FcγRIIa requires lipid rafts, but not co-localization into rafts, for effector function
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- Vieth, J.A., Kim, M., Glaser, D. et al. Inflamm. Res. (2013) 62: 37. doi:10.1007/s00011-012-0548-1
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To determine if receptor localization into lipid rafts, or the lipid rafts themselves, are important for FcγRIIa effector functions.
Wild-type FcγRIIa or mutant FcγRIIa(C208A) that does not translocate to lipid rafts were transfected into Chinese hamster ovary (CHO) cells which have been shown to be reliable cells for studying FcγR function.
Cells were treated with buffer or methyl-β-cyclodextrin (MβCD) to deplete cholesterol and dissolve the structure of lipid rafts.
To evaluate lipid raft association, transfected CHO cells were lysed and centrifuged over a sucrose gradient. Fractions were run on SDS-PAGE and blotted for FcγRIIa or sphingolipid GM1 to illustrate the lipid raft fractions. Lateral mobility of GFP-tagged wild-type or mutant FcγRIIa was assessed using fluorescence recovery after photobleaching (FRAP) microscopy. Internalization of IgG-opsonized erythrocytes was assessed by fluorescence microscopy and uptake of heat-aggregated IgG (haIgG) was measured using flow cytometry.
We observed that FcγRIIa(C208A) did not localize into lipid rafts. However, the mutant FcγRIIa retained lateral mobility and effector function similar to wild-type FcγRIIa. However, mutant FcγRIIa function was abolished upon treatment with MβCD.
Lipid rafts provide an essential component required for effector activities independent of receptor localization.
KeywordsLipid raftEndocytosisPhagocytosisFc receptors
Lipid rafts are membrane microdomains enriched with glycophospholipids (such as ganglioside GM1), high concentrations of cholesterol, and GPI-linked proteins. Receptor localization into lipid rafts has been shown to be important in the process of ligand binding and elicitation of a biological effect for a number of different receptors, including FcγRs [1–8]. Lipid rafts are thought to serve as accumulation sites for cytoplasmic signaling molecules, allowing for efficient and ordered initiation of signaling following receptor ligation [6, 9, 10]. Because lipid rafts are rich in cholesterol, they can be perturbed using cholesterol chelating agents such as MβCD which has been used extensively to disrupt lipid rafts and assess their functional significance in various processes (reviewed in ). For example, previous reports have shown that FcγRIIa requires lipid rafts for ligand binding and for initiating intracellular signaling (Ca2+, NF-κB, p-ERK, etc.) [2, 4–7]. FcγRIIa has also recently been shown to require lipid rafts for internalization of large targets through phagocytosis, but not for endocytosis of small targets .
Activation of FcγRIIa by IgG-coated pathogens results in cross-linking of receptors and lipid raft localization. FcγRIIa is then phosphorylated on tyrosine residues in the ITAM-like sequence by members of the Src family of tyrosine kinases which are known to be associated with lipid rafts [8–10, 12]. Phosphorylation of the ITAM-like region creates two SH2 (Src Homology 2) binding sites, together being able to bind spleen tyrosine kinase (Syk) . This signaling leads to reorganization of actin filaments, driving the formation of a phagosome and the initiation of signaling events which result in the destruction of pathogens and release of pro-inflammatory mediators such as cytokines and reactive oxygen species [14–16]. Although the ITAM is essential for function, residues outside the ITAM have been reported to be essential for proper receptor function [17–19].
Recruitment of FcγRIIa to lipid rafts has been shown to be dependent on transmembrane residues or on the palmitoylation of a juxtamembrane cysteine (cysteine 208) [2, 4]. Furthermore, these mutations (which impair the localization of FcγRIIa within lipid rafts) have been shown to have a negative influence on intracellular signaling, including calcium signaling and transcription factor activation [2, 4]. Interestingly, one report showed that although p-ERK was diminished, phagocytosis of large latex beads was not inhibited, suggesting that lipid raft localization is not necessary for effector function [2, 4]. Because of these observations, we wanted to examine whether lipid rafts are required for FcγRIIa function independent of the receptor localizing into them. To do this, we constructed mutant FcγRIIa containing a single substitution at amino acid 208 from cysteine to alanine [FcγRIIa(C208A)]. We confirm that the mutant FcγRIIa does not localize into lipid rafts, the lateral diffusion of the receptor is not changed, and that phagocytosis takes place normally. However, we demonstrate that treatment with MβCD inhibits phagocytosis of both wild-type and mutant FcγRIIa, which suggests that the lipid rafts themselves (or components associated with them) are important for FcγRIIa effector activity while localization into rafts is not required. Since internalization of a recognized target is one of the most important activities associated with FcγRIIa-mediated immune responses, we are interested in determining the role of receptor–raft interactions during internalization mechanisms.
Materials and methods
Chinese hamster ovary (CHO) cells expressing human wild-type FcγRIIa or mutant FcγRIIa(C208A) (or C-terminus GFP-tagged versions of either construct) were generated similar to previous reports and maintained in Ham’s F-12 (BioWhittaker, Walkersville, MD, USA) supplemented with 10 % fetal bovine serum (Summit Biotechnology, Ft. Collins, CO, USA) . Expression was maintained by selection in G-418 (HyClone, Logan, UT, USA) and evaluated by flow cytometry .
Measurement of cellular cholesterol
Total cellular cholesterol was measured as previously described . Briefly, cellular cholesterol was measured using Amplex Red Cholesterol Assay Kit (Invitrogen, Carlsbad, CA, USA) or labeling with Filipin complex from Streptomyces filipinensis (Sigma, St. Louis, MO, USA). Filipin staining was performed at a working concentration of 0.5 mg/ml on ice for 30 min, then washed away, and the remaining filipin bound to cholesterol was quantitated (355 nm excitation, 460 nm emission) using a FLUOstar Omega microplate reader (BMG Labtech, Offenburg, Germany).
IgG-opsonized sheep erythrocytes (EA) were used to investigate FcγRIIa-mediated activities. EA were prepared as described previously by opsonizing sheep erythrocytes with rabbit anti-sheep erythrocyte IgG at the highest sub-agglutinating concentration, then washed and resuspended . EA were added to effector cells at a ratio of 10:1 (targets to cells). For internalization assays, six-well plates were floated on the surface of a 37 °C water bath and allowed to internalize for 30 min. Following internalization, the cells were cooled on ice, cold phosphate-buffered saline (PBS) was added to each well, and non-internalized beads were labeled with goat anti-human IgG F(ab´)2 fragments conjugated with phycoerythrin (Jackson Immunoresearch, Gilbertsville, PA, USA) in a 2.5 μg/ml solution for 20 min. Cells were washed again with ice-cold PBS and fixed in 2 % paraformaldehyde. Binding and phagocytosis were assessed by counting the number of bound beads per cell as well as the number internalized, determined by lack of staining with the secondary antibody. After treatments were complete, samples were placed on an Axiovert 200 fluorescence microscope (Carl Zeiss, Thornwood, NY, USA). Cells were visualized using differential interference contrast or fluorescence microscopy. Images were observed using an Orca ER-AG (Hamamatsu, Japan) CCD camera connected to a Dell Optiplex 620 Workstation (Round Rock, TX, USA). Metamorph software (Molecular Devices, Downingtown, PA, USA) was used to acquire and process images.
Data are expressed as the number of internalized beads per effector cell. Each experiment was performed in triplicate wells and repeated at least three times on separate days.
Heat-aggregated IgG (haIgG) complexes
Solutions of 10 mg/ml FITC-conjugated human IgG (Sigma) was aggregated at 62 °C for 20 min. Large aggregates were cleared by centrifugation (10,000g for 10 min). The remaining soluble complexes have been reported to contain from two to six IgG molecules per complex . The IgG complexes (concentration 100 μg/ml) were allowed to bind to cells for 45 min on ice. Excess IgG was removed by washing twice with PBS (containing calcium and magnesium). Internalization assays were performed by floating six-well plates containing the cells on the surface of a 37 °C water bath and allowed the cells to internalize for 30 min. Following internalization, the cells were returned to ice and non-internalized IgG complexes were labeled with a 2.5 μg/ml solution of goat anti-human IgG F(ab´)2 fragments conjugated with phycoerythrin (PE) for 20 min. Excess secondary antibody was removed by washing with ice-cold PBS. The cells were detached and fixed in 2 % paraformaldehyde overnight, then analyzed on a BD-FACSCalibur (Becton–Dickinson, San Jose, CA, USA) using Cell Quest software.
