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*
Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands;
Sir William Dunn School of Pathology, Oxford, United Kingdom; and
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN-
had little or no effect. Using inhibitory
mAbs, the relevance of MARCO for the clearance of circulating bacteria
in vivo was determined. Although the overall elimination of live
Escherichia coli and Staphylococcus
aureus from the blood did not appear to be affected by
treatment with these Abs, the capturing of heat-killed bacteria by
macrophages in the marginal zone areas of the spleen was clearly
inhibited. This study suggests a role for MARCO in the host
antibacterial defense. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)2 play an important
role in the first line of host defense against bacterial and viral
pathogens. M possess a wide range of receptors, including pattern
recognition receptors (1, 2), that are involved in the detection,
phagocytosis, and destruction of pathogens and initiate an immune
response by B and T cells (3, 4). An important family of receptors
involved in these processes are the class A M scavenger receptors
(SR-A). The SR-A are trimeric membrane proteins with a collagenous
structure (5) and can bind a broad range of polyanionic ligands,
including modified lipoproteins, polyribonucleotides, and
polysaccharides (6, 7). Initial studies of the SR-A have been focused
on their role in atherosclerosis (8). More recently, however, the
importance of these receptors in the immune response against bacterial
and viral pathogens has been established (9, 10, 11, 12). The SR-AI and SR-AII,
which are derived from one gene by alternative splicing (13), can bind
both Gram-positive and Gram-negative bacteria by interaction with cell
wall components, such as lipoteichoic acid (14). Importantly, recent
studies showed that SR-AI/II knockout mice have a higher susceptibility
to infection with the bacteria Listeria monocytogenes (11)
and have a lower resistance against endotoxic shock (12). Also, the in
vivo clearance of bacterial LPS from the circulation was found to be
inhibited by an SR-A competitor (9). Recently, a novel member of the class A scavenger receptor family has been identified in mice and man, named MARCO (macrophage receptor with collagenous structure) (15, 16). The molecular structure of this receptor resembles that of SR-AI, containing a triple-helical collagenous domain and a scavenger receptor cysteine-rich (SRCR) domain at the C terminus. Like the other members of the SR-A, MARCO can bind Gram-positive and Gram-negative bacteria (15). Mutagenesis studies with human MARCO have revealed that the N-terminal side of the cysteine-rich domain is important for ligand binding (16).
Under normal, noninflamed conditions, the expression of MARCO in situ
is restricted to distinct populations of M in the spleen and lymph
nodes (15), whereas SR-AI/II have a broader distribution (17, 18, 19). So
far, most of the studies considering the regulation of the SR-A have
been focusing on atherosclerosis (20). However, it has also been shown
that bacterial endotoxins can up-regulate expression of SR-AI/II in
vivo (21). Despite structural similarities between SR-AI and MARCO, the
different distribution and the possible differences in regulation and
ligand binding specificity of these receptors might suggest functional
dissimilarities in vivo. In the spleen, MARCO is expressed on M
located in the marginal zone area. The marginal zone is a unique
lymphoid compartment positioned at a location where the bloodstream
changes from a closed arterial circulation into the sinusoidal venous
circulation of the red pulp (22). These areas contain different types
of highly phagocytic M (23, 24) that are very effective in capturing
particles from the passing bloodstream (19. 25). These cells have been
implicated in processing of Ags and induction of an immune response
against particulate bacterial Ags and T cell-independent type 2 Ags in
mice (26, 27, 28). In line with the findings of animal experiments, studies
of splenectomy patients showed a high incidence of severe bacterial
infections, suggesting an important role for the splenic marginal zone
in bacterial clearance (29).
In this study we generated a panel of mAbs directed against different
domains of MARCO, and these were used to investigate the regulation and
function of this receptor. The regulation of MARCO was determined in
vivo, during the course of BCG infection and systemic bacterial sepsis,
and in vitro with the mouse M cell line J774.2. We also investigated
the relevance of bacterial binding by MARCO in vivo by studying the
effect of the inhibitory Abs on the clearance of bacteria from the
bloodstream by splenic M. Our results support the notion that MARCO
has a role in antibacterial host defense mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the generation of mAbs, WAG/Rij rats were used (Netherlands Cancer Institute, Amsterdam, The Netherlands). The tissue distribution of MARCO was examined in embryonic or adult BALB/c (Netherlands Cancer Institute), Swiss (Harlan CPB, Zeist, The Netherlands), and C57BL/6 mice (Harlan). To this, tissue samples were snap frozen in liquid nitrogen and stored at -70°C until later use. BCG infection studies used C57BL/6 mice at 8–10 wk of age and were performed at the Sir William Dunn School of Pathology (Oxford, U.K.). Livers from uninfected and infected mice for immunohistochemical analysis were frozen in OCT compound (BDH-Merck, Dorset, U.K.) and cooled in isopentane over dry ice. Bacterial clearance experiments were performed with BALB/c mice (20–30 g).
Transfections
The cDNA encoding the full-length murine MARCO was engineered into a pSG5 vector (Stratagene, La Jolla, CA) using the BglII restriction sites. Chinese hamster ovary (CHO) cells were obtained from the European Collection of Animal Cell Cultures (ECACC, Salisbury, U.K.) and transfected with the pSG5-MARCO construct and a plasmid containing the neo gene for neomycin resistance. Stable transfected cells were cultured in DMEM/Glutamax medium (Life Technologies, Paisley, U.K.) containing 100 IU/ml penicillin, 50 mg/ml streptomycin (Pen/Strep), and 10% FCS and selected using neomycin analogue G418 (0.8 mg/ml). Only 10–20% of the cells express the receptor on the cell surface.
Different mutant forms of MARCO were generated by a site-directed
insertion of a stop codon using PCR as described elsewhere (16).
Truncations were made at the C terminus, deleting the entire
cysteine-rich domain (from residue 419 till the C terminus) or leaving
the 22 N-terminal residues of this domain (deleting from residue 441
onwards) (Fig. 1
). Four types of
cytoplasmic (N-terminal) truncations were made, respectively lacking
residues 2–7, 2–13, 2–27, and 2–37 (Fig. 1). Stably transfected CHO
cells expressing these mutant receptors were cultured on glass
coverslips, fixed, and permeabilized with ice-cold methanol (10 min),
and stained with anti-MARCO mAbs (see below). Extensive analysis of
these mutants will be described elsewhere (T. Pikkarainen et al.,
manuscript in preparation).
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For the generation of mAbs against MARCO, two rats were
immunized by s.c. injection of 1 x 107
MARCO-expressing CHO cells suspended in CFA. The animals were boostered
twice, 4 and 8 wk after primary immunization, by i.p. injection of
1 x 107 transfected cells suspended in incomplete
Freund’s adjuvant. Three days after the final booster, animals were
sacrificed, and the spleens were removed. Splenocytes were suspended
and fused with the myeloma cell line Sp2/0 by the polyethyleneglycol
(PEG) method and plated in 10 24-well plates containing rat peritoneal
feeder cells. Primary selection of hybridomas was based on
immunoreactivity with cryostat sections of mouse spleen and with
MARCO-expressing CHO cells as analyzed by flow cytometry. Positive
hybridomas were subcloned twice by limiting dilution. This resulted in
seven clones producing specific Abs directed against MARCO (Table I).
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Ags; FA11 (IgG2a), recognizing
the pan-M marker macrosialin (30, 31); TIB 120 (IgG2b), anti-MHC
class II (32); 2.4G2 (IgG2b), anti-FcRII/III (PharMingen, San
Diego, CA); and MECA-367 (IgG2a), anti-mucosal addressin
cell adhesion molecule (MAdCAM)-1 (PharMingen). The irrelevant control
IgG1, GL113 (rat anti-E. coli ß-galactosidase), was
provided by Dr. van Halteren (VU, Amsterdam). For standard immunohistochemistry, 8-µm cryostat sections of mouse tissue were fixed with acetone for 10 min, air dried, and incubated with primary Ab diluted in PBS (pH 7.6) at a final concentration of 5 µg/ml of purified and/or biotinylated IgG or 1:3 dilution of hybridoma supernatant. After 60 min incubation at room temperature (RT), sections were washed twice with an excess of PBS and incubated with peroxidase-conjugated rabbit anti-rat IgG (Dakopatts, Copenhagen, Denmark) or, in the case of biotinylated primary mAb, with avidin-peroxidase (Vector, Burlingame, CA) diluted in PBS containing 1% mouse serum for 45 min at RT. For isotyping of the ED mAbs, isotype-specific biotinylated rabbit-Abs were used (Dakopatts). Peroxidase activity was demonstrated with the substrate 3,3'-diaminobenzidine-tetrahydrochloride (DAB; Sigma, St. Louis, MO; 0.5 mg/ml) dissolved in 0.05 M Tris-buffer (pH 7.6) containing 0.03% H2O2. Sections were counterstained with hematoxylin. For analysis of BCG experiments, 5-µm frozen sections of liver were cut 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 with 10-2 M glucose, 10-3 M sodium azide, and 0.4 U/ml glucose oxidase in PBS by incubating for 15 min at 37°C. Avidin-biotin blocking agents were used (Vector, Peterborough, U.K.). Sections were incubated for 60 min with primary Ab in PBS containing 10% FCS and 5% normal rabbit serum at RT. Sections were washed, and then biotinylated secondary mAb was added for 30 min, followed by incubation with avidin-biotin-peroxidase complex (ABC elite, Vector) for 30 min. The presence of Ag was revealed by incubation with 0.5 mg/ml diaminobenzidine (Polysciences, Northampton, U.K.) and 0.03% H2O2 in 10 mM PBS imidazole. Sections were counterstained with cresyl violet acetate and mounted in DPX (BDH-Merck). All reagents were used according to the supplier’s instructions.
The regulation of MARCO in vivo
Live BCG (Pasteur strain) was kindly provided by Dr. Genevieve Milon (Pasteur Institute, Paris, France). The BCG stocks were stored at -70°C, thawed, and sonicated immediately before use. Mice were inoculated i.p. with approximately 107 CFU in 0.2 ml PBS. Organs were removed from the mice at day 42 following inoculation and analyzed by immunohistochemistry. BALB/c mice were injected i.v. with two doses of purified LPS (O111; Sigma) and sacrificed after 6 or 24 h. Expression of MARCO was determined in spleen, liver, lung, thymus, and lymph nodes by immunohistochemistry using mAb ED31. Short-term up-regulation of MARCO was determined in vivo after injection of living Staphylococcus aureus bacteria (107 CFU/mouse i.p., 5 h) or fluorescein-labeled, heat-killed Escherichia coli or S. aureus (Molecular Probes Europe, Leiden, The Netherlands; 4 x 107/mouse i.v., 45–60 min).
MARCO regulation in vitro
The in vitro regulation of MARCO expression was investigated
using the mouse M cell line J774.2, obtained from the ECACC. Cells
were cultured in 24-well plates (1 x 105/well) in
RPMI 1640 medium (Life Technologies) containing glutamine,
Pen/Strep, and 10% FCS in the presence or absence of different
stimulating agents. After stimulation for 1–48 h, cells were detached
from the culture plates by incubation with PBS containing 5 mM EDTA at
4°C, and MARCO expression was determined by flow cytometry using
biotinylated ED31 Ab (5 µg/ml). To prevent Fc-mediated binding, cells
were preincubated with mAb 2.4G2 directed against mouse FcRII/III.
Binding of ED31 was detected by avidin-phycoerythrin (PE)
(Vector) using a FACScan flow cytometer (Becton Dickinson, San Jose,
CA). Stimuli used were LPS (O111; Sigma), IFN- (a gift
from Dr. van der Meide, TNO Rijswijk, The Netherlands), TNF- (Pepro
Tech, Rocky Hill, NJ), IL-1ß (ImmunoSource, Zoersel-Halle, Belgium),
and IL-6 (ImmunoSource).
AcLDL and bacteria binding in vitro
For in vitro binding assays, CHO cells transfected with the full-length MARCO cDNA construct were used. Cells were cultured in DMEM/Glutamax medium containing 10% FCS and Pen/Strep. For the binding assays, cells were detached with trypsin/EDTA, seeded in 24-well plates (2 x 105/well), and cultured for at least 18 h before use. Before the start of the experiment, the cells were placed in new culture medium and incubated for 30 min at 37°C with blocking or control Abs (20 µg/ml purified IgG or 1:3 diluted supernatant), polyguanylic or polycytidylic acid (polyG or polyC; Boehringer, Mannheim, Germany; 400 µg/ml in DEPC-treated aquadest). After preincubation, fluorescent AcLDL (final concentration 6 µg/ml; Molecular Probes) labeled with 1.1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) or fluorescein-labeled, heat-killed bacteria (1 x 107/ml E. coli or S. aureus; Molecular Probes) were directly added to the wells and incubated at 37°C and 5% CO2 for 90 min or 45 min, respectively. Unbound particles were removed by washing the cells three times with PBS. For analysis by flow cytometry, cells were detached from the culture plate with PBS containing 5 mM EDTA by incubation at 4°C.
Clearance of bacteria in vivo
Fluorescein-labeled, heat-killed bacteria (10–50 x 106 in PBS) were injected in the tail vein of mice previously treated for 30 min with mAb ED31, ED25, or GL113 (all 250 µg IgG injected i.p.) or PBS (200 µl, i.p.). After 45–60 min, mice were killed by cervical dislocation, and spleen, liver, and lung tissue sections were examined by fluorescence microscopy. Sections were scored by two independent observers without knowledge of the object. For studies with live bacteria, E. coli (K12 strain) or S. aureus (human isolate) were cultured overnight in 10 ml Luria-Bertani (LB)-medium at 37°C. The number of bacteria was assessed by OD and adjusted with sterile PBS. Thirty minutes before giving the bacteria, mice were treated i.p. with mAb (250 µg) or PBS (200 µl). Bacteria (2–17 x 107/mouse) were injected either i.p. or i.v., and the CFU of bacteria in the blood was determined after certain time points by collecting blood from the tail vein. After 4 h, animals were anesthetized by i.p. injection of Nembutal, and 200 µl of blood was isolated by heart puncture with a heparin-filled injection needle. The blood was serially diluted in 10-fold steps in sterile PBS, and suspensions were plated on Luria-Bertani agar plates. After overnight culture at 37°C, the number of colonies was counted and the number of CFU in the blood was calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain more insight into the distribution, regulation, and
function of the mouse scavenger receptor MARCO, a panel of mAbs was
generated by inducing an immune response against MARCO-expressing CHO
cells in rats. A panel of seven mAbs directed against MARCO was
derived. The specificities of these mAbs are summarized in Table I
.
Using stably transfected CHO cells expressing truncated forms of MARCO,
the location of the epitopes of the mAbs was determined. As shown in
Fig. 1, three of the mAbs (ED28, ED29, and ED31) recognize the
cysteine-rich C-terminal domain of the receptor, and the other four
mAbs recognize the cytoplasmic N-terminal domain. The epitopes of ED23,
ED25, and ED27 are located in the first 7 amino acid residues of the
cytoplasmic tail, whereas mAb ED26 binds between residues 7 and 13. Two
of the mAbs that recognize the cysteine-rich domain, ED29 and ED31,
bind between residue 441 and the C terminus, whereas the epitope of
ED28 is located at the N-terminal part of the cysteine-rich domain
(between residues 419 and 441). The presence of the epitopes for mAbs
ED23, ED26, and ED27 within the cytoplasmic domain was confirmed by
Western blot analysis of a glutathione S-transferase
(GST)-fusion protein of the MARCO N-terminal domain (not shown).
To determine whether these Abs could interfere with the ligand binding
activity of MARCO, transfected CHO cells expressing MARCO were
incubated with fluorescent-labeled AcLDL or heat-killed bacteria
(E. coli or S. aureus). Both mAbs ED29 and ED31
completely blocked the MARCO-mediated uptake of these ligands, whereas
the other anti-MARCO mAbs and control mAbs had no effect (Fig. 2). MARCO-mediated uptake could be
inhibited by polyG but not by the control polyribonucleotide polyC
(Fig. 2B). The transfectants also bound living E.
coli (K12) bacteria but not to the neutral polysaccharide Ficoll
(not shown).
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The tissue distribution of MARCO observed with the mAbs was in
line with the earlier findings (15) showing restricted localization to
M in the spleen marginal zone area and the medulla and subcapsular
sinus of lymph nodes. Some nonspecific staining was observed with the
IgM ED28 in MARCO-negative tissues such as the liver (not shown).
Resident and thioglycollate-elicited peritoneal M were also found to
be positive for MARCO, as was demonstrated by flow cytometry (not
shown). During mouse embryonic development, MARCO is expressed in the
spleen from day E17 onwards as detected by immunohistochemistry.
However, using Northern blot analysis, MARCO mRNA expression could
already be detected by day E15 (O. Elomaa, unpublished results). The
M markers ER-TR9 and MOMA-1 could be detected only much later in
ontogeny, when the compartmentalization of the spleen and the
architecture of the marginal zone have been established (R. Mebius,
manuscript in preparation). The results are summarized in Table II.
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. Treatment of
adult mice with clodronate liposomes, which specifically eliminate M
in direct contact with the blood (27), resulted in the complete loss of
positive staining in the spleen marginal zone area (not shown).
However, with anti-MARCO mAbs, some dendritic staining pattern was
also observed near the white pulp B cell follicles, which remained
present after liposome treatment. This MARCO staining did not appear to
be associated with staining for the M markers ER-TR9, MOMA-1, and
the lysosomal enzyme acid phosphatase, or with the follicular dendritic
cell (FDC)-surface marker FDC-8, as determined by double
immunohistochemistry (data not shown). The expression of MARCO during inflammation
To investigate whether the expression of MARCO is altered during
M activation in vivo, the livers of BCG-infected and noninfected
mice were compared. In normal uninfected liver, the resident M,
Kupffer cells, express macrosialin (Fig. 3a) but are not activated and
therefore do not express MHC class II (Fig. 3c). During BCG
infection, macrosialin-positive M are recruited in large numbers to
sites of infection (Fig. 3b) and undergo a process of
activation mediated by IFN- (33, 34). These recruited cells, located
in granulomata, and the resident Kupffer cell population express MHC II
(Fig. 3d), suggesting a state of activation. When stained
for MARCO, expression was observed on a population of M in the
granulomata (Fig. 3f). MARCO was detected from as early as
day 6 of BCG infection (not shown). In addition to the MARCO expression
on recruited M, expression was also observed on Kupffer cells (Fig. 3f), which had not previously expressed the receptor (Fig. 3e). Acute endotoxic shock induced by Klebsiella
pneumoniae instillation in the lung also results in a dramatic
up-regulation of MARCO on subpopulations of M that do not normally
express this receptor, including Kupffer cells and alveolar M (not
shown).
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in the liver and the spleen red pulp. Living E.
coli or S. aureus also induced MARCO expression (data
not shown). To see whether the up-regulation observed after injection
of E. coli is mediated by bacterial LPS, mice were given
different doses of purified LPS, and MARCO expression was examined.
With a dose of 25 µg LPS, MARCO-positive M were observed in the
liver, lung, and the spleen red pulp after 6 h. More positive
cells were observed with a higher dose of LPS (100 µg), or at a later
time point (24 h), as shown in Fig. 4. At
this time point, the spleen marginal zone structure starts to
dissociate and becomes discontinuous as a result of endotoxic shock.
The induction of MARCO expression by live or heat-killed bacteria
appears to be faster than the up-regulation observed after exposure to
LPS. This, together with the fact that Gram-positive as well as
Gram-negative bacteria are able to induce expression, suggests that
other bacterial components apart from LPS contribute to activation.
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The regulation of MARCO was studied in vitro using the mouse M
cell line J774.2. Nonstimulated J774.2 cells express low but detectable
levels of MARCO on their cell surface (Fig. 5). This was confirmed by Northern blot
analysis, showing the presence of MARCO mRNA in these cells (O. Elomaa,
unpublished observation). After 24 h stimulation with LPS, surface
expression of MARCO was substantially increased. The response was
dependent on the dose of LPS given but reached a plateau with a
concentration of 0.1–1.0 µg/ml (Fig. 6A). The induction of MARCO
was time dependent, showing no up-regulation after 1 h or 4 h
but a high level of expression after 24 h (Fig. 6B) and
48 h of stimulation (not shown). Stimulation of J774.2 cells with
M-CSF and the proinflammatory cytokines TNF-, IFN- (Fig. 5),
IL-1, and IL-6 (not shown) had little or no effect on MARCO expression.
LPS-induced MARCO up-regulation was dependent on the presence of serum
and was increased in combination with IFN- (not shown).
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. Involvement of MARCO in clearance of bacteria in vivo
A series of bacterial clearance experiments was conducted to
determine the relevance of MARCO to the binding of bacteria in vivo.
Mice were treated with inhibitory or noninhibitory anti-MARCO Abs
or control mAbs, after which the clearance of fluorescently labeled
heat-killed bacteria by the spleen and liver was determined. Capturing
of bacteria from the bloodstream by M appears to be very fast.
Thirty minutes after the bacteria were injected i.v. (4–5 x
107), they were abundantly found in the liver and the
spleen, particularly in the marginal zone area. The uptake of
circulating S. aureus and E. coli by marginal
zone M was substantially reduced in mice treated with ED31 as
compared with controls (Fig. 7). ED31 had
no apparent effect on the uptake of bacteria by M in the liver (not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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play a key role in the innate immune response against
bacterial and viral pathogens. This first line of defense is also
important in inducing an appropriate response by the B and T cells of
the acquired immune system (1, 3, 4). A group of receptors expressed by
M have been classified as pattern-recognition receptors based on
their intrinsic ability to recognize specific elements unique and
essential to microbial and viral pathogens. These receptors form the
basis of the essential self vs nonself recognition (4). The M SR-A
are such pattern-recognition receptors, and their importance has been
confirmed in a recent study of SR-AI/II-deficient mice (11). It was
shown that these mice had a reduced resistance to bacterial and viral
infections. In this paper we provide both direct and indirect evidence
that MARCO also plays a role in the antibacterial host defense. A panel of seven mAbs was raised against different domains of the murine MARCO receptor. Four of these Abs recognized the cytoplasmic tail and three recognized the cysteine-rich (SRCR) domain. The two mAbs that interfered with the ligand binding of MARCO both recognized the SRCR domain.
Mutagenesis studies with human MARCO have now shown that the N-terminal
part of the SRCR domain (residues 432–442) is essential for binding of
bacteria (16). Whether this is also true for the murine MARCO remains
to be determined. Alternatively the ligand-binding site may reside
within the collagenous domain, as found for human and bovine SR-AI and
SR-AII (35, 36). Although both inhibitory Abs ED29 and ED31 against
MARCO seem to bind to the carboxyl-terminal part of the SRC (Fig. 1),
the inhibitory effects could have been mediated indirectly by steric
hindrance of adjacent ligand-binding sites.
In the present study we have demonstrated that in mice MARCO expression
is induced on several populations of tissue M in response to various
inflammatory conditions. The rapid induction of MARCO could be found on
cells that do not normally express the receptor, like Kupffer cells in
the liver. In normal nondiseased mice, no RNA message can be detected
for MARCO in the liver (37), suggesting that Kupffer cells have no
apparent mRNA pool for this receptor. This implies that the rapid
expression of MARCO, at least in the liver, is a result of activation
of transcription of the MARCO gene and de novo synthesis of the
receptor and that this can take place in less than 45 min. For the
up-regulation observed in the lung and spleen red pulp after systemic
administration of bacteria or LPS, it was difficult to tell whether
MARCO expression was induced on the resident M or whether positive
cells had been recruited from the blood. The BCG infection model,
however, provided some useful clues to this. With this infection model,
it was demonstrated that MARCO is not only up-regulated on resident
tissue M but is also expressed on M freshly recruited to the
granulomata. The fact that thioglycollate-elicited peritoneal M have
high MARCO expression could suggest that M recruited during
inflammation are in general positive for this receptor. An intriguing
observation made in the BCG model is that, although the vast majority
of M in the granulomata are activated and express high levels of MHC
class II, only a subset of these cells express MARCO, suggesting that
the expression of this receptor is tightly regulated and not per se
associated with cell activation. The function of MARCO on the activated
M during BCG infection may be quite different from its role on cells
in lymphoid organs. For example, the receptor may be involved in the
clearance of apoptotic cells. Cell turnover in the granulomata is
rapid, and other scavenger receptors, such as SR-A, have been shown to
be involved in the uptake of apoptotic cells (38). MARCO may also
contribute to the recognition and killing of the mycobacteria.
The induction of MARCO on M cell line J774.2 was slower than
observed in vivo, with up-regulation seen only after 24 h of
stimulation. Of all the stimuli tested, LPS gave the highest level of
induction. The different proinflammatory cytokines had little or no
effect on expression. Only IFN- seemed to further increase the
LPS-induced elevation. It is known that the J774.2 cell line expresses
CD14, which mediates LPS-induced activation (2) and that IFN- can
boost the M responsiveness to LPS. It cannot be excluded that the
LPS-induced up-regulation of MARCO is a result of autocrine activation
via the cytokines produced by activated M, as the slower induction
might suggest. Such a regulation mechanism has been described for the
LPS-induced down-regulation of SR-AI/II on monocytes, which is mediated
by autocrine stimulation of TNF- (39).
Using the inhibitory Ab ED31, we have shown that MARCO is functionally
involved in the binding and phagocytosis of bacteria by M located in
the spleen marginal zone. This finding confirms that MARCO functions as
a receptor for bacteria in vivo, as was suggested by the binding of
bacteria by MARCO-expressing transfectants. Several lines of evidence
indicate that M in the marginal zone are important for the capturing
and processing of bacterial Ags. Particulate Ags are processed
particularly efficiently by these M and presented as Ag in an MHC
class II-restricted fashion, resulting in the induction of an
appropriate humoral immune response (27, 28). In a recent report, it
was shown that an immune response against their own serum albumin is
induced in mice when this protein is modified in a way that makes it a
ligand for M SR-A (40, 41). Targeting of Ags to SR-A seems to be
sufficient to disrupt tolerance against self Ags and is able to induce
an immune response in the absence of additional immunological
adjuvants. Whether MARCO is involved in the immune induction remains to
be determined, but it could be speculated that it functions in the
immune response against circulating Ags.
The overall clearance and killing of circulating living bacteria did
not appear to be affected by treatment with ED31. This confirms that
there is a redundancy in the pathways by which bacteria are cleared
from the blood. Several M receptors have been identified that can
either directly or indirectly mediate the binding and phagocytosis of
bacteria (2, 4, 14). In the case of the SR-A family, our study
indicates that the ligand-binding characteristics of MARCO are similar
to those of SR-AI/II. This suggests that, if one of the SR-A is blocked
in vivo, the other members can take over part of its function. On the
level of organs, there also may be an overlap in bacterial clearance
activity. Although MARCO-mediated capturing of bacteria in the spleen
is inhibited by ED31, the remainder of circulating bacteria could be
cleared in the liver in a MARCO-independent fashion. It is known that
the liver contributes to the bacterial clearance, involving both
activated Kupffer cells and recruited neutrophilic granulocytes and
M (42).
In conclusion, we have demonstrated in the present study that MARCO,
like the other SR-A (12), is involved in the host response and
indicated to have a role in the capturing of circulating bacteria from
the blood by M in the spleen. In response to inflammatory
conditions, MARCO expression is rapidly induced on M subpopulations
that, similar to cells in lymphoid organs, may utilize this receptor
for recognition and clearance of bacterial pathogens. Additional
experiments will be required to elucidate the function of MARCO and to
determine the relevance of expression on activated M. MARCO knockout
mice are currently being developed and should provide more information
about the role of this receptor in bacterial clearance in vivo.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: M, macrophage; SR-A, class A M scavenger receptor; MARCO, M receptor with collagenous structure; SRCR, scavenger receptor cysteine-rich; BCG bacillus Calmette-Guérin; CHO, Chinese hamster ovary; RT, room temperature; AcLDL, acetylated low density lipoprotein; DiI, 1.1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate; PP, postpartum; M-CSF, macrophage-CSF.
Received for publication April 10, 1998. Accepted for publication September 30, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Immunol. 148:112.[Abstract]
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 N. J. Rogers, M. J. Lees, L. Gabriel, E. Maniati, S. J. Rose, P. K. Potter, and B. J. Morley A Defect in Marco Expression Contributes to Systemic Lupus Erythematosus Development via Failure to Clear Apoptotic Cells J. Immunol., February 15, 2009; 182(4): 1982 - 1990. [Abstract] [Full Text] [PDF]
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Y. Miyazaki, M. Bunting, D. M. Stafforini, E. S. Harris, T. M. McIntyre, S. M. Prescott, V. S. Frutuoso, F. C. Amendoeira, D. de Oliveira Nascimento, A. Vieira-de-Abreu, et al. Integrin {alpha}D 2 Is Dynamically Expressed by Inflamed Macrophages and Alters the Natural History of Lethal Systemic Infections J. Immunol., January 1, 2008; 180(1): 590 - 600. [Abstract] [Full Text] [PDF]
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S. Kanno, A. Furuyama, and S. Hirano A Murine Scavenger Receptor MARCO Recognizes Polystyrene Nanoparticles Toxicol. Sci., June 1, 2007; 97(2): 398 - 406. [Abstract] [Full Text] [PDF]
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 Y. Chen, M. Sankala, J. R. M. Ojala, Y. Sun, A. Tuuttila, D. E. Isenman, K. Tryggvason, and T. Pikkarainen A Phage Display Screen and Binding Studies with Acetylated Low Density Lipoprotein Provide Evidence for the Importance of the Scavenger Receptor Cysteine-rich (SRCR) Domain in the Ligand-binding Function of MARCO J. Biol. Chem., May 5, 2006; 281(18): 12767 - 12775. [Abstract] [Full Text] [PDF]
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S. Jozefowski, M. Arredouani, T. Sulahian, and L. Kobzik Disparate Regulation and Function of the Class A Scavenger Receptors SR-AI/II and MARCO J. Immunol., December 15, 2005; 175(12): 8032 - 8041. [Abstract] [Full Text] [PDF]
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Y. Chen, T. Pikkarainen, O. Elomaa, R. Soininen, T. Kodama, G. Kraal, and K. Tryggvason Defective Microarchitecture of the Spleen Marginal Zone and Impaired Response to a Thymus-Independent Type 2 Antigen in Mice Lacking Scavenger Receptors MARCO and SR-A J. Immunol., December 15, 2005; 175(12): 8173 - 8180. [Abstract] [Full Text] [PDF]
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 Q. Li, Q. Lu, V. Bottero, G. Estepa, L. Morrison, F. Mercurio, and I. M. Verma Enhanced NF-{kappa}B activation and cellular function in macrophages lacking I{kappa}B kinase 1 (IKK1) PNAS, August 30, 2005; 102(35): 12425 - 12430. [Abstract] [Full Text] [PDF]
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R. Alarcon, C. Fuenzalida, M. Santibanez, and R. von Bernhardi Expression of Scavenger Receptors in Glial Cells: COMPARING THE ADHESION OF ASTROCYTES AND MICROGLIA FROM NEONATAL RATS TO SURFACE-BOUND {beta}-AMYLOID J. Biol. Chem., August 26, 2005; 280(34): 30406 - 30415. [Abstract] [Full Text] [PDF]
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 S. Mukhopadhyay, L. Peiser, and S. Gordon Activation of murine macrophages by Neisseria meningitidis and IFN-{gamma} in vitro: distinct roles of class A scavenger and Toll-like pattern recognition receptors in selective modulation of surface phenotype J. Leukoc. Biol., September 1, 2004; 76(3): 577 - 584. [Abstract] [Full Text] [PDF]
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 M. Arredouani, Z. Yang, Y. Ning, G. Qin, R. Soininen, K. Tryggvason, and L. Kobzik The Scavenger Receptor MARCO Is Required for Lung Defense against Pneumococcal Pneumonia and Inhaled Particles J. Exp. Med., July 19, 2004; 200(2): 267 - 272. [Abstract] [Full Text] [PDF]
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 A. G. Tempone, D. Perez, S. Rath, A. L. Vilarinho, R. A. Mortara, and H. F. de Andrade Jr Targeting Leishmania (L.) chagasi amastigotes through macrophage scavenger receptors: the use of drugs entrapped in liposomes containing phosphatidylserine J. Antimicrob. Chemother., July 1, 2004; 54(1): 60 - 68. [Abstract] [Full Text] [PDF]
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Y.-S. Kang, J. Y. Kim, S. A. Bruening, M. Pack, A. Charalambous, A. Pritsker, T. M. Moran, J. M. Loeffler, R. M. Steinman, and C. G. Park The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen PNAS, January 6, 2004; 101(1): 215 - 220. [Abstract] [Full Text] [PDF]
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 F. Granucci, F. Petralia, M. Urbano, S. Citterio, F. Di Tota, L. Santambrogio, and P. Ricciardi-Castagnoli The scavenger receptor MARCO mediates cytoskeleton rearrangements in dendritic cells and microglia Blood, October 15, 2003; 102(8): 2940 - 2947. [Abstract] [Full Text] [PDF]
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A. Grolleau, D. E. Misek, R. Kuick, S. Hanash, and J. J. Mule Inducible Expression of Macrophage Receptor Marco by Dendritic Cells Following Phagocytic Uptake of Dead Cells Uncovered by Oligonucleotide Arrays J. Immunol., September 15, 2003; 171(6): 2879 - 2888. [Abstract] [Full Text] [PDF]
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I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
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M. C.I. Karlsson, R. Guinamard, S. Bolland, M. Sankala, R. M. Steinman, and J. V. Ravetch Macrophages Control the Retention and Trafficking of B Lymphocytes in the Splenic Marginal Zone J. Exp. Med., July 21, 2003; 198(2): 333 - 340. [Abstract] [Full Text] [PDF]
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L.-H. Bin, L. D. Nielson, X. Liu, R. J. Mason, and H.-B. Shu Identification of Uteroglobin-Related Protein 1 and Macrophage Scavenger Receptor with Collagenous Structure as a Lung-Specific Ligand-Receptor Pair J. Immunol., July 15, 2003; 171(2): 924 - 930. [Abstract] [Full Text] [PDF]
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T. Ichiba, T. Teshima, R. Kuick, D. E. Misek, C. Liu, Y. Takada, Y. Maeda, P. Reddy, D. L. Williams, S. M. Hanash, et al. Early changes in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays Blood, July 15, 2003; 102(2): 763 - 771. [Abstract] [Full Text] [PDF]
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 E. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]
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 R. LaCourse, L. Ryan, and R. J. North Expression of NADPH Oxidase-Dependent Resistance to Listeriosis in Mice Occurs during the First 6 to 12 Hours of Liver Infection Infect. Immun., December 1, 2002; 70(12): 7179 - 7181. [Abstract] [Full Text] [PDF]
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M. Sankala, A. Brannstrom, T. Schulthess, U. Bergmann, E. Morgunova, J. Engel, K. Tryggvason, and T. Pikkarainen Characterization of Recombinant Soluble Macrophage Scavenger Receptor MARCO J. Biol. Chem., August 30, 2002; 277(36): 33378 - 33385. [Abstract] [Full Text] [PDF]
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D. Mason, P. Andre, A. Bensussan, C. Buckley, C. Civin, E. Clark, M. de Haas, S. Goyert, M. Hadam, D. Hart, et al. CD antigens 2002 Blood, May 15, 2002; 99(10): 3877 - 3880. [Full Text] [PDF]
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 K. L. Tyson, P. L. Weissberg, and C. M. Shanahan Heterogeneity of gene expression in human atheroma unmasked using cDNA representational difference analysis Physiol Genomics, May 10, 2002; 9(2): 121 - 130. [Abstract] [Full Text] [PDF]
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Y. Zhang, L. L. McCormick, S. R. Desai, C. Wu, and A. C. Gilliam Murine Sclerodermatous Graft-Versus-Host Disease, a Model for Human Scleroderma: Cutaneous Cytokines, Chemokines, and Immune Cell Activation J. Immunol., March 15, 2002; 168(6): 3088 - 3098. [Abstract] [Full Text] [PDF]
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S.-H. Su, H.-i. Chen, and C. J. Jen C57BL/6 and BALB/c Bronchoalveolar Macrophages Respond Differently to Exercise J. Immunol., November 1, 2001; 167(9): 5084 - 5091. [Abstract] [Full Text] [PDF]
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S.-H. Su, H.-i. Chen, and C. J. Jen Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages J. Leukoc. Biol., January 1, 2001; 69(1): 75 - 80. [Abstract] [Full Text]
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 V. Terpstra, E. S. van Amersfoort, A. G. van Velzen, J. Kuiper, and T. J. C. van Berkel Hepatic and Extrahepatic Scavenger Receptors : Function in Relation to Disease Arterioscler Thromb Vasc Biol, August 1, 2000; 20(8): 1860 - 1872. [Full Text] [PDF]
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L. Peiser, P. J. Gough, T. Kodama, and S. Gordon Macrophage Class A Scavenger Receptor-Mediated Phagocytosis of Escherichia coli: Role of Cell Heterogeneity, Microbial Strain, and Culture Conditions In Vitro Infect. Immun., April 1, 2000; 68(4): 1953 - 1963. [Abstract] [Full Text] [PDF]
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A. Palecanda, J. Paulauskis, E. Al-Mutairi, A. Imrich, G. Qin, H. Suzuki, T. Kodama, K. Tryggvason, H. Koziel, and L. Kobzik Role of the Scavenger Receptor MARCO in Alveolar Macrophage Binding of Unopsonized Environmental Particles J. Exp. Med., May 3, 1999; 189(9): 1497 - 1506. [Abstract] [Full Text] [PDF]
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T. Pikkarainen, A. Brannstrom, and K. Tryggvason Expression of Macrophage MARCO Receptor Induces Formation of Dendritic Plasma Membrane Processes J. Biol. Chem., April 16, 1999; 274(16): 10975 - 10982. [Abstract] [Full Text] [PDF]
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 S.-I. Hagiwara, M. Takeya, H. Suzuki, T. Kodama, L. J. W. van der Laan, G. Kraal, N. Kitamura, and K. Takahashi Role of Macrophage Scavenger Receptors in Hepatic Granuloma Formation in Mice Am. J. Pathol., March 1, 1999; 154(3): 705 - 720. [Abstract] [Full Text] [PDF]
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Z. Pancer Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin PNAS, November 21, 2000; 97(24): 13156 - 13161. [Abstract] [Full Text] [PDF]
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419-Ct) or leaving the
N-terminal 22 amino acids of this domain (
0.05
(Wilcoxon).
