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Production of Chemokines, Interleukin-8 and Monocyte Chemoattractant
Protein-l, During Monocyte:Endothelial Cell Interactions
By Nicholas W. Lukacs, Robert M. Strieter, Victor Elner, Holly L. Evanoff, Marie D. Burdick, and Steven L. Kunkel
The extravasationof leukocytesfrom the
lumen of the vessel
to.a site of inflammation requires specific binding events.
The interaction of leukocytes with endothelium, via specific
receptors, may provide intracellular signals that activate extravasating cells. In thepresent study, we have investigated
the production of chemokines, interleukin-8 (IL-8). and
monocyte chemoattractant protein-l (MCP-1) during monocyte:endothelial cell interactions. Both unstimulated and interferon-? (IFN-?)-prestimulated human umbilical vein endothelial cells (HUVEC) produced low constitutive levels of
IL-8 and MCP-1. The addition of enriched monocytes with
unstimulated HUVEC resulted in synergistic increases in production of both IL-8 and MCP-1. Monocytes cultured with
IFN-y-preactivated HUVECs demonstrated little additional
increase in IL-8 and MCP-1 production in coculture assays
compared with unstimulated HUVEC. Northern blot analysis
paralleled the protein data, demonstrating upregulated expression of IL-8 andMCP-1 mRNAin stimulated and unstimulated coculture assays. Culture of enrichedmonocytes and
endothelial cells in transwells demonstrated no increases in
IL-8 or MCP-1, indicating the necessity for cellular contact
for chemokine production. In previous investigations,
we have demonstrated that increased monocyte-derived
MIP-la production was induced by intracellular adhesion
molecule-l (ICAM-1) interactions on activated HUVECs. In
contrast, addition of anti-CAM-l monoclonal antibodies
(MoAbs) did notdiminish the production of IL-8 and MCP-1
in the present study. Furthermore,neither antibodies t o IL1 nor tumor necrosisfactor (TNF) diminished the production
of either IL-8 or MCP-1. However, when soluble matrix proteins were added to the coculture t o block cellular interactions, the chemokine protein and mRNA levels were significantly decreased.IL-8 production was decreased by both
soluble collagen and fibronectin, whereas MCP-1 was decreased by only soluble collagen, suggestingdifferential activation pathways. These results indicate that IL-8 andMCP1 production are increasedduring monocyte and endothelial
cell interactions in part due t o matrix protein binding mechanisms. This mechanism may servea role in cell activation,
production of chemokines, as well as extravasation and recruitment of additional leukocytes during inflammatory responses.
0 1995 by The American Societyof Hematology.
T
strated that monocyte interaction with intracellular adhesion
molecule-l (ICAM-l), but not vascular cell adhesion molecule-l (VCAM-l), on the surface of activated endothelial
cells, induced the expression of macrophage inflammatory
protein-la (MIP-~cI).’~ These results support the concept
that adhesion events not only provide a method of cellular
interaction, but also may aid in the activation of the cells
and induce the production of chemokines.
In the present study, we have determined that monocyte:human umbilical vein endothelial cells (HUVEC) interactions resulted in the increased expression of MCP-1 and
IL-8. This increased expression was neither dependent on
the early response cytokine cascades (IL-l, tumor necrosis
factor [TNF]) nor expression of adhesion molecules, ICAM1 or VCAM-1, which were upregulated during endothelial
cell activation. However, chemokine expression was significantly reduced during the cell-to-cell interactions by addition
of soluble matrix proteins before the coculture. These results
suggest that once leukocytes adhere they may be activated
HE INTERACTION of leukocytes with vascular endothelium is a primary event that occurs during cellular
extravasation into inflamed tissue.’.’This interaction requires
the expression of adhesion molecules on the endothelium to
initiate the leukocyte adherence e ~ e n t .The
~ . ~currently held
concept is that adhered leukocytes undergo migration into
the tissue through a series of detachmentheadherence events
typified by the polar expression of integrins specific for the
adhesion molecules on the surface of mesenchymal-derived
cells.’ The movement of cells into the tissue is mediated via
chemotactic gradients with the highest concentration at the
site of inflammation. Specific chemotactic factors have been
isolated and classified into a supergene family of chemotactic
cytokines (chemokines). The chemokines have been divided
into two subgroups based on the juxtaposition of the first
two cysteine residues in their amino acid sequences. The CX-C family, typified by interleukin-8 (IL-8), is primarily
chemotactic for neutrophils, while the C-C chemokine family, typified by monocyte chemoattractant protein-l (MCPl), is primarily chemotactic for mononuclear leukocytes.’
