| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Liver Research Laboratories, University of Birmingham Institute of Clinical Science, Medical Research Council Center for Immune Regulation, Edgbaston, Birmingham, United Kingdom; and
Medicity Research Laboratories, University of Turku, Turku, Finland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-stimulated HSEC was
dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow,
blocking VAP-1 reduced lymphocyte adhesion to TNF--treated HSEC by
50% and significantly reduced the proportion of adherent lymphocytes
that transmigrated across cytokine or LPS-activated endothelium. In
addition, inhibition of the amine oxidase activity of VAP-1 reduced
both adhesion and transmigration of lymphocytes to a level similar to
that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial
migration as well as adhesion, and both functions are dependent on its
enzymatic activity. In the absence of selectins and CD31, VAP-1 may
play a specific role in lymphocyte recruitment via hepatic sinusoidal
endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium
in response to inflammation, its ability to support lymphocyte
transendothelial migration may be an important systemic function of
VAP-1. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lymphocytes recirculate continuously between blood and tissues as part
of the process of immune surveillance, and recent evidence suggests
that specialized subsets of T cells exhibit distinct tissue-specific
patterns of recirculation in vivo (11, 12). These cells
are directed to particular tissues by combinations of adhesion
molecules and chemokines that control lymphocyte recognition of and
adhesion to endothelium. For example, memory T cells that recirculate
to the gut lamina propria express the integrin
4
7 and bind to an
endothelial ligand, mucosal cell adhesion molecule-1, found mainly in
gut endothelium (13), whereas T cells that migrate to the
skin do not express
47, but do express
high levels of the cutaneous lymphocyte Ag that binds to E-selectin on
dermal endothelium (14). The liver is a major site of Ag
exposure and contains large numbers of lymphocytes even under normal
conditions. Although some of these are terminally differentiated
lymphocytes that are removed by apoptosis, the liver also contains
functional lymphocytes recruited as part of the process of immune
surveillance (15, 16, 17, 18). It is thus possible that
tissue-specific signals will regulate lymphocyte recruitment to the
liver (19).
The hepatic sinusoids are lined by specialized endothelium that supports lymphocyte adhesion and recruitment in a unique low shear environment (20). Hepatic sinusoidal endothelium has a distinct phenotype compared with endothelium from other vascular beds. In vivo sinusoidal endothelial cells express low levels of CD31 and show little, if any, expression of selectins, which are the most common capture receptors in other tissues (21, 22). Moreover, functional studies in selectin-deficient animals suggest a minimal role for selectins in leukocyte recruitment via the sinusoids (23). However, human hepatic sinusoids constitutively express the endothelial adhesion molecule vascular adhesion protein-1 (VAP-1),3 which is largely absent from noninflamed vessels in extralymphoid organs (24, 25). The ability of VAP-1 to mediate sialic acid-dependent adhesion suggests that it could have a particular function in the liver by mediating shear-dependent adhesion in the absence of selectins (23). To date, the sequence of events governing lymphocyte recruitment by human liver endothelial cells under conditions of shear stress is unknown. We have previously shown (25, 26) that VAP-1 supports T cell adhesion to human hepatic endothelium in tissue sections, but in the present study we have determined in vitro the detailed nature of VAP-1-mediated adhesion of lymphocytes to human hepatic endothelium.
The molecular characterization and cloning of VAP-1 show it to be a 170-kDa homodimeric sialoglycoprotein consisting of two 90-kDa subunits with close sequence homology to the copper-dependent semicarbazide-sensitive amine oxidases (SSAO). Both the transmembrane and soluble forms of VAP-1 exhibit monoamine oxidase activity (27, 28, 29), and two of us (M. Salmi and S. Jalkanen) have recently proposed that VAP-1 on endothelium supports lymphocyte adhesion by binding to and oxidatively deaminating a primary amino group presented on the lymphocyte surface, resulting in the formation of a transient covalent bond between the two cell types (30).
