©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Region of Human CD14 Required for Lipopolysaccharide Binding (*)

(Received for publication, August 10, 1994; and in revised form, October 26, 1994)

Suganya Viriyakosol (1) Theo N. Kirkland (1) (2)(§)

From the  (1)Department of Pathology and (2)Medicine, Department of Veterans Affairs Medical Center, University of California, San Diego, San Diego, California 92161

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). CD14 binding of LPS is enhanced by serum proteins, especially lipopolysaccharide binding protein. The serum-dependent binding of LPS to CD14 stimulates macrophages to make cytokines, which can cause septic shock in humans and animals. Here, we identify a region in human CD14 which is important in serum-dependent LPS binding and LPS-induced cellular activation. Four small regions (4-5 amino acids long) within the N-terminal 65 amino acids of CD14 were deleted singly or in combination. The deletion mutants were stably expressed in Chinese hamster ovary (CHO) cells. The mutants were characterized in three assays: reactivity with anti-CD14 monoclonal antibody, serum-dependent LPS binding, and LPS-induced activation of NF-kappaB. Some of the mutants selectively lost reactivity with the anti-CD14 monoclonal antibody that inhibited serum-dependent LPS binding and cellular activation. All of the mutants bound much less LPS than wild type CD14 in the presence of serum. None of the mutants bound more LPS than control CD14-CHO cells in the absence of serum. CD14-CHO cells respond to LPS by activation of NF-kappaB. All of the deletion mutants were less active LPS receptors than wild type CD14-CHO cells. The DeltaAVEVE mutant, the DeltaDDED and DeltaPQPD double mutant, and the DeltaDDED, DeltaPQPD, DeltaAVEVE, and DeltaDPRQY quadruple deletion mutants were essentially inactive LPS receptors in CHO cells. These studies suggest that the 65 N-terminal amino acids of CD14 are critical for serum-dependent binding of LPS to CD14 and subsequent signal transduction in CHO cells.


INTRODUCTION

Bacterial lipopolysaccharide (LPS), (^1)or endotoxin, is one of the most potent natural stimulators of the inflammatory response. In patients with Gram-negative sepsis, LPS can cause a syndrome of shock and multi-organ failure. For several years, it has been thought that cytokines made by macrophages in response to LPS, such as tumor necrosis factor, interleukin-1, and a host of others, are critical in the development of the shock syndrome(1, 2, 3) . Therefore, understanding the interaction of LPS with receptor(s) such as CD14 is an important step in elucidating the molecular mechanisms of LPS activation of macrophages.

CD14 is a glycophosphatidylinositol-anchored (GPI) protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes (4) , which binds LPS(5, 6, 7) . CD14 binding of LPS is enhanced by the serum protein, LPS-binding protein (LBP)(5, 6, 8) . Experiments in a number of laboratories have shown that CD14 binding to LPS leads to activation of macrophages (5, 9, 10) and pre-B lymphocytes genetically engineered to express CD14(6) . One unanswered question is which domain or domains of the CD14 molecule are required for LPS binding. In this manuscript we begin to address this question.

CD14 was identified quite some time ago as a monocyte/macrophage differentiation antigen so a number of monoclonal antibodies (mAbs) to CD14 are available. A subset of these anti-CD14 mAbs block LPS binding; other anti-CD14 mAbs do not block LPS binding(6, 8, 9, 11, 12) . We have used the approach of mapping the epitopes of mAbs which inhibit LBP-enhanced LPS binding by making 4-5 amino acid deletions in CD14. We have identified regions within the 65 N-terminal amino acids of CD14 which are required for binding of the inhibitory mAbs and for serum-dependent binding of LPS. Some of the deletions in the N-terminal region had a profound effect on LPS-induced activation of CHO cells.


EXPERIMENTAL PROCEDURES

Anti-CD14 Antibodies

Table 1lists the mAbs used in this study. The antibodies were purified except for 23bf and 121n, which were used as ascites. The ability of mAbs to inhibit [^3H]LPS binding to CHO cells stably transfected with the hCD14 gene (hCD14-CHO) was assayed at an [^3H]LPS concentration of 150 ng/ml and an antibody concentration of 10 µg/ml, using conditions which allowed [^3H]LPS binding without internalization(8) . 10% human serum was required for significant [^3H]LPS binding to CD14. All inhibition studies have been done multiple times, and some have previously been published(8) . The antibodies which were partially inhibitory were tested at a variety of concentrations of mAb and [^3H]LPS and found to consistently inhibit 50-80%.



