The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barabé, F.
Right arrow Articles by Naccache, P. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barabé, F.
Right arrow Articles by Naccache, P. H.
The Journal of Immunology, 2002, 168: 4042-4049.
Copyright © 2002 by The American Association of Immunologists

Early Events in the Activation of Fc{gamma}RIIA in Human Neutrophils: Stimulated Insolubilization, Translocation to Detergent-Resistant Domains, and Degradation of Fc{gamma}RIIA1

Frédéric Barabé, Emmanuelle Rollet-Labelle, Caroline Gilbert, Maria J. G. Fernandes, Samia N. Naccache and Paul H. Naccache2

Centre de Recherche en Rhumatologie et Immunologie, Canadian Institutes for Health Research Group on the Molecular Mechanisms of Inflammation, Centre de Recherche du Centre Hospitalier de l’Université Laval, and Department of Medicine, Faculty of Medicine, Laval University, Sainte-Foy, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signal transduction mechanisms associated with the ligation of Fc{gamma}RIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of Fc{gamma}RIIA following its cross-linking. The results obtained indicate that cross-linking of Fc{gamma}RIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive Fc{gamma}RIIA in the cells. The stimulated degradation of Fc{gamma}RIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked Fc{gamma}RIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by {beta}-octylglucoside. Sucrose gradient analysis of the distribution of Fc{gamma}RIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of Fc{gamma}RIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed Fc{gamma}RIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated Fc{gamma}RIIA and provide new insights into the early events following Fc{gamma}RIIA cross-linking.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The role polymorphonuclear neutrophils play in host defense and in inflammatory responses is tightly coupled to their phagocytic ability. At least two distinct classes of receptors, the complement and Fc{gamma}Rs, are present on the surface of neutrophils and participate in this function (1, 2). Human neutrophils constitutively express two activating members of the Fc{gamma}R family, namely Fc{gamma}RIIA (CD32A) and Fc{gamma}RIIIB (CD16B), both of which bind the Fc portion of IgG Igs with low affinity. Fc{gamma}RIIIB is a GPI-anchored receptor while Fc{gamma}RIIA is a single chain transmembrane receptor that possesses an immunoreceptor tyrosine-based activation motif (ITAM)3 on its cytoplasmic tail (3). While the mechanisms by which Fc{gamma}RIIIB transmits its signals through the plasma membrane remain to be clarified, it is known that the engagement of Fc{gamma}RIIA leads to the tyrosine phosphorylation of its ITAM (4). Following this event, several physiological responses are initiated: intracellular calcium mobilization, degranulation, activation of the respiratory burst, and phagocytosis (5, 6, 7, 8, 9).

The structure of Fc{gamma}RIIA is well known (10), and the stimulation of its phosphorylation has been repeatedly described (4, 11, 12, 13). The postulated protein tyrosine kinases involved in the phosphorylation of Fc{gamma}RIIA are the Src kinases, namely Lyn, Fgr, and Hck (11, 13, 14, 15, 16, 17). Additional data suggest that the ligation of Fc{gamma}RIIA leads to the activation and the Triton X-100 insolubility of Fgr in human neutrophils (18). Furthermore, cross-linked Fc{gamma}RIIA and lyn were recently found in detergent-resistant membranes in the monocytic cell line U937 (17). These results are reminiscent of the observations concerning other receptors possessing ITAMs which have also been reported to migrate to specialized membrane microdomains insoluble in nonionic detergents following their activation in various cell types (17, 19, 20, 21, 22, 23, 24). These membrane domains rich in cholesterol and sphingolipids, named lipid rafts (25, 26), have been shown to concentrate signaling molecules such as tyrosine kinases (17, 27, 28), phospholipase C (29), and adapter proteins (30) and are believed to be involved in the activation of signaling cascades.

The present study was initiated to further examine the early events that follow the cross-linking of Fc{gamma}RIIA in human neutrophils. Evidence was obtained indicating that Fc{gamma}RIIA was rapidly degraded following its activation in a Src kinase-dependent manner. Before its degradation, Fc{gamma}RIIA migrated to a nonionic detergent-insoluble fraction. The insolubilization of Fc{gamma}RIIA did not appear to be required for the initiation of the activation of the signaling sequences leading to the stimulation of the tyrosine phosphorylation pattern and the mobilization of calcium. Saponin and {beta}-octylglucoside solubilized the activated receptor, suggesting that cholesterol-rich membranes were involved in the insolubilization of the receptor. However, sucrose gradient characterization of the detergent-resistant membranes revealed that the receptor migrated to high-density fractions that were distinct from those in which low-density lipid rafts were found. These results describe novel elements in the early events that follow the activation of Fc{gamma}RIIA in human neutrophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

The anti-Fc{gamma}RIIA mAb IV.3 was purified from ascites of mice inoculated with hybridoma HB 217 obtained from the American Type Culture Collection (Manassas, VA). CT10 is an IgG fraction of a polyclonal rabbit serum against the cytoplasmic domain of Fc{gamma}RIIA raised against the synthetic peptide whose sequence was published by Ibarrola et al. (11). The anti-phosphotyrosine Ab 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). F(ab')2 directed against mouse F(ab')2 used for cross-linking were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

F(ab')2 of Ab IV.3 (Pierce, Rockford, IL) were prepared essentially as described in the manufacturer’s catalog. Briefly, the Abs were digested with pepsin (as pepsin beads) and the undigested Abs were eliminated by adding protein A and protein G beads. The integrity and the purity of the F(ab')2 were verified by their ability to label intact human neutrophils as determined by flow cytometry, as well as by their ability to activate neutrophils.

Reagents

Diisopropylfluorophosphate (DFP), filipin III, Triton X-100, {beta}-octylglucoside, wortmannin, cytochalasin B, and saponin were purchased from Sigma-Aldrich (St. Louis, MO). Nonidet P-40 (NP40) was obtained from Boehringer Mannheim (Laval, Québec, Canada). ST638 and fura 2-AM were purchased from Calbiochem (San Diego, CA) and from Molecular Probes (Junction City, OR), respectively. Piceatannol and PP1 were obtained from Biomol (Plymouth Meeting, PA).

Neutrophil purification

Venous blood was collected in isocitrate anticoagulant from healthy adult volunteers and neutrophils were purified sterilely as previously described (31). Neutrophils at 4 x 107 cells/ml were resuspended in HBSS containing 1.6 mM CaCl2, but no magnesium, and pretreated with 1 mM DFP for 10 min at room temperature before any additional manipulation (except for the mobilization of calcium experiments).

Cell stimulation

Neutrophils, at the indicated concentrations, were incubated with 2.5 µg/ml F(ab')2 of Ab IV.3 for 10 min at 4°C. The cells were then transferred to 37°C and cross-linking of Fc{gamma}RIIA was initiated upon the addition of 25 µg/ml goat F(ab')2 against mouse F(ab')2 for the time indicated. At the desired time intervals, 100 µl of the cell suspensions were added to an equal volume of boiling 2x Laemmli sample buffer (1x is 62.5 mM Tris-HCl (pH 6.8), 4% SDS, 5% 2-ME, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitrophenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol blue) and boiled for 7 min. The samples were then subjected to 7.5–20% SDS-PAGE gradients and then transferred to Immobilon PVDF membranes (Millipore, Bedford, MA). Immunoblotting was performed using either the 4G10 antiphosphotyrosine (final dilution 1/4000) or the CT10 (final dilution 1/1000) Abs and revealed using the ECL detection system as previously described (32).