In the present paper we show that human hepatic sinusoidal endothelial cells maintain a unique morphology and phenotype in culture and use VAP-1 in conjunction with ICAM-1 to support lymphocyte adhesion under flow. VAP-1 was not expressed by non-hepatic endothelial cells and could not be induced on these cells by cytokine treatment. Under physiologically relevant levels of shear stress, VAP-1 made a minor contribution to rolling adhesion of lymphocytes on hepatic endothelium, but mediated levels of stable adhesion equivalent to those supported by ICAM-1. In addition, blockade of the adhesive or enzymic capacity of VAP-1 consistently inhibited lymphocyte transendothelial migration through hepatic endothelial monolayers. Thus, we suggest that VAP-1 on hepatic endothelium can mediate adhesion and transendothelial migration of lymphocytes and thereby play an important role in directing T cell recirculation to the liver.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Liver endothelial cells were isolated from 
150 g human liver
tissue obtained from donor tissue surplus to surgical requirements as
described previously (16) using a modified collagenase
perfusion technique. Nonparenchymal cells were separated by density
gradient centrifugation over metrizamide (Sigma, Poole, U.K.), and
endothelial cells were isolated from the resultant heterogeneous cell
mixture by positive immunomagnetic selection using Abs against CD31
(Dako, Ely, U.K.; M823, 10 µg/ml) and Dynabeads conjugated
with goat-anti mouse mAb (Dynal Biotech, Wirral, U.K.) according to the
manufacturer’s protocol. HUVEC, isolated using standard methods, were
used as a control endothelial cell line.
Maintenance and culture of endothelial cells
Following isolation, hepatic endothelial cells were cultured in complete medium composed of human endothelial basal growth medium (Life Technologies, Paisley, U.K.), 10% AB human serum (HD Supplies, Glasgow, U.K.), 10 ng/ml vascular endothelial growth factor (VEGF) and 10 ng/ml HGF (R&D Systems, Abingdon, U.K.). The cells were placed in collagen-coated culture flasks and were maintained at 37°C in a humidified 5% CO2 incubator until confluent. HUVEC were maintained in the same medium, except the HGF and VEGF were replaced by 10 ng/ml EGF (R&D Systems) and 1 µg/ml hydrocortisone (Sigma). These cells were grown to confluence in culture flasks coated with 1% type B bovine skin gelatin dissolved in PBS (Sigma).
Expression of cell surface adhesion molecules on endothelial cells
Endothelial cells were grown to confluence in collagen-coated
96-well plates and fixed with methanol before performing ELISA. On some
occasions endothelial cells were stimulated with cytokines (10 ng/ml
recombinant human TNF-, 10–50 ng/ml recombinant human TNF-,
10–100 ng/ml recombinant human IFN-
, 10 ng/ml recombinant human
IL-1; all from PeproTech, London, U.K.), LPS (1–10 µg/ml; Sigma),
bile salts (ursodeoxycholic acid and chenodeoxycholic acid, 50–200
µM; Sigma), or 50% heat-inactivated serum from patients with
alcoholic hepatitis for 24 h before ELISA development. Nonspecific
binding of mAb to endothelial cells was prevented by preincubation of
the cells with 4% goat serum (Sigma) for 1 h. The endothelial
cells were then incubated with mouse anti-human primary mAb
(ICAM-1: BBA3, 1 µg/ml; VCAM-1: BBA5, 1 µg/ml (both from R&D
Systems); CD31: M823, 4 µg/ml; E-selectin: M7105, 3 µg/ml (both
from Dako); and VAP-1: 1B2, 1 µg/ml) for 45 min at 37°C. The cells
were washed thoroughly and incubated with a peroxidase-conjugated goat
anti-mouse secondary Ab (P0447, 1/5000; Dako) for 45 min at 37°C.
The ELISA was developed using O-phenylenediamine
substrate (Dako) according to the manufacturer’s instructions, and the
absorbance values were determined at 490 nm. All treatments were
performed in triplicate for each experiment.
Static adhesion assays
Peripheral blood lymphocytes were isolated from EDTA
anti-coagulated venous blood by centrifugation over
Ficoll (Histopaque; Sigma) as previously described (5).
