Correspondence to: Patricia A. Detmers, Merck Research Laboratories, 126 E. Lincoln Ave., RY80W-250, Rahway, NJ 07065. Tel:732-594-1431 Fax:732-594-4620 E-mail:patricia_detmers{at}merck.com.
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Abstract |
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Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14EGFP) was used to follow trafficking of mCD14 and BODIPYLPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complexlike structure. mCD14EGFP was functional in mediating binding of and responses to LPS. BODIPYLPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14EGFP on the cell surface. However, within 510 min, the BODIPYLPS distributed to intracellular vesicles that did not contain mCD14EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.
Key Words: enhanced green fluorescent protein, U373 cells, intracellular trafficking
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Introduction |
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Cells that express plasma membranebound (m)CD14,1 either naturally or through transfection, bind bacterial LPS. Monomeric LPS is removed from LPS aggregates and reaches mCD14 by a two-step process in serum (1). The serum protein LPS binding protein (LBP) binds to LPS aggregates and transfers monomers to a soluble (s) form of CD14, also present in serum. sCD14, in turn, may transfer LPS either to serum lipoprotein particles, causing LPS neutralization, or to cells, leading to production of cytokines and other responses. Transfer of LPS between CD14 molecules is a rapid process (2), suggesting that the movement of LPS from sCD14 to mCD14 on mCD14-bearing cells is likely to be more rapid than the movement of LPS from sCD14 to the surfaces of cells that do not express CD14.
The importance of mCD14 to the binding of LPS and the cellular responses that follow is underscored by a variety of observations. Blockade of LPS binding to mCD14 on leukocytes with mAbs (3) abrogates responses such as cytokine production by monocytes (4) and enhanced integrin-dependent adhesion by neutrophils (2) (5). In addition, paroxysmal nocturnal hemoglobinuria, a disease characterized by a variable deficiency in the expression of mCD14 and other glycosylphosphatidyl inositol (GPI)-linked proteins on hemopoietic cells, renders monocytes less sensitive to LPS (6) (7) (8). Perhaps the strongest evidence for the primacy of mCD14 in mediating responses to LPS is the observation that monocytes from CD14-deficient mice show a strongly attenuated cytokine response to LPS (9). Moreover, transfection with mCD14 endows various cell types that do not otherwise express CD14 with the ability either to become responsive to LPS (10) or to become sensitive to much lower concentrations of LPS (11) (12). Thus, mCD14 serves as the initial site of interaction between LPS and the surfaces of CD14-bearing cells.
Several lines of evidence suggest that after binding to mCD14, LPS must be internalized to initiate intracellular signaling leading to cellular responses. Recently, we and others have observed that fluorescently labeled LPS is rapidly endocytosed when it is presented to neutrophils or cultured human monocytes as monomeric LPS complexed with sCD14 (13) (14). In neutrophils, integrin-mediated adhesion in response to LPS exhibits a lag of 1020 min and is completely blocked by phosphatidyl inositol 3 kinase inhibitors or by lowering the temperature to 19°C, two treatments that prevent vesicular transport (13). Thieblemont and Wright demonstrated that macrophages from mice with the LPS-hyporesponsive (Lpsd) trait are defective in vesicular transport of LPS (14). Furthermore, LPS antagonists block both the transport of LPS inside the cell (15) and cellular responses to LPS. Internalization of LPS may therefore be a key process for eliciting responses from cells expressing mCD14.
Although LPS internalization has been directly observed under a variety of circumstances, the precise role of mCD14 in this process is not known. The ability of CD14 to transfer LPS to high density lipoprotein particles (16) and phospholipid micelles (17) suggests that mCD14 might participate in the catalytic transfer of LPS to the lipid bilayer of the plasma membrane. LPS may then move within the cell independently of mCD14 or in a vesicle also containing mCD14. Alternatively, LPS may remain bound to mCD14 while being internalized, exhibiting the behavior of a classic ligandreceptor complex.