Percent internalization is calculated by comparing the mean fluorescence intensity (MFI) of PE (external immune complex only, internalization value) at each concentration of MβCD and normalizing to untreated control samples. Each experiment was performed in triplicate and repeated at least three times on separate days. 10,000 cellular events were assessed for each test sample.
Lipid raft disruption
Disruption of lipid rafts by plasma-membrane cholesterol depletion was accomplished through treatment with 8 mM MβCD (Sigma Aldrich, St. Louis, MO, USA) as previously described . To determine the effect of lipid raft disruption on internalization, binding of target was allowed to occur on ice for 45 min, and then excess non-bound target was removed by washing with PBS. Lipid raft disruption was achieved by incubation with MβCD for 45 min (still on ice) prior to warming cells to 37 °C for stimulation of internalization. Previous work in our laboratory has shown that treatment with 8 mM MβCD for 30 min is sufficient to deplete membrane cholesterol at either 37 or 4 °C .
Membrane fractionation and Western blotting
FcγRIIa-expressing CHO cells were exposed to opsonized targets for 30 min on ice then treated with MβCD (8 mM) for 30 min on ice, then warmed to 37 °C for an additional 30 min. Cells were washed twice with cold PBS to remove unbound targets and incubated in TNE buffer (0.05 % TX-100 and protease inhibitor) for 30 min on ice for lysis. Lysates were processed as previously described [8, 23]. Briefly, lysates were suspended in a final concentration of 40 % sucrose, loaded onto a sucrose step gradient (10–80 %), and centrifuged overnight. Fractions of 1 ml were collected, sucrose was removed by MeOH/chloroform precipitation, and protein loaded onto an SDS-PAGE gel. Following gel electrophoresis, samples were transferred to a PVDF membrane and blotted with an anti-MYC FcγRIIa antibody (Santa Cruz, Santa Cruz, CA, USA). Fractions were compared for the presence of FcγRIIa following exposure to either target or MβCD drug treatment. Samples were blotted for GM1 using cholera toxin B (Sigma) to confirm rafts in lanes 7–9. Blots were imaged and quantitated on an Omega 12iC (Ultra-Lum, Claremont, CA, USA) using UltraQuant 6.0 software (Ultra-Lum).
Fluorescence recovery after photobleaching (FRAP)
Treated samples were mounted on glass coverslips and imaged with an inverted Leica SP5 laser scanning confocal microscope (Leica Microsystems, Mannheim, Germany) using FRAP wizard. Excitation was accomplished using laser lines at 488 nm for FITC/GFP and fluorescence emission was collected at 530 nm. Acquisition and processing of FRAP experiments was accomplished using Leica Applications Suite Advanced Fluorescence (LAS AF) v.2.0.2 software (Leica Microsystems).
Significance values were determined using the two-tailed unpaired equal variance Student’s t test.
FcγRIIa translocates to lipid rafts in response to target binding
FcγRIIa(C208A) mutants show similar rates of membrane diffusion
To understand the role of lipid rafts in regulating the movement of FcγRIIa through the membrane, we used fluorescence recovery after photobleaching (FRAP) to measure the lateral diffusion of the receptor. Green fluorescent protein (GFP) was fused to the C-terminus of wild-type FcγRIIa and mutant FcγRIIa(C208A), and the function was tested in the presence and absence of MβCD to deplete lipid rafts. A representative example of FRAP studies of wild-type FcγRIIa is shown (Fig. 2a). Both constructs were analyzed for the ability to diffuse through the membrane (Fig. 2b). Previous studies from our and other laboratories show no difference in the ability of FcγRIIA-GFP to bind and internalize IgG aggregates or IgG-coated targets (data not shown) . Under steady state conditions, we observed that both FcγRIIa and FcγRIIa(C208A) exhibited similar membrane mobility that was not affected by MβCD treatment (Fig. 2b). Interestingly, after ligation with haIgG, the receptors appear to congregate, indicating association and possibly intercalation into lipid rafts (Fig. 2a, row 3). Calculation of immobile fractions shows that ligation significantly limits receptor mobility in both wild-type and Cys-Ala mutant FcγRIIa, as indicated by an increase in the immobile fraction after ligation with haIgG (Fig. 2b). Interestingly, receptor mobility is restored to unligated levels upon treatment with MβCD. Plasma membrane cholesterol content and cell viability was measured by filipin staining and Trypan Blue exclusion, respectively, as previously described with similar results (Fig. 2c, d). These data suggest that the decrease in receptor mobility following ligation is lipid-raft-dependent, though not reliant on an actual interaction between the receptor and lipid rafts.
