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Caspase-3 is a main executioner of apoptotic cell death. The general notion is that, in viable cells, caspase-3 is found as a cytosolic inactive proenzyme and that caspase-3 activation is largely confined to processes associated with cell death. In this study, we challenge this notion by showing that enzymatically active caspase-3 is stored in viable mast cells. The enzymatically active caspase-3 was undetectable in the cytosol of viable cells, but was recovered in subcellular fractions containing secretory granule-localized proteases. Moreover, active caspase-3 was rapidly released into the cytosolic compartment after permeabilization of the secretory granules. Using a cell-permeable substrate for caspase-3, the presence of active caspase-3-like activity in granule-like compartments close to the plasma membrane was demonstrated. Moreover, it was shown that mast cell activation caused release of the caspase-3 to the cell exterior. During the course of mast cell differentiation from bone marrow cells, procaspase-3 was present in cells of all stages of maturation. In contrast, active caspase-3 was undetectable in bone marrow precursor cells, but increased progressively during the process of mast cell maturation, its accumulation coinciding with that of a mast cell-specific secretory granule marker, mouse mast cell protease 6. Together, the current study suggests that active caspase-3 can be stored within secretory compartments of viable mast cells.

Exocytosis is a key event in mast cell functions. By this process, mast cells release inflammatory mediators, contained in secretory granules (SGs), which play important roles in immunity and wound healing but also provoke allergic and inflammatory responses. The mechanisms underlying mast cell exocytosis remained poorly understood. An essential step toward deciphering the mechanisms behind exocytosis is the identification of the cellular components that regulate this process. Because Rab GTPases regulate specific trafficking pathways, we screened 44 Rabs for their functional impacts on exocytosis triggered by the FcεRI or combination of Ca ²⁺ ionophore and phorbol ester. Because exocytosis involves the continuous reorganization of the actin cytoskeleton, we also repeated our screen in the presence of cytochalasin D that inhibits actin polymerization. In this paper, we report on the identification of 30 Rabs as regulators of mast cell exocytosis, the involvement of 26 of which has heretofore not been recognized. Unexpectedly, these Rabs regulated exocytosis in a stimulus-dependent fashion, unless the actin skeleton was disrupted. Functional clustering of the identified Rabs suggested their classification as Rabs involved in SGs biogenesis or Rabs that control late steps of exocytosis. The latter could be further divided into Rabs that localize to the SGs and Rabs that regulate transport from the endocytic recycling compartment. Taken together, these findings unveil the Rab networks that control mast cell exocytosis and provide novel insights into their mechanisms of action.

Human β-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. β-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human β-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature β-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.

Ischemia with subsequent reperfusion (IR) injury is a significant clinical problem that occurs after physical and surgical trauma, myocardial infarction, and organ transplantation. IR injury of mouse skeletal muscle depends on the presence of both natural IgM and an intact C pathway. Disruption of the skeletal muscle architecture and permeability also requires mast cell (MC) participation, as revealed by the fact that IR injury is markedly reduced in c-kit defective, MC-deficient mouse strains. In this study, we sought to identify the pathobiologic MC products expressed in IR injury using transgenic mouse strains with normal MC development, except for the lack of a particular MC-derived mediator. Histologic analysis of skeletal muscle from BALB/c and C57BL/6 mice revealed a strong positive correlation (R(2) = 0.85) between the extent of IR injury and the level of MC degranulation. Linkage between C activation and MC degranulation was demonstrated in mice lacking C4, in which only limited MC degranulation and muscle injury were apparent. No reduction in injury was observed in transgenic mice lacking leukotriene C(4) synthase, hemopoietic PGD(2) synthase, N-deacetylase/N-sulfotransferase-2 (enzyme involved in heparin biosynthesis), or mouse MC protease (mMCP) 1. In contrast, muscle injury was significantly attenuated in mMCP-5-null mice. The MCs that reside in skeletal muscle contain abundant amounts of mMCP-5 which is the serine protease that is most similar in sequence to human MC chymase. We now report a cytotoxic activity associated with a MC-specific protease and demonstrate that mMCP-5 is critical for irreversible IR injury of skeletal muscle.

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE(-/-)) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE(-/-) mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood eosinophilia and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the small intestine, serum levels of mouse MC protease-1 also were lower in parasite-infected IgE(-/-) animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE(-/-) mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo.

Sec14p homology domains are found in a large number of proteins from plants, yeast, invertebrates, and higher eukaryotes. We report that the N-terminal Sec14p homology domain of the human protein tyrosine phosphatase PTP-MEG2 binds phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in vitro and colocalizes with this lipid on secretory vesicle membranes in intact cells. Point mutations that prevented PtdIns(3,4,5)P(3) binding abrogated the capacity of PTP-MEG2 to induce homotypic secretory vesicle fusion in cells. Inhibition of cellular PtdIns(3,4,5)P(3) synthesis also rapidly reversed the effect of PTP-MEG2 on secretory vesicles. Finally, we show that several different phosphoinositide kinases colocalize with PTP-MEG2, thus allowing for local synthesis of PtdIns(3,4,5)P(3) in secretory vesicle membranes. We suggest that PTP-MEG2 through its Sec14p homology domain couples inositide phosphorylation to tyrosine dephosphorylation and the regulation of intracellular traffic of the secretory pathway in T cells.

Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells.