratt, 1979: Hall et al.. 1980, Kauffman et al., 1980), alcoholic liver disease (Penner et al., 1978) and idiopathic glomerulonephritis (Cairns et al., 1982). However, the techniques available do not distinguish between polymeric

To ~hom correspondence and requests for reprints should be sent. 0022-1759,83, $03.00 '~, 1983 Elsevier Science Publishers B.V.

and monomer~c IgA immt.ne complexe,, and the pathogenet~c r,~lc ,4 th¢,c 2 cl,t-.,c, of ~mmune comple\e,~ remains, unexplored. in an experimental model of lgA nephropath?., the e\p, tcnce ,,f p,,l,,mcnc I&A-contaimng immune complexe.,, ~a~, o,m,idered critical for the inducDon ,~1 nephritic histological changes. In coqtra.,,t, monomeric I&-~ immune c~mlph.'\c, f.~lle,,t to produce glomerular deposits (Rifai et al.. 1979). Our recent demon,tr,flu)n ,)1 high le`.els of polymeric lgA iq the serum and kidne,,,, of patient, ~ith primart, [g~ nephropath3 IBerger',, di.,,ease)(L6pez-Trasca,a et al.. IgS0). Hen,)ch Schd, nlcln .,xndrome tEgido el al.. Igg0). and alcoholic lixer di,,ea,,e (Sancho c t a l . . I~,)S2) .,uggested that this might also be true in human,. In .,,tudie,, with ,ucrose den.,it,, gra&ent ultracentnfugation pol~,menc IgA-conraining immt.ne complexe., ~`.ere found 1.1 patient,, ~`.ith alcoh~qic h~cr d~,ea-e ISancho et al.. 1992). Howe~er. tin,, technique p, not appropriate I,,r large ,tale .,,tudie, and c,ther simpler a,,a\~, are needed. VCe ha~e de\eloped a ,,imple and reproducible technique capable of detecting polsmeric I&-k immune comple\e~. The a,,,.,ax i~ ba,,ed upon the ,,pecific binding affinit,, of secretory component for pol 5 merit Ig.-k. In the fir.,,t ,,tep. IgA ~m,,nomeric and p o l s m e r i c ) i m m u n e complexe,, are determined bx the ,tandard Ra.li cell ,~,,a', adapted for the lgA i,,ot?,pe. Then. after immunoadsorpt~o.~ of IgM th,tt nnght interfere in the test. labeled ,ecretor3 component IS(')i,~ added to the a,,,a\. Thi, SC bind.,, to the polsmeric lgA immune comple\es pre'`lOu,,l) fixed on Rail cell, With thp, procedure, po13 mer~c igA ~mnmne complexe, `.,,ere detected in patient., xx~th IgA nephropath~, or alcoholic li'`er disease, a,, well a., in normal ,ubject, after meal,, s~~th a high protein content.

Material and l~lethods Human .sera

Sera from 21 health~ adult.,,. 23 pauents `.~ith IgA nephropath?, t Berger', di.,,e,t,e~ and 7 pat.ents '`~ith alcoholic li'`er disease
SC wa_, isolated from '``.he,,' by affinit~ chromatograph 3 on IgM-Sepharo,e lUnderdov, n et al.. 19771. Briefly. the IgM-Sepharose ,..,as .ncubated for 2 h at 4:'(. v,'ith diluted vchey (6-16 mg I g M / m l of neat whey). The mixture ~a,, poured a:, a chromatograph~ column and ~`.ashed extensively u, ith phosphate-buffered _,,aline ( P B S ) to remo','e u n a d s o r b e d p r o t e i n . T h e adsorbed protein ,.,,'as eluted x`.ith 1.0 M KSCN. 0.1 M potassium phosphate, pH 7.0. dialyzed against PBS and filtered on an Uhrogel AcA34 column. Two peaks '`'`'ere obtained, the second containing SC. The resuhing material gave one line b~ immunoelectrophoresis against a.m,~erum to human whole milk proteins (Nordic Laboratories, Tilburgl and one band b,, SDS-PAGE, showing minimal lactoferrin contamination b~ double immuuodJffu_-,ion anal.~ .,,is.

307

lsohaion of polymeric human lgA lgA myeloma serum containing a high level of pol3meric lgA was diluted I/"2 ~ith PBS and precipitated with 50% (NH,~)_,SO4 ( ~ / v l . The precipitated proteins were centrifuged, redissol~ed and dial3zed against 0.015 M phosphate buffer, pH 7.5. The protein solution was chromatographed on a DEAE-cellulose column (DE-52 Whatman) equilibrated with the same buffer. Unbound protein, mostl3 of IgG class, ~as discarded. Monomeric lgA was eluted ~ith 0.5 M phosphate buffer, pH 7.0 and polynaeric IgA b3 gradient fractionation using 0.05 M phosphate to 0.2 M NaCI, in 0.05 M phosphate buffer, pH 7.0. Protein solutions ~ere thereafter dialyzed against PBS, concentrated and chromatographed on a column ~2.6 x 70 cm: Pharmacia. Upp_,,ala) of Llltrogel AcA22 ~LKB lnstrunaent,,, Ltd.. Bromma). The 2 purified preparations were concentrated at 10 nag., ml and analyzed by immunoelectropboresis. The molecular weights of pol.,,meric and monomeric lgA ~ere estimated by gel filtration on Ultrogel AcA22 or AcA34 columns previously calibrated with marker proteins. .,-I t l l l 3 e r a

