Lectin site ligation of CR3 induces conformational changes and signaling

Abstract

Neutrophils provide an innate immune response to tissues infected with fungal pathogens such as Candida albicans. This response is tightly regulated in part through the interaction of integrins with extracellular matrix ligands that are distributed within infected tissues. The β(2) integrin, CR3 (CD11b/CD18), is unique among integrins in containing a lectin-like domain that binds the fungal pathogen-associated molecular pattern β-glucan and serves as the dominant receptor for recognition of fungal pathogens by human granulocytes. β-Glucan, when isolated in soluble form, has been shown to be a safe and effective immune potentiator when administered therapeutically. Currently a pharmaceutical grade preparation of β-glucan is in several clinical trials with an anti-cancer indication. CR3 binding of extracellular matrix, carbohydrate, or both ligands simultaneously differentially regulates neutrophil function through a mechanism not clearly understood. Using FRET reporters, we interrogated the effects of soluble β-glucan on intracellular and extracellular CR3 structure. Although the canonical CR3 ligand fibrinogen induced full activation, β-glucan alone or in conjunction with fibrinogen stabilized an intermediate conformation with moderate headpiece extension and full cytoplasmic tail separation. A set of phosphopeptides differentially regulated by β-glucan in a CR3-dependent manner were identified using functional proteomics and found to be enriched for signaling molecules and proteins involved in transcriptional regulation, mRNA processing, and alternative splicing. These data confirm that CR3 is a signaling pattern recognition receptor for β-glucan and represent the first direct evidence of soluble β-glucan binding and affecting a signaling-competent intermediate CR3 conformation on living cells.

FIGURE 1.

CR3 binds extracellular matrix components and yeast β-glucan. A, shown is a schematic of CR3 and representative binding sites within CR3 that permit dual occupancy of protein ligands in the I-domain and β-glucan in the lectin site. ICAM, intercellular adhesion molecule. B, shown is in vitro staining of β-glucan (FITC, top left) on live yeast hyphae interacting with neutrophils stained for CR3 (Texas Red, top right). The merged image indicates colocalization of CR3 to areas with immuno-accessible β-glucan in yellow (bottom right). Shown is a differential interference contrast (DIC) image (bottom left). FITC, Texas Red, and differential interference contrast images were acquired with a Nikon Eclipse TE2000-U epifluorescence microscope coupled to a CoolSNAP HQ CCD camera using NIS elements software. Nikon B-2E/C and Y-2E/C cubes were used for FITC and Texas Red imaging, respectively. Cells were visualized at 37 °C in L-15 medium with 2 mg/ml glucose. A xenon lamp was used to illuminate the cells that were viewed through a 40× Plan APO objective lens.

FIGURE 2.

The use of FRET to report integrin activation; development of a K562 cell line that stably expresses the CR3-FRET activation reporter; loss of FRET in activation reporter with native ligand. A, the integrin activation FRET reporter is shown. In the inactive, bent conformation (left), the cytoplasmic domains of the α and β subunits are closely apposed, allowing for efficient FRET between mCFP and mYFP fused to these C termini. In the active, extended conformation (right), the α and β subunits lose their interactions in the tailpiece leading to a separation greater than 100 Å between the mCFP and mYFP, thereby abolishing FRET. See also supplemental Figs. S1 and S2 for reporter construction and functional characterization. B, shown are bright field (BF), CFP, and YFP images of the K562 cell line stably expressing the CR3-FRET activation construct (K562:CR3-FRETa) acquired with a Nikon Eclipse TE2000-U epifluorescence microscope coupled to a CoolSNAP HQ CCD camera using NIS elements software. Nikon CFP HQ and YFP HQ cubes without emission filters were used for CFP and YFP imaging, respectively. Cells were visualized at 37 °C in L-15 medium with 2 mg/ml glucose. A xenon lamp was used to illuminate the cells through a 33-mm ND4 filter, and the cells were viewed through a 60× oil immersion Plan APO objective lens. Exposure time was 500 ms for both CFP and YFP. C, FRET efficiencies: basal FRET, non-activating treatment with 1 mm Mn²⁺ FRET, activation with 1 mm Mn²⁺ plus 25 μg/ml FgnD or 20 nm phorbol 12-myristate 13-acetate (PMA). Each sample represents analysis of at least 25 cells in three separate experiments. *, p < 0.01.

