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Idiotypic interactions in type II mixed cryoglobulins
Blood Transfusion and I m m u n o h a e m a t o l o g y T o m e X X V I I . - - N ° 6. - - 1984
737
Idiotypic interactions in type II mixed cryoglobulins b y J . C . R e n v e r s e z • , S. R o u s s e l * , M . J . V a l l e e * , G. B r i g h o u s e ** a n d P . H . L a m b e r t ** * Laboratoire de Biochimie A, Centre Hospitalier RGgional et Universitaire de Grenoble, FRANCE. ** WHO Immunology Research and Training Centre, Department of Medicine and Centre of Transfusion, H6pital Cantonal Universitaire, Gen~ve, SWITZERLAND.
INTRODUCTION the a u t o i m m u n e diseases those associated w i t h the proA MONG duction of antibodies directed against imrnunoglobulins (Ig.), for e x a m p l e diseases with r h e u m a t o i d f a c t o r (RF) m a y be anticipated in the d i s t u r b a n c e of the regulation of the i m m u n e r e s p o n s e [1]. I n t e r a c t i o n s b e t w e e n i m m u n o g l o b u l i n molecules certainly play a m a j o r p a r t in the f o r m a t i o n of the i m m u n e complexes like those consisting of IgM RF a n d IgG p r e s e n t in p a t i e n t s w i t h r h e u m a t o i d arthritis [2, 3]. The m o s t c o m m o n of these anti-imrnunoglobulin auto-antibodies, such as RF, are directed against antigenic determ i n a n t s localized on the Fc f r a g m e n t of IgG [4]. Other anti-immunoglobulin auto-antibodies r e a c t with d e t e r m i n a n t s on the constant regions of i m m u n o g l o b u l i n s [4, 5] b u t a p a r t i c u l a r attention has b e e n recently given to antibodies reacting with the variable region of i m m u n o g l o b u l i n molecules (anti-idiotypes) [6, 7]. An internal
738
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network of idiotypes and anti-idiotypes has been hypothesized to modulate antibody production against exogenous antigens [8]. It seems possible that a similar mechanism may play some role in controlling the synthesis of RF autoantibodies [9]. A disease associated with a high incidence of anti-immunoglobulin antibodies is cryoglobulinemia in which monoclonal IgM possess an antibody reactivity against polyclonal IgG (mixed type II cryoglobulins) [10]. A broad pattern of reactivities and serological specificities have been demonstrated from this material, but the distribution of the antigenic determinants recognized by the IgM is not as complete [11, 12]. The question of whether the IgM cryoantiglobulins select certain subspecies of IgG from the seric heterogeneous IgG pool has been postulated in the reactivities of some monoclonal or mixed cryoglobulins [13, 14]. The purpose of this study was to investigate the nature of immunoglobulin interactions involved in 18 patients with type II mixed cryoglobulinemia and to evaluate the role of idiotypic interactions in the formation of those cryoglobulins. First, the spectrotype of the cryoprecipitating IgG was analyzed to determine whether the formation of the IgM-IgG complex would involve a selection of IgG molecules produced by particular B cell clones. Secondly, the specificity o f the reaction of each of the 18 cryoprecipitating IgG with each of the cryoprecipitating IgM was studied in solid phase assays. This specificity was then better defined in some selected cases using absorption or inhibition experiments.
MATERIAL AND M E T H O D S Patients s e l e c t i o n
Eigtheen patients with mixed cryoglobulinemia unassociated with any underlying disease were choosen. Most of them had necrotizing vasculitis and purpura or arthritis. Eleven had renal biopsy proven membrano-proliferative glomerulonephritis. All had significant amounts of cryoglobulins in their serum. As controls monoclonal IgG of the four subclasses were obtained from the sera of myeloma patients; monoclonal IgM were purified from the serum of Waldenstr6m macroglobulinemic patients. The source of IgM rheumatoid factor (RF) was the serum of a patient with rheumatoid arthritis and a very high titer of R.F. ( 1 : 2 000).
I D I O T Y P I C I N T E R A C T I O N S IN C R Y O G L O B U L I N S
739
Mixed cryoglobulins 50 ml blood were kept at 370 C until complete clot retraction, t h e r e a f t e r the s e r u m was stored at 4 °C for 7 days. A series of centrifugations and sterile washes were used to separate the cryoprecipitates f r o m the remaining serum [15]. The overall concentration of proteins was taken f r o m the Biuret m e t h o d after redissolution of an aliquot of the cryoprecipitates in 0,1 M NaOH. Concentrations of the G, A, M immunoglobulins and C3 c o m p o n e n t were d e t e r m i n e d by radial immunodiffusion at 37°C (Tripartigen, Behring, Marburg, Germany).
Separation of IgG and IgM from cryoglobulins Separation of the c r y o c o m p o n e n t s was done by ultracentrifugation in linear 0-40% sucrose density gradients [16]. Tubes were spun at 290,000 g for 12 hours at 10°C in a MSE-75 Ultracentrifuge using a swinging b u c k e t rotor. After the run, 0,5 ml fraction samples were collected. After dialyzing in 0,15 M phosphate Saline b u f f e r (PBS) the two IgG and IgM fractions were c o n c e n t r a t e d u n d e r positive pressure (Y M-10 membranes-Amicon Corp., Lexington, USA) and quantified by radial immunodiffusion as described above. F u r t h e r identifications were carried out using 1,5% agar gel i m m u n o e l e c t r o p h o r e s i s with the specific gamma, mu, kappa, lambda and whole h u m a n s e r u m antisera (Organon, Tecknika b.v. Oss, Holland). Isolated IgM were all found w i t h o u t any o t h e r detectable protein. Some IgG containing traces of albumin were t h e r e a f t e r passed t h r o u g h a DEAE ion-exchange c h r o m a t o g r a p h y column with a 0,015 M to 1 M linear Na C1 gradient. Pure IgG were eluted in 0,015 M Na C1. Purity of the two fractions was assessed b y electrophoresis on 6-20% polyacrylamide slab gels containing 0,1% sodium dodecyl sulfate (SDS) with a discontinuous b u f f e r system [17]. Determination of the subclasses present in the IgG fractions was done using the specific 1, 2, 3, 4 IgG antisera (Organon) by double diffusion in 1% agar. IgG heavy" and light chains V region subgroups were d e t e r m i n e d also with the same technique using monospecific VH 1, 2, 3; VK 1, 2, 3; VL 1, 2, 3, 4, 5 antisera kindly provided by the Blood Transfusion Centre in Rouen (France). Allotypic Gm and K m m a r k e r s were checked using specific antisera f r o m the same source using an hemagglutination inhibition method.
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Papain digestion of the IgG fractions To p r e p a r e Fab and Fc fragments f r o m the cryo IgG fractions, papain (Worthington Biochemical Corp., Freehold, USA) was added to IgG at an enzyme substrat ratio of 1:10 during 2 h o u r s at 370 C [18]. The proteolytic fragments w e r e separated f r o m undigested IgG immediately by gel filtration on a Sephadex G-100 c o l u m n ( P h a r m a c i a Fine Chemicals, Uppsala, Sweden). Fab and Fc fragments w e r e isolated with the same salt gradient ion exchange c h r o m a t o g r a p h y as described above; Fab was d e s o r b e d in 0,25 M Na C1 and Fc in 0,300 M Na C1. Fab were finally purified f r o m residual impurities by affinity c h r o m a t o g r a p h y p e r f o r m e d with an anti g a m m a chain i m m u n o s o r b e n t (Dako a n t i s e r u m - - Dakopatts ab., HSgersterl, Sweden), p r e p a r e d by insolubilization [19]. Purity of the fragments was controled by i m m u n o e l e c t r o p h o r e s i s with the specific Fab, Fc and whole h u m a n s e r u m antisera (Organon). SDS slab gel electrophoresis was used to verify the molecular weight of the fragments.
Purification of control immunoglobulins 7 Monoclonal IgG were isolated f r o m serum samples by ion exchange c h r o m a t o g r a p h y . 3 Monoclonal IgM and a polyclonal R.F. positive IgM were purified by gel filtration on Biogel A 1,5 m (Biorad Laboratories, Richmond, USA) followed b y affinity c h r o m a t o g r a p h y p e r f o r m e d with an anti-mu chain i m m u n o s o r b e n t (Dako antiserum). After adjusting the concentrations to 10 m g / m l the isolated proteins w e r e all found to be devoid of any o t h e r detectable protein b y i m m u n o e l e c t r o p h o r e s i s against whole h u m a n atiserum.
Isoelectric focusing procedure The isolated cryo IgM fractions were focused on agarose slab gels containing 0,8% agarose ( I E F agarose u Pharmacia) and 10% (W/V) sorbitol [20]. An ampholine mixture of 2% final c o n c e n t r a t i o n was used as follow: pH 3,5-10, 71,5%; p H 9-11, 9,5%; p H 7-9, 9,5%; pH 3,5-5, 9,5%. Polyclonal IgM and Pi m a r k e r s (Pharmacia) were r u n in parallel. The isolated cryo IgG fractions were isofocused on 5% polyacrylamide slab gels [21]. N o r m a l polyclonal IgG and Pi calibration m a r k e r s (Pharmacia) were r u n in parallel. After fixing the proteins, the gels were stained using a silver staining [22].
