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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 328-335
RED CELLS
Altered control of self-reactive IgG by autologous IgM in
patients with warm autoimmune hemolytic anemia
Dorothea Stahl,
Sébastien Lacroix-Desmazes,
Didier Heudes,
Luc Mouthon,
Srini V. Kaveri, and
Michel D. Kazatchkine
From INSERM U430 and Université Pierre et Marie Curie,
Hôpital Broussais, Paris, France.
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Abstract |
Warm autoimmune hemolytic anemia (WAIHA) is characterized by an
accelerated clearance of red blood cells (RBCs) associated with the
presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the
present study, we analyzed the self-reactive IgG and IgM antibody
repertoires of patients with WAIHA using a technique of quantitative
immunoblotting on a panel of whole tissue extracts as sources of
self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC
eluates do not differ between healthy donors and patients with WAIHA,
whereas autoreactive repertoires of IgM from patients exhibit broadly
altered patterns of reactivity as compared with those of healthy
controls. We further demonstrate that IgG purified from eluates of RBCs
of healthy donors induces agglutination of RBCs in an indirect Coombs
assay to a similar extent as IgG purified from eluates of RBCs of
patients with WAIHA. The capability of IgG to induce agglutination of
RBCs is suppressed in unfractionated eluates of healthy donors' cells,
whereas it is readily found in unfractionated eluates of patients'
RBCs. IgM is an essential factor in controlling the ability of IgG in
unfractionated RBC eluates to induce agglutination of RBCs. These
observations indicate that anti-RBC IgG autoantibodies of patients with
WAIHA share extensive similarity with natural antiRBC
autoantibodies of healthy donors and suggest that defective control
of IgG autoreactivity by autologous IgM is an underlying mechanism
for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)
© 2000 by The American Society of Hematology.
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Introduction |
Warm autoimmune hemolytic anemia (WAIHA) is
characterized by an accelerated clearance of red blood cells
(RBCs) associated with the presence of polyclonal anti-RBC
immunoglobulin (Ig)G autoantibodies that optimally bind to erythrocytes
at 37°C.1 Anti-RBC IgG of patients with WAIHA reacts
with a variety of blood group-related RBC antigens and other membrane
components of autologous and homologous RBCs.2 The most
common target antigens are the band-3 anion transporter, glycophorin A,
and Rh-related proteins.3
Autoimmunity in WAIHA is related to a breakdown in tolerance mechanisms
toward RBC antigens. The basis for such failure is not understood.
Current concepts on the understanding of self-/nonself discrimination
focus on the central induction of self-tolerance in thymus and bone
marrow by deleting, receptor-editing, or anergizing self-reactive T- and B-cell clones, and on the active
maintenance of self-tolerance in the periphery.4,5 However,
self-reactive T cells,6 self-reactive B cells, which have
been positively selected and are maintained on the basis of their
autoreactivity,7 and natural self-reactive autoantibodies
of the IgG, IgM, and IgA isotypes8 are present in healthy
human individuals. Thus, self-reactive T cells with specificity for the
Rhesus polypeptide9 and naturally occurring autoantibodies
to RBC band 3,10 spectrin,11 and ABO blood
group antigens12 are found in healthy individuals. Under
normal conditions, autoreactivity of IgG is largely masked in serum by
idiotypic interactions between self-reactive IgG and autologous
IgM.13,14 The inhibitory activity of autologous IgM toward
autoreactive IgG is altered in patients with active Hashimoto's
thyroiditis14 and systemic lupus erythematosus
(SLE).15 Such a failure in the peripheral control of
autoreactivity also has been suggested as an underlying mechanism of
hemolytic anemia in NZB mice.16
In the present study, we analyzed the self-reactive IgG and IgM
antibody repertoires of patients with WAIHA during acute hemolysis using a technique that allows comparative assessment of antibody reactivities of different individuals toward a wide range of
self-antigens by means of quantitative immunoblotting with
multiparametric statistical analysis.17,18 This method has
been used to characterize the self-reactive antibody repertoires in
healthy individuals and in various pathologic
conditions.15,18-23 Our observations in patients with WAIHA
indicate that anti-RBC IgG autoantibodies of patients share extensive
similarity with natural anti-RBC autoantibodies of healthy blood donors
and suggest that defective peripheral control of IgG autoreactivity by
a broadly altered autologous IgM repertoire is an underlying mechanism
for autoimmune hemolysis in patients with WAIHA.
