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IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human
Journal of Reproductive Immunology, 20 ( 1991 ) 129--140 Elsevier Scientific Publishers Ireland Ltd.
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129
JRI 00725
IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human Ileana Malan Borel a, Teresa Gentile a, Juana Angelucci a, Jos6 Pividori a, Maria del Carmen Guala a, Ruben A. Binaghi b and Ricardo A. Margni a IDEHU-lnstituto de Estudios de la lnmunMad Humoral (CONICET-UBA). Departamento de Microbiologia. lnmunologla y Bioteenologia. Faeultud de Farmacia y Bioquimica. Universitkul de Buenos Aires, Junin 956. l l l3-Buenos Aires (Argentina) and hCentre National de la Recherche Scient(fique. Paris (France) (Accepted for publication 5 March 1991)
Summary The proportion of symmetric and asymmetric IgG molecules was studied in 10 mothers at delivery. IgG was obtained from peripheral blood and placental blood sera and by elution at 4 M KCI from placenta cell membranes. The percentage of symmetric and asymmetric molecules was determined in the IgG and in their corresponding F(ab')2 fragments by absorption to Con A-Sepharose. The presence of antipaternal antibodies was investigated by IIF and MC tests using paternal lymphocytes. The average percentage of asymetric IgGmolecules in the sera was 24.4, which is about double the value of that found in normal subjects. In the IgG eluted from the placenta, the proportion of asymmetric IgG was much higher, averaging 44.4%. Antipaternal antibodies were detected in 5 mothers by IIF and MC and in two mothers only by IIF. In three mothers no antibodies could be detected. It was found that the concentration of antipaternal antibodies was about three times higher in the asymmetric lgG fraction than in the symmetric one. Considering the percentage of asymmetric IgG molecules with antipaternal antigen specificity eluted from placenta and the possibility that they function as blocking antibodies, their participation in fetal protection is suggested. Correspomlenee to." Prof. R.A. Margni, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956. I113-Buenos Aires, Argentina. 0165-0378/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
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Key words: pregnancy; asymmetric antibodies; blocking antibodies; placenta;
fetus. Introduction The survival of the semiallogeneic mammaliam fetus in the presence of an alloreactive maternal environment remains still to be explained. However it is known that the immune system suffers several changes during pregnancy that are useful for the embryonic development (Deer et al., 1979, 1981; Bell et al., 1983; Chaouat, 1986). Recent work suggests that the most important protection for the fetus is provided by the placenta and decidua although the detailed mechanisms through which the tissues at the maternal-fetal interface accomplish this function needs clarification. Moreover, there is evidence that antipaternal MHC antibodies may confer protection from spontaneous fetal resorption. It is well known that pregnancy serum often contains leukocytotoxic antibodies with paternal specificity depending on the number of pregnancies a woman has had. These antibodies have been demonstrated by microcytotoxicity using leukocytotoxic assays with complement. However, there appears to be no correlation between the presence or absence of these antibodies and the success or failure of pregnancy. It is now well documented that in most immune sera a population of antibodies exists that is unable to form precipitates with the antigen, although they can coprecipitate in the presence of precipitating antibodies of the same specificity. These "non-precipitating" or "coprecipitating" antibodies were first obtained by Heidelberger and Kendall (1935) and in recent years a number of studies have been performed in our laboratories in order to elucidate their physicochemical and biological properties (Ronco et al., 1984; Leoni et al., 1986; Labeta et al., 1986; Morelli et al., 1989; Malan Borel et ai., 1990). These studies have been recently reviewed (Margni and Binaghi, 1988). It was demonstrated that non-precipitating antibodies possess an asymmetric structure due to an oligosaccharide moiety, present only in one of the two Fab regions of the molecules. The combination of the corresponding antibody site with the antigen is sterically hindered by the oligosaccharide group and as a consequence the molecule behaves functionally as univalent. The term of "asymmetric" has been employed to designate these molecules in opposition to symmetric precipitating antibodies which are not glycosylated in the two Fab regions. The antibody site present in the Fab region of asymmetric antibody devoid of the prosthetic carbohydrate group acts normally, and therefore the
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molecule can be firmly combined with its corresponding antigen. Asymmetric antibodies are generally unable to activate effector functions, such as complement fixation, phagocytosis, cytotoxicity, etc. However, since they combine with the antigen, they act in a competitive way when mixed with precipitating antibodies of the same specificity. In fact, asymmetric igG antibodies act as blocking antibodies. Asymmetric antibodies are present in practically all mammalian sera and in all lgG subclasses. They constitute about 10% of the total population, but this proportion can increase strikingly in some cases (Margni et al., 1983, 1986). The asymmetric molecules are equally present, in about the same proportion, in all non-immune sera (Malan Borel et al., 1989). Symmetric and asymmetric IgG antibodies are synthesized by the same cellular clone (Morelli et al., 1989). The existence of asymmetric antibodies in all animal species studied so far suggests that they could play same important physiological role (Margni, 1989). Some observations indicate that this may be the case in allergic processes and in chronic infections. Taking into account the fact that they are functioning as blocking antibodies, it can be speculated that they may participate in the immunological aspects of the complex mechanism controlling the equilibrium between mother and foetus. In order to gain more information on this subject we have studied the production of asymmetric lgG molecules during human pregnancy, and we have determined their proportion in the maternal serum and in the placenta, as well as their specificity against paternal antigens. Materials and Methods
Isolation and purification of lgG from sera of pregnant and non-pregnant women IgG was obtained from normal pregnant women sera by precipitation at 50% saturation of (NH4)2SO4 followed by DEAE-callulosa chromatography. The IgG was eluted by 0.01 M phosphate buffer (pH 7.4). Isolation and purification of lgG from non-pregnant women sera was achieved in a similar way. Isolation and purification of IgG from placental blood Placenta were frozen at -20"C immediately after delivery and processed as soon as possible, within a few days maximum. After thawing, they were covered with a piece of wood, a weight of about 10 kg was applied and were left 2 h in the cold room. The exudate obtained was clarified by centrifugation, precipitated at 50% ammonium sulphate and the IgG was purified as indicated above.
