Gabriela Ma ClaudiaTiago

IVIG is a blood product administered intravenously

It contains the pooled IgG extracted from the plasma of over one thousand blood

donors

Immunoglobulin products from human plasma were first used in 1952 to treat immune

deficiency. It was initially shown to be effective in ITP in 1981.

Treatment for: Immune deficiencies (plasma protein replacement therapy)

Autoimmune Diseases (anti-inflammatory at a high dose ≈1-2 g/Kg)

Acute Infections

IVIG cost is climbing and well over $50/g. ($8,000 for a 80 kg person at 2g/kg)

IVIG's effects last between 2 weeks and 3 months

primary immune dysfunction: 100 to 400 mg/kg of body weight every 3 to 4 weeks.

autoimmune diseases: 2 grams per kilogram of body weight for three to six months over a five day

course once a month. Then maintenance therapy of 100 to 400 mg/kg of body weight every 3 to 4

weeks follows.

Rational basis

IVIG: pool of IgGImmunoglobulins: effector molecules of immune defense

Igs properties:

Variable domains

Constant domainFc

DiversitySpecificity

Cellular effector pathways

Fc

immune complex

IVIG mechanism of action

Neutralization ???

Target: harmful antibodies

Idiotypic Network TheoryIdiotypic Network Theory

A B C

A e B = IDIOTYPEA e C = ISOTYPE

Vaz & Pordeus. Visita à imunologia. Arq. Bras. Cardiol. vol.85 no.5 São Paulo Nov. 2005

OK

Antibody Feedback: Cross-linking between BCR and Antibody Feedback: Cross-linking between BCR and FcFcIIR IIR B cell blocked B cell blocked

Fc mechanism of IVIG action

IVIG mechanism of actionThe precise mechanism by which IVIG suppresses harmful inflammation has not been definitively established

BUT….is believed to involve the Fc receptor…

How? Which one(s)?

FcγR

FcαR

FcεR

FcγRI

FcγRIIA FcγRIIB1

FcγRIIB2

FcγRIIIA

FcγRIIIB

FcεRI FcεRII

FcαRI Fcα/μR

FcRn

IVIG mechanism action

Fc Receptors (FcyR)

a protein found on the surface of NK cells, macrophages, neutrophils, mast cells and

others

FcγRs are the most important Fc receptors for inducing phagocytosis of opsonized

microbes

Glycoforms of IgG (Asn297)

Carbohydrate whit terminal sugar residues such as galactose, sialic acid, N-acetylglucosamine, and fucose

more than 30 different antibody glycovariants have been detected in human serum, with about 25%–30% of them in the IgG glycoform.

Thus, these variants, multiplied by the four different IgG subclasses, result in more than 120 different glycoproteins in the IVIG preparation that could contain the active anti-inflammatory component

IVIG has anti-inflammatory effect at a high dose ≈1-2 g/Kg

120 different glycoproteins in the IVIG preparation terminal sugar residues of sialic acid confers anti-inflammatory property

1-3% of IgGs in IVIG have sFc (sialylation)

recombinant sFc: enhanced 35 fold of action in vivo

Carbohydrate

Carbohydrate-Binding Proteins

C-Type Lectins

Siglecs

Galectins

CD1

DC-SIGN is a C-type lectin receptor

binds to mannose type carbohydrates.

Phagocytosis

Cell rolling interactions (ICAM) and activation of CD4+ T cells

•Binds sFc anti-inflammatory responses

-Population of regulatory macrophage-Splenic Marginal Zone

Maria Claudia

Tiago

Objective:

• To define the mechanism by which the 2,6-sialylated Fc mediates an anti- inflammatory

response

• To identify the properties of the regulatory macrophage population

• To identify the receptor required for initiating this pathway in response to 2,6-sialylated Fc.

ResultsAre the splenic marginal zone macrophage necessary for IVIG-mediated immune

suppression?

Defined defects in specific immune cell populations

IVIG1 hour after Arthritis inducing sera

(K/BxN)Clinical score

analysis

Specific macrophage populations in the splenic marginal zone might be required for the anti-

inflammatory effect of the 2,6 sialylated Fc found in IVIG

ResultsWhich receptor expressed in macrophages is required for IVIG protection?

Interacting with glycopeptides:Scavenger receptor (MARCO) – bacteriasSialoadhesin receptor (CD169) – sialic acidC-type lectin receptor (SIGN-R1) – polysaccharide dextran

Blocking antibodies

1 hour after Arthritis inducing sera(K/BxN)

1 hour afterIVIG

TKO-SIGNR1 - antibody that results in the transient down-regulation of SIGN-R1 expression

ResultsWhich receptor expressed in macrophages is required for IVIG protection?

C57BL/6 and SIGN-R1-/-

IVIG (2,6 Fc)

1 hour after Arthritis inducing sera(K/BxN)

Clinical score analysis

The c-type lectin, SIGN-R1, is required for IVIG protection

Ankle bones

ResultsDid SIGN-R1 able to bind to the 2,6-sialyted Fc?

Transfected macrophage

(RAW-SIGN-R1)

Pulsed with flourochrome-labeded

2,6-Fcs (red)

SIGN-R1 binds 2,6-sialylated Fc.

ResultsDid SIGN-R1 able to bind to the 2,6-sialyted Fc and asialylated Fcs?

