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) (
)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 LPS
sCD14 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 LPS
sCD14 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 (pDSR
2, (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 pDSR
2
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
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
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
non-essential amino acids (Life Technologies, Inc.), and 1
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
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
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
H-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
10
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)
-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
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
H-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
H-LPS with sCD14 dramatically increased the
amount of
H-LPS
sCD14
and
H-LPS
sCD14
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.
H-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. (
)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
(
RU of 400 between steps 6 and 8) and MEM-18 (
RU 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.