To determine whether mCD14 traffics along with LPS from the plasma membrane to intracellular compartments, we constructed a GPI-anchored fusion protein of CD14 and enhanced green fluorescent protein (EGFP) and used it to follow the location of mCD14 in the astrocytoma cell line U373. The fusion protein was expressed on the cell surface in a fully functional form. Expression of mCD14EGFP endowed U373 cells with the ability to internalize detectable amounts of fluorescently labeled LPS (BODIPYLPS). Using confocal microscopy, we observed that BODIPYLPS presented in a monomeric form colocalized with mCD14 at the cell surface but distributed to intracellular locations without mCD14 as soon as internalization of LPS could be detected. Our observations suggest that monomeric LPS is rapidly transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. Additional studies indicate that LPS aggregates are trafficked differently from monomers. LPS aggregates are known to be slowly internalized and directed to lysosomes (18), and we find that mCD14 remains associated with LPS aggregates during internalization.
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Materials and Methods |
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Construction of the mCD14EGFP and mEGFP Chimeras.
pEGFP-N1 (Clontech) encodes EGFP, a bright redshifted variant of GFP containing the amino acid substitutions phenylaline-64leucine and serine-65
threonine of GFPmut1 (19). An mCD14EGFP gene fusion was constructed in pEGFP-N1 in two steps. First, a 1.2-kb BamHI fragment coding for the entire CD14 protein minus the last eight COOH-terminal residues was inserted into BamHI-digested pEGFP-N1, yielding pCD14EGFP. Second, a 116-bp BsrGINotI fragment, encoding the 36 COOH-terminal residues of decay accelerating factor (DAF) and a stop codon, was inserted into BsrGI- plus NotI-digested pCD14EGFP, yielding pCD14EGFPGPI. The amino acid sequence of the fusion protein coded by this construct is shown (see Figure 1 A).
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Similarly, an mEGFP gene fusion was constructed by first inserting a 116-bp BsrGINotI fragment, encoding the 36 COOH-terminal residues of DAF and a stop codon, into BsrGI- plus NotI-digested pEGFP-N1, yielding pEGFPGPI. Then, a 134-bp SalIBamHI fragment coding for the first 19 NH2-terminal residues, the signal peptide (SP) of CD14, was inserted into SalI- plus BamHI-digested pEGFPGPI, yielding pSPEGFPGPI. The sequence of the fusion protein coded by this construct is shown (see Figure 1 B). These constructs placed the mCD14EGFP and mEGFP chimeras under the control of the cytomegalovirus promoter/enhancer and permitted the selection of stable clones using geneticin.
The 1.2-kb BamHI fragment encoding most of CD14 was synthesized by PCR using pcDNAI-neo-CD14 as a template (20) and the primers 5'-GAG atg gat cca cca tgg agc gcg cgt cct gc-3' and 5'-GAG ATG GAT CCA GCA CCA GGG TTC CCG A-3'. The 116-bp BsrGINotI fragment encoding part of DAF was synthesized by RT-PCR using total RNA from human monocytes as a template and the primers 5'-aat atg tac aat aaa gga agt gga acc ac-3' and 5'-taa agc ggc cgc taa gtc agc aag ccc at-3'. The 134-bp SalIBamHI fragment was synthesized by RT-PCR using total RNA from human neutrophils as a template and the primers 5'-ACG CGT CGA CGC CGC TGT GTA GGA AAG-3' and 5'-CGC GGA TCC GCA GAG ACG TGC ACC Aat-3'. All syntheses were followed by digestion with the appropriate restriction enzymes and gel purification. Both RT-PCR amplifications were performed using the Gene Amp RNA PCR kit purchased from Perkin-Elmer Corp. The PCR insertions were sequenced to confirm the absence of PCR synthesis errors.
U373 Cell Lines.
U373 cells were grown as monolayers in RPMI (BioWhittaker, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Inc.), penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively), and 2 mM glutamine. For making stable transfectants, 105 cells from a confluent culture of U373 cells were seeded on a 35-mm cell culture dish and grown to subconfluence for 2448 h before transfection with either pCD14EGFPGPI or pSPEGFPGPI. For each dish, 12 µg of highly purified expression plasmid was used for transfection with 6 µl lipofectamine (GIBCO BRL) according to the manufacturer's instructions. The DNAlipofectamine mixture remained on the cells for 6 h at 37°C and was then replaced by RPMI with 10% FBS and 2 mM glutamine without antibiotics. 72 h after transfection, the cells were trypsinized, plated at clonal density, and selected with 0.5 mg/ml geneticin (GIBCO BRL). After 3 wk, surviving cell colonies were visually screened for fluorescence. Several positive clones were identified, isolated using cloning rings, and expanded into cell lines for further analysis. U373CD14 cells were obtained by selecting clones of U373 cells stably transfected with pcDNAI-neo-CD14 as described elsewhere (20).
sCD14 and LPS.
Recombinant human sCD14 was purified from conditioned medium of Schneider-2 insect cells transfected with cDNA encoding human CD14 as previously described (21). LPS from Salmonella minnesota R595 was purchased from List Biological Labs. The fluoroprobe BODIPY 558/568 (Molecular Probes, Inc.) was conjugated to unlabeled LPS micelles as previously described (22).
LPSsCD14 and BODIPYLPSsCD14 complexes were formed by incubating LPS or BODIPYLPS (20 µg/ml), respectively, with sCD14 (500 µg/ml) overnight at 37°C in Dulbecco's PBS (BioWhittaker, Inc.) with 0.5% pyrogen-free human serum albumin (Centeon, Armour, and Berring Pharmaceutical Co.). Previous work has shown that under these conditions all of the LPS forms stoichiometric complexes with monomeric sCD14 and that these complexes efficiently stimulate cells and deliver LPS to the plasma membrane (2) (3).
BODIPYLPS aggregates were prepared by incubating BODIPYLPS at 1 µg/ml in FBS for 10 min at 37°C. The aggregation state of LPS was verified by monitoring its fluorescence emission at 568 nm before and after the addition of detergent, as described elsewhere (22). Adding 2% SDS to the BODIPYLPS aggregates led to a 10-fold increase in fluorescence due to the loss of self-quenching as monomers were released from the aggregates. In addition, aggregates observed directly by fluorescence microscopy exhibited a pointillistic pattern of fluorescence, rather than the very diffuse fluorescence seen with BODIPYLPSsCD14 complexes.
Confocal Microscopy.
U373 transfectants were cultured for 2448 h before experiments in RPMI without phenol red (BioWhittaker, Inc.) supplemented with 10% FBS, antibiotics, and 2 mM glutamine on glass chamber slides (Nunc, Inc.) precoated with 0.5% gelatin (Sigma Chemical Co.). The cells were washed twice in HAP buffer (Dulbecco's PBS, 0.05% human serum albumin, and 3 mM D-glucose, containing 0.5 U/ml of aprotinin) and incubated in HAP at 37°C with or without LPSsCD14, BODIPYLPSsCD14, or BODIPYLPS aggregates. Slides were washed twice with HAP and further incubated at 37°C. At the end of the incubation, the plastic chamber and silicone gasket were removed, and the slide was mounted in HAP for immediate microscopic observation. For removal of cell surface mCD14EGFP with phosphatidyl inositol phospholipase C (PI-PLC), cells on slides were incubated for 1 h in 20 mM Hepes, pH 7.4, and 150 mM NaCl on ice with 5 U/ml of PI-PLC (Boehringer Mannheim).
When anti-CD14 mAb 26ic or 60b (23) was used, cells were incubated with the antibody at 10 µg/ml in HAP buffer at 4°C for 30 min and washed twice with ice-cold HAP and once with HAP at 37°C just before adding the BODIPYLPSsCD14 complexes.
Confocal scanning laser microscopy was performed using a Nikon Optiphot-2 microscope with a x60 objective (NA 1.4) and Bio-Rad MRC 1024 instrumentation with a krypton/argon laser. Unless otherwise noted, each image represents a single Kalman averaged (610 scans) optical section collected with a 23-mm-diameter iris aperture. Optical sections were collected digitally and analyzed using LaserSharp software (Bio-Rad Labs.). For two-color images, each color was acquired sequentially. This was necessary because EGFP has a broad peak of fluorescence, and some signal bleedthrough was observed in the BODIPYLPS channel when simultaneous collection was attempted.
Intracellular Fluorescence.
Quenching of cell surface fluorescence by trypan blue was employed to both quantitate and observe the distribution of intracellular mCD14EGFP and mEGFP. U373 transfectants were grown to confluence in a 96-well culture plate and, after the experiment, the total fluorescence associated with the cells was measured using a Cytofluor 4000 (PE Biosystems) (excitation 485 nm, emission 530 nm). Trypan blue (200 µg/ml, ambient temperature) was added to the wells to quench fluorescence from cell surface EGFP, and the remaining fluorescence from intracellular mCD14EGFP or mEGFP was immediately measured. Intracellular fluorescence is expressed as a percent of total fluorescence from triplicate samples. Trypan blue was also used to quench cell surface EGFP on U373 transfectants before observing the cells by confocal microscopy. After the experimental manipulation, U373 transfectants cultured on glass chamber slides were washed once and mounted in HAP containing 200 µg/ml trypan blue before immediate observation.
IL-6 Production by U373 Cells.
U373 transfectants grown in 96-well cell culture plates were washed extensively with AIM-V serumfree medium (GIBCO BRL) and incubated as indicated in AIM-V medium containing 0.5 mg/ml human serum albumin. After 16 h at 37°C, the overlying medium was collected from each well and assayed for IL-6 by ELISA as described (24).
SDS-PAGE and Western Blotting.
For each condition, 106 cells were lysed by incubation on ice for 20 min in 300 µl of 100 mM Tris/HCl, pH 8.0, 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.3 U/ml aprotinin, 2 mM PMSF, 3 mM diisopropyl fluorophosphate, 50 µg/ml benzamidine, and 5 µg/ml each of antipain, leupeptin, chymostatin, and pepstatin A. Lysates were centrifuged for 15 min at 12,000 g, and the supernatants were prepared for SDS-PAGE under reducing conditions. SDS-PAGE was run on a 420% gradient Trisglycine gel (Novex). Proteins were electrotransferred to nitrocellulose membranes and detected with either an anti-EGFP rabbit pAb (Clontech) or an anti-CD14 pAb (25). Horseradish peroxidaseconjugated goat antirabbit IgG was used as the secondary antibody, and the enzymatic reaction was detected with an ECL kit (Amersham Corp.). Purified rEGFP was obtained from Clontech.
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Results |
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EGFP-tagged mCD14 Is Expressed on the Surfaces of U373 Cells.
To generate a fluorescently tagged CD14 that was attached to the membrane via a GPI anchor, we fused a sequence coding for CD14 to one coding for EGFP and added the sequence for the 36 COOH-terminal residues of DAF. The chimeric protein resulting from the gene fusion pCD14EGFPGPI, shown in Figure 1, contains the NH2-terminal signal peptide of CD14 and the COOH-terminal signal peptide of DAF. Thus, when it was expressed and processed, it would translocate normally into the endoplasmic reticulum and have a GPI anchor attached. For a control, we also engineered a chimeric EGFP with a GPI anchor that contained the NH2-terminal signal peptide (SP) of CD14 and the COOH-terminal signal peptide of DAF. This second chimera was used to compare the distribution of EGFP-tagged CD14 (mCD14EGFP) with a generic GPI-anchored protein (mEGFP).
The two gene fusion products were transfected into the U373 astrocytoma cell line, which does not express mCD14. Stable transfectants for mCD14EGFP and mEGFP were designated U373CD14EGFP and U373EGFP, respectively. One clone of U373CD14EGFP was used for the results presented, but a second clone gave identical results.
Expression of mCD14EGFP as an intact polypeptide in U373CD14EGFP was tested in Western blots of cell lysates run in parallel with rsCD14 and EGFP. Antibodies against either CD14 or EGFP recognized the appropriate control protein and the same single band at 80 kD in U373CD14EGFP (Figure 2). The band corresponded to a protein of the expected mass for mCD14EGFP and was not present in cell lysates of untransfected U373. Thus, mCD14EGFP was expressed in U373 cells as an intact polypeptide, and we can be confident that by observing EGFP fluorescence we are also observing the location of mCD14.
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We further confirmed by confocal microscopy that both mCD14EGFP and mEGFP were expressed on the surfaces of U373 cells. Optical sections of live U373CD14EGFP revealed fluorescence associated with the plasma membrane that was relatively uniform in distribution (Figure 3). Fluorescence was also observed on fine, filamentous projections from the cell surface. Intracellular mCD14EGFP was observed in a juxtanuclear reticulum, a structure characteristic of the Golgi apparatus. A similar labeling pattern was observed with U373EGFP (data not shown), suggesting that this is a normal distribution of GPI-anchored proteins in U373 cells.
