This information is current as
of June 17, 2017.
Selective Localization of Recognition
Complexes for Leukotriene B4 and
Formyl-Met-Leu-Phe within Lipid Raft
Microdomains of Human Polymorphonuclear
Neutrophils
Robert G. Sitrin, Sarah L. Emery, Timothy M. Sassanella, R.
Alexander Blackwood and Howard R. Petty
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 177:8177-8184; ;
doi: 10.4049/jimmunol.177.11.8177
http://www.jimmunol.org/content/177/11/8177
The Journal of Immunology
Selective Localization of Recognition Complexes for
Leukotriene B4 and Formyl-Met-Leu-Phe within Lipid Raft
Microdomains of Human Polymorphonuclear Neutrophils1
Robert G. Sitrin,2* Sarah L. Emery,* Timothy M. Sassanella,* R. Alexander Blackwood,†
and Howard R. Petty‡
L
ipid raft microdomains are generally envisioned as small
dynamic regions within plasma membranes with characteristically high concentrations of sphingolipids and cholesterol. Lipid rafts, although heterogeneous, are enriched in many
proteins engaged in neutrophil activation and migration, including
TLR, chemokine receptors, multiple signaling molecules (Lyn and
other src kinases, PI3K, Rho GTPases, G proteins, Src homology
region 2 domain-containing phosphatase 1), and cytoskeleton-related proteins (1–7). The highly saturated sphingolipids generally
pack tightly together in cholesterol-stabilized gel-like states
termed liquid-ordered (Lo)3 arrays. This physical state is conducive to concentrating and constraining the mobility of multiple
proteins to facilitate assembly of competent signaling complexes.
Lipid rafts distribute uniformly throughout the plasma membranes
of quiescent leukocytes but reorganize dramatically as cells polarize to form large aggregates at the leading edges and uropods, with
*Pulmonary and Critical Care Medicine Division, Department of Internal Medicine,
†
Department of Pediatrics and Communicable Diseases, and ‡Department of Ophthalmology, University of Michigan, Ann Arbor, MI 48109
Received for publication June 29, 2006. Accepted for publication September 8, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants HL58283,
AI060983, and AI51789.
2
Address correspondence and reprint requests to Dr. Robert G. Sitrin, 6301 MSRB
III, BOX 0642, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642. E-mail
address: [email protected]
3
Abbreviations used in this paper: Lo, liquid ordered; LTB4, leukotriene B4; FRET,
fluorescence resonance energy transfer; [Ca2⫹]i, intracellular calcium concentration;
M␤CD, methyl-␤-cyclodextrin; ␣-CD, ␣-cyclodextrin; BLT-1, B leukotriene receptor-1; FPR, formyl peptide receptor.
Copyright © 2006 by The American Association of Immunologists, Inc.
aggregates at each location possessing highly distinct ganglioside
and protein content (2, 8 –10).
The dynamic polarization of lipid rafts and their capacity to
regulate activation signaling and cytoskeletal rearrangement foster
the hypothesis that lipid rafts are critically important to the mechanisms governing cellular locomotion and, in particular, directional migration. This may occur at many levels. First, lipid rafts
may be the locus of chemotactic receptors that initiate cellular
movement. CXCR1, CXCR4, and CCR5 chemokine receptors
have been shown to localize in lipid rafts (2, 3, 11). Further, chemotaxin receptors characteristically couple to regulatory G proteins, and G proteins concentrate in lipid rafts (2, 3, 11). Lipid rafts
may organize ion channels and other signaling elements within the
plasma membrane to translate initial activation signaling into properly oriented signaling waves. We have shown that lipid raft disruption completely disables cells from propagating traveling Ca2⫹
waves along the plasma membrane of spontaneously polarized
neutrophils (10). Not to the exclusion of important effects on upstream signaling, lipid rafts may also spatially couple the signaling
apparatus to actin cytoskeleton and microtubules to achieve effective movement (3, 12). The observations that chemokine receptors
and signaling intermediates localize within lipid rafts has raised
the possibility that it may be a general property that chemotaxin
recognition systems are fully functional only in lipid raft environments. Defining the extent to which chemotaxin recognition systems necessarily colocalize in lipid rafts would be of great value in
understanding not only lipid raft function but also the potential for
physical interplay between different chemotaxin recognition complexes within the same microdomains. In this study, we sought to
determine whether receptors for two major nonchemokine neutrophil chemotaxins, leukotriene B4 (LTB4) and the bacterial peptide
0022-1767/06/$02.00
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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-␤-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca2ⴙ 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. The Journal of Immunology, 2006, 177: 8177– 8184.