IDIOTYPIC INTERACTIONS IN CRYOGLOBULINS
741
Solid phase RF ELISA test A solid p h a s e RF enzyme linked i m m u n o s o r b e n t assay (ELISA) was used first to d e t e r m i n e the global activity of the s e p a r a t e d cryo IgM fractions against pooled h u m a n or r a b b i t IgG. The s a m e test was also done to analyze the reactivity of each cryo IgM against m o n o c l o n a l IgG, each cryo IgG fraction and proteolytic IgG fragments. Controls included a pool of n o r m a l h u m a n sera f r o m b l o o d donors, an RF positive polyclonal IgM and 7 monoclonal IgM. The test was p e r f o r m e d in f l a t - b o t t o m m i c r o t i t e r plates specially t r e a t e d for the ELISA (Titerteck-Flow Laboratories, Richmond, USA) and was done in duplicate. I n t a c t IgG or IgG f r a g m e n t s (100 microlit.) w e r e usually used to coat the m i c r o p l a t e s f o r 16 h o u r s at 370 C. Pooled h u m a n and r a b b i t IgG w e r e checked at 1 or 10 m i c r o g r . / m l concentrations; m o n o c l o n a l IgG, cryo IgG a n d IgG f r a g m e n t s were tested at 1, 5 and 10 m~crogr./ml concentrations, always diluted in PBS. After coating, plates w e r e rinced 3 times in PBS and the wells w e r e filled with 1% bovine s e r u m a l b u m i n (BSA) (220 microlit.) during 2 hours. After r e m o v a l of the albumin, the various cryo or control IgM (100 microlit.) w e r e a d d e d at the s a m e concentrations in the wells. After incubation 1 h 30 at 37 ° C, plates w e r e w a s h e d and an alkaline p h o s p h a t a s e c o n j u g a t e d mu-chain a n t i s e r u m was added in each well (100 microlit.) and allowed to i n c u b a t e for 2 hours. After washing, 100 microlit, of a nitro-phenyl-phosphate (NPP) s u b s t r a t e (1 m g / m l ) w e r e added. After 15 min. incubation 100 microlit, of 0,5 M s o d i u m h y d r o x y d e w e r e a d d e d and the optical densities in the wells were r e a d with an a u t o m a t i c s p e c t r o p h o t o m e t e r (Multiscan-Titertek) at 405 nln.
Absorption experiments Anti-immunoglobulin reactivities and cross-reactivities of the cryo IgM w e r e tested in a b s o r p t i o n e x p e r i m e n t s using the s a m e solid p h a s e assay described above. Plates w e r e coated w i t h polyclonal or cryo IgG Fab a n d Fc f r a g m e n t s . After incubation of control or cryo IgM in the wells, these s a m p l e s w e r e t r a n s f e r r e d in a second series of plates coated w i t h Fab o r Fc f r a g m e n t s to test their residual reactivities. The p e r c e n t a g e of residual binding was calculated f r o m the optical densities o b t a i n e d w i t h or w i t h o u t absorption. A fluid p h a s e inhibition e x p e r i m e n t was done for 2 cryo IgM b y mixing the IgM (10 m i c r o g r . / m l ) to different cryo IgG Fab (1 to 20 m i c r o g r . / m l ) in glass tubes. After 2 h o u r s of incubation the fluid p h a s e was t r a n s f e r r e d in plates coated w i t h polyclonal Fc (5 m i c r o g r , /
R E N V E R S E Z J.C. et coil.
742
TABLE I
General characteristics of the studied cryoglobulins.
IgM
IgG Cryo
Gm factors
11o
I /s el
V.L.
V.H.
L.C.
SUBTYPES
/s cl
V.H. V.L. S LIBIYPES
1,2
K
]II
K I
L.C.
I
K L K L
1,2
K
I
KI
1,2
K
III
K II
1 ZAH
5, 10, 11, 23
K + L [I, I I I
2 SAV
1, 2, 10, 13
K+L
I, I I I II, I I I II I, I I I
]
Km factors --I
3 DEN
5, I0, 11, 14
K+L
II, I I I
K I, I I I L I, II, I I I
4 MED
5, 10, 11, 13, 14
K+L
I, I I I
K III L II, I I I
1,2
K
I
KI
I
K III L II
1,2
K
I
K III
1,2
K
III
K I
1,2
K
II
iK II
III
L II
5 MAN
10, 11
6 AUD
1,2,4,10, 11, 14
K+L
7 DAS
5, 10, 11, 13
K+L
10, 11, 13, 15
K+L
I
KO
L II, I I I
1,2
L
K+L
II, I I I
K I
L II, I I I
3
K
10, 11, 13, 15
K+L
I
K III L II, I I I
1,2
K
1, 2, 4, 5, 3 (+1) 10, 11
K+L
I
K II L II, I I I
1,2
L
I
II' I I I
K III L IV K III L I , II
1,2
K
I
1,2
L
I
8 MAS 9 KOS 10 COC 11 MAT
3
3 (+1) 1 , 2 , 4 , 5 , 1 0 3
K+L
12 MASS
3
5, 10, 11
K+L
13 CHA
3
5, 11, 14
K+L
14 BOD
3
5, 11, 13, 14
K+L
15 VEY
3
5, 10, 11, 13, 14
K+L
16 MON
3
5, 10, 11, 13, 14
K+L
17 HUG
3
5, 10, 11
K+L
18 BAZ
3
10, i1, 21
K+L
1,2
1,2,4,5,10
3,4
11, 13, 14, 15, 21, 23
Normal polyclonal IgG
III
I, I I , I I I [
K+L III
K II L II, I I I K I , II L III
K I
L II K II, I I I L II K If, I I I L II KI L II, I I I
II
K II
K III I i
3
K
III
IK I
1,2
K
II
I]L I I I
3
K
I
IlK
1,2!
~
I
KI
If
t KI L lII
II
K I , II, I I I - I L I , II, I I I 1,2 IV,
1,2
K ~ U
polyclonal
-----
K L 7-
I
II III
--
1DIOTYPIC INTERACTIONS IN CRYOGLOBUL1NS
743
ml) and the anti-Fc reactivity was m e a s u r e d by ELISA as described above.
RESULTS General characteristics of the studied cyroglobulins All 18 cryoglobulins have b e e n first analyzed by conventional m e t h o d s . All precipitates contained b o t h IgG a n d IgM while in 10 cases, traces of C3 w e r e detected. All c r y o p r e c i p i t a t e s displayed a r h e u m a t o i d f a c t o r (R.F.) activity w h e n tested in a latex R.F. slide test. After s e p a r a t i o n of IgG f r o m the IgM c o m p o n e n t it w a s shown, as p r e s e n t e d in T a b l e I t h a t the IgM contained only one type of light chains, either k a p p a (15 cases) or l a m b d a (3 cases), one type of VH and VL s u b g r o u p s w i t h a p a r t i c u l a r f r e q u e n c y of V H 1 (12 cases) and VL K 1 (9 cases). I n all cases the IgG fractions c o n t a i n e d b o t h k a p p a and l a m b d a chains. The IgG a subclass was p r e s e n t in all IgG fractions; it was associated with traces of IgG 1 in 4 cases or IgG 2 in 1 case, b u t I g G 3 was the only detected subclass in 13 cases. The m a j o r allotypic m a r k e r s of these cryo IgG w e r e consistant w i t h the n a t u r e of IgG subclasses. T h e r e w a s not a p a r t i c u l a r f r e q u e n c y of any G m o r K m as c o m p a r e d with n o r m a l h u m a n IgG f r o m blood donors. I n 9 cases, only the IgG VH s u b g r o u p I was detected. In those patients, the c r y o p r e c i p i t a t e d IgM was also of the V H I subgroup. Such a restriction w a s not o b s e r v e d f o r VL subgroups.
Isoelectric focusing analysis of the IgM and IgG fractions of the cryoglobuHns The s p e c t r o t y p e s of the 18 IgM fractions w e r e analyzed b y agarose gel isoelectrofocusing. Only one m a j o r b a n d was o b s e r v e d in 14 samples, while s o m e additional m i n o r b a n d s w e r e also seen in 4 samples (n o 2-4-8-17), Fig. 1. The s p e c t r o t y p e s of the 18 IgG fractions s e p a r a t e d by polyacrylamide gel isoelectric focusing w e r e quite different f r o m each others (Fig. 2 a , 2b). I n m a r k e d c o n t r a s t to the large heterogeneity of the pooled n o r m a l h u m a n IgG used as control the m a j o r i t y of the IgG f r o m cryoglobulins p r e s e n t e d a r e s t r i c t e d profile w i t h a very limited n u m b e r of bands. I n d e e d in 10 cryo IgG only 2 to 6 bands, often on the cathodic side of the gel w e r e seen. The o t h e r 8 IgG fractions w e r e m o r e heterogeneous.
744
RENVERSEZ J.C. et coil.
FIG. 1. -- Isoelectrofocusing spectrotypes of some of the cryo IgM (C.) compared to reference markers (Pi ref.) and to pooled normal IgM (N). In 4/18 samples (N° 2, 4, 8, 17) minor bands were seen in addition to one major band.
Interaction of IgM fractions w i t h intact IgM m o l e c u l e s The interaction of the 18 IgM fractions was studied with each one of the 18 IgG fractions obtained f r o m the precipitates. I n a first series of test the IgG was used to coat the plastic microplates at a c o n c e n t r a t i o n Of 1 microgr./ml. The results, s u m m a r i z e d on Fig. 3, indicate that there is often a high degree of restriction in the IgM-IgG interactions, p r o b a b l y reflecting m a r k e d differences in avidity. I n general m o s t IgM fractions reacted very well with a significant binding (O:D. > 200) with the IgG fraction isolated f r o m the same cryoprecipitate. I n addition some of the IgM fractions (n o 1-2-3-4-5-6) reacted only which one to three IgG fractions f r o m o t h e r cryoprecipitates. Others IgM fractions (n o 7-8-9-10-11-12) reacted with four to eight IgG fractions from o t h e r cryoprecipitates. Only six IgM fractions (n o 13-14-15-16-17-18) reacted with m o r e t h a n eight IgG fractions. I n similar experiments, there was no binding of m o n o c l o n a l WaldenstrSm IgM to any of the solid phase cryo IgG.
Fro. 2 (a and b): Isoelectrofocusing spectrotypes of some cryo IgG (C.) and of n o r m a l h u m a n IgG (N) c o m p a r e d to reference m a r k e r s (Te).
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RENVERSEZ J.C. et coll.
I n a s e c o n d series of test, IgM f r a c t i o n s w e r e u s e d to coat the m i c r o p l a t e s a n d the b i n d i n g of e a c h IgG f r a c t i o n w a s s t u d i e d . The same results with the same restricted reactivities were obtained but n o t as m a r k e d as in t h e r e v e r s e e x p e r i m e n t . IgM 1 2 3 4
0 87
IgG
"
5
6
I-
7 8 9 10 11 12 13 14 15 16 17 18 RF
I
.....
10F i: i_-.]I_ji
FI6. 3 . - Interactions of each cryo IgM fraction and of one RF IgM with each cryo IgG fraction. Dark areas represent strongly positive reactions (OD over 600); hatched areas represent positive reactions (OD over 200).
