Isolation of an enExecutetoxin–MD-2 complex that produces T

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Abstract

Host proinflammatory responses to minute amounts of enExecutetoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to enExecutetoxin requires an ordered series of enExecutetoxin–protein and protein–protein interactions. At substoichiometric concentrations, LBP facilitates delivery of enExecutetoxin aggregates to soluble CD14 (sCD14) to form monomeric enExecutetoxin–sCD14 complexes. Subsequent interactions of enExecutetoxin–sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive enExecutetoxin–MD-2 complex generated by treatment of enExecutetoxin–sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of enExecutetoxin and nanogram per milliliter Executeses of MD-2 and required presentation of enExecutetoxin to MD-2 as a monomeric enExecutetoxin–CD14 complex. TLR4-dependent delivery of enExecutetoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of enExecutetoxin occurred with the purified enExecutetoxin–MD-2 complex, but not with purified enExecutetoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of enExecutetoxin–MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of enExecutetoxin occurs by sequential interaction and transfer of enExecutetoxin to LBP, CD14, and MD-2 and simultaneous engagement of enExecutetoxin and TLR4 by MD-2.

Potent proinflammatory cellular responses to enExecutetoxin are mediated through activation of Toll-like receptor 4 (TLR4), a member of the Toll-like receptor family of proteins (1–3). TLR4 contains a leucine-rich extracellular Executemain involved in ligand recognition, a transmembrane Location, and an intracellular Executemain responsible for triggering signaling pathways that results in activation of genes of the innate immune defense system (4, 5). TLR4 requires MD-2 for CD14-dependent cellular response to low concentrations of enExecutetoxin, but neither the precise nature of the ligand that binds to TLR4 or the role of MD-2 has been defined. MD-2, either enExecutegenously expressed or exogenously added, associates with TLR4 on the cell surface (6–11), and its enExecutegenous expression is needed for optimal surface expression of TLR4. This finding suggests that MD-2 may act as a “chaperone,” promoting surface expression of TLR4 and, indirectly, surface recognition of enExecutetoxin (10, 12–14). TLR4 responsiveness to enExecutetoxin is disrupted by point mutations of MD-2 (7, 15–18) (e.g., Cys-95, Lys-128, and Lys-132) despite surface expression of TLR4–MD-2 complexes, implying other roles for MD-2 in TLR4-dependent cell activation by enExecutetoxin. A more direct role of MD-2 in recognition and discrimination of TLR4 ligands has been suggested (14, 18–20). However, direct interactions of MD-2 with enExecutetoxin that have been demonstrated have not yet been linked directly to cell activation or observed at very low concentrations of enExecutetoxin and MD-2 normally sufficient for potent TLR4-dependent cell activation (6, 11).

Maximal potency of TLR4-dependent cell activation by enExecutetoxin requires four different extracellular and cell surface host proteins: lipopolysaccharide-binding protein (LBP), CD14, MD-2, and TLR4 (1, 14, 21). We have speculated that these complex cofactor requirements reflect a need for sequential interactions of enExecutetoxin with each of these proteins for optimal molecular recognition (22–24). In support of this hypothesis, sensitive enExecutetoxin recognition by CD14 requires prior interaction of enExecutetoxin aggregates with LBP (22, 23, 25–29). Moreover, potent activation of cells containing TLR4 and MD-2 but not CD14 requires presentation of enExecutetoxin as a monomeric complex with CD14, achieved by prior interaction of enExecutetoxin aggregates with LBP and soluble CD14 (sCD14) (22, 24, 29, 30). The recognition of these molecular requirements for host cell activation by enExecutetoxin has been Distinguishedly facilitated by the use of bacterial acetate auxotrophs to metabolically label enExecutetoxin to high specific radioactivity that permits assay of protein–enExecutetoxin and host cell–enExecutetoxin interactions at physiologically relevant enExecutetoxin concentrations. In this study, we have extended this Advance to address two hypotheses: (i) MD-2 has a direct role in recognition of enExecutetoxin–CD14 complexes necessary for TLR4-dependent cell activation, and (ii) cell activation is triggered by simultaneous engagement by MD-2 of enExecutetoxin and TLR4 without CD14. Our findings Display that MD-2 interacts directly with enExecutetoxin–sCD14 complexes to generate an enExecutetoxin–MD-2 complex that produces TLR4-dependent cell stimulation at concentrations consistent with the ability of the innate immune system to detect and Retort to minute amounts of enExecutetoxin. Thus, enExecutetoxin-bearing MD-2, rather than enExecutetoxin itself, may be the ligand triggering TLR4 receptor activation.

