1. Art and Diseño

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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
By T. Carlos, N. Kovach, 6.Schwartz, M. Rosa, B. Newrnan, E. Wayner, C. Benjamin, L. Osborn, R. Lobb, and J. Harlan
Vascular cell adhesion molecule-1 (VCAM-1) and endothelialleukocyte adhesion molecule-1 (ELAM-I) are adhesive proteins induced on endothelium by cytokines. We examined the
contribution of these adhesive proteins to human peripheral
blood monocyte adherence to endothelium using transfected
Chinese hamster ovary (CHO) cells stably expressing these
proteins and monoclonal antibodies (MoAbs) to ELAM-I,
VCAM-1, or CD49d/CD29 (VLA-4). the leukocyte receptor for
VCAM-1. Monocytes bound to CHO cells transfected with
cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was
inhibited by MoAb to VCAM-1 or the a-chain of very late
activation antigen-4 ( V U - 4 ) (CD49d). Additive inhibition of
adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocyteswere pretreated
with both MoAb to CD49d and MoAb to CD18, the common
@-chainof the leukocyte p2 integrin receptors. Adherence of
monocytes to HUVE stimulated by recombinant human tumor necrosis factor-a was not reduced by MoAbs to CD18,
CD49d. or ELAM-1 when used singly, but combinations of
these MoAbs produced significant inhibition. We conclude
that multiple receptor-ligand systems are involved in monocyte adherence to endothelium.
0 1991 by The American Society of Hematology.
T
studies have suggested that monocytes also bind to VCAM1" but the binding of monocytes to ELAM-1 has been
difficult to demonstrate clearly.'-'
In this report we demonstrate that human monocytes
adhere to ELAM-1 and VCAM-1 using Chinese hamster
ovary (CHO) cells that have been transfected with cDNA of
ELAM-1 or VCAM-1 and are stably expressing these
adhesive proteins. We show that this binding is inhibited by
monoclonal antibodies (MoAbs) that are specific for
ELAM-1, VCAM-1, or the leukocyte receptor for VCAM-1,
very late activation antigen-4 (VLA-4, CD49d/CD29).'33'4
Finally, we demonstrate that these endothelial cell ligands
are involved in the adherence of human monocytes to
human umbilical vein endothelium (HUVE).
HE SEQUENCE OF EVENTS leading to monocyte
emigration in response to extravascular inflammatory
stimuli can be summarized as follows. Signals are generated
at the inflammatory site that activate the circulating monocyte and the adjacent endothelium. As a consequence of
activation, the circulating monocytes adhere to the endothelium, migrate along the endothelial surface, diapedese, and
finally emigrate through subendothelial matrix to participate in the inflammatory reaction.'
Neutrophils are initially the predominant leukocyte at
inflammatory sites with the peak of emigration occurring
within the first several hours after the onset of inflammation.* However, within 12 to 24 hours mononuclear phagocytes become the most abundant cell in the inflammatory
The different kinetics of emigration and subsequent accumulation in tissue of neutrophils and monocytes
may be explained in part by differences in expression or
configuration of adhesion proteins on the phagocyte or the
endothelium.
Endothelial-leukocyte adhesion molecule-1 (ELAM-1)
and vascular cell adhesion molecule-1 (VCAM-1) are
surface proteins that are induced on endothelial cells and
are involved in leukocyte adhesion. Neutrophils, but not
peripheral blood lymphocytes (PBL), have been reported
to bind to ELAM-l.'-9 Conversely, VCAM-1 has been
shown to bind PBL, but not neutrophils.'0," Preliminary
From the Department of Medicine (Hematology), University of
Washington, Seattle, WA; Biogen, Inc, Cambridge,MA; and Oncogen,
Seattle, WA.
Submitted May 14,1990; accepted January 11, 1991.
Supported by US Public Health Services Grant HL 18645. T.C. is
the recipient of a Clinician-ScientistAward from the American Heart
Association. J.H. is the recipient of an Established Investigator Award
from the American Heart Association.
Address reprint requests to Timothy Carlos, MD, Division of
Hematology, ZA-34, Harborview Medical Center, 325 Ninth Ave,
Seattle, WA 98104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971191/7710-0010$3.00i0
2266
MATERIALS AND METHODS
Generation of CHO cell lines that stably express ELAM-I and
VCAM-I. The animal cell expression vectors pBG341JOD.ELAM
and .VCAM were constructed as follows: vector pBG312" was
modified by insertion of a Notl site into the polylinker sequence.
The resulting plasmid, pBG341, was cleaved by Notl and Aat2.
