© 1997 American Heart Association, Inc.
Articles |
Molecular Determinants of Oxidized Low-Density Lipoprotein–Induced Leukocyte Adhesion and Microvascular Dysfunction
From the Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, and Alkermes, Inc, Cambridge, Mass.
Correspondence to Dr D. Neil Granger, Department of Physiology and Biophysics, LSU Medical Center, 1501 Kings Hwy, PO Box 33932, Shreveport, LA 71130-3932. E-mail dgrang{at}lsumc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Oxidized low-density lipoproteins (oxLDL) have been implicated in the leukocyte recruitment and microvascular dysfunction associated with atherosclerosis. The objectives of this study were to define the adhesion molecules that mediate oxLDL-induced leukocyte–endothelial cell adhesion and to determine whether leukocyte–endothelial cell adhesion contributes to the endothelial barrier dysfunction elicited by oxLDL. Leukocyte–endothelial cell adhesion and emigration, albumin extravasation, and mast cell degranulation were monitored in rat mesentery in response to native LDL (nLDL) or copper-oxidized LDL (oxLDL). Intra-arterial infusion of oxLDL but not nLDL elicited increases in leukocyte adherence and emigration, mast cell degranulation, and albumin leakage. The oxLDL-induced leukocyte adherence/emigration was attenuated by pretreatment with monoclonal antibodies directed against CD11/CD18, intercellular adhesion molecule-1, P-selectin, and L-selectin but not by pretreatment with a nonbinding monoclonal antibody. The albumin leakage and mast cell degranulation responses were attenuated by all of the same monoclonal antibodies except L-selectin. In addition, a peptide previously shown to inhibit leukocyte–endothelial cell adhesion in vitro also attenuated leukocyte adherence and mast cell degranulation in this model. These findings implicate CD11/CD18, L-selectin, intercellular adhesion molecule-1, and P-selectin in the leukocyte recruitment elicited by oxLDL and invoke a role for adherent leukocytes in the accompanying increase in mast cell degranulation and albumin leakage.
Key Words: vascular permeability • selectins • ß2 integrins • mast cells
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Oxidized LDLs have been implicated in the development of atherosclerotic lesions in large arterial vessels and in the microvascular dysfunction associated with hypercholesterolemia.1 2 3 Intravital microscopic studies of tissues exposed to copper-oxidized human LDLs have revealed an acute inflammatory response that is characterized by enhanced albumin leakage and an increased adherence and emigration of leukocytes in postcapillary venules.4 5 It has been proposed that leukocyte–endothelial cell adhesion may be a critical step in the development of atherosclerotic lesions in large vessels and in the more subtle alterations in microvessel function elicited by hypercholesterolemia.2 6 These observations have raised the level of interest in defining the leukocyte and endothelial cell adhesion glycoproteins that mediate oxLDL-induced leukocyte adherence and emigration in the microcirculation. Although the contribution of CD11b/CD18 and P-selectin to oxLDL-induced leukocyte–endothelial cell adhesion in arterioles and postcapillary venules has been addressed in different reports,7 8 a systematic analysis of the relative roles of different leukocyte and endothelial cell adhesion molecules to this inflammatory process has not been undertaken to date. Thus, a major objective of the present study was to assess the contributions of CD11/CD18, L-selectin, P-selectin, and ICAM-1 to oxLDL-induced leukocyte–endothelial cell adhesion in rat mesenteric venules.
We also examined the effect on oxLDL-induced inflammatory responses of a novel bacterial peptide (herein called F20) derived from the FHA of Bordetella pertussis.9 FHA promotes bacterial adherence to human monocytes/macrophages through an interaction with CD11b/CD18.10 11 12 This complex bacterial adhesin, which contains the eukaryotic cell adhesion motif Arg-Gly-Asp (RGD), appears to structurally and functionally mimic one or more cell adhesion molecules. Some evidence suggests that the RGD sequence on FHA mediates the aforementioned bacterial adherence to monocyte CD11b/CD18 through an interaction with a leukocyte signal-transduction complex.11 12 This receptor pair, called LRI and IAP, recognizes RGD and regulates some integrin functions.13 14 15 A 20-mer FHA peptide containing the RGD triplet (F20) has been shown to modulate ligand binding by CD11b/CD18 on leukocytes in a manner similar to that seen with antibodies against LRI/IAP.9 11 This bacterial peptide inhibits leukocyte–endothelial cell adhesion in vitro and impedes recruitment of leukocytes into cerebrospinal fluid of animals with experimental meningitis.9
Previous studies have shown that the oxLDL-induced recruitment of rolling, firmly adherent, and emigrated leukocytes is accompanied by an enhanced rate of albumin extravasation from postcapillary venules as well as an increased number of degranulated mast cells in the adjacent perivenular interstitium.5 On the basis of experiments demonstrating an attenuating action of nitric oxide–donating agents on oxLDL-induced leukocyte–endothelial cell adhesion, albumin leakage, and mast cell degranulation, it has been suggested that these temporally related intravascular and extravascular inflammatory reactions may be mechanistically linked. Hence, a second objective of the present study was to determine whether inhibiting the recruitment of adherent and emigrated leukocytes with MABs directed against adhesion molecules leads to an attenuation of the enhanced albumin leakage and mast cell degranulation elicited by oxLDL.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
LDL Isolation and Oxidation
Fresh blood was obtained from nonfasted, healthy, normolipidemic male and female human subjects and placed into tubes containing 1.5 mg EDTA (Na2) per milliliter of blood. LDL fractions (d=1.019 to 1.063 g/mL) were isolated by density gradient ultracentrifugation16 from pooled plasma. The LDL sample was extensively dialyzed against PBS at 4°C without EDTA, at a final concentration of 1 mg LDL protein/mL. Protein was determined by the method of Lowry et al,17 with bovine serum albumin used as a standard. Oxidation of LDL was initiated by addition of CuSO4 to a final concentration of 10 µmol/L and incubated at 37°C for 24 hours. The reaction was terminated by the addition of EDTA (final concentration, 0.01 mmol/L). The mixture was then dialyzed against PBS with EDTA. nLDL was prepared by dialyzing the sample against PBS with EDTA (final concentration, 0.01 mmol/L). All lipoprotein fractions were stored at 4°C and used within 3 weeks. We determined the efficacy of the oxidation procedure by analyzing the presence of thiobarbituric acid–reactive substances, expressed as MDA equivalents,18 and by agarose gel electrophoresis.19 In a typical experiment, nLDL contained 0.4±0.1 nmol MDA Eq/mg protein and oxLDL contained 15.0±1.6 nmol MDA Eq/mg protein. The ratio of the relative electrophoretic motility of oxLDL to nLDL was 1.8:1.0. Previous studies that used the limulus assay have ruled out the possibility of endotoxin contamination of the so-prepared nLDL and oxLDL.8 The ability of copper-oxidized LDL to promote leukocyte–endothelial cell adhesion, albumin leakage, and mast cell degranulation in rat mesenteric venules was recently compared with that of other forms of oxLDL.20
Surgical Procedure
Seventy-five male Sprague-Dawley rats (200 to 250 g) were
maintained on a purified laboratory diet and fasted for 24 hours before
each experiment. The animals were initially anesthetized with an
intraperitoneal injection of 140 mg thiobutabarbital (Inactin) per
kilogram of body weight. A tracheotomy was performed on each rat to
facilitate breathing throughout the experiment. The right carotid
artery was cannulated, and systemic arterial pressure was measured with
a Statham P23A pressure transducer connected to the carotid artery
cannula. Systemic blood pressure and heart rate were continuously
recorded with a Grass Instruments physiological recorder. A midline
abdominal incision was made to allow a section of mesentery from the
small intestine to be exteriorized. The aorta was cannulated with the
tip of the cannula placed at the bifurcation of the SMA. The left
jugular vein was also cannulated for drug administration. All exposed
tissue was moistened with saline-soaked gauze to minimize evaporation
and tissue damage.
Intravital Microscopy
Rats were placed in a supine position on an adjustable acrylic
plastic microscope stage, and the mesentery was prepared for
microscopic observation as described previously.21
Briefly, the mesentery was draped over a nonfluorescent coverslip that
allowed for observation of a 2-cm2 segment of tissue. The
exposed bowel wall was covered with Saran Wrap (Dow Chemical Co), then
the mesentery was superfused (bathed at a constant rate) with BBS
(37°C, pH 7.4) that was bubbled with a mixture of 5%
CO2, 95% N2, which exposes mesenteric tissue
to an oxygen tension of 
40 mm Hg.
An inverted microscope (Diaphot 300, Nikon) with a 40x objective lens (Fluor; Nikon) was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a 12-V, 100-W direct current–stabilized light source. A video camera (VK-C150; Hitachi) mounted on the microscope projected the image onto a color monitor (PMV-2030; Sony), and the image was recorded by use of a videocassette recorder (BR-S601MU; JVC). A video time-date generator (WJ810; Panasonic) projected the time, date, and stopwatch functions onto the monitor.
Single unbranched venules with diameters ranging between 25 and 35 µm and length >150 µm were selected for study. Venular diameter was measured either on-line or off-line with a video caliper (Microcirculation Research Institute, Texas A&M University). RBC centerline velocity was measured in venules with an optical Doppler velocimeter (Microcirculation Research Institute). The velocimeter was calibrated against a rotating glass disk coated with RBCs. Venular blood flow was calculated from the product of mean RBC velocity (Vmean=Centerline Velocity/1.6)22 and microvascular cross-sectional area, assuming cylindrical geometry. Wall shear rate (¥) was calculated on the basis of the newtonian definition: ¥=8(Vmean/D), where D is diameter.
The number of adherent leukocytes was determined off-line during
playback of the videotaped images. A leukocyte was considered to be
adherent to venular endothelium if it remained stationary for a period
30 seconds.23 Adherent leukocytes were expressed as the
number per 100 µm length of venule. The number of emigrated
leukocytes was also determined off-line during playback of videotaped
images. Any interstitial leukocytes present in the mesentery at the
onset of the experiment were subtracted from the total number of
leukocytes that accumulated during the course of the experiment.
Leukocyte emigration was expressed as the number per microscopic field
(2.12x10-2 mm2). Rolling
leukocytes were defined as those white blood cells that moved at a
velocity less than that of RBCs in the same stream of blood. The flux
of rolling leukocytes (FWBC) was determined by the number
of rolling leukocytes that crossed a point within a given period of
time. FWBC was expressed as the number of leukocytes per
second. Leukocyte rolling velocity (VWBC) was determined
from the time required for a leukocyte to roll a given distance along
the length of the venule. The number of rolling leukocytes per 100
µm venule length was calculated as
FWBC/VWBC.24 To visualize mast
cells surrounding the mesenteric microvasculature, 0.1 g% toluidine
blue was added onto the mesentery at the end of each
experiment.25 The number of intact and degranulated mast
cells was determined, and the percentage of degranulated mast cells was
calculated.
To quantify albumin leakage across mesenteric venules, 50 mg/kg of FITC-labeled bovine albumin (Sigma Chemical Co) was administered intravenously to the animals 15 minutes before each experiment.5 26 Fluorescence intensity (excitation wavelength, 420 to 490 nm; emission wavelength, 520 nm) was detected with a silicon-intensified target camera (C2400-08, Hamamatsu Photonics). The fluorescence intensity of FITC-albumin within three segments of the venule under study and in three contiguous areas of perivenular interstitium within 10 to 50 µm of the venular wall was measured at various times after administration of FITC-albumin with a computer-assisted digital imaging processor (NIH Image 1.35 on a Macintosh computer, Quadra 840AV). An index of vascular albumin leakage was determined from the ratio of interstitial to venular intensity of FITC-albumin fluorescence at specific intervals (10 minutes) after infusion of oxLDL. All data presented for albumin leakage represent this ratio at 30 and 60 minutes after injection of oxLDL.5 26
Experimental Protocols
After all parameters measured on-line were in a steady state,
images from the mesenteric preparation were recorded on videotape for
10 minutes. Immediately thereafter, either nLDL or oxLDL was infused
into the SMA at a rate of 1 mg LDL
protein·kg-1·min-1
for 5 minutes with the mesentery superfused with BBS (2 mL/min), and
repeat measurements were obtained at regular intervals for 60 minutes.
In some experiments, the same protocol was used but the animals were
pretreated (15 minutes before control measurements) with an MAB
directed against either CD18 (CL26, 100 µg per rat),27
ICAM-1 (IA29, 2 mg/kg),28 29 L-selectin (HRL3, 1
mg/kg),30 P-selectin (PB1.3, 2 mg/kg),31 or a
nonbinding antibody for P-selectin (P-23, 2 mg/kg).32 At
the doses used, none of the MABs caused leucopenia. Another group of
experiments tested peptide F20 (RGDPHQGVLAQGDIIMDAKG, corresponding to
residues 1097 to 1116 of the FHA of Bordetella
pertussis9 ; custom synthesized at QCB, Inc) and
peptide F23 (IDNPQHGGAMRDLGIAGGKD, scrambled version of F20; QCB, Inc).
A single peptide was added to the superfusate at the beginning of the
experiment, and the concentration was maintained at 50 µmol/L
throughout the entire experiment. In addition, a bolus (200 µg) of
either F20 or F23 was injected into the rat SMA 5 minutes after the
infusion of oxLDL. Otherwise, the same protocol as used with the MABs
was followed. All mean values presented herein represent data derived
from 6 animals, except for the control and oxLDL-treated groups, which
consisted of 12 animals each, and the nLDL-treated group, which
consisted of 9 animals.
Statistics
The data obtained in this study are expressed as mean±SE. The
data were analyzed by the use of standard statistical analysis, ie,
one-way ANOVA with Scheffé's post hoc test and Student's
t test. Statistical significance was set at
P<.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Fig 1
summarizes the effects of MABs directed
against different leukocyte (CD18 and L-selectin) and endothelial cell
(ICAM-1 and P-selectin) adhesion molecules on oxLDL-induced adherence
of leukocytes in rat mesenteric venules. The recruitment of adherent
leukocytes induced by oxLDL was significantly attenuated by
pretreatment with different MABs; ie, MABs against the leukocyte
adhesion molecules CD18 and L-selectin reduced adherence by 83% and
59%, respectively, whereas MABs against the endothelial cell adhesion
molecules ICAM-1 and P-selectin reduced adherence by 78% and 56%,
respectively. However, no significant changes in leukocyte adherence
were noted in animals receiving a nonbinding MAB (P-23).
|
A similar pattern of effectiveness in reducing leukocyte emigration was
observed with the different MABs (Fig 2); ie, MABs
against leukocyte adhesion molecule CD18 and L-selectin reduced
emigration by 81% and 58%, respectively, whereas MABs against
endothelial cell adhesion molecule ICAM-1 and P-selectin reduced
emigration by 71% and 55%, respectively. The nonbinding MAB had no
effect.
|
Table 1 summarizes the effects of the different MABs and
peptides on the recruitment of rolling leukocytes elicited by oxLDL.
Although infusion of either nLDL or oxLDL increased the number of
rolling leukocytes, only oxLDL elicited a significant response. The
oxLDL-induced recruitment of rolling leukocytes was significantly
reduced by pretreatment with MABs against either CD18 (85% at both 30
and 45 minutes after oxLDL infusion) or ICAM-1 (81% and 80%,
respectively) but not P-selectin, L-selectin, or a nonbinding antibody.
Peptides F20 and F23 failed to alter the rolling response to oxLDL.
|
Fig 3 illustrates that the oxLDL-induced increase in
albumin leakage across postcapillary venules was significantly
attenuated by MABs directed against CD18 (74% to 76%), ICAM-1 (57%
to 80%), and P-selectin (69% to 76%) at both 30 and 60 minutes after
the infusion of oxLDL. The L-selectin and nonbinding MABs did not
reduce oxLDL-induced albumin leakage.
|
Fig 4 summarizes the effects of different MABs on the
mast cell degranulation response observed 60 minutes after infusion of
oxLDL. The oxLDL-induced mast cell degranulation was significantly
attenuated by pretreatment with MABs against CD18, ICAM-1, or
P-selectin (by 83%, 37%, and 62%, respectively). However, no
significant changes in mast cell degranulation were noted in animals
pretreated with either an L-selectin–specific MAB or a nonbinding
MAB.
|
Table 2 summarizes the effect of peptides F20 and F23 on
leukocyte adhesion, emigration, and mast cell degranulation induced by
oxLDL. Peptide F20 significantly reduced oxLDL-induced leukocyte
adherence (53%) and mast cell degranulation (59%) at 60 minutes after
oxLDL infusion, whereas the scrambled control peptide F23 had no
effect. Neither peptide significantly affected oxLDL-induced leukocyte
emigration.
|
Venular diameter, RBC velocity, and wall shear rate were not
significantly altered by any treatment (Table 3),
indicating that the protective effects exerted by some of the different
MABs and by peptide F20 were not secondary to microhemodynamic changes.
Systemic blood pressure was also unaffected by infusion of either nLDL
or oxLDL.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
There is growing evidence that confers a key role for both leukocytes and oxLDL in the vascular dysfunction associated with hypercholesterolemia and atherosclerosis.2 3 It is widely believed that leukocytes, which are activated and become adherent to endothelial cells in response to oxLDL, mediate the vascular injury (fatty streaks) and microvessel dysfunction that are characteristic features of atherogenesis. The microvascular dysfunction in atherosclerosis and hypercholesterolemia is manifested in both arterioles and postcapillary venules, with arterioles exhibiting an impaired endothelium-dependent vasodilation, whereas venules leak excessive quantities of plasma proteins.33 In both instances, it is assumed that endothelial cell injury mediated by firmly adherent leukocytes accounts for the altered microvascular function. Although this view has gained considerable support from studies4 5 that focus on endothelium-dependent arterial vessel abnormalities, there is little direct evidence to support the contribution of such a mechanism in the venular dysfunction elicited by oxLDL and atherogenesis. To better define the contributions of leukocytes to oxLDL-induced alterations in the function of postcapillary venules, we systematically evaluated the contribution of different leukocyte and endothelial cell adhesion molecules to the leukocyte recruitment, mast cell degranulation, and albumin leakage responses elicited in rat mesenteric venules by local intra-arterial infusion of oxLDL.
The leukocyte and endothelial cell–surface glycoproteins that mediate
the adhesive interactions induced by oxLDL have been studied using
endothelial cell monolayers exposed to oxLDL. oxLDL elicits an
increased adherence of neutrophils to endothelial cell monolayers that
is inhibited by MABs directed against either the 
- (CD11a
and CD11b) or ß-subunits (CD18) of the leukocyte adhesion
glycoprotein complex CD11/CD18.34 The inhibitory effects
of the individual CD11a- and CD11b-specific MABs were less than that
observed after immunoneutralization of the common ß-subunit. It has
also been shown that an MAB directed against CD11b/CD18 largely
abolishes the oxLDL-induced leukocyte rolling and adherence observed in
hamster dorsal skinfold arterioles and postcapillary
venules.8 Our findings with a CD18-specific MAB in rat
mesenteric venules confirm these published in vitro and in vivo
observations with oxLDL and support the view that the
ß2-integrins on leukocytes are an important determinant
of the leukocyte adherence and emigration elicited by oxLDL.
Along with the antibodies directed against CD11/CD18, we tested the synthetic peptide F20 that was derived from the FHA of B. pertussis.9 This peptide contains the cell adhesion motif RGD, which on FHA is believed to interact with a leukocyte signal-transduction complex consisting of LRI and IAP.10 11 12 13 14 15 Some evidence suggests that this interaction modulates CD11b/CD18 activity and facilitates FHA-mediated bacterial binding to monocytes via this ß2-integrin.10 11 12 Recently, we9 demonstrated in vitro that peptide F20 could inhibit neutrophil adherence and transendothelial migration in response to tumor necrosis factor stimulation of endothelial cells. In experimental meningitis in rabbits, in which the CD18 integrins have been shown to be critically involved in leukocyte recruitment into the central nervous system,35 F20 administration significantly reduced cerebrospinal fluid leukocytosis.9 Additionally, in a rat model of transient middle cerebral artery occlusion,36 F20 administration significantly reduced ischemic cell damage and inhibited neutrophil influx into the ischemic brain lesion, with no detectable effect on the number of circulating leukocytes. Our finding in the present study that peptide F20 significantly inhibits leukocyte adherence to endothelial cells supports the notion that CD11b/CD18 plays a role in the leukocyte inflammatory response provoked by oxLDL.
The results of the present study also invoke a role for another leukocyte adhesion glycoprotein in oxLDL-induced leukocyte recruitment, ie, L-selectin. We observed that an L-selectin MAB reduced leukocyte adherence and emigration by 55% to 60%, which was less than the inhibition (80% to 85%) afforded by a CD18 MAB, suggesting that CD11/CD18 makes a larger contribution to this recruitment process. The available data in the literature suggest that L-selectin likely contributes to oxLDL-induced leukocyte recruitment by mediating the low-affinity leukocyte rolling that precedes the firm attachment of leukocytes to endothelial cells.23 It has been shown37 that soon after activation of leukocytes, L-selectin mediates rolling and then is shed from the leukocyte surface. Indeed, it has been shown38 that incubation of isolated neutrophils with oxLDL results in the shedding of L-selectin from the neutrophil surface. The observation that the L-selectin MAB does not significantly attenuate the oxLDL-induced leukocyte rolling and does not reduce leukocyte adherence/emigration to the same level (assuming rolling is a prerequisite for firm adherence and emigration) as a CD18-specific MAB suggests that additional adhesion molecules are likely to participate in oxLDL-induced leukocyte rolling.
Our findings indicate that two endothelial cell–associated adhesion molecules contribute to oxLDL-induced leukocyte recruitment in postcapillary venules, ie, P-selectin and ICAM-1. P-selectin, which mediates leukocyte rolling in vivo,39 is present in Weibel-Palade bodies internalized within endothelial cells and can be rapidly (5 to 10 minutes) translocated to the cell surface, where it can exert an adhesive action. Recently, it has been reported40 that oxLDL causes a prolonged (>1 hour) increase in P-selectin expression on cultured human umbilical vein endothelial cells. Our observation that P-selectin MABs are effective in attenuating oxLDL-induced leukocyte adherence and emigration but not leukocyte rolling in postcapillary venules are consistent with previous studies7 suggesting that leukocytes may roll on unidentified molecules other than P-selectin and L-selectin. Nevertheless, the results of the present study support a role for increased P-selectin expression in leukocyte adhesion and emigration in microvessels exposed to oxLDL.
The level of constitutive expression of ICAM-1 on endothelial cells in most tissues is quite high.41 This adhesion glycoprotein, which serves as a counterreceptor for CD11/CD18 on leukocytes, has been implicated in the firm adhesion and emigration of leukocytes observed in several models of inflammation.26 42 43 The present study provides the first evidence that invokes a role for ICAM-1 in oxLDL-induced leukocyte rolling and adhesion in postcapillary venules. Because ICAM-1 requires 2 to 3 hours for significant increases in surface expression after endothelial cell activation,44 it is likely that constitutively expressed ICAM-1 participates in oxLDL-induced leukocyte recruitment by serving as a ligand for the CD11/CD18 that is expressed on the surface of activated leukocytes. Although it remains unclear whether oxLDL can elicit an increased expression of ICAM-1 on endothelial cells, preliminary studies in our laboratory indicate that oxLDL does not increase ICAM-1 expression on endothelial cells in the gut up to 5 hours after systemic administration, which is consistent with the responses noted for cultured endothelial cells exposed to minimally oxidized LDL.45 However, it has also been reported that exposure of porcine coronary arteries to oxLDL induces ICAM-1 expression, with an accompanying increase in neutrophil adhesion.46 Furthermore, in another study,47 it was shown that oxLDL enhances tumor necrosis factor–induced ICAM-1 expression in cultured human aortic endothelial cells.
Previous work in our laboratory5 demonstrated that local intra-arterial infusion of oxLDL elicits an increased albumin leakage in postcapillary venules of rat mesentery. Our findings with MABs directed against different leukocyte and endothelial cell adhesion molecules provide new insight concerning the contribution of leukocyte adhesion to the endothelial barrier dysfunction elicited by oxLDL. The results of the present study clearly demonstrate that the vascular leakage response to oxLDL is significantly blunted by treatment with MABs directed against either CD18, ICAM-1, or P-selectin but not L-selectin or a nonbinding MAB. Overall, these observations are consistent with previous reports25 showing a strong positive correlation between the magnitude of the albumin leakage response and the number of adherent and emigrated leukocytes in mesenteric venules. Hence, our findings indicate that the firm adhesion and/or emigration of leukocytes across venular endothelium is a requirement for the enhanced albumin extravasation elicited by oxLDL.
Although most of the binding MABs to adhesion molecules were effective in reducing both leukocyte adherence/emigration and albumin leakage, the MAB directed against L-selectin only affected the adhesion responses. Although a definitive explanation for the inability of L-selectin to inhibit the albumin leakage response is not readily available, it may be related to oxLDL-induced mast cell degranulation. An interesting and novel observation in the present study was the ability of different adhesion molecule–specific MABs and peptide F20 to attenuate oxLDL-induced mast cell degranulation. Mast cells are known to release a variety of substances, including platelet-activating factor, histamine, leukotrienes, and superoxide anion, each of which can elicit leukocyte–endothelial cell adhesion and vascular protein leakage.48 The demonstration that adhesion molecule–specific MABs and peptide F20, which is believed to modulate CD11b/CD18 activity, all blunt mast cell degranulation suggests that adherent or emigrated leukocytes release substances that promote mast cell degranulation. Therefore, it is conceivable that products of mast cell degranulation that are released in response to leukocyte recruitment account for the increased albumin leakage. This could explain why the L-selectin MAB, which did not attenuate oxLDL-induced mast cell degranulation, also does not blunt the albumin leakage response.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by a grant from the National Heart, Lung, and Blood Institute (HL-26441). The authors wish to thank Dr Scott Putney for helpful discussions.
Dr Starzyk's current address is Genetics Institute, Inc, 87 Cambridge Park Dr, Cambridge, MA 02140.
Received March 31, 1996; accepted July 12, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Harrison DG. Endothelial dysfunction in atherosclerosis. Basic Res Cardiol. 1994;89:87-102.
- Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]
- Steinberg D, Parthasarathy TE, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modification of low-density lipoprotein that increases its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]
- Lehr HA, Hubner C, Nolte D, Finckh B, Beisiegel U, Kohlschutter A, Messmer K. Oxidatively modified human low-density lipoprotein stimulates leukocyte adherence to the microvascular endothelium in vivo. Res Exp Med. 1991;191:85-90. [Medline] [Order article via Infotrieve]
-
Liao L, Granger DN. Modulation of oxidized
low-density lipoprotein-induced microvascular dysfunction by nitric
oxide. Am J Physiol. 1995;268:H1643-H1650.
[Abstract/Free Full Text] -
Gauthier TW, Scalia R, Murohara T, Guo JP, Lefer
AM. Nitric oxide protects against leukocyte-endothelium
interactions in the early stages of hypercholesterolemia.
Arterioscler Thromb Vasc Biol. 1995;15:1652-1659.
[Abstract/Free Full Text] - Lehr HA, Olofsson AM, Carew TE, Vajkoczy P, Andrian UHV, Hubner C, Berndt MC, Steinberg D, Messmer K, Arfors KE. P-selectin mediates the interaction of circulating leukocytes with platelets and microvascular endothelium in response to oxidized lipoprotein in vivo. Lab Invest. 1994;71:380-386. [Medline] [Order article via Infotrieve]
- Lehr HA, Krober M, Hubner C, Vajkoczy P, Menger MD, Nolte D, Kohlschutter A, Messmer K. Stimulation of leukocyte/endothelium interaction by oxidized low-density lipoprotein in hairless mice: involvement of CD11b/CD18 adhesion receptor complex. Lab Invest. 1993;68:388-395. [Medline] [Order article via Infotrieve]
- Rozdzinski E, Spellerberg B, van der Flier M, Bhattacharyya C, Hoepelman AI, Moran MA, Jarpe A, Putney SD, Starzyk RM, Tuomanen E. Peptide from a prokaryotic adhesin blocks leukocyte migration in vitro and in vivo. J Infect Dis. 1995;172:785-793. [Medline] [Order article via Infotrieve]
-
Relman D, Tuomanen E, Falkow S, Golenbock DT,
Saukkonean K, Wright SD. Recognition of a bacterial adhesin by
an integrin: macrophage CR3 (m,ß2, CD11b/CD18) binds filamentous
hemagglutinin of Bordetella pertussis.
Cell. 1990;61:1375-1382. [Medline]
[Order article via Infotrieve]
- Van Strijp JA, Russell DG, Tuomanen E, Brown EJ, Wright SD. Ligand specificity of purified complement receptor type three (CD11b/CD18, alpha m beta 2, Mac-1): indirect effects of an Arg-Gly-Asp (RGD) sequence. J Immunol. 1993;151:3324-3336. [Abstract]
-
Ishibashi Y, Claus S, Relman DA.
Bordetella pertussis filamentous hemagglutinin interacts
with a leukocyte signal transduction complex and stimulates bacterial
adherence to monocyte CR3 (CD11b/CD18). J Exp Med. 1994;180:1225-1233.
[Abstract/Free Full Text] -
Gresham HD, Goodwin JL, Allen PM, Anderson DC, Brown
EJ. A novel member of the integrin receptor family mediates
Arg-Gly-Asp-stimulated neutrophil phagocytosis. J Cell
Biol. 1989;108:1935-1943.
[Abstract/Free Full Text] -
Brown EJ, Hooper L, Ho T, Gresham H.
Integrin-associated protein: a 50 kd plasma membrane antigen physically
and functionally associated with integrins. J Cell
Biol. 1990;111:2785-2794.
[Abstract/Free Full Text] -
Zhou M, Brown EJ. Leukocyte response integrin
and integrin-associated protein act as a signal transduction unit in
generation of a phagocyte respiratory burst. J Exp
Med. 1993;178:1165-1174.
[Abstract/Free Full Text] - Redgrave TG, Roberts DCK, West CE. Separation of plasma lipoproteins by density-gradient ultracentrifugation. Anal Biochem. 1975;65:42-49. [Medline] [Order article via Infotrieve]
-
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent. J
Biol Chem. 1951;193:265-275.
[Free Full Text] - Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;30:302-310.
- Joris I, Zand T, Nunnari JJ, Krolikowski FJ, Majno G. Studies on the pathogenesis of atherosclerosis, I: adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats. Am J Pathol. 1983;113:341-358. [Abstract]
-
Liao L, Aw TY, Kvietys PR, Granger DN. Oxidized
low-density lipoprotein–induced microvascular dysfunction: dependence
on oxidation procedure. Arterioscler Thromb Vasc
Biol. 1995;15:2305-2311.
[Abstract/Free Full Text] - Asako H, Kurose I, Wolf R, DeFree S, Zheng Z, Phillips ML, Paulson JP, Granger DN. Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules. J Clin Invest. 1994;93:1508-1515.
- Davis MJ. Determination of volumetric flow in capillary tubes using an optical Doppler velocimeter. Microvasc Res. 1987;34:223-230. [Medline] [Order article via Infotrieve]
- Granger DN, Kubes P. The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion. J Leukocyte Biol. 1994;55:662-675. [Abstract]
-
Firrell JC, Lipowsky HH. Leukocyte margination
and deformation in mesenteric venules of rat. Am J
Physiol. 1989;256:H1667-H1674.
[Abstract/Free Full Text] -
Kurose I, Wolf R, Grisham MB, Granger DN.
Modulation of ischemia/reperfusion-induced microvascular
dysfunction by nitric oxide. Circ Res. 1994;74:376-382.
[Abstract/Free Full Text] - Kurose I, Kubes P, Wolf R, Anderson DC, Paulson J, Miyasaka M, Granger DN. Inhibition of nitric oxide production: mechanisms of vascular albumin leakage. Circ Res. 1993;73:164-171. [Abstract]
- Mulligan MS, Varani J, Warren JS, Till GO, Smith CW, Anderson DC, Todd RD, Ward PA. Roles of beta 2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury. J Immunol. 1992;148:1847-1857. [Abstract]
- Tamatani T, Kotani M, Miyasaka M. Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies. Eur J Immunol. 1991;21:627-633. [Medline] [Order article via Infotrieve]
-
Tamatani T, Miyasaka M. Identification of
monoclonal antibodies reactive with the rat homolog of ICAM-1, and
evidence for a differential involvement of ICAM-1 in the adherence of
resting versus activated lymphocytes to high endothelial cells.
Int Immunol. 1990;2:165-171.
[Abstract/Free Full Text] - Tamatani T, Kitamura F, Kuida K, Shirao M, Mochizuki M, Suematsu M, Schmid SG, Watanabe K, Tsurufuji S, Miyasaka M. Characterization of rat LECAM-1 (L-selectin) by the use of monoclonal antibodies and evidence for the presence of soluble LECAM-1 in rat sera. Eur J Immunol. 1993;23:2181-2188. [Medline] [Order article via Infotrieve]
- Mulligan MS, Polley MJ, Bayer RJ, Nunn MF, Paulson JC, Ward PA. Neutrophil-dependent acute lung injury: requirement for P-selectin (GMP-140). J Clin Invest. 1992;90:1600-1607.
-
Ma L, Raycroft D, Asa D, Anderson DC, Geng JG. A
sialoglycoprotein from human leukocytes functions as a ligand for
P-selectin. J Biol Chem. 1994;269:27739-27746.
[Abstract/Free Full Text] -
Schuschke DA, Joshua IG, Miller FN. Comparison
of early microcirculatory and aortic changes in hypercholesterolemic
rats. Arterioscler Thromb. 1991;11:154-160.
[Abstract/Free Full Text] - Liao L, Takemura T, Kvietys PR, Granger DN. Oxidized low density lipoproteins induced neutrophil-endothelial cell adhesion: dependence on oxidation procedure. FASEB J. 1994;8:A1983.
-
Tuomanen E, Saukkonen K, Sande S, Cioffe C, Wright
SD. Reduction of inflammation, tissue damage and mortality in
bacterial meningitis in rabbits treated with monoclonal antibodies
against adhesion-promoting receptors of leukocytes. J Exp
Med. 1989;170:959-969.
[Abstract/Free Full Text] - Zhang RL, Chopp M, Tang WX, Zhang ZG, Putney SD, Starzyk RM. A synthetic peptide derived from the Bordetella pertussis bacterium reduces infarct volume after transient middle cerebral artery occlusion in rat. Neurology. In press.
-
von Andrian UH, Hansell P, Chambers JD, Berger EM,
Filho IT, Butcher EC, Arfors KE. L-selectin function is required
for ß2-integrin-mediated neutrophil adhesion at
physiological shear rates in vivo. Am J
Physiol. 1992;263:H1034-H1044.
[Abstract/Free Full Text] - Lehr HA, Krombach F, Munzing S, Bodlaj R, Glaubitt SI, Seiffge D, Hubner C, von Andrian UH, Messmer K. In vitro effects of oxidized low density lipoprotein on CD11b/CD18 and L-selectin presentation on neutrophils and monocytes with relevance for the in vivo situation. Am J Pathol. 1995;146:218-227. [Abstract]
-
Dore M, Korthuis RJ, Granger DN, Entman ML, Smith
CW. P-selectin mediates spontaneous leukocyte rolling in
vivo. Blood. 1993;82:1308-1316.
[Abstract/Free Full Text] - Gebuhrer V, Murphy JF, Bordet J-C, Reck M-P, McGregor JL. Oxidized low-density lipoprotein induces the expression of P-selectin (GMP 140/PADGEM/CD62) on human endothelial cells. Biochem J. 1995;306:293-298.
-
Panes J, Perry MA, Anderson DC, Manning A, Leone B,
Cepinskas G, Rosenbloom CL, Miyasaka M, Kvietys PR, Granger DN.
Regional differences in constitutive and induced ICAM-1 expression in
vivo. Am J Physiol. 1995;269:H1955-H1964.
[Abstract/Free Full Text] -
Kurose I, Anderson DC, Miyasaka M, Tamatani T, Paulson
JC, Todd RF, Rusche JR, Granger DN. Molecular determinants of
reperfusion-induced leukocyte adhesion and vascular protein
leakage. Circ Res. 1994;74:336-343.
[Abstract/Free Full Text] -
Zimmerman BJ, Holt JW, Paulson JC, Anderson DC,
Miyasaka M, Tamatani T, Todd RF, Rusche JR, Granger DN.
Molecular determinants of lipid mediator-induced leukocyte adherence
and emigration in rat mesenteric venules. Am J
Physiol. 1994;266:H847-H853.
[Abstract/Free Full Text] - Kishimoto TK, Anderson DC. The role of integrins in inflammation. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: Basic Principles and Clinical Correlates. 2nd ed. New York, NY: Raven Press; 1992:353-406.
-
Kim JA, Territo MC, Wayner E, Carlos TM, Parhami
F, Smith CW, Haberland ME, Fogelman AM, Berliner JA. Partial
characterization of leukocyte binding molecules on endothelial cells
induced by minimally oxidized LDL. Arterioscler
Thromb. 1994;14:427-433.
[Abstract/Free Full Text] -
Sugiyama S, Kugiyama K, Ohgushi M, Fujimoto K, Yasue
H. Lysophosphatidylcholine in oxidized low-density lipoprotein
increases endothelial susceptibility to polymorphonuclear
leukocyte-induced endothelial dysfunction in porcine coronary arteries:
role of protein kinase C. Circ Res. 1994;74:565-575.
[Abstract/Free Full Text] - Khan BV, Parthasarathy S, Alexander RW, Medford RM. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995;95:1262-1270.
- Kubes P, Granger DN. Leukocyte-endothelial cell interactions invoked by mast cells. Cardiovasc Res. In press.
This article has been cited by other articles:
 |
|
 |
|
  O. Quehenberger Thematic Review Series: The Immune System and Atherogenesis. Molecular mechanisms regulating monocyte recruitment in atherosclerosis J. Lipid Res., August 1, 2005; 46(8): 1582 - 1590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Sedgwick, Y. S. Hwang, H. A. Gerbyshak, H. Kita, and W. W. Busse Oxidized Low-Density Lipoprotein Activates Migration and Degranulation of Human Granulocytes Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 702 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. A. van Haaren, E. VanBavel, H. Vink, and J. A. E. Spaan Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2848 - H2856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Constantinescu, H. Vink, and J. A.E. Spaan Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface Arterioscler. Thromb. Vasc. Biol., September 1, 2003; 23(9): 1541 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Alvarez and M.-J. Sanz Reactive oxygen species mediate angiotensin II-induced leukocyte-endothelial cell interactions in vivo J. Leukoc. Biol., August 1, 2001; 70(2): 199 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Chavakis, E. Dernbach, C. Hermann, U. F. Mondorf, A. M. Zeiher, and S. Dimmeler Oxidized LDL Inhibits Vascular Endothelial Growth Factor-Induced Endothelial Cell Migration by an Inhibitory Effect on the Akt/Endothelial Nitric Oxide Synthase Pathway Circulation, April 24, 2001; 103(16): 2102 - 2107. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Y. Stokes, E. C. Clanton, J. M. Russell, C. R. Ross, and D. N. Granger NAD(P)H Oxidase-Derived Superoxide Mediates Hypercholesterolemia-Induced Leukocyte-Endothelial Cell Adhesion Circ. Res., March 16, 2001; 88(5): 499 - 505. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Mori, Y. Horie, Y. Nimura, R. Wolf, and D. N. Granger Hepatic microvascular responses to ischemia-reperfusion in low-density lipoprotein receptor knockout mice Am J Physiol Gastrointest Liver Physiol, December 1, 2000; 279(6): G1257 - G1264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Mehrabi, H. Sinzinger, C. Ekmekcioglu, F. Tamaddon, K. Plesch, H. D Glogar, G. Maurer, T. Stefenelli, and I. M. Lang Accumulation of oxidized LDL in human semilunar valves correlates with coronary atherosclerosis Cardiovasc Res, March 1, 2000; 45(4): 874 - 882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Johnson, C. L. Jackson, G. D. Angelini, and S. J. George Activation of Matrix-Degrading Metalloproteinases by Mast Cell Proteases in Atherosclerotic Plaques Arterioscler. Thromb. Vasc. Biol., November 1, 1998; 18(11): 1707 - 1715. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||