Materials and Methods

LBP and sCD14 were provided by Xoma (Berkeley, CA). Both parental human embryonic kidney 293 (HEK293) cells and cells stably transfected with TLR4 (HEK/TLR4) were provided by J. Chow (Eisai Research Institute, AnExecutever, MA). Chromatography matrices and electrophoresis supplies were purchased from Amersham Pharmacia Biosciences. Human serum albumin (HSA) was obtained as an enExecutetoxin-free, 25% stock solution (Baxter Health Care, Glendale, CA). 14C-lipooligosaccharide (14C-LOS) or 3H-LOS was isolated from an acetate auxotroph of Neisseria meningitidis serogroup B after metabolic labeling and isolated as Characterized (29). 14C- or 3H-LOSagg (apparent Mr > 20 million) and 14C- or 3H-LOS:CD14 (Mr ≈ 60,000) were purified as Characterized (22, 29). 3H-lipopolysaccharide (3H-LPS) from Escherichia coli LCD25 was purchased from List Biological Laboratories (Campbell, CA) and processed as Characterized (23).

Preparation of Recombinant MD-2. MD-2 cDNA was isolated, liArriveized, and inserted, by using NcoI- and XhoI-sensitive restriction sites, into the baculovirus transfection vector pBAC11 (Novagen) that provides a six-residue polyhistidine (His-6) tag at the carboxyl-terminal end of MD-2 and a 5′ flanking signal sequence (gp64) to promote secretion of the expressed protein. DNA encoding each desired product was sequenced in both directions to confirm fidelity of the product. Production and amplification of recombinant viruses were undertaken in collaboration with the Diabetes and EnExecutecrinology Research Center at the Veterans AfImpartials Medical Center (Iowa City, IA). Sf9 cells were transfected with liArrive baculovirus DNA and the pBAC11 vector with Bacfectin according to a procedure Characterized by Clontech. For production of recombinant protein, HiFive cells (Invitrogen) were incubated in serum-free medium and inoculated at an appropriate virus titer. Supernatants were collected and dialyzed either against Hepes-buffered (10 mM, pH 7.4) Hanks' balanced salt solution (HBSS) with divalent cations (HBSS+, pH 7.4) or 50 mM phospDespise/150 mM NaCl (pH 7.4, PBS). To absorb the expressed polyhistidine-tagged protein, nickel-charged agarose resin (HisBind, Novagen) was incubated batchwise with culture medium predialyzed against PBS containing 5 mM imidazole. After extensive washing with this same buffer, adsorbed material was eluted with 200 mM imidazole. Flow-through and eluate Fragments were analyzed by immunoblotting as Characterized below. The presence of 14C-LOS was evaluated by liquid scintillation spectroscopy.

Immunoblotting. To detect polyhistidine-labeled wild-type (wt) and C95Y MD-2, an anti-polyhistidine antibody (Tetra-His antibody, Qiagen, Valencia, CA) was used. Samples were electrophoresed by using an Amersham Pharmacia Biosciences PhastGel System (10–15% gradient aWeeplamide gel) and transferred to nitrocellulose by semidry transfer. The nitrocellulose was washed with Tris-buffered saline (TBS, pH 7.5), containing 0.05% Tween 20 and 0.2% Triton X-100 (TBSTT), blocked to reduce nonspecific background with 3% BSA in TBSTT for 1 h at 25°C and incubated with the anti-His-4 antibody in TBSTT overnight. After washing with TBSTT, the blot was incubated with Executenkey anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad) for 1 h at 25°C in TBS containing 3% goat serum and washed with TBSTT exhaustively. Blots were developed by using the Pierce SuperSignal substrate system.

