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,
Departments of
*
Immunology,
Pediatrics, and
Surgery, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated porcine aortic endothelium were
examined under static and physiologic flow conditions. L-selectin and
E-selectin had overlapping functions in neutrophil capture and rolling,
whereas Ab blockade of E-selectin and the ß2 integrins
inhibited firm arrest of rolling neutrophils. Combined blockade of
selectins and ß2 integrins resulted in negligible human
neutrophil attachment to pig endothelium. Lymphocyte attachment to
porcine endothelium was primarily L-selectin mediated, whereas
ß2 integrin and VCAM-1/very late Ag-4 (VLA-4)
interactions promoted static adhesion. Concurrent ß2
integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited
lymphocyte attachment. Thus, interactions between leukocyte-endothelial
cell adhesion receptor pairs remained remarkably intact across the
human-porcine species barrier. Moreover, disrupting the adhesion
cascade may impair the ability of human leukocytes to infiltrate a
transplanted porcine organ during rejection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recruitment of circulating leukocytes into inflamed tissues depends on interactions between adhesion molecules on leukocytes and vascular endothelial cells (11, 12, 13, 14, 15). The initial capture of flowing leukocytes and their rolling along the vascular endothelium is mediated by the selectins. L-selectin is constitutively expressed by most classes of leukocytes, whereas P-selectin and E-selectin are expressed on activated endothelial cells and facilitate neutrophil and monocyte rolling as well as attachment or engagement of the endothelial surface (12). Human neutrophils and lymphocyte subsets express a P-selectin ligand termed P-selectin glycoprotein ligand-1 (PSGL-1)3 that is also bound by L- and E-selectin (16, 17). P- and L-selectin binding to PSGL-1 is inhibited by the anti-PSGL-1 mAb, PL1, under most conditions, but this mAb does not block the binding of E-selectin under most circumstances (16, 18). Subsequent to selectin-mediated rolling, interactions between integrins, including LFA-1 (CD11a/CD18) expressed by neutrophils and lymphocytes, and Ig superfamily members, such as ICAM-1, arrest leukocyte rolling by inhibiting their forward progression and promoting firm adhesion or "sticking" to the endothelial surface. In addition, lymphocytes and monocytes constitutively express the ß1 integrin very late Ag-4 (VLA-4; CD49d/CD29) that binds to VCAM-1 (CD106) expressed by cytokine-activated endothelium. The process culminates in diapedesis of the leukocyte into the injured tissue.
Adhesion molecules clearly play a role in allograft rejection (19, 20, 21, 22, 23, 24, 25, 26, 27).
Less clear is the involvement of adhesion molecules in xenograft
rejection. E-selectin expression is induced in vascularized guinea pig
hearts placed into rats (28); in this model, treatment with an
anti-P-selectin mAb improves cardiac graft survival (29). Porcine
endothelial cells express VCAM-1 after in vitro activation with human
IL-1, LPS, or TNF- (30). Results using in vitro assays also
suggest interactions between porcine adhesion molecules and their
corresponding human ligands. Expression of porcine E-selectin in monkey
COS cells promotes human neutrophil binding (31). Immobilization of a
recombinant soluble form of porcine VCAM-1 on plastic also supports
attachment of a human lymphoid cell line (32). Despite these studies,
however, the extent to which adhesion molecules support human leukocyte
interactions with porcine vascular endothelium remains generally
uncharacterized.
To assess how leukocyte and vascular adhesion molecules might contribute to leukocyte entry into tissues following transplantation, the quantitative ability of human leukocytes to bind to porcine vascular endothelium was examined. In addition, the degree to which the selectins, integrins, and their ligands contribute to human leukocyte adhesion to pig endothelium was assessed using specific function-blocking mAbs. Remarkably, interactions between human adhesion molecules and their corresponding porcine counterreceptors remained strikingly intact across the human-porcine species barrier.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Porcine aortic endothelial cells (PAEC) were harvested and
characterized as previously described (33). PAEC were passaged serially
and maintained in DMEM (Life Technologies, Gaithersburg, MD)
supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO),
penicillin (100 U/ml), streptomycin (100 µg/ml), 2 mM
L-glutamine, and 25 mM HEPES (all from Life Technologies).
