Classical monocytes, the non-inflammatory phenotype, display the most robust pro-inflammatory response to LPS in vitro after immediate cytometric analysis

BACKGROUND: Circulating monocytes have been classified according to expression of certain membrane markers. CD14 (Monocyte identifying Toll-Like Receptor) and CD16 (FcyRIII co-receptor, marker of inflammatory monocytes) were used to define 3 subpopulations of circulating monocytes with different attributes in terms of inflammatory and phagocytic behavior. There are contradictory reports regarding response of circulating monocytes to pro-inflammatory or non-inflammatory stimuli in vitro. Here we aimed to analyze the phenotypic changes in circulating monocytes when stimulated with pro and non-inflammatory stimuli. METHODS: Whole blood from 9 healthy donors was extracted and studied within 90 minutes using erythrocyte lysis protocol. Monocyte subpopulations were directly measured using flow cytometry without PBMC Ficoll extraction method. Pro-inflammatory interleukin IL-1β was measured by intracellular cytometry. Whole blood- extracted monocytes were stimulated using LPS and IL-4 as previously described. Changes against non-stimulated (N-S) populations were statistically analyzed. RESULTS: Compared to N-S, LPS-stimulated monocytes display a singular behavior, peaking intracellular IL-1β in parallel with CD14+CD163-/CD14+CD163+ ratio. CD163 shows positive correlation with levels of IL-1β. In t-SNE (T-distributed Stochastic Neighbor Embedding) analysis, after LPS stimulation, subpopulation CD14+CD16-CD163-, containing classical monocytes, show a higher number of IL-1β+ cells. CONCLUSION: Whole blood extracted early monocyte analysis avoids previously described artifacts in membrane protein expression seen when using PBMC Ficoll protocol. Using this method, we were able to show that classical monocytes display the most intense response to LPS. Additionally, CD163 appears to be a suitable addition to CD14-CD16 classification to improve its performance.