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To confirm that the mCD14EGFP was attached to the plasma membrane via a GPI anchor, we digested U373CD14EGFP cells with PI-PLC, which cleaves GPI-anchored proteins from their GPI anchors, before observing them by confocal microscopy. After 1-h treatment at 4°C with PI-PLC, cell surface fluorescence on U373CD14EGFP was barely detectable (not shown), suggesting that most of the cell surface associated mCD14EGFP was removed.
EGFP-tagged mCD14 Enhances Responses of U373 Cells to LPS.
U373 astrocytoma cells do not respond to LPS alone, but when LPS is added in the presence of sCD14, they produce IL-6 (26). Transfection of U373 cells with mCD14 allows responses to LPS in the absence of sCD14. More importantly, it greatly increases the sensitivity of these cells to LPSsCD14 complexes and allows more rapid responses to LPSsCD14.2 This is consistent with observations that mCD14 on neutrophils and monocytes is necessary for responses to LPS in the absence of sCD14 and greatly increases the sensitivity of their responses to LPSsCD14 complexes (2) (9).
To show that the mCD14EGFP chimera was functional, we measured secretion of IL-6 by U373CD14EGFP and U373EGFP cells in response to either LPS or LPSsCD14 complexes. The U373EGFP cells did not respond to LPS alone up to 100 ng/ml (Figure 4 A). In contrast, expression of mCD14EGFP in U373 cells led to IL-6 production in response to concentrations of LPS of 1 ng/ml or higher. Similarly, U373CD14EGFP cells responded to concentrations of LPSsCD14 ~100-fold lower than those required to elicit the same response in U373EGFP cells (Figure 4 A). The enhancement of sensitivity by mCD14EGFP was quantitatively similar to that previously observed with mCD14.2 Thus, the presence of EGFP does not affect the function of mCD14EGFP on cells.
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To determine the time of exposure required to elicit a response to LPS, U373CD14EGFP and U373EGFP cells were given a fixed concentration of LPS (40 ng/ml), either alone or in sCD14 complexes, for increasing intervals of time before washing and incubation to allow synthesis of IL-6. U373EGFP cells were essentially unresponsive to LPS alone after up to 2 h of exposure (Figure 4 B). As previously seen with mCD14,2 the presence of mCD14EGFP dramatically enhanced the magnitude and speed of cellular responses to LPS. In particular, after as little as 5 min of exposure to LPS, U373CD14EGFP cells responded with IL-6 production to both LPSsCD14 complexes (Figure 4 B) and LPS aggregates (data not shown).
Taken together, these results suggest that mCD14EGFP was able to bind LPS and mediate cellular responses to it. All of the properties exhibited by U373CD14EGFP are the same as those of mCD14 expressed in U373,2 indicating that mCD14 expressed as a chimera with EGFP was still a functional entity. These results further suggest that the distribution and trafficking pattern of mCD14EGFP in U373 would reflect that of a fully functional protein.
LPS Binds to U373CD14EGFP and Colocalizes with mCD14 on the Cell Surface.
The association of LPS with mCD14EGFP was confirmed by confocal microscopy. BODIPYLPS in the form of monomeric complexes with sCD14 was incubated with U373CD14EGFP for 23 min at 37°C before washing and observation. Fluorescence from BODIPYLPS was seen associated with the plasma membrane of U373CD14EGFP (Figure 5 A, no Ab, red panel), indicating that it was successfully transferred from sCD14. There was colocalization of BODIPYLPS with mCD14EGFP on the cell surface, as demonstrated by the overlap in fluorescence signals (Figure 5 A, no Ab, merge panel). The same cell surface distribution of BODIPYLPS was observed with U373 transfectants expressing mCD14 without EGFP attached (Figure 5 B, panel d). In contrast, U373EGFP cells incubated briefly with BODIPYLPSsCD14 complexes did not have detectable BODIPYLPS fluorescence associated with their cell surfaces (Figure 5 B, panels a and b).
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To confirm that the transfer of BODIPYLPS from sCD14 was mCD14 dependent, we pretreated U373CD14EGFP cells with a blocking anti-CD14 mAb, 60b (3). The antibody prevented binding of BODIPYLPS, and no colocalization with mCD14EGFP was observed (Figure 5 A). Colocalization of BODIPYLPS and mCD14EGFP was still observed when the cells were pretreated with 26ic, a nonblocking anti-CD14 antibody (3) (Figure 5 A). Thus, colocalization of BODIPYLPS and mCD14 required binding of LPS to mCD14.