Rabbit antisera to whole human serum proteins and monospecific antisera for the hea~3 chain of human IgM were purchased from Operon IZaragoza). Antisera to human lgA (specific for a-chainl, secretory component and lactoferrin ~ere purchased from Behring Diagnostics I F . R . G . ) o r Dakopatts ISweden). Anti-IgA a a s tested for immunospecificity by immunoelectrophoresis and Ouchterlony double diffusion against human lgG, lgM. lgA, Bence Jones kappa and lambda, and free SC. A single precipitation line ~as observed only ~ith lgA. To further ensure specificit 3 at the level required for Raji cell assay, cross-reactivity ~ith IgG and lgM was tested by radioimmunoassav. Non-specific binding was alwa3s less than 10~.

lgM mmmnoadsorption Rabbit "y-globulin fractions, obtained b~ 50% ammonium sulfate precipitation, of an antiserum to human lgM ispecific for ~ chain) (Operon. Zaragoza) were co~alentl.~ coupled to CNBr-activated Sepharose 4B (Cuatrecasas and Anfinsen, 19711 at a concentration of 10-15 nag of protein per g of dry gel. The cross-reacti~it~ of anti-lgM Sepharose 4B immunoadsorbent was tested with labeled lgG, lgA, lgM and free SC at different concentrations. The non-specific binding ~as less than 10~. This immunoadsorbentWas stored at 4°C at a concentration of 200 nag Sepharose/ml of PBS. FiR3 ~1 of protein-Sepharose conjugate were added to an equal volume of serial dilutions of pooled normal sera. The mixture was incubated in I 1 mm x 70 mm polystyrene tubes at room temperature for 2 h with gentle stirring and centrifuged at 200 x g for 5 min at room temperature. The supernatant was analyzed by double immunodiffusion against a rabbit antiserum to human lgM. Under these conditions (I :2 diluted sera) all lgM from the pooled normal sera (150 m g / d l ) was adsorbed. To be sure that even in patients with elevated levels of lgM this immunoglobulin was completely adsorbed, in further assays a I: 10 dilution of serum was employed. The amount of immunoadsorbent employed (I mg of protein

Sepharose conJugate) ``~a,~ capable of binding 55 i.~g of IgM. 3-fold tugher than the upper limit v a l u e o f lgM in the pooled normal ~,erumlrange 12 I.'7, ggl.

Radtohtbehtsg A n t i i h u m a n l g ~ Ispecific for ~-challll rabbit lgG x``a, labeled ~ l t h J ' : l b,, the c h l o r a m i n e - [ m e t h o d ( M c ( ' o n a h e ) and Di\on, 1966} to a ,,pecific acuxit', of u.3 I).5 p.Ci/.~8. Free secretor\ c o m p o n e n t (S('} x'`a, labeled b,, the , o h d - p h a , e l a c t o p e r o x i d a s e method ( H u b b a r d and Cohn. 19721 to a specific acm~t', of Q.5 I p.Ci p.g. Both protein., ``~ere diluted x'`ith I).15 M T n s - b u f f e r e d ,,ahne. p H "7..4. '`~ith i).5°~ BSA at a c o n c e n t r a t i o n of 100 ,ug ml and stored at 4-'(" until u,ed.