The ability of mAb to inhibit LPS activation of CD14-70Z/3 cells (6) was determined as follows. CD14-70Z/3 cells were incubated with or without 10 µg/ml mAb in RPMI 1640 tissue culture media with 5% fetal calf serum (FCS) for 30 min. Escherichia coli Re LPS was added (at concentrations from 0.1 to 100 ng/ml) and the cells incubated at 37 °C for 24h. At that point the cells were harvested, stained with fluorescent goat anti-mouse Ig, and analyzed by flow microfluorometry, exactly as described previously(13) . The percent inhibition is calculated by comparing the amount of LPS required to achieve half-maximal activation in the presence and absence of antibody. 90% inhibition indicates that 10-fold more LPS was required to achieve half-maximal activation in the presence of antibody, compared with the control.

The goat polyclonal anti-CD14 was prepared by repeatedly immunizing a goat with purified soluble recombinant CD14 (expressed in CHO cells) and was given to us by Peter Tobias (The Scripps Research Institute). The antisera reacted exclusively with CD14 in Western blots of hCD14-CHO cells.

Construction of hCD14 Deletion Mutants

Deletion mutants of the hCD14 gene were generated by overlap extension using the polymerase chain reaction (PCR)(14) . PCR was done over a small portion of the gene in order to minimize the possibility of inadvertent nucleotide changes introduced by the amplification reactions. hCD14 cDNA (a gift from J. D. Lee) subcloned into the XbaI/SmaI site of pSP64(poly(A)) (Promega) was used as the template DNA. To generate the DeltaDDED mutant lacking the amino acids 28-31, four oligonucleotide primers were commercially synthesized (Operon Tech. Inc.). For the first pair the forward primer A is

the sequence 5` of the multiple cloning site of pSP64. The reverse primer B is

corresponding to the complementary sequence to 20 bp immediately 3` to the region coding for the amino acids DDED followed by 10 bp 5` to the region coding for the DDED. For the second pair, the forward primer C is

corresponding to the sequence 20 bp 5` of the DDED coding region followed immediately by the sequence 10 bp after the desired deletion. The reverse primer D is

corresponding to the complementary sequence of the nucleotides 635-660 which located downstream from the DDED coding region and 3` of the unique SacII site. PCR was carried out using Taq polymerase (Perkin Elmer). Amplification was done by adding 100 ng of hCD14/pSP64, 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1 mM MgCl(2), 0.01% (w/v) gelatin, 200 mM of each dNTP, 1 mM of each primer, and 1 unit of Taq polymerase in a final volume of 50 µl. The samples were amplified for 30 cycles at 65 °C annealing temperature using a DNA Thermal Cycler (Perkin Elmer).

PCR products were analyzed on a 4% NuSieve 3:1 agarose gel (FMC) in 40 mM Tris acetate, 1 mM EDTA, pH 8.0, 0.5 µg/ml EtdBr. 10 µl of the products AB and CD were gel-purified using the Geneclean Kit (BIO 101). The equivalent of 1 µl each of PCR products AB and CD were mixed, and the second round of amplification was carried out as above using the primers A and D. The final product was restriction-digested with HindIII/SacII and gel-purified. The fragment which has deletion in DDED was subcloned to replace the wild-type HindIII/SacII fragment of hCD14 in pSP64. To confirm that the mutated genes contain the correct deletions, double-stranded plasmid DNA was sequenced by dideoxy chain-termination method using Sequenase (U. S. Biochemical Corp.) throughout the amplified regions.

Other deletion mutants were constructed in the similar manner. The A and D primers of the DeltaPQPD, DeltaDDED + DeltaPQPD, and DeltaAVEVE mutants were the same as described above for the DeltaDDED. The B and C primers for the DeltaPQPD are

and

and

and

for the DeltaAVEVE. To construct the DeltaDDED + DeltaPQPD mutant, primer B of the DeltaDDED and primer C of the DeltaPQPD were used. For the construction of the DeltaDPRQY, primer A is the same as in the DeltaDDED, primer B is

primer (C) is

and primer D is

The final PCR product of this deletion was digested with HindIII/NsiI and subcloned to replace the wild type HindIII/NsiI fragment. The quadruple mutant which contains all the four single deletions was made by using the DeltaDDED + DeltaPQPD DNA as a template in the PCR reaction instead of the wild type DNA. The DeltaAVEVE mutant was generated by PCR using the primers for DeltaAVEVE outlined above. The DNA containing the three deletions was then used as a template to generate the deletion in the DeltaDPRQY region by PCR using the DeltaDPRQY primers described above.

hCD14 gene containing the GPI anchor sequence of the decay accelerating factor (DAF) gene (CD14DAF) subcloned into the vector pRc/RSV was a gift from J. D. Lee, at The Scripps Research Institute(15) . Deletion mutants containing the GPI anchor sequence from the DAF gene were made by replacing the HindIII/NheI fragment of the hCD14DAF with the same fragment containing the deletion from the mutants.

