Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Natural anti-Siglec autoantibodies mediate potential immunoregulatory mechanisms: implications for the clinical use of intravenous immunoglobulins (IVIg)

Human intravenous immunoglobulins (IVIg) contain natural autoantibodies against the inhibitory lectin-receptors Siglec-8 and Siglec-9. These two members of the Siglec family are known to mediate both inhibitory and death signals. Here, we discuss recent findings regarding the cytotoxic effects of natural anti-Siglec autoantibodies on both neutrophils and eosinophils, and present the concept of a novel regulatory mechanism exhibiting anti-inflammatory properties. Consequently, IVIg may amplify this regulatory pathway by increasing the concentration of natural anti-Siglec autoantibodies in blood and tissues.

Active regulatory T-cells contribute to broadened T-cell repertoire diversity in ivIg-treated SLE patients

Octapharma.

Broadened T-cell Repertoire Diversity in ivIg-treated SLE Patients is Also Related to the Individual Status of Regulatory T-cells

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4 + Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density.

DC-SIGN and alpha 2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells

Intravenous immunoglobulin (IVIg) is widely used to treat autoimmune diseases. Several mutually nonexclusive mechanisms are proposed to explain the beneficial effects of IVIg in patients (1, 2). Lately, Ravetch and colleagues (3) demonstrate that anti-inflammatory activity of IVIg is mediated mainly by antibodies that contain terminal _2,6-sialic acid linkages at the Asn297-linked glycan of Fc region.

Intravenous immunoglobulin expands regulatory T cells via induction of cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) play a critical role in the maintenance of immune tolerance. Intravenous immunoglobulin (IVIg), a therapeutic preparation of normal pooled human IgG, expands Tregs in various experimental models and in patients. However, the cellular and molecular mechanisms by which IVIg expands Tregs are relatively unknown. As Treg expansion in the periphery requires signaling by antigen-presenting cells such as dendritic cells (DCs) and IVIg has been demonstrated to modulate DC functions, we hypothesized that IVIg induces distinct signaling events in DCs that subsequently mediate Treg expansion. We demonstrate that IVIg expands Tregs via induction of cyclooxygenase (COX)-2-dependent prostaglandin E2 (PGE(2)) in human DCs. However, costimulatory molecules of DCs such as programmed death ligands, OX40 ligand, and inducible T-cell costimulator ligands were not implicated. Inhibition of PGE(2) synthesis by COX-2 inhibitors prevented IVIg-mediated Treg expansion in vitro and significantly diminished IVIg-mediated Treg expansion in vivo and protection from disease in experimental autoimmune encephalomyelitis model. IVIg-mediated COX-2 expression, PGE(2) production, and Treg expansion were mediated in part via interaction of IVIg and F(ab('))(2) fragments of IVIg with DC-specific intercellular adhesion molecule-3-grabbing nonintegrin. Our results thus uncover novel cellular and molecular mechanism by which IVIg expands Tregs.

The anti-inflammatory properties of intravenous immunoglobulin in a murine model of allergic airway disease ; effects on the development of regulatory T-cells

Les immunoglobulines intraveineuses (IVIg) constituent une préparation polyclonale d’IgG isolée et regroupée à partir du plasma sanguin de multiples donneurs. Initialement utilisé comme traitement de remplacement chez les patients souffrant d’immunodéficience primaire ou secondaire, les IVIg sont maintenant largement utilisées dans le traitement de plusieurs conditions auto-immunes, allergiques ou inflammatoires à une dose élevée, dite immunomodulatrice. Différents mécanismes d’action ont été postulés au fil des années pour expliquer l’effet thérapeutique des IVIg dans les maladies auto-immunes et inflammatoires. Entre autre, un nombre grandissant de données issues de modèles expérimentaux chez l’animal et l’humain suggère que les IVIg induisent l’expansion et augmentent l’action suppressive des cellules T régulatrices (Tregs), par un mécanisme qui demeure encore inconnu. Également, les patients atteints de maladies auto-immunes ou inflammatoires présentent souvent un nombre abaissé de Tregs par rapport aux individus sains. Ainsi, une meilleure compréhension des mécanismes par lesquels les IVIg modulent les cellules T régulatrices est requise afin de permettre un usage plus rationnel de ce produit sanguin en tant qu’alternative thérapeutique dans le traitement des maladies auto-immunes et inflammatoires. Par le biais d’un modèle expérimental d’allergie respiratoire induite par un allergène, nous avons démontré que les IVIg diminuaient significativement l’inflammation au niveau des voies aériennes ce, en association avec une différenciation des Tregs à partir des cellules T non régulatrices du tissu pulmonaire. Nous avons également démontré qu’au sein de notre modèle expérimental, l’effet anti-inflammatoire des IVIg était dépendant des cellules dendritiques CD11c+ (CDs) pulmonaires, puisque cet effet pouvait être complètement reproduit par le transfert adoptif de CDs provenant de souris préalablement traitées par les IVIg. À cet effet, il est déjà établi que les IVIg peuvent moduler l’activation et les propriétés des CDs pour favoriser la tolérance immunitaire et que ces cellules seraient cruciales pour l’induction périphérique des Tregs. C’est pourquoi, nous avons cherché à mieux comprendre comment les IVIg exercent leur effet sur ces cellules. Pour la première fois, nous avons démontré que la fraction d’IgG riche en acide sialique (SA-IVIg) (constituant 2-5% de l’ensemble des IgG des donneurs) interagit avec un récepteur dendritique inhibiteur de type lectine C (DCIR) et active une cascade de signalement intracellulaire initiée par la phosphorylation du motif ITIM qui est responsable des changements observés en faveur de la tolérance immunitaire auprès des cellules dendritiques et des Tregs. L’activité anti-inflammatoire de la composante SA-IVIg a déjà été décrite dans des études antérieures, mais encore une fois le mécanisme par lequel ce traitement modifie la fonction des CDs n’a pas été établi. Nous avons finalement démontré que le récepteur DCIR facilite l’internalisation des molécules d’IgG liées au récepteur et que cette étape est cruciale pour permettre l’induction périphérique des Tregs. En tant que produit sanguin, les IVIg constitue un traitement précieux qui existe en quantité limitée. La caractérisation des mécanismes d’action des IVIg permettra une meilleure utilisation de ce traitement dans un vaste éventail de pathologies auto-immunes et inflammatoires.

Self-reactivity in the dimeric intravenous immunoglobulin fraction

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.

Intravenous immunoglobulin contains a broad repertoire of anticarbohydrate antibodies that is not restricted to the IgG2 subclass

BACKGROUND: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. OBJECTIVE: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. METHODS: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. RESULTS: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. CONCLUSIONS: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non-pathogen-associated prokaryotic glycans.

Immunoglobulin M-enriched intravenous immunoglobulin inhibits classical pathway complement activation, but not bactericidal activity of human serum

Acute or even hyperacute humoral graft rejection, mediated by classical pathway complement activation, occurs in allo- and xenotransplantation due to preformed anti-graft antibodies. Intravenous immunoglobulin (IVIg) preparations can prevent complement-mediated tissue injury and delay hyperacute xenograft rejection. It is known that IgM-enriched IVIg (IVIgM) has a higher capacity to block complement than IVIgG. Different IVIgs were therefore tested for specificity of complement inhibition and effect on anti-bacterial activity of human serum. IVIgM-I (Pentaglobin), 12% IgM), IVIgM-II (IgM-fraction of IVIgM-I, 60% IgM), and three different IVIgG (all >95% IgG) were used. The known complement inhibitor dextran sulfate was used as control. Hemolytic assays were performed to analyze pathway-specificity of complement inhibition. Effects of IVIg on complement deposition on pig cells and Escherichia coli were assessed by flow cytometry and cytotoxicity as well as bactericidal assays. Complement inhibition by IVIgM was specific for the classical pathway, with IC50 values of 0.8 mg/ml for IVIgM-II and 1.7 mg/ml for IVIgM-I in the CH50 assay. Only minimal inhibition of the lectin pathway was seen with IVIgM-II (IC50 15.5 mg/ml); no alternative pathway inhibition was observed. IVIgG did not inhibit complement in any hemolytic assay. Classical pathway complement inhibition by IVIgM was confirmed in an in vitro xenotransplantation model with PK15 cells. In contrast, IVIgM did not inhibit (mainly alternative pathway mediated) killing of E. coli by human serum. In conclusion, IgM-enriched IVIg is a specific inhibitor of the classical complement pathway, leaving the alternative pathway intact, which is an important natural anti-bacterial defense, especially for immunosuppressed patients.

Intravenous immunoglobulins--understanding properties and mechanisms

High-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcgammaR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled 'IVIg--Understanding properties and mechanisms' at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26-28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcgammaRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy.

In vitro comparison of the complement-scavenging capacity of different intravenous immunoglobulin preparations

BACKGROUND AND OBJECTIVES: Complement inhibition is considered important in the mechanism of action of intravenous immunoglobulin (IVIG) in a number of inflammatory and autoimmune disorders. The capacity of different IVIG preparations to 'scavenge' activated C3 and thereby inhibit complement activation was assessed by a new in vitro assay. MATERIALS AND METHODS: Diluted human serum as a complement source, with or without addition of different concentrations of IVIG, was incubated in microtitre plates coated with heat-aggregated human IgG. Complement scavenging was measured by detecting reduced C3 binding and determining fluid phase C3b-IgG complex formation. Complement activation induced by the IVIG preparations was measured as C5a formation. RESULTS: All IVIG preparations exhibited a dose-dependent inhibition of C3b deposition, correlating strongly with binding of C3b to fluid-phase IgG, but the extent of complement scavenging varied considerably between different IVIG preparations. At an IVIG concentration of 0.9 mg/ml, the inhibition of C3b deposition ranged from 72 +/- 16% to 22 +/- 4.1%. The reduction of C3b deposition on the complement-activating surface was not due to IVIG-induced complement activation in the fluid phase, as shown by the low C5a formation in the presence of serum. CONCLUSION: In vitro analysis allows comparison of the complement-inhibitory properties of IVIG preparations. The extent of complement scavenging varies between the products.

