www.sciencemag.org/cgi/content/full/313/5787/670/DC1

Supporting Online Material for

Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation

Yoshikatsu Kaneko, Falk Nimmerjahn, Jeffrey V. Ravetch*

*To whom correspondence should be addressed. E-mail: ravetch@rockefeller.edu

Published 4 August 2006, Science 313, 670 (2006)

DOI: 10.1126/science.1129594

This PDF file includes: Materials and Methods Figs. S1 to S11 References

Kaneko et al.

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Supporting online material

Materials and methods

Mice

C57BL/6 and NOD mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

FcγRIIB-/- (S1) mice were generated in our laboratory and backcrossed for 12 generations

to the C57BL/6 background. KRN TCR transgenic mice on a C57BL/6 background

(K/B) were gifts from D. Mathis and C. Benoist (Harvard Medical School, Boston, MA)

and were bred to NOD mice to generate K/BxN mice (S2). Female mice at 8-10 weeks of

age were used for all experiments and maintained at the Rockefeller University animal

facility. All experiments were done in compliance with federal laws and institutional

guidelines and have been approved by the Rockefeller University (New York, NY).

Antibodies and soluble Fc receptors

6A6 antibody switch variants were produced by transient transfection of 293T cells

followed by purification via protein G as described (6, 15). Sialic acid rich antibody

variants were isolated from these antibody preparations by lectin affinity chromatography

with Sambucus nigra agglutinin (SNA) agarose (Vector Laboratories, Burlingame, CA).

Enrichment for sialic acid content was verified by lectin blotting (see below). Human

intravenous immune globulin (IVIG, 5% in 10% maltose, chromatography purified) was

purchased from Octapharma (Herndon, VA). Digestion of human IVIG was performed as

described (20). Briefly, IVIG was digested by 0.5 mg/ml papain for 1 hr at 37 oC, and

stopped by the addition of 2.5 mg/ml iodoacetamide. Fab and Fc resulting fragments

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were separated from non-digested IVIG on a HiPrep 26/60 S-200HR column (GE

Healthcare, Piscataway, NJ), followed by purification of Fc and Fab fragments with a

Protein G column (GE Healthcare) and a Protein L column (Pierce, Rockford, IL).

Fragment purity was checked by SDS-PAGE, followed by coomassie brilliant blue

staining and immunoblotting using anti-human IgG Fab or Fc-specific antibodies

(Jackson ImmunoResearch, West Grove, PA). Purity was judged to be greater than 90%.

The F4/80 antibody was from Serotec (Oxford, UK). The Ly 17.2 antibody was from

Caltag (Burlingame, CA). Sheep anti-glomerular basement membrane (GBM) antiserum

(nephrotoxic serum, NTS) was a gift from M. P. Madaio (University of Pennsylvania,

Philadelphia, PA). Soluble Fc receptors containing a C-terminal hexa-hisitidine tag were

generated by transient transfection of 293T cells and purified from cell culture

supernatants with Ni-NTA agarose as suggested by the manufacturer (Qiagen).

Surface plasmon resonance analysis

Surface plasmon resonance analysis was performed as described (6, 15). Briefly, 6A6

antibody variants containing high or low levels of sialic acid residues in their sugar side

chains were immobilized on the surface of CM5 sensor chips. Soluble Fcγ-receptors

were injected at 5 different concentrations through flow cells at room temperature in

HBS-EP running buffer (10mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and

0.005% surfactant P20) at a flow rate of 30µl/min. Soluble Fc-receptors were injected for

3 minutes and dissociation of bound molecules was observed for 7 minutes. Background

binding to control flow cells was subtracted automatically. Control experiments were

performed to exclude mass transport limitations. Affinity constants were derived from

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sensorgram data using simultaneous fitting to the association and dissociation phases and

global fitting to all curves in the set. A 1:1 Langmuir binding model closely fitted the

observed sensorgram data and was used in all experiments.

Immunothrombocytopenia (ITP) model

ITP was induced by intravenous injection of 4 µg of 6A6 antibody variants as described

(6, 15). Platelet counts before and 4 hours after injection were determined by blood

collection (40 µl) from the retro-orbital plexus and measuring platelet counts of a 1:10

dilution in PBS/5%BSA in an Advia 120 haematology system (Bayer).

