By
From the * Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford
University, Stanford, California 94305, and Center for Molecular Biology and Medicine, Veterans
Affairs Health Care System, Palo Alto, California 94304; Department of Cardiovascular Medicine,
Stanford University, Stanford, California 94305; § Department of Pathology, Center for the Health
Sciences, University of California at Los Angeles, Los Angeles, California 90024
Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.
Adherence and accumulation of monocytes in discrete
segments of arterial endothelium is among the earliest
detectable events in atherogenesis and is a central feature of
the pathogenesis of atherosclerosis (1). The recruitment of
monocytes from the blood is directed in vivo by selective
monocyte-endothelial cell recognition. This process supports regional immune responses by targeting cells to particular organs and tissues as a function of the local microenvironment and inflammatory state. Monocytes display a
number of potentially relevant adhesion molecules (2) and endothelial cells overlying atherosclerotic lesions express a number of vascular ligands (5); however, the
identity of the molecules required for monocyte recruitment associated with early lesion formation remains unclear
(6, 8, 9).
Cybulsky and Gimbrone (6) showed that the rabbit homologue of vascular cell adhesion molecule (VCAM)1-1 is
highly expressed in lesion areas. Monocytes express the A potential requirement for multiple adhesion mechanisms is not unexpected in light of current models of leukocyte-endothelial interaction. Recruitment of lymphocytes from the blood has been separated into multiple
sequential steps characterized as contact initiation ("tethering"), rolling, pertussis toxin-sensitive G We have identified a 50-kD molecule participating in a
novel adhesion pathway involved in monocyte binding to
activated endothelium. Antibody blocking studies implicate
this novel molecule in monocyte adhesion in atherogenesis.
Cells and Reagents.
bEnd3 cells, a mouse EC line derived
from primary cultured mouse brain endothelial cells transformed
by polyoma virus middle T antigen (16; provided by W. Risau,
Max Plank Institute, Bad Neuheim, Germany), at passage 21-28
were maintained in cDMEM (DMEM [BioWhittaker, Inc.,
Walkersville, MD] supplemented with 5% fetal bovine serum
[endotoxin <10 pg/ml; Gemini Scientific, Calabasas, CA] and 5%
Fetal Clone [endotoxin <10 pg/ml; Hyclone Labs, Logan, UT]).
4
1 integrin receptor for VCAM-1, and VCAM-1 has
been shown to be upregulated focally in lesion-prone areas
of the rabbit aorta as early as 1 wk after initiation of an
atherogenic diet in rabbit (6). Indeed, in this model, upregulation of VCAM-1 precedes accumulation of monocytes
and macrophages (10) suggesting that expression of VCAM-1
may participate in initiation of diet-induced atherosclerosis
in rabbits. However, although antibodies to
4 or VCAM-1
inhibit monocyte binding to activated aortic endothelial cells in culture by ~50% in assays performed at 4°C, no
blocking by anti-
4 or VCAM-1 mAbs is observed when assays are performed at physiological temperatures (6). These
results suggest that while VCAM-1 may play a role in
monocyte accumulation in the cholesterol-fed rabbit model,
additional adhesion mechanisms must operate as well.
i-mediated activation, and activation-dependent integrin triggering and
arrest. Each step may be mediated by different adhesion or
activation receptors, allowing specificity through use of
unique combinations of receptors to create specific homing
pathways (11). This model suggests that several adhesion and activation pathways may work in concert to
achieve recruitment of monocytes into the vessel wall in vivo.
Monocyte-Endothelial Binding Assay.
bEnd3 (mouse endothelial) cells were passaged using a 1:3 split by growth area into 1-cm2
wells of 8-well Lab-Tek® chamber slides (Nunc Inc., Naperville,
IL) and allowed to grow to confluence for 2-3 d. Some wells were
treated with IL-1 (10 U/ml; R&D Sys., Inc., Minneapolis, MN),
TNF- (1 ng/ml), or LPS (1 µg/ml; 0111B12; Sigma Chemical
Co., St. Louis, MO) for 18 h, washed once with assay buffer, and
preincubated with 50 µl of 30 µg/ml blocking or negative control IgM
mAbs (OZ42, LM5.9, LM137.3, and LM142.12) for
20 min at 4°C or RT. WEHI 78/24 or U937 (human monocytoid) cells (5 × 105 for 4°C assays, 2 × 105 for RT assays) were
added in 50 µl for a final volume of 100 µl. After a 30 min incubation at 4°C or RT with continuous rocking to allow binding,
the top portion of the chambers and the gasket were removed
and the slide was dipped twice in Hepes-buffered saline to remove unbound cells and placed in 2% gluteraldehyde in PBS
containing 1 mM Ca2+ and 1 mM Mg2+. Slides were visualized
by light microscopy and the mean number of cells bound in 10 fields (representing 6.3 mm2) in triplicate wells was determined.
mAb Production.
mAbs LM151, LM141, LM92, and LM13.13
were produced as follows. Fisher F344 rats (Charles River, Hollister, CA) were immunized with TNF- stimulated bEnd3 (mouse
endothelial) cells. In brief, confluent cultures (350 cm2 surface
area) of bEnd3 cells were stimulated with TNF-
for 18 h,
washed with copious volumes of HBSS to reduce serum protein contamination, harvested with a rubber policeman, and injected subcutaneously into a Fisher F344 rat. Rats received two subcutaneous boosts and a final intraperitoneal boost with identically prepared antigen at 3-wk intervals. 3 d after the final boost, rats were killed by CO2 asphyxiation, spleens removed aseptically, and hybridomas prepared following standard fusion protocols using SP2/0
cells (American Type Culture Collection, Rockville, MD) as the
myeloma fusion partner. Hybridomas were plated in 24-well plates
resulting in formation of multiple independent clones (~50)
forming colonies in each well.
Biochemical Characterization.
Molecular weight determination
was performed by SDS-PAGE and Western blot analysis of reduced and nonreduced samples. In brief, untreated or cytokinetreated confluent cultures of bEnd3 (mouse endothelial) cells or
untreated MM-LDL-treated confluent cultures of RAEC were
solubilized in 10% NP-40 (Boehringer Mannheim, Indianapolis, IN) in 150 mM NaCl, 15 mM Tris, 1 mM CaCl2, 1 mM MgCl2
containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 × 107 M
pepstatin A, 1 mM PMSF, and 10 mM N-ethylmalemide, pH
7.0. Lysates were ultracentrifuged at 100,000 g for 45 min to remove insoluble material. Soluble material was diluted with SDS
sample buffer with or without
-mercaptoethanol, separated by
SDS-PAGE, and electrotransferred to nitrocellulose membrane.
Blots were probed with control mAb MECA79 (IgM
; reference
21), LM151, LM141, or LM92 followed by alkaline phosphataseconjugated anti-rat IgM (Jackson ImmunoResearch Labs., West
Grove, PA) and developed using NBT-BCIP (GIBCO BRL) or
horseradish peroxidase-conjugated goat anti-rat IgM (Zymed
Labs., Inc., South San Francisco, CA) and visualized using the
chemiluminescence detection reagent ECL (Amersham, Arlington Heights, IL).
Immunohistochemistry, Immunofluorescence, and Flow Cytometry.
Frozen sections from multiple tissues from normal and TNF--
injected mice were prepared using standard avidin-biotin immunohistochemical protocols. Alternatively, mice were injected with 150 µg of LM151 or control IgM mAb, killed after 10 min, perfused
with 20 ml of HBSS, and frozen section of tissues stained with
PE-conjugated mouse anti-rat IgG (Chomoprobe, Mountain
View, CA).
En Face Rabbit Aortic Endothelial-Monocyte Assay. Male New Zealand white rabbits were fed either normal rabbit chow or rabbit chow enriched with 1% cholesterol (ICN Biomedicals, Inc., Costa Mesa, CA). 1 d before they were killed (13 d after initiation of diet), animals were lightly sedated with a 3-mg subcutaneous injection of acepromazine maleate solution (Ayerst Labs., Philadelphia, PA) and blood samples were collected in EDTA. Total plasma cholesterol levels as well as high density lipoprotein (HDL) were enzymatically measured (Sigma Chemical Co.). Total cholesterol and HDL levels (mg/dl) of the control animals were 53.8 ± 11.1 and 28.7 ± 3.7, respectively, whereas cholesterol feeding for 2 wk resulted in 803 ± 113 and 25.8 ± 3.8 mg/ dl of total cholesterol and HDL, respectively.
2 wk after initiating the high cholesterol diet, rabbits were killed by intravenous injection of sodium pentobarbitol (35 mg/kg; Ayerst Labs.). Thoracic aortae were removed and placed in cold, oxygenated phosphate-buffered saline, excess adventitial fat was removed, and a 15-mm segment of thoracic aorta was excised immediately distal to the left subclavian artery. Segments were opened longitudinally and placed into 35-mm culture dishes previously coated with a solid layer of 3% agarose and equilibrated with binding buffer (HBSS supplemented with 2 mM Ca2+, 2 mM Mg2+, and 20 mM Hepes, pH 7.0). The aortic segments were pinned to the dish to expose the endothelial surface to the medium and preincubated with LM151, control IgM mAb OZ42, or binding control IgM LM13.13, each at 50 µg/ml, for 20 min at RT. Culture dishes were then placed on a rocking platform and 106 tetramethyl rhodamine isothiocyanate-labeled WEHI78/24 cells were added, co-incubated for 30 min with constant rocking. The dishes were rotated 120° every 10 min to facilitate even binding. After the co-incubation period, the medium was aspirated and replaced with 2 ml of fresh binding buffer and allowed to incubate with rocking for 5 min to remove unbound cells. The washing procedure was repeated three times and then the aortic segment was placed endothelial side up on a glass slide. Adherent cells from at least 30 sites/segment were counted under epifluorescent microscopy. The data are expressed as a percentage of the number of adherent cells on LM151 treated versus control antibody-treated aortic segments.WEHI 78/24 is a mouse
monocyte-like cell line (17) that expresses L-SELECTIN, 4
integrin, LFA-1, and MAC-1 (25). It binds poorly to confluent, unstimulated bEnd3 endothelial cells under the conditions used here. After stimulation of bEnd3 cells for 4-20 h
with IL-1
(10 U/ml), TNF-
(1 ng/ml), or LPS (1 µg/
ml), however, a dramatic increase in WEHI 78/24 cell binding is observed. mAbs were produced against 18 h TNF
-stimulated bEnd3 cells and screened initially for their ability
to block WEHI78/24 binding to TNF-
-stimulated bEnd3
cells. Three inhibitory mAbs LM151, LM141, and LM92
(all IgM
) were isolated. All three blocked WEHI78/24 adhesion to LPS (not shown) and TNF-
-stimulated (Fig. 1
A) bEnd3 cells by >50% at both 4°C and at RT. They also
inhibited binding of the human monocyte-like cell line
U937, previously used to model involvement of "atheroELAMs" (6) in monocyte adhesion (Fig. 1 B). Mouse neutrophil and lymphocyte binding to LPS-stimulated bEnd3 cells
were not affected by mAbs LM151, LM141, and LM92
(Fig. 1 C). Binding of WEHI78/24 cells to TNF-
-stimulated bEnd3 cells was not influenced by the irrelevant
IgM
mAb OZ42 (Fig. 1 A) or by LM5.9, LM137.2, or
LM142.12, IgM
mAbs that stain bEnd3 cells with intensities greater or equal to LM151 by FACS® analysis (not
shown).
Inhibitory mAbs Define a Common 50-kD Antigen, VMAP-1.
Western blots of NP-40 lysates of stimulated bEnd3 cells
were probed with the three blocking antibodies. Each recognized an identical pattern with a dominant species at 50 kD under reducing and nonreducing conditions (Fig. 2
A), indicating that the antigen does not exist as a disulfidelinked dimer or multimer. Silver staining of LM151 affinity-isolated material revealed a single band at 50 kD (Fig. 2
B). LM151, LM141, and LM92 (all IgM), but not control
rat IgM
mAb MECA79, recognize LM151 affinity-isolated material (Fig. 2 C ) confirming that all three mAbs react with the same 50 kD species, termed VMAP-1. The antibody LM151 was selected for all subsequent studies.
To determine if VMAP-1 is GPI anchored (23), bEnd3 cells were treated with PI-PLC before immunostaining and FACS® analysis. PI-PLC failed to cleave VMAP-1 from the surface of bEnd3 cells (not shown), indicating that VMAP-1 in not GPI anchored.
Expression of VMAP-1 on Arterial Endothelium In Vivo.To ask if VMAP-1 were displayed by aortic and other large
vessel endothelium in vivo, frozen sections of heart, kidney, lung, and lymphoid tissues were stained with LM151
or control IgM mAbs MECA79 or OZ42. As illustrated in
Fig. 3, anti-VMAP-1 mAb LM151 revealed focal staining
of the endothelial lining of valves in the aortic root (Fig. 3 A)
and the heart ventricle (Fig. 3 B) as well as of subsets of arteries and arterioles from many tissues including the heart
and kidney (Fig. 3 C). No reactivity was observed in capillary endothelium or with postcapillary high endothelial venules in peripheral lymph nodes or PP. VMAP-1 was
not restricted to vascular endothelium; LM151 also stained
subsets of bronchial and intestinal epithelial cells and stromal elements in lymphoid tissues, but did not stain any leukocytes (lymphocytes, monocytes, and neutrophils) isolated
from lymph nodes, spleen, or bone marrow, as assessed by
FACS® analysis (not shown).