All of the plasmid constructs containing the mutant genes were sequenced to confirm the desired mutation by double-stranded dideoxy chain-termination method using sequenase (U. S. Biochemical Corp.).

In Vitro Translation of hCD14 Gene

To screen for the expression of CD14 before transfecting cell lines, we translated the DNA in vitro and assayed expression by immunoprecipitation. The deletion mutants and wild-type CD14 gene were subcloned into the vector pSP64(poly(A)) (Promega) so that sense mRNA was transcribed from the SP6 promotor. Minipreps DNA was made using the Magic Miniprep DNA purification kit (Promega). 1-5 µg of the DNA was used in the in vitro transcription and translation reaction with rabbit reticulocyte lysate (TNT Coupled Reticulocyte Lysate System; Promega) to make [S]methionine-labeled protein. Canine pancreatic microsomal membranes were supplemented in the reaction in order to facilitate glycosylation and post-translational processing. These were used according to the manufacturer's protocol (Promega). Correct formation of some of the intrachain disulfide bonds was achieved by the addition of 2 mM oxidized glutathione (Sigma) after adding the DNA to the reaction.

Immunoprecipitation of the Translation Products

Five µl of the [S]methionine-labeled translation products were solubilized in 100 µl of immunoprecipitation buffer (50 mM Tris-HCl, pH 8, 0.4 M NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) and incubated with 0.5 µg of purified mAb (Table 1), 1 µl of ascitic fluid, or 3 µl of goat anti-CD14 at 4 °C overnight.

The immune complex was captured by the addition of 50 µl of 50% protein G-agarose (Pharmacia Biotech Inc.) with gentle mixing on a rotator at 4 °C for 1.5 h. The agarose beads were washed five times in the immunoprecipitation buffer and resuspended in 30 µl of 2 times sample buffer. The samples were analyzed by SDS-polyacrylamide gel electrophoresis and fluorograpy.

Expression of hCD14 in CHO K1 Cells

The wild type and mutant hCD14 genes were subcloned into the HindIII/NotI site of the eukaryotic vector pRc/RSV (Invitrogen) containing a Rous sarcoma virus long terminal repeat promotor of transcription initiation in front of the inserted DNA and a neomycin resistance gene for the selection of stable transformants. The transfer of DNA into CHO cells was achieved by lipofection using Lipofectamine (Life Technologies, Inc.). Plasmid DNA was prepared using Plasmid Maxi Kit (Quiagen). The ratio of DNA and lipid used was 1 µg of DNA to 10 µl of Lipofectamine reagent. The DNA-lipid complex was incubated with CHO K1 cells (grown to 50-70% confluence) for 6 h. Stable transformants were selected with 1 mg/ml G418 (Life Technologies, Inc.) 48 h after the addition of the DNA.

Analysis of hCD14 Surface Expression in CHO K1 Cells

The transfected cells were stained with mAbs (Table 1) using FITC-(Fab)`(2) goat anti-mouse IgG (Zymed) as a second antibody and analyzed in a cytofluorograph on a four decade log scale (FACStar Plus, Beckman). The data were analyzed using Multiplus software (Phoenix Flow, San Diego, CA). All deletion mutants were positively selected by FACS with mAb 63D3 two to three times before the data on mAb reactivity was obtained. The wild type hCD14-CHO cells have been described previously(8) . The polyclonal goat anti-CD14 antisera was used at a 1:50 dilution with an FITC protein G (Zymed) diluted as suggested by the manufacturer.

LPS Binding Assays

Three different types of assays were done. The first was photoaffinity labeling, using I-ASD-Re LPS, as described previously(16) . 2 times 10^6 transfected CHO cells were suspended in 0.5 ml of 0.15 M NaCl, 20 mM HEPES, 1 mM EDTA and 0.03% bovine serum albumin. In some reactions 10% human serum was included. 0.5 µg/ml I-ASD-Re595 LPS was added to all tubes, and the reactions were incubated at 10 °C for 30 min. The reactions were photolysed, washed twice, and the total cell pellet treated with SDS sample buffer and electrophoresed on a 10-20% gradient SDS-polyacrylamide gel electrophoresis gel as described previously(16) .

The second assay was binding of [^3H]LPS, using previously described methods(8) . These binding assays were done under conditions which inhibited internalization of LPS(8) , as well as at 37 °C, without metabolic inhibitors, to measure total cell-associated [^3H]LPS.

The association of fluorescein-Re595 LPS (FITC-Re595) with transfected CHO cells was determined as follows. FITC-Re595 was prepared as decribed previously(17) . 1 times 10^6 transfected CHO cells were suspended in 200 µl of RPMI 1640 containing 10 mM HEPES. 10% human serum was added to some of the tubes and the FITC-Re595 was added. The cells were incubated at 37 °C for 30 min and then put on ice until analysis. The samples were analyzed on a four decade log scale using a Beckman FACStar Plus. These data were analyzed using Multiplus software (Phoenix Flow, San Diego, CA).

LPS-induced Activation of NF-kappaB in CD14-CHO Cells

Transfected CHO cells were split into 75-cm^2 flasks 24 h before testing. The medium (Dulbecco's modified Eagle's medium/F-12 with 10% FCS) was replaced with 2.5 ml of fresh medium, containing 1% FCS and the activating LPS. In some experiments, the cells were stimulated with 20 ng/ml human recombinant tumor necrosis factor(11) . The cells were incubated with E. coli Re LPS (D31m4) complexed to bovine serum albumin (13) at 37 °C for 30 min, the media aspirated, and the cells washed twice with ice-cold phosphate-buffered saline. 2.5 ml of phosphate-buffered saline containing 1 mM EDTA was added and the cells incubated for 15 min on ice, to remove the cells from the flask. The cells were removed from the flask and pelleted.

Experiments in the absence of serum were done as follows. Transfected CHO cells were harvested, washed extensively, and plated in Opti-MEM I media (Life Technologies, Inc.). The following day the cells were harvested by scraping (they adhered poorly in this medium, but grew well) and pelleted in a 50-ml centrifuge tube. The cells were resuspended in 2.5 ml of Opti-MEM I (with or without FCS or human serum) and the LPS added. The remainder of the experiment was done as above.

The cell pellets were resuspended in 400 µl of nuclear extraction buffer A consisting of 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DDT, and 1 mM phenylmethylsulfonyl fluoride and incubated on ice for 15 min. Then 23 µl of 10% Nonidet P-40 was added, and the cells were mixed. The cells were spun at 15,000 RPM for 5 s in a refrigerated microcentrifuge, and the supernatant was removed. The pellet was resuspended in 120 µl of nuclear extraction buffer B, consisting of 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, and 1 mM EGTA, vortexed, and incubated on ice for 30 min. The nuclei were spun at 15,000 rpm for 10 s in a refrigerated microcentrifuge and the supernatant stored at -70 °C.

The protein was determined using the Coomassie Plus protein assay (Pierce). The gel shift was run using 6 µg (protein) of nuclear extract for each sample, using a previously described technique(18) . The gels were dried and autoradiographed or scanned with the PhosphorImager (Molecular Dynamics).

The sequence of double-stranded NF-kappaB oligonucleotide was as follows.

The kappaB motif is underlined; the bold letters indicate the region that was changed to ATCT in the mutant oligonucleotide. The retarded band was thought to be due to NF-kappaB activity, since a 3-fold excess of unlabeled kappaB oligonucleotide completely inhibited retardation and a 10-fold excess of the mutant oligonucleotide did not.


RESULTS

Antibody Binding

Computer prediction of the secondary structure of CD14 revealed that the N-terminal region was hydrophilic and, therefore, might be exposed on the surface of the molecule and elicit an antibody response. We made four 4-5-amino acid deletion mutants, each of which eliminated the most hydrophilic regions in the first 65 amino acid residues (Fig. 1). In addition, molecules with multiple deletions were constructed. One consisted of DeltaDDED and DeltaPQPD, another contained all four deletions (see Fig. 1). These molecules could be translated in vitro and immunoprecipitated with several anti-CD14 mAbs, indicating that the mutant molecules retained some CD14 epitopes.


Figure 1: The N-terminal region of CD14. Amino acids in bold type within brackets were deleted.