Serum transfer, arthritis scoring and histology

Serum was prepared as described previously (19). Arthritis was induced by one

intravenous injection of 1.5x diluted K/BxN serum (4 µl of pooled K/BxN serum per

gram of mouse). Arthritis was scored by clinical examination as described (19). Index of

all four paws was added: 0 [unaffected], 1 [swelling of one joint], 2 [swelling of more

than one joint], and 3 [severe swelling of the entire paw]. IVIG, fetuin (Sigma-Aldrich),

or transferrin (Sigma-Aldrich) was injected 1 hr before K/BxN serum injection. For

histological examination, ankle joints were fixed in 10% formalin, decalcified for 48 hr in

Decal solution, and embedded in paraffin. Two micrometer sections were stained with

hematoxylin/eosin.

Measurement of circulating human IgG levels in mice serum

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Ninety-six-well ELISA plates coated with 5 µg/ml of F(ab’)2 goat anti-human IgG Fc

antibody (Serotec) were incubated with diluted test sera (1/1000) after blocking with 5%

bovine serum albumin. After washing with PBS containing 0.05% Tween 20, the plates

were incubated with HRP-conjugated mouse anti-human IgG CH2 domain monoclonal

antibody (Serotec). For the color development, 3,3’,5,5’-tetramethylbenzidine was used.

Human IgG was used as a standard.

In vitro de-glycosylation and de-sialylation of IVIG and Fc

For in vitro de-glycosylation, 100 mg of IVIG in 1 ml of 0.2M sodium phosphate buffer

(pH 8.5) was treated with 25,000 units of Peptide:N-Glycosidase F (PNGaseF) purified

from Flavobacterium meningosepticum (New England BioLabs) at 37 oC for 40 h. For in

vitro de-sialylation, 100 mg of IVIG or Fc fragment of IVIG in 1 ml of 0.05 M sodium

citrate buffer (pH 6.0) was incubated with 700 units of recombinant α2,3/ α2,6

neuraminidase cloned from Clostridium perfringens (New England BioLabs) at 370C for

20h. IVIG or Fc preparations were purified by Protein G affinity chromatography. The

monomeric composition of the de-glycosylated and de-sialylated IVIG or Fc preparations

was confirmed by SDS-PAGE.

Lectin affinity column purification

Agarose-bound SNA lectin (Vector Laboratories) was used for purification of human or

mouse IgG containing sialic acid. Forty milligrams of IVIG or 10-15 mg of the 6A6-

IgG1/IgG2b antibodies in 4 ml of Tris-buffered saline (TBS, pH 7.5) with 0.1 mM CaCl2

were applied to 4ml of SNA column resin. Since the polyclonal IgG in IVIG may also

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contain N-linked glycans on the variable regions of the light and heavy chains that can be

sialylated, we confirmed that the increase in anti-inflammatory activity of the SNA-

enriched IgG preparation resulted from increased sialylation of the N-linked

glycosylation site on the Fc. For this Fc fragments were generated from unfractionated

IVIG first by papain digestion. As for IVIG, 40mg of this purified Fc preparation were

applied to 4ml of SNA column resin. After incubation at room temperature for 10 min,

the SNA column was washed with 8 ml of TBS and the flow-through fraction was

collected and defined as the non SNA-binding fraction. After additional washing with

8ml of TBS, IVIG, Fc fragment of IVIG, or 6A6 antibody isotypes bound to SNA were

eluted in 4 ml of 0.5 M lactose in TBS for 10 min, followed by the same amount of 0.5 M

lactose in 0.2 M acetic acid for another 10 min. The eluted IVIG fraction was purified in

the same way a second time to increase enrichment. After buffer exchange into PBS by

using a PD-10 desalting column (GE Healthcare) this preparation was used as the SNA-

enriched fraction.

