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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2054-2061
Antibody to Granulocyte-Macrophage Colony-Stimulating Factor Is a
Dominant Anti-Cytokine Activity in Human IgG Preparations
By
Morten Svenson,
Morten Bagge Hansen,
Christian Ross,
Marcus Diamant,
Klaus Rieneck,
Henrik Nielsen, and
Klaus Bendtzen
From Lab Med Immunol, and Lab Clin IFN research, Institute for
Inflammation Research, and Dept Clin Immunol, Rigshospitalet University
Hospital, Copenhagen, Denmark.
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ABSTRACT |
Pharmaceutical preparations of normal human immunoglobulin (IgG) are
known to contain high-avidity and neutralizing antibodies (Ab) to the
cytokines interleukin (IL)-1 , IL-6, and interferon (IFN). To test
for other cytokine Ab, 23 batches of IgG were tested for saturable
binding to eight 125I-labeled recombinant
cytokines. All batches bound granulocyte-macrophage colony-stimulating
factor (GM-CSF) with high avidity (Kav  10 pmol/L) and capacities
of up to 5 µmol GM-CSF/mol IgG. Only 1 of 15 batches bound IL-5, also
with high avidity, whereas 13 of 15 batches bound to IL-10 but with
lower capacities and avidities. None of the IgG preparations bound IL-1
receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, or G-CSF. Cross-binding
and absorption analyses revealed identical or slightly stronger binding
of recombinant GM-CSF, IL-5, and IL-10 than their native counterparts.
GM-CSF-IgG complexes did not bind to cellular GM-CSF receptors, but
Fc-dependent binding occurred to blood polymorphonuclear cells.
Increased binding of GM-CSF to patient sera correlated positively with
the binding capacities of infused IgG preparations. Patient and normal
sera did not interfere with the binding of Ab to GM-CSF. From these and
previous experiments, we conclude that pools of normal human IgG
contain variable amounts of specific and high-avidity Ab to some
cytokines, and that Ab to GM-CSF constitute a dominant anti-cytokine activity in these preparations. These Ab are available for reaction in vivo following IgG therapy.
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INTRODUCTION |
NORMAL HUMAN immunoglobulin suitable for
intravenous use (IgG) are used for treatment of an increasing number of
infectious and immunoinflammatory diseases.1 These
preparations usually contain IgG from several thousand individuals and
hence a broad range of binding specificities and idiotypes. The
mechanisms by which pooled IgG influence immunoinflammatory reactions
are incompletely understood but may include antigen neutralization,
Fc-receptor blockade, attenuation of complement activation,
anti-idiotypic interactions, and binding to other molecules involved in
immunoinflammatory processes.2
Cytokines constitute a large group of signal peptides produced during
infections and other immunoinflammatory conditions. Several reports
have described the presence of antibodies (Ab) against cytokines in
patients and in healthy individuals.3,4 Pharmaceutically
prepared IgG from normal donors has also been shown to contain specific
and high-avidity Ab against the cytokines interleukin (IL)-1, IL-6,
and interferon (IFN), and these Ab block receptor binding of the
respective cytokines.5-7 Human natural Ab to IL-1 have
recently been cloned and expressed, and their high-affinity binding has
been confirmed.8
In the present study, we tested for binding of granulocyte-macrophage
colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), IL-2, IL-3, IL-4,
IL-5, and IL-10 to several batches of pooled human IgG from three
manufacturers. High-affinity Ab against GM-CSF were present in all
batches, whereas Ab against IL-5 and IL-10 appeared less frequently
and/or with lower activity. The other cytokines bound in a
nonsaturable manner. The anti-GM-CSF Ab blocked receptor binding of
GM-CSF by simple competition. These observations strengthen the notion
that anti-cytokine Ab contained in pharmaceutically prepared human IgG
may contribute to the immunomodulatory activities of these
preparations.
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MATERIALS AND METHODS |
Sera, Plasma, and Human IgG Preparations
Sera and citrate plasma samples obtained from healthy adults were from
the Blood Bank (Rigshospitalet, Copenhagen, Denmark). Serum samples
from two patients with systemic lupus erythematosus and mixed
connective tissue disease were obtained immediately before and after 3 days of infusions with pooled IgG. Informed consent was received from
the patients.
