(Received for publication, August 10, 1994; and in revised form, October 26, 1994)
From the
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-B. 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-
B. All of the deletion mutants were
less active LPS receptors than wild type CD14-CHO cells. The
AVEVE
mutant, the
DDED and
PQPD double mutant, and the
DDED,
PQPD,
AVEVE, and
DPRQY 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.
Bacterial lipopolysaccharide (LPS), ()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.
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.
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, 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 PQPD,
DDED +
PQPD, and
AVEVE mutants were the same as described above for the
DDED.
The B and C primers for the
PQPD
are
and
and
and
for the AVEVE. To construct the
DDED +
PQPD
mutant, primer B of the
DDED and primer C of the
PQPD were
used. For the construction of the
DPRQY, primer A is the same as
in the
DDED, 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 DDED
+
PQPD DNA as a template in the PCR reaction instead of the
wild type DNA. The
AVEVE mutant was generated by PCR using the
primers for
AVEVE outlined above. The DNA containing the three
deletions was then used as a template to generate the deletion in the
DPRQY region by PCR using the
DPRQY 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.).
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
sample buffer. The samples were analyzed by SDS-polyacrylamide
gel electrophoresis and fluorograpy.
The second assay was binding of
[H]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
[
H]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
10
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).
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-B oligonucleotide was as
follows.
The B 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-
B activity, since a
3-fold excess of unlabeled
B oligonucleotide completely inhibited
retardation and a 10-fold excess of the mutant oligonucleotide did not.
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 DDED,
PQPD, and
the
DDED,
PQPD double mutant all had similar levels of
fluorescence with median channel number (MCN) of fluorescence of
2.64-2.84. The
DPRQY mutant was stained more brightly (MCN
8.66). The
AVEVE 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
AVEVE-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
AVEVE-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, DDED stained with
FITC-PG alone; 2,
DDED stained with goat anti-CD14 and
FITC-PG (very similar results were obtained with
PQPD and the
DDED,
PQPD double mutants); 3,
DPRQY stained
with goat anti-CD14 and FITC-PG; 4, CD14 stained with goat
anti-CD14 and FITC-PG. B: 1,
AVEVE stained with
FITC-PG alone; 2,
AVEVE 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,
AVEVE-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 DDED mutant is not
recognized by six of seven inhibitory mAb. FMC17 recognized the
DDED mutant poorly, and Cris-6 recognized the
DDED 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
DDED mutant. The double mutant
DDED +
PQPD, had
exactly the same reactivity pattern as the
DDED mutant, as would
be predicted since the
PQPD mutant lost no additional epitopes.
The
AVEVE mutant has the same pattern of reactivity with mAbs as
the
DDED (except for a small difference in reactivity with FMC17),
despite the distance in the linear amino acid sequence between the two
regions. The
AVEVE-DAF mutant had exactly the same pattern of
reactivity as the
AVEVE mutant (data not shown).
The DPRQY
mutant, like the
PQPD mutant, is recognized by most of the
anti-CD14 mAbs. One mAb which does not recognize
DPRQY mutant,
Cris-6, is capable of inhibiting LPS binding to CD14. Cris-6 is also
the only inhibitory mAb that recognized the
DDED and
AVEVE
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.
Figure 3:
Binding of [H]LPS by
CD14 and mutant CD14 expressed on CHO cells. The cells were incubated
with 200 ng/ml [
H]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
[H]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
AVEVE-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,
DDED,
PQPD double mutant.
Figure 5:
Activation of NF-B in CHO cells by E. coli Re LPS. A, demonstrates the retarded
B
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-B 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-B 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-B 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.
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
-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 AVEVE mutant, the
DDED,
PQPD double mutant, and
the
DDED,
PQPD,
AVEVE,
DPRQY 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
DDED mutant had the same pattern of reactivity with mAbs
as the
AVEVE mutant, and none of the mutants bound LPS in the
assays used. Some mutants (especially
DDED and
PQPD) were
expressed in much lower numbers than wild type CD14. However, the
AVEVE-DAF mutant was expressed at very high levels, and it was no
more responsive to LPS than
AVEVE. 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. (
)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 DDED and the
AVEVE 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
DDED and the
AVEVE deletion mutants, but does not bind the
DPRQY 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 for LPS of CD14 expressed on CHO cells is
approximately 3
10
M, 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 DDED mutant do not
respond to LPS at all in the absence of serum,
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.