Measurement of subclass distribution of IVIG

Subclass distribution of untreated IVIG or SNA-enriched IVIG was determined by

ELISA. Ninety-six-well ELISA plates coated with 10 µg/ml of subclass-specific mouse

anti-human IgG1 (clone HP6001), IgG2 (clone HP6014), IgG3 (clone HP6050) or IgG4

(clone HP6023) antibody were incubated with diluted IVIG after blocking with 5%

bovine serum albumin. After washing with PBS containing 0.05% Tween 20, the plates

were incubated with an HRP-conjugated mouse anti-human IgG CH2 domain

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monoclonal antibody (Serotec). For the color development, 3,3’,5,5’-

tetramethylbenzidine was used.

MALDI-TOF-MS analysis

Monosaccharide composition analysis was performed by UCSD GRTC Glycotechnology

Core Resource (San Diego, CA). Glycoprotein samples were denatured with SDS and 2-

mercaptoethanol, and digested with PNGase F. The released mixed N-glycans were

purified by reversed-phase HPLC and solid-phase extraction, and then all exposed

hydroxyl groups of the N-glycans were permethylated to protect from the loss of sialic

acid (S3 and Fig. S10). The resulting derivatized saccharides were purified again by

reversed-phase HPLC and subjected to MALDI-TOF-MS. Glycan standards were

purchased from QA-Bio (Palm Desert, CA). Note that this method does not allow to

determine the precise arm structure and linkage of the sugar residues.

Lectin blot

The indicated amounts of 6A6 antibody variants, IVIG, or purified serum IgG or IgM

from untreated or NTS treated mice were resolved by SDS-PAGE using polyacrylamide

gel under reduced of non-reduced conditions. Proteins were transferred to a

polyvinylidene difluoride (PVDF) membrane (Milipore), blocked with Western Blocking

Reagent (Roche), followed by incubation with biotinylated SNA lectin (2 µg/ml, Vector

Laboratories) and alkaline phosphatase (AP-conjugated goat anti-biotin antibody

(Sigma). Bound antibody was visualized with 4-nitro blue tetrazolium chloride/5-bromo-

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4-chloro-3-indolyl phosphate (Roche). The relative intensity of bands was quantified

using the NIH Image software. Specificity of SNA lectin blotting was verified by hapten

inhibition with 0.5M N-acetylneuraminic acid (Fig. S11).

Immunoprecipitation

Mouse serum (1ml) was preincubated with 50 µl protein G sepharose (GE Healthcare) for

2 hours at 40C. After removal of the protein G sepharose, 1 µg of anti-transferrin

antibody (Bethyl laboratories) was added to the serum and incubated for 4 hours with

shaking, followed by the addition of fresh protein G beads. After 2 hours the beads

containing immunoprecipitated transferrin were recovered by centrifugation and washed

two times with PBS before SDS PAGE gel analysis.

Platelet Elisa with 6A6 antibody isotypes and glycosylation variants

Platelets were isolated from peripheral blood by several consecutive centrifugation steps.

To collect plasma, cells were pelleted by centrifugation at 300xg for 5 minutes, followed

by three washing steps with PBS (spin down platelets at 1500xg for 15 minutes). Finally,

red blood cells were removed by centrifugation at 100xg for 10 minutes and the platelet

containing supernatant was directly used for coating 96 well Elisa plates (Nunc

Maxisorb). Platelets were coated overnight at 40C in PBS. The next day platelets were

fixed by addition of 2% paraformaldehyde (PFA) for 5 minutes. After washing three

times with PBS, wells were blocked with PBS/1%BSA for 2 hours at room temperature,

followed by incubation with the platelet specific 6A6 antibodies in PBS/1%BSA at the

indicated concentrations for one hour at room temperature. After three more washes

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bound antibodies were detected by addition of a horseradish-peroxidase (HRP) coupled

mouse IgG specific antibody (Bethyl laboratories) and addition of HRP substrate solution

(KPL laboratories). When using SNA-rich antibody preparations we additionally

included a control to evaluate the effect of sialic acid on secondary antibody (anti-mouse

HRP conjugate) binding to sialic acid enriched antibody variants. For this, 6A6

antibodies or 6A6 SNA rich variants were coated directly to Elisa wells followed by

detection with the secondary antibody. The difference that was obtained in this assay was

used to correct the values obtained in the platelet Elisa.

Flow cytometry

Spleen and bone marrow cells were prepared and resuspended in PBS. After red blood

cell lysis, cells were stained with the indicated monoclonal antibodies, and subjected to

flow cytometric analysis.