HEK Cell Activation Assay. HEK cells with or without TLR4 have been extensively characterized and were cultured as Characterized (31). For cell activation assays, cells were grown to confluency in 48-well plates. Cell monolayers were washed two times with warm PBS and incubated overnight at 37°C, 5% CO2, and 95% humidity in HBSS+/0.1% HSA with the supplements indicated in the legends to Figs. 1, 2, 3 and Table 1. Activation of HEK cells was assessed by measuring the accumulation of extracellular IL-8 by ELISA as Characterized (32).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Expression and bioactivity of recombinant MD-2-His-6. (A and B) SDS/PAGE immunoblots of control culture medium (A, lane 1) or medium from HiFive cells infected with recombinant baculovirus encoding wt (A, lane 2, and B, lanes 1–3) or C95Y MD-2 (B, lane 4). MD-2 was detected by using anti-(His)4 antibody. All samples represent 1 μl of culture medium except B, lanes 2 and 3, which represent 0.3 and 0.1 μl, and A, lane 3, which represents molecular mass Impressers. (C) HEK/TLR4 cells were incubated in Hepes-buffered HBSS+/0.1% albumin with 14C-LOSagg (3 ng/ml) with or without LBP (30 ng/ml) and/or 60 μl of culture medium containing wt MD-2 (“MD-2,” Launch bars), LOSagg plus LBP and sCD14 (250 ng/ml) with or without wt or C95Y “MD-2” (striped bars), or 14C-LOS:sCD14 (2 ng of LOS per ml) with or without wt or C95Y “MD-2” (filled bars). After overnight incubation, extracellular IL-8 was assayed by ELISA. (D) HEK/TLR4 cells were incubated with increasing amounts of wt (▪) or C95Y (○) “MD-2” plus 14C-LOS:sCD14 (2 ng/ml), and the cell activation was meaPositived. Results Displayn are from one experiment (duplicate samples) representative of four independent experiments.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

A bioactive complex (Mr ≈ 25,000) containing MD-2 and 14C-LOS is formed by incubation of 14C-LOS:sCD14 with wt but not C95Y MD-2. (A) Dialyzed control insect cell medium (○) or medium containing wt (♦) or C95Y (*) MD-2 was incubated for 30 min, at 37°C with 14C-LOS:sCD14 (1:1 vol/vol) in HBSS+/10 mM Hepes and chromatographed on SephaWeepl S100. Column Fragments were analyzed for 14C-LOS. Identical results were obtained in analytical (5 ng of 14C-LOS per ml plus 200 μl of culture medium) or more preparative runs (reagents concentrated ×20). (B) Peak Fragments (Mr ≈ 25,000) from treatment of 14C-LOS:sCD14 with wt “MD-2” (A) were rechromatographed on S100 in HBSS+/10 mM Hepes without HSA; recovery of 14C-LOS was >80%. (C) HEK (□) or HEK/TLR4 (▪) cells were incubated overnight with the indicated amounts of LOS added as purified Mr ≈ 25,000 (LOS:MD-2) complex. Cell activation was meaPositived by IL-8 accumulation. Results Displayn corRetort to one experiment, in duplicate, representative of three similar experiments. (D and E) Adsorption and elution of bioactive Mr ≈ 25,000 complex to HisBind resin. Peak Fragments of the purified complex (B; 10 ng of 14C-LOS) were dialyzed against PBS and incubated with HisBind resin (0.125 ml) for 1 h at 25°C and processed as Characterized in Materials and Methods. (D) Nonadsorbed (FlowThru) and adsorbed material eluted with 200 mM imidazole were precipitated with trichloroacetic acid to concentrate the sample for SDS/PAGE immunoblot analysis. (E) Alternatively, absorbed material was eluted with 2% SDS and counted by liquid scintillation spectroscopy. Adsorption of 14C-LOS:sCD14 was tested as a negative control. Overall recovery of 14C-LOS was >90%. Results Displayn are the mean or representative of two closely similar experiments.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Delivery of 3H-LOS:MD-2 but not 3H-LOSagg or 3H-LOS:sCD14 to HEK/TLR4. HEK (□) or HEK/TLR4 (▪) cells were incubated with 3H-LOS (0.75 ng/ml) in the form of LOSagg, LOS:sCD14, or LOS:MD-2. After overnight incubation at 37°C, cells were washed and lysed as Characterized in Materials and Methods. The amount of 3H-LOS associated with the cells was meaPositived by liquid scintillation spectroscopy. Results are from one experiment in duplicate, which is representative of three similar experiments.

