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.
Highlights
Replacement therapy with plasma-derived immunoglobulin G (IgG) is the standard of care to treat primary and secondary immunodeficiency
If the FT was re-applied to an Sambucus nigra agglutinin (SNA) column no additional material was recovered in the binding fractions, indicating that the affinity column had not been overloaded, but rather that the remaining sialic acid might not be accessible for SNA
Treatment with neuraminidase did not influence binding of intravenous immunoglobulin (IVIG) to human erythrocytes. These findings demonstrate that the composition of specific antibodies was skewed in sialicacid enriched IgG fractions, supporting the claim that lectin affinity chromatography fractionation of IVIG is mainly driven by Fab-interactions
Summary
Replacement therapy with plasma-derived immunoglobulin G (IgG) is the standard of care to treat primary and secondary immunodeficiency. For this purpose IgG is applied either intravenously (IVIG) or subcutaneously (SCIG). Over the years IVIG/SCIG has been increasingly used for immunomodulation of acute and chronic autoimmune diseases (for an overview see ref [1]). A number of possible non-exclusive mechanisms have been proposed to explain the immunomodulatory effects of IVIG. They include interference with complement components and the cytokine network, modulation of B and T cell function, Fc receptor blockage and effects on the anti-idiotype network. There are multiple pathways operating in parallel [7,8,9,10,11]
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