When Presented in a Monomeric Form, LPS Is Internalized Separately from mCD14.
To determine whether LPS remained bound to mCD14 after internalization, we observed the trafficking of mCD14EGFP and BODIPYLPS simultaneously in live cells. U373CD14EGFP cells were incubated with BODIPYLPSsCD14 complexes for 23 min at 37°C, the complexes were removed by washing, and the cells were incubated further at 37°C before observation. Particular attention was devoted to observations made 510 min after the incubation began. At the earliest time points, BODIPYLPS was clearly visible in small intracellular vesicles (Figure 6, 10 min, red panel). These vesicles were visible at times as early as 5 min (data not shown). However, the vesicles containing BODIPYLPS did not contain mCD14EGFP, as evidenced by the lack of overlap in fluorescent signals (Figure 6, 10 min, merge panel). This indicates that in the brief time required for LPS to reach a vesicular location, LPS had separated from mCD14.
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At later times, vesicles containing BODIPYLPS accumulated predominantly in the perinuclear area (Figure 6 and 60 min, red panels). Although by 60 min a punctate localization of mCD14EGFP in the same vicinity was observed (Figure 6, 60 min, green panel), repeated observations with LPS concentrations between 10 and 200 ng/ml demonstrated that colocalization of BODIPYLPS and mCD14EGFP in this area did not occur (not shown). LPS concentration therefore had no effect on its localization. Thus, not only did LPS leave mCD14 upon entering the cells, but it also did not reassociate with mCD14 in any intracellular compartment.
The kinetics of BODIPYLPS internalization in U373CD14EGFP were identical to those in U373 expressing mCD14 without EGFP (data not shown). Furthermore, the distribution of intracellular BODIPYLPS in vesicles and their subcellular localization was the same in both cell types (data not shown). This indicates that the presence of EGFP on mCD14 did not disturb the normal trafficking pattern of LPS in U373 cells.
Intracellular CD14 Is Exocytosed in Response to LPS.
Although there was no apparent change in the distribution of mCD14EGFP in response to LPS in the colocalization studies, the bright cell surface fluorescence prevented observation of any changes in the distribution of intracellular mCD14EGFP. To better observe the distribution of intracellular mCD14EGFP, we quenched the cell surface fluorescence on U373CD14EGFP cells with trypan blue (Figure 7A and Figure B). In addition to the bright fluorescence emanating from the Golgi apparatus area, numerous fluorescent vesicles of ~50100-nm average diameter were distributed throughout the cytoplasm. The vesicles were particularly evident near the basal aspects of the cells (Figure 7 B). A similar intracellular localization of mEGFP in the Golgi complex and cytoplasmic vesicles was also observed (data not shown). Thus, the vesicular compartment may represent either a component of the secretory pathway en route to the cell surface or a recycling compartment for GPI-anchored proteins.
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The effect of LPS on the intracellular distribution of mCD14EGFP was examined by adding LPSsCD14 complexes to U373CD14EGFP cells and observing mCD14EGFP localization by confocal microscopy after trypan blue quenching. The localization of mCD14EGFP in the Golgi complex was unaffected by LPS stimulation (Figure 7 C). However, after addition of LPS, we observed a steady decrease in the number of mCD14EGFP-containing vesicles from the earliest times observed (25 min), with almost no mCD14EGFP-containing vesicles remaining after 4560 min (Figure 7 D), suggesting that the compartments containing mCD14EGFP were exocytosed upon exposure of the cells to LPS. Although the resolution of these experiments does not allow us to rule out the possibility that a small fraction of mCD14EGFP was internalized, the decline in the number of intracellular vesicles suggests that exocytosis was the preferred route. A similar loss of fluorescent vesicles was observed in U373EGFP cells exposed to LPSsCD14 complexes for 4560 min (data not shown), indicating that the vesicle pool in question is not defined by the presence of CD14 per se. These results suggest that LPS either induces the release of a compartment in U373 cells that contains GPI-anchored proteins or that it prevents the reinternalization of a recycled pool of membrane containing GPI-anchored proteins. Thus, rather than being internalized with LPS from the cell surface, mCD14 apparently moved to the cell surface from an intracellular store in response to LPS.
