The localization of phospholipase A2 in the secretory granule.

A heat-resistant phospholipase A2 has been detected in the secretory granules of the mast cell [Chock, Rhee, Tang and Schmauder-Chock (1991) Eur. J. Biochem. 195, 707-713]. By using ultrastructural immunocytochemical techniques, we have now localized this enzyme to the matrix of the secretory granule. Like the cyclo-oxygenase [Schmauder-Chock and Chock (1989) J. Histochem. Cytochem. 37, 1319-1328], this enzyme also adheres tightly to the ribbon-like granule matrix components. The results from Western-blot analysis suggest that it has a molecular mass of about 14 kDa. The localization of the phospholipase A2, the presence of a phospholipid store with millimolar concentrations of calcium and the localization of the enzymes of the arachidonic acid cascade make the secretory granule a natural site for lipid-mediator synthesis. The packaging of phospholipase A2, together with its substrate and the components of the arachidonic acid cascade, in the secretory granule represents a physical arrangement by which the initiation of the cascade and the release of mediators can be directly linked to the stimulation of cell-surface receptors.

Ultrastructural localization of tumour necrosis factor-alpha.

The application of an antibody against tumour necrosis factor-alpha (TNF) to thin sections of plastic-embedded mouse tissue has identified sites of TNF activity in normal and endotoxin-treated C3N/HeN mice. Prior to endotoxin treatment, TNF was observed in the secretory granules of the antibacterial Paneth cell and one type of crypt endocrine cell. Four hours after endotoxin treatment, these two types of intestinal cell were found to have degranulated. In addition, endotoxin treatment resulted in the appearance of TNF in the secretory granules of all eosinophils, neutrophils and monocytes in the bone marrow, spleen, lung and the proximal intestine. TNF was also observed in the internal elastic lamina (IEL) of arterioles. These results suggest that the process of TNF induction specifically targets the immune system and the vasculature. An invasive stimulus, such as circulating endotoxin, can provoke the immune cells to be armed with TNF. That same stimulus may cause arteriole smooth muscle cells to secrete TNF. TNF secretion in the presence of arteriole smooth muscle cells may play a role in the adjustment of arteriole tone. In the venules, TNF may be responsible for platelet and neutrophil accumulation which leads to embolism formation.

Mast cell growth factor enhances multilineage hematopoietic recovery in vivo following radiation-induced aplasia.

Based on in vitro studies, mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has been implicated as an important hematopoietic regulator, especially in the presence of additional hematopoietic cytokines. Since hematopoietic regeneration follows sublethal radiation-induced hematopoietic injury and is thought to be mediated by endogenously produced cytokines, the ability to accelerate recovery from radiation-induced hematopoietic hypoplasia was used to evaluate in vivo effects of MGF administration. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of 60Co radiation followed by subcutaneous administration of either saline or 100, 200, or 400 micrograms/kg/d recombinant murine MGF on days 1 to 17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC), and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF accelerated hematopoietic recovery at the 100 and 200 micrograms/kg/d doses. The 100 micrograms/kg/d dose accelerated recovery of only GM-CFC, while the 200 micrograms/kg/d dose accelerated CFU-S, GM-CFC, WBC, and PLT recoveries. In contrast, hematopoietic recovery was delayed in mice receiving the 400 micrograms/kg/d dose. These studies demonstrate the in vivo dose-dependent ability of MGF to accelerate multilineage hematopoietic regeneration following radiation-induced hematopoietic hypoplasia. They also document detrimental effects of providing "supraoptimal" doses of this growth factor and suggest caution in dose-escalation trials in humans.

Prostaglandin E2 localization in the rat ileum.

The application of anti-prostaglandin E2 immunoglobulin to plastic-embedded thin sections of the rat ileum has permitted the localization of prostaglandin E2 in this tissue. In agreement with the published data (Chock & Schmauder-Chock (1988), Schmauder-Chock & Chock (1989)), the results also suggest the presence of an arachidonic acid cascade in the granules of various secretory cells of the gut. Since antibody labelling was found within the secretory granules of connective tissue mast cells, goblet cells, and Paneth cells, the presence of the arachidonic acid cascade in these granules is implied. The appearance of prostaglandin E2 over the non-cellular internal elastic lamina of arterioles suggests that it may have been secreted along with the elastin. The even distribution of prostaglandin throughout the cytoplasm of the erythrocyte is consistent with the concept that this cell scavenges the eicosanoid from the circulation. These data further link the secretory granule to the production of eicosanoids and therefore illustrate the potential sources of prostaglandins in the rat ileum.

Linking phospholipase A2 to phospholipid turnover and prostaglandin synthesis in mast cell granules.

Rapid incorporation of exogenous arachidonic acid into phospholipid has been detected in conjunction with eicosanoid synthesis by purified mast cell granules [Chock, S. P. & Schmauder-Chock, E. A. (1988) Biochem. Biophys. Res. Commun. 156, 1308-1315]. The species of phospholipid formed has now been identified primarily as phosphatidylinositol. A calcium-dependent phospholipase A2 has also been detected in the secretory granule. This enzyme, like the cyclooxygenase [Schmauder-Chock, E. A. & Chock, S. P. (1989) J. Histochem. Cytochem. 37, 1319-1328], appears to bind tightly to the granule matrix components. It is heat resistant and requires millimolar concentrations of calcium for optimal activity. It prefers phosphatidylinositol over phosphatidylcholine as substrate. Since the granule contains a large amount of phospholipid, the action of this phospholipase A2 can provide the required substrate for the arachidonic acid cascade. These findings provide the basis for linking phospholipase A2 to the production of eicosanoids during granule exocytosis. Since the granule also contains both an active acylating system that can rapidly reacylate lysophosphatidylinositol to form phosphatidylinositol, and an active phospholipase A2 which hydrolyzes phosphatidylinositol, a rapid turnover involving the fatty acid at the sn-2 position of phosphatidylinositol may occur. These findings are consistent with our postulation that the secretory granule is the source and/or the cause of many of the early biochemical events associated with the process of stimulus-secretion coupling.

A new model for the mechanism of stimulus-secretion coupling.

By combining ultrastructural techniques with a biochemical approach to study the mechanism of mast cell stimulus-secretion coupling and by using purified secretory granules to confirm those early biochemical events which originate from within the secretory granule, a new model for the mechanism of secretory granule exocytosis has emerged. This model not only provides the mechanism by which an activated granule can achieve fusion with the plasma membrane, but it also provides the rationale for the linking of the various early biochemical events to the process of granule activation and thus to exocytosis. Although we still do not understand how the 'activating signal', which results from the stimulation of cell surface receptors, can be conveyed to the granule to cause its activation, we are certain that this 'signal' must cause an influx of water into the matrix of the target granule. This influx of water is what initiates the granule activation process. The major intragranular events which are triggered by this water influx include: (i) de novo membrane assembly; (ii) protein proteolysis; (iii) release of arachidonic acid from matrix-bound phospholipid by phospholipase A2; (iv) initiation of the arachidonic acid cascade and the synthesis of eicosanoids; (v) rapid phospholipid turnover; and (vi) the discharge of matrix materials into the cytoplasm of the activated cell via the fusion of de novo generated vesicles with the perigranular membrane. The ejection of some matrix contents which may include histamine, Ca2+, calmodulin, protease, the products of the arachidonic acid cascade and the products of phospholipid turnover into the cytosole, may serve to turn on the various metabolic machineries needed to initiate a cellular recovery phase.

New membrane assembly in IgE receptor-mediated exocytosis.

The presence of excess membrane has been observed in the secretory granules of mast cells activated via the physiological mechanism of IgE receptor-mediated exocytosis. This excess membrane is the result of a de novo assembly from phospholipid, cholesterol, and other membrane components stored in the matrix of the quiescent granule. Following receptor stimulation, membrane bilayer structures of varying size and shape can be seen in the subperigranular membrane space where the perigranular membrane has lifted away from the granule matrix. Vesicles as small as 25 nm in outer diameter are frequently found beneath the perigranular membrane at the site of granule fusion. Membrane in the form of elongated vesicles, tubes, or sheets has also been observed. The wide variation in size and shape of the newly assembled membrane may reflect the spontaneity of the entropy-driven membrane generation process and the fluid characteristic of the biological membrane in general. Fusion of the newly assembled membrane with the perigranular membrane enables the activated granule to enlarge. This rapid expansion process of the perigranular membrane may be the principal mechanism by which an activated granule can achieve contact with the plasma membrane in order to generate pore formation. The fact that new membrane assembly also occurs in the IgE receptor-mediated granule exocytosis, supports our observation that de novo membrane generation is an inherent step in the mechanism of mast cell granule exocytosis. Whether new membrane assembly is a common step in the mechanism of secretory granule exocytosis in general, must await careful reinvestigation of other secretory systems.

Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell.

The application of anti-cyclo-oxygenase and anti-prostaglandin E2 immunoglobulins to A23187-stimulated rat connective tissue mast cells has permitted the localization of cyclooxygenase activity (prostaglandin H2 synthetase) and the site of prostaglandin E2 (PGE2) formation in the secretory granules. Because binding was carried out after stimulation but before dehydration and embedding, we have limited the loss of these antigens due to normal degradation and to aqueous and solvent washes. As this method permits labeling of exposed cell surfaces, only granules that have been exteriorized can be labeled. Contrary to what might have been expected, no labeling was associated with plasma membranes or with any portion of damaged cells. Antibodies to PGE2 were bound evenly over the surface of the granule matrix, whereas antibodies to cyclo-oxygenase appeared to be bound to strands of proteo-heparin projecting from the surface of the granule matrix. Where granule matrix had become unraveled and dispersed, label appeared to adhere throughout the ribbon-like proteo-heparin strands. These results support our previous conclusion that the secretory granule is the site of the arachidonic acid cascade during exocytosis.

Phospholipid storage in the secretory granule of the mast cell.

A spontaneous membrane assembly process has been postulated to account for the rapid perigranular membrane enlargement which occurs during mast cell secretory granule activation. This process requires the presence of a phospholipid store in the quiescent granule. By using purified granules with intact membranes we have determined the total phospholipid content of the average quiescent granule. The results suggest that the average quiescent granule contains sufficient phospholipid to sustain at least a trebling of its perigranular membrane surface area during activation. As much as two-thirds of the total cellular phospholipid is found in the granules, and since a large portion of this phospholipid is extruded into the extracellular space along with the granule matrix during exocytosis, it is implied that this phospholipid can serve as the substrate for the formation of the lipid-derived mediators of inflammation.

Synthesis of prostaglandins and eicosanoids by the mast cell secretory granule.

The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of the eicosanoid production with the release of histamine have prompted us to examine whether the secretory granule may also serve as both the source as well as the site of prostaglandin synthesis during exocytosis. By exposing the contents of purified granules to exogenous arachidonic acid at neutral pH, we observed the rapid formation of many eicosanoids. The presence of prostaglandins E2, D2 and F2a were identified. The kinetics of E2 formation was also followed. The localization of the arachidonic acid cascade to the secretory granule explains why the production of eicosanoids is so intimately tied to the process of granule exocytosis.

Selective association and transport of Campylobacter jejuni through M cells of rabbit Peyer's patches.

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.

Mechanism of secretory granule exocytosis: can granule enlargement precede pore formation?

Secretory granules have been observed to swell during the process of exocytosis. Swelling is an indication of osmotic stress. The probable role of osmotic pressure in facilitating membrane fusion makes it necessary to determine whether granule membrane 'swelling' can occur prior to its fusion with the plasma membrane (pore formation) in the process of exocytosis. By subjecting adjacent thin and semi-thin sections of an activated granule to ultrastructural examination for membrane enlargement, and to metachromatic staining for verification of pore formation it is concluded that the perigranular membrane can indeed enlarge prior to pore formation. However, the degree of membrane enlargement can far exceed the limit of 2-3% stretching allowed under normal osmotic stress for a membrane bilayer. Such an extensive membrane enlargement, which takes place in the mechanism of exocytosis, cannot be achieved without being accompanied by the insertion of additional membrane.

Evidence of de novo membrane generation in the mechanism of mast cell secretory granule activation.

Evidence which suggests the occurrence of a rapid new membrane assembly has been observed in the secretory granules of the rat peritoneal mast cell during the early stage of granule activation. The rapid insertion of these newly generated vesicles enables the perigranular membrane of the activated granule to enlarge and expand prior to fusion with the plasma membrane and/or with the neighboring granule membranes. If the newly inserted membrane represents "specialized fusogenic membrane patches", then the presence of de novo membrane generation as an integral step in the mechanism of mast cell granule exocytosis would constitute a fail-safe mechanism in histamine release.