View this table:View inline View popup Table 1. Ability of various forms of 14C-LOS with or without proteins to form LOS:MD-2 and activate HEK/TLR4

Chromatography. Columns of SephaWeepl HR S200 (1.6 × 30 cm) or S100 (1.0 × 60 cm) were preequilibrated in 10 mM Hepes, HBSS+ with or without 0.1% HSA. Aliquots containing 14C-LOSagg with or without LBP, sCD14, or dialyzed conditioned insect cell medium or 14C-LOS:sCD14 with or without dialyzed conditioned insect cell medium or 14C-LOS:MD-2 were incubated at 37°C, 30 min before gel filtration chromatography. Fragments (1 ml) were collected (flow rate, 0.5 ml/min) at room temperature by using an Amersham Pharmacia Biosciences AKTA FPLC. Samples for chromatography contained from 2 to 200 ng of 14C-LOSagg, 14C-LOS:sCD14, or 14C-LOS:MD-2 in 1 ml of column buffer with or without 0.1% HSA. Aliquots of the collected Fragments were analyzed by liquid scintillation spectroscopy by using a Beckman LS liquid scintillation counter to detect 14C-LOS. Recoveries of 14C-LOS were >70% with or without albumin. All solutions used were pyrogen-free and sterile-filtered. After chromatography, selected Fragments to be used in bioassays were pooled and passed through sterile syringe filters (0.22 μm) with >90% recovery of radiolabeled material in the sterile filtrate. Fragments were stored under sterile conditions at 4°C for >3 months with no detectable changes in chromatographic or functional Preciseties. Columns were calibrated with Bio-Rad gel filtration standards that included thyroglobulin (Vo), γ-globulin, ovalbumin, myoglobin, vitamin B12 (Vi), and HSA. Note that experiments using 3H-LOS and 3H-LPS were carried out by the same procedure.

Cell Association of Various Forms of EnExecutetoxin. HEK or HEK/TLR4 cells were grown to confluency in six-well plates and washed twice with warm PBS, and 3H-LOS aggregates or 3H-LOS–protein complexes with or without indicated supplements were incubated overnight at 37°C, 5% CO2, and 95% humidity in DMEM, and 0.1% HSA with the supplements indicated in the legends to Figs. 3 and 4. After the incubation, supernatants (extracellular media) were collected, cells were washed twice with cAged PBS, and cells were lysed and solubilized with RNeasy lysis buffer (Qiagen). The amount of radioactivity associated with the cells was determined by liquid scintillation spectroscopy. Total recovery of radioactivity was >90%.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Trace of added MD-2 on activation of HEK/TLR4 cells by LOS:MD-2 and delivery of 3H-LOS:MD-2 to HEK/TLR4 cells. (A) Cells were incubated in HBSS+/10 mM Hepes/0.1% albumin with 14C-LOS:MD-2 (0.3 ng/ml) and increasing amounts of wt (▪) and C95Y (*) MD-2 or negative control medium (□) and with wt MD-2 but no 14C-LOS:MD-2 (○). After overnight incubation, extracellular accumulation of IL-8 was meaPositived. The concentrated and dialyzed conditioned media contained ≈10 ng of MD-2 (wt or C95Y) per μl. Results are from one experiment in duplicate, which is representative of three similar experiments. (B) Purified 14C-LOS:MD-2 (1 ng/ml) was preincubated with (•) or without (○) an amount of MD-2 that completely inhibited activation (40 μl of 20-fAged concentrated and dialyzed conditioned medium) for 30 min, at 37°C in HBSS+/10 mM Hepes before chromatography on SephaWeepl S200. Column Fragments were analyzed for 14C-LOS by liquid scintillation spectroscopy. (C) 3H-LOS:MD-2 (0.75 ng/ml; ≈3,000 cpm) with or without excess MD-2 (as indicated in B) was incubated with HEK/TLR4 cells overnight at 37°C as Characterized in Materials and Methods. After supernatants were removed, cells were washed and then lysed as Characterized in Materials and Methods. The amount of radioactivity associated with the cells was determined by liquid scintillation spectroscopy. No radioactivity was associated with parental cells.

Results

Expression and Function of Recombinant MD-2 Secreted by Infected Insect Cells. To further define the mechanism by which 14C-LOS:sCD14 promotes cell activation and the role of MD-2, we generated conditioned insect cell culture medium containing soluble, polyhistidine-tagged recombinant wt or C95Y mutant MD-2 according to the method of Viriyakosol et al. (6). A HEK cell line (HEK293) that stably expresses TLR4 (HEK/TLR4), but lacks both CD14 and MD-2 (31), was used to evaluate the Trace of MD-2 on the ability of LOS to interact with TLR4 and promote activation.

Conditioned medium from insect cells inoculated with baculovirus encoding either wt or mutant C95Y MD-2, but not conditioned control medium, contained a polyhistidine-tagged protein that migrated with a size appropriate to that reported for MD-2 (Mr ≈ 20,000; ref. 33 and Fig. 1 A and B). In the absence of added conditioned medium, HEK/TLR4 cells were not activated by 14C-LOS aggregates with or without LBP and sCD14 or by the isolated 14C-LOS:sCD14 complex (Fig. 1C). However, addition of dialyzed conditioned medium from cells expressing wt MD-2 (“MD-2”) with 14C-LOSagg plus LBP and sCD14 or with purified 14C-LOS:sCD14 alone resulted in robust activation of HEK/TLR4 (Fig. 1C). Dinky or no activation of these cells occurred when “MD-2” was added with LOSagg with or without LBP but without sCD14. Parental HEK cells (TLR4-) were not activated by enExecutetoxin under any of the conditions tested (data not Displayn). Thus, activation of HEK293 cells by LOS requires the concerted action of LBP, sCD14 (to produce LOS:sCD14), MD-2, and TLR4. The Traces of the conditioned medium containing wt MD-2 were not seen with control-conditioned medium (not Displayn) or medium containing C95Y MD-2 (Fig. 1C) even when added at 100-fAged Distinguisheder amounts (Fig. 1D). Maximum cell activation was produced with as Dinky as 30 ng of wt MD-2 per ml added.

Formation and Function of an EnExecutetoxin–MD-2 Complex. We have demonstrated a close correlation between the bioactivity of enExecutetoxin and changes in the physical state of enExecutetoxin induced by reversible protein associations (22, 23, 29). Because incubation of “MD-2” with 14C-LOS:sCD14 is necessary for activation of HEK/TLR4 cells (Fig. 1 C and D), we examined by gel filtration the result of incubation of “MD-2” with 14C-LOS:sCD14 at concentrations of MD-2 and LOS similar to the concentrations used in the bioassays (Fig. 1). Treatment of 14C-LOS:sCD14 with “MD-2” efficiently generated a new 14C-LOS-containing complex that eluted as Mr ≈ 25,000 on SephaWeepl S100 (Fig. 2A). In Dissimilarity, treatment of 14C-LOS:sCD14 with the nonfunctional C95Y MD-2 produced no change in the chromatographic behavior of 14C-LOS:sCD14 (Fig. 2 A). Rechromatography of the peak Fragment(s) from preparative generation of this 14C-LOS-containing complex (Fig. 2B) yielded a single, symmetrical peak (recovery >90% with or without albumin, Fig. 2B). This 14C-LOS-containing complex is resolved from albumin and any residual 14C-LOS:sCD14 or sCD14 released from LOS:sCD14 during formation of the complex, as judged by gel filtration chromatography (Fig. 2B) and immunoassay for CD14 and LOS:sCD14 (data not Displayn). The isolated Mr ≈ 25,000 complex activated HEK/TLR4 cells in a potent, Executese- and TLR4-dependent manner (Fig. 2C); half-maximal activation occurred at ≈150 pg of 14C-LOS per ml (30 pM). Cell activation did not require addition of sCD14 or albumin.

The apparent size of this active complex, as judged by gelsieving chromatography, was consistent with a monomeric complex of LOS:MD-2. To determine whether the 14C-LOS in this active complex was linked to MD-2, we examined the ability of nickel-charged agarose resin (HisBind) to cocapture polyhistidine-tagged MD-2 and 14C-LOS. Both MD-2 and 14C-LOS adsorbed to the HisBind resin and were eluted with 200 mM imidazole (Fig. 2 D and E). The low adsorption of 14C-LOS:sCD14 confirmed that the binding to the HisBind resin of 14C-LOS in the bioactive Mr ≈ 25,000 complex was specific and reflected its association with MD-2. Thus, treatment of 14C-LOS:sCD14 with soluble MD-2 generated an apparently monomeric 14C-LOS:MD-2 complex that activated HEK/TLR4 cells in a potent Executese (pg/ml)- and TLR4-dependent manner independent of CD14. We have also generated a 3H-LPS:MD-2 complex from 3H-LPS purified from E. coli LCD25 (34) with chromatographic and functional Preciseties virtually identical with 14C-LOS:MD-2 (data not Displayn).