To confirm luminal display of VMAP-1, mice were injected with LM151 or control IgM, killed, and perfused. Frozen sections of tissues were stained by immunofluorescence to detect retained antibody. Luminal, endothelial VMAP-1 reactivity was observed focally in the thoracic and abdominal aorta, heart ventricle, and in arterioles in kidney, heart and other tissues (not shown).
Constitutive and LPS or Cytokine Upregulated Expression of VMAP-1 by bEnd3 Cells.To assess the regulation of VMAP-1
in vitro, the relative amounts of VMAP-1 in lysates of unstimulated and LPS-stimulated bEnd3 cells were compared
by Western analysis (normalized to cell number). There
was significant superinduction of VMAP-1 after 24 h LPS
stimulation (Fig. 4 A). In addition, the surface expression of
LM151 by normal and stimulated bEnd3 cells was evaluated by flow cytometry; LM151 is constitutively expressed
by bEnd3 cells and variable increases in expression are observed after 4, 24, and 48 h of LPS stimulation (Fig. 4 B) as
well as IL-1 and TNF-
stimulation (data not shown). Results of a representative experiment illustrating superinduction of cell surface expression are shown in Fig. 4 B; however, the extent of superinduction assessed by flow cytometry
was variable, perhaps reflecting differences in endothelial
cell responsiveness or preinduction of VMAP-1 by the conditions of culture.
LM151 Blocks Binding of Monocytes to MM-LDL-stimulated Rabbit Aortic Endothelium In Vitro.
The fat-fed New Zealand
white rabbit is a widely used animal model in atherosclerosis research. The three anti-VMAP-1 mAbs cross-react with
rabbit aortic endothelium as illustrated immunohistologic staining with LM151 in Fig. 5 A. Furthermore, Western
blot analysis of RAEC indicate that anti-VMAP-1 mAb
LM151 recognizes a constitutively expressed 50-kD protein in
the rabbit (Fig. 5 B). The rabbit homologue of VMAP-1
can be superinduced in RAEC by MM-LDL stimulation, as shown by Western analysis (Fig. 5 B) and flow cytometric
analysis (Fig. 5 C), which further reveals that increased expression after MM-LDL stimulation is due to an increase in
surface expression by all cells including a subpopulation of
RAEC with extremely high expression.
This fortuitous cross-reactivity with rabbit endothelium
allows the evaluation of the role of VMAP-1 in well characterized in vitro assays relevant to atherogenesis. Berliner
et al. have previously shown that pretreatment of RAEC
with MM-LDL induces selective adhesiveness for monocytes with no increase in neutrophil binding, for example
(26). Monocyte adhesion in this model does not involve
E-selectin, VCAM-1, or intracellular adhesion molecule (ICAM)-1 (8). In contrast, incubation of MM-LDL-stimulated RAEC with LM151 blocks binding of human monocytes by ~95% (Fig. 6). Irrelevant control IgM mAb OZ42
and LM13.13 (an IgM
-binding negative control mAb)
both failed to block monocyte binding to control or MMLDL-stimulated RAEC (Fig. 6). Thus, VMAP-1 plays an
important role in monocyte binding to MM-LDL-stimulated endothelial cells, inhibiting an interaction that is independent of known adhesion pathways.
Blockade of Binding to Cholesterol-fed Rabbit Aorta Ex Vivo.
To extend these in vitro observations to an assay system
better reflecting the complex nature of the prelesional vessel, an ex vivo system was developed. The binding of
WEHI78/24 (mouse monocytoid) cells to intact aortic endothelium from control or fat-fed rabbits was compared.
After 2 wk on a high cholesterol diet, a threefold increase
in monocytoid cell binding was observed (Fig. 7). Preincubation of the aortic segments with LM151 dramatically inhibited WEHI78/24 binding, reducing adhesion nearly to
control levels (Fig. 7). LM13.13 (an IgM-binding negative control) failed to block WEHI78/24 binding to aortic
segments (n = 2; data not shown).
We have identified and characterized a novel vascular molecule, VMAP-1, involved in monocyte adhesion to stimulated mouse and rabbit aortic endothelium. Three independent mAbs against this molecule, LM151, LM141, and LM92, block binding of WEHI78/24 mouse monocytoid cells (but not neutrophils or lymphocytes) to cytokine- or LPS-stimulated mouse endothelial cells by >50% at both 4°C and RT. Anti-VMAP-1 mAbs abrogate enhanced binding of monocytes to MM-LDL-stimulated RAEC and cholesterol-enhanced binding to rabbit aortic endothelium. The ability of anti-VMAP-1 mAbs to block monocyte binding in these models of atherogenesis is unique, as mAbs to other known adhesion pathways display little or no effect on monocyte or monocytoid cell binding under conditions similar to those used here (RT or 37°C) to LPS-stimulated RAEC (6), to cytokine-stimulated bEnd3 (mouse endothelial) cells (our unpublished observations), or to MM-LDL-stimulated RAEC (8). These data suggest that VMAP-1 may play a critical role in monocyte adhesion to vascular endothelium under pathophysiological conditions such as hypercholesterolemia.
VMAP-1 is clearly distinct from known vascular adhesion molecules VCAM-1 (110 kD), the GPI-linked variant
of VCAM-1 (VCAM-GPI; 47 kD), ICAM-1 (95 kD),
ICAM-2 (55 kD), mucosal addressin cell adhesion molecule (MAdCAM)-1 (58-66 kD), E-selectin (115 kD), P-selectin (140 kD), IG9 antigen (105 kD; reference 27), and CD31 (platelet-endothelial cell adhesion molecule 1, 130 kD). First, the molecular weight (50 kD) distinguishes it
clearly from that of all but VCAM-GPI (28, 29), MAdCAM-1
(30), and ICAM-2 (31). Second, the expression pattern of
VMAP-1 in vivo (determined by immunohistochemistry
and immunofluorescence) is quite distinct from that of any
known adhesion molecule including MAdCAM-1 (21, 30)
and ICAM-2 (31). VMAP-1 is expressed by the endothelium on a subset of arteries and arterioles, but is not detectable on capillary endothelium or high endothelial venules
(HEV) in peripheral lymph nodes or PP, whereas ICAM-2
is constitutively expressed by almost all endothelial cells, including HEV in the human (32) and in the mouse (McEvoy,
L.M., and E.C. Butcher, unpublished observation), and vascular MAdCAM-1 expression is highly and selectively expressed by HEV in PP and mesenteric lymph node. Third,
VCAM-GPI is GPI-linked (29), whereas VMAP-1 is not.
Fourth, ICAM-1 and -2 are expressed by lymphocytes and
leukocyte cell lines (31), whereas anti-VMAP-1 mAbs fail
to stain mouse leukocytes by FACS® analysis. Fifth, the expression and superinduction of VMAP-1 on bEnd3 cells contrasts with the patterns of endothelial regulation of VCAM-1,
VCAM-GPI, ICAM-2, MAdCAM-1, the IG9 antigen, P-,
and E-selectin. VMAP-1 is constitutively expressed by bEnd3
cells and can be superinduced by TNF- and LPS while
VCAM-1 (9), VCAM-GPI (29), MAdCAM-1 (33), and
E-selectin (9, and Hubbe, M. and L.M. McEvoy, unpublished observation) are only expressed after stimulation by
LPS or cytokines. ICAM-2 is constitutively expressed and
is not upregulated by TNF-
stimulation of mouse endothelioma cell lines (31) including bEnd3 cells (McEvoy, L.M., unpublished observation). Furthermore, induced expression of E-selectin in mouse endotheliomas returns to
baseline levels 24 h after stimulation (9), unlike the sustained expression of VMAP-1. In contrast to the sustained
superinduced expression of VMAP-1 after cytokine stimulation, P-selectin is rapidly upregulated and subsequently
lost after cytokine stimulation by other mouse endothelioma cells (9). It is unlikely that anti-VMAP-1 mAbs recognize the mouse homologue of the IG9 antigen (27) since VMAP-1 is constitutively expressed by cultured RAEC,
whereas IG9 antigen is not, and the kinetics of induction of
the IG9 antigen (27) are distinct from those of VMAP-1.
Sixth, blocking mAbs to
4, the monocyte receptor for
VCAM-1 and CS-1-containing fibronectin, and
2 integrins, the monocyte receptors for ICAM-1 and -2, have no
inhibiting effect on induced WEHI78/24 binding. Seventh,
anti-VMAP-1 mAbs LM151, LM141, and LM92 fail to
stain mouse ICAM-1, VCAM-1, or MAdCAM-1 transfected Chinese hamster ovary and CD31 transfected COS
cells (McEvoy, L.M., and E.C. Butcher, personal observation). Finally, although several vascular adhesion receptors
are widely expressed by other cell types (especially ICAM-1
and VCAM-1), as is VMAP-1, here also the pattern of cell
type-specific staining with anti-VMAP-1 mAbs are distinct
as assessed immunohistologically. Together, these considerations indicate that VMAP-1 represents a novel element involved in monocyte vascular adhesion.
The fortuitous cross-reactivity of LM151 with rabbit VMAP-1 allowed evaluation of the role of VMAP-1 in monocyte binding to endothelium in several well-characterized models of atherosclerosis. As described above, Kim et al. have demonstrated that treatment of rabbit (and human) aortic endothelial cells with MM-LDL results in a monocyte-selective increase in adhesiveness without upregulation or involvement of VCAM-1, E-selectin, or ICAM-1 (8). Anti-VMAP-1 mAb LM151 abrogates binding of human monocytes to MM-LDL-stimulated RAEC. Furthermore, the enhanced binding of WEHI78/24 cells to the intact aortic endothelium after cholesterol feeding of New Zealand white rabbits is also abrogated by LM151 pretreatment of the endothelium in ex vivo binding assays. These data indicate that VMAP-1 plays a role in the monocyte-selective adhesiveness stimulated by MM-LDL or cholesterol feeding, suggesting that VMAP-1 may play a role in enhanced monocyte recruitment in the rabbit models studied here. Additional studies are required to identify a potential VMAP-1 homologue in humans.
Monocytes and monocytoid cells bind poorly in our assays to unstimulated endothelial cells in vitro and to normal aortic endothelium ex vivo. Significant binding is only observed after "activation" of the endothelium by LPS, cytokine, or MM-LDL stimulation in vitro, or by fat feeding in vivo. Superinduction of VMAP-1 by these studies may contribute to the upregulation of monocyte binding; however, significant constitutive expression of VMAP-1 by endothelium suggests that VMAP-1 is not likely the sole determinant of monocyte binding, but instead must function in conjunction with other adhesion and/or signaling molecules in regulation of monocyte interactions. This concept is consistent with our current model of leukocyte-endothelial interaction as a multistep process in which involvement of several adhesion and activating molecules in sequence is required for firm adhesion to endothelium and successful recruitment from the blood. In this context, it is relevant that stimulation of endothelium can induce expression of elements that can influence monocyte activation, adhesion, and/or diapedesis. For example, MM-LDL has been shown to induce expression of monocyte chemoattractant protein 1 (34), macrophage CSF (35), tissue factor (36), and a GRO homologue in RAEC (37), as well as a 105-kD adhesion protein for monocytes recognized by mAb IG9 (27). Many monocyte chemoattractants (including macrophage CSF, monocyte chemoattractant protein 1 [2, 38] and platelet activating factor [reviewed in reference 39]) are expressed or displayed by binding to extracellular matrix molecules in atherosclerotic lesions. Thus, through regulated constitutive or induced expression of adhesion molecules and accumulation and display of chemotactic or activating factors derived from the endothelium or other local cells (including smooth muscle cells and previously accumulated monocytes/macrophages), the endothelium over a lesion may become decorated by a combination of adhesion, activation, and chemotactic molecules that can act in concert to recruit monocytes. Our results suggest that a monocyte-selective role of VMAP-1, in combination with other adhesion pathways and chemotactic factors, may contribute to the inducible multistep cascade controlling monocyte-selective adhesion and extravasation in atherogenesis.
Address correspondence to Leslie M. McEvoy, Department of Pathology, L235, Stanford University, Stanford, CA 94305.
Received for publication 15 January 1997 and in revised form 31 March 1997.
L.M. McEvoy was a Senior Fellow of the American Heart Association, California Division, and the National Multiple Sclerosis Society during part of this work. H. Sun is supported by Public Health Service grant No. CAO9302 and a predoctoral award from the National Cancer Institute. P.S. Tsao was the recipient of a National Service Research Award. J.P. Cooke was a recipient of the Vascular Academic Award from the National Heart, Lung, and Blood Institute. This work was supported by grants from the National Institutes of Health and the Core Facilities of the Stanford Digestive Disease Center under DK38707.The authors thank Evelyn Resurrecion, Jean Jang, and June Twelves for technical assistance and Dr. M. Hubbe for comments on the manuscript.
1. | Ross, R.. 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (Lond.). 362: 801-809 [Medline]. |
2. | Valente, A.J., M.M. Rozek, E.A. Sprague, and C.J. Schwartz. 1992. Mechanisms in intimal monocyte-macrophage recruitment. A special role for monocyte chemotactic protein-1. Circulation. 86(Suppl.):III20-III25. |
3. | Carlos, T., N. Kovach, B. Schwartz, M. Rosa, B. Newman, E. Wayner, C. Benjamin, L. Osborn, R. Lobb, and J. Harlan. 1991. Human monocytes bind to two cytokine-induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1. Blood. 77: 2266-2271 [Abstract]. |
4. |
Carlos, T.M., and
J. M. Harlan.
1994.
Leukocyte-endothelial
adhesion molecules.
Blood.
84:
2068-2101
|
5. | Poston, R., D. Haskard, J. Coucher, N. Gall, and R. Johnson-Tidey. 1992. Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. Am. J. Pathol. 140: 665-673 [Abstract]. |
6. | Cybulsky, M.I., and M.A. Gimbrone Jr.. 1991. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science (Wash. DC). 251: 788-791 [Medline]. |
7. | Davies, M.J., J.L. Gordon, A.J. Gearing, R. Pigott, N. Woolf, D. Katz, and A. Kyriakopoulos. 1993. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J. Pathol. 171: 223-229 [Medline]. |
8. | Kim, J.A., M.C. Territo, E. Wayner, T.M. Carlos, F. Parhami, C.W. Smith, M.E. Haberland, A.M. Fogelman, and J.A. Berliner. 1994. Partial characterization of leukocyte binding molecules on endothelial cells induced by minimally oxidized LDL. Arterioscler. Thromb. 14: 427-433 [Abstract]. |
9. | Hahne, M., U. Jager, S. Isenmann, R. Hallmann, and D. Vestweber. 1993. Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes. J. Cell Biol 121: 655-664 [Abstract]. |
10. | Li, H., M.I. Cybulsky, M.A. Gimbrone Jr., and P. Libby. 1993. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler. Thromb. 13: 197-204 [Abstract]. |
11. | Butcher, E.C.. 1991. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 67: 1033-1036 [Medline]. |
12. | Butcher, E.C., and L.J. Picker. 1996. Lymphocyte homing and homeostasis. Science (Wash. DC). 272: 60-66 [Abstract]. |
13. | Shimizu, Y., W. Newman, Y. Tanaka, and S. Shaw. 1992. Lymphocyte interactions with endothelial cells. Immunol. Today. 13: 106-112 [Medline]. |
14. | Imhof, B.A., and D. Dunon. 1995. Leukocyte migration and adhesion. Adv. Immunol. 58: 345-416 [Medline]. |
15. | Springer, T.A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 76: 301-314 [Medline]. |
16. | Montesano, R., M.S. Pepper, U. Mohle-Steinlein, W. Risau, E.F. Wagner, and L. Orci. 1990. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell. 62: 435-445 [Medline]. |
17. |
Walker, E.B.,
L.L. Lanier, and
L.L. Warner.
1982.
Characterization and functional properties of tumor cell lines in accessory cell replacement assays.
J. Immunol.
128:
852-859
|
18. | Berliner, J.A., M. Territo, L. Almada, A. Carter, E. Shafonsky, and A.M. Fogelman. 1986. Monocyte chemotactic factor produced by large vessel endothelial cells in vitro. Arteriosclerosis. 6: 254-258 [Abstract]. |
19. | Watson, A.D., J.A. Berliner, S.Y. Hama, B.N. La, Du, K.F. Faull, A.M. Fogelman, and M. Navab. 1995. Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein. J. Clin. Invest. 96: 2882-2891 [Medline]. |
20. | Parhami, F., Z.T. Fang, A.M. Fogelman, A. Andalibi, M.C. Territo, and J.A. Berliner. 1993. Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J. Clin. Invest. 92: 471-478 [Medline]. |
21. | Streeter, P.R., B.T. Rouse, and E.C. Butcher. 1988. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J. Cell Biol 107: 1853-1862 [Abstract]. |
22. | Picker, L.J., S.A. Michie, L.S. Rott, and E.C. Butcher. 1990. A unique phenotype of skin-associated lymphocytes in humans. Preferential expression of the HECA-452 epitope by benign and malignant T cells at cutaneous sites. Am. J. Pathol. 136: 1053-1068 [Abstract]. |
23. | Low, M.G., and A.R. Saltiel. 1988. Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science (Wash. DC). 239: 268-275 [Medline]. |
24. |
Roberts, W.L.,
J.J. Myher,
A. Kuksis,
M.G. Low, and
T.L. Rosenberry.
1988.
Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to
phosphatidylinositol-specific phospholipase C.
J. Biol. Chem.
263:
18766-18775
|
25. | Jutila, M.A., D.M. Lewinsohn, E.L. Berg, and E.C. Butcher. 1988. Homing receptors in lymphocyte, neutrophil, and monocyte interactions with endothelial cells. In Leukocyte Adhesion Molecules: Structure, Function and Regulation. T.A. Springer, editor. Springer-Verlag, New York. 227-235. |
26. | Berliner, J.A., M.C. Territo, A. Sevanian, S. Ramin, J.A. Kim, B. Bamshad, M. Esterson, and A.M. Fogelman. 1990. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J. Clin. Invest. 85: 1260-1266 [Medline]. |
27. | Calderon, T.M., S.M. Factor, V.B. Hatcher, J.A. Berliner, and J.W. Berman. 1994. An endothelial cell adhesion protein for monocytes recognized by monoclonal antibody IG9. Expression in vivo in inflamed human vessels and atherosclerotic human and Watanabe rabbit vessels. Lab. Invest. 70: 863-849 . |
28. | Kinashi, T., Y. St. Pierre, and T.A. Springer. 1995. Expression of glycophosphatidylinositol-anchored and -non-anchored isoforms of vascular cell adhesion molecule 1 in murine stromal and endothelial cells. J. Leukocyte Biol. 57: 168-173 [Abstract]. |
29. | Terry, R.W., L. Kwee, J.F. Levine, and M.A. Labow. 1993. Cytokine induction of an alternatively spliced murine vascular cell adhesion molecule (VCAM) mRNA encoding a glycosylphosphatidylinositol-anchored VCAM protein. Proc. Natl. Acad. Sci. USA. 90: 5919-5923 [Abstract]. |
30. | Streeter, P.R., E.L. Berg, B.T.N. Rouse, R.F. Bargatze, and E.C. Butcher. 1988. A tissue-specific endothelial cell molecule involved in lymphocyte homing. Nature (Lond.). 331: 41-46 [Medline]. |
31. |
Xu, H.,
J.K. Bickford,
E. Luther,
C. Carpenito,
F. Takei, and
T.A. Springer.
1996.
Characterization of murine intercellular
adhesion molecule-2.
J. Immunol.
156:
4909-4914
|
32. | de Fougerolles, A.R., S.A. Stacker, R. Schwarting, and T.A. Springer. 1991. Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. J. Exp. Med. 174: 253-267 [Abstract]. |
33. |
Sikorski, E.E.,
R. Hallmann,
E.L. Berg, and
E.C. Butcher.
1993.
The Peyer's patch high endothelial receptor for lymphocytes, the mucosal vascular addressin, is induced on a murine endothelial cell line by tumor necrosis factor-alpha and
IL-1.
J. Immunol.
151:
5239-5250
|
34. | Navab, M., S.S. Imes, S.Y. Hama, G.P. Hough, L.A. Ross, R.W. Bork, A.J. Valente, J.A. Berliner, D.C. Drinkwater, H. Laks, et al . 1991. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J. Clin. Invest. 88: 2039-2046 [Medline]. |
35. | Rajavashisth, T.B., A. Andalibi, M.C. Territo, J.A. Berliner, M. Navab, A.M. Fogelman, and A.J. Lusis. 1990. Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins. Nature (Lond.). 344: 254-257 [Medline]. |
36. | Drake, T.A., K. Hannani, H.H. Fei, S. Lavi, and J.A. Berliner. 1991. Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells. 138:601-607. |
37. | Schwartz, D., A. Andalibi, L. Chaverri-Almada, J.A. Berliner, T. Kirchgessner, Z.T. Fang, P. Tekamp-Olson, A.J. Lusis, C. Gallegos, A.M. Fogelman, et al . 1994. Role of the GRO family of chemokines in monocyte adhesion to MM-LDL- stimulated endothelium. J. Clin. Invest. 94: 1968-1973 [Medline]. |
38. | Yla-Herttuala, S., B.A. Lipton, M.E. Rosenfeld, T. Sarkioja, T. Yoshimura, E.J. Leonard, J.L. Witztum, and D. Steinberg. 1991. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc. Natl. Acad. Sci. USA. 88: 5252-5256 [Abstract]. |
39. | Yla-Herttuala, S.. 1992. Gene expression in atherosclerotic lesions. Herz. 17: 270-276 [Medline]. |