Mast cells are activated by antigen crosslinking of IgE-bound high-affinity receptor for IgE (FcεRI) receptors, and aggregation of these receptors results in rapid phosphorylation of tyrosine residues in the ITAMs of β and γ chains by lyn kinase, which leads to recruitment and activation of spleen tyrosine kinase (syk) and fyn. Selleck MLN0128 Both fyn and syk phosphorylate downstream targets, leading to calcium mobilization,

degranulation, arachidonic acid metabolization, and cytokine and chemokine gene transcription 9, 10. As opposed to activation, desensitization is a process in which mast cells are rendered hypo-responsive to an activating challenge, either by exposure to low antigen doses in calcium-depleted conditions 11 or by exposure to incremental

doses of antigen, in the presence of calcium 12, 13. Calcium-depleted conditions cannot be applied to human desensitizations, and few studies have addressed physiological desensitizations, since events occurring in the absence of extracellular calcium may not reflect the same pathways DAPT in vivo as those occurring in the presence of calcium 14. Internalization of FcεRI through progressive crosslinking at low levels of antigen has been postulated as the likely mechanism for cell-surface depletion of IgE and cellular unresponsiveness to specific activating doses of allergen 12. Depletion of molecular targets of activation such as syk has been shown in prolonged antigen desensitization, indicating a universal rather than specific desensitization 15. Based on our previous study 16, we report here a model of mouse BM-derived mast cell (BMMCs) specific rapid desensitization to DNP and OVA antigens in the presence of physiologic levels of calcium. Increasing doses of antigen delivered at fixed time intervals induced a highly specific and prolonged hypo-responsiveness to triggering doses of the desensitizing antigen.

Mast cells desensitized to DNP or OVA demonstrated almost complete inhibition of β-hexosaminidase and pre-formed TNF-α release, calcium flux and arachidonic acid metabolization. They did not release significant amounts of newly generated IL-6 or TNF-α and failed to phosphorylate STAT6 and p38 MAPK. When sensitized to both DNP and OVA antigens, DNP-desensitized cells responded fully to OVA and vice versa. Most importantly, Lepirudin specific rapid desensitization targeted the internalization of antigen/IgE/FcεRI complexes since antigen-specific IgE bound to the α chain of the FcεRI remained at the membrane level. This model may provide support for the specificity and effectiveness of human desensitizations. In order to compare single-dose antigen delivery (activation) with sequential cumulative doses (rapid desensitization), we first assessed the dose response curve to DNP-human serum albumin conjugated (DNP-HSA) antigen, by β-hexosaminidase release, with cells sensitized with anti-DNP IgE (see Fig. 1A).