Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Abstract

SummaryEosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34 + cell GM-CSFRa expression. Functionally, CB CD34 + cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

IL-4 and IL-13 Differentially Regulate TLR-Induced Eosinophil-Basophil Differentiation Of Cord Blood CD34+ Progenitor Cells

“…+ cells, mononuclear cells from the centrifuged pellet were selected using the EasySep Negative Selection Human Progenitor Cell Enrichment Cocktail with CD41 depletion (Stem Cell Technologies) as previously described [11]. The purity of CD34 + cells was between 85-90%.…”

“…+ at a plating concentration of 2610 4 cells/35 mm610 mm culture dish (Falcon Plastics, CA) in duplicate [11]. In addition to growth factors IL-3 (1 ng/ml), IL-5 (1 ng/ml) and GM-CSF (10 ng/ml) and the endotoxin LPS (10 mg/ml), cultures were supplemented with IL-4 or IL-13 (50 ng/mL) to assess their role in Eo/B CFU formation.…”

“…+ cells stimulated with LPS from our previous kinetic evaluations [11]. For receptor neutralization studies, cells were pre-incubated with anti-IL-4Ra or IL-13Ra1 (R&D Systems) for 15 min before the addition of IL-4 or IL-13 and LPS.…”

“…For receptor neutralization studies, cells were pre-incubated with anti-IL-4Ra or IL-13Ra1 (R&D Systems) for 15 min before the addition of IL-4 or IL-13 and LPS. Following the incubation, cells were analyzed using phosphoflow cytometry [11] with a sequential multiparameter gating strategy that has previously been shown to accurately enumerate CD34 + progenitor cells ( Figure 1A) [7][8][9][10]. CD34 + blast cells (R4), are identified as cells with high expression of CD34, low to intermediate expression of CD45, and scatter properties characteristic of lymphoblastic cells.…”

“…+ cells via autocrine activation of MAPK [11] and atopic at-risk infants have elevated T H 2 cytokines in their CB [12,13], we were interested in what effect these cytokines may have on LPS-induced Eo/B CFU [10]. Relatedly, maternal cytokines (which can be transferred to the CB) have been shown to play instructive roles in fetal immune development.…”