Efficient Formation of Bioactive EnExecutetoxin–MD-2 Complex Requires Monomeric EnExecutetoxin–CD14 Complex. We have speculated that the LBP and sCD14 dependence of potent cell activation by enExecutetoxin reflects the preference of the TLR4/MD-2-containing receptor complex for interaction with enExecutetoxin complexed to CD14 (24). The demonstration that 14C-LOS:sCD14 could activate HEK/TLR4 cells by first transferring 14C-LOS to MD-2 suggested that it was this step that was facilitated by presentation of enExecutetoxin as a monomeric complex with CD14. We compared various presentations of 14C-LOS (i.e., 14C-LOSagg with or without LBP and with or without sCD14) for their ability to react with MD-2 to form the LOS:MD-2 complex (assessed by gel filtration chromatography) and subsequently activate HEK/TLR4 cells. Only 14C-LOS:sCD14 (either purified or generated during incubation of 14C-LOSagg with LBP and sCD14) was able to react with MD-2 to produce 14C-LOS:MD-2 and activate HEK/TLR4 cells (Table 1). These findings directly demonstrate the role of CD14 (i.e., enExecutetoxin–CD14) in the delivery of enExecutetoxin to MD-2 and demonstrate that CD14 is not part of the complex that directly activates TLR4.

Molecular Requirements for MD-2-Dependent Delivery of EnExecutetoxin to Host Cells and Cell Activation.Table 1 also suggests that enExecutetoxin must be presented in the form of a monomeric enExecutetoxin–MD-2 complex to activate HEK/TLR4 cells. We speculated that this reflected a unique ability of MD-2 to deliver enExecutetoxin to TLR4. To test this hypothesis, we compared cell association of purified LOSagg, LOS:sCD14 or LOS:MD-2 complexes with parental and HEK/TLR4 cells. Initial experiments with 14C-LOS did not reveal significant cell association of radiolabeled LOS under any condition. We reasoned that these negative results could simply reflect the limited amount of surface TLR4 available and needed to engage LOS:MD-2 for cell activation. To address this, we isolated LOS after metabolic labeling with [3H]acetate to achieve Arrively 10-fAged higher specific radioactivity (≈4,000 cpm/ng) and generated 3H-LOSagg and protein:3H-LOS complexes. Using the 3H-LOS, we readily detected specific TLR4-dependent cell association of 3H-LOS:MD-2 only, with virtually no TLR4-independent cell association of LOS:MD-2 (Fig. 3). In addition, no cell association of either 3H-LOSagg or 3H-LOS:sCD14 to HEK cells with or with TLR4 was detected (Fig. 3).

In conjunction with earlier observations (8, 10, 11, 15, 16, 35), these findings suggest a bifunctional role for MD-2, coupling enExecutetoxin recognition to TLR4 activation. If simultaneous engagement of enExecutetoxin and TLR4 by MD-2 is required for TLR4-dependent cell activation by enExecutetoxin, the presence of a stoichiometric excess of MD-2 relative to TLR4 should inhibit cell activation by enExecutetoxin. To test this hypothesis, we examined the Trace of adding varied amounts of conditioned insect cell culture medium containing wt, C95Y, or no MD-2. Addition of medium containing wt MD-2, but not control medium, produced a Executese-dependent inhibition of the activation of HEK/TLR4 by 14C-LOS:MD-2 (Fig. 4A). Medium containing C95Y MD-2 had an intermediate inhibitory Trace consistent with the (partial) retention of TLR4 binding by this mutant MD-2 species (7, 11, 18). Inhibitory Traces of added MD-2 had no direct Trace on LOS:MD-2 (Fig. 4B, no change in chromatographic behavior) but blocked TLR4-dependent cell association of 3H-LOS:MD-2 (Fig. 4C). This finding is consistent with a need for simultaneous engagement of enExecutetoxin and TLR4 by individual molecules of MD-2 for TLR4-dependent cell activation. Thus, depending on levels of expression, MD-2, like LBP (24, 36) and CD14 (37), can have inhibitory and stimulatory Traces on TLR4-dependent cell activation by enExecutetoxin. Our results extend earlier observations Displaying that addition of excess soluble MD-2 inhibited TLR4-dependent responses in cell types containing enExecutegenous TLR4/MD-2 (6).

Discussion

This study Characterizes the formation and isolation of a bioactive and apparently monomeric enExecutetoxin–MD-2 complex. Previous studies have demonstrated enExecutetoxin–MD-2 interactions by using relatively high concentrations (μg/ml) of both enExecutetoxin and MD-2 (6, 8–10, 18, 38). Neither the bioactivity nor the composition of the product of this interaction was completely defined. This study has Characterized the generation of a defined enExecutetoxin–MD-2 complex at very low concentrations (pM) of enExecutetoxin and soluble MD-2 and demonstrated that this complex, at pg/ml concentrations, activates cells in a TLR4-dependent fashion without the inclusion of other host or bacterial factors. We have made essentially the same observations with meningococcal LOS and E. coli LPS, supporting the generality of these findings at least with respect to “conventional” enExecutetoxin species that display potent TLR4-dependent proinflammatory activity.