Anti-GBM glomerulonephritis

Anti-GBM glomerulonephritis was induced as described previously (20). Briefly, mice

were pre-immunized intraperitoneally with 200 µg of sheep IgG (Serotec) in CFA,

followed by intravenous injection of 2.5 µl of NTS serum (S4) per gram body weight four

days later. Blood was collected from non-treated control mice or anti-GBM nephritis

mice 4 days after the anti-GBM antiserum injection, and serum IgG was purified by

Protein G (GE Healthcare) and sepharose-bound sheep IgG column, generated by

covalently coupling sheep IgG on NHS-activated sepharose column (GE Healthcare),

affinity chromatography.

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Lectin immunohistochemistry and immunofluorescence

Four micrometer frozen sections were fixed in acetone and incubated with 10 µg/ml of

biotinylated SNA lectin after blocking with Western Blocking Reagent (Roche) and

biotin blocking (DakoCytomation, Carpinteria, CA). After blocking of endogenous

peroxidase, HRP-conjugated rabbit anti-biotin antibody (DakoCytomation) was used as a

secondary antibody and 3-3’-diaminobenzidine for visualization. Immunofluorescence

experiments were performed as previously described (20) using FITC-conjugated anti-

sheep IgG (Jackson ImmunoResearch) or FITC-conjugated anti-mouse IgG2b (BD

Biosciences).

Statistical analysis

Statistical differences of clinical scores were calculated with Mann-Whitney’s U test.

Repeated measure ANOVA-test was used for difference of human IgG levels in mouse

serum. Statistical differences of the ratio of F4/80+ Ly 17.2 + cells and between

experimental groups in the ITP model were determined with Student’s t test. P < 0.05

was considered significant.

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Supporting online figures

Figure S1

Fig. S1. Composition of the carbohydrate moieties released from N297 IgG Fc. The core

sugar structure linked to the asparagine residue 297 in the antibody heavy chain is

composed of N-acetylglucosamine (GlcNAc) and mannose (Man). Individual glycoforms

vary with respect to attachment of one or two terminal galactose (Gal) residues,

attachment of terminal sialic acid (N-acetylneuraminic acid or Neu5Ac for human and N-

glycolylneuraminic acid or Neu5Gc for mouse) residues, and/or the attachment of

bisecting GlcNAc or fucose (Fuc). Numbers indicate the molecular weight of the

different sugar compositions as determined by MALDI-TOF MS. The mass for the

glycan structures are indicated for human (black) and mouse (red).

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Figure S2

Fig. S2. Carbohydrate spectra of 6A6-IgG antibody isotypes. N-glycans derived from

6A6-IgG1, IgG2a and IgG2b were analyzed by MALDI-TOF MS. Peaks containing sialic

acid residues are indicated (red bracket). Recombinant 6A6 antibody switch variants

produced by transient transfection of 293T cells contained minimal levels of sialic acid

residues in their Asn-297 attached carbohydrates.

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Figure S3

Fig. S3. Antibody activity in vivo is modulated by sialic acid. 6A6-IgG1 was enriched for

sialic acid by affinity chromatography with SNA-agarose. A fraction of this SNA-

enriched preparation was treated with neuraminidase (SNA-enriched + Neuraminidase).

(A) Sialic acid content in antibody preparations was determined by lectin blotting with

SNA. (B) In vivo antibody activity was tested by monitoring platelet depletion induced by

injection of 4 µg of the respective antibody preparations (n=4-5 mice per group).

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Figure S4:

0

0.2

0.4

0.6

0.8

1

1.2

8.75

4.37

5

2.18

75

1.09

3

0.54

6

0.27

34 0

antibody per well (ng)

OD

450

nm6A6-IgG1 6A6-IgG1-SNA

Fig. S4. Influence of Fc-sialylation on antibody specificity. Platelets were immobilized to

96 well plates and the indicated amounts of 6A6-IgG1 or 6A6-IgG1 enriched for sialic

acid (6A6-IgG1-SNA) were added followed by detection of bound antibody by a

secondary horseradish peroxidase coupled antibody specific for mouse IgG1. Shown are

the values corrected for secondary antibody binding to 6A6-IgG1 or 6A6-IgG1-SNA,

respectively.