Exocytosis of the mCD14-containing vesicles was confirmed by quantitative measurements of the time course. U373CD14EGFP cells were grown in 96-well tissue culture plates, and both total and intracellular fluorescence was measured before and at various times after exposure to LPSsCD14 complexes (see Materials and Methods). Stimulation with LPS did not induce any change in total fluorescence associated with the cells (data not shown) but did induce a rapid decrease in intracellular mCD14EGFP (Figure 8). A decrease of ~20% in the intracellular fluorescence was observed after a 15-min incubation of U373CD14EGFP cells with LPSsCD14 complexes. A similar decrease in intracellular fluorescence associated with U373EGFP cells was observed in response to LPSsCD14 complexes (Figure 8), indicating that the compartments released contained GPI-anchored proteins in addition to mCD14EGFP.
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LPS Aggregates and mCD14 Colocalize on the Cell Surface and Are Cointernalized.
LPS aggregates are formed when LPS micelles and LBP are incubated with little or no sCD14. These LPSLBP aggregates can bind to mCD14 on the surfaces of cells, and they are subsequently internalized (27) (28) (29). However, binding of LPSLBP aggregates by mCD14 differs from binding of monomeric LPS presented as a complex with sCD14. Aggregates bind at 4°C, but monomeric LPS cannot be transferred from sCD14 to mCD14 at this temperature. In addition, although internalization of monomeric LPS correlates with intracellular signaling (13) (14), internalization of LPS aggregates can be dissociated from the generation of signals (27). These results suggest that LPS aggregates may have a pathway for internalization that is distinct from that of monomeric LPS.
To determine whether mCD14 traffics with LPS aggregates during internalization, we incubated U373CD14EGFP with BODIPYLPS aggregates at concentrations between 40 and 100 ng/ml and followed both BODIPYLPS and mCD14EGFP fluorescence by confocal microscopy. After incubation for 510 min at 37°C with BODIPYLPS aggregates, the cells were washed and incubated further at 37°C. The aggregates bound to the cell surface and colocalized with mCD14EGFP (Figure 9). At least 15 min at 37°C was required to detect BODIPYLPS in intracellular vesicles, suggesting that internalization of LPS aggregates was somewhat slower than internalization of LPS monomers in U373CD14EGFP. The mCD14EGFP colocalized with BODIPYLPS in intracellular vesicles detected at the earliest times, although not all of the vesicles that contained BODIPYLPS also contained mCD14EGFP. These results suggest that, in contrast with LPS monomers, LPS aggregates can remain bound to mCD14 during internalization. This supports the idea that there is more than one pathway for internalization of LPS.
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Discussion |
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Here we have used a chimeric construct of mCD14 with EGFP transfected into the astrocytoma cell line U373 as a probe for observing the distribution of mCD14 in living cells. Labeling mCD14 directly with a fluorescent tag avoided any perturbation of the distribution of mCD14, a GPI-anchored protein, that might be caused by antibody cross-linking (30). A variety of functional studies confirmed that the mCD14EGFP chimera was an intact and functional protein that was attached to the cell surface via a GPI anchor.
Using this method, we observed that mCD14 was not only expressed on the cell surface but was also present intracellularly both in the perinuclear area and in small vesicles near the basal aspect of the cells. These vesicles might represent compartments in which mCD14, and perhaps other GPI-anchored proteins, traffic from the Golgi apparatus to the plasma membrane. They may also represent a recycling compartment for GPI-linked proteins that are internalized from the plasma membrane and then returned to it (31). Most of these vesicles were exocytosed when U373CD14EGFP cells were exposed to LPSsCD14 complexes, indicating that they are capable of fusion with the plasma membrane.
The rapid exocytosis of the vesicles containing mCD14EGFP upon stimulation of U373CD14EGFP cells with LPS was reminiscent of the exocytosis of secretory vesicles containing mCD14 and other GPI-anchored proteins upon stimulation of neutrophils with an agonist (25). Secretory vesicles are thought to be an endocytic compartment, as they also contain the serum proteins albumin and tetranectin (32) (33). In response to formyl peptide, secretory vesicles are brought rapidly to the neutrophil surface, augmenting the expression of GPI-anchored proteins on the plasma membrane (25). Additional studies will be required to determine whether any of the mCD14-containing vesicles in U373CD14EGFP cells represent a recycling compartment for GPI-anchored proteins similar to the secretory vesicles of neutrophils.