Analysis of the pellets and supernatants

Neutrophils (4 x 107cells/ml) were preincubated and stimulated as described above. A total of 500 µl of the cell suspensions were transferred to 2x cold lysis buffer (1x is 20 mM Tris-HCl (pH 7.3), 137 mM NaCl, 10% glycerol, 2 mM orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) containing the indicated detergent(s) (NP40, Triton X-100, {beta}-octylglucoside, or saponin). Lysates were kept on ice for 10 min and then centrifuged (13,000 rpm) for 10 min at 4°C. Aliquots of the supernatants (100 µl) were added to an equal volume of boiling sample buffer and boiled for 7 min. The remaining supernatants were removed and the pellets were resuspended in 200 µl of 2x lysis buffer. After addition of the same volume of 2x sample buffer, the pellets were vortexed and boiled until complete dissolution. The samples were then subjected to SDS-PAGE as described above. Cell equivalent amounts of supernatants and pellets were loaded onto the gels.

The following protocol was followed in those experiments in which the effects of PP1 were monitored. After stimulation, the reactions were rapidly stopped by transferring the cell suspensions in precooled (-20°C) 1.5-ml microcentrifuge tubes. The cells were then centrifuged for 5–10 s at 6000 x g in a microcentrifuge and the pellets were resuspended at a final concentration of 40 x 106 cells/ml in a hypotonic lysis buffer (HLB) (final concentrations: 0.1% NP40, 20 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA, 2 mM orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM PMSF, 50 µg/ml trypsin inhibitor soybean, and 3 mM DFP). After a 5-min incubation at 4°C, the lysates were centrifuged at 600 x g for 10 min at 4°C. Aliquots of the supernatants were added to an equal volume of boiling 2x sample buffer. The pellets were resuspended in HLB and then diluted in the same volume of 2x sample buffer and processed for immunoblotting.

Calcium mobilization

Neutrophils (107cells/ml) were incubated at 37°C for 30 min with 1 µg/ml filipin or its solvent (ethanol) and with 1 µM fura-2/AM. The cells were washed twice to remove filipin and the extracellular probe and resuspended at 5 x 106cells/m in HBSS with 1.6 mM CaCl2. Neutrophils were then kept at 37°C and transferred to the thermostatted cuvette compartment of a spectrofluorometer (SLM 8000C; Aminco, Urbana, IL). The cells were incubated for 2 min with 2.5 µg/ml F(ab')2 of Ab IV.3 and Fc{gamma}RIIA was cross-linked upon addition of 25 µg/ml goat F(ab')2 against mouse F(ab')2. The fluorescence of the cells was monitored at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. The internal calcium concentrations were calculated as described by Grynkiewicz et al. (33).

Sucrose gradients

Neutrophils at 4 x 107cells/ml were stimulated by cross-linking Fc{gamma}RIIA as described above. After a rapid centrifugation and the removal of the supernatants, the reactions were stopped by adding 500 µl of cold lysis buffer (20 mM Tris-HCl (pH 7.3), 137 mM NaCl, 1% Triton X-100, 2 mM orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mM PMSF) to the pellets of the cells. After a 15-min incubation on ice, aliquots of the lysates (400 µl) were mixed with an equal volume of 80% sucrose, homogenized with a Dounce homogenizer, and transferred to SW60 centrifuge tubes. Aliquots (100 µl) of the cells lysates were also transferred to boiling sample buffer and analyzed by immunoblotting to ensure that equal amounts of Fc{gamma}RIIA were deposited on sucrose gradients for both unstimulated and stimulated cells. The sample-sucrose mixtures were overlaid with 2.8 ml of 30% sucrose and then with 0.4 ml of lysis buffer. The samples were centrifuged at 4°C at 43,000 rpm for 18–20 h. Following centrifugation, the samples were divided into 15 fractions of 270 µl and the proteins were chloroform/methanol-precipitated as previously described (34). Because fractions 7 and 8 were routinely devoid of proteins, these fractions were not analyzed (data not shown; see also Ref. 21). The precipitates were resuspended and boiled in sample buffer and the totality of each fraction was subjected to SDS-PAGE as described above. The pellets of the sucrose gradients were resuspended in 200 µl of 2x sample buffer and boiled until complete dissolution before being analyzed by SDS-PAGE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulated degradation of Fc{gamma}RIIA

A rapid and transient pattern of tyrosine phosphorylation was induced following the cross-linking of Fc{gamma}RIIA. The time course of this response is illustrated in Fig. 1GoA. A maximum level of tyrosine phosphorylation was consistently observed ~30 s following the cross-linking of Fc{gamma}RIIA, and this was followed by a return to the baseline level of phosphorylation within the next 30 min. At the maximum of tyrosine phosphorylation, prominent tyrosine phosphorylated bands at ~40, 55–70, and 120 kDa were observed, with minor bands at 95 and 130 kDa. When the same membrane, after stripping, was immunoblotted with CT10, a serum directed against the cytoplasmic tail of Fc{gamma}RIIA, a decrease of the amount of Fc{gamma}RIIA was observed after 2 min of stimulation which persisted for up to 30 min. This effect was not observed in unstimulated cells in which equal amounts of Fc{gamma}RIIA were observed at 15 s and after 60 min of incubation. It should also be pointed out that aliquots of the whole cell suspensions (cells and suspending buffer) were directly transferred to the tubes containing boiling sample buffer. Therefore, the decrease in CT10 immunoreactivity cannot be explained by a release of intact Fc{gamma}RIIA to the extracellular medium.



View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 1. Kinetics of degradation of Fc{gamma}RIIA. Fc{gamma}RIIA was cross-linked on human neutrophils as described in Materials and Methods for the indicated times. A, Samples were probed with an anti-phosphotyrosine Ab (4G10). B, After a stripping, the membrane was reblotted with an anti-Fc{gamma}RIIA Ab (CT10). The data shown are representative of at least three different experiments.

 
Inhibition of the stimulated degradation of Fc{gamma}RIIA by PP1

The activation of Fc{gamma}RIIA is associated with an extensive increase in the level of tyrosine phosphorylation (see Fig. 1GoA). Previous investigations have implicated, albeit indirectly, Src kinases in this response (11, 13, 16). The relationship between the stimulation of the tyrosine phosphorylation and the apparent degradation of Fc{gamma}RIIA was thus tested using one of the most selective Src kinase inhibitors presently available, namely PP1 (35). As shown in Fig. 2GoA, a 10-min preincubation with 5 µM PP1 significantly reduced the tyrosine phosphorylation response observed following the cross-linking of Fc{gamma}RIIA. A reblot of the same membrane with CT10 (Fig. 2GoB) provided evidence that the stimulated degradation of Fc{gamma}RIIA (Fig. 2GoB, first six lanes) was also significantly inhibited by PP1 for up to 10 min. The densitometric data, derived from three experiments, summarized in Fig. 2GoC confirmed that PP1 significantly inhibited the stimulated degradation of Fc{gamma}RIIA.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. Inhibition of the pattern of tyrosine phosphorylation and of the degradation of Fc{gamma}RIIA by the Src kinase inhibitor PP1. Neutrophils (15 x 106/ml) were incubated for 10 min with 5 µM PP1 following which Fc{gamma}RIIA was cross-linked as described in Materials and Methods. A, Samples were probed with an anti-phosphotyrosine Ab (4G10). B, After a stripping, the membrane was reblotted with an anti-Fc{gamma}RIIA Ab (CT10). The data shown are representative of at least three different experiments. C, The densitometric data of the amount of Fc{gamma}RIIA, expressed as the percentage of control cells, derived from three independent experiments. The measurements were done at 0.5 and 2 min. *, Statistically significant data comparing PP1-treated cells to control cells at 2 min of stimulation (p < 0.05, Wilcoxon rank sum test).

 
Other inhibitors of signaling pathways have been tested for their effects on the degradation of Fc{gamma}RIIA. Previous investigations have reported an inhibitory effect of the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin on the calcium mobilization induced by cross-linking Fc{gamma}RIIA (36, 37). Additionally, the protein tyrosine kinase Syk and the tyrosine phosphorylation of several proteins have been involved in Fc{gamma}R activation (11, 38, 39, 40, 41, 42, 43, 44, 45). Neutrophils were preincubated with 50 nM wortmannin (PI3K inhibitor), 10 µM piceatannol (Syk inhibitor) (46), 50 µM ST638 (tyrosine kinase inhibitor), or 10 µM cytochalasin B (cytoskeleton inhibitor) for 10 min before cross-linking Fc{gamma}RIIA. None of these inhibitors prevented the degradation of Fc{gamma}RIIA, as monitored 10 min after its cross-linking (Fig. 3Go). Different concentrations of wortmannin were tested (50, 100, and 300 nM), and none had any significant effect on the degradation of Fc{gamma}RIIA induced under the same conditions or on the pattern of tyrosine phosphorylation observed subsequent to Fc{gamma}RIIA cross-linking. In contrast, the mobilization of calcium was totally inhibited by 50 nM wortmannin (data not shown). Piceatannol also failed to prevent the degradation of the receptor and had little, if any, effect on the overall pattern of tyrosine phosphorylation (47). ST638, a more general tyrosine kinase inhibitor, and cytochalasin B were similarly without effect on the stimulated degradation of Fc{gamma}RIIA. Different times of incubation with the inhibitors, as well as varying cell concentrations, were tested with similar results.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of wortmannin, piceatannol, ST638, and cytochalasin B on the degradation of Fc{gamma}RIIA. Neutrophils (15 x 106/ml) were incubated at 37°C with 50 nM wortmannin, 10 µM piceatannol, 50 µM ST638, or 10 µM cytochalasin B for 10 min. Fc{gamma}RIIA was cross-linked for 10 min and the samples were processed for immunoblotting as described in Materials and Methods. The membrane was revealed with the anti-Fc{gamma}RIIA Ab (CT10). This experiment is representative of at least three independent experiments.

 
Insolubilization of cross-linked Fc{gamma}RIIA and lack of inhibition by PP1

Previous studies have shown that Src kinases translocate to insoluble fractions in human neutrophils in which Fc{gamma}RIIA was cross-linked (18, 48). The data described in Fig. 2Go indicate that PP1 inhibited the degradation and the pattern of tyrosine phosphorylation induced by the cross-linking of Fc{gamma}RIIA. This prompted us to test the potential translocation of the receptor to insoluble fractions and the effect of PP1 on the insolubilization and degradation of Fc{gamma}RIIA. Hypotonic lysates with 0.1% NP40 were tested because these conditions maximized the preservation of proteins. Neutrophils were preincubated with 5 µM PP1 for 10 min and then stimulated and lysed in NP40 buffer. Before stimulation almost all the Fc{gamma}RIIA was present in the soluble fraction (Fig. 4Go). After 30 s of cross-linking, Fc{gamma}RIIA translocated from the supernatants to the pellets. At 2 min (Fig. 4Go, lane 3), the amounts of Fc{gamma}RIIA in the supernatants as well as in the pellets decreased. These results are in accord with those obtained in the whole cell lysates (Figs. 1Go and 2Go). PP1 did not prevent the translocation of the stimulated Fc{gamma}RIIA to the pellets; the same amounts of Fc{gamma}RIIA translocated to the pellets in control and PP1-treated cells after 30 s, while equivalent decreases were observed in the supernatants. Two minutes after cross-linking, a decrease in the total amount of Fc{gamma}RIIA is observed in the untreated cells while PP1 preserved Fc{gamma}RIIA from degradation, in accord with the results illustrated in Fig. 2Go. It should be noted that the distribution of Fc{gamma}RIIA in PP1-treated neutrophils between the supernatants and the pellets was similar after 2 min of stimulation to what it was after 30 s of stimulation.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 4. Insolubilization of the receptor and effect of PP1. Neutrophils (4 x 107cells/ml) were preincubated for 10 min with 5 µM PP1. After cross-linking of Fc{gamma}RIIA for the indicated times, the reactions were rapidly stopped, the cells were lysed in a HLB, and soluble and insoluble fractions were prepared as described in Materials and Methods. The samples were subjected to SDS-PAGE and immunoblotted with the anti-Fc{gamma}RIIA Ab CT10. This experiment is representative of at least three different experiments.

 
Translocation of the Fc{gamma}RIIA to detergent-insoluble fractions after its cross-linking

To confirm the translocation of cross-linked Fc{gamma}RIIA to detergent-insoluble fractions, we tested its insolubility in classical 1% Triton X-100 isotonic buffers as described by Cheng et al. (61), Lang et al. (24), Field et al. (20), Janes et al. (27), Ilangumaran and Hoessli (no. 6962; Ref. 58) for other cell surface receptors. Neutrophils were stimulated and then lysed in isotonic lysis buffers containing different nonionic detergents. After centrifugation at 13,000 rpm, the supernatants and the pellets were analyzed. Fig. 5Go shows a CT10 immunoblot of the supernatants (Fig. 5Go, upper panel) and the pellets (Fig. 5Go, lower panel) of unstimulated cells and 0.5 and 2 min after Fc{gamma}RIIA cross-linking in three different buffers. The first detergent tested was NP40, at a final concentration of 1% (Fig. 5Go, first three lanes). Before stimulation, almost all the Fc{gamma}RIIA was present in the soluble fraction. After 30 s of activation, there was a drastic shift of the receptor to the pellets with a concomitant loss from the supernatants. At 2 min (Fig. 5Go, lane 3), the amounts of Fc{gamma}RIIA in the supernatants as well as in the pellets decreased. These results are similar to those obtained in hypotonic lysates containing 0.1% NP40 (Fig. 4Go). Similar results were obtained using Triton X-100-containing buffer (Fig. 5Go, lanes 4–6). The third buffer tested contained NP40 as well as saponin, both at a final concentration of 1% (Fig. 5Go, lanes 7–9). Before cross-linking, the distribution of the Fc{gamma}RIIA was the same as in the two other buffers; i.e., most of the Fc{gamma}RIIA was found in the soluble fraction. However, after 30 s of cross-linking in the combined presence of NP40 and saponin, most of the Fc{gamma}RIIA was solubilized and remained in the supernatant, in contrast to what was found in the NP40 and Triton X-100 buffers (compare lane 8 to lanes 2 and 5 in Fig. 5Go), while only a small amount was found in the pellet. The nondegraded Fc{gamma}RIIA that was still detectable after 2 min of cross-linking was found in the soluble fraction, as in the two other buffers. In experiments not reported here, it was also observed that {beta}-octylglucoside (60 mM) was also able to solubilize Fc{gamma}RIIA, even at 30 s of stimulation (data not shown). Saponin alone, without NP40 or Triton X-100, was not sufficient to solubilize Fc{gamma}RIIA after cross-linking, nor was a combination of NP40 and Triton X-100 (each at 1%) (data not shown).



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 5. Translocation of the Fc{gamma}RIIA to insoluble fractions after its activation. After cross-linking of Fc{gamma}RIIA for the indicated times as previously described, cells (4 x 107/ml) were lysed in buffers containing either NP40, Triton X-100, or NP40 with saponin as described in Materials and Methods. The final concentration of detergents was 1%. Amounts of the supernatants and the pellets corresponding to the same number of cells were run on SDS-PAGE. The membranes were blotted with the anti-Fc{gamma}RIIA Ab (CT10). Results are representative of at least three different experiments.

 
The translocation of Fc{gamma}RIIA is to a high-density detergent-insoluble fraction

The translocation of Fc{gamma}RIIA to a nonionic detergent-insoluble fraction following its activation and its solubilization in saponin and {beta}-octylglucoside buffers suggested a stimulated translocation of the receptor to lipid rafts (49). To test this hypothesis, we characterized the distribution of Fc{gamma}RIIA on sucrose gradients. Neutrophils were stimulated as described above by cross-linking Fc{gamma}RIIA for 30 s, lysed in 1% Triton X-100 cold buffer, and submitted to ultracentrifugation over sucrose gradients (see Materials and Methods). Tris buffers were chosen because they preserved Fc{gamma}RIIA better than MES-containing buffers (data not shown). After gradient fractionation and protein precipitation, the fractions were boiled in sample buffer, loaded onto SDS-PAGE, and transferred to PVDF membranes. The low-density lipid raft fractions corresponded to fractions 11–15 and were identified visually as well as from their content of the Src family kinase Hck (data not shown). Fractions 1–5 contained the Triton-soluble proteins. Fig. 6Go shows the distribution of Fc{gamma}RIIA in the sucrose gradients derived from unstimulated (Fig. 6Go, upper panel) and Fc{gamma}RIIA-cross-linked (Fig. 6Go, lower panel) neutrophils. At rest, Fc{gamma}RIIA was almost entirely located in the Triton X-100-soluble phase of the gradient (fractions 1–5), with only a minor fraction of the total amount of Fc{gamma}RIIA found in the low-density lipid raft fractions (fractions 11–15). After 30 s of stimulation, a decrease in the amounts of Fc{gamma}RIIA in the Triton X-100-soluble phase was noted (fractions 1–5; Fig. 6Go, lower panel). In contrast, no increase of Fc{gamma}RIIA was found in the low-density lipid rafts fractions (fractions 11–15; Fig. 6Go, lower panel). However, a shift in the distribution of Fc{gamma}RIIA from the upper (fraction 15) to the lower (fractions 11 and 12) fractions of the low-density fractions was observed in most of the experiments following its cross-linking.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 6. Analysis of the sucrose gradients. Neutrophils (40 x 106/ml) were stimulated by cross-linking Fc{gamma}RIIA for 30 s. Cell lysates were then subjected to ultracentrifugation on sucrose gradients as described in Materials and Methods. Following the centrifugation, the samples were divided in 15 fractions of 270 µl and the proteins were precipitated as described in Materials and Methods. After precipitation, the totality of each fraction was subjected to SDS-PAGE gradient and fractions were blotted with the anti-Fc{gamma}RIIA Ab CT10. Upper panel, Unstimulated cells; lower panel, stimulated cells. Lanes 11–15 represent fractions containing lipid rafts while lanes 1–5 contain the Triton X-100-soluble fractions. Data shown are representative of three different experiments.

 
The lack of evidence of a translocation of Fc{gamma}RIIA to the low-density lipid rafts following its cross-linking, coupled to the important decrease of the total amount of Fc{gamma}RIIA in stimulated cells at a time (30 s) where the receptor is not yet degraded, suggested that an analysis of the pellets of the sucrose gradients was warranted. Before the analysis of the pellets, the total cell lysates were tested to ensure that the same amounts of Fc{gamma}RIIA were present in unstimulated and stimulated cells before the ultracentrifugation step on sucrose gradients. The results of these experiments are illustrated in the first two lanes of Fig. 7Go, upper panel. They demonstrate, first, that equal amounts of Fc{gamma}RIIA were preserved in both control and stimulated cells, thereby ruling out the possibility that degradation of Fc{gamma}RIIA during the 30-min incubation that precedes the ultracentrifugation step could explain the decrease of the total amount of the receptor in the sucrose gradient fractions upon stimulation. CT10 immunoblots of the sucrose gradient pellets derived from control and Fc{gamma}RIIA cross-linked neutrophils are shown in Fig. 7Go, upper panel (rightmost two lanes). The pellet of the unstimulated cells contained a small amount of Fc{gamma}RIIA, while an important translocation of Fc{gamma}RIIA to that fraction was detected following cross-linking of the receptor. Stimulation of the cells with an unrelated agonist, the chemotactic peptide fMet-Leu-Phe, did not alter the distribution of Fc{gamma}RIIA (data not shown).



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 7. Analysis of the pellets of the sucrose gradients. Cell stimulation and sucrose gradients were prepared as described in Materials and Methods. Thirty minutes after the cold lysis, before sucrose, aliquots (100 µl) of total cell lysates were transferred to the same volume of boiling 2x Laemmli sample buffer. After ultracentrifugation, the sucrose gradients were removed and analyzed, and 200 µl of 2x Laemmli sample buffer were added to the pellets and boiled to complete dissolution. Samples were blotted with the anti-Fc{gamma}RIIA Ab CT10 (upper panel) or the anti-phosphotyrosine Ab 4G10 (lower panel). This experiment is representative of at least three different experiments.

 
As signaling molecules and tyrosine phosphorylated substrates are known to concentrate in detergent-insoluble microdomains, the sucrose gradients pellets were probed with anti-phosphotyrosine Abs. The patterns of tyrosine phosphorylation of the cell lysates before ultracentrifugation were also monitored. Anti-phosphotyrosine immunoblots of these lysates demonstrated that the patterns of tyrosine phosphorylation were well preserved under these conditions in both buffers (Fig. 7Go, lower panel) when compared with the patterns obtained by direct transfer of cell aliquots to denaturing Laemmli sample buffer (see Fig. 1Go). Analysis of the tyrosine phosphorylation in the pellets showed that an important quantity of tyrosine-phosphorylated proteins was present in the pellets of Fc{gamma}RIIA-stimulated neutrophils as compared with resting cells. The pattern of tyrosine phosphorylation found in the sucrose gradient pellets preserved some elements of that of whole cells, in particular bands at 40 (possibly Fc{gamma}RIIA itself), 50–55 (possibly Src family kinases; preliminary experiments provided evidence for both Lyn and Hck in the pellets of the gradients (data not shown)), and 70 kDa (possibly Syk).

Inhibition of the degradation of Fc{gamma}RIIA by filipin

The ability of saponin and {beta}-octylglucoside to solubilize Fc{gamma}RIIA after its cross-linking suggested a migration of the receptor to cholesterol-rich membranes upon stimulation (50, 51), although that stimulated Fc{gamma}RIIA did not translocate to classical lipid rafts as demonstrated with sucrose gradients. To corroborate these findings, we next tested the effects of filipin, a cholesterol-sequestering agent (52, 53), on the degradation of Fc{gamma}RIIA. Neutrophils were preincubated at 37°C with 1 µg/ml filipin or its solvent (ethanol at 0.1% final) for 30 min at 15 x 106 cells/ml and then washed once in HBSS. Following Fc{gamma}RIIA cross-linking, the reactions were stopped by transferring aliquots of the cell suspensions to boiling sample buffer. The results shown in Fig. 8Go demonstrate that the initial levels of Fc{gamma}RIIA after incubation with filipin or with ethanol were the same (Fig. 8Go, compare lanes 1 and 4). The treatment with filipin resulted in a significant inhibition of the degradation of Fc{gamma}RIIA at 2 and 10 min in comparison to untreated cells. We also tested the effects of filipin on the pattern of tyrosine phosphorylation and on the mobilization of calcium induced by the activation of Fc{gamma}RIIA. Treatment of neutrophils with filipin had little effect on the stimulated pattern of tyrosine phosphorylation as shown in Fig. 9Go: not only was no inhibition detected, but a slight increase in the intensity of the pattern of tyrosine phosphorylation was consistently detected following filipin treatment. Monitoring different times of incubation with filipin or of Fc{gamma}RIIA cross-linking gave the same results. The next set of experiments was conducted to evaluate the effects of filipin on the mobilization of calcium induced by the activation of Fc{gamma}RIIA. The cells were incubated for 30 min with 1 µg/ml filipin or its solvent and the mobilization of calcium that resulted from the cross-linking of Fc{gamma}RIIA was monitored (Fig. 10Go). The addition of the cross-linking Abs induced a rapid and transient increase in the concentration of cytoplasmic free calcium that was similar in control and filipin-treated cells, reaching the same peak of intracellular calcium with superimposable kinetics.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of filipin on the degradation of Fc{gamma}RIIA. Cells (15 x 106/ml) were incubated at 37°C with 1 µg/ml filipin or its solvent as control (ethanol). After 30 min of incubation, the cells were washed once with HBSS and Fc{gamma}RIIA was cross-linked as described in Materials and Methods. Whole cell aliquots were then processed for SDS-PAGE and Ab CT10 was used to probe the membrane. The data are representative of at least three independent experiments.

 


View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 9. Effect of filipin on the pattern of tyrosine phosphorylation. Cells (15 x 106/ml) were incubated at 37°C with 1 µg/ml filipin or its solvent (ethanol, 0.1% final). After 30 min of incubation, the cells were washed once with HBSS and Fc{gamma}RIIA was cross-linked as described in Materials and Methods. Whole cell aliquots were then processed for SDS-PAGE. Anti-phosphotyrosine Ab 4G10 was used to probe the membrane. The data shown are representative of three different experiments.

 


View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 10. Effect of filipin on the mobilization of calcium. Neutrophils were isolated as described in Materials and Methods but were not incubated with DFP. Cells (10 x 106/ml) were incubated at 37°C with 1 µM fura-2 AM and with 1 µg/ml filipin or its solvent for 30 min and treated as described in Materials and Methods. The cells were washed twice and resuspended at 5 x 106/ml and incubated for 2 min with 2.5 µg/ml F(ab')2 of Ab IV.3, which was then cross-linked at the time indicated by the arrow by the addition of F(ab')2 against mouse F(ab')2. The data are from a single experiment representative of three other independent determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ligation of Fc{gamma}RIIA on neutrophils results in the activation of several, mostly tyrosine phosphorylation-dependent, signaling pathways and in the initiation of various functional responses leading to the performance of the defense-oriented phagocytic function of these cells. In this study, we provide evidence that the early events that follow Fc{gamma}RIIA cross-linking include a translocation to a detergent-insoluble fraction that is rapidly followed by a degradation of the receptor. These events occur in parallel to those leading to signal transduction through Fc{gamma}RIIA.

Cross-linking of Fc{gamma}RIIA results in a time-dependent, biphasic stimulation of the cells’ pattern of tyrosine phosphorylation and in a significant decrease in the amount of Fc{gamma}RIIA. The latter was evident within 2 min of cross-linking and was maintained for up to 60 min following receptor ligation (the longest time tested). This decreased immunoreactivity was not secondary to lack of recognition of the phosphorylated form of Fc{gamma}RIIA by the Abs, because the peak of phosphorylation of Fc{gamma}RIIA occurs at 30 s (data not shown) and, at this time, no decrease in the amount of Fc{gamma}RIIA was observed (54). Moreover, the levels of Fc{gamma}RIIA remained depressed for up to 60 min, while the tyrosine phosphorylation of Fc{gamma}RIIA declined after 2 min (data not shown). The degradation of Fc{gamma}RIIA was a specific response to its own activation because other phagocytic particles that do not interact with Fc{gamma}RIIA (monosodium urate crystals) (55) and chemotactic factors (fMLP) did not induce its degradation (data not shown). The presently available tools do not allow determination of whether the receptor was totally degraded or if only its cytoplasmic tail (the region against which the antiserum was raised) was cleaved. The other commercially available Abs against Fc{gamma}RIIA do not immunoblot adequately enough to resolve this question. However, it is possible to conclude that for up to 60 min after cross-linking there is no recycling or re-expression of intact Fc{gamma}RIIA in the total cell lysates. It should also be pointed out that the results cannot be explained by a shedding of intact Fc{gamma}RIIA to the extracellular milieu, because whole cell suspension aliquots were directly transferred to sample buffer and analyzed. The lack of blotting Abs against the extracellular domain of Fc{gamma}RIIA prevents testing to determine whether the latter is cleaved (shed) upon cross-linking. The functional significance of the degradation of Fc{gamma}RIIA remains to be clearly identified. However, it is tempting to speculate that it may play a role in signal modulation and, in particular, in signal termination of Fc{gamma}RIIA-mediated activation. To the best of our knowledge, this rapid process of degradation of Fc{gamma}RIIA has not been described in other cell types (monocytes, platelets) where the receptor is present, although it is reminiscent of work of Paolini and Kinet (56) in which the activated {beta}- and {gamma}-chains of the Fc{epsilon}RI are ubiquitinated and degraded.

The data obtained with the Src kinase inhibitor PP1 indicate that the degradation of Fc{gamma}RIIA is secondary to the initiation of the tyrosine phosphorylation cascade, as PP1 inhibited the pattern of tyrosine phosphorylation induced upon the cross-linking of Fc{gamma}RIIA as well as its degradation. The specificity of the inhibitory effects of PP1 is highlighted by the lack of effects of ST638 and piceatannol, representatives of two classes of tyrosine kinase inhibitors with selectivities different from that of PP1. These observations suggest a specific role of the tyrosine phosphorylation pathway and of one or more Src kinase(s) in the initiation of the events leading to the stimulated degradation of the receptor. Several publications identified Lyn as being involved in the phosphorylation of Fc{gamma}RIIA (11, 13, 14, 15), while other Src kinases, namely Fgr and Hck, have also been postulated to be involved in the mediation of the signal transduction pathways associated with this receptor (15, 16). Whether it is the tyrosine phosphorylation of Fc{gamma}RIIA itself or that of another cellular element which signals its degradation is presently not known.

Inhibitors of other pathways known to be involved in the activation of Fc{gamma}RIIA have also been tested to identify other possible critical steps in the degradation of the receptor. Inhibition of the activation of PI3K and of the rearrangement of actin cytoskeleton failed to prevent the degradation of the receptor. It should be pointed out that while wortmannin was without effect on the stimulated degradation of Fc{gamma}RIIA it, in contrast, completely inhibited the mobilization of calcium observed under the same conditions (36). These results suggest that the induction of the degradation of the receptor is independent of the mobilization of intracellular calcium. Syk has been identified as a crucial element in Fc{gamma}R-mediated phagocytosis (39, 43). The lack of effect of the Syk inhibitor, piceatannol, indicates that the regulation of the degradation of the receptor is regulated independently from the internalization itself and depends on upstream, Src kinase-dependent events. This interpretation is consistent with the results of Bonnerot et al. (38), who showed that the internalization of the {gamma}-chain of the Fc{gamma}R in T cells was independent of Syk.

Several lines of evidence suggest that cross-linking of Fc{gamma}RIIA leads to its rapid translocation to detergent-resistant membrane microdomains. The stimulated insolubility of Fc{gamma}RIIA in NP40- and Triton X-100-containing buffers is consistent with this interpretation. The latter is reinforced by the ability of saponin and {beta}-octylglucoside to solubilize Fc{gamma}RIIA. These two detergents have previously been shown to extract membrane proteins from lipid rafts (50, 57, 58, 59). Sucrose gradient analysis showed that a very small amount of Fc{gamma}RIIA is present at rest in the buoyant fraction classically associated with the lipid rafts. However, no significant movements of Fc{gamma}RIIA from the soluble fractions to the low-density lipid rafts fractions were observed upon receptor stimulation. Unexpectedly, Fc{gamma}RIIA shifted to the pellets of the sucrose gradients following its cross-linking. This translocation of Fc{gamma}RIIA was a specific response to its activation, because stimulation of the cells by fMLP did not induce it (data not shown). The behavior of Fc{gamma}RIIA in neutrophils is thus different from that recently described in the human monocytic cell line U937. In that study, which examined the behavior of the fraction of the receptor present in high-m.w. complexes in the supernatants of a 10,000 x g centrifugation, cross-linking of Fc{gamma}RIIA led to its accumulation in low-buoyant density detergent-resistant fractions (17). In contrast, other surface receptors of the Ig superfamily present on various leukocytes have previously been shown to behave similarly to Fc{gamma}RIIA. For example, a translocation of the B cell receptor (BCR), as well as of the Fc{gamma}RIIB following BCR engagement, to high-density detergent-insoluble fractions derived from B cells has recently been observed (23).

It should be noted that the translocation of Fc{gamma}RIIA to the high-density detergent-resistant fraction is associated with that of several other tyrosine-phosphorylated proteins. This result may be related to the previously reported insolubilization of Fgr upon cross-linking of Fc{gamma}RIIA (18). Although the identification of these proteins was not, and in fact goes beyond, the aim of the present study, the stimulated presence of these proteins in the pellets of the sucrose gradients underlines the importance of further studies of the identity and functional significance of this fraction in Fc{gamma}RIIA-initiated signal transduction.

The cholesterol-sequestering agent filipin inhibited Fc{gamma}RIIA degradation. This observation suggests that the translocation of the receptor into cholesterol-rich membrane domains is critical to this process, which is also dependent on the initiation of the tyrosine phosphorylation cascade. In contrast, the integrity of the cholesterol-rich microdomains does not appear to be crucial for Fc{gamma}RIIA-mediated signal transduction, because filipin did not affect the stimulated pattern of tyrosine phosphorylation and did not have a significant effect on the mobilization of calcium induced upon cross-linking of the receptor.

The fraction of the total Fc{gamma}RIIA that is degraded upon stimulation correlates with that which becomes insoluble. We can hypothesize that the nondegraded fraction of Fc{gamma}RIIA is either soluble (because a subpopulation of Fc{gamma}RIIA remained in the soluble fractions (Figs. 4Go and 5Go)) or not phosphorylated. This interpretation is consistent with our observations that phosphorylated Fc{gamma}RIIA was present in the insoluble fraction (60). While PP1 inhibited the degradation of the receptor, it failed to inhibit the translocation of the receptor to the insoluble fraction. These observations suggest that the insolubility phenomenon was independent of the stimulation of the tyrosine phosphorylation pathway and that the clustering of the receptors by cross-linking is sufficient to induce its insolubility. In a recent publication, PP2, another Src kinase inhibitor, was similarly found to fail to block the translocation of the BCR into lipid rafts (61).

In summary, the results of this study describe novel elements of the regulation of Fc{gamma}RIIA signaling. They provide evidence that stimulated Fc{gamma}RIIA translocates to a high-density detergent-insoluble fraction before being degraded. Importantly, the presently reported observations indicate that the translocation of Fc{gamma}RIIA is not a prerequisite for signal transduction. In contrast, our results suggest a mechanism of regulation or termination of the activation of Fc{gamma}RIIA dependent on its insolubilization and degradation. These data provide important novel information concerning the regulation of activated Fc{gamma}RIIA in human neutrophils.


   Acknowledgments
 
We thank Sylvain Levasseur and Guillaume Paré for their expert help with the preparation of the F(ab')2 and the calcium measurements, respectively.


   Footnotes
 
1 This work was supported in part by grants from the Canadian Institutes for Health Research and the Arthritis Society of Canada. C.G. is supported by fellowships from the K. M. Hunter Charitable Foundation, the Canadian Institutes for Health Research, and the Fonds de la Recherche en Santé du Québec. Back

2 Address correspondence and reprint requests to Dr. Paul H. Naccache, Centre Hospitalier de l’Université Laval, Room T1-49, 2705 Boulevard Laurier, Sainte-Foy, Québec, G1V 4G2, Canada. E-mail address: paul.naccache{at}crchul.ulaval.ca Back

3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; DFP, diisopropylfluorophosphate; HLB, hypotonic lysis buffer; NP40, Nonidet P-40; BCR, B cell receptor; PI3K, phosphatidylinositol 3-kinase. Back