Preparations of normal human IgG, pharmaceutically prepared for
intravenous use, were as follows: 9 batches of Sandoglobulin (Sandoz,
Basel, Switzerland), 13 batches of Gammagard (Baxter, Allerød,
Denmark), and 5 batches of Nordimmun (Novo Nordisk, Bagsværd, Denmark). Sandoglobulin and Gammagard were from plasma pools of over
8,000 Swiss and North American donors, respectively. Both were produced
by alcohol precipitation, and the IgG of Sandoglobulin was further
treated at pH 4 with traces of pepsin. Nordimmun was prepared by
polyethylene glycol precipitation of a plasma pool of at least 2,000 Danish donors and contains no chemically or enzymatically modified
immunoglobulins. The IgG contents of the stock solutions of
pharmaceutic IgG and of patients sera were determined by turbidimetry
using rabbit Ab to human IgG, code Q 331 (Dako, Copenhagen, Denmark)
and HOO-03 reference serum from Janssen Biochemical (Beerse, Belgium).
Cytokines, Soluble Receptors and Ab
The nonglycosylated human cytokines were all expressed in
Escherichia coli.
The following were used: IL-1 (Dainippon, Osaka,
Japan), IL-1ra (Upjohn, Kalamazoo, MI), IL-2 (Kendall A. Smith, Cornell Medical College, New York, NY), IL-3 (Genzyme/Bie & Berntsen, Rødovre,
Denmark), IL-4 (DNAX, Palo Alto, CA), IL-5 (Genzyme), IL-6 (Sandoz),
IL-10 (Schering-Plough, Kennilworth, NJ), GM-CSF (PeproTech/TriChem,
Virum, Denmark, and Sandoz/Schering-Plough, Copenhagen, Denmark
[Leucomax]), G-CSF (Hoffmann La-Roche, Basel, Switzerland
[Neupogen]), and IFN2A (Hoffmann La-Roche [Roceron-A]).
Glycosylated human recombinant cytokines.
IL-5 expressed in T ni cells (Pharmingen, San
Diego, CA), or in Sf-21 insect cells (R&D Systems/TriChem), or
in Sw 25 murine myeloma cells (NIBSC, Potters Bar, Hertfordshire,
England), and GM-CSF expressed in CHO cells (Sandoz/Schering-Plough),
or in yeast (Genzyme).
Native human cytokines.
IL-5 and GM-CSF, generated in vitro by Ficoll-Hypaque (Nycomed,
Oslo, Norway)-isolated blood mononuclear cells (MNC) stimulated over 4 days with 5 µg/mL of phytohemagglutinin (PHA)-P (Difco, Detroit, MI).
IL-10 was generated by MNC challenged over 2 days with PHA-P plus 1 µg/mL of E coli endotoxin Westphal method 055:B5 (Difco).
Human soluble cytokine receptors.
The IL-1 receptor type I (IL-1RI) and the IL-4 receptor (IL-4R) were
both expressed in COS cells (Immunex Corp, Seattle, WA).
Ab to human cytokines and GM-CSF receptor.
Neutralizing and monospecific Ab against E coli-derived
recombinant IL-2, IL-3, and IL-4 were generated by immunizing rabbits with the purified cytokines, similarly as described.9
Monoclonal Ab (MoAb) against IL-10 (MoAb 9D7 and MoAb 12G8) were from
Schering-Plough. A blocking MoAb to the -chain of the human GM-CSF
receptor (GM-CSF-R) was from Genzyme (code 80-2977-01).
Radiolabeled Cytokines
All recombinant cytokines labeled with 125I were expressed
in E coli, except IL-5, which was expressed in Sf-21
cells. Radiolabeled IL-2 (code IM 227), IL-3 (code IM 220), IL-4 (code
IM 242), IL-5 (code IM 265), GM-CSF (code IM 224), and G-CSF (code IM
262) were kindly donated by Amersham (Birkerød, Denmark). IL-1ra was
radioiodinated using Bolton Hunter reagent, and IL-10 and GM-CSF were
radioiodinated using chloramine-T, as previously
described.5,10
All radioiodinated cytokines were separated by molecular size
chromatography on Sephadex G-75 superfine columns (Pharmacia, Uppsala,
Sweden) and refractionated if necessary to obtain higher purity of the
tracer, see below. RPMI 1640 or 0.02 mol/L phosphate buffer, 0.125 mol/L NaCl, pH 7.4 (PBS), both containing 0.5% to 1% (vol/vol) bovine
serum albumin (BSA)(Sigma, St Louis, MO), were optimal for purification
of 125I-labeled IL-2, IL-10, and G-CSF. When purifying the
other radiolabeled cytokines, BSA was substituted by 0.025% to 0.05%
(vol/vol) gelatin type A: from porcine skin (Sigma) plus 0.1%
(vol/vol) Triton X-100 (Sigma) without influence on the chromatographic
behavior or binding activities of the cytokines.
Validation of radiolabeled cytokines.
The 125I-labeled cytokines were validated by their ability
to bind to specific Ab and/or to soluble and cellular
receptors. Briefly, monomeric 125I-GM-CSF obtained from
Amersham and E coli-derived GM-CSF labeled in our laboratory
both expressed binding capacities >90% and specific activities of 1 to 2 × 105 cpm/ng when tested with specific Ab contained
in pharmaceutic IgG. Specific binding of monomeric
125I-G-CSF to human polymorphonuclear granulocytes (PMN)
showed a binding capacity >50% with specific activity 0.7 to 2 × 105 cpm/ng. Monomeric 125I-IL-1ra expressed the
highest binding capacity >90%, to soluble IL-1RI with specific
activity 1 to 2 × 105 cpm/ng. Maximal binding capacity,
80% to 90%, was obtained for monomeric 125I-IL-2 to a
rabbit Ab with specific activity 0.3 to 1 × 105 cpm/ng.
Monomeric 125I-IL-3 expressed binding capacity >90% to
rabbit Ab and specific activity 0.4 to 0.8 × 105 cpm/ng.
Highest binding capacity, 80% to 90%, was obtained with monomeric
125I-IL-4 to IL-4R and rabbit Ab with specific activities 1 to 3 × 105 cpm/ng. Dimeric 125I-IL-5 had a
binding capacity >90% to Ab identified in IgG and specific activity
3 to 5 × 104 cpm/ng. Dimeric 125I-IL-10
reacted stronger than monomeric 125I-IL-10 with MoAb 9D7
and MoAb 12G8, binding capacity >90% (specific activity 0.5 to
1 × 105 cpm/ng).
Chromatographic Experiments
All separations were carried out at 4°C. Molecular size
chromatography was performed in 0.9 × 32 cm columns containing
Sephadex G-75 superfine or Sephacryl 300 HR (Pharmacia). Samples, 300 µL, were separated at a flow rate of 2.6 mL/h with collection of
fractions every 9 minutes. The columns were calibrated with a mixture
of molecular weight markers (Bio-Rad, Richmond, CA) in PBS. Elution buffers were RPMI 1640 with 0.15% BSA (natural cytokines) or PBS with
0.025% gelatin and 0.1% Triton X-100 (recombinant cytokines). Columns
of 0.9 × 10 cm containing Sephadex G-75 superfine were used for
purification of the 125I-labeled cytokines and for binding
assays. As running buffers RPMI with 0.5% BSA or PBS with 0.05%
gelatin and 0.1% Triton X-100 were used, depending on the tracer (see
above).
Affinity chromatography for determination of 125I-cytokine
bound to IgG was carried out in columns containing 0.5 mL protein G or
protein A Sepharose (Pharmacia). A maximum of 300 µL was applied
followed by 100 µL washing buffer and 15 minutes of incubation. The
columns were then washed with 3 mL PBS with 0.5% BSA, and the bound
material was eluted with 3 mL 0.1 mol/L glycine, pH 2.4.
Binding of Cytokines to IgG and Serum
If not otherwise stated, the samples were incubated at 4°C for 18 hours before evaluating the amounts of bound (B) and free (F)
125I-cytokine by chromatographic separations, as described
above. Nonspecific binding was assessed in the presence of 200 to 1,000 ng/mL of unlabeled cytokine. For screening purposes, 0.1 to 0.2 ng/mL
of 125I-labeled cytokine was used.
Saturation binding analyses of the individual cytokines to different
batches of IgG were performed as described.4 The results were expressed as Kav in pmol/L and the binding capacity as bound cytokine in µmol/mol IgG, calculated from molecular weights of 14 kD
for GM-CSF, 26 kD for IL-5, 38 kD for IL-10, and 150 kD for IgG.
Recovery analyses.
Mixtures of equal amounts of two to three batches of IgG, along with
the individual batches, were incubated at 37°C or 4°C for 18 hours.
Serial two-fold dilutions from a total of 4 mg IgG/mL were made in
duplicates and added 3,000 cpm/100 µL of 125I-GM-CSF
followed by incubation and detection of bound tracer by protein G
affinity chromatography. The binding activities of the samples were
expressed relative to the IgG batch expressing the highest binding.
Recovered binding was calculated as:
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{M}A similar procedure was followed for detection of recovered
IL-5 binding to mixtures of batches of IgG.
Binding of GM-CSF to sera was assessed by molecular size
chromatography. Duplicates of 100 µL containing 66 µL serum, 3,000 cpm of 125I-GM-CSF with or without excess unlabeled E
coli GM-CSF were tested. Binding of GM-CSF to Ab added to the
individual sera was measured by protein G affinity chromatography. The
test was performed as above but with added IgG capable of 30% and 70%
specific binding of the 125I-GM-CSF, respectively. Ten sera
were analyzed with two batches of IgG. In addition, five sera were
tested by preincubation of 80% of the single serum with IgG for 18 hours at 37°C followed by 1.4 times dilution and coincubation with
125I-GM-CSF and unlabeled GM-CSF as above.
In a plasma pool of equal volumes of the individual plasma samples, the
recovered anti-GM-CSF IgG binding activity was analyzed by the
principles described above.
Estimation of recovered anti-GM-CSF Ab in sera from patients treated
with IgG were carried out in the following way. Serial two-fold
dilutions of serum drawn before or after IgG therapy, along with a
preparation of the infused IgG, were added to a constant amount of
125I-GM-CSF. The binding activities of the sera were
expressed relative to the activity of the infused IgG with the
dimension mgeq/mL. The influence of sera collected before and after IgG
infusion on the activity of the corresponding IgG batch was tested by
addition of serum (60% final concentration) to 20 mg/mL, 15 mg/mL, and 10 mg/mL of the infused IgG, followed by serial dilutions and addition
of tracer plus/minus unlabeled GM-CSF.
Papain and Pepsin Treatment of IgG
Papain-agarose (Sigma), 20 mg, was preincubated at 37°C in PBS,
containing 10 mmol/L L-cysteine and 2 mmol/L EDTA. After 2 hours, the
agarose was washed and incubated for 18 hours at 37°C with 20 mg IgG
in 1 mL PBS, 2 mmol/L EDTA. The supernatants were stored at 4°C until
use.
Pepsin digestion of IgG was carried out by incubating 10 mg IgG in 1 mL
0.2 mmol/L acetate buffer, pH 4.1, with 0.2 mg/mL of pepsin (Sigma) at
37°C for 20 hours. The sample was then dialyzed against PBS and
applied on protein A columns using 0.5 mL dialyzed material/mL protein
A-Sepharose and PBS as washing buffer. The unabsorbed material was
placed in a dialysis bag and concentrated on powdered polyethylene
glycol 20,000 (Merck, Darmstadt, Germany) and stored at 4°C.
ELISA and RIA of Cytokines
Duplicates were made of serial two-fold dilutions of the reference
cytokines and at least three consecutive dilutions of the samples.
Parallel runs of the curves for the native and the reference recombinant cytokines were always obtained in semi log plots.
ELISAs for human GM-CSF (DGM00) and IL-5 (D5000) were from R&D; their
detection limits were 5 to 10 pg/mL. IL-10 was measured by double
sandwich ELISA using monospecific polyclonal rabbit Ab to purified
recombinant human IL-10, as described.9 The assay was
calibrated with an IL-10 international standard (NIBSC). The inter- and
intraassay coefficients of variation for the concentration range
between 30 pg/mL and 2 ng/mL were <15%, and the sensitivity limit
was 30 pg/mL.
RIAs for GM-CSF, IL-5, and IL-10 were carried out with the specific Ab
contained in pooled human IgG and 0.1 to 0.2 ng/mL of the respective
125I-labeled cytokine. The assays were performed as
described above for the determination of cytokine binding to IgG. The
intra- and interassay variations were <10% and the sensitivity
limits 50 to 100 pg/mL.
As a supplement to the RIA measurements, the same Ab were also used for
absorption of the native and reference cytokines. The relative content
of a native cytokine was estimated from a plot of the amount of free
cytokine (F) divided by the total amount of the cytokine (T) as a
function of T (F/T versus T). Generally, the two assays are
complementary in that the same relative value for the sample will be
estimated by the tests only when the test ligand and the reference
ligand show identical binding. If the reference ligand binds stronger,
the RIA will underestimate the cytokine level, whereas the F/T versus T
analysis will overestimate the concentration.
In the F/T versus T assay, the following procedure was used for
quantitation of GM-CSF, IL-5, and IL-10: 200 µL samples of preincubated constant amount of Ab with variable concentrations of MNC
supernatant or reference cytokine were applied on columns containing
0.5 mL protein G Sepharose. Identical samples, but without Ab, were run
in parallel. Two × 100 µL washing buffer were added at intervals of
8 minutes, unabsorbed material was washed out in 1.6 mL of buffer, and
the eluted cytokine was quantitated by ELISA. From use of
125I-labeled cytokines alone, 0.98 ± 0.02 (N = 20) were
recovered in the fraction of unabsorbed material.
Cell Receptor Assays
A human promyelocytic cell line, HL-60 (ATCC, Rockville, MD), was grown
in RPMI 1640 with 20% (vol/vol) heat-inactivated fetal calf serum
(FCS). Human blood PMN were purified as described11 and
resuspended in RPMI 1640 with 1% (vol/vol) BSA, 8 mmol/L EDTA, 0.1%
NaN3. Receptor assays were made over 18 hours at 4°C in
duplicate samples of 250 µL containing 0.7 to 1.1 × 107
cells/mL, 125I-labeled cytokine, variable concentrations of
human IgG, F(ab )2 fragments of IgG, with or without
unlabeled cytokine (200-1,000 ng/mL) or MoAb to GM-CSF-R (5-10 µg/mL). Cell-bound cytokine was separated by centrifugation of 200 µL of the cell suspension on dibutyl phthalate and bis
(2-ethylhexyl)phthalate (ratio 1:1) (Merck). One hundred microliters of
the supernatant were aspirated for evaluation of free and complexed
tracer (cpmfree and cpmbound) by molecular size
chromatography. Cell-bound cytokine was measured in the pellet after
cutting the tube. The activities were counted with errors below 2%.
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RESULTS |
Screening for Specific IgG to Human Cytokines
There was no saturable binding of 125I-labeled E
coli-derived IL-1ra, IL-2, IL-3, IL-4, or G-CSF to 10 mg/mL of at
least 15 different batches of IgG from three manufacturers. In
contrast, GM-CSF, IL-5, and IL-10 bound at variable degrees to
individual batches of IgG (Fig 1 and Table
1). Binding of IL-5 was only detected in 1 of 15 IgG preparations.
Papain or pepsin treatment of IgG showed that the binding of GM-CSF,
IL-5, and IL-10 occurred predominantly or exclusively to the Fab part
of IgG, because cytokines bound to intact IgG but not to enzymatically
treated IgG were retained on protein A columns.
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| Fig 1.
Saturable binding of GM-CSF, IL-5, and IL-10 to IgG. Five
to 9 batches of Gammagard (G), Sandoglobulin (S), and Nordimmun (N)
were tested in parallel at 4 mg/mL IgG and 0.1 to 0.2 ng/mL 125I-cytokines: E coli-derived GM-CSF and IL-10,
and Sf-21 cell-derived IL-5. The results are shown as median
percentage (ranges) of displaceable binding divided by the total amount
of 125I-cytokine in the assay. The background binding was
<3%.
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Binding Avidities and Capacities
As shown in Table 1, all IgG batches bound
GM-CSF with high activities (Kav 10 pmol/L) but with variable
binding capacities. IL-5 bound with similar avidity to the single
positive IgG preparation, and IL-10 bound with over ten times lower
avidities. The highest binding capacities, 2 to 5 µmol/mol IgG, were
obtained with GM-CSF, the lowest with IL-10.
It is unlikely that the IgG preparations contained molecules that
interfered with the cytokine Ab. Thus, when mixing low activity batches, or low and high activity batches, GM-CSF bound as the sum of
activities contributed by the individual IgG preparations. There was
also no effect on the binding of IL-5 when mixing the positive batch
with either of seven negative ones. Taken together, this suggests that
individual batches of IgG contained highly variable amounts of cytokine
Ab.
Binding Specificity
Using 1,000 to 10,000 times excess amounts of unlabeled cytokines,
there was no cross-binding to IgG between the eight cytokines tested,
and IL-1, IL-6, or IFN2A. Hence, IgG bound specifically to
GM-CSF, IL-5, and IL-10, respectively.
Effect of cytokine glycosylation.
Since nonglycosylated and variously glycosylated recombinant GM-CSF and
IL-5 competed differently for their respective Ab (Table
2), we investigated the binding of native
cytokines in supernatants of in vitro stimulated human MNC. Complete
suppression of binding to 125I-labeled recombinant
cytokines was obtained with supernatants containing RIA reactivity for
GM-CSF at 35 kD and IL-5 at 50 kD (in agreement with the reported sizes
of the native, glycosylated cytokines12,13).
We also quantitated the supernatant contents of native GM-CSF and IL-5
using the same IgG batch for RIA and for absorption of both native,
recombinant, and reference recombinant cytokines (Fig 2 and Table
2). Native GM-CSF tested with only 40%
higher values in the F/T versus T assay compared with those in RIA
(Table 2), and the relative binding of native and E coli GM-CSF
(the reference preparation) was independent of the IgG preparation used. The table also shows similar binding of native IL-5 and recombinant IL-5 expressed by the insect cell line Sf-21 (the reference preparation), but not by T ni cells or E
coli-derived IL-5. Absorption experiments with 20 mg/mL IgG and 3 ng/mL of native and E coli IL-10 (the reference preparation)
showed <30% binding even to maximum capacity IgG batches (binding of
0.6 to 2 ng IL-10/20 mg IgG, N = 3). Because of the sensitivity
limit, it was not possible to analyze lower concentrations of IL-10. Since the reference preparations bound identically or only slightly stronger than the native cytokines, it is likely that the results shown
in Fig 1 and Table 1 are valid approximations of the binding characteristics of IgG to native GM-CSF, IL-5, and IL-10.
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| Fig 2.
Binding of native and recombinant GM-CSF to IgG. F/T
versus T plots obtained by absorption to IgG (2 mg/mL) of native ( ) and E coli-derived ( ) GM-CSF. Total and free GM-CSF were
quantitated by ELISA. Results are shown as means of duplicates.
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Characterization of GM-CSF Binding to IgG
Because of their prevalence and strong binding, the Ab to GM-CSF were
further characterized.
GM-CSF-IgG complex formation and binding stability.
Complexes with molecular weights  600 kD were formed when
125I-GM-CSF reacted with increasing concentrations of IgG
(Fig 3). These complexes were highly
stable. Using IgG levels that bound approximately 40 pmol/L GM-CSF,
more than 85% of bound 125I-GM-CSF was retained after 8 hours at 37°C in presence of excess unlabeled cytokine. Similar
results were obtained at 4°C.
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| Fig 3.
Sephacryl S-300 HR molecular size elution profile of
125I-GM-CSF bound at variable IgG concentrations.
125I-GM-CSF, 0.25 ng/mL, was incubated at 37°C for 18 hours with 10 mg/mL ( ), 5 mg/mL (---),
and 0.5 mg/mL (-------) of the same IgG batch. Similar findings were
obtained with IgG from all three manufacturers.
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Inhibition of binding to GM-CSF-R+ cells by IgG.
In contrast to what was seen using the human cell line HL-60, variable
amounts of 125I-GM-CSF associated to PMN when incubated
with different IgG batches, but with constant levels of
125I-GM-CSF and anti-GM-CSF-R MoAb. This variation
disappeared if F(ab)2 fragments of IgG were used (Table
3). Consequently, there was a significant
binding of GM-CSF-IgG complexes to PMN and this binding depended on
the Fc part of the IgG molecules (possibly to Fc -RII and Fc-RIII
on PMN14,15).
To avoid misinterpretation of IgG-induced interference with GM-CSF-R
binding, background binding to PMN was assessed in the presence of
excess anti-GM-CSF-R MoAb. Now, IgG suppressed the binding of
125I-GM-CSF to both HL-60 cells and human PMN in a dose
dependent manner, and complete blockade could be achieved with the IgG
preparations (data not shown).
To further evaluate the interaction of IgG with cellular binding of
GM-CSF, the amounts of cytokine bound to GM-CSF-R were plotted as a
function of the free cytokine concentrations in the absence or presence
of 15 different batches of IgG. As shown in Fig
4, simple binding competition was observed
with different IgG preparations, because the amount of specifically
bound 125I-GM-CSF depended solely on the free concentration
of the cytokine.
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| Fig 4.
Effect of IgG on the binding of GM-CSF to GM-CSF-R. Data
are expressed as specific 125I-GM-CSF bound to cells versus
free cytokine in the absence of IgG (X), or presence of Gammagard
(), Sandoglobulin ( ), or Nordimmun ( ). Five different batches
of IgG from each manufacturer were tested in parallel. HL-60 cells were
incubated with 3,900 cpm/200 µL and 1.25 mg/mL of IgG, or with
decreasing concentrations of the tracer alone. PMN were treated as the
HL-60 cells, except that they were coincubated with 4 mg/mL of IgG at
2,900 cpm/200 µL. The background binding was assessed with excess Ab
to GM-CSF-R. Data are means of duplicates and representatives of two to
five experiments.
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Low prevalence of anti-GM-CSF IgG in healthy individuals.
Less than 15% of total 125I-GM-CSF bound in a nonsaturable
manner to sera of 50 healthy individuals. Because of recoveries
exceeding 95% in individual sera (N = 15), there was no interference
with the GM-CSF binding activity of IgG. Consequently, 1,258 plasma samples were tested for GM-CSF binding to IgG; 4 were positive. The
Kavs were from 11 to 70 pmol/L and the Bmax values were from 5 to 320 µmol GM-CSF/mol IgG. The anti-GM-CSF activities of these plasma
samples were fully recovered in the total plasma pool.
Recovery of infused anti-GM-CSF IgG in vivo.