]tllDlltllOaX,~lF ~I'{]( cdlo'e3 Since Raji cells nlight attach pol)menc IgA b~ a h)pothetical Fc receptor ,pecJfic for that isotype. '`~e studied the binding and affinit,, of pol)meric l~-k to the.,,e cell, b,, the a.,,sa,, described b,, Segal and Hur``~itz (19"77) v, lth some modification.,,. Bnell',. 2 ," I0" cells in minimum essential medium containing 0.5r'; BSA ~ M E M BSA} ``'`ere added to II mm x. "70 mm poly~t,,rene tubes containing radiolabeled polymeric lgA with and v.Jthout excess of unlabeled protein. The mixture~ x``ere incubated for 4.5 rain at 37°C. After incubation, the contents of each tube uere centrifuged and v, ashed 3 times with MEIM-BSA, and the radioactp.']t'~ in the cell pellet determined in a gamma counter. Non-~pecific binding x``as determined from the ~amples containing excess unlabeled polymeric lgA. Scatchard ( 1949} plot, ``~ere constructed from the data to calculate the apparent association constant. Correction for non-specific binding in Scatchard plot~ ``'`as made according to Chamnes,, and M c G u i r e (19751. C i r c u l a t i n g m u h i m e r i c ( m o n o m e r i c and pol)meric} i&@ i m m u n e c o m p l e \ e , , (l(.} '`'`ere d e t e r m i n e d b,, the Raji cell r a d i o i m m u n e a,~a~ ~Thenfilopoulo, and Dixon. ] 9761 as modified b~ Hall et al. I 19801. Briefl,,. 2 x 10 ~' Raft cell~ in 51)/.LI of I M EM ) ````ere incubated 145 rain. 370( "} with 25 p.I of the test >erum diluted I: 4 in ,,aline. A f t e r 3 ',,.ashes m M E M , a previously s t a n d a r d i z e d a m o u n t of LZ~l-radiolabeled fraction of a n t i - h u m a n lgA { - 21) p.g) in M E M - B S A ````a~ added, and incubation t~a~ c o n t i n u e d for 30 rain at 4 o ( ". The cells ``,,ere then ``,,ashed and the radJoactix it,, in the cell pellet u a s d e t e r m i n e d in a g a m m a counter. Specific p o b m e r i c IgA IC ``'`'ere d e t e r m i n e d a,, follows: 2 x IU ~ Rail cell,; t~ere mixed 145 rain, 37"C1 with 25 gl of the tebt serum previousl) lgM a d s o r b e d as d e s c r i b e d above. After 3 ````ashes in M E M - B S A , a s t a n d a r d a m o u n t of I2"l-labeled SC, previously d e t e r n u n e d . ``~as a d d e d and incubation ````as c o n t i n u e d for 2 h at room temperature. The cells u e r e then u a s h e d and the radioacti``ih in the cell pellet d e t e r m i n e d in a g a m m a counter. The results of IC assa) ````ere expressed a c c o r d i n g to the statistical anal)si~ e m p l o ) e d by Hall et al. (1980). p a r t i a l l y modified. Sera from 21 normal subjects were used to d e t e r m i n e the 95% c o n f i d e n c e limits for normal serum in each assay. In o r d e r to o b v i a t e the d a y - t o - d a ) variation in the binding of control sera, 4 s t a n d a r d control sera, selected to represent statistically the d i s t r i b u t i o n of the total control group, were tested in each d a y ' s assa~,, ( W o o d r o f f e et al.. 19771. The u p p e r 95%.

309 confidence limit (Ui) was calculated by means of the formula: Ui = Ci + 2.2627~,,'SE~ + SE~ where Ci = mean cpm bound in control group, S E = standard error of control group, SEp = standard error of the test sera. The t value ~as 2.2627 ( P = 0.05) for the 9 degrees of freedom in the analysis. The mean of the duplicate values of the test sera (Ti) ~as then divided b3 Ui. If this value T i / U i was greater than I, the test serum ~as judged abnormal.

Sucrose gradient fractionation Three sera from selected patients with alcoholic liver disease were fractionated by linear (5-40%) sucrose density gradient in Tris-buffered saline (TBS), pH 7.4 and gl3cine-HCI 0.1 M, pH 2.8, as described (Lopez-Trascasa et al., 1980). Fractions of 200 #1 were collected by piercing the tubes from below, and these were assayed for the presence of IgA bv double antibod~ radioimmunoassay (L6pez-Trascasa et al., 1980). The percentage and absolute values of dissociable serum IgA at pH 2.8, as well as the presence of true polymeric lgA IC, were calculated as previousl 3 described (Sancho et al., 1982).

Results

Spec~l'ic binding of polymeric lgA to Raji cells Fig. I (left) shows the anaount of polymeric lgrA bound to Raji cells. The binding is non-specific because Raji cells bind labeled polymeric IgA to about the same extent in the presence or absence of a large excess (I-1000-fold excess) of cold protein (data not shown). The results were confirmed by Scatchard plot of the data (Fig. 1, right). The asymptote corresponding to non-specific binding was subtracted from the measured binding according to Chamness and McGuire (1975) and non-specific binding accounted for all the radioactivity.

Binding spec(ficitv of SC to Raji cells The binding specificity of SC to Raji cells was studied in the same way. As seen in Fig. 2, SC was poorly bound to Raji cells because by Scatchard plots the asymptote corresponding to non-specific binding was higher than the specific binding (Fig. 2, right).

Effect o[ polymeric lgA on binding of ':~l-labeled SC to Raft cells As sho~n in Fig. 3, the incubation of Raji cells with various concentrations of polymeric lgA did not increase significantly the uptake of ~-'Sl-labeled SC by Raji cells over that observed with buffer alone confirming the inefficient binding of polymeric lgA observed in Fig. 1.

?.Ill

10

5

8~ b

E.6 ~2-

~4 o

I~mr 1 . . . . . . . . . .

