Fig 1.
Time Course of Mast Cell Association of AgNPs
(A) Representative Transmission Electron Microcopy (TEM) image demonstrating AgNP shape and size. (B) Representative TEM images of mast cells following exposure to 20 nm AgNPs over time. Mast cells were treated with AgNPs (25 μg/ml) for 10, 20, 30 and 60 min and AgNPs uptake by mast cells was assessed. Arrow indicates AgNPs that were being taken up by a mast cell (inset). A representative image was obtained from at least 5 different images.
Table 1.
Characterization of 20nm citrate-coated AgNPs in water and HEPES buffer
Fig 2.
Mast cell degranulation following exposure to AgNPs
Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. (A) Cells were pre-treated with the SR-B1 inhibitor Blt-2 (50 μM), SR-B1 specific antibodies (1:100 dilution) 30 min prior to AgNP (25 μg/ml) exposure for 1h and release of β-hexosaminidase was assessed. (B) Cells were sensitized with anti-DNP IgE overnight and then exposed to AgNPs (25 μg/ml) for 1 h and release of β-hexosaminidase was assessed. (C) Cells were pre-treated with Blt-2 (50 μM) for 30 min then activated with either DNP (30 min) or AgNP (1 h) and release of β-hexosaminidase was assessed. (D) Representative immunoblots for global p-Tyr and p-Ser/Thr following DNP (100 ng/ml) or AgNPs (25 μg/ml) exposure. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)
Fig 3.
Calcium signal in mast cell following exposure to AgNPs
Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. (A) Mast cells degranulation was measured following exposure to AgNPs in the presence and absence of calcium. (B–left panel) Cells were stained with Fluo-4 AM (5 μM) and mean fluorescence intensity was assessed before (baseline NT control, solid line) and after exposure to ionomycin (1 μM) or AgNPs (50 μg/ml) (dotted line) for 2 min. (B–right panel) A representative graph of 3 independent experiments showing fold change of mean fluorescence intensity relative to NT control. (C) Cells were pre-treated with the CRAC calcium channels inhibitor Synta (10 μM) 30 min prior to AgNP (25 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from indicated groups (p≤0.05)
Fig 4.
PLC and PI3K signaling in response to AgNPs
Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. Cells were pre-treated with (A) the PLCγ inhibitor U73122 (1 μM) or (B) the PI3K inhibitor wortmannin (100 nM) 30 min prior to AgNP (25 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed. (C) Representative immunoblots for p-PI3K in samples pretreated with or without Blt-2 (50 μM) and followed by AgNP exposure for 1 h. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)
Fig 5.
Involvement of other signaling pathways in response to AgNPs
Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. Cells were pre-treated with (A) the sphingosine kinase inhibitor DMS (1 μM) or (B) the PKC inhibitor Ro31-8220 (10 μM) for 30 min prior to AgNP (25 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)
Fig 6.
Confirmation of BMMCs results in RBL-2H3
(A) Cells were pre-treated with Blt-2 (50 μM) 30 min prior to AgNP (50 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed into supernatants. (B) Mast cells degranulation was measured following exposure to AgNPs (50 μg/ml) in the presence and absence of calcium. (C) Cells were pre-treated with the indicated inhibitors 30 min prior to AgNP (50 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed into supernatants. (D) Cells were treated with AgNPs (25 or 50 μg/ml) for 1, 6, and 24 h and cell viability was assessed by measuring the conversion of MTS into formazan. (E) Representative immunoblots for p-PLCγ and p-PI3K of mast cell in the presence or absence of DNP (100 ng/ml) for 5 min or AgNPs (25 μg/ml) for 5 and 30 min. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)
Fig 7.
A schematic representation of proposed signaling pathway involved in activation of mast cells by AgNPs
We propose that AgNPs interact with SR-B1 leading to recruitment of PDZK1 (SR-B1 adaptor protein), which activates downstream signaling cascade involving PI3K and PLCγ. Inositol 1,4,5-triphosphate (IP3), which is released following activation of PLCγ, interacts with its receptor IP3R on smooth endoplasmic reticulum (SER) leading to the release of Ca2+ from ER stores. As a result of a drop in Ca2+ levels in SER, the CRAC Ca2+ channels (cell membrane) are activated leading to influx of extracellular Ca2+. Increasing intracellular Ca2+ levels ([Ca2+]i) is ultimately culminated in mast cell degranulation. PKC: Protein kinase C; IP3 (InsP3): inositol triphosphate; PtdIns(4,5)P2: phosphatidylinositol-4,5-bisphosphate (PIP2); PtdIns(3,4,5)P3: phosphatidylinositol-3,4,5-Triphosphate (PIP3); PIP2 and PIP3 are membrane phospholipids; DAG: diacylglycerol. cSrc: cellular sarcoma (protein tyrosine kinase); TRPC: transient receptor potential cation channels.