8178
LIPID RAFT DISTRIBUTION OF NEUTROPHIL LTB4, fMLP RECEPTORS
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FIGURE 1. Top, Partitioning of BLT-1 and FPR in detergent-extracted and fractionated membranes of unstimulated neutrophils. Unstimulated neutrophils were extracted with 1% Brij-58 and applied to discontinuous sucrose gradients, as described in Materials and Methods. Virtually all BLT-1 was
distributed in lower density fractions, corresponding closely to the distribution of a lipid raft-associated protein, flotillin-1. By contrast, all detectable FPR
was found in the highest density fraction where it codistributed with Cdc42, a protein enriched in nonraft membrane. The Western blots shown are
representative of at least three independent experiments using different donor neutrophils. Bottom, Partitioning of BLT-1 and FPR in detergent-extracted
and fractionated membranes of LTB4- and fMLP-stimulated neutrophils. Neutrophils were stimulated with LTB4 (10⫺8 M) for 20 s (A) or 5 min (C) or
fMLP (5 ⫻ 10⫺7 M) for 90 s (B) or 5 min (D) and then extracted with 1% Brij-58 and applied to discontinuous sucrose gradients, as described in Materials
and Methods. The distribution of BLT-1 in lower density fractions and FPR in the highest density fractions closely resembled their respective distributions
in unstimulated neutrophils. The corresponding distributions of lipid raft and non-raft markers (flotillin-1 and Cdc42, respectively) are also shown. Western
blots shown are representative of at least three independent experiments using different donor neutrophils.
fMLP, partition to lipid rafts either constitutively or with stimulation and whether lipid raft integrity is necessary for activation
signaling through these receptors.
Materials and Methods
ject Research. Briefly, citrate-anticoagulated blood was sedimented with
6% dextran (0.9% NaCl), the erythrocytes were removed by hypotonic
lysis, and neutrophils were isolated by density gradient centrifugation on a
10% Ficoll-Hypaque cushion. The resulting cells (⬎95% neutrophils) were
washed in PBS and transferred to the appropriate buffer for further studies.
Purification and stimulation of human neutrophils
Fractionation of lipid raft and nonraft plasma membranes
Neutrophils were isolated using the method of Boyum (13) from peripheral
blood obtained from healthy volunteers according to a protocol approved
by the University of Michigan Institutional Review Board for Human Sub-
Fractionation of neutrophil membranes into lipid raft and nonraft fractions
was performed using standard methods, with minor modifications (14, 15).
Neutrophils were stimulated as described (⬃2 ⫻ 108 cells per preparation),
The Journal of Immunology
8179
and immediately collected by centrifugation in buffer A (25 mM HEPES,
150 mM NaCl, 1 mM EDTA (pH 7.0), with 1.4 ␮g/ml pepstatin A, 1.4
␮g/ml leupeptin, 100 U/ml aprotinin, 4 mM iodoacetic acid, and 100 ␮M
PMSF) at 4°C. The cells were then lysed in buffer A with 2.5 mM diisopropylfluorophosphate, 1 mM sodium orthovanadate, and 1.0% Brij-58
(Sigma-Aldrich) for 30 min at 4°C. The lysates were then layered on
discontinuous sucrose gradients of 40-30-10%. After centrifugation at
180,000 ⫻ g for 20 h, the fractions were removed in 1-ml volumes. Protein
concentrations were determined with a microBCA assay (Pierce). Samples
of each fraction, adjusted to equal protein content, were examined by Western blotting as above, using the following primary Abs: rabbit anti-human
B leukotriene receptor-1 (BLT-1) (120111; Cayman Chemical); and goat
anti-human formyl peptide receptor (FPR) (sc-13193), rabbit anti-human
flotillin-1 (sc-25506), and rabbit anti-human Cdc42 (sc-87X), all from
Santa Cruz Biotechnology.
