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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 942-951
Immunoglobulin M-Enriched Human Intravenous Immunoglobulin Prevents
Complement Activation In Vitro and In Vivo in a Rat Model of Acute
Inflammation
By
Robert Rieben,
Anja Roos,
Yvonne Muizert,
Caroline Tinguely,
Arnout F. Gerritsen, and
Mohamed R. Daha
From the Departments of Cardiology and Hematology, Bern University
Hospital, Bern, Switzerland; and the Department of Nephrology, Leiden
University Medical Center, Leiden, The Netherlands.
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ABSTRACT |
An important antiinflammatory mechanism of intravenous
immunoglobulin preparations (IVIG) is their ability to block complement activation. The purpose of this study was to compare the
complement-inhibitory activity of four IVIG preparations differing in
isotype composition. The preparations were: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2) Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L
IgA); (3) IVIgM (35 g/L IgM, 12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG), all from Biotest Pharma GmbH, Dreieich, Germany.
Their complement inhibitory activity was assessed in vitro by
measurement of the blocking of C1q-, C4-, and C3
deposition on solid-phase aggregated rabbit IgG by enzyme-linked
immunosorbent assay (ELISA). Complement inhibition in this ELISA was
best for IVIgM, followed by Pentaglobin and IVIgG; IVIgA did not
exhibit an inhibitory effect. Control experiments with excess
concentrations of C1q as well as with C1q-depleted serum showed that
the inhibitory effects of IVIG were not caused by complement activation
and thus, consumption, but that C4 and C3 were scavenged by IgM and to
a lesser extent by IgG. These results were confirmed in vivo in the rat
anti-Thy 1 nephritis model, in which a single dose of 500 mg/kg of
IVIgM prevented C3-, C6-, and C5b-9 deposition in the rat glomeruli,
whereas the effect of IVIgG was much less pronounced. Reduction of
complement deposition was paralleled by a diminished albuminuria, which
was completely absent in the IVIgM-treated rats. IVIgM and to a lesser
extent IVIgG also prevented rat C3 deposition on cultured rat
glomerular mesangial cells in vitro, but did not influence anti-Thy 1 binding. Neither IVIgM nor Pentaglobin nor IVIgG negatively affected in
vitro phagocytosis of Escherichia coli (E coli)
by human granulocytes. In conclusion, we have shown that IgM enrichment
of IVIG preparations enhances their effect to prevent the inflammatory
effects of complement activation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTRAVENOUS IMMUNOGLOBULIN
preparations (IVIG) exert a beneficial effect in many clinical
situations that are characterized or accompanied by a dysfunction or
dysregulation of the immune system. Among others, these include
immunodeficiencies, autoimmune diseases, sepsis, and severe trauma.
Besides the effect of IgG substitution in case of a deficiency, several
other mechanisms have been postulated for the beneficial effect of
IVIGs: blocking of Fc receptors on proinflammatory cells,1
antiidiotypic neutralization of autoantibodies,2 modulation
of cytokine production by lymphocytes,3,4 and effects on
the complement system.5 The interaction of IVIG with the
complement system was reported to take place at least at three
different stages: Basta et al6,7 reported a binding of the
activated complement components C4b and C3b to the Ig molecules and
Mollnes et al8 found binding of C1q to the Ig molecules in
an in vitro system. Both of these mechanisms divert the attack of
complement from the target by scavenging active complement components.
A third mechanism was recently shown by Lutz et al,9 who
described an enhancement of the physiologic cleavage of C3b-Ig complexes. This latter effect of IVIG prevents damage by C3b-carrying immune complexes in circulation by accelerating their decay. It was
recently suggested that isotype differences exist concerning the
capacity of human Ig to scavenge C4b and C3b. Miletic et
al10 showed in vitro that human IgM and IgA have a higher
capacity to bind C4b and C3b than IgG.
The aim of the present study was to compare the complement-inhibitory
capacities of different IVIG preparations using in vitro and in vivo
models of complement activation and to obtain further insight in the
complement components involved in this process. Besides pure
IgG-containing IVIG (IVIgG), we had the opportunity to include in our
tests two different IVIGs, which were enriched in IgM, content as well
as one IgA-enriched preparation.
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MATERIALS AND METHODS |
IVIG preparations.
Ig fractions of large pools of human plasma were obtained from Biotest
Pharma GmbH (Dreieich, Germany). Four preparations were compared in the
different assays: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2)
Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L IgA); (3) IVIgM (35 g/L IgM,
12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG). The IVIG
preparations were dissolved to a protein concentration of 50 g/L in a
buffer containing 26 g/L of glucose, pH 6.6. Intraglobin F and
Pentaglobin are commercially available, whereas IVIgM and IVIgA were
laboratory preparations.
Antibodies.
Antisera to human and rat C1q, C4, C3, and C9, respectively, were
raised in goats or rabbits by repeated subcutaneous injections of the
purified components and their specificity was checked by enzyme-linked
immunosorbent assay (ELISA) and Western blotting. The IgG were purified
from the antisera by ammonium sulfate precipitation and diethyl
aminoethyl (DEAE) Sepharose column chromatography (Pharmacia Biotech, Uppsala, Sweden). Labeling of these antibodies with
digoxygenin (DIG, Boehringer Mannheim GmbH, Mannheim, Germany), biotin
(Pierce Chemical Co, Rockford, IL), and fluorescein isothiocyanate (FITC, Koch-Light Laboratories Ltd, Colnbrook, Bucks, UK) was performed
according to the instructions of the suppliers. Horseradish peroxidase
(HRP)-labeled sheep F(ab')2 to DIG was purchased from Boehringer Mannheim. Hybridomas secreting mouse monoclonal antibody (MoAb) specific for human IgM and IgG (clones HB57 and HB43) were obtained from the American Type Culture Collection (Rockville, MD) and
the antibodies purified from cell supernatant according to standard
techniques. HRP- and FITC-labeled goat anti-mouse IgG antibodies were
from Southern Biotechnology Associates (SBA, Birmingham, AL). For
fluorescence-activated cell sorting (FACS) analysis, the following
antibodies and conjugates were used: a biotinylated mouse MoAb directed
against rat C3 (C3-1), described previously,11
phycoerythrin (PE)-labeled goat anti-mouse antibodies (Dako, Glostrup,
Denmark), PE-conjugated streptavidin (Becton Dickinson, San Jose, CA),
FITC-conjugated goat anti-human IgM (Nordic Immunologic Laboratories,
Tilburg, The Netherlands), and FITC-conjugated goat anti-human IgG (De
Beer Medicals, Diessen, The Netherlands).
Analysis of the C1q-, C4-, and C3 scavenging activity in vitro.
An ELISA system was set up in which the inhibition of C1q-,
C4-, and C3 deposition from normal human serum (NHS) to aggregated rabbit IgG by the different IVIG preparations was measured.
Heat-aggregated rabbit IgG (aIgG, 10 mg/mL, 20 minutes at 63°C,
insoluble aggregates were removed by centrifugation for 10 minutes at
3,000g) was coated to microtiter plates (NUNC Maxisorp, NUNC
AB, Roskilde, Denmark) at 10 µg/mL in 0.1 mol/L carbonate buffer pH
9.6 for 2 hours at 37°C or overnight at room temperature. After
washing with phosphate-buffered saline (PBS) containing 0.05% Tween
20, the plates were saturated with PBS containing 0.05% Tween 20 and
2% caseine (Sigma-Aldrich Co, St Louis, MO) for 1 hour at 37°C.
Human serum was diluted 1:200 in half-isotonic veronal-buffered saline
containing 0.05% gelatin, 0.15 mmol/L CaCl2, 0.5 mmol/L
MgCl2, 0.05% Tween 20 (GVB++), mixed with a
serial dilution (25 to 0.03 mg/mL) of IVIG and then put on the coated
plates for 1 hour at 37°C. DIG-labeled goat antibodies to human
C1q, C4, or C3, HRP-labeled sheep anti-DIG F(ab')2
and ABTS substrate (Sigma) were used to reveal the deposition of C1q,
C4, or C3 on the plate. The absorption at 415 nm was measured with a
microplate reader.
Two different variations of this technique were used to assess whether
the inhibitions of C4- and C3-binding were independent of the
inhibition of C1q-binding to the aIgG coat: (1) the human serum was
diluted in C4-deficient guinea pig serum (end-concentration in the
assay: NHS 1:200, guinea pig serum 1:2) instead of GVB++,
ensuring an excess of all complement components besides C4. (2)
Purified human C1q (produced at our own laboratory as described earlier)12 was added to the aIgG coated plates at a
concentration of 7 µg/mL in GVB++, the plates washed, and
subsequently 1:200 diluted C1q-depleted NHS used for the assay. The C1q
depletion of this serum was performed by absorption on an IgG-Sepharose
column and the levels of all complement components besides C1q were in
the normal range, as assessed by hemolytic assays. Bound C1q, C4, and
C3 to the aIgG/C1q-coated plate was detected as described above.
Assessment of in vitro C3a generation by IVIgG and IVIgM in human
serum.
Aliquots of fresh human serum were incubated with serial dilutions of
either IVIgG, Pentaglobin, or IVIgM (100 µL serum + 100 µL of IVIG
diluted in veronal-buffered saline containing 0.15 mmol/L
CaCl2 and 0.5 mmol/L MgCl2 (VBS++)
from 25 to 0.03 mg/mL) for 1 hour at 37°C. After the incubation, the C3a contents of the samples were immediately quantitated using an
ELISA (Quidel, San Diego, CA). As controls, serum was also incubated
with heat aggregated IVIgG (20 minutes, 63°C) and VBS++ alone.
Binding of C4 to human Ig.
After preincubation of the IVIG with human serum and incubation on the
plate coated with aIgG, binding of C4 to human IgM and IgG was analyzed
by sandwich ELISA. Microtiter plates (NUNC Maxisorp) were coated with
goat anti-human C4 at 2 µg/mL in 0.1 mol/L carbonate buffer pH 9.6. After saturation and washing of the plates, the supernatant of the
above-described test (hemolytically active human serum 1:200 with a
serial dilution of IVIG) was incubated for 1 hour at 37°C. After
washing, human IgM and IgG, which were bound to the plate were revealed
isotype-specifically by MoAb and goat anti-mouse HRP conjugate.
IVIG in the rat anti-Thy 1 nephritis model.
The induction of acute glomerular nephritis in the rat by IV injection
of mouse IgG2a MoAb (ER4G) against the rat Thy 1.1 antigen has been
described earlier.13 We used this model to investigate the
potential of IVIG to prevent complement activation and inflammation in
vivo. Inbred male Wistar rats, weighing approximately 200 g, were
divided into three groups of four rats each. All rats received an IV
injection of 2 mL of either PBS (group 1, control group), IVIgG (500 mg/kg, group 2), or IVIgM (500 mg/kg, group 3); all IV injections were
given in the tail vein. Thirty minutes after the injection with PBS or
IVIG, the rats were administered IV 0.5 mg/kg of ER4G in 0.5 mL PBS.
Kidney biopsies were taken after 10 minutes, 30 minutes, and 24 hours,
and the animals were killed after the last biopsy. All tissue samples
were snap-frozen in precooled isobutanol and stored in liquid nitrogen
until analyzed further. Blood samples were taken before the injection
of IVIG (time =  30 minutes), before injection of ER4G (t = 0 min), and at the same time as the biopsies (t = 10 minutes, 30 minutes, and 24 hours). The blood samples were kept on ice during the
experiment, centrifuged, and the sera stored at 80°C. The
sera were used later to immunochemically determine the levels of rat
C1q, C3, C6, and C5b-9, as well as human IgM and IgG.
For the assessment of albuminuria, another three groups of three Wistar
rats each were injected according to the same scheme as above, but no
biopsies were performed. In addition, one rat each was injected with
500 mg/kg IVIgG and IVIgM at t = 30 minutes followed by 0.5 mL
of PBS instead of ER4G to detect any albuminuria caused by IVIG. Urine
was collected from 0 to 4 hours and 4 to 24 hours after injection of
the anti-Thy 1 MoAb with  1% merthiolate (Koch-Light Laboratories)
and stored frozen at 20°C until the albumin contents were measured.
Detection of deposition of complement components, as well as human
Ig in kidney tissue.
Snap-frozen renal tissue was cut into 2-µm sections, air dried, and
acetone fixed for 10 minutes at 22°C. Slides were washed twice for
7 minutes in PBS and immunofluorescence was performed either directly
using FITC-conjugated antibodies or DIG-conjugates followed by
HRP-labeled sheep anti-DIG F(ab')2 (Boehringer
Mannheim) and tyramide-FITC conjugate (NEN Life Science, DuPont de
Nemours B.V., Dordrecht, The Netherlands). The following antibodies
were used: (1) detection of bound anti-Thy 1 MoAb: goat anti-mouse IgG2a-FITC (Nordic, Tilburg, The Netherlands); (2) detection of rat
C1q: rabbit F(ab')2 anti-rat C1q (produced at our own
laboratory) followed by HRP-conjugated goat anti-rabbit IgG (Boehringer
Mannheim); (3) detection of rat C3: rabbit anti-rat C3-FITC (produced
at our own laboratory); (4) detection of rat C6: DIG-labeled mouse MoAb
specific for rat C6 (produced at our own laboratory); and (5) detection
of rat C5b-9: DIG-labeled mouse MoAb specific for a rat C5b-9
neoantigen (courtesy of W.G. Couser, see Schulze et al14).
Deposition of human IgG was visualized by DIG-labeled HB43 MoAb,
followed by FITC-labeled sheep anti-DIG F(ab')2
(Boehringer Mannheim), and human IgM was detected by biotinylated HB57
MoAb and streptavidin-FITC conjugate (Amersham).
Measurement of complement components in rat serum.
Rat C4- and C3 concentrations were determined by rocket-electrophoresis
using monospecific antisera as described earlier.15 In
brief, rabbit anti-rat C4 and C3, respectively, were diluted 1:30 in
1% agarose solution. Subsequently, the agarose gels were poured onto
glass plates and allowed to set. Wells were punched in the agarose and
filled with 1:4 diluted samples and a serial dilution of pooled normal
rat serum as a standard. The samples were subjected to electrophoresis
for 4 hours at 30 mA and the plates then rinsed in PBS containing 2 mmol/L EDTA. Precipitation arcs were stained with amido black (Sigma)
and the C4 and C3 concentrations of the samples calculated by
comparison of their rocket heights with the ones of the standards.
Measurement of albuminuria in rat urine.
Proteinuria was assessed by quantitation of rat albumin in a rocket
electrophoresis. Rabbit anti-rat albumin antiserum for the rocket
electrophoresis (produced at our own lab) was used at a dilution of
1:100 and, otherwise, the test was performed as described above for the
rat C4 and C3 determination.
Quantitation of human IgM and IgG in rat serum.
The concentrations of human IgM and IgG were measured in the serum
samples of the rats treated with IVIgM. ELISA plates (NUNC maxisorp)
were coated with either rabbit anti-human IgM or IgG (DAKO A/S,
Glostrup, Denmark). After washing and saturation of the plates, the
sera were incubated at a 1:16,000 dilution for 1 hour at 37°C.
Biotinylated mouse MoAbs against human IgM (HB 57) and IgG (HB 43),
followed by streptavidin-HRP conjugate (Amersham) and ABTS substrate,
were used to show bound human IgM and IgG antibodies. A human serum
with known concentrations of IgM and IgG was included in the test as a standard.
Binding of anti-Thy 1 MoAb and rat C3 to cultured rat glomerular
mesangial cells.
Mesangial cells (RMC) were isolated from glomeruli of Sprague Dawley
rats and cultured in vitro as described.16 IVIgM and IVIgG
were serially diluted in culture medium (RPMI containing penicillin,
streptomycin, and 10% fetal calf serum [FCS]) from 10 to 0.6 mg/mL
and added to RMC grown in 24-well plates to subconfluence. After a
30-minute incubation at 37°C, anti-Thy 1 MoAb ER4G was added at a
final concentration of 1 µg/mL, representing a nonsaturating concentration of ER4G. After another incubation for 30 minutes, rat
serum (Wistar) was added to a final concentration of 10%, followed by
incubation for 60 minutes. These conditions were chosen to minimize
cell lysis during the incubation period. The cells were then harvested
using 20 mmol/L EDTA in PBS and binding of ER4G and C3, as well as
human IgM and IgG, were analyzed by flow cytometry (FACScan, Becton
Dickinson, Franklin Lakes, NJ). Five minutes before measurement,
propidium iodide (PI) was added at 1 µg/mL, and only cells being
impermeable for PI were gated for analysis.
Granulocyte phagocytosis assay.
The phagocytosis of E coli K12 bacteria by human granulocytes
was investigated using the commercially available Phagotest from
OrpegenPharma (Heidelberg, Germany). The test was performed as
described previously,17 using blood from healthy volunteer donors and nonopsonized, FITC-labeled E coli. Heparinized whole blood was centrifuged to separate plasma from cells, and the latter were subsequently washed three times with PBS pH 7.4. IVIG preparations were mixed with an equal volume of the fresh plasma, resulting in an
IVIG concentration of 25 mg/mL. After addition of the washed, unfractionated blood cells and the labeled E coli bacteria, the reaction mixture was incubated for 10 minutes at 37°C. The ratio of
E coli to white blood cells was 25:1. Internalization of E coli by granulocytes was visualized by FACS (Becton Dickinson) and
analysis of these measurements performed with the CELL Quest software
version 3.0.1 (Becton Dickinson). Phagocytosis activity was expressed
as the percentage of granulocytes that had internalized FITC-labeled
E coli.
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RESULTS |
Prevention of complement deposition on solid-phase aggregated rabbit
IgG in vitro.
In the ELISA system with hemolytically active human serum, a
dose-dependent inhibition of deposition of the complement components C1q, C4, and C3 on aIgG could be observed for IVIgM, Pentaglobin, and
IVIgG. IVIgM was most effective in preventing complement deposition, followed by Pentaglobin and IVIgG, whereas the IVIgA preparation did
not show an inhibitory effect (Table 1).
Dose response curves of the inhibition experiments with IVIgM and IVIgG
are given in Fig 1. About the same
concentrations of IVIG were needed for 50% inhibition of C4 and C3
binding (Table 2), but with the difference that binding of C4 could be blocked to greater than 90% with the IgM-containing preparations (IVIgM and Pentaglobin), whereas C3 binding
could only be blocked to a maximum of 75%. Higher concentrations of
IVIG were needed to block C1q binding in the ELISA system and the
difference in inhibitory capacity between IgM-containing preparations and IVIgG was not as pronounced in this case as for blocking C4 and C3
binding.
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| Fig 1.
Inhibition of complement deposition on solid-phase bound
rabbit aIgG by IVIG I. Dose response curves of the effect of IVIgM and
IVIgG on in vitro complement deposition. Human serum diluted 1:200 in
GVB++ was incubated for 1 hour at 37°C in
aIgG-coated wells together with a serial dilution of IVIgM or IVIgG.
Solid-phase bound C1q, C4, and C3 were then detected with specific
DIG-labeled goat antibodies, sheep F(ab')2 anti-DIG
HRP conjugate and ABTS substrate. A representative experiment of three
with similar results is shown.
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Table 2.
