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Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils
International Immunopharmacology 3 (2003) 1775 – 1790 www.elsevier.com/locate/intimp
Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils Florin Tuluc, John Meshki, Satya P. Kunapuli * Department of Physiology, Temple University Medical School, 3420 N. Broad Street, Philadelphia, PA 19140, USA Received 29 June 2003; received in revised form 14 July 2003
Abstract The integrity of lipid microdomains is disrupted after cell treatment with cholesterol-depleting reagents, such as methyl-hcyclodextrin (MCD). We investigated the roles of lipid microdomains in the regulation of intracellular signaling events and functional responses in isolated human neutrophils. Treatment of neutrophils with MCD caused inhibition of intracellular calcium increase evoked by interleukin-8 (IL-8) or low concentrations of formyl-Met-Leu-Phe (fMLP). No significant decrease of the initial peak of the calcium response was measured when neutrophils were stimulated with 100 nM or higher concentrations of fMLP. MCD inhibited the phosphorylation of extracellular signal-regulated kinase (Erk) induced by IL-8 or lower concentrations of fMLP. However, Erk phosphorylation evoked by higher concentrations of fMLP was only slightly affected. MCD treatment increased phosphorylation of p38 MAP kinase and caused strong up-regulation of both CD11b and CD66b in resting neutrophils. Cholesterol depletion greatly inhibited IL-8-induced elastase release but had little effect of fMLPinduced degranulation. Our study brings evidence suggesting that lipid microdomains are critically required for the signaling events triggered by IL-8. Calcium mobilization and elastase release induced by WKYMVM, a selective agonist for formyl peptide receptor-like 1 (FPRL1), were significantly inhibited by MCD, suggesting that the resistance of fMLP-mediated responses to MCD is not related to the partition of receptor subtypes to lipid microdomains. It is more probable that cholesterol depletion interferes with the ability of different G proteins to couple to their corresponding receptors and this might account for the differential effects of MCD treatment on chemoattractant-induced effects in human neutrophils. D 2003 Elsevier B.V. All rights reserved. Keywords: Lipid rafts; Neutrophil; Interleukin-8; Formyl-Met-Leu-Phe; MAP kinases; Degranulation
1. Introduction Polymorphonuclear leukocytes have a key role in host defense against invading microorganisms and tissue injury. Neutrophils possess multiple receptors
* Corresponding author. Tel.: +1-215-707-4615; fax: +1-215707-4003. E-mail address: [email protected] (S.P. Kunapuli). 1567-5769/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2003.08.002
for chemoattractants and chemokines which are able to cause multiple responses such as chemotaxis, generation of superoxide radicals, and degranulation. Chemoattractant receptors are coupled to pertussis toxin-sensitive heterotrimeric G proteins [1]. The bacterial tripeptide fMLP is the most extensively studied chemoattractant, and it appears to be able to mediate the activation of several pivotal intracellular signaling molecules such as phospholipase C h2 and h3 [2], phosphoinositide 3-kinase [3], Src-family
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protein kinases and mitogen-activated protein kinase (MAPK) [4]. IL-8 is a chemokine with similar binding affinities for two receptor subtypes (CXCR1 and CXCR2), which are major chemokine receptors expressed on neutrophils [5]. The effects of fMLP on human neutrophils are mediated by the classical formyl peptide receptor, which is sensitive to fMLP concentrations in nanomolar range and also by the formyl peptide receptor-like 1 (FPRL1), which is sensitive to concentrations of fMLP in micromolar range [6– 8]. The relationship between various intracellular signal transduction events triggered by chemoattractants or chemokines and subsequent functional responses is incompletely understood. For many years, the lipid bilayer of the cell membrane was considered a two-dimensional ‘‘fluid mosaic’’ [9]. In this model, the membrane proteins are uniformly dispersed in the lipid solvent, and were often compared to ‘‘icebergs in a sea of lipids’’. However, in the last decade, many independent laboratories provided evidence for the existence in the cellular membranes of microdomains enriched in sphingolipids and cholesterol that make them more ordered and less fluid than the rest of the membrane. Sphingolipids contain long, mostly saturated acyl chains that allow them to pack tightly together while phospholipids are rich in unsaturated, kinked acyl chains [10]. These membrane domains are resistant to non-ionic detergents at low temperatures [11,12] and to other detergents from the Brij series [13,14]. The lipid microdomains from the cellular membranes are most often referred to as lipid rafts, but many other names have been used in the literature: glycolipidenriched membranes (GEMs), detergent-insoluble glycolipid-enriched microdomains (DIGs), detergentresistant membranes (DRMs), low-density Triton-insoluble (LDTI) complexes (for reviews, see Refs. [15,16]). The lipid microdomains are able to incorporate different classes of proteins: glycosylphosphatidyl inositol (GPI)-anchored proteins, doubly acylated peripheral membrane proteins, cholesterol-linked proteins, and palmitoylated transmembrane proteins. It is now clear that lipid rafts have a role in cellular functions such as vesicular trafficking and signal transduction [17 –20]. Cell membranes contain two major types of liquidordered microdomains: the caveolae and lipid rafts. Both types of liquid ordered microdomains show a
light buoyant density on sucrose gradients and contain the ganglioside GM1, which is the molecular target for the subunit B of cholera toxin. The presence of other subtypes of lipid microdomains in living cells has been suggested [21 –23] but strong evidence for their existence is still missing. The main feature of caveolae is that they contain specific proteins called caveolins, which are able to dramatically change the spatial conformation of the liquid ordered microdomains to form caveolae. Commonly described as flask- or omega-shaped structures, caveoli appear like invaginations of the cell membrane that are able to detach from the plasma membrane and form plasmalemmal vesicles. Lipid rafts are generally considered as flat microdomains that are devoid of caveolins. One hypothesis is that lipid rafts may represent precursors of caveolae that facilitate the insertion of caveolins into membranes [15]. However, human neutrophils seem to be devoid of caveolins [24] but the lipid microdomains are still identifiable in their plasma membranes [25], suggesting that lipid rafts may play well-defined physiological roles in the absence of caveolins. This view is supported by several reports [25 –27] showing that cholesterol depletion from the plasma membrane prevents isolation of lipid raft by centrifugation on sucrose gradients and at the same time selectively interferes with certain intracellular signaling events such as cytosolic free calcium increase [28] or phosphorylation of Src-family tyrosine kinases [25 – 27]. The involvement of lipid rafts in regulating neutrophil functions is also suggested by reports showing that certain functional responses in neutrophils can be selectively altered by cholesterol depletion. Non-opsonic phagocytosis of Mycobacterium kansasii [29] and superoxide production induced by FcgRIIa activation [25] are inhibited after neutrophil treatment with MCD but fMLP-induced superoxide production is increased [25,28] after the same treatment. Increasing evidence supports the view that lipid rafts might play a key role in the processes involved in the infection with the human immunodeficiency virus (HIV). It has been found that treatment of peripheral blood lymphocytes with MCD reduced their susceptibility to membrane fusion with cells expressing HIV1 Env that utilize CXCR4 or CCR5 [30]. Although cholesterol does not appear to be required for HIV-1 Env-mediated membrane fusion per se, its depletion
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from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion [30]. Moreover, cholesterol depletion of HIV-1 and simian immunodeficiency virus with MCD inactivates and permeabilizes the virions [31], and topical application of MCD seems to block transmission of cell-associated HIV-1 by interfering with cell migration and budding of virus from lipid rafts [32]. It has been shown that cholesterol depletion does not affect the initial peak of calcium mobilization but shortens the duration of the calcium spike that follows the addition of fMLP. Consistent with the idea that cholesterol depletion affects calcium channels, cell treatment with MCD completely abrogated the intracellular calcium increase caused by 10 10 M plateletactivating factor (PAF), which depends mostly on the influx of calcium [28]. In our study, we show for the first time that cholesterol depletion dramatically inhibits IL-8-induced intracellular signaling events and physiological responses in human neutrophils. Only some of fMLP-induced responses in neutrophils are blocked after cholesterol depletion, suggesting that fMLP activates multiple intracellular signaling pathways and some of them are dependent on lipid rafts. In addition, we show that phosphorylation of p44/42 MAP kinases is inhibited whereas phosphorylation of p38 MAP kinases is stimulated by MCD treatment, suggesting that lipid microdomains play important regulatory roles for certain intracellular signaling pathways in neutrophils. Thus, our results provide the first evidence that signaling events downstream of some Gprotein-coupled receptors strongly rely on the integrity of lipid microdomains in neutrophil membranes.
2. Materials and methods 2.1. Reagents Methyl-h-cyclodextrin, N-(methoxysuccinyl)-AlaAla-Pro-Val p-nitroanilide, filipin III, SB203580, and bovine serum albumin (fraction V) were obtained from Sigma (St. Louis, MO). Dextran T500, Ficol-Paque, and enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ). Fura-2 AM and 5-chloromethylfluorescein were from Molecular Probes (Eugene, OR). The monoclonal anti-
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CD11b R-phycoerythrin-conjugated and monoclonal anti-CD66b FITC-conjugated antibodies were from BD Biosciences (San Jose, CA). Polyclonal anti phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-p44/ 442 MAP kinase, anti p38 MAP kinase (Thr180/ Tyr182) antibodies, and anti p38 MAP kinase were from Cell Signaling Technology (Beverly, MA). 2.2. Neutrophil isolation Venous blood was collected from healthy subjects in polypropylene tubes containing ACD anticoagulant (1.5% citric acid, 2.5% sodium citrate, 2% dextrose). Blood was mixed with an equal volume of 3% dextran T500 in saline. Erythrocytes were allowed to sediment for 20 min, then the leukocyte-rich plasma was subjected to centrifugation on Ficol-Paque at 400 g for 45 min. The pellet was collected and the contaminating erythrocytes were removed by hypotonic lysis. Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS) containing 0.2% bovine serum albumin (BSA). Neutrophils were counted using a Reichert –Jung hemacytometer (Hausser Scientific, Horsham, PA). Cell viability was checked by the Trypan blue exclusion method and was routinely found greater than 96%. 2.3. Cholesterol depletion from the outer leaflet of the neutrophil membrane Neutrophils were incubated for 1 h with rocking at room temperature in medium containing 10 mM MCD. Neutrophils were washed twice in HBSS containing 0.2% BSA by centrifugation at 200 g for 5 min. For control experiments, neutrophils were subjected to incubation in identical conditions but in the absence of MCD. For some experiments, cholesterol depletion was performed by incubating the cells for 15 min with 3 AM filipin III. 2.4. Isolation of lipid rafts in sucrose gradients and GM1 detection Neutrophils were isolated and subjected to cholesterol depletion with MCD as described above. Each sample was incubated with 100 ng/ml peroxidaseconjugated cholera toxin subunit B for 30 min on ice, then cells were washed three times in HBSS + 1% BSA.