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Figure S5

Fig. S5. Serum half-life and protein integrity of de-sialylated IVIG. (A) The level of

human IgG in the serum of IVIG treated mice on the indicated days was measured by

ELISA (N = 4, mean +/- SEM). There was no significant difference in half-life of IVIG

and de-sialylated IVIG. Significance was determined by the repeated measure ANOVA-

test. Despite the suggestion that the neonatal Fc-receptor (FcRn) might be involved in

mediating the anti-inflammatory activity of IVIG (S5-S7), the unchanged half-life of de-

sialylated IVIG strongly argues against an important role of FcRn for the loss of the anti-

inflammatory activity of desialylated IVIG. (B) Ten microgram of IVIG or de-sialylated

IVIG were resolved by SDS-PAGE using an 8% polyacrylamide gel under non-reduced

conditions and visualized with coomassie brilliant blue staining. The monomeric

composition and structural integrity of IVIG were not affected by neuraminidase

treatment.

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Figure S6

Fig. S6. Serum half-life and subclass composition of SNA-enriched IVIG. (A) The level

of human IgG in the serum of IVIG treated mice at the indicated days was measured by

ELISA (N = 4, mean +/- SEM). There was no significant difference in half-life of IVIG

and the SNA-enriched IVIG fraction, arguing against differential binding to FcRn (5).

Significance was determined by the repeated measure ANOVA test. (B) IgG subclasses

in untreated and SNA-enriched IVIG were determined by ELISA. No differences were

observed.

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Figure S7

Fig. S7. Sialylated proteins with similar carbohydrate structures do not protect mice from

K/BxN serum induced arthritis. Equivalent molar amounts (6.7 µmol per kilogram) or

equal weight (1 g per kilogram) of IVIG or sialo-proteins fetuin and transferrin were

administered 1 hr before K/BxN serum injection, and clinical scores were examined on

day 4 (N = 4, mean +/- SEM). PBS was used as an additional control. Compared to IgG,

fetuin or transferrin had no statistically significant anti-inflammatory activity at

equivalent molar concentrations. Significance was calculated with the Mann-Whitney’s U

test.

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Figure S8

Fig. S8. Anti-inflammatory activity of SNA enriched IVIG requires FcγRIIB. (A)

Unfractionated IVIG (1 g/kg mouse weight), SNA-enriched IVIG (0.1 g/kg mouse

weight), or PBS as a control were injected into FcγRIIB-deficient mice 1 hr before

K/BxN serum injection, and clinical scores were examined on day 4 (N = 4, mean +/-

SEM). There were no significant differences in clinical scores of arthritis. Significance

was calculated with the Mann-Whitney’s U test. (B) In vivo accumulation of FcγRIIB+

monocytes by SNA-enriched IVIG. Wild type mice were injected with 1 g/kg, 0.1 g/kg

IVIG or 0.1 g/kg of SNA-enriched IVIG, or PBS as a control. Bone marrow (left panel)

and spleen cells (right panel) were collected and analyzed by flow cytometry 1 day after

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the injection (N = 4). F4/80+ FcγRIIB+ cells accumulated significantly after treatment

with 1 g/kg of IVIG or 0.1 g/kg of SNA-enriched IVIG. Significance was calculated with

the Student’s t test.

Figure S9

Fig. S9. Sialic acid content in serum transferrin. Serum transferrin from pooled serum

(n=7-10) of preimmune mice or mice with nephrotoxic nephritis (NTN) was immuno-

precipitated and sialic acid content was detected by lectin-blotting with SNA. Band

intensity was quantified using the Scion-Software.

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Figure S10

Fig. S10. Standardization of MALDI-TOF-MS. A Di-sialylated or asialo-, core-

fucosylated bi-antennary complex -type N-glycan was used as a glycan standard for

MALDI-TOF-MS.

.

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Figure S11

Fig. S11. Specificity of SNA lectin blotting. Two hundred nanogram of IVIG or

α2,3/α2,6 neuraminidase-treated IVIG (NA IVIG) was blotted with SNA (2 µg/ml) with

or without 0.5 M N-acetylneuraminic acid for hapten inhibition.

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