The smaller of the two fragments was ligated to the larger of the
two fragments generated by Notl and Aat2 cleavage of vector
pJOD-S.l6 The resulting vector, pBG341JOD, contains an expression cassette for the dihydrofolate reductase cDNA gene'' and a
unique Notl site downstream from the adenovirus major late
promoter. The cDNA sequences for either ELAM-1' or for
VCAM-1" were inserted into the vector Notl site to generate the
animal cell expression vectors pBG341JOD.ELAM and .VCAM,
respectively.
To generate cell lines stably expressing ELAM-1 and VCAM-1,
CHO-dihydrofolate reductase folato (CHO-DHFR-) cells18were
electroporated with 200 pg pBG341JOD.ELAM or .VCAM as
described.16 Before electroporation the plasmid was linearized
using Aat2. Following transfection the cells were cultured in
selective medium (minimal essential medium [MEM] a minus;
GIBCO, Grand Island, NY) lacking ribonucleosides and deoxynucleotides, supplemented with 200 nmol/L methotrexate, 4 mmol/L
glutamine, and 10% dialysed fetal bovine serum (FBS) to select for
methotrexate-resistant cells. Individual colonies were picked, plated
into 96-well cluster plates, and grown to confluence. Clones
expressing sufficient ELAM-1 or VCAM-1 to bind HL60 cells or
Ramos cells, respectively, were detected by adhesion assay as
described.'
CHO cells expressing ELAM-1, but not control CHO cells, bind
Blood, Vol77, No 10 (May 15), 1991: pp 2266-2271
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2267
HUMAN MONOCYTES BIND TO VCAM-1 AND ELAM-1
the ELAM-1-specific MoAb BBll and MoAb BBll immunoprecipitates a protein of about 130 Kd from CHO.ELAM cell lines."
MoAb BB11, but not MoAb 4B9, binds to CHO.ELAM-1 while
MoAb 4B9, but not MoAb BB11, binds CHO.VCAM-1" (T.
Carlos, unpublished observation, March, 1990). MoAb 4B9 immunoprecipitates a protein of about 100 Kd from CHO.VCAM-1 but
not control CHO."
Untransfected CHO cells were maintained in MEM a medium
supplemented with 4 m m o m glutamine and 10% FBS. Transfected CHO cells (ELAM-1 and VCAM-1) were maintained in
MEM a-minus medium supplemented with 4 mmol/L glutamine,
500 nmol/L methotrexate, and 10% FBS.
Nonadherent cells. PB monocytes were purified from heparinized whole blood by the method of Recalde?' Preparations were
greater than 85% pure monocytes as determined by morphology,
nonspecific esterase staining, and reactivity with a CD14 MoAb on
fluorescence-activated cell sorter (FACS). The leukemic cell lines,
HL60 and Molt 4, were obtained from the American Type Culture
Collection (Rockville, MD) and were maintained in MEM (Flow
Labs, McLean, VA) supplemented with 10% Cellect fetal calf
serum (FCS; Flow Labs). Leukocytes were labeled with "Cr as
described" and were resuspended in RPMI medium (M.A. Bioproducts, Walkersville, MD).
Cell culture. HUVE were harvested by collagenase treatment
of vessels as previously published?' Cells were maintained in RPMI
1640 medium supplemented with 10% adult bovine serum (Hyclone Sterile Systems, Logan, UT), 10% normal calf serum (NCS;
Armour Pharmaceutical Co, Kanakee, IL), heparin (90 &mL;
Sigma Chemical Co, St Louis, MO), and endothelial growth factor
(50 p,g/mL) as described by Thorton et al." Endothelial growth
factor was prepared from bovine hypothalamus as described by
Maciag et al.=
MoAbs. MoAb 4B9 is a murine IgG, that recognizes a 100-Kd
antigen on cytokine-activated endothelium. MoAb 4B9 binds to
COS cells transfected with VCAM-1 cDNA9 and inhibits PBL
adherence to cytokine-stimulated HUVE." MoAb 60.3, a murine
IgG,, that recognizes the common P-subunit of the CDlUCD18
complex," and MoAb 60.5, a murine IgG, that recognizes class I
HLA framework antigen, were provided by Dr Patrick Beatty, Fred
Hutchinson Cancer Research Center, Seattle, WA. MoAb P4C2 is
a murine IgG, that recognizes an epitope on the a-chain polypeptide (CD49d) of VLA-4 (CD49diCD29):' MoAb P4C2 does not
recognize CHO cells or HUVE as determined by enzyme-linked
immunosorbent assay (ELISA). MoAb BBll is a murine IgG, that
binds to an epitope on ELAM-1.I9 Anti-Leu-M3 (CD14) was
purchased from Becton Dickinson Immunocytometry Systems
(Mountain View, CA).
Adherence assay. CHO cells were plated onto gelatin-coated
48-well tissue culture plates (Costar Corp, Cambridge, MA) and
allowed to reach confluence. The medium was decanted and the
wells were washed twice with RPMI containing 2% NCS (RPMI/
NCS). Subsequently, 0.2 mL of control medium (RPMINCS) or
medium containing MoAbs BB11,4B9, or P4C2 was added to the
monolayer 30 minutes before assay. MoAb P4C2 does not bind to
either CHO or HUVE by ELISA assay,I4but binds to monocytes by
FACS analysis. Untreated leukocytes or leukocytes that were
pretreated for 30 minutes with MoAb 60.3 (40 p,g/mL) were added
to each well. Experiments involving monocyte adherence to HUVE
were performed in a similar manner. For these experiments, some
wells of the 48-well plates were pretreated with 10 ng/mL recombinant human tumor necrosis factor-a (rhTNF-a; Biogen Inc, Cambridge, MA) for 4 hours. Following 30 minutes of incubation at
37"C, nonadherent cells were discarded and the wells were washed
once with 0.5 mL phosphate-buffered saline (PBS). Adherent cells
were lysed with 1N NH,OH and the lysate was counted in a gamma
spectrophotometer. Percent adherence was calculated by the
formula:
% adherence =
"Cr cpm in lysate
x 100
total 5'Cr cpm added
Statistics. Levels of statistical significance were calculated by
paired t-test using Statview (Brainpower, Inc, Calabasas, CA).
RESULTS
Monocytes bind to ELAM-l-tramfected CHO cells. The
adherence of the leukemic cell line HL60 to control CHO
cells or CHO cells stably expressing ELAM-1 was examined
in three separate experiments (Fig 1A). The adherence of
HL60 to control CHO was moderate (33% f 5%) and was
not reduced by MoAb 60.3 (28% ? 9%), MoAb BBll
(31% 2 8%), MoAb P4C2 (27% f 6%), or MoAb 4B9
(31% f 6%). The adherence of HL60 to ELAM-1transfected CHO was 89% f 2%. MoAb BBll completely
blocked the binding of HL60 cells to levels observed in
control CHO (26% 2 4%, P < .001). MoAb 60.3, MoAb
P4C2, and MoAb 4B9 did not significantly inhibit monocyte
..
.I*
Treatmentof
HL60:
CHO:
-
60.3
-
-
-
-
loorB
Q,
-
-
489 8811
P4C2 60.3 60.3 60.3 60.3+
P4C2
- - 489 BBll
-
T
T
80
V
C
60
Q,
c
2
is
40
20
Treatmentof
monocytes :
CHO:
- 60.3
-
-
-
-
-
-
P4C2 60.3 60.360.3 60.3+
P4C2
489 8811
- 489 8811
-
-
Fig 1. Inhibition of HL60 (A) and blood-derived human monocyte
(B) binding to CHO cells stably expressing ELAM-1. "Cr-labeled HL-60
leukemic cells or monocytes were incubated in control medium
(RPMI) or medium containing MoAb 60.3 (40 pg/mL) for 30 minutes.
Leukocytes were then added to 48-well plates containing untransfected ( 0 ) or ELAM-1-transfected [D) CHO cells that had been
pretreated for 30 minutes with control medium (RPMI/P%NCS) or
control medium containing anti-VCAM-1 MoAb, 489 (20 pg/mL),
CD49d P4C2 (1:lo0 dilution of hybridoma supernatant), or antiELAM-1 MoAb BE11 (20 pg/mL). Values represent means ? SEM of
three experiments with triplicate wells in each experiment. (§P < .01
Y control CHO. *P < .01 Y control, **P < .01 v MoAb 60.3-control
CHO, tP < .01Y ELAM-1/CHO, ttP < .01 Y MoAb 60.3-ELAM-l/CHO.)
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2268
CARLOS ET AL
§
W
T
u 30
C
z
2
2
is?
20
IO
0
Treoimentof
Molt-4
-
603
-
-
-
CHO:
-
-
-
489
eeii
B
~ 4 ~ 2 6 06 033 6 0 3 6 0 3 +
P4C2
- - 489 mi
-
9
60
s
0
Treatment of
monocyies:
CHO:
-
603
-
-
-
-
-
-
489
mi
P4C2603 6 0 3 6 0 3 6 0 3 +
P4C2
- 489 eeii -
Fig 2. Inhibition of Molt-4 (A) and blood-derived human monocyte
(6) binding t o CHO cells stably expressing VCAM-1. "Cr-labeled
Molt-4 leukemic cells or monocytes were incubated in control medium (RPMI) or medium containing MoAb 60.3 (40 pg/mL) for 30
minutes. Leukocytes were then added t o 48-well plates containing
untransfected ( 0 )or VCAM-1-transfected (D)CHO cells that had been
pretreated for 30 minutes with control medium (RPMI/P%NCS) or
control medium containing anti-VCAM-1 MoAb 489 (20 pg/mL),
CD49d P4C2 (1:100 dilution of hybridoma supernatant), or antiELAM-1 MoAb B B l l (20 pg/mL). Values represent means 2 SEM of
three experiments with triplicate wells in each experiment. (§P< .01
v control CHO, *P < .01 v control CHO, **P < .05 v VCAM-l/CHO,
tP < .01 vVCAM-l/CHO, ttP < .01 v MoAb 60.3-VCAM-l/CHO.)
Molt-4 cells to control CHO was low (1.5% f 0.3%). The
adherence of Molt4 cells to CHO cells that stably express
VCAM-1 was significantly higher (26% f 6%). MoAbs 4B9
or P4C2 completely inhibited this binding ( < 1%) while the
anti-ELAM-1 MoAb BBll was without significant effect
(19% f 4%). The binding of monocytes to CHO cells that
had been transfected with VCAM-1 cDNA was also examined in a series of experiments (Fig 2B). The binding of
MoAb 60.3-pretreated monocytes to VCAM-1 CHO cells
was 49%
5%. The anti-ELAM-1 MoAb BBll did not
reduce this binding (50% f 7%). The CD49d MoAb P4C2
and the anti-VCAM-1 MoAb 4B9 significantly inhibited
the adherence of MoAb 60.3-pretreated monocytes to
VCAM-1-transfected CHO cells (9% 2 2% and 19% f 2%,
respectively; P < .05). The combination of MoAb P4C2
and MoAb 4B9 produced no greater inhibition of monocyte
binding than that observed with MoAb P4C2 alone (data
not shown).
Monocyte adherence to unstimulated or rh TNF-a-stimulated HUVE. The binding of monocytes to untreated
HUVE or HUVE that were stimulated with rhTNF-a for 4
hours was examined (Fig 3). Because the CD49d MoAb was
more effective than the anti-VCAM-1 MoAb in blocking
monocyte binding to VCAM-1-transfected CHO cells (Fig
2B), MoAb P4C2 rather than MoAb 4B9 was used to inhibit
monocyte binding to VCAM-1 expressed on HUVE.
Monocyte adherence to unstimulated HUVE was 57% 2
-I
A
*
binding to ELAM-1-transfected CHO cells (92% 9%,
84% f 4%, and 88% f 4%, respectively).
The binding of monocytes to control CHO cells was high
(50% f 17%), but was nearly abolished by the CD18
MoAb 60.3 (4% f 3%, mean f SE of three experiments).
Using MoAbs specific for C D l l a and CDllb, binding to
CHO cells was found to be mediated by CDllaKD18 (data
not shown). To detect specific binding to ELAM-1 or
VCAM-1, monocytes were pretreated with the CD18 MoAb
60.3 in subsequent experiments.
The binding of MoAb 60.3-pretreated monocytes to
ELAM-1-transfected CHO cells was 41% 2 6% (Fig 1B).
Preincubation of ELAM-1-transfected CHO cells with the
anti-ELAM-1 MoAb BBll reduced binding of MoAb
60.3-pretreated monocytes to 8%
4% (P < .01). No
effect was observed when MoAb 60.3-pretreated monocytes
were added to ELAM-1-transfected CHO cells that had
been were preincubated with the CD49d MoAb P4C2 or
the anti-VCAM-1 MoAb 4B9 (32% f 9% and 39% 2 8%
binding, respectively).
Monocytes bind to VCAM-1-transfected CHO cells. The
binding of the Molt-4 leukemic cell line to control CHO
cells and to CHO cells that stably express the adhesive
protein VCAM-1 was examined (Fig 2A). The binding of
*
\
Y
BO-.
Treatmentof
monocytes
-
603
P4C2
-
603
+
603
P4C2
P4C2
HUVE:
-
-
mi
-
603
+
P4C2
m i 1
BBI~
eeii
Fig 3. Inhibition of human blood-derived monocyte binding t o
unstimulated (A) or rhTNF-a-stimulated HUVE (B). *'Cr-labeled monocytes were incubated in control medium (RPMI) or control medium
containing the CD18 MoAb, 60.3 (40 pg/mL), for 30 minutes. Labeled
monocytes were then added t o 48-well plates containing confluent
HUVE that had either been untreated (A) or pretreated for 4 hours
with rhTNF-a (10 ng/mL) (6). Triplicate wells of the HUVE plate were
pretreated for 30 minutes with control medium (RPMI/P%NCS) or
with control medium containing the anti-ELAM-1 MoAb B B l l (20
pg/mL), or the CD49d MoAb P4C2 (1:lOO dilution of hybridoma
supernatant). Values represent means 2 SEM of nine experiments.
(*P< .01 MoAb 60.3 v control, tP < . O l MoAbs 60.3 and P4C2, or
MoAbs P4C2 and B B l l v MoAb 60.3 alone, §P < .01 MoAbs 60.3,
P4C2, and B B l l v MoAbs 60.3 and P4C2.)
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HUMAN MONOCYTES BIND TO VCAM-1 AND ELAM-1
3% and was significantly reduced by the pretreatment of
monocytes with the CD18 MoAb 60.3 (37% 3%, P < .Ol).
While the CD49d MoAb P4C2 alone did not significantly
inhibit monocyte adherence to unstimulated HUVE
(49% f 2%), a statistically significant additive inhibitory
effect was seen when MoAb P4C2 was used in conjunction
with MoAb 60.3 (23% f 3%, P < .01). The further addition of the anti-ELAM-1 MoAb BBll did not affect
adherence to unstimulated HUVE (25% 3%).
In a separate set of experiments, the addition of the
CD14 MoAb Leu-M3 did not produce additive inhibition of
binding of MoAb 60.3-pretreated monocyte to unstimulated HUVE. In these studies the adherence of untreated
monocytes was significantly reduced by MoAb 60.3
(57% f 2% v 18% -C 2%, P < .05). The addition of MoAb
P4C2 to MoAb 60.3-pretreated monocytes produced further significant inhibition of binding (12% f l%, P < .05)
in contrast to the addition of Leu-M3 (19% & 1%, P = not
significant [NS]).
The adherence of monocytes to HUVE that had been
treated with rhTNF-a was significantly greater than monocyte binding to unstimulated HUVE (69% f 2%, P < .OS).
In contrast to the inhibitory effect of the CD18 MoAb 60.3
on binding to untreated HUVE, no inhibition of monocyte
adherence to rhTNF-treated HUVE was produced by the
CD18 MoAb (71% f 2%). The addition of the CD49d
MoAb P4C2 to MoAb 60.3-pretreated monocytes significantly reduced binding (53% f 2%, P < .Ol). However,
pretreatment of rhTNF-pretreated HUVE with the antiELAM-1 MoAb BBll and P4C2 followed by the addition of
monocytes that were pretreated with MoAb 60.3 produced
the most significant reduction of binding (38% f 2%,
P < .01 versus the combination of MoAbs 60.3 and P4C2).
In a separate set of experiments, the addition of the
HLA-class I MoAb 60.5 to rhTNF-a-stimulated HUVE did
not produce additive inhibition of binding of monocytes
that were pretreated with MoAbs 60.3 and P4C2. In these
experiments the binding of untreated monocytes to rhTNFa-stimulated HUVE was 67% f 2%. Pretreatment of the
monocytes with MoAbs 60.3 and P4C2 significantly inhibited this binding (26% f 6%,P < .05). Pretreatment of the
rhTNF-a-pretreated HUVE with MoAb BBll produced
further significant inhibition of adherence (15% f 2%,
P < .05) while pretreatment with MoAb 60.5 was ineffectual (36% f 3%, P = NS).
*
DISCUSSION
Monocytes adhere avidly to unstimulated HUVE. This
basal adherence is mediated in part by the CD18 complex
on the monocyte." Pretreatment of HUVE with lipopolysaccharide (LPS) produces an additional increase in monocyte
adherence21.26
that is CD18-independent." In this report we
demonstrate that human PB monocytes bind to two endothelial adhesion proteins, ELAM-1 and VCAM-1, that are
induced on endothelial cells by LPS or cytokine treatment
and that these ligands are involved in monocyte adherence
to HUVE.
The capacity of monocytes to bind to ELAM-1 and
VCAM-1 was first established using transfected CHO cells
2269
stably expressing these proteins. Because of the significant
basal monocyte adherence to CHO cells, it was necessary to
pretreat monocytes with a CD18 MoAb. By using CDlla
(lymphocyte function-associated antigen-1 [LFA-11) and
CDllb (Mac-1) MoAbs in our initial studies, this basal
binding to CHO cells was determined to be mediated by
CDlla. CDlla is a receptor for both intercellular adhesion
molecules-1 and -2, ICAM-1 (CD54)27and ICAM-2.w The
surface expression of ICAM-1 was not detected on CHO
cells by ELISA with three MoAbs to human ICAM-1.
Because our human CD54 MoAbs did not bind to CHO
ICAM-1, we cannot determine whether ICAM-1 or ICAM-2
is involved in this basal monocyte adherence to CHO cells.
To detect specific binding to ELAM-1- or VCAM-1transfected CHO cells, in subsequent experiments monocytes were pretreated with CDl8 MoAb to eliminate
CDlla/CD18-dependent binding.
Previous reports have demonstrated that ELAM-1 binds
neutrophils and some leukemic cell lines (HL60,U937), but
is not involved in the adherence of lymphocytes or lymphocytic cell line^.^!^,^ An initial report suggested that ELAM-1
also bound monocytes: but this result was questioned by
the same investigators in subsequent studies.628
Our results
demonstrate that HL60 cells and human monocytes bind to
ELAM-1 expressed in CHO cells and that this binding is
inhibited by the anti-ELAM-1 MoAb BB11. MoAb BBll
also binds to ELAM-1 induced on HUVE and inhibits
binding of HL-60 cells to TNF-treated HUVE.19This result
suggests that the inhibition of monocyte binding to ELAM1-transfected CHO cells is not a selective property of
ELAM-1 expressed in CHO cells.
VCAM-1 is a recently described adhesion protein that is
induced on endothelium by cytokines (TNF-a or interleukin-1 [IL-11) and LPS.9z'o The leukocyte receptor for
VCAM-1 is VLA-4.'3s14
COS cells transfected with VCAM-1
cDNA bind leukemic cell lines (Ramos, HL60, THP-1) that
express VLA-4 but not neutrophils that lack VLA-4.29
MoAb 4B9 binds to CHO cells transfected with cDNA for
VCAM-1 but not ELAM-1 or ICAM-1 and inhibits binding
of lymphocytic cell lines, PBL, and leukocyte adhesion
deficient (LAD)-lymphoblasts to rhTNF-a-treated
HUVE."s'4 Our results show that the human T-cell line
Molt-4 and PB monocytes bind to transfected CHO cells
stably expressing VCAM-1 and that this binding is inhibited
by the CD49d MoAb P4C2 and the anti-VCAM-1 MoAb
4B9.
Having established that monocytes are capable of binding to ELAM-1 and VCAM-1, we next examined the role of
these endothelial ligands in monocyte adherence to untreated and rhTNF-a-treated HUVE using various combinations of MoAbs to the adhesion proteins. An MoAb to
CD18 (60.3) was used to inhibit binding to ICAM-1" or
ICAM-2, another possible ligand for CDlla/CD18.'' An
MoAb to the a-chain polypeptide (CD49d) of VLA-4 was
used to inhibit monocyte binding to VCAM-l.L32'4
Binding
to ELAM-1 was inhibited by an anti-ELAM-1 MoAb
(~~11).19
As previously described," monocyte adherence to untreated HUVE was high and a significant portion of this
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CARLOS E T A L
2270
adherence was CD18-dependent. The CD49d MoAb alone
had little effect on monocyte adherence to untreated
HUVE, but produced additive inhibition when used in
combination with CD18 MoAb. The anti-ELAM-1 MoAb
was without effect on monocyte binding to unstimulated
HUVE either when used alone or in combination with
CD18 and CD49d MoAbs.
The adherence of monocytes was significantly increased
by pretreatment of the HUVE with rhTNF-a. Each of the
MoAbs alone produced no significant inhibition of adherence. However, combinations of the MoAbs significantly
inhibited the adherence of monocytes to rhTNF-stimulated
HUVE. The need to use multiple MoAbs to detect inhibition of adherence may explain the previous difficulty in
demonstrating that monocytes bind to ELAM-1. While
MoAbs against one or even two adhesion proteins that are
involved in the binding of monocytes to stimulated HUVE
may inhibit some portion of adherence, the remaining
adhesion system(s) appears to compensate so that little if
any effect is observed.
The adherence of monocytes to multiple adhesive ligands
induced on endothelium by cytokines or LPS may explain
the continued recruitment of tissue phagocytes to the
inflammatory ~ite.3.~
Monocytes express the pan-leukocyte
integrin CDllaKD18 that is the receptor for the endothelial ligands ICAM-1 and ICAM-2. ICAM-1 is constituitively
expressed in vivoM and both proteins are detected on
unstimulated endothelial cells in c ~ l t u r e .An
~ ~increase
~~'
in
the surface expression of ICAM-1 is observed for more than
48 hours after cytokine ~timulation.~'
Because the ICAMs
are constituitively expressed, they may be involved in the
normal transmigration of monocytes as they exit the vasculature to become tissue macrophage^.^^ An increase in
surface expression of ICAM-1 at sites of inflammation
could facilitate leukocyte emigration, although it would
lack selectivity because its receptor, CDlla, is present on
all leukocytes.
The induction of ELAM-1 and VCAM-1 on endothelium
by cytokines may contribute to prolonged emigration of
monocytes at sites of chronic inflammation. The induction
of ELAM-1 could augment monocyte as well as neutrophil
emigration during the initial period of inflammation. Cytokine-induced expression of VCAM-1, an endothelial protein that does not bind neutrophils, persists for at least 48
hours in vitro.lO~"If expression of VCAM-1 is similarly
maintained at sites of inflammation, monocyte emigration,
mediated by the interaction of VLA-4 and VCAM-1, may
persist throughout the inflammatory response and lead to
the accumulation of mononuclear leukocytes that characterizes later lesion^.^^^
We acknowledge that the adherence of isolated PB
monocytes to cultured endothelium may not reproduce the
sequence of events that are involved during monocyte
adherence and emigration in vivo. However, these in vitro
experiments have identified two inducible endothelial surface proteins that are potentially involved in this heterotypic adhesive process. In the present study we have also
pointed out the complexity of this process. Clearly, the
relative contribution of the various adhesion systems to
monocyte emigration in inflammatory disorders can only be
determined by in vivo studies. It will be important to
examine the time course of expression of the endothelial
adhesive ligands (ICAM-1, ELAM-1, and VCAM-1) during
the inflammatory response and, particularly, to determine
the effect of MoAbs to the leukocyte receptors (eg, CD18,
VLA-4, MEL-14) or endothelial ligands on monocyte
accumulation at inflammatory sites.
ACKNOWLEDGMENT
We acknowledge Dr Russell Ross for helpful suggestions during
the course of this work. We thank Signe Annet Anderson for her
invaluable assistance.
REFERENCES
1. Harlan JM: Leukocyte-endothelial interactions. Blood 65:
513,1985
2. Issekutz AC, Movat HZ: The in vivo quantitation and kinetics
of rabbit neutrophil leukocyte accumulation in the skin in response
to chemotactic agents and Escherichia coli. Lab Invest 42:310,1980
3. Issekutz TB,Issekutz AC, Movat H Z The in vivo quantitation
and kinetics of monocyte migration into acute inflammatory tissue.
Am J Pathol 103:47,1981
4. Paz RA, Spector WG: The mononuclear-cell response to
injury. J Pathol Bacteriol84:85, 1962
5. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone MA: Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes,
monocytes, and related leukocytes cell lines. J Clin Invest 76:2003,
1985
6. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone MA Jr: Identification of an inducible endothelial-leukocyte
adhesion molecule. Proc Natl Acad Sci USA 84:9238,1987
7. Bevilacqua MP, Stengelin S, Gimbrone MA Jr, Seed B:
Endothelial leukocyte adhesion molecule 1: An inducible receptor
for neutrophils related to complement regulatory proteins and
lectins. Science 243:1160, 1989
8. Bevilacqua MP, Gimbrone MA Jr: Identification and characterization of endothelial-leukocyte adhesion molecule 1, in Springer
TA, Anderson DA, Rosenthal AS, Rothlein R (eds): Leukocyte
Adhesion Molecules. New York, NY, Springer-Verlag, 1989, p 215
9. Hession C, Osborn L, Goff D, Chi-Rosso G, Vassallo C,
Pasek M, Pittack C, Tizard R, Goelz S, McCarthy K, Hopple S,
Lobb R: Endothelial leukocyte adhesion molecule 1:Direct expression cloning and functional interactions. Proc Natl Acad Sci USA
87:1673,1990
10. Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S,
Chi-Rosso G, Lobb R: Direct expression cloning of vascular cell
adhesion molecule 1, a cytokine-induced endothelial protein that
binds to lymphocytes. Cell 59:1203, 1989
11. Carlos TM, Schwartz BR, Kovach NL, Yee E, Rosa M,
Osborn L, Chi-Rosso G, Newman B, Lobb R, Harlan JM: Vascular
cell adhesion molecule-1 (VCAM-1) mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood
76:965,1990
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
HUMAN MONOCYTES BIND TO VCAM-1 AND ELAM-I
12. Carlos TM, Harlan JM: Membrane proteins involved in
phagocyte adherence to endothelium. Immunol Rev 1145,1990
13. Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyj S,
Hemler M, Lobb RR: VCAM-1 on activated endothelium interacts
with the leukocyte integrin VLA-4 at a site distinct from the
VLA-4Kibronectin binding site. Cell 60577, 1990
14. Schwartz BR, Wayner EA, Carlos TM, Ochs HD, Carter
WG, Harlan JM: Identification of adherence molecules modulating adhesion of CDlliCD18-deficient lymphoblastoid cells to
cultured human endothelium. J Clin Invest 85:2019,1990
15. Cate RL, Mattalino RJ, Hession C, Tizard R, Farber NM,
Cheung A, Ninfa EG, Frey AZ, Gash DJ, Chow EP, Fisher RA,
Bertonis JM, Torres G, Wallner BP, Ramachandran KL, Ragin
RC, Manganaro TF, MacLaughlin DT, Donahoe P K Isolation of
the bovine and human genes for mullerian inhibitory substance and
expression of the human gene in animal cells. Cell 45685,1986
16. Barsoum J: Introduction of stable high copy number DNA
into Chinese hamster ovary cells by electroporation. DNA Cell Biol
9293,1990
17. Subramani S, Mulligan R, Berg P: Expression of the mouse
dihydrofolate reductase complementary deoxyribonucleic acid in
simian virus 40 vectors. Mol Cell Biol 1:854,1981
18. Urlaub G, Chasin L A Isolation of Chinese hamster cell
mutants deficient in dihydrofolate reductase activity. Proc Natl
Acad Sci USA 77:4216,1980
19. Benjamin C, Dougas I, Chi-Rosso G, Luhowskyj S, Rosa M,
Osborn L, Vassallo C, Hession C, Goelz S, McCarthy K, Lobb R A
blocking monoclonal antibody to endothelial-leukocyte adhesion
molecule-1 (ELAM-1). Biochem Biophys Res Commun 171:348,
1990
20. Recalde HR: A simple method of obtaining monocytes in
suspension. J Immunol Methods 6971,1984
21. Carlos TM, Dobrina A, Ross R, Harlan JM: Multiple
receptors on human monocytes are involved in adhesion to
cultured human endothelial cells. J Leukoc Biol48:451,1990
22. Thornton SC, Mueller SN, Levine EM: Human endothelial
2271
cells: Use of heparin in cloning and long-term serial cultivation.
Science 222623, 1983
23. Maciag T, Ceruadolo J, Ilsley S, Kelley PR, Forand R: An
endothelial cell growth factor from bovine hypothalamus: Identification and partial characterization. Proc Natl Acad Sci USA
765674,1979
24. Beatty PG, Ledbetter JA, Martin PJ, Price TH, Hansen J A
Definition of a common leukocyte cell-surface antigen (GP95-150)
associated with diverse cell-mediated immune functions. J Immuno1 131:2913,1983
25. Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald
JA, Carter WG: Identification and characterization of the T
lymphocyte adhesion receptor for an alternative cell attachment
domain (CS-1) in plasma fibronectin. J Cell Biol109:1321,1989
26. Downs EC, Cornwell DG, Proctor VK, Whisler R L IL-1
and bacterial lipopolysaccharide increase the ability of human
endothelial cells to bind peripheral blood monocytes. Lymphokine
Res 6:351,1987
27. Kishimoto TK, Larson RS, Corbi AL, Dustin ML, Staunton
DE, Springer TA: The leukocyte integrins. Adv Immunol 46:149,
1989
28. Staunton DE, Dustin ML, Springer T A Functional cloning
of ICAM-2, a cell adhesion ligand for LFA-1 homologous to
ICAM-1. Nature 339:61,1989
29. Hemler ME: VLA proteins in the integrin family: Structures, functions, and their roles on leukocytes. Annu Rev Immunol
8:365,1990
30. Munro JM, Pober JS, Cotran RS: Tumor necrosis factor and
interferon-y induce distinct patterns of endothelial activation and
associated leukocyte accumulation in skin of Papio anubis. Am J
Pathol 135:121, 1989
31. Pober JS, Gimbrone MA Jr, Lapierre LA, Mendrick DL,
Fiers DL, Rothlein R, Springer TA Overlapping patterns of
activation of human endothelial cells by interleukin 1, tumor
necrosis factor, and immune interferon. J Immunol 1371893, 1986
32. Johnston RB: Monocytes and macrophages. N Engl J Med
318:747, 1988
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1991 77: 2266-2271
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
T Carlos, N Kovach, B Schwartz, M Rosa, B Newman, E Wayner, C Benjamin, L Osborn, R Lobb
and J Harlan
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