| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Institute for Virus Research and
Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan; and
Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
increased LOX-1-dependent adhesion of E. coli, but not
that of S. aureus, suggesting that S.
aureus adhesion to BAEC may require additional molecules, which
cooperate with LOX-1 and suppressed by TNF-. Taken together, LOX-1
can work as a cell surface receptor for Gram-positive and Gram-negative
bacteria, such as S. aureus and E. coli,
in a mechanism similar to that of class A scavenger receptors; however,
other unknown molecules may also be involved in the adhesion of
E. coli to BAEC, which is enhanced by
poly(I). |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Scavenger receptor family molecules have been implicated in bacterial adhesion to macrophages. Scavenger receptor class A (SR-A)2 can bind Gram-positive (4) and Gram-negative (5) bacteria, including S. aureus and Escherichia coli, as well as lipoteichoic acid from Gram-positive bacteria (4) and LPS from Gram-negative bacteria (6). Studies with SR-A knockout mice revealed that SR-A plays important roles in host defense against bacterial infection; it enhances sensitivities for S. aureus (7) and Listeria infection (8) as well as for LPS-mediated septic shock (9). MARCO, another member of SR-A, can also adhere to Gram-positive and Gram-negative bacteria (10, 11, 12).
We have identified lectin-like oxidized low density lipoprotein
receptor 1 (LOX-1), a 48- to 50-kDa type II membrane glycoprotein with
C-type lectin-like structure in the extracellular domain, which acts as
an endocytosis receptor for oxidized low density lipoprotein in
cultured bovine aortic endothelial cells (BAEC) (13).
LOX-1 is synthesized as a 40-kDa precursor protein with
N-linked high mannose carbohydrate chains and is
subsequently further glycosylated and processed into a 48-kDa mature
form (14). LOX-1 has a broad spectrum of physiological
ligands, including oxidized or aged erythrocytes, apoptotic cells
(15), and activated platelets (16). In vivo,
LOX-1 is highly expressed in endothelial cells, macrophages, and smooth
muscle cells in atherosclerotic lesions of humans (17) and
hypercholesterolemic rabbits (18). Interestingly, LOX-1
can be induced by proinflammatory stimuli, such as TNF-
(19), TGF-
(20), and LPS
(21), as well as by fluid shear stress (21, 22), suggesting its roles in the settings of inflammatory
diseases and vascular injury.
In the present study, we studied whether LOX-1 can support adhesion of bacteria, such as S. aureus and E. coli, in both cultured vascular endothelial cells and Chinese hamster ovary-K1 (CHO-K1) cells stably expressing LOX-1.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DMEM and Ham’s F-12 medium were obtained from Nissui (Tokyo, Japan). FCS were purchased from Sanko Junyaku (Tokyo, Japan). Poly(I) and poly(C) were purchased from Sigma (St. Louis, MO).
Cell culture
BAEC were isolated from bovine aortas by scraping the inner surface with glass coverslips and were cultured in DMEM containing 10% heat-inactivated FCS in an atmosphere of 95% air/5% CO2 at 37°C. Wild-type CHO-K1 cells were maintained in F-12/10% FCS. CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) were maintained in F-12/10% FCS supplemented with 10 µg/ml of blasticidin S (Funakoshi, Tokyo, Japan) as previously described (13).
DNA transfection into COS-7 cells
COS-7 cells were prepared by cotransfection of expression vectors encoding LOX-1 and truncated Aic2A with extracellular and transmembrane regions (23), and cells expressing Aic2 were collected using the panning method with anti-Aic2 mAb (24). Finally, LOX-1 was expressed in >80% of the collected cells.
Flow cytometry
BLOX-1-CHO or BAEC (1 x 106 cells/50 µl of buffer A; PBS with 5% FCS and 0.05% sodium azide) were incubated with anti-LOX-1 mAb (20 µg/ml) or control IgG (20 µg/ml) on ice for 1 h. After washing with buffer A, the cells were incubated with FITC-conjugated anti-mouse IgG mAb (20 µg/ml; PharMingen, San Diego, CA) for 1 h on ice, washed, and applied to EPICS Elite (Coulter, Hialeah, FL).
Bacterial adhesion assay
After BLOX-1-CHO or BAEC were incubated with FITC-labeled S. aureus or E. coli (3 x 106 cells/ml unless otherwise indicated; Molecular Probes, Eugene, OR) in the culture medium at 37°C for 1 h, BLOX-1-CHO or BAEC were washed with the culture medium twice and thereafter with PBS twice to remove unbound bacteria, then subjected to fluorescence microscopy (Axiophoto2; Zeiss, New York, NY). Cells were treated with trypsin for 5 min, and then detached cells were subjected to flow cytometry by use of EPICS Elite (Coulter). Numbers of BLOX-1-CHO or BAEC that bound FITC-labeled bacteria were calculated. In some experiments cells were pretreated with poly(I), poly(C) (100 or 500 µg/ml), a neutralizing anti-LOX-1 mAb (JTX20, 30 µg/ml) (16), or control IgG (30 µg/ml) for 15 min before addition of FITC-labeled bacteria. After BLOX-1-CHO or BAEC were incubated with FITC-labeled S. aureus or E. coli (3 x 106 cells/ml unless otherwise indicated; Molecular Probes) in the culture medium at 37°C for 1 h, BLOX-1-CHO or BAEC were washed with the culture medium twice and thereafter with PBS twice to remove unbound bacteria, then treated with trypsin for 5 min. After detached cells were chilled to 4°C, the cells were incubated with rhodamine-labeled Con A (20 µg/ml; Vector Laboratories, Burlingame, CA) for 60 min at 4°C to visualize the plasma membrane. After washing three times with PBS, the cells were immediately fixed with 10% (v/v) formaldehyde and observed under a confocal laser microscope (Bio-Rad, Hercules, CA).
To examine adhesion of bacteria to LOX-1 under nonstatic conditions,
FITC-labeled bacteria (250-µl suspension in DMEM/10% FCS) were
incubated with BLOX-1-CHO or BAEC cultured in 12-well plates on a
seesaw shaker (Biocraft, Elmond Park, NY) at a frequency of 36
cycles/min at 37°C for 1 h. In some experiments, BAEC had been
pretreated with or without TNF- (10 ng/ml) for 24 h before the
bacteria adhesion assay was performed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We first examined levels of LOX-1 expression in BLOX-1-CHO and
BAEC. As shown in Fig. 1
, both BLOX-1-CHO
and BAEC expressed significant levels of LOX-1 on the cell surface,
which was shown by flow cytometry, although the level of LOX-1
expression was slightly higher and was heterologous in BLOX-1-CHO. In
addition, as we have shown previously (19), a
proinflammatory cytokine, TNF-, induced cell surface expression of
LOX-1 (Fig. 1, C and D).
|
, B and D, BLOX-1-CHO
showed prominent adhesion of FITC-labeled S. aureus and
E. coli. In contrast, untransfected CHO-K1 cells did not
show any significant binding of FITC-labeled S. aureus or
E. coli (Fig. 2, A and C). Confocal
microscopy showed that FITC-labeled S. aureus (Fig. 2E) and E. coli (Fig. 2F) were
engulfed by BLOX-1-CHO. The numbers of cell that bound FITC-labeled
S. aureus were quantified by flow cytometry (Fig. 2, G–J). Bar graphs indicate that 
10% of BLOX-1-CHO bound
both FITC-labeled S. aureus and E. coli, although
these bacteria adhered to <3% of untransfected CHO-K1 cells (Fig. 2, K and L).
|
, the results were almost identical with those observed in complete
culture medium with 10% serum (Fig. 2
, K and L),
indicating that neither divalent cations nor serum factors are
necessary for the adhesion of S. aureus and E.
coli to LOX-1.
|
Previous studies have shown that poly(I) inhibits bacterial
adhesion to MARCO, a member of class A scavenger receptors
(12). In addition, poly(I) blocked binding of oxidized
low-density lipoprotein to LOX-1 (25). We, therefore,
examined whether poly(I) can inhibit binding of S. aureus
and E. coli to BLOX-1-CHO. As shown in Fig. 4, A and B,
adhesion of FITC-labeled S. aureus and E. coli to
BLOX-1-CHO was remarkably suppressed by poly(I), but not by poly(C). An
mAb directed to bovine LOX-1 also blocked binding of FITC-labeled
S. aureus and E. coli to BLOX-1-CHO (Fig. 4, C and D).
|
In addition to BLOX-1-CHO, BAEC can bind and engulf FITC-labeled
S. aureus and E. coli (Fig. 5, A–D). Quantification of
BAEC binding S. aureus and E. coli are also shown
(Fig. 5, E–G). A functional blocking anti-bovine LOX-1
mAb significantly reduced adhesion of FITC-labeled S. aureus
and E. coli to BAEC (Fig. 6, A and C), although the inhibition was less than
that observed in BLOX-1-CHO (Fig. 4, C and D).
Binding of S. aureus to BAEC was inhibited by
poly(I), but not by poly(C) (Fig. 6B), as shown in
BLOX-1-CHO (Fig. 4A). In contrast, E. coli
adhesion was not suppressed by poly(I), but, rather, was enhanced (Fig. 6D), although E. coli binding was inhibited by
poly(I) in BLOX-1-CHO (Fig. 4B). These results indicate that
LOX-1 on the cell surface of BAEC can bind S. aureus and
E. coli, although additional molecules on BAEC may also be
involved, especially in the case of E. coli adhesion to
BAEC. In BAEC, E. coli may adhere to molecules other than
LOX-1 expressed on the cell surface, which can be enhanced by poly(I).
These molecular mechanisms, however, remain to be clarified.
|
|
on S. aureus and E.
coli adhesion to BAEC
TNF- can induce cell surface expression of LOX-1 (Fig. 1, C and D). Therefore, we determined whether
TNF--treated BAEC show enhanced adhesion of S. aureus or
E. coli. As shown in Fig. 6C, total adhesion of
E. coli as well as LOX-1-dependent E. coli
adhesion (none minus anti-LOX-1) was significantly enhanced by
TNF- treatment. In contrast, neither total adhesion nor
LOX-1-dependent adhesion (none minus anti-LOX-1) of S.
aureus was significantly enhanced by TNF- (Fig. 6A).
These results suggest that LOX-1 alone can support the adhesion of
E. coli, but may not support that of S. aureus.
Other molecules that can be down-regulated or functionally suppressed
by TNF- may act in cooperation with LOX-1 to support adhesion of
S. aureus in BAEC, although further studies are needed to
clarify this point. These unidentified molecules might be
constitutively expressed and functionally active in CHO-K1 cells.
LOX-1 can support adhesion of S. aureus and E. coli under nonstatic conditions
To determine whether LOX-1 can support adhesion of bacteria only
under static conditions, adhesion assay was performed under nonstatic
conditions using a seesaw shaker. Both FITC-labeled S.
aureus (Fig. 7A) and
E. coli (Fig. 7B) were bound to BLOX-1-CHO, but
not control CHO-K1 cells, on a seesaw shaker. Poly(I), but not poly(C),
inhibited adhesion of S. aureus and E. coli to
BLOX-1-CHO (Fig. 7, C and D). A neutralizing
anti-LOX-1 Ab significantly reduced the number of BAEC that bind
both FITC-labeled S. aureus and E. coli under
nonstatic conditions (Fig. 7, E and F). These
results are in parallel with those found under static conditions, as
demonstrated in Fig. 4. Taken together, LOX-1 appears to support
adhesion of S. aureus and E. coli under both
static and nonstatic conditions.
|
C, both total and
LOX-1-dependent (none minus anti-LOX-1) adhesion of FITC-labeled
E. coli was significantly increased after TNF- treatment.
In contrast, TNF--induced increases in both total and
LOX-1-dependent (none minus anti-LOX-1) adhesion of FITC-labeled
S. aureus were modest (Fig. 8A). Poly(I), but not
poly(C), inhibited adhesion of S. aureus, but did not
significantly inhibit that of E. coli, to BAEC under
nonstatic conditions (Fig. 8, B and D), which
appeared similar to that observed under the static condition (Fig. 6B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As previously shown in SR-A, poly(I) suppressed adhesion of S. aureus and E. coli to LOX-1, suggesting a similar ligand specificity of LOX-1 to these scavenger receptors, although LOX-1 does not significantly bind acetylated low-density lipoprotein (25), which is a high-affinity ligand for SR-A. In BAEC, in contrast, poly(I) inhibited binding of S. aureus, but not E. coli, suggesting that molecules other than LOX-1 which can specifically bind E. coli but not S. aureus, through some interactions with poly(I), may also be involved, although this point remains to be further clarified.
Although bacteria can bind to multiple molecules, including extracellular matrix glycoproteins (26, 27, 28, 29), binding of bacteria to endothelial membrane proteins at the primary infection site might prevent bacteria from dissemination to the bloodstream and other organs. This appears to be an alternative hypothesis concerning the roles of LOX-1 in bacterial infection; however, studies with suitable animal experimental models may be required to elucidate the exact roles of LOX-1.
As well as MARCO and SR-A, LOX-1 is also expressed by tissue macrophages (20, 30, 31). Interestingly, proinflammatory stimuli dramatically induced the expression of LOX-1 (20, 30), although these stimuli down-regulate SR-A expression (32, 33) in macrophages. Adhesion and subsequent phagocytosis of bacteria in macrophages appear to play important roles in Ag presentation and immune responses after bacterial infection. In fact, in CHO-K1 cells stably expressing LOX-1 and BAEC, bacteria can be engulfed after the adhesion to the cell surface LOX-1. Therefore, bacteria may also be engulfed or phagocytosed in macrophages after the adhesion to LOX-1 as well as class A scavenger receptors on their cell surface.
In summary, the present report for the first time provides evidence that LOX-1, alone or in cooperation with other molecules, can support adhesion of Gram-positive and Gram-negative bacteria. Further studies would elucidate the pathophysiological roles of LOX-1 in bacterial infection in vivo.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: SR-A, scavenger receptor class A; LOX-1, lectin-like oxidized low-density lipoprotein receptor 1; BAEC, bovine aortic endothelial cells; CHO, chinese hamster ovary.
Received for publication August 9, 2000. Accepted for publication February 15, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 increases the expression of lectin-like oxidized low density lipoprotein receptor-1. Biochem. Biophys. Res. Commun. 272:357.[Medline]
. FEBS Lett. 440:29.[Medline]
is transcriptionally and post-transcriptionally regulated. J. Biol. Chem. 271:7767.1 inhibits scavenger receptor activity in THP-1 human macrophages. J. Biol. Chem. 266:22866.This article has been cited by other articles:
|
|
 |
|
  Y. Fujita, A. Kakino, N. Nishimichi, S. Yamaguchi, Y. Sato, S. Machida, L. Cominacini, Y. Delneste, H. Matsuda, and T. Sawamura Oxidized LDL Receptor LOX-1 Binds to C-Reactive Protein and Mediates Its Vascular Effects Clin. Chem., February 1, 2009; 55(2): 285 - 294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Herczenik and M. F. B. G. Gebbink Molecular and cellular aspects of protein misfolding and disease FASEB J, July 1, 2008; 22(7): 2115 - 2133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Murphy, R. S. Vohra, S. Dunn, Z. G. Holloway, A. P. Monaco, S. Homer-Vanniasinkam, J. H. Walker, and S. Ponnambalam Oxidised LDL internalisation by the LOX-1 scavenger receptor is dependent on a novel cytoplasmic motif and is regulated by dynamin-2 J. Cell Sci., July 1, 2008; 121(13): 2136 - 2147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Esen and T. Kielian Central Role for MyD88 in the Responses of Microglia to Pathogen-Associated Molecular Patterns. J. Immunol., June 1, 2006; 176(11): 6802 - 6811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-i. Hinagata, M. Kakutani, T. Fujii, T. Naruko, N. Inoue, Y. Fujita, J. L. Mehta, M. Ueda, and T. Sawamura Oxidized LDL receptor LOX-1 is involved in neointimal hyperplasia after balloon arterial injury in a rat model Cardiovasc Res, January 1, 2006; 69(1): 263 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Inoue, Y. Arai, H. Kurihara, T. Kita, and T. Sawamura Overexpression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Induces Intramyocardial Vasculopathy in Apolipoprotein E-Null Mice Circ. Res., July 22, 2005; 97(2): 176 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Park, F. G. Adsit, and J. C. Boyington The 1.4 A Crystal Structure of the Human Oxidized Low Density Lipoprotein Receptor Lox-1 J. Biol. Chem., April 8, 2005; 280(14): 13593 - 13599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Prevo, S. Banerji, J. Ni, and D. G. Jackson Rapid Plasma Membrane-Endosomal Trafficking of the Lymph Node Sinus and High Endothelial Venule Scavenger Receptor/Homing Receptor Stabilin-1 (Feel-1/Clever-1) J. Biol. Chem., December 10, 2004; 279(50): 52580 - 52592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G F Ferraccioli and E Gremese Autoantibodies and thrombophilia in RA: TNF{alpha} and TNF{alpha} blockers Ann Rheum Dis, June 1, 2004; 63(6): 613 - 615. [Full Text]
|
|
|
|
|
|
A. S. J. Marshall, J. A. Willment, H.-H. Lin, D. L. Williams, S. Gordon, and G. D. Brown Identification and Characterization of a Novel Human Myeloid Inhibitory C-type Lectin-like Receptor (MICL) That Is Predominantly Expressed on Granulocytes and Monocytes J. Biol. Chem., April 9, 2004; 279(15): 14792 - 14802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shimaoka, T. Nakayama, N. Kume, S. Takahashi, J. Yamaguchi, M. Minami, K. Hayashida, T. Kita, J. Ohsumi, O. Yoshie, et al. Cutting Edge: SR-PSOX/CXC Chemokine Ligand 16 Mediates Bacterial Phagocytosis by APCs Through its Chemokine Domain J. Immunol., August 15, 2003; 171(4): 1647 - 1651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Rogers, J. Thornton, K. S. Barker, D. O. McDaniel, G. S. Sacks, E. Swiatlo, and L. S. McDaniel Pneumolysin-Dependent and -Independent Gene Expression Identified by cDNA Microarray Analysis of THP-1 Human Mononuclear Cells Stimulated by Streptococcus pneumoniae Infect. Immun., April 1, 2003; 71(4): 2087 - 2094. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Honjo, K. Nakamura, K. Yamashiro, J. Kiryu, H. Tanihara, L. M. McEvoy, Y. Honda, E. C. Butcher, T. Masaki, and T. Sawamura Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation PNAS, February 4, 2003; 100(3): 1274 - 1279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Coutant, L. Perrin-Cocon, S. Agaugue, T. Delair, P. Andre, and V. Lotteau Mature Dendritic Cell Generation Promoted by Lysophosphatidylcholine J. Immunol., August 15, 2002; 169(4): 1688 - 1695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Minami, N. Kume, T. Shimaoka, H. Kataoka, K. Hayashida, Y. Akiyama, I. Nagata, K. Ando, M. Nobuyoshi, M. Hanyuu, et al. Expression of SR-PSOX, a Novel Cell-Surface Scavenger Receptor for Phosphatidylserine and Oxidized LDL in Human Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1796 - 1800. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
