Modification of the degree of branching of a beta-(1,3)-glucan affects aggregation behavior and activity in an oxidative burst assay.

Scleroglucan is a ß-(1,3)-glucan which is highly branched at the 6-position with a single glucose residue. Acid hydrolysis of a high molecular weight scleroglucan gave a medium molecular weight, freely soluble material. Linkage analysis by the partially methylated alditol acetate method showed that the solubilized material had 30% branching. When the material was subjected to partial Smith degradations, the percent branching was reduced accordingly to 12% or 17%. After the percent branching was reduced, the average molecular weight of the samples increased considerably, indicating the assembly of higher ordered aggregate structures. An aggregate number distribution analysis was applied to confirm the higher aggregated structures. These aggregated structures gave the material significantly enhanced activity in an in vitro oxidative burst assay compared to the highly branched material.

Differential regulation of oxidative burst by distinct ß-glucan-binding receptors and signaling pathways in human peripheral blood mononuclear cells.

ß-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known ß-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived ß-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble ß-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of ß-glucan.

Binding of Soluble Yeast ß-Glucan to Human Neutrophils and Monocytes is Complement-Dependent.

The immunomodulatory properties of yeast ß-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate ß-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble ß-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast ß-glucan, this study evaluated and characterized the binding of soluble ß-glucan to human neutrophils and monocytes. The results demonstrated that soluble ß-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble ß-glucan in these cells. Binding of soluble ß-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble ß-glucan was demonstrated by detection of iC3b, the complement opsonin on ß-glucan-bound cells, as well as by the direct binding of iC3b to ß-glucan in the absence of cells. Binding of ß-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.