2

6

r~C

1

10 1/. 18 C0r~c (M).10 -8

3

5

7

r(.10-19M)

F~g ] Binding ol p~l~,merJc Ig.k to Rail cell,, at 37':( (lel'tl Binding a,,,a,.~ ~ere carried out ~tl~ medL~ containing 0.05q azide t,.~ mimm~ze mternahzau<,n. Scatch,,rd pl,,t fright) [ h e h m m n g r c r.,th, ..~.,, m u h i p h e d b,, the free hgand concentration I,..'p at each p,~mt t,~ determine non-,peofic binding. The, ~,,, then subtracted from the mea_,,ured total b m d m g r t<, find the ,pet[tic bmdm~ r r In t h ~ , , J , c r r ~1~

4 o'~

"5"

03 ¸

~2

02

rh~ll

-6

"

~

E 0 L

i

3

-5

.,

7

C0nc {M), 10-12

2

3

r{'10 -19)

Fig. 2. Binding of secretor~ c o m p o n e n t to Rajt cells at 37:L" tleft). Scatchard plot qrtght) A.,.,a'.,, ~ere performed as m Fig I. The specific binding r,v '~a', 0 at each point after .,ubtr,Jctmg the non-,.pec[fl~: binding.

Opttmal concentratum oI ": ~l-laheled S C The optimal

concentration

first calculated

",'5

S

'

~"

theoreticall',

of ]-"l-labeled

SC for use m the SC binding

In the sera studied,

the maximal

amount

a~,~,a', v.a-,

<,f Ic,.~ I(" ~ a , ,

;

e

,

' 12 5

r

"~

,

SI",

1C(,

plgA ~,,J~edllJg I

Fig. 3 Effect of e,,cess pol>mertc [gA on bindm8 of ~Z~l-labeled SC to Rajt cells. Each point represent', D. ,,f the Ra.II uptake ,,1

mean + S.D. for 2 experiments. Shaded area L, the range ,,f le,.els bet,aeen 2 S i _"~ I-labeled SC v. ith<,ut pre'. tou.~ incubation v, ith pol~, merle Ig.M

311

about 7 mg,'ml (Table I). If all these immune complexes were formed by polymeric IgA, they ~ould represent 17.5 p,g polymeric lgA at the dilution employed. Therefore the dose of ~25I-labeled SC needed to obtain optimal binding (molar ratio SC: lgA = I : 7.5) (Brandtzaeg, 1974) ~ould be 455 ng. Since in prelimary studies we ne~er obtained SC binding by Raji cells higher than 80 ng of ~2-~l-labeled SC, in further experiments 300 ng of ~2-~l-labeled SC were used. The effect of serum dilution on the binding of ;-Vl-&heled SC to Rajt cells 1-he uptake of ~-'Sl-labeled SC (300 ng) by Raft cells is sho~n in Fig. 4. It is ex ident that the amount of x25I-[abeled SC taken up by the cells was directly related to the anaount of serum present. Therefore. these results show that Raji cells can be used efficientlx for the detection of immune complexes with affinity for SC. The e~l]~'ct of IgM immunoadsorption ./horn sera on the binding o1 SC to Raft ceils In preliminary experiments significant SC binding to Raji cells was observed in ~erum from some patients, that had no multimeric lgA IC binding capacity for SC. Taking into account that it could be due to the presence of IgM IC, we studied the SC binding assa~ to Raji cells (SC-BA) in sera depleted or not of lgM..,ks sho~vn in Fig. 5 (top) prior to lgM adsorption (SC-BA: lgM ÷) 6 out of 8 sera lacked muhimeric lgA IC (IgA-BA) but showed positive SC binding (Ti/Ui/> I), that became negative ( T i / U i < I) after IgM adsorption (SC-BA: lgM-). Furthermore. the positive SC binding in 2 sera ~ith multimeric lgA IC (IgA-BA) became negative after lgM adsorption, which indicates the presence in these sera of both monomeric lgA IC and IgM IC. In the other 10 sera, with or without lgA IC (Fig. 5, bottomk there ssas no change in the SC binding assay (SC-BA) after lgM depletion presumabl,, because of the absence of lgM 1C. These results suggest the presence of IgM IC in some patients ~sith lgA nephropaTABLE I P O L Y M E R I C IgA IMMLrNE COMPLEXES IN PATIENTS WITH ALCOHOLIC LIVER C I R R H O SIS Pauents

SC-Rajt assa?,

lga~-Raji assay

IgA(9-13S~ h

E.J. E.M. A.R. M.'F. R.M. SG

1.06 " 1.54 0.80 0.91 0.70 0.86

1.08 1.01 0.86 0 76 0.90 I.'70

7.14 4.10 0.03 N.D. ~ N.D. N.D.

Ti Lh rauO. representmg the cpm L2Sl-labeled SC bound to Raji cells incubated v,'tth the patient's >erum d~slded by the upper limit of confidence obtained wtth 4 normal human sera. The result ~as considered positive if Ti ,'Lil ~ I. b Concentration m rag. ml of I~A ~mmune complexes detected bs sucrose gradtent ultracentrifugation after treatment of the samples at pH 2.80, alues found in normal subJeCtS: 0.03+_001 mg 'mlp. • N.D." not detern'uned.

SC-BA ~,#A.BA ~gM* IgM:-

16.,t ......

P <000, ,5

J 08 .

.

.

.

.

.

.

(J

r~s 1

~-0~ 1'-08

.....