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Placental eluates The whole placenta from one woman was minced with scalpel blades into fine slices and suspended in phosphate buffered saline (PBS). It was washed with the same buffer and this procedure was repeated until the O.D. of the supernatant fell below 0.10. After the final wash the tissue was centrifuged for 10 min at 12,000 x g, the pellet resuspended in 100 ml of 4 M KC1, mixed with a magnetic stirrer for 60 rain, centrifuged and the supernatant dialyzad against PBS; then concentrated to 10 ml using a pressure concentration device (Amicon Corp., MA). The purification of IgG was made as indicated above.
F(ab' )2 fragment This was obtained by pepsin digestion as described by Turner et al. (1970). The IgG was dialyzed against 0.1 M sodium acetate buffer (pH 4.5) and digested with papain (Sigma), enzyme/substrate ratio 1/100, for 18 h at 37°C. The digestion was stopped by the addition of Tris to obtain a pH of 8. Purification of the F(ab')2 fragment from the mixture was achieved by Sephadex G-200 gel filtration. Purity of the F(ab')2 fragment was checked by immunoelectrophoresis using rabbit anti-human serum and by SDS-PAGE.
Determination of symmetric and asymmetric lgG and F(ab' )2./i'agnlents by the concanavalin A (Con A) test Separation of both, symmetric and asymmetric IgG molecules was acchieved by Con A-Sepharose chromatography, taking into account that this lectin binds molecules containing o~-D-mannopyranosyl, oz-D-glucopyranosyl and sterically related residues (Malan Borel et al., 1990). Briefly: Con A-Sepharose was washed with the elution buffer (Tris--HCl, 0.025 M; NaCI 0.2 M; CaCI_,, MgCI2, MnCI2, 0.003 M each; Na azide 0.02%, pH 7.2). The protein samples were dialyzed against the same buffer. Equal volumes (approx. 1 ml) of 50% packed Con A-Sepharose and protein (about 1 mg/ml were mixed in small tubes and gently shaken for 1 h at room temperature, then overnight in the cold. After centrifugation the pellets were repeatedly washed with the elution buffer and the protein in the collected supernates was estimated by measuring the optical density at 280 nm. This value corresponds to the symmetric IgG, not bound by the Con A. The asymmetric IgG was eluted from the pellets by repeated washing with 0.15 M o~methyl-mannoside in the elution buffer and estimated by measuring the optical density. All tests were made in triplicate. Recovery of protein (not retained plus retained by Con A) was generally not less than 95% of the initial sample. Separation of symmetric and asymmetric F(ab')2 fragments by Con ASepharose was achieved as indicated for IgG molecules.
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In some cases when small amounts of IgG eluted from the placenta, or their respective F(ab')2 fragments, were chromatographed on Con ASepharose, the proteins were previously labelled with esI as described by Greenwood et al. (1963). The radioactivity was measured in a gamma radiation counter to determine the Con A bound and eluted protein.
Indirect immunofluorescence (IIF) Symmetric and asymmetric IgG from placental eluates and peripheral blood ware analyzed by indirect immunofluorescence, using as target paternal lymphocytes and non-paternal lymphocytes (negative control), Ten microliters of 106 cells/ml were put on a glass-slide and air dried for 30 min at room temperature. After that the cells were successively washed with a 0.1 M glycine-HCl buffer (pH 3), PBS and water. Fc receptors were blocked with 10 #1 of aggregated rabbit IgG (4 mg/ml) (Dickler et al., 1972) for 30 min at room temperature and then washed with PBS. 10/~1 of the sample, containing about 8 #g protein was added and incubated for 30 min at room temperature, then washed twice with PBS. Finally, fluoresceinconjugated rabbit F(ab')2 anti-human IgG was added and after 30 min the slides were washed with PBS. For visualization of the stained cells a Zeiss epifluorescent microscope was employed.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 7.5% polyacrylamide gels were poured as vertical slabs and run according to the method of Laemmli (1970). Intact and digested immunoglobulins were visualized by staining with 0.1% (w/w) Coomassie Blue R-250 (Mallinckrodt) in methanol/glacial acetic acid/water (5:2:5 by vol.). Staining patterns were compared with those of standard molecular weight markers (Sigma).
Purification of lymphocytes Human peripheral blood lymphocytes were obtained from the paternal heparinized blood by centrifugation on Ficoll-Hypaque (density 1.077 0.001 g/cm 3) at 300 x g for 20 min. After isolation they were washed three times in RPMI 1640, counted and resuspended to a concentration of 5 x 10 6 cells/ml. The percentage of viable white blood cells was determined by the trypan blue exclusion test.
Microcytotoxicity test ( MC) This was done in microplates and the method described by Terasaki (1973) was employed with minor modifications. One microliter of sample and 1 t~l of paternal lymphocytes (2 x 10 6 cells/ml) were added to each well. After incubation for 30 min at room temperature 5 #1 of previously titrated fresh rabbit sera (complement source)
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were added and the plates incubated again at the same temperature for 1 h and 30 min. After that 5 #1 o f 5°/,, eosin in water was added and 3 min later 5 #1 o f non-diluted formalin was incorporated into the walls. The percentage of lysed cells was determined by counting dead (stained) and viable (unstained) cells using an inverted microscope. Results Ten pregnant women were studied at delivery. Six of the mothers were in their first pregnancy, and four (1 4 in the Tables) had three or more pregnancies with the same partner. Placentae were collected and treated, as described in Methods, to obtain placental blood and placental eluates. Maternal serum was simultaneously obtained. IgG was purified from these samples and from sera of nonpregnant woman and their F(ab')2 fragments were prepared. TABLE
I
Percentage o f C o n A f i x a t i o n a n d s p e c i f i c a c t i v i t y f o r paternal lymphocytes of IgG and ( F b ' ) 2 fragments purified from peripheral b l o o d o f pregnant women (a) and f r o m b l o o d o b t a i n e d b y mechanical pressure o f their placenta (b). Samples l I--16 are controls (non-pregnant women). Sample
igG
F(ab)2
No.
Fixation
MC
IIF
Fixation
to C o n A (%) a
IIF
to Con A a
b
a
b
b
(%1 a
a b
I
20
20
+
+
++
++
19
20
++
++
2
25
24
+
+
++
++
24
23
++
++
3
26
25
+
+
++
++
25
25
++
++
4
30
30
+
+
++
++
30
30
++
++
++
++
++
5
26
26
+/-
26
25
++
6
24
24
.
.
+ .
.
24
24
--
7
30
30
.
.
.
.
29
30
--
8
22
22
.
.
.
.
22
22
--
++
+
22
21
++
++
--
++
+
22
22
++
++
9
22
22
10
22
22
II
12
--
I1
12
10
--
13
13 14
15 13
---
15
14
--
--
14
--
16
9
--
--
11
--
--
--
15 12
---
M C r e s u l t s : ( + ) p e r c e n t a g e o f l y s e d cells more than 3 0 % ; ( - - ) p e r c e n t a g e o f l y s e d cells less than 3~Y'/.. I I F results: (+) percentage of fluorescent cells between 5% and 20"/,,. ( + + ) more than 2 0 % . ( - - ) less than 5"/.
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All the samples were studied by the Con A test, in order to estimate their proportion of asymmetric molecules. Anti-paternal antibodies ware investigated by indirect fluorescence using paternal lymphocytes as target cells. The lgG samples were also tested by the microcytotoxicity test. The results are reported in Tables 1 to 4. Table 1 shows the results obtained using IgG or F(ab')2 fragments isolated from maternal blood and from blood obtained by mechanical extrusion of the placenta. It can be seem that both results are strictly coincident. The percentage of IgG combining to Con A was from 19 to 30%. (average 24.4%). This value is significantly higher than that of 12.1% observed in nonpregnant women sera, indicating that the production of asymmetric IgG increases significantly during pregnancy. The IIF test using paternal lymphocytes and maternal serum was positive in seven out of ten mothers but the microcytotoxicity test was positive only in five of these seven mothers. In three cases IIF and MC were both negative (mothers 6-7-8) and they corresponded to mothers having their first pregnancy. Adequate controls were made using non-paternal lymphocytes from a third party. When IgG and F(ab')2 fragments, obtained from the placenta by elution with high ionic concentration, were analyzed (Table 2), the positivity observed in IIF and MC was the same as the one observed in the maternal sera. However, the percentage of asymmetric IgG was strongly increased, with values ranging from 32 to 62% (average 44.4%). TABLE 2 P e r c e n t a g e o f C o n A f i x a t i o n a n d specific a c t i v i t y f o r p a t e r n a l l y m p h o c y t e s o f I g G a n d F ( a b ' )2 f r a g m e n t s eluted f r o m p l a c e n t a . S y m b o l s a r e the s a m e as i n d i c a t e d in T a b l e I. Sample No.
Total lgG
Total F(ab')2
Fixation to C o n A ("/,,)
MC
IIF
Fixation
ilF
to Con A (,'/,,)
1
32
+
2
55
+
++ ++
31 55
++ ++
3 4 5 6
54 62 56 36
+ + + --
++ ++ ++ --
54 61 55 36
++ ++ ++ --
7 8 9 10
39 36 38 39
-----
--++ ++
38 35 38 38
--++ ++
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The IgG eluted from the placenta at high ionic concentration represents the population of IgG which is fixed to the cells, either because it is combined with membrane antigens, presumbly paternal antigens of the fetus against which the mother has produced antibodies, or directly fixed via Fc receptors. It was of interest to study the specificity of the population by IIF and MC against paternal antigens, after fractionation into symmetric and asymmetric molecules. This fractionation was done by chromatography with Con A Sepharose using five of the maternal samples. The results are presented in Table 3. It is seen that in all cases antipaternal antibodies were present in both symmetric and asymmetric molecules, as evidenced by IIF, the asymmetric fraction being apparently more active. The MC test was positive in three samples of the symmetric molecules and was negative with all five asymmetric molecules as expected, since they do not fix complement. In an attempt to evaluate the relative proportion of asymmetric antibody molecules, placental IgG was further investigated in some cases (mothers 9 and 10). After fractionation by Con A-Sapharose, both populations were tested by IIF. When equal concentrations were used, it was found that the antibody activity of the asymmetric fraction was higher than that of the symmetric one. The results obtained are presented in Table 4. From the values obtained it can be estimated that the concentration of antipaternal antibodies is about three times higher in the asymmetric fraction than in the symmetric one. Discussion The studies described in the present paper reveal that, during pregnancy,
TABLE 3 A n t i p a t e r n a l l y m p h o c y t e specificity o f I g G s y m m e t r i c a n d a s y m m e t r i c m o l e c u l c s eluted f r o m p l a c e n t a . S y m b o l s are the s a m e as i n d i c a t e d in T a b l e I. Sample No.