CHO cells were transfected with the mutant CD14 molecules and stable cell lines were made. The reactivity of the mutants with polyclonal goat anti-CD14 is shown in Fig. 2. The DeltaDDED, DeltaPQPD, and the DeltaDDED,DeltaPQPD double mutant all had similar levels of fluorescence with median channel number (MCN) of fluorescence of 2.64-2.84. The DeltaDPRQY mutant was stained more brightly (MCN 8.66). The DeltaAVEVE and the quadruple mutant had an intermediate level of reactivity (MCN 3.40 and 5.83, respectively). Because all of these mutants expressed less CD14 than wild type CD14, we also constructed the DeltaAVEVE-DAF mutant. The CD14-DAF construct, which has the C-terminal region of DAF(15) , was expressed at higher levels than wild-type CD14 (MCN 24.58 and 12.4, respectively). The DeltaAVEVE-DAF mutant was also stained somewhat more brightly than the CD14-DAF mutant (compare peaks 4 and 5, Fig. 2B).


Figure 2: Flow microfluorometry tracings of transfected CHO cells stained with goat anti-CD14 and fluorescein Protein G (FITC-PG). A: 1, DeltaDDED stained with FITC-PG alone; 2, DeltaDDED stained with goat anti-CD14 and FITC-PG (very similar results were obtained with DeltaPQPD and the DeltaDDED,DeltaPQPD double mutants); 3, DeltaDPRQY stained with goat anti-CD14 and FITC-PG; 4, CD14 stained with goat anti-CD14 and FITC-PG. B: 1, DeltaAVEVE stained with FITC-PG alone; 2, DeltaAVEVE stained with goat anti-CD14 and FITC-PG; 3, the quadruple mutant stained with goat anti-CD14 and FITC-PG; 4, CD14-DAF stained with goat anti-CD14 and FITC-PG; 5, DeltaAVEVE-DAF stained with goat anti-CD14 and FITC-PG.



The patterns of reactivity with the mAbs are shown in Table 2. mAb which inhibit both serum-enhanced LPS binding to CD14-CHO cells and LPS activation of CD14-70Z/3 cells by 50% or more were considered to be inhibitory. The DeltaDDED mutant is not recognized by six of seven inhibitory mAb. FMC17 recognized the DeltaDDED mutant poorly, and Cris-6 recognized the DeltaDDED mutant well. In contrast, seven of nine mAbs which inhibited LPS binding to CD14 poorly (i.e. less than 50%) reacted relatively well with the DeltaDDED mutant. The double mutant DeltaDDED + DeltaPQPD, had exactly the same reactivity pattern as the DeltaDDED mutant, as would be predicted since the DeltaPQPD mutant lost no additional epitopes. The DeltaAVEVE mutant has the same pattern of reactivity with mAbs as the DeltaDDED (except for a small difference in reactivity with FMC17), despite the distance in the linear amino acid sequence between the two regions. The DeltaAVEVE-DAF mutant had exactly the same pattern of reactivity as the DeltaAVEVE mutant (data not shown).



The DeltaDPRQY mutant, like the DeltaPQPD mutant, is recognized by most of the anti-CD14 mAbs. One mAb which does not recognize DeltaDPRQY mutant, Cris-6, is capable of inhibiting LPS binding to CD14. Cris-6 is also the only inhibitory mAb that recognized the DeltaDDED and DeltaAVEVE mutants. Some mAbs, such as 63D3, CLB301, 18E12, 5G3, 23bf, and 121n, recognize all the mutants. These antibodies do not inhibit LBP-enhanced LPS binding to CD14 (Table 1). 18E12 is a unique antibody because it inhibits LPS activation without inhibiting LPS binding. Because all of the mutants tested react with 18E12, we do not know what region of CD14 it recognizes. The quadruple mutant lost reactivity with all the mAbs capable of inhibiting LPS binding, as well as MO2 and RPA. This is what would be expected based on the mAb reactivity pattern of the single mutants.

LPS Binding

LPS binding was measured using three techniques: photoaffinity labeling cells with an I-labeled photoaffinity derivative of Re LPS(14) , a quantitative [^3H]LPS binding assay(8) , and by FACS analysis of cells exposed to FITC-Re595 LPS. In all these assays, significant LPS binding requires the presence of serum or purified LBP(8) . Fig. 3shows a typical [^3H]LPS binding assay with CD14-CHO cells and all of the mutants. As described previously, CD14-CHO cells bind [^3H]LPS in the presence of serum or LBP(8) . The binding is saturable and has an apparent K(D) of approximately 3times10M(8) . In contrast, CHO cells expressing the mutant CD14 molecules did not bind LPS in the presence of serum. In fact, there was more LPS associated with the cells expressing mutant CD14 in the absence of serum than in its presence. The amount of LPS bound by CHO cells expressing mutant CD14 in the absence of serum was the same as vector-transfected CHO cells. These experiments were initially done using conditions that prevented transfer of LPS to structures other than CD14(8) . To assay total cell-associated LPS, we repeated the assays at 37 °C, without any metabolic inhibitors. Once again, we could detect no serum-dependent binding or uptake in the mutants (data not shown).


