Published online before print August 31, 2006
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Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
1 Correspondence: University of Massachusetts Medical School, NRB, 370L, 364 Plantation Street, Worcester, MA 01605, USA. E-mail: alberto.visintin{at}umassmed.edu
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Key Words: innate immunity • LPS • pathogen recognition
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25-kDa-secreted glycoprotein that binds to the extracellular domain of TLR4 and is indispensable for proper LPS signaling [12
]. The apparent Kd of the interaction of MD-2 with TLR4 has been determined experimentally to be 12 nM in humans [13
] and 63 nM in mice [14
]. Human MD-2 (hMD-2) can bind directly to LPS [8
, 9
, 14
15
16
17
], thus serving as an extracellular-activating adaptor linking LPS to the signaling component of the receptor complex TLR4. Upon LPS ligation, MD-2 and TLR4 probably undergo a conformational change [18
], which induces the homotypic aggregation responsible for the initiation of the signaling cascade [19
20
21
]. A direct interaction between TLR4 and LPS has yet to be reported experimentally in literature, despite genetic evidence suggesting that this might occur [22
, 23
]. Nagai and colleagues [12 ] have demonstrated that the genetic deletion of the MD-2 gene generates a phenocopy of the TLR4-deficient mouse. Most, if not all, LPS-induced responses are absent in MD-2–/– mice [12 ]. In this report, the authors also presented evidences supporting their hypothesis that MD-2 expression is required for cell surface distribution of TLR4. This is in agreement with the notion that TLR4 and MD-2 are assembled in the Golgi apparatus when coexpressed [24 ]. In this respect, MD-2 was said to resemble MD-1 (Ly86), a MD-2-related protein, which is required for transporting RP105 on the cell surface [25 ]. The functional implication of these results is that the activity of TLR4, which is strictly dependent on its cell surface localization [19 ], can be modulated by the levels of MD-2 protein and therefore, by its promoter [26 27 28 29 ]. Studies based on the IL-3-dependent mouse B cell line Ba/F3 suggested that surface localization of functional mouse TLR4 (mTLR4) depends on fully glycosylated mMD-2 [18 , 20 ]. The importance of these observations is that as a result, MD-2 might be considered to have a specific chaperone function that has the potential to regulate host responses to bacterial endotoxin and greatly influence the outcome of serious Gram-negative infections.
However, additional evidence challenges this unique and attractive hypothesis that surface expression of TLR4 is regulated by MD-2 coexpression.
First, most TLR4 gain-of-function studies are based on the ectopic expression of functional TLR4 cDNA in MD-2 null cells (most notably, 293 cells, which are a human, embryonic, epithelial kidney line; ref. [30 ]). We have found that these cells can express a properly folded form of TLR4, suggesting that the molecule undergoes trans-Golgi maturation in the absence of hMD-2 [13 , 14 ]. Second, by FACS analysis, confocal microscopy, surface biotinylation, and surface cross-linking with an anti-hTLR4 antibody, it was demonstrated that hTLR4 is functional and localized to the cell surface of these cells [8 , 19 , 31 ]. Likewise, LPS-nonresponsive cell lines, which are TLR4+/MD-2–, bind to recombinant soluble MD-2 (sMD-2; derived from transfected 293 and Chinese hamster ovary cells) [8 , 15 ] and become LPS-responsive when supplemented with recombinant sMD-2 [9 , 16 , 24 , 31 ]. Thus, LPS-ligated hMD-2 can function as a coreceptor or perhaps, a true endogenous ligand for hTLR4. One final consideration is that at least one human cell line, the human corneal epithelial cells (HCEC), expresses endogenous TLR4 and responds to LPS in the presence of sMD-2 [9 ].
Thus, the "helper" role of MD-2 appeared to be restricted to the mouse model. We therefore wished to determine whether the biology of mTLR4 was different from its human counterpart, as far as the requirement for MD-2 coexpression is concerned [32 ]. We conclude that functional hTLR4 and mTLR4 are inserted on the cell surface in the absence of MD-2. It is interesting that mMD-2, which was produced in 293 cells, stably interacts with LPS, only when in the presence of mTLR4. In contrast, hMD-2 forms detergent-stable complexes with LPS in the absence of hTLR4 [8 , 15 ].
