LOX-1 Supports Adhesion of Gram-Positive
and Gram-Negative Bacteria
This information is current as
of June 17, 2017.
Takeshi Shimaoka, Noriaki Kume, Manabu Minami,
Kazutaka Hayashida, Tatsuya Sawamura, Toru Kita and Shin
Yonehara
J Immunol 2001; 166:5108-5114; ;
doi: 10.4049/jimmunol.166.8.5108
http://www.jimmunol.org/content/166/8/5108
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References
LOX-1 Supports Adhesion of Gram-Positive and
Gram-Negative Bacteria
Takeshi Shimaoka,* Noriaki Kume,1† Manabu Minami,† Kazutaka Hayashida,†
Tatsuya Sawamura,‡ Toru Kita,† and Shin Yonehara*
A
dhesion of bacteria to vascular endothelial cells has been
suggested as one of the initial steps in the invasion of
blood-borne bacteria to other organs. In particular, adhesion of Staphylococcus aureus may be important, because S.
aureus often causes bacterial endocarditis in the absence of preexisting vascular injury (1). A previous report has suggested that
50-kDa (2) and 130-kDa (3) cell surface molecules on cultured
vascular endothelial cells can support binding of S. aureus; however, molecular identification has not been accomplished. In addition, adhesion and subsequent engulfment of Gram-positive and
Gram-negative bacteria by macrophages appear crucial for host
defense and immune responses after bacterial infection.
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 Gramnegative 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 Grampositive and Gram-negative bacteria (10 –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 (CHOK1) cells stably expressing LOX-1.
*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
Materials and Methods
Received for publication August 9, 2000. Accepted for publication February 15, 2001.
Reagents
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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).
1
Address correspondence and reprint requests to Dr. Noriaki Kume, Department of
Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: nkume@
kuhp.kyoto-u.ac.jp
2
Abbreviations used in this paper: SR-A, scavenger receptor class A; LOX-1, lectinlike oxidized low-density lipoprotein receptor 1; BAEC, bovine aortic endothelial
cells; CHO, chinese hamster ovary.
Copyright © 2001 by The American Association of Immunologists
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
0022-1767/01/$02.00
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Adhesion of bacteria to vascular endothelial cells as well as mucosal cells and epithelial cells appears to be one of the initial steps
in the process of bacterial infection, including infective endocarditis. We examined whether lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), a member of scavenger receptor family molecules with C-type lectin-like structure, can support
adhesion of Gram-positive and Gram-negative bacteria. Chinese hamster ovary-K1 (CHO-K1) cells stably expressing LOX-1 can
support binding of FITC-labeled Staphylococcus aureus and Escherichia coli, which was suppressed by poly(I) and an anti-LOX-1
mAb. Adhesion of these bacteria to LOX-1 does not require divalent cations or serum factors and can be supported under both
static and nonstatic conditions. Cultured bovine aortic endothelial cells (BAEC) can also support adhesion of FITC-labeled S.
aureus, which was similarly suppressed by poly(I) and an anti-LOX-1 mAb. In contrast, binding of FITC-labeled E. coli to BAEC
was partially inhibited by the anti-LOX-1 mAb, and poly(I) did not block FITC-labeled E. coli adhesion to BAEC, but, rather,
enhanced it under a static condition. TNF-␣ 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). The Journal of Immunology, 2001, 166: 0000 – 0000.
The Journal of Immunology
5109
expressing bovine LOX-1 (BLOX-1-CHO) were maintained in F-12/10%
FCS supplemented with 10 ␮g/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 ⫻ 106 cells/50 ␮l of buffer A; PBS with 5%
FCS and 0.05% sodium azide) were incubated with anti-LOX-1 mAb (20
␮g/ml) or control IgG (20 ␮g/ml) on ice for 1 h. After washing with buffer
A, the cells were incubated with FITC-conjugated anti-mouse IgG mAb
(20 ␮g/ml; PharMingen, San Diego, CA) for 1 h on ice, washed, and
applied to EPICS Elite (Coulter, Hialeah, FL).
Bacterial adhesion assay
Results
FIGURE 1. Expression of LOX-1 in BLOX-1-CHO and BAEC. Flow
cytometry was performed to measure levels of LOX-1 expression in control CHO-K1 cells (A), BLOX-1-CHO (B), BAEC without activation (C),
and BAEC treated with TNF-␣ (D).
E. coli, although these bacteria adhered to ⬍3% of untransfected
CHO-K1 cells (Fig. 2, K and L).
In addition to BLOX-1-CHO, COS-7 cells transiently transfected with human LOX-1 cDNA similarly bound both FITC-labeled S. aureus and E. coli (data not shown). Furthermore, BLOX1-CHO did not significantly bind zymosan (data not shown) as in
the case of MARCO (10). To explore the requirement of serum
factors and divalent cation, adhesion assay was performed in PBS
without magnesium, calcium, or serum. As shown in Fig. 3, 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.
S. aureus and E. coli are bound to cells expressing LOX-1
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-1CHO. In addition, as we have shown previously (19), a proinflammatory cytokine, TNF-␣, induced cell surface expression of
LOX-1 (Fig. 1, C and D).
To determine whether LOX-1 can bind Gram-positive and
Gram-negative bacteria, we examined FITC-labeled S. aureus and
E. coli binding to BLOX-1-CHO and control CHO-K1 cells under
usual culture conditions. As shown in Fig. 2, 3B and D, BLOX-1CHO 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
Poly(I) inhibits S. aureus and E. coli binding 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 BLOX1-CHO (Fig. 4, C and D).
BAEC bind S. aureus and E. coli depending upon LOX-1
In addition to BLOX-1-CHO, BAEC can bind and engulf FITClabeled 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
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After BLOX-1-CHO or BAEC were incubated with FITC-labeled S. aureus or E. coli (3 ⫻ 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 ␮g/ml), a neutralizing
anti-LOX-1 mAb (JTX20, 30 ␮g/ml) (16), or control IgG (30 ␮g/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 ⫻ 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
␮g/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-␮l 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.
5110
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.
FIGURE 2.
Continued
Effects of TNF-␣ 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-1dependent 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
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FIGURE 2. Adhesion of FITC-labeled S. aureus and E. coli to LOX-1.
FITC-labeled S. aureus (A and B) and E. coli (C and D) was incubated with
BLOX-1-CHO (B and D) or untransfected CHO-K1 cells (A and C), for
1 h, washed, and subjected to fluorescence microscopy. Confocal microscopy shows that FITC-labeled S. aureus (E) and E. coli (F) were engulfed
by BLOX-1-CHO. The numbers of cells binding FITC-labeled S. aureus
(G and H) and E. coli (I and J) in BLOX-1-CHO (H and J) and untransfected CHO-K1 cells (G and I) were measured by flow cytometry. The
numbers of BLOX-1-CHO (percentage of total) that bind FITC-labeled S.
aureus (K) and E. coli (L) are also indicated as bar graphs.
BACTERIA BINDING TO LOX-1
The Journal of Immunology
5111
FIGURE 3. Adhesion of FITC-labeled S. aureus and E. coli to LOX-1
in PBS without calcium or magnesium. FITC-labeled S. aureus (A) and E.
coli (B) were incubated with BLOX-1-CHO or untransfected CHO-K1
cells for 1 h in PBS with 0.25% BSA and washed, and the numbers of
BLOX-1-CHO (percentage of total) binding FITC-labeled S. aureus (A) or
E. coli (B) are indicated as bar graphs.
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nonstatic conditions using a seesaw shaker. Both FITC-labeled S.
aureus (Fig. 7A) and E. coli (Fig. 7B) were bound to BLOX-1CHO, 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
FIGURE 4. Poly(I) and an anti-LOX-1 Ab inhibit adhesion of S. aureus
and E. coli to LOX-1. FITC-labeled S. aureus (A) and E. coli (B) were
incubated with BLOX-1-CHO in the presence or the absence of poly(I) or
poly(C) (100 ␮g/ml) as indicated. Relative numbers of bacteria adherent
cells were calculated compared with those without poly(I) or poly(C).
FITC-labeled S. aureus (C) and E. coli (D) were incubated with BLOX1-CHO in the presence or the absence of an anti-LOX-1 Ab or control IgG
(30 ␮g/ml) as indicated. Relative numbers of bacteria adherent cells were
calculated compared with those without the Ab.
FIGURE 5. Adhesion of S. aureus and E. coli to BAEC. FITC-labeled
S. aureus (A) and E. coli (B) were incubated with BAEC for 1 h, washed,
and subjected to fluorescence microscopy. Confocal microscopy indicates
that FITC-labeled S. aureus (C) and E. coli (D) were engulfed by BAEC.
The numbers of BAEC that bind FITC-labeled S. aureus (E) and E. coli (F)
were measured by flow cytometry. Bar graphs indicate the numbers (percentage of total cells) of BAEC that bind FITC-labeled bacteria as indicated (G).
5112
BACTERIA BINDING TO LOX-1
BLOX-1-CHO (Fig. 7, C and D). A neutralizing anti-LOX-1 Ab
significantly reduced the number of BAEC that bind both FITClabeled 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.
Bacterial adhesion to BAEC was also examined under nonstatic
conditions. As shown in Fig. 8C, 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
Adhesion of bacteria to vascular endothelial cells has been suggested as one of the crucial steps in the dissemination of bacteria
to other organs via the bloodstream, including infective endocarditis. In macrophages, adhesion and subsequent engulfment of bacteria appear to play important roles in self-defense and immune
responses after bacterial infection. The present study for the first
time provides evidence that LOX-1, one of the scavenger receptor
family members, expressed by vascular endothelial cells and macrophages, can support adhesion of Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli. In addition to bacteria,
FIGURE 7. Adhesion of S. aureus and E. coli to LOX-1 under nonstatic
condition. FITC-labeled S. aureus (1.2 ⫻ 107 cells/ml; A, C, and E) and E.
coli (1.2 ⫻ 107 cells/ml; B, D, and F) were incubated with BLOX-1-CHO
on a seesaw shaker at 37°C for 1 h in the presence or the absence of an
anti-LOX-1 Ab (30 ␮g/ml; E and F), control IgG (30 ␮g/ml; E and F),
poly(I) (500 ␮g/ml; C and D), or poly(C) (500 ␮g/ml; C and D) as indicated, and then washed to remove nonadherent bacteria. Relative numbers
of bacteria adherent cells were calculated compared with those without
inhibitors (C–F).
bacterial endotoxin, LPS, is a ligand of SR-A, a member of the
scavenger receptor family; however, LOX-1 did not significantly
bind LPS (data not shown).
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.
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FIGURE 6. Effects of an anti-LOX-1 Ab and poly(I) on adhesion of S.
aureus and E. coli to BAEC. FITC-labeled S. aureus (A) and E. coli (C)
were incubated with BAEC, which had been pretreated with or without
TNF-␣ (1 ng/ml) for 24 h, in the presence or the absence of an anti-LOX-1
Ab or control IgG (30 ␮g/ml) as indicated. Relative numbers of bacteria
adherent cells were calculated compared with those without Abs. FITClabeled S. aureus (B) and E. coli (D) were incubated with BAEC in the
presence or the absence of poly(I) or poly(C) (500 ␮g/ml) as indicated are
indicated.
The Journal of Immunology
5113
References
Although bacteria can bind to multiple molecules, including extracellular matrix glycoproteins (26 –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
We thank Central Pharmaceutical Research Institute, Japan Tobacco Co.
Ltd. (Osaka, Japan), for kindly providing us with a neutralizing anti-LOX-1
mAb (JTX20).
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FIGURE 8. Adhesion of S. aureus and E. coli to BAEC under nonstatic
conditions. FITC-labeled S. aureus (A; 6 ⫻ 106 cells/ml) and E. coli (C;
6 ⫻ 106 cells/ml) were incubated with BAEC, which had been pretreated
with or without TNF-␣, on a seesaw shaker at 37°C for 1 h in the presence
or the absence of an anti-LOX-1 Ab or control IgG (30 ␮g/ml). BAEC
were incubated with FITC-labeled S. aureus (B; 6 ⫻ 106 cells/ml) and E.
coli (D; 6 ⫻ 106 cells/ml) on a seesaw shaker at 37°C for 1 h in the
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