Analysis and Functional Consequences of Increased Fab-Sialylation of Intravenous Immunoglobulin (IVIG) after Lectin Fractionation

Abstract

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab’)2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

“…Both α2,6-sial and -asial F241A Fc bound to DC-SIGN, induced IL-33 expression, and transferred antiinflammatory activity with stimulated DC-SIGN + BMMΦs to K/BxN serum-challenged mice, recapitulating the antiinflammatory activity of IVIG and sialylated IgG (29). Recently, reports have been published that question the essential role of Fc sialylation for modulating immune responses (37)(38)(39). Our data, and that of several other groups, have confirmed the antiinflammatory role of sialylated Fc in multiple models of antibody-mediated inflammation (9, 12-14, 16, 40).…”

The Emerging Importance of IgG Fab Glycosylation in Immunity

“…Recent studies emphasized the importance of glycosylation in the therapeutic effects of IVIg (73). In certain mouse models (74,75), but not in others (13,(76)(77)(78), the anti-inflammatory activity of IVIg depends mainly on a fraction of sialylated Abs. These sialylated Abs may interact with C-type lectins (like DC-SIGN, DCIR) and sialic acid-binding Ig-type lectins, such as CD22, at the surface of immune cells (79,80).…”

“…These studies used Sambucus nigra agglutinin (SNA) lectin-affinity chromatography to fractionate IVIg, thereby predominantly enriching for Fab sialylation rather than Fc sialylation (13,76). Fab-sialylated IVIg downregulated CD54 expression and the MCP-1 secretion of monocytes, whereas neither IVIg without sialylated Fab nor a highly sialylated Fc could achieve these effects (13). SNA-enriched IVIg also was shown to induce upregulation of PGE 2 by monocytes, thereby enabling inhibition of IFN-a production by plasmacytoid dendritic cells (82).…”

“…Altogether, the available literature suggests that the anti-inflammatory effects of IVIg are mediated, at least in part, by its Fab arms, with a potential role for Ig variable domain sialylation (73). Differential effects by SNA-enriched and -depleted fractions might reflect, in part, differences in their respective Ab specificities and not be a direct result of the presence or absence of a glycan (13). However, the fact that removal of sialic acid using neuraminidase can ameliorate the effects of IVIg suggests otherwise (75).…”

IVIG pluripotency and the concept of Fc-sialylation: challenges to the scientist

“…However, two of the cited articles do not include functional experiments but rather focus on aspects of human IgG sialylation and lectin fractionation. The other articles report that in a human whole blood inflammation assay, the anti-inflammatory activity of IVIG was associated with Fab sialy lation and not Fc sialylation 5 , and that in a murine model of passive-immune thrombo cytopenia no increase in platelet count was observed in response to the administration of IVIG that was enriched for sialylated IgG 6 . Together, the data presented in these publications do not support the proposed Fc-sialylation concept -by contrast, they rather challenge it.…”