The movement of specific populations of leukocytes into
tissue is thought to be mediated by concentration gradients
of chemokines that appear to be chemotactic for specific
groups of leukocytes. Therefore, the extravasation of leukocytes into inflamed tissue relies on the coordination of endothelial expressed adhesion molecules, leukocyte adherence,
and specific chemotactic gradients.
The expression of chemokines can be induced by a variety
of cytokines in a variety of immune and nonimmune cells.
These cell types include fibroblasts, epithelial, endothelial,
and smooth muscle cells, as well as mononuclear leukocytes
and gran~locytes.~
In addition, recent studies have demonstrated the expression of cytokine mRNAon adhesion of
monocytes to plastic or tissue matrix, thus indicating that
adherence events alone were sufficient for the activation of
cytokine
Recently our laboratory has demonBlood, Vol 86,No 7 (October l ) , 1995:pp 2767-2773
From the Departments of Pathology and Internal Medicine, the
Division of Pulmonary and Critical Care, Medicine, University of
Michigan Medical School, Ann Arbor.
Submitted March 6, 1995; accepted May 31, 1995.
Supported in part by National Institutes of Health Grants No.
HL02401, HL31963, and HL35276, and the American Heart Association of Michigan.
Address reprint requests to Nicholas W . Lukacs, PhD, University
of Michigan Medical School, Department of Pathology, 1301 Catherine, Ann Arbor, MI 48109-0602.
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 1734 solely to
indicate this fact.
0 I995 by The American Society of Hematology.
0006-4971/95/8607-~20$3.00/0
2767
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2768
LUKACSET AL
by mechanisms in conjunction with extravasation, resulting
in increased production of IL-8 and MCP- 1.
MATERIALS AND METHODS
Mononuclear cell isolation. Peripheral bloodwas drawn into a
heparinized syringe from healthy volunteers, diluted 1:l in normal
saline and mononuclear cells separated by density gradient centrifugation. The recovered cells were washed three times withRPM1
1640. The PBMs were then layered onto a density gradient (1.068
g/mL) for the enrichment of monocytes (Fico-lite, Atlanta Biologics,
Atlanta, GA). The isolated cells were then washed, cytospun onto
and difa glass slide, stained with Diff-Quik (Baxter, McGaw, L),
ferentially counted. The purity of the monocytes from the gradient
was consistently between 75% and 80% monocytes with the remainder lymphocytes.
HUVEC culture. HUVEC were isolated and established in culture as previously described? Briefly, primary cultures were passaged and maintained in endothelial cell growth medium (modified
MCDB 131; Clonetics, San Diego, CA). For experiments, the cells
were subcultured (passage 3) into 6-well plates (Costar, Kennebunkport, ME), grown to confluency, and used in monocyte adherence
assays.
HUVECtmonocyte cocultures. Enriched monocyte populations
(5 X lo5cells/mL) were layered onto unstimulated or 24 hour stimulated (IFN-y, 1.000 UlmL) HUVEC monolayers in 6-well plates.
Before the addition of the monocytes, HUVECs were gently washed
free of IFN-7. The monocyte enriched cells were then added to the
activated HUVECs in a total of 1 mL of media in the 60-mm culture
dishes. Culture supernatants were collected at specific times (4, 12,
24, and 48 hours) after coculture. Peak MCP-1 and IL-8 production
was detected at 24 hours of coculture. To detect the source of the
chemokines, monocytes were separated from the endothelial cells
after 4 hours of incubation using cold (4°C) Ca", Mgz'-free saline
containing EDTA (3-minute incubation). In addition, monocytes
were added to transwells (Costar) in the HUVEC cultures to detect
whether cell contact was obligatory for chemokine production.
