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Differential behavior of sPLA2-V and sPLA2-X in human neutrophils
I Solodkin-Szaingurten1, R Levy, N Hadad
1Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva 84105, Israel.
Abstract:
Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.
Insights
Secreted phospholipase A(2) (sPLA(2)) types V and X in neutrophils behave differently upon stimulation. sPLA(2)-V is secreted, while sPLA(2)-X localizes to the plasma membrane, and neither is regulated by cPLA(2)alpha.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Neutrophils and PLB-985 cells express multiple phospholipase A(2) (PLA(2)) enzymes, including cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)), and secreted PLA(2)s (sPLA(2)s).
- Understanding the distinct roles and regulation of these PLA(2) isoforms is crucial for comprehending inflammatory and immune responses.
Purpose of the Study:
- To investigate the behavior and regulation of specific secreted PLA(2) (sPLA(2)) isoforms, namely sPLA(2)-V and sPLA(2)-X, within neutrophils and PLB cells.
- To determine the relationship between sPLA(2) activity and cPLA(2)alpha in these cell types.
Main Methods:
- Differential analysis of sPLA(2)-V and sPLA(2)-X localization and secretion in neutrophils and PLB cells following stimulation with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ), or A23187.
- Assessment of sPLA(2) activity in neutrophil supernatants and on plasma membranes.
- Utilizing granulocyte-like cPLA(2)alpha-deficient PLB cells to examine the regulatory role of cPLA(2)alpha on sPLA(2) activity and secretion.
Main Results:
- Neutrophils contain sPLA(2)-V and sPLA(2)-X in azurophil granules, exhibiting differential responses to stimuli: sPLA(2)-V is secreted extracellularly, while sPLA(2)-X is detected on the plasma membrane.
- Stimulation kinetics of sPLA(2) secretion varied depending on the stimulus used (fMLP, OZ, A23187).
- Priming neutrophils with inflammatory cytokines or LPS enhanced fMLP-induced sPLA(2) activity in supernatants, primarily due to increased sPLA(2)-V secretion.
- sPLA(2) in PLB cells showed characteristics similar to neutrophils, with comparable activity and optimal pH of 7.5.
- Secretion and activity of sPLA(2) in PLB cells were unaffected by the presence or absence of cPLA(2)alpha, indicating no cPLA(2)alpha-mediated regulation.
Conclusions:
- Neutrophil sPLA(2)-V and sPLA(2)-X exhibit distinct subcellular localization and secretion patterns upon stimulation.
- Neutrophil priming enhances sPLA(2)-V secretion, contributing to increased extracellular sPLA(2) activity.
- cPLA(2)alpha does not regulate the secretion or activity of sPLA(2) in granulocyte-like PLB cells, suggesting independent regulatory pathways.