T o t a l I g G eluted f r o m p l a c e n t a Symmetric IgG
Asymmetric lgG
MC
IIF
MC
IIF
2 3
+ +
+ ++
~
++ ++
4 9 10
+
+ + +
--
--
++ ++ ++
137 TABLE 4 Antipaternal lymphocyte activity of lgG fixed to placenta. The % of IIF expressed are the average of the results obtained with samples Nos. 9 and 10. In each case 400 cells were counted. Sample
#g IgG/tube
lgG symmetric molecules
25 50 100
IgG asymmetric molecules
25 50 100
5 14 26
Total lgG from non-immune human sera
25 50 100
<4 <4 <4
PBS (control)
% of fluorescent cells 2 2.5 7.5
<4
the production of asymmetric lgG molecules is considerably increased. The average percentage found in the sera of the 10 mothers studied was 24.4; this is the double of the percentage present in non-pregnant women sera and in non-immune subjects as already reported. Exactly the same value was found in the serum obtained from the placenta by mechanical pressure, as could be expected since it is indeed maternal serum. On the contrary, when IgG was obtained from the placenta itself after careful washing and subsequent treatment at a high ionic concentration (4 M KCI), the percentage of the asymmetric molecules was much higher, with an average of 44.4% and values reaching up to 62%. The IgG so obtained is of maternal origin and fixed to the membrane of the placental cells. This IgG is fixed to the cellular membrane by two different machanisms: through the Fc receptor or by binding to paternal antigens. Since asymmetric and symmetric molecules have the same Fc structure, the proportion of symmetric and asymmetric molecules fixed to Fc receptors must be the same as that present in the serum. On the other hand, the proportion of symmetric and asymmetric molecules bound to paternal antigens must depend on the relative concentration of antibodies in each population. The fact that the proportion of fixed asymmetric molecules is twice as high as the one present in the serum, strongly suggests that the concentration of asymmetric antipaternal antibodies is higher than that of symmetric ones.
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Exactly the same results were obtained using IgG or their F(ab')z fragment. The reason why F(ab')2 was studied was to eliminate the posibility that IgG molecules attach to Con A via eventual carbohydrate groups present in the Fc regions, that are not relevant to the asymmetry of the molecule. This possibility exists in the case of IgG from same animal species, but in the case of human IgG practically no Con A-binding carbohydrate is present in the Fc region. The present results confirm previous observations (Leoni et al., 1986; Labeta et al., 1986). Antibodies against paternal antigens were investigated using two different techniques: IIF and MC assays. By IIF, both antibody types, symmetric and asymmetric should be detected. The cytotoxic assay, on the contrary, can only be positive with complement-fixing antibodies, that is, only with symmetric ones. It was found that 7 mothers gave a positive IIF test either with serum IgG or with placenta-eluted IgG. The cytotoxic test was positive in five of these seven mothers. In three mothers no antipaternal antibody specificity could be detected either by IIF or by MC. The absence of antipaternal antibodies is probably due to a weak antigenic stimulation, since mothers 5--10 were in their first pregnancy. It cannot be excluded, however, that the negative results simply reflect a low sensitivity of the test employed. Five of the placental IgG samples were further studied. They were processed by chromatography on Con A-Sepharose, to obtain the asymmetric and symmetric populations, which were then tested by IIF and MC with paternal lymphocytes. IIF was positive in all the fractions, the reactions being the strongest with the asymmetric populations. The MC assay was negative in all the asymmetric fractions, as expected, since this assay is complementdependent and asymmetric IgG does not fix complement. In the symmetric fractions, the MC assay gave positive results in three cases and was negative in mothers 9 and 10. A semi-quantitative IIF assay was performed with the symmetric and asymmetric fractions by evaluation of the percentage of fluorescence obtained at various concentrations. The asymmetric fractions proved to be, about three times more active than the symmetric fractions indicating that the antipaternal antibodies were in the majority of the asymmetric type. From the results reported in the present paper, two conclusions can be drawn. First, it is clear that the production of asymmetric IgG molecules increases strikingly during pregnancy. Since the increase is observed in the total IgG population, out of which antibodies directed to paternal antigens are only a minimal fraction, this non-specific increase seems to be induced by pregnancy itself, perhaps by factors secreted by the placenta. This possibility is now under study. Secondly, it was found that antipaternal an-
139
tibodies synthesized by the mother are, in the majority, of the asymmetric type. This is not the usual finding when immunizations are performed with soluble antigens, but it is in line with previous observations showing a preferential synthesis of asymmetric antibodies when particulate antigens are used, as well as in some chronic infections (Hajos et al., 1982; Margni et al., 1983, 1986; Carbonetto et al., 1986). Since asymmetric antibodies do not fix complement and do not activate effector immune mechanisms, and may further act as blocking antibodies, it is worth asking whether they play a role in the protection of the fetus against cytotoxic symmetric, complement fixing antipaternal antibodies produced by the mother. A blocking effect has already been demostrated in other systems, such as complement fixation (Margni et al., 1980) and phagocytosis of opsonized bacteria (Perdig6on et al., 1982). In all cases, it was found that asymmetric antibodies compete with their symmetric counterpart by binding the same antigen. What appears to be determinant for the blocking effect is not the absolute quantity of asymmetric antibodies but the relative proportion of asymmetric to symmetric antibodies, acting simultaneously. In the case of complement fixation, the blocking effect is already perceptible when 20"/,, of the total antibodies are asymmetric, and the complement fixation is completely abolished at 80%. Incidentally, these observations must be taken into account when performing tests to detect antibody activity. When the assay is solely based in the primary interaction between antibody and antigen, as is the case with immunofluorescence, both symmetric and asymmetric antibody molecules are detected, but when the assay implies an effector mechanism, as in the case of complement fixation or complement-dependent cytotoxic reactions, a negative result may be obtained because of a competitive effect due to asymmetric antibodies. Although the present observations do not allow to draw any conclusions concerning a possible protective effect upon the fetus, the high proportion of asymmetric molecules found in the IgG fixed to the placenta is highly suggestive in this respect.
Acknowledgement This work was supported in part by a scientific cooperation agreement between CONICET (Argentina) and INSERM (France).
References Bell, S.C. and Billington, W.D. (1983) Humoral immune response in murine pregnancy. III - - Relationship between antipaternal alloantibody levels in maternal serum, placenta and fetus. J. Reprod. Immunol. 5, 299--310.
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JRI 00725
IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human Ileana Malan Borel a, Teresa Gentile a, Juana Angelucci a, Jos6 Pividori a, Maria del Carmen Guala a, Ruben A. Binaghi b and Ricardo A. Margni a IDEHU-lnstituto de Estudios de la lnmunMad Humoral (CONICET-UBA). Departamento de Microbiologia. lnmunologla y Bioteenologia. Faeultud de Farmacia y Bioquimica. Universitkul de Buenos Aires, Junin 956. l l l3-Buenos Aires (Argentina) and hCentre National de la Recherche Scient(fique. Paris (France) (Accepted for publication 5 March 1991)
Summary The proportion of symmetric and asymmetric IgG molecules was studied in 10 mothers at delivery. IgG was obtained from peripheral blood and placental blood sera and by elution at 4 M KCI from placenta cell membranes. The percentage of symmetric and asymmetric molecules was determined in the IgG and in their corresponding F(ab')2 fragments by absorption to Con A-Sepharose. The presence of antipaternal antibodies was investigated by IIF and MC tests using paternal lymphocytes. The average percentage of asymetric IgGmolecules in the sera was 24.4, which is about double the value of that found in normal subjects. In the IgG eluted from the placenta, the proportion of asymmetric IgG was much higher, averaging 44.4%. Antipaternal antibodies were detected in 5 mothers by IIF and MC and in two mothers only by IIF. In three mothers no antibodies could be detected. It was found that the concentration of antipaternal antibodies was about three times higher in the asymmetric lgG fraction than in the symmetric one. Considering the percentage of asymmetric IgG molecules with antipaternal antigen specificity eluted from placenta and the possibility that they function as blocking antibodies, their participation in fetal protection is suggested. Correspomlenee to." Prof. R.A. Margni, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956. I113-Buenos Aires, Argentina. 0165-0378/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
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Key words: pregnancy; asymmetric antibodies; blocking antibodies; placenta;
fetus. Introduction The survival of the semiallogeneic mammaliam fetus in the presence of an alloreactive maternal environment remains still to be explained. However it is known that the immune system suffers several changes during pregnancy that are useful for the embryonic development (Deer et al., 1979, 1981; Bell et al., 1983; Chaouat, 1986). Recent work suggests that the most important protection for the fetus is provided by the placenta and decidua although the detailed mechanisms through which the tissues at the maternal-fetal interface accomplish this function needs clarification. Moreover, there is evidence that antipaternal MHC antibodies may confer protection from spontaneous fetal resorption. It is well known that pregnancy serum often contains leukocytotoxic antibodies with paternal specificity depending on the number of pregnancies a woman has had. These antibodies have been demonstrated by microcytotoxicity using leukocytotoxic assays with complement. However, there appears to be no correlation between the presence or absence of these antibodies and the success or failure of pregnancy. It is now well documented that in most immune sera a population of antibodies exists that is unable to form precipitates with the antigen, although they can coprecipitate in the presence of precipitating antibodies of the same specificity. These "non-precipitating" or "coprecipitating" antibodies were first obtained by Heidelberger and Kendall (1935) and in recent years a number of studies have been performed in our laboratories in order to elucidate their physicochemical and biological properties (Ronco et al., 1984; Leoni et al., 1986; Labeta et al., 1986; Morelli et al., 1989; Malan Borel et ai., 1990). These studies have been recently reviewed (Margni and Binaghi, 1988). It was demonstrated that non-precipitating antibodies possess an asymmetric structure due to an oligosaccharide moiety, present only in one of the two Fab regions of the molecules. The combination of the corresponding antibody site with the antigen is sterically hindered by the oligosaccharide group and as a consequence the molecule behaves functionally as univalent. The term of "asymmetric" has been employed to designate these molecules in opposition to symmetric precipitating antibodies which are not glycosylated in the two Fab regions. The antibody site present in the Fab region of asymmetric antibody devoid of the prosthetic carbohydrate group acts normally, and therefore the
131
molecule can be firmly combined with its corresponding antigen. Asymmetric antibodies are generally unable to activate effector functions, such as complement fixation, phagocytosis, cytotoxicity, etc. However, since they combine with the antigen, they act in a competitive way when mixed with precipitating antibodies of the same specificity. In fact, asymmetric igG antibodies act as blocking antibodies. Asymmetric antibodies are present in practically all mammalian sera and in all lgG subclasses. They constitute about 10% of the total population, but this proportion can increase strikingly in some cases (Margni et al., 1983, 1986). The asymmetric molecules are equally present, in about the same proportion, in all non-immune sera (Malan Borel et al., 1989). Symmetric and asymmetric IgG antibodies are synthesized by the same cellular clone (Morelli et al., 1989). The existence of asymmetric antibodies in all animal species studied so far suggests that they could play same important physiological role (Margni, 1989). Some observations indicate that this may be the case in allergic processes and in chronic infections. Taking into account the fact that they are functioning as blocking antibodies, it can be speculated that they may participate in the immunological aspects of the complex mechanism controlling the equilibrium between mother and foetus. In order to gain more information on this subject we have studied the production of asymmetric lgG molecules during human pregnancy, and we have determined their proportion in the maternal serum and in the placenta, as well as their specificity against paternal antigens. Materials and Methods
Isolation and purification of lgG from sera of pregnant and non-pregnant women IgG was obtained from normal pregnant women sera by precipitation at 50% saturation of (NH4)2SO4 followed by DEAE-callulosa chromatography. The IgG was eluted by 0.01 M phosphate buffer (pH 7.4). Isolation and purification of lgG from non-pregnant women sera was achieved in a similar way. Isolation and purification of IgG from placental blood Placenta were frozen at -20"C immediately after delivery and processed as soon as possible, within a few days maximum. After thawing, they were covered with a piece of wood, a weight of about 10 kg was applied and were left 2 h in the cold room. The exudate obtained was clarified by centrifugation, precipitated at 50% ammonium sulphate and the IgG was purified as indicated above.
132
Placental eluates The whole placenta from one woman was minced with scalpel blades into fine slices and suspended in phosphate buffered saline (PBS). It was washed with the same buffer and this procedure was repeated until the O.D. of the supernatant fell below 0.10. After the final wash the tissue was centrifuged for 10 min at 12,000 x g, the pellet resuspended in 100 ml of 4 M KC1, mixed with a magnetic stirrer for 60 rain, centrifuged and the supernatant dialyzad against PBS; then concentrated to 10 ml using a pressure concentration device (Amicon Corp., MA). The purification of IgG was made as indicated above.
F(ab' )2 fragment This was obtained by pepsin digestion as described by Turner et al. (1970). The IgG was dialyzed against 0.1 M sodium acetate buffer (pH 4.5) and digested with papain (Sigma), enzyme/substrate ratio 1/100, for 18 h at 37°C. The digestion was stopped by the addition of Tris to obtain a pH of 8. Purification of the F(ab')2 fragment from the mixture was achieved by Sephadex G-200 gel filtration. Purity of the F(ab')2 fragment was checked by immunoelectrophoresis using rabbit anti-human serum and by SDS-PAGE.
Determination of symmetric and asymmetric lgG and F(ab' )2./i'agnlents by the concanavalin A (Con A) test Separation of both, symmetric and asymmetric IgG molecules was acchieved by Con A-Sepharose chromatography, taking into account that this lectin binds molecules containing o~-D-mannopyranosyl, oz-D-glucopyranosyl and sterically related residues (Malan Borel et al., 1990). Briefly: Con A-Sepharose was washed with the elution buffer (Tris--HCl, 0.025 M; NaCI 0.2 M; CaCI_,, MgCI2, MnCI2, 0.003 M each; Na azide 0.02%, pH 7.2). The protein samples were dialyzed against the same buffer. Equal volumes (approx. 1 ml) of 50% packed Con A-Sepharose and protein (about 1 mg/ml were mixed in small tubes and gently shaken for 1 h at room temperature, then overnight in the cold. After centrifugation the pellets were repeatedly washed with the elution buffer and the protein in the collected supernates was estimated by measuring the optical density at 280 nm. This value corresponds to the symmetric IgG, not bound by the Con A. The asymmetric IgG was eluted from the pellets by repeated washing with 0.15 M o~methyl-mannoside in the elution buffer and estimated by measuring the optical density. All tests were made in triplicate. Recovery of protein (not retained plus retained by Con A) was generally not less than 95% of the initial sample. Separation of symmetric and asymmetric F(ab')2 fragments by Con ASepharose was achieved as indicated for IgG molecules.
133
In some cases when small amounts of IgG eluted from the placenta, or their respective F(ab')2 fragments, were chromatographed on Con ASepharose, the proteins were previously labelled with esI as described by Greenwood et al. (1963). The radioactivity was measured in a gamma radiation counter to determine the Con A bound and eluted protein.
Indirect immunofluorescence (IIF) Symmetric and asymmetric IgG from placental eluates and peripheral blood ware analyzed by indirect immunofluorescence, using as target paternal lymphocytes and non-paternal lymphocytes (negative control), Ten microliters of 106 cells/ml were put on a glass-slide and air dried for 30 min at room temperature. After that the cells were successively washed with a 0.1 M glycine-HCl buffer (pH 3), PBS and water. Fc receptors were blocked with 10 #1 of aggregated rabbit IgG (4 mg/ml) (Dickler et al., 1972) for 30 min at room temperature and then washed with PBS. 10/~1 of the sample, containing about 8 #g protein was added and incubated for 30 min at room temperature, then washed twice with PBS. Finally, fluoresceinconjugated rabbit F(ab')2 anti-human IgG was added and after 30 min the slides were washed with PBS. For visualization of the stained cells a Zeiss epifluorescent microscope was employed.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 7.5% polyacrylamide gels were poured as vertical slabs and run according to the method of Laemmli (1970). Intact and digested immunoglobulins were visualized by staining with 0.1% (w/w) Coomassie Blue R-250 (Mallinckrodt) in methanol/glacial acetic acid/water (5:2:5 by vol.). Staining patterns were compared with those of standard molecular weight markers (Sigma).
Purification of lymphocytes Human peripheral blood lymphocytes were obtained from the paternal heparinized blood by centrifugation on Ficoll-Hypaque (density 1.077 0.001 g/cm 3) at 300 x g for 20 min. After isolation they were washed three times in RPMI 1640, counted and resuspended to a concentration of 5 x 10 6 cells/ml. The percentage of viable white blood cells was determined by the trypan blue exclusion test.
Microcytotoxicity test ( MC) This was done in microplates and the method described by Terasaki (1973) was employed with minor modifications. One microliter of sample and 1 t~l of paternal lymphocytes (2 x 10 6 cells/ml) were added to each well. After incubation for 30 min at room temperature 5 #1 of previously titrated fresh rabbit sera (complement source)
134
were added and the plates incubated again at the same temperature for 1 h and 30 min. After that 5 #1 o f 5°/,, eosin in water was added and 3 min later 5 #1 o f non-diluted formalin was incorporated into the walls. The percentage of lysed cells was determined by counting dead (stained) and viable (unstained) cells using an inverted microscope. Results Ten pregnant women were studied at delivery. Six of the mothers were in their first pregnancy, and four (1 4 in the Tables) had three or more pregnancies with the same partner. Placentae were collected and treated, as described in Methods, to obtain placental blood and placental eluates. Maternal serum was simultaneously obtained. IgG was purified from these samples and from sera of nonpregnant woman and their F(ab')2 fragments were prepared. TABLE
I
Percentage o f C o n A f i x a t i o n a n d s p e c i f i c a c t i v i t y f o r paternal lymphocytes of IgG and ( F b ' ) 2 fragments purified from peripheral b l o o d o f pregnant women (a) and f r o m b l o o d o b t a i n e d b y mechanical pressure o f their placenta (b). Samples l I--16 are controls (non-pregnant women). Sample
igG
F(ab)2
No.
Fixation
MC
IIF
Fixation
to C o n A (%) a
IIF
to Con A a
b
a
b
b
(%1 a
a b
I
20
20
+
+
++
++
19
20
++
++
2
25
24
+
+
++
++
24
23
++
++
3
26
25
+
+
++
++
25
25
++
++
4
30
30
+
+
++
++
30
30
++
++
++
++
++
5
26
26
+/-
26
25
++
6
24
24
.
.
+ .
.
24
24
--
7
30
30
.
.
.
.
29
30
--
8
22
22
.
.
.
.
22
22
--
++
+
22
21
++
++
--
++
+
22
22
++
++
9
22
22
10
22
22
II
12
--
I1
12
10
--
13
13 14
15 13
---
15
14
--
--
14
--
16
9
--
--
11
--
--
--
15 12
---
M C r e s u l t s : ( + ) p e r c e n t a g e o f l y s e d cells more than 3 0 % ; ( - - ) p e r c e n t a g e o f l y s e d cells less than 3~Y'/.. I I F results: (+) percentage of fluorescent cells between 5% and 20"/,,. ( + + ) more than 2 0 % . ( - - ) less than 5"/.
135
All the samples were studied by the Con A test, in order to estimate their proportion of asymmetric molecules. Anti-paternal antibodies ware investigated by indirect fluorescence using paternal lymphocytes as target cells. The lgG samples were also tested by the microcytotoxicity test. The results are reported in Tables 1 to 4. Table 1 shows the results obtained using IgG or F(ab')2 fragments isolated from maternal blood and from blood obtained by mechanical extrusion of the placenta. It can be seem that both results are strictly coincident. The percentage of IgG combining to Con A was from 19 to 30%. (average 24.4%). This value is significantly higher than that of 12.1% observed in nonpregnant women sera, indicating that the production of asymmetric IgG increases significantly during pregnancy. The IIF test using paternal lymphocytes and maternal serum was positive in seven out of ten mothers but the microcytotoxicity test was positive only in five of these seven mothers. In three cases IIF and MC were both negative (mothers 6-7-8) and they corresponded to mothers having their first pregnancy. Adequate controls were made using non-paternal lymphocytes from a third party. When IgG and F(ab')2 fragments, obtained from the placenta by elution with high ionic concentration, were analyzed (Table 2), the positivity observed in IIF and MC was the same as the one observed in the maternal sera. However, the percentage of asymmetric IgG was strongly increased, with values ranging from 32 to 62% (average 44.4%). TABLE 2 P e r c e n t a g e o f C o n A f i x a t i o n a n d specific a c t i v i t y f o r p a t e r n a l l y m p h o c y t e s o f I g G a n d F ( a b ' )2 f r a g m e n t s eluted f r o m p l a c e n t a . S y m b o l s a r e the s a m e as i n d i c a t e d in T a b l e I. Sample No.
Total lgG
Total F(ab')2
Fixation to C o n A ("/,,)
MC
IIF
Fixation
ilF
to Con A (,'/,,)
1
32
+
2
55
+
++ ++
31 55
++ ++
3 4 5 6
54 62 56 36
+ + + --
++ ++ ++ --
54 61 55 36
++ ++ ++ --
7 8 9 10
39 36 38 39
-----
--++ ++
38 35 38 38
--++ ++
136
The IgG eluted from the placenta at high ionic concentration represents the population of IgG which is fixed to the cells, either because it is combined with membrane antigens, presumbly paternal antigens of the fetus against which the mother has produced antibodies, or directly fixed via Fc receptors. It was of interest to study the specificity of the population by IIF and MC against paternal antigens, after fractionation into symmetric and asymmetric molecules. This fractionation was done by chromatography with Con A Sepharose using five of the maternal samples. The results are presented in Table 3. It is seen that in all cases antipaternal antibodies were present in both symmetric and asymmetric molecules, as evidenced by IIF, the asymmetric fraction being apparently more active. The MC test was positive in three samples of the symmetric molecules and was negative with all five asymmetric molecules as expected, since they do not fix complement. In an attempt to evaluate the relative proportion of asymmetric antibody molecules, placental IgG was further investigated in some cases (mothers 9 and 10). After fractionation by Con A-Sapharose, both populations were tested by IIF. When equal concentrations were used, it was found that the antibody activity of the asymmetric fraction was higher than that of the symmetric one. The results obtained are presented in Table 4. From the values obtained it can be estimated that the concentration of antipaternal antibodies is about three times higher in the asymmetric fraction than in the symmetric one. Discussion The studies described in the present paper reveal that, during pregnancy,
TABLE 3 A n t i p a t e r n a l l y m p h o c y t e specificity o f I g G s y m m e t r i c a n d a s y m m e t r i c m o l e c u l c s eluted f r o m p l a c e n t a . S y m b o l s are the s a m e as i n d i c a t e d in T a b l e I. Sample No.
