Selective Localization of Recognition Complexes for Leukotriene B4 and Formyl-Met-Leu-Phe within Lipid Raft Microdomains of Human Polymorphonuclear Neutrophils

Abstract

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, coisolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, coisolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-β-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca2+ mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.

Migrating Human Neutrophils Exhibit Dynamic Spatiotemporal Variation in Membrane Lipid Organization

“…Large foci of persistently high GP were found at the uropod. In many cells, high-GP areas were also found near the leading edge (images [13][14][15][16][17][18], in keeping with earlier findings demonstrating concentrated LRM markers at lamellipodia (2,13,23). Lower GP values were generally found over the body of the cells, suggesting a predominance of non-LRM membrane, but transient foci of high GP were identified here as well.…”

“…The addition to living cells of bulky markers, such as antibodies and fluorescent labels, which are generally more massive than the supporting lipids of the LRMs, may have been another source of artifacts in this area of research. LRMs are clearly enriched in many receptors and downstream signaling intermediates important for cell movement (CD87, CD59, leukotriene B 4 receptor-1 [BLT-1], Rac1, phosphatidylinositol bisphosphate, phosphatidylinositol 3,4,5-trisphosphate [PIP 3 ], and transient receptor potential channel 1) as well as membrane proteins that link to the cytoskeleton (talin, ezrin/radixin/ moesin, and focal adhesion kinase) (2,(11)(12)(13)(14)(15)(16). Moreover, LRM markers polarize in motile PMNs, forming aggregates near the uropod and the lamellipodium (2, 13).…”

FcγRI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions

“…By contrast, Sitrin et al previously reported that BLT1 was constitutively present in LRs of human neutrophils. 30 Differences in BLT1 localization to LRs between these 2 phagocyte populations remain to be explained, but could reflect differences in fractionation protocols, in the activation states of the 2 cell types, or in their LR compositions. That BLT1 translocation to LRs is a specific consequence of Fc␥R engagement, rather than a universal finding during phagocytosis, was evidenced by the fact that it failed to redistribute to LRs upon AM stimulation with Candida albicans (data not shown), whose ingestion is mediated by pathogen recognition receptors such as the mannose and dectin receptors.…”

“…That M␤CD in fact disrupted LR integrity was evidenced by the facts that CD45 and flotillin-1 were now distributed among both LR and non-LR fractions (supplemental Figure 3A-B), and that both GM1 and BLT1 were colocalized with CD45 in IgG-RBC-stimulated AMs (data not shown). To investigate the functional consequences of LR disruption, cyclodextrin compounds were added at 5 mM for 5 minutes 30 before addition of LTB 4 (10 nM), after which phagocytosis of IgG-RBCs was determined. With intact LR integrity, LTB 4 ( Figure 3A) increased phagocytosis, as expected on the basis of our previous reports.…”

Signalling in Neutrophils: A Retro Look

“…In view of the inhibitory effects of pertussis toxin on the activation of Src kinases by chemotactic factors, it is highly likely that these kinases are responding either directly or indirectly to activated (GTP-bound, dissociated) heterotrimeric G proteins. G proteins and Src family kinases, as well as some chemoattractant receptors (e.g., BLT1 but not FPR [79]), have been observed in lipid rafts which may provide the proximity required for the activation of the latter.…”