Acidic pH is essential for maintaining mast cell secretory granule homeostasis

Abstract

It has been recognized for a long time that the secretory granules of mast cells are acidic, but the functional importance of maintaining an acidic pH in the mast cell granules is not fully understood. Here we addressed this issue by examining the effects of raising the pH of the mast cell secretory granules. Mast cells were incubated with bafilomycin A1, an inhibitor of the vacuolar-type ATPase proton pump. Supporting a role of vacuolar-type ATPase in mast cell granule acidification, bafilomycin A1 treatment caused a robust increase in granule pH. This was accompanied by marked effects on mast cell granules, including swelling and acquisition of vacuole-like morphology. Moreover, bafilomycin A1 caused extensive, yet selective effects on the granule content. These included aberrant processing of pro-carboxypeptidase A3 and a reduction in the level of intracellular histamine, the latter being accompanied by an increase in extracellular histamine. In contrast, the storage of β-hexosaminidase, a prototype lysosomal hydrolase known to be stored in mast cell granules, was not affected by abrogation of granule acidification. Moreover, bafilomycin A1 caused a reduction of tryptase enzymatic activity and appearance of tryptase degradation products. Tryptase inhibition prevented the formation of such degradation products, suggesting that the pH elevation causes tryptase to undergo autoproteolysis. Taken together, our findings reveal that mast cell secretory granule homeostasis is critically dependent on an acidic milieu.

Figure 1

Effect of bafilomycin A1 on mast cell viability and granule acidification. (a) Mast cells (bone marrow-derived mast cells; BMMCs), 10 000 cells/well, were incubated in the absence or presence of bafilomycin A1 at the indicated concentrations and time periods, followed by assessment of viability using a fluorogenic viability assay test. Residual viability is given relative to vehicle-treated control cells. (b) BMMCs (0.44 × 10⁶ cells) were incubated for 3 h in the absence or presence of bafilomycin A1 at the indicated concentrations. Next, Lysosensor Blue DND-167 (1 μM) was added to the cells followed by flow cytometry analysis. Results are representative of two individual experiments. Results are given as mean values±S.D. (n=3). *P<0.05, **P<0.01, ***P<0.001

Figure 2

Impaired acidification causes aberrant granule morphology in mast cells. Mast cells (BMMCs; 0.5 × 10⁶ cells) were incubated in the absence or presence of 20 nM bafilomycin A1 at the indicated time periods, followed by preparation of cytospin slides (50 000 cells/slide) and May Grünwald/Giemsa staining. Images representative of three independent experiments are shown

Figure 3

Transmission electron microscopy (TEM) analysis of the effects of bafilomycin A1 on mast cell morphology. Mast cells (4 × 10⁶ cells) were treated for 48 h with 20 nM bafilomycin A1 or vehicle, followed by TEM analysis. The upper panels show representative overviews of cells, whereas the lower panels represent enlarged images. Original magnifications: 5000 × (upper panels), 9000 × (middle panels), 40 000 × (lower panels)

Figure 4

Effect of bafilomycin A1 on β-hexosaminidase storage and immunological release. (a) Mast cells (0.5 × 10⁶ cells) were incubated either in the absence or presence of bafilomycin A1 at the indicated concentrations and during the indicated time periods. Cells were pelleted and intracellular content of β-hexosaminidase activity was measured. (b) Mast cells (1 × 10⁶ cells/ml) were incubated for 24 h with the indicated concentrations of bafilomycin A1. Cells were then washed twice with PBS and further incubated in presence of either IgE alone (0.1 μg/ml) or IgE (0.1 μg/ml) + DNP (0.5 μg/ml). After an additional incubation period (60 min), the amount of released β-hexosaminidase activity was measured in the cell culture supernatant to monitor the extent of mast cell degranulation. Results are representative of three individual experiments. Results are given as mean values±S.D. (n=3). **P<0.01, ****P<0.001

Figure 5

Histamine storage in mast cells is dependent on acidic granule pH. Mast cells (1 × 10⁶ cells/ml) were incubated for 24 or 48 h with 0, 5 or 15 nM bafilomycin A1. Histamine content in the cell pellets (a) and in the supernatants (b) was measured by ELISA. (c) Mast cells were incubated with bafilomycin A1 at the concentrations and time periods indicated. Cells were pelleted by centrifugation, followed by RNA isolation and qPCR analysis for content of mRNA coding for Hdc. The results are representative of two individual experiments. Results are given as mean values±S.D. (n=4). **P<0.01, ***P<0.001, ****P<0.001; ns, not significant

Figure 6

Aberrant processing of CPA3 in bafilomycin A1-treated mast cells. Mast cells (1 × 10⁶ cells/ml) were incubated with bafilomycin A1 at the concentrations and time periods indicated. (a) Cells were then recovered by centrifugation, followed by preparation of cell protein extracts and western blot analysis for CPA3 processing products using an anti-CPA3 antibody. The migration position of proCPA3 and the fully processed form (active) of CPA3 are indicated. Note the appearance of an intermediate processing form of CPA3 (Int) in cells treated with bafilomycin A1. β-actin was used as loading control. (b) Cell extracts from non-treated and bafilomycin A1-treated mast cells were analyzed for levels of CPA3 activity using a chromogenic substrate. Results are representative of five individual experiments. Results are given as mean values±S.D. (n=4). (c) RNA from non-treated and bafilomycin A1-treated mast cells was analyzed by qPCR for content of mRNA coding for the CPA3 gene (Cpa3). Results are given as mean±S.D. (n=3). The results are representative of three individual experiments

Figure 7

Impaired granule acidification reduces the granule content of enzymatically active tryptase. Mast cells (1 × 10⁶ cells/ml) were incubated with bafilomycin A1 at the concentrations and time periods indicated. (a) Cell extracts were prepared and analyzed for tryptase activity using a chromogenic substrate. Results are representative of five individual experiments. Results are given as mean values±S.D. (n=3). (b) Cell extracts were prepared and subjected to western blot analysis using an anti-tryptase (mMCP6) antibody. β-actin was used a loading control. (c) RNA from vehicle-treated, bafilomycin A1-treated and nafamostat-treated mast cells (0.5 × 10⁶ cells) was analyzed by qPCR for content of mRNA coding for the tryptase gene (Mcpt6). Results are given as mean±S.D. (n=3). Results are representative of three individual experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.001; ns, not significant

Figure 8

In situ detection of tryptase activity after bafilomycin A treatment. Cytospin slides were prepared from cultures of wild-type (a) and tryptase-deficient (mMCP6^−/−) and were stained with fast garnet for detection of trypsin-like activity. Mast cells were either non-treated (control) or incubated with 10 nM bafilomycin A1 for various time periods as indicated, followed by preparation of cytospin slides and fast garnet staining

Figure 9

Impaired granule acidification causes autoproteolysis of tryptase. Mast cells (1 × 10⁶ cell/ml) were incubated with bafilomycin A1, either alone or in combination with Nafamostat mesylate (tryptase inhibitor) at the concentrations and time periods indicated. (a) Cell extracts were prepared and analyzed for tryptase activity using a chromogenic substrate. Results are representative of three individual experiments. Results are given as mean values±S.D. (n=3). (b) Cell protein extracts were prepared and subjected to western blot analysis using an anti-tryptase (mMCP6) antibody. β-actin was used a loading control. (c,d) Quantification of the intensities of bands representing intact (c) and cleaved (d) mMCP6. The results are representative of three individual experiments. Results are given as mean±S.D. (n=3). **P<0.01, ****P<0.001; ns, not significant