By
From the * Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United
Kingdom; and The Third Department of Internal Medicine, University of Tokyo and Tokyo
University Hospital, Tokyo 113, Japan
During gram-negative bacterial infections, lipopolysaccharide (LPS) stimulates primed macrophages (M) to release inflammatory mediators such as tumor necrosis factor (TNF)-
,
which can cause hypotension, organ failure, and often death. Several different receptors on M
have been shown to bind LPS, including the type A scavenger receptor (SR-A). This receptor
is able to bind a broad range of polyanionic ligands such as modified lipoproteins and lipoteichoic acid of gram-positive bacteria, which suggests that SR-A plays a role in host defense. In
this study, we used mice lacking the SR-A (SRKO) to investigate the role of SR-A in acquired immunity using a viable bacillus Calmette Guérin (BCG) infection model. We show that activated M
express SR-A and that this molecule is functional in assays of adhesion and endocytic
uptake. After BCG infection, SRKO mice are able to recruit M
to sites of granuloma formation where they become activated and restrict BCG replication. However, infected mice lacking the SR-A are more susceptible to endotoxic shock and produce more TNF-
and interleukin-6 in response to LPS. In addition, we show that an antibody which blocks TNF-
activity
reduces LPS-induced mortality in these mice. Thus SR-A, expressed by activated M
, plays a
protective role in host defense by scavenging LPS as well as by reducing the release by activated M
of proinflammatory cytokines. Modulation of SR-A may provide a novel therapeutic approach to control endotoxic shock.
The macrophage (M The range of ligands recognized by SR-A is wide, including LPS of gram-negative and lipoteichoic acid of
gram-positive bacteria (9, 10). SR-A types I and II exhibit
similar binding properties, specifically binding a large selection of polyanionic ligands with high affinity. This broad
ligand specificity has suggested that SR-A may play a role
in a wide range of M Recently the repertoire of SR-A functions has been extended. Work in our laboratory, using a monoclonal antibody (2F8) which recognizes the mouse type I and II SR-A,
has established that SR-A mediates a component of adhesion of M This study was designed to further our understanding of
the role of SR-A in host defense. In a model of cell-mediated immunity, we identify activated M Animals.
Mice deficient in type I and II SR-A were produced by disruption of exon 4 of the SR-A gene that codes for
the Media and Reagents.
RPMI 1640 (GIBCO BRL, Paisley, UK)
was supplemented with 10% heat-inactivated fetal bovine serum
(GIBCO BRL), 10 mM Hepes, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (R10). All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise
specified.
Antibodies.
The following rat anti-mouse mAbs were prepared
in our laboratory and were used as diluted hybridoma supernatants unless specified; F4/80, specific for an M Microorganisms and Determination of Bacillus Calmette Guérin
(BCG) CFU.
Live BCG (Pasteur strain) was provided by Dr.
G. Milon (Pasteur Institut, Paris, France). BCG stocks were
stored at )1 scavenger receptor type A (SR-A) is a trimeric integral membrane glycoprotein which
exists in two forms, type I and II, generated by alternative
splicing of a single gene product (1). This family of SRs
has recently been extended through the discovery of several additional SR genes and now includes at least three independent SR classes (5). SRs are defined according to
their ability to bind and mediate uptake of modified low
density lipoproteins (LDL), such as acetylated (Ac) LDL. The recent detection of SR-A in atheromatous plaques, and its
ability to mediate uptake of modified LDL by arterial wall
M
, has implicated the molecule in the pathogenesis of
atherosclerosis (6).
-associated physiological and pathophysiological processes (11). For example, Janeway has
suggested that such receptors may have arisen early in the evolution of host defense systems and could enable self/
nonself discrimination (14). SR-A is expressed on a wide
range of tissue M
and also on the sinusoidal endothelium
of the liver (15). This tissue distribution is consistent with a
pattern recognition function for SR-A and also suggests
that it may play a role in host defense by recognizing and
mediating the clearance of pathogens (16).
in vitro (17). SR-A might therefore function as
an adhesion molecule in vivo and act to retain M
within
ligand-rich tissues. Support for this theory has come from
observations, using physiological ligands, that SR-A can
mediate in vitro adhesion of rodent microglia and human monocytes to
-amyloid fibril-coated surfaces, implicating
SR-A in the pathogenesis of Alzheimers disease (18). An
additional role for SR-A may be as a receptor used in the
phagocytosis of apoptotic cells in the thymus (19).
and examine
whether SR-A is required for M
recruitment to sites of
granuloma formation. Previous studies have shown that
M
can bind, internalize, and partially break down LPS,
lipid A, and its bioactive precursor lipid IVa (9). This binding and subsequent metabolism to a less active form by M
-like RAW 264.7 cells is mediated by the SR-A. SR-A
ligands greatly inhibit uptake of lipid IVa in mice (9). Taken
together, these observations suggested that SR-A may have
a role in the uptake and degradation of endotoxin in animals. Using wild-type and SR-A-deficient (SRKO) mice,
we investigate an in vivo role for SR-A in the body's response to LPS. These results provide the first evidence that
SR-A acts to prevent the development of endotoxic shock.
-helical coiled coil domain, which is essential for the formation of functional trimeric receptors (4). These mice were bred
onto a 129/ICR background and are described here as SRKO.
Wild-type 129 mice were cross-bred on to an identical 129/ICR
background and are described here as 129 mice. Brother-sister
matings were used to generate homozygous SRKO and 129 mice
on an identical genetic background. Mice used in these experiments were housed at the Dunn School of Pathology and were
used between 6 and 10 wk of age.
plasma membrane antigen (20, 21); FA11, which recognizes macrosialin (22,
23); 2F8, which recognizes class I and II SR-A (24); and TIB
120, which recognizes class II MHC (25). The hamster mAb
TN3 19.12 used in vivo was a gift of Dr. R. Schreiber (Washington University, St. Louis, MO) and was used at 250 µg/mouse.
The IgG2b isotype-matched controls, CAMPATH (CP)-1G (rat
anti-human) and anti-Thy-1 (rat anti-canine) were a gift of Dr. S. Cobbold (University of Oxford, Oxford, UK; reference 26).
70°C, thawed, and sonicated immediately before use.
Mice were inoculated with ~107 CFU in 0.2 ml of PBS by intraperitoneal injection. Organs were removed from the mice at various time points after inoculation with BCG. A sample of the organ (0.2 g) was homogenized in 5 ml of sterile PBS containing
0.02% BSA and 0.05% Tween 20. 10-fold serial dilutions of the
homogenate were prepared and 25 µl was plated onto a quadrant
of a plate containing Middlebrook 7A10 agar with OADC enrichment (Difco, Detroit, MI). Plates were incubated at 37°C and
colonies of BCG were counted 10-14 d later.
Flow Cytometry.
SRKO and 129 mice were injected intraperitoneally with 107 CFU of BCG. 5 d later, peritoneal cells were
harvested, resuspended in R10, and plated onto tissue culture plastic (TCP) dishes. 5 h later nonadherent cells were removed by repeated washing with PBS. Lidocaine (4 mg/ml) and EDTA (5 mM)
in PBS were used to lift the M from the plate. The cells were
fixed on ice for 40 min using 4% paraformaldehyde/250 mM Hepes
in PBS. The cells were then permeabilized for 30 min at 4°C using saponin (0.1%) in PBS. Cells were resuspended in FACS®
buffer (0.1% saponin, 0.1% BSA, 1% normal mouse serum, and
10 mM sodium azide) and incubated with primary antibodies
(e.g., 10 µg/ml purified 2F8 or CP) for 1 h, washed three times,
and incubated for 1 h with FITC-conjugated mouse anti-rat second Ab (Jackson ImmunoResearch Labs., West Grove, PA) at a
1:500 dilution. The cells were washed and analyzed on a FACScan® using Cellquest software.
Adhesion Assays.
BCG peritoneal cells were harvested as described for flow cytometry. Cells were plated in R10 at 3 × 105
M/well in a 96-well plate in the presence of various antibodies and/or EDTA. Plates were incubated at 4°C for 30 min, then at 37°C for 90 min before washing to remove nonadherent cells.
Adherent cells were fixed in methanol and stained with 40% Giemsa for 1 h. The level of adhesion was quantified by solubilizing
the dye in methanol and reading the OD at 450 nm. Adhesion
(mean ± SD) is represented as the percentage of that obtained in
medium alone, and is the result of quadruplicate well assays.
Endocytic Uptake of DiIAcLDL. (Biogenesis, Bournemouth, UK).
SRKO and 129 mice were injected intraperitoneally with 107
CFU of BCG. 7 d later, peritoneal cells were harvested and resuspended in R10. Cells were plated at 3 × 105 M/well of a 96-well
plate and cultured overnight. Nonadherent cells were removed
by washing with PBS. Medium (R10) was added containing either
the 2F8 or isotype control (CP) antibodies, and the plate was
placed on ice for 30 min. DiIAcLDL was added to each well and
the plate was incubated at 37°C for 5 h. The cells were washed,
resuspended using 5 mM EDTA/4 mg/ml lidocaine, and the dye
was solubilized with Butan-1-ol. Fluorescence was read on a Fluoroskan II plate reader (Titertek; ICN, Costa Mesa, CA). Uptake
of DiIAcLDL is expressed as units of fluorescence (mean ± SD)
of four replicate wells.
Immunohistochemistry.
At day 25 of BCG infection, livers
were harvested from 129 and SRKO mice, frozen in OCT compound (BDH-Merck, Dorset, UK), and cooled in isopentane over
dry ice. Frozen sections were cut at 5 µm and fixed for 10 min in
2% paraformaldehyde before staining. Sections were washed in
0.1% Triton X-100 in PBS . Endogenous peroxidase activity was
quenched by incubation of sections with 102 M glucose, 10
3 M
sodium azide, and 40 U glucose oxidase in 100 ml PBS for 15 min at 37°C. Avidin/biotin blocking agents (Vector Labs., Peterborough, UK) were used according to the supplier's recommendation. Fetal bovine serum (10%) and normal rabbit serum (5%) were
then used to block irrelevant binding sites and sections were incubated for 60 min in hybridoma supernatant or isotype-matched
control Ab. Sections were washed, affinity-purified, and then biotinylated second Ab was added at 1% for 30 min. The sections
were washed and avidin-biotin-peroxidase complex (ABC elite;
Vector Labs.) was used for 30 min according to the supplier's recommendation. The presence of antigen was revealed by incubation with 0.5 mg/ml diaminobenzidine (Polysciences Inc.,
Northampton, UK) and 0.024% hydrogen peroxide in 10 mM
PBS imidazole. Sections were counterstained with cresyl violet
acetate and mounted in DPX (BDH-Merck).
Assessment of Biological Response to LPS.
Mice (age-matched
wild-type and SRKO) were injected intraperitoneally with 107
CFU of BCG. At 14 d after infection, mice were injected with various doses of LPS from Salmonella typhimurium (Westphal
strain; Difco) in nonpyrogenic saline intraperitoneally. To block
the activity of TNF-, 250 µg of TN3 19.12 mAb was given to
the mice 10 h before administration of the LPS. The condition of
the mice was monitored regularly over 5 d and those suffering
significant morbidity were killed.
Cytokine Assays.
SRKO and 129 mice were injected intraperitoneally with ~107 CFU of BCG. At 14 d after infection,
mice were injected with 10 µg of LPS intraperitoneally. The
concentration of TNF- and IL-6 in serum at various time points
after injection was determined using capture ELISAs. In brief, for
the TNF-
assay, 50 µl of TN3 19.12 mAb (4 µg/ml) in carbonate buffer (pH 9.6) were added per well of a 96-well plate (Sterilin, Stone, UK) and left at 4°C overnight. Plates were washed
twice with PBS/0.05% Tween 20 and twice with PBS alone, and
then samples of the sera (1:10 dilution in R10) were added along
with a dilution series of recombinant murine TNF-
(Serotec,
Kidlington, Oxford, UK). Plates were incubated overnight at
4°C, washed as above, and then 100 µl/well of rabbit anti-
murine TNF-
(Serotec; 1:1000 in PBS) was added and left at
room temperature for 90 min. Plates were washed as above, donkey anti-rabbit horseradish peroxidase (Chemikon International,
Inc., Temecula, CA) at 1:1,000 in PBS/Tween (0.05%)/BSA
(0.1%) was added and incubated at 20°C for 1 h. Plates were
washed and then 100 µl of reaction mix was added as recommended by supplier. The reaction was stopped by the addition of
50 µl of 3 M sulfuric acid and plates were read at 492 nM. A similar protocol was adopted for measurement of IL-6 using appropriate antibodies (PharMingen, San Diego, CA).
Statistics. Parametric data for cytokine assays and DiIAcLDL endocytic uptake were compared using Student's t test with the Welch modification to compensate for different levels of variance for data sets. Survival data were compared using a likelihood ratio test (27).
4-6 d after intraperitoneal injection of BCG, a mixed leukocyte population which is rich in M can be harvested
(28). These M
have undergone a process of activation mediated by IFN-
, resulting in changes in both secreted
products and cell surface receptor expression. Using immunostaining with FA11, a pan-M
marker which recognizes
the intracellular protein macrosialin, the M
in the peritoneal population were identified (Fig. 1, c and d; reference 22).
Similar numbers of peritoneal M were harvested from
wild-type and SRKO mice (~2 × 107 M
/mouse). Using
an electronic gate to select FA11 positive cells, BCG-activated M
from the wild-type mice were shown to express SR-A, whereas BCG-activated peritoneal M
from the
SRKO mouse lacked the SR-A (Fig. 1, a and b). The TIB
120 mAb was used to demonstrate that despite lacking SR-A,
BCG-primed M
from SRKO mice were activated and
expressed MHC class II (Fig. 1, e and f). Supporting evidence for activation was provided for wild-type and SRKO
BCG peritoneal M
since both produce high levels of nitric oxide spontaneously when harvested after day 4 of intraperitoneal infection (results not shown).
Previous work has demonstrated a role for SR-A in mediating cation-independent adhesion of thioglycollate broth-
elicited peritoneal M (29) to TCP in the presence of serum (17). To examine whether activated M
from the
SRKO mouse showed an altered adhesion phenotype in
vitro, BCG-elicited peritoneal cells were harvested and their
adhesion to TCP was examined in the presence of serum
(Fig. 2 A). In the presence of medium (R10) alone or control antibody (anti-Thy-1), the adhesion of wild-type and
SRKO cells was identical. However, in the presence of
EDTA, which chelates divalent cations, significant adhesion of the wild-type cells remained, whereas adhesion of
the SRKO cells was completely inhibited. In the additional
presence of the anti-SR-A blocking mAb 2F8, wild-type
adhesion was further reduced to the level of the SRKO cells.
SR-A mediates endocytosis of a wide range of ligands
(9). The uptake of DiIAcLDL in vitro has provided a relatively convenient assay for this activity (30). The SRKO
mouse now provides us with a model system in which to
investigate both the role of SR-A and the contribution of
other potential scavenger molecules in this uptake. We observed that BCG-activated M from the SRKO mouse, when plated in vitro, endocytosed only 30-40% of the
amount of DiIAcLDL taken up by control cells (Fig. 2 B).
When the 2F8 mAb was added to the medium, the uptake
by the wild-type cells was reduced to the level obtained with
the SRKO cells, whereas addition of an isotype-matched
control mAb (CP) had no effect. Thus the 2F8 mAb can
completely inhibit the component of DiIAcLDL uptake attributable to SR-A. However, this SR-A independent uptake can be reduced further in the presence of the polyanionic inhibitor Poly G, suggesting the involvement of other
cell surface molecules in the endocytic uptake of modified
lipoproteins (not shown).
To investigate
whether SR-A is involved in the adhesion and recruitment
of M to sites of infection in vivo, wild-type and SRKO
mice were injected intraperitoneally with BCG. In this
model of cellular immunity, M
are recruited to sites of infection (granulomata) and undergo the process of activation
mediated by IFN-
. Using immunohistochemistry of the
liver, we discovered that SR-A is expressed not only on resident Kupffer cells and endothelium but also on activated
M
in granulomata of wild-type mice (Fig. 3). In addition,
staining with F4/80 and FA11 established that, despite lacking SR-A, M
are efficiently recruited to sites of infection in vivo (Fig. 3, b and d). These M
fail to stain with 2F8
since they lack SR-A, but become activated and upregulate
MHC class II (Fig. 3, f and h). Examination of BCG CFU
for up to 2 wk of infection in liver, spleen, and lung showed
that SRKO mice were able to limit mycobacterial replication (e.g., liver BCG CFU at day 8 of infection: wild-type,
6.20 ± 1.23 × 107; SRKO, 6.26 ± 3.19 × 107).
Examination of a Role for SR-A in a Model of Endotoxic Shock.
BCG-infected mice contain large numbers of activated M which can produce high levels of proinflammatory cytokines (e.g., TNF-
, IL-1) in response to LPS challenge (31). Unlike signaling caused by ligation of the CD14
receptor, SR-A is thought to be a neutral receptor for LPS (9).
SRKO mice provide a powerful tool to test whether SR-A
binding to LPS in vivo reduces the risk of endotoxic shock
and were used in the following model of endotoxic shock.
Wild-type and SRKO mice were infected with BCG,
resulting in the production of numerous M-rich granulomas in parenchymatous organs. Groups of BCG-infected
mice were challenged with a range of LPS doses by intraperitoneal injection at days 12-14 of infection. The significant discovery was that the SRKO mice showed approximately sevenfold higher morbidity and mortality than wild-type mice (Fig. 4). In preliminary studies, similar results were
obtained when an inactivated preparation of C. parvum was
used to prime the mice before LPS challenge (e.g., LPS
dose 10 µg: mortality of 129, 0%; of SRKO, 75%; n = 4). In
addition, increased susceptibility to LPS challenge of the
SRKO mice can be demonstrated even in uninfected mice
which have received no stimulus for IFN-
priming of
M
, although doses of LPS required are two orders of
magnitude higher (e.g., LPS dose 500 µg: mortality of 129, 0%; of SRKO, 66%; n = 3).
In Vivo Cytokine Production After LPS Challenge.
During
the course of serious gram-negative bacterial infections,
LPS induces stimulation of macrophages, which results in the release of inflammatory mediators (32). The activity of these mediators, which include prostaglandins and cytokines (e.g., TNF-, IL-6, and IL-1
) is, in turn, responsible for hypotension, organ failure, and often death. TNF-
is produced in large amounts by activated M
in response
to LPS (33). We reasoned that this molecule was a candidate mediator of the difference in mortality observed between wild-type and SRKO mice. Therefore, we assayed plasma cytokine levels in BCG infected mice after LPS challenge. Significantly higher levels of TNF-
were present in
the plasma of SRKO mice relative to wild-type mice (Fig.
5 A). To confirm that the measured differences in this cytokine were not confounded by differences in response to
viable microorganisms, mice were injected with inactivated
C. parvum and subsequently challenged with LPS. When
serum was assayed 3 h after LPS challenge, levels of TNF-
were also higher in those samples from mice deficient in
SR-A (Fig. 5 B). In addition, levels of serum IL-6 were assayed at the same time points and similar differences between the wild-type and SRKO were observed (Fig. 5 C).
In contrast, serum levels of IL-10 showed no significant
difference at these time points (results not shown).
Use of a Blocking mAb to TNF-
To test whether TNF-
played a key role in the pathogenesis of endotoxic shock in
this model, we injected SRKO mice with a blocking antibody to TNF-
(TN3 19.12) before LPS challenge. In contrast to mice pretreated with PBS, those receiving TN3
19.12 showed lower and delayed levels of mortality (Table
1). However, this treatment offered only partial protection.
Therefore other proinflammatory cytokines, such as IL-1
,
IL-12, and IL-6, probably also play a role in this mechanism.
Our data provide the first direct evidence that SR-A acts
to protect the host against endotoxin. Specifically, we show
that SRKO mice suffer higher mortality in a model of endotoxic shock. After challenge with LPS, serum levels of
proinflammatory cytokines are higher in SRKO relative to
wild-type mice. We establish that TNF- is an important
mediator in the mechanism of shock in SRKO mice,
through use of a blocking antibody which provides partial protection.
In addition, we demonstrate, for the first time, that activated M express high levels of SR-A, which is functional
in mediating both cell adhesion and endocytic uptake.
Thus, activated M
from SRKO mice have an altered adhesion phenotype and express no compensatory mechanism for mediating adhesion in the presence of EDTA. In
contrast, activated M
from SRKO mice are still able to
mediate uptake of significant amounts of the ligand DiIAcLDL. One of the candidate molecules that may account
for this remaining endocytic function is MARCO, a third
class of SR-A, which has previously been shown to mediate binding of DiIAcLDL (34).
The development of granulomata in response to mycobacterial infection presents a model system for investigating
molecules involved in cell adhesion and leukocyte recruitment. Recently, cytokines such as TNF- (35) and IFN-
(36) have been demonstrated to play a role in the development of granulomata. However, studying the key cell surface molecules involved in the adhesion and recruitment of
M
to granulomata has proved more difficult. For example, blockade of the type 3 complement receptor (CR3) prevents formation of M
-rich granulomata in response to Listeria, but has no effect on the recruitment of M
to sites of
BCG infection (37). In this laboratory, frozen section assays
have been used to show that SR-A can mediate adhesion
of M
to tissue ligands (15). Thus, SR-A is a candidate adhesion receptor involved in the M
recruitment process.
Previous in vivo studies examining this hypothesis have been
frustrated by the short plasma half-life of the 2F8 mAb (our
unpublished results). In the model of BCG infection investigated here, M
lacking SR-A were able to migrate to sites
of granuloma formation and become activated. Therefore,
to evaluate any adhesion component attributable to SR-A,
further studies will need to involve the blockade of additional adhesion receptors on SRKO and wild-type cells.
This model of mycobacterial infection also enabled us to
test the immunocompetence of SRKO mice. Recent work
has suggested that SRKO mice are more susceptible to Listeria monocytogenes and Herpes simplex virus, but the mechanism remains unclear (4). However, we found that levels
of mycobacteria within the liver, lung, and spleen of wild-type and SRKO mice were not significantly different in this
study. This result may reflect that M are known to use a
wide range of receptors to bind mycobacteria, including CR3 and the mannose receptor (38). Therefore, additional
experiments whereby other cell surface receptors are
blocked on M
already lacking SR-A are required to determine if SR-A plays a role in the binding and uptake of mycobacteria.
In addition to its potential role in adhesion, SR-A binds
the gram negative cell wall component, LPS, with high affinity (39). SR-A is expressed widely by many M popuations, such as those in the gut and Kupffer cells in the
liver, and thus is well placed to mediate uptake of LPS in
both physiological and pathological conditions. Results presented in this paper establish that mice lacking the SR-A
are more susceptible to endotoxic shock. Thus, SR-A plays
a protective role in the uptake and cellular response to LPS.
Furthermore, we demonstrate that SRKO mice produce
more TNF-
and IL-6 in response to LPS challenge than
do wild-type mice. Therefore, to explain why mice deficient in SR-A are more susceptible to endotoxic shock, one
must explore the mechanism that gives rise to higher levels
of proinflammatory cytokines in these mice.
There are at least three possible explanations for these
findings. The first is that there are simply greater numbers
of activated M in BCG-infected SRKO compared to wild-type mice. This could result in increased plasma levels of
TNF-
after LPS challenge simply by summation of secreted products from a larger number of M
. However,
our results indicate that similar numbers of activated M
are recruited to the peritoneal cavity in the SRKO and
wild-type mice, and also to sites of granuloma formation
(Fig. 3). Also, the differential response to LPS challenge occurs whether the mice are infected with BCG, injected
with inactivated C. parvum, or left uninfected. Therefore,
differences in numbers of M
cannot explain these results.
A second explanation is that SRKO and wild-type mice
respond differently to LPS at the single cell level. For example, this might result from differences in relative levels of
those receptors which trigger release of cytokines upon LPS
binding, e.g., CD14, and those which bind LPS but are not
thought to trigger a response, e.g., SR-A. Several receptors, such as CD14 and CD11c/CD18, are involved in the
recognition and signaling of events after LPS binding (40,
41). Our unpublished observations indicate no difference in
the level of expression of CD14 on wild type and SRKO
BCG-elicited peritoneal cells. The data support the hypothesis that the response of activated M to LPS depends
to some extent on the relative levels of surface expression
of SR-A and CD14. The smaller difference in LPS susceptibility between wild-type and SRKO mice compared with
the extensive resistance of CD14 KO mice may reflect the
fact that other scavenger receptors may also be involved in
this protective role (42).
A third possibility is that SR-A expressed on M and
hepatic endothelium clears the LPS more rapidly from the
plasma after challenge. If this theory is correct, then one might
predict a longer plasma half-life of LPS in SRKO mice, resulting in continued stimulation of primed M
through
CD14, and higher levels of proinflammatory cytokines. Recent studies have reported that in uninfected wild-type and
SRKO mice, there is no detectable difference in the plasma half-life of LPS (43). However, if the mice are pretreated
with liposomes to eliminate Kupffer cells and other M
populations, then the investigators are able to demonstrate
a longer plasma half-life for LPS in the SRKO mice. This
result may indicate that SR-A expressed on hepatic endothelium plays a key role in LPS clearance. In addition, recent work failed to show a difference in the clearance of
modified LDL (DiIAcLDL) from the circulation of SRKO
relative to wild-type mice (4).
However, there are several important differences from
the BCG infection model described here. Previously we established, using in situ hybridization, that the activated M
in the granuloma are the main sources of both TNF-
and
IL-1
after LPS challenge (44). Thus, activated M
in this
model are not only able to secrete cytokines in response to
LPS binding to CD14, but may also bind and mediate uptake of LPS through SR-A without signaling. This raises
the intriguing possibility of a built-in defense mechanism whereby, under appropriate circumstances, the activated M
may bind and take up LPS through SR-A, and thus reduce
the amount of TNF-
produced from the same cell through
LPS-mediated CD14 signaling. Indeed, this system may be
more complex, since the net result of exposure of an activated M
to LPS is likely to depend not only on the relative expression levels of CD14, SR-A, and other receptors,
but also on interactions between signaling pathways within
the cell. Binding of LPS-LBP complexes to certain domains
of CD14 has been shown to result in activation of c-Jun NH2-terminal kinase, which results in transcription of genes
encoding for proinflammatory regulators of the immune
response (45). By comparison, the downstream events after
engagement of SR-A with its ligands remain to be established.
Further studies are required to determine the relative importance of SR-A in both plasma clearance and in the cellular response to LPS. However, it seems clear that this work establishes SR-A as an effective protective mechanism in the host response to endotoxin. Our results indicate that the modulation of SR-A may provide a novel therapeutic approach to control of endotoxic shock in humans.
Address correspondence to Professor Siamon Gordon, Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford OX1 3RE, UK. Phone: 01-865-275534; FAX: 01-865-275501.
Received for publication 27 January 1997 and in revised form 11 July 1997.
1 Abbreviations used in this paper: Ac, acetylated; BCG, bacillus Calmette-Guérin; CP, CAMPATH; DiI, 1,1-dioctadecyl-1,3,3,3We thank Dr. S. Cobbold for help with the FACS® equipment, Dr. G. Milon for generously providing the BCG organisms, and Mr. L. Tomlinson for photography. Dr. M. Cortina-Borja provided helpful advice on statistical analysis.
This work was supported by grants from the Medical Research Council, UK. Richard Haworth is supported by a Veterinary Research Training Scholarship, awarded by the Wellcome Trust.
1. |
Kodama, T.,
M. Freeman,
L. Rohrer,
J. Zabrecky,
P. Matsudaira, and
M. Krieger.
1990.
Type I macrophage scavenger
receptor contains ![]() |
2. | Rohrer, L., M. Freeman, T. Kodama, M. Penman, and M. Krieger. 1990. Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type II. Nature (Lond.). 343: 570-572 [Medline]. |
3. | Freeman, M., J. Ashkenas, D.J.D. Rees, D.M. Kingsley, N.G. Copeland, N.A. Jenkins, and M. Krieger. 1990. An ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type I and type II murine macrophage scavenger receptors. Proc. Natl. Acad. Sci. USA. 87: 8810-8814 [Abstract]. |
4. | Suzuki, H., Y. Kurihara, M. Takeya, N. Kamada, M. Kataoka, K. Jishage, O. Ueda, H. Sakaguchi, T. Higashi, T. Suzuki, et al . 1997. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature (Lond.). 386: 292-296 [Medline]. |
5. | Acton, S., P. Scherer, H. Lodish, and M. Krieger. 1994. Expression cloning of SR-B1, a CD36-related class B scavenger receptor. J. Biol. Chem. 269: 21005-21009 . |
6. | Freeman, M., Y. Ekkel, L. Rohrer, M. Penman, N.J. Freedman, G.M. Chisolm, and M. Krieger. 1991. Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein. Proc. Natl. Acad. Sci. USA. 88: 4931-4935 [Abstract]. |
7. | Steinberg, D., S. Parthasarathy, T.E. Carew, J.C. Khoo, and J.L. Witztum. 1989. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320: 915-924 [Medline]. |
8. | Krieger, M.. 1992. Molecular flypaper and atherosclerosis: structure of the macrophage scavenger receptor. Trends Biochem. Sci. 17: 141-146 [Medline]. |
9. | Hampton, R.Y., D.T. Golenbock, M. Penman, M. Krieger, and C.R.H. Raetz. 1991. Recognition and plasma clearance of endotoxin by scavenger receptors. Nature (Lond.). 352: 342-344 [Medline]. |
10. | Dunne, W.D., D. Resnick, J. Greenburg, M. Krieger, and K.A. Joiner. 1994. The type I macrophage scavenger receptor binds to Gram-positive bacteria and recognizes lipoteichoic acid. Proc. Natl. Acad. Sci. USA. 91: 1863-1867 [Abstract]. |
11. | Hoffman, J.. 1995. Innate immunity of insects. Curr. Opin. Immunol. 7: 4-10 [Medline]. |
12. | Abrams, J.M., A. Lux, H. Steller, and M. Krieger. 1992. Macrophages in Drosophila embryos and L2 cells exhibit scavenger receptor-mediated endocytosis. Proc. Natl. Acad. Sci. USA. 89: 10375-10379 [Abstract]. |
13. |
Pearson, A.,
A. Lux, and
M. Krieger.
1995.
Expression cloning of dSR-C1, a class C macrophage-specific scavenger receptor from Drosophila melanogaster.
Proc. Natl. Acad. Sci.
USA.
92:
4056-4060
|
14. | Janeway, C.A.. 1992. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today. 13: 11-16 [Medline]. |
15. | Hughes, D.A., I.P. Fraser, and S. Gordon. 1995. Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and non-lymphoid organs. Eur. J. Immunol. 25: 466-473 [Medline]. |
16. | Pearson, A.M.. 1996. Scavenger receptors in innate immunity. Curr. Opin. Immunol. 8: 20-28 [Medline]. |
17. | Fraser, I., D. Hughes, and S. Gordon. 1993. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature (Lond.). 364: 343-346 [Medline]. |
18. |
Khoury, J.E.,
C.A. Thomas,
S.E. Hickman,
C.A. Thomas,
L. Cao,
S.C. Silverstein, and
J.D. Loike.
1996.
Scavenger receptor mediated adhesion of microglia to ![]() |
19. |
Platt, N.,
H. Suzuki,
Y. Kurihara,
T. Kodama, and
S. Gordon.
1996.
Role for the class a macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.
Proc.
Natl. Acad. Sci. USA.
93:
12456-12460
|
20. | Austyn, J.M., and S. Gordon. 1981. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11: 805-815 [Medline]. |
21. |
McKnight, A.J.,
A.J. MacFarlane,
P. Dri,
L. Turley, and
S. Gordon.
1996.
Molecular cloning of F4/80, a murine macrophage-restricted cell-surface glycoprotein with homology
to the G-protein-linked transmembrane 7 hormone receptor
family.
J. Biol. Chem.
271:
486-489
|
22. | Rabinowitz, S., and S. Gordon. 1991. Macrosialin, a macrophage-restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli. J. Exp. Med. 174: 827-836 [Abstract]. |
23. | Smith, M.J., and G.L.E. Koch. 1987. Differential expression of murine macrophage surface glycoprotein antigens in intracellular membranes. J. Cell Sci. 87: 113-119 [Abstract]. |
24. | Fraser, I., D. Hughes, and S. Gordon. 1993. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature (Lond.). 364: 343-346 [Medline]. |
25. |
Bhattacharya, A.,
M.E. Dorf, and
T.A. Springer.
1981.
A
shared alloantigenic determinant on Ia antigens encoded by
the I-A and I-E subregions: evidence for I region gene duplication.
J. Immunol.
127:
2488-2495
|
26. | Hale, G., S.P. Cobbold, H. Waldmann, G. Easter, P. Matejtschuk, and R.R. Coombs. 1987. Isolation of low-frequency class-switch variants from rat hybrid myelomas. J. Immunol. Methods. 103: 59-67 [Medline]. |
27. | Snedecor, G.W., and W.G. Cochran. 1980. Statistical Methods. 7th ed. Iowa State University Press, Ames, Iowa. |
28. | Gordon, S., S. Keshav, and M. Stein. 1994. BCG-induced granuloma formation in murine tissues. Immunobiology. 1994: 369-377 . |
29. |
Johnson, R.B.J.,
C.A. Godzik, and
Z.A. Cohn.
1978.
Increased superoxide anion production by immunologically activated and chemically elicited macrophages.
J. Exp. Med.
148:
115-127
|
30. |
Penman, M.,
A. Lux,
N.J. Freedman,
L. Rohrer,
Y. Ekkel,
H. Mckinstry,
D. Resnick, and
M. Krieger.
1991.
The type I
and type II bovine scavenger receptors expressed in Chinese
hamster ovary cells are trimeric proteins with collagenous triple helical domains comprising noncovalently associated
monomers and Cys83-disulfide-linked dimers.
J. Biol. Chem.
266:
23985-23993
|
31. |
Wysocka, M.,
M. Kubin, and
G. Trinchieri.
1995.
Interleukin 12 is required for ![]() |
32. | Raetz, C.R.H.. 1990. Biochemistry of endotoxins. Annu. Rev. Biochem. 59: 129-170 [Medline]. |
33. | Kusunoki, T., E. Hailman, and S.D. Wright. 1995. Molecules from Staphlococcus aureus that bind CD14 and stimulate innate immune responses. J. Exp. Med. 182: 1673-1682 [Abstract]. |
34. | Elomaa, O., M. Kangas, and K. Tryggvason. 1995. Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages. Cell. 80: 603-609 [Medline]. |
35. | Kindler, V., A.-P. Sappino, G.E. Grau, P.-F. Piguet, and P. Vassalli. 1989. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell. 56: 731-740 [Medline]. |
36. |
Kamijo, R.,
J. Le,
D. Shapiro,
E.A. Havell,
S. Huang,
M. Aguet,
M. Bosland, and
J. Vilcek.
1993.
Mice that lack the
interferon-![]() |
37. | Rosen, H., S. Gordon, and R.J. North. 1989. Exacerbation of murine listeriosis by a monoclonal antibody specific for the type 3 complement receptor of myelomonocytic cells. J. Exp. Med. 170: 27-37 [Abstract]. |
38. | Schlesinger, L.S., C.G. Bellinger-Kawahara, N.R. Payne, and M.A. Horwitz. 1990. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J. Immunol. 144: 2711-2780 . |
39. |
Krieger, M.,
S. Acton,
J. Ashkenas,
A. Pearson,
M. Penman, and
D. Resnick.
1993.
Molecular flypaper, host defense, and
atherosclerosis.
J. Biol. Chem.
268:
4569-4572
|
40. | Wright, S.D., R.A. Ramos, P.S. Tobias, R.J. Ulevitch, and J.C. Mathison. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science (Wash. DC). 249: 1431-1433 [Medline]. |
41. | Ingalls, R.R., and D.T. Golenbock. 1995. CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. J. Exp. Med. 181: 1473-1479 [Abstract]. |
42. | Haziot, A., E. Ferrero, and S.M. Goyert. 1996. Resistance to endotoxin shock and reduced dissemination of gram negative bacteria in CD-14 deficient mice. Immunity. 4: 407-414 [Medline]. |
43. | Amersfoort, E.S.V., T.J.C. Van Berkel, and J. Kuiper. 1996. Clearance of lipopolysaccharide in scavenger receptor knock out mice. J. Leukocyte Biol. 210(Suppl):48. |
44. | Keshav, S., M. Stein, L.P. Chung, and S. Gordon. 1992. Cytokine gene expression in situ: differential expression of lysozyme, IL-1, and TNF mRNA in murine liver during BCG infection. In Mononuclear Phagocytes. R.V. Furth, editor. Kluwer Academic, Dordrecht, The Netherlands. 366-374. |
45. |
Hambleton, J.,
S.L. Weinstein, and
A.L. Defranco.
1996.
Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages.
Proc. Natl. Acad. Sci. USA.
93:
2774-2778
|