HSEC and HUVEC were grown to confluence in 48-well tissue culture
plates either without stimulation or with 10 ng/ml TNF- for 24
h before use. Lymphocytes at a final concentration of 1 x
106 cells/ml were incubated with confluent
monolayers of endothelial cells for 30 min at 37°C. The wells were
then washed and fixed in methanol, and the number of adherent
lymphocytes was determined by counting 10 replicate fields (x100
magnification) of cells in each of two replicate wells per experiment;
adhesion was expressed as adherent cells per field.
Flow-based adhesion assays
To determine the effects of physiological blood flow on lymphocyte adhesion, cytokine-stimulated HSEC were cultured to confluence in glass capillary tubes and connected to the flow system previously described (5). Lymphocytes (1 x 106 cells/ml) were perfused through the microslide over the endothelial cells within physiologically relevant ranges of shear stress between 0.05 and 0.2 Pa. Adherent lymphocytes were observed microscopically or via video monitor. Adhesion was converted to cells per square millimeter and corrected for the number of lymphocytes perfused (i.e., adherent cells per square millimeter per 106 perfused). Phase contrast video recordings made during lymphocyte perfusion were analyzed off-line to determine the percentage of rolling cells, statically adherent cells, and transmigrated cells. Rolling cells moved slowly, but steadily, over the endothelial surface during 5–10 s of observation, while stationary adherent cells did not make any detectable movement over the same period. Migrated cells appeared phase dark and were clearly distinguishable from polarized cells migrating across the luminal endothelial surface, which remained phase bright.
Ab interventions
To determine which molecules were involved in lymphocyte adhesion to HSEC, confluent monolayers of cytokine-treated endothelial cells or peripheral blood lymphocytes were treated with function-blocking mAb for 20 min before perfusion of lymphocytes. The mAb used were against CD11a (R3.1.E2; gift from A. Wayne, Boehringer Ingleheim, Hartford, CT), CD18 (R15.7/H4; gift from A. Wayne), P-selectin (Mab2154; Chemicon, Hartford, CT), VCAM-1 (BBA5; R&D Systems), ICAM-1 (BBA3; R&D Systems), VAP-1 (1B2), E-selectin (BBA2; R&D Systems), and CD31 (mAb 1398z; Chemicon). All mAb were used at saturating concentrations.
Inhibition of the enzymic capacity of VAP-1
To elucidate whether the monoamine oxidase activity of VAP-1 had a role in the adhesive function of this molecule when presented by hepatic endothelia we pretreated endothelial cells with semicarbazide (200–500 µM; Sigma) or hydroxylamine (1–10 µM; Sigma) for 20 min before perfusion of lymphocytes.
Statistical analysis
Comparisons between treatments were analyzed by ANOVA with MiniTab software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cultured HSEC demonstrated a typical endothelial cobblestone
morphology after 1 wk in culture, bound Ulex europaeus
lectin, and displayed characteristics of sinusoidal endothelium, in
that they took up labeled acetylated low density lipoprotein with a
cytoplasmic staining pattern (31) (data not shown). In
addition, although TNF- treatment stimulated substantial P-selectin
expression on HUVEC we were unable to induce HSEC to express similar
levels of P-selectin under the same conditions. Lack of P-selectin
expression is another characteristic feature of sinusoidal endothelium
(21). ELISA revealed constitutive expression of ICAM-1,
CD31, and low levels of VAP-1 (Fig. 1
A), induction of VCAM-1 and
E-selectin, and increased levels of ICAM-1 following treatment with
TNF-. CD31 expression was consistently lower than that seen on HUVEC
in keeping with the reported reduced expression of CD31 on hepatic
sinusoidal endothelium in vivo. Compared with HSEC, HUVEC expressed
similar profiles of most endothelial adhesion molecules (Fig. 1B), but could not be induced to express VAP-1. It was noted
that basal VAP-1 expression on HSEC varied between donors and tended to
be lost after five or six passages in culture (for example, the VAP-1
ELISA absorbance values for two typical isolate of cells treated
identically were reduced from 0.214 and 0.115 to 0.093 and 0.058,
respectively, after one passage in culture).