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The neutrophils were finally resuspended in TNE buffer (25 mM Tris –HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5), then lysed by adding an equal volume of 2 lysis buffer containing a standard mixture of protease and phosphatase inhibitors. The final concentrations of reagents in the lysis medium were as follows: 25 mM Tris –HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X100, 10 Ag/ml leupeptin, 10 Ag/ml aprotinin, 1 mM sodium vanadate, 0.1 AM caliculyn B. The lysates were mixed with an equal volume of 80% sucrose in TNE buffer and layered on the bottom of a 3.5-ml ultracentrifuge tube. Layers of 30% (1.8 ml) and 5% (0.9 ml) sucrose were added sequentially and the tubes were sealed. The centrifugation was performed on a Beckman ultracentrifuge TL-100 using a rotor model TLA100.3 at 53,000 rpm for 17– 18 h. Ten fractions were collected from each tube starting from the top of the gradient. The amount of peroxidase in each fraction was assayed by a chromogenic reaction using o-phenylenediamine dihydrochloride as a substrate. 2.5. Measurement of cytoplasmic free Ca2+ concentration Isolated neutrophils were incubated for 45 min at room temperature with 1 AM fura-2 AM (Molecular Probes). The cells were washed twice and then resuspended at a concentration of 3 106/ml in HBSS containing 0.2% BSA. Some cells were treated with MCD 10 mM for 60 min to deplete cell membranes of cholesterol while control cells were incubated in identical conditions with medium only. Cells were then washed and resuspended in fresh medium and aliquots of 0.5 ml were placed in a quartz cuvette maintained at 37 jC. In some experiments, cells were pelleted and then resuspended in medium containing 0.5 mM EGTA. The intracellular calcium concentrations were assayed during agonist stimulation using excitation wavelengths of 340 and 380 nm and the emission was monitored at 510 nm using an AB2 spectrofluorometer (Spectronic Instruments, Rochester, NY). The cytoplasmic concentrations of calcium were calculated according to Tsien’s ratiometric method [33]. 2.6. Measurement of manganese influx Neutrophils were loaded with fura-2, then treated with 10 mM MCD or control buffer as described. The
cells were washed and resuspended in HBSS without calcium and placed in a quartz cuvette in an AB2 spectrofluorometer (Spectronic Instruments). Manganese influx was measured by quenching of intracellular fura-2 fluorescence. Wavelengths of 360 and 560 nm were used for excitation and emission, respectively. Fluorescence was recorded for 30 s then 200 AM MnCl2 was added to the cuvette; after another 30 s, the stimulus was applied and fluorescence tracings were recorded for 4 min. Manganese influx is reported in arbitrary fluorescence units. 2.7. Western blotting Cells were lysed as described above for GM1 detection experiments. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane and incubated for 1 h in TBS containing 1% BSA. Primary antibodies were diluted in TBS (20 mM Tris –HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) containing 2% BSA and membranes were incubated overnight at 4 jC in the presence of the indicated primary antibody. Membranes were washed four times for 5 min at room temperature. The appropriate secondary antibody conjugated with horseradish peroxidase was diluted (1:5000) in TBS containing 1% BSA. The membranes were incubated with the secondary antibody for 1 h at room temperature. Chemiluminescent ECL reagents from Amersham Biosciences were used to visualize the reactive proteins. 2.8. Elastase release Cell suspensions were treated with medium or MCD (10 mM) for 60 min at room temperature, then washed and resuspended in fresh medium containing 2 mM calcium chloride, 1 mM magnesium chloride and 10 AM cytochalasin B. Some cells were treated for 15 min with the cholesterol-sequestering reagent filipin III (3 AM). Aliquots of 150 Al cell suspension were stimulated with agonists for 20 min at 37 jC in a polypropylene 96 well plate. The plate was centrifuged for 3 min at 250 g, then supernatants were collected and the elastase activity was assayed using N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide as a chromogenic substrate. The absorbance for each sample was determined at 405 nM every 5 min using a
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Polarstar Galaxy microplate reader (BMG Labtechnologies, Bristol, RI). The elastase activity was expressed as the rate of cleavage of the chromogenic substrate and normalized for the maximum degranulating effect induced by fMLP.
curves were compared, two-way analysis of variance (ANOVA) was performed.
2.9. Chemotaxis
3.1. Effect of MCD on the lipid rafts in human neutrophils
Cell suspensions were incubated for 30 min with 2 AM 5-chloromethylfluorescein diacetate, then cells were treated with medium or MCD (10 mM) for 60 min at room temperature, then washed and resuspended in fresh HBSS containing 2 mM calcium chloride, 1 mM magnesium chloride. Cell concentration was adjusted to 1.2 106/ml. Chemotaxis was measured using ChemoTx 96 well plates (Neuroprobe, Gaithersburg, MD), with pore size of 5 Am and site diameter of 5.7 mm. Each site was loaded with 25 Al cell suspension (30,000 cells/site) and the assay was performed according to manufacturer’s instructions, with 90 min incubation at 37 jC, 5% CO2. The number of migrated cells in each well was determined by measuring the fluorescence in the lower compartment of the ChemoTx system on a Polarstar Galaxy microplate reader (BMG Labtechnologies) with excitation and emission wavelengths of 490 and 520 nm, respectively.
3. Results
MCD has been shown to disrupt the membrane lipid microdomains by cholesterol depletion [10,19,20]. To demonstrate that our conditions of MCD treatment disrupt lipid rafts in isolated human neutrophils, we labeled the ganglioside GM1 with peroxidase-conjugated cholera toxin subunit B. Cholera toxin has a high affinity for GM1, which has been previously shown to be naturally associated with lipid rafts [34,35]. In the samples derived from intact cells, GM1 was detected at significantly higher concentrations in fractions 3 and 4 ( p < 0.05), which were close to the interface between 5% and 30% sucrose layers (Fig. 1). Samples derived from MCD-treated cells showed a different pattern of distribution for GM1, with elevated concentrations of peroxidase detectable only in the high-density fractions. These findings suggest that in our experimental conditions the treatment of neutrophils with MCD disrupts the lipid rafts.
2.10. Flow cytometry Neutrophils were isolated and the treatment with 10 mM MCD was performed for 30 or 60 min. Cells were then fixed in 1% paraformaldehyde for 20 min. After three washes, cells were incubated for 30 min on ice in HBSS containing 1% bovine serum albumin, then they were incubated for 1 h with antibodies against CD11b conjugated with R-phycoerythrine and with antibodies against CD66b conjugated with fluorescein isothiocyanate. Cells were washed once in HBSS containing 1% BSA and analyzed on an Epics flow cytometer (Coulter, Hialeah, FL). 2.11. Statistical analysis The probability of a statistically significant difference between the mean values of two data sets was determined a nonparametric two-tailed Student’s ttest; when two or more concentration – response
Fig. 1. Neutrophil treatment with MCD prevents lipid rafts isolation on sucrose gradients. The cells were incubated for 1 h at room temperature without (opened circles) or with 10 mM MCD (closed circles), then the ganglioside GM1 was labeled with cholera toxin subunit B conjugated with peroxidase. Cells were lysed and subjected to ultracentrifugation on sucrose gradients, as described under Materials and methods. Ten fractions were collected and the amount of peroxidase was detected in each fraction. Lipid rafts can be detected in the lysates obtained from intact cells but not after cell treatment with MCD.
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3.2. Effect of MCD on IL-8- or fMLP-induced intracellular calcium increase Cholesterol depletion by MCD treatment has been shown to inhibit fMLP-induced calcium influx in human neutrophils without affecting mobilization of calcium from the intracellular stores [28]. We evaluated the effect of cholesterol depletion on intracellular signaling mediated by fMLP and IL-8. Addition of fMLP to a suspension of neutrophils causes a concentration-dependent increase in cytoplasmic free calcium with an EC50 value of 10 nM (Fig. 2A), as monitored using fura-2 as a fluorescent calcium probe. The increase was rapid and transient with the cytosolic free
calcium coming back to basal levels in about 2 min. The treatment of neutrophils with MCD had little effect on the amplitude of the initial spike of calcium increase caused by 100 nM fMLP or higher concentrations. However, it reduced the duration of the calcium peak, apparently affecting the maintenance of elevated calcium levels. These data are consistent with a recent study [28] wherein it has been suggested that MCD treatment interferes with the activity of calcium channels responsible for maintaining elevated cytoplasmic calcium levels. However, in our study, we found that the intracellular calcium peaks caused by lower concentrations of fMLP were significantly decreased after cholesterol depletion ( p < 0.05; Fig. 2A,C,E).
Fig. 2. Effect of MCD on cytosolic calcium increase induced by fMLP (panels A, C – E) or IL-8 (panels B, F – H). Neutrophils were loaded with fura-2 AM (1 AM) for 45 min at room temperature, then washed and preincubated with MCD (10 mM) or control buffer for 1 h. Cells were washed twice in HBSS, then fluorescence was monitored as described under Materials and methods during stimulation with IL-8 or fMLP. The peaks of intracellular calcium increase were determined. Averages of three or more independent experiments and the corresponding standard errors of the mean (S.E.M.) are shown in panels A and B for IL-8 and fMLP, respectively. Empty circles correspond to controls; closed circles correspond to MCD-treated samples. Representative tracings for fMLP- or IL-8-induced calcium increases are shown in panels C – E and F – H, respectively. Thin tracings correspond to controls; bold tracings correspond to MCD-treated samples.
