Dectin-1 is a major beta-glucan receptor on macrophages

Abstract

Zymosan is a beta-glucan- and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a beta-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the beta-glucan receptor mediating this activity. To address the role of the recently described beta-glucan receptor, Dectin-1, we generated a novel anti-Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the beta-glucan-dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte beta-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of beta-glucans for therapeutic drug design.

Figure 1.

A β-glucan receptor is a major receptor for unopsonized zymosan. The β-glucan, laminarin, specifically inhibits the recognition of unopsonized FITC-labeled zymosan by BMDM (white bars), Tg- (striped bars), and Biogel-elicited macrophages (black bars). The addition of carbohydrates which block the mannose receptor (mannan) and the lectin domain of CR3 (methyl glucoside) had no effect on zymosan binding. The data shown have been normalized to the uninhibited control and expressed as percent relative fluorescence.

Figure 2.

CR3 is not a receptor for unopsonized zymosan on macrophages. (A) The β-glucan dependent recognition of unopsonized zymosan is unaffected in CR3-deficient (CD11b^−/−) BMDM. (B) CR3 deficiency does affect the binding of serum opsonized FITC-labeled zymosan, although recognition of opsonized zymosan in both types of macrophage retains a β-glucan–dependent component. The data are shown as the directly measured fluorescence units and have not been normalized, so as to allow a direct comparison between the macro-phages. Note the difference in scale between the panels (A and B). The data shown are the average of triplicates and error bars indicate the standard deviation.

Figure 3.

The mAb 2A11 recognizes Dectin-1 and detects its expression on the surface of primary macrophages. (A) The mAb 2A11, but not an IgG2b control mAb, recognizes Dectin-1, and can specifically immunoprecipitate soluble recombinant hemagglutinin (HA)-tagged Dectin-1. (B) The mAb 2A11 stains Dectin-1 transductants (shaded histogram), but not nontransduced cells (unshaded histogram), by flow cytometry. (C) 2A11 detects Dectin-1 expression on the surface of freshly isolated F4/80⁺CR3⁺ Tg macrophages. The gate shown in the left panel denotes the population examined in the right panels. (D) Dectin-1 is expressed at a similar level on the surface of both wild-type and CD11b^−/− bone marrow–derived macrophages.

Figure 4.

Dectin-1 is a major macrophage receptor for unopsonized zymosan. (A) 2A11 (anti-Dectin-1), but not 5C6 (anti-CR3), specifically inhibits the recognition of unopsonized FITC-labeled zymosan by Tg macrophages. The level of inhibition with 2A11 is similar to that obtained with the exogenous β-glucan, glucan phosphate (GluP), or laminarin (unpublished data). Samples have been normalized to their respective controls, indicated by similar shading, and the data shown are from a representative experiment and are the average of triplicates with error bars indicating the standard deviation. (B) Both 2A11 and 5C6 can inhibit the binding of opsonized FITC-labeled zymosan, although the absolute values vary depending on the level of opsonization. The simultaneous addition of both antibodies, however, always had an additive inhibitory effect. To demonstrate the variation in the level of opsonization and its effects on the inhibition obtained with the antibodies, the data shown are the average of the means of triplicates from three independent experiments, and the error bars indicate the standard error of the mean.

Figure 5.

Soluble β-glucans block the ability of 2A11 to detect Dectin-1 on Tg-elicited peritoneal macrophages. Flow cytometric analysis of the surface levels of Dectin-1 and CR3 before and after the addition of glucan phosphate (GluP) or laminarin (Lam), with or without brief warming at 37°C to induce receptor internalization. Cells were stained with isotype-control (IgG2b) or receptor-specific mAbs, in the presence and absence of the exogenous glucans, as indicated above the panels. No difference in 2A11 staining was observed when the cells were warmed without glucan phosphate or laminarin (unpublished data).