We finally investigated Ab to GM-CSF in sera of two patients, who over
several years received monthly injections of IgG at high dosage. As
shown in Fig 5, IgG infusion increased the
binding of 125I-GM-CSF to serum. Furthermore, serum samples
did not interfere with 125I-GM-CSF binding to the infused
IgG. To estimate the in vivo recovery, we calculated the
increase in total serum IgG following infusion. Similar results were
obtained when calculated on the basis of specific Ab to GM-CSF
(14 ± 3 mg/mL, N = 6) and total IgG (13 ± 2 mg/mL). These
data strongly suggest that the infused Ab to GM-CSF were fully
available and functional in vivo.
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| Fig 5.
GM-CSF binding to serum of patients treated with IgG. Two
patients with systemic lupus erythematosus and mixed connective tissue
disease, respectively, received 60 g of either Gammagard or
Sandoglobulin over 3 days at intervals of 1 month. Sera were collected
immediately before and after three consecutive series of infusions.
Data are means of duplicates and expressed as percent saturable binding
of 125I-GM-CSF to IgG relative to the total amount of
tracer using 4% (vol/vol) serum and 2,500 cpm/100 µL of
125I-GM-CSF.
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DISCUSSION |
The occurrence of IgG Ab against certain cytokines in healthy and
diseased individuals is increasingly realized.4,16-22
Natural Ab have been reported against recombinant human IL-1 , IL-2,
IL-8 and tumor necrosis factor (TNF) by some23-30 but
not by others.4,18,31 Besides differences in assay
sensitivities, the discrepancies in detecting cytokine Ab may be
related to the different assay methods. Saturation binding analysis is
generally the preferred method of testing these Ab, and the binding
must be shown to occur exclusively to the Fab part of the
Ab.4,32 These criteria are fulfilled in previous studies of
IL-1, IL-6, and IFN binding to human IgG,5-7,33 and
they were used in the present study for the detection of anti-cytokine
Ab in serum or plasma samples, and in pooled human IgG.
Specific Ab against GM-CSF, IL-5, and IL-10 were detected in pooled
human IgG, and none were found against five other cytokines, including
IL-2. In agreement with our findings of Ab to GM-CSF in 4 of 1,258 plasma samples (this study) and none against IL-10 in 50 sera,4 other studies have shown less than 2% of sera weakly positive for anti-GM-CSF Ab or anti-IL-10 Ab.16,20
In contrast, IgG Ab directed against IL-1 and IL-6 have been
reported in up to 30% and 20% of healthy individuals,
respectively.4
Despite a much less frequent occurrence in sera of healthy individuals,
the avidities of anti-GM-CSF Ab in pharmaceutically prepared IgG were
in the same range as those reported for IL-1 Ab; the binding
capacities were generally one to five times higher.5 This
observation makes the Ab binding of GM-CSF the dominant cytokine binding activity presently identified in pooled IgG.5,7
Higher activities of anti-IFN Ab than those detected in individual
sera have also been observed in human IgG.7 Simple addition
of Ab activities contributed by individual donors to a pool of IgG may not be found in situations where increased clonality of an Ab specificity or interference with anti-idiotypic Ab has
developed.3 There is, however, no evidence of
anti-idiotypic Ab blocking the activity of any anti-cytokine Ab. In
this study, for example, none of several individual sera or IgG
preparations interfered with the binding of GM-CSF to IgG. Ab to
GM-CSF, administered to patients during high-dose IgG therapy, were
also fully recovered and available for reaction, in agreement with
similar observations for Ab to IL-1, IL-6, and IFN infused with
pooled IgG.34 Finally, a similar degree of polyclonality
was detected by molecular size chromatography of GM-CSF-IgG complexes
from different IgG batches containing variable amounts of these Ab.
We found 0.3% of individual plasma samples positive for anti-GM-CSF
Ab, but these samples had binding capacities up to 100 times higher
than, and avidities similar to, the ones usually detected in the IgG
preparations. In a pool of nearly 1,000 plasma samples, the
anti-GM-CSF Ab activity was the sum of the activities contributed by
these few positive specimens. Hence, the anti-GM-CSF Ab activities in
pharmaceutical IgG preparations are most likely contributed by only a
few highly positive donors. This would explain the variable levels of
anti-GM-CSF Ab activities in different preparations of pooled normal
IgG; differences between the donor populations and manufacturing
procedures might contribute to this.
Recombinant proteins may differ from their native counterparts in their
binding characteristics.10,35,36 The naturally occurring Ab
identified with 125I-labeled recombinant cytokines in the
present and previous studies have all been shown to cross-bind the
corresponding native cytokine.6,7,37 To evaluate the
relative binding to Ab of recombinant and native cytokine, we compared
the results obtained by RIA with those from absorption experiments
using the same Ab. This showed that IgG bound similarly to native
(glycosylated) and to nonglycosylated GM-CSF. On the other hand, IgG
bound in a similar fashion to native (glycosylated) and to glycosylated
recombinant IL-5, but not to nonglycosylated IL-5.