I~

18 Set ij rr,

lit [,iI u t ,or,

F~g 4 Raji cell uptake of ~;'[-laheled S(" incubated ~,.~th dd'ferent ddut~,m, of 2 dH'l'erent ,.era that had been pre',w, usl'. depleted ol Ig,M Each p,,;tnt repre~,ent- the mean __+.";.D. ,,1' duph,.:ate ,ample, Fig.. 5 Effect of IgM mm]unoadsorpuon of ,,era on the binding ol ~-'~l-labeled S(" to Rail cell, Top. T',~.o patient, ,.,,ith a pc,~m~.e ITi Lh . I) IgA.B.A tbmding a,-,,a,,p and SC-BA II~[k]+~ became neg.atl~e tTi til .¢- II after I~M mmmnoad,,orphc.n. The ,,ame occurred m 6 other pauent> '.~lth po-Jti~e S(-Ig.M * '.~.llhoul [g,'~-I(.. Tht, experm~ent t,, m fa'.or of the e'.,i,,tence of IgM ~mmune complexes, m the,¢ patient, Bottom: t'.~.o patient,, vqth po~ltl'.e lgA-BA remmmng po>Ht'.e after IgM mm~unc.ad,,orpt~on .-Xnother patient ha, onl,. monomenc Ig.A in)murte complo.e,

t h \ . a n d ~ndicate a r e q u i r e m e n t for l g M a d s o r p t i o n b e f o r e the e x i s t e n c e o f s p e c i f i c p o l y m e r i c I~A i m m u n e complexe,,, c a n b e a c c e p t e d .

Reprmhwtbtlitv . t the pol.wnertc !~.4-1C a~sav W h e n q u a d r u p l i c a t e s a m p l e s o f 7 p o s i t i v e .,,era ~ e r e t e s t e d o n t h e >ame d a x , t h e i n t r a - a ~ a ~ r e p r o d u c i b i l i t 3 e x p r e s s e d a~ t h e s t a n d a r d d e v i a t i o n d i d n o t e x c e e d 2 15% ( m e a n 7.4%). W h e n t h e s a m e sera v, ere t e s t e d c o n s e c u t i v e [ , , for 3 da,,s t h e inter-assay ~ariabilit~ ~a~0.1 12% ( m e a n 5 , 5 ~ ) . N o s i g n i f i c a n t e f f e c t o n I(5" le~el~ v, as o b s e r v e d a f t e r 3 c 3 c l e s o f f r e e z i n g ~ - 7 0 ° C ) a n d t h a w i n g ( 2 2 ° C L F o r d u p l i c a t e s a m p l e s , t h e s t a n d a r d d e v i a t i o n o f the m e a n d i d n o t e x c e e d 15%.

Detection ql polymeric l.g,-1 IC in the serum of patients with lgA nephropathy m" al¢ohoh¢ hrer (hsease (.4 LD) S i n c e Raji cells efficientb,, b i n d c o m p l e m e n t - f i x i n g m u l t i m e r i c IgA IC ( H a l l et al., 1980) a n d IC w i t h a f f i n i t y to SC d u e to the p r e s e n c e o f p o l y m e r i c IgA o r I g M . t h e s e

313

cells were used in a radioimmunoassay system for the detection of circulating polymeric IgA IC in humans (Fig. 6). Accordingly, cells were incubated with test sera previously depleted of IgM and then reacted with an optimal amount of either radioactive antibody to human IgA or ~2-~I-labeled SC. Subsequent uptake was determined and compared with the uptake for 4 healthy volunteers" sera. Only sera exceeding the 95% confidence limits for normal sera in the 2 assays were considered positive. In IgA nephropathy (Berger's disease) the Raji cell assa'< detected multimeric IgA IC in 12 out of 23 patients (Fig. 7). Seven of these 12 patients also had IC with affiniw to SC. Because the IgM had been previously adsorbed, these results distinguish the presence of true polymeric lgA IC. To demonstrate further the specificity of the assay, we determined the [2s I-labeled SC uptake by cells incubated with sera from patients with ALD containing high lexels of polymeric IgA IC detected by sucrose density gradient uhracentrifugation (Sancho et al., 1982). As shown in Table 1, EJ and EM sera ~ere positive in both the IgA-Raji and SC-Raji assays. A third serum (AR) seemed to have a positive SC-Raji CONTROLS n:.,

1.8

IgA NEPHEq~'JPATHY n::, 7,, 12 = 58"•.

1.6 14 1.2 oplgZ,- ¢:

b

1.0 ', CELL J

,_~s,_~,g. 3~'C

,'" h . . . . ~ ,

.'d RA.,, '~

~,-s,: 2,, RT \ sc . c

scJ R.~.,, 1,sc

X:> L "~' IgA-B,nClw,g ASso~

08

0.6 SC SC-B,ncl,ng Asso?

sc4~ ",gA'4~'sc-~ 'IgA~

Fig. 6. Schematic representation companng (he Raj, IgA (muldmeric lgA IC) and the Raji SC (pol~mer,c IgA IC) radioimmunoassa',. (C') pol,,meric IgA IC; ( r ) nluhimeric IgA IC: ( 1 ) 1:51-labeled rabbit an(~-human IgA; (SC) secreto~ component F,g. 7. Levels of multlmene and pol)meric IgA immune complexes in sera of patients v.ith lgA nephropathy (Berger's disease) and in normal controls after ,mmunoadsorption of IgM. Values greater (hart 1.0 are abnormal; SC-BA = secretor>, component binding assay (pobmenc IgA ,mmune complexes). Twelve out of 23 pat,ents v.ith IgA nephropathy had multimeric IC (IgA-BA): 7 out of 12 pat,ents had polymenc IC.

: i ..I a,>a\ tTi Lli = 1.21 and negative lgA-Rajl a,,ax and I g A - I ( detected t+,, ultracentr,ft.gauon. Ho~ever. the binding t+f labeled S ( to Rail cell, decrea,ed t,, normal after ad,orptlcm of lgM. ~TI Lii=0.14~) indicating that the patient had IgM I( Another serum t g o ~ txa> positive in the IgA-Rall ,t>,ax but n o t ill the .q(-Rall a , , u \ . suggesting the pre~ence of montm~erlc I g A - l ( . Tt~o other patient, had neither le-\ nor SC-I(" b; the Rali cell a,>ax,.

Discussion Despite the e\istence of a large number of non-specific a s , a \ , for immune complexes, it is only recentl~ that methods for detecting lgA imnlune c o m p l e \ e ha~e been described (Hall et al.. 198(i): Kat, ffman et al.. 19801. B', using sucro,e densitx gradient ultracentrifugation, a techmque unsuitable for routine a,,ax,. ~e have found that patients ~,.ith Berger's disease (Ldpez-Tra>ca,a et al.. IqgOi. H e n o c h - S c h o n l e i n s \ n d r o m e i~ of the,e diseases (Egido el al.. 1OS2cl and dermatLti, herpetil'orml, ~U n , ~ o r t h et al.. 19821. as t~ell a, in an experimental model of IgA uephropathx qRH'ai et al.. 197t.)1. development of a ne~. ~pecil'ic and ,imple technique for tile detectl~m c~f pol>meric lgA immune comple\es ~ a , tht~ught nece,,ar\. In tills paper ~e describe ,l ne~ a<~ax f~r pc, l.', merle IgA illlnlUll¢ comple\e,. Ttle as..,a,, is based upon tile ~peciflc binding of ,ecretor'+ c o m D m e n t for pol 7 merle IgA. In a first step. lg.~ immune c, mlplexe, are determined b\ the Ra.ll cell ,l<,a; ~hlch detects cotnplement-contaitling breakdo~,.n product> through the receptor, on Raji cells for C3-C3b and C3d ITheofilopoulo~ and Di\,m. 19761 and to a lesser extent for C l q ( G u p t a et al.. 197g}. The inmmnoglobull.1 i>~t~.pe c o m p o n e n t of the,e IC~ i, established b~ the u,e of appropriate ,pecific radiolabeled anti-hea~ x chain anti,era. In a second step. labeled S(" i, added to the cell, perm,tting recogniticm of the e\istence of p o b m e r i c IgA immune comple\e,. Several points deserve comment. Raj~ celia haxe >url'ace membrane Fc receptor, for lgG but not. to our know, ledge. Fc receptors for lgA. IgA receptor, ~ere not initially described in cells such as monoc,,te, ~L,mrence et al.. 1'-)751. but ha~e ~t.bsequentl 3 been reported i Fanger et al.. 1980). It i, po,,ible that polTmeric [gA Ior aggregated lg+%l x~ould ea,il~ attach tt~ such h3pothetical receptor,. Hinderer. the pre~ence of increasing amounts of polymeric IgA t Fig. 31 did not pr~,,luce ,ignil'icant changes in the binding of labeled S(' to Rali cell, a, compared ~ith normal human >erum. Our data not bind to Ra.li cell, dlrectl~ through Fc receptors, but rather through complement breakdo~l~ products. as occurs '.xith lgG IC ITheofilopoulos and Dixon. 10~61. The presence of complement-fl\ing IgM immune c~m~plexe, in the ,erum might al,o produce po, itixe result, becauqe pol>meric igM al,~ ha> afl'inlt~, I'~r S('. For thi,

