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Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.

Käsermann F, Boerema DJ, Rüegsegger M, Hofmann A, Wymann S, Zuercher AW, Miescher S - PLoS ONE (2012)

Bottom Line: 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.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.The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

View Article: PubMed Central - PubMed

Affiliation: Research & Development, CSL Behring AG, Bern, Switzerland. fabian.kaesermann@cslbehring.com

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.

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Whole blood stimulation assay.A: Whole blood was stimulated with LPS (left panels) or PHA (right panel). Upper panels: concentration dependent inhibition of CD54 (ICAM-1) expression on monocytes by the indicated IVIG fractions was monitored by flow cytometry. MFI: mean fluorescence intensity. Lower panels: concentration dependent inhibition of MCP-1 (CCL2) release by IVIG was quantified by ELISA in the supernatant of stimulated blood cultures. Mean values from representative experiments using blood from single donors are shown. neg: no stimulation, pos: stimulation without IVIG, FT: flow through, E1/E2 SNA binding IVIG fractions, NAase: neuraminidase treated. B: Inhibition of PHA mediated MCP-1 release by Fc and F(ab')2 fragments. Left panel: Mean MCP-1 concentration of stimulated minus non stimulated samples from a representative experiment is shown. Right panel: PHA stimulated whole blood was treated with 40 µM IgG, Fc, F(ab’)2 or highly sialylated Fc (S+Fc). Normalized values from experiments with blood from two donors are shown. S+ Fc: SNA binding Fc fraction (highly sialylated Fc).
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pone-0037243-g005: Whole blood stimulation assay.A: Whole blood was stimulated with LPS (left panels) or PHA (right panel). Upper panels: concentration dependent inhibition of CD54 (ICAM-1) expression on monocytes by the indicated IVIG fractions was monitored by flow cytometry. MFI: mean fluorescence intensity. Lower panels: concentration dependent inhibition of MCP-1 (CCL2) release by IVIG was quantified by ELISA in the supernatant of stimulated blood cultures. Mean values from representative experiments using blood from single donors are shown. neg: no stimulation, pos: stimulation without IVIG, FT: flow through, E1/E2 SNA binding IVIG fractions, NAase: neuraminidase treated. B: Inhibition of PHA mediated MCP-1 release by Fc and F(ab')2 fragments. Left panel: Mean MCP-1 concentration of stimulated minus non stimulated samples from a representative experiment is shown. Right panel: PHA stimulated whole blood was treated with 40 µM IgG, Fc, F(ab’)2 or highly sialylated Fc (S+Fc). Normalized values from experiments with blood from two donors are shown. S+ Fc: SNA binding Fc fraction (highly sialylated Fc).

Mentions: Potential anti-inflammatory effects of IVIG and sialic acid-enriched fractions were assessed in a functional in vitro assay (Fig. 5). Whole blood was stimulated with LPS (Fig. 5A left panels) or PHA (Fig. 5A right panels) and cell surface expression of CD54 (ICAM-1) on monocytes as well as MCP-1 secretion into the cell culture supernatant were quantified. Under the chosen conditions the results were most pronounced for MCP-1, but other cytokines were measured as well (IL-6, IL-8, MIP-1β and IL-1ra), yielding similar results (data not shown). LPS induced upregulation of CD54 expression on monocytes; this effect was down-regulated by E2 in a dose-dependent manner (Fig. 5A top left) and could be abrogated by treatment with neuraminidase (NAase E2). Similarly, PHA-induced CD54 expression was inhibited by E2 slightly more efficiently than with all other tested fractions. Again neuraminidase treatment abolished the anti-inflammatory effect (Fig. 5A top right). MCP-1 release into the supernatant was inhibited by E1 and E2 in a dose dependent fashion; the effect was more pronounced than with IVIG (Fig. 5A, bottom panels). Similar as for CD54 expression, the inhibitory effect was lost when E2 was desialylated. Control experiments showed that IVIG and sialic acid-enriched fractions contained anti-LPS antibodies, however, the measured amounts did not correlate with the observed anti-inflammatory effects, suggesting additional mechanisms at work (E2 showed a lower anti-LPS compared to IVIG, data not shown). No antibodies against the PHA used in these experiments were detected. Whereas, in agreement with recently reported data using PHA-L [19], antibodies to PHA from different providers were found in IVIG. Interestingly, inhibition of PHA-induced MCP-1 secretion by IVIG was dependent on the F(ab’)2 whereas the Fc region did not appear to contribute to anti-inflammatory effects in this system (Fig. 5B left panel). Similarly, highly sialylated Fc fragments (S+ Fc) tested at 40 µM did not show an inhibitory effect on the PHA-mediated MCP-1 release. This result supports the finding, that IVIG effects are sialic acid, in particular Fc-sialylation independent in this system (Fig. 5B right panel).


Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.

Käsermann F, Boerema DJ, Rüegsegger M, Hofmann A, Wymann S, Zuercher AW, Miescher S - PLoS ONE (2012)

Whole blood stimulation assay.A: Whole blood was stimulated with LPS (left panels) or PHA (right panel). Upper panels: concentration dependent inhibition of CD54 (ICAM-1) expression on monocytes by the indicated IVIG fractions was monitored by flow cytometry. MFI: mean fluorescence intensity. Lower panels: concentration dependent inhibition of MCP-1 (CCL2) release by IVIG was quantified by ELISA in the supernatant of stimulated blood cultures. Mean values from representative experiments using blood from single donors are shown. neg: no stimulation, pos: stimulation without IVIG, FT: flow through, E1/E2 SNA binding IVIG fractions, NAase: neuraminidase treated. B: Inhibition of PHA mediated MCP-1 release by Fc and F(ab')2 fragments. Left panel: Mean MCP-1 concentration of stimulated minus non stimulated samples from a representative experiment is shown. Right panel: PHA stimulated whole blood was treated with 40 µM IgG, Fc, F(ab’)2 or highly sialylated Fc (S+Fc). Normalized values from experiments with blood from two donors are shown. S+ Fc: SNA binding Fc fraction (highly sialylated Fc).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366990&req=5

pone-0037243-g005: Whole blood stimulation assay.A: Whole blood was stimulated with LPS (left panels) or PHA (right panel). Upper panels: concentration dependent inhibition of CD54 (ICAM-1) expression on monocytes by the indicated IVIG fractions was monitored by flow cytometry. MFI: mean fluorescence intensity. Lower panels: concentration dependent inhibition of MCP-1 (CCL2) release by IVIG was quantified by ELISA in the supernatant of stimulated blood cultures. Mean values from representative experiments using blood from single donors are shown. neg: no stimulation, pos: stimulation without IVIG, FT: flow through, E1/E2 SNA binding IVIG fractions, NAase: neuraminidase treated. B: Inhibition of PHA mediated MCP-1 release by Fc and F(ab')2 fragments. Left panel: Mean MCP-1 concentration of stimulated minus non stimulated samples from a representative experiment is shown. Right panel: PHA stimulated whole blood was treated with 40 µM IgG, Fc, F(ab’)2 or highly sialylated Fc (S+Fc). Normalized values from experiments with blood from two donors are shown. S+ Fc: SNA binding Fc fraction (highly sialylated Fc).
Mentions: Potential anti-inflammatory effects of IVIG and sialic acid-enriched fractions were assessed in a functional in vitro assay (Fig. 5). Whole blood was stimulated with LPS (Fig. 5A left panels) or PHA (Fig. 5A right panels) and cell surface expression of CD54 (ICAM-1) on monocytes as well as MCP-1 secretion into the cell culture supernatant were quantified. Under the chosen conditions the results were most pronounced for MCP-1, but other cytokines were measured as well (IL-6, IL-8, MIP-1β and IL-1ra), yielding similar results (data not shown). LPS induced upregulation of CD54 expression on monocytes; this effect was down-regulated by E2 in a dose-dependent manner (Fig. 5A top left) and could be abrogated by treatment with neuraminidase (NAase E2). Similarly, PHA-induced CD54 expression was inhibited by E2 slightly more efficiently than with all other tested fractions. Again neuraminidase treatment abolished the anti-inflammatory effect (Fig. 5A top right). MCP-1 release into the supernatant was inhibited by E1 and E2 in a dose dependent fashion; the effect was more pronounced than with IVIG (Fig. 5A, bottom panels). Similar as for CD54 expression, the inhibitory effect was lost when E2 was desialylated. Control experiments showed that IVIG and sialic acid-enriched fractions contained anti-LPS antibodies, however, the measured amounts did not correlate with the observed anti-inflammatory effects, suggesting additional mechanisms at work (E2 showed a lower anti-LPS compared to IVIG, data not shown). No antibodies against the PHA used in these experiments were detected. Whereas, in agreement with recently reported data using PHA-L [19], antibodies to PHA from different providers were found in IVIG. Interestingly, inhibition of PHA-induced MCP-1 secretion by IVIG was dependent on the F(ab’)2 whereas the Fc region did not appear to contribute to anti-inflammatory effects in this system (Fig. 5B left panel). Similarly, highly sialylated Fc fragments (S+ Fc) tested at 40 µM did not show an inhibitory effect on the PHA-mediated MCP-1 release. This result supports the finding, that IVIG effects are sialic acid, in particular Fc-sialylation independent in this system (Fig. 5B right panel).

Bottom Line: 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.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.The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

View Article: PubMed Central - PubMed

Affiliation: Research & Development, CSL Behring AG, Bern, Switzerland. fabian.kaesermann@cslbehring.com

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.

Show MeSH
Related in: MedlinePlus