Lipid rafts are more important than receptor localization for effector functions
To elucidate the differences in lipid raft requirement related to FcγRIIa function, internalization assays were performed using EA and haIgG. As binding of both EA and haIgG are dependent on lipid rafts, binding was allowed to occur on ice prior to MβCD treatment and samples were evaluated by microscopy and flow cytometry to be certain that displacement of targets did not occur. Following binding, cells were treated with 8 mM MβCD on ice for 45 min. After PBS washes (0 °C) to remove any unbound target, the samples were heated to 37 °C for 30 min to initiate internalization. Phagocytosis (EA) and endocytosis (haIgG) was arrested by returning the cells to ice. In order to discriminate between internal and external targets, phycoerythrin (PE) tagged secondary antibody against EA or haIgG was employed. Fluorescence microscopy was performed and the total number of internalized EA (PE-negative) was determined by subtracting the PE-labeled (external) targets from the total EA associated with the cell.
Similar to published reports, phagocytosis by FcγRIIa is significantly inhibited in the presence of MβCD (Fig. 3a), while endocytosis is unaffected (Fig. 3b). Interestingly, FcγRIIa(C208A)-mediated phagocytosis was also inhibited by MβCD similar to wild-type FcγRIIa, while having no affect on endocytosis. This suggests that, as with changes in mobility following ligation, the function of FcγRIIa during phagocytosis requires the presence of lipid rafts, but not necessarily interaction with them.
FcγRs are known for the utilization of lipid rafts to mediate effector activities [3, 5, 8, 24–28]. Importantly, FcγRIIA requires lipid rafts for binding of IgG-containing targets [1, 3]. Previous reports suggest that essential residues of FcγRIIa are needed to translocate to lipid rafts, including a cytoplasmic cysteine (C208A) and a transmembrane domain alanine (A224S) [2–4]. Mutation of either of these residues resulted in loss of FcγRIIa translocation into lipid rafts and a decrease in signaling events including intracellular calcium, NF-κB activation, and p-ERK, but had no effect on phagocytosis. Consistent with these studies, we previously reported that lipid rafts are essential for internalization by phagocytosis, but that endocytosis was not impacted by loss of lipid raft integrity by MβCD treatment . These results are consistent with previous observations suggesting that entry of small ligands is through clathrin-coated pits versus phagocytosis, which is actin-dependent [29, 30]. In all, these observations suggest that lipid rafts are necessary for efficient FcγRIIa activity but that localization of FcγRIIa into lipid rafts is not needed.
The series of experiments in this manuscript and in previous reports raises an interesting question about the true function of lipid rafts. It is well known that lipid rafts are rich in signaling molecules such as Src family members (reviewed in ). These cytosolic proteins all contain cationic C-2 (PKC Conserved-2) domains which associate with positively-charged phospholipids such as phosphatidylserine that are commonly found in abundance in lipid rafts [32, 33]. Combined with our data, these observations suggest that lipid rafts may act as a signaling scaffold that is necessary for inducing large signals responsible for cellular responses such as phagocytosis.
Taking the current studies into account (as well as previous data regarding the role of lipid rafts), it is becoming increasingly clear that lipid rafts play an important regulatory role in communication between surface receptors and downstream effector functions. This places lipid rafts at the cusp of the two most important events on the recognition of an immune target: (1) internalization and neutralization of that target; and (2) downstream signaling and overall cellular response through activation and cytokines. Furthermore, it suggests that lipid rafts may play the role of a “bridge” during FcγRIIa-mediated events, bringing together multiple signaling cascades which can be cross-activated when coalesced in the raft environment. If this holds true, it implicates lipid rafts as an exciting and extremely powerful regulator of the various intricate immunological signaling pathways, one which can be explored in the future as a target for direct manipulation of the extensive signaling which occurs upon the activation of an immune receptor.
This work was supported by an Arthritis Foundation Investigator Award (to R.G.W.) and National Institutes of Health grant HL-28207 (to A.D.S.). The authors would like to thank Dr. Andrea Kalinoski in the University of Toledo Advanced Microscopy and Imaging Center (AMIC) and the Flow Cytometry Core facility for technical assistance and use of the equipment.