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IL-8 increased intracellular calcium in human neutrophils in a concentration-dependent manner similar to fMLP. The duration of the peaks caused by IL-8 was slightly shorter than in the case of fMLP, the basal cytoplasmic calcium levels being restored after less than 1 min upon the addition of IL-8. However, IL8 showed a significantly higher potency (EC50 = 0.5 nM) than fMLP. MCD treatment dramatically inhibited both the amplitude and the duration of the intracellular calcium responses to IL-8 (Fig. 2B,F,H), even at IL8 levels causing maximal responses and considered to be above physiological concentrations. In order to determine whether MCD-induced inhibition of intracellular calcium increase can be explained by an alteration of calcium influx, we determined the calcium increases caused by IL-8 or fMLP in suspensions of neutrophils in calcium-free medium in the presence of 0.5 mM EGTA. We found that calcium peaks elicited by fMLP or IL-8 were not inhibited after chelation of calcium in the extracellular medium (Fig. 3). 3.3. Effect of MCD on IL-8- or fMLP-induced manganese influx As MCD treatment was suggested to affect the calcium channels in the neutrophil membrane [28], we investigated the effect of MCD on manganese influx
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as a measure of calcium channel activity. Manganese influx was determined during neutrophil stimulation with fMLP or IL-8 by quenching of the fluorescence of intracellular fura-2. As shown in Fig. 4, both IL8 and fMLP caused a detectable manganese influx in human neutrophils. Treatment of neutrophils with MCD abolished manganese influx induced by IL8 or fMLP, indicating that, unlike the intracellular calcium mobilization, the effect of MCD treatment is affecting the channel activity in a stimulus-independent manner. 3.4. Effect of MCD on IL-8- or fMLP-induced MAP kinase phosphorylation in human neutrophils As MCD treatment selectively altered IL-8-induced intracellular calcium increases, we investigated whether MCD affects other intracellular signaling events induced by chemokines. In human neutrophils, chemokines activate MAP kinase pathways [4]. We investigated the effect of MCD treatment on IL-8- or fMLP-induced Erk 1/2 and p38 MAP kinase phosphorylation in human neutrophils. IL-8 caused rapid Erk phosphorylation in a time-dependent manner which was maximal after 2 min of agonist treatment. The effect of fMLP appeared to be slower but reached the maximum in less than 5 min (data not shown). In MCD-treated human neutrophils, Erk phosphorylation
Fig. 3. Extracellular calcium removal does not affect the peak of cytosolic calcium increase induced by fMLP or IL-8. Neutrophils were loaded with fura-2 AM (1 AM) for 45 min at room temperature, then washed and maintained in medium containing CaCl2 (1 mM). Cells were pelleted and then resuspended in calcium-free medium containing EGTA 0.5 mM (bold tracings) or in medium containng 1 mM CaCl2 (thin tracings). Fluorescence was monitored during stimulation with fMLP (panel A) or IL-8 (panel B) as described under Materials and methods. Representative tracings for separate experiments performed on three donors are shown.
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Fig. 4. Effect of MCD on Mn2 + influx stimulated by fMLP or IL-8. MnCl2 influx was determined by quenching the fluorescence of intracellular fura-2 as described under Materials and methods. The addition of MnCl2, fMLP or IL-8 is indicated in each panel. Thin tracings correspond to controls; bold tracings correspond to MCD-treated cells. Representative tracings for separate experiments performed on two or more donors are shown.
caused by all concentrations of IL-8 tested was strongly inhibited (Fig. 5). MCD had a similar effect on Erk phosphorylation induced by 1 or 10 nM fMLP. However, the inhibitory effect of MCD was less prominent but still detectable after cell stimulation with 100 nM fMLP. No detectable levels of phosphorylated Erk were present in resting neutrophils, even after MCD treatment. Interestingly, MCD caused detectable levels of p38 MAP kinase phosphorylation in unstimulated cells (Fig. 5C); however, the phosphorylation levels of p38 MAP kinase induced by IL8 or fMLP were not affected by MCD treatment (data
not shown). For each experiment, equal loading of lanes was established by detecting total amount of the corresponding MAP kinase. 3.5. Effect of cholesterol depletion on chemoattractant-induced release of neutrophil primary granule contents One of the important physiological responses of neutrophils is elastase release from the azurophilic or primary granules. IL-8 and fMLP are known to cause primary granule release from human neutrophils [36].
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Fig. 5. Effect of MCD on p44/42 MAP kinase (Erk1/2) and p38 MAP kinase phosphorylation. Neutrophils were incubated for 1 h with MCD (10 mM) or control buffer, then stimulated for 5 min with different concentrations of IL-8 (panel A) or fMLP (panel B). Cells were lysed with a buffer containing 1% Triton X-100 and a standard mixture of protease and phosphatase inhibitors, as described under Materials and methods. Protein separation by SDS-PAGE followed by transfer of proteins on PVDF membranes was performed. The membranes were probed with antibodies raised against phosphorylated or against nonphosphorylated MAP kinases, as indicated in each panel.
The effect of fMLP on the release of primary granule contents in the extracellular medium was assessed in aliquots of cell suspensions pretreated with cytochalasin B, using elastase as a marker. As previously shown by Borregaard [36], in the absence of cytochalasin B, a negligible amount of elastase was released from neutrophils (data not shown). Cells stimulated with fMLP released very small amounts of elastase when no calcium was added to the neutrophil suspensions (data not shown). When calcium chloride (2
mM) was added to the medium, fMLP caused concentration-dependent release of elastase from neutrophils (Fig. 6A). In order to deplete cell membranes of cholesterol, we used two different agents: MCD (10 mM) or filipin III (3 AM). Cholesterol depletion caused no significant inhibition of primary granule release induced by fMLP ( p>0.1). IL-8 was able to induce a concentration-dependent release of elastase, with potency higher than fMLP but with significantly lower efficacy (Fig. 6B); the maximum concentration
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integrity of lipid rafts. The effect of MCD treatment on calcium increase and primary granule release induced by WKYMVM, a FPRL-1 selective agonist, was determined (Fig. 7B). MCD inhibited both calcium elevation and elastase release caused by WKYMVM ( p < 0.05).
Fig. 6. Effect of cholesterol depletion on fMLP- and IL-8-induced elastase release from human neutrophils. Neutrophils were incubated with MCD (10 mM; diamonds) for 1 h or with filipin III (3 AM; triangles) for 15 min. Cells were stimulated for 20 min at 37 jC with fMLP or IL-8 in the presence of cytochalasin B (10 AM). Elastase activity was detected in the supernatants collected after centrifugation of the cell suspensions using a chromogenic substrate specific for elastase. Results were normalized for the maximal response measured in cells treated with 1 AM fMLP. Averages for three or more experiments performed in duplicates with the corresponding S.E.M. are shown. Time matched controls are shown as opened circles.
of IL-8 tested caused only about 50% of the maximum fMLP response. Cholesterol depletion of neutrophil membranes significantly inhibited the degranulating response caused by IL-8. MCD treatment almost abolished IL-8-induced elastase release, whereas filipin III produced a dramatic inhibition of primary granule release ( p < 0.05). Since fMLP is able to simultaneously activate the ‘‘classical’’ formyl peptide receptor (FPR) and the formyl peptide receptor-like 1 (FPRL1), we hypothesized that the resistance of certain fMLP-induced effects to MCD treatment might be due to the fact that FPRL-1-mediated effects are not dependent on the
Fig. 7. The effect of MCD on WKYMVM-induced calcium increase and elastase release from isolated human neutrophils. Cytosolic calcium recordings and elastase release measurements were performed as described under Materials and methods. Panel A: Cells were prepared for calcium measurements and stimulated with 10 nM WKYMVM. The thin tracing corresponds to control; the bold tracing corresponds to MCD-treated sample. Representative tracings for three independent experiments are shown. Panel B: Concentration – response curve for elastase release induced by WKYMVM. Experiments were performed as described in the legend for Fig. 6. Empty symbols correspond to controls. Filled symbols correspond to MCD-treated cells. Averages for three or more experiments performed in duplicates with the corresponding S.E.M. are shown.
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3.6. Effect of MCD on IL-8- or fMLP-induced chemotaxis It has been previously shown that cholesterol depletion interferes with chemoattractant-stimulated lamellipod extension and actin polymerization in neutrophils and lipid organization is critical for human neutrophil polarization and cell migration [37]. We determined the effect of MCD treatment on fMLP- or IL-8-induced chemotaxis (Fig. 8). Both agonists caused concentration-dependent migration of neutrophils which was significantly inhibited by MCD treatment ( p < 0.01).
Fig. 9. Effect of MCD on CD11b and CD66b expression on neutrophil membrane. Neutrophils were treated with 10 mM MCD (shaded bars) or control buffer (empty bars) for 30 min or 1 h, then they were fixed with paraformaldehyde (1%) for 20 min on ice. For some samples, 10 AM SB203580 were added to the medium during the incubation with MCD (black bars). Cells were incubated with PE-conjugated monoclonal antibodies against CD11b and with FITC-conjugated monoclonal antibodies against CD66b, and then analyzed by flow cytometry. Averages for the mean fluorescence intensities (MFI) with the corresponding standard errors of the means are shown; *p < 0.05 versus corresponding untreated controls.
3.7. Cholesterol depletion promotes up-regulation of CD11b and CD66b on neutrophil membranes Fig. 8. The effect of MCD on fMLP- or IL-8-induced chemotaxis. Chemotaxis induced by IL-8 (panel A) or fMLP (panel B) was measured as described under Materials and methods. Empty symbols correspond to controls. Filled symbols correspond to MCD-treated cells. Averages for three or more experiments performed in duplicates with the corresponding S.E.M. are shown.
CD11b is known to be present in secretory vesicles as well as in secondary (specific) and tertiary (gelatinase) granules, whereas CD66b is found in secondary granules only. Upon neutrophil stimulation, the gran-
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ules fuse with the plasma membrane to release their content in the extracellular medium and some of the proteins found in granules are incorporated in the neutrophil membrane. The up-regulations on CD11b and CD66b are commonly used as markers of neutrophil activation. We investigated the effect of cholesterol depletion on chemokine-induced secondary and tertiary granule release. As shown in Fig. 9, after treatment for 1 h with MCD, both CD11b and CD66b were dramatically up-regulated even in the absence of any agonist ( p < 0.05). Treatment of neutrophils with 10 AM SB203580, a p38 MAP kinase inhibitor, did not affect membrane up-regulation of CD11b and CD66b, suggesting that although MCD treatment causes both p38 MAP kinase phosphorylation and up-regulation of CD11b and CD66b, these two events could be unrelated.
4. Discussion Lipid rafts have been known to modulate a number of cellular functions and intracellular signaling events [16,18,34,35]. In the present study, we investigated the effects of lipid rafts disruption on intracellular signaling events and functional responses mediated by chemoattractants in isolated human neutrophils. In order to disrupt the lipid rafts functionality, we used MCD and filipin III, two reagents that are widely used to deplete cholesterol from cellular membranes[18,20,38 – 40]. The concentrations of MCD and filipin III used in this study were similar to those previously shown in other cell types to effectively reduce plasma membrane cholesterol levels with minimal overall cell damage [20,38 – 40]. There are several indicators showing that the effect of this type of reagents is not due to a nonspecific effect on cell viability [28]. We did not detect significant changes in neutrophil morphology as seen by optical microscopy, and cell viability assessed by Trypan blue method was not affected by cholesterol depletion. Cytoplasmic calcium concentrations in resting cells were not significantly affected by MCD treatment. In addition, cell integrity after MCD treatment is supported by the observation that calcium responses were selectively affected by cholesterol depletion. The initial peaks of calcium response caused by 100 nM or higher con-
centrations of fMLP were not altered while the peak caused by IL-8 was drastically decreased at all concentrations tested. MCD treatment seems to have mixed stimulatory and inhibitory effects on neutrophils as shown by the up-regulation of CD11b and CD66b, which are markers of neutrophil activation, as opposed to the strong inhibition of elastase release, chemotaxis and intracellular calcium responses caused by IL-8. Finally, Erk phosphorylation was inhibited by MCD, while agonist-induced p38 MAP kinase phosphorylation was not significantly affected (data not shown). Our results show that MCD treatment blocks almost completely the manganese influx induced by either fMLP or IL-8; however, cytosolic calcium increase is differently affected by cholesterol depletion. MCD treatment appears to inhibit the activity of calcium channels in the membrane and thereby affecting agonist-induced calcium influx. This effect on calcium channels in human neutrophils has been demonstrated with fMLP and PAF as agonists [28]. Low doses of IL-8 are unable to elicit any calcium response after MCD treatment, although in intact cells the same doses are able to evoke robust responses. Calcium increase caused by higher concentrations of fMLP is obviously more resistant to the effect of cholesterol-depleting reagents, suggesting that the mechanisms involved in IL-8-induced calcium increase are dependent on the integrity lipid microdomains. It is known that after addition of EGTA to the extracellular medium to prevent calcium influx, IL-8 is still able to cause a strong calcium response in isolated human neutrophils [41]. Hence the influx and the mobilization of calcium from intracellular stores are both responsible for the calcium increase. Since our experiments were performed in medium containing calcium, we determined whether the inhibition of calcium increase after cholesterol depletion is due to an alteration of calcium influx. Hence, we measured calcium increase in cells resuspended in calcium-free medium containing 0.5 mM EGTA (Fig. 3). Under such conditions, MCD treatment did not cause significant alterations of the calcium peaks elicited by low concentrations of IL-8 or fMLP. However, cytosolic calcium concentrations returned to basal levels faster, suggesting that calcium influx plays no role in generating the peaks of intracellular calcium; however, calcium influx is required for maintaining elevated
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cytosolic concentrations for a longer time. Therefore, since MCD appears to block cation influxes induced by either fMLP or IL-8, the difference observed for MCD effects on the response mediated by the two stimuli can be explained only at the level of calcium mobilization from the intracellular stores. The exact mechanism involved is yet to be determined; however, it does not seem to involve the two formyl peptide receptor subtypes present on neutrophils. Since only the responses elicited by high concentrations of fMLP are resistant to cholesterol depletion, we hypothesized that the low affinity FPRL1 confers the resistance to MCD effect, while FPR is highly sensitive. To test this hypothesis we used the hexapeptide WKYMVM, which has been previously shown to selectively activate FPRL1 [42,43]. Calcium mobilization and elastase release induced by WKYMVM were significantly inhibited by MCD (Fig. 7), suggesting that the resistance of fMLP-mediated responses to MCD is not related to the ability of different G-protein-coupled receptors to partition to lipid microdomains. It is more probable that cholesterol depletion interferes with the ability of G proteins to couple to their corresponding receptors and this might account for the differential effects of MCD treatment on chemoattractant-induced effects in human neutrophils. Different Gi-coupled receptors, including IL-8 or fMLP receptors, can signal qualitatively different functions in human neutrophils [44,45] but the mechanisms responsible for these differences are not fully understood. IL-8 receptors (CXCR1 and CXCR2) are physically associated with Gai2 [46] but other intracellular signaling mechanisms such as coupling with the pertussis toxin-insensitive Ga16 may also account for some of the IL-8-mediated responses [47]. Gaq activation is detected after agonist stimulation in COS-7 or HEK293 cells transfected with the MCP-1 receptor. However, the same cell lines transfected with CXCR1 receptors showed no coupling with Gaq but they are able to signal through Ga16 [47]. The receptors for fMLP have been shown to couple selectively to Ga16 causing PLC activation [48], without being able to couple to Gaq [43], similar to CXCR1 receptor. Despite these similarities in G protein coupling, some differences were noticed between the functional responses caused by IL-8 and fMLP in neutrophils. Receptors for IL-8, but not for fMLP, are cross-desensitized by other peptide chemo-
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attractants [45]. Cytochalasin B, despite its potent enhancing effect on superoxide release triggered by fMLP, significantly inhibited the superoxide release triggered by IL-8 in human neutrophils [49]. Some of these discrepancies between IL-8- and fMLP-induced responses might be due to a regulatory role of lipid microdomains on signaling events mediated by the two receptors. Inhibitory effects of cholesterol-depleting reagents on human neutrophils were previously reported; for example, MCD was shown to inhibit phagocytosis of M. kansasii [29] in isolated human neutrophils. By the other side, Katsumata et al. [25] have shown that the treatment of neutrophils with MCD interferes with cross-linking-dependent initiation of tyrosine phosphorylation pathways and it potentiates superoxide generation caused by the activation of formyl peptide receptor. Barabe et al. [28] confirmed that fMLPinduced superoxide production is significantly enhanced by MCD treatment; moreover, fMLP-induced phospholipase D (PLD) activation paralleled superoxide production, suggesting a relationship between the two events. Alteration of Erk activation by cholesterol depletion has been previously reported [50]. Treatment of cultured fibroblasts with MCD caused decreased levels of Erk2 in cholesterol-depleted caveolae fraction, but endothelial growth factor (EGF) caused hyperactivation of the remaining Erk isoenzymes from caveolae; even cholesterol depletion by itself stimulated Erk activation. We found that unlike the fibroblasts, human neutrophils do not show increased levels of Erk phosphorylation after cholesterol depletion, but agonist-induced Erk1 and Erk2 activation is inhibited. However, p38 MAP kinase is phosphorylated subsequent to cholesterol depletion, suggesting that lipid rafts can selectively modulate MAP kinase activity, but this role may differ greatly from one cell type to another. Erk phosphorylation is affected by cholesterol depletion in an agonist-independent manner. Hence, it appears that a common intracellular mechanism leading to Erk phosphorylation is activated by fMLP and IL-8 receptors; however, the signaling pathways involved in primary granule release appear to be different for fMLP and IL-8, since the fMLP-induced degranulation is much more resistant to cholesterol depletion as compared to the IL-8 effect. Neutrophil treatment with MAP kinase inhibitors has been previously shown to
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interfere with degranulation. SB203580, an inhibitor of p38 MAPK, has been shown to decrease the release of primary and secondary granules, but not that of secretory vesicles [4]. Blocking ERK pathway with PD98059 had no effect on any of exocytic responses induced by fMLP [4]; however, it enhanced IL-8induced degranulation [51]. Hence, although MCD treatment altered MAP kinase phosphorylation in human neutrophils, it appears that cholesterol depletion interferes with granule release by mechanisms independent of p44/42 or p38 MAP kinases. We found that cholesterol depletion inhibits primary granule release from human neutrophils in an agonist-dependent manner. It appears that there is a good correlation between the effect of MCD treatment on calcium levels and on elastase release, suggesting that the effect of MCD on degranulation is mainly related to the interference with cytosolic calcium increase. Cholesterol depletion caused also a significant inhibition of neutrophils chemotaxis induced by either IL-8 or fMLP. However, the effect of either agonist appeared to be equally inhibited by MCD treatment, in contrast to the data obtained for primary granule release, where fMLP was more resistant than IL-8. In our study, we showed that the effects of chemoattractants on human neutrophils are highly dependent on the integrity of lipid microdomains and cholesterol depletion has mixed stimulatory and inhibitory effects on neutrophils. Moreover, IL-8- and fMLP-mediated signaling pathways are differentially affected by lipid raft disruption with cholesteroldepleting reagents. The selectivity of MCD on chemoattractant-induced effects in neutrophils seems to be dependent on the intracellular signaling pathways activated at G protein level. Our findings support the view that cholesterol depletion has selective effects at the intracellular level and suggest that lipid rafts may be involved in the modulation of a number of intracellular signaling pathways and functional responses in neutrophils, most likely due to alterations in the ability of G proteins to partition to lipid microdomains. Therefore, lipid rafts may possess important regulatory functions on chemoattractant-mediated responses in leukocytes and understanding their roles in leukocytes might lead to the development of new lipid rafts targeting drugs for the treatment of inflammatory diseases.
Acknowledgements This work was supported by research grant HL63933 from the National Institutes of Health.
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Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils Florin Tuluc, John Meshki, Satya P. Kunapuli * Department of Physiology, Temple University Medical School, 3420 N. Broad Street, Philadelphia, PA 19140, USA Received 29 June 2003; received in revised form 14 July 2003
Abstract The integrity of lipid microdomains is disrupted after cell treatment with cholesterol-depleting reagents, such as methyl-hcyclodextrin (MCD). We investigated the roles of lipid microdomains in the regulation of intracellular signaling events and functional responses in isolated human neutrophils. Treatment of neutrophils with MCD caused inhibition of intracellular calcium increase evoked by interleukin-8 (IL-8) or low concentrations of formyl-Met-Leu-Phe (fMLP). No significant decrease of the initial peak of the calcium response was measured when neutrophils were stimulated with 100 nM or higher concentrations of fMLP. MCD inhibited the phosphorylation of extracellular signal-regulated kinase (Erk) induced by IL-8 or lower concentrations of fMLP. However, Erk phosphorylation evoked by higher concentrations of fMLP was only slightly affected. MCD treatment increased phosphorylation of p38 MAP kinase and caused strong up-regulation of both CD11b and CD66b in resting neutrophils. Cholesterol depletion greatly inhibited IL-8-induced elastase release but had little effect of fMLPinduced degranulation. Our study brings evidence suggesting that lipid microdomains are critically required for the signaling events triggered by IL-8. Calcium mobilization and elastase release induced by WKYMVM, a selective agonist for formyl peptide receptor-like 1 (FPRL1), were significantly inhibited by MCD, suggesting that the resistance of fMLP-mediated responses to MCD is not related to the partition of receptor subtypes to lipid microdomains. It is more probable that cholesterol depletion interferes with the ability of different G proteins to couple to their corresponding receptors and this might account for the differential effects of MCD treatment on chemoattractant-induced effects in human neutrophils. D 2003 Elsevier B.V. All rights reserved. Keywords: Lipid rafts; Neutrophil; Interleukin-8; Formyl-Met-Leu-Phe; MAP kinases; Degranulation
1. Introduction Polymorphonuclear leukocytes have a key role in host defense against invading microorganisms and tissue injury. Neutrophils possess multiple receptors
* Corresponding author. Tel.: +1-215-707-4615; fax: +1-215707-4003. E-mail address: [email protected] (S.P. Kunapuli). 1567-5769/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2003.08.002
for chemoattractants and chemokines which are able to cause multiple responses such as chemotaxis, generation of superoxide radicals, and degranulation. Chemoattractant receptors are coupled to pertussis toxin-sensitive heterotrimeric G proteins [1]. The bacterial tripeptide fMLP is the most extensively studied chemoattractant, and it appears to be able to mediate the activation of several pivotal intracellular signaling molecules such as phospholipase C h2 and h3 [2], phosphoinositide 3-kinase [3], Src-family
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protein kinases and mitogen-activated protein kinase (MAPK) [4]. IL-8 is a chemokine with similar binding affinities for two receptor subtypes (CXCR1 and CXCR2), which are major chemokine receptors expressed on neutrophils [5]. The effects of fMLP on human neutrophils are mediated by the classical formyl peptide receptor, which is sensitive to fMLP concentrations in nanomolar range and also by the formyl peptide receptor-like 1 (FPRL1), which is sensitive to concentrations of fMLP in micromolar range [6– 8]. The relationship between various intracellular signal transduction events triggered by chemoattractants or chemokines and subsequent functional responses is incompletely understood. For many years, the lipid bilayer of the cell membrane was considered a two-dimensional ‘‘fluid mosaic’’ [9]. In this model, the membrane proteins are uniformly dispersed in the lipid solvent, and were often compared to ‘‘icebergs in a sea of lipids’’. However, in the last decade, many independent laboratories provided evidence for the existence in the cellular membranes of microdomains enriched in sphingolipids and cholesterol that make them more ordered and less fluid than the rest of the membrane. Sphingolipids contain long, mostly saturated acyl chains that allow them to pack tightly together while phospholipids are rich in unsaturated, kinked acyl chains [10]. These membrane domains are resistant to non-ionic detergents at low temperatures [11,12] and to other detergents from the Brij series [13,14]. The lipid microdomains from the cellular membranes are most often referred to as lipid rafts, but many other names have been used in the literature: glycolipidenriched membranes (GEMs), detergent-insoluble glycolipid-enriched microdomains (DIGs), detergentresistant membranes (DRMs), low-density Triton-insoluble (LDTI) complexes (for reviews, see Refs. [15,16]). The lipid microdomains are able to incorporate different classes of proteins: glycosylphosphatidyl inositol (GPI)-anchored proteins, doubly acylated peripheral membrane proteins, cholesterol-linked proteins, and palmitoylated transmembrane proteins. It is now clear that lipid rafts have a role in cellular functions such as vesicular trafficking and signal transduction [17 –20]. Cell membranes contain two major types of liquidordered microdomains: the caveolae and lipid rafts. Both types of liquid ordered microdomains show a
light buoyant density on sucrose gradients and contain the ganglioside GM1, which is the molecular target for the subunit B of cholera toxin. The presence of other subtypes of lipid microdomains in living cells has been suggested [21 –23] but strong evidence for their existence is still missing. The main feature of caveolae is that they contain specific proteins called caveolins, which are able to dramatically change the spatial conformation of the liquid ordered microdomains to form caveolae. Commonly described as flask- or omega-shaped structures, caveoli appear like invaginations of the cell membrane that are able to detach from the plasma membrane and form plasmalemmal vesicles. Lipid rafts are generally considered as flat microdomains that are devoid of caveolins. One hypothesis is that lipid rafts may represent precursors of caveolae that facilitate the insertion of caveolins into membranes [15]. However, human neutrophils seem to be devoid of caveolins [24] but the lipid microdomains are still identifiable in their plasma membranes [25], suggesting that lipid rafts may play well-defined physiological roles in the absence of caveolins. This view is supported by several reports [25 –27] showing that cholesterol depletion from the plasma membrane prevents isolation of lipid raft by centrifugation on sucrose gradients and at the same time selectively interferes with certain intracellular signaling events such as cytosolic free calcium increase [28] or phosphorylation of Src-family tyrosine kinases [25 – 27]. The involvement of lipid rafts in regulating neutrophil functions is also suggested by reports showing that certain functional responses in neutrophils can be selectively altered by cholesterol depletion. Non-opsonic phagocytosis of Mycobacterium kansasii [29] and superoxide production induced by FcgRIIa activation [25] are inhibited after neutrophil treatment with MCD but fMLP-induced superoxide production is increased [25,28] after the same treatment. Increasing evidence supports the view that lipid rafts might play a key role in the processes involved in the infection with the human immunodeficiency virus (HIV). It has been found that treatment of peripheral blood lymphocytes with MCD reduced their susceptibility to membrane fusion with cells expressing HIV1 Env that utilize CXCR4 or CCR5 [30]. Although cholesterol does not appear to be required for HIV-1 Env-mediated membrane fusion per se, its depletion
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from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion [30]. Moreover, cholesterol depletion of HIV-1 and simian immunodeficiency virus with MCD inactivates and permeabilizes the virions [31], and topical application of MCD seems to block transmission of cell-associated HIV-1 by interfering with cell migration and budding of virus from lipid rafts [32]. It has been shown that cholesterol depletion does not affect the initial peak of calcium mobilization but shortens the duration of the calcium spike that follows the addition of fMLP. Consistent with the idea that cholesterol depletion affects calcium channels, cell treatment with MCD completely abrogated the intracellular calcium increase caused by 10 10 M plateletactivating factor (PAF), which depends mostly on the influx of calcium [28]. In our study, we show for the first time that cholesterol depletion dramatically inhibits IL-8-induced intracellular signaling events and physiological responses in human neutrophils. Only some of fMLP-induced responses in neutrophils are blocked after cholesterol depletion, suggesting that fMLP activates multiple intracellular signaling pathways and some of them are dependent on lipid rafts. In addition, we show that phosphorylation of p44/42 MAP kinases is inhibited whereas phosphorylation of p38 MAP kinases is stimulated by MCD treatment, suggesting that lipid microdomains play important regulatory roles for certain intracellular signaling pathways in neutrophils. Thus, our results provide the first evidence that signaling events downstream of some Gprotein-coupled receptors strongly rely on the integrity of lipid microdomains in neutrophil membranes.
2. Materials and methods 2.1. Reagents Methyl-h-cyclodextrin, N-(methoxysuccinyl)-AlaAla-Pro-Val p-nitroanilide, filipin III, SB203580, and bovine serum albumin (fraction V) were obtained from Sigma (St. Louis, MO). Dextran T500, Ficol-Paque, and enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ). Fura-2 AM and 5-chloromethylfluorescein were from Molecular Probes (Eugene, OR). The monoclonal anti-
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CD11b R-phycoerythrin-conjugated and monoclonal anti-CD66b FITC-conjugated antibodies were from BD Biosciences (San Jose, CA). Polyclonal anti phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-p44/ 442 MAP kinase, anti p38 MAP kinase (Thr180/ Tyr182) antibodies, and anti p38 MAP kinase were from Cell Signaling Technology (Beverly, MA). 2.2. Neutrophil isolation Venous blood was collected from healthy subjects in polypropylene tubes containing ACD anticoagulant (1.5% citric acid, 2.5% sodium citrate, 2% dextrose). Blood was mixed with an equal volume of 3% dextran T500 in saline. Erythrocytes were allowed to sediment for 20 min, then the leukocyte-rich plasma was subjected to centrifugation on Ficol-Paque at 400 g for 45 min. The pellet was collected and the contaminating erythrocytes were removed by hypotonic lysis. Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS) containing 0.2% bovine serum albumin (BSA). Neutrophils were counted using a Reichert –Jung hemacytometer (Hausser Scientific, Horsham, PA). Cell viability was checked by the Trypan blue exclusion method and was routinely found greater than 96%. 2.3. Cholesterol depletion from the outer leaflet of the neutrophil membrane Neutrophils were incubated for 1 h with rocking at room temperature in medium containing 10 mM MCD. Neutrophils were washed twice in HBSS containing 0.2% BSA by centrifugation at 200 g for 5 min. For control experiments, neutrophils were subjected to incubation in identical conditions but in the absence of MCD. For some experiments, cholesterol depletion was performed by incubating the cells for 15 min with 3 AM filipin III. 2.4. Isolation of lipid rafts in sucrose gradients and GM1 detection Neutrophils were isolated and subjected to cholesterol depletion with MCD as described above. Each sample was incubated with 100 ng/ml peroxidaseconjugated cholera toxin subunit B for 30 min on ice, then cells were washed three times in HBSS + 1% BSA.
