Spatiotemporally and mechanically controlled triggering of mast cells using atomic force microscopy - PubMed (original) (raw)

Spatiotemporally and mechanically controlled triggering of mast cells using atomic force microscopy

Kenneth K Hu et al. Immunol Res. 2014 May.

Abstract

Mast cells are thought to be sensitive to mechanical forces, for example, coughing in asthma or pressure in "physical urticarias." Conversion of mechanical forces to biochemical signals could potentially augment antigenic signaling. Studying the combined effects of mechanical and antigenic cues on mast cells and other hematopoietic cells has proven difficult. Here, we present an approach using a modified atomic force microscope cantilever to deliver antigenic signals to mast cells while simultaneously applying mechanical forces. We developed a strategy to concurrently record degranulation events by fluorescence microscopy during antigenic triggering. Finally, we also measured the mechanical forces generated by mast cells while antigen receptors are ligated. We showed that mast cells respond to antigen delivered by the atomic force microscopy cantilever with prompt degranulation and the generation of strong pushing and pulling forces. We did not discern any relationship between applied mechanical forces and the kinetics of degranulation. These experiments present a new method for dissecting the interactions of mechanical and biochemical cues in the signaling responses of immune cells.

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Figures

Fig. 1

Fig. 1

a) Wide field time-lapse imaging of IgE-sensitized C57 mast cells loaded with calcium sensitive dye Fluo-4 stimulated with soluble DNP-HSA at 100 ng/mL. Antigen was added at 30 s. b) Fluo-4 versus time for two independent cells (red and blue) after stimulation with DNP-HSA. Fluorescence was normalized to intensity at time 0. c) Same plot showing responses of two un-sensitized C57 cells after stimulation with DNP-HSA.

Fig. 2

Fig. 2

Wide-field imaging of IgE-sensitized C57 cells transduced with pHluorin-LAMP1 construct. Cells stimulated with 100 ng/mL DNP-HSA. Fluorescence intensity of pHluorin reporter is shown in false-color.

Fig. 3

Fig. 3

a) Schematic illustrating setup of AFM cantilever for localized stimulation of mast cells and for monitoring responses. b) Wide-field time lapse imaging of IgE sensitized C57 cells transduced with pHluorin-LAMP1 construct (false colored). DNP-HSA coated AFM cantilever tip was brought into contact with the mast cell surface in the middle of the cell area with a force trigger of 600 pN and held with constant z-position for 10 min.

Fig. 4

Fig. 4

Summarization of the kinetics of degranulation for cells contacted at force triggers of a) 200 pN, b) 600 pN, and c) 2 nN. Degranulation is measured in accumulated “blips” (pHluorin dequenching events). Each color is an independent mast cell.

Fig. 5

Fig. 5

Representative traces of the mechanical forces generated by mast cells following forceful contact with antigen-coated AFM cantilever. Degranulation is also shown, as measured in accumulated “blips” (pHluorin dequenching events). Three different force triggers when contacting the cells with antigen were tested to assess the sensitivity of mast cells to mechanical forces while undergoing receptor ligation. a and b) 200 pN; c and d) 600 pN; e and f) 2 nN.

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