gabriela m a claudia tiago. ivig is a blood product administered intravenously it contains the...
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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