Taken together, the data show that native GM-CSF binds to IgG with high
avidity (Kav 10 pmol/L) and with capacities of 2 to 5 µmol/mol
IgG. Hence, IgG at 10 mg/mL would have the potential to bind >90% of
GM-CSF, if present at concentrations up to 2 ng/mL (calculated
according to Svenson et al5). Native IL-10 cannot be
expected to bind significantly to IgG, whereas strong binding of IL-5
may occur in sporadic IgG preparations.
It is uncertain from previous studies whether anti-GM-CSF Ab induced
by treatment of patients with E coli- or yeast-expressed GM-CSF
react with endogenous GM-CSF.14,36,38 In contrast, our
study demonstrates binding of native GM-CSF to anti-GM-CSF Ab in
pooled normal IgG. Any differences in this regard between therapy-induced and naturally occurring anti-GM-CSF Ab would have to
be resolved by binding and cross-binding analyses.
An agonistic effect in vivo of cytokine Ab has been
proposed.39 This has been underlined by experience with in
vitro neutralizing MoAb to cytokines, which have shown both inhibition
and enhancement of cytokine activities in vivo.40 The
agonistic effect of in vitro neutralizing MoAb has been explained by a
decreased clearance of the cytokine if present in monomeric immune
complexes, combined with low concentrations of the MoAb that favor
redistribution of the cytokine from the immune complexes to cellular
cytokine receptors. A carrier function of anti-GM-CSF Ab in pooled IgG is considered unlikely, however, because of their high avidity, stability, and capacity to form large immune complexes with GM-CSF and
block binding to GM-CSF receptors.
GM-CSF stimulates the formation and function of PMN and
monocytes.41 Maturation of monocytes to macrophages and
dendritic cells and MHC class II expression are augmented by GM-CSF,
and it is a strong adjuvant during immunizations with weak immunogens such as tumor cells and soluble proteins and peptides.41-45
GM-CSF is induced by and potentiates the release of cytokines such as IL-1 and TNF, both known to be centrally involved in acute and chronic inflammatory reactions.19,41 Indeed, treatment with Ab to GM-CSF increases the survival of endotoxin-challenged mice and,
in combination with Ab to IL-3, mice with experimental cerebral malaria.46,47 Therefore, Ab to GM-CSF in pooled human IgG
may be of benefit in the treatment of immunoinflammatory disorders, but
their presence could reduce the efficacy of IgG preparations used in
the prevention of infection. Isolation of different, naturally occurring anti-cytokine Ab and the use of IgG preparations selectively enriched or depleted of Ab to specific cytokines are attractive ways to
further analyze the therapeutic potential of natural Ab to cytokines.
| |
FOOTNOTES |
Submitted August 19, 1997;
accepted October 31, 1997.
Supported by Rigshospitalet University Hospital, the Danish Cancer
Society, the Danish Biotechnology Program, the Danish Rheumatism Association.
Address reprint requests to Klaus Bendtzen, MD, DMSc, IIR 7521, Rigshospitalet, Tagensvej 20, DK-2200 N, Copenhagen, Denmark.
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.
| |
ACKNOWLEDGMENT |
Drs Børge Thing Mortensen (Rigshospitalet, Copenhagen), Kendall A. Smith (Cornell Medical College, New York, NY), Satwant Narula
(Schering-Plough, Kennilworth, NJ), Jan de Vries (DNAX, Palo Alto, CA),
and Steven Gillis (Immunex Corporation, Seattle, WA) are thanked for
generous gifts of cytokines and cytokine receptors. Amersham DK and
Novo-Nordisk kindly donated radiolabeled cytokines and Nordimmun,
respectively. Dr Charlotte Lundsgaard (Copenhagen County Hospital in
Glostrup) helped with the sampling of patient sera, and Susanne
Meldgaard provided excellent technical help.
| |
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[Full Text]
[PDF]
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S. E. Epstein, Y. F. Zhou, and J. Zhu
Infection and Atherosclerosis : Emerging Mechanistic Paradigms
Circulation,
July 27, 1999;
100
(4):
e20 - e28.
[Abstract]
[Full Text]
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D. G. McNeel, K. Schiffman, and M. L. Disis
Immunization With Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor as a Vaccine Adjuvant Elicits Both a Cellular and Humoral Response to Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor
Blood,
April 15, 1999;
93(8):
2653 - 2659.
[Abstract]
[Full Text]
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Abstract
Introduction
Abstract