Received for publication October 23, 2001. Accepted for publication February 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Daeron, M.. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203.[Medline]
  2. Indik, Z. K., J. G. Park, S. Hunter, A. D. Schreiber. 1995. The molecular dissection of Fc{gamma} receptor mediated phagocytosis. Blood 86:4389.[Abstract/Free Full Text]
  3. Isakov, N.. 1998. Role of immunoreceptor tyrosine-based activation motif in signal transduction from antigen and Fc receptors. Adv. Immunol. 69:183.[Medline]
  4. Dusi, S., M. Donini, V. Della Bianca, G. Gandini, F. Rossi. 1994. In human neutrophils the binding to immunocomplexes induces the tyrosine phosphorylation of Fc{gamma}RII but this phosphorylation is not an essential signal for Fc-mediated phagocytosis. Biochim. Biophys. Acta 201:30.
  5. Huizinga, T. W., F. van Kemenade, L. Koenderman, K. M. Dolman, A. E. von dem Borne, P. A. Tetteroo, D. Roos. 1989. The 40-kDa Fc{gamma} receptor (FcRII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis. J. Immunol. 142:2365.[Abstract]
  6. Anderson, C. L., L. Shen, D. M. Eicher, M. D. Wewers, J. K. Gill. 1990. Phagocytosis mediated by three distinct Fc{gamma} receptor classes on human leukocytes. J. Exp. Med. 171:1333.[Abstract/Free Full Text]
  7. Indik, Z., C. Kelly, P. Chien, A. I. Levinson, A. D. Schreiber. 1991. Human Fc{gamma}RII, in the absence of other Fc{gamma} receptors, mediates a phagocytic signal. J. Clin. Invest. 88:1766.
  8. Rosales, C., E. J. Brown. 1991. Two mechanisms for IgG Fc-receptor-mediated phagocytosis by human neutrophils. J. Immunol. 146:3937.[Abstract]
  9. Hundt, M., R. E. Schmidt. 1992. The glycosylphosphatidylinositol-linked Fc{gamma} receptor III represents the dominant receptor structure for immune complex activation of neutrophils. Eur. J. Immunol. 22:811.[Medline]
  10. Brooks, D. G., W. Q. Qiu, A. D. Luster, J. V. Ravetch. 1989. Structure and expression of human IgG FcRII(CD32): functional heterogeneity is encoded by the alternatively spliced products of multiple genes. J. Exp. Med. 170:1369.[Abstract/Free Full Text]
  11. Ibarrola, I., P. J. Vossebeld, C. H. Homburg, M. Thelen, D. Roos, A. J. Verhoeven. 1997. Influence of tyrosine phosphorylation on protein interaction with Fc{gamma}RIIa. Biochim. Biophys. Acta 1357:348.[Medline]
  12. Mitchell, M. A., M. M. Huang, P. Chien, Z. K. Indik, X. Q. Pan, A. D. Schreiber. 1994. Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc{gamma}RIIA: effect on receptor tyrosine phosphorylation and phagocytosis. Blood 84:1753.[Abstract/Free Full Text]
  13. Bewarder, N., V. Weinrich, P. Budde, D. Hartmann, H. Flaswinkel, M. Reth, J. Frey. 1996. In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (Fc{gamma}RII) phosphorylation. Mol. Cell. Biol. 16:4735.[Abstract]
  14. Gulle, H., A. Samstag, M. M. Eibl, H. M. Wolf. 1998. Physical and functional association of Fc{alpha}R with protein tyrosine kinase Lyn. Blood 91:383.[Abstract/Free Full Text]
  15. Ghazizadeh, S., J. B. Bolen, H. B. Fleit. 1994. Physical and functional association of Src-related protein tyrosine kinases with Fc{gamma}RII in monocytic THP-1 cells. J. Biol. Chem. 269:8878.[Abstract/Free Full Text]
  16. Hamada, F., M. Aoki, T. Akiyama, K. Toyoshima. 1993. Association of immunoglobulin G Fc receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils. Proc. Natl. Acad. Sci. USA 90:6305.[Abstract/Free Full Text]
  17. Kwiatkowska, K., A. Sobota. 2001. The clustered Fc{gamma} receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner. Eur. J. Immunol. 31:989.[Medline]
  18. Zhou, M. J., D. M. Lublin, D. C. Link, E. J. Brown. 1995. Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc{gamma}RII or Fc{gamma}RIIIB ligation in human polymorphonuclear leukocytes: implications for immune complex activation of the respiratory burst. J. Biol. Chem. 270:13553.[Abstract/Free Full Text]
  19. Sheets, E. D., D. Holowka, B. Baird. 1999. Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of Fc{epsilon}RI and their association with detergent-resistant membranes. J. Cell Biol. 145:877.[Abstract/Free Full Text]
  20. Field, K. A., D. Holowka, B. Baird. 1997. Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains. J. Biol. Chem. 272:4276.[Abstract/Free Full Text]
  21. Guo, B., R. M. Kato, M. Garcia-Lloret, M. I. Wahl, D. J. Rawlings. 2000. Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Immunity 13:243.[Medline]
  22. Cheng, P. C., M. L. Dykstra, R. N. Mitchell, S. K. Pierce. 1999. A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J. Exp. Med. 190:1549.[Abstract/Free Full Text]
  23. Petrie, R. J., P. P. Schnetkamp, K. D. Patel, M. Awasthi-Kalia, J. P. Deans. 2000. Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation. J. Immunol. 165:1220.[Abstract/Free Full Text]
  24. Lang, M. L., L. Shen, W. F. Wade. 1999. {gamma}-Chain-dependent recruitment of tyrosine kinases to membrane rafts by the human IgA receptor Fc{alpha}R. J. Immunol. 163:5391.[Abstract/Free Full Text]
  25. Simons, K., E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387:569.[Medline]
  26. Brown, D. A., E. London. 2000. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275:17221.[Free Full Text]
  27. Janes, P. W., S. C. Ley, A. I. Magee. 1999. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J. Cell Biol. 147:447.[Abstract/Free Full Text]
  28. Montixi, C., C. Langlet, A. M. Bernard, J. Thimonier, C. Dubois, M. A. Wurbel, J. P. Chauvin, M. Pierres, H. T. He. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334.[Medline]
  29. Aman, M. J., K. S. Ravichandran. 2000. A requirement for lipid rafts in B cell receptor induced Ca2+ flux. Curr. Biol. 10:393.[Medline]
  30. Plyte, S., M. B. Majolini, S. Pacini, F. Scarpini, C. Bianchini, L. Lanfrancone, P. Pelicci, C. T. Baldari. 2000. Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts. Oncogene 19:1529.[Medline]
  31. Gaudry, M., C. J. Roberge, R. de Médicis, A. Lussier, P. E. Poubelle, P. H. Naccache. 1993. Crystal-induced neutrophil activation. III. Inflammatory microcrystals induce a distinct pattern of tyrosine phosphorylation in human neutrophils. J. Clin. Invest. 91:1649.
  32. al-Shami, A., S. G. Bourgoin, P. H. Naccache. 1997. Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. I. Tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters. Blood 89:1035.[Abstract/Free Full Text]
  33. Grynkiewicz, G., M. Poenie, R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440.[Abstract/Free Full Text]
  34. Wessel, D., U. I. Flugge. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141.[Medline]
  35. Hanke, J. H., J. P. Gardner, R. L. Dow, P. S. Changelian, W. H. Brissette, E. J. Weringer, B. A. Pollok, P. A. Connelly. 1996. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor: study of Lck- and FynT-dependent T cell activation. J. Biol. Chem. 271:695.[Abstract/Free Full Text]