Monoclonal antibodies to either ICAM-l, or VCA"1 (R 8r D
Systems, Minneapolis, MN) were added at a concentration of 5 pg/
mL to the HUVECmonocyte interactions. This concentration has
been shown in previous studies to be optimal for inhibiting monocyte:endothelial cell interaction^.'^
Northern blot analysis. Total RNA from either monolayer of
cells or from homogenized tissue was isolated using a modification
of the method of Chirgwin" andJonas.16Briefly, samples were
dispersed in a solution of 25 mmoVL Tris Ph 8.0 containing 4.1
m o m guanidine isothiocyanate, 0.5% sarkosyl, and 0.1 m o m beta
mercaptoethanol. The RNA was further extracted with chloroformphenol and then alcohol precipitated. The separated RNA was transblotted to nitrocellulose, baked, prehybridized, and hybridized with
'*P-5' end-labeled oligonucleotide antisense probes for IL-8,and
MCP-l. In addition, a labeled oligonucleotide for actin served as a
control. Autoradiographs were quantitated using a computer image
analysis system. Equivalent amounts of RNA/gel were monitored
by assessing 28s and 18s rRNA or beta actin mRNA.
Blockage of cellular interactions. Previous results have indicated a role of matrix protein binding for activation of cytokine
expression in monocytes. To interfere with the interaction of monocytes with endothelial cells, the addition of soluble matrix proteins,
fibronectin (Chemicon, Temecula, CA) from human serum or basement membrane component collagen type 1(30 pg/mL), to the coculture assays was used toelucidate the role of these interactions during
cell-to-cell contact. Monocytes were preincubated with the matrix
proteins for 30 minutes at 37°C before the addition to the HUVEC
monolayers. Culture supematants were harvested at various time
Table 1. Production of IL-8 and MCP-1 During
Monocyte:EndothelialCell Interaction
IL-8 Production
(ng/mL)
Group
Nonstimulated HUVEC
Nonstimulated monocytes
Nonstimulated HUVEC + monocytes
IFN-stimulated HUVEC
IFN-stimulated monocytes
IFN-stimulated HUVEC + monocytes
2.5
1.5
28.7
4.3
1.0
37.5
i 0.72
-t 0.20
i 4.4
1.10
2 0.02
i 4.1
MCP-1
Production
(nglmL)
1.4 2 0.16
1.01
5.9 2 0.66
2.7 2 0.62
1.01
6.2 i 0.38
points and assayed for the production of IL-8and MCP- 1 and compared with an intraassay control culture.
IL-8 and MCP-I enzyme-linked immunosorbent assay. Extracellular immunoreactive L-8 and MCP-l was quantitated using a modification of a double-ligand method as previously described." Briefly,
flat-bottomed 96-well microtiter plates were coated with 50 pL/well
of rabbit anticytokine antibodies (1 pg/mL in 0.6 mom NaCl, 0.26
moVL H'BO,, and 0.08 N NaOH, pH 9.6) for 16 hours at 4°C and
then washed with phosphate-buffered saline (PBS) pH 7.5, 0.05%
Tween 20 (wash buffer). Nonspecific binding sites on microtiter
plates were blocked with 2% bovine serum albumin (BSA) in PBS.
Plates were rinsed with wash buffer, and samples added, followed
by incubation for 1 hour at 37°C. Plates were washed, then 50 pL/
well of biotinylated rabbit anti-IL-l0 was added and incubated for
30 minutes at 37°C. After washing of plates, chromogen substrate
was added. The plates were incubated at room temperature, and the
reaction terminated with 50 pL/well of 3 m o m H3S04solution, and
read at 490 nm in an enzyme-linked immunosorbent assay (ELISA)
reader. Standards were '/* log dilutions of recombinant IL-8 and
MCP-l from 1 pg/mL to 100 ng/mL. This ELISA method consistently detected concentrations above 10 pglmL and did not crossreact with MIP-lP, RANTES, IL-la/P, TNFa, ENA-78 (epithelialderived neutrophil activating protein-78), MIP-la, growth-related
oncogene a (GROa), GROP, GRO-y, or neutrophil activating protein-2 (NAP-2).
Statistical analysis. Data are expressed as means ? standard
error ofmean (SEM). Data that appeared statistically significant
were compared by analysis of variance (ANOVA) for comparing
the means of multiple groups and considered significant if P values
were less than .05.
RESULTS
Production of IL-8 and MCP-l during monocyte:HUVEC
interactions. To determine whether HUVEC:monocyte interactions induced increased production of the chemokines,
supernatants from a time course of cocultures were assayed.
Both HUVEC and monocytes individually produced E-8.
However, when monocytes were added onto monolayers of
HUVEC cultures, a synergistic increase in IL-8 production
was observed (P < .OOl). Additional experiments demonstrated that 12 to 18 hours' preactivation of HUVEC monolayers with IFN-y did not significantly increase the level of
IL-8 in either the HUVEC alone or during coculture conditions (Table 1). MCP-1 was produced by unstimulated cultures of HUVEC, but not by monocytes. On HUVEC:monocyte coculture, there was an increase in MCP-1 production
( P < .001). The preactivation of HUVEC monolayers significantlyincreased MCP-l production in HUVEC alone,
but not in monocyte cocultures (Table 1). The addition of
enrichedmonocytesontoHUVECmonolayersreleased
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2769
PRODUCTION OF CHEMOKINES
1-mstimalated HWEC
Fig l.TheexpressionofIL-8
(A) and MCP-1 (B) mRNA during
monocyte:endothelial cell interactions. Enriched monocytes (5
x 105/mLIwere plated onto unstimulated and IFN-y-stimulated (1.000 UlmL) HUVEC
monolayer cultures. Total cellular RNA was isolated from 8hour
cultures
(time of
peak
mRNA expression). Northern
blot analysis
was
performed
using 11-8and MCP-1specific P’*labeled oligo-probes. Repeat exp e r i m e n t ~showed
~
similar results.
2-IFW-HWEC +
3-unsti mutated H W E C
+ t l o no:,
.c
i
W
0
1
2
1
3
2
3
18s
chemokines, IL-8, and MCP- 1 as early as 4 hours’ postcombination and peaked after 24 hours. Furthermore, when enriched monocytes were incubated with HUVEC monolayers
for 4 hours and separated for an additional 24 hours, MCP1 production was derived from the HUVEC monolayers and
not the separated monocytes (data not shown). In contrast,
IL-8 was produced by both the HUVEC monolayer and the
separated monocytes, with the majority (70% to 75%) produced by the HUVEC monolayer (data not shown). These
results indicate that both cell populations were activated during coculture. Neither the coculture of enriched lymphocyte
( S O % ) nor neutrophil (>95%) populations induced the production of either IL-8 or MCP-I, indicating leukocyte specificity for the response. Furthermore, in experiments using
transwell plates to separate the two cell populations, no increased expression of chemokine was observed (data not
shown), indicating a requirement for cell-to-cell contact.
The expression of IL-8 and MCP-I mRNA was analyzed
by Northern blot analysis. Little constitutive expression of
IL-8 or MCP-I mRNA was detected in unstimulated HUVEC cultures. Addition of monocytes to the HUVEC monolayers significantly increased the expression of bothIL-8
(Fig 1A) and MCP-l (Fig IB) mRNA. As with the results
of the protein data, prestimulation of the HUVEC cultures
with IFN-.)I did not increase chemokine mRNA expression
over unstimulated HUVEC cultures. In fact, the cultures that
were not prestimulated with IFN-y appeared to express a
higher level of the chemokine mRNA.
Inability of Ab to either adhesion molecules or inJlammatory cytokines to inhibit IL-8 or MCP-I production. Our
laboratory has demonstrated that the ability to induce MIPla, a C-C chemokine, during HUVEC:monocyte coculture
was dependent on ICAM-l interactions.’3 To determine
whether IL-8 and MCP-I were also induced by adhesion
molecules, unstimulated and IFN-y-stimulated HUVEC
were layered with enriched monocytes in the presence or
absence of anti-ICAM-I MoAb. The results showed that no
inhibition of IL-8 (Fig 2) or MCP-I (Fig 3) production was
observed. In addition, Northern blot analysis of IL-8 and
MCP- 1 mRNA also demonstrated no decrease in the mRNA
T
50
A
A
I
I
.
E
.-
0
-30
V
U
10
0
P>
3
X
Unstimulated
C
m
0
Y
E
>
3
X
IFN-g Pre-stimulated
Fig 2. Blockage of adhesion molecule, ICAM-1, interaction does
not inhibitIL-8 production during monocyte:endothelial cell interactions. Enriched monocytes (5 x 105/mL)were plated onto unstimulated and IFN-y-stimulated (1.000 UlmL) HUVEC monolayer cultures.
MoAbs (5 pg/mL) directed against CAM-l were added to the cocultures and24-hoursupernatantswereharvestedandassayedby
ELlSA for IL-8.
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2770
LUKACS ET AL
‘I
.I
t
m
T
T
T
E
v
ti
0
I
1
+
8
>
C
m
3
I
unStirnulated
1 production was inhibited only by the addition of soluble
collagen (P < .05), butnot significantly withfibronectin
(Fig 5 ) , possibly suggesting differences inthe activation
pathway for these two chemokines. The examination of the
mRNA expression during the coculture also demonstrated
similar findings.When the monocytes were preincubated
withmatrix proteins, the expression of IL-8 andMCP-I
mRNAwas decreased (Fig 6). Similar to the results for
protein levels of IL-8 and MCP-1, IL-8 mRNA levels were
diminished when monocytes were incubated with either soluble collagen or fibronectin, whereas MCP-1 demonstrated
that only collagen was effective in decreasing the mRNA
expression. There appeared to be some specificity for the
matrix protein used, as laminin did not inhibit either IL-8 or
MCP-1 (data not shown). Altogether, the latter data suggest
that IL-8 and MCP-I production during cell-to-cell contact
may be induced, in part, via matrix protein binding events.
IFNg Pre-Stimulated
Fig 3. Blockage of adhesionmolecule, CAM-1, interaction does
not inhibit MCP-1 productionduring monocyte:endothelial cell interactions. Enrichedmonocytes(5 x 106/mL)were plated onto unstimulated and IFN-y-stimulated (1.000 UlmL) HUVEC monolayer cultures.
MoAbs (5 pg/mLl directed against ICAM-1 were added to thecocultures and 24-hour supernatants were harvested andassayed by
ELISA for MCP-1.
expression by treatment of cocultures with anti-ICAM-11
VCAM-I molecule MoAbs (data not shown).
In additional experiments, we were also interested in
whether IL-1 and TNF played a role in the production of
IL-8 and MCP- 1. These two potent inflammatory cytokines
have previously been shown to induce chemokines in most
cell types. In coculture supernatants from different time
points ( 1 , 4, 8, 12, and 24 hours) little IL-I (< 100 pg/
mL) and no TNF was detected. Subsequently, addition of
neutralizing antibodies to the coculture assays directed
against IL- 1 and TNF had no effect on either IL-8 or MCPl production (data not shown). Altogether, these studies
show that neither adhesion molecule nor inflammatory cytokine cascades were responsible for the upregulation of IL8 and MCP-1.
Inhibition of chemokine production by blocking matrix
protein interactions. To determine whether additional cellto-cell interactions were influencing the production of IL-8
and MCP-I, we examined the role of matrix protein interactions. Previous studies have identified matrixprotein interactions as inducing IL-8 production.” To identify therole
matrix proteins played in the HUVEC:monocyte interactions, the mononuclear cells were preincubated with either
soluble fibronectin or type I collagen (30 pg/mL) for 15
minutes and then layered onto unstimulated HUVEC monolayers. These studies demonstrated a significant inhibition in
chemokine production during the cell-to-cell interactions,
(Figs 4 and 5). IL-8 production during cell-to-cell interactions was inhibited by the addition of either soluble fibronectin or collagen to the culture media (Fig 4) ( P <c .05). MCP-
DISCUSSION
The extravasation of cells from the lumen of a vessel
into inflamed tissue depends on leukocyte interaction with
activated endothelium.’,’ The initial adhesion relies on interactions between P-integrins on the surface of leukocytes and
30-
20-
10
-
P
0
S
+
U
W
2
I
o +
W
>
+ +
3
I
I:
Fig 4. Inhibition of IL-8 productionby solublematrix proteinsduring monocyte:endothelialcell interactions. H U M C and monocyte
populations were preincubated with soluble collagen or fibronectin
for 30 minutes before coculture. Enriched monocytes 15 x 1O5/mL1
were plated onto unstimulated HUVEC monolayer cultures. Cell-free
culture supernatants were harvested after 24 hours and analyzedfor
IL-8 production. Data represent the mean of three repeat experiments.
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PRODUCTION
OF CHEMOKINES
277 1
T
Fig 5. Inhibition of MCP-1 production by soluble matrix proteins
during monocyte:endothelial cell interactions. HUVEC and monocyte
populations were preincubated with soluble collagen or fibronectin
for 30 minutes before coculture. Enriched monocytes l5 x 105/mL)
were plated onto unstimulated HUVEC monolayer cultures. Cell-free
culture supernatants were harvested after
24 hours and analyzedfor
MCP-1 production. Data represent the mean of three repeat experiments.
adhesion molecules on the activated endothelium. Additional
interactions between the endothelium and adhered leukocytes, which may include binding via matrix proteins as well
as other specific receptor-ligand interactions, also appear to
be ~perative.'"'~
In the present study, our results demonstrate
activation of IL-8 and MCP-I expression during monocyte:HUVEC interactions. The upregulation of the two
chemokines was dependent on cell-to-cell contact andnot
soluble mediators, as the addition of enriched monocytes to
HUVEC cultures in transwell separated culture dishes did
not induce the expression of the chemokines. Because monocyte adherence and stretching over the endothelium is a reversible and dynamic event,*').*'the interaction of monocytes
with the HUVEC must be transient. Interestingly, the increased production of the two chemokines was notdependent
on activation of the endothelium, as IFN-y pretreatment of
HUVEC monolayers had little effect on IL-8 or MCP-I production. In addition, the interaction between cells leading to
the production of IL-8 and MCP-l wasnot dependent on
ICAM- 1 or VCAM- 1 interactions, as MoAbs to these molecules also had no effect on the chemokine production. These
findings are in contrast to our previous study that examined
MIP-la production during monocyte:HUVEC interaction^.'^
In the previous study, we showed that MIP-la production
was dependent on ICAM-I expression and interactions of
monocytes with activated endothelial cells. The present
study also investigated production of potentinflammatory
cytokines, IL-I, and TNF, which could potentially induce
the increased IL-8 and MCP-I production through cytokine
cascades. These results demonstrated little detectable IL-I
and TNF production at any time throughout the coculture
assay. In addition, antibodies directed to IL-I and TNF did
not inhibit the production of either IL-8 or MCP-I. These
data suggest that the increased production of the two chemokines is independent of traditional inflammatory cytokine
ACTIN
IL-8
MCP-1
A
Fig 6. Inhibition of K-8 and MCP-1 mRNA expression during monocyte:endothelial cell interactions
by soluble matrix proteins. RNA was analyzed from
HUVEC alone (A) or HUVEC and monocyte populations preincubated with BSA (B), solublefibronectin
IC), or soluble collagentype I ID) for 30 minutes be(5 x 105/mL)
fore coculture.Enrichedmonocytes
were plated onto unstimulated HUMC monolayer
cultures. Total cellular RNA was isolatedfrom 8-hour
cultures (time of peak mRNA expression). Northern
blot analysis was performed using IL-8 and MCP-1
specific
P3*-labeled
oligo-probes.
Repeat
experiments showed similar results.
B
C
c
A
B
C
D
D
5-
A
B
C
D
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2772
cascades. However, the production of IL-8 and MCP-1 was
dependent on the interaction of monocytes with matrix proteins, possibly via p-integrins." This latter mechanism was
verified using soluble matrix proteins to inhibit chemokine
production. The fact that both soluble fibronectin and collagen decreased IL-8, while only soluble collagen diminished
MCP-1 production, suggests differential regulation during
the cell-to-cell interactions. Interestingly, IL-8 was produced
by both cell types, whereas MCP-1 was produced by only
the HUVEC monolayer. This discrepancy in chemokine production may explain the differential regulation by the soluble
matrix proteins. Because the preincubation of cells before
coculture only partially decreased the production of chemokines, it is likely that other mechanisms are operative during
the binding events that facilitate the upregulation of the
chemokine production other than matrix binding. Altogether,
these results may be indicative of regulational events that
occur during cellular interactions, adhesion events, and
chemokine production.
The induction of cytokines during monocyte adhesion has
previously been shown in other studies."." In addition, the
upregulation of L - 8 protein in monocytes during fibronectin
adherence events has demonstrated a relationship of cellular
adhesion to chemokine production." The mechanism of
these interactions during leukocyte extravasation under flow
conditions would be dependent on the initial binding of
monocytes to endothelial expressed selectin and adhesion
molecules, thus allowing secondary adhesive events to conAs previously demonstrated, the specific binding to
ICA"1 was required to upregulate the production of MIPla from monocyte^.'^ The binding of monocytes under nonflow conditions to other ligands on endothelium,'"20as demonstrated in the present study, can upregulate the expression
of IL-8 and MCP-1. However, in vivo, the secondary interactions under flow conditions would always be dependent on
the initial requirement of selectin and ICAM-1 expression
on the endothelial cell layer','and subsequent monocyte
binding to additional ligands, such as matrix proteins. Therefore, the limiting step for upregulation of IL-8 and MCP-1
under the shear forces of flow conditions would always be
inflammation-induced expression of adhesion molecules on
the endothelium. This would give the system the fidelity
needed to control the induction and release of the chemokines during homeostatic and inflammatory events. These
interactions may be a contributing step in exacerbation of
vascular diseases, such as atherosclerosis, in which there is
prolonged and intimate interaction of monocytes and endothelial cells correlated with chemokine expression within the
Iesion.22-25
The significance of the increased production of chemokines during extravasational events may be multifunctional.
The activation of leukocytes as they extravasate into inflamed tissue may allow deposition of chemokines into the
surrounding tissue, intensifying the chemotactic gradient,
and increasing the accumulation and activational status of
the infiltrate. Additionally, these chemokines may also function to activate not only the infiltrating leukocytes, but also
the surrounding structural cells, therefore, maintaining the
inflammatory milieu. Finally, the increased production of
IL-8 and MCP-l may aid in roles other than recruitment of
LUKACS ET AL
leukocytes. For example, IL-8 has recently been described
as a potent angiogenic
whereas MCP-1 (JE) wasfirst
described as a competence factor for fibroblast activation and
proliferation." Both of these understudied areas of chemokine functions could also contribute to the maintenance of
an inflammatory response.
The production of cytokines during cell-to-cell interactions may be an important mechanism for maintenance and
regulation of an inflammatory response. Increased IL-8 and
MCP-1 production during monocyte:endothelial cell interaction was not dependent on inflammatory cytokine (IL-1 or
TNF) cascades or expression of adhesion molecules (ICAM1 or VCAM-l), but, in part, on interactions with matrix
proteins. These studies suggest that interactions of leukocytes with endothelial cells can induce XL-8 and MCP-I
production, possibly influencing the intensity of the inflammatory infiltrate and maintenance of the response in vivo.
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1995 86: 2767-2773
Production of chemokines, interleukin-8 and monocyte
chemoattractant protein-1, during monocyte: endothelial cell
interactions
NW Lukacs, RM Strieter, V Elner, HL Evanoff, MD Burdick and SL Kunkel
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