Interactions of I g M fractions w i t h IgG f r a g m e n t s The r e a c t i v i t y of t h e 18 IgM f r a c t i o n s w a s t h e n s t u d i e d w i t h t h e F a b a n d Fc f r a g m e n t s o b t a i n e d f r o m t h e 18 cryo IgG f r a c t i o n s . These f r a g m e n t s w e r e u s e d to c o a t t h e m i c r o p l a t e s (1 or 5 m i c r o g r . / ml). E a c h IgM f r a c t i o n f r o m c r y o g l o b u l i n s w e r e t e s t e d w i t h e a c h of t h e s e 18 F a b or Fc p r e p a r a t i o n s . M o s t p o s i t i v e r e a c t i o n s p r e v i o u s l y o b s e r v e d w i t h solid p h a s e IgG f r a c t i o n s w e r e also seen using F a b c o a t e d p l a t e s w i t h a s i m i l a r r e s t r i c t e d p r o f i l e (Fig. 4). The b i n d i n g of IgM to F a b f r a g m e n t s w a s u s u a l l y l o w e r t h a n to i n t a c t IgG f r a c t i o n s . M o s t of t h e IgM f r a c t i o n s e x h i b i t e d a m a x i m a l b i n d i n g to F a b f r a g m e n t s o b t a i n e d f r o m the s a m e c r y o p r e c i p i t a t e . W h e n Fc f r a c t i o n s w e r e u s e d for c o a t i n g (1 m i c r o g r . / m l ) i n s t e a d of F a b f r a c t i o n s , t h e p a t t e r n of r e a c t i v i t y w a s d i f f e r e n t f r o m t h a t o b t a i n e d
IGM 1 7. 3 4 5 6
7
8 9 10 11 12 13 14 15 16 17 18 RF
1 2 3 4 5 6 7 8 Fab
9 10 11 12 13
14 15 16 17 18 FzG. 4. - - I n t e r a c t i o n s of e a c h cryo IgM f r a c t i o n s a n d one RF IgM w i t h Fab f r a g m e n t s f r o m each cryo IgG fractions. Repres e n t a t i o n s i m i l a r to figure 4.
1
Fc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 11 18
2 3 4
5 6
IgM 7 8 g 10 11 12 13 14 15 16 17 18 RF
i
FIG. 5. - - I n t e r a c t i o n s of each cryo IgM f r a c t i o n s a n d one RF IgM w i t h Fc f r a g m e n t s for e a c h cryo IgG fractions. Repres e n t a t i o n s i m i l a r to figure 4.
748
RENVERSEZ 1.C. et coll.
w i t h i n t a c t I g G f r a c t i o n s (Fig. 5). T h i s b i n d i n g w a s a l w a y s r a t h e r l o w a c o m p a r e d to t h a t o f t h e R F I g M u s e d as c o n t r o l . T h i s R F did not react with any of the Fab fractions tested.
Absorption of anti-immunoglobulin activities with Fab or Fc fragments Absorption experiments were done by incubating first cryo IgM samples with solid phase Fab or Fc preparations, then by testing the residual anti-Fab and anti-Fc activities. In a first series of experiments, Fc from normal pooled IgG was u s e d a s a s o l i d p h a s e a b s o r b a n t f o r t h e 18 I g M s a m p l e s (1 m i c r o g r . / ml). These ones were then tested for their reactivity with Fc from polyclonal IgG or from the corresponding cryo IgG as well as with F a b f r o m t h e c o r r e s p o n d i n g c r y o IgG. F o r a l l IgM, t h e r e w a s a n extensive absorption of the anti-Fc activity. A similar decrease was observed for a polyclonal RF IgM after absorption on Fc from polyc l o n a l I g G . T h e r e w a s a l s o a s i g n i f i c a n t d e c r e a s e ( m o r e t h a n 50%) o f t h e b i n d i n g of 10/18 I g M t o t h e c o r r e s p o n d i n g c r y o F a b ( T a b l e I I ) . H o w e v e r , f o r o t h e r c r y o I g M (e.g.; n o 4, 14, 17), t h e r e a c t i o n w i t h Fab was only slightly decreased.
TABLE II Absorption of anti-Fc or anti-Eab activities of cryo IgM. % RESIDUAL BINDING OF IgM 'to CRYO IgM
ABSORBANT
Fc
polyclonal IgG cryo 2 SAV 4 MED 14 BOD 12 MASS 17 HUG 18 BAZ
RF
Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab poly
17 24 19 14 5 21 7 35 16 58 12 56
22 27 38 22 59 21 7 23 20 12 23 13
Fc poly Fab cryo
23 100
20 80
Fc cryo
Fab cryo
25 31 30 25 21 20
2 1 8 3 3 4 20 12 65 16 54 5
23 58 20 63
749
IDIOTYPIC INTERACTIONS IN CRYOGLOBUL1NS
E
,,.
",.
~,
~,-~ l ~~ . ~
~.~ ~ u~
~',, -
. \.\.
O,.4
;
,-,. ",,
~.~.
.~ .;~ ~
e~ 4\\ '\
'i
No
= ~'~ o ~
\i',
~ . -'-z. ~ ~
o-~'g o
\i' \i~
~oC~
]
LD~ •
I
,d @4I
A
.:-.
umgO~ O0
750
RENVERSEZ
J.C. et coll.
In a second series of experiments, Fab fragments from each cryo IgG were used to absorb the corresponding IgM fraction and the results were compared with the absorption with polyc!onal Fab from normal pooled IgG. With all IgM fractions, there was an extensive decrease of the reactivity with Fab after the incubation with the corresponding solid phase Fab (4 to 18% of residual activity) (Table II). This absorption with Fab also led to a variable but often very significant decrease of the anti-Fc activity of the IgM fractions (less than 50% residual activity) in 10/18 IgM. A similar absorption of a polyclonal RF IgM on Fab from polyclonal IgG did not lead to any decrease of the anti-Fc activity (Table II). These results were confirmed using 3 cryo IgM at various concentrations (Fig. 6). Absorption experiments confirmed a more efficient depletion of the reactivity with Fab after absorption on solid phase Fab, than on solid phase Fc. Inversely the reactivity with Fc is better absorbed on solid phase Fc.
DISCUSSION
The main point of this work is the demonstration of a frequent restriction in the clonotypes of the IgG molecules involved in the formation of cryoprecipitates with monoclonal IgM in type II mixed cryoglobulinemia It also confirms the possibility of idiotypic interactions in many of these cryoprecipitates. By definition the IgM of mixed type II cryoglobulins is a monoclonal immunoglobulin. In the present study, each IgM fraction was found homogeneous by immunoelectrophoresis and by isoelectrofocusing with a very restricted pattern of bands and a well defined Pi. Furthermore, there was a prevalence of VH I and VK I variable region subgroups, which suggests that some physicochemical characteristics may be common to some of these IgM, although they differed from those analyzed by other authors who found a prevalence of VK III in their series of monoclonal RF IgM [23], or from polyclonal RF IgM [24]. The homogeneity of the sPectrotypes in IgM from mixed cryoglobulins has been previously described in 4 cases using liquid isoetectrofocusing [25]. Monoclonal IgM antiglobulins have also been studied with respect to their idiotypes and cross idiotypic reactions. Specific common antigen among IgM antigammaglobulins were found using a precipitation assay [26]. A cross- idiotypic specificity was clearly demonstrated among a group of monoelonal IgM antiglobulins, using antisera prepared against single monoclonal IgM [27, 28].
