The impact of neutrophil extracellular trap from patients with systemic lupus erythematosus on the viability, CD11b expression and oxidative burst of healthy neutrophils

Abstract

Background: NET (neutrophil extracellular trap) has been shown to directly influence inflammation; in SLE (systemic lupus erythematosus), it is reportedly a plausible cause for the broken self-tolerance that contributes to this pathology. Meanwhile, the role of NET is not easily explicable, and there is a serious discrepancy in the role of NET in SLE pathology and generally inflammation; in particular, the interactions of neutrophils with NET have been rarely inspected. This study evaluates the effect of NET on neutrophils in the context of SLE. The neutrophils were incubated by the collected NET (from SLE patients and healthy controls) and their expression of an activation marker, viability and oxidative burst ability were measured.

Results: The level of cell mortality, CD11b expression and the oxidative burst capacity were elevated in NET-treated neutrophils. Also, the elevation caused by the SLE NET was higher than that produced by the healthy NET.

Conclusion: The decreased neutrophil viability was not due to the increase in apoptosis; rather, it was because of the augmentation of other inflammatory cell-death modes. The upregulation of CD11b implies that NET causes neutrophils to more actively contribute to inflammation. The increased oxidative burst capacity of neutrophils can play a double role in inflammation. Overall, the effects induced by NET on neutrophils help prolong inflammation; accordingly, the NET collected from SLE patients is stronger than the NET from healthy individuals.

Keywords: Damage-associated molecular patterns (DAMP); Inflammation; Neutrophil extracellular trap (NET); Neutrophils; Systemic lupus erythematosus (SLE).

Fig. 1

The detection of neutrophil-derived NET by immunofluorescence microscopy. To observe NET, activated (A photographs) PMNs or resting PMNs (B photographs) were fixed and immune-stained with specific anti-NE antibody followed by a fluorescent secondary antibody (red, middle photographs). The samples were stained with DAPI to counter stain the nucleus and extracellular DNA in blue (upper photographs). The overlay of the two channels is shown in the photographs at the bottom. All photos presented neutrophils from a control sample. NE: neutrophil elastase

Fig. 2

The Detection of dead cells by flow cytometry. The blood granulocytes isolated were cultured with the NET of either the healthy subjects or the patients for about 4 h, then stained with FITC-annexin-V and PI to differentiate between the viable, necrotic, early and late apoptotic cells. Plots B and C represent the same sample double-stained after coculture with normal (b) and SLE neutrophil-derived NET (c); plot A shows the same sample unstained. An-V: annexin-V, PI: Propidium Iodide

Fig. 3

The effects of NET on the death of granulocytes. The death of neutrophils was evaluated after 4 h-contact with the NET of both healthy subjects and patients. In the case of early apoptosis, no significant differences were observed between the coculture with NET, NCM or nothing. Regarding late apoptosis, a significant increase was detected in the cocultures with either normal or SLE NET in comparison to the control cocultures (the cocultures contained no NET or NCM). The level of late apoptosis was also significantly higher in the cocultures of SLE NET than the cocultures of normal NET. The percentage of necrotic neutrophils after contact with the NET of both healthy subjects and patients for about 4 h increased significantly compared to the neutrophils in contact with no NET. The difference in the percentage of necrotic cells was statistically insignificant between the SLE NET-contained cocultures and the normal NET-contained cocultures. NCM: NET control medium, which refers to the medium that was collected from the unstimulated healthy and SLE neutrophils in the NET-inducing experiments

Fig. 4

The upregulated CD11b expression following exposure to NET. Plot A shows the overlaid histogram of the unstimulated (as the negative control) and the LPS-stimulated neutrophils plus the normal-NET-treated and SLE-NET-treated LPS-stimulated neutrophils of the same sample, and their corresponding MFIs (Mean Fluorescence Intensities) are also presented in the table above the figure. The isotype control histogram is also shown in black. Plots B-D represent the unstimulated, LPS-stimulated, and normal NET-treated neutrophils of the same sample

Fig. 5

The impact of NET on the activation of granulocytes. After the designated treatments (with or without healthy and SLE NET), the upregulation of CD11b expression on the cell surface of LPS-challenged neutrophils was compared. Although the differences between the untreated and either the patient or healthy NET-treated neutrophils were significant, the difference between CD11b expression in untreated and NCM-treated granulocytes was not significant. NET pretreatment alone (without LPS stimulation) did not change the CD11b expression on the neutrophils. NCM: NET control medium; refers to the collected medium from the unstimulated healthy and SLE neutrophils in the NET-inducing experiments. MFI: Mean Fluorescence Intensity

Fig. 6

The effect of NET on the respiratory burst of neutrophils. a Following treatments with or without NET, PMA-stimulated granulocytes were stained by DHR and run on the flow cytometer to determine their OB capacity. The OB ability of the incubated neutrophils with either healthy and SLE NET was enhanced compared to the unincubated cells; the difference between the the two latter groups was also significant. The OB capacity of neutrophils did not change with NET pretreatment alone (with no stimulation). b The mean fluorescence of the activated neutrophils stained by DHR (details in the Methods section) increased because of ROS production as a result of OB. The plot shows the overlaid histograms of the unstimulated, PMA-stimulated, NET-treated unstimulated, normal-NET-treated PMA-stimulated, and SLE-NET-treated PMA-stimulated neutrophils from the same sample plus their corresponding OBI (Oxidative Burst Index) in the table. The unstained neutrophils are also shown. NCM or NET control medium refers to the medium that was collected from the unstimulated healthy and SLE neutrophils in the NET-inducing experiments