Fluorescence resonance energy transfer (FRET)
FITC-conjugated murine anti-human BLT-1 Ab (FAB099F) NAS obtained
from R&D Systems. Anti-Cdc42 and anti-flotillin-1 Abs (Santa Cruz Biotechnology) were conjugated with FITC or tetramethylrhodamine isothiocyanate
with standard kits (Invitrogen Life Technologies/Molecular Probes) and purified by gel chromatography. Fluorescein-conjugated formyl-Nle-Leu-Phe-NleTyr-Lys (F1314) was obtained from Molecular Probes. Washed neutrophils
were labeled with the appropriate Ab or fMLP analog, washed thoroughly, and
fixed with 4% paraformaldehyde without being permeabilized. An Axiovert-135 inverted fluorescence microscope with HBO-100 mercury
illumination (Zeiss) interfaced to a Dell 410 workstation via a Scion SG-7
video card (Vay Tek) was used. A narrow bandpass-discriminating filter set
was used with excitation at 485DF20 nm and emission of 530DF30 nm for
FITC. For tetramethylrhodamine isothiocyanate, an excitation of 540DF20
nm and an emission of 590DF30 nm were used (Omega Optical). Long
pass dichroic mirrors at 510 and 560 nm were used for FITC and rhodamine, respectively. Single-cell spectra were obtained using an imaging
spectrophotometer system. Labeled cells were illuminated with an excitation filter at 485DF22 nm and a 510LP dichroic mirror for resonance energy transfer experiments (16, 17). The emission spectra were obtained
with an Acton-150 imaging spectrophotometer fiberoptically coupled to a
microscope. The exit port of the spectrophotometer was attached to a
Gen-II intensifier coupled with an I-MAX-512 camera (Princeton Instru-
ments). Spectra collection was controlled by a high speed Princeton ST-133
interface and a Stanford Research Systems DG-535 delay gate generator and
analyzed with Winspec software (Princeton Instruments).
ERK 1/2 tyrosine kinase activation
After polymorphonuclear neutrophils were treated as indicated, lysates
were prepared for Western blots according to a method adapted from the
work of Gilbert et al. (18). Polymorphonuclear neutrophils were washed
and resuspended in buffer A with protease inhibitors and mixed 1:1 (v/v)
with boiling lysis buffer (62.5 mM Tris-HCl (pH 6.8) with 4% SDS, 8.5%
glycerol, 5% 2-ME, 2 mM orthovanadate, 10 ␮g/ml aprotinin, 10 mM
p-nitrophenylphosphate, and 0.025% bromphenol blue) and boiled for another 7 min. These lysates were then mixed 3:1 with four times sample
buffer (0.25 M Tris (pH 6.8), 31% glycerol, and 8% SDS), boiled for 5 min,
and electrophoresed on 8 –16% gradient polyacrylamide gels, transferred to
polyvinylidene difluoride membranes, labeled as indicated, and developed
by ECL. Polyclonal goat anti-human Abs to total and phosphorylated
ERK1/2, and HRP-conjugated donkey anti-goat secondary Ab, were obtained from Santa Cruz.
Measurement of intracellular calcium concentration ([Ca2⫹]i )
Cells were loaded (5 ⫻ 106/ml) with the Ca2⫹-sensitive fluorescent dye fluo3-acetoxymethyl ester (2 ␮M; Molecular Probes) at 30°C for 30 min in 145
mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 4 mM probenecid, and
10 mM HEPES, pH 7.4. After pretreatments as indicated, 2.5 ⫻ 106 cells were
suspended in 1 ml of incubation buffer and prewarmed to 37°C. Fluorescence
intensities were then measured with a SLM8000 spectrofluorometer equipped
with SLM Spectrum Processor v3.5 software (SLM Instruments), using a 1-cm
light path cuvet at an excitation wavelength of 505 nm and an emission wavelength of 530 nm. Fluorescence intensities were acquired at 2-s intervals with
continuous stirring of the cell suspension. These measurements were converted
to nanomolar concentrations of [Ca2⫹]i by the calibration method of
Grynkiewicz et al. (19), using a Kd for fluo-3 of 864 nM (20).
Statistical analysis
Comparisons of means were performed with t tests using a p value of 0.05
to determine significance (GraphPad Prism version 3.00 for Windows;
GraphPad).
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FIGURE 2. FRET between BLT-1,
FPR, and markers of lipid raft and
nonraft membrane domains. Representative emission spectra of labeled neutrophils stained with FITC-conjugated
(donor) and tetramethylrhodamine isothiocyanate-conjugated (acceptor) labels. Proteins were labeled with fluorescent Abs except for FPR, which was
detected with fluoroscein-conjugated
formyl-Nle-Leu-Phe-Nle-Tyr-Lys. A,
Substantial resonance energy transfer
(arrow) between flotillin-1 (the lipid
raft marker) and anti-BLT-1 Ab on a
dual-labeled cell; B, No evidence of
FRET between the nonraft marker,
Cdc42, and BLT-1. C, No demonstrable FRET on a cell dual-labeled with
anti-flotillin-1 and formyl-Nle-LeuPhe-Nle-Tyr-Lys. D, FRET (arrow)
on a cell dual-labeled with antiCdc42 and formyl-Nle-Leu-PheNle-Tyr-Lys. The emission spectra
are representative of at least three
independent experiments performed
with different donor neutrophils. The
sharp spike seen at 605 nm in all spectra represents an internal calibration
standard.
8180
LIPID RAFT DISTRIBUTION OF NEUTROPHIL LTB4, fMLP RECEPTORS
Results
Partitioning characteristics of BLT-1 and FPR in fractionated
membranes of unstimulated neutrophils
FRET analysis of associations between BLT-1 or FPR and lipid
raft markers
FRET was performed to determine whether BLT-1 on unstimulated neutrophils could be found in association with flotillin-1. As
shown in Fig. 2A, there is substantial FRET with this labeling
scheme, indicating molecular proximity (within a few nanometers)
FIGURE 3. Effects of lipid raft disruption on LTB4- and fMLP-mediated
activation signaling: [Ca2⫹]i mobilization. Fluo-3-loaded unstimulated neutrophils were pretreated with M␤CD (5 mM, 5 min) to destabilize the Lo state of
lipid rafts. Cells were subsequently stimulated with fMLP (5 ⫻ 10⫺7 M) or
LTB4 (5 ⫻ 10⫺8 M). M␤CD had no effect on initial mobilization of [Ca2⫹]i
in response to fMLP (A) but significantly inhibited the same response to LTB4
(B). The effect of M␤CD on LTB4-mediated signaling could not be duplicated
with ␣-CD, which binds cholesterol poorly. The tracings show the average
change in [Ca2⫹]i measured from at least three independent experiments using
different donor neutrophils. ⴱ, p ⬍ 0.03.
between flotillin-1 and BLT-1. In control experiments, there was
no 590 nm emission in the presence of donor fluorochrome alone,
and there was no FRET between flotillin-1 and Cdc42, the respective lipid raft and nonraft markers (not shown). By contrast,
formyl-Nle-Leu-Phe-Nle-Tyr-Lys, used to detect FPR, produced
robust FRET with Cdc42 (Fig. 2D), but none with flotillin-1 (Fig.
2C). This pattern agrees entirely with the membrane fractionation
studies (Fig. 1), placing FPR predominantly in nonraft fractions. In
preliminary experiments (not shown), we also evaluated GM1 and
GM3 gangliosides as lipid raft markers and found very strong concordance with the results obtained with flotillin-1.
Effects of lipid raft disruption on LTB4- and fMLP-induced
mobilization of intracellular Ca2⫹
The association of BLT-1, but not FPR, with lipid raft markers
suggests that lipid raft integrity may selectively influence the
capacity for signal transduction through these receptors. To address this question, previously unstimulated human neutrophils
were loaded with fluo-3 to measure [Ca2⫹]i changes in response
to chemotaxins. The cells were pretreated with methyl-␤-cyclodextrin (Sigma-Aldrich) (M␤CD; 5 mM for 5 min), a cyclic
heptasaccharide that binds cholesterol avidly and extracts it
from the plasma membrane, thereby destabilizing the Lo state
of lipid rafts (22, 23). Preliminary experiments established that
under these conditions, M␤CD reduced plasma membrane cholesterol content by 17.4 ⫾ 0.9% ( p ⬍ 0.01), using a cholesterol
oxidase assay (24). We have also shown that this protocol reduces the liquid ordering of neutrophil lipid raft aggregates
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Although every method for assessing whether specific proteins
associate with lipid rafts is susceptible to artifact and misinterpretation, the most widely used of these has exploited two general properties of lipid rafts: their relatively low buoyant density; and their resistance to non-ionic detergents (14, 15, 21).
Membrane fractionation experiments were performed to examine the association of high affinity LTB4 and fMLP receptors
(BLT-1 and FPR, respectively) with lipid rafts, using a relatively nonselective detergent extraction protocol with 1.0%
Brij-58. In unstimulated neutrophils (Fig. 1, top), virtually all
the membrane BLT-1 is distributed in lower density lipid raft
fractions where it coisolates with flotillin-1, a protein highly
enriched in lipid rafts (7). By contrast, FPR partitions almost
exclusively to the dense nonraft fractions where it codistributes
with Cdc42, a protein largely excluded from lipid rafts in unstimulated neutrophils (22). In preliminary studies, we saw virtually identical distributions of BLT-1 and FPR when the cells
were extracted with 0.5% Brij-58 (not shown). Similar results
were also obtained when the cells were extracted with 1% Triton X-100, the detergent usually used for lipid raft fractionation, although in these experiments the yield of BLT-1 was
compromised (not shown). This seemed to be a function of poor
recovery rather than BLT-1 being partially soluble in 1% Triton
X-100, given that BLT-1 did not appear in dense fractions with
other soluble proteins. Experiments were next performed to determine whether cellular stimulation affects the partitioning of
BLT-1 or FPR within membrane fractions. Neutrophils stimulated with LTB4 (5 ⫻ 10⫺8 M) for 20 s (corresponding to the
timing of the peak in ERK 1/2 phosphorylation; see below) or
for 5 min resembled unstimulated cells, because BLT-1 continued to partition to the lipid raft fractions whereas FPR remained
in the nonraft fractions (Fig. 1, A and C, bottom panels). Likewise, stimulating neutrophils with fMLP for 90 s (corresponding to the timing of the peak in ERK 1/2 phosphorylation) or for
5 min did not change the partitioning of either BLT-1 or FPR
(Fig. 1, B and D, bottom panels). To guarantee an adequate
yield of lipid raft fractions for these experiments, all the neutrophils obtained from each donor were dedicated to a single
stimulation protocol and membrane fractionation. For this reason, and also because the Western blots were not standardized
between experiments, it is not possible to conclude from the
results in Fig. 1 that stimulation with LTB4 or fMLP affected
the total amounts of any of these proteins in the plasma membrane. Our findings do indicate, however, that the receptor/signaling complexes for fMLP and LTB4 are physically segregated
from one another within neutrophil plasma membranes, both
constitutively and after stimulation. Further, the disparity in the
distributions of BLT-1 vs FPR is relatively robust with respect
to the conditions of membrane fractionation. Still, it is possible
that proteins could be selectively lost or redistributed during
detergent extraction, so we sought corroboration by using FRET
to examine the lipid raft localization of these proteins in intact
cells.
The Journal of Immunology
8181
FIGURE 4. Effects of lipid raft disruption on
LTB4- and fMLP-mediated activation signaling: ERK 1/2 phosphorylation. Unstimulated
(UNSTIM) neutrophils were pretreated with
M␤CD (5 mM for 5 min) to destabilize the Lo
state of lipid rafts. Cells were subsequently
stimulated with LTB4 (10⫺8 M for 20 s; A) or
fMLP (5 ⫻ 10⫺7 M for 90 s; B). M␤CD effectively blocked ERK 1/2 phosphorylation in
response to LTB4 but had no effect on the
response to fMLP. C, The effect of M␤CD on
LTB4-mediated signaling was duplicated with
2-␤HP-CD, which binds cholesterol with
roughly the same affinity as M␤CD, but not
␣-CD, which has poor affinity for cholesterol.
Western blots shown are representative of at
least three independent experiments using different donor neutrophils.
fMLP disables subsequent responsiveness to LTB4, even while
they maintain separate partitioning within the plasma membrane
All the preceding experiments have described the relationship between lipid rafts and activation signaling through fMLP and LTB4
in previously unstimulated neutrophils. It is well established that
neutrophil activation signaling pathways are highly interactive,
with some agonists capable of selectively desensitizing cells to
subsequent stimulation by other agonists. The underlying mechanisms for this, however, are not fully defined. The following experiments were performed to determine whether the organization
Effects of lipid raft disruption on LTB4- and fMLP-induced
phosphorylation of ERK 1/2 tyrosine kinase
It is possible that some of the effects of M␤CD on intracellular
Ca2⫹ mobilization could be considerably distanced from the initial
engagement between BLT-1 and/or FPR with signaling kinases.
Accordingly, we sought to assess the role of lipid raft integrity in
another immediate signal transduction event. Fig. 4 shows that
M␤CD pretreatment completely abrogates the increased phosphorylation of ERK 1/2 tyrosine kinase in response to stimulation by
10⫺8 M LTB4. The effect was completely agonist specific, given
that M␤CD had no effect on fMLP (5 ⫻ 10⫺7 M)-induced ERK
1/2 phosphorylation. To confirm that the effect of M␤CD is not
a cyclodextrin-related artifact, we demonstrated that ␣-CD had
no effect on ERK 1/2 phosphorylation. In addition, the effects of
M␤CD were duplicated with 2-␤-hydroxypropylcyclodextrin
(Sigma-Aldrich; 5 mM, 5 min), a cyclodextrin with cholesterol
affinity that is comparable with that of M␤CD.
FIGURE 5. Cross-desensitization of fMLP- and LTB4-mediated [Ca2⫹]i
mobilization A, Fluo-3-loaded neutrophils were stimulated with LTB4
(5 ⫻ 10⫺8 M), either without prior stimulation or 5 min after stimulation
with fMLP (5 ⫻ 10⫺7 M). Neutrophils stimulated first by LTB4
(5 ⫻ 10⫺8 M) were fully able to response to a second stimulation 5 min
later with fMLP (5 ⫻ 10⫺7 M). B, Prior stimulation with fMLP negated
subsequent responsiveness to LTB4. The tracings show the average change
in [Ca2⫹]i measured from at least three independent experiments using
different donor neutrophils.
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(10). Following pretreatment, the cells were stimulated with
fMLP (5 ⫻ 10⫺7 M) or LTB4 (5 ⫻ 10⫺8 M). Experiments
confirmed that M␤CD under these conditions did not adversely
affect cell viability, as determined by trypan exclusion and by
confirming a stable baseline [Ca2⫹]i (data not shown). Also,
flow cytometry confirmed that M␤CD pretreatment did not significantly affect the levels of BLT-1 on the plasma membrane
(not shown). As shown in Fig. 3, pretreatment with M␤CD did
not significantly affect the magnitude of the initial increase in
[Ca2⫹]i in response to fMLP, although the duration of the recovery phase was shortened. It has been demonstrated previously that M␤CD has no direct effect on intracellular Ca2⫹
mobilization, but it does directly inhibit capacitative Ca2⫹ entry
through the plasma membrane, thereby accounting for a hastened recovery of [Ca2⫹]i even when the initial [Ca2⫹]i flux is
unperturbed (25). By contrast, pretreatment with M␤CD under
identical conditions substantially reduced initial Ca2⫹ mobilization in response to LTB4, in keeping with the localization of
BLT-1 in lipid rafts. To exclude a nonspecific effect of cyclodextrins, cells were pretreated with ␣-cyclodextrin (␣-CD;
Sigma-Aldrich), which has poor affinity for cholesterol. ␣-CD
(5 mM, 5 min), did not suppress the [Ca2⫹]i response to LTB4.