  36. Vossebeld, P. J., C. H. Homburg, R. C. Schweizer, I. Ibarrola, J. Kessler, L. Koenderman, D. Roos, A. J. Verhoeven. 1997. Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca2+-signalling induced by Fc{gamma} receptor cross-linking in human neutrophils. Biochem. J. 323:87.
  37. Chuang, F. Y., M. Sassaroli, J. C. Unkeless. 2000. Convergence of Fc{gamma} receptor IIA and Fc{gamma} receptor IIIB signaling pathways in human neutrophils. J. Immunol. 164:350.[Abstract/Free Full Text]
  38. Bonnerot, C., V. Briken, V. Brachet, D. Lankar, S. Cassard, B. Jabri, S. Amigorena. 1998. syk protein tyrosine kinase regulates Fc receptor {gamma}-chain-mediated transport to lysosomes. EMBO J. 17:4606.[Medline]
  39. Crowley, M. T., P. S. Costello, C. J. Fitzer-Attas, M. Turner, F. Meng, C. Lowell, V. L. Tybulewicz, A. L. DeFranco. 1997. A critical role for Syk in signal transduction and phagocytosis mediated by Fc{gamma} receptors on macrophages. J. Exp. Med. 186:1027.[Abstract/Free Full Text]
  40. Ghazizadeh, S., J. B. Bolen, H. B. Fleit. 1995. Tyrosine phosphorylation and association of Syk with Fc{gamma}RII in monocytic THP-1 cells. Biochem. J. 305:669.
  41. Hunter, S., N. Sato, M. K. Kim, Z. Y. Huang, D. H. Chu, J. G. Park, A. D. Schreiber. 1999. Structural requirements of Syk kinase for Fc{gamma} receptor-mediated phagocytosis. Exp. Hematol. 27:875.[Medline]
  42. Johnson, S. A., C. M. Pleiman, L. Pao, J. Schneringer, K. Hippen, J. C. Cambier. 1995. Phosphorylated immunoreceptor signaling motifs (ITAMs) exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases. J. Immunol. 155:4596.[Abstract]
  43. Kiefer, F., J. Brumell, N. Al-Alawi, S. Latour, A. Cheng, A. Veillette, S. Grinstein, T. Pawson. 1998. The Syk protein tyrosine kinase is essential for Fc{gamma} receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18:4209.[Abstract/Free Full Text]
  44. Raeder, E. M., P. J. Mansfield, V. Hinkovska-Galcheva, J. A. Shayman, L. A. Boxer. 1999. Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis. J. Immunol. 163:6785.[Abstract/Free Full Text]
  45. Marcilla, A., O. M. Rivero-Lezcano, A. Agarwal, K. C. Robbins. 1995. Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fc{gamma} receptors. J. Biol. Chem. 270:9115.[Abstract/Free Full Text]
  46. Oliver, J. M., D. L. Burg, B. S. Wilson, J. L. McLaughlin, R. L. Geahlen. 1994. Inhibition of mast cell Fc{epsilon}R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697.[Abstract/Free Full Text]
  47. Desaulniers, P., M. Fernandes, C. Gilbert, S. G. Bourgoin, P. H. Naccache. 2001. Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals. J. Leukocyte Biol. 70:659.[Abstract/Free Full Text]
  48. Yan, S. R., L. Fumagalli, G. Berton. 1996. Activation of SRC family kinases in human neutrophils. Evidence that p58C-FGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity. FEBS Lett. 380:198.[Medline]
  49. Simons, K., D. Toomre. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1:31.[Medline]
  50. Cerneus, D. P., E. Ueffing, G. Posthuma, G. J. Strous, A. van der Ende. 1993. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis: role of cholesterol. J. Biol. Chem. 268:3150.[Abstract/Free Full Text]
  51. Solomon, K. R., M. A. Mallory, R. W. Finberg. 1998. Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells. Biochem. J. 334:325.
  52. Norman, A. W., R. A. Demel, B. de Kruyff, L. L. van Deenen. 1972. Studies on the biological properties of polyene antibiotics: evidence for the direct interaction of filipin with cholesterol. J. Biol. Chem. 247:1918.[Abstract/Free Full Text]
  53. Schnitzer, J. E., P. Oh, E. Pinney, J. Allard. 1994. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J. Cell Biol. 127:1217.[Abstract/Free Full Text]
  54. Vely, F., N. Gruel, J. Moncuit, O. Cochet, H. Rouard, S. Dare, J. Galon, C. Sautes, W. H. Fridman, J. L. Teillaud. 1997. A new set of monoclonal Abs against human Fc{gamma}RII (CD32) and Fc{gamma}RIII (CD16): characterization and use in various assays. Hybridoma 16:519.[Medline]
  55. Barabé, F., C. Gilbert, N. Liao, S. G. Bourgoin, P. H. Naccache. 1998. Crystal-induced neutrophil activation VI. Involvement of Fc{gamma}RIIIB (CD16) and CD11b in response to inflammatory microcrystals. FASEB J. 12:209.[Abstract/Free Full Text]
  56. Paolini, R., J. P. Kinet. 1993. Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors. EMBO J. 12:779.[Medline]
  57. Draberova, L., M. Amoui, P. Draber. 1996. Thy-1-mediated activation of rat mast cells: the role of Thy-1 membrane microdomains. Immunology 87:141.[Medline]
  58. Ilangumaran, S., D. C. Hoessli. 1998. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem. J. 335:433.
  59. van’t Hof, W., M. D. Resh. 1999. Dual fatty acylation of p59Fyn is required for association with the T cell receptor {zeta} chain through phosphotyrosine-Src homology domain-2 interactions. J. Cell Biol. 145:377.[Abstract/Free Full Text]
  60. Gilbert, C., E. Rollet-Labelle, and P. H. Naccache. Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling. J. Immunol. Methods In press.
  61. Cheng, P. C., B. K. Brown, W. Song, S. K. Pierce. 2001. Translocation of the B cell antigen receptor into lipid rafts reveals a novel step in signaling. J. Immunol. 166:3693.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Bournazos, S. P. Hart, L. H. Chamberlain, M. J. Glennie, and I. Dransfield
Association of Fc{gamma}RIIa (CD32a) with Lipid Rafts Regulates Ligand Binding Activity
J. Immunol., June 15, 2009; 182(12): 8026 - 8036.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Marois, M. Vaillancourt, S. Marois, S. Proulx, G. Pare, E. Rollet-Labelle, and P. H. Naccache
The Ubiquitin Ligase c-Cbl Down-Regulates Fc{gamma}RIIa Activation in Human Neutrophils
J. Immunol., February 15, 2009; 182(4): 2374 - 2384.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. V. Kondadasula, J. M. Roda, R. Parihar, J. Yu, A. Lehman, M. A. Caligiuri, S. Tridandapani, R. W. Burry, and W. E. Carson III
Colocalization of the IL-12 receptor and Fc{gamma}RIIIa to natural killer cell lipid rafts leads to activation of ERK and enhanced production of interferon-{gamma}
Blood, April 15, 2008; 111(8): 4173 - 4183.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
F. R. Sheppard, M. R. Kelher, E. E. Moore, N. J. D. McLaughlin, A. Banerjee, and C. C. Silliman
Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation
J. Leukoc. Biol., November 1, 2005; 78(5): 1025 - 1042.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
E. Krautkramer, S. I. Giese, J. E. Gasteier, W. Muranyi, and O. T. Fackler
Human Immunodeficiency Virus Type 1 Nef Activates p21-Activated Kinase via Recruitment into Lipid Rafts
J. Virol., April 15, 2004; 78(8): 4085 - 4097.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. A. Rarok, P. C. Limburg, and C. G. M. Kallenberg
Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies
J. Leukoc. Biol., July 1, 2003; 74(1): 3 - 15.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barabé, F.
Right arrow Articles by Naccache, P. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barabé, F.
Right arrow Articles by Naccache, P. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS