- Email: [email protected]
Antiidiotypic suppression of autoantibodies with normal polyspecific immunoglobulins
© ELSEVIER Paris 1989
Res. Immunol. 1989, 140, 19-31
ANTIIDIOTYPIC SUPPRESSION OF AUTOANTIEgODIES WITH NORMAL POLYSPECIFIC IMMUNOGLOBULINS F. Rossi, Go Dietrich and M.D. Kazatchkine INSERA4 U28, HOpitai Broussais, 75014 Paris
SUMMARY A mechanism by which therapeutic normal polyspecific immunoglobulins (IVIg) may suppress autoimmune responses in vivo is that of antiidiotypic suppression of autoantibodies mediated by anti-idiotypes present in IVIg. In vitro incubation with iVig of either the plasma or the IgG fraction from plasma of patients with autoantibodies against procoagulant factor VIII (VIII:C), DNA, thyroglobulin, peripheral nerve and intrinsic factor resulted in dosedependent inhibition of autoantibody activity. The pattern of inhibition curves showed a prozone phenomenon. Maximal inhibition was achieved at a ratio of patient's IgG to .twt . . . . :,:.. ,,,, ~,. . . ., ~., , . . ~ ,,., antibody tested. Inhibi• l~. . . . , , , , .k:..t. ..,.,., was ~,~,,11,. tion wa~ dependent on idiotypic/antiidiotypic interactions between autoantibodies and IVIg since: 1) F(ab')2 from IVIg inhibited autoantibody activity in F(ab')2 fragments from patients' IgG; 2) IVIg contained no antigen-like activity and no antibodies against the commonest allotypes expressed in F(ab')2 fragments of human IgG; 3)autoantibody activity in F(ab')2 fragments from patients' IgG was specifically retained on affinity columns of Sepharose-bound F(ab')2 fragments from IVIg. The presence of antiidiotypes against autoantibodies in pooled normal IgG supports the concept of a functional idiotypic network regulating autoimmune responses in man. Ir~Y-WORDS" Therapeutic immunoglobu'in, Idiotype, Immunosuppression, Autoimmunity; Antiidiotypic suppression, Polyspecific Ig.
Received September 8, 1988. This manuscript was originally scheduled for publication in the November 1988 special issue on ~Idiotypy 1988, (P.-A. Cazenave).
20
F. R O S S I A N D COLL.
The evidence that autoantibodies are encoded by genes derived from a limited number of V germ-line genes and may arise from perturbed idiotypic regulation of self-reactive clones [1] has opened the prospect for antiidiotypic therapeutic intervention in organ-specific autoimmune diseases. Downregulation of autoantibody responses has been obtained with autologous or syngeneic anti-idiotypes in adult animals in several experimental models of autoimmune aiseases including autoimmune thyroiditis [2], myasthenia gravis [3], autoimmune interstitial nephritis [4] and murine systemic lupus erythematosus (SLE) [5]. Clinical observations indicate that spontaneously occurring anti-idiotypes in patients with autoimmune diseases may regulate the production of rheumatoid factors [6, 7] and of autoantibodies to DNA [8], the acetylcholine receptor [9, 10] and procoagulant factor VIII (VIII:C) [11]. This manuscript reviews some of the in vitro evidence indicating that therapeutic preparations of normal polyspecific immunoglobulins for intravenous use (IVIg) contain anti-idiotypes against alitoantibodies [11, 12-14]. Results from these studies may explain the beneficial effect that has been observed with IVIg in some autoimmune diseases [15] and support the concept of a functional idiotypic network regulating autoimmune responses in man. IVIg preparations were ~ Sandoglobulin, (Sandoz SA, Basel, Switzerland) and immunoglobulins from the Central Laboratory of the Dutch Red Cross Blood Transfusion Service (Amsterdam, The Netherlands), two preparations of intact IgG obtained from large plasma pools of normal donors and ~ Veinoglobuline, (Institut Merieux, Lyon, France), a preparation containing intact IgG and F(ab) fragments obtained from a pool of placental blood from a large number of donors. Anti-VIII:C antibody titre was measured by the method of Kasper et al. [16] and expressed in Bethesda Units (BU). VIII:C activity was measured in ou~-~tag~ . . . . . a ~ y '--oy ~'m~ acuvmeu -~"..... -' paIxlm thrombo astin time method, with plasma of a severe haemophiliac as substrate. Antithyroglobulin antibodies and anti-DNA antibodies were quantitated by ELISA with purified porcine thyroglobulin and calf thymus DNA as antigens and a revealing peroxidaselabelled goat anti-human Fc antibody (Cappel Laboratories, Cochranville, PA). Anti-thyroid microsome antibodies and antibodies against the 108ccl 5 neuroblastoma cell line antigen (Van Doorn, P.A., Rossi, F., Brand, A., Lint, M.V., Vermeulen, M., and Kazatchkine, M.D., Treatment of chronic inflammatory demyelinating polyneuropathy with high dose intravenous immunoglobulins, submitted) were determined using semi-quantitative indirect immunofluorescence assays. Anti-intrinsic factor activity was measured using a radioimmunoassay in which the autoantibody was competing with radiolabelled vitamin B12 for binding to human gastric intrinsic factor [17]. -
-
-
BU = Bethesda unit. C I D P = chronic inflammatory demyelinating polyneuropathy. Ig = immunoglobulin.
--~'-'
pl
IVIg = normal polyspecific imrnunoglobulin for intravenous use. SLE = systemic lupus erythematosus.
POL YSPECIFIC Ig SUPPRESS A UTOANTIBODIES
21
The IgG fraction from patients' plasma was prepared by ammonium sulphate precipitation and chromatography on DEAE Trisacryl (IBF, Villeneuve la Garenne, France). F(ab')2 fragments which were free of detectable Fc fragments were prepared from IgG by pepsin digestion (2 % W/W) and chromatography on protein A Sepharose. Initial studies concerned two patients with autoantibodies against VIII:C in where intravenous administration of 0.4 g/kg/body weight of Sandogiobulin for five consecutive days resulted in a 95 °70decrease in circulating autom~tibody titre [12] (fig. 1). The decrease in autoantibody titre occurred within 24-48 h of infusion of IVIg. The autoantibody remained at its lowest titre in plasma for 2 to 3 weeks before it slightly increased for 2 weeks and then decreased again. A similar cycle occurred after three weeks in a pattern that resembled the cyclical appearance of Ab3 antibodies in experimental animals immunized with Abl [18]. In both patients, the autoantibody titre has remained between 5 and 10 070of preIVIg levels for over four years now. Similar effects of iVIg on anti-VIII:C autoantibody titre in vivo have been reported by others [19].
I£
__w~300_,
'
=, >
mOO0,
)
~
i)
'is
'~
" ~z
a~ ' ~'0
a~
'fo
dayu
FIO. 1. - - Time course o f decrease in autoantibody titre following infusion o f IVIg (arrows) in a patient with an anti-VIII:C autoantibody ( f r o m [12]).
22
F. R O S S I A N D COLL.
loo] o - - o Patient 1 "~
e
~ Patient 2
=
'=-
so
g
0
1
W/W ratio of patient's
10
IgG to therapeutic IgG
FIG. 2. - - Neutralization o f anti- VIII:C activity in the plasma o f two patients with anti- VIII:C autoantibodies upon incubation with IVlg ( f r o m [12]).
The rapid suppressive effect of IVIg on autoantibody titre in vivo suggested that IVIg directly interacted with circulating anti-VIII:C artoantibodies. The prolonged effect of IVIg, far beyond the half life of infused IgG, indicated that infused IVIg interfered with autoantibody synthesis. In vitro incubation of the patients' plasma with IVIg for 60 min at 37°C and overnight at 4°C resulted in a dose-dependent neutralization of autoantibody activity [12] (fig. 2). Maximal inhibition was observed at a specific molar ratio between IVIg and patient's IgG in each patient. An increase or a decrease in the optimal inhibitory ratio resulted in less or no inhibition of autoantibody activity, in an analogous fashion to the prozone phenomenon observed in precipitation curves. Several lines of evidence suggested that the inhibitory effect of IVIg on anti-VIII:C activity was mediated by anti-idiotypes against anti-VIII:C autoantibodies, present in the,' therapeutic immunoglobulin preparation: 1) IVIg contained no antigen-like (VIII:C) activity; 2) IVIg contained no detectable rheumatoid factor activity; a"~" . inl r~'-'-'" t,a o J2 fragments from t. .v. .lg hibited autoantibody activity in the IgG fraction and in F(ab')2 fragments from IgG of patients with anti-VIII:C autoantibodies; 4) IVIg contained no antiaUotypic antibodies directed against the G m l (3), G m l (4), G m l (17), G m l (1) and Km (1) allotypes which are most commonly expressed in the
~'C POL YSPEC!F!C !g S ~T.D. D D,,,-.~o Af
T'7"f~ A
,,~
~r-rrD~.~ r ~ r ~ o
F(ab')2 region of human IgG [13]. Inhibition by F(ab')2 fragments from IVIg of anti-VIII:C activity in F(ab')2 fragments prepared from the plasma of patients with autoantibodies is shown in table I. Inhibition curves followed the pattern of precipitation curves with a maximum of inhibition being achieved at a specific molar ratio between patient's F(ab')2 and F(ab')2 from IVlg. It is interesting to note that anti-VIII:C autoantibody activity from one patient (patient Gra.) was inhibited by one preparation of IVIg but not by the other, indicating that IVIg preparations may differ in their respective quantitative and qualitative content in specific anti-idiotypes.
TABLE I. - - Inhibition of anti-VIII:C activity in F(ab')z fragments obtained from patients with anti-VIII:C autoantibodies by F(ab') 2 fragments from IVIg.
IVIg F(ab:)2
Patient Gra. Patient Bes. Patient Esk.
SAGL
VGL
070 inhibition
070 in~bition
0.45 (0.45-1.80) 1.60 (0.40-3.20) 2.60 (0.60-5.20)
100 °70 76 070 100 070
0 070 (0.24-3.90) 3.00 (0.75-12.2) N.D.
75 070 N.D.
Indicated are the maximal inhibition of anti-VIII:C activity observed and the molar ratio between F(ab') 2 from patients' containing anti-VIII:C activity and F(ab')2 from IVIg at which maxima~ inhibition was achieved. Parentheses indicate the range of molar ratios between F(ab') 2 containing anti-VIII:C antibodies and F(ab')2 from IVIg that were tested. SAGL = <>; VGL = <~.
Table II (upper panel) shows the results of experiments similar to those described in table I which were performed with F(ab')2 fragments prepared from prerecovery and postrecovery plasma of a patient (patient Esk.) who spontaneously recovered from anti-VIII:C autoimmune disease [11]. F(ab')2 fragments from samples obtained 6 weeks and 4 years after recovery of the patient inhibited anti-VIII:C activity in F(ab')2 fragments from the patient's prerecovery plasma. Similar amounts of F(ab')2 fragments from late pn~t,,ecovery_~.. (4 years) ~,,,,~,,,,,"! . . . . . . ,,,u-~ ~,,"¢F(ab')2 from IVIg were required to achieve 100 070 inhibition of autoantibody activity in F(ab')2 fragments from prerecovery plasma, suggesting that the content in anti-idiotype of IVIg is equivalent to that which prevents expression of anti-VIII:C autoantibodies x'1,ao /2 f r o m postrecovery plasma in postrecovery plasma from the patient. ~,.k,~ .~ of patient Esk. also neutralized anti-VIII:C activity of F(ab')2 fragments
24
F. R O S S I A N D COLL. TABLE II. - - Inhibition of anti-Vlll:C activity in F(ab')2 fragments obtained from patients with anti-VIH:C autoantibodies by F(ab')2 fragments from patient Esk.'s post-recovery plasma (upper panel) and inhibition of anti-neuroblastoma cell line activity in the serum of a patient with CIDP by F(ab')2 fragments from patient B.'s post-recovery plasma (lower panel).
F(ab')2 from IVlg(*) F(ab')2 fragments from patient Esk.'s postrecovery plasma 6 weeks 4 years Ratio ~/o inhibition Ratio 070inhibition Ratio 07oinhibition 100 070 50 070 100 070
Patient Gra. F(ab')2 0.72 Patient Bes. F(ab')2 5.48 Patient Esk. F(ab')2 4.80
0.~9 5.12 2.40
100 070 54 070 100 070
fragments from patient B.'s postrecovery plasma Ratio 070inhibition
F(ab')2
Patient B. (serum) Patient with CIDP (serum)
0.28
100 070
0.019
100 070
0.45 1.60 2.60
100 070 76 070 100 070
F(ab')2 from IVlg Ratio 07oinhibition N.D. 0.021
94 070
Indicated are maximal inhibition of autoantibody activity observed and molar ratio between F(ab')2 or IgG in serum containing the autoantibody and F(ab')2 from postrecovery plasma or IVIg at which maximal inhibition was achieved. (*) Sandoglobu!in.
prepared from the plasma of two other patients with anti-VIII:C autoantibodies. These results suggest that spontaneous recovery of patient Esk. from autoimmune disease was associated with the ~eneration of anti-idintvneg against prerecov~y antibodies and that anti-VIII:C autoantibodies from different patients share cross-reactive idiotypes. The lower panel of table II depicts analogous results obtained with postrecovery serum and F(ab')2 fragments from the serum of a patient who recovered from Guillain Barr6 syndrome. Postrecovery serum from this patient (patient B.) inhibited the binding of antiperipheral nerve autoantibodies in the patient's prerecovery serum to the 108cc 15 neuroblastoma cell line. The neuroblastoma cells express a membrane antigen that cross-reacts with human peripheral nerve [20]. Postrecovery antibodies from patient B. also inhibited, although less efficiently, autoantibody activity in the serum of a patient with chronic inflammatory demyelinating polyneuropathy (CIDP), the chronic form of Guillain Barr6 syndrome. F(ab')2 prepared from IVIg had an equivalent capacity to that of F(ab')2 fragments from the serum of patient B. to inhibit autoantibody activity in the serum of the patient with CIDP. Results shown in table II indicate that anti-idiotypes against prerecovery antibodies which may occur in sera from patients who recovered from autoimmune disease are more efficient in inhibiting antibody activity of the patient's own (prerecovery) autoantibodies than the activity of other patients' autoantibodies. This is probably due to -
~
J
F
25
POL YSPECIFIC Ig SUPPRESS A UTOANTIBODIES
the fact that spontaneously generated postrecovery anti-idiotypes are directed against both shared and private idiotypic specificities expressed by patient's prerecov~-y autoantibodies. The results also indicate that IgG from postrecovery plasma and IVIg have an equivalent inhibitory capacity towards autoantibodies from other patients. IVIg and F(ab')2 fragments from IVIg were also found to inhibit autoantibody activity in the serum and F(ab')2 fragments prepared from the IgG fraction of patients with other autoantibodies, including antibodies to thyroglobulin, thyroid microsomes, DNA, peripheral nerve and intrinsic factor (table III) [14]. As for autoantibodies to VIII:C, autoantibodies of various specificities which were inhibited by IVIg in vitro represented 40 to 80 °70 of all antibodies tested. In contrast, we have thus far never observed an inhibitory effect of IVIg on cold anti-erythrocytic antibody activity.
TABLE III. ~ Inhibition of autoantibody activity in the IgG fraction or serum from individual patients with autoimmune diseases by F(ab')2 fragments from IVlg.
Autoantibody specificity Thyroglobulin
F ( a b ' ) 2 fragments from SAGL VGL Ratio °70 inhibition Ratio 070 inhibition
0.090 (0.090-5.730) Thyroid microsomes 0.867 (0.430-5.780) DNA 0.280 (0.035-0.560) Peripheral nerve 0.015 (108cc15 neuroblastoma (0.005-0.023) cell line) Intrinsic factor 6.670 (0.417-13.25)
61 07o 100 070 76 °70 100 070 36 070
0.045 37 070 (0.045-1.440) N.D. 23.04 32 070 (0.720-46.08) N.D.
(0.126-8.08)
0 070
Indicated are the maximal inhibition of autoantibody activity found and the molar ratio between IgG containing autoantibodies and F(ab')2 from IVIg at which maximal inhibition was achieved. Parentheses indicate the range of molar ratios between IgG containing autoantibodies and F(ab') 2 from Wig which are tested. SAGL = Sandoglobulin; VGL = Veinoglobuline.
