The Differential Role of Extracellular Signal-Regulated Kinases and p38 Mitogen-Activated Protein Kinase in Eosinophil Functions

Abstract

The activation of eosinophils by cytokines is a major event in the pathogenesis of allergic diseases. We have investigated the activation of mitogen-activated protein (MAP) kinases and their functional relevance in eosinophil differentiation, survival, degranulation, and cytokine production. IL-5 induced phosphorylation and activation of extracellular signal-regulated kinases (ERK) and p38 MAP kinases in eosinophils. PD98059, a MAP/ERK kinase inhibitor, blocked phosphorylation of ERK1/2 in a dose-dependent manner. SB202190, a p38 inhibitor, blocked p38-dependent phosphorylation of activating transcription factor-2. To study the importance of the MAP kinases on eosinophil differentiation, we cultured mouse bone marrow cells with IL-3 and IL-5 in the presence of the inhibitors. SB202190 dramatically inhibited eosinophil differentiation by 71%. PD98059 was less potent and reduced eosinophil differentiation by 28%. Both inhibitors marginally inhibited eosinophil survival only at the highest doses. Prolonged incubation of eosinophils with IL-5 induced significant eosinophil-derived neurotoxin release. Both PD98059 and SB202190 nearly completely inhibited (87% and 100% inhibition, respectively) IL-5-stimulated eosinophil-derived neurotoxin release in a dose-dependent manner. Next, we examined the effect of the MAP kinase inhibitors on eosinophil production of the cytokine macrophage-inflammatory protein (MIP)-1α. PD98059 blocked C5a- but not ionomycin-induced MIP-1α production (59% inhibition at 50 μM concentration). In contrast, SB202190 nearly completely inhibited (99%) C5a-induced MIP-1α production. Further, it blocked ionomycin-stimulated production by 66%. Our results suggest that both p38 and ERK1/2 MAP kinases play an important role in eosinophil differentiation, cytokine production, and degranulation. The p38 MAP kinase plays a greater role than ERK1/2 in eosinophil differentiation and cytokine production.

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

“…Although inhibition of p38 MAPK resulted in overall decreases in Eo/B CFU derived from stimulation by three hematopoietic cytokines-findings observed by others [22] -it was the inhibition of ERK 1/2 in the cultures that resulted in the specific inhibition of GM-CSF-and IL-3-responsive Eo/B CFU. Our colony data (Figure 4) not only reinforce the importance of MAPK signalling in eosinophil differentiation [21], but also corroborate our phospho-flow analysis illustrating reduced ERK 1/2 expression after IL-4 stimulation ( Figure 1C). The addition of IL-4 to the cultures not only reduced both GM-CSFand IL-3-responsive Eo/B CFU in the presence of LPS (Figure 2A-B), but reduced LPS-induced ERK 1/2 signalling ( Figure 1C), a finding supported by others [24].…”

“…As shown in Figure 4A-B, the addition of PD98059, a specific pharmacological inhibitor of ERK 1/2 signalling [21], resulted in reduced GM-CSF-(p = 0.009) and IL-3-responsive (p = 0.007) Eo/B CFU formation in the presence of LPS. No differences were observed with IL-5-responsive Eo/B CFU ( Figure 4C).…”

“…Eosinophilopoiesis is a highly regulated process that has been demonstrated to be dependent on ERK signalling of murine BM CD34 + cells [21]. Since we found that IL-4 down-regulated LPS induction of ERK 1/2 ( Figure 1C), we next assessed the effects of blocking ERK 1/2 in CB CD34…”

“…The role of LPS-induced signalling on Eo/B CFU formation was investigated by adding the specific inhibitors to the methylcellulose culture assay: 5 or 50 mM PD98059 (ERK 1/2 inhibitor) [21], or 2 or 20 mM SB203580 (p38 MAPK inhibitor) [22] (Calbiochem, Cambridge, MA) or DMSO vehicle control. These concentrations were found to be non-toxic to cells [11].…”

The Crucial Role of GATA-1 in CCR3 Gene Transcription: Modulated Balance by Multiple GATA Elements in the CCR3 Regulatory Region

“…In addition to chromatin remodeling, BA and/or IL-5 treatment might activate signals that are necessary for CCR3 mRNA induction. Activation of p38 MAPK is known to be involved in BA-mediated differentiation of K562 cells to erythroid cells (45) and also occurs during IL-5-induced differentiation of bone marrow-derived eosinophils (46). Given the intimate relationship between the differentiation of K562 cells and eosinophils and CCR3 expression, MAPK signaling, which is unlikely to be activated by the mere presence of GATA-1 and other transcription factors that influence the expression of eosinophil-specific genes, may be necessary for the expression of CCR3 mRNA to be induced.…”