Mast cell activation on patterned lipid bilayers of subcellular dimensions

Abstract

T Cells, B cells, and other specialized cells in the immune system are activated by clustering of antigenic receptors on their plasma membrane surfaces; rat basophilic leukemia (RBL) mast cells which express cell surface immunoglobulin E (IgE) receptors and operate in the allergic response serve as a prototypic model. To investigate signaling pathways of the cellular response, a versatile system is being developed for presenting antigens on micron scale patterns of lipid bilayers that are prepared with a polymer-based wet lift-off method. A supported lipid bilayer is formed on a silicon substrate after photolithographic patterning of a polymer and before the polymer is mechanically peeled away in solution. This forms well-defined bilayer patterns that can contain haptenated and fluorescent lipids and can be used to simulate a biological substrate for cellular receptor engagement and response. For this model system, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino] hexanoyl] (ammonium salt) (DNP-cap-DPPE) was used for specific binding of anti-DNP IgE. IgE's bound to mast cell surface receptors (FcεRI) aggregate and cluster over the patterned features if they are significantly smaller than the cells. For patterned features that are cell sized or larger, cells adhere to the silicon surface with receptors polarizing toward the edge of the patterned lipid. RBL cells with specifically engaged IgE-receptors are activated to undergo striking morphological changes that can be analyzed with fluorescence or scanning electron microscopy. This novel method for patterning antigen-functionalized lipids provides spatial control down to micron resolution for the antigenic stimulus while retaining the features of a plasma membrane substrate, including dynamics and variable composition. This method offers an alternative to microcontact printing with some enhanced capabilities for cellular immunological studies.