|
We attempted to increase cell surface VAP-1 expression on HSEC by
treating the cells with a variety of factors, including TNF-,
IL-1, IFN-, bile acids, and LPS. None of these factors
consistently increased the expression of VAP-1, although levels of
ICAM-1 were elevated appropriately (Fig. 2). Interestingly, serum from patients
with alcoholic hepatitis who demonstrated high levels of circulating
soluble VAP-1 (32) did appear to cause a modest, but
significant, elevation of VAP-1. However, serum had no effect on the
level of ICAM-1 expression by HSEC.
|
The detection of constitutive expression of VAP-1 on the cell
surface of HSEC allowed us to investigate the adhesive function of
VAP-1 in detail. In static adhesion assays using peripheral blood
lymphocytes, adhesion to unstimulated HSEC was inhibited by mAb against
CD11a, CD18, or VAP-1, whereas inhibiting VCAM-1, ICAM-1, or the
selectins alone had little effect. This suggests that both VAP-1 and
ICAM-2 have a role in adhesion in this system. When TNF--activated
HSEC were used, adhesion was inhibited by mAb against CD11a, CD18,
ICAM-1, VCAM-1, and VAP-1. Anti-E-selectin and anti-P-selectin had
no effect. VAP-1 Abs had no effect on lymphocyte adhesion to HUVEC
(Fig. 3B) or the dermal
endothelial cell line HMEC-1 (not shown).
|
, TNF-, and LPS for
24 h before lymphocyte perfusion greatly increased the number of
adherent cells (Fig. 4A), and
the level of this adhesion was shear stress dependent (Fig. 5 shows representative data for
TNF-treated endothelial cells (EC)). Offline frame-by-frame analysis
of the experimental video record was performed to quantify the number
of adherent lymphocytes that exhibited sustained, rolling adhesion. The
percentage of lymphocytes that underwent sustained rolling adhesion on
HSEC varied according to which cytokine was used to stimulate the
endothelial cells (Fig. 4B). However, on average, <10% of
the total number of adherent lymphocytes rolled persistently over the
HSEC, and the majority immediately arrested upon contact with EC. This
finding was particularly interesting and suggested that in the unique
rheological environment present within the hepatic sinusoid, classical
sustained rolling adhesion is not a prerequisite for lymphocyte capture
and arrest.
|
|
-stimulated HSEC.
Since TNF- treatment of HSEC resulted in the highest levels of
lymphocyte adhesion, we decided to use this cytokine for subsequent
experiments designed to elucidate whether VAP-1 has a role in
lymphocyte adhesion to hepatic endothelium under conditions of shear
stress. Treatment of TNF--stimulated HSEC with anti-VAP-1 mAb
consistently reduced the total number of lymphocytes adhering by
50% (Fig. 6A). The degree
of adhesion blockade varied between different cell preparations from
different livers, but was related to the level of VAP-1 expression seen
on each cell isolate. Treatment of TNF--stimulated HUVEC
withanti-VAP-1 had no effect on lymphocyte adhesion (for example,
in one representative experiment adhesion of lymphocytes to HUVEC at
0.05 Pa was 818 and 850
cells/mm2/106 cells
perfused for control and VAP-1 Ab-treated cells, respectively). A
blocking mAb raised against ICAM-1 reduced lymphocyte adhesion to both
HSEC (Fig. 6A) and HUVEC. Combinations of both VAP-1 and
ICAM-1 Abs reduced adhesion to HSEC more than either Ab alone, but no
additional inhibitory effect on rolling adhesion was observed under
these circumstances (Fig. 6). An isotype-matched control Ab had no
effect (not shown). To totally inhibit lymphocyte adhesion to HSEC
under shear, it was necessary to treat the endothelium with a
combination of VAP-1, E-selectin, VCAM-1, and ICAM-1 Abs (not
shown).
|
-stimulated HSEC (Fig. 6
B), but the effect was small
and was not statistically significant. However, treatment of
cytokine-activated HSEC with anti-E-selectin mAb significantly
reduced the percentage of adherent cells that rolled (Fig. 7). Although little if any E-selectin is
expressed on hepatic sinusoidal endothelium in vivo, isolated cultured
HSEC expressed E-selectin following TNF- treatment (see Fig. 1A). Therefore, to reproduce the adhesive environment that
exists in vivo, we repeated our adhesion experiments in the continuous
presence of anti-E-selectin mAb. Under these conditions the
addition of anti-VAP-1 mAb-1 reduced the total adhesion further,
but did not reduce the number of rolling cells (Fig. 7). These data
suggest that even in the absence of selectins, VAP-1 does not act as a
classical rolling receptor.
|
-stimulated endothelial cells, but only 10% migrated through
TNF--stimulated HSEC (Fig. 8).
