Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function.

Our objective was to establish an experimental model of a self-sustained and bistable extracellular signal-regulated kinase 1/2 (ERK1/2) signaling process. A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 4 h. Repeated stimulation leads to sustained activation of ERK1/2 but not Jun N-terminal protein kinase (JNK), p38, or STAT6. The ERK1/2 activation lasts for 3 to 7 days and depends upon a positive-feedback mechanism involving Sprouty 2. Overexpression of Sprouty 2 induces, and its genetic deletion abrogates, ERK1/2 bistability. Sprouty 2 directly activates Fyn kinase, which then induces ERK1/2 activation. A genome-wide microarray analysis shows that the bistable phospho-ERK1/2 (pERK1/2) does not induce a high level of gene transcription. This is due to its nuclear exclusion and compartmentalization to Rab5+ endosomes. Cells with sustained endosomal pERK1/2 manifest resistance against growth factor withdrawal-induced cell death. They are primed for heightened cytokine production. Epithelial cells from cases of human asthma and from a mouse model of chronic asthma manifest increased pERK1/2, which is associated with Rab5+ endosomes. The increase in pERK1/2 was associated with a simultaneous increase in Sprouty 2 expression in these tissues. Thus, we have developed a cellular model of sustained ERK1/2 activation, which may provide a mechanistic understanding of self-sustained biological processes in chronic illnesses such as asthma.

Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma.

BACKGROUND: Existing asthma models develop tolerance when chronically exposed to the same allergen. OBJECTIVE: We sought to establish a chronic model that sustains features of asthma long after discontinuation of allergen exposure. METHODS: We immunized and exposed mice to a combination of single, double, or triple allergens (dust mite, ragweed, and Aspergillus species) intranasally for 8 weeks. Airway hyperreactivity (AHR) and morphologic features of asthma were studied 3 weeks after allergen exposure. Signaling effects of the allergens were studied on dendritic cells. RESULTS: Sensitization and repeated exposure to a single allergen induced tolerance. Sensitization to double and especially triple allergens broke through tolerance and established AHR, eosinophilic inflammation, mast cell and smooth muscle hyperplasia, mucus production, and airway remodeling that persisted at least 3 weeks after allergen exposure. Mucosal exposure to triple allergens in the absence of an adjuvant was sufficient to induce chronic airway inflammation. Anti-IL-5 and anti-IL-13 antibodies inhibited inflammation and AHR in the acute asthma model but not in the chronic triple-allergen model. Multiple allergens produce a synergy in p38 mitogen-activated protein kinase signaling and maturation of dendritic cells, which provides heightened T-cell costimulation at a level that cannot be achieved with a single allergen. CONCLUSIONS: Sensitivity to multiple allergens leads to chronic asthma in mice. Multiple allergens synergize in dendritic cell signaling and T-cell stimulation that allows escape from the single allergen-associated tolerance development.

A phosphosite screen identifies autocrine TGF-beta-driven activation of protein kinase R as a survival-limiting factor for eosinophils.

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase Cepsilon, and glycogen synthase kinase 3 alpha and beta. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2alpha are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-beta in eosinophils. TGF-beta directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-beta Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phospho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-beta and that this could be modulated by a Th2 microenvironment in vivo.