In all experiments, PAEC were used between passages 4 and 9. Cells to
be used in adhesion assays were plated on glass microscope slides (Gold
Seal, Portsmouth, NH) within a 22-mm2 area demarcated with
a hydrophobic slide marker and grown to confluence. Confluent PAEC
monolayers were stimulated for 6 h in culture medium containing
200 U/ml of recombinant human TNF- (Genzyme, Cambridge, MA).
Leukocyte isolation
Neutrophils were isolated by density gradient centrifugation of heparinized human blood from normal volunteers using Mono-Poly Resolving Medium (ICN Biomedicals, Costa Mesa, CA). Human lymphocytes were isolated by density gradient centrifugation using Lymphoprep (Nycomed Pharma, Oslo, Norway). All cells were kept on ice until utilized for adhesion assays. All protocols were approved by the Human Use Committee of Duke University.
Reagents and Abs
Abs used in these studies included: purified leukocyte adhesion molecule (LAM)1–3 mAb (IgG1) directed against the lectin domain of human L-selectin (34); purified LAM1–14 mAb (IgG1) directed against the consensus repeat domain of human L-selectin (35); the 4B4 anti-human CD29 mAb (VLA ß-chain, IgG1) (36); the H52 anti-human CD18 mAb (LFA-1 ß-chain, IgG1, Developmental Studies Hybridoma Bank, Iowa City, IA) (37); purified PL-1 anti-human PSGL-1 mAb (IgG1) (16, 18); the HAE2d anti-human and porcine VCAM-1 mAb (38); and purified MAB2124 anti-human and porcine E-selectin mAb (Chemicon International, Temecula, CA) (39). Purified mAbs were used at a final concentration of 10 µg/ml whereas other mAbs were used as ascites fluid diluted to final concentrations found to be at least fivefold in excess of saturation (usually 1/100 dilutions). To identify the presence of a ligand for L-selectin, a human L-selectin/murine IgM fusion protein (L'IgM), consisting of the extracellular domains of human L-selectin fused with the CH2, CH3, and CH4 domains of murine IgM heavy chain, was employed (L.T., M. Delahunty, and T.F.T., unpublished data). Supernatant fluid from L'IgM cDNA-transfected COS cells was concentrated 50-fold and used at 50 µl added to 106 PAEC for staining. Secondary Abs used in these studies included goat anti-mouse IgM Ab (Southern Biotechnology Associates, Birmingham, AL), goat anti-mouse IgG Ab (Caltag, South San Francisco, CA), and horseradish peroxidase-conjugated rabbit anti-mouse Ig Ab (Dako, Carpinteria, CA), used at final concentrations 40 µg/ml, 14 µg/ml, and 1/100 dilutions, respectively.
Flow cytometry
PAEC were stimulated for 6 h with TNF-, then lifted from
the culture flask using 1.5 mM EDTA in Dulbecco’s PBS (Life
Technologies). Cells were washed in PBS containing 0.9 mM
CaCl2, 0.5 mM MgCl2, and 2% bovine calf serum
(PBS-FCS). Diluted HAE2d and MAB2124 mAbs and L'IgM were added to the
cells before they were incubated at 4°C for 20 min. Cells were
washed, then incubated with goat anti-mouse IgG or IgM Abs for 20
min at 4°C. Cells were washed, resuspended in 2% formaldehyde in
PBS-FCS, and analyzed by flow cytometry using a FACScan flow cytometer
(Becton Dickinson, San Jose, CA). At least 10,000 cells were analyzed
for each sample, with fluorescence intensity shown on a three-decade
log scale.
