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The characterization and quantification of antigen-induced Ca2+ oscillations in a rat basophilic leukaemia cell line
J Narenjkar1, S J Marsh, E S Assem
1Department of Pharmacology, University College London, UK.
Insights
Antigenic stimulation causes calcium (Ca2+) oscillations in RBL-2H3 cells, mimicking physiological responses. While not essential for secretion initiation, these Ca2+ oscillations likely enhance mast cell signaling during immune responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Mast cells and basophils play crucial roles in allergic reactions.
- Intracellular calcium concentration ([Ca2+]i) is a key regulator of cellular processes, including mediator release.
- Understanding antigen-induced signaling pathways is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the role of intracellular calcium ([Ca2+]i) oscillations in antigen-induced responses in RBL-2H3 cells.
- To quantify the relationship between antigen concentration and Ca2+ oscillations.
- To compare Ca2+ signaling patterns induced by various stimuli.
Main Methods:
- Utilized the ratiometric Ca2+ indicator, indo-1, for real-time measurement of intracellular Ca2+.
- Quantified Ca2+ oscillations using the area under the curve (AUC) method.
- Assessed responses to various stimuli including DNP-HSA, concanavalin A (Con-A), A23187, thapsigargin, and NECA.
Main Results:
- Antigenic stimulation (DNP-HSA) induced concentration-dependent, asynchronous Ca2+ oscillations.
- The dose-response curve of AUC correlated with antigen-induced mediator release.
- Ca2+ oscillations were dependent on external Ca2+ and mimicked by Con-A, but not by A23187, thapsigargin, or NECA.
Conclusions:
- Calcium oscillations are likely integral to the physiological response of mast cells and basophils to antigenic challenges.
- While not essential for initiating secretion, Ca2+ oscillations may enhance the efficacy of the calcium signal.
- The study provides insights into the complex signaling mechanisms underlying allergic responses.
Abstract:
Using the ratiometric Ca2+ indicator, indo-1, the antigen-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was measured in individual RBL-2H3 cells which had been passively sensitized with monoclonal antibody to the dintrophenyl (DNP) haptenic group. Antigenic stimulation using DNP-human serum albumin conjugate (DNP-HSA) induced concentration-dependent asynchronous Ca2+ oscillations, or irregular spikes. To achieve a quantitative comparison of the effects of different concentrations of antigen on changes in Ca2+[i, the area under the curve (AUC) of Ca2+ oscillations in each cell was calculated. The dose-response curve of the calculated AUC is consistent with the bell-shaped dose-response curve for antigen-induced mediator release, depolarization and 86Rb(+)-efflux. Ca2+ oscillations induced by antigenic stimulation were abolished by removal of external Ca2+ and the subsequent reintroduction of external Ca2+ caused their resumption. To investigate the role of Ca2+ oscillations in the secretory response, changes in [Ca2+]i induced by concanavalin A (Con-A), A23187, thapsigargin and NECA were also monitored. Con-A mimicked the response induced by antigen, whilst A23187 and thapsigargin induced a large transient non-oscillatory response. NECA, an adenosine receptor agonist, induced only a small transient rise in Ca2+[i without oscillatory behaviour. Since all these stimuli accept NECA-induced degranulation in these cells, it is suggested that, although Ca2+ oscillations are not essential for the initiation of secretion, they probably underlie the in-vivo physiological response of mast cells and basophils to an antigenic challenge. They also seem to enhance the efficacy of the Ca2+ signal.
