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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4418-4424
Normal Human Serum Contains Natural Antibodies Reactive With Autologous
ABO Blood Group Antigens
By
Sergio H. Spalter,
Srini V. Kaveri,
Emmanuelle Bonnin,
Jean-Claude Mani,
Jean-Pierre Cartron, and
Michel D. Kazatchkine
From INSERM U430 and Université Pierre et Marie Curie, Hopital
Broussais, Paris, France; CNRS UMR 9921, Montpellier, France; and
INSERM U76 and Institut National de Transfusion Sanguine, Paris,
France.
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ABSTRACT |
It is widely accepted that the serum of healthy individuals contains
natural antibodies only against those blood group A or B antigens that
are not expressed on the individual's red blood cells. The mechanisms
involved in tolerance to autologous blood group antigens remain
unclear. In the present study, we show that IgM and IgG antibodies
reactive with autologous blood group antigens are present in the
immunoglobulin fraction of normal human serum. Natural IgG anti-A
antibodies purified by affinity chromatography from IgG of individuals
of blood group A exhibited an affinity for A trisaccharide antigen in
the micromolar range and agglutinated A red cells at sixfold higher
concentrations than those required for agglutination with
affinity-purified anti-A IgG of individuals of blood group B. Whereas
autoantibodies reactive with self A and B antigens are readily detected
in purified IgG and IgM fractions, their expression is restricted in
whole serum as a result of complementary interactions between variable
regions of antibodies. These observations suggest that tolerance to
autologous ABO blood group antigens is dependent on peripheral control
of antibody autoreactivity.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PRESENCE OF antibodies directed
against A and B alloantigens pre-existing in the serum of naive
individuals before transfusion has been recognized for several
decades.1 It has been considered that because ABO
histo-blood group antigens are oligosaccharides commonly occurring in
nature, individuals of blood group A become immunized to type-B
carbohydrate antigen present in the bacterial environment, although
they remain tolerant to A antigens.2 Thus, anti-A
antibodies are found in the serum of group O and B individuals and
anti-B antibodies are found in the serum of group O and A individuals.
Group AB individuals are believed not to have anti-A nor anti-B
antibodies because they express both antigens on their red cells.
Natural antibodies, ie, antibodies produced in the absence of overt
antigenic stimulation, have been recognized in the serum of healthy
individuals.3 Natural autoantibodies consist of immunoglobulinM (IgM), IgG, and IgA immunoglobulins encoded by germline
genes, and react with a wide range of self-antigens including nuclear
antigens, intracellular and membrane components, and circulating plasma
proteins.4 Natural autoantibodies are often polyreactive, exhibit idiotypic cross-reactivity and variable (V) region
connectivity.5-8 Complementary interactions between V
regions regulate autoreactivity of natural autoantibodies in whole
serum, thus contributing to the maintenance of immune homeostasis under
physiological conditions.9,10
We now show that IgG and IgM antibodies reactive with autologous A and
B antigens are present in normal serum immunoglobulin. Our results
indicate that autoantibody activity to A and B antigens is controlled
in whole serum by antibodies complementary to the V regions of the
antibodies to autologous blood group antigens. These observations
suggest that "tolerance" to autologous ABO blood group antigens
as observed in serum, is dependent on peripheral control of antibody autoreactivity.
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MATERIALS AND METHODS |
Antibodies.
Serum was obtained from 32 healthy adult blood donors of blood groups
A1 (n = 8), B (n = 8), AB (n = 8), and O (n = 8). All donors were RhD-negative. IgG was purified from serum by chromatography on protein G-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden). When required, F(ab')2 fragments were prepared from
IgG by pepsin digestion (2% wt/wt) (Sigma Chemical Co, St Louis, MO)
in acetate buffer pH 4.1 for 18 hours at 37°C and chromatography on
protein A-Sepharose (Pharmacia). Concentrations of purified IgG and
F(ab')2 fragments were determined
spectrophotometrically at 280 nm using an extinction coefficient of
1.4. The concentration of IgG in serum was determined by ELISA. IgM was
quantitated in the flow-through of the protein G-Sepharose column by
ELISA and by using the BCA protein assay (Pierce, Rockford, IL). The
concentration of IgG in the IgG-depleted fraction of serum was below 20 µg/mL.
Anti-A antibodies were purified from pooled serum of six donors of
blood group A1 and of six donors of blood group B by
affinity chromatography on a macroporous glass matrix covalently coated with poly[N-(2-hydroxyethyl)acrylamide] (PAA)-bound synthetic A
trisaccharide (Atri-PAA-MPG) (Syntesome, Moscow, Russia)
and elution of the column at pH 3.0 followed immediately by
neutralization of the eluted fractions using Tris 2.0 mol/L.
Affinity-purified anti-A IgG antibodies were then separated from anti-A
IgM by chromatography on protein G-Sepharose. Concentrations of
purified anti-A IgM were determined using the BCA protein assay. The
purity of the PAA-conjugates was determined by initial saccharides.
Synthetic w-aminoalkyl glycosides used for coupling with the polymer
were more than 95% pure as evidenced by high-performance liquid
chromatography (HPLC) and 1H-NMR. M06 is a human monoclonal IgG
directed against pp65 antigen of cytomegalovirus,11 a kind
gift of Dr M. Ohlin (Lund, Sweden).
ABO antigens.
PAA-bound A trisaccharide glycoside (GalNAc 1-3(Fuc1-2)Gal 1)
and B trisaccharide (Gal1-3(Fuc1-2)Gal1) were obtained from Syntesome. The A and B antigen conjugates contained 0.77µmol/mg (15%) and 0.36µmol/mg (5%) of saccharide, respectively.
Enzyme immunoassays.
Anti-A and anti-B antibody activities were quantitated by ELISA. Ninety
six-well polystyrene flat-bottom microtiter ELISA plates (Nunc,
Roskilde, Denmark) were coated with PAA-bound A (10 µg/mL) or B (10 µg/mL) trisaccharides in carbonate buffer pH 9.0 for 2 hours at
37°C and, subsequently, for 12 hours at 4°C. The plates were
then incubated with 1.0% bovine serum albumin (BSA) (Sigma) in
phosphate-buffered saline (PBS) for 1 hour at room temperature. After
washing with PBS, the plates were incubated with various dilutions of
the antibodies to be tested in PBS containing 1.0% BSA for 1 hour at
37°C. The plates were then washed with PBS and incubated with
peroxidase-labeled goat antihuman Fc (Jackson Laboratories, West
Grove, PA) or peroxidase-labeled goat antihuman Fcµ antibodies
(Southern Biotechnology, Birmingham, AL) for 1 hour at 37°C. Bound
IgG or IgM were revealed using the enzyme substrate OPD. The reaction
was stopped after 4 minutes in all assays. Absorbance at 495 nm was
determined using an Emax ELISA reader (Molecular Devices, Menlo Park,
CA). Anti-A and anti-B monoclonal antibodies (MoAbs) (clones A003
anti-A; B005 anti-B; Biotest Pharma, Dreieich, Germany) were used as
positive controls for standardization of ELISA. Optical density values
in ELISA < 0.3 were considered "negative."
Agglutination assays.
Blood group A1 and B red blood cells for use in agglutination tests
were prepared from freshly drawn venous blood of healthy volunteers.
Indirect antiglobulin tests were performed by incubating 50 µL of a
3.0% saline suspension of indicator red cells with 100 µL of 7.0%
BSA and 100 µL of the source of antibody to be tested diluted in PBS,
for 45 minutes at 37°C. After incubation, the cells were washed
three times with PBS, and blotted dry. One drop of antihuman globulin
Coombs reagent (Organon Teknika BV, Boxtel, Holland) was then added.
The tubes were mixed, centrifuged, and examined for agglutination
microscopically. Agglutination was graded from  to ++++, using a
conventional grading system. Direct agglutination assays were performed
by incubating indicator cells with antibodies in V-bottom wells for 1 hour at 37°C. Agglutination was recorded on inclination of the
plates and by microscopic examination.
Biosensor measurement of antibody activity.
Real-time analysis of antigen-antibody complex formation between A and
B trisaccharides and anti-A and anti-B antibodies was performed using
the BIAcore system (Pharmacia). Biotinylated PAA-antigen A
(Atri-PAA-biotin) and antigen B
(Btri-PAA-biotin) (Mr~30 kD) (Syntesome) were
immobilized on streptavidin-coated sensor chip SA5
(Pharmacia) in HEPES complete buffer pH 7.4 (10 mmol/L HEPES, 150 mmol/L NaCl, 3.4 mmol/L EDTA, 0.05% Surfactant P20) (HBS) at a
continuous flow rate of 5 µL/minute. One hundred microliters of HBS
containing 1.0 mg/mL of purified IgG or 0.3 mg/mL of affinity-purified anti-A IgG to be tested were injected at a continuous flow rate of 5 µL/minute allowing a total contact time with the antigens of 6 minutes. Kinetic analysis and calculations of association and
dissociation constants were performed using the BIAevaluation Software (Pharmacia).
Statistics.
When appropriate, statistical analysis was performed using analysis of
variance (ANOVA), Fisher's test, and the StatView software (SAS
Institute Inc, Cary, NC).
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RESULTS |
IgG and IgM antibodies to autologous ABO antigens in normal human
serum.
The reactivity of IgG in the serum and in the purified IgG fraction of
the serum of four healthy adults of each of the blood group categories
A1, B, AB, and O, with A and B antigens, was measured by means of ELISA
(Fig 1). As expected, IgG in whole serum of
individuals of blood groups B and O bound to A antigen in a
dose-dependent fashion. The binding of serum IgG of individuals of
blood groups A1 and AB was low and differed significantly from that of
serum IgG of individuals B and O (P < .0001 in all
instances). The mean binding of serum IgG of individuals of blood group
A1 did not differ from that of individuals of blood group AB.
Similarly, IgG in whole serum of individuals of blood groups A1 and O
exhibited a significantly higher reactivity with B antigen than did
serum IgG of individuals of blood groups B and AB (P < .0001 in all instances). The mean binding to B antigen of IgG in whole serum of individuals of blood group O was significantly higher than that of
serum IgG of individuals of blood group A1 (P < .0001). In
contrast to results obtained using whole serum, we observed no
difference in the mean binding activity of IgG purified from the serum
of donors of blood groups A1, B, AB, and O to A and B antigens. Thus,
there was no difference in the mean reactivity of purified IgG of
donors of blood group A1 and individuals of blood group B with antigen
A (P = .8698) and antigen B (P = .3439) (Fig 1). The
specificity of interaction of natural antibodies to blood group
antigens was validated by using MoAbs. Thus, we showed that MoAbs
directed against A antigen bound to A antigen and not to B antigen and
conversely MoAbs directed against B antigen bound to B antigen and not
to A antigen (data not shown).
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| Fig 1.
Natural IgG antibodies to autologous A and B blood group
antigens in normal human serum. Upper panels: reactivity of IgG in
whole serum of individuals of blood group A ( ), B ( ), O ( ),
and AB ( ) with immobilized A (left panel) and B (right panel)
antigens, as assessed by ELISA. Lower panels: reactivity of IgG
purified from the serum of individuals of blood groups A (), B
(), O (), and AB () with A (left panel) and B ( right panel)
antigens. Each data point represents mean OD ± SD obtained on testing
IgG of four individuals in each blood group category.
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The binding of IgM in the serum of individuals of blood groups B and O
to A antigen was higher than that of serum IgM of individuals of blood
group A1 and AB (P < .0001 in all cases). Similarly, the
binding of serum IgM of individuals of blood group A1 and O to B
antigen was higher than that of serum IgM of individuals of blood group
B and AB (P < .0001 in all cases). We then prepared the
IgG-depleted fraction of serum of the four donors by chromatography on
protein G Sepharose and used it as a source of IgM. IgM thus purified
from the serum of A1 and B donors bound to autologous A and B antigens,
although no or little such binding was observed on testing of IgM in
whole serum (Fig 2). Thus, the difference in reactivity of purified IgM with allo- and with autologous A and B
antigens was small (Fig 2).