Comparison of the Inhibitory Capacity (ID 50) of the
Different IVIG Preparations on Complement Deposition in an In Vitro
Assay
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Two sets of experiments were performed to check whether complement
activation by the IVIG preparations was involved in the observed
inhibitions of C1q, C4, and C3 binding to aIgG: first, C4-deficient
guinea pig serum was used for the 1:200 dilution of the human serum to
have an excess of all complement components available besides C4, the
binding of which was measured on the aIgG-coated plate. In this
experiment, the inhibition curves for C4-binding were similar to the
ones observed without guinea pig serum (Fig 1), but higher amounts of
IVIG were needed to obtain 50% inhibition (Table 2). Second,
another modification of the inhibition ELISA was used in which purified
human C1q was added to the aIgG-coated plate and C1q-depleted, 1:200
diluted human serum used as the complement source. Also, in this
system, a dose-dependent inhibition of C4 and C3 binding was observed,
which was best for IVIgM and Pentaglobin, followed by IVIgG and IVIgA
(Fig 2). In contrast to the experiment with
whole, hemolytically active human serum, the inhibitory capacities of
IVIgM and Pentaglobin were almost equal and also IVIgA showed some
inhibitory effect. In addition, the in vitro complement activation by
IVIgG, Pentaglobin, and IVIgM was assessed by quantitating C3a
concentrations after a 30-minute incubation of the IVIGs with human
serum. As shown in Fig 3, all tested IVIG
preparations were equally active to induce C3a in human serum. Only at
the highest concentration of IVIG used in this test (12.5 mg/mL), the
C3a levels were significantly elevated (P < .01, Student's
t-test) as compared with serum incubated for 30 minutes with
VBS++ buffer alone. Heat-aggregated IVIgG, used as a
complement-activating control, already led to elevated C3a levels at a
concentration of 0.15 mg/mL.
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| Fig 2.
Inhibition of complement deposition on solid-phase bound
rabbit aIgG by IVIG II. After coating of the microtiter plates with
aIgG, purified C1q was added as a second step, the plates washed, and
the inhibition experiments with IVIgM and IVIgG as shown in Fig 1 for
C4 and C3 deposition were then performed in 1:200 diluted, C1q-depleted
human serum. A representative experiment of three with similar results
is shown.
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| Fig 3.
C3a generation in human serum by IVIG. A total of 100 µL of serial dilutions of the IVIG preparations in
VBS++ was incubated for 1 hour at 37°C with 100 µL of fresh human serum and the C3a contents of this incubation
mixture immediately quantitated by a commercial ELISA. As controls, the
baseline C3a content of the used serum and the C3a generation by
heat-aggregated IVIgG are also depicted in the figure. Average values
of duplicates are given with indication of the standard deviations
(SD).
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Binding of C4 to human IgM and IgG.
After incubation on the aIgG-coated plates, the supernatants of an
inhibition test (human serum diluted 1:200 plus a serial dilution of
IVIG) were analyzed for binding of C4 to human IgM and IgG. For IVIgG,
a binding of C4 to IgG was observed, which mirrored the decrease of C4
deposited on the aIgG-coated plate. The higher inhibitory capacities of
the IgM-containing IVIG preparations Pentaglobin and IVIgM on
C4-deposition to aIgG were associated with binding of C4 to IgM, which
was most pronounced in the case of IVIgM. For IVIgA, which did not
measurably inhibit the deposition of C4 on aIgG, some binding of C4 to
IgG was observed, which is its main constituent besides IgA. A column
graph representation of the results obtained with an IVIG concentration
of 2.8 mg/mL is given in Fig 4.
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| Fig 4.
Binding of C4 to human IgG and IgM in the IVIG
preparations. Inhibition experiments for C4 binding to coated aIgG were
performed as described for Fig 1. Percents of inhibition of C4 binding
to the aIgG coat at a dose of 2.8 mg/mL IVIG are given in the top panel
( , representative experiment of three with similar results). The
supernatants of the incubation mixtures were then removed and incubated
on a plate coated with goat anti-C4 antibody. Human IgG ( ) and IgM
( ) bound to this anti-C4 coat were then shown by MoAb and goat
anti-mouse HRP conjugate. The OD 415 values (averages of
duplicates) of the latter assay are given in the bottom panel.
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Inhibition of complement deposition in the rat anti-Thy 1 nephritis
model.
IVIgG and IVIgM were injected IV at a concentration of 500 mg/kg into
rats 30 minutes before injection of 0.5 mg/kg anti-Thy 1.1 MoAb ER4G.
Whereas in the rats that received PBS instead of IVIG deposition of
complement components (C1q, C3, C6, and C5b-9) in the glomeruli could
be observed by immunofluorescence as early as 10 minutes after the
injection of the ER4G MoAb, this deposition was virtually absent in
rats that received IVIgM (Fig 5,
Table 3). A diminished complement
deposition, as compared with the control group receiving PBS instead of
IVIG, was observed in the case of IVIgG-treated rats. In the control
group, the complement deposition was visible in all biopsy samples, ie,
after 10 minutes, 30 minutes, and 24 hours. The protection from
complement deposition by the IVIG preparations did not change with time
during the 24-hour observation period; IVIgM almost completely
prevented complement deposition as assessed by immunofluorescence, and
IVIgG treatment led to a staining intermediate between the one seen in
the PBS control and the IVIgM-treated rats (Table 3). No change of
fluorescence intensity for ER4G MoAb binding by IVIG could be observed
(Fig 5, top row).
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| Fig 5.
