A flow cytometry technique to study intracellular signals NF-kappaB and STAT3 in peripheral blood mononuclear cells

Abstract

Background: Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-kappaB (nuclear factor kappaB) and STAT (signal transducer and activator of transcription) families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-kappaB and STAT rely on specific ELISAs, Western Blots and--most recently described--flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification.

Results: The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells). To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1beta, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA); the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-kappaB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry.

Conclusion: In response to IL1beta, or IL10, the levels of phosphorylated NF-kappaB and STAT3--respectively--increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages--but, interestingly, not T-lymphocytes (in the context of PBMCs)--responded significantly to sCD40L by increasing phosphorylated NF-kappaB.

Figure 1

Western blot and ELISA analysis of phosphorylated NF-κB and STAT3 in PBMCs. Western Blot of pNF-κB (A, C) and pSTAT3 (E) in PBMCs nuclear extract was observed with or without stimuli: IL1β (50 ng/mL) (A), sCD40L (50 ng/mL) (C) or IL10 (100 ng/mL) (E) for various periods (0 to 30 min). Data were representative of five experiments. pNF-κB and pSTAT3 were detected by ELISA (n = 5) in activated PBMCs nuclear extracts and the time dependant effect of the PBMCs stimulus was observed. PBMCs were stimulated for various periods of time (0 to 30 min) with or without IL1β (B), sCD40L (D) or IL10 (F). Statistical significance (wilcoxon paired test; p < 0.05) was represented by an asterisk (*). Data represented the mean (± SD) of five experiments.

Figure 2

Flow cytometry of simultaneous phosphorylated NF-κB and STAT3 expression from B-cells, T-lymphocytes and monocytes/macrophages stimulated by IL1β (control). Cytogram (A and C) depicted one experiment and showed the isotype control (A1 and C1 for B-cells, A4 and C4 for T-lymphocytes and A7 and C7 for monocytes/macrophages). Cytogram (A) showed the pNF-κB translocation of B-cells (A2, A3), T-lymphocytes (A5, A6) and monocytes/macrophages (A8, A9) with (A3, A6, A9) or without (A2, A5, A8) stimulus IL1β (50 ng/mL) for 30 min. Cytogram (C) showed the pSTAT3 translocation of B-cells (C2, C3), T-lymphocytes (C5, C6) and monocytes/macrophages (C8, C9) with (C3, C6, C9) or without (C2, C5, C8) stimulus IL1β (50 ng/mL) for 30 min. Data were representative of seven experiments. Summary of flow cytometry analysis (n = 7) of percentage of phosphorylated NF-κB (B) and phosphorylated STAT3 (D) activation (versus untreated) from B-cells, T-lymphocytes and monocytes/macrophages. The graphs represent the difference in percentage of phosphorylated nuclear factor between stimulated and untreated cells. Statistical significance (wilcoxon paired test; p < 0.05) was represented by an asterisk (*). Data represented the mean (± SD) of seven experiments.

Figure 3

Flow cytometry of simultaneous phosphorylated NF-κB and STAT3 expression from B-cells, T-lymphocytes and monocytes/macrophages. Summary of flow cytometry analysis (n = 7) of percentage of phosphorylated NF-κB (A) and phosphorylated STAT3 (B) activation (versus untreated) from B-cells, T-lymphocytes and monocytes/macrophages. PBMCs were stimulated for the appropriate time and concentration of sCD40L (A) and IL10 (B) (as identified previously). The graphs represent the difference in percentage of phosphorylated nuclear factor between stimulated and untreated cells. Statistical significance (wilcoxon paired test; p < 0.05) was represented by an asterisk (*). Data represented the mean (± SD) of seven experiments.