An obligate role for membrane-associated neutral sphingomyelinase activity in orienting chemotactic migration of human neutrophils.

For polymorphonuclear neutrophils (PMNs) to orient migration to chemotactic gradients, weak external asymmetries must be amplified into larger internal signaling gradients. Lipid mediators, associated with the plasma membrane and within the cell, participate in generating these gradients. This study examined the role in PMN chemotaxis of neutral sphingomyelinase (N-SMase), a plasma membrane-associated enzyme that converts sphingomyelin to ceramide. A noncompetitive N-SMase inhibitor, GW4869 (5 mM, 5 minutes), did not inhibit PMN motility (as percentage of motile cells, or mean cell velocity), but it abrogated any orientation of movement toward the source of the chemotaxin, formylmethionylleucylphenylanaline (FMLP) (net displacement along the gradient axis in micrometers, or as percentage of total migration distance). This defect could be completely reversed by treatment with lignoceric ceramide (5 µg/ml, 15 minutes). Immunolocalization studies demonstrated that N-SMase (1) distributes preferentially toward the leading edge of some elongated cells, (2) is associated with the plasma membrane, (3) is more than 99.5% localized to the cytofacial aspect of the plasma membrane, (4) is excluded from pseudopodial extensions, and (5) increases rapidly in response to FMLP. Morphologically, the inhibition of N-SMase limited cellular spreading and the extension of sheet-like pseudopods. Elongated PMNs demonstrated a polarized distribution of GTPases, with Rac 1/2 accumulated at, and RhoA excluded from, the front of the cell. This polarity was negated by N-SMase inhibition and restored by lignoceric ceramide. We conclude that N-SMase at the cytofacial plasma membrane is an essential element for the proper orientation of PMNs in FMLP gradients, at least in part by polarizing the distribution of Rac 1/2 and RhoA GTPases.

Migrating human neutrophils exhibit dynamic spatiotemporal variation in membrane lipid organization.

Highly ordered sphingolipid-enriched lipid raft microdomains (LRMs) within plasma membranes purportedly function as specialized signaling platforms. Leukocyte migration is believed to entail LRM redistribution, but progress in studying LRMs in situ during cell movement has been limited. By using an improved method for imaging the spectral shift of the environmentally sensitive probe, laurdan (expressed as a generalized polarization function), the plasma membrane order (i.e., tight packing of membrane bilayer lipids) of human polymorphonuclear neutrophils (PMNs) was mapped in real time during migration. Morphologically polarized PMNs exhibited prominent LRM clusters at the uropod, where in every instance membrane order was found to oscillate with mean periodicities of 37.0 ± 1.46 and 149.9 ± 9.0 seconds (P < 0.01). LRM aggregates were also demonstrated in punctate and clustered distributions of nonpolarized cells and transiently at the lamellipodia of polarized PMNs. Cellular polarization was not accompanied by an overall increase in membrane order. LRM disorganization with methyl-ß-cyclodextrin had small negative effects on cell velocity, but it abrogated directionally biased migration toward chemotactic gradients of FMLP or leukotriene B(4). LRMs disruption also caused redistribution of Rac 1/2 GTPase and GM3 ganglioside away from the lamellipodium, as well as extension of multiple pseudopods simultaneously or in rapid succession, rather than formation of a defined leading edge. Thus, we demonstrate that the plasma membrane order of migrating PMNs changes dynamically, with prominent oscillations consistently seen at the uropod. These findings solidify the existence of rapidly reorganizing LRMs in situ and support a role for LRMs in chemotaxin responsiveness.

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils.

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, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating 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-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) 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.

Direct observation of lipid bilayer disruption by poly(amidoamine) dendrimers.

Atomic force microscopy (AFM) is employed to observe the effect of poly(amidoamine) (PAMAM) dendrimers on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers. Aqueous solutions of generation 7 PAMAM dendrimers cause the formation of holes 15-40 nm in diameter in previously intact bilayers. This effect is observed for two different branch end-groups--amine and carboxyl. In contrast, carboxyl-terminated core-shell tectodendrimer clusters do not create holes in the lipid membrane but instead show a strong affinity to adsorb to the edges of existing bilayer defects. A possible mechanism for the formation of holes in the lipid bilayer is proposed. The dendrimers remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules. Dynamic light scattering (DLS) measurements as well as 31P NMR data support this explanation. The fact that tectodendrimers behave differently suggests that their cluster-like architecture plays an important role in their interaction with the lipid bilayer.