Application of SILAC Labeling to Primary Bone Marrow-Derived Dendritic Cells Reveals Extensive GM-CSF-Dependent Arginine Metabolism

Abstract

Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the quantitative proteomic technique of metabolic labeling (SILAC) into the field of DC research would therefore be highly beneficial. To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of (13)C/(15)N-labeled amino acids. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, ∼20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of (15)N from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of ≥90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.

The Early Dendritic Cell Signaling Induced by Virulent Francisella tularensis Strain Occurs in Phases and Involves the Activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 In the Later Stage

“…Suspension cells were harvested on the day 9 of cultivation. SILAC labeling of BMDCs was performed as previously described (12). All experiments using mice were performed in accordance with guidelines of the Animal Care and Use Ethical Committee of the Faculty of Military Heath Sciences, University of Defense, Czech Republic (project no.…”

Systems-level effects of ectopic galectin-7 reconstitution in cervical cancer and its microenvironment

“…4b). Unlabeled L-proline was added to a final concentration of 2.61 mM (300 mg/L) to prevent arginine-proline conversion [20]. …”

Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells

“…Since human monocyte-derived dendritic cells exhibit high inter-donor variation, BMDCs from inbred C57BL/6 mice were used. The cells were labelled ex vivo with stable isotopes (SILAC) 12 and advantage was taken of the use of a non-enzymatic CyaA-AC - toxoid as negative control. CyaA-AC - is unable to elevate cAMP in cells 28 , 29 but retains the full capacity to bind the cell surface receptor CR3, to permeabilize cellular membrane and to elicit Ca 2+ influx into cells and K + efflux from cells, like intact CyaA.…”

“…The generation and SILAC labelling of C57BL/6 murine BMDCs was carried out as previously described 12 and outlined in detail in the Supplementary methods section. BMDCs were generated from bone marrow progenitors isolated from femurs and tibias of 6- to 8-week-old female C57BL/6 mice.…”

“…GM-CSF induces catabolism of 13 C/ 15 N-labelled arginine, yielding heavy arginine-derived proline and 15 N isotope incorporation, thus skewing estimation of SILAC ratios. Therefore an optimized SILAC-labeling medium was used to suppress these effects, as described earlier 12 .…”