Resident peritoneal m were harvested

1. C57BL/6 mice2. Lack all IgG Fc receptors3. SIGN-R1-/-

Pulsed with 2,6-Fcs or asialylated Fcs

The amount of bound Fcs were

determined

The 2,6-sialylation of the IgG Fc converts the molecule to a species that acquires the ability to engage a mSIGN-R1 and mediate an antiinflammatory response.

Results

CRD - carbohydrate recognition domains

Yellow – Identical amino acidsGreen – Similar amino acids

Human DC-SIGN expressed on dendritic cells

ResultsDid DC-SIGN able to bind to the 2,6-sialyted Fc?

CHO cells expressing SIGN-R1, hDC-SIGN or

hFcRIIb

Pulsed with 2,6-Fcs

Mannan = ligand for DC-SIGNFibrinogen = similar to Fc linked glycanHuman DC-SIGN, binds 2,6-sialylated Fc

Results

2,6-sialylation Fc

antiinflammatoryresponse

FcRIIb

FcR binding

mSIGN-R1, hDC-SIGN binding

1 hour after Arthritis inducing sera(K/BxN)

1 hour afterIVIGC57Bl/6

SIGN-R1-/-

FcγRIIb-/-

Results

Did FcRIIb involve in the mechanism by which the 2,6-Fc mediates an anti-inflammatory response?

The absence of FcRIIb in the recipient prevented the protection afforded by these splenocytes

K/BxN

Conclusion

Objective: To study hDC-SIGN in the context of IVIG anti-inflammatory activity in expressing-hDC-SIGN mice.

Could hDC-SIGN mediate anti-inflammatory protection by IVIG?

hDC-SIGN+/SIGN-R1-/-

WT SIGN-R1-/-

Treated with sFc

Challenged with

arthritogenic K/BxN serum

Clinical score assessement

hDC-SIGN substitutes for SIGN-R1 in mediating IVIG anti-inflammatory protection

BMMФ

Were hDC-SIGN+ macrophages sufficient to induce an anti-inflammatory response?

hDC-SIGN+

WT

30minsFc or

asyaloFc

+

WT

Transfered to WT mice

hDC-SIGN+ Macrophages treated with sFC showed reduced joint inflammation

Challenged with K/BxN

Clinical score

assessement

Is FcγRIIB required to the anti-inflammatory property induced hDC-SIGN+ macrophages?

hDC-SIGN+-BMMФ

hDC-SIGN+

30min

sFc or PBS

+

SIGN-R1-/-

Transfered to

FcγRIIB-/-

The anti-inflammatory property induced by hDC-SIGN+ macrophages depends on FcγRIIB

Challenged with K/BxN

Clinical score

assessement

Was IL-4 responsable for mediating IVIG anti-inflammatory activity?

BMMФ

hDC-SIGN+

30min

sFc or PBS

+

WT

Transfered to

IL-4-/-

IL-4 is crucial for mediating IVIG anti-inflammatory activity

Challenged with K/BxN

Clinical score

assessement

Could Th2 cytokines supress K/BxN-induced inflammation?

WT FcγRIIB-/-

Treated with IL-4, IL-13 or IL-3

K/BxN

Clinical score evaluation

Inflammation was attenuated after Th2 cytokines administration

Did sFc administration increase Th2 cytokines production?

SIGN-R1-/-

WT

Treated with sFc (1h)

Splenic cells were

removed

Quantification of IL-4, IL-33 and IL-25 mRNA expression

(qPCR)

IL-33 mRNA was upregulated in WT mice after sFc administration

WT

Treated with PBS, IL-33, IL-25

or TSLP

K/BxN

Clinical score evaluation and analyses of IL-4 levels

Can IL-33 induce IL-4 production?

IL-33 reverts K/BxN-induced inflammation by increasing IL-4 levels

hDC-SIGN+/SIGN-R1-/-

Treated with sFc or sFc+anti-IL-33Rα

K/BxN

Clinical score evaluation

Does Anti-IL-33Rα ablate the sFc protection?

The IL-33Rα blocking increases joint inflammation

Did IL-33 and IL-4 increase FcγRIIB expression on monocytes?

hDC-SIGN+-Monocytes

(CD11b+Ly6G+)

+

PBS or IL-4 or IL-33 or IL-25

24h

FcγRIIB expression by

FACS

FcγRIIB expression on monocytes was increased after IL-33 and IL-4 treatment

Are basophils involved with reduced joint inflammation?

hDC-SIGN+/SIGN-R1-/-

Treated with sFc or sFc+anti-FcεRI

K/BxN

Clinical score evaluation

Basophils contribute for IVIG anti-inflammatory activity

IL-4-GFP mice

Treated with PBS or sFc

K/BxN

Clinical score evaluationand quantification of IL-4-producing basophils

Are basophils the main source of IL-4 production during sFc treatment?

Increased IL-4-producing basophils were induced during sFC treatment

Were basophils associated with anti-inflammatory activity induced by sFc?

WT or FcγRIIB-/-

Basophils (DX5+FcεRI+c-Kit-)

PBS, IVIG or IL-33

+

Transfered to

WT Challenged with K/BxN

IL-33-treated basophils also increased anti-inflammatory activity in a FcγRIIB-dependent manner

CONCLUSION