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Gabriela Ma Claudia Tiago 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 IgG Immunoglobulins: effector molecules of immune defense Igs properties: immune complex Variable domains Constant domain Fc Fc Diversity Specificity Cellular effector pathways IVIG mechanism of action Neutralization ??? OK Target: harmful antibodies Idiotypic Network Theory AeB= IDIOTYPE A e C = ISOTYPE A B C Vaz & Pordeus. Visita à imunologia. Arq. Bras. Cardiol. vol.85 no.5 São Fc mechanism of IVIG action Antibody Feedback: Cross-linking between BCR and FcIIR B cell blocked IVIG mechanism of action The 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)? FcαR FcαRI FcεR FcεRI FcγR Fcα/μR FcεRII FcγRI FcγRIIIA FcγRIIA FcγRIIB1 FcγRIIB2 FcRn FcγRIIIB 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 p 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. Results Are the splenic marginal zone macrophage necessary for IVIG-mediated immune suppression? 1 hour after IVIG Arthritis inducing sera (K/BxN) Clinical score analysis Defined defects in specific immune cell populations Specific macrophage populations in the splenic marginal zone might be required for the antiinflammatory effect of the 2,6 sialylated Fc found in IVIG Results Which receptor expressed in macrophages is required for IVIG protection? 1 hour after IVIG Blocking antibodies Interacting with glycopeptides: Scavenger receptor (MARCO) – bacterias Sialoadhesin receptor (CD169) – sialic acid C-type lectin receptor (SIGN-R1) – polysaccharide dextran 1 hour after Arthritis inducing sera (K/BxN) TKO-SIGNR1 - antibody that results in the transient down-regulation of SIGN-R1 expression Results Which receptor expressed in macrophages is required for IVIG protection? C57BL/6 and SIGN-R1-/- IVIG 1 hour after (2,6 Fc) Clinical score analysis Arthritis inducing sera (K/BxN) Ankle bones The c-type lectin, SIGN-R1, is required for IVIG protection Results Did 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. Results Did SIGN-R1 able to bind to the 2,6-sialyted Fc and asialylated Fcs? 1. 2. 3. C57BL/6 mice Lack all IgG Fc receptors SIGN-R1-/- Resident peritoneal m were harvested 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 Human DC-SIGN expressed on dendritic cells Yellow – Identical amino acids Green – Similar amino acids CRD - carbohydrate recognition domains Results Did DC-SIGN able to bind to the 2,6-sialyted Fc? CHO cells expressing SIGN-R1, hDC-SIGN or hFcRIIb Mannan = ligand for DC-SIGN Fibrinogen = similar to Fc linked glycanHuman Pulsed with 2,6-Fcs DC-SIGN, binds 2,6-sialylated Fc Results 2,6-sialylation Fc 1 hour after C57Bl/6 SIGN-R1-/FcγRIIb-/- FcR binding mSIGN-R1, hDCSIGN binding antiinflammatory response FcRIIb IVIG 1 hour after Arthritis inducing sera (K/BxN) Results Did FcRIIb involve in the mechanism by which the 2,6-Fc mediates an anti-inflammatory response? K/BxN The absence of FcRIIb in the recipient prevented the protection afforded by these splenocytes 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? WT SIGN-R1- hDC-SIGN+/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 Were hDC-SIGN+ macrophages sufficient to induce an anti-inflammatory response? WT hDCSIGN+ BMMФ + sFc or asyaloFc 30min Transfered to WT mice WT Challenged with K/BxN Clinical score assessemen t hDC-SIGN+ Macrophages treated with sFC showed reduced joint inflammation Is FcγRIIB required to the anti-inflammatory property induced hDC-SIGN+ macrophages? hDC-SIGN+BMMФ + hDCSIGN+ 30min sFc or PBS Transfered to SIGN-R1/- FcγRIIB-/- Challenged with K/BxN Clinical score assesseme nt The anti-inflammatory property induced by hDC-SIGN+ macrophages depends on FcγRIIB Was IL-4 responsable for mediating IVIG antiinflammatory activity? hDCSIGN+ BMMФ + 30min sFc or PBS Transfered to WT IL-4-/- Challenged with K/BxN Clinical score assessemen t IL-4 is crucial for mediating IVIG anti-inflammatory Could Th2 cytokines supress K/BxN-induced inflammation? Treated with IL4, IL-13 or IL-3 FcγRIIB-/- WT K/BxN Clinical score evaluation Inflammation was attenuated after Th2 cytokines administration Did sFc administration increase Th2 cytokines production? WT SIGN-R1/- 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 Can IL-33 induce IL-4 production? Treated with PBS, IL-33, IL-25 or TSLP WT K/BxN Clinical score evaluation and analyses of IL-4 levels IL-33 reverts K/BxN-induced inflammation by increasing IL-4 levels Does Anti-IL-33Rα ablate the sFc protection? Treated with sFc or sFc+anti-IL-33Rα hDC-SIGN+/SIGNR1-/- K/BxN Clinical score evaluation 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? Treated with sFc or sFc+anti-FcεRI hDC-SIGN+/SIGNR1-/- K/BxN Clinical score evaluation Basophils contribute for IVIG anti-inflammatory activity Are basophils the main source of IL-4 production during sFc treatment? Treated with PBS or sFc IL-4-GFP mice K/BxN Clinical score evaluation and quantification of IL4-producing basophils Increased IL-4-producing basophils were induced during sFC treatment Were basophils associated with antiinflammatory activity induced by sFc? WT or FcγRIIB-/Basophils + (DX5+FcεRI + c-Kit ) PBS, IVIG or IL33 Transfered to WT Challenged with K/BxN IL-33-treated basophils also increased anti-inflammatory activity in a FcγRIIBdependent manner CONCLUSION