PAEC whole cell ELISA
Cell surface expression of E-selectin and VCAM-1 by activated
PAEC was assessed by growing PAEC to confluence in gelatin-coated
96-well plates (Costar, Cambridge, MA). PAEC were activated with 200
U/ml TNF- for 0.5, 2, 4, 6, 14, or 24 h before the medium was
decanted and the plates were gently washed with cold DMEM containing
10% FCS. The cells were incubated for 45 min at room temperature with
HAE2d or MAB2124 mAb in DMEM containing 10% rabbit serum (Sigma) and
5% FCS. The plates were then washed three times with cold DMEM
containing 10% FCS, and incubated for 30 min at room temperature with
a secondary horseradish peroxidase-conjugated rabbit anti-mouse IgG
Ab. Plates were washed five times with PBS containing 1% BSA and once
with PBS. The developing solution, containing hydrogen peroxide and
o-phenylenediamine in citrate buffer, was added and the
optical density of the reaction product was quantified at 492 nm light.
In vitro adhesion assays
Leukocyte adhesion assays were performed as previously described
(38). Briefly, PAEC were grown to confluence on glass slides,
stimulated with TNF-, washed, and incubated for 20 min at 37°C
with medium or HAE2d or MAB2124 mAbs. Neutrophils or lymphocytes
(106) were resuspended in 200 µl of culture medium either
alone or containing mAb at the appropriate dilutions. For assays done
at 37°C, neutrophils and lymphocytes were maintained at room
temperature until use, whereas for experiments done at 4°C,
leukocytes were kept on ice after isolation. The leukocyte suspension
was added to the endothelial monolayer under either static (gentle
rocking at 10 cycles/min) or horizontal rotating (64 rpm) conditions.
After a 30-min incubation period, the medium was tipped off, and the
slides were fixed vertically in 2.4% glutaraldehyde (Polysciences,
Warrington, PA) in PBS, pH 7.4. The number of leukocytes adherent to
the endothelial monolayer was determined by counting between 4 and 12
x400 microscopic fields that were located at half-radius distances
from the center of the monolayers. Results were expressed as mean
values ± SEM. Endothelial cell monolayers were confluent both at
the beginning and at the end of the experiments.
Hydrodynamic flow chamber experiments
Adhesion of human neutrophils to TNF--stimulated PAEC under
physiologic flow conditions was assessed as described (40). Briefly,
PAEC were grown to confluence on 25-mm round glass coverslips (Fisher
Scientific, Pittsburgh, PA), activated with TNF- for 6 h,
placed in a parallel plate laminar flow chamber, and mounted on the
stage of an inverted phase-contrast microscope (Olympus, Lake Success,
NY). Neutrophils were stored at 4°C until just before use, then
resuspended in PBS containing 0.75 mM CaCl2, 0.75 mM
MgCl2, and 0.5% BSA at a cell density of 106
cells/ml. In experiments involving blocking mAb, either neutrophils or
endothelium were preincubated for 5–20 min with various combinations
of mAb. The neutrophil suspensions were drawn through the chamber for a
10-min period at a wall shear stress of 1.85 dynes/cm2 via
a syringe pump (Harvard Apparatus, South Natick, MA).
Neutrophil-endothelial interactions were recorded using a
charged-coupled device (CCD) videocamera (Hitachi Denshi, Tokyo, Japan)
and a Sony SuperVHS videorecorder. At the end of the 10-min neutrophil
perfusion period, multiple x100 fields were recorded for at least
30 s. The total number of adherent neutrophils (rolling + firmly
adherent) was determined by analyzing the videotapes. Stable adhesion
was defined as firm attachment for a minimum of 30 s. The rolling
fraction was calculated by dividing the number of rolling neutrophils
in any given field by the total number of adherent neutrophils. A
minimum of ten 0.16-mm2 fields were analyzed for each
experiment. The data from multiple experiments were pooled.
Statistical analysis
Results are expressed as mean values ± SEM unless indicated otherwise. Student’s t test was used to determine the level of significance of differences in mean values between treatment groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of TNF--activated PAEC to express E-selectin,
VCAM-1, and vascular ligand(s) for L-selectin was assessed. L-selectin
ligand expression by PAEC was assessed using a fusion protein,
consisting of the extracellular domains of human L-selectin and the
constant region of mouse IgM, termed L'IgM. Quiescent PAEC bound L'IgM
at low levels as determined by flow cytometry analysis relative to
staining with an unreactive mouse IgM control mAb (Fig. 1
A). However,
TNF--activated PAEC bound L'IgM at significantly higher levels.
L'IgM binding was reduced to background levels by treating quiescent or
activated PAEC with EDTA to block L-selectin binding (data not shown).
Abs to E-selectin and VCAM-1 failed to label quiescent PAEC, but
TNF- activation up-regulated surface expression of these receptors
(Fig. 1, B–D). Maximal E-selectin expression
occurred at 4 h of activation and decreased thereafter (Fig. 1D). VCAM-1 expression was maximal at 6 h and remained
at that level for 24 h (Fig. 1D). Therefore, adhesion
molecule expression by TNF--activated PAEC mimics what has been
shown for human vascular endothelium (38, 41, 42).
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The binding of human neutrophils to PAEC monolayers was assessed
under both static and rotating conditions. Leukocyte attachment in
static assays mimics the firm adhesive interactions that occur
subsequent to initial leukocyte capture and rolling. In contrast, the
shear force induced by 64 rpm rotation optimizes the assay to visualize
selectin-mediated leukocyte binding to vascular endothelium which may
mimic the in vivo conditions whereby circulating leukocytes are
initially captured by the endothelium (38). Quiescent PAEC supported
minimal human neutrophil adhesion at 37°C under both static and
rotating conditions (Fig. 2 and Fig. 3, A and B).
However, PAEC activation with TNF- enhanced neutrophil binding by
greater than 10-fold (Figs. 2 and 3).
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A).
Blocking endothelial E-selectin or neutrophil PSGL-1 functions under
static conditions reduced neutrophil binding by 53% and 41%,
respectively (Figs. 2 and 3A). It is likely that P-selectin
plays a role in neutrophil adhesion in these assays since the PL-1
(anti-PSGL-1) mAb used in these experiments predominantly inhibits
P-selectin and L-selectin binding to neutrophils rather than E-selectin
function under these conditions. Blocking neutrophil CD18 function
resulted in a 44% reduction in adhesion, whereas blockade of all
selectin-ligand interactions resulted in a 67% decrease in binding of
neutrophils. Inhibiting selectin and CD18 function reduced neutrophil
binding to the level seen with quiescent endothelium (91% inhibition;
Fig. 3A, p < 0.01).
When adhesion assays were performed under rotating conditions at
37°C, L-selectin-mediated neutrophil binding to PAEC was revealed, as
LAM1–3 mAb inhibited neutrophil binding by 71% (Fig. 3B).
E-selectin mAb blockade inhibited neutrophil binding to 23% of control
levels (Fig. 3B). Under rotating conditions, PSGL-1 was less
important for neutrophil binding to PAEC, as mAb against this structure
inhibited binding by only 21% (Fig. 3B). Although blocking
CD18 function under both rotating and static conditions resulted in a
41% reduction in adhesion (Fig. 3, A and B), the
contribution of this molecule relative to that of other individual
adhesion receptors was greater in static assays. Inhibition of the
selectins and their ligands resulted in nearly complete inhibition of
neutrophil binding, with modest additional benefit conferred by CD18
blockade (Fig. 3B, p < 0.05).
To further dissect the function of the three selectins during different
phases of leukocyte recruitment, additional assays were performed at
4°C. At this temperature, L-selectin endoproteolytic release from the
cell surface is minimal and ß2 integrins are
nonfunctional (38). When the assays were performed with rotation at
4°C, blocking L-selectin function inhibited neutrophil attachment by
95% (Fig. 3D), compared with only 53% inhibition under
static conditions (Fig. 3C). mAb reactive with E-selectin
did not reduce neutrophil binding under static conditions at 4°C
(Fig. 3C), but diminished binding by 31% during rotation
(Fig. 3D). mAb reactive with PSGL-1 blocked neutrophil
binding under both static and rotating conditions at 4°C (46% and
56%; Fig. 3, C and D). Not surprisingly,
anti-CD18 mAb did not inhibit human neutrophil binding to porcine
endothelium under either static or rotating conditions at 4°C (Fig. 3, C and D).
Human lymphocyte adhesion to activated PAEC
Lymphocyte binding to PAEC, like neutrophil binding, was
dramatically enhanced after PAEC activation with TNF- (Figs. 4 and 5).
When the binding assays were performed at 37°C under static
conditions, blocking L-selectin function inhibited lymphocyte binding
by 32% (Fig. 5A). Blocking CD29, VCAM-1, and CD18 function
reduced lymphocyte binding by 61%, 43%, and 39%, respectively (Fig. 5A). Simultaneous blockade of CD18, VLA-4, and VCAM-1
reduced lymphocyte binding to baseline levels, with L-selectin blockade
conferring some additional benefit (Fig. 5A;
p < 0.01). Blocking L-selectin function inhibited
lymphocyte binding by 63% under rotating conditions (Fig. 5B). Under rotating conditions, blocking CD29, VCAM-1, and
CD18 function inhibited adhesion by 60%, 50%, and 43%, respectively
(Fig. 5B). Blockade of CD29, VCAM-1, and CD18 diminished
lymphocyte binding by 89%, whereas additional L-selectin blockade
further reduced binding to 5% of control levels under rotating
conditions (Fig. 5B, p < 0.05).
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, C and D).
Blocking CD18 and CD29 function did not affect binding under static or
rotating conditions. The fact that blocking VCAM-1 function had no
effect on adhesion at 4°C under rotating conditions (Fig. 5D), despite the functional ability of VCAM-1 to bind to
VLA-4 at 4°C, suggests that L-selectin is the primary mediator of
initial lymphocyte capture in these assays. The ability of
anti-L-selectin mAb to also diminish binding by 50% under static
conditions (Fig. 5C) suggests that initial
L-selectin-mediated capture is prerequisite to establishment of
subsequent integrin-mediated stable adhesion in this assay system. Human neutrophil adhesion to PAEC monolayers under physiologic flow
The ability of human neutrophils to bind PAEC under physiologic
flow conditions was assessed by passing human neutrophil suspensions
through a parallel-plate flow chamber over adherent PAEC monolayers at
37°C. A physiologic wall shear stress of 1.85 dynes/cm2
was used for all experiments. In these assays, the total number of
adherent neutrophils (rolling + firmly adherent cells) was determined
after a 10-min period of chamber perfusion. The rolling fraction was
determined by dividing the number of rolling neutrophils by the total
number of firmly adherent neutrophils. In these assays, TNF-
activation of PAEC increased neutrophil adhesion by 200-fold; 208
± 14 neutrophils/0.16 mm2 vs 1.1 ± 0.2
neutrophils/0.16 mm2 for unstimulated PAEC monolayers
(p < 0.01, Fig. 6). Rolling neutrophils accounted for
14% of the attached cells. Although incubating neutrophils with a
control nonfunction blocking L-selectin mAb did not significantly
affect the total number of adherent cells, the rolling fraction fell.
However, the absolute number of rolling cells was small, and the
control mAb may have exerted steric effects, spatially interfering with
selectin-binding sites involved in rolling.
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) and reduced the rolling
fraction to 2.1% (p < 0.01). Blocking
E-selectin function diminished neutrophil attachment by 53% (Fig. 6)
and reduced the rolling fraction to 2.4% (p <
0.01). Blocking PSGL-1 function reduced attachment by 42% (Fig. 6) and
reduced the rolling fraction to 7.9% (p <
0.01). Combined blockade of L-selectin, E-selectin, and PSGL-1 reduced
binding to 23% of control levels (Fig. 6). Blocking neutrophil CD18
function in these assays had a less pronounced effect on neutrophil
adhesion (25% inhibition; Fig. 6), but markedly increased the rolling
fraction (36%; p < 0.01). Blocking E-selectin and
CD18 function resulted in 78% fewer bound neutrophils (Fig. 6, p < 0.01), but in this case, the majority of
neutrophils were rolling (62%; p < 0.01). The lower
number of bound neutrophils seen with blockade of CD18 alone or a
combination of E-selectin and CD18 reflects decreased stable arrest,
with many interacting cells rolling past the field being counted.