Deposition of ER4G, rat C1q, C3, C6, and C5b-9 in kidney
biopsy specimens. Groups of four rats each were injected IV 500 mg/kg
of IVIG and 30 minutes later 0.5 mg/kg anti-Thy 1 MoAb, ER4G. Kidney
biopsies were performed 10 minutes, 30 minutes, and 24 hours after ER4G
injection and analyzed by immunofluorescence for the presence of ER4G
and rat complement components. The extraglomerular staining observed
for C3 is frequently encountered in kidney sections of adult rats and
not related to the anti-Thy 1 injection. Magnification was 250:1 for
all micrographs.
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Table 3.
Detection of ER4G, Rat Complement Components, and
Human Ig on Kidney Biopsy Samples by Immunofluorescence
Staining
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In the IVIgG-treated group, deposition of human IgG could be observed
on the glomeruli, as well as in the interstitial area on all biopsy
samples. Staining for both human IgG and IgM was weakly positive on
biopsy specimens of the IVIgM-treated rats (Table 3).
Influence of IVIG treatment on anti-Thy 1-induced proteinuria.
Albuminuria was measured in groups of three rats each receiving either
PBS, IVIgG, or IVIgM 30 minutes before injection of ER4G. Significant
albuminuria was found in the 4- to 24-hour samples, and the mean total
albumin contents are depicted in Fig 6. We found that injection of IVIgM prevented the anti-Thy 1-induced albuminuria and that IVIgG treatment led to an approximately two thirds
reduction as compared with the control group receiving PBS. Control
rats that were treated with IVIG only, without injection of anti-Thy 1 MoAb, showed albuminuria of 80 to 140 µg during the 4- to 24-hour
period after the injection, which is not significantly different from
healthy Wistar rats (results not shown).
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| Fig 6.
Anti-Thy 1-induced albuminuria of rats treated with PBS
(control), IVIgG, or IVIgM. Groups of three rats each were injected 500 mg/kg of IVIgG, IVIgM, or 2 mL of PBS, followed after 30 minutes by 0.5 mg/kg ER4G. Urine was collected from 4 to 24 hours after ER4G injection
and the albumin content quantitated by rocket electrophoresis. Single
determinations for each of the rats were performed, mean values per
group and SD are shown. Differences between groups were statistically
significant (P < .05) by Student's t-test.
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C4 and C3 levels in sera of rats treated with IVIG and anti-Thy 1 MoAb.
To assess whether complement consumption was involved in the effects of
IVIG in the anti-Thy 1 model, the serum levels of rat C4 and C3 were
quantitated. Serum levels of C4 decreased to approximately 50% at t = 30 minutes in the PBS control group (P < .05 by
t-test), whereas no significant change of circulating C4 was
found in the IVIgG and IVIgM groups (Fig
7). For C3, a similar picture was seen, but with a less pronounced
decrease of C3 in the PBS control group as compared with C4 (results
not shown).
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| Fig 7.
Circulating C4 levels in rats after IVIG treatment and
subsequent injection of anti-Thy 1 MoAb. Sera of the rats used in the
experiment described in Fig 5 were used for quantitation of circulating
complement levels. The concentration of C4 was quantitated by rocket
electrophoresis using a monospecific rabbit anti-rat C4 antiserum. The
asterisk (*) indicates a significant reduction of C4 as compared with t
= 30 minutes (Student's t-test, P < .05). Columns are mean values of duplicate measurements with indication
of the SD.
|
|
Concentrations of human IgM and IgG in rat sera after injection of
IVIgM and IVIgG.
The circulating levels of human IgM and IgG in the sera of the rats
that received 500 mg/kg of either IVIgM or IVIgG were measured by
sandwich ELISA. At the time of anti-Thy 1 injection (ie, 30 minutes
after IVIgM infusion), human IgM concentrations were at 8 mg/mL and
then decreased to 5 mg/mL within another 30 minutes and reached 2 mg/mL after 24 hours. Serum levels of human IgG were at 2 mg/mL 30 minutes after IVIgG infusion and decreased linearly to 1 mg/mL
within 24 hours, resulting in approximate circulation half-lives of 12 hours for IgM and 24 hours for IgG (results not shown).
Influence of IVIG on binding of anti-Thy 1 MoAb and rat C3 to
cultured rat mesangial cells.
To check the possibility that IVIG might compete with the ER4G MoAb for
binding sites on glomerular mesangial cells, we used an in vitro assay
with cultured RMC. The binding of ER4G, as well as human IgM and IgG on
RMC, was quantitated by FACS analysis. ER4G bound to RMC at a high
level, showing mean fluorescence intensities (MFI) >1,500.
Furthermore, a dose-dependent binding of the human Igs could be
detected. As shown on the upper panel of
Fig 8, preincubation of RMC with IVIG did
not inhibit binding of ER4G. In the same experiment, binding of rat C3
to RMC was also measured. Similar to the experiments with aggregated
IgG, a dose-dependent inhibition of C3 binding to RMC was observed,
which was more pronounced for IVIgM than for IVIgG (Fig 8, lower
panel).
View larger version (15K):
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| Fig 8.