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and transgenes
All mammalian cells were maintained in DMEM (BioWhittaker, Walkersville, MD), supplemented with 10% FBS (complete medium, Hyclone, Logan, UT) and antibiotics under standard conditions. 293 Cells (American Type Culture Collection, Manassas, VA, CRL-1573) were obtained from Dr. Jesse Chow (EISAI, Andover, MA) and were TLR4–/MD-2– by RT-PCR [34
]. HCEC were obtained from Dr. Kottarappat Dileepan (University of Kansas Medical Center, Kansas City) and were TLR4+/MD-2– by RT-PCR (not shown). mMD-2 and hMD-2 were cloned in the pCMV1FLAG vector using the NotI and XhoI restriction sites. The native leader sequences, amino acids 1–16, were replaced by the vector-provided pre-pro trypsin leader, which introduces an N-terminal FLAG tag in the mature proteins. Full-length cDNA for mTLR4 (NM_021297), a gift from Dr. Bruce Beutler (The Scripps Institute, La Jolla, CA), was cloned into pcDNA3-YFP by the BamHI restriction site and SalI/XhoI fusion. C-terminally YFP-tagged mTLR4 was subcloned into the HindIII/NotI sites of a modified pCL retroviral vector [35
, 36
], which was packed, and the target cells were transduced as described [24
]. In some experiments, the indicated recombinant DNA was transiently transfected into 293 cells using Genejuice per the manufacturer’s instructions (Novagen-EMD Biosciences, La Jolla, CA). Transiently transfected cells were used 48 h post-transfection.
Peritoneal and bone marrow-derived macrophages (BM
)
All the mice were housed and handled in accordance with the Institutional Animal Care and Use Committee at the University of Massachusetts Medical School (Worcester). Mice deficient in MD-2 were a gift from Dr. K. Miyake and Japan Science and Technology Agency, and TLR4–/– mice were provided by Dr. Shizuo Akira (Osaka University, Osaka, Japan). Peritoneal macrophages were obtained from peritoneal lavage of thioglycollate-injected mice 3 days postinjection (3 ml/mouse, Remel, Lenexa, KS). Mouse bone marrows were collected from femurs and plated in 10 cm tissue-culture dishes at a density of 1 x 107 cells/ml RPMI, 10% FCS. Adherent cells were maintained in complete RPMI supplemented with 20% of L929-conditioned medium as a source of mouse M-CSF [37
]. After 10 days, the resulting BM were plated in 96-well plates at a density of 105 cells/well. Stimulations were performed overnight in a final volume of 50 or 100 µl/well.
Immunofluorescence analysis
Adherent cells were detached mechanically from plastic. When mouse macrophages were used, FcRs were blocked by preincubating the cells for 15 min on ice with Fc block (Southern Biotechnologies, Birmingham, AL) at 10 µg/ml in FACS buffer (1% FBS in PBS), plus 50 µg total rat IgG/ml (Jackson Immunoresearch Labs, West Grove, PA). Specific antibodies were then added for an additional 30 min at 5 µg/ml, followed by a wash step in PBS. When required, labeled secondary reagents were used at 3 µg/ml, incubated with cells for 30 min, and washed. Samples (10,000 total events) were acquired using a LSR 2 cytofluorimeter (BD Biosciences, San Jose, CA). The FACS profiles shown in each panel were acquired in the same experiment and are representative of at least three unrelated experiments.