Different IVIG glycoforms affect in vitro inhibition of anti-ganglioside antibody-mediated complement deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.

Protection from experimental autoimmune encephalomyelitis by polyclonal IgG requires adjuvant-induced inflammation

BACKGROUND Intravenous immunoglobulin (IVIG) proved to be an efficient anti-inflammatory treatment for a growing number of neuroinflammatory diseases and protects against the development of experimental autoimmune encephalomyelitis (EAE), a widely used animal model for multiple sclerosis (MS). METHODS The clinical efficacy of IVIG and IVIG-derived F(ab')2 fragments, generated using the streptococcal cysteine proteinase Ide-S, was evaluated in EAE induced by active immunization and by adoptive transfer of myelin-specific T cells. Frequency, phenotype, and functional characteristics of T cell subsets and myeloid cells were determined by flow cytometry. Antibody binding to microbial antigen and cytokine production by innate immune cells was assessed by ELISA. RESULTS We report that the protective effect of IVIG is lost in the adoptive transfer model of EAE and requires prophylactic administration during disease induction. IVIG-derived Fc fragments are not required for protection against EAE, since administration of F(ab')2 fragments fully recapitulated the clinical efficacy of IVIG. F(ab')2-treated mice showed a substantial decrease in splenic effector T cell expansion and cytokine production (GM-CSF, IFN-γ, IL-17A) 9 days after immunization. Inhibition of effector T cell responses was not associated with an increase in total numbers of Tregs but with decreased activation of innate myeloid cells such as neutrophils, monocytes, and dendritic cells. Therapeutically effective IVIG-derived F(ab')2 fragments inhibited adjuvant-induced innate immune cell activation as determined by IL-12/23 p40 production and recognized mycobacterial antigens contained in Freund's complete adjuvant which is required for induction of active EAE. CONCLUSIONS Our data indicate that F(ab')2-mediated neutralization of adjuvant contributes to the therapeutic efficacy of anti-inflammatory IgG. These findings might partly explain the discrepancy of IVIG efficacy in EAE and MS.

Immunorégulation de la réaction du greffon contre l'hôte (GvHD) dans un modèle murin xénogénique : le rôle des immunoglobulines intraveineuses (IVIG)

La réaction du greffon contre l’hôte (GvHD) est une complication majeure de la transplantation de cellules souches hématopoïétiques (HSCT). Les traitements de prophylaxie contre le développement de la GvHD reposent essentiellement sur l’utilisation d’agents immunosuppresseurs, ce qui contribue à ralentir la reconstitution immunitaire post-greffe et à prolonger la durée de l’état immunosupprimé des patients. Le développement de prophylaxie pour la GvHD à base d’agents immunomodulateurs est ainsi privilégié. À l’aide d’un modèle murin xénogénique chez les souris NOD/scid-IL2rγ-/- (NSG), on a étudié le potentiel immunomodulateur des immunoglobulines intraveineuses (IVIG) dans la prévention de la GvHD, ainsi que leurs effets sur la qualité et la cinétique de la reconstitution immunitaire. On a déterminé qu’un traitement hebdomadaire d’IVIG peut effectivement réduire l’incidence de la GvHD, ainsi que la mortalité qui y est reliée, avec une efficacité similaire à celle obtenue avec la cyclosporine A, un immunosuppresseur couramment utilisé dans la prophylaxie de la GvHD. Par ailleurs, on a déterminé que le mécanisme d’action des IVIG dans la réduction de la GvHD est distinct de celui des immunosuppresseurs. De plus, on a démontré que les IVIG induisent l’expansion et l’activation des cellules NK présentes au sein du greffon, lesquelles sont nécessaires pour l’obtention de l’effet protecteur des IVIG contre le développement de la GvHD, et sont dépendantes de la présence de lymphocytes T activés. Grâce à un modèle murin humanisé, on a également démontré que le traitement hebdomadaire d’IVIG induit un délai transitoire de la reconstitution humorale, ce qui n’affecte toutefois pas la qualité globale de la reconstitution immunitaire. Ces résultats mettent cependant en doute la pertinence de l’utilisation des IVIG dans les protocoles cliniques de prophylaxie de la GvHD, puisque les immunosuppresseurs seront toujours utilisés, et qu’on a démontré que les IVIG ont besoin de lymphocytes T activés afin de prévenir efficacement le développement de la GvHD.