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| Fig 2.
Natural IgM antibodies to autologous A and B blood group
antigens in normal human serum. Upper panels: reactivity of IgM in
whole serum of individuals of blood group A (), B (), O (),
and AB () with immobilized A (left panel) and B (right panel)
antigens, as assessed by ELISA. Lower panels: reactivity of IgM
(IgG-depleted serum) of individuals of blood groups A (), B (), O
(), and AB () with A (left panel) and B (right panel) antigens.
Each data point represents mean OD ± SD obtained on testing IgG of
four individuals in each blood group category.
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Inhibition of the binding of IgG to autologous blood group antigens
by autologous IgG-depleted serum.
IgG purified from the serum of individuals of blood groups A1, B, AB,
and O was coincubated with IgG-depleted serum, used as a source of
autologous IgM, at a ratio of IgG/IgM (wt/wt) of 1:10, for 1 hour at
37°C before assessing the reactivity of IgG with A and B antigens
by ELISA. IgM (IgG-depleted serum) of individuals of blood group A1
inhibited the reactivity of autologous IgG with A antigen but not with
B antigen (Fig 3). In a similar fashion, IgM (IgG-depleted serum) of individuals of blood group B inhibited the
reactivity of autologous IgG with B antigen but not with A antigen. IgM
of individuals O did not affect the binding of the corresponding IgG to
both antigens. IgM (IgG-depleted serum) of AB blood group individuals
inhibited the reactivity of autologous IgG to both antigens. These
results suggest that the absence of reactivity of IgG with autologous A
and B antigens in the serum of individuals of blood groups A and B, is
dependent on inhibition of self-reactive IgG by factors present in
IgG-depleted fraction of serum, most likely by autologous IgM.
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| Fig 3.
IgM inhibits the reactivity of purified autologous IgG
with autologous blood group antigens. IgG was purified from serum of
individuals of blood groups A1 (), B (), O (), and AB ()
and incubated with the respective autologous IgG-depleted fractions of
serum used as sources of IgM at a ratio of IgG to IgM (wt/wt) of 1:10
for 1 hour at 37°C. The reactivity of IgG with A (left panel) and B
antigens (right panel) was then assessed by ELISA. Each data point
represents the mean OD ± SD of binding of IgG from four individuals
of each blood group category. The binding of IgG of individuals of
blood group A to A antigen in the presence of autologous IgM was
significantly lower than the binding of IgG of individuals of blood
group B coincubated with autologous IgM (P < .0001).
Similarly, the binding of B IgG to B antigen in the presence of B IgM
was significantly lower than that of A IgG in the presence of A IgM
(P < .0001).
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We then affinity-purified anti-A antibodies from pooled serum of six
individuals belonging to blood groups A1 and B, respectively, using a
macroporous glass matrix covalently coated with
poly[N-(2-hydroxyethyl)acrylamide] (PAA)-bound synthetic A
trisaccharide (Atri-PAA-MPG). Anti-A IgG was then isolated
from the non-IgG containing fraction by chromatography on protein G
Sepharose. We then assessed the reactivity of anti-A IgG of individuals
of the two blood groups with A antigen by ELISA. As shown in
Fig 4, we found no difference in the mean
binding activity to A antigen of purified anti-A IgG of donors of blood groups A1 and B. However, the non-IgG fraction obtained from
individuals of blood group B bound with higher avidity to A antigen
than the non-IgG fraction of individuals of blood group A1 (Fig 4). The specificity of the binding of affinity-purified anti-A IgG to immobilized A antigen was confirmed by two additional experiments. First, we increased the concentration of BSA, used as a blocking agent
from 1.0% to 4.0% and observed a dose-dependent binding of
affinity-purified anti-A IgG of an individual A to the A antigen bound
to an ELISA plate. Second, we showed a dose-dependent inhibition of the
binding of affinity-purified anti-A IgG of individuals A and B to
immobilized A antigen, by soluble A blood group antigen in an ELISA
(Fig 5). We further observed that
coincubation of the non-IgG fraction obtained as described above, of
donors of blood group A1 with anti-A IgG, inhibited the binding of
anti-A IgG of A1 donors to A antigen. In contrast, the addition of the non-IgG fraction obtained from blood group B donors did not inhibit the
binding of anti-A IgG of individuals of blood group A1 to A antigen
(Fig 6). Taken together, these data
indicate that normal circulating IgG expresses antibody activity
against autologous ABO blood group antigens and that autoreactivity of
IgG to ABO antigens is masked in serum by interactions of IgG with
autologous complementary molecules. A rough estimate indicates that for
every antibody molecule that binds to A antigen, there are
approximately 30 complementary inhibitory molecules. Conversely, it is
likely that the reactivity of circulating IgM with autologous ABO
antigens is masked by complementary V region-dependent interactions
with autologous IgG.
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| Fig 4.
Reactivity with A antigen of affinity-purified anti-A
antibodies isolated from the serum of donors of group A1 and of group
B. Anti-A antibodies were affinity-purified from pooled serum of six
individuals belonging to each blood group category by chromatography on
a macroporous glass matrix covalently coated with synthetic A
trisaccharide. Anti-A IgG was then separated from anti-A IgM by
chromatography on protein G Sepharose. Left panel: Binding of anti-A
IgG of A1 () and B ( ) individuals to A antigen, as assessed by
ELISA. Right panel: Binding of anti-A IgM of A1 () and B ()
individuals to A antigen, as assessed by ELISA.
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| Fig 5.
Inhibition of the binding of anti-A IgG of an individual
of blood group A and of an individual of blood group B, to A antigen by
soluble A antigen. Anti-A IgG of an individual of blood group A (left
panel) and anti-A IgG of an individual of blood group B (right panel)
were coincubated with varying concentrations of inhibitors soluble A
antigen, B antigen, and H substance. Antibody binding was determined
using an ELISA.
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| Fig 6.
IgM (IgG-depleted fraction of the serum) of donors of
blood group A but not of donors of blood group B inhibits the
reactivity of affinity-purified autologous anti-A IgG with A antigen.
Affinity-purified anti-A IgG were obtained as described in the legend
to Fig 3. Increasing concentrations of IgM (IgG-depleted fraction of
the serum) of donors of blood group A1 ( ) and of blood group B ()
were coincubated with affinity-purified, biotinylated anti-A IgG of
individuals of blood group A1 before assessing the binding of IgG to A
antigen by ELISA.
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Agglutination of red cells by natural anti-ABO antibodies.
We examined the capacity of purified IgG to agglutinate red cells
expressing self- or allo-ABO antigens, by means of an indirect antiglobulin Coombs test. Whereas normal IgG purified from serum agglutinated cells expressing allo-ABO antigens at concentrations above
0.25 mg/mL, IgG did not agglutinate red cells expressing self-ABO
antigens under the same experimental conditions
(Table 1). The lack of self-agglutinating
ability of purified serum IgG, even if cross-linked, suggests that
natural IgG autoantibodies to autologous ABO antigens are present in
small amounts and/or exhibit low affinity toward the self antigens.
However, agglutination of A red cells was observed when anti-A IgG of
individuals A had been affinity-purified and used instead of
unfractionated IgG (Table 2). The
concentration of anti-A IgG, necessary for agglutination was 3.25 mg/mL. Similar to findings with IgG, we observed that IgM in purified
form (IgG-depleted fraction of serum) agglutinated allo-ABO
antigen-bearing red cells but did not agglutinate autologous ABO
antigen-bearing cells at similar inputs in the assay (Table 1);
however, affinity-purified anti-A IgM of A individuals agglutinated A1
red cells (Table 2).
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Table 2.
Agglutination of Red Blood Cells of A1 Individuals by
Affinity-Purified Natural Anti-A IgG and IgM Antibodies*
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The agglutinating ability of affinity-purified anti-A IgG of A
individuals was further investigated by means of a direct agglutination assay. As shown in Table 3,
affinity-purified anti-A IgG of A individuals agglutinated A red cells
at a concentration of IgG of 1.8 mg/mL. At an equivalent concentration,
no agglutination occurred in the presence of the human monoclonal MO6
IgG directed against pp65 antigen of cytomegalovirus. Additional
negative controls consisted of antibodies present in the effluent
(pass-through) from the affinity column of (PAA)-bound synthetic A
trisaccharide, onto which IgG of individual of blood group A had been
loaded. The effluent that was checked for depletion of anti-A activity by ELISA, did not agglutinate the red blood cells of individuals of
blood group A. Affinity-purified anti-A IgG of B individuals agglutinated the cells at concentrations of 400 and 200 µg/mL, in a
dose-dependent fashion. Affinity-purified anti-A IgG of A individuals
did not agglutinate red cells of individuals of blood groups B and O.
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Table 3.
Agglutination of Red Blood Cells of Individuals of Blood
Groups A, B, and O by Affinity-Purified Natural Anti-A IgG of an A
Individual, as Assessed by Means of a Direct Agglutination
Assay*
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Analysis of real time complex formation between natural IgG
antibodies and autologous ABO antigens.
We used the BIAcore technology to further analyze the
interaction of natural IgG antibodies with autologous blood group
antigens. Synthetic A antigen was immobilized onto the sensor chip (1.8 ng per mm3). IgG was purified from pooled serum of six
individuals of each blood group A1, B, AB, and O. The binding constants
measured at equilibrium (KE) of the binding of purified IgG
to A antigen were calculated from the on- and off-rate constants to be
2.60, 0.64, 3.22, and 0.88 × 106 mol/L for IgG of
individuals A1, B, AB, and O, respectively
(Fig 7). We then affinity-purified anti-A
IgG antibodies from pooled serum of six A1 and six B individuals. The
binding constants for the binding of affinity-purified anti-A IgG to A
antigen were 127 and 28 × 106 mol/L for IgG of
individuals A1 and B, respectively (Fig 7). We then deduced mole:mole
ratios of bound antibody to antigen from the amount of Resonance Units
(RU) bound at equilibrium, using the BIAevaluation software that allows
the calculation of theoretical amounts of bound antibody when
equilibrium is reached. The results indicate that,
although their association constant for A antigen is higher, much fewer
molecules of specific anti-A IgG are present in IgG of individuals of
blood group A than in IgG of individuals of blood group B.
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| Fig 7.
Real time analysis of the binding of anti-A antibodies to
immobilized A antigen. Top panels: apparent equilibrium constants
(KA) for the binding of purified IgG from serum of
individuals A1, B, AB, and O (left panel) and for the binding of
affinity-purified anti-A IgG from individuals A1 and B (right panel) to
A antigen. Lower panels: mole to mole ratios of bound antibody to
antigen for the binding to A antigen of IgG of individuals A1, B, AB,
and O (left panel) and for the binding to A antigen of
affinity-purified anti-A IgG from individuals A1 and B (right panel).
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DISCUSSION |
It is widely accepted that the serum of healthy individuals contains
natural antibodies only against those blood group A or B antigens that
are not expressed on the individual's red blood cells. The
trisaccharidic terminal structures recognized by anti-ABO blood group
antigen antibodies result from the sequential activity of H, A, and/or
B glycosyltransferases. In individuals of blood groups A, B, O, and AB,
the H transferase adds a fucose residue to a terminal galactose. In
individuals of blood groups A, B, or AB, the A or B transferase then
adds either an N-acetylgalactosamine (blood group A) or galactose
(blood group B). The mechanisms involved in tolerance to autologous
blood group antigens remain unclear. It is generally believed that
healthy individuals are exposed to ABO trisaccharides expressed on
bacteria of the intestinal flora, and, as a result, produce antibodies
to allo-ABH antigens although they remain unresponsive to autologous
ABH antigens. Natural anti-ABO blood group antigen antibodies have long
been considered to be of the IgM isotype, in contrast with immune
antibodies of the IgG isotype that occur after alloimmunization in
pregnancy or ABO incompatible transfusions. It had been proposed by
Landsteiner12 that natural antibodies had a dual origin,
antigen-induced and spontaneous. Natural anti-A and anti-B antibodies
believed to be heteroagglutinins are produced as a response to
substances in the environment. Antierythrocyte antibodies were
considered to be of spontaneous origin.13 Anti-ABO
alloantibodies of the four IgG subclasses have, however, been
characterized in normal serum.14 In the present study, we
show that natural IgM and IgG autoantibodies reactive with self blood
group antigens with affinities in the micromolar range are present in
normal serum immunoglobulin. Whereas these antibodies are readily
detected in purified IgG and IgM fractions, their expression is
restricted in whole serum as a result of complementary interactions
between V regions of antibodies.
We observed that purified IgG and IgM of healthy individuals expressed
similar reactivity toward A and B trisaccharides, irrespective of the
ABO blood group of the donor, when assessed by ELISA. Thus, IgG and IgM
antibodies reactive with autologous A and B blood group antigens are
present in serum immunoglobulin of healthy individuals. Assays using
whole serum have previously detected low levels of self reactivity of
natural anti-A and anti-B antibodies although the results were
interpreted as being nonspecific.14 When we tested IgG and
IgM in whole serum at the same concentrations as those used for testing
purified IgG and IgM, little if no reactivity was observed with
autologous A and B antigens. As expected, high reactivity of IgG and
IgM was observed with allo-A or B antigens. The results indicate that
IgG reactivity with self A and B antigens is controlled in whole serum
by non-IgG serum factors. The results suggest that, under the
conditions of the assay, inhibition by autologous IgM of the reactivity
of anti-A IgG of individuals A1 with A antigen is dependent on
complementary V region interactions between IgG and IgM rather than on
a competition between immunoglobulins for the binding to the antigen.
We and others have shown previously that autoreactivity of IgG in
normal human and mouse serum is regulated by complementary interactions
between V regions of IgG and V regions of autologous IgM
antibodies.9,10 This is one of the mechanisms of peripheral
control of autoreactivity that has been widely documented, both under
physiological conditions9,10,15,16 and in patients in
remission of autoimmune disease.17-20 Thus, the addition of
autologous IgM dose-dependently inhibited the reactivity of purified
human IgG with a panel of homologous antigens.10 The
regulatory capacity of IgM is selective in that certain IgG self
reactivities are more stringently regulated than others, as shown by
using a semiquantitative immunoblotting technique using solubilized
proteins from tissue extracts as sources of self
antigens.21 Selectivity of control of IgG autoreactivity by
IgM differs from one individual to another,22 which has led to the concept of "antibody-finger printing" referring to the individual patterns of self reactivity of IgG in serum as opposed to
the highly homogeneous patterns of reactivity of purified IgG with the
same antigen panels.23 Thus, the paratopic repertoire of
self-reactive IgG in serum is highly dependent on the idiotypic repertoire displayed by autologous IgM. No control by IgM of IgG reactivity was found when binding of IgG to foreign antigens rather than to self antigens, was examined.10 As we added
autologous IgM to purified IgG, we observed a dose-dependent inhibition
of the binding of IgG to autologous A and B trisaccharides. The
inhibitory effect of IgM was restricted to the autologous combination
of antibodies, ie, IgM of individuals A inhibited the reactivity of IgG
of individuals A with A antigen, whereas IgM of individuals B or O
failed to inhibit IgG anti-A reactivity of individuals A. The
inhibitory effect of IgM was not dependent on paratopic competition
between IgG and IgM for the binding to the blood group antigen because
the reactivity of IgM of individuals A with A antigen was much lower
than that of IgM of individuals B. The results strongly suggest that,
as in the case of other natural self-reactive IgG antibodies, the
reactivity of natural anti-self ABO blood group antigen IgG antibodies
in serum is restricted by V-region complementary interactions with
autologous IgM. We speculate that, as a corollary, self reactivity of
IgM with A and B blood group antigens is controlled in serum by
complementary V regions expressed by autologous IgG. An additional
degree of complexity determines the outcome of the binding reaction of
self-reactive IgG antibodies in whole serum, in that the IgG and IgM
fractions contain both autoantibodies and antiautoantibodies that are
not readily detectable when the reactivity of purified IgG or IgM is
tested, although these may dissociate from complexes and exert a
regulatory effect when IgG, IgM, and the source of self antigen are
allowed to interact in the solid phase-binding assay. Furthermore, soluble A and B (ABO) antigens may also contribute to the
neutralization of autologous anti-ABO blood group antibodies in AB serum.
To further characterize natural antibodies reactive with self ABO
antigens, we isolated anti-A IgG and IgM antibodies from the serum of A
individuals by affinity chomatography. We observed that the binding of
anti-A IgG autoantibodies to A antigen was similar to that of
affinity-purified anti-A antibodies of individuals of blood group B. The calculated affinity constant for the binding of anti-A IgG
autoantibodies to the A trisaccharide was in the micromolar range,
similar to that of allo-anti-A IgG antibodies of B individuals. Thus,
natural self-reactive anti-ABO blood group antigen antibodies are of
significant affinity, as previously reported in the case of natural IgG
autoantibodies to eg, cytokines and cytoskeletal
proteins.4,24 The data further argues against the
hypothesis that natural alloreactive antibodies exhibit the characteristics of immune antibodies that would result as a response to
foreign A and B substances on microorganisms. Although autoantibodies and alloantibodies exhibited similar association constants, less autoantibody bound to the A antigen as compared with allo antibody when
the mole to mole ratio was calculated from real time binding experiments using the Biacore. Furthermore, the autoantibody was found
not to agglutinate A red blood cells at concentrations similar to those
of allo-anti-A IgG. This may be explained by the high dissociation rate
of autoantibodies as compared with that of alloantibodies and may
provide a means for protecting self tissues from the potential deleterious effects of autoantibodies. However, using high
concentrations of affinity-purified anti-A IgG of A individuals, we
could show agglutination of A red cells, in a direct agglutination
assay. The agglutinating capacity of the antibodies was specific in
that equivalent concentrations of an irrelevant human MoAb had no effect.
Our results imply that the lack of self reactivity of IgG and IgM with
A and B blood group antigens in serum is not dependent on central
tolerance or peripheral B/T-cell anergy, in that equal amounts of
autoantibodies and alloantibodies are found in the IgG and IgM
fractions of normal serum. The results agree with the findings of
Rieben et al,14,25 showing that B cells capable of
generating antibodies to autologous blood group antigens are present in
healthy subjects. However, by using a limiting dilution technique,
these authors found a higher frequency of precursor B cells producing
alloreactive than that of cells producing autoreactive anti-ABO antigen
antibodies. Our observations further imply that normal human plasma
should also contain natural IgG autoantibodies to substance H and the
corresponding regulatory IgM molecules.
Taken together, our observations suggest that tolerance to autologous
ABO blood group antigens is dependent on peripheral control rather than
on clonal deletion or anergy at the B- or T-cell level. In this
respect, autoreactivity to A and B blood group antigens appears to be
controlled in a similar fashion to autoreactivity with DNA,
thyroglobulin, Factor VIII, and neutrophil cytoplasmic antigens that
are also subject to tight regulation by V region-connected antibodies
in serum.26
| |
FOOTNOTES |
Submitted May 11, 1998; accepted February 12, 1999.
Supported by Institut National de la Santé et de la Recherche
Médicale (INSERM), France, and the Central Laboratory of the Swiss Red Cross, Bern, Switzerland. S.H.S. is a recipient of a grant
from INSERM.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Michel D. Kazatchkine, MD, INSERM U430,
Hopital Broussais, 96 Rue Didot, 75014 Paris, France; e-mail:
michel.kazatchkine{at}brs.ap-hop-paris.fr.
| |
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