Fig. 1. Mast cell responses differ following DNP or 2NP stimulation of FceRI. (A) Degranulation (as measured by b-hexosaminidase
release) of wild-type (WT) BMMCs after stimulation with indicated
concentrations of DNP or 2NP. ***P < 0.001; two-way analysis of
variance (ANOVA). (B) Leukotriene B4 (LTB4) secretion from BMMCs
induced by treatment with 2NP (3000 ng/ml) is less than that with
DNP (30 ng/ml). **P < 0.01, ***P < 0.001; one-way ANOVA. (C)
Tumor necrosis factor– a (TNFa), interleukin-6 (IL6), and IL13 are
similarly affected at the concentrations of DNP and 2NP indicated
in (B). *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA. (D)
Release of CCL2, CCL3, and CCL4 is increased after stimulation
with 2NP relative to DNP [conditions as in (B)]. **P < 0.01, ***P <
0.001; one-way ANOVA. Data were collected from four to eight individual experiments.
Fig. 2. Characteristics of FceRI clusters differ with
2NP or DNP stimulation of BMMCs. (A) Kymographs
of a mast cell labeled with Alexa Fluor 568–labeled IgE
(as an FceRI marker) as captured by TIRF microscopy.
Static view of IgE-FceRI microcluster dynamics (trajectory)
captured upon cell contact with DNP or 2NP imbedded
in a planar supported lipid bilayer (see supplementary
materials) is shown in two dimensions (x and y) of movement. Movement of receptors clusters with time (t) is also
shown. Sequential image sections for 0.5 to 450 s are
shown in chronological order. Scale bars, 5 mm. (B) The
average number of IgE-FceRI clusters on DNP- or 2NP-
imbedded planar supported lipid bilayer is different.
Red bars are means T SE from 24 individual cells. ***P <
0.001, Student’s t test. (C) Average area of IgE-FceRI clusters on DNP- or 2NP-imbedded planar supported lipid bilayers differs. Red bars are means T SE from 24 individual
cells. ***P < 0.001, Student’s t test. (D) Cumulative probability distribution of the diffusion coefficient of individual
FceRI clusters at any given time from analysis of 7835 to
11,198 FceRI clusters on 24 different cells for DNP and
2NP. (E) Tyrosine phosphorylation with IgE-FceRI microclusters in DNP- or 2NP-treated cells as determined by
intracellular staining with antibodies to phosphotyrosine.
Fluorescence intensity in cross section (white dotted line)
is shown. Localization was analyzed for more than 40 cells
in each condition. Scale bars, 5 mm. *P < 0.05, **P < 0.01,
***P < 0.001; one-way ANOVA. Data are representative
of at least three independent experiments.