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Our analysis of the IgG c o m p o n e n t of m i x e d type I I cryoglobulins indicated a p a r t i c u l a r selection of IgG molecules during cryoprecipitation. All these fractions contained m o s t l y IgG a subclass molecules. Although b o t h k a p p a and l a m b d a light chains w e r e represented, t h e r e was a very r e s t r i c t e d charge heterogeneity in the IgG and a very h o m o g e n e o u s profile was seen for s o m e of them. The VH I subg r o u p was p r e d o m i n a n t since it w a s f o u n d alone in 9/18 IgG and in association with V H I t a n d / o r VH I I I in 6 o t h e r IgG fractions. There was not such restriction for the light chain V subgroups. The distribution of G m and K m allotypic m a r k e r s did not indicate a p a r t i c u l a r selection differing f r o m n o r m a l polyclonal IgG. A n u m b e r of o b s e r v a t i o n s have suggested that t h e r e is a p r e d o m i n a n c e of the IgGa subclass in cryoglobulins [29, 30, 31]. A restricted isoelectrofocusing profile for one IgG out of five isolated f r o m mixed cryoglobulins has b e e n also previously described [32]. Our results s u p p o r t the hypothesis of a selection of certain subspecies of IgG f r o m the heterogeneous IgG pool during cryoprecip i t a t i o n and are consistant with a r e s t r i c t e d reactivity b e t w e e n the two c o m p o n e n t s in the IgG-IgM cryoglobulins. Similarly a r e s t r i c t e d reactivity profile was exhibited for 6 cryo IgM with antiglobulin activity against autologous or isologous h u m a n IgG or their subunits [33]. In a p o p u l a t i o n of 11 m o n o c l o n a l IgM f r o m type I I cryoglobulins, multiple antiglobulin specificities w e r e shown [14], S o m e of the IgM w e r e reactive with Fc f r a g m e n t s ; s o m e others reacted w i t h IgG F(ab')2 f r a g m e n t s . S o m e of the anti-Fab antibodies w e r e directed against IgG idiotypes. These a u t h o r s suggested t h a t the IgM anti-Fab m a y react as anti-idiotypic antibodies with IgG. I n o u r investigations, we have c o n f i r m e d t h a t all of the 18 cryo IgM studied reacted with autologous a n d isologous cryo IgG. However this reaction was often of relatively low avidity since, for m o s t of the cryo IgM, it was only d e m o n s t r a t e d w h e n at least 10 m i c r o g r . / ml of IgG w e r e used for coating in the solid p h a s e assay. At lower c o n c e n t r a t i o n s of IgG (e.g. 1 m i c r o g / m l ) , m o s t cryo IgM did r e a c t w i t h the cryo IgG f r o m the c o r r e s p o n d i n g cryoglobulin b u t only w i t h a limited n u m b e r of cryo IgG f r o m o t h e r cryoglobulins. The p a t t e r n o b s e r v e d suggests a p r e f e r e n t i a l reactivity of the cryo IgM for s o m e IgG p r e p a r a t i o n s . This does not a p p e a r related to the IgG subclasses, since m o s t cryo IgG w e r e I g Q , n o r to be allotypic m a r k e r s of IgG. The fact t h a t m o s t of the cryo IgM react with b o t h Fab and Fc f r a g m e n t s of the reacting IgG suggests two types of interactions b e t w e e n cryo IgM and cryo IgG. Indeed, we can exclude the possibility t h a t the reactivity of cryo IgM with Fab would be due to c o n t a m i n a t i n g Fc since (a) m a n y cryo IgM react m o r e with
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Fab t h a n w i t h Fc, (b) each cryo IgM exhibits a p r o p e r p a t t e r n of reactivity w i t h the Fab f r a g m e n t s f r o m the 18 cryo IgG a n d (c) polyclonal RF did not r e a c t with a n y of the Fab p r e p a r a t i o n s used. The reaction b e t w e e n cryo IgM and Fab is not due to a reactivity of those Fab w i t h the c o n s t a n t p a r t of IgM since t h e r e was no binding to those Fab of o t h e r m o n o c l o n a l IgM f r o m p a t i e n t s with W a l d e n s t r 6 m m a c r o g l o b u l i n e m i a . One should n o t e t h a t the absorption of cryo IgM on solid p h a s e Fc did lead to a decrease of their binding to Fc b u t w i t h 8/18 cryo IgM, t h e r e was not a parallel decrease of their binding to Fab f r a g m e n t s f r o m the c o r r e s p o n d i n g IgG. Inversely, although the a b s o r p t i o n of cryo IgM w i t h Fab f r o m the c o r r e s p o n d i n g cryo IgG did inhibit c o m p l e t e l y the binding of all cryo IgM to Fab, t h e r e was only a partial parallel depletion of the anti-Fc activity. These d a t a suggest the existence of a certain heterogeneity in the m o n o c l o n a l IgM molecules and c o n f i r m the involv e m e n t of 2 different sites of reaction. S o m e cryo IgG a p p e a r to i n t e r a c t w i t h cryo IgM similarly to anti-idiotypic antibodies directed against the p a r a t o p e of the IgM RF. Thus, all these d a t a are consistant, w i t h the idea t h a t in a b o u t two thirds of the studied cryoglobulins, the IgM RF would p r e f e r e n t i a l l y r e a c t with anti-idiotypic IgG. S o m e of these anti-idiotypes are probably directed against d e t e r m i n a n t s outside the p a r a t o p e , b u t in s o m e cases, they would r e a c t w i t h the anti-Fc p a r a t o p e . I n s o m e cryoglobulins, it is likely t h a t the avidity of the IgM RF w o u l d b e sufficient by itself to lead to the f o r m a t i o n of stable IgM-IgG complexes. However, the coexistence of 2 reactions, anti-Fc IgM w i t h IgG-Fc and anti-idiotype IgG with IgM idiotypes, should confer a higher stability to IgM-IgG complexes. One m a y w o n d e r w h e t h e r the cross-reactives idiotypes which have been identified on m o n o c l o n a l or polyclonal RF [27, 9] are involved in the recognition of IgM by auto-anti-idiotypic IgG. The i n v o l v e m e n t of anti-idiotypic antibodies in m i x e d c r y o g l o b u l i n e m i a allows for s o m e hypothesis concerning the pathogenesis of the disease. I t is possible t h a t the a b n o r m a l p r o l i f e r a t i o n of one clone of IgM-RF p r o d u c i n g B cells would b e followed in s o m e patients b y a relatively r e s t r i c t e d anti-idiotypic response. The equilibrium b e t w e e n these two processes m i g h t d e t e r m i n e s p o n t a n e o u s variations in the expression of the disease. As it was previously suggested [14], such o b s e r v a t i o n s are consistant w i t h the existence of idiotypic interactions in man. Besides a physiological role in the regulation of the i m m u n e r e s p o n s e [8] one should consider the possibility t h a t idiotype anti-idiotype complexes w o u l d r e p r e s e n t a m a j o r source of cryo-
1DIOTYPIC INTERACTIONS IN CRYOGLOBULINS
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precipitating i m m u n e complexes and m a y be involved in h u m a n glomerulonephritis, vasculitis or arthritis. SUMMARY An analysis of interactions b e t w e e n i m m u n o g l o b u l i n molecules within cryoglobulins has b e e n carried out in 18 patients w i t h type I I mixed cryoglobulinemia. I n this series, t h e r e was a prevalence of VH I and VK I variable regions s u b g r o u p s in the m o n o c l o n a l IgM c o m p o n e n t . Our analysis of the IgG c o m p o n e n t indicated a p a r t i c u l a r selection of I g G molecules during cryoprecipitation. T h e r e was a prevalence of IgG3 and of the VH I s u b g r o u p and the isoelectrofocusing p a t t e r n revealed a very r e s t r i c t e d s p e c t r o t y p e in two thirds of these IgG. These results w h i c h suggested a r e s t r i c t e d reactivity b e t w e e n cryo-IgM and IgG fractions w e r e c o n f i r m e d b y the analysis of the interaction b e t w e e n each IgG and each IgM f r o m the cryoprecipitates. All IgM r e a c t e d w i t h intact IgG or Fc f r a g m e n t s b u t a n o t h e r reaction was o b s e r v e d b e t w e e n cryo-IgM and Fab f r a g m e n t s f r o m a limited n u m b e r of cryo-IgG, with a p a t t e r n suggestive of idiotypic specificity. Results of the a b s o r p t i o n of each cryo-IgM on Fc or on Fab fragm e n t s f r o m the c o r r e s p o n d i n g cryo-IgG also suggested the existence of a reaction b e t w e e n IgM a n d IgG Fab in addition to t h a t involving IgM Fab a n d IgG Fc. The coexistence of the 2 reactions should confer a higher stability to the IgM-IgG complex. Therefore, it is possible t h a t the p r o l i f e r a t i o n of one clone of IgM-RF p r o d u c i n g B cells w o u l d be followed in certain cases b y a relatively r e s t r i c t e d anti-idiotypic IgG response. The IgM-RF w o u l d preferentially r e a c t w i t h these anti-idiotypic IgG. Request reprints from: Jean-Charles RENVERSEZ, M.D., Laboratoire de Biochimie A Centre Hospitalier R6gional et Universitaire de Grenoble 38043 GRENOBLECedex, France.
REFERENCES
[1] ZUBLER R.H., NYDEGGER U., PERRIN L.H., FEHR K., Mc CORMICK J., LAMBERT P.H. and MIZSCnER P.A. - - Circulating immune complexes in patients with rheumatoid arthritis. J. Clin. lnvest., 57, 1308, 1976.
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E.C., HOLMAN H.R., Mi,iLLER-EBERHARD H.J. a n d KUNKEL H.G. - - An u n u s u a l p r o t e i n c o m p o n e n t of high molecular weight i n the s e r u m of certain patients with r h e u m a t o i d arthritis. ]. Exp. Med., 105, 425, 1957. [3] KUNKEL H.G., MiiLLER-EBERHARD H.J., FUDENBERGH.H. a n d TOMASI T.B. - - G a m m a g l o b u l i n complexes in r h e u m a t o i d arthritis a n d certain other conditions. J. Clin. Invest., 40, 117, 1961.
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[4] SCHROH~NLOHER R.E., KUNKEL H.G. a n d TOMASI T.B. - - Activity of dissociated and reassociated 19 S anti-gammaglobulillS. J. Exp. Med., 120, 1215, 1964. [5] KUNKEL H.G., MANNIK M. a n d WILLIAMS R.C. - - Individual antigenic specificity of isolated antibodies. Science, 140, 1218, 1963. [6] KUNKEL H.G., AGNELLOV., JOSLIN F.G., WINCHESTER R.J. a n d CAPRA J.D. - - Cross idiotypic specificity a m o n g m o n o c l o n a l IgM proteins with antig a m m a g l o b u l i n activity. J. Exp. Med., 137, 331, 1973. [7] BONA C.A., FINLEY S., WATERS S. a n d KUNKEL H.G. - - Anti-immunoglobulin antibodies. I I I properties of sequential anti-idiotypic antibodies to heterologous anti-gammaglobulins. Detection of reactivity of antiidiotype antibodies with epitopes of Fc fragments a n d with epitopes and idiotypes. J. Exp. Med., 156, 986, 1982.
[8] JERNE N.K. - - Towards a n e t w o r k theory of the i m m u n e system. Ann. Immunol. (Inst. Pasteur), 125, 373, 1974. [9] FONGS., GILBERTSON T.A. and CARSON D. - - The i n t e r n a l image of IgG in cross-reactive anti-idiotypic antibodies against h u m a i n r h e u m a t o i d factors. Jo Immunol., 131; 719, 1983. [10] FRANKLIN E . C . -
Cryoglobulinemia. Am. J. Med. Sci., 262, 50, 1971.
[11] GREY H.M., KOHLER P.H., TERRY W.D. a n d FRANKLIN E.G. - - H u m a n monoclonal g a m m a M cryoglobulin with anti-gammaglobulin activity. J. Clin. Invest., 47, 1875, 1968. [12] ALLEN J.C. a n d KUNKEL H.G. - - Hidden r h e u m a t o i d factors with specificity for native gammaglobulillS. Arthr. and Rheum., 9, 758, 1966. [13] ABRAHAMG.N., PODELLD.N., WEILCH E.H. a n d JOHNSTON S.L. - - Idiotypic relatedness of h u m a n m o n o c l o n a l IgG cryoglobulins. Immunology, 48, 315, 1983. [14] GELTNER D., FRANKLIN E.C. a n d FRANGIONEB. - - Anti-idiotypic activity
in the IgM fractions of mixed cryoglobulins. J. ImmunoI., 125, 1530, 1980. [15] RENVERSEZ J.C., VIALTEL P., SEIGNEURIN J.M. and CORDONNIER D. - Cryoglobulins as i m m u n e complexes: a n i m m u n o c h e m i c a l study of 376 cryoprecipitates. In: e Proticles o[ the Biological Fluids~, H. Peters, Editor, p. 391, 1979. P e r g a m o n Press, Oxford. [16] RENVERSEZ J.C., GROSLAMBERTP., VIALTEL P., CORDONNIER D. a n d GROULADE J. - - Analyse des cryoglobulines p a r ultracelltrifugation ell gradient de densit6 stabilis6 suivie d ' i m m u n o d i f f u s i o n . Ann. Biol. Clin., 37, 163, 1979. [17] LAEMMLI U.K. - - Cleavage of s t r u c t u r a l proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680, 1970.
