Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs) (1)
Excessive NET production or a dysregulation of NET clearance have been negatively implicated in an ever growing number of diseases, many of them associated with considerable morbidity and socioeconomic impact such as chronic inflammatory diseases like rheumatoid arthritis, systemic lupus erythematosus, chronic obstructive pulmonary disease (COPD) and psoriasis, ischemia-reperfusion injury after myocardial infarction, thrombosis, impaired wound healing, preeclampsia, and cancer.
NET research heavily relies on the isolation of fresh neutrophils from donors and their ex vivo stimulation. For murine neutrophils, the most frequently used supplements are FCS and BSA, though some activators of NETosis such as CaI and LPS are largely being studied in serum-free medium. These differences may greatly influence the outcome of NET-experiments, as albumin may bind proteins like lipopolysaccharides (LPS). Serum-free culture conditions may allow a certain degree of spontaneous NET formation.
Isolation of Mouse Neutrophils
Mouse neutrophils were isolated from 6 to 10-week-old wild type C57BL/6J mice (male and female mice, equally distributed between groups). The blood was collected from the retroorbital venous plexus under full anesthesia with isoflurane and collected into the 15mM EDTA containing BSA/PBS-solution. The isolation was performed according to previously published standard protocols. After centrifugation, the cells were layered on top of a 10:1-diluted percoll gradient consisting of 78, 69, and 52% layers in PBS. Afterwards, erythrocytes were lysed with deionized water followed by a washing-step. Then, cells were resuspended in HBSS and cellular identity as well as purity >95% were confirmed as described above.
Addition of BSA 0.5% as well as HSA 0.5% to RPMI/ HEPES significantly inhibited spontaneous NETosis and completely abolished NET formation mediated by CaI and LPS at 10, 25, and 100μg/ml, whereas stimulation in pure RPMI/ HEPES led to a robust induction of NETs. The release of DNA-bound NE after stimulation with CaI or LPS (100μg/ml) was inhibited by addition of 0.5% HSA, but was not reduced in response to PMA. When 10μg/ml LPS were used for the induction of NETosis, 0.5% hiFCS were sufficient to reduce NET formation dramatically from 95 to 5%. When 100μg/ml LPS were employed, a significant reduction of NETosis to 22% could only be reached by adding 2% hiFCS.
Stimulation of neutrophils by 100nM PMA was not influenced by either BSA or HSA.
For LPS from Pseudomonas aeruginosa, a minimum concentration of 25 μg/ml was needed to induce significant NET formation in murine neutrophils (23%) even without addition of media supplements. In general, LPS-induced NET formation was lower compared to that of human neutrophils at the same LPS concentration. Similar to human neutrophils, addition of 0.5% BSA, or 2% hiFCS sufficed to completely inhibit LPS-induced NETosis.
A dose-dependent increase of anisotropy with LPS, indicating that both BSA and HSA do indeed bind LPS. Even at concentrations of HSA and BSA that were lower than those used in our NETosis experiments, a clear binding between partners could be observed, which would be higher at higher HSA/BSA concentrations.
Approximately half of the experiments in human (51 %) and mouse (56 %) neutrophils were performed without any supplement.
NETosis induction by LPS relies heavily on integrin receptors such as MAC-1 and this outside-in signaling may be influenced by the addition of large proteins and certainly also of serum components which contain a mixture of active components that can engage integrin receptors. In contrast, PMA is a direct activator of protein kinase C (PKC), a vital and relatively far downstream step in most pathways leading to NETosis. Therefore, outside-in signaling could be expected to be less crucial during the activation cascade, at least for human neutrophils.
The used anticoagulant can significantly influence pre-activation, calcium levels, and number of obtained neutrophils. EDTA would be a better anticoagulant.