Blood, 15 February 2003, Vol. 101, No. 4, pp. 1660-1660
CORRESPONDENCE
To the editor:
Autoimmune complexes in human serum in presence of therapeutic
amounts of intravenous immunoglobulins
Intravenous immunoglobulins (IVIGs) are widely used in the
supportive therapy of immunodeficient patients and in the treatment of
a wide variety of chronic autoimmune and inflammatory
diseases.1,2 The proposed mechanisms of action of IVIG in
these diseases include the inhibition of phagocytosis and
immunomodulatory effects. Inhibition of phagocytosis has been
observed in diseases such as immune thrombocytopenia purpura (ITP), in
which platelets opsonized by the pathogenic autoantibodies are no
longer phagocytosed shortly after IVIG infusion.3 Several
mechanisms of inhibition of phagocytosis by IVIG have been proposed and
include the direct competitive blockade of the Fc receptors I and III
(FcRI and III)4,5 and the interaction with the inhibitory
FcRIIB.6 However, the nature and origin of the immune
complexes (ICs) that can interact with FcRs have remained unclear.
Previous work has focused on the presence of IgG aggregates in IVIG
that could be formed during the manufacturing process7 or
through anti-idiotypic interactions.8,9
Healthy individuals continuously produce self-reactive IgM and
IgG. The production and reactivity of serum IgG autoantibodies are
tightly regulated in order to avoid formation of immune complexes, which could result in inflammation.10 It has been shown
that the reactivity of autoreactive IgG was constantly inhibited by id-anti-id interactions with IgM and IgG antibodies.10,11
IVIG preparations contain mostly IgG (> 98%), with only trace amounts of IgM, IgA, and other plasma proteins. In the present work, we have
tested the possibility that the injection of large amounts of IVIG
could oversaturate the normal mechanisms of control of autoreactive IgG
present in human plasma and result in the formation of soluble
autoimmune complexes (auto-ICs).
It is known that purified IgGs are much more polyreactive than serum
IgGs,12 and we confirmed this observation with human ferritin. IVIGs reacted strongly with ferritin in enzyme-linked immunosorbent assay (ELISA), while serum exhibited a very low antiferritin reactivity at 25 µg/mL of IgGs (OD < 0.1;
data not shown). Using this differential reactivity, we determined the ability of a fixed volume of human serum to inhibit the reactivity of
increasing concentrations of IVIG. As shown in Figure
1A,
optical density (OD) results indicated that the addition of a fixed
amount of serum resulted in a significant reduction of the IVIG
antiferritin reactivity. The inhibition was high (> 75%) up to a dose
of about 50 µg/mL of IVIG and then gradually decreased. These results
indicated that the ability of serum to inhibit the autoreactivity of
exogenously added IgG is saturable. The amount of serum IgG can be
increased by a factor of about 3 (25 µg/mL of endogenous IgG and 50 µg/mL of exogenous IgG) before a significant in vitro autoreactivity of the serum-IVIG blend can be detected. Similar IgG increases can be
observed in the plasma of IVIG-treated patients, since it has been
estimated that the infusion of 100 mg/kg of IVIG increases the IgG
plasma level by 2.5 mg/mL.13
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| Figure 1.
Analysis by ELISA of the ferritin reactivity and IgG
content of various fractions.
(A) Antiferritin reactivity of IVIG in presence of human serum.
Reactivity was measured in ELISA in presence ( ) or absence ( ) of
a fixed volume of human serum corresponding to a final IgG
concentration of 25 µg/mL. (B) Amount of total IgG in PEG
precipitates. Quantification of IgG was done by ELISA. (C) Proportion
of total biotin-labeled IVIG in PEG precipitates. Biotin-IgG was
quantified by ELISA using streptavidin-HRP conjugate and the starting
biotin-IVIG solution as standard. (D) Proportion of ferritin-reactive
biotin-IVIG in the PEG precipitates. Ferritin reactivity of serial
dilutions of the PEG-precipitated fractions was measured by ELISA and
detected using a streptavidin-HRP conjugate. One unit of ferritin
reactivity was defined as the volume of each fraction, which produced
an OD of 0.4.