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Materials and methods |
Patients and controls
Heparin-plasma samples were obtained from 20 patients with
WAIHA (10 men and 10 women) aged 33 to 82 years (mean SEM,
65 ± 13 years) and from 20 healthy sex-matched blood
donors aged 20 to 50 years (mean, 31 ± 8 years). WAIHA was
idiopathic in 4 patients and secondary to malignant disease in 14 patients (B-cell chronic lymphocytic leukemia [B-CLL] in 8 patients,
myeloproliferative syndrome in 2 patients, osteomyelofibrosis with
secondary acute myelocytic leukemia in 1 patient, heavy-chain disease
in 1 patient, angioimmunoblastic lymphoma in 1 patient,
and B-cell non-Hodgkin's lymphoma in 1 patient). Whether autoimmune
hemolysis was idiopathic or secondary was unknown in 2 patients.
Samples were obtained at the time of acute hemolysis, as defined by
hemoglobin levels < 12 g/dL, haptoglobin concentrations < 50
mg/dL, lactate dehydrogenase activity > 250 U/L, and serum bilirubin
levels > 1.0 mg/dL. None of the patients had received
intravenous immunoglobulin (IVIg) before the collection of blood
samples. In 2 patients, plasma samples were also obtained during
clinical remission, and in 1 patient, a plasma sample was
available 1 year before disease onset. Plasma was aliquoted and stored
at  80°C until use.
Immunohematologic procedures were performed in accordance with the
guidelines of the American Association of Blood Banks
(AABB).24 Antisera from Biotest (Biotest Diagnostics,
Dreieich, Germany), Gull Laboratories (Bad Homburg, Germany), and
DiaMed (Cressier sur Morat, Switzerland) were used for RBC typing. Test
cells for antibody screening and identification were obtained from
DiaMed. Immunohematologic investigations, including the direct
antiglobulin test (DAT) with polyspecific as well as with monospecific
antisera, were performed using the DiaMed Micro Typing ID gel system.
The DAT was positive in all of the patients. The DAT was positive with
anti-IgG in 19 patients, positive with both anti-IgG and anti-C3d
in 11 patients, positive with anti-IgG and anti-IgM in 2 patients, and positive with both anti-IgG and anti-IgA in 1 patient. In
1 patient, the DAT was negative with anti-IgG and positive with
anti-IgM and anti-C3d; the RBC eluate of the latter patient contained,
however, small amounts of IgG in addition to IgM. IgG eluted from
patients' RBCs reacted broadly with homologous RBCs without detectable
specificity of anti-RBC IgG. In 1 patient, however, we found a
stronger reactivity of eluted anti-RBC IgG with Rh(D)-positive RBCs
than with Rh(D)-negative RBCs. Titers of warm autoantibodies in plasma
ranged between undetectable and > 4.000. Anti-Lea,
anti-E, anti-Jka, anti-Cw, or anti-S
alloantibodies were present in the plasma of 5 of the patients. Healthy
blood donors exhibited a negative DAT. Alloantibodies against RBC
antigens were not detectable in their plasma.
Immunoglobulins in plasma and in RBC eluates
The concentrations of plasma IgM and IgG in patients' samples were
(mean ± SD) 1.39 ± 2.10 g/L (range, 0.23-9.92 g/L) and 8.74 ± 6.20 g/L (range, 2.67-25.40 g/L), respectively, as
determined by nephelometry. The concentrations of plasma IgM and IgG in
healthy controls were (mean ± SD) 1.23 ± 0.63 g/L (range,
0.34-2.64 g/L) and 9.21 ± 1.94 g/L (range, 5.45-12.40 g/L),
respectively. IgG was purified from plasma by affinity chromatography
on protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden). The
concentration of IgG was determined spectrophotometrically at 280 nm.
Purity of IgG was assessed by enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IgM was purified from the serum of 3 donors and 3 patients
by affinity chromatography using anti-human IgM coupled to
CNBr-activated Sepharose (Pharmacia). The concentration of IgM was
determined spectrophotometrically. Normal human IgG for therapeutic
intravenous use (IVIg) (Sandoglobulin; a gift of the Central Laboratory
of the Swiss Red Cross, Bern, Switzerland) was used as a standard for
IgG. A reference preparation of normal IgM (IVIgM) was obtained by
submitting Pentaglobin (Biotest Pharma, Dreieich, Germany), an
IgM-enriched therapeutic preparation of normal human immunoglobulin, to
size-exclusion chromatography on Sephacryl HR S-300.
F(ab')2 fragments were prepared from purified IgG and
from IVIg by pepsin digestion and affinity chromatography on Protein
G-Sepharose (Pharmacia). The purity of F(ab')2
fragments was assessed by SDS-PAGE.
Immunoglobulins were eluted on ice, using citric acid,25
from extensively washed patients' and donors' RBCs
(5 × 105 RBCs/individual). The washing procedures
were performed with phosphate-buffered saline (PBS), pH 7.4, at
37°C, and the final wash solutions were shown to be free of IgG and
IgM by ELISA and SDS-PAGE. The eluates were immediately neutralized to
a pH of 7.0 and dialyzed against PBS containing 0.01%
NaN3. IgG was purified from eluates by affinity
chromatography on protein G-Sepharose (Pharmacia). Purified IgG was
free of IgM as assessed by ELISA. The concentrations of IgG and IgM in
the eluates and in purified IgG fractions from the eluates were
determined by ELISA. The capability of eluted IgG to induce
agglutination of RBCs in an indirect Coombs assay was assessed using
the DiaMed-ID Micro Typing System and the test cell reagents ID-DiaCell
I+II (homologous RBCs; DiaMed) in the case of patients and of healthy
donors. Briefly, RBCs were incubated with eluted anti-RBC IgG. The
binding of anti-RBC IgG antibodies to RBCs was visualized by an
agglutination reaction using rabbit anti-human IgG as the agglutinating
reagent. In addition to homologous test cells, autologous RBCs were
used for agglutination assays in the case of healthy donors. Homologous
and autologous RBCs were used suspended in LISS (low ionic strength
solution, ID-diluent 2; DiaMed) as well as in PBS at pH 7.4.
Analysis of antibody repertoires by quantitative immunoblotting
To analyze antibody repertoires, we used a quantitative
immunoblotting technique that allows for the simultaneous investigation of reactivities of different sources of antibodies with a large number
of antigens in normal homologous tissue extracts.17,18 Sources of self-antigens were extracts of histologically normal human
liver, kidney, and stomach obtained during surgical procedures; extracts of pooled RBCs of 5 healthy individuals (blood group 0 Rh[D+]); pooled RBC membranes (ghosts) of 5 healthy individuals (blood group 0 Rh[D+]); pooled RBC membranes of 3 patients with WAIHA
(blood group 0 Rh[D+]); and F(ab')2 fragments of
IVIg. Extracts were prepared from tissues and RBCs in 2% SDS, 1.45 mol/L 2-mercaptoethanol, and 125 mmol/L Tris/HCl, pH 6.8, containing
1.0 mg/mL aprotinin, 1.0 mg/mL pepstatin A, and 1.0 mmol/L
ethylenediaminetetra-acetic acid (EDTA) on ice. The extracts were
centrifuged, boiled for 5 minutes, and dialyzed against PBS, pH 7.4. RBC membranes were prepared according to Lutz et al26;
briefly, leukocyte-free RBC suspensions were submitted to hypotonic
lysis in 5 mmol/L phosphate, 1 mmol/L EDTA, pH 7.4, on ice. The RBC
membranes were then incubated in the presence of 1 mmol/L
phenylmethylsulfonyl fluoride, 1.0 mg/mL aprotinin, and 1.0 mg/mL
pepstatin A on ice; washed; aliquoted; and stored at 80°C
until use in the presence of 1% SDS and 5 mmol/L
N-ethylmaleimide. The amount of solubilized tissue proteins
subjected to electrophoresis ranged between 200 and 600 µg/gel,
depending on the tissue extract, and was maintained constant for a
given tissue in all experiments. Low amounts of carbohydrates were
detected in solubilized protein samples that represented approximately
1:10 to 1:20 of protein on a weight basis. Proteins were subjected to
preparative SDS-PAGE in 10% polyacrylamide. The proteins were then
transferred onto nitrocellulose (Schleicher & Schuell, Dassel, Germany)
for 1 hour at 0.8 mA/cm2 using a Semi Dry Electroblotter
model A (Ancos, H jby, Denmark). Membranes were blocked for 1 hour at
room temperature with PBS containing 0.2% Tween 20. The antibodies to
be tested were incubated with the membranes after the addition of 1 sample per slot in a Cassette Miniblot system (Immunetics Inc.,
Cambridge, MA). The membranes were incubated for 4 hours at room
temperature, washed, and revealed with µ-chain-specific secondary
rabbit anti-human IgM antibody or  -chain-specific secondary rabbit
anti-human IgG antibody coupled to alkaline phosphatase (Dako,
Glostrup, Denmark) for 90 minutes at room temperature.
Immunoreactivities were revealed using the NBT/BCIP (nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) substrate (Sigma, St.
Louis, MO). Antibodies were tested at concentrations of 20 µg/mL and
200 µg/mL of plasma or purified plasma immunoglobulin in the case of
IgM and IgG, respectively. The concentration of IgG was adjusted to 30 µg/mL in experiments aimed at analyzing the reactivity profiles of
IgG purified from RBC eluates. Quantitation of immunoreactivities was
performed by scanning the membranes (SnapScan 600; Agfa Gevaert, New
York, NY). Blotted proteins were then stained with
colloidal gold (Protogold; BioCell, Cardiff, Wales) and subjected to a
second densitometric analysis to record the protein profile and
to quantitate transferred proteins. Data were analyzed using a
Power Mac G3 computer (Apple Computer Inc., Cupertino, CA) and IGOR
software (Wavemetrics, Lake Oswego, OR). Densitometric profiles of
immunoreactivity were compared by referring to their corresponding
protein profile after correction of the migration defects by
superimposition of protein peaks. A sample of the reference IgM (IVIgM)
or IgG (IVIg) preparations was included in each blot, allowing us to
rescale the different membranes transferred with a given protein
extract and to adjust for the intensity of staining of different
membranes. Migration distance (X-axis) and optical density (Y-axis)
were expressed as arbitrary units (AU). Migration distances of 200, 600, and 1000 AU corresponded to apparent molecular weights of 200, 65, and 20 kDa, respectively.21
The assay was reproducible with a 10% variation coefficient. The 95%
confidence interval of the mean area under the curve corresponding to
each peak of immunoreactivity was 30% in the case of IgM18
and 25% in the case of IgG,19 as calculated using
Student t test.
Statistical analysis
Multivariate statistical treatment of the data was performed using
IGOR software, including specially written software packages, and
StatView software. Densitometric profiles of reactivity of IgM and IgG
of patients and healthy donors with antigens in each tissue extract
were divided into sections corresponding to individual peaks of
immunoreactivity. Respective peak areas under the curve were calculated
for each section. To discriminate between groups of individuals, areas
corresponding to sections were submitted to principal component
analysis (PCA).27 The repertoire of reactivities of each
individual in a given sample was represented as a single symbol in a
2-dimensional linear subspace accounting for between 60% and 90% of
the data. Discrimination between repertoires of groups of individuals
was investigated by submitting the PCA data to linear discriminant
analysis (LDA) and by subsequently comparing factors 1 of the LDA by
means of a Mann-Whitney U test. Differences were considered
statistically significant if P values were < .05 as assessed
by the Mann-Whitney U test. The PCA of repertoires of antibody
reactivities performed individually for each group of subjects allowed
the calculation of respective variances. Variances were compared using
the F test.
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Results |
Self-reactive antibody repertoires of plasma IgG
We analyzed the patterns of reactivity of IgG purified from plasma
and of IgG in whole unfractionated plasma of patients with WAIHA and
healthy blood donors toward antigens in liver, kidney, stomach, and RBC
extracts, and in RBC membranes. The patterns of reactivity of purified
IgG were highly homogeneous among patients and among controls in the
case of all tissue extracts that we tested (Figure
1). IgG of patients recognized the same
antigenic bands in the tissue extracts as did IgG of healthy donors,
although with a higher intensity of reactivity (Figure
2). Enhanced reactivity of patients' IgG
toward self-antigens accounted for the fact that multiparametric
analysis of the data discriminated between repertoires of patients and
controls (.0002 < P < .02, depending on the tissue extract). These results were substantiated by the observation that the
intensity of reactivity of IgG obtained from 3 patients at the time of
acute disease was higher than that of IgG obtained at the time of
remission or before acute hemolysis, although the antibody repertoires
did not differ in these samples in terms of the nature of the antigenic
bands that were recognized (data not shown). We also found no
difference in antibody repertoires of purified IgG of patients and
healthy blood donors when analyzed toward pooled membranes prepared
from RBCs of 3 patients with WAIHA (data not shown). The specificity of
reactivity was confirmed in experiments showing that the same antigenic
bands in tissues were recognized by F(ab')2 fragments
of IgG and by uncleaved IgG, and that the binding of
F(ab')2 fragments of IgG to RBC extracts was
inhibited by soluble RBC extracts, but not by soluble liver antigens
(data not shown).
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| Fig 1.
Western blotting of IgG with extracts of normal human
RBCs.
IgG purified from plasma of patients with WAIHA and healthy blood
donors was immunoblotted at 200 µg/mL with a soluble extract of
pooled RBCs of healthy donors, as described in Materials and Methods.
Lanes 1 to 10: IgG of patients with WAIHA; lanes 15 to 24: IgG of
healthy blood donors; lanes 12 and 13: normal reference IgG (IVIg);
lanes 11 and 14: secondary anti-Fc antibody alone.
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| Fig 2.
Patterns of reactivity of IgG purified from plasma of
patients and healthy blood donors with self-antigens.