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[18] NISONOFF A., MARKUS G. a n d WISSLER F.C. - - Separation of u n i v a l e n t fragments of r a b b i t a n t i b o d y by reduction of a single labile disulfide bond. Nature (Lond.), 4761, 293, 1961. [19] AVRAMEASS. a n d TERNYNCKT. - - Coupling of enzyme to proteins with glutaraeldehyde. Use of the conjugates for the detection of antigens a n d antibodies. Immunochem., 6, 43, 1969. [20] ROSEN A., EK K. a n d ANN P. - - Agarose isoelectric focusing of native h u m a n i m m u n o g l o b u l i n M a n d alpha-2 macroglobulin. J. Immunol. Methods, 28, 1, 1979. [21] BRENDEL S., MULI)ER J. a n d VERHAAR M.A. - - Heterogeneity of monoclonal i m m u n o g l o b u l i n G proteins studied by isoelectric focusing. Clin. Chim. Acta., 54, 243, 1974. [22] OAKLEY B.R., KIRSCH D.R. and MORRIS N.R. - - A simplified ultrasensitive silver strain for detecting proteins in polyacrylamide gels. AnaIyt. Biochem., 105, 361, 1980. [23] KUNKELH.G., WINCHESTERJ., JOSLIN F.C. and CAPRA J.D. - - Similarities in the light chains of anti-gammaglobulins showing cross-reactive specificities. J. Exp. Med., 149, 128, 1974. [24] FORRE O.T., JOHNSON P.M. a n d NATVIG J.B. - - A study of the variable heavy chain (VH r e g i o n s ) i n h u m a n polyclonal IgM r h e u m a t o i d factors. Scand. J. RheumatoI., 6, 113, 1977. [25] TRIESI-IMANNH.W., ABRAHAM G.N. and SANTUCCI E.A. - - The characterization of h u m a n anti-IgG auto-antibodies by liquid isoelectric focusing. J. Immunol., 114, 176, 1975. [26] FRANKLIN E.C. a n d FRANGIONEB. - - C o m m o n s t r u c t u r a l a n d antigenic properties of h u m a n g a m m a M anti-gammaglobulins. J. ImmunoI., 107, 1527, 1971. [27] KUNKELH.G., AGNELLOV., JOSLIN F.G. a n d CAPRA J.C. - - Cross idiotypic specificity among monoclonal IgM proteins with anti-gammaglobulin activity. ]. Exp. Med., 137, 331, 1973. [28] FORRE O.T., DOBLOUGJ.H., MlCHAELSENT.E. and NATVlGJ.B. - - Evidence of similar idiotypic d e t e r m i n a n t s on different r h e u m a t o i d factor populations. Scand. J. Immunol., 9, 281, 1979. [29] VIRELLA G. and HOBBS J.R. - - Heavy chain typing in IgG monoclonal g a m m a p a t h i e s with special reference to cases of serum hyperviscosity and cryoglobulinaemia. Clin. Exp. Immunol., 8, 973, 1971. [30] CREAM J.J., HOWARD A. a n d VIRELLA G. - - IgG heavy chain subclasses in mixed cryoglobulins. Immunology, 23, 405, 1972. [31] BROUET J.C., CLAUVEL J.P., DANON F., KLEIN M. and SELIGMANN M. - Biological a n d clinical significance of cryoglobulins: a report of 86 cases. Am. J. Med., 57, 775, 1974. [32] JOHNSTON S.L., JORIZZO J.R., LIBER~aAND:F. and ABRAHAMG.N. - - Studies of h u m a n IgM anti-IgG cryoglobulins. II. Isoelectric focusing characteristics of the reactive antigen a n d residual serum IgG pools. Immunology, 36, 707, 1979. [33] JOHNSTON S.L. a n d ABRAHAM G.N. - - Studies of h u m a n IgM anti-IgG cryoglobulins. I. Patterns of reactivity with autologous a n d isologous h u m a n IgG a n d its subunits. Immunology, 36, 671, 1979.
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Idiotypic interactions in type II mixed cryoglobulins b y J . C . R e n v e r s e z • , S. R o u s s e l * , M . J . V a l l e e * , G. B r i g h o u s e ** a n d P . H . L a m b e r t ** * Laboratoire de Biochimie A, Centre Hospitalier RGgional et Universitaire de Grenoble, FRANCE. ** WHO Immunology Research and Training Centre, Department of Medicine and Centre of Transfusion, H6pital Cantonal Universitaire, Gen~ve, SWITZERLAND.
INTRODUCTION the a u t o i m m u n e diseases those associated w i t h the proA MONG duction of antibodies directed against imrnunoglobulins (Ig.), for e x a m p l e diseases with r h e u m a t o i d f a c t o r (RF) m a y be anticipated in the d i s t u r b a n c e of the regulation of the i m m u n e r e s p o n s e [1]. I n t e r a c t i o n s b e t w e e n i m m u n o g l o b u l i n molecules certainly play a m a j o r p a r t in the f o r m a t i o n of the i m m u n e complexes like those consisting of IgM RF a n d IgG p r e s e n t in p a t i e n t s w i t h r h e u m a t o i d arthritis [2, 3]. The m o s t c o m m o n of these anti-imrnunoglobulin auto-antibodies, such as RF, are directed against antigenic determ i n a n t s localized on the Fc f r a g m e n t of IgG [4]. Other anti-immunoglobulin auto-antibodies r e a c t with d e t e r m i n a n t s on the constant regions of i m m u n o g l o b u l i n s [4, 5] b u t a p a r t i c u l a r attention has b e e n recently given to antibodies reacting with the variable region of i m m u n o g l o b u l i n molecules (anti-idiotypes) [6, 7]. An internal
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network of idiotypes and anti-idiotypes has been hypothesized to modulate antibody production against exogenous antigens [8]. It seems possible that a similar mechanism may play some role in controlling the synthesis of RF autoantibodies [9]. A disease associated with a high incidence of anti-immunoglobulin antibodies is cryoglobulinemia in which monoclonal IgM possess an antibody reactivity against polyclonal IgG (mixed type II cryoglobulins) [10]. A broad pattern of reactivities and serological specificities have been demonstrated from this material, but the distribution of the antigenic determinants recognized by the IgM is not as complete [11, 12]. The question of whether the IgM cryoantiglobulins select certain subspecies of IgG from the seric heterogeneous IgG pool has been postulated in the reactivities of some monoclonal or mixed cryoglobulins [13, 14]. The purpose of this study was to investigate the nature of immunoglobulin interactions involved in 18 patients with type II mixed cryoglobulinemia and to evaluate the role of idiotypic interactions in the formation of those cryoglobulins. First, the spectrotype of the cryoprecipitating IgG was analyzed to determine whether the formation of the IgM-IgG complex would involve a selection of IgG molecules produced by particular B cell clones. Secondly, the specificity o f the reaction of each of the 18 cryoprecipitating IgG with each of the cryoprecipitating IgM was studied in solid phase assays. This specificity was then better defined in some selected cases using absorption or inhibition experiments.
MATERIAL AND M E T H O D S Patients s e l e c t i o n
Eigtheen patients with mixed cryoglobulinemia unassociated with any underlying disease were choosen. Most of them had necrotizing vasculitis and purpura or arthritis. Eleven had renal biopsy proven membrano-proliferative glomerulonephritis. All had significant amounts of cryoglobulins in their serum. As controls monoclonal IgG of the four subclasses were obtained from the sera of myeloma patients; monoclonal IgM were purified from the serum of Waldenstr6m macroglobulinemic patients. The source of IgM rheumatoid factor (RF) was the serum of a patient with rheumatoid arthritis and a very high titer of R.F. ( 1 : 2 000).
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Mixed cryoglobulins 50 ml blood were kept at 370 C until complete clot retraction, t h e r e a f t e r the s e r u m was stored at 4 °C for 7 days. A series of centrifugations and sterile washes were used to separate the cryoprecipitates f r o m the remaining serum [15]. The overall concentration of proteins was taken f r o m the Biuret m e t h o d after redissolution of an aliquot of the cryoprecipitates in 0,1 M NaOH. Concentrations of the G, A, M immunoglobulins and C3 c o m p o n e n t were d e t e r m i n e d by radial immunodiffusion at 37°C (Tripartigen, Behring, Marburg, Germany).
Separation of IgG and IgM from cryoglobulins Separation of the c r y o c o m p o n e n t s was done by ultracentrifugation in linear 0-40% sucrose density gradients [16]. Tubes were spun at 290,000 g for 12 hours at 10°C in a MSE-75 Ultracentrifuge using a swinging b u c k e t rotor. After the run, 0,5 ml fraction samples were collected. After dialyzing in 0,15 M phosphate Saline b u f f e r (PBS) the two IgG and IgM fractions were c o n c e n t r a t e d u n d e r positive pressure (Y M-10 membranes-Amicon Corp., Lexington, USA) and quantified by radial immunodiffusion as described above. F u r t h e r identifications were carried out using 1,5% agar gel i m m u n o e l e c t r o p h o r e s i s with the specific gamma, mu, kappa, lambda and whole h u m a n s e r u m antisera (Organon, Tecknika b.v. Oss, Holland). Isolated IgM were all found w i t h o u t any o t h e r detectable protein. Some IgG containing traces of albumin were t h e r e a f t e r passed t h r o u g h a DEAE ion-exchange c h r o m a t o g r a p h y column with a 0,015 M to 1 M linear Na C1 gradient. Pure IgG were eluted in 0,015 M Na C1. Purity of the two fractions was assessed b y electrophoresis on 6-20% polyacrylamide slab gels containing 0,1% sodium dodecyl sulfate (SDS) with a discontinuous b u f f e r system [17]. Determination of the subclasses present in the IgG fractions was done using the specific 1, 2, 3, 4 IgG antisera (Organon) by double diffusion in 1% agar. IgG heavy" and light chains V region subgroups were d e t e r m i n e d also with the same technique using monospecific VH 1, 2, 3; VK 1, 2, 3; VL 1, 2, 3, 4, 5 antisera kindly provided by the Blood Transfusion Centre in Rouen (France). Allotypic Gm and K m m a r k e r s were checked using specific antisera f r o m the same source using an hemagglutination inhibition method.