Blocking E-selectin function alone results in fewer adherent
neutrophils either due to less efficient neutrophil capture or to
impaired transition from capture to rolling. Combined blockade of
selectin function and CD18 dramatically reduced the number of adherent
neutrophils to 8% of control values (Fig. 6). Overall, these data
indicate that initial attachment of neutrophils to PAEC, as well as
subsequent rolling, occur via collaborative interactions among the
selectins. The ensuing development of stable adhesive bonds between
human neutrophils and PAEC occurs through the concerted actions of both
the ß2 integrins and E-selectin. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation of porcine
vascular endothelium enhances surface expression of E-selectin, VCAM-1,
and L-selectin ligand(s) and 2) human neutrophils and lymphocytes bind
extensively to cytokine-activated PAEC in an adhesion
receptor-dependent manner. In fact, the pattern and kinetics of
E-selectin, VCAM-1, and L-selectin ligand expression by PAEC (Fig. 1)
were similar to those described previously for cultured human umbilical
vein endothelial cells (38, 39, 43). Under static and flow conditions,
binding of both human neutrophils and lymphocytes to porcine
endothelial monolayers was inhibited by mAbs directed against pig or
human adhesion molecules (
Figs. 2–6), demonstrating that interactions
between specific human adhesion receptors and their porcine
counterreceptors are well conserved. Although not directly compared in
these studies, human neutrophil binding to activated porcine vascular
endothelium under physiologic flow conditions (Fig. 6) and in static
and nonstatic adhesion assays (Fig. 3) was of roughly the same order of
magnitude as previously described in studies of human neutrophil
attachment to TNF--activated human vascular endothelium under
corresponding conditions (38, 44). The same appears to be true for the
binding of human lymphocytes to both activated porcine and human
vascular endothelium (38). That the selectins and integrins may mediate
human leukocyte adhesion to PAEC on a scale of the same order of
magnitude as that observed for human leukocytes interacting with human
endothelial cells (38) suggests that these interactions may be
well preserved in vivo following xenotransplantation.
The current studies significantly extend previous studies examining the
molecular events that occur during human leukocyte interactions with
porcine endothelial cells (30, 31, 32, 45, 46). As shown in the current
studies, interactions between human or porcine L-, E-, and P-selectins
and their ligands are consistent with the highly conserved nature of
selectin binding across species (35, 45, 47, 48). Previous studies by
two groups have shown that porcine E-selectin is 71–75% homologous
with human E-selectin and that when overexpressed in COS cells it would
mediate some level of human neutrophil adhesion (31, 45). In addition,
a chimeric molecule consisting of the lectin domain of porcine
E-selectin and the epidermal growth factor domain of human
E-selectin fused to the human IgG constant region would bind porcine
granulocytes and a subpopulation of porcine lymphocytes (45). The
ability of human leukocyte integrins to bind porcine Ig superfamily
molecules has also been described (30, 32, 46). Although mouse LFA-1
does not bind to human ICAM-1 (49), human LFA-1 interacts efficiently
with porcine ICAMs (Figs. 3, 5, and 6). Others have shown
VLA-4-dependent binding of a human lymphoid cell line to both
recombinant porcine VCAM-1 bound to plastic as well as VCAM-1 expressed
by cytokine-stimulated PAEC (32, 46). In those studies, an
anti-VLA-4 mAb blocked adhesion of human T cells to stimulated PAEC
by 60–65% in static assays. Therefore, the current studies provide
qualitative evidence for the compatibility of human and porcine
adhesion receptors and are the first to quantitate the sequential
contributions of individual adhesion molecules in a physiologic flow
setting.
The selectins act in concert to mediate the initial capture of
free-flowing leukocytes from the circulation under shear conditions
(12). Under nonstatic or hydrodynamic flow conditions, preincubation of
human neutrophils with anti-L-selectin mAb reduced by 56–71% the
total number of neutrophils adherent to porcine endothelium (Figs. 3 and 6). In a similar fashion, exposure of the endothelium to an
anti-E-selectin mAb reduced neutrophil binding by 53–77% (Figs. 3 and 6), and reduced rolling by 83%. Although P-selectin function was
not examined directly because function-blocking anti-porcine
P-selectin mAbs were not identified, P-selectin function was assessed
by treating human neutrophils with a mAb that blocks the P-selectin
binding site of PSGL-1. This anti-PSGL-1 mAb reduced neutrophil
binding less dramatically, a 21–42% decrease (Figs. 3 and 6),
although P-selectin function is likely to be more critical at early
time points following PAEC activation as occurs with human endothelium
(50). Nonetheless, the combined blockade of L-selectin, E-selectin, and
PSGL-1 function produced the greatest effect, decreasing total
neutrophil adhesion by 77–96% (Figs. 3 and 6). By contrast,
lymphocyte attachment under shear conditions was predominantly
L-selectin-mediated (Fig. 5). These results suggest that the three
selectins act in concert to promote the initial capture of circulating
leukocytes and their subsequent rolling on activated porcine
endothelium as occurs with human endothelium at sites of inflammation.
