Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells

Abstract

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

FIGURE 1.

Syt2 is expressed in mature MCs. Peritoneal MCs purified from P16 Syt2^+/+ and Syt2^−/− mice. A, immunoblot of cell lysates with anti-Syt2 antibody (αSyt2). GAPDH immunoreactivity was used as a loading control. B, immunohistochemistry of cytospins using the same antibody (bar, 4 μm). We demonstrated the presence of Syt2 in mature wild type MCs and the absence of Syt2 in Syt2^−/− MCs.

FIGURE 2.

Syt2 deficiency impairs the response in passive cutaneous anaphylaxis. Ears of MC-deficient W^sh/W^sh mice reconstituted intradermally with Syt2^+/+ or Syt2^−/− BMMCs were sensitized with anti-DNP IgE (experimental ear) or polyclonal IgE (control ear). After intravenous challenge with DNP-HSA in Evans Blue, the amount of extravasated dye was measured as absorbance at 610 nm. Nonreconstituted MC-deficient animals failed to respond, and there was a marked difference between mice reconstituted with Syt2^−/− and Syt2^+/+ BMMCs. (Error bars represent S.E.; n = 9; *, p < 0.01.)

FIGURE 3.

Syt2 deficiency does not alter MC development, migration into tissues, or morphology. Representative images of samples obtained from P16 Syt2^+/+ and Syt2^−/− mice are shown. An in-depth morphometric analysis can be found in Table 1. A, fluorescent microscopy of ear sections (bar, 50 μm). MCs were labeled with FITC-avidin (green) and cell nuclei with Hoechst (blue). Background tissue was visualized as autofluorescence in the red channel. Syt2^+/+ and Syt2^−/− mice had similar densities of MCs in the dermis of their ears. B, cytospins of peritoneal lavages stained with Wright-Giemsa (bar, 100 μm). Insets depict higher power magnifications of representative MCs (bar, 5 μm). Syt2^+/+ and Syt2^−/− mice had similar numbers of MCs with similar gross morphology in their peritoneal cavities. C, transmission EM of peritoneal MCs (bar, 1 μm). MCs from Syt2^+/+ and Syt2^−/− mice had similar size and surface complexity, and similar number, size, and density of electron-dense granules. D, flow cytometry of BMMCs. After 6 weeks in medium supplemented with SCF and IL-3, BMMCs from Syt2^+/+ and Syt2^−/− mice had the same proportion of Kit⁺/FcϵRIα⁺ cells (% in brackets), and similar surface expression of both receptors (gray lines near both axes represent mean ± S.D. of fluorescence intensity). E, transmission EM of BMMCs (bar, 1 μm). Syt2^+/+ and Syt2^−/− BMMCs were indistinguishable.

FIGURE 4.

Syt2 deficiency affects MC-regulated exocytosis. BMMCs from Syt2^+/+ and Syt2^−/− mice were incubated with anti-DNP IgE and then exposed to DNP-HSA. Secreted products were measured in supernatants before and 1 h after stimulation. Total β-hexosaminidase enzymatic activity was also measured in cell lysates. A, secretion of β-hexosaminidase with different doses of DNP-HSA. Values are expressed as the difference in enzymatic activity in supernatants between stimulated and unstimulated cells in percent of total activity in cell lysates. The maximal response and greatest difference were found at 100 ng/ml DNP-HSA (n = 3). This concentration was used in all subsequent experiments. B, secretion of β-hexosaminidase at 100 ng/ml DNP-HSA (n = 8). C, histamine in supernatants before (open bars) and after (black bars) challenge (n = 5). Release of products stored in MC secretory granules and secreted via regulated exocytosis was significantly decreased in Syt2^−/− BMMCs. (Error bars represent S.E.; *, p < 0.01.)

FIGURE 5.

Syt2 deficiency does not affect other MC secretory mechanisms. Secreted products were measured in supernatants of BMMCs from Syt2^+/+ and Syt2^−/− mice incubated with anti-DNP IgE before and at different time points after addition of DNP-HSA. A, TNF-α before (open bars) and after (6 h, black bars) challenge (n = 5; color legend applies also to C and D). B, time course of IL-4 secretion (n = 3 per time point). C, PGD₂ and D, LTC₄ 30 min after challenge (n = 4 for both). Secretion dependent on constitutive exocytosis or independent of exocytosis was not affected in Syt2^−/− BMMCs. (Error bars represent S.E.)

FIGURE 6.

Syt2 deficiency does not affect [Ca²⁺]_i transients or MC exocytic competence. A, representative [Ca²⁺]_i traces of isolated peritoneal MCs incubated with anti-DNP IgE and stimulated by addition of DNP-HSA (arrows). Gray lines represent the mean baseline and maximal [Ca²⁺]_i used to calculate Δ[Ca²⁺]_i. B, average Δ[Ca²⁺]_i showed no difference between the two genotypes (n = 9). C, representative C_m traces (upper panels) measured in peritoneal MCs dialyzed with 5 μm GTPγS via the patch pipette. The [Ca²⁺]_i in both cells was buffered to 700 nm (lower panels). D, average ΔC_m demonstrated that degranulation can be induced in Syt2^−/− MCs under these conditions (n = 8; error bars represent S.E.).