315 reason a prior lgM immuno-adsorption was necessary. This is easily performed as described, and is not time-consuming. Its use allowed simultaneous study of a larger number of sera. The fact that preformed soluble IgA immune complexes often do not activate complement (Egido et al., 1982b" Pfaffenbach et al., 1982), makes it difficult to assess the true sensitivit~ of our method. Although some investigators have attempted to plot dose-dependent curves with lgA and lgG aggregates simultaneously, ~ hich under such conditions seem capable of activating complement, results are not convincing (Zone et al., 1980): it is for this reason that ~e ha~e preferred to express our results by using the value Ti./Ui, as described b~ Hall et al. (1980). Furthermore, on a molar basis, I mole of polymeric lgA is capable of binding 1 mole of SC (Weicker and Underdown, 1975) so that we ma} calculate theoretically the range of sensitivity of our assay. Thus, the lowest specific binding of ~-'-~l-labeled SC to Raji cells was in the region of 7 × 10 i_~ M / 1 × 10 ° cells, which represents 250 ng of dimeric IgA (mol. x~t. 350,000) and 25%. of the total polymeric IgA added to the cells. We found a high incidence of polymeric IgA immune complexes in patients with lgA nephropathy and alcoholic liver disease. Since we recently detected polymeric lgA immune complexes in the latter condition by sucrose densit~ ultracentrifugation at pH 7.4 and acid pH. followed by SC-affinity tests in IgA fractions bet~een 9 and 13S (Sancho et al., 1982), we have compared the 2 techniques. Although the number of patients studied was small there was a good correlation between them (Table I). One patient (AR) had positixe IgA-SC Raji IC that became negatixe after IgM adsorption, probabl,, oxsing to the presence of complement-fixing IgM IC. The increased levels of SC reported in the sera of patients ssith a sariet5 of liver diseases (Thompson et al., 1973: Delacroix and Vaerman, 19811 might in principle bind IgA and IgM complexes and thus cause looser apparent levels of polymeric lgA immune complexes. There is hossever, some opposing evidence: (I) in the patients studied, the proportion of polymeric lgA forming part of the immune complexes is lower than that in uncomplexed form (L6pez-Trascasa et al., 1980: Sancho et al.. 1982): (2) the serum SC circulates for a large part bound to IgM as well as to pol.,,meric IgA (Delacroix and Vaerman, 1982) and IgM is completely adsorbed in our assay: (3) the levels of secretor~ lgA as assessed b5 quantitative radioimmunoassa 5 I Delacroix and Vaerman, 1981) represent about 0.3% of the whole polymeric IgA (9-13S fractions) of patients with alcoholic cirrhosis and 2%. of that of normal subjects (Sancho et al., 1982). These data indicate that levels of SC, even in pathological conditions, cannot saturate the binding sites of polymeric IgA immune complexes and in this ssa5 interfere with our assav. The possibility of false positive resuhs due to anti-15 mphoc.~te antibodies, such as occur in some patients with systenfic lupu~ ervthematosus ~hen Rail cells are used to detect immune complexes, seems improbable, since mo~t such antibodies belong to the lgG class and, to our knossledge, no antilsmphocste polymeric IgA antibodies have been described. The use of Raji cells requires expensive culture methods, but ~e think that the principle of our assay, i.e. the binding of SC to polymeric I~,-X, could be adapted

31~-,

v, lth minor change~ to other method,~ capable of detecting multimeric lg-~ inlMtll'le comple\es, such as the conglutinln binding a~.,,a', ( ( o p p o et al.. 19S(11 o r the antl-lg.-k inhibition a~,,,a~ I Kauffman et al.. 198()). The ph?,~iopathological role of pol,,meric IgA I(" in hunlan,, ],, ilot kllov~tl. Recentl~ Jt has been demon.,,trated that. at lea~t in rodent.,,, p¢-,13mer~c IgA pla,,,, a role in the transport of absorbed antigens from blood to bile IPeppard et al.. 19~1: Ru,-,,,,ell et al.. 1981: Sockem et al.. 1981 ). We ha~e recentl~ ob~,erved that thi> Ma,, also be true in man. Thu,, some normal .,,ubject~ form pol 3 merit ,,eruru lgA I(" ,ffter meal.,, of high protein content, disappearing from the circulation after -,e~eral hour~, tin preparation). The higher prevalence and per,,,i,,,tence of these I ( ~n the ,,era of patient.,, v, ith IgA nephropathies (Berger'a di,,,ea~,e, or alcoholic h~er d~.,,ea,,e~, together ~,~ith their pre~ence in the kidney IEgJdo et al.. 1980: Sancho et al.. 1982 ~uggest a pathogenetic role for the~e IC when the\ are not promptl 3 eliminated from the circulation (Egido et al.. 1982c). It is possible that a defect in removal of lgA I¢,_" b~ the liver exist,,, in these diseases. Sera from the~e patients sJgnificantl 3 decrease neutrophil migration in normal controls, a p h e n o m e n o n clo,~el~ linked to pol,, merit lgA fraction~ (Egido et al., 1982a). Thi~ together ~ t h impaired handhng of lgA aggregate~ (akin to IgA-IC) b5 the mononuclear phagoc3tic s~,~tem a~ compared with lgG aggregates, might result in the persistence of immune complete,, in the circulation and deposition at sites susceptible to in.lur 5 (Egido et al.. 1982b). Further at'tal,,~i~ of these IC ma3 help to discriminate betv, een ph3~iolog~cal and pathological pol 3 merit lgA IC.

Aekno~ledgements This ~ork v, as supported b~ a grant from the Instituto Nacional de la Salud (lnsalud). Dr. Jaime Sancho i~ the recipient of a grant from the Jim,~nez Diaz Foundation. \\'e thank Liselotte Gulliksen for secretarial assistance. Dr. Muela,, kindly provided the Rali cells.

References Brandtzaeg. P.. 1974. Scand. J. Immunol. 3, 707. Cairns. S.,~.. R..A. London and N.P. Malhck. 1982, KJdne,, Int. 21. 5()"7. Chamne,,~.. G.C. and W,L. McGulre. 1975. Steroids 26. 538. C,~ppo. R, M. De Marctu, A. Aprato. D. Rocatello, M. Messma. G. Segc,lom, G Camu,~l. S..AIh',atl. F Elacchinc,, .A.O. Carbonara, A. Vercellone and A. Piccoli. 19,q0, Proc. Eur Dial. Transplant A,~,, 17. 667. Cuatrecasas. P. and C B..Anfinsen, 1971, .Ann Re',. B~.~hem. 40. 25tL Delacrotx. D.L. and J P. Vaermafl. I981. Chn. Exp. Immunol. 43. 633 Delacrolx. D.L. and J.P Vaerman, Iq82. Chn. Exp. Immunol. 4q.-',17. Egldo, J., J. Sancho. F Iktampa~o. M. L~,pez-Trac,casa. M.. Sanchez-Crespc,. R. Blasco and L Hernando 1980. Proc. Europ. Dial Transplant..Assoc. 17. 660. Egido. J . J Sancho. F. Lorente and E. Fontan. I982a. Chn. E,~p Immunol. 49. 71)',-I.

317 Egndo, J., J. Sancho, F. I~vera and M. Sanchez-Crespo, 1982b. Immunolog} 46. I. Egido. J.. J. Sancho, R. Blasco. F. Ri~era and L. Hernando, 1982c. in: Actualites Nephrologaques de I'H6pital Necker. eds. J. Hamburger, J. Crosnier and J. Grunfeld ( F l a m m a n a n . Paris) p. 147. Fanger. M.W., k. Shen, J. Pugh and G.M. Bernier, 1980. Proc. Natl. Acad. Scu. U.S.A. 77, 3640 Gupta, R.C., F.C. McDuffie, G. Tappeiner and R.E. Jordan. 1978. Immunology, 34. 75. Hall. R.P., "l-.J. kawle.~, J.A. Heck and S.|. Katz. 1080, Chn. Exp. lmmunol 40. 431. Hubbard. A i . and Z.A. Cohn. 1972, J. Cell Biol. 55, 390. Kauffman. R.H. and W.A. Herrmann, C.J.L.M Me0er. M R . Daha and L.A. Van E,,. 1980. Chn. Exp. |mmunol. 41. 459. La~rence, D.A., W.A. Weigle and H.L. Spnegelberg. 1975. J. Ckm. Inxest 55. 368. kexmsk.~. R.J. and T.M. Barratt, 1979, Lancet ii, 1100. Le'.msky, R.J. and J.F. Soothdl. 1977. Clin. Exp. Immunol. 29. 428. Lopez-Trascasa, M.. J. Egido. J. Sancho and L. Hernando. 1980, Clin. Exp. Immunol. 42. 247. McConahey. P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29. 185. Penner, E.. B. Albm] and F. Milgrom. 1978, Clin. Exp. Immunol. 34. 28. Peppard, J . E. Orlans, A.W.R. Payne and E. Andre~, 1981. Immunolog.~ 42. 83. Pfaffenbach, G.. M.E. Lamm and 1. GIgli, 1982, J. Exp. Med. 155, 231. Rifai. A.. P.A. Small. P.O. Teague and E. A}oub. 1979, J. Exp. Med. 150. 1161. Russell. M.W.. T.A. Brov, n and J. Mesteck} 1981, J. Exp. Med. 153, 1981. Sancho. J., J. Egido, M. Sanchez-Crespo and R. Blasco, 1982, Clin. E,~p. Immunol. 47. 327. Scatchard, G., 1949, Ann. N.Y, Acad. Sci. 51,660. Segal. D.M. and E. Hurwttz, 1977. lmmunol. J. 118. 1338. Sockem. D.J.. E.S. Simms. B. Nag}. M.M. Fisher and B.J. Underdo~n. 1981, Mol. Immunol. 18. 345. Theofilopoulos, A.N. and F.J. Dixon, 1976. J. Clin. Inxest. 57, 160. Thompson, R.A., R. Carter. R,P. Stokes, A.M. Geddes and J.A.D. Goodall. 1973, Chn. Exp. Immunol 14. 335. Underdown. B.J., J. DeRose, K. Koczekank, D. Socken and J. Weucker. 1977. Immunochemistr3. 14, II I. Linsworth. D.J.. A.W. Pa,sne. J.N. Leonard and E.J. Holborow, 1982. Lancet i. 478. Weicker. J. and B.J. Underdown. 1975. J. Immunol. 114. 1337. Woodroffe, A.J., W.A. Border. A.N. Theofilopoulos, O. GL_',tze. R.J. Glassock. F.J. Do~.on and C B. Wilson, 1977, Kidney Int. 12, 268. Zone, J.J., B.A. La Salle and T.T. Provost. 1980. J. Invest. Dermatol. 75, 152.