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

Abstract

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.

Figure 1.

Effects of inhibiting neutral sphingomyelinase (N-SMase) on formylmethionylleucylphenylalanine (FMLP) induced PMN chemotaxis. PMNs pretreated with buffer (NT) or GW4869 (5 mM) were loaded onto Dunn chambers charged with FMLP (5 × 10⁻⁷M), and cell tracks were assembled from serial images over 30 minutes. GW4869 did not significantly affect the percentage of motile cells (A), and it slightly increased mean cell velocity (B). (C) However, the net displacement along the axis of the chemotactic gradient was negated, with net movement directed away from the FMLP source (P < 0.05). (D) Accordingly, the chemotactic index (displacement along the gradient axis as percentage of total path length) was reduced to near-zero (mean ± SEM, n = 12, ≥ 60 cell tracks per experiment). The effects of N-SMase inhibition on the net displacement of individual PMNs migrating in FMLP gradients are shown in scatterplots (E) (three off-scale tracks in each group are not shown) and histograms (F), using the same pool of cells as in A–D. GW4869 negated the group progress toward the FMLP source (Kruskal–Wallis post hoc test applied to one-way ANOVA, P < 0.0001). (G) A frequency analysis (χ²) of the number of cell tracks displaced at least 60 μm toward or away from the FMLP source (data are shown as percentages of total cell tracks).

Figure 2.

Exogenous ceramide (CER) reverses the effects of N-SMase inhibition on FMLP-induced PMN chemotaxis. PMNs were pretreated with buffer, GW4869 (5 mM), or GW4869 with lignoceric ceramide (5 μg/ml), as described in Materials and Methods. PMNs were then loaded onto Dunn chambers charged with FMLP (5 × 10⁻⁷ M). Data are expressed as in Figure 1. Exogenous ceramide completely restored the mean displacement along the FMLP gradient (C) and the chemotactic index (D) to levels equivalent to control cells, reversing the inhibitory effects of GW4869. P values refer to the results of Bonferroni post hoc tests applied to one-way ANOVA. Values are given as mean ± SEM (n = 5, ≥ 60 cell tracks per experiment).

Figure 3.

Effects of exogenous ceramide and sphingomyelin (SM) on FMLP-induced chemotaxis. (A–D) Exogenous ceramide (5 μg/ml) had no effects on motility, displacement along the gradient, or chemotactic index, relative to untreated control samples. Exogenous sphingomyelin (5 μg/ml) did not affect the percentage of motile cells (E) or cell velocity (F), but partly suppressed displacement along the axis of the FMLP gradient (D_axis) (G) (P < 0.02) and the chemotactic index (H) (P < 0.01) (n = 4). Data are expressed as in Figure 1 (n = 6, ≥ 60 cell tracks per experiment).

Figure 4.

Subcellular localization of N-SMase. PMNs were stimulated with FMLP (5 × 10⁻⁷ M), fixed, permeabilized, and immunolabeled, as described in Materials and Methods. Images of N-SMase immunofluorescence were merged with differential interference contrast microscopy images to highlight cell outlines. (A) N-SMase was distributed uniformly in round PMNs. Some elongated PMNs (B) demonstrated no front-to-back asymmetry in the distribution of N-SMase, whereas others (C) demonstrated preferential expression toward the front of the cell, and little at the tail. Confocal images in each row (far right) demonstrate that immunostaining was associated with the plasma membrane, with virtually no immunostaining in the cell interior. (D) N-SMase was virtually excluded from any pseudopodial extensions (arrows) anywhere on the cells.

Figure 5.

N-SMase expression according to flow cytometry. (A) Flow cytometry was performed on fixed/permeabilized PMNs labeled with Oregon Green–conjugated anti-SMase antibody (Ab). Compared with untreated control samples, FMLP (5 × 10⁻⁷ M for 15 minutes) significantly increased N-SMase immunoreactivity, whereas GW4869 had no effect, either on untreated or FMLP-treated cells. Data are expressed as mean fluorescence intensity (MFI) of at least 10,000 cells per experiment (mean ± SEM, n = 4). (B) Flow cytometry was performed on PMNs that were labeled to measure external immunoreactivity. Aliquots of these cells were then treated with anti–Oregon Green 488 Ab to quench external labeling, followed by fixation/permeabilization and relabeling with anti–N-SMase Ab to measure internal N-SMase immunoreactivity, as described in Materials and Methods. Data are expressed as MFI of at least 10,000 cells per experiment (mean ± SEM, n = 4). External MFI was 0.4% of internal MFI; *P < 0.05.

Figure 6.

Effects of N-SMase inhibition and ceramide reconstitution on PMN morphology. Scanning electron microscopy was performed on PMNs treated with buffer, GW4869 (5 mM), or GW4869 with lignoceric ceramide (5 μg/ml), as described in Materials and Methods, followed by FMLP (5 × 10⁻⁷ M). Compared with cells treated with FMLP alone (NT), N-SMase inhibition with GW4869 caused elongated PMNs to limit their spreading, partially detach from the surface, and limit pseudopodial extensions to a few narrow spikes. Lignoceric ceramide restored spreading, adhesion, and the formation of extended, sheet-like pseudopodia to FMLP/GW4869–treated cells.

Figure 7.

Effects of N-SMase inhibition and lignoceric ceramide on the distribution of Rac 1/2 and RhoA GTPases. Immunofluorescence microscopy of fixed/permeabilized FMLP-treated PMNs was performed as described in Materials and Methods. Two cells representative of each treatment condition are shown. (A) Rac 1/2 was concentrated at the leading edge of elongated PMNs, with weak expression at the tail (arrowheads), whereas GW4869 (5 mM) caused the distribution to be uniform. Lignoceric ceramide (5 μg/ml) added to GW4869-treated cells restored the concentration of Rac 1/2 at the leading edge, with relative exclusion at the tail (arrowheads). (B) RhoA was excluded from the leading edge in elongated PMN (arrowheads). GW4869 (5 mM) caused RhoA to redistribute to the leading edge, rendering a uniform distribution over the cell. Lignoceric ceramide (5 μg/ml) added to GW4869-treated cells restored the exclusion of RhoA from the leading edge (arrowheads). Dotted lines indicate cell outlines.