Mast cells (MC) are critical effector cells in asthma and allergy. They develop in situ from bone marrow-derived committed progenitors (PrMC), which traffic from the circulation to various tissues. Mature MC can rapidly redistribute to mucosal surfaces and to secondary lymphoid tissues in response to inflammatory stimuli. Neither the homing of PrMC nor the redistribution of mature MC are well-understood, but both processes are pertinent to a variety of immune responses and diseases. An in vitro culture system permits the first derivation of virtually pure human (h)PrMC from cord blood mononuclear cells, using the triad of recombinant human stem cell factor (SCF), IL-6, and IL-10. These hPrMC express 4 functional cell surface chemokine receptors; CXCR2, CXCR4, CCR3, and CCR5. With continued culture, the hPrMC develop into mature hMC that are functionally and immunochemically similar to their in vivo counterparts. These hMC express CCR3, but lack CXCR2, CXCR4, or CCR5 on their surface. Preliminary study now reveals that both mature cultured hMC and nasal polyp hMC posses intracellular stores of both CXCR4 and CCR3. CCR3 redistributes to the hMC plasma membrane within 1 h of IgE-dependent activation, while strong de novo steady-state expression of mRNA encoding CCR7 is induced by 6 h. Thus hMC sequentially alter both their expression and distribution of chemokine receptors in response to activation signals. Based on a yeast 2-hybrid assay to detect interacting proteins, both CCR3 and CXCR4 associate with WAIT (WD-repeat protein associated with integrin tails)-1, a protein that also appears to localize to the granules of hMC under basal conditions. Finally, co-stimulation of hMC with both IgE receptor cross-linkage and the recombinant CCR3 ligand eotaxin-1 selectively amplifies the generation of IL-13 and induces IL-4 production de novo.
Specific Aim 1 focuses on defining the storage site(s) of pre-formed CCR3 and CXCR4 in hPrMC and hMC derived in vitro, modulation of the distribution and function of these receptors in response to IgE-dependent and non-IgE- dependent activation signals, and the functional relevance of the inducible CCR7, which may facilitate the movement of hMC to regional lymph nodes.
Specific Aim 2 focuses on the potential functions of WAIT-1 in controlling the localization and function(s) of CCR3 and CXCR4.
Specific Aim 3 explores the mechanisms by which CCR3 controls cytokine generation by hMC. These studies, carried out in non-transformed hPrMC and hMC, have potential implications for the control of hMC distribution and effector function in asthma.
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