Real-time imaging of mast cell degranulation in vitro and in vivo.

Mast cells undergo degranulation in response to various stimuli and rapidly release pre-formed mediators present in secretory granules, leading to immediate-type allergic reactions. Mast cell degranulation is commonly detected and quantified in vitro by measuring histamine or ß-hexosaminidase released to culture medium. However, this type of assay cannot monitor degranulation of individual cells in real time, and it is not suitable for in vivo detection of degranulation. At the aim of real time imaging of mast cell degranulation at single cell level, we here developed a fluorescent protein-based indicator of degranulation, designated immuno-pHluorin (impH). When expressed in mast cells, impH is located in the membrane of secretory granules and non-fluorescent under homeostatic conditions while it turns fluorescent following degranulation, due to the pH change inside of granules during exocytosis. impH enabled us to detect polarized degranulation within one single cell when mast cells were stimulated via the small area of cell surface. Transplantation of impH-expressing mast cells into mast cell-deficient mice demonstrated that impH could function as a real-time indicator of degranulation in vivo. Thus, impH is a useful tool for imaging of mast cell activation and degranulation in vitro and in vivo, and may be applied for screening of reagents regulating mast cell degranulation.

Large particulate allergens can elicit mast cell-mediated anaphylaxis without exit from blood vessels as efficiently as do small soluble allergens.

Anaphylaxis is a rapid-onset, life-threatening allergic reaction in that IgE, mast cells and histamine are commonly involved. It can be experimentally induced in IgE-sensitized animals by intravenous injection of corresponding allergens, and the sign of anaphylactic reaction can be detected within minutes after allergen challenge. However, it remains puzzling why the anaphylactic reaction can be initiated in vivo so quickly, considering that allergens are delivered into the blood circulation while mast cells reside within peripheral tissues but not in the blood circulation. To address this issue, we compared two different forms of the same allergen, small soluble and large particulate ones, in their ability to induce anaphylaxis in IgE-sensitized mice. In contrast to our expectation, particulate allergens could induce anaphylaxis as quickly and efficiently as did soluble allergens, even though they remained inside of blood vessels. In vivo imaging analysis suggested the direct interaction of intravascular particulate allergens and perivascular mast cells across the capillary wall. Taken together with previous report that perivascular mast cells can capture IgE in the blood circulation by extending cell processes across the vessel wall, our findings imply that blood-circulating allergens, regardless of their size, can stimulate mast cells without exit from blood vessels, by means of cross-linking IgE on mast cell processes inserted into the vessel lumen, and hence initiate anaphylactic reaction so quickly.