Korean J Physiol Pharmacol.  2022 May;26(3):175-182. 10.4196/kjpp.2022.26.3.175.

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils

• Ham HY
• Kang SH
• Song DK

• KMID: 2529400
• DOI: http://doi.org/10.4196/kjpp.2022.26.3.175

Abstract

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

Keyword

Azurophil granule; Lysophosphatidylcholine; Neutrophil; Translocation

Figure

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  Fig. 1 Translocation of azurophil granule in response to LPC in human neutrophils. (A, B) Time- (at 3 μM) and concentration- (at 15 min) dependency of LPC-induced CD63 surface expression. After fixation, cells were stained with FITC-conjugated antibody against CD63, an azurophil granule marker for 1 h on ice, and were analyzed with flow cytometry. An average ± SEM of more than three experiments is shown. LPC, lysophosphatidylcholine; MFI, mean fluorescence intensity. *p < 0.05 and **p < 0.01.

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  Fig. 2 LPC-induced CD63 surface expression via fibrous (F)-actin polymerization in human neutrophils. (A) Representative FACS histograms of CD63 surface expression in response to cytochalasin B (CB), fMLP, CB + fMLP, LPC, and CB + LPC. Isolated neutrophils stabilized 30 min were treated with CB (2.5 μg/ml, 5 min), fMLP (1 μM, 15 min), or LPC (3 μM, 15 min). When combined, neutrophils were pretreated with CB and then treated with either fMLP or LPC. (B) Bar graph shows mean fluorescence intensity (MFI) of more than three independent experiments. (C) FITC-conjugated phalloidin, a marker of F-actin, was measured after exposure of neutrophils to LPC for 15 min. An average ± SEM of more than three experiments is shown. LPC, lysophosphatidylcholine; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine. *p < 0.05, **p < 0.01, and ***p < 0.001.

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  Fig. 3 Rho is involved in LPC-induced CD63 surface expression in human neurophils. (A–C) Neutrophils were pretreated with NSC23766 (50 μM), Y27632 (10 μM), Tat-C3 exoenzyme (2.5–5 μg/ml), or vehicle for 30 min, and further stimulated with LPC (3 μM) for 15 min. (D) Rho activation in response to LPC in human neutrophils determined by pull-down assay. An average ± SEM of more than three experiments is shown. LPC, lysophosphatidylcholine; MFI, mean fluorescence intensity. *p < 0.05 and **p < 0.01.

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  Fig. 4 ROCK is involved in LPC-induced fibrous (F)-actin polymerization in human neutrophils. (A, B) Neutrophils were pretreated with Y27632 (10 μM) or vehicle for 30 min, and further stimulated with LPC (3 μM) for 15 min, and then were stained with FITC-conjugated phalloidin for F-actin polymerization. Neutrophils were analyzed with FACS (A) and immunofluorescence microscopy (bar, 2 μm) with a × 100 (scan zoom 3) objectives (B). An average ± SEM of more than three experiments is shown. ROCK, Rho kinase; LPC, lysophosphatidylcholine. **p < 0.01 and ***p < 0.001.

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  Fig. 5 Two distinct signaling pathways in LPC-induced azruophil granule translocation. (A) Anti-G2A Ab blocks LPC-induced CD63 surface expression. Neutrophils were pretreated with anti-G2A Ab (1 μg/ml) for 1 h and further stimulated with LPC (3 μM) for 15 min. Then neutrophils were fixed and stained with FITC-conjugated anti-CD63 Ab for 1 h on ice. (B, C) SB203580 blocks LPC-induced CD63 surface expression, but not F-actin polymerization. Neutrophils were pretreated with SB203580 (10 μM) for 30 min, and further stimulated with LPC (3 μM) for 15 min. After fixation, cells were stained with FITC-conjugated anti-CD63 Ab (B) or FITC-conjugated phalloidin (C) for 1 h. (D) Y27632 does not affect LPC-induced [Ca2+]i increase. Neutrophils were loaded with Fluo-3 AM (4 μM) in HEPES for 1 h and, then pretreated with Y27632 (10 μM) for 30 min before LPC stimulation. [Ca2+]i changes in Fluo-3 AM-loaded neutrophils were expressed as the relative fluorescence intensity over the resting fluorescence value (F/Fo). An average ± SEM of more than three experiments is shown. LPC, lysophosphatidylcholine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MFI, mean fluorescence intensity. *p < 0.05, **p < 0.01, and ***p < 0.001.

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  Fig. 6 Model for two distinct signaling pathways for LPC-induced azurophil granule translocation. LPC, lysophosphatidylcholine; ROCK, Rho kinase; F-actin, fibrous-actin.

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