The combined blockade of L-selectin, E-selectin, PSGL-1, and CD18
function drastically decreased human neutrophil adhesion to activated
PAEC by >90% (Figs. 3 and 6). Lymphocyte static adhesion under shear
conditions was predominantly promoted by VCAM-1/VLA-4 and
ß2 integrin interactions (Fig. 5). Combined mAb blockade
of CD18, VLA-4, VCAM-1, and L-selectin essentially attenuated all
(85–95%) lymphocyte attachment to PAEC (Fig. 5). Surprisingly, CD18
blockade alone only partially inhibited neutrophil attachment in
parallel plate flow chamber experiments (Fig. 6). However, E-selectin
may function synergistically with the ß2 integrins to
stabilize human neutrophil attachment to porcine endothelium under
these conditions, since E-selectin blockade significantly impaired
neutrophil attachment to PAEC in static binding assays (Fig. 3). By
contrast, E-selectin blockade did not significantly inhibit neutrophil
attachment at 4°C, a temperature at which CD18 is nonfunctional. This
finding suggests that a component of E-selectin-mediated attachment is
dependent on CD18 function. Therefore, it is likely that E-selectin and
CD18 ligands cooperate to mediate firm leukocyte adhesion. This would
explain why the simultaneous blockade of both E-selectin and CD18
significantly impaired the ability of rolling neutrophils to stably
arrest in hydrodynamic flow assays (Fig. 6). This result is also in
agreement with the recent finding that a specific E-selectin mAb can
block neutrophil recruitment into an inflamed peritoneum without
affecting neutrophil capture or rolling in vitro or in vivo (51). In
addition, mice lacking E-selectin show normal rolling but reduced
arrest of leukocytes on cytokine-activated microvascular endothelium
(52). Thus, E-selectin may participate in steps of the adhesion cascade
that extend well beyond initial rolling.
Many questions regarding the role of adhesion molecules in allograft rejection remain unanswered, and still less is known about their involvement in xenograft rejection. The current studies lay some important groundwork in this area. Importantly, human leukocyte binding to porcine endothelium appears to be primarily mediated by specific interactions between adhesion molecules and their counterreceptors, interactions that are remarkably conserved across species. Moreover, these results suggest that xenografted tissues are likely to support the physiologic entry of human leukocytes binding to xenogeneic vascular endothelium. In addition, interfering with sequential steps of the adhesion cascade, leukocyte capture, rolling and firm arrest, through appropriate mAb treatments, may impair the ability of the human host’s leukocytes to infiltrate a transplanted porcine organ. Further studies are needed to determine whether such interventions may translate into prolonged xenograft survival, and ultimately, may help to make the placement of porcine organs into patients awaiting transplants a practical reality.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Thomas F. Tedder, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address:
3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; VLA-4, very late Ag-4; PAEC, porcine aortic endothelial cells; L'IgM, L-selectin/murine IgM fusion protein.
Received for publication May 5, 1998. Accepted for publication August 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N. C. Kirkiles-Smith, D. A. Tereb, R. W. Kim, J. M. McNiff, J. S. Schechner, M. I. Lorber, J. S. Pober, and G. Tellides Human TNF Can Induce Nonspecific Inflammatory and Human Immune-Mediated Microvascular Injury of Pig Skin Xenografts in Immunodeficient Mouse Hosts J. Immunol., June 15, 2000; 164(12): 6601 - 6609. [Abstract] [Full Text] [PDF]
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