8182
LIPID RAFT DISTRIBUTION OF NEUTROPHIL LTB4, fMLP RECEPTORS
of lipid rafts influences the interaction between LTB4- and fMLPmediated activation signaling. The ability of prior stimulation with
LTB4 or fMLP to modulate subsequent responsiveness to the other
agonist was examined, using initial intracellular Ca2⫹ mobilization
as evidence of a signaling response (Fig. 5). As shown in Fig. 5B,
prior stimulation with fMLP completely negates responsiveness to
LTB4 within 1 min (the recovery time for fMLP-induced intracellular Ca2⫹ transients). This desensitization lasts up to 10 min (data
not shown). By contrast, prior stimulation with LTB4 had no effect
on subsequent transients in response to fMLP stimulation (Fig.
5A). The corresponding membrane fractionation experiments (Fig.
1) showed that neutrophils stimulated with LTB4 or fMLP did not
change the partitioning characteristics of either BLT-1 or FPR.
Collectively, these data demonstrate that 1) BLT-1 retains its physical separation from FPR within distinct microdomains in stimulated neutrophils and 2) heterologous desensitization of BLT-1 by
FPR does not require that the two receptors be physically located
within the same plasma membrane microdomains.
The Singer-Nicholson model of the plasma membrane describing
a homogeneous protein-imbedded phospholipid bilayer has been
replaced by a model incorporating heterogeneous lipid raft microdomains, enriched in sphingolipids and cholesterol and with
distinctive and varied protein content (reviewed in Refs. 1 and
26 –28). Many signaling proteins, including heterotrimeric G proteins and tyrosine kinases, concentrate at the cytofacial aspect of
lipid rafts (28). For this reason, it has been proposed that lipid rafts
provide the physical infrastructure for concentrating multiple elements to form signaling assemblies (29, 30). This engenders lateral
organization among signaling cascades, potentially increasing their
efficiency by keeping key elements in close proximity and by
avoiding inappropriate cross-talk between pathways. Presently,
there is relatively little information as to how lipid rafts regulate
activation signaling in neutrophils.
Chemotaxin receptors, although diverse in their ligand specificity, share certain structural features such as seven membrane-spanning domains and binding to regulatory G proteins (i.e., G proteincoupled receptors) (11). It has been shown that the chemotaxin
receptors CXCR1, CXCR4, and CCR5 localize to lipid rafts either
constitutively or after activation by their ligands (2, 3, 11). Prior
work has raised the possibility that most, if not all, chemotaxin
recognition systems involving G protein-coupled receptors are located in lipid rafts and require lipid raft integrity to interact effectively with G proteins and downstream signaling elements. Our
data show that this is not necessarily so. As shown in Figs. 3 and
4, fMLP is fully capable of activating at least some neutrophil
signaling pathways (Ca2⫹ mobilization, ERK 1/2 phosphorylation)
despite lipid raft disruption with M␤CD. The existing literature
regarding putative lipid raft associations of fMLP receptors illustrates the difficulties of establishing whether a protein is associated
with lipid rafts. Our data agree with those of Pierini et al. (31) who
showed that fMLP-induced up-regulation of ␤2 integrins was not
sensitive to M␤CD. Our findings also agree with those of Barabé
et al. (25), who found that M␤CD did not affect the peak Ca2⫹
response to fMLP but did suppress the later sustained response due
to a direct effect of M␤CD on capacitative Ca2⫹ entry through the
plasma membrane. Pierini et al. (31) found only partial suppression of the initial fMLP-induced increase in intracellular Ca2⫹ and
a greater effect on the sustained response. Tuluc et al. (32) reported
that M␤CD could disrupt Ca2⫹ influx and ERK 1/2 phosphorylation in response to ⱕ10 nM fMLP, but the effect diminished with
concentrations similar to those used in the present study. It is difficult to reconcile their findings with ours, partly because we did
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Discussion
not examine suboptimal concentrations of fMLP, and Tuluc et al.
