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Stimulus-dependent leukotriene release from human basophils: a comparative study of C5a and Fmet-leu-phe
1Johns Hopkins Asthma and Allergy Center, Division of Clinical Immunology, Baltimore, Maryland 21224.
Abstract:
Previous studies of human basophil mediator release have noted that the bacterial peptide fmet-leu-phe and the anaphylatoxin C5a induce comparable levels of histamine release while only fmet peptide induces leukotriene release. Since 5-lipoxygenase metabolism of arachidonic acid is calcium dependent, we examined the characteristics of the human basophil [Ca++]i response which follows its activation by either fmet peptide or C5a. While the peak [Ca++]i response was essentially identical for these two stimuli, fmet peptide induced a prolonged increase in [Ca++]i while C5a stimulated only a transient increase in [Ca++]i that was essentially over within 2 minutes of adding the stimulus. Simultaneous addition of EDTA with fmet peptide revealed the two phases of the [Ca++]i response and demonstrated that leukotriene release was dependent on an elevated [Ca++]i level in the 2-5 minutes following challenge. Enhancement of leukotriene release induced by C5a by agents such as staurosporine and interleukin-3 also produced a [Ca++]i kinetic curve which resembled fmet peptide. Single cell studies of the [Ca++]i response could detect no subpopulations of cells which responded preferentially to fmet peptide or C5a, eliminating the possibility that the ability of fmet peptide to induce leukotriene was a result of its action on a functionally distinct population of basophils.
Insights
Bacterial peptides and C5a trigger similar histamine release in human basophils, but only peptides induce leukotriene release. This difference is linked to prolonged calcium signaling, crucial for leukotriene production.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Human basophil activation by bacterial peptide fmet-leu-phe and anaphylatoxin C5a results in comparable histamine release.
- Only fmet peptide, not C5a, induces significant leukotriene release from basophils.
- 5-lipoxygenase metabolism, essential for leukotriene synthesis, is calcium-dependent.
Purpose of the Study:
- To investigate the characteristics of intracellular calcium ([Ca++]i) response in human basophils upon activation by fmet peptide versus C5a.
- To determine the role of calcium signaling kinetics in differential leukotriene release induced by these stimuli.
Main Methods:
- Measurement of intracellular calcium ([Ca++]i) levels in human basophils stimulated with fmet peptide or C5a.
- Assessment of leukotriene release in conjunction with calcium flux analysis, including experiments with EDTA.
- Evaluation of the impact of enhancing agents (staurosporine, interleukin-3) on C5a-induced responses.
- Single-cell analysis of basophil calcium responses to identify potential subpopulations.
Main Results:
- Both fmet peptide and C5a elicited identical peak intracellular calcium ([Ca++]i) responses.
- Fmet peptide induced a prolonged elevation of [Ca++]i, whereas C5a caused a transient increase lasting less than 2 minutes.
- Leukotriene release was dependent on elevated [Ca++]i levels observed between 2-5 minutes post-stimulation with fmet peptide.
- Enhancement of C5a-induced leukotriene release by other agents resulted in a prolonged [Ca++]i kinetic similar to fmet peptide.
- Single-cell studies did not reveal distinct basophil subpopulations responsible for differential responses.
Conclusions:
- The prolonged intracellular calcium ([Ca++]i) increase following fmet peptide stimulation, but not the transient rise induced by C5a, is critical for leukotriene release in human basophils.
- The differential kinetic pattern of calcium signaling, rather than the peak response magnitude or distinct cell subpopulations, underlies the differing abilities of fmet peptide and C5a to induce leukotriene production.
- Modulating calcium signaling kinetics can influence mediator release, offering potential therapeutic insights.