JNK signaling during IL-3-mediated differentiation contributes to the c-kit-potentiated allergic inflammatory capacity of mast cells.

Mast cells are leukocytes that mediate various aspects of immunity and drive allergic hypersensitivity pathologies. Mast cells differentiate from hematopoietic progenitor cells in a manner that is largely IL-3 dependent. However, molecular mechanisms, including the signaling pathways that control this process, have yet to be thoroughly investigated. Here, we examine the role of the ubiquitous and critical mitogen-activated protein kinase signaling pathway due to its position downstream of the IL-3 receptor. Hematopoietic progenitor cells were harvested from the bone marrow of C57BL/6 mice and differentiated to bone marrow-derived mast cells in the presence of IL-3 and mitogen-activated protein kinase inhibitors. Inhibition of the JNK node of the mitogen-activated protein kinase pathway induced the most comprehensive changes to the mature mast cell phenotype. Bone marrow-derived mast cells differentiated during impaired JNK signaling expressed impaired c-kit levels on the mast cell surface, first detected at week 3 of differentiation. Following 1 wk of inhibitor withdrawal and subsequent stimulation of IgE-sensitized FcεRI receptors with allergen (TNP-BSA) and c-kit receptors with stem cell factor, JNK-inhibited bone marrow-derived mast cells exhibited impediments in early-phase mediator release through degranulation (80% of control), as well as late-phase secretion of CCL1, CCL2, CCL3, TNF, and IL-6. Experiments with dual stimulation conditions (TNP-BSA + stem cell factor or TNP-BSA alone) showed that impediments in mediator secretion were found to be mechanistically linked to reduced c-kit surface levels. This study is the first to implicate JNK activity in IL-3-mediated mast cell differentiation and also identifies development as a critical and functionally determinative period.

Attenuation of allergen-mediated mast cell activation by rosemary extract (Rosmarinus officinalis L.).

Mast cells are immune sentinels and a driving force in both normal and pathological contexts of inflammation, with a prominent role in allergic hypersensitivities. Crosslinking of FcεRI by allergen-bound IgE Abs leads to mast cell degranulation, resulting in an early-phase response and release of newly synthesized pro-inflammatory mediators in the late-phase. The MAPK and NF-κB pathways are established as critical intracellular mechanisms directing mast cell-induced inflammation. Rosemary extract (RE) has been shown to modulate the MAPK and NF-κB pathways in other cellular contexts in vitro and in vivo. However, the effect of RE on mast cell activation has not been explored, and thus we aim to evaluate the potential of RE in modulating mast cell activation and FcεRI/c-kit signaling, potentially via these key pathways. Primary murine mast cells were sensitized with anti-TNP IgE and stimulated with cognate allergen (TNP-BSA) under stem cell factor (SCF) potentiation while treated with 0-25 µg/ml RE. RE treatment inhibited phosphorylation of p38 and JNK MAPKs while also impairing NF-кB transcription factor activity. Gene expression and mediator secretion analysis showed that RE treatment decreased IL-6, TNF, IL-13, CCL1, and CCL3, but major component polyphenols do not contribute to these effects. Importantly, RE treatment significantly inhibited early phase mast cell degranulation (down to 15% of control), with carnosic acid and carnosol contributing. These findings indicate that RE is capable of modulating mast cell functional outcomes and that further investigation of the underlying mechanisms and its potential therapeutic properties in allergic inflammatory conditions is warranted.