Siglec-E promotes β2-integrin-dependent NADPH oxidase activation to suppress neutrophil recruitment to the lung

Abstract

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.

Keywords: Lipopolysaccharide (LPS); Lung Injury; NADPH Oxidase; Neutrophil; Reactive Oxygen Species (ROS); Siglec-E.

FIGURE 1.

Selective defect in β2-integrin-dependent ROS in siglec-E-KO mice. A, ROS-dependent chemiluminescence shown as relative light units (RLU) using bone marrow cells from WT and siglec-E KO mice stimulated as indicated. Solid traces, WT; dashed traces, siglec-E KO; gray traces in the fibrinogen (Fb)-treated group indicate TNF-α priming. B, luminescence responses in siglec-E KO cells expressed as a percentage of values seen with WT cells; mean ± S.D. from triplicate wells from at least two independent experiments. IC, immune complex; OPS zym, serum-opsonized zymosan.

FIGURE 2.

Siglec-E-dependent activation of β2-integrin-triggered ROS production requires sialic acid recognition. A, luminescence responses of TNF-α-primed WT and siglec-E KO cells plated onto fibrinogen (A) or unprimed cells plated onto poly-RGD (B)-coated wells both pretreated with or without sialidase in sodium acetate buffer, pH 5. Luminescence responses are expressed as the percentage of TNF-α-primed WT cells plated onto buffer-treated fibrinogen (A) or WT response to buffer-treated poly-RGD (B). Data show means ± S.D. from triplicate wells from seven independent experiments.

FIGURE 3.

β2-Integrin-triggered Akt activation is promoted by siglec-E and is required for ROS production. A, Western blots were prepared from WT and siglec-E KO bone marrow cells plated on wells precoated with fibrinogen for 5 or 20 min at 37 °C and probed with the indicated antibodies. Data are representative of two independent experiments. B, ROS-dependent chemiluminescence using bone marrow cells from WT mice pretreated with the Akt inhibitor, MK2206, or DMSO as a control. Luminescence responses are expressed as the percentage of DMSO-treated controls from MK2206-treated, TNF-α-primed cells plated onto fibrinogen (Fb) or stimulated with PDBu. Data show means ± S.D. from triplicate wells from three to four independent experiments.

FIGURE 4.

Siglec-E-dependent production of neutrophil ROS in lungs of LPS-exposed mice. Groups of mice were exposed to aerosolized LPS. 3 h later, mice were injected intravenously with L-012 and anesthetized, and excised lungs were bioimaged from 5 to 10 min. A, chemiluminescent signals from representative lungs of five mice per genotype. Color bar depicts luminescent light emission intensity (photons/s/cm²/sr). B, upper panel shows luminescence signals normalized to neutrophil counts obtained from collagenase-digested lung (lower panel). Data are expressed as means ± S.E., n = 14 per group. p values were calculated using the Mann Whitney U test.

FIGURE 5.

Blockade of NADPH oxidase in vivo reverses siglec-E-dependent suppression of neutrophil recruitment. A, mice were pretreated with apocynin or DMSO vehicle as control and then exposed to aerosolized LPS. After 3 h, lung tissue was collagenase-digested, and total cells and neutrophils were enumerated. Data are expressed as scatterplots with the bars depicting the means, n = 9 per group from two independent experiments. p values were calculated using the Mann Whitney U test. B, WT mice were treated as in A. Left panel shows luminescence signals from in vivo bioimaging after normalizing for neutrophil counts obtained from collagenase-digested lung tissue (right panel). Data are expressed as means ± S.E., n = 4 per group. p values were calculated using the Mann Whitney U test. C, bone marrow cells were pretreated with the indicated concentrations of apocynin or with DMSO vehicle control for 20 min, and luminescence was measured using wells precoated with fibrinogen. Data are presented as means ± S.D. representative accumulated light emission (relative light units (RLU))over time from triplicate wells from duplicate samples.