©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Soluble CD14 Truncated at Amino Acid 152 Binds Lipopolysaccharide (LPS) and Enables Cellular Response to LPS (*)

(Received for publication, September 20, 1994 )

Todd S.-C. Juan Michael J. Kelley David A. Johnson Leigh A. Busse Eric Hailman (1) Samuel D. Wright (1) Henri S. Lichenstein (§)

From the From Amgen, Inc., Thousand Oaks, California 91320 and the Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14 and purified the protein to homogeneity. sCD14 bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14 was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14. We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild-type bioactivity in vitro.


INTRODUCTION

CD14 has been identified as the principal receptor that enables leukocytes to produce inflammatory cytokines and up-regulate integrin function in response to picomolar levels of lipopolysaccharide (LPS) (1, 2) . On leukocytes, CD14 exists as a glycosylphosphatidylinositol-anchored protein having a molecular weight of approximately 55,000(3, 4, 5) . CD14 also exists as a soluble form found in serum at a concentration of 2-6 µg/ml(6, 7) . Soluble CD14 (sCD14) (^1)has been shown to enable responses of cell types that do not express membrane-bound CD14, such as endothelial cells, astrocytes, and epithelial cells(8, 9, 10, 11) .

We have recently shown that sCD14 forms stoichiometric complexes with LPS and that LPSbulletsCD14 complexes stimulate human neutrophils and endothelial cells(12) . An acute phase plasma protein known as LPS-binding protein (LBP; 13) acts catalytically to facilitate the binding of LPS to sCD14, but is not a part of LPSbulletsCD14 complex. These studies have focused attention on the LPS-binding properties of sCD14.

Characterization of the CD14 cDNA (14, 15) has revealed that CD14 is a member of a family of proteins containing leucine-rich repeats(16) . In other proteins, leucine-rich repeats have been proposed to mediate protein-lipid or protein-protein interactions(17, 18, 19, 20) . Here, we report on the isolation of a sCD14 truncation mutant that lacks the seven C-terminal leucine-rich repeats. The mutant sCD14 bound LPS and this binding was facilitated by rLBP. In addition, the mutant sCD14 enabled LPS-dependent biological responses in both CD14-expressing and CD14-negative cells.


MATERIALS AND METHODS

Reagents

Recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP) were constructed and purified as described(12) . Concentrations of all recombinant proteins were determined with a Micro BCA protein kit (Pierce) according to manufacturer's specification. Since full-length rsCD14 terminates at position 348 of the mature protein(12) , we herein refer it as sCD14. The anti-CD14 mAbs used were 3C10 (purified by chromatography on Protein G from the conditioned medium (CM) of ATCC TIB 228) and MEM-18 (purchased from SANBIO, The Netherlands). Rabbit polyclonal antiserum was raised against human rsCD14 and prepared by Antibodies, Inc. (Davis, CA). Horseradish peroxidase-conjugated donkey anti-rabbit IgG antisera was purchased from Amersham. Enzymes for DNA manipulation and polymerase chain reaction (PCR) were purchased from Boehringer Mannheim.