IgG purified from plasma of patients with WAIHA and healthy blood
donors was interacted at 200 µg/mL with self-antigens, as described
in Materials and Methods. The figure depicts the mean densitometric
profiles of reactivity of purified IgG (i.e., the arithmetic mean of
the recorded intensities constituting the reactivity profile of each
individual) of 20 patients with WAIHA (black area) and 20 healthy blood
donors (gray area) with antigens in soluble extracts of normal RBCs (A)
and normal stomach (B). White areas depict the pattern observed in the
presence of secondary anti-Fc antibody alone. Optical densities and
migration distances are expressed as arbitrary units (AU). Migration
distances of 200, 600, and 1000 AU correspond to molecular weights of
200, 65, and 20 kDa, respectively.
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In contrast to the results obtained with purified IgG, the patterns of
reactivity of IgG in unfractionated plasma were heterogeneous among
patients and among healthy controls. Self-reactive antibody repertoires
of IgG in plasma of patients differed from those of healthy donors in
terms of both intensity of reactivity and the nature of the antigenic
bands that were recognized, and were discriminated by PCA
(P = .001) (Figure 3).
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| Fig 3.
Comparative analysis of the patterns of reactivity of IgG
in unfractionated plasma of patients and healthy blood donors with
self-antigens.
IgG in unfractionated plasma of patients with WAIHA and healthy blood
donors was interacted at 200 µg/mL with self-antigens in extracts of
kidney, liver, stomach, and RBCs, as described in Materials and
Methods. For each individual, the densitometric profile of reactivity
with a given tissue extract was divided into sections corresponding to
peaks of reactivity. The areas under the curve of each peak were
calculated in the case of each tissue extract. The data were subjected
to PCA and fitted within a 2-dimensional linear subspace (factor
1/factor 2). The figure shows the result of a cumulative PCA of the
repertoires of reactivities of IgG in unfractionated plasma toward the
antigens in all of the self-tissues tested. Percentages of variance
accounted for by factor 1 and factor 2 are indicated on the abscissa
and ordinate, respectively. Each symbol represents the reactivity of
IgG of a single individual for the group of patients with WAIHA ( )
and healthy blood donors ( ). PCA discriminated between repertoires
of patients and controls (P = .001 by the Mann-Whitney U
test).
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Self-reactive antibody repertoires of plasma IgM
The patterns of reactivity of plasma IgM toward antigens in
extracts of liver, kidney, stomach, RBCs, and in RBC membranes differed among the patients and differed from the homogeneous patterns
of reactivity observed in the case of healthy blood donors (Figures
4 and 5).
Self-reactive antibody repertoires of IgM of patients and
controls were discriminated by PCA (.0001 < P < .0005, depending on the tissue extract; Figure 6).
The individual variances of repertoires of self-reactivities of plasma
IgM of patients and controls, calculated in the case of each tissue
extract and tested by PCA, did not differ significantly between
patients and healthy donors as assessed by the F test, indicating that
self-reactive repertoires of IgM were homogenous within the 2 groups of
individuals (Table 1). The intensity of
reactivity of IgM of WAIHA patients toward F(ab')2
fragments of normal IgG was significantly lower than the intensity of
reactivity of IgM of healthy controls (P = .0001, data not
shown).
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| Fig 4.
Western blotting of IgM with extracts of normal human
kidney.
IgM in the plasma of patients with WAIHA and healthy blood donors was
interacted at 20 µg/mL with a soluble extract of kidney antigens, as
described in Materials and Methods. Lanes 1 to 10: IgM of patients;
lanes 15 to 24: IgM of healthy blood donors; lanes 12 and 13: normal
reference IgM (IVIgM); lanes 11 and 14: secondary anti-Fcµ antibody
alone.
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| Fig 5.
Patterns of reactivity of IgM of patients and healthy
blood donors with self-antigens.
The densitometric profiles of reactivity of IgM with antigens in kidney
(A,B) and RBC (C,D) extracts are depicted as individual profiles (black
curves) in the case of 20 patients with WAIHA (A,C) and as individual
profiles (black curves) in the case of 20 healthy donors (B,D). Gray
areas show the densitometric pattern observed in the presence of the
secondary anti-Fcµ antibody alone. Optical densities and migration
distances are expressed as arbitrary units (AU). Migration distances of
200, 600, and 1000 AU correspond to apparent molecular weights of 200, 65, and 20 kDa, respectively.
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| Fig 6.
Comparative analysis of the patterns of reactivity of IgM
of patients and healthy blood donors with self-antigens.
IgM in the plasma of patients with WAIHA and healthy blood donors was
interacted at 20 µg/mL with self-antigens, as described in Materials
and Methods. For each individual, the densitometric profile of
reactivity with a given tissue extract was divided into sections
corresponding to peaks of reactivity. Respective peak areas were
calculated in the case of each tissue extract. The data were subjected
to PCA within a 40- to 56-dimension vector space, depending on the
tissue extract, and fitted within a 2-dimensional linear subspace
(factor 1/factor 2). PCA discriminated between repertoires of patients
and controls in the case of all tissue extracts tested
(.0001 < P < .001, depending on the tissue extract, by
the Mann-Whitney U test). The figure shows the results of the
comparative analyses in the case of kidney antigens (A) and RBC
extracts (B). Percentages of variance accounted for by factor 1 and
factor 2 are indicated on the abscissa and ordinate, respectively. Each
symbol represents the reactivity of IgM of a single individual for the
group of patients with WAIHA () and healthy blood donors ().