Expression of mCD14EGFP on the plasma membrane enabled uptake of BODIPYLPS (Figure 5 and Figure 6) and cellular responses to LPS (Figure 4 A). CD14 binds LPS (34) and, not surprisingly, added LPS was found to colocalize with plasma membrane mCD14EGFP (Figure 5 and Figure 6). After association with the membrane, monomeric LPS is known to be rapidly internalized (13) (14). Here we show that when LPS was internalized, it moved into intracellular vesicles that did not contain mCD14. Thus, we have demonstrated in living cells that LPS, once it is bound by mCD14 on the cell surface, is internalized without being accompanied by its receptor. Together with our previous observations that each mCD14 on the surfaces of human monocytes enables the uptake of 15 LPS molecules in 30 min (3) and that this uptake depends on an additional cell surface protein (3), these observations support a model for LPS trafficking that involves transfer of LPS from mCD14 to another cell surface protein or to the lipid bilayer of the plasma membrane.
In addition to our studies with monomeric LPS, we have also observed the trafficking of LPS aggregates. Aggregated LPS may engage multiple copies of mCD14 on the cell surface at the same time, and it may be more difficult for mCD14 to transfer LPS from aggregates to the plasma membrane. Using the mCD14EGFP chimera, we observed that mCD14 was internalized with aggregated LPS in U373 cells. This behavior is opposite to that of LPS monomers. It is, however, consistent with a variety of other studies documenting different fates of monomeric and aggregated LPS. After internalization, primarily through noncoated structures (35), LPS aggregates move over the course of several hours into a compartment that is likely to be lysosomal in nature. There, acyloxyacyl hydrolase deacylates and thus detoxifies LPS (18). Internalization of aggregates can be disassociated from signaling (27) (29) and thus appears more relevant to the detoxification and clearance of LPS rather than signaling.
Several observations indicate a close correlation between signaling and LPS transit to the Golgi complex. For example, inactive structural analogues of LPS are not transported to the Golgi complex (15), and cells from Lpsd mice, which exhibit a defect in LPS signaling, fail to transport LPS to the Golgi complex (14). Recent work has shown that Lpsd mice are defective in Toll-like receptor (TLR)4, a member of the IL-1 receptor family (36). In this regard, it is interesting to note that the ligated IL-1 receptor type I (IL-1RI) may require trafficking to an intracellular compartment to generate signals (37). A thymocyte cell line has been identified that is defective in its responses to IL-1 and also does not internalize IL-1RI. The defect can be overcome by intracellular delivery of IL-1 (38) or by transfection with IL-1R accessory protein (IL-1RAcP), which restores both IL-1RI internalization (39) and IL-1 responses (39). Interaction of IL-1 with IL-1RI and IL-1RAcP triggers a cascade of signaling events, including activation of the stress-activated, mitogen-activated protein (MAP) kinase pathways and transcription factor NF-B. Of interest in this regard is that TLR4 shares homology in its cytoplasmic domain with the IL-1R family (40), suggesting the possibility that it may also share similar directions of intracellular trafficking. Whether TLR4 colocalizes with LPS before or after transport to the Golgi complex, however, will have to await further studies.
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A preliminary version of this work was presented at the Fifth Conference of the International Endotoxin Society, Santa Fe, NM, September 1215, 1998.
2 Le Grand, C.B., N. Lamping, T. Sugiyama, S.D. Wright, and R. Thieringer, manuscript submitted for publication.
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Acknowledgements |
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We thank Drs. Samuel Wright, Nathalie Thieblemont, and Norbert Lamping for critical reading of this manuscript.
Submitted: February 12, 1999; Revised: June 11, 1999; Accepted: June 22, 1999.
1used in this paper: BODIPY, boron dipyrromethane; DAF, decay accelerating factor; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GPI, glycosylphosphatidyl inositol; LBP, LPS binding protein; m, membrane-bound; PI-PLC, phosphatidyl inositol phospholipase C; s, soluble; SP, signal peptide; TLR, Toll-like receptor
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