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J.C. et coll.
Papain digestion of the IgG fractions To p r e p a r e Fab and Fc fragments f r o m the cryo IgG fractions, papain (Worthington Biochemical Corp., Freehold, USA) was added to IgG at an enzyme substrat ratio of 1:10 during 2 h o u r s at 370 C [18]. The proteolytic fragments w e r e separated f r o m undigested IgG immediately by gel filtration on a Sephadex G-100 c o l u m n ( P h a r m a c i a Fine Chemicals, Uppsala, Sweden). Fab and Fc fragments w e r e isolated with the same salt gradient ion exchange c h r o m a t o g r a p h y as described above; Fab was d e s o r b e d in 0,25 M Na C1 and Fc in 0,300 M Na C1. Fab were finally purified f r o m residual impurities by affinity c h r o m a t o g r a p h y p e r f o r m e d with an anti g a m m a chain i m m u n o s o r b e n t (Dako a n t i s e r u m - - Dakopatts ab., HSgersterl, Sweden), p r e p a r e d by insolubilization [19]. Purity of the fragments was controled by i m m u n o e l e c t r o p h o r e s i s with the specific Fab, Fc and whole h u m a n s e r u m antisera (Organon). SDS slab gel electrophoresis was used to verify the molecular weight of the fragments.
Purification of control immunoglobulins 7 Monoclonal IgG were isolated f r o m serum samples by ion exchange c h r o m a t o g r a p h y . 3 Monoclonal IgM and a polyclonal R.F. positive IgM were purified by gel filtration on Biogel A 1,5 m (Biorad Laboratories, Richmond, USA) followed b y affinity c h r o m a t o g r a p h y p e r f o r m e d with an anti-mu chain i m m u n o s o r b e n t (Dako antiserum). After adjusting the concentrations to 10 m g / m l the isolated proteins w e r e all found to be devoid of any o t h e r detectable protein b y i m m u n o e l e c t r o p h o r e s i s against whole h u m a n atiserum.
Isoelectric focusing procedure The isolated cryo IgM fractions were focused on agarose slab gels containing 0,8% agarose ( I E F agarose u Pharmacia) and 10% (W/V) sorbitol [20]. An ampholine mixture of 2% final c o n c e n t r a t i o n was used as follow: pH 3,5-10, 71,5%; p H 9-11, 9,5%; p H 7-9, 9,5%; pH 3,5-5, 9,5%. Polyclonal IgM and Pi m a r k e r s (Pharmacia) were r u n in parallel. The isolated cryo IgG fractions were isofocused on 5% polyacrylamide slab gels [21]. N o r m a l polyclonal IgG and Pi calibration m a r k e r s (Pharmacia) were r u n in parallel. After fixing the proteins, the gels were stained using a silver staining [22].
IDIOTYPIC INTERACTIONS IN CRYOGLOBULINS
741
Solid phase RF ELISA test A solid p h a s e RF enzyme linked i m m u n o s o r b e n t assay (ELISA) was used first to d e t e r m i n e the global activity of the s e p a r a t e d cryo IgM fractions against pooled h u m a n or r a b b i t IgG. The s a m e test was also done to analyze the reactivity of each cryo IgM against m o n o c l o n a l IgG, each cryo IgG fraction and proteolytic IgG fragments. Controls included a pool of n o r m a l h u m a n sera f r o m b l o o d donors, an RF positive polyclonal IgM and 7 monoclonal IgM. The test was p e r f o r m e d in f l a t - b o t t o m m i c r o t i t e r plates specially t r e a t e d for the ELISA (Titerteck-Flow Laboratories, Richmond, USA) and was done in duplicate. I n t a c t IgG or IgG f r a g m e n t s (100 microlit.) w e r e usually used to coat the m i c r o p l a t e s f o r 16 h o u r s at 370 C. Pooled h u m a n and r a b b i t IgG w e r e checked at 1 or 10 m i c r o g r . / m l concentrations; m o n o c l o n a l IgG, cryo IgG a n d IgG f r a g m e n t s were tested at 1, 5 and 10 m~crogr./ml concentrations, always diluted in PBS. After coating, plates w e r e rinced 3 times in PBS and the wells w e r e filled with 1% bovine s e r u m a l b u m i n (BSA) (220 microlit.) during 2 hours. After r e m o v a l of the albumin, the various cryo or control IgM (100 microlit.) w e r e a d d e d at the s a m e concentrations in the wells. After incubation 1 h 30 at 37 ° C, plates w e r e w a s h e d and an alkaline p h o s p h a t a s e c o n j u g a t e d mu-chain a n t i s e r u m was added in each well (100 microlit.) and allowed to i n c u b a t e for 2 hours. After washing, 100 microlit, of a nitro-phenyl-phosphate (NPP) s u b s t r a t e (1 m g / m l ) w e r e added. After 15 min. incubation 100 microlit, of 0,5 M s o d i u m h y d r o x y d e w e r e a d d e d and the optical densities in the wells were r e a d with an a u t o m a t i c s p e c t r o p h o t o m e t e r (Multiscan-Titertek) at 405 nln.
Absorption experiments Anti-immunoglobulin reactivities and cross-reactivities of the cryo IgM w e r e tested in a b s o r p t i o n e x p e r i m e n t s using the s a m e solid p h a s e assay described above. Plates w e r e coated w i t h polyclonal or cryo IgG Fab a n d Fc f r a g m e n t s . After incubation of control or cryo IgM in the wells, these s a m p l e s w e r e t r a n s f e r r e d in a second series of plates coated w i t h Fab o r Fc f r a g m e n t s to test their residual reactivities. The p e r c e n t a g e of residual binding was calculated f r o m the optical densities o b t a i n e d w i t h or w i t h o u t absorption. A fluid p h a s e inhibition e x p e r i m e n t was done for 2 cryo IgM b y mixing the IgM (10 m i c r o g r . / m l ) to different cryo IgG Fab (1 to 20 m i c r o g r . / m l ) in glass tubes. After 2 h o u r s of incubation the fluid p h a s e was t r a n s f e r r e d in plates coated w i t h polyclonal Fc (5 m i c r o g r , /
R E N V E R S E Z J.C. et coil.
742
TABLE I
General characteristics of the studied cryoglobulins.
IgM
IgG Cryo
Gm factors
11o
I /s el
V.L.
V.H.
L.C.
SUBTYPES
/s cl
V.H. V.L. S LIBIYPES
1,2
K
]II
K I
L.C.
I
K L K L
1,2
K
I
KI
1,2
K
III
K II
1 ZAH
5, 10, 11, 23
K + L [I, I I I
2 SAV
1, 2, 10, 13
K+L
I, I I I II, I I I II I, I I I
]
Km factors --I
3 DEN
5, I0, 11, 14
K+L
II, I I I
K I, I I I L I, II, I I I
4 MED
5, 10, 11, 13, 14
K+L
I, I I I
K III L II, I I I
1,2
K
I
KI
I
K III L II
1,2
K
I
K III
1,2
K
III
K I
1,2
K
II
iK II
III
L II
5 MAN
10, 11
6 AUD
1,2,4,10, 11, 14
K+L
7 DAS
5, 10, 11, 13
K+L
10, 11, 13, 15
K+L
I
KO
L II, I I I
1,2
L
K+L
II, I I I
K I
L II, I I I
3
K
10, 11, 13, 15
K+L
I
K III L II, I I I
1,2
K
1, 2, 4, 5, 3 (+1) 10, 11
K+L
I
K II L II, I I I
1,2
L
I
II' I I I
K III L IV K III L I , II
1,2
K
I
1,2
L
I
8 MAS 9 KOS 10 COC 11 MAT
3
3 (+1) 1 , 2 , 4 , 5 , 1 0 3
K+L
12 MASS
3
5, 10, 11
K+L
13 CHA
3
5, 11, 14
K+L
14 BOD
3
5, 11, 13, 14
K+L
15 VEY
3
5, 10, 11, 13, 14
K+L
16 MON
3
5, 10, 11, 13, 14
K+L
17 HUG
3
5, 10, 11
K+L
18 BAZ
3
10, i1, 21
K+L
1,2
1,2,4,5,10
3,4
11, 13, 14, 15, 21, 23
Normal polyclonal IgG
III
I, I I , I I I [
K+L III
K II L II, I I I K I , II L III
K I
L II K II, I I I L II K If, I I I L II KI L II, I I I
II
K II
K III I i
3
K
III
IK I
1,2
K
II
I]L I I I
3
K
I
IlK
1,2!
~
I
KI
If
t KI L lII
II
K I , II, I I I - I L I , II, I I I 1,2 IV,
1,2
K ~ U
polyclonal
-----
K L 7-
I
II III
--
1DIOTYPIC INTERACTIONS IN CRYOGLOBUL1NS
743
ml) and the anti-Fc reactivity was m e a s u r e d by ELISA as described above.
RESULTS General characteristics of the studied cyroglobulins All 18 cryoglobulins have b e e n first analyzed by conventional m e t h o d s . All precipitates contained b o t h IgG a n d IgM while in 10 cases, traces of C3 w e r e detected. All c r y o p r e c i p i t a t e s displayed a r h e u m a t o i d f a c t o r (R.F.) activity w h e n tested in a latex R.F. slide test. After s e p a r a t i o n of IgG f r o m the IgM c o m p o n e n t it w a s shown, as p r e s e n t e d in T a b l e I t h a t the IgM contained only one type of light chains, either k a p p a (15 cases) or l a m b d a (3 cases), one type of VH and VL s u b g r o u p s w i t h a p a r t i c u l a r f r e q u e n c y of V H 1 (12 cases) and VL K 1 (9 cases). I n all cases the IgG fractions c o n t a i n e d b o t h k a p p a and l a m b d a chains. The IgG a subclass was p r e s e n t in all IgG fractions; it was associated with traces of IgG 1 in 4 cases or IgG 2 in 1 case, b u t I g G 3 was the only detected subclass in 13 cases. The m a j o r allotypic m a r k e r s of these cryo IgG w e r e consistant w i t h the n a t u r e of IgG subclasses. T h e r e w a s not a p a r t i c u l a r f r e q u e n c y of any G m o r K m as c o m p a r e d with n o r m a l h u m a n IgG f r o m blood donors. I n 9 cases, only the IgG VH s u b g r o u p I was detected. In those patients, the c r y o p r e c i p i t a t e d IgM was also of the V H I subgroup. Such a restriction w a s not o b s e r v e d f o r VL subgroups.