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The neutrophils were finally resuspended in TNE buffer (25 mM Tris –HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5), then lysed by adding an equal volume of 2 lysis buffer containing a standard mixture of protease and phosphatase inhibitors. The final concentrations of reagents in the lysis medium were as follows: 25 mM Tris –HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X100, 10 Ag/ml leupeptin, 10 Ag/ml aprotinin, 1 mM sodium vanadate, 0.1 AM caliculyn B. The lysates were mixed with an equal volume of 80% sucrose in TNE buffer and layered on the bottom of a 3.5-ml ultracentrifuge tube. Layers of 30% (1.8 ml) and 5% (0.9 ml) sucrose were added sequentially and the tubes were sealed. The centrifugation was performed on a Beckman ultracentrifuge TL-100 using a rotor model TLA100.3 at 53,000 rpm for 17– 18 h. Ten fractions were collected from each tube starting from the top of the gradient. The amount of peroxidase in each fraction was assayed by a chromogenic reaction using o-phenylenediamine dihydrochloride as a substrate. 2.5. Measurement of cytoplasmic free Ca2+ concentration Isolated neutrophils were incubated for 45 min at room temperature with 1 AM fura-2 AM (Molecular Probes). The cells were washed twice and then resuspended at a concentration of 3 106/ml in HBSS containing 0.2% BSA. Some cells were treated with MCD 10 mM for 60 min to deplete cell membranes of cholesterol while control cells were incubated in identical conditions with medium only. Cells were then washed and resuspended in fresh medium and aliquots of 0.5 ml were placed in a quartz cuvette maintained at 37 jC. In some experiments, cells were pelleted and then resuspended in medium containing 0.5 mM EGTA. The intracellular calcium concentrations were assayed during agonist stimulation using excitation wavelengths of 340 and 380 nm and the emission was monitored at 510 nm using an AB2 spectrofluorometer (Spectronic Instruments, Rochester, NY). The cytoplasmic concentrations of calcium were calculated according to Tsien’s ratiometric method [33]. 2.6. Measurement of manganese influx Neutrophils were loaded with fura-2, then treated with 10 mM MCD or control buffer as described. The
cells were washed and resuspended in HBSS without calcium and placed in a quartz cuvette in an AB2 spectrofluorometer (Spectronic Instruments). Manganese influx was measured by quenching of intracellular fura-2 fluorescence. Wavelengths of 360 and 560 nm were used for excitation and emission, respectively. Fluorescence was recorded for 30 s then 200 AM MnCl2 was added to the cuvette; after another 30 s, the stimulus was applied and fluorescence tracings were recorded for 4 min. Manganese influx is reported in arbitrary fluorescence units. 2.7. Western blotting Cells were lysed as described above for GM1 detection experiments. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane and incubated for 1 h in TBS containing 1% BSA. Primary antibodies were diluted in TBS (20 mM Tris –HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) containing 2% BSA and membranes were incubated overnight at 4 jC in the presence of the indicated primary antibody. Membranes were washed four times for 5 min at room temperature. The appropriate secondary antibody conjugated with horseradish peroxidase was diluted (1:5000) in TBS containing 1% BSA. The membranes were incubated with the secondary antibody for 1 h at room temperature. Chemiluminescent ECL reagents from Amersham Biosciences were used to visualize the reactive proteins. 2.8. Elastase release Cell suspensions were treated with medium or MCD (10 mM) for 60 min at room temperature, then washed and resuspended in fresh medium containing 2 mM calcium chloride, 1 mM magnesium chloride and 10 AM cytochalasin B. Some cells were treated for 15 min with the cholesterol-sequestering reagent filipin III (3 AM). Aliquots of 150 Al cell suspension were stimulated with agonists for 20 min at 37 jC in a polypropylene 96 well plate. The plate was centrifuged for 3 min at 250 g, then supernatants were collected and the elastase activity was assayed using N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide as a chromogenic substrate. The absorbance for each sample was determined at 405 nM every 5 min using a
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Polarstar Galaxy microplate reader (BMG Labtechnologies, Bristol, RI). The elastase activity was expressed as the rate of cleavage of the chromogenic substrate and normalized for the maximum degranulating effect induced by fMLP.
curves were compared, two-way analysis of variance (ANOVA) was performed.
2.9. Chemotaxis
3.1. Effect of MCD on the lipid rafts in human neutrophils
Cell suspensions were incubated for 30 min with 2 AM 5-chloromethylfluorescein diacetate, then cells were treated with medium or MCD (10 mM) for 60 min at room temperature, then washed and resuspended in fresh HBSS containing 2 mM calcium chloride, 1 mM magnesium chloride. Cell concentration was adjusted to 1.2 106/ml. Chemotaxis was measured using ChemoTx 96 well plates (Neuroprobe, Gaithersburg, MD), with pore size of 5 Am and site diameter of 5.7 mm. Each site was loaded with 25 Al cell suspension (30,000 cells/site) and the assay was performed according to manufacturer’s instructions, with 90 min incubation at 37 jC, 5% CO2. The number of migrated cells in each well was determined by measuring the fluorescence in the lower compartment of the ChemoTx system on a Polarstar Galaxy microplate reader (BMG Labtechnologies) with excitation and emission wavelengths of 490 and 520 nm, respectively.
3. Results
MCD has been shown to disrupt the membrane lipid microdomains by cholesterol depletion [10,19,20]. To demonstrate that our conditions of MCD treatment disrupt lipid rafts in isolated human neutrophils, we labeled the ganglioside GM1 with peroxidase-conjugated cholera toxin subunit B. Cholera toxin has a high affinity for GM1, which has been previously shown to be naturally associated with lipid rafts [34,35]. In the samples derived from intact cells, GM1 was detected at significantly higher concentrations in fractions 3 and 4 ( p < 0.05), which were close to the interface between 5% and 30% sucrose layers (Fig. 1). Samples derived from MCD-treated cells showed a different pattern of distribution for GM1, with elevated concentrations of peroxidase detectable only in the high-density fractions. These findings suggest that in our experimental conditions the treatment of neutrophils with MCD disrupts the lipid rafts.
2.10. Flow cytometry Neutrophils were isolated and the treatment with 10 mM MCD was performed for 30 or 60 min. Cells were then fixed in 1% paraformaldehyde for 20 min. After three washes, cells were incubated for 30 min on ice in HBSS containing 1% bovine serum albumin, then they were incubated for 1 h with antibodies against CD11b conjugated with R-phycoerythrine and with antibodies against CD66b conjugated with fluorescein isothiocyanate. Cells were washed once in HBSS containing 1% BSA and analyzed on an Epics flow cytometer (Coulter, Hialeah, FL). 2.11. Statistical analysis The probability of a statistically significant difference between the mean values of two data sets was determined a nonparametric two-tailed Student’s ttest; when two or more concentration – response
Fig. 1. Neutrophil treatment with MCD prevents lipid rafts isolation on sucrose gradients. The cells were incubated for 1 h at room temperature without (opened circles) or with 10 mM MCD (closed circles), then the ganglioside GM1 was labeled with cholera toxin subunit B conjugated with peroxidase. Cells were lysed and subjected to ultracentrifugation on sucrose gradients, as described under Materials and methods. Ten fractions were collected and the amount of peroxidase was detected in each fraction. Lipid rafts can be detected in the lysates obtained from intact cells but not after cell treatment with MCD.
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3.2. Effect of MCD on IL-8- or fMLP-induced intracellular calcium increase Cholesterol depletion by MCD treatment has been shown to inhibit fMLP-induced calcium influx in human neutrophils without affecting mobilization of calcium from the intracellular stores [28]. We evaluated the effect of cholesterol depletion on intracellular signaling mediated by fMLP and IL-8. Addition of fMLP to a suspension of neutrophils causes a concentration-dependent increase in cytoplasmic free calcium with an EC50 value of 10 nM (Fig. 2A), as monitored using fura-2 as a fluorescent calcium probe. The increase was rapid and transient with the cytosolic free
calcium coming back to basal levels in about 2 min. The treatment of neutrophils with MCD had little effect on the amplitude of the initial spike of calcium increase caused by 100 nM fMLP or higher concentrations. However, it reduced the duration of the calcium peak, apparently affecting the maintenance of elevated calcium levels. These data are consistent with a recent study [28] wherein it has been suggested that MCD treatment interferes with the activity of calcium channels responsible for maintaining elevated cytoplasmic calcium levels. However, in our study, we found that the intracellular calcium peaks caused by lower concentrations of fMLP were significantly decreased after cholesterol depletion ( p < 0.05; Fig. 2A,C,E).
Fig. 2. Effect of MCD on cytosolic calcium increase induced by fMLP (panels A, C – E) or IL-8 (panels B, F – H). Neutrophils were loaded with fura-2 AM (1 AM) for 45 min at room temperature, then washed and preincubated with MCD (10 mM) or control buffer for 1 h. Cells were washed twice in HBSS, then fluorescence was monitored as described under Materials and methods during stimulation with IL-8 or fMLP. The peaks of intracellular calcium increase were determined. Averages of three or more independent experiments and the corresponding standard errors of the mean (S.E.M.) are shown in panels A and B for IL-8 and fMLP, respectively. Empty circles correspond to controls; closed circles correspond to MCD-treated samples. Representative tracings for fMLP- or IL-8-induced calcium increases are shown in panels C – E and F – H, respectively. Thin tracings correspond to controls; bold tracings correspond to MCD-treated samples.