However, in each experiment, regardless of which cytokine was used to
stimulate the endothelium, treatment of HSEC with anti-VAP-1 mAb
reduced the proportion of transmigrating cells. Anti-ICAM-1 also had a
significant effect on lymphocyte transendothelial migration, whereas
isotype-matched control Ab, anti-VCAM-1, and anti-E-selectin
Abs had no effect.
|
shows
that when HSEC were activated for 24 h with TNF- and then
treated with either hydroxylamine or semicarbazide before
perfusion of lymphocytes, adhesion was significantly reduced. We noted
that the magnitude of the inhibitory effect of these agents was
sometimes greater than that of the anti-VAP-1 mAb 1B2. We also
analyzed the percentage of adherent lymphocytes that migrated through
HSEC after treatment with inhibitors of monoamine oxidase activity and
noted that migration was almost abolished after treatment (Fig. 9B). We also noted that the effects of these inhibitors were
rapidly reversible. If the monoamine oxidase inhibitors were not
maintained in our wash solution, adhesion and transmigration of
lymphocytes returned to those levels seen in the absence of
inhibitor.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Functional characterization of VAP-1 has been difficult because it is not expressed on the cell surface of previously studied human endothelial cell lines (36). Our finding of significant levels of cell surface VAP-1 on liver endothelial cells in vitro allowed us to report for the first time the detailed function of VAP-1 using primary endothelial cells. VAP-1 is constitutively expressed on human liver endothelium in vivo, and by isolating endothelial cells from human liver and culturing them in a combination of HGF and VEGF we were able to establish primary cultures that had morphological and functional characteristics of sinusoidal endothelium. We refer to these cells as human HSEC. Although rabbit cardiac endothelial cells have been shown to express VAP-1 in culture, we have failed to induce VAP-1 expression on non-hepatic human endothelial cells in vitro, suggesting that microenvironmental factors are critical for VAP-1 expression (34, 36). Compared with HUVEC cultured under the same conditions, HSEC had a distinctive phenotype-expressing cell surface VAP-1, low levels of CD31, and little or no P-selectin even under conditions in which P-selectin was detected on HUVEC. This is consistent with the phenotype in vivo where sinusoidal endothelial cells lack Weibel-Palade bodies, express little CD31 or P-selectin, but constitutively express VAP-1 (37, 38, 39), demonstrating that these cells maintain tissue-specific characteristics in vitro. HSEC could be induced to express functional E-selectin by treatment with pro-inflammatory cytokines, which contrasts with the situation in vivo where little or no E-selectin expression is detected even on inflamed sinusoidal endothelium, suggesting that E-selectin up-regulation is suppressed in vivo (21, 22).
We attempted to increase cell surface expression of VAP-1 with a
variety of factors, including pro-inflammatory cytokines, LPS, and bile
acids. None of these factors consistently increased cell surface VAP-1,
suggesting that VAP-1 expression is tightly regulated and is not
responsive to factors that increase the expression of broadly expressed
adhesion molecules such as ICAM-1, VCAM-1, and the selectins. However,
we observed that serum from patients with alcoholic liver disease who
had high circulating soluble levels of VAP-1 modestly elevated
endothelial expression of VAP-1 (Fig. 2A). Since we did not
see a concurrent elevation in ICAM-1 levels (Fig. 2B) on the
same endothelial isolates, we suspect that the VAP-1 response is
probably not due to a classical proinflammatory cytokine, but as yet we
have not identified the factor responsible. Thus, VAP-1 shows
restricted tissue expression in vivo and is up-regulated by a limited
number of signals in vitro, suggesting that it might play a specific
role in lymphocyte recruitment to the liver. This is supported by our
previous observations that the circulating form of VAP-1 is derived
from the hepatic vascular bed and is elevated in chronic inflammatory
liver diseases (32, 40).
