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Published on: June 23, 2017
Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils
Jens Martin Herrmann1, John Bernardo, Heidi J Long
1Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA.
Abstract:
Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.
Insights
Human neutrophils (PMN) signaling pathways were investigated using fluorescent indicators. Different stimuli like fMLP and immune complexes (IC) elicit distinct calcium and pH responses, impacting subsequent cellular functions.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Human polymorphonuclear neutrophils (PMN) are crucial in innate immunity, responding to foreign entities through chemotaxis and phagocytosis.
- The initial signaling events, including calcium (Ca2+) and pH dynamics, are critical for initiating bactericidal functions in PMN.
- Understanding these early signaling mechanisms is key to comprehending neutrophil activation and immune responses.
Purpose of the Study:
- To explore the initial signaling events in naïve, unprimed PMN.
- To investigate the role of cytoplasmic calcium ([Ca(2+)](i)) and pH (pH(i)) dynamics in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC).
- To determine how the order, dose, and nature of stimuli affect subsequent PMN responses.
Main Methods:
- Observing naïve, unprimed PMN in suspension using fluorescent indicators for [Ca(2+)](i), pH(i), oxidative species, and elastase.
- Stimulating PMN with varying concentrations and combinations of fMLP and IC.
- Analyzing the transient and sustained changes in [Ca(2+)](i) and pH(i) and their correlation with bactericidal functions.
Main Results:
- Both fMLP and IC rapidly increase [Ca(2+)](i) from intracellular stores and decrease pH(i), followed by restoration of pH(i) via Na(+)/H(+) antiport.
- The response to a second stimulus depends on the initial stimulus's nature, dose, and the restoration of [Ca(2+)](i).
- Low-dose fMLP (10(-7) M) induced [Ca(2+)](i) rise but no bactericidal functions, while high-dose fMLP (10(-5) M) did, suggesting involvement of low-affinity receptors and different Ca(2+) sources.
Conclusions:
- Sequential stimulation of PMN with fMLP and IC reveals complex signaling cross-talk and desensitization.
- The initial signaling threshold and receptor affinity (high- vs. low-affinity) dictate the subsequent functional outcomes, including bactericidal activity.
- PMN activation is a finely tuned process where stimulus characteristics critically influence the cellular response and effector functions.
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