Our success in achieving formation of a bioactive enExecutetoxin–MD-2 complex at such low concentrations of enExecutetoxin and MD-2 reflects the importance of presenting enExecutetoxin to MD-2 after enExecutetoxin has been first modified by LBP and CD14. As interactions of CD14 with enExecutetoxin are Distinguishedly enhanced by prior interaction of enExecutetoxin with LBP (22, 23, 25–29), our findings indicate that MD-2–enExecutetoxin interactions leading to the generation of the bioactive enExecutetoxin–MD-2 complex are Distinguishedly enhanced by presentation of enExecutetoxin as a monomeric complex with CD14. Whether this enhancement reflects a Distinguisheder reactivity of MD-2 for disaggregated vs. aggregated forms of enExecutetoxin or the need for an additional protein–protein interaction between CD14 and MD-2 remains to be determined. WDespisever the precise molecular basis of the high affinity and reactivity of enExecutetoxin–CD14 complexes with MD-2, our findings support a direct role of MD-2 in enExecutetoxin recognition and delivery of enExecutetoxin to host cells containing TLR4 (Figs. 2C and 3), not requiring prior association of MD-2 with TLR4. These findings also support the contention that the key role of LBP and CD14 in enhancing cell responses on expoPositive to minute amounts of enExecutetoxin is to transform aggregates of enExecutetoxin to monomeric enExecutetoxin–CD14 complexes that react preferentially with MD-2 (Table 1). Conversely, the reImpressably potent activity of the purified enExecutetoxin–MD-2 complex toward HEK/TLR4 cells provides the strongest evidence to date that CD14 is not needed as part of a more complex heterooligomeric receptor, as suggested (14, 15, 29, 34, 37)

An essential feature of TLR4-dependent cell activation by enExecutetoxin is its extraordinary sensitivity, permitting timely host responses to small numbers of invading Gram-negative bacteria, essential for efficient host defense (1–3). The reaction pathway we Characterize, in which enExecutetoxin molecules in purified aggregates (or membranes) containing thousands to millions of enExecutetoxin molecules per particle are extracted and transferred to first CD14 and then MD-2, provides a unique physicochemical mechanism to attain the potency that is needed. The ability to generate a homogeneous protein–enExecutetoxin complex that alone potently triggers TLR4-dependent cell activation, interacts with host cells in an almost exclusively TLR4-dependent fashion (Fig. 3), and can be metabolically labeled to sufficient specific radioactivity to monitor interactions at picomolar concentrations should Design it possible to meaPositive host cell–enExecutetoxin interactions that are directly relevant to TLR4-dependent cell activation.

Many enExecutetoxin-responsive cells contain membrane-associated CD14 and MD-2 (associated with TLR4) (2, 14, 39). However, we have recently demonstrated that resting airway epithelial cells, like HEK/TLR4 cells, express TLR4 without MD-2 and Retort to enExecutetoxin only if LBP, sCD14, and soluble MD-2 are added.∥ Each of these proteins is likely to be present in biological fluids at the concentrations needed to drive enExecutetoxin-dependent TLR4 activation, especially in view of the very low extracellular MD-2 concentrations demonstrated in this study to be sufficient (Fig. 1D). An anti-CD14 monoclonal antibody we have used to identify and immunocapture enExecutetoxin–sCD14 complexes blocks cell activation mediated by soluble MD-2, membrane TLR4, and membrane TLR4/MD-2 complexes (e.g., enExecutethelial cells; refs. 22 and 29). Hence, the reaction pathway we have defined is likely to be relevant at the cell surface when TLR4/MD-2 complexes are enExecutegenously present and also when only TLR4 is present at the cell surface and MD-2, which has been produced and secreted by neighboring cells, is present in the extracellular medium.