Isoelectric focusing analysis of the IgM and IgG fractions of the cryoglobuHns The s p e c t r o t y p e s of the 18 IgM fractions w e r e analyzed b y agarose gel isoelectrofocusing. Only one m a j o r b a n d was o b s e r v e d in 14 samples, while s o m e additional m i n o r b a n d s w e r e also seen in 4 samples (n o 2-4-8-17), Fig. 1. The s p e c t r o t y p e s of the 18 IgG fractions s e p a r a t e d by polyacrylamide gel isoelectric focusing w e r e quite different f r o m each others (Fig. 2 a , 2b). I n m a r k e d c o n t r a s t to the large heterogeneity of the pooled n o r m a l h u m a n IgG used as control the m a j o r i t y of the IgG f r o m cryoglobulins p r e s e n t e d a r e s t r i c t e d profile w i t h a very limited n u m b e r of bands. I n d e e d in 10 cryo IgG only 2 to 6 bands, often on the cathodic side of the gel w e r e seen. The o t h e r 8 IgG fractions w e r e m o r e heterogeneous.
744
RENVERSEZ J.C. et coil.
FIG. 1. -- Isoelectrofocusing spectrotypes of some of the cryo IgM (C.) compared to reference markers (Pi ref.) and to pooled normal IgM (N). In 4/18 samples (N° 2, 4, 8, 17) minor bands were seen in addition to one major band.
Interaction of IgM fractions w i t h intact IgM m o l e c u l e s The interaction of the 18 IgM fractions was studied with each one of the 18 IgG fractions obtained f r o m the precipitates. I n a first series of test the IgG was used to coat the plastic microplates at a c o n c e n t r a t i o n Of 1 microgr./ml. The results, s u m m a r i z e d on Fig. 3, indicate that there is often a high degree of restriction in the IgM-IgG interactions, p r o b a b l y reflecting m a r k e d differences in avidity. I n general m o s t IgM fractions reacted very well with a significant binding (O:D. > 200) with the IgG fraction isolated f r o m the same cryoprecipitate. I n addition some of the IgM fractions (n o 1-2-3-4-5-6) reacted only which one to three IgG fractions f r o m o t h e r cryoprecipitates. Others IgM fractions (n o 7-8-9-10-11-12) reacted with four to eight IgG fractions from o t h e r cryoprecipitates. Only six IgM fractions (n o 13-14-15-16-17-18) reacted with m o r e t h a n eight IgG fractions. I n similar experiments, there was no binding of m o n o c l o n a l WaldenstrSm IgM to any of the solid phase cryo IgG.
Fro. 2 (a and b): Isoelectrofocusing spectrotypes of some cryo IgG (C.) and of n o r m a l h u m a n IgG (N) c o m p a r e d to reference m a r k e r s (Te).
746
RENVERSEZ J.C. et coll.
I n a s e c o n d series of test, IgM f r a c t i o n s w e r e u s e d to coat the m i c r o p l a t e s a n d the b i n d i n g of e a c h IgG f r a c t i o n w a s s t u d i e d . The same results with the same restricted reactivities were obtained but n o t as m a r k e d as in t h e r e v e r s e e x p e r i m e n t . IgM 1 2 3 4
0 87
IgG
"
5
6
I-
7 8 9 10 11 12 13 14 15 16 17 18 RF
I
.....
10F i: i_-.]I_ji
FI6. 3 . - Interactions of each cryo IgM fraction and of one RF IgM with each cryo IgG fraction. Dark areas represent strongly positive reactions (OD over 600); hatched areas represent positive reactions (OD over 200).
Interactions of I g M fractions w i t h IgG f r a g m e n t s The r e a c t i v i t y of t h e 18 IgM f r a c t i o n s w a s t h e n s t u d i e d w i t h t h e F a b a n d Fc f r a g m e n t s o b t a i n e d f r o m t h e 18 cryo IgG f r a c t i o n s . These f r a g m e n t s w e r e u s e d to c o a t t h e m i c r o p l a t e s (1 or 5 m i c r o g r . / ml). E a c h IgM f r a c t i o n f r o m c r y o g l o b u l i n s w e r e t e s t e d w i t h e a c h of t h e s e 18 F a b or Fc p r e p a r a t i o n s . M o s t p o s i t i v e r e a c t i o n s p r e v i o u s l y o b s e r v e d w i t h solid p h a s e IgG f r a c t i o n s w e r e also seen using F a b c o a t e d p l a t e s w i t h a s i m i l a r r e s t r i c t e d p r o f i l e (Fig. 4). The b i n d i n g of IgM to F a b f r a g m e n t s w a s u s u a l l y l o w e r t h a n to i n t a c t IgG f r a c t i o n s . M o s t of t h e IgM f r a c t i o n s e x h i b i t e d a m a x i m a l b i n d i n g to F a b f r a g m e n t s o b t a i n e d f r o m the s a m e c r y o p r e c i p i t a t e . W h e n Fc f r a c t i o n s w e r e u s e d for c o a t i n g (1 m i c r o g r . / m l ) i n s t e a d of F a b f r a c t i o n s , t h e p a t t e r n of r e a c t i v i t y w a s d i f f e r e n t f r o m t h a t o b t a i n e d
IGM 1 7. 3 4 5 6
7
8 9 10 11 12 13 14 15 16 17 18 RF
1 2 3 4 5 6 7 8 Fab
9 10 11 12 13
14 15 16 17 18 FzG. 4. - - I n t e r a c t i o n s of e a c h cryo IgM f r a c t i o n s a n d one RF IgM w i t h Fab f r a g m e n t s f r o m each cryo IgG fractions. Repres e n t a t i o n s i m i l a r to figure 4.
1
Fc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 11 18
2 3 4
5 6
IgM 7 8 g 10 11 12 13 14 15 16 17 18 RF
i
FIG. 5. - - I n t e r a c t i o n s of each cryo IgM f r a c t i o n s a n d one RF IgM w i t h Fc f r a g m e n t s for e a c h cryo IgG fractions. Repres e n t a t i o n s i m i l a r to figure 4.
748
RENVERSEZ 1.C. et coll.
w i t h i n t a c t I g G f r a c t i o n s (Fig. 5). T h i s b i n d i n g w a s a l w a y s r a t h e r l o w a c o m p a r e d to t h a t o f t h e R F I g M u s e d as c o n t r o l . T h i s R F did not react with any of the Fab fractions tested.
Absorption of anti-immunoglobulin activities with Fab or Fc fragments Absorption experiments were done by incubating first cryo IgM samples with solid phase Fab or Fc preparations, then by testing the residual anti-Fab and anti-Fc activities. In a first series of experiments, Fc from normal pooled IgG was u s e d a s a s o l i d p h a s e a b s o r b a n t f o r t h e 18 I g M s a m p l e s (1 m i c r o g r . / ml). These ones were then tested for their reactivity with Fc from polyclonal IgG or from the corresponding cryo IgG as well as with F a b f r o m t h e c o r r e s p o n d i n g c r y o IgG. F o r a l l IgM, t h e r e w a s a n extensive absorption of the anti-Fc activity. A similar decrease was observed for a polyclonal RF IgM after absorption on Fc from polyc l o n a l I g G . T h e r e w a s a l s o a s i g n i f i c a n t d e c r e a s e ( m o r e t h a n 50%) o f t h e b i n d i n g of 10/18 I g M t o t h e c o r r e s p o n d i n g c r y o F a b ( T a b l e I I ) . H o w e v e r , f o r o t h e r c r y o I g M (e.g.; n o 4, 14, 17), t h e r e a c t i o n w i t h Fab was only slightly decreased.
TABLE II Absorption of anti-Fc or anti-Eab activities of cryo IgM. % RESIDUAL BINDING OF IgM 'to CRYO IgM
ABSORBANT
Fc
polyclonal IgG cryo 2 SAV 4 MED 14 BOD 12 MASS 17 HUG 18 BAZ
RF
Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab cryo Fc poly Fab poly
17 24 19 14 5 21 7 35 16 58 12 56
22 27 38 22 59 21 7 23 20 12 23 13
Fc poly Fab cryo
23 100
20 80
Fc cryo
Fab cryo
25 31 30 25 21 20
2 1 8 3 3 4 20 12 65 16 54 5
23 58 20 63
749
IDIOTYPIC INTERACTIONS IN CRYOGLOBUL1NS
E
,,.
",.
~,
~,-~ l ~~ . ~
~.~ ~ u~
~',, -
. \.\.
O,.4
;
,-,. ",,
~.~.
.~ .;~ ~
e~ 4\\ '\
'i
No
= ~'~ o ~
\i',
~ . -'-z. ~ ~
o-~'g o
\i' \i~
~oC~
]
LD~ •
I
,d @4I
A
.:-.
umgO~ O0
750
RENVERSEZ
J.C. et coll.
In a second series of experiments, Fab fragments from each cryo IgG were used to absorb the corresponding IgM fraction and the results were compared with the absorption with polyc!onal Fab from normal pooled IgG. With all IgM fractions, there was an extensive decrease of the reactivity with Fab after the incubation with the corresponding solid phase Fab (4 to 18% of residual activity) (Table II). This absorption with Fab also led to a variable but often very significant decrease of the anti-Fc activity of the IgM fractions (less than 50% residual activity) in 10/18 IgM. A similar absorption of a polyclonal RF IgM on Fab from polyclonal IgG did not lead to any decrease of the anti-Fc activity (Table II). These results were confirmed using 3 cryo IgM at various concentrations (Fig. 6). Absorption experiments confirmed a more efficient depletion of the reactivity with Fab after absorption on solid phase Fab, than on solid phase Fc. Inversely the reactivity with Fc is better absorbed on solid phase Fc.