The constitutive expression of VAP-1 and the minimal role for selectins in the sinusoids (23) together with the distinct rheological features of this vascular bed (41) suggest that some of the elements of the adhesion cascade will differ from those operating in postcapillary venules. To assess the role of VAP-1 under conditions comparable to those seen within the liver in vivo, we established a flow-based adhesion system in which we replicated the low levels of shear stress detected within hepatic sinusoids. This model allowed us to study the nature of the adhesion mediated by hepatic endothelial VAP-1 under flow for the first time and thereby to elucidate the adhesive function of this unique receptor.
Under conditions of low shear stress that reflect those found in
sinusoids in vivo, only a small proportion of lymphocytes exhibited
sustained rolling, and most of the adherent cells underwent rapid
arrest without prior rolling. This may reflect the comparatively low
levels of E-selectin detected on these cells in vitro as well as a
reduced dependency on rolling interactions to capture lymphocytes
within the low shear environment of the sinusoids. Inhibition of VAP-1
had a consistent inhibitory effect on the adhesion of lymphocytes to
HSEC under flow, although the magnitude of the effect varied between
cell isolations and with the activating stimulus. When
TNF--activated HSEC were used, the inhibition seen with
anti-VAP-1 was comparable to that seen with an Ab to ICAM-1, but
the ability of VAP-1 Ab to block adhesion to LPS- or TNF--activated
endothelium was less marked despite similar levels of VAP-1 on all
preparations. This may reflect increased expression of other molecules,
because TNF--activated endothelium consistently supported higher
total adhesion than TNF-- or LPS-activated endothelium.
A role for VAP-1-mediated adhesion under shear stress is consistent with previous studies showing VAP-1-dependent lymphocyte adhesion to rat peripheral lymph node endothelial cells transfected with human VAP-1 and recent intravital studies where it has been shown to act as a brake for neutrophils on rabbit mesenteric vessels (34). The precise nature of the adhesion mediated by VAP-1 is unclear. It did not act as a classic rolling receptor because the proportion of rolling cells was unaffected by VAP-1 blockade in most of our experiments. Furthermore, although the total number of adherent cells was consistently reduced when inhibition of VAP-1 was combined with inhibition of E-selectin, the number of rolling cells was the same as that seen with E-selectin blockade alone, suggesting that these receptors are acting at different points in the adhesion cascade.
In addition to inhibiting adhesion, blockade of VAP-1 had a marked
inhibitory effect on lymphocyte transendothelial migration that was
independent of its ability to support adhesion and was seen with
TNF-- and LPS-activated HSEC as well as TNF--treated cells. This
effect was specific for VAP-1 or ICAM-1 Abs and did not occur with
control Ab or Abs against VCAM-1 or E-selectin. Comparatively little is
known about the molecules that regulate the transendothelial migration
of adherent lymphocytes. There is evidence that
2 integrins and their ligands are involved
(42, 43), although other studies suggest that they act
predominantly at the firm adhesion step rather than directly regulating
transmigration (44). Several molecules detected
preferentially at endothelial cell junctions, including CD31 and JAM-1,
have been proposed to mediate transmigration of adherent leukocytes
(45). The role of CD31 in lymphocyte transmigration is
contentious, and because CD31 and its putative receptor
v3 are not expressed
on all lymphocyte subsets, it cannot provide a general mechanism for
transmigration (46, 47). Moreover, mice that are deficient
in CD31 exhibit normal lymphocyte migration (48). In the
present study we found that both anti-ICAM-1 and anti-VAP-1
inhibited lymphocyte transmigration under conditions of flow. We were
careful to assess transendothelial migration as a proportion of
adherent cells to allow an accurate assessment of transmigration
independently of the contact and arrest steps, allowing us to state
that both ICAM-1 and VAP-1 are directly involved in transendothelial
migration in our model. These results support a role for VAP-1 and
ICAM-1 in lymphocyte recruitment to the liver and also suggest that
VAP-1 could be involved in transmigration at other sites where it is
up-regulated during inflammation (24).
VAP-1 is a monoamine oxidase belonging to the subfamily of semicarbizide-sensitive amine oxidases. We have recently shown that the previously reported increased monoamine oxidase activity in serum of patients with chronic liver disease (49, 50, 51) is, in fact, SSAO activity derived from soluble VAP-1 protein (40). Recent reports suggest that the adhesive and enzymic functions of VAP-1 are intimately linked, and we therefore investigated whether this was the case in our system. We used specific inhibitors to block enzymic activity of VAP-1 and found that the specific semicarbazide-sensitive amine oxidase inhibitor (semicarbazide) and a broad-acting monoamine oxidase inhibitor (hydroxylamine) both markedly reduced lymphocyte adhesion and transmigration to levels comparable to those observed with Ab blockade. These data differ from those from our previous studies (33), which found no role for the enzymic capacity of VAP-1 in the adhesion of lymphocytes to VAP-1-transfected cells. However, the transfected cells have relatively low monoamine oxidase activity (S. Jalkanen, unpublished observations), and there may clearly be other differences between transfectants and our primary endothelial cells. We conclude that native VAP-1 expressed by hepatic endothelial cells has both adhesive and enzymic functions.
This effect was specific for hepatic endothelial cells that express VAP-1, since adhesion and transmigration of lymphocytes on HUVEC were not decreased by either treatment. Other molecules have been reported to share adhesive and enzymatic properties, including CD26, CD73, and the adamalysins, but VAP-1 is the only adhesion molecule with amine oxidase activity (30, 52). We have not yet elucidated a mechanism by which enzymic catalysts can support adhesion, but two of us (M. Salmi and S. Jalkanen) have recently proposed that the active site of SSAO in VAP-1 may mediate adhesion via interactions with immobilized amine residues on the lymphocyte surface (30). The fact that the inhibitors had a similar effect on transendothelial migration is the first demonstration of enzymic regulation of this process.
In summary, we have shown that VAP-1 is expressed on human hepatic endothelial cells in culture and that its expression is regulated by a limited number of factors. The ability of VAP-1 to support lymphocyte adhesion and transendothelial migration under conditions of shear stress comparable to those found in the hepatic sinusoids in vivo suggest that it is functionally important in regulating lymphocyte recruitment to the human liver.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. David H. Adams, Liver Research Laboratories, Institute for Clinical Research, Queen Elizabeth Hospital, Edgbaston, Birmingham, U.K. B15 2TT. E-mail address: d.h.adams{at}bham.ac.uk
3 Abbreviations used in this paper: VAP-1, vascular adhesion protein-1; EC, endothelial cells; HSEC, human hepatic sinusoidal endothelial cells; SSAO, semicarbazide-sensitive amine oxidase; VEGF, vascular endothelial growth factor.
Received for publication November 6, 2001. Accepted for publication May 10, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4 integrins mediate lymphocyte attachment and rolling under physiological flow. Cell 80:413.[Medline]
1 and 2 integrins on T lymphocytes. Immunity 4:179.[Medline]
. Nature 361:79.[Medline]
47 integrin mediates binding to the mucosal vascular addressin MAdCAM-1. Cell 74:185.[Medline]
4 integrins/VCAM-1 and LFA-1/ICAM-1 in the binding and transendothelial migration of T lymphocytes and T lymphoblasts across high endothelial venules. Int. Immunol. 12:241.2 integrin LFA-1 involved in transendothelial migration of leukocytes. Nat. Immunol. 3:151.[Medline]
2 integrin activation. J. Immunol. 156:1515.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  N. Anderson and J. Borlak Molecular Mechanisms and Therapeutic Targets in Steatosis and Steatohepatitis Pharmacol. Rev., September 1, 2008; 60(3): 311 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D H Adams, B Eksteen, and S M Curbishley Immunology of the gut and liver: a love/hate relationship Gut, June 1, 2008; 57(6): 838 - 848. [Full Text] [PDF]
|
|
|
|
 |
|