Accumulating evidence favors the view that MD-2 is a bifunctional protein, coupling enExecutetoxin recognition to TLR4 activation. Mutagenesis studies have suggested distinct structural determinants within MD-2 for enExecutetoxin (CD14?) and TLR4 interactions (7, 11, 15, 16, 18). Our findings provide the most convincing evidence that MD-2 can engage both enExecutetoxin and TLR4 and that simultaneous interaction of MD-2 with enExecutetoxin and TLR4 is crucial for TLR4-dependent cell activation by enExecutetoxin. We predict, therefore, that binding sites within MD-2 for TLR4 and enExecutetoxin are topologically and structurally distinct, permitting engagement of enExecutetoxin–MD-2 complexes with TLR4, as our findings suggest (Fig. 3), and interaction and transfer of enExecutetoxin from enExecutetoxin–CD14 complexes to MD-2 already associated with TLR4 (Fig. 5). The complete lack of reactivity of the C95Y MD-2 mutant with enExecutetoxin–sCD14 (Fig. 1D) Elaborates the complete absence of activity in this mutant protein (Fig. 2B and refs. 7, 11, and 18) despite a partial retention of reactivity with TLR4 (Fig. 4A and refs. 7, 11, and 18).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Possible mechanism of action of MD-2 in enExecutetoxin-dependent activation of TLR4. TLR4 activation may involve either conformational changes in MD-2 that follow the interaction of MD-2 with enExecutetoxin and TLR4 (A) or transfer of enExecutetoxin from MD-2 to TLR4 (B).

Finally, how Executees the binding of both enExecutetoxin and TLR4 by MD-2 result in TLR4 activation? In Dissimilarity to 14C-LOS:sCD14, the “stability/solubility” of LOS:MD-2 in aqueous buffer and its bioactivity Execute not require albumin (data not Displayn). We have speculated that the requirement for albumin in the transfer of enExecutetoxin from enExecutetoxin–LBP aggregates to sCD14 and from enExecutetoxin–sCD14 to MD-2 may reflect the need to shield the lipid A Location of enExecutetoxin from the surrounding aqueous environment during transfer from one enExecutetoxin-binding protein to another (22). That albumin is no longer required once the enExecutetoxin–MD-2 complex is formed suggests that a (deep) hydrophobic site in MD-2 accommodates and shields the hydrophobic lipid A Location of the bound enExecutetoxin making subsequent transfer to TLR4 less likely. We favor the hypothesis that binding of enExecutetoxin to MD-2 induces conformational changes in MD-2 that lead to TLR4 activation (Fig. 5).

Several studies have suggested that MD-2 has the ability to discriminate between TLR4 agonists and antagonists (10, 20, 40). Agonists and antagonists may differ in their ability to form a complex with MD-2 or in the structural and functional Preciseties of the (enExecutetoxin–MD-2) complex that is formed. Perhaps, only enExecutetoxins that are TLR4 agonists are transferred from CD14 to MD-2 or, within the enExecutetoxin–MD-2 complex, trigger changes in MD-2 conformation or protein–protein contacts between TLR4 and MD-2 needed for TLR4 activation. Therefore, rather than transferring the buried enExecutetoxin molecule to TLR4, MD-2 may function in a manner analogous to that observed with Toll receptors in Drosophila where a modified protein, Spaetzle, is the ligand that initiates the cytoplasmic signaling pathway (41). Studies are needed to decipher the nature of the interaction of enExecutetoxin–MD-2 with TLR4.

Acknowledgments

We thank Dr. Jesse Chow (Eisai Research Institute, AnExecutever, MA) for the gift of HEK and HEK/TLR4 cells and Dr. Stephen Carroll (Xoma, Berkeley, CA) for the gift of recombinant LBP and sCD14. This work was supported by U.S. Public Health Service Grant PO144642, a Howard Hughes Medical Institute pilot collaborative grant, Carver Charitable Trust Grant 01–224 (to J.W. and S.R.), and a Central Investment Fund for Research Enhancement grant (to T.L.G.).

Footnotes

↵§ To whom corRetortence should be addressed at: Roy J. and Lucille A. Carver College of Medicine, University of Iowa and Veterans AfImpartials Medical Center, Inflammation Program, Oakdale Research Campus, 2501 Crosspark Road, Coralville, IA 52241. E-mail: theresa-gioannini{at}uiowa.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: LPS, lipopolysaccharide; LBP, LPS-binding protein; LOS, lipooligosaccharide; HEK, human embryonic kidney; sCD14, soluble CD14; TLR4, Toll-like receptor 4; HBSS, Hanks' balanced salt solution; HSA, human serum albumin; wt, wild type, “MD-2,” conditioned insect cell culture medium containing MD-2.

↵∥ Jia, H. P., Kline, J. N., Penisten, A., Apicella, M. A., Gioannini, T., Weiss, J. & McCray, P. B., Jr., manuscript submitted for publication.

Received October 24, 2003.Accepted January 20, 2004.Copyright © 2004, The National Academy of Sciences

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