Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4343-4346
Inverse Association Between IgG-Anti-
and Antierythrocyte
Autoantibodies in Patients With Cold Agglutination
By
Peter Terness,
Dan Navolan,
Gerhard Opelz, and
Dieter Roelcke
From the Institute of Immunology, University of Heidelberg,
Heidelberg, Germany; and the Ministry of Health, Bucharest, Romania.
 |
ABSTRACT |
It has been known for a long time that
IgG-anti-F(ab')2 antibodies (Abs) are able to
suppress the B-cell response. We showed that natural
IgG-anti-F(ab')2 autoantibodies appear in the serum of patients with cold agglutination. If the anti-F(ab')2 Ab
suppresses cold agglutinin (CA)-producing B cells, one would expect an
inverse correlation between the titers of these two Abs. Our study
confirmed this correlation. Subsequent experiments showed that some
anti-F(ab')2 Abs bind to the hinge region of IgG. It
was difficult to explain how this Ab suppresses CA-producing B cells,
which are of IgM isotype. Here we show that patients with cold
agglutination have an IgG-anti-
light chain autoantibody in their
serum. This is another member of the anti-F(ab')2 Ab
group. Because the vast majority of CAs are IgM- Abs, the anti-
Ab might suppress CA-producing B cells. If this is the case, there
should be an inverse association between the titer of anti- Ab and
CA. In a group of 302 patients, we found that high titers of the
anti- Ab correlate with low titers of CA and vice versa (P
= .009). Interestingly, this association is found only in patients
whose disease is caused by noninfectious agents, including mainly
B-cell proliferations (P = .0058). Our data show that the
inverse correlation is not confined to a particular CA autoantibody
specificity. The results are discussed in the light of recent findings
showing that anti-IgM Abs may either inactivate or kill tumoral B cells
by apoptosis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IgG-anti-F(ab')2
AUTOANTIBODIES were shown to strongly suppress the B-cell response
including that of autoreactive cells.1-3 If these
antibodies (Abs) were also involved in the suppression of
antierythrocyte autoantibody producing B cells, there should be an
inverse correlation between anti-F(ab')2 and antierythrocyte autoantibody titer in patients with autoimmune hemolytic anemia. We studied a group of patients with cold
agglutination, a disease caused by cold reactive antierythrocyte
autoantibodies (cold agglutinins [CA]),4 and found an
inverse association between the 2 Abs.5 The vast majority
of previously described human anti-F(ab')2
autoantibodies were antiidiotypes, ie, they bound to epitopes located
in the variable region of the immunoglobulin molecule. In previous
studies, we showed that some anti-F(ab')2 autoantibodies recognize a constant region epitope located in the hinge
region of the IgG molecule. Subsequent clinical studies showed that the
previously defined inverse correlation between CA and
anti-F(ab')2 Abs also applies to the anti-hinge
Ab.6 However, it was difficult to explain how an Ab
directed against the hinge region of IgG regulates CA-producing B
cells, which belong to the IgM subclass. We speculated that the
anti-hinge Ab might cross-react with an homologous epitope on IgM, that
IgM-producing B cells coexpress a small number of IgG molecules, or
that other mechanisms are involved. All of those explanations, however,
were not satisfactory.
Meanwhile, our studies have shown that anti-F(ab')2
autoantibodies contain, in addition to the anti-hinge, other
specificities. One of them is an Ab binding to light chains. It is
known that the vast majority of CAs are IgM Abs with light
chains.4 Therefore, anti- Abs interact with CAs and thus
might regulate their activity. If they had an inhibitory effect on
CA-producing B cells, there should be an inverse association between
IgG-anti- and CA titer. In the present study, we analyze the
association between the anti- and the CA autoantibodies in a group
of 302 patients with cold agglutination.
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MATERIALS AND METHODS |
Detection of CA.
Sera of 302 patients with cold agglutination were tested. CA were
detected and their specificities defined by titration of patients'
sera against untreated, protease-, and sialidase-treated human adult
red blood cells, as well as newborn cells in the standard tube technique.
Detection of IgM antibodies against mycoplasma pneumoniae,
Epstein-Barr virus (EBV), and rubella virus.
IgM Abs against infectious agents were determined to show fresh
infections. Anti-I containing sera were tested for antimycoplasma, anti-i sera for anti-EBV, and anti-Pr sera for antirubella virus Abs
using standard kits (Virotech, Rüsselsheim and Abbott GmbH Diagnostika, Wiesbaden, Germany, respectively).
Detection of IgG-anti- antibody.