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IL-8 increased intracellular calcium in human neutrophils in a concentration-dependent manner similar to fMLP. The duration of the peaks caused by IL-8 was slightly shorter than in the case of fMLP, the basal cytoplasmic calcium levels being restored after less than 1 min upon the addition of IL-8. However, IL8 showed a significantly higher potency (EC50 = 0.5 nM) than fMLP. MCD treatment dramatically inhibited both the amplitude and the duration of the intracellular calcium responses to IL-8 (Fig. 2B,F,H), even at IL8 levels causing maximal responses and considered to be above physiological concentrations. In order to determine whether MCD-induced inhibition of intracellular calcium increase can be explained by an alteration of calcium influx, we determined the calcium increases caused by IL-8 or fMLP in suspensions of neutrophils in calcium-free medium in the presence of 0.5 mM EGTA. We found that calcium peaks elicited by fMLP or IL-8 were not inhibited after chelation of calcium in the extracellular medium (Fig. 3). 3.3. Effect of MCD on IL-8- or fMLP-induced manganese influx As MCD treatment was suggested to affect the calcium channels in the neutrophil membrane [28], we investigated the effect of MCD on manganese influx
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as a measure of calcium channel activity. Manganese influx was determined during neutrophil stimulation with fMLP or IL-8 by quenching of the fluorescence of intracellular fura-2. As shown in Fig. 4, both IL8 and fMLP caused a detectable manganese influx in human neutrophils. Treatment of neutrophils with MCD abolished manganese influx induced by IL8 or fMLP, indicating that, unlike the intracellular calcium mobilization, the effect of MCD treatment is affecting the channel activity in a stimulus-independent manner. 3.4. Effect of MCD on IL-8- or fMLP-induced MAP kinase phosphorylation in human neutrophils As MCD treatment selectively altered IL-8-induced intracellular calcium increases, we investigated whether MCD affects other intracellular signaling events induced by chemokines. In human neutrophils, chemokines activate MAP kinase pathways [4]. We investigated the effect of MCD treatment on IL-8- or fMLP-induced Erk 1/2 and p38 MAP kinase phosphorylation in human neutrophils. IL-8 caused rapid Erk phosphorylation in a time-dependent manner which was maximal after 2 min of agonist treatment. The effect of fMLP appeared to be slower but reached the maximum in less than 5 min (data not shown). In MCD-treated human neutrophils, Erk phosphorylation
Fig. 3. Extracellular calcium removal does not affect the peak of cytosolic calcium increase induced by fMLP or IL-8. Neutrophils were loaded with fura-2 AM (1 AM) for 45 min at room temperature, then washed and maintained in medium containing CaCl2 (1 mM). Cells were pelleted and then resuspended in calcium-free medium containing EGTA 0.5 mM (bold tracings) or in medium containng 1 mM CaCl2 (thin tracings). Fluorescence was monitored during stimulation with fMLP (panel A) or IL-8 (panel B) as described under Materials and methods. Representative tracings for separate experiments performed on three donors are shown.
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Fig. 4. Effect of MCD on Mn2 + influx stimulated by fMLP or IL-8. MnCl2 influx was determined by quenching the fluorescence of intracellular fura-2 as described under Materials and methods. The addition of MnCl2, fMLP or IL-8 is indicated in each panel. Thin tracings correspond to controls; bold tracings correspond to MCD-treated cells. Representative tracings for separate experiments performed on two or more donors are shown.
caused by all concentrations of IL-8 tested was strongly inhibited (Fig. 5). MCD had a similar effect on Erk phosphorylation induced by 1 or 10 nM fMLP. However, the inhibitory effect of MCD was less prominent but still detectable after cell stimulation with 100 nM fMLP. No detectable levels of phosphorylated Erk were present in resting neutrophils, even after MCD treatment. Interestingly, MCD caused detectable levels of p38 MAP kinase phosphorylation in unstimulated cells (Fig. 5C); however, the phosphorylation levels of p38 MAP kinase induced by IL8 or fMLP were not affected by MCD treatment (data
not shown). For each experiment, equal loading of lanes was established by detecting total amount of the corresponding MAP kinase. 3.5. Effect of cholesterol depletion on chemoattractant-induced release of neutrophil primary granule contents One of the important physiological responses of neutrophils is elastase release from the azurophilic or primary granules. IL-8 and fMLP are known to cause primary granule release from human neutrophils [36].
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Fig. 5. Effect of MCD on p44/42 MAP kinase (Erk1/2) and p38 MAP kinase phosphorylation. Neutrophils were incubated for 1 h with MCD (10 mM) or control buffer, then stimulated for 5 min with different concentrations of IL-8 (panel A) or fMLP (panel B). Cells were lysed with a buffer containing 1% Triton X-100 and a standard mixture of protease and phosphatase inhibitors, as described under Materials and methods. Protein separation by SDS-PAGE followed by transfer of proteins on PVDF membranes was performed. The membranes were probed with antibodies raised against phosphorylated or against nonphosphorylated MAP kinases, as indicated in each panel.
The effect of fMLP on the release of primary granule contents in the extracellular medium was assessed in aliquots of cell suspensions pretreated with cytochalasin B, using elastase as a marker. As previously shown by Borregaard [36], in the absence of cytochalasin B, a negligible amount of elastase was released from neutrophils (data not shown). Cells stimulated with fMLP released very small amounts of elastase when no calcium was added to the neutrophil suspensions (data not shown). When calcium chloride (2
mM) was added to the medium, fMLP caused concentration-dependent release of elastase from neutrophils (Fig. 6A). In order to deplete cell membranes of cholesterol, we used two different agents: MCD (10 mM) or filipin III (3 AM). Cholesterol depletion caused no significant inhibition of primary granule release induced by fMLP ( p>0.1). IL-8 was able to induce a concentration-dependent release of elastase, with potency higher than fMLP but with significantly lower efficacy (Fig. 6B); the maximum concentration
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integrity of lipid rafts. The effect of MCD treatment on calcium increase and primary granule release induced by WKYMVM, a FPRL-1 selective agonist, was determined (Fig. 7B). MCD inhibited both calcium elevation and elastase release caused by WKYMVM ( p < 0.05).
Fig. 6. Effect of cholesterol depletion on fMLP- and IL-8-induced elastase release from human neutrophils. Neutrophils were incubated with MCD (10 mM; diamonds) for 1 h or with filipin III (3 AM; triangles) for 15 min. Cells were stimulated for 20 min at 37 jC with fMLP or IL-8 in the presence of cytochalasin B (10 AM). Elastase activity was detected in the supernatants collected after centrifugation of the cell suspensions using a chromogenic substrate specific for elastase. Results were normalized for the maximal response measured in cells treated with 1 AM fMLP. Averages for three or more experiments performed in duplicates with the corresponding S.E.M. are shown. Time matched controls are shown as opened circles.
of IL-8 tested caused only about 50% of the maximum fMLP response. Cholesterol depletion of neutrophil membranes significantly inhibited the degranulating response caused by IL-8. MCD treatment almost abolished IL-8-induced elastase release, whereas filipin III produced a dramatic inhibition of primary granule release ( p < 0.05). Since fMLP is able to simultaneously activate the ‘‘classical’’ formyl peptide receptor (FPR) and the formyl peptide receptor-like 1 (FPRL1), we hypothesized that the resistance of certain fMLP-induced effects to MCD treatment might be due to the fact that FPRL-1-mediated effects are not dependent on the
Fig. 7. The effect of MCD on WKYMVM-induced calcium increase and elastase release from isolated human neutrophils. Cytosolic calcium recordings and elastase release measurements were performed as described under Materials and methods. Panel A: Cells were prepared for calcium measurements and stimulated with 10 nM WKYMVM. The thin tracing corresponds to control; the bold tracing corresponds to MCD-treated sample. Representative tracings for three independent experiments are shown. Panel B: Concentration – response curve for elastase release induced by WKYMVM. Experiments were performed as described in the legend for Fig. 6. Empty symbols correspond to controls. Filled symbols correspond to MCD-treated cells. Averages for three or more experiments performed in duplicates with the corresponding S.E.M. are shown.
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3.6. Effect of MCD on IL-8- or fMLP-induced chemotaxis It has been previously shown that cholesterol depletion interferes with chemoattractant-stimulated lamellipod extension and actin polymerization in neutrophils and lipid organization is critical for human neutrophil polarization and cell migration [37]. We determined the effect of MCD treatment on fMLP- or IL-8-induced chemotaxis (Fig. 8). Both agonists caused concentration-dependent migration of neutrophils which was significantly inhibited by MCD treatment ( p < 0.01).
Fig. 9. Effect of MCD on CD11b and CD66b expression on neutrophil membrane. Neutrophils were treated with 10 mM MCD (shaded bars) or control buffer (empty bars) for 30 min or 1 h, then they were fixed with paraformaldehyde (1%) for 20 min on ice. For some samples, 10 AM SB203580 were added to the medium during the incubation with MCD (black bars). Cells were incubated with PE-conjugated monoclonal antibodies against CD11b and with FITC-conjugated monoclonal antibodies against CD66b, and then analyzed by flow cytometry. Averages for the mean fluorescence intensities (MFI) with the corresponding standard errors of the means are shown; *p < 0.05 versus corresponding untreated controls.
3.7. Cholesterol depletion promotes up-regulation of CD11b and CD66b on neutrophil membranes Fig. 8. The effect of MCD on fMLP- or IL-8-induced chemotaxis. Chemotaxis induced by IL-8 (panel A) or fMLP (panel B) was measured as described under Materials and methods. Empty symbols correspond to controls. Filled symbols correspond to MCD-treated cells. Averages for three or more experiments performed in duplicates with the corresponding S.E.M. are shown.
CD11b is known to be present in secretory vesicles as well as in secondary (specific) and tertiary (gelatinase) granules, whereas CD66b is found in secondary granules only. Upon neutrophil stimulation, the gran-
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ules fuse with the plasma membrane to release their content in the extracellular medium and some of the proteins found in granules are incorporated in the neutrophil membrane. The up-regulations on CD11b and CD66b are commonly used as markers of neutrophil activation. We investigated the effect of cholesterol depletion on chemokine-induced secondary and tertiary granule release. As shown in Fig. 9, after treatment for 1 h with MCD, both CD11b and CD66b were dramatically up-regulated even in the absence of any agonist ( p < 0.05). Treatment of neutrophils with 10 AM SB203580, a p38 MAP kinase inhibitor, did not affect membrane up-regulation of CD11b and CD66b, suggesting that although MCD treatment causes both p38 MAP kinase phosphorylation and up-regulation of CD11b and CD66b, these two events could be unrelated.
4. Discussion Lipid rafts have been known to modulate a number of cellular functions and intracellular signaling events [16,18,34,35]. In the present study, we investigated the effects of lipid rafts disruption on intracellular signaling events and functional responses mediated by chemoattractants in isolated human neutrophils. In order to disrupt the lipid rafts functionality, we used MCD and filipin III, two reagents that are widely used to deplete cholesterol from cellular membranes[18,20,38 – 40]. The concentrations of MCD and filipin III used in this study were similar to those previously shown in other cell types to effectively reduce plasma membrane cholesterol levels with minimal overall cell damage [20,38 – 40]. There are several indicators showing that the effect of this type of reagents is not due to a nonspecific effect on cell viability [28]. We did not detect significant changes in neutrophil morphology as seen by optical microscopy, and cell viability assessed by Trypan blue method was not affected by cholesterol depletion. Cytoplasmic calcium concentrations in resting cells were not significantly affected by MCD treatment. In addition, cell integrity after MCD treatment is supported by the observation that calcium responses were selectively affected by cholesterol depletion. The initial peaks of calcium response caused by 100 nM or higher con-
centrations of fMLP were not altered while the peak caused by IL-8 was drastically decreased at all concentrations tested. MCD treatment seems to have mixed stimulatory and inhibitory effects on neutrophils as shown by the up-regulation of CD11b and CD66b, which are markers of neutrophil activation, as opposed to the strong inhibition of elastase release, chemotaxis and intracellular calcium responses caused by IL-8. Finally, Erk phosphorylation was inhibited by MCD, while agonist-induced p38 MAP kinase phosphorylation was not significantly affected (data not shown). Our results show that MCD treatment blocks almost completely the manganese influx induced by either fMLP or IL-8; however, cytosolic calcium increase is differently affected by cholesterol depletion. MCD treatment appears to inhibit the activity of calcium channels in the membrane and thereby affecting agonist-induced calcium influx. This effect on calcium channels in human neutrophils has been demonstrated with fMLP and PAF as agonists [28]. Low doses of IL-8 are unable to elicit any calcium response after MCD treatment, although in intact cells the same doses are able to evoke robust responses. Calcium increase caused by higher concentrations of fMLP is obviously more resistant to the effect of cholesterol-depleting reagents, suggesting that the mechanisms involved in IL-8-induced calcium increase are dependent on the integrity lipid microdomains. It is known that after addition of EGTA to the extracellular medium to prevent calcium influx, IL-8 is still able to cause a strong calcium response in isolated human neutrophils [41]. Hence the influx and the mobilization of calcium from intracellular stores are both responsible for the calcium increase. Since our experiments were performed in medium containing calcium, we determined whether the inhibition of calcium increase after cholesterol depletion is due to an alteration of calcium influx. Hence, we measured calcium increase in cells resuspended in calcium-free medium containing 0.5 mM EGTA (Fig. 3). Under such conditions, MCD treatment did not cause significant alterations of the calcium peaks elicited by low concentrations of IL-8 or fMLP. However, cytosolic calcium concentrations returned to basal levels faster, suggesting that calcium influx plays no role in generating the peaks of intracellular calcium; however, calcium influx is required for maintaining elevated
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cytosolic concentrations for a longer time. Therefore, since MCD appears to block cation influxes induced by either fMLP or IL-8, the difference observed for MCD effects on the response mediated by the two stimuli can be explained only at the level of calcium mobilization from the intracellular stores. The exact mechanism involved is yet to be determined; however, it does not seem to involve the two formyl peptide receptor subtypes present on neutrophils. Since only the responses elicited by high concentrations of fMLP are resistant to cholesterol depletion, we hypothesized that the low affinity FPRL1 confers the resistance to MCD effect, while FPR is highly sensitive. To test this hypothesis we used the hexapeptide WKYMVM, which has been previously shown to selectively activate FPRL1 [42,43]. Calcium mobilization and elastase release induced by WKYMVM were significantly inhibited by MCD (Fig. 7), suggesting that the resistance of fMLP-mediated responses to MCD is not related to the ability of different G-protein-coupled receptors to partition to lipid microdomains. It is more probable that cholesterol depletion interferes with the ability of G proteins to couple to their corresponding receptors and this might account for the differential effects of MCD treatment on chemoattractant-induced effects in human neutrophils. Different Gi-coupled receptors, including IL-8 or fMLP receptors, can signal qualitatively different functions in human neutrophils [44,45] but the mechanisms responsible for these differences are not fully understood. IL-8 receptors (CXCR1 and CXCR2) are physically associated with Gai2 [46] but other intracellular signaling mechanisms such as coupling with the pertussis toxin-insensitive Ga16 may also account for some of the IL-8-mediated responses [47]. Gaq activation is detected after agonist stimulation in COS-7 or HEK293 cells transfected with the MCP-1 receptor. However, the same cell lines transfected with CXCR1 receptors showed no coupling with Gaq but they are able to signal through Ga16 [47]. The receptors for fMLP have been shown to couple selectively to Ga16 causing PLC activation [48], without being able to couple to Gaq [43], similar to CXCR1 receptor. Despite these similarities in G protein coupling, some differences were noticed between the functional responses caused by IL-8 and fMLP in neutrophils. Receptors for IL-8, but not for fMLP, are cross-desensitized by other peptide chemo-
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attractants [45]. Cytochalasin B, despite its potent enhancing effect on superoxide release triggered by fMLP, significantly inhibited the superoxide release triggered by IL-8 in human neutrophils [49]. Some of these discrepancies between IL-8- and fMLP-induced responses might be due to a regulatory role of lipid microdomains on signaling events mediated by the two receptors. Inhibitory effects of cholesterol-depleting reagents on human neutrophils were previously reported; for example, MCD was shown to inhibit phagocytosis of M. kansasii [29] in isolated human neutrophils. By the other side, Katsumata et al. [25] have shown that the treatment of neutrophils with MCD interferes with cross-linking-dependent initiation of tyrosine phosphorylation pathways and it potentiates superoxide generation caused by the activation of formyl peptide receptor. Barabe et al. [28] confirmed that fMLPinduced superoxide production is significantly enhanced by MCD treatment; moreover, fMLP-induced phospholipase D (PLD) activation paralleled superoxide production, suggesting a relationship between the two events. Alteration of Erk activation by cholesterol depletion has been previously reported [50]. Treatment of cultured fibroblasts with MCD caused decreased levels of Erk2 in cholesterol-depleted caveolae fraction, but endothelial growth factor (EGF) caused hyperactivation of the remaining Erk isoenzymes from caveolae; even cholesterol depletion by itself stimulated Erk activation. We found that unlike the fibroblasts, human neutrophils do not show increased levels of Erk phosphorylation after cholesterol depletion, but agonist-induced Erk1 and Erk2 activation is inhibited. However, p38 MAP kinase is phosphorylated subsequent to cholesterol depletion, suggesting that lipid rafts can selectively modulate MAP kinase activity, but this role may differ greatly from one cell type to another. Erk phosphorylation is affected by cholesterol depletion in an agonist-independent manner. Hence, it appears that a common intracellular mechanism leading to Erk phosphorylation is activated by fMLP and IL-8 receptors; however, the signaling pathways involved in primary granule release appear to be different for fMLP and IL-8, since the fMLP-induced degranulation is much more resistant to cholesterol depletion as compared to the IL-8 effect. Neutrophil treatment with MAP kinase inhibitors has been previously shown to
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interfere with degranulation. SB203580, an inhibitor of p38 MAPK, has been shown to decrease the release of primary and secondary granules, but not that of secretory vesicles [4]. Blocking ERK pathway with PD98059 had no effect on any of exocytic responses induced by fMLP [4]; however, it enhanced IL-8induced degranulation [51]. Hence, although MCD treatment altered MAP kinase phosphorylation in human neutrophils, it appears that cholesterol depletion interferes with granule release by mechanisms independent of p44/42 or p38 MAP kinases. We found that cholesterol depletion inhibits primary granule release from human neutrophils in an agonist-dependent manner. It appears that there is a good correlation between the effect of MCD treatment on calcium levels and on elastase release, suggesting that the effect of MCD on degranulation is mainly related to the interference with cytosolic calcium increase. Cholesterol depletion caused also a significant inhibition of neutrophils chemotaxis induced by either IL-8 or fMLP. However, the effect of either agonist appeared to be equally inhibited by MCD treatment, in contrast to the data obtained for primary granule release, where fMLP was more resistant than IL-8. In our study, we showed that the effects of chemoattractants on human neutrophils are highly dependent on the integrity of lipid microdomains and cholesterol depletion has mixed stimulatory and inhibitory effects on neutrophils. Moreover, IL-8- and fMLP-mediated signaling pathways are differentially affected by lipid raft disruption with cholesteroldepleting reagents. The selectivity of MCD on chemoattractant-induced effects in neutrophils seems to be dependent on the intracellular signaling pathways activated at G protein level. Our findings support the view that cholesterol depletion has selective effects at the intracellular level and suggest that lipid rafts may be involved in the modulation of a number of intracellular signaling pathways and functional responses in neutrophils, most likely due to alterations in the ability of G proteins to partition to lipid microdomains. Therefore, lipid rafts may possess important regulatory functions on chemoattractant-mediated responses in leukocytes and understanding their roles in leukocytes might lead to the development of new lipid rafts targeting drugs for the treatment of inflammatory diseases.
Acknowledgements This work was supported by research grant HL63933 from the National Institutes of Health.
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