 B. Petri, M. Phillipson, and P. Kubes The Physiology of Leukocyte Recruitment: An In Vivo Perspective J. Immunol., May 15, 2008; 180(10): 6439 - 6446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Elvevold, B. Smedsrod, and I. Martinez The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G391 - G400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jalkanen and M. Salmi VAP-1 and CD73, Endothelial Cell Surface Enzymes in Leukocyte Extravasation Arterioscler. Thromb. Vasc. Biol., January 1, 2008; 28(1): 18 - 26. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-Y. Jung, S.-Y. Park, and I.-S. Kim Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with {alpha}M 2 integrin J. Leukoc. Biol., November 1, 2007; 82(5): 1156 - 1165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Koskinen, S. Nevalainen, M. Karikoski, A. Hanninen, S. Jalkanen, and M. Salmi VAP-1-Deficient Mice Display Defects in Mucosal Immunity and Antimicrobial Responses: Implications for Antiadhesive Applications J. Immunol., November 1, 2007; 179(9): 6160 - 6168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jalkanen, M. Karikoski, N. Mercier, K. Koskinen, T. Henttinen, K. Elima, K. Salmivirta, and M. Salmi The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and P-selectins and leukocyte binding Blood, September 15, 2007; 110(6): 1864 - 1870. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Salmi and S. Jalkanen Developmental regulation of the adhesive and enzymatic activity of vascular adhesion protein-1 (VAP-1) in humans Blood, September 1, 2006; 108(5): 1555 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. K. Lai, P. J. Sun, J. Zhang, A. Jennings, P. F. Lalor, S. Hubscher, J. A. McKeating, and D. H. Adams Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium: A Role for Capturing Hepatitis C Virus Particles Am. J. Pathol., July 1, 2006; 169(1): 200 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Curbishley, B. Eksteen, R. P. Gladue, P. Lalor, and D. H. Adams CXCR3 Activation Promotes Lymphocyte Transendothelial Migration across Human Hepatic Endothelium under Fluid Flow Am. J. Pathol., September 1, 2005; 167(3): 887 - 899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Heydtmann, P. F. Lalor, J. A. Eksteen, S. G. Hubscher, M. Briskin, and D. H. Adams CXC Chemokine Ligand 16 Promotes Integrin-Mediated Adhesion of Liver-Infiltrating Lymphocytes to Cholangiocytes and Hepatocytes within the Inflamed Human Liver J. Immunol., January 15, 2005; 174(2): 1055 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Salmi, K. Koskinen, T. Henttinen, K. Elima, and S. Jalkanen CLEVER-1 mediates lymphocyte transmigration through vascular and lymphatic endothelium Blood, December 15, 2004; 104(13): 3849 - 3857. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Eksteen, A. J. Grant, A. Miles, S. M. Curbishley, P. F. Lalor, S. G. Hubscher, M. Briskin, M. Salmon, and D. H. Adams Hepatic Endothelial CCL25 Mediates the Recruitment of CCR9+ Gut-homing Lymphocytes to the Liver in Primary Sclerosing Cholangitis J. Exp. Med., December 6, 2004; 200(11): 1511 - 1517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Martelius, V. Salaspuro, M. Salmi, L. Krogerus, K. Hockerstedt, S. Jalkanen, and I. Lautenschlager Blockade of Vascular Adhesion Protein-1 Inhibits Lymphocyte Infiltration in Rat Liver Allograft Rejection Am. J. Pathol., December 1, 2004; 165(6): 1993 - 2001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. John and I. N. Crispe Passive and Active Mechanisms Trap Activated CD8+ T Cells in the Liver J. Immunol., May 1, 2004; 172(9): 5222 - 5229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Koskinen, P. J. Vainio, D. J. Smith, M. Pihlavisto, S. Yla-Herttuala, S. Jalkanen, and M. Salmi Granulocyte transmigration through the endothelium is regulated by the oxidase activity of vascular adhesion protein-1 (VAP-1) Blood, May 1, 2004; 103(9): 3388 - 3395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Choudhury, C. L. Russell, S. Randhawa, L. S. Young, D. H. Adams, and S. C. Afford Differential Induction of Nuclear Factor-kappa B and Activator Protein-1 Activity after CD40 Ligation Is Associated with Primary Human Hepatocyte Apoptosis or Intrahepatic Endothelial Cell Proliferation Mol. Biol. Cell, April 1, 2003; 14(4): 1334 - 1345. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or
TNF-
) by treatment with 10 ng/ml TNF-