Influence of IVIG on anti-Thy 1 binding and rat C3
deposition on rat glomerular mesangial cells (RMC) in vitro. Cultured
RMC were incubated with serial dilutions of IVIgM and IVIgG followed by
addition of anti-Thy 1 MoAb, ER4G (1 µg/mL) and 10% normal rat
serum. Binding of ER4G (top) and rat C3 (bottom) was analyzed in
parallel by FACS. A representative experiment of two performed is
shown.
|
|
Effect of IVIG on in vitro phagocytosis of E coli by human
granulocytes.
A commercially available phagocytosis test was used to assess the
phagocytic function of human granulocytes at a high concentration of
IVIG. Compared with the effect of heat-inactivation of the plasma, only
a minimal inhibitory effect on phagocytosis could be observed with the
tested IVIG preparations. The results, expressed as the percentage of
granulocytes that had ingested FITC-labeled E coli bacteria,
are represented in Table 4.
| |
DISCUSSION |
The results presented in this study show that IgM enrichment of IVIG
preparations leads to an enhanced complement-inhibitory capacity both
in vitro and in vivo as compared with pure IVIgG. We developed an ELISA
in which the inhibitory effect of IVIG on deposition of the components
involved in the classical pathway of complement activation could be
quantitated. The 70% pure IVIgM preparation was clearly the best
inhibitor of C4 and C3 deposition in this ELISA system, followed by
Pentaglobin, which contains 12% IgM, and standard,  95% pure IVIgG
(Intraglobin). In contrast to earlier reports by others,10
we could not find an enhanced complement-inhibitory activity of IVIgA
as compared with IVIgG.
The scavenging of C4b and C3b was reported by some investigators to be
independent of C1q, the recognition phase of the classical complement
cascade,18 whereas others challenged this finding by
showing that the complement-inhibitory capacity of IVIG was mainly
dependent on competitive binding of C1q to Ig molecules and only to a
lesser extent on C4 and C3 binding.8 In our ELISA system,
we found both inhibition of C1q and C4/C3 binding, and we therefore
designed three experiments to see whether binding of C1q to the Ig
molecules in the IVIG preparations led to complement activation and,
thus, consumption: (1) A 100-fold excess of C1q over C4 as compared
with the relations in serum was used by diluting the human serum in
C4-deficient guinea pig serum. (2) By adding purified C1q to the coated
aIgG and then using C1q-depleted human serum as the source of C4 and
C3, the effects of scavenging and complement activation were locally
separated. (3) The C3a generation by IVIG in human serum was
quantitated by an ELISA.
For experiment (1), the resulting concentrations necessary for 50%
inhibition (ID 50) of C4-binding were only twofold to sixfold higher
than with 1:200 diluted human serum alone. As in this system, a
100-fold excess of C1q was present as compared with the original experiment, a strict dependency of the inhibition of C4-binding to aIgG
on C1q-dependent complement activation by IVIG seems unlikely. Experiment (2) was designed in such a way that the absence of C1q in
solution only allowed complement activation on the solid phase, whereas
scavenging of C4 and C3 is a fluid phase event. As compared with the
experiment with whole, hemolytically active serum, the ID 50 was 10
times higher for IVIgM, whereas the values were not significantly
elevated for Pentaglobin and IVIgG. Interestingly, IVIgA showed
inhibition of complement deposition under these conditions, although
its action was the weakest of all tested IVIGs. Finally, with
experiment (3) we showed that generation of C3a on incubation of IVIG
with normal human serum was equal for IVIgG and the IgM-containing preparations Pentaglobin and IVIgM. For all of the preparations we
could only find a significant generation of C3a at a concentration of
12.5 mg/mL, indicating that the in vitro complement-activating capacity
of the IgM-containing preparations is not higher than the one of IVIgG.
That binding of complement components to the Ig molecules, scavenging,
was the mechanism by which IVIG prevented their deposition on the
activating surface was directly shown for C4. All inhibitory active
IVIG preparations (IVIgM, Pentaglobin, and IVIgG) showed binding of C4
to IgM and/or IgG after incubation with serum in the
aIgG-coated microtiter plate. The stronger inhibitory effect of the
IgM-containing preparations was associated with a higher amount of C4
bound to IgM as compared with IgG (Fig 4). Taken together, the in vitro
experiments showed that (1) IVIgM and IVIgG were able to block
deposition of C1q, C4, and C3 on a surface activating the classical
complement cascade, (2) a higher concentration of IgM in the
preparation enhanced this effect, (3) scavenging of complement
components rather than complement activation and consumption was the
underlying mechanism of the inhibitory effect.
