Degranulation

Peter M Henson, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado, USA

Many cells, particularly those involved in inflammatory and immunological reactions, exhibit morphologically distinct intracytoplasmic granules. Degranulation is usually taken to describe the process by which these granules or their contents are discharged to the outside of the cell. Accordingly the term is often used synonymously with secretion, discharge or release. Strictly, however, degranulation represents a state in which the granules are no longer visible microscopically, only presumptively a result of discharge, and this discharge might as well be into a phagosome as to the extracellular environment.

In vivo the phenomenon is generally assessed by histological (or ultrastructural) determination of the granularity of the cell. Quantitation here has to involve the techniques of stereology. In isolated cell populations in vitro it is more usually measured by secretion of the granule contents to the outside of the cell. This has the advantage of being amenable to more accurate quantitative assessment and is generally much faster. With mixed cell populations it is clearly important to use a marker that is unique to the cell or granule in question. However, the concept of degranulation came originally from the response of professional phagocytes such as neutrophils or eosinophils to the process of phagocytosis, wherein elegant cinematographic studies in the 1950s showed the dramatic loss of phase-dense cell granules as they were discharged into the phagosome. Subsequent cell fractionation analysis placed this upon a quantitative footing.

Granules are membrane-bounded cytoplasmic storage structures in which the contents (for example, enzymes) are generally thought to exist in a state of latency, requiring changes in pH, release from a matrix, and/or hydration for optimal activity changes that accompany degranulation (into an endosome or to the outside). Cells involved in inflammation and immunological reactions tend to have at least two types of granules, those specific for the given cell type and those that represent lyso-somes, although these latter may contain unique components in given cell types, for example myeloperoxidase and elastase in the 'azurophil' granule of the neutrophil. The granules that are unique to these inflammatory cells include the amine- and protease-containing granules of the mast cell and basophil, the characteristic crystal-containing granule of the eosinophil, the dense granule of the platelet and the 'specific' granules of the neutrophil. Characteristic staining properties of these granules were generally used to identify and name the particular cell - hence neutrophil, basophil and eosinophil. Similarly, the mast cell granules exhibit the characteristic meta-

chromasia with stains such as toluidine blue. Loss of this staining is used to indicate degranulation. It has been suggested that these cell-specific granules are true secretory structures, whose contents are directly released by exocytosis to the cell exterior (as well as into phagosomes in phagocytic cells). They would therefore be analogous to the secretory granules of chromaffin, pancreatic and neurosecretory cells.

By contrast, the modified lysosomal granules in this simplified concept are primarily involved in fusion with endosomes (including phagosomes). However, it is clear that in stimulation of macrophages or neutrophils, lysosomal contents are released to the exterior, although the mechanisms remain obscure. Certainly degranulation of the two major granule types of the neutrophil (whether to the outside or into the phagosome) is under separate control. The 'specific' granules are discharged earlier, leading to teleologic speculation that their largely neutral pH-effective contents have time to act before the pH of the phagosome drops and the acid-optimum contents of the azurophil granule take precedence. Calcium ionophores stimulate specific granule release, sodium or potassium ionophores secretion of the azurophil granules.

Direct exocytosis involves the movement of the granule to the plasma membrane followed by fusion of the membranes and discharge of the contents. A modification of this process is seen in mast cells in which there is an explosive degranulation involving fusion of a small section of the plasma membrane with many granules at once, either expelling the granule contents to the outside or exposing them to the extracellular milieu through a network of channels. Granule contents such as histamine are liberated from the proteoglycan matrix by ion exchange mechanisms (Figure 1). 'Piecemeal' degranulation is a process described for basophils in which the granule contents are apparently transported to the plasma membrane in vesicles. These two cell types can also regranulate, a property which may not be shared with neutrophils or eosinophils. In professional phagocytes, including macrophages, the mechanisms of secretion are less clear. Probably some granules fuse directly with the plasma membrane as they have been clearly demonstrated to do with the phagosome, some extracellular discharge occurs as a result of phagosome-granule fusion before the phagosome is closed to the extracellular environment, and some granule contents may be released as a result of vesicle transport and recycling, particularly from the modified lysosomes. In all these cell types, the degranulation appears to occur only at the sites on the plasma membrane that are stimulated. Interaction with stimuli on surfaces too large to be internalized

Explosive degranulation (e,g. in mast cells)

Piecemeal degranulation

Explosive degranulation (e,g. in mast cells)

Discharge into phagosomes

Piecemeal degranulation

Release due to discharge into developing phagosomes

Figure 1 Suggested mechanisms of degranulation in inflammatory cells.

Discharge into phagosomes

Release due to discharge into developing phagosomes

Figure 1 Suggested mechanisms of degranulation in inflammatory cells.

therefore results in release to the outside along the points of contact with that surface, a process termed colloquially, frustrated phagocytosis. These sites represent a protected environment under the cell which may allow secreted constituents to act on the underlying substrata even in the presence of inhibitors in the surrounding tissue fluid.

The signal transduction pathways involved in degranulation are by no means fully identified but do not seem to differ radically from those involved in other responses of these cells and include phospholipid metabolism, calcium mobilization, G proteins and kinase activation. The fungal metabolites cytochalasins B and D significantly enhance secretion in these cells by mechanisms that are not completely understood. However, the effect is employed widely to study secretion of granule contents and caution should be employed in interpreting mechanistic conclusions when these agents are present.

The accumulation of inflammatory cells in a tissue is not by itself enough to provide host defense or induce injury; it is the secretory responses of these cells that are critical, whether into a bacteria-laden phagosome or into the surrounding tissue structures. The early concept that release of contents resulted from cell lysis is now known to be significantly limited; all these cells undergo active, stimulated and highly regulated secretion of their granule contents, as well as of other materials such as newly synthesized proteins and lipids. The granule contents in particular often include highly destructuve materials, including nonspecific proteases and phospholipases as well as potent toxic cations. Their release is commonly associated with tissue destruction and disease.

See also: Basophils; Eosinophils; Exocytosis; Mast cells; Neutrophils; Phagocytosis; Serotonin.

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