Figure 3: Binding of [^3H]LPS by CD14 and mutant CD14 expressed on CHO cells. The cells were incubated with 200 ng/ml [^3H]LPS (80 nM) as described under ``Experimental Procedures.'' Open bars represent the amount of LPS bound to cells incubated with LPS in the absence of serum; shaded bars represent he amount of LPS bound to cells incubated with LPS in the presence of serum. Error bars represent standard deviations of triplicate determinations.



The [^3H]LPS binding experiments require washing to separate bound from free LPS. To try to detect lower affinity binding, we used a FITC-Re595 LPS binding assay. Because the detection is done by FACS, the cells can be analyzed in the presence of FITC-Re595 in the fluid phase. Representative data are shown in Table 3. Once again, wild type CD14-CHO cells bound significantly more FITC-Re595 in the presence of serum than in the absence of serum, but the mutants did not. Similar results were obtained with all the mutant CD14 proteins.



An autoradiograph of CD14-CHO and most of the mutant CD14-CHO cells incubated with I-ASD-Re LPS is shown in Fig. 4. In the presence of serum (or purified LBP, not shown), a doublet band at 60-62 kDa is labeled in the CD14-CHO cells. This band has been shown to be CD14 by immunoprecipitation (data not shown). The 65-kDa band probably represents LBP(19) . In the mutant CD14-CHO cells incubated with I-ASD-Re LPS and serum, no band consistent with CD14 or LBP was labeled. Identical results were obtained with all the mutant CD14 transfectants, including the DeltaAVEVE-DAF construct (data not shown).


Figure 4: Photoaffinity labeling pattern of the indicated cells with 0.5 µg/ml I-ASD-Re595 LPS. 10% H.S., 10% human serum; DOUBLE MUTANT, DeltaDDED, DeltaPQPD double mutant.



LPS Activation

Golenbock et al. (20) have reported that hCD14-CHO cells can respond to LPS with activation of the DNA binding protein NF-kappaB. We found that hCD14-CHO cells activated NF-kappaB at concentrations of LPS as low as 0.1 ng/ml; control RSV-transfected CHO cells did not respond to concentrations of LPS as high as 1 µg/ml (Fig. 5). CHO cells expressing mutant CD14 molecules did not respond as well to LPS as cells expressing wild type CD14. Cells expressing the mutants required at least 10-fold more LPS to elicit a response, compared with cells expressing wild type CD14 (data not shown). The NF-kappaB responses of cells expressing wild type and mutant CD14 to 10 ng/ml LPS are shown in Fig. 6. The DeltaDDED,PQPD double mutant, the DeltaAVEVE mutant, and the quadruple mutant all responded very poorly to 10 ng/ml LPS. The DeltaDDED, DeltaPQPD, and DeltaDPRQY mutants were more responsive to LPS than the other mutants or the RSV control, but were less responsive than the wild type CD14. The DeltaDPRQY mutant made a more variable response than the other mutants. The NF-kappaB responses of some of the mutants and wild type CD14-CHO cells over a range of LPS concentrations is shown in Fig. 7. Even at concentrations of LPS as high as 1 µg/ml, there was minimal activation of the cells expressing these two mutant CD14 types. To evaluate the effect of receptor density on activation, the DeltaAVEVE and the DeltaAVEVE-DAF mutants were compared. Neither of the transfectants responded well to LPS, despite the difference in level of expression (Fig. 8). We know that the DAF substitution is not functionally important, because CD14-CHO cells and CD14-DAF CHO cells have identical dose response curves to LPS (data not shown). All the mutant CD14-expressing CHO cells respond as well as hCD14-CHO to human tumor necrosis factor alpha, indicating that they are capable of making a normal NF-kappaB response to an unrelated stimulus (data not shown).


Figure 5: Activation of NF-kappaB in CHO cells by E. coli Re LPS. A, demonstrates the retarded kappaB probe in gel retardation assays as described under ``Experimental Procedures.'' B, the intensity of radiation in the bands shown in A was measured by the PhosphorImager (Molecular Dynamics). The intensity of the retarded band seen with nuclear extracts of cells not exposed to LPS was subtracted.




Figure 6: Activation of NF-kappaB in CHO cells expressing mutant CD14 by E. coli Re LPS. The cells were exposed to 10 ng/ml LPS for 30 min. These data were derived from duplicate determinations in two separate experiments (a total of four determinations). The intensity of the retarded band is normalized to the intensity of the retarded band seen with CD14-CHO cells. The means ± S.D. are shown.




Figure 7: Activation of NF-kappaB in CHO cells expressing mutant or wild type CD14 by E. coli Re LPS. The intensity of the retarded band is plotted on the y axis; the intensity of the retarded band in the absence of LPS is subtracted.




Figure 8: Activation of NF-kappaB in CHO cells expressing mutant or wild type CD14 by E. coli Re LPS. The intensity of the retarded band is plotted on the y axis; the intensity of the retarded band in the absence of LPS is subtracted.




DISCUSSION

In this paper, we have begun to define a region of the CD14 molecule which has three properties: 1) it contains epitopes for many anti-CD14 mAbs which inhibit serum-dependent LPS binding; 2) it is required for the binding of LPS by CD14; and 3) it is required for maximal cellular activation by LPS. This region consists of at least 65 amino acid residues in the N-terminal region of CD14. Much of the region is highly hydrophilic and some critical regions, such as the AVEVE region, are negatively charged. These properties are what would be expected for antibody epitopes, because this region of the molecule would be exposed on the surface of the molecule. However, these are unexpected properties for regions critical for the binding of LPS.

LPS is an amphipathic molecule with both hydrophobic and hydrophilic domains. Lipid A, the minimal structure which has complete LPS activity, consists of six C12-14 fatty acids linked to a diglucosamine headgroup. The most hydrophilic moieties are the phosphate groups at the 1 and 6` position of the diglucosamine. This region contributes multiple negative charges to lipid A at neutral pH. From the structure of lipid A, one would predict that a hydrophobic, positively charged region of CD14 would be required for LPS binding. In fact, the three-dimensional crystal structure of Limulus anti-LPS factor, an LPS-binding protein, has recently been solved(21) . One region was identified as a possible LPS binding structure which was also found in LBP and bactericidal/permeability increasing protein. This proposed LPS binding loop is an alternating series of positive charged and hydrophobic residues in an extended beta-conformation(21) . There is no region similar to this in the N-terminal portion of CD14. We suggest that this apparent paradox is due to the involvement of serum components in the binding interaction. The N-terminal region of CD14 may be critical for binding of LPS:serum protein complexes in addition to LPS, as discussed below.

The DeltaAVEVE mutant, the DeltaDDED,DeltaPQPD double mutant, and the DeltaDDED,DeltaPQPD,DeltaAVEVE,DeltaDPRQY quadruple deletion mutants in CHO cells respond very poorly to LPS, in comparison with the other mutants. These results suggest that the AVEVE region is particularly critical for LPS binding and cellular activation. The importance of this region could not have been predicted from the quantitative LPS binding assays or the pattern of reactivity with mAbs, since the DeltaDDED mutant had the same pattern of reactivity with mAbs as the DeltaAVEVE mutant, and none of the mutants bound LPS in the assays used. Some mutants (especially DeltaDDED and DeltaPQPD) were expressed in much lower numbers than wild type CD14. However, the DeltaAVEVE-DAF mutant was expressed at very high levels, and it was no more responsive to LPS than DeltaAVEVE. In previous studies, we have shown that substituting the C-terminal region of DAF has no effect on CD14 LPS receptor function in 70Z/3 cells (15) and CD14-CHO cells and CD14DAF-CHO cells respond equally well to LPS. (^2)These results suggest that the difference in receptor density between the mutants and the wild type CD14 is unlikely to be responsible for the loss of LPS binding and the poor receptor activity of the CD14 deletion mutants.

Five of seven inhibitory mAb lost reactivity with both the DeltaDDED and the DeltaAVEVE deletion mutants. This result suggests that most of the inhibitory mAb tested bind epitopes which are clustered together. We presume that the epitopes of these mAb must be complex and nonlinear because the DDED and AVEVE regions are 22 amino acids apart. One inhibitory mAb, Cris-6, has a different reactivity pattern. Cris-6 binds the DeltaDDED and the DeltaAVEVE deletion mutants, but does not bind the DeltaDPRQY mutant. Therefore, mAb with a least two distinct epitopes are capable of inhibiting LPS binding. mAb 18E12 is an unusual antibody which inhibits LPS-induced activation of cells(15) , without effecting LPS binding to CD14. 18E12 binds well to all the mutants in this study, so it must bind to some region of CD14 other than deleted regions in the N terminus.

Despite our inability to detect serum-dependent binding of LPS by the CD14 deletion mutants, three of the single deletion mutants are capable of transmitting LPS-induced signals in CHO cells, although they are less effective receptors than the wild type. Therefore, these molecules must bind LPS, even though we have not been able to demonstrate that binding. This discrepancy could be explained by several factors. One is the difference in sensitivity of the binding assay compared with the activation assay. The molar ratio of LPS:CD14 at saturation appears to be 8-18(8) , and if the molar ratio of LPS:mutant CD14 was 1:1 or less, the binding assays probably would not be able to detect specific binding. Similarly, the K(D) for LPS of CD14 expressed on CHO cells is approximately 3times10M, and if the mutant molecules had more than a 10-fold drop in affinity for LPS, the binding assays would not be able to detect LPS binding. In contrast, it is probable that the binding of LPS to a small fraction of the CD14 molecules expressed on CHO cells might be sufficient to trigger activation. A similar discrepancy between binding (and uptake) and activation was seen by Kitchens in his study with deacylated LPS (9) . Taken together, these studies suggest that LPS activation of cells via CD14 may occur at extremely low receptor occupancy.

None of the assays we have used is able to detect CD14-dependent serum-independent LPS binding to cells since vector-transfected cells bind as much LPS in the absence of serum as the CD14-CHO cells. Nevertheless, we know that CD14 binds LPS in the absence of serum, since LPS is able to activate CD14-expressing cells via CD14 in the absence of serum(6) . In addition, Wright has presented physical evidence that LPS can bind directly to soluble CD14, although LBP acts as a catalyst for this process(22) . It is possible that CD14 contains one LPS binding domain which is independent of LBP and another which is LBP-dependent. Although our data do not exclude this possibility, CHO cells expressing the DeltaDDED mutant do not respond to LPS at all in the absence of serum,^2 although they do make a significant response in serum. Therefore, this mutant is not as responsive to LPS in the absence of serum as is wild type CD14, arguing that both LBP-dependent and -independent LPS binding have been effected by this mutation.

In this study we have found that segments of the N-terminal 65 amino acid region are critical for serum-enhanced binding of LPS and LPS-dependent activation of CD14-CHO cells. Although other segments of the 65-amino acid region may be important, these studies indicate the overall importance of this region. Not all regions of CD14 are important for functional activity. Lee et al.(15) have previously shown that large substitutions can be made in the C-terminal region of the molecule, without effecting LPS binding or cellular activation, so we know that some regions of the CD14 molecule are not functionally critical. It is certainly possible, however, that other regions of the CD14 molecule are as important for LPS binding and LPS signal transduction as the ones we have identified so far. The properties of the mAb 18E12 indicate that there are other structural features outside the 65 amino acid region studied here that are also essential. Information about the three-dimensional structure of CD14 would be extraordinarily helpful in understanding the critical domains for LPS binding and cellular activation.


FOOTNOTES

*
This work was supported by the R. W. Johnson Pharmaceutical Research Institute, United States Public Health Service Grants GM-37696 and T32 AI07036, and by the Research Service of the Department of Veterans Affairs. 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: 111-F, VA Medical Center, 3350 La Jolla Village Dr., San Diego, CA 92161. Tel.: 619-552-7446; Fax: 619-552-4398.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; CHO cells, Chinese hamster ovary cells; CD14-CHO, human CD14-transfected Chinese hamster ovary cells; RSV-CHO, Chinese hamster ovary cells transfected with the pRC/RSV vector without inserted DNA; DAF, decay accelerating factor; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; FITC-PG, fluorescein isothiocyanate conjugated to protein G; FITC-Re595, fluorescein isothiocyanate conjugated S. minnesota Re595 LPS; GPI, glycophosphatidylinositol; LBP, lipopolysaccharide binding protein; I-ASD-Re595, I-labeled 2-(p-azidosalicylamido)1,3`-dithioproprionamide S. minnesota Re595 LPS; mAb, monoclonal antibody; MCN, median channel number; FACS, fluorescence-activated cell sorter.

(^2)
S. Viriyakosol and T. N. Kirkland, unpublished observations.


ACKNOWLEDGEMENTS

We appreciate the gifts of reagents from Robert Munford, Peter Tobias, Vladamir Kravchenko, Ann Moriarty, and Didier Leturcq. The technical assistance of Frances Multer and Fred Finley was invaluable.


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