|
|
The above findings and the observation that IVIG reacted with multiple
plasma proteins in Western blot experiments (data not shown) suggested
the formation of auto-ICs in serum in the presence of IVIG. This
possibility was tested directly by precipitating the IC in the presence
of 2.5% polyethylene glycol (PEG).14 The results of a
representative experiment (Figure 1B) showed that the amount of
PEG-precipitated IgG in the serum-IVIG blend was significantly higher
than the sum of precipitated IgG obtained with the 2 isolated
fractions. To determine if the added IVIG contributed to this
synergistic effect, the experiment was repeated with biotin-labeled
IVIGs, which were specifically detected using a
streptavidin-horseradish peroxidase (HRP) conjugate. The
result (Figure 1C) confirmed the involvement of IVIG, since 60% more biotin-labeled IVIGs were present in the PEG precipitate of the serum-IVIG blend compared to the PEG precipitate of IVIG alone. To
determine if autoantibodies were involved, the precipitated ICs were
tested for ferritin reactivity, which was also detected with a
streptavidin-HRP conjugate. The PEG precipitate of IVIG contained about
10% of the initial ferritin reactivity. But the PEG precipitate of the
serum-IVIG blend contained almost 3 times more ferritin-reactive
antibodies than the starting blend. This unexpected result indicated
first that ferritin-reactive autoantibodies are found mostly in the IC
fraction. The higher ferritin reactivity of the isolated IC is
reminiscent of the higher polyreactivity of purified IgG compared to
serum,12 and suggested that the ferritin reactivity of the
soluble IC is inhibited by undefined serum components, which are not
precipitated by PEG.
The results support the hypothesis that therapeutic doses of IVIG can
oversaturate the normal mechanisms of inhibition of natural
autoantibodies and result in the formation of soluble IC. Soluble
plasma ICs have been observed in a number of autoimmune diseases,15 but the possible formation of soluble IC
containing IVIG and normal plasma proteins has not been specifically
studied so far. It is possible that these ICs are rapidly cleared from the circulation after interaction with FcR-bearing cells, thus making
them difficult to detect in the plasma of IVIG-treated patients.
However, there is evidence that auto-IC may be involved in the
therapeutic effects of IVIG in some diseases. The reactive macrophage
activation syndromes are characterized by a massive increase in plasma
ferritin levels.16 It was recently reported that the
successful treatment of this disease by injection of large doses of
IVIG was associated with immune clearance of ferritin, which could be
detected in plasma IC one day after IVIG injection.16 Further characterization of the IC formed in human serum following IVIG
addition is under way in our laboratory and will permit better definition of their composition and biological activity. The work may
permit the defining of ways to purify the IgG responsible for the
formation of IC starting from currently used IVIG preparations. Such a
procedure could result in the preparation of different subpreparations
of IVIG for use in different diseases, and may thus permit
treatment of more patients with the available amounts of IVIG.
For precipitation of IC, PEG 6000 (Sigma, Oakville, ON, Canada) was
added to a final concentration of 2.5% to the diluted IVIG and serum
solutions containing about 5 mg/mL of IgG as described previously.14 After overnight incubation at 4°C,
precipitates were collected by centrifugation at 1500g for
20 minutes, washed twice in 2.5% PEG, and dissolved by incubation for
30 minutes at 37°C in phosphate buffered saline (PBS)
containing 0.05% Tween 20, 10 mM EDTA (ethylenediaminetetraacetic
acid), and 0.01% thimerosal.
For ELISA, microplate wells were coated with human ferritin
(Calbiochem, La Jolla, CA) or anti-human IgG (Fc-specific; Jackson ImmunoResearch Laboratories, West Grove, PA) in 100 mM carbonate buffer, pH 9.7. After overnight incubation at 4°C, wells were blocked
with 5% bovine serum albumin (BSA) in 0.05% Tween 20-PBS for one
hour at 37°C. After washing with 0.85% NaCl, samples diluted in 1%
BSA-0.05% Tween 20-PBS were distributed into triplicate wells, and
the plate was incubated for 1 hour at 37°C. After washing, bound
antibodies were detected with peroxidase-labeled goat anti-human IgG
(Fc specific; Jackson ImmunoResearch Laboratories; Figure 1A-B) or
peroxidase-labeled streptavidin (Jackson ImmunoResearch Laboratories;
Figure 1C-D) and revealed with the ortho-phenylenediamine (OPD) substrate (Abbott Laboratories; Abbott Park, IL).
Optical densities were read at 490 nm with a reference at 630 nm.
Josée Lamoureux, Éric Aubin, and Réal Lemieux
Correspondence: Réal Lemieux,
HÉMA-QUÉBEC, Research and Development, 2535, Laurier Blvd, Ste-Foy, Quebec, Canada, G1V 4M3; e-mail:
real.lemieux{at}hema-quebec.qc.ca
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