Generation of Truncation Mutants

A modified version of a mammalian expression vector (pDSRalpha2, (12) ) containing the cDNA for sCD14 was used as template for PCRs that generated 10 different CD14 cDNAs encoding CD14 truncated at amino acids 98, 124, 152, 176, 204, 231, 258, 279, 301, and 312. Each PCR used a common oligonucleotide (5`- GTCCCTCTAGACCACCATGGAGCGCGCGTCCTGC-3`) generated from the 5` end of CD14 which was paired with 10 different oligonucleotides (5`-AACTTCCAGTCGACTTAGCGGGAGTACGCTAGCACACGC-3`, 5`-AACTTCCAGTCGACTTATGCAAGTCCTGTGGCTTCCAGAG-3`, 5`-AACTTCCAGTCGACTTAGCCTGGCTTGAGCCACTGCTGC-3`, 5`-AACTTCCAGTCGACTTAGGCCGGGAAGGCGCGAACCTG-3`, 5`-AACTTCCAGTCGACTTAGGCCGGGAACTTGTGGGGACAG-3`, 5`-AACTTCCAGTCGACTTACTGCACACCTGCCGCCGCCAG-3`, 5`-AACTTCCAGTCGACTTAGGCGCTGGACCACATGCATCTCG-3`, 5`-AACTTCCAGTCGACTTACTTGGCTGGCAGTCCTTTAGGCACC-3`, 5`-AACTTCCAGTCGACTTACTCGGGCAGCTCGTCAGGCTGC-3`, and 5`-AACTTCCAGTCGACTTACAGGAAGGGATTCCCGTCCAGTG-3`) specifying the 3` ends of the truncated CD14 cDNAs indicated above. The PCR products were then digested with XbaI and SalI and ligated to pDSRalpha2 linearized with XbaI and SalI. All mutant constructs were sequenced to confirm the mutation.

Transient Expression of Mutant CD14 cDNAs in COS-7 Cells

To express mutant sCD14 proteins, mammalian expression vectors containing sCD14 cDNA were introduced into COS-7 (ATCC CRL 1651) cells by electroporation. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT). For each transfection, five million cells were electroporated with 20 µg of plasmid DNA using a Gene pulser electroporator (Bio-Rad). After electroporation, cells were maintained in Dulbecco's modified Eagle's medium + 5% fetal bovine serum for 24 h. Cells were then washed three times with 1 times Hank's balanced salt solution (Life Technologies, Inc.) and incubated 4 days with serum-free AIM-V (Life Technologies, Inc.) medium. To test for sCD14 expression, 20 µl of CM were electrophoresed on 4-20% SDS-polyacrylamide gradient gels (Noval Experimental Technologies, San Diego, CA), and proteins were electrophoretically transferred to nitrocellulose membranes. The membranes were incubated with anti-CD14 polyclonal antisera in TBS buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG antisera in TBS buffer for 1 h. For detection of immune complexes, enhanced chemiluminesence (ECL kit, Amersham, Arlington Heights, IL) was performed as described by the manufacturer.

Quantitation of sCD14 in COS-7 Conditioned Medium

Concentrations of sCD14 were determined with the aid of a BIAcore biosensor instrument (Pharmacia Biotech Inc., Piscataway, NJ). Briefly, mAb 3C10 (200 µg/ml) was immobilized on a sensor chip at an injection rate of 5 µl/min for 10 min. A standard curve was then generated relating the change in response unit (RU) to varying dilutions of purified sCD14 of known concentration. Conditioned medium from transfected COS-7 cells was injected onto the sensor chip containing immobilized 3C10 and sCD14 concentrations were calculated by measuring the change in RU and comparing it to the standard curve.

Purification of sCD14

The expression vector containing the cDNA encoding sCD14 was stably transfected into Chinese hamster ovary cells deficient in dihydrofolate reductase as described(12) . A single clone was grown without serum to generate CM containing sCD14. A 2.5 times 9-cm Q-Sepharose column (Pharmacia) was equilibrated with 50 mM Tris-HCl, pH 8.0, 10% glycerol. Four liters of CM, to which glycerol had been added to a final concentration of 10%, was then passed over the column. After loading, the column was then washed with the above buffer, and protein was eluted with a 280-ml gradient of 0-1 M NaCl in 50 mM Tris-HCl, pH 8.0, 10% glycerol. Western blot analysis using rabbit anti-CD14 polyclonal antiserum was used to identify fractions containing sCD14. The fractions were then pooled and diluted 4-fold with phosphate-buffered saline (PBS), 10% glycerol and loaded onto an affinity column of mAb 3C10 which had been equilibrated with PBS, 10% glycerol. After washing with the same buffer, the bound protein was eluted with 100 mM glycine-HCl, pH 2.7, 10% glycerol. Fractions were neutralized with 0.5 M sodium phosphate, pH 8.0. The buffer of the eluted protein was then exchanged to PBS by passing through a Sephadex G-25 (Pharmacia) gel filtration column. For deglycosylation, 0.25 µg of sCD14 was treated with 0.25 units of N-glycanase (Genzyme, Cambridge, MA), 0.01 units of neuraminidase (Calbiochem, La Jolla, CA) in 0.02% sodium azide, 0.1% SDS, 10 mM Tris-HCl, pH 7.5, for 16 h at 37 °C. Purity of the sample was checked by SDS-PAGE followed by silver staining or Coomassie Blue staining.

U373 Bioassays

Human astrocytoma U373 cells were obtained from the American Type Culture Collection (ATCC HTB 17, Rockville, MD) and maintained in minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 1 times non-essential amino acids (Life Technologies, Inc.), and 1 times sodium pyruvate (Life Technologies, Inc.). U373 cells were plated in 24-well plates at a density of 100,000 cells/well 24 h before stimulation. The cells were washed three times with 1 times Hank's balanced salt solution and then incubated with 0.5 ml of COS-7 CM or varying concentrations of purified sCD14. After 24 h, the CM was harvested and IL-6 levels were quantitated by ELISA (R& Systems, Minneapolis, MN) according to the manufacturer's specification. LPS prepared from Salmonella minnesota strain Re595 (List Biological Laboratories, Campbell, CA) was used at a concentration of 10 ng/ml in those assays performed in the presence of LPS.

Inhibition of LPS-mediated Limulus Amebocyte Lysate (LAL) Reaction

Both sCD14 and sCD14 were tested for their ability to inhibit an LAL reaction. Purified sCD14 or sCD14 were added at various concentrations directly to Costar (no. 3596, Cambridge, MA) 96-well plates. rLBP (0.17 nM) and LPS (2 ng/ml in PBS) from Escherichia coli strain 055:B5 (Endosafe, Charleston, SC) were then added to yield a final volume of 50 µl/well. After incubation at 37 °C for 1 h, 50 µl of LAL reagent (Biowhittaker QCL-1000 kit, Walkersville, MD) were added and the mixture was allowed to incubate at room temperature for 25 min. One hundred µl of chromogenic substrate from the kit were then added and the reaction was stopped 20 min later with 100 µl of 25% acetic acid. Optical density at 405 nm was measured with a V(max) microplate reader (Molecular Devices, Menlo Park, CA). In our laboratory, this assay yields a maximal response with 2 ng/ml LPS and a half-maximal response with 1 ng/ml LPS. Thus, a 50% reduction in A signifies neutralization of 1 ng/ml LPS.

Native PAGE Gel Shift Assay

To analyze binding of sCD14 or sCD14 to LPS, a gel shift assay was performed as described(12) . Briefly, sCD14 or sCD14 were incubated at various concentrations (0, 101, 303, and 909 nM) with 3 µg/ml ^3H-LPS prepared from E. coli K12 strain LCD25 (provided by Dr. Robert Munford, University of Texas, Southwestern Medical Center, Dallas, TX) in the presence or absence of 16.7 nM rLBP. The reaction was incubated at 37 °C for 30 min and then electrophoresed on native 4-20% polyacrylamide gels. Gels were prepared for fluorography by fixing for 45 min in 40% methanol, 10% acetic acid. Enlightening solution (DuPont NEN, Boston, MA) was then added for 45 min and the gel was dried and exposed to X-Omat (Kodak, Rochester, NY) film for 48 h.

Activation of Polymorphonuclear Leukocytes (PMN) by LPS and sCD14

The adhesive capacity of leukocyte integrins can be induced by LPS with LBP (2) or by complexes of LPS and sCD14(12, 21) . Here we used conditions under which both LBP and sCD14 are required to induce adhesion. Freshly isolated PMN were fluorescently labeled with 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester as described(22) . Mixtures containing smooth LPS (from E. coli O111:B4, List Biological Laboratories), rLBP (1 µg/ml), and sCD14 or sCD14 at various concentrations were diluted in 40 µl of Dulbecco's PBS with Ca and Mg and 0.5% human serum albumin. Ten µl of PMN (2 times 10^7 cells/ml in HAP buffer (Dulbecco's PBS with 0.5 unit/ml aprotinin, 0.05% human serum albumin, and 3 mMD-glucose)) were then added, and the mixtures were allowed to incubate for 10 min at 37 °C. PMN were then washed with HAP buffer and added to 72-well Terasaki plates pre-coated with fibrinogen. After 15 min incubation at 37 °C, adhesion of PMN to the plate was quantitated. The fluorescence in each well was measured using a Cytofluor 2300 microplate reader (Millipore, Bedford, MA) to estimate the total number of cells per well. The plate was then washed with PBS and fluorescence was measured again. Binding is expressed as the percentage of cells remaining in the well after the washing step.

BIAcore Analyses of Interactions between sCD14 and Anti-CD14 mAbs

Recognition of purified sCD14 preparations by anti-CD14 mAbs was measured with a BIAcore biosensor instrument. The instrument, CM5 sensor chips, and amine coupling kit were purchased from Pharmacia Biosensor (Piscataway, NJ). mAb 3C10 (200 µg/ml in 20 mM sodium acetate, pH 3.4) was immobilized on a CM5 sensor chip by amine coupling according to the manufacturer's specifications. To assess sCD14 recognition by mAbs 3C10 and MEM-18, the flow cell immobilized with 3C10 was incubated in succession with 5 solutions as detailed in the following steps: step 1, 10 µg/ml sCD14 for 2 min; step 2, HBS wash buffer (10 mM Hepes, pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0.005% (v/v) surfactant P20 (Pharmacia Biosensor)) for 2 min; step 3, 50 µg/ml MEM-18 (in HBS buffer) for 2-3 min; step 4, HBS wash buffer for 2 min; step 5, 10 mM HCl for 2 min. All solutions were injected at a flow rate of 5 µl/min. To assess recognition of sCD14 by mAb 3C10 and MEM-18, after step 5, the chip was incubated with 5 µg/ml sCD14 at 5 ml/min for 2 min and steps 2-5 were repeated.


RESULTS

Truncated sCD14 Expressed in COS-7 Is Biologically Active

The CD14 protein possesses 10 leucine-rich repeats spanning amino acids 67-312(14) . In order to assess the significance of these repeats relative to the biological activity of CD14, we systematically deleted increasing numbers of repeats and attempted to express the mutant CD14 molecules in COS-7 cells. From the 10 mutants generated, only four (sCD14, sCD14, sCD14, and sCD14) were found to be expressed by COS-7 cells at detectable levels (Fig. 1). To test whether the truncated sCD14s were biologically active, we utilized a U373 bioassay in which production of IL-6 by U373 cells is induced by the presence of LPS and sCD14. A representative experiment using CM containing sCD14 is shown in Fig. 2. The data illustrate that CM containing sCD14 is active in stimulating IL-6 production from U373 cells in the presence of LPS. In this experiment, the concentrations of sCD14 and sCD14 in COS-7 CM were 0.37 and 0.59 nM, respectively. We also observed that CM from the other three mutants was able to induce normal levels of IL-6 in the presence of LPS (data not shown). Since sCD14 was the most severely truncated mutant that retained biological activity, we chose to characterize this truncation mutant in greater detail.


Figure 1: Transient expression of sCD14 truncation mutants in COS-7 cells. Individual sCD14 mutants are designated as sCD14 where ``A'' refers to the first amino acid of mature sCD14 and ``B'' refers to the last amino acid encoded in the mutant. The first line shows full-length sCD14 with 10 leucine-rich repeats (boxes with numbers) organized as described(14) . Expression of sCD14 mutants in COS-7 cells was determined by Western blot (see ``Materials and Methods''), and ``+'' indicates detectable sCD14 expression.




Figure 2: COS-7 CM containing sCD14 or sCD14 induces IL-6 secretion from U373 cells. Transfection, collection of CM, and treatment of U373 cells are described under ``Materials and Methods.'' MOCK refers to CM from COS-7 cells electroporated in the absence of DNA. Levels of IL-6 are determined by IL-6 ELISA as described under ``Materials and Methods'' and are presented as picograms/ml of CM.



Characterization of Purified sCD14

To produce large quantities of purified protein, a stable Chinese hamster ovary clone expressing sCD14 was generated and the mutant protein was purified from the serum-free CM of this cell line. Fig. 3(lane 2) shows that purified sCD14 ran with an apparent molecular weight ranging from 26,000 to 31,000 when analyzed by reducing SDS-PAGE. All bands of purified sCD14 reacted in a Western blot with the rabbit anti-CD14 polyclonal serum (data not shown). After deglycosylation and desialylation (Fig. 3, lane 3), purified sCD14 ran as a single band with an apparent molecular weight of 22,000. Thus, multiple bands in our sCD14 preparation (lane 2) could represent alternative glycosylation forms of sCD14.


Figure 3: Analysis of purified sCD14. Purified sCD14 was deglycosylated with N-glycanase and neuraminidase as described under ``Materials and Methods.'' The samples were analyzed by SDS-PAGE on a 4-20% gel followed by silver staining. All samples were heated at 90 °C for 3 min in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 0.005% bromphenol blue, 10% glycerol, 10% (v/v) beta-mercaptoethanol) prior to loading. Lane 1, 10 µg of Mark-12 molecular weight markers (Noval Experimental Technologies, San Diego, CA); lane 2, 0.25 µg of purified sCD14; lane 3, 0.25 µg of deglycosylated sCD14; lane 4, negative control with 0.25 units of N-glycanase and 0.01 units of neuraminidase.



sCD14 Inhibits an LPS-Induced LAL Response

An LAL assay was used to determine whether sCD14 or sCD14 interacts with LPS. In an LAL reaction, LPS binds to the proenzyme Factor C (23) and initiates a protease cascade whose activity can be quantitated upon addition of chromogenic substrate. Inhibition of the LAL response by a protein may thus signify that the protein binds LPS and prevents activation of the proenzyme. In pilot experiments, we determined that at concentrations less than 100 nM, sCD14 did not inhibit the LAL reaction. However, addition of 0.17 nM rLBP (a concentration previously determined not to inhibit in the LAL reaction) enabled strong CD14-dependent inhibition of the LAL reaction. Further studies showed that in the presence of rLBP, but not in the absence of rLBP, both sCD14 and sCD14 completely inhibited the LAL response at a concentration of 100 nM (Fig. 4). The IC for CD14 was 30 nM while sCD14 had an IC of 40 nM.


Figure 4: Inhibition of LAL reaction by sCD14 or sCD14. Various concentrations of sCD14 or sCD14 were added to the LAL reaction as described under ``Materials and Methods.'' The reactions were performed in the presence of rLBP at a concentration of 0.17 nM. After incubation, the reaction was stopped with 25% acetic acid and optical density at 405 nm was measured with a V(max) microplate reader. Experiments were performed three times, and values expressed are the means ± S.D. from one representative experiment.



rLBP Facilitates the Formation of Stable Complexes between LPS and sCD14

The previous experiment suggested that sCD14 interacts with LPS. To directly address whether sCD14 is capable of binding LPS, we utilized a native PAGE assay (12) that detects complexes of sCD14 with ^3H-LPS. In the absence of rLBP, both 909 nM sCD14 or sCD14 formed complexes with LPS after 30 min incubation (Fig. 5A). The complex of sCD14 migrated faster than the sCD14 complex due to the smaller size of the sCD14 protein. Addition of rLBP during the incubation of ^3H-LPS with sCD14 dramatically increased the amount of ^3H-LPSbulletsCD14 and ^3H-LPSbulletsCD14 formed. Under these conditions, complexes with LPS were readily seen with 101 nM and 303 nM sCD14 (Fig. 5B). Thus, rLBP facilitates the binding of LPS to both sCD14 and sCD14.


Figure 5: rLBP facilitates the formation of stable complexes between sCD14 and LPS. ^3H-LPS (3 µg/ml) was incubated with increasing amounts of sCD14 or sCD14 without rLBP (A) or with 16.7 nM rLBP (B) for 30 min at 37 °C, then run on a native 4-20% PAGE gel. Fluorography was performed as described under ``Materials and Methods.'' Lane 1 contains LPS in the absence of additional protein. Positions of uncomplexed LPS and complexes between LPS and sCD14 or sCD14 are indicated.



sCD14 Increases PMN Adhesion in Response to LPS

To confirm that sCD14 can interact with LBP and enable cellular responses to LPS, LPS-induced adhesion of PMN to fibrinogen was measured. We have previously shown that PMN express a low amount of cell surface CD14 (2) and that rough LPS (Re 595) with LBP stimulates the adhesivity of these cells in a CD14-dependent fashion(2, 12) . In contrast, when smooth LPS (such as 0111:B4) is used, the cell surface CD14 of PMN appears insufficient, and a sensitive adhesive response is only observed upon addition of sCD14. (^2)We asked whether mutant sCD14 could enable LBP-dependent responses of PMN to smooth LPS. Addition of either sCD14 or sCD14 enabled a strong adhesive response of PMN to smooth LPS and LBP (Fig. 6). The dose-response relationships for the two proteins were similar, suggesting that sCD14 retains full activity in this assay. No response was seen to LPS and sCD14 or sCD14 in the absence of rLBP, suggesting that both sCD14 and sCD14 are able to directly interact with rLBP. These data confirm that sCD14 is biologically active in vitro and can interact with both LPS and rLBP.


Figure 6: sCD14 and sCD14 mediate responses of PMN to LPS and LBP. Freshly isolated PMN were incubated with ``smooth'' LPS (30 ng/ml), LBP (1 µg/ml), and the indicated concentrations of sCD14 or sCD14. Adhesion of PMN to fibrinogen-coated wells was measured as described under ``Materials and Methods.'' Error bars indicate standard deviations of triplicate determinations.



Induction of LPS-dependent IL-6 Secretion in U373 Cells by sCD14

Our initial experiments performed with COS-7 CM containing sCD14 (Fig. 2) suggested that the mutant protein could activate U373 cells. To rule out the possibility that this activation was caused by irrelevant proteins in COS-7 CM, we performed a dose-response experiment comparing the ability of purified sCD14 or sCD14 to induce IL-6 production from U373 cells. Both sCD14 and sCD14 induced similar levels of IL-6 from U373 cells at equivalent concentration (Fig. 7). Neither protein activated U373 cells in the absence of LPS. These results confirm the ability of sCD14 to interact with LPS and cells in a fashion identical with sCD14.


Figure 7: sCD14 induces a dose-dependent production of IL-6 in U373 cells. U373 cells were treated with various concentrations of sCD14 or sCD14 with or without LPS. IL-6 levels were determined by ELISA as described under ``Materials and Methods.'' Values presented are amounts of IL-6 expressed in serum-free CM from U373 cells. Data presented are means ± S.D. from four readings.



sCD14 Is Recognized by Blocking Anti-CD14 mAbs

Anti-CD14 mAbs 3C10 and MEM-18 have been previously characterized as specific blockers of CD14 function(1, 2, 8, 11, 12, 24, 25) . A BIAcore biosensor instrument was used to assess whether these mAbs recognize epitopes within sCD14. Fig. 8shows that sCD14 recognized 3C10 (DeltaRU of 400 between steps 6 and 8) and MEM-18 (DeltaRU of 1000 between steps 7 and 9). This result was confirmed in the U373 bioassay in which both 3C10 and MEM-18 completely inhibited IL-6 production induced by sCD14 and LPS (data not shown).


Figure 8: mAbs 3C10 and MEM-18 recognize sCD14. Immobilization of mAb 3C10 to a sensor chip is described under ``Materials and Methods.'' Injection of solutions at various ``steps'' are marked on the sensorgram. ``Wash'' indicates a washing step using HBS buffer as described under ``Materials and Methods.'' Binding of purified proteins to mAbs was assessed by measuring change in RU. The experiments were performed three times and the results of one experiment are shown.




DISCUSSION

Here we provide evidence that the N-terminal 152 amino acids of CD14 is sufficient to mediate all of its known biological properties. sCD14 binds LPS, and studies measuring both inhibition of LAL (Fig. 4) and interaction with radiolabeled LPS (Fig. 5) indicate that this binding is quantitatively similar to that exhibited by sCD14. Furthermore, in both assays, sCD14 or sCD14 complexes with LPS were formed more efficiently in the presence of rLBP. This suggests that not only is the LPS-binding domain contained within sCD14, but sites for interaction with LBP must also be present. Presumably, CD14 interaction with LBP leads to an accelerated transfer of LPS into CD14.

Another important biological property attributed to sCD14 is its ability to enable LPS-dependent signaling in a variety of cells. Here, we show that sCD14 is just as effective as sCD14 in initiating LPS-dependent responses in PMN (up-regulation of adhesion) and epithelial cells (up-regulation of IL-6 production). It has been hypothesized that CD14 possesses domains for binding LPS and for cell signaling through an interaction with an unidentified transmembrane signaling protein(8, 26) . Our results suggest that both of these putative domains must be contained within the N-terminal 152 amino acids of CD14, since this protein can bind LPS and can mediate signaling by cells.

CD14 contains 10 leucine-rich repeats spanning amino acids 67 to 312. In other proteins, it has been proposed that leucine-rich repeats play a role in protein-protein or protein-membrane interactions. The fact that we produced a CD14 mutant lacking repeats 4 through 10 which still had wild-type function suggests that these repeats are not critical for LPS binding or CD14 bioactivity. The biological functions of these repeats remain to be determined.

In this study, sCD14 truncation mutants lacking leucine-rich repeats 1-3 could not be expressed in COS-7 cells, thus it was not possible to distinguish whether LPS-binding domains or cell-signaling domains are localized to leucine-rich repeats 1-3 or to amino acids preceding this region. A recent hypothesis suggests that other LPS-binding proteins such as Limulus anti-LPS factor(27) , bactericidal/permeability-increasing protein(28) , and LBP (6) interact with LPS through a specific amphipathic domain proposed to exist in each of these proteins(29) . Since amphipathic regions do exist in CD14 before leucine-rich repeats 1-3, it is possible that the amphipathic regions in CD14 are also involved in LPS binding. We are currently using the technique of site-directed mutagenesis to test whether the amphipathic regions in CD14 are involved in LPS recognition.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 805-447-3064; Fax: 805-499-9452.

(^1)
The abbreviations used are: sCD14, soluble CD14; CM, conditioned medium; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; LAL, Limulus amebocyte lysate; LBP, LPS-binding protein; LPS, lipopolysaccharide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMN, polymorphonuclear leukocyte; r, recombinant; RU, response unit; mAb, monoclonal antibody.

(^2)
E. Hailman and S. D. Wright, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Mark Zukowski for critical reading of the manuscript.


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