Microtiter plates were coated with 0.25 µg/well of human
light chains (ARP, Belmont, MA). Remaining active groups were
blocked with phosphate-buffered saline (PBS) + 0.4% gelatine. The test serum was diluted 1:20 and applied to precoated plates (50 µL/well, duplicates). A reference serum with known anti- activity was used as
positive and a serum without anti- activity, as well as PBS, as
negative controls. After incubation, 50 µL of alkaline phosphatase-conjugated goat antihuman IgG(Fc) (Jackson Immunoresearch Lab, West Grove, PA) was added. Each step was followed by extensive washing with PBS + 0.05% Tween. Substrate (250 µg p-nitrophenyl phosphate disodium/well) (Sigma Chemical Co, St Louis, MO) was added
and the extinction was measured at 405 nm every minute up to 90 minutes
and values were multiplied by 103. The test was stopped at
an extinction of 1,000 optical density (OD) 405 nm in the
positive control.
Statistical analysis.
Data are expressed as the median ± MAD (median absolute deviation).
Patients with identical CA titer were included in the same group.
Correlations were calculated according to the Spearman rank correlation
test corrected for ties (rho) and corresponding P values determined.
| |
RESULTS |
Sera of patients with cold agglutination contain an IgG-anti- light
chain autoantibody.
Our previous studies showed that sera of patients with cold
agglutination contain IgG-anti-F(ab')2 Abs whose
concentrations show an inverse correlation with CA titers.5
This study has identified an IgG-anti- light chain autoantibody in
the same group of patients. Because light chains are expressed in
most F(ab')2 fragments, anti- Abs bind to
F(ab')2 and therefore belong to the
anti-F(ab')2 Ab group. The first experiment addressed
the question of whether the titer of anti- Ab parallels that of
anti-F(ab')2 Ab. As shown in
Fig 1, a strong positive correlation
between the 2 Ab titers is found in CA patients (P < .0001).
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| Fig 1.
Direct association between the IgG-anti- and
IgG-anti-F(ab')2 autoantibody in patients with cold
agglutination. In the serum of 204 patients with cold agglutination,
the IgG-anti- and IgG-anti-F(ab')2 autoantibody
titers were measured by enzyme-linked immunosorbent assay (ELISA).
Antibody titers (median ± MAD) are given in OD 405 nm (ordinate).
|
|
IgG-anti- antibody titers show an inverse correlation with CA
antibody titers.
The question arose whether similar to anti-F(ab')2,
the anti- autoantibody correlates with the CA titers. In a group of
302 patients, both anti- and CA titers were measured and
statistically analyzed (Fig 2). A weak, but
significant (P = .009), inverse association between the 2 Abs
was detected. Increasing CA titers were associated with decreasing
anti- titers.
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| Fig 2.
Inverse association between IgG-anti- autoantibody
and CA in patients with cold agglutination. In a group of 302 patients,
the titers of IgG-anti- and CA were determined (left and right
ordinate). Sera with identical CA titers were included into the same
group (abscissa). Anti- titers are given in OD 405 nm (median ± MAD).
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The inverse correlation appears only in cold agglutination of
noninfectious etiology.
According to its etiology, the cold agglutination can be subdivided
into a postinfection and noninfection group. The latter is a
heterogenous entity including mainly patients with lymphoproliferative diseases. Because previous data5 showed that the
anti-F(ab')2 Ab's role is related to the disease's
etiology, in the present study, the patients were subdivided into 2 groups, 1 with recent infections (IgM Abs against mycoplasma pneumonia,
EBV, or rubella virus) and 1 without demonstrable infections.
Interestingly, the inverse association between CA and anti- Ab
appeared only in the noninfection group (P = .0058)
(Fig 3).
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| Fig 3.
Inverse association between IgG-anti- autoantibody
and CA in cold agglutination caused by noninfectious agents. In
patients (A) without (n = 165) and (B) with (n = 120) recent
infections, the anti- and CA autoantibody were determined (left and
right ordinate). Sera with identical CA titers were included in the
same group (abscissa). Anti- titers are given in OD 405 nm (median ± MAD).
|
|
The inverse correlation appears in all noninfection patients
regardless of the CA's specificity.
Noninfection patients were further subdivided according to the
specificity of their CA (Fig 4). Both
anti-I and anti-i patients presented the above-described inverse
association (P = .03 and P = .02). Although the same
correlation also appeared in the anti-Pr group, it was not
statistically significant.
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| Fig 4.
Inverse association between IgG-anti- autoantibody
and CA in noninfection patients with anti-I and anti-i autoantibodies.
In patients with cold agglutination of noninfectious origin caused by
(A) anti-I (n = 128), (B) anti-i (n = 16), and (C) anti-Pr (n = 22) autoantibody, the titers of IgG-anti- and CA were determined
(left and right ordinate). Sera with identical CA titers were included
into the same group (abscissa). Anti- titers are given in OD 405 nm
(median ± MAD).