Immunoprecipitation and Western blotting
To study the association between TLR4YFP and MD-2FLAG, cells ectopically expressing both proteins were grown to confluence in 10 cm tissue-culture dishes and solubilized in 1 ml lysis buffer (20 mM Tris, pH 8, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10% glycerol, and freshly added protease inhibitors) for 10 min on ice. Lysates were cleared by centrifugation, and postnuclear supernatants were immunoprecipitated with 2 µg anti-GFP polyclonal antibody and 20 µl packed protein A sepharose beads (CL4B, Amersham-Biosciences)/sample. The immunocomplexes were resolved in 10% SDS-reducing precast gels (Gradipore Inc., Frenchs Forest, Australia) and transferred to nitrocellulose filter paper (Hybond-C, Amersham-Biosciences). The upper portion of the membrane was probed with an anti-GFP antibody (to detect YFP-tagged TLR4) and the lower portion with a HRP-labeled anti-FLAG mAb (to detect FLAG-tagged MD-2). In all Western blot procedures, the proteins were revealed by ECL (Amersham-Pharmacia). Biotinylation of surface proteins was obtained using the membrane-impermeable normal human serum (NHS)-biotin, as per the manufacturer’s instructions (Pierce, Rockford, IL). Biotinylated proteins were Western-blotted using a HRP-conjugated, antibiotin antiserum. All the gels shown are representative of three independent repeats unless otherwise stated.
LPS-binding assay
The LPS-binding assay was performed exactly as described in ref. [8
]. Briefly, hydrazide-biotin (Pierce) was used to biotin-label LPS in its glycan moiety. After mixing, complexes of MD-2 and biotinylated LPS (LPSbiotin) were captured using 20 µl packed streptavidin beads/condition for at least 1 h at 4°C. MD-2FLAG was revealed by Western blotting using an anti-FLAG antibody (Sigma Chemical Co.). To purify TLR4YFP/MD-2FLAG/LPSbiotin complexes, adherent monolayers of the indicated transfectants were incubated with 1 µg LPSbiotin/ml for 1 h at 37°C. Cells were washed in HBSS and solubilized in 1 ml lysis buffer. LPSbiotin-containing complexes were captured using streptavidin beads for 1 h at 4°C. Precipitates were analyzed by Western blot, and a mouse anti-GFP mAb was used to reveal LPS-bound TLR4YFP.
NF-
B luciferase (KB-Luc) reporter assays
293 Cells stably expressing TLR4cyan fluorescent protein or transiently transfected with mTLR4YFP or hTLR4YFP plus or minus MD-2FLAG (20 ng/well for each plasmid) were transiently transfected with 10 ng of a KB-Luc reporter plasmid/5 x 104 cells/well. In some experiments, recombinant sMD-2 was provided to the system by adding a 1:1 dilution of conditioned medium from cells stably secreting MD-2 [24
] or as a purified protein. Adherent cells were treated in the 96-well format. Stimulations with LPS were performed in duplicate wells at the indicated concentrations for 4–16 h. Luciferase activity was determined using a luciferase kit per the supplier’s instructions (Promega, Madison, WI), and the readings were divided by the average reading of unstimulated controls [relative luciferase units (RLU)]. Values are shown as average ± SD of duplicate readings.
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Figure 1. mTLR4 is expressed on the surface of 293 cells. (A) 293 Cells stably transduced with a retrovirus encoding mTLR4YFP (green panels) were stained with the anti-mTLR4 mAb Sa15-21. Alexa647-labeled antirat IgG was used as a secondary reagent. Dots represent the red and green fluorescence (y- and x-axis, respectively) of the counted events. Nontransfected 293 cells were stained with the same antibody combinations (red panel) as a negative control. (B) Cells expressing mTLR4YFP and hTLR2YFP (in the absence or presence of transiently transfected mMD-2) were subjected to surface biotinylation, lysed, and immunoprecipitated with an anti-GFP polyclonal antiserum. Biotinylated proteins were revealed by Western blotting using a HRP-labeled biotin polyclonal antiserum followed by ECL. Anti-GFP immunoblots were performed on whole cell lysates (WCL) to control for TLR mobility. No differences are observed in TLR4 or TLR2 surface expression as a result of MD-2 transgene expression. Note that TLR2 coprecipitated a high molecular weight protein, possibly corresponding to hyperglycosylated TLR1 or TLR6 (white asterisk). These experiments were repeated twice with similar results.
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derived from the wild-type and MD-2 KO mice showed a clear, positive staining when labeled with the anti-mTLR4 antibody Sa15-21. The patterns were almost identical. Peritoneal macrophages from the C57BL/6, TLR4–/–, and MD-2–/– mice were stained using the same antibody and an isotype-matching mAb to confirm specificity of staining (Fig. 2C)
. As expected, peritoneal macrophages from TLR4 null animals were negative, whereas the wild-type and MD-2–/– mice had similar levels of staining.
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Figure 2. mMD-2 is expressed on the cell surface of mouse macrophages. BM from the wild-type (C57BL/6; A) and MD-2-deficient mice (B) were stained with the Alexa647-labeled anti-mTLR4 mAb Sa15-21. (C) Sa15-21 mAb was used to stain thioglycollate-elicited peritoneal macrophages from wild-type (blue line), MD-2 (cyan line), and TLR4 (green line) gene-deleted mice. A labeled isotype-matched rat IgG gave profiles similar to the control (CTRL) mAb (red line) in all the tested macrophages.
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did not respond to soluble LPS or lipid A but were similar to wild-type cells as far as the cytokine response to other TLR ligands was concerned (Fig. 3
).
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Figure 3. MD-2–/– macrophages do not respond to LPS. BM from wild-type (A) and MD-2-deficient mice (B) were stimulated overnight with Pam2 (1 ng/ml), pIC (50 µg/ml), R848 (20 µM), LPS (10 ng/ml), and Lipid A (10 ng/ml), and secreted RANTES and IL-6 were detected in the cell supernatants by indirect ELISA. Shown are the average absorbance readings at 450 nm ± SD of duplicates. This phenotypical characterization is representative of one of many routine analyses we perform on cells derived from KO mice.
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Figure 4. mMD-2 forms LPS-stable complexes only when it is bound to TLR4. (A) Supernatants (10 ml) of conditioned medium containing FLAG-tagged sm/hMD-2 (from transfected 293 cells) were subjected to anti-FLAG immunoprecipitation (IP) using the anti-FLAG mAb (Total and right panels) or LPSbiotin (LPS bound panel). Immunocomplexes were captured with protein A beads, and MD-2:LPSbiotin complexes were captured using streptavidin beads. Pellets were resolved in reducing (left panel) or nonreducing SDS-PAGE (right panel), and MD-2FLAG was revealed by anti-FLAG Western blot (WB). (B) Adherent 293 cells expressing the indicated combinations of TLR4YFP and MD-2FLAG were incubated with biotinylated LPS (LPSbiotin; 1 µg/ml) for 1 h at 37°C. After extensive washing, the cells were solubilized and bio-LPS captured using streptavidin beads. The pellets were resolved by reducing SDS-PAGE and Western blotted using an anti-GFP pAb to reveal YFP-tagged TLR4 (arrowhead). A nonspecific band below TLR4 is marked with an asterisk. This experiment is representative of two repeats. (C) 293 Cells expressing the indicated combinations of h/mTLR4 and h/mMD-2 or TLR2 and MD-2 were subjected to immunoprecipitation with an anti-GFP pAb and Western blotted for YFP-tagged TLR4s (upper panel,  GFP) and MD-2FLAG (lower panel, FLAG). Note that TLR4 coprecipitated multiple immature glycoforms of MD-2, which generate the complex pattern in the lower portion of the gel. The (negative) coprecipitation of TLR2YFP and MD-2 was used as a specificity control. (D) 293 Cells stably expressing hTLR4YFP and a KB-Luc reporter plasmid were transfected simultaneously with hMD-2, mMD-2, or the empty vector. After an overnight stimulation with titrated amounts of LPS, luciferase activity was assessed by luminometry (left panel). In a similar experiment (right panel), mTLR4 and mMD-2 were transiently transfected along with the KB-Luc reporter plasmid. LPS-dependent NF-B activation was assessed as described above. Luciferase values were normalized by the average value of nontreated wells (RLU). Shown is the average of duplicate reading ± SD from one representative experiment out of three.
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To establish whether TLR4 expressed on the surface in the absence of MD-2 is functional, we developed an assay in which TLR4-expressing cells were allowed to interact with sMD-2 or sMD-2 + LPS for defined periods of time, washed, and rested for 4 h. Activation of the KB-Luc reporter plasmid indicated that surface TLR4 was able to capture sMD-2 and LPS in the allowed time frames, thus producing a functional interaction. In the experiment shown in figure Figure 5A
, TLR4-expressing reporter cells were pulsed for 10 or 30 min with MD-2- and LPS-containing medium. Cells were then washed extensively in HBSS, and after 4 h incubation in HBSS, NF-B activation was assessed by luminometry. A similar experiment was performed by pulsing the cells with MD-2-conditioned medium, followed by a 4-h chase in LPS containing HBSS (Fig. 5B)
. In both conditions, incubation of TLR4 reporter cells with MD-2 for as little as 10 min was sufficient to confer full LPS responsiveness in these cells (Fig. 5C)
. These experiments indicate that TLR4 expressed in the absence of MD-2 is capable of capturing MD-2 from the supernatant, thus implying that no intracellular preassociation of TLR4 with MD-2 appears to be necessary to initiate signal transduction.
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Figure 5. Surface-expressed TLR4 is functional. 293 Cells stably expressing TLR4YFP along with a NF-B reporter plasmid were pulsed for the indicated times (10 or 30 min) with (A) MD-2 and LPS (0, 10, and 100 ng/ml) or (B) MD-2 alone. MD-2 was provided as a 1:1 dilution of conditioned medium from cells stably secreting shMD-2. After three washes in HBSS, cells were chased in HBSS (A) or HBSS plus LPS at the indicated concentrations (B). Luciferase activity was measured as in Figure 4
. As a control, cells stimulated for 4 h in MD-2-conditioned medium plus LPS are shown (C). Purified baculoviral hMD-2 activates mTLR4. 293 Cells transiently expressing mTLR4 and a KB-Luc reporter plasmid were stimulated with the indicated amounts of LPS in complete medium supplemented with baculoviral hMD-2 (bMD-2; 10 nM; D). Luciferase activity was determined as in Figure 4
. The experiments shown in these panels are representative of at least three independent repeats.
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MD-2–/– BM express functional TLR4 on the cell surface
Previous studies suggested that MD-2 is important for generating a correctly glycosylated TLR4 on the cell surface [40
]. To determine whether cell-surface TLR4 from the MD-2–/– mouse is functional (i.e., with the correct post-translational modifications), add-back experiments were performed in mouse KO cells using baculoviral hMD-2. BM from the MD-2–/– mouse were stimulated with LPS in the presence or absence of exogenous sMD-2. Cellular activation was assessed by quantification of secreted RANTES (Fig. 6A
) and IL-6 (Fig. 6B)
in the cell culture supernatants. These experiments demonstrated that hMD-2 could functionally complement mMD-2–/– cells, implying that TLR4 is present on the cell surface, and it is functional. It would be unreasonable to hypothesize that exogenous MD-2 interacts with immature TLR4 after internalization and undergoes trans-Golgi maturation, thus leading to the formation of a functional receptor on the cell surface. It is noteworthy that when MD-2 was added in large excess, LPS responses were inhibited, in accordance with data previously reported by Viriyakosol et al. [15
].
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Figure 6. Baculoviral hMD-2 confers LPS responses to MD-2–/– macrophages. BM from the MD-2–/– mouse were treated with the indicated combinations of baculoviral hMD-2 and LPS. After 16 h, the concentration of RANTES (A) and IL-6 (B) was determined by ELISA and calculated using standard reagents provided by the manufacturer. Shown are the averages of duplicate readings ± SD. This experiment is representative of five with similar results.
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B-dependent luciferase gene, only when MD-2 was transiently transfected (left panel) or when it was added to the system as a soluble molecule (in the form of MD-2-conditioned medium, right panel). These results confirm what was previously reported by Kennedy et al. [9
] and further support the notion that TLR4 reaches the cell surface in the absence of MD-2.
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Figure 7. HCEC express a functional TLR4 on the cell surface. (A) HCEC were transiently transfected with hMD-2 ( ) or mock-transfected ( ), along with a KB-Luc reporter plasmid and plated in 96-well plates. Forty-eight hours later, the cells were stimulated with the indicated amounts of LPS and Luciferase activity, measured after 8 h, as described in Figure 2
(left panel). (B) HCEC were transfected with the KB-Luc reporter plasmid and treated with 10 ng LPS/ml in complete medium (open bars) or MD-2-conditioned supernatants (CM; solid bars). Results are the average of duplicate wells and representative of three independent experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, the universal readout of TLR activation. The IL-1 receptor and ectopically expressed TLRs and in particular, TLR4 [4
, 30
] are functional in 293 cells, thus implying that they possess an intact signaling machinery that links the receptors to NF-B. It also implies that 293 cells can place these receptors in the correct subcellular compartment and produce the correct post-translational modifications required for physiological triggering (e.g., glycosylation). We reported here that ectopic expression of mTLR4 in 293 cells induces the expression of a receptor, which is localized on the cell surface and is capable of triggering a signaling event to NF-B when supplemented with MD-2 of human or mouse derivation. We have shown that primary immune cells from the MD-2-deleted mouse express detectable amounts of TLR4 on their surfaces and respond to exogenously added MD-2. The findings presented here are in contrast with the report of Nagai et al. [12 ], which proposed that lack of MD-2 expression prevents surface localization of TLR4. If Nagai’s conclusion is correct, then there are a number of explanations that can elucidate this discrepancy. One possibility is that different MD-2-negative cell lines, such as 293, process mTLR4 in aberrant yet functionally sound ways. However, the fact that even the S2 insect cells can secrete a recombinant form of the extracellular domain of mTLR4 suggests that this molecule normally undergoes trans-Golgi maturation in different cell types [41 ]. It is noteworthy that the genome of Drosophila does not appear to contain MD-2-like molecules [42 ]. A second possibility is that the mouse embryo fibroblasts (MEFs) from the MD-2–/– mouse have lost the ability of correctly processing TLR4. The pleiotropic effects of a protein-like MD-2, which is involved in differentiation and maintenance of several different cell types [43 ], may not be fully appreciated yet. For example, gp96 [44 ] has been reported to be required for properly assembling the LPS receptor, i.e., correctly fold TLR4, MD-2, or both. Konno and collaborators [45 ] have recently cloned two additional proteins, PRAT4A and PRAT4B, which appear to be required for surface localization of hTLR4 and mTLR4. The fact that MEFs failed to place TLR4 on the cell surface in the absence of MD-2 might simply reflect limiting amounts of such proteins in the MD-2–/– MEFs.
Another possibility is that the experiments suggesting a role of MD-2 as a chaperone-like molecule for TLR4 [12 , 46 , 47 ] are inconclusive. In those experiments, a retrovirus encoding a FLAG-tagged mTLR4 was used to transduce wild-type and MD-2–/– MEFs. These cells were fixed in 3.7% formaldehyde, permeabilized using 0.2% Triton X-100, and stained with an anti-FLAG mAb, followed by staining with a fluorescently tagged secondary reagent. This approach may not be optimal for discriminating the surface from intracellular staining.
The findings reported here will help correct a somewhat rooted misconception about the biology of TLR4. We showed that TLR4 is a cell surface receptor whose plasma membrane distribution does not depend on MD-2 in mice or humans. Although TLR4 and MD-2 can interact promiscuously with each other, only the combinations mTLR4 + h/mMD-2 and hTLR4 + hMD-2 generate stable surface complexes with LPS, which elicit a signal. In our preliminary experiments of ectopic expression in 293 cells, among a panel of five different MD-2 orthologs (that will be reported elsewhere), we observed that under the experimental conditions used in this paper, i.e., biotinylated LPS pull-down, only rabbit and shMD-2 interact with LPS in solution. These findings may provide hints on a molecular basis, explaining some of the species-specific differences in LPS recognition between humans and mice.
Nevertheless, Mus musculus is the elective animal model for a growing list of diseases and in particular, in infectious diseases. To draw educated conclusions from this animal model, the analogies and differences that exist between the human and the mouse models should be fervently sought and studied in the deepest detail. TLR4 represents a major potential target for pharmacological intervention in LPS-related human diseases. In this report, we confirmed that the biology of mTLR4 closely resembles its human counterpart, which allows us to proceed with renewed confidence when designing experimental, therapeutical approaches in this animal model.
Received June 9, 2006; revised July 18, 2006; accepted July 23, 2006.
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