T o t a l I g G eluted f r o m p l a c e n t a Symmetric IgG
Asymmetric lgG
MC
IIF
MC
IIF
2 3
+ +
+ ++
~
++ ++
4 9 10
+
+ + +
--
--
++ ++ ++
137 TABLE 4 Antipaternal lymphocyte activity of lgG fixed to placenta. The % of IIF expressed are the average of the results obtained with samples Nos. 9 and 10. In each case 400 cells were counted. Sample
#g IgG/tube
lgG symmetric molecules
25 50 100
IgG asymmetric molecules
25 50 100
5 14 26
Total lgG from non-immune human sera
25 50 100
<4 <4 <4
PBS (control)
% of fluorescent cells 2 2.5 7.5
<4
the production of asymmetric lgG molecules is considerably increased. The average percentage found in the sera of the 10 mothers studied was 24.4; this is the double of the percentage present in non-pregnant women sera and in non-immune subjects as already reported. Exactly the same value was found in the serum obtained from the placenta by mechanical pressure, as could be expected since it is indeed maternal serum. On the contrary, when IgG was obtained from the placenta itself after careful washing and subsequent treatment at a high ionic concentration (4 M KCI), the percentage of the asymmetric molecules was much higher, with an average of 44.4% and values reaching up to 62%. The IgG so obtained is of maternal origin and fixed to the membrane of the placental cells. This IgG is fixed to the cellular membrane by two different machanisms: through the Fc receptor or by binding to paternal antigens. Since asymmetric and symmetric molecules have the same Fc structure, the proportion of symmetric and asymmetric molecules fixed to Fc receptors must be the same as that present in the serum. On the other hand, the proportion of symmetric and asymmetric molecules bound to paternal antigens must depend on the relative concentration of antibodies in each population. The fact that the proportion of fixed asymmetric molecules is twice as high as the one present in the serum, strongly suggests that the concentration of asymmetric antipaternal antibodies is higher than that of symmetric ones.
138
Exactly the same results were obtained using IgG or their F(ab')z fragment. The reason why F(ab')2 was studied was to eliminate the posibility that IgG molecules attach to Con A via eventual carbohydrate groups present in the Fc regions, that are not relevant to the asymmetry of the molecule. This possibility exists in the case of IgG from same animal species, but in the case of human IgG practically no Con A-binding carbohydrate is present in the Fc region. The present results confirm previous observations (Leoni et al., 1986; Labeta et al., 1986). Antibodies against paternal antigens were investigated using two different techniques: IIF and MC assays. By IIF, both antibody types, symmetric and asymmetric should be detected. The cytotoxic assay, on the contrary, can only be positive with complement-fixing antibodies, that is, only with symmetric ones. It was found that 7 mothers gave a positive IIF test either with serum IgG or with placenta-eluted IgG. The cytotoxic test was positive in five of these seven mothers. In three mothers no antipaternal antibody specificity could be detected either by IIF or by MC. The absence of antipaternal antibodies is probably due to a weak antigenic stimulation, since mothers 5--10 were in their first pregnancy. It cannot be excluded, however, that the negative results simply reflect a low sensitivity of the test employed. Five of the placental IgG samples were further studied. They were processed by chromatography on Con A-Sepharose, to obtain the asymmetric and symmetric populations, which were then tested by IIF and MC with paternal lymphocytes. IIF was positive in all the fractions, the reactions being the strongest with the asymmetric populations. The MC assay was negative in all the asymmetric fractions, as expected, since this assay is complementdependent and asymmetric IgG does not fix complement. In the symmetric fractions, the MC assay gave positive results in three cases and was negative in mothers 9 and 10. A semi-quantitative IIF assay was performed with the symmetric and asymmetric fractions by evaluation of the percentage of fluorescence obtained at various concentrations. The asymmetric fractions proved to be, about three times more active than the symmetric fractions indicating that the antipaternal antibodies were in the majority of the asymmetric type. From the results reported in the present paper, two conclusions can be drawn. First, it is clear that the production of asymmetric IgG molecules increases strikingly during pregnancy. Since the increase is observed in the total IgG population, out of which antibodies directed to paternal antigens are only a minimal fraction, this non-specific increase seems to be induced by pregnancy itself, perhaps by factors secreted by the placenta. This possibility is now under study. Secondly, it was found that antipaternal an-
139
tibodies synthesized by the mother are, in the majority, of the asymmetric type. This is not the usual finding when immunizations are performed with soluble antigens, but it is in line with previous observations showing a preferential synthesis of asymmetric antibodies when particulate antigens are used, as well as in some chronic infections (Hajos et al., 1982; Margni et al., 1983, 1986; Carbonetto et al., 1986). Since asymmetric antibodies do not fix complement and do not activate effector immune mechanisms, and may further act as blocking antibodies, it is worth asking whether they play a role in the protection of the fetus against cytotoxic symmetric, complement fixing antipaternal antibodies produced by the mother. A blocking effect has already been demostrated in other systems, such as complement fixation (Margni et al., 1980) and phagocytosis of opsonized bacteria (Perdig6on et al., 1982). In all cases, it was found that asymmetric antibodies compete with their symmetric counterpart by binding the same antigen. What appears to be determinant for the blocking effect is not the absolute quantity of asymmetric antibodies but the relative proportion of asymmetric to symmetric antibodies, acting simultaneously. In the case of complement fixation, the blocking effect is already perceptible when 20"/,, of the total antibodies are asymmetric, and the complement fixation is completely abolished at 80%. Incidentally, these observations must be taken into account when performing tests to detect antibody activity. When the assay is solely based in the primary interaction between antibody and antigen, as is the case with immunofluorescence, both symmetric and asymmetric antibody molecules are detected, but when the assay implies an effector mechanism, as in the case of complement fixation or complement-dependent cytotoxic reactions, a negative result may be obtained because of a competitive effect due to asymmetric antibodies. Although the present observations do not allow to draw any conclusions concerning a possible protective effect upon the fetus, the high proportion of asymmetric molecules found in the IgG fixed to the placenta is highly suggestive in this respect.
Acknowledgement This work was supported in part by a scientific cooperation agreement between CONICET (Argentina) and INSERM (France).
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