DISCUSSION
The main point of this work is the demonstration of a frequent restriction in the clonotypes of the IgG molecules involved in the formation of cryoprecipitates with monoclonal IgM in type II mixed cryoglobulinemia It also confirms the possibility of idiotypic interactions in many of these cryoprecipitates. By definition the IgM of mixed type II cryoglobulins is a monoclonal immunoglobulin. In the present study, each IgM fraction was found homogeneous by immunoelectrophoresis and by isoelectrofocusing with a very restricted pattern of bands and a well defined Pi. Furthermore, there was a prevalence of VH I and VK I variable region subgroups, which suggests that some physicochemical characteristics may be common to some of these IgM, although they differed from those analyzed by other authors who found a prevalence of VK III in their series of monoclonal RF IgM [23], or from polyclonal RF IgM [24]. The homogeneity of the sPectrotypes in IgM from mixed cryoglobulins has been previously described in 4 cases using liquid isoetectrofocusing [25]. Monoclonal IgM antiglobulins have also been studied with respect to their idiotypes and cross idiotypic reactions. Specific common antigen among IgM antigammaglobulins were found using a precipitation assay [26]. A cross- idiotypic specificity was clearly demonstrated among a group of monoelonal IgM antiglobulins, using antisera prepared against single monoclonal IgM [27, 28].
IDIOTYP1C I N T E R A C T I O N S I N C R Y O G L O B U L I N S
751
Our analysis of the IgG c o m p o n e n t of m i x e d type I I cryoglobulins indicated a p a r t i c u l a r selection of IgG molecules during cryoprecipitation. All these fractions contained m o s t l y IgG a subclass molecules. Although b o t h k a p p a and l a m b d a light chains w e r e represented, t h e r e was a very r e s t r i c t e d charge heterogeneity in the IgG and a very h o m o g e n e o u s profile was seen for s o m e of them. The VH I subg r o u p was p r e d o m i n a n t since it w a s f o u n d alone in 9/18 IgG and in association with V H I t a n d / o r VH I I I in 6 o t h e r IgG fractions. There was not such restriction for the light chain V subgroups. The distribution of G m and K m allotypic m a r k e r s did not indicate a p a r t i c u l a r selection differing f r o m n o r m a l polyclonal IgG. A n u m b e r of o b s e r v a t i o n s have suggested that t h e r e is a p r e d o m i n a n c e of the IgGa subclass in cryoglobulins [29, 30, 31]. A restricted isoelectrofocusing profile for one IgG out of five isolated f r o m mixed cryoglobulins has b e e n also previously described [32]. Our results s u p p o r t the hypothesis of a selection of certain subspecies of IgG f r o m the heterogeneous IgG pool during cryoprecip i t a t i o n and are consistant with a r e s t r i c t e d reactivity b e t w e e n the two c o m p o n e n t s in the IgG-IgM cryoglobulins. Similarly a r e s t r i c t e d reactivity profile was exhibited for 6 cryo IgM with antiglobulin activity against autologous or isologous h u m a n IgG or their subunits [33]. In a p o p u l a t i o n of 11 m o n o c l o n a l IgM f r o m type I I cryoglobulins, multiple antiglobulin specificities w e r e shown [14], S o m e of the IgM w e r e reactive with Fc f r a g m e n t s ; s o m e others reacted w i t h IgG F(ab')2 f r a g m e n t s . S o m e of the anti-Fab antibodies w e r e directed against IgG idiotypes. These a u t h o r s suggested t h a t the IgM anti-Fab m a y react as anti-idiotypic antibodies with IgG. I n o u r investigations, we have c o n f i r m e d t h a t all of the 18 cryo IgM studied reacted with autologous a n d isologous cryo IgG. However this reaction was often of relatively low avidity since, for m o s t of the cryo IgM, it was only d e m o n s t r a t e d w h e n at least 10 m i c r o g r . / ml of IgG w e r e used for coating in the solid p h a s e assay. At lower c o n c e n t r a t i o n s of IgG (e.g. 1 m i c r o g / m l ) , m o s t cryo IgM did r e a c t w i t h the cryo IgG f r o m the c o r r e s p o n d i n g cryoglobulin b u t only w i t h a limited n u m b e r of cryo IgG f r o m o t h e r cryoglobulins. The p a t t e r n o b s e r v e d suggests a p r e f e r e n t i a l reactivity of the cryo IgM for s o m e IgG p r e p a r a t i o n s . This does not a p p e a r related to the IgG subclasses, since m o s t cryo IgG w e r e I g Q , n o r to be allotypic m a r k e r s of IgG. The fact t h a t m o s t of the cryo IgM react with b o t h Fab and Fc f r a g m e n t s of the reacting IgG suggests two types of interactions b e t w e e n cryo IgM and cryo IgG. Indeed, we can exclude the possibility t h a t the reactivity of cryo IgM with Fab would be due to c o n t a m i n a t i n g Fc since (a) m a n y cryo IgM react m o r e with
752
R E N V E R S E Z Y.C. et coll.
Fab t h a n w i t h Fc, (b) each cryo IgM exhibits a p r o p e r p a t t e r n of reactivity w i t h the Fab f r a g m e n t s f r o m the 18 cryo IgG a n d (c) polyclonal RF did not r e a c t with a n y of the Fab p r e p a r a t i o n s used. The reaction b e t w e e n cryo IgM and Fab is not due to a reactivity of those Fab w i t h the c o n s t a n t p a r t of IgM since t h e r e was no binding to those Fab of o t h e r m o n o c l o n a l IgM f r o m p a t i e n t s with W a l d e n s t r 6 m m a c r o g l o b u l i n e m i a . One should n o t e t h a t the absorption of cryo IgM on solid p h a s e Fc did lead to a decrease of their binding to Fc b u t w i t h 8/18 cryo IgM, t h e r e was not a parallel decrease of their binding to Fab f r a g m e n t s f r o m the c o r r e s p o n d i n g IgG. Inversely, although the a b s o r p t i o n of cryo IgM w i t h Fab f r o m the c o r r e s p o n d i n g cryo IgG did inhibit c o m p l e t e l y the binding of all cryo IgM to Fab, t h e r e was only a partial parallel depletion of the anti-Fc activity. These d a t a suggest the existence of a certain heterogeneity in the m o n o c l o n a l IgM molecules and c o n f i r m the involv e m e n t of 2 different sites of reaction. S o m e cryo IgG a p p e a r to i n t e r a c t w i t h cryo IgM similarly to anti-idiotypic antibodies directed against the p a r a t o p e of the IgM RF. Thus, all these d a t a are consistant, w i t h the idea t h a t in a b o u t two thirds of the studied cryoglobulins, the IgM RF would p r e f e r e n t i a l l y r e a c t with anti-idiotypic IgG. S o m e of these anti-idiotypes are probably directed against d e t e r m i n a n t s outside the p a r a t o p e , b u t in s o m e cases, they would r e a c t w i t h the anti-Fc p a r a t o p e . I n s o m e cryoglobulins, it is likely t h a t the avidity of the IgM RF w o u l d b e sufficient by itself to lead to the f o r m a t i o n of stable IgM-IgG complexes. However, the coexistence of 2 reactions, anti-Fc IgM w i t h IgG-Fc and anti-idiotype IgG with IgM idiotypes, should confer a higher stability to IgM-IgG complexes. One m a y w o n d e r w h e t h e r the cross-reactives idiotypes which have been identified on m o n o c l o n a l or polyclonal RF [27, 9] are involved in the recognition of IgM by auto-anti-idiotypic IgG. The i n v o l v e m e n t of anti-idiotypic antibodies in m i x e d c r y o g l o b u l i n e m i a allows for s o m e hypothesis concerning the pathogenesis of the disease. I t is possible t h a t the a b n o r m a l p r o l i f e r a t i o n of one clone of IgM-RF p r o d u c i n g B cells would b e followed in s o m e patients b y a relatively r e s t r i c t e d anti-idiotypic response. The equilibrium b e t w e e n these two processes m i g h t d e t e r m i n e s p o n t a n e o u s variations in the expression of the disease. As it was previously suggested [14], such o b s e r v a t i o n s are consistant w i t h the existence of idiotypic interactions in man. Besides a physiological role in the regulation of the i m m u n e r e s p o n s e [8] one should consider the possibility t h a t idiotype anti-idiotype complexes w o u l d r e p r e s e n t a m a j o r source of cryo-
1DIOTYPIC INTERACTIONS IN CRYOGLOBULINS
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precipitating i m m u n e complexes and m a y be involved in h u m a n glomerulonephritis, vasculitis or arthritis. SUMMARY An analysis of interactions b e t w e e n i m m u n o g l o b u l i n molecules within cryoglobulins has b e e n carried out in 18 patients w i t h type I I mixed cryoglobulinemia. I n this series, t h e r e was a prevalence of VH I and VK I variable regions s u b g r o u p s in the m o n o c l o n a l IgM c o m p o n e n t . Our analysis of the IgG c o m p o n e n t indicated a p a r t i c u l a r selection of I g G molecules during cryoprecipitation. T h e r e was a prevalence of IgG3 and of the VH I s u b g r o u p and the isoelectrofocusing p a t t e r n revealed a very r e s t r i c t e d s p e c t r o t y p e in two thirds of these IgG. These results w h i c h suggested a r e s t r i c t e d reactivity b e t w e e n cryo-IgM and IgG fractions w e r e c o n f i r m e d b y the analysis of the interaction b e t w e e n each IgG and each IgM f r o m the cryoprecipitates. All IgM r e a c t e d w i t h intact IgG or Fc f r a g m e n t s b u t a n o t h e r reaction was o b s e r v e d b e t w e e n cryo-IgM and Fab f r a g m e n t s f r o m a limited n u m b e r of cryo-IgG, with a p a t t e r n suggestive of idiotypic specificity. Results of the a b s o r p t i o n of each cryo-IgM on Fc or on Fab fragm e n t s f r o m the c o r r e s p o n d i n g cryo-IgG also suggested the existence of a reaction b e t w e e n IgM a n d IgG Fab in addition to t h a t involving IgM Fab a n d IgG Fc. The coexistence of the 2 reactions should confer a higher stability to the IgM-IgG complex. Therefore, it is possible t h a t the p r o l i f e r a t i o n of one clone of IgM-RF p r o d u c i n g B cells w o u l d be followed in certain cases b y a relatively r e s t r i c t e d anti-idiotypic IgG response. The IgM-RF w o u l d preferentially r e a c t w i t h these anti-idiotypic IgG. Request reprints from: Jean-Charles RENVERSEZ, M.D., Laboratoire de Biochimie A Centre Hospitalier R6gional et Universitaire de Grenoble 38043 GRENOBLECedex, France.
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