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Table 1.
Total variances of the self-reactive antibody
repertoires of IgM of patients with WAIHA and healthy blood donors
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In contrast to a generally enhanced reactivity of purified IgG of
patients toward the antigenic bands in all the tissue extracts during
acute disease, we found a higher reactivity of IgM toward certain
selected antigenic bands and decreased reactivities toward other
antigenic bands during acute disease when compared with the reactivity
of IgM in plasma samples obtained at the time of disease remission or
before acute hemolysis (data not shown). We verified in the case of 3 patients and 3 healthy donors that, as demonstrated
previously,18 reactivities of IgM purified from plasma did
not differ from those of IgM tested in unfractionated plasma
(data not shown).
Self-reactive antibody repertoires of IgG in eluates of RBCs
Cell-bound immunoglobulins were eluted from RBCs of patients with
WAIHA and healthy blood donors. The mean numbers of IgM and IgG
molecules per patients' RBCs in the eluates were 37 (< 10-136) and
3300 (14-10 000), respectively. Numbers were 18 (< 10-28) and 173 (< 10-330)/RBCs in the case of healthy donors. We
purified IgG from the RBC eluates before testing it for self-reactivity toward antigens in RBC extracts and in RBC membranes. The patterns of
reactivity were homogeneous among patients and controls and did not
differ between patients and controls (Figure
7). The patterns of reactivity with
self-antigens of IgG purified from RBC eluates did not differ from
those of IgG purified from plasma in the case of patients and healthy
donors (data not shown).
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| Fig 7.
Patterns of reactivity of IgG purified from RBC eluates
with self-antigens in RBC membranes.
IgG purified from RBC eluates of patients' and blood donors' RBCs was
incubated (30 µg/mL) with antigens in RBC membranes, as described in
Materials and Methods. The mean densitometric profiles of reactivity of
purified IgG (i.e., the arithmetic mean of the recorded intensities
constituting the reactivity profile of each individual) of 4 patients
with WAIHA (black area) and 4 healthy blood donors (gray curve) with
antigens in RBC membranes are depicted. White areas show the
densitometric pattern observed in the presence of the secondary
antiFc antibody alone. Optical densities and migration distances are
expressed as arbitrary units.
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In vitro binding to RBCs of anti-RBC IgG eluted from RBCs
We investigated the ability of anti-RBC IgG in unfractionated RBC
eluates and purified from RBC eluates to bind to RBCs by using an
indirect Coombs assay. At similar inputs, IgG in unfractionated eluates
of patients' RBCs and IgG purified from eluates of patients' RBCs
induced agglutination of unselected homologous cells to the same
extent. In contrast, IgG in unfractionated eluates of healthy donors'
RBCs did not induce agglutination of unselected homologous RBCs,
whereas IgG purified from eluates of healthy donors' RBCs did induce
agglutination. The extent of agglutination of unselected homologous
RBCs induced by IgG purified from eluates of healthy donors' RBCs was
similar to that induced by IgG purified from eluates of patients' RBCs
(Table 2). In the case of healthy donors, we further observed that IgG purified from RBC eluates induced agglutination of autologous RBCs to a similar extent as of homologous RBCs.
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Table 2.
In vitro binding of IgG in unfractionated RBC eluates
and of IgG purified from RBC eluates to unselected homologous RBCs
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The coincubation of IgG purified from RBC eluates of healthy donors
with normal plasma at a mol/mol ratio of purified eluted anti-RBC IgG
to plasma IgM of 1:350 inhibited the agglutination of homologous RBCs
induced by natural anti-RBC IgG. A similar inhibition of agglutination
was observed using a 1:1 mixture (wt/wt) of purified plasma IgG and
purified plasma IgM of healthy donors instead of whole plasma.
Inhibition was not observed when purified IgG or purified IgM of
healthy donors was used alone. Agglutination of homologous RBCs induced
by natural anti-RBC IgG was not inhibited when a 1:1 mixture of
purified plasma IgG of healthy donors and purified plasma IgM of
patients with WAIHA was used. Agglutination of homologous RBCs induced
by anti-RBC IgG of patients was also inhibited by either healthy
donors' plasma or a mixture of purified plasma IgG and purified plasma
IgM of healthy donors. Agglutination of homologous RBCs induced by
patients' IgG was not inhibited by a mixture of purified IgG of
healthy donors and purified IgM of patients or by purified IgG or
purified IgM from healthy donors alone. Agglutination of unselected
homologous RBCs induced by anti-RBC IgG purified from RBC eluates of
healthy donors or of patients was not inhibited by coincubating either
with PBS or with serum albumin (Table 3).
| |
Discussion |
WAIHA is associated with the presence of IgG autoantibodies directed
against surface antigens expressed on RBCs. Bound antibodies mediate
the adherence of RBCs to phagocytes, resulting in an accelerated clearance of erythrocytes.1 Investigations in WAIHA so far have focused on the identification of disease-associated
autoantigens2,3 or on the mechanisms of Fc/FcR-mediated RBC
clearance.28 In contrast, the present study investigates
the overall IgG and IgM self-reactive antibody repertoires of patients
with WAIHA. The data provide evidence for a normal IgG but broadly
distorted IgM autoantibody repertoire and suggest that defective
peripheral control of IgG autoreactivity by altered autologous IgM is
an underlying mechanism for autoimmune hemolysis in patients with WAIHA.
Complementary idiotypic interactions occur between self-reactive IgG
and autologous IgM in normal human serum,14,29-31 providing a mechanism by which IgG autoreactivity is largely "masked" in unfractionated normal serum13,14,19,32 and by which natural autoantibodies may be prevented from expressing pathogenic
potential.33-35 There is evidence that such peripheral
control of self-reactive IgG by IgM is defective in antineutrophil
cytoplasmic autoantibody-positive vasculitis,36 Hashimoto's
thyroiditis,14 SLE,15 and in the context of the
hemolytic anemia occurring in NZB mice.16 In the present
study, we observed that self-reactive antibody repertoires of IgG
purified from plasma do not differ between healthy donors and patients
with WAIHA when tested on tissue extracts and RBCs from healthy donors.
In addition, no difference was observed when tested on RBCs obtained
from patients with WAIHA, in which the protein composition may be
altered as compared with that of normal RBCs.37 In
contrast, the autoreactive antibody repertoires of plasma IgM of
patients with WAIHA are distorted and differ significantly from those
of healthy controls on multiparametric analysis. These differences were
not dependent on heterogeneity between individual patients because the
calculated individual variances of repertoires of self-reactivities of
IgM did not differ significantly between patients and healthy donors.
These observations suggest that altered patterns of self-reactivity of
IgG in the unfractionated plasma of patients with WAIHA are related to
defective control of IgG autoreactivity by autologous IgM. The
hypothesis of defective peripheral control of autoreactive anti-RBC IgG
by autologous IgM is consistent with observations in patients with
WAIHA secondary to B-CLL, indicating that anti-RBC IgG autoantibodies
in these cases are not the product of the malignant clone, which
typically expresses IgM, but originate from remnant B
lymphocytes.38
Consistent with the results obtained upon testing of purified plasma
IgG, the patterns of reactivity of anti-RBC IgG purified from RBC
eluates of DAT-positive patients with antigens in homologous RBC
extracts and RBC membranes did not differ from those of DAT-negative healthy blood donors. In addition, natural anti-RBC IgG purified from
the RBC eluates of healthy blood donors was capable of inducing agglutination of homologous and autologous RBCs in an indirect Coombs
assay to a similar extent as IgG purified from eluates of RBCs of
patients with WAIHA. Because the LISS buffer used in the standard
indirect Coombs assay could favor to some extent a nonspecific binding
of IgG to RBCs and enhance the binding of specific low-affinity
antibodies,39 we verified that agglutination of RBCs
induced by IgG purified from RBC eluates was also observed using PBS
instead of LISS in the indirect Coombs assay. The capability of IgG in
RBC eluates to induce agglutination of RBCs was suppressed in
unfractionated eluates of healthy donors' cells, whereas it was
readily found in unfractionated eluates of patients' RBCs. The data
suggest that non-IgG molecules, including IgM, inhibit the binding
capability of IgG in unfractionated eluates of healthy donors' RBCs
and that such inhibitory activity is defective or absent in eluates of
RBCs of patients with WAIHA. Indeed, coincubating IgG purified from RBC
eluates of healthy donors and of patients with WAIHA with plasma or
with a 1:1 mixture of purified plasma IgG and purified plasma IgM
of healthy donors inhibited the agglutination induced by natural and by
disease-associated anti-RBC IgG, whereas coincubation with plasma IgM
of patients instead of plasma IgM of healthy blood donors did not.
These results indicate that reconstituting the "regulatory
IgM" prevents in vitro anti-RBC IgG autoantibodies in patients with
WAIHA from binding to RBCs.
Consistent with data in the literature,40 we observed
higher amounts of IgG bound to RBCs in patients as compared with
healthy donors. Such an increase in RBC-bound IgG could be due to an
increased clonal frequency of RBC-specific B cells or to an increased
binding ability of IgG toward RBC antigens. To our knowledge, there is no evidence of an increased frequency of single RBC-specific
B-cell clones in WAIHA. In contrast, it has been documented that
anti-RBC antibodies in WAIHA due to B-CLL usually show a polyclonal
light-chain distribution.38,41 It may be speculated that a
dysregulated inhibitory IgM control may influence the binding ability
(avidity) of anti-RBC IgG antibodies.
The observation of a lower reactivity of plasma IgM of patients with
WAIHA toward F(ab')2 fragments of normal IgG, as
compared with that of IgM of healthy individuals, suggests altered
idiotypic interactions between IgM and IgG in patients with WAIHA. Our
results are consistent with previous observations showing that RBC
eluates of DAT-positive blood donors contain both anti-RBC IgG
autoantibodies and IgG anti-idiotypic antibodies directed against the
RBC autoantibody42 and suggest that, in addition to IgG
anti-idiotypes, IgM anti-idiotypes may participate in the regulation of
anti-RBC IgG autoreactivity. At this stage, we provide evidence for a
distortion in the functional equilibrium between IgG and IgM in eluates
of patients' RBCs as compared with healthy controls. We have no
quantitative data on the relative amounts of idiotypes and
anti-idiotypes in the eluates, on the precise binding site of the
anti-idiotypic IgM on anti-RBC IgG, or on the relative affinities of
these antibodies for their respective antigens, as yet.
Anti-F(ab')2 antibodies interact with the variable
region of the target antibody in multiple ways and may recognize
idiotypic determinants within or outside the paratope43-48
or conserved domains of the F(ab')2.49-52
A subpopulation of anti-F(ab')2 antibodies has been
shown to cover a part of the physiologically active Fc domain and
nevertheless is able to immobilize the Fab arms, thus exerting
immunoregulatory functions.51,52 Thus, IgM
anti-(Fab')2 antibodies recovered in RBC eluates are likely to be heterogeneous with regard to binding sites on the target
anti-RBC IgG and, therefore, also with regard to the ability to
interfere with the specific binding of anti-RBC IgG antibodies to RBC
antigens. It could be expected that such IgG-IgM complexes are
copurified during affinity chromatography and thus are found in the
fraction of purified IgG, which we observed to be free of IgM. It is
possible that complexes undergo dissociation during the washing steps
or acid treatment. More direct evidence for the existence of anti-RBC
IgG-IgM complexes comes from another set of experiments, in which we
adsorbed the anti-RBC IgG autoantibody in patients' plasma to
homologous native RBCs before comparing the patterns of reactivity of
IgG and IgM in nonabsorbed and absorbed plasma with RBC antigens in the
immunoblot assay. We found a highly significant change in the patterns
of IgG toward RBC antigen extracts after absorption of the anti-RBC IgG
autoantibodies from the plasma. The same change in reactivity was
observed in the case of IgM, indeed suggesting that anti-RBC IgG in
WAIHA is complexed with IgM (Stahl et al, unpublished observation).
Taken together, the results suggest that defective peripheral control
of IgG autoreactivity by autologous IgM is an underlying mechanism for
autoimmune hemolysis in patients with WAIHA. The molecular defects in
such regulatory IgM are still unclear. Our observations may open the
prospect of therapeutic strategies aimed at reconstituting the
physiologic regulatory interactions between autologous IgM and
self-reactive IgG, rather than nonspecifically suppressing autoantibody production.
| |
Acknowledgments |
We thank Constantin Fesel and Matthias Haury, Institut Pasteur, Paris,
for advice and discussion; and Jacques Blouin, Marie-Françoise Bloch, and Stéphanie Rose for technical assistance. We also thank many colleagues in the Department of Hematology/Oncology, Heidelberg, and the blood banks of Heidelberg and Hôpital Broussais, Paris, for helping in collecting and processing blood samples.
| |
Footnotes |
Submitted April 29, 1999; accepted August 31, 1999.
Supported by the Institut National de la Santé et de la
Recherche Médicale (INSERM) and the Centre National de la
Recherche Scientifique (CNRS), France; by the Central Laboratory of the Swiss Red Cross, Bern, Switzerland; and by the German Society for
Transfusion Medicine and Immunohaematology (DGTI), Germany.
Reprints: Michel D. Kazatchkine, INSERM U430 and
Université Pierre et Marie Curie, Hôpital Broussais, 96, rue Didot, 75014 Paris, France; e-mail:
michel.kazatchkine{at}brs.ap-hop-paris.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction