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Updated: Oct 5, 2026
Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
Published on: July 4, 2018
Recovery of purification-associated reduction in antigen-induced histamine release from rat peritoneal mast cells
1Department of Pharmacology, Gifu Pharmaceutical University, Japan. inagakin@gifu-pu.ac.jp
Abstract:
Immunoglobulin E (IgE)-dependent histamine release from purified rat peritoneal mast cells (PMC) is very low in comparison to that from a non-purified preparation (PEC). The reduced histamine release from PMC is recovered or potentiated by reconstitution with separated non-mast cells (NMC). In the present study, further characterization was undertaken to elucidate the mechanisms involved. Sensitized mast cells were recovered from peritoneal cavities of rats, and purified by density gradient centrifugation with Percoll. Effects of NMC reconstitution, membrane fraction of NMC, NMC incubation supernatant, adhesion molecules and extracellular matrix proteins on IgE-dependent histamine release from PMC were examined. IgE-dependent histamine release was significantly potentiated by NMC reconstitution to PMC. The potentiation was dependent on the concentration of NMC reconstituted and reached a plateau after 30 min incubation. Increasing concentration of PMC did not affect the histamine release. Membrane fraction prepared from NMC also potentiated PMC histamine release in a dose-dependent manner. The potentiation reached a plateau in 5 min. Furthermore, incubation supernatant of NMC potentiated PMC histamine release. Antibodies against intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-1, very late activation antigen (VLA)-1, VLA-4 and VLA-6, and fibronectin did not affect the potentiation of PMC histamine release by NMC reconstitution. Fibronectin, laminin and collagen failed to potentiate PMC histamine release. These results indicate that the membrane component(s) of NMC in the rat peritoneal cavity seems to modulate IgE-dependent histamine release from peritoneal mast cells of rats, and that the active molecule(s) may be released from NMC. Adhesion molecules such as ICAM-1, LFA-1 and VLA are not involved.
Insights
Non-mast cells (NMC) from rat peritoneal cavities enhance Immunoglobulin E (IgE)-dependent histamine release from purified mast cells (PMC). This potentiation involves NMC membrane components or released factors, not adhesion molecules.
Area of Science:
- Immunology
- Cell Biology
- Allergy Research
Background:
- Immunoglobulin E (IgE)-mediated histamine release from purified rat peritoneal mast cells (PMC) is significantly lower than from non-purified preparations (PEC).
- Reconstitution of purified PMC with non-mast cells (NMC) restores or enhances histamine release, suggesting a modulatory role for NMC.
Purpose of the Study:
- To elucidate the mechanisms by which non-mast cells (NMC) modulate IgE-dependent histamine release from purified rat peritoneal mast cells (PMC).
- To investigate the involvement of NMC membrane components, released factors, adhesion molecules, and extracellular matrix proteins in this modulation.
Main Methods:
- Purification of rat peritoneal mast cells (PMC) using Percoll density gradient centrifugation.
- Reconstitution of purified PMC with varying concentrations of NMC or NMC-derived fractions (membrane, supernatant).
- Assessment of IgE-dependent histamine release following incubation.
- Testing the effect of antibodies against specific adhesion molecules (ICAM-1, LFA-1, VLA-1, VLA-4, VLA-6) and extracellular matrix proteins (fibronectin, laminin, collagen).
Main Results:
- NMC reconstitution significantly potentiated IgE-dependent histamine release from PMC in a concentration-dependent manner, reaching a plateau after 30 minutes.
- NMC membrane fractions and NMC incubation supernatant also dose-dependently potentiated PMC histamine release, with membrane fractions acting rapidly (plateau in 5 minutes).
- Antibodies against ICAM-1, LFA-1, VLA-1, VLA-4, VLA-6, and fibronectin did not inhibit the potentiation; fibronectin, laminin, and collagen alone did not enhance release.
Conclusions:
- Membrane-bound components and/or soluble factors released from non-mast cells (NMC) modulate IgE-dependent histamine release from rat peritoneal mast cells (PMC).
- The observed potentiation is not mediated by the tested adhesion molecules (ICAM-1, LFA-1, VLAs) or major extracellular matrix proteins.