FIGURE 3.

Soluble β-glucan increases homotypic CR3 interactions and induces CR3 activation. A, K562:CR3-FRETa were incubated with 50 μg/ml sβglu plus or minus 1 mm Mn²⁺ in the presence or absence of 25 μg/ml FgnD. Large clumps, likely due to homotypic interactions, rendered FRET analysis difficult. Bright field images were acquired for each condition with a Nikon Eclipse TE2000-U epifluorescence microscope coupled to a CoolSNAP HQ CCD camera using NIS Elements software. Cells were visualized at 37 °C in L-15 medium with 2 mg/ml glucose. A xenon lamp was used to illuminate the cells through a 33-mm ND4 filter, and the cells were viewed through a 20× Plan APO objective lens. See also supplemental Figs. S1 and S2 for reporter construction and functional characterization. B, shown is the graphic comparison of FRET efficiencies of K562:CR3-FRETa (gray bars) and K562:LFA1-FRETa (white bars) for each treatment, performed in the presence of human serum and in the absence of Mn²⁺. *, p < 0.01 versus untreated.

FIGURE 4.

Measuring exposure of an activation-specific epitope of CR3 in response to stimulation. A, shown is a schematic indicating epitope binding of specific mAbs on inactive and fully extended CR3. B, freshly isolated neutrophils in the presence of donor serum were treated with either 10 nm fMLP, 25 μg/ml FgnD, 50 μg/ml sβglu, or combinations for 15 min. Treated and control cells were labeled with FITC-ICRF44 (which binds to both inactive and extended conformations of CR3) FITC-CBRM1/5 (which binds to an epitope revealed upon activation and extension) (C) or FITC-Fgn (a CR3 I-domain ligand) (D). Data are plotted as the fraction of FITC mean channel fluorescence (MCF) in untreated neutrophils and are from at least four independent experiments and blood donors, with each condition run in duplicate. *, p < 0.01 versus untreated; †, p < 0.01 10 mm fMLP versus 50 μg/ml sβglu.

FIGURE 5.

Measuring extension of the extracellular domain of CR3 by loss of FRET in response to stimulation. A, shown is a schematic demonstrating the loss of FRET between ORB membrane dye and FITC-conjugated mAbs upon the extension of the extracellular domain. B–D, freshly isolated neutrophils in the presence of donor serum were treated with either 10 nm fMLP or 50 μg/ml sβglu for 15 min. Treated and control cells were labeled with FITC-ICRF44 (B), FITC-CBRM1/5 (C), or FITC-Fgn (D). Neutrophils were then incubated with 0, 75, 200, or 400 nm ORB and then analyzed by FACS. Representative data are plotted as the fraction of donor mean fluorescence intensity in the absence of acceptor fluorophore (F_D) to that in the presence of acceptor fluorophore (F_DA) on the y axis (F_D/F_DA) versus ORB mean fluorescence on the x axis to normalize for CR3 expression levels. Plots represent a single blood donor, with duplicate samples for each condition. E, the slope of each line is combined with those of biological replicates to calculate distance ratios used to quantitatively compare relative distance between the FITC-labeled donor on the CR3 receptor and the ORB acceptor in the cell membrane between experimental conditions. *, p < 0.01 versus untreated or Fgn alone; **, p < 0.01 between treatment conditions.

FIGURE 6.

Affinity regulation of integrins. Electron microscopy defined low, intermediate, and high affinity conformers and hypothetical intermediates where the headpiece-tailpiece and α tailpiece-β tailpiece interfaces are destabilized in outside-in or inside-out signaling. Also schematized are the predicted data in response to cytoplasmic domain and extracellular domain FRET reporter systems.

FIGURE 7.

Set of phosphopeptides differentially regulated in response to sβglu in a CR3-dependent manner. Schematic of the phosphopeptides found using SILAC in K562 and K562:CR3 cells to be differentially regulated in response to sβglu in a CR3-dependent manner, organized by their likely cellular functions. See also supplemental Tables S1 and S2 and Fig. S3.