The interactions between antiidiotypic antibodies present in IVIg and autoantibodies were further investigated by examining the binding of the IgG fraction from patients with autoantibodies to F(ab')2 from IVIg and the specific binding of F(ab')2 containing autoantibody activity to affinity columns of Sepharose-bound F(ab')2 fragments from Wig [11, 13]. Microtitre plates were coated with F(ab')2 from IVIg and interacted with anti-VIII:Ccontaining IgG before addition of peroxidase-labeUed anti-human Fc gamma
26
F. R O S S I A N D C O L L .
antibody. As shown in figure 3, IgG from the three patients bound to F(ab')2 from IVlg, including the IgG from patient Gui., in which anti-VIII:C activity was not neutralized by IVIg in functional experiments. IgG from another patient, patient Gra., was found to bind to F(ab')2 from Sandoglobulin and F(ab')2 from Veinoglobuline although anti-VIII:C activity in the IgG preparation was only inhibited by one IVIg preparation (Sandoglobuiin) (table I). IgG from a normal donor also bound to insolubilized F(ab')2 from IVIg, further indicating that a number of idiotype/anti-idiotype interactions unrelated to anti-VIII:C/anti-anti-VIII:C interactions are likely to be involved in the binding of IgG from patients with anti-VIII:C antibodies to IVIg. Using the ELISA described above, we have recently found that F(ab')2 from two IVIg preparations (Sandoglobulin and Veinoglobuline) overall express a similar antiidiotypic repertoire [13]. As mentioned earlier, different preparations of IVIg may, however, differ in their relative content in anti-idiotypes against specific autoantigens (e.g. VIII:C) (table I). In order to demonstrate that IVIg contain antibody species that can specifically bind autoantibodies, affinity chromatography experiments were performed using columns of Sepharose to which F ( a b ' ) 2 from IVIg had been bound [11, 13]. Results of affinity experiments with anti-VIII:C antibodies are shown in table IV. Columns were loaded with buffer containing F ( a b ' ) 2 fragments with autoanti-VIII:C activity, washed until no protein could be detected in the effluent and then eluted at acidic pH. Specific binding of
0.~-. o
E c
qP 0 u c 9 JD ,0
4( 0.1 •
i
O~
~
O5
2
IgG
(mo~l)
FIG. 3. - - Binding of IgG from patients Gra. ( I ) , Bes. (0), Gui. (&) and from a normal individual (~) to insolubilized F(ab')2fragments from ~ Sandoglobulin ~ (open symbols)
and ~ Veinoglobuline~> (clo~ed symbols) as asseised by ELISA.
POL YSPECIFIC Ig SUPPRESS A UTOANTIBOD!ES
27
F(ab')2 anti-VIII:C antibodies to the column was assessed by measuring antibody activity in the acid eluate from the column. A specific enrichment in autoantibody activity in the acid-eluted fractions was calculated by comparing the specific antibody activity (BU/mg) loaded onto the column with that in the acid eluate. In the case of patients in whom anti-VIII:C autoantibodies were neutralized by IVIg in vitro (i.e. patients Bes., Gra. and Cer. presented in table IV), the acid-eluted F(ab')2 antibodies contained an anti-VIII:C specific activity that was enriched 1.6- to 40-fold as compared with the specific activity of the material loaded on the column. This indicated that at least some anti-VlII:C autoantibodies were specifically recognized in a high-affinity binding interaction by some antibody (anti-idiotype) species in IV!g. Sandoglobulin-sensitive anti-VlII:C F(ab')2 from patient Gra. were only recovered from a Sepharose-Sandoglobulin column, but not following affinity chromatography on Sepharose-Veinoglobuline. In a similar fashion~ no antiVIII:C activity was recovered in the acid-eluted fractions upon chromatography on Sepharose-Sandoglobulin and Sepharose-Veinoglobuline of F(ab')2 prepared from the plasma of patient Gui., in whom anti-VIII:C activity was not inhibited by either preparation of IVIg. Thus, the ability of an IVIg preparation to neutralize the anti-VIII:C activity of autoantibodies is strongly correlated with the presence of demonstrable anti-idiotypes against anti-VlIl:C antibodies by affinity chromatography. Analogous experiments were also performed using Sepharose-bound F(ab')2 fragments from postrecovery serum of patient Esk. as a soerce of immobilized anti-idiotypes [11]. Affinity chromatography of F(ab')2 fragments prepared from the patient's prerecovery serum resulted in an 80-fold enrichment in specific anti-VIII:C activity in the acid-eluted fractions from the column. The relatively high ability of patient Esk.'s postreccwery antibodies to recognize idiotypes expressed by the patient's prerecovery antibodies is conceivable if one considers that recovery from autoimmune disease is associated with generation of anti-idiotypes against both public and private idiotypes
TABLE IV. - - Affinity chromatography of F(ab')2 fragments containing anti-VIll:C activity on Sepharose-bonnd F(ab')z fragments from SAGL and VGL.
Sepharose-boundF(ab')2 from SAGL Sepharose-boundF(ab')2 from VGL Loaded Eiuted Loaded Eluted Amount Specific Amount Specific Amount Specific Amount Specific Anti-VlIl:C (ttg) anti-Vlll:C (ttg) anti-VlIl:C (ttg) anti-Vill:C (~tg) a.nti-Vlll:C F(ab')2 (BO/mg) (BU/mg) (BU/mg) (BU/mg) Patient Bes. 4250 Patient Gra. 5670 Patient Cer. 9000 Patient Gui. 10.71
3 2.38
45 315
5 11.80
4 4
0.98 98
73 0.00
38.09 1680
10.71
S A G L -- S a n d o g l o b u l i n ; V G L = V e i n o g l o b u l i n e .
3 10.20 N.D. 56
945 78 0.00
12.5 0.00
28
F. R O S S I A N D COLL.
expressed by the patient's autoantibodies. Columns of Sepharose-bound F(ab')2 from patient Esk.'s postrecovery plasma were less efficient in specifically retaining anti-VIII:C antibodies from another patient [11], indicating that anti-VIII:C antibodies from that patient and from patient Esk. express cross-reactive idiotypes that only account for part of the idiotypic specificities recognized by patient Esk.'s postrecovery IgG. Specific binding of F(ab')2 from autoantibodies to Sepharose-bound F(ab')2 from IVIg has also been recently documented with antithyroglobulin and with anti-DNA antibodies (F. Rossi and M.D. Kazatchkine, unpublished results), thus strengthening the general concept that IVIg contain anti-idiotypes against autoantibodies. It is unclear at the present time whether all or only a few of the donors contribute to the IVIg pool with anti-idiotypes. Based on the network theory [21] it may be speculated that all (or most) donors' plasma contain antiidiotypic antibodies directed against private and/or some cross-reactive idiotypes expressed by autoantibodies. The presence of a potent antiidiotypic activity in pooled IVIg may represent the sum of multiple antiidiotypic specificities. Recent studies by Tankersley et al. [22] indicate that, as the number of donors increases in an IgG preparation, an increasing amount of dimers formed between idiotypes and anti-idiotypes is found in the pool. Likewise, the probability of expression of a specific anti-idiotype in a therapeutic IgG preparation should increase with the size of the donor pool. Intravenous infusion of high doses of IVIg has resulted in clinical improvement and/or a decrease in autoantibody titre in a number of autoimm_une diseases including idiopathic thrombocytopenic purpura [23], autoimmune neutropenia [24], autoimmune red cell aplasia [25]~ n-iyasthenia gravis [26], CIDP [27] and in patients with autoantibodies to VIII:C [12]. Several mechanisms may account for the be~:eficial effect of IVig [15]. In addition to anti-VIII:C autohr.rn-une disease, there is in vitro evidence that the beneficial effect of IVIg in patients with autoimmune red cell aplasia [25] and chronic autoimmune inflammatory demyelinating polyneuropathy (Van Doom et al., ibid., submitted) is also dependent on antiidiot~qaic neutralization of autoantibodies. Furthermore, and as mentioned earlier, the suppressive effect of IVIg in some patients with anti-VIII:C autoantibodies has been prolonged far beyond the half life of infused IVIg, suggesting that, in addition to shortterm inhibitory effects, anti-idiotypes in IVIg may alter the expression of the patients' repertoire by autoantibody-producing cells. Results summarized in this manuscript may open prospects for further investigation of therapeutic anti-idiotype suppression of autoantibodies with IVIg. The demonstration of anti-idiotypes in normal immunoglobulins supports the concept of a functional idiotypic network regulating autoimmune responses in man.