The anti-Thy 1-induced nephritis model in the rat was chosen to
validate our in vitro data in an in vivo system. In this model, binding
of the anti-Thy 1 MoAb, ER4G, to its respective antigen on glomerular
mesangial cells leads to complement activation via the classical
pathway and subsequently inflammation, which is associated
with proteinuria. At an ER4G dose of 1 mg/kg, this induced
proteinuria is transient, reaching maximum levels at day 3 after
injection and then slowly regressing to normal levels after 3 weeks.19 The ER4G concentration of 0.5 mg/kg, which we used
in our study, was chosen to result in a clear-cut complement deposition, as well as albuminuria, but to be able to detect and compare beneficial effects of IVIGs without having an excess of inflammatory stimulus available. A comparison of IVIgG and IVIgM in
this system showed a much better complement inhibitory and antiinflammatory effect of the latter. Both complement deposition and
albuminuria were almost completely prevented by IVIgM, whereas only
partial protection from these effects was provided by IVIgG.
Quantitation of C4 and C3 levels in the rat sera showed that neither
PBS nor IVIgG- or IVIgM injection by itself led to a consumption of C4
or C3 (Fig 7, t = 0 minutes). However, after injection of ER4G, the
expected consumption of these components (especially of C4) could be
observed in the control group receiving PBS, whereas the rats treated
with IVIgG or IVIgM were protected from ER4G-induced complement
consumption. These data are in accordance with the results on
complement deposition on the biopsies, showing a reduction of C4 and C3
deposition in the IVIgG, and notably the IVIgM, but not the PBS group.
To determine whether IVIG might compete with ER4G for Thy 1 antigen on
the glomerular mesangial cells, we performed in vitro experiments with
cultured rat mesangial cells. Neither IVIgM nor IVIgG influenced the
binding of the ER4G MoAb to RMC as assessed by FACS analysis, whereas a
dose-dependent inhibition of rat C3 binding was also observed in this
in vitro model. As in the experiments with aggregated IgG, the
inhibition of C3 binding was better for IVIgM as compared with IVIgG.
As in the rat experiments, the dose of ER4G (1 µg/mL) that was used
in this assay was titrated to be nonsaturating and the highest IVIG
concentration (10 mg/mL) was equal to or higher than the maximum serum
levels for human Ig that were achieved in vivo (8 mg/mL for human IgM
and 2 mg/mL for IgG). Besides proving in vitro that binding of ER4G to
RMC was not affected by IVIG, these results also indicate that human IVIG has a similar effect on rat complement activation in vitro as it
has on the activation of human complement. A potent inhibition of rat
complement activation by the different human IVIG preparations, best
again for IVIgM, was also observed in an assay for total complement
activity (Autokit CH50, Wako Pure Chemical Industries, Osaka, Japan;
results not shown).
One important reason for the clinical use of IVIG today is its
antibacterial activity. The observation of IVIG-mediated complement inhibition, therefore, prompted investigations into the influence of
IVIG on complement-bacteria interactions. It was recently shown by
Wagner et al20 that IVIgG did not inhibit complement
deposition on a number of different bacterial strains. Granulocyte
phagocytosis is a major physiologic consequence of bacterial
opsonization by Ig and complement; therefore, we decided to measure the
impact of our IVIG preparations on in vitro phagocytosis of E coli
K12 by human granulocytes. In contrast to heat inactivation of the used plasma, addition of IVIG at 25 mg/mL had only a minimal effect on
phagocytosis and no difference between IVIgG and the IgM-enriched preparations could be observed (Table 4). Although more experiments in
this direction with different types of bacteria and phagocytes of
different healthy donors and patients will have to be performed, we
judge these results as preliminary evidence that IgM enrichment of IVIG
will not negatively influence phagocytosis of bacteria by human granulocytes.
Today, in fact, IgM enrichment of IVIG is mainly looked at
as an improvement of its antibacterial activity, primarily because of
the opsonizing effect of IgM.21 Clinical studies with bone marrow transplant recipients22,23 and also experience with intensive care patients24-27 have proven the safety and
efficacy of the IgM-enriched preparation Pentaglobin. In view of our
results and the ones of others,10, 28 we can think of an
extension of the indications of IgM-enriched IVIG, especially of
preparations with a high concentration (50%) of IgM. The use of such
an IVIgM might be beneficial in many clinical situations in which the
blocking of complement activation is crucial, ranging from autoimmunity
to vascular allograft or xenograft rejection. One could speculate that
in the future IgM-enriched preparations will be used as a
complementation of other antiinflammatory treatments.
| |
ACKNOWLEDGMENT |
We thank Daniëlle van Gijlswijk-Jansen and Ria Faber-Krol
(Leiden) and Katja Dzelalija (Bern) for their excellent technical assistance. The continuing support of this research project by Dr Paul
J. Mohacsi is gratefully acknowledged, and we also thank Prof Urs E. Nydegger for valuable discussions.
| |
FOOTNOTES |
Submitted May 8, 1998; accepted September 23, 1998.
Supported by Grant No. 823A-040153 from the Swiss National Science
Foundation (to R.R.) and Biotest Pharma GmbH, Dreieich, Germany. M.R.D.
and R.R. are partners in the EU Biotechnology Project on
Xenotransplantation No. BIO4-CT97-2242/Swiss Federal Office for
Education and Science No. 97.0369.
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 Robert Rieben, PhD, Department of
Cardiology, University Hospital, CH-3010 Bern, Switzerland; e-mail:
rieben{at}webshuttle.ch.
| |
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Abstract
Introduction