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|
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DISCUSSION |
The data analyzed in this study rely on the largest group of cold
agglutination patients described so far. Therefore, they offer an
excellent opportunity to study the role of various factors involved in
the pathogenesis of this disease.
Our statistical analyses showed an inverse correlation between the
anti- and CA autoantibody titer. As in the case of
anti-F(ab')2 autoantibody,5 this
association applied only to the subgroup of patients whose disease was
caused by noninfectious agents, mainly by B-cell proliferation. Of
course, the described association does not allow us to conclude that
the anti- Ab suppresses CA production. However, this finding becomes
interesting in the light of many previous observations showing that
anti-F(ab')2 Abs strongly suppress the B-cell
response, including that of autoreactive cells.1-3,7-14
Although it was not the objective of this report to elucidate the
mechanism by which the anti- Ab might suppress CA-producing B cells,
it is tempting to speculate on it. Previous experiments showed that
murine B-cell lymphoma cells undergo apoptosis in response to
cross-linking of membrane immunogobulin (mIg) with anti-Ig
reagents.7 A similar effect was described with regard to
human tumoral B cells. Cross-linking of surface IgM by Abs induced
apoptosis in Burkitt lymphoma, an effect enhanced by transforming growth factor (TGF)-beta 18 or the Fc
IIB
receptor.9 Cold agglutination of noninfectious origin is
mainly caused by B-cell proliferative disorders. We speculate that the
anti- autoantibody induces apoptosis of tumoral B cells and thus
suppresses the production of the deleterious antierythrocyte
autoantibody. This would be one mechanism of suppression.
In a series of experiments in mice, it was shown that Abs to IgM induce
a state of dormancy in B-cell lymphoma.10,11 Tumor dormancy
is an operational term used to describe a prolonged quiescent state in
which tumor cells are present but do not proliferate. If this mechanism
is also operative in humans, one would expect that high titers of
anti- autoantibodies render CA-producing tumoral B cells dormant.
This could be another control mechanism of CA-producing tumor cells.
It has been known for a long time that anti-Ig Abs are able to suppress
the B-cell response by cross-linking the mIg with the FcII receptor
on the B-cell membrane.12-14 The first model of
FcR-mediated suppression of B-cell response was advanced by Chan and
Sinclair.15 The biochemical basis of the FcR-inhibitory effect has been partially elucidated. It is known that an influx of
extracellular Ca+2 is necessary for B-cell proliferation
and differentiation. Receptor cross-linking prevents the influx of
extracellular Ca+2 by closing the plasma membrane
Ca+2 channels.16,17 A 13-amino acid motif in
the cytoplasmatic tail of the FcRII receptor is necessary and
sufficient for this effect.16,18 Tyrosine at residue 309 is
phosphorylated on cross-linking, and mutation of this residue aborts
the inhibitory effect.16 Our previous experiments in rats
showed that IgG-anti-Ig autoantibodies exert a B-cell suppressive
effect by mIg-FcII cross-linking, and that suppression is induced
only if the B cells' mIg is occupied by its ligand.1,2
That means that only antigen-occupied B cells are susceptible to
suppression. One lymphocyte subpopulation whose antigen is always
available is autoreactive B cells. Therefore, they might be
preferentially downregulated by anti-Ig autoantibodies. Suppression of
antierythrocyte autoantibody-producing B cells by the anti- Ab
through mIg-FcR cross-linking might be another immunosuppressive
mechanism in cold agglutination. This latter possibility, however, does
not provide an explanation for the preferential suppression of tumoral
B cells as indicated by our results.
In conclusion, our data show a highly significant inverse association
between CA and anti- autoantibody, a biomolecule with a potential
B-cell suppressive function, in patients with cold agglutination caused
by noninfectious agents.
| |
ACKNOWLEDGMENT |
D.N. is deeply indebted to Prof Dr Irinel Popescu, Prof Dr 
tefan
Drãgulescu, and Dr Francise Bárányi for their
continuous support that enabled him to participate in this project.
| |
FOOTNOTES |
Submitted April 22, 1999; accepted August 20, 1999.
P.T. and D.N. contributed equally to this work and should both be
regarded as the first author.
Supported in part by the Ministry of Education (Bucharest, Romania) by
an award to D.N.
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 reprint requests to Peter Terness, MD, Department
of Transplantation Immunology, Institute of Immunology, University of
Heidelberg, INF-305, 69120 Heidelberg, Germany.
| |
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ABSTRACT
INTRODUCTION