P O L Y S P E C I F I C Ig S U P P R E S S A U T O A N T I B O D I E S
29
RI~SUMt~ SUPPRESSION ANTIIDIOTYPIQUEDES AUTOANTICORPS A L'AIDE D'IMMUNOGLOBULINESPOLYSPI~CIFIQUESNORMALES
L'un des m6canismes par lesquels les immunoglobulines polysp6cifiques th6rapeutiques (IVIg) peuvent inhiber in vivo certaines r6ponses autoimmunes chez l'homme, est la suppression antiidiotypique des autoanticorps par des antiidiotypes pr6sents dans les IVIg. Nous avons observ6 que l'activit6 autoanticorps dans le plasma ou celle des IgG provenant de patients avec des anticorps anti-facteur VIII (VIII:C), anti-DNA, anti-thyroglobuline, anti-nerf p6riph6rique ou anti-facteur _intrins6que, est inhib6e in vitro lors de leur incubation avec des IVIg. Les courbes d'inhibition montrent un ph6nom6ne de prozone. L'inhibition maximale survient pour un rapport molaire - - entre les IgG du patient et les IVIg - - sp6cifique de chaque anticorps test6. Un certain nombre de raisons impliquent que l'inhibition de l'activit6 autoanticorps est d6pendante d'interactions entre idiotypes et antiidiotypes: 1) les fragments F(ab')2 des IVIg inhibent l'activit6 autoanticorps dans des fragments F(ab')2 pr6par6s/t partir des IgG contenant l'activit6 autoanticorps; 2) les IVIg ne contiennent pas d'activit6 biologique ressemblant/t celle de l'antig6ne, ni d'anticorps dirig6s contre les allotypes les plus commun6ment exprim6s dans les fragments F(ab')2 des IgG humaines; 3) l'activit6 autoanticorps pr6sente dans les fragments F(ab')2 pr6par6s /t partir des IgG des patients est sp6cifiquement retenue sur des colonnes de chromatographie d'affinit6 de fragments F(ab')2 d'IVIg. La pr6sence d'antiidiotypes dirig6s contre les autoanticorps dans des pools d'IgG provenant d'individus normaux est en faveur de l'existence d'un r6seau idiotypique physiologique r6gulant la r6ponse autoimmune chez rhomme. MOTS-CLi~S: Immunoglobuline th6rapeutique, Idiotype, Immunosuppression, Autoimmunit6; Ig polysp6cifiques, Suppression antiidiotypique.
A/'~V hJ('~[/I .i ~ . , z ~ . L ~,....i
]~" ~
vv
't.[~'k 4 ]t.t.t.t.t.t.t.t.I~.T ~" T ~
JL..,I...aZ.J~.~I-,I~II...,I~
•
This work was supported by INSERM, Sandoz SA and Institut M6rieux.
REFERENCES
[1] ROITT, I.M. & COOKE,A., Idiotypes and autoimmunity, in ~ Progress in im[2]
[3] [4] [5]
munology V I , (B. Cinader & R.G. Miller) (pp. 512-535). Academic Press, London, New York, 1986. ZANETTI, M. & BIGAZZI,P.E., Antiidiotypic immunity a::d autoimmunity. - I. In vitro and in vivo effects of antiidiotypic antibodies to spontaneously occurring autoantibodies to rat thyroglobulin. Europ. J. lmmunol., 1981, 11, 187-195. SOUROUJON,M.C., BERCHON,D. & FUCHS,S., Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes. Immunol. letters., 1985, 9, 331-336. NEILSON,E.G. & PHILLIPS,S.M., Suppression of interstitial nephritis by autoantiidiotypic immunity. J. exp. Med., 1982, 155, 179-189. HAHN, B.H. & EBLING, F.M., Suppression of murine lupus nephritis by administration of an antiidiotypic antibody to anti-DNA. J. Immunol., 1984, 132, 187-190.
30
F. R O S S I A N D C O L L .
[6] KOOPMAN,W.J., SCHROHENLOHER,R.E., BARTON, J.C. • GREENLEAF,E.C., Suppression of in vitro monoclonal human rheumatoid factor synthesis by antiidiotypic antibodies. J. clin. Invest., 1983, 72, 1410-1419. [7] GELTNER,D., FRANKLIN,E.L. & FRANGIONE,B., Antiidiotypic activity in the IgM fractions of mixed cryoglobulins. J. Immunol., 1980, 125, 1530-1535. [8] ABr,ou, N.I., WALL, H., LINDSEY,H.B., HALSEY,J.F. & SuzuKi, T., Network theory in autoimmunity: in vitro suppression of serum anti-DNA antibody binding to DNA by antiidiotypic antibody in systemic lupus erythematosus. J. din. Invest., 1981, 67, 1297-1301. [9] LEVVERT,A.K., Antiidiotypic antibodies against the receptor antibodies in myasthenia gravis. Scand. J. Immunol., 1981, 13, 493-497. [10] DWYER,D.S., BRADLEY,R.J., URQUnART, C.K. & KBARNEY, J.F., Naturally occurring antiidiotypic antibodies in myasthenia gravis patients. Nature (Lond.), 1983, 301, 611-614. [111 SULTAN,Y., ROSSI,F. & KAZATCtmU~B,M.D., Recovery from anti-VIII:C (antihemophilic factor) autoimmune disease is dependent on generation of antiidiotypes against anti-VIII:C autoantibodies. Proc. nat. Acad. Sci. (Wash.), 1987, 84, 828-831. [12] SULTAN, Y., KAZATCnrmCB, M.D., MAISO~EUVB, P. & NYDEGGER, U.E., Antiidiotypic suppression of autoantibodies to factor VIII by high-dose intravenous gammaglobulin. Lancet, 1984, If, 765-768. [13] Rossl, F., SULTAN,Y. & KAZATCHKXNB,M.D., Anti-idiotypes against autoantibodies and alloantibodies to VIII:C (anti-haemophilic factor) are present in therapeutic polyspecific normal immunoglobulins. Clin. exp. Immunol., 1988, 74, 311-316. [14] K A Z A T C ~ , M.D., Rossl, F., DmTRICH,G. & SULTAN,Y., Clinical relevance of antiidiotypic antibodies during treatment with high doses of intravenous immunoglobulins (IVIg), in t~Immunoglobulins)) (H.N. Krijnen). Elsevier, Amsterdam, 1988 (in press). [15] MORELL,A. & NYDEGGER,U.E., Clinical use of intravenous immunoglobulins. Academic Press, London, New York, 1986. [16] gASPER,C.K., ALEDORT,L.M., COUNTS,R.B., EDSON, J.R., FRATANTONE,J., GREEN,D., HEMPTON,J.W., HILGARTNER,M.N., LAZARSON,J., LEVIN,P.H., MACmLLAN,C.N., POOL, J.C., SHAPIRO,S.S., SCHULMAN,N.R. & EVES, J., A more uniform measurement of factor VIII inhibitors. Thrombos. Diathes. r~aemorrn, ( s t uttg. ), 1~13, .... ~4~ 809-81Z. [17] Rorrr, I.M. & DONIACH,D., Assay for the estimation of autoantibodies to gastric intrinsic factor in pernicious anaemia. W.H.O., Geneva, 1974. [18] COURAUD,P.O., Lu, B.Z. & STROSBERG,A.D., Cyclical antiidiotypic response to anti-hormone antibodies due to neutralization by autologous antiidiotypic antibodies that bind hormone. J. exp. Med., 1983, 157, 1369-1378. [19] Z~4ERMAN, R., KOMMERELL,]~., HARENBERG,J., EICH, W., ROTHER, K. & SCmMpS, K.Lo, Intravenous IgG for patients with spontaneous inhibitor to factor VIII. Lancet. 1985, I, 27a-2/4." "¢ [20] VAN DOORN,P.A., BRAND,A. & YERMEULEN,M., Anti-neuroblastoma cell line antibodies in inflammatory demyelinating polyneuropathy. Neurology, 1988 (in press). [211 JERNE, N.K., Towards ~ network ~heory of the immune response. Ann. lmmunol. (Inst. Pasteur), 1984, 125 C, 373-389. [22] TANKERSLEY,D.L., PRESTON, M.S. & FINLAYSON,J.S., Immunoglobulin G dimer: an idiotype-anti-idiotype complex. Mol. Immunol., 1988, 25, 41-48. [23] IMUAC,, P., BARA~g~UN, S., D'Apuzzo, V., BAUMGARTNER,C., HIRT, A., MORELL,A., RossI, E., SC8ONI,M., VEST,M. & WAGNER,H.P., High-dose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood. Lancet, 1981, I, 1228-1230. .
.
.
.
.
.
.
.
.
POL YSPECIFIC Ig S U P P R E S S A U T O A N T I B O D I E S
31
[24] BUSSEL, J.B., LALEZARI, P., HILGARTNER, M., PARTIN, J., FRIKRIG, S.,
O'MALLEY, J. ,~ BARANDUN,S., Reversal of neutropenia with intravenous gammaglobulin in autoimmune neutropenia of infancy. Blood, 1983, 62, 398-400. [25] McGUIRE, W.A., YANG, H.H., BRUNO, E., BRANDT, J., BRIDELL, R., COA~ES, T.D. & HOFFMAN,R., Treatment of antibody-mediated pure red cell aplasia with high dose intravenous gammaglobulin. New EngL J. Med., 1987, 317, 1004-1008. [26] GAJDOS,Ph., OUTIN, H., ELKARRAT,D., BRUNET, D., DE ROHAN-CHABOT,P., RAPHAEL,J.C. 8£ GOULON,M., High-dose intravenous immunoglobulin for myasthenia gravis. Lancet, 1984, I, 406-407. [27] VERMEULEN, M., VAN DER MECHE, F.G.A., SPEELMAN,J.D., WEBER, A. BUSCH, H.F.M., Plasma and gammag!obulin infusion in chronic inflammatory polyneuropathy. J. NeuroL Sci., i985, 70, 317-326.
Res. Immunol. 1989, 140, 19-31
ANTIIDIOTYPIC SUPPRESSION OF AUTOANTIEgODIES WITH NORMAL POLYSPECIFIC IMMUNOGLOBULINS F. Rossi, Go Dietrich and M.D. Kazatchkine INSERA4 U28, HOpitai Broussais, 75014 Paris
SUMMARY A mechanism by which therapeutic normal polyspecific immunoglobulins (IVIg) may suppress autoimmune responses in vivo is that of antiidiotypic suppression of autoantibodies mediated by anti-idiotypes present in IVIg. In vitro incubation with iVig of either the plasma or the IgG fraction from plasma of patients with autoantibodies against procoagulant factor VIII (VIII:C), DNA, thyroglobulin, peripheral nerve and intrinsic factor resulted in dosedependent inhibition of autoantibody activity. The pattern of inhibition curves showed a prozone phenomenon. Maximal inhibition was achieved at a ratio of patient's IgG to .twt . . . . :,:.. ,,,, ~,. . . ., ~., , . . ~ ,,., antibody tested. Inhibi• l~. . . . , , , , .k:..t. ..,.,., was ~,~,,11,. tion wa~ dependent on idiotypic/antiidiotypic interactions between autoantibodies and IVIg since: 1) F(ab')2 from IVIg inhibited autoantibody activity in F(ab')2 fragments from patients' IgG; 2) IVIg contained no antigen-like activity and no antibodies against the commonest allotypes expressed in F(ab')2 fragments of human IgG; 3)autoantibody activity in F(ab')2 fragments from patients' IgG was specifically retained on affinity columns of Sepharose-bound F(ab')2 fragments from IVIg. The presence of antiidiotypes against autoantibodies in pooled normal IgG supports the concept of a functional idiotypic network regulating autoimmune responses in man. Ir~Y-WORDS" Therapeutic immunoglobu'in, Idiotype, Immunosuppression, Autoimmunity; Antiidiotypic suppression, Polyspecific Ig.
Received September 8, 1988. This manuscript was originally scheduled for publication in the November 1988 special issue on ~Idiotypy 1988, (P.-A. Cazenave).
20
F. R O S S I A N D COLL.
The evidence that autoantibodies are encoded by genes derived from a limited number of V germ-line genes and may arise from perturbed idiotypic regulation of self-reactive clones [1] has opened the prospect for antiidiotypic therapeutic intervention in organ-specific autoimmune diseases. Downregulation of autoantibody responses has been obtained with autologous or syngeneic anti-idiotypes in adult animals in several experimental models of autoimmune aiseases including autoimmune thyroiditis [2], myasthenia gravis [3], autoimmune interstitial nephritis [4] and murine systemic lupus erythematosus (SLE) [5]. Clinical observations indicate that spontaneously occurring anti-idiotypes in patients with autoimmune diseases may regulate the production of rheumatoid factors [6, 7] and of autoantibodies to DNA [8], the acetylcholine receptor [9, 10] and procoagulant factor VIII (VIII:C) [11]. This manuscript reviews some of the in vitro evidence indicating that therapeutic preparations of normal polyspecific immunoglobulins for intravenous use (IVIg) contain anti-idiotypes against alitoantibodies [11, 12-14]. Results from these studies may explain the beneficial effect that has been observed with IVIg in some autoimmune diseases [15] and support the concept of a functional idiotypic network regulating autoimmune responses in man. IVIg preparations were ~ Sandoglobulin, (Sandoz SA, Basel, Switzerland) and immunoglobulins from the Central Laboratory of the Dutch Red Cross Blood Transfusion Service (Amsterdam, The Netherlands), two preparations of intact IgG obtained from large plasma pools of normal donors and ~ Veinoglobuline, (Institut Merieux, Lyon, France), a preparation containing intact IgG and F(ab) fragments obtained from a pool of placental blood from a large number of donors. Anti-VIII:C antibody titre was measured by the method of Kasper et al. [16] and expressed in Bethesda Units (BU). VIII:C activity was measured in ou~-~tag~ . . . . . a ~ y '--oy ~'m~ acuvmeu -~"..... -' paIxlm thrombo astin time method, with plasma of a severe haemophiliac as substrate. Antithyroglobulin antibodies and anti-DNA antibodies were quantitated by ELISA with purified porcine thyroglobulin and calf thymus DNA as antigens and a revealing peroxidaselabelled goat anti-human Fc antibody (Cappel Laboratories, Cochranville, PA). Anti-thyroid microsome antibodies and antibodies against the 108ccl 5 neuroblastoma cell line antigen (Van Doorn, P.A., Rossi, F., Brand, A., Lint, M.V., Vermeulen, M., and Kazatchkine, M.D., Treatment of chronic inflammatory demyelinating polyneuropathy with high dose intravenous immunoglobulins, submitted) were determined using semi-quantitative indirect immunofluorescence assays. Anti-intrinsic factor activity was measured using a radioimmunoassay in which the autoantibody was competing with radiolabelled vitamin B12 for binding to human gastric intrinsic factor [17]. -
-
-
BU = Bethesda unit. C I D P = chronic inflammatory demyelinating polyneuropathy. Ig = immunoglobulin.
--~'-'
pl
IVIg = normal polyspecific imrnunoglobulin for intravenous use. SLE = systemic lupus erythematosus.
POL YSPECIFIC Ig SUPPRESS A UTOANTIBODIES
21
The IgG fraction from patients' plasma was prepared by ammonium sulphate precipitation and chromatography on DEAE Trisacryl (IBF, Villeneuve la Garenne, France). F(ab')2 fragments which were free of detectable Fc fragments were prepared from IgG by pepsin digestion (2 % W/W) and chromatography on protein A Sepharose. Initial studies concerned two patients with autoantibodies against VIII:C in where intravenous administration of 0.4 g/kg/body weight of Sandogiobulin for five consecutive days resulted in a 95 °70decrease in circulating autom~tibody titre [12] (fig. 1). The decrease in autoantibody titre occurred within 24-48 h of infusion of IVIg. The autoantibody remained at its lowest titre in plasma for 2 to 3 weeks before it slightly increased for 2 weeks and then decreased again. A similar cycle occurred after three weeks in a pattern that resembled the cyclical appearance of Ab3 antibodies in experimental animals immunized with Abl [18]. In both patients, the autoantibody titre has remained between 5 and 10 070of preIVIg levels for over four years now. Similar effects of iVIg on anti-VIII:C autoantibody titre in vivo have been reported by others [19].
I£
__w~300_,
'
=, >
mOO0,
)
~
i)
'is
'~
" ~z
a~ ' ~'0
a~
'fo
dayu
FIO. 1. - - Time course o f decrease in autoantibody titre following infusion o f IVIg (arrows) in a patient with an anti-VIII:C autoantibody ( f r o m [12]).
22
F. R O S S I A N D COLL.
loo] o - - o Patient 1 "~
e
~ Patient 2
=
'=-
so
g
0
1
W/W ratio of patient's
10
IgG to therapeutic IgG
FIG. 2. - - Neutralization o f anti- VIII:C activity in the plasma o f two patients with anti- VIII:C autoantibodies upon incubation with IVlg ( f r o m [12]).
The rapid suppressive effect of IVIg on autoantibody titre in vivo suggested that IVIg directly interacted with circulating anti-VIII:C artoantibodies. The prolonged effect of IVIg, far beyond the half life of infused IgG, indicated that infused IVIg interfered with autoantibody synthesis. In vitro incubation of the patients' plasma with IVIg for 60 min at 37°C and overnight at 4°C resulted in a dose-dependent neutralization of autoantibody activity [12] (fig. 2). Maximal inhibition was observed at a specific molar ratio between IVIg and patient's IgG in each patient. An increase or a decrease in the optimal inhibitory ratio resulted in less or no inhibition of autoantibody activity, in an analogous fashion to the prozone phenomenon observed in precipitation curves. Several lines of evidence suggested that the inhibitory effect of IVIg on anti-VIII:C activity was mediated by anti-idiotypes against anti-VIII:C autoantibodies, present in the,' therapeutic immunoglobulin preparation: 1) IVIg contained no antigen-like (VIII:C) activity; 2) IVIg contained no detectable rheumatoid factor activity; a"~" . inl r~'-'-'" t,a o J2 fragments from t. .v. .lg hibited autoantibody activity in the IgG fraction and in F(ab')2 fragments from IgG of patients with anti-VIII:C autoantibodies; 4) IVIg contained no antiaUotypic antibodies directed against the G m l (3), G m l (4), G m l (17), G m l (1) and Km (1) allotypes which are most commonly expressed in the
~'C POL YSPEC!F!C !g S ~T.D. D D,,,-.~o Af
T'7"f~ A
,,~
~r-rrD~.~ r ~ r ~ o
F(ab')2 region of human IgG [13]. Inhibition by F(ab')2 fragments from IVIg of anti-VIII:C activity in F(ab')2 fragments prepared from the plasma of patients with autoantibodies is shown in table I. Inhibition curves followed the pattern of precipitation curves with a maximum of inhibition being achieved at a specific molar ratio between patient's F(ab')2 and F(ab')2 from IVlg. It is interesting to note that anti-VIII:C autoantibody activity from one patient (patient Gra.) was inhibited by one preparation of IVIg but not by the other, indicating that IVIg preparations may differ in their respective quantitative and qualitative content in specific anti-idiotypes.
TABLE I. - - Inhibition of anti-VIII:C activity in F(ab')z fragments obtained from patients with anti-VIII:C autoantibodies by F(ab') 2 fragments from IVIg.
IVIg F(ab:)2
Patient Gra. Patient Bes. Patient Esk.
SAGL
VGL
070 inhibition
070 in~bition
0.45 (0.45-1.80) 1.60 (0.40-3.20) 2.60 (0.60-5.20)
100 °70 76 070 100 070
0 070 (0.24-3.90) 3.00 (0.75-12.2) N.D.
75 070 N.D.
Indicated are the maximal inhibition of anti-VIII:C activity observed and the molar ratio between F(ab') 2 from patients' containing anti-VIII:C activity and F(ab')2 from IVIg at which maxima~ inhibition was achieved. Parentheses indicate the range of molar ratios between F(ab') 2 containing anti-VIII:C antibodies and F(ab')2 from IVIg that were tested. SAGL = <
Table II (upper panel) shows the results of experiments similar to those described in table I which were performed with F(ab')2 fragments prepared from prerecovery and postrecovery plasma of a patient (patient Esk.) who spontaneously recovered from anti-VIII:C autoimmune disease [11]. F(ab')2 fragments from samples obtained 6 weeks and 4 years after recovery of the patient inhibited anti-VIII:C activity in F(ab')2 fragments from the patient's prerecovery plasma. Similar amounts of F(ab')2 fragments from late pn~t,,ecovery_~.. (4 years) ~,,,,~,,,,,"! . . . . . . ,,,u-~ ~,,"¢F(ab')2 from IVIg were required to achieve 100 070 inhibition of autoantibody activity in F(ab')2 fragments from prerecovery plasma, suggesting that the content in anti-idiotype of IVIg is equivalent to that which prevents expression of anti-VIII:C autoantibodies x'1,ao /2 f r o m postrecovery plasma in postrecovery plasma from the patient. ~,.k,~ .~ of patient Esk. also neutralized anti-VIII:C activity of F(ab')2 fragments
24
F. R O S S I A N D COLL. TABLE II. - - Inhibition of anti-Vlll:C activity in F(ab')2 fragments obtained from patients with anti-VIH:C autoantibodies by F(ab')2 fragments from patient Esk.'s post-recovery plasma (upper panel) and inhibition of anti-neuroblastoma cell line activity in the serum of a patient with CIDP by F(ab')2 fragments from patient B.'s post-recovery plasma (lower panel).
F(ab')2 from IVlg(*) F(ab')2 fragments from patient Esk.'s postrecovery plasma 6 weeks 4 years Ratio ~/o inhibition Ratio 070inhibition Ratio 07oinhibition 100 070 50 070 100 070
Patient Gra. F(ab')2 0.72 Patient Bes. F(ab')2 5.48 Patient Esk. F(ab')2 4.80
0.~9 5.12 2.40
100 070 54 070 100 070
fragments from patient B.'s postrecovery plasma Ratio 070inhibition
F(ab')2
Patient B. (serum) Patient with CIDP (serum)
0.28
100 070
0.019
100 070
0.45 1.60 2.60
100 070 76 070 100 070
F(ab')2 from IVlg Ratio 07oinhibition N.D. 0.021
94 070
Indicated are maximal inhibition of autoantibody activity observed and molar ratio between F(ab')2 or IgG in serum containing the autoantibody and F(ab')2 from postrecovery plasma or IVIg at which maximal inhibition was achieved. (*) Sandoglobu!in.
prepared from the plasma of two other patients with anti-VIII:C autoantibodies. These results suggest that spontaneous recovery of patient Esk. from autoimmune disease was associated with the ~eneration of anti-idintvneg against prerecov~y antibodies and that anti-VIII:C autoantibodies from different patients share cross-reactive idiotypes. The lower panel of table II depicts analogous results obtained with postrecovery serum and F(ab')2 fragments from the serum of a patient who recovered from Guillain Barr6 syndrome. Postrecovery serum from this patient (patient B.) inhibited the binding of antiperipheral nerve autoantibodies in the patient's prerecovery serum to the 108cc 15 neuroblastoma cell line. The neuroblastoma cells express a membrane antigen that cross-reacts with human peripheral nerve [20]. Postrecovery antibodies from patient B. also inhibited, although less efficiently, autoantibody activity in the serum of a patient with chronic inflammatory demyelinating polyneuropathy (CIDP), the chronic form of Guillain Barr6 syndrome. F(ab')2 prepared from IVIg had an equivalent capacity to that of F(ab')2 fragments from the serum of patient B. to inhibit autoantibody activity in the serum of the patient with CIDP. Results shown in table II indicate that anti-idiotypes against prerecovery antibodies which may occur in sera from patients who recovered from autoimmune disease are more efficient in inhibiting antibody activity of the patient's own (prerecovery) autoantibodies than the activity of other patients' autoantibodies. This is probably due to -
~
J
F
25
POL YSPECIFIC Ig SUPPRESS A UTOANTIBODIES
the fact that spontaneously generated postrecovery anti-idiotypes are directed against both shared and private idiotypic specificities expressed by patient's prerecov~-y autoantibodies. The results also indicate that IgG from postrecovery plasma and IVIg have an equivalent inhibitory capacity towards autoantibodies from other patients. IVIg and F(ab')2 fragments from IVIg were also found to inhibit autoantibody activity in the serum and F(ab')2 fragments prepared from the IgG fraction of patients with other autoantibodies, including antibodies to thyroglobulin, thyroid microsomes, DNA, peripheral nerve and intrinsic factor (table III) [14]. As for autoantibodies to VIII:C, autoantibodies of various specificities which were inhibited by IVIg in vitro represented 40 to 80 °70 of all antibodies tested. In contrast, we have thus far never observed an inhibitory effect of IVIg on cold anti-erythrocytic antibody activity.
TABLE III. ~ Inhibition of autoantibody activity in the IgG fraction or serum from individual patients with autoimmune diseases by F(ab')2 fragments from IVlg.
Autoantibody specificity Thyroglobulin
F ( a b ' ) 2 fragments from SAGL VGL Ratio °70 inhibition Ratio 070 inhibition
0.090 (0.090-5.730) Thyroid microsomes 0.867 (0.430-5.780) DNA 0.280 (0.035-0.560) Peripheral nerve 0.015 (108cc15 neuroblastoma (0.005-0.023) cell line) Intrinsic factor 6.670 (0.417-13.25)
61 07o 100 070 76 °70 100 070 36 070
0.045 37 070 (0.045-1.440) N.D. 23.04 32 070 (0.720-46.08) N.D.
(0.126-8.08)
0 070
Indicated are the maximal inhibition of autoantibody activity found and the molar ratio between IgG containing autoantibodies and F(ab')2 from IVIg at which maximal inhibition was achieved. Parentheses indicate the range of molar ratios between IgG containing autoantibodies and F(ab') 2 from Wig which are tested. SAGL = Sandoglobulin; VGL = Veinoglobuline.
The interactions between antiidiotypic antibodies present in IVIg and autoantibodies were further investigated by examining the binding of the IgG fraction from patients with autoantibodies to F(ab')2 from IVIg and the specific binding of F(ab')2 containing autoantibody activity to affinity columns of Sepharose-bound F(ab')2 fragments from Wig [11, 13]. Microtitre plates were coated with F(ab')2 from IVIg and interacted with anti-VIII:Ccontaining IgG before addition of peroxidase-labeUed anti-human Fc gamma
26
F. R O S S I A N D C O L L .
antibody. As shown in figure 3, IgG from the three patients bound to F(ab')2 from IVlg, including the IgG from patient Gui., in which anti-VIII:C activity was not neutralized by IVIg in functional experiments. IgG from another patient, patient Gra., was found to bind to F(ab')2 from Sandoglobulin and F(ab')2 from Veinoglobuline although anti-VIII:C activity in the IgG preparation was only inhibited by one IVIg preparation (Sandoglobuiin) (table I). IgG from a normal donor also bound to insolubilized F(ab')2 from IVIg, further indicating that a number of idiotype/anti-idiotype interactions unrelated to anti-VIII:C/anti-anti-VIII:C interactions are likely to be involved in the binding of IgG from patients with anti-VIII:C antibodies to IVIg. Using the ELISA described above, we have recently found that F(ab')2 from two IVIg preparations (Sandoglobulin and Veinoglobuline) overall express a similar antiidiotypic repertoire [13]. As mentioned earlier, different preparations of IVIg may, however, differ in their relative content in anti-idiotypes against specific autoantigens (e.g. VIII:C) (table I). In order to demonstrate that IVIg contain antibody species that can specifically bind autoantibodies, affinity chromatography experiments were performed using columns of Sepharose to which F ( a b ' ) 2 from IVIg had been bound [11, 13]. Results of affinity experiments with anti-VIII:C antibodies are shown in table IV. Columns were loaded with buffer containing F ( a b ' ) 2 fragments with autoanti-VIII:C activity, washed until no protein could be detected in the effluent and then eluted at acidic pH. Specific binding of
0.~-. o
E c
qP 0 u c 9 JD ,0
4( 0.1 •
i
O~
~
O5
2
IgG
(mo~l)
FIG. 3. - - Binding of IgG from patients Gra. ( I ) , Bes. (0), Gui. (&) and from a normal individual (~) to insolubilized F(ab')2fragments from ~ Sandoglobulin ~ (open symbols)
and ~ Veinoglobuline~> (clo~ed symbols) as asseised by ELISA.
POL YSPECIFIC Ig SUPPRESS A UTOANTIBOD!ES
27
F(ab')2 anti-VIII:C antibodies to the column was assessed by measuring antibody activity in the acid eluate from the column. A specific enrichment in autoantibody activity in the acid-eluted fractions was calculated by comparing the specific antibody activity (BU/mg) loaded onto the column with that in the acid eluate. In the case of patients in whom anti-VIII:C autoantibodies were neutralized by IVIg in vitro (i.e. patients Bes., Gra. and Cer. presented in table IV), the acid-eluted F(ab')2 antibodies contained an anti-VIII:C specific activity that was enriched 1.6- to 40-fold as compared with the specific activity of the material loaded on the column. This indicated that at least some anti-VlII:C autoantibodies were specifically recognized in a high-affinity binding interaction by some antibody (anti-idiotype) species in IV!g. Sandoglobulin-sensitive anti-VlII:C F(ab')2 from patient Gra. were only recovered from a Sepharose-Sandoglobulin column, but not following affinity chromatography on Sepharose-Veinoglobuline. In a similar fashion~ no antiVIII:C activity was recovered in the acid-eluted fractions upon chromatography on Sepharose-Sandoglobulin and Sepharose-Veinoglobuline of F(ab')2 prepared from the plasma of patient Gui., in whom anti-VIII:C activity was not inhibited by either preparation of IVIg. Thus, the ability of an IVIg preparation to neutralize the anti-VIII:C activity of autoantibodies is strongly correlated with the presence of demonstrable anti-idiotypes against anti-VlIl:C antibodies by affinity chromatography. Analogous experiments were also performed using Sepharose-bound F(ab')2 fragments from postrecovery serum of patient Esk. as a soerce of immobilized anti-idiotypes [11]. Affinity chromatography of F(ab')2 fragments prepared from the patient's prerecovery serum resulted in an 80-fold enrichment in specific anti-VIII:C activity in the acid-eluted fractions from the column. The relatively high ability of patient Esk.'s postreccwery antibodies to recognize idiotypes expressed by the patient's prerecovery antibodies is conceivable if one considers that recovery from autoimmune disease is associated with generation of anti-idiotypes against both public and private idiotypes
TABLE IV. - - Affinity chromatography of F(ab')2 fragments containing anti-VIll:C activity on Sepharose-bonnd F(ab')z fragments from SAGL and VGL.
Sepharose-boundF(ab')2 from SAGL Sepharose-boundF(ab')2 from VGL Loaded Eiuted Loaded Eluted Amount Specific Amount Specific Amount Specific Amount Specific Anti-VlIl:C (ttg) anti-Vlll:C (ttg) anti-VlIl:C (ttg) anti-Vill:C (~tg) a.nti-Vlll:C F(ab')2 (BO/mg) (BU/mg) (BU/mg) (BU/mg) Patient Bes. 4250 Patient Gra. 5670 Patient Cer. 9000 Patient Gui. 10.71
3 2.38
45 315
5 11.80
4 4
0.98 98
73 0.00
38.09 1680
10.71
S A G L -- S a n d o g l o b u l i n ; V G L = V e i n o g l o b u l i n e .
3 10.20 N.D. 56
945 78 0.00
12.5 0.00
28
F. R O S S I A N D COLL.
expressed by the patient's autoantibodies. Columns of Sepharose-bound F(ab')2 from patient Esk.'s postrecovery plasma were less efficient in specifically retaining anti-VIII:C antibodies from another patient [11], indicating that anti-VIII:C antibodies from that patient and from patient Esk. express cross-reactive idiotypes that only account for part of the idiotypic specificities recognized by patient Esk.'s postrecovery IgG. Specific binding of F(ab')2 from autoantibodies to Sepharose-bound F(ab')2 from IVIg has also been recently documented with antithyroglobulin and with anti-DNA antibodies (F. Rossi and M.D. Kazatchkine, unpublished results), thus strengthening the general concept that IVIg contain anti-idiotypes against autoantibodies. It is unclear at the present time whether all or only a few of the donors contribute to the IVIg pool with anti-idiotypes. Based on the network theory [21] it may be speculated that all (or most) donors' plasma contain antiidiotypic antibodies directed against private and/or some cross-reactive idiotypes expressed by autoantibodies. The presence of a potent antiidiotypic activity in pooled IVIg may represent the sum of multiple antiidiotypic specificities. Recent studies by Tankersley et al. [22] indicate that, as the number of donors increases in an IgG preparation, an increasing amount of dimers formed between idiotypes and anti-idiotypes is found in the pool. Likewise, the probability of expression of a specific anti-idiotype in a therapeutic IgG preparation should increase with the size of the donor pool. Intravenous infusion of high doses of IVIg has resulted in clinical improvement and/or a decrease in autoantibody titre in a number of autoimm_une diseases including idiopathic thrombocytopenic purpura [23], autoimmune neutropenia [24], autoimmune red cell aplasia [25]~ n-iyasthenia gravis [26], CIDP [27] and in patients with autoantibodies to VIII:C [12]. Several mechanisms may account for the be~:eficial effect of IVig [15]. In addition to anti-VIII:C autohr.rn-une disease, there is in vitro evidence that the beneficial effect of IVIg in patients with autoimmune red cell aplasia [25] and chronic autoimmune inflammatory demyelinating polyneuropathy (Van Doom et al., ibid., submitted) is also dependent on antiidiot~qaic neutralization of autoantibodies. Furthermore, and as mentioned earlier, the suppressive effect of IVIg in some patients with anti-VIII:C autoantibodies has been prolonged far beyond the half life of infused IVIg, suggesting that, in addition to shortterm inhibitory effects, anti-idiotypes in IVIg may alter the expression of the patients' repertoire by autoantibody-producing cells. Results summarized in this manuscript may open prospects for further investigation of therapeutic anti-idiotype suppression of autoantibodies with IVIg. The demonstration of anti-idiotypes in normal immunoglobulins supports the concept of a functional idiotypic network regulating autoimmune responses in man.
P O L Y S P E C I F I C Ig S U P P R E S S A U T O A N T I B O D I E S
29
RI~SUMt~ SUPPRESSION ANTIIDIOTYPIQUEDES AUTOANTICORPS A L'AIDE D'IMMUNOGLOBULINESPOLYSPI~CIFIQUESNORMALES
L'un des m6canismes par lesquels les immunoglobulines polysp6cifiques th6rapeutiques (IVIg) peuvent inhiber in vivo certaines r6ponses autoimmunes chez l'homme, est la suppression antiidiotypique des autoanticorps par des antiidiotypes pr6sents dans les IVIg. Nous avons observ6 que l'activit6 autoanticorps dans le plasma ou celle des IgG provenant de patients avec des anticorps anti-facteur VIII (VIII:C), anti-DNA, anti-thyroglobuline, anti-nerf p6riph6rique ou anti-facteur _intrins6que, est inhib6e in vitro lors de leur incubation avec des IVIg. Les courbes d'inhibition montrent un ph6nom6ne de prozone. L'inhibition maximale survient pour un rapport molaire - - entre les IgG du patient et les IVIg - - sp6cifique de chaque anticorps test6. Un certain nombre de raisons impliquent que l'inhibition de l'activit6 autoanticorps est d6pendante d'interactions entre idiotypes et antiidiotypes: 1) les fragments F(ab')2 des IVIg inhibent l'activit6 autoanticorps dans des fragments F(ab')2 pr6par6s/t partir des IgG contenant l'activit6 autoanticorps; 2) les IVIg ne contiennent pas d'activit6 biologique ressemblant/t celle de l'antig6ne, ni d'anticorps dirig6s contre les allotypes les plus commun6ment exprim6s dans les fragments F(ab')2 des IgG humaines; 3) l'activit6 autoanticorps pr6sente dans les fragments F(ab')2 pr6par6s /t partir des IgG des patients est sp6cifiquement retenue sur des colonnes de chromatographie d'affinit6 de fragments F(ab')2 d'IVIg. La pr6sence d'antiidiotypes dirig6s contre les autoanticorps dans des pools d'IgG provenant d'individus normaux est en faveur de l'existence d'un r6seau idiotypique physiologique r6gulant la r6ponse autoimmune chez rhomme. MOTS-CLi~S: Immunoglobuline th6rapeutique, Idiotype, Immunosuppression, Autoimmunit6; Ig polysp6cifiques, Suppression antiidiotypique.
A/'~V hJ('~[/I .i ~ . , z ~ . L ~,....i
]~" ~
vv
't.[~'k 4 ]t.t.t.t.t.t.t.t.I~.T ~" T ~
JL..,I...aZ.J~.~I-,I~II...,I~
•
This work was supported by INSERM, Sandoz SA and Institut M6rieux.
REFERENCES
[1] ROITT, I.M. & COOKE,A., Idiotypes and autoimmunity, in ~ Progress in im[2]
[3] [4] [5]
munology V I , (B. Cinader & R.G. Miller) (pp. 512-535). Academic Press, London, New York, 1986. ZANETTI, M. & BIGAZZI,P.E., Antiidiotypic immunity a::d autoimmunity. - I. In vitro and in vivo effects of antiidiotypic antibodies to spontaneously occurring autoantibodies to rat thyroglobulin. Europ. J. lmmunol., 1981, 11, 187-195. SOUROUJON,M.C., BERCHON,D. & FUCHS,S., Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes. Immunol. letters., 1985, 9, 331-336. NEILSON,E.G. & PHILLIPS,S.M., Suppression of interstitial nephritis by autoantiidiotypic immunity. J. exp. Med., 1982, 155, 179-189. HAHN, B.H. & EBLING, F.M., Suppression of murine lupus nephritis by administration of an antiidiotypic antibody to anti-DNA. J. Immunol., 1984, 132, 187-190.
30
F. R O S S I A N D C O L L .
[6] KOOPMAN,W.J., SCHROHENLOHER,R.E., BARTON, J.C. • GREENLEAF,E.C., Suppression of in vitro monoclonal human rheumatoid factor synthesis by antiidiotypic antibodies. J. clin. Invest., 1983, 72, 1410-1419. [7] GELTNER,D., FRANKLIN,E.L. & FRANGIONE,B., Antiidiotypic activity in the IgM fractions of mixed cryoglobulins. J. Immunol., 1980, 125, 1530-1535. [8] ABr,ou, N.I., WALL, H., LINDSEY,H.B., HALSEY,J.F. & SuzuKi, T., Network theory in autoimmunity: in vitro suppression of serum anti-DNA antibody binding to DNA by antiidiotypic antibody in systemic lupus erythematosus. J. din. Invest., 1981, 67, 1297-1301. [9] LEVVERT,A.K., Antiidiotypic antibodies against the receptor antibodies in myasthenia gravis. Scand. J. Immunol., 1981, 13, 493-497. [10] DWYER,D.S., BRADLEY,R.J., URQUnART, C.K. & KBARNEY, J.F., Naturally occurring antiidiotypic antibodies in myasthenia gravis patients. Nature (Lond.), 1983, 301, 611-614. [111 SULTAN,Y., ROSSI,F. & KAZATCtmU~B,M.D., Recovery from anti-VIII:C (antihemophilic factor) autoimmune disease is dependent on generation of antiidiotypes against anti-VIII:C autoantibodies. Proc. nat. Acad. Sci. (Wash.), 1987, 84, 828-831. [12] SULTAN, Y., KAZATCnrmCB, M.D., MAISO~EUVB, P. & NYDEGGER, U.E., Antiidiotypic suppression of autoantibodies to factor VIII by high-dose intravenous gammaglobulin. Lancet, 1984, If, 765-768. [13] Rossl, F., SULTAN,Y. & KAZATCHKXNB,M.D., Anti-idiotypes against autoantibodies and alloantibodies to VIII:C (anti-haemophilic factor) are present in therapeutic polyspecific normal immunoglobulins. Clin. exp. Immunol., 1988, 74, 311-316. [14] K A Z A T C ~ , M.D., Rossl, F., DmTRICH,G. & SULTAN,Y., Clinical relevance of antiidiotypic antibodies during treatment with high doses of intravenous immunoglobulins (IVIg), in t~Immunoglobulins)) (H.N. Krijnen). Elsevier, Amsterdam, 1988 (in press). [15] MORELL,A. & NYDEGGER,U.E., Clinical use of intravenous immunoglobulins. Academic Press, London, New York, 1986. [16] gASPER,C.K., ALEDORT,L.M., COUNTS,R.B., EDSON, J.R., FRATANTONE,J., GREEN,D., HEMPTON,J.W., HILGARTNER,M.N., LAZARSON,J., LEVIN,P.H., MACmLLAN,C.N., POOL, J.C., SHAPIRO,S.S., SCHULMAN,N.R. & EVES, J., A more uniform measurement of factor VIII inhibitors. Thrombos. Diathes. r~aemorrn, ( s t uttg. ), 1~13, .... ~4~ 809-81Z. [17] Rorrr, I.M. & DONIACH,D., Assay for the estimation of autoantibodies to gastric intrinsic factor in pernicious anaemia. W.H.O., Geneva, 1974. [18] COURAUD,P.O., Lu, B.Z. & STROSBERG,A.D., Cyclical antiidiotypic response to anti-hormone antibodies due to neutralization by autologous antiidiotypic antibodies that bind hormone. J. exp. Med., 1983, 157, 1369-1378. [19] Z~4ERMAN, R., KOMMERELL,]~., HARENBERG,J., EICH, W., ROTHER, K. & SCmMpS, K.Lo, Intravenous IgG for patients with spontaneous inhibitor to factor VIII. Lancet. 1985, I, 27a-2/4." "¢ [20] VAN DOORN,P.A., BRAND,A. & YERMEULEN,M., Anti-neuroblastoma cell line antibodies in inflammatory demyelinating polyneuropathy. Neurology, 1988 (in press). [211 JERNE, N.K., Towards ~ network ~heory of the immune response. Ann. lmmunol. (Inst. Pasteur), 1984, 125 C, 373-389. [22] TANKERSLEY,D.L., PRESTON, M.S. & FINLAYSON,J.S., Immunoglobulin G dimer: an idiotype-anti-idiotype complex. Mol. Immunol., 1988, 25, 41-48. [23] IMUAC,, P., BARA~g~UN, S., D'Apuzzo, V., BAUMGARTNER,C., HIRT, A., MORELL,A., RossI, E., SC8ONI,M., VEST,M. & WAGNER,H.P., High-dose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood. Lancet, 1981, I, 1228-1230. .
.
.
.
.
.
.
.
.
POL YSPECIFIC Ig S U P P R E S S A U T O A N T I B O D I E S
31
[24] BUSSEL, J.B., LALEZARI, P., HILGARTNER, M., PARTIN, J., FRIKRIG, S.,
O'MALLEY, J. ,~ BARANDUN,S., Reversal of neutropenia with intravenous gammaglobulin in autoimmune neutropenia of infancy. Blood, 1983, 62, 398-400. [25] McGUIRE, W.A., YANG, H.H., BRUNO, E., BRANDT, J., BRIDELL, R., COA~ES, T.D. & HOFFMAN,R., Treatment of antibody-mediated pure red cell aplasia with high dose intravenous gammaglobulin. New EngL J. Med., 1987, 317, 1004-1008. [26] GAJDOS,Ph., OUTIN, H., ELKARRAT,D., BRUNET, D., DE ROHAN-CHABOT,P., RAPHAEL,J.C. 8£ GOULON,M., High-dose intravenous immunoglobulin for myasthenia gravis. Lancet, 1984, I, 406-407. [27] VERMEULEN, M., VAN DER MECHE, F.G.A., SPEELMAN,J.D., WEBER, A. BUSCH, H.F.M., Plasma and gammag!obulin infusion in chronic inflammatory polyneuropathy. J. NeuroL Sci., i985, 70, 317-326.