pretreated neutrophils with M␤CD under conditions exceeding
those that we have found to be injurious to the cells. Also, in their
study, lipid raft associations were only inferred from the effects of
M␤CD. Demonstrating cholesterol dependence alone is insufficient to prove that a signaling apparatus is located within lipid
rafts. Cholesterol binds to many receptors and modulates their
function, so it does not necessarily follow that effects of cholesterol extraction must be attributed to lipid raft destabilization (33).
Moreover, cholesterol binding/extraction does not have entirely
predictable effects on lipid raft structure, and the possibility of
cholesterol binding agents acting nonspecifically often cannot be
excluded (21, 28). As an example, Xue et al. (34) found that
M␤CD disrupted FPR-mediated Ca2⫹ signaling, but the presence
of FPR in lipid rafts was inconclusive because a general colocalization with GM1 ganglioside could not be corroborated by colocalization with flotillin-1 or by FPR partitioning with lipid rafts in
fractionated membranes. Also, this was demonstrated in FPR⫹
U937 cells, so their observations may be a function of cell type or
signaling through a transfected FPR. Although it is possible that
some signaling events initiated by FPR could be tied to lipid raft
organization, our study shows that FPR functions independently of
lipid rafts in engaging at least two downstream signaling pathways.
Further, we also demonstrated the physical exclusion of FPR from
lipid rafts, both by membrane fractionation and by FRET. FPR
could be present in a previously described high density subset of
lipid rafts (7), and our membrane fractionation scheme would not
have recognized this, but this subset would have to lack flotillin-1
and ganglioside raft markers, and also be insensitive to M␤CD, to
conform with our results. By contrast, an identical experimental
approach showed that BLT-1 is found virtually exclusively in lipid
rafts. This was demonstrated by a complete concordance between
its partitioning characteristics in membrane fractionation experiments, molecular coupling with lipid raft markers by FRET, and
by the dependence of LTB4-mediated signaling on membrane cholesterol. The association of BLT-1 with lipid rafts was constitutive
and persists after LTB4 or fMLP stimulation. To our knowledge,
this is the first demonstration that BLT-1 is a lipid raft-associated
protein. The segregation of BLT-1 from FPR within the plasma
membrane is particularly striking, considering that many of the
downstream signaling events and complex effector functions (i.e.,
chemotaxis) closely resemble those elicited by fMLP.
Neutrophils responding to a chemotaxin assume a polarized
morphology with a distinct group of proteins, including receptors
and downstream signaling intermediates, redistributing in or near
the lamellipodium (reviewed in Ref. 3). It appears that lipid raft
reorganization has much to do with maintaining this polarity.
Some proteins redistribute within GM1 ganglioside-enriched lipid
raft aggregates at the tail end of polarized cells within or near the
uropod, while a distinct population of GM3 ganglioside-enriched
lipid rafts, containing an equally distinct profile of proteins,
aggregate in or near lamellipodia. The nonraft membrane of polarized
cells predominates over the remaining cell body. This redistribution was demonstrated mostly by monitoring lipid raft-associated
proteins and ganglioside markers, but we have also demonstrated
the polarization of lipid rafts by directly assessing the degree of
liquid ordering in the neutrophil plasma membrane (10). Lipid
raft-associated proteins implicated in cell movement and found at
the leading edges include GPI-linked proteins (CD87, CD59), and
many signaling intermediates (2, 9, 22, 35–39). Lipid rafts at the
rear contain CD44, CD43, PSGL-1, ICAM adhesion proteins, and
ezrin-radixin-moesin actin-binding proteins (8, 9). This evidence
certainly implicates lipid rafts in the formation of asymmetrical,
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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