Fluid shear- and time-dependent modulation of molecular
interactions between PMNs and colon carcinomas
Sameer
Jadhav and
Konstantinos
Konstantopoulos
Department of Chemical Engineering, The Johns Hopkins
University, Baltimore, Maryland 21218
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ABSTRACT |
This study compares the effects of
fluid shear on the kinetics, adhesion efficiency, stability, and
molecular requirements of polymorphonuclear leukocyte (PMN) binding to
two colon adenocarcinoma cell-lines, the
CD54-negative/sLex-bearing LS174T cells and the
CD54-expressing/sLex-low HCT-8 cells. The efficiency of
PMN-colon carcinoma heteroaggregation decreases with increasing shear,
with PMNs binding HCT-8 more efficiently than LS174T cells at low shear
(50-200 s
1). In the low shear regime, CD11b is
sufficient to mediate PMN binding to LS174T cells. In contrast, both
CD11a and CD11b contribute to PMN-HCT-8 heteroaggregation, with CD54 on
HCT-8 cells acting as a CD11a ligand at early time points. At high
shear, only PMN-LS174T heteroaggregation occurs, which is initiated by
PMN L-selectin binding to a sialylated, O-linked, protease-sensitive
ligand on LS174T cells. PMN-LS174T heteroaggregation is primarily
dependent on the intercellular contact duration (or shear rate),
whereas PMN-HCT-8 binding is a function of both the intercellular
contact duration and the applied force (or shear stress). Cumulatively, these studies suggest that fluid shear modulates the kinetics and
molecular mechanisms of PMN-colon carcinoma cell aggregation.
CD11a/CD18; CD11b/CD18; L-selectin; polymorphonuclear leukocytes; LS174T cells; HCT-8 cells
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INTRODUCTION |
DURING BLOOD-BORNE
METASTASIS cancerous cells undergo extensive interactions with
various host cells, including polymorphonuclear leukocytes (PMNs), in
the circulatory system before they eventually colonize a distant
tissue. Several lines of evidence suggest that these adhesive
interactions may affect the ability of tumor cells to metastasize. Some
studies indicate that PMNs may exert a direct cytotoxic effect on tumor
cells (7), and their tumoricidal ability relies on
intimate contact between PMNs and tumor cells (6, 19).
However, several independent studies have shown that PMNs may enhance
tumor metastasis. This has been demonstrated by experiments showing
that PMNs facilitate tumor cell extravasation in vitro (35,
38) and promote the arrest and deposition of tumors in the
microvasculature of target organs in animal models (3, 11,
35). Light and electron microscopic studies also reveal close
association of PMNs with metastatic cancer cells in vivo
(5). Hence, whether PMNs abet hematogenous metastasis or
are cytotoxic to tumor cells remains controversial. Nevertheless, PMN-tumor cell binding appears to be critical to both these processes, and the molecular mechanisms mediating these cell-cell interactions require further investigation.
Published data indicate that PMNs can bind certain melanoma,
neuroblastoma, and colon adenocarcinoma cells through the CD18 (
2) integrin receptor on PMN surface in the absence of
any selectin contribution under static conditions (16, 22,
26). Alternatively, PMNs have been reported to interact with
certain colon carcinomas via PMN CD62L (L-selectin) in a
CD18-independent manner at 4°C, a temperature that renders integrins
inactive (20). However, static binding assays fail to
replicate the hydrodynamic shear environment of the vasculature in
which PMN-tumor cell binding typically occurs and, thus, may not be
sufficient to determine the precise molecular requirements of this
adhesion process. It is now well established that fluid shear affects
the binding kinetics and receptor specificity of PMN homotypic
aggregation (37) as well as PMN heterotypic interactions
with platelets (15) and endothelial cells (13,
34). Furthermore, we recently showed that CD11b (Mac-1) is
sufficient to mediate binding of chemotactically stimulated PMNs to
CD54 (ICAM-1)-negative/sLex-bearing LS174T colon carcinoma
cells at low shear (100 s
1) (12). In marked
contrast, L-selectin, CD11a (lymphocyte function-associated antigen
LFA-1), and CD11b are all requisite for optimal PMN-LS174T binding
under high shear conditions (800 s
1) (12).
Along these lines, the CD54-expressing/sLex-low HCT-8 colon
carcinoma cells fail to aggregate with PMNs at 800 s
1.
However, a detailed quantitative analysis of PMN binding to LS174T vs.
HCT-8 colon carcinoma cells as well as the relative contributions of
L-selectin, CD11a, CD11b, and their respective ligands in mediating
these heterotypic adhesive interactions as a function of fluid shear
and time have yet to be investigated.
In the present study, we utilized a rheometric-flow cytometric
methodology to compare the aggregation kinetics, stability, and
molecular requirements of PMN binding to LS174T cells with that to
HCT-8 cells under defined shear conditions in the presence of the
chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (fMLP). To quantify cell-cell interactions independent of physical parameters such as shear rate, cell size, and initial cell
concentration, we calculated the efficiency of PMN homotypic and
PMN-colon carcinoma heterotypic aggregation by using a mathematical
model based on Smoluchowski's two-body collision theory (33, 36,
37). Moreover, the contributions of intercellular contact
duration (dictated by shear rate) and tensile forces (exerted by shear
stress) on receptor-ligand bonds mediating PMN-colon carcinoma
heteroaggregation were differentiated by varying the viscosity of the
cell suspension (10, 29, 37).
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MATERIALS AND METHODS |
Reagents.
The IgG murine monoclonal antibodies (MAbs) 6.7(blocking anti-CD18),
HI111 (blocking anti-CD11a), and ICRF44(44) (blocking anti-CD11b) were purchased from BD Pharmingen (San Diego, CA). The
blocking F(ab')2 anti-CD54 MAb MHCD54F was from Caltag
(Burlingame, CA). Red-out and PMN isolation media were obtained from
Robbins Scientific (Sunnyvale, CA). Citrate-phosphate-dextrose (CPD)
solution, fMLP, chymotrypsin, trypsin,
benzyl-2-acetamido-2-deoxy-
-D-galactopyranoside (Bzl
GalNAc), tunicamycin, dextran sulfate, and fucoidan were from
Sigma-Aldrich (St. Louis, MO). Heparin was from Elkins-Sinn (Cherry
Hill, NJ). Dulbecco's phosphate-buffered saline (D-PBS) and
trypsin/EDTA were acquired from Life Technologies (Gaithersburg, MD).
Phosphatidylinositol-specific phospholipase C (PI-PLC) was purchased
from Glyko (Novato, CA), and Vibrio cholerae neuraminidase was from Roche Molecular Biochemicals (Indianopolis, IN).
d,l-Threo-1-phenyl-2-amino3-morpholino-1-propanol hydrochloride (threo-PPPP) was procured from Matreya (State College, PA). CellTracker orange dye CMTMR
[5(6)-(4-chlormethylbenzoylamino)tetramethylrhodamine] and green dye CFDA-SE [5(6)-carboxyfluorescein
diacetate-succinimidyl ester] were purchased from Molecular Probes
(Eugene, OR). CMTMR and CFDA-SE are excited at 488 nm by the argon
laser of a flow cytometer, and their emission spectra are well
separated (570 nm for CMTMR and 515 nm for CFDA-SE), thereby allowing
simultaneous two-color immunofluorescence measurements.
Colon carcinoma cell culture and staining.
The LS174T and HCT-8 human colon adenocarcinoma cell lines were
obtained from American Type Culture Collection (Manassas,VA) and
cultured in the recommended media. Before each experiment, LS174T or
HCT-8 cells were detached from the tissue culture substrate by mild
trypsinization (0.25% trypsin/EDTA for 2 min at 37°C). The colon
carcinoma cells were resuspended at a concentration of 1 × 107 cells/ml in the recommended media and incubated for
2 h at 37°C to regenerate surface glycoproteins (12, 20,
21). During this period, the cells were incubated with 1 µM
CMTMR (orange dye) at 37°C for 1 h. The cells were washed once
to remove excessive dye and resuspended at a concentration of 2.5 × 106 cells/ml in D-PBS containing
Ca2+/Mg2+/0.1% BSA (Sigma-Aldrich) and
maintained at 4°C for no longer than 3 h before being used in
binding assays or flow cytometry.
PMN isolation and labeling.
Human blood was drawn by venipuncture from healthy volunteers into a
sterile syringe containing CPD solution (2.8 ml CPD/20 ml blood). A red
blood cell agglutinating agent (Red-out; 1/100 volume) was added to the
blood to minimize erythrocyte contamination in PMNs (12,
14). The blood was gently mixed, layered over the PMN isolation
medium, and centrifuged to allow for density separation of cell
populations. The PMN layer was carefully aspirated and washed once, and
the PMNs were resuspended in Ca2+/Mg2+-free
D-PBS at a concentration of 1 × 107 cells/ml. The
PMNs were then incubated with 0.1 µM CFDA-SE at 4°C for 1 h,
washed once to remove excess dye, resuspended in Ca2+/Mg2+-free D-PBS containing 0.1% BSA at a
concentration of 0.5 × 107 cells/ml, and maintained
at 4°C for no longer than 3 h before being used in aggregation
assays. L-selectin was preserved during PMN isolation and staining with
CFDA-SE as assessed by flow cytometry (12).
PMN-colon carcinoma cell aggregation assay.
CFDA-SE-labeled PMNs and CMTMR-stained colon carcinoma cells were mixed
at final cell concentrations of 1 × 106 and 2 × 106 cells/ml, respectively, and allowed to equilibrate at
37°C for 2 min. This mixed suspension (500 µl) was then placed on
the plate of a cone-and-plate rheometer (RS150, 60-mm-diameter plate,
0.5°Cone angle; Haake, Paramus, NJ) and stimulated with 1 µM fMLP
1 s before initiation of shear at 37°C. The suspension was
subjected to well-defined hydrodynamic conditions with shear rates
ranging from 50 to 1,200 s
1 for durations of 10, 30, 60, 120, 180, and 300 s. Upon termination of shear, 100-µl aliquots
were immediately fixed with 1% formaldehyde (final concentration) at
room temperature (RT) and subsequently analyzed in a FACSCalibur flow
cytometer (Becton Dickinson, San Jose, CA), as previously described
(12). The fixative was kept in the specimens during the
flow cytometric analysis to preserve aggregate integrity
(12). In selected experiments, 6% Ficoll was used to
increase the viscosity of the cell suspension (10, 37).
Cell treatment with MAbs and enzymes.
For some blocking experiments, PMNs (1 × 107
cells/ml) were pretreated for 10 min at 37°C with anti-CD11a and/or
anti-CD11b MAbs (20 µg/ml), which were kept present during the
aggregation assays. In some studies, the HCT-8 cells were incubated
with F(ab')2 anti-CD54 MAb (10 µg/ml) for 10 min at
37°C before being mixed with PMNs in the cone-and-plate rheometer.
For selected inhibition studies, PMNs were incubated with chymotrypsin
(1 U/ml) for 20 min at RT to cleave L-selectin [and
P-selectin-glycoprotein ligand-1 (PSGL-1)] from the PMN surface and
washed once before being used in aggregation assays (12).
For others, heparin (100 U/ml) (25), dextran sulfate (100 µg/ml) (32), or fucoidan (10 µg/ml) (8) were added to the suspension just before the application of shear. For
experiments with biosynthetic inhibitors, LS174T cells were cultured
for 48 h in the presence of either Bzl GalNAc (2 mM) to inhibit
O-linked glycosylation or tunicamycin (200 ng/ml) to inhibit N-linked
glycosylation of surface glycoproteins before being used in aggregation
assays (30). In other studies, LS174T cells were cultured
with 5 µM threo-PPPP for 96 h, which blocks the transfer of
UDP-glucose to ceramide, thereby blocking the biosynthesis of
glycosphingolipids having a glucosylceramide core (1).
Colon carcinoma cells treated with the aforementioned biosynthetic
inhibitors were washed twice before being used in aggregation assays
(22). In selected experiments, LS174T cells were incubated
at 37°C for 30 min with either 20 µg/ml trypsin to cleave cell
surface glycoproteins or 0.1 U/ml Vibrio cholerae neuraminidase to cleave terminal cell surface sialic acid
residues, or for 1 h with 1 U/ml PI-PLC, which cleaves glycosyl
phosphatidylinositol (GPI)-linked surface glycoproteins
(21), and washed once (twice in the case of trypsin)
before being mixed with PMNs. Cell viability was consistently >97%
after treatment with each of the aforementioned enzymes and inhibitors,
as detected by the trypan blue exclusion assay.
Quantitation of receptor expression.
For indirect immunofluorescence, colon carcinoma cells were incubated
with saturating concentrations of primary MAbs directed against either
sLex or CD54 for 30 min at 4°C and then washed once with
D-PBS/0.1%BSA. After an additional 30-min incubation with 15 µg/ml
FITC-labeled IgM or phycoerythrin-labeled IgG (Vector Laboratories,
Burlingame, CA), the specimens were washed again, fixed with
1% formaldahyde, and analyzed by flow cytometry (21).
Appropriate isotype-matched MAbs were also included for background
fluorescence determination.
Flow cytometric analysis of PMN-colon carcinoma aggregation.
A dual-color flow cytometric methodology (FACSCalibur flow cytometer)
was employed to analyze the sheared samples for size distribution and
cellular composition of formed aggregates. The PMN and colon carcinoma
cell populations were identified on the basis of their forward-scatter,
side-scatter, and fluorescence profiles. Single PMN (P) and single
tumor cell (T) populations were identified on the green (FL1) vs.
orange (FL2) fluorescence dot plots, and the aggregates were resolved
as integral multiples of the mean fluorescence values of single P and T
populations (12). The homotypic PMN aggregates were
resolved into doublets (P2) and higher order aggregates
(P3+), whereas heterotypic aggregates consisted of a single
colon carcinoma cell adhering to one (TP), two (TP2), and
three or more (TP3+) PMNs. The aggregation was quantified
as the fraction of total PMN in homotypic or heterotypic aggregates as
described previously (12).
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Aggregates with more than three PMN were <10% of the total
neutrophil population and resulted in <3% error in estimation of
percent aggregation.
Estimation of PMN homotypic and PMN-colon carcinoma cell
heterotypic adhesion efficiencies.
PMN homotypic and PMN-colon carcinoma heterotypic aggregation observed
in the cone-and-plate rheometer result from intercellular collisions
induced by subjecting cell suspensions to fluid shear. The aggregation
process may be expressed as a system of chemical reactions, where
single PMN and colon carcinoma cells represent two monomer species and
(k,l) denotes a general aggregate comprised of
k PMN and l colon carcinoma cells. Each
interaction between (k,l) and another aggregate
(i,j) represents an elementary reaction step, and
K[(i,j),(k,l)] is the aggegation rate coefficient for this reaction. The two-species aggregation kinetics may then be expressed in the form of a population balance equation (PBE) (17)
where C(k,l) is the concentration of the
aggregate and
i,j is the
Kronecker delta function. The first term on the right-hand side of the
PBE represents the formation of the aggregate
(k,l) by the combination of two smaller aggregates, whereas the second term represents the consumption of the
aggregate (k,l), resulting in the formation of a
larger aggregate. kmax and
lmax denote the maximum number of PMNs and colon
carcinoma cells, respectively, that can be found in an aggregate. For
our system, lmax = 1 because colon
carcinoma cells do not aggregate homotypically under the experimental
conditions of this study, and kmax = 3 because aggregates containing more than three PMNs are rare events.
The rate of aggregate formation depends on the intercellular collision
frequency as well as the adhesion efficiency of these collisions. The
collision frequency per unit volume
f[(i,j),(k,l)] for particles of radii r(i,j)
and r(k,l) with concentrations
C(i,j) and C(k,l),
respectively, at a shear rate of G (s
1) is
given by (33)
The radius of PMNs was taken to be 3.75 µm (36).
The radii of LS174T and HCT-8 cells were measured by light microscopy and found to be 6.0 and 6.65 µm, respectively.
The adhesion efficiency is defined as the fraction of collisions
resulting in the formation of stable aggregates and is given by
If j = l = 0, then
E[(i,j),(k,l)]
is the homotypic adhesion efficiency for PMNs,
EPP. If i = k = 0, then
E[(i,j),(k,l)] is the homotypic adhesion efficiency for colon carcinoma cells, ETT (ETT = 0, because tumor
cells do not aggregate homotypically in this study); otherwise,
E[(i,j),(k,l)] is the heterotypic adhesion efficiency, EPT.
Here, we assume that heterotypic aggregation results from the adhesion
of PMN or PMN homotypic aggregates to colon carcinoma cells and not to
PMN already bound to colon carcinoma cells. This assumption is a good
approximation when the concentration of TP1 is much larger
than that of heterotypic aggregates containing more than one PMN
(TP2 and TP3+), as observed at early time points in this study. Specifically, the fraction of colon carcinoma cells with more than one adherent PMNs was <3% after 30 s of
shear. The PBE represents a set of ordinary differential equations with two unknown parameters, EPP and
EPT, and is integrated by using the fourth-order
Runge-Kutta-Gill method (9). The PMN homotypic and
PMN-colon carcinoma cell heterotypic adhesion efficiencies (EPP and EPT) are
obtained by minimizing the objective function given below by using the
Nelder and Mead simplex method (2). C(k,l)exp and
C(k,l)cal are the experimentally
observed concentrations and those calculated by the integration of the
PBE, respectively.
For pure PMN suspensions, a single-species PBE was numerically
integrated by using the Runge-Kutta-Gill method, and the homotypic adhesion efficiency was estimated by fitting the experimental data
using the Golden section search method (2).
Statistics.
Data are expressed as means ± SE. Statistical significance of
differences between means was determined by one-way ANOVA. Posttests were performed by using the Tukey method. Values of P < 0.05 were selected to be statistically significant.
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RESULTS |
Comparative efficiency of heteroaggregation of LS174T
and HCT-8 colon carcinoma cells with fMLP-stimulated PMNs under
hydrodynamic shear.
At low shear rates (50-200 s
1), PMN-LS174T
heteroaggregation increased with time until 120 s, when as many as
60% PMNs were in heterotypic aggregates (Fig.
1A). Partial disaggregation of these heterotypic aggregates was observed at longer periods
(180-300 s) of shear exposure for all shear rates examined.
Similarly, HCT-8 cells aggregated extensively with PMNs under these low
shear conditions, with maximal values
75% after 120 s of shear
exposure (Fig. 1B). Partial disaggregation of heterotypic
aggregates followed at later times for shear rates of 100-200
s
1. In marked contrast to LS174T cells, PMN-HCT-8 cell
heteroaggregates did not undergo disaggregation at 50 s
1
(Figs. 1, A and B).

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Fig. 1.
Kinetics of polymorphonuclear leukocyte (PMN)-colon carcinoma cell
and PMN-PMN aggregation. CMTMR (orange dye)-labeled colon carcinoma
cells (LS174T or HCT-8, 2 × 106 cells/ml) were
incubated with CFDA-SE (green dye)-labeled PMNs (1 × 106 cells/ml) at 37°C for 2 min, stimulated with 1 µM
fMLP, and sheared at prescribed shear rates and times. Sheared
specimens were immediately fixed with 1% formaldehyde and analyzed
with a FACSCalibur flow cytometer. A and C:
percentage of total PMNs in heterotypic and homotypic aggregates in the
presence of LS174T cells, respectively. B and D:
percentage of total PMNs in heterotypic and homotypic aggregates in the
presence of HCT-8 cells, respectively. Values are means ± SE
(n = 3-13).
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In the low-shear regime (50-200 s
1), PMN homotypic
aggregation in the presence of colon carcinoma cells was ~15% at
early times (10-30 s) and either remained unchanged (PMN-LS174T
cell suspensions, Fig. 1C) or showed a slight decrease
(PMN-HCT-8 cell suspensions, Fig. 1D) at longer shear
exposure times. The low percentage of PMNs in homotypic aggregates is
attributed to the extensive recruitment of PMNs by colon carcinoma
cells at low shear.
PMN homotypic aggregation was observed to increase with increasing
shear in PMN-HCT-8 cell suspensions, reaching maximal values of ~40
and 65% at 400 and 800 s
1, respectively, after 60 s
of shear (Fig. 1D). The percent PMNs in homotypic aggregates
was observed to decrease subsequently on exposure to longer duration
(
120 s) of shear. The decrease in the extent of PMN homotypic
aggregation at 800 s
1 is entirely a disaggregation
process, because the incorporation of PMNs into PMN-HCT-8 cell
heterotypic aggregates at this level of shear is negligible (Fig.
1D). However, at 400 s
1, the heterotypic
aggregation was observed to increase with time to ~40% after
300 s of shearing (Fig. 1D) and could be partially responsible for the low PMN homotypic aggregation observed at later
time points. This is also corroborated by previous observations showing
the absence of any appreciable disaggregation of PMN homotypic aggregates when pure PMN suspensions were subjected to a shear rate of
400 s
1 (37).
PMN homotypic aggregation was also found to increase with increasing
shear in PMN-LS174T cell suspensions with ~35-40% PMNs in
homotypic aggregates at high shear rates (800-1,200
s
1, Fig. 1C). In distinct contrast, PMN-LS174T
heteroaggregation decreased with increasing shear, and only ~25%
PMNs were recruited by LS174T cells in the high-shear regime (Fig.
1A). Partial disaggregation of the PMN-LS174T aggregates was
observed at later times (120-300 s) for all shear rates.
To further investigate how PMN homotypic aggregation is affected
by the presence of colon carcinoma cells, we compared homotypic aggregation in pure PMN suspensions with that observed in PMN-colon carcinoma cell suspensions. Because disaggregation is insignificant at
early times (37), the extents of aggregation occurring
over the first 30 s were determined. Moreover, to quantify
cell-cell interactions independent of physical parameters such as
aggregate size, cell concentration, and intercellular collision
frequency, we estimated PMN homotypic and PMN-colon carcinoma
heterotypic adhesion efficiencies by fitting the aggregation data over
the first 30 s to a mathematical model based on Smoluchowski's
two-body collision theory. After 30 s of shear, the extent of PMN
homotypic aggregation in the presence LS174T cells was significantly
less than that observed when PMNs were sheared alone over the entire range of shear rates examined (Fig.
2A). However, PMN homotypic aggregation in the presence of HCT-8 cells was significantly lower than
that observed for pure PMN suspensions only at low shear rates
(50-200 s
1), whereas no appreciable change was
detected in the high-shear regime (
400 s
1).
Nevertheless, our data indicate that PMN homotypic adhesion efficiency
in the presence of either LS174T or HCT-8 cells is similar to that
observed for pure PMN suspensions over the entire range of shear rates
examined (Fig. 2B). Cumulatively, these data suggest that
PMN homotypic aggregation is reduced in the presence of tumor cells
because of PMN recruitment by tumor cells and not because of the
release of any inhibitory enzymes by tumor cells. In all three cases,
the homotypic adhesion efficiency decreased sharply from 0.12 to 0.04 with increasing shear rate between 50 and 200 s
1, after
which it was almost constant till 1,600 s
1 (Fig.
2B).

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Fig. 2.
PMN homotypic and PMN-colon carcinoma heterotypic adhesion
efficiencies as a function of shear rate. The percentages of PMNs in
homotypic (A) and heterotypic (C) aggregates
after 30 s of shear exposure of either pure PMN or PMN-colon
carcinoma cell suspensions were determined as a function of
hydrodynamic shear by a dual-color flow cytometric methodology. The
adhesion efficiencies for homotypic (B) and heterotypic
(D) aggregation were calculated by using a mathematical
model based on Smoluchowski's theory. The experimental protocol was
similar to that mentioned in the legend of Fig. 1. Values are
means ± SE (n = 3-6).
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At low shear rates, initial PMN recruitment by HCT-8 cells was
significantly higher than that by LS174T cells (Fig. 2C).
Consequently, the PMN-HCT-8 cell heterotypic adhesion efficiency was
higher than the PMN-LS174T cell adhesion efficiency (0.09 for HCT-8 and 0.06 for LS174T at 50 s
1). However, in the high-shear
regime, LS174T cells displayed significant PMN recruitment, whereas
PMN-HCT-8 cell heteroaggregation was close to background levels. Along
these lines, at shear rates
400 s
1, heterotypic
adhesion efficiency for HCT-8 cells fell below that for LS174T cells.
At 800 s
1, the heterotypic adhesion efficiencies for
LS174T and HCT-8 cells were 0.0033 and 0.0008, respectively.
Relative contribution of CD11a, CD11b, and CD54 to PMN-colon
carcinoma cell adhesion.
Function-blocking MAbs were used against PMN CD11a and CD11b to
investigate their relative roles in PMN-LS174T heteroaggregation in the
shear range of 100-800 s
1. Blocking CD11b function
abrogated PMN binding to LS174T cells under all shearing conditions
(Fig. 3A). In distinct
contrast, use of an anti-CD11a MAb did not appreciably affect
PMN-LS174T heteroaggregation in the low-shear regime (100-200
s
1). However, PMN binding to LS174T cells was reduced by
40 and >60% in the presence of anti-CD11a MAb relative to control
values at shear rates of 400 and 800 s
1, respectively.
Together, our data illustrate that CD11b is requisite for PMN-LS174T
binding over the entire range of shear rates examined, whereas CD11a
contribution becomes evident only in the high-shear regime.

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Fig. 3.
The relative contributions of CD11a, CD11b, CD54, and L-selectin to
PMN-colon carcinoma cell heteroaggregation. CFDA-SE-labeled PMNs
(1 × 106 cells/ml) were incubated with anti-CD11a (20 µg/ml) and/or anti-CD11b (20 µg/ml) function-blocking MAbs at
37°C for 10 min before being mixed with CMTMR-labeled colon carcinoma
cells (2 × 106 cells/ml) at 37°C for 2 min. The
cell mixture was then stimulated with 1 µM fMLP 1 s before
application of shear. Sheared samples were immediately fixed with 1%
formaldehyde and analyzed by flow cytometry. A: the extent
of PMN-LS174T heteroaggregation after shearing for 30 s at
prescribed shear rates. B: the extent of PMN-HCT-8 cell
heteroaggregation after 30 s of shearing at 100 s 1.
C: the extent of PMN-HCT-8 cell heterotypic aggregation
after 300 s of shear exposure at 100 or 400 s 1. In
selected experiments, PMNs were treated with 1 U/106 cells
chymotrypsin for 20 min at room temperature before being mixed with
LS174T cells and subjected to shear. In some experiments, HCT-8 cells
were incubated with an anti-CD54 function-blocking MAb at 37°C for 10 min before being mixed with PMNs. Values are means ± SE
(n = 4-10). *P < 0.05 with
respect to control (untreated) samples. #P < 0.05 with
respect to CD54-treated samples.
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The relative contributions of CD11a and CD11b in mediating PMN binding
to HCT-8 cells were assessed at 100 s
1 after 30 s of
shear exposure. In distinct contrast to PMN-LS174T heteroaggregation,
PMN attachment to HCT-8 cells was partially inhibited by either
anti-CD11a (30%) or anti-CD11b (50%) (Fig. 3B). However,
simultaneous blockade of CD11a and CD11b completely abrogated PMN-HCT-8
cell binding after 30 s of shear at 100 s
1 (Fig.
3B). Subsequent studies aimed to identify the
counterreceptor(s) for CD11a and CD11b. Our flow cytometric data
indicate that CD54, a potential ligand to CD11a and CD11b, is expressed
on HCT-8 cells (Table 1). To assess its
potential involvement in this process, HCT-8 cells were treated
with anti-CD54 F(ab')2 MAb before the cells were sheared
with PMNs. This treatment significantly reduced PMN binding to HCT-8
cells by ~35% after 30 s of shear at 100 s
1 (Fig.
3B). The extent of heteroaggregation of anti-CD54-treated HCT-8 cells with anti-CD11a-treated PMNs was similar to the binding of
anti-CD54-treated HCT-8 cells to untreated PMNs (Fig. 3B). However, simultaneous treatment of HCT-8 cells with anti-CD54 and PMNs
with anti-CD11b was more effective than anti-CD11b treatment alone,
although it did not completely abrogate adhesion (Fig. 3B).
Cumulatively, these results indicate that, under the above conditions
(100 s
1, 30 s), CD54 preferentially binds CD11a,
whereas CD11b appears to interact with a yet unidentified
counterreceptor on the HCT-8 cell surface.
We next examined the contribution of CD18 integrins in mediating
PMN-HCT-8 cell heteroaggregation at longer shear exposure (300 s) at
both 100 and 400 s
1. Use of either anti-CD11a or
anti-CD11b partially inhibited PMN-HCT-8 cell heteroaggregation by 40 and 60%, respectively, at 100 s
1 (Fig. 3C).
It is noteworthy that under high-shear conditions (400 s
1, 300 s), PMN binding to HCT-8 cells was
completely abrogated by anti-CD11b, whereas it was also dramatically
inhibited by anti-CD11a. Interestingly, use of an anti-CD54 MAb failed
to significantly reduce PMN adhesion to HCT-8 cells on longer shear
exposure at both 100 and 400 s
1. Together, our data
suggest that at longer exposure to hydrodynamic shear, CD54 does not
play a significant role in PMN-HCT-8 adhesion, suggesting that both
CD11a and CD11b bind other yet unidentified ligands on the HCT-8 cells.
CD54 is minimally expressed on LS174T cells (Table 1) and, therefore,
does not appear to be involved in mediating PMN-LS174T cell adhesion
(12). The ligand on LS174T binding PMN CD18 integrins has
yet to be characterized.
Role of L-selectin in PMN-LS174T adhesion and characterization of
L-selectin ligand on colon carcinoma cells.
We next wanted to systematically investigate the relative contribution
of L-selectin to the PMN-LS174T adhesion process over a wide range of
shear rates (100-800 s
1) and to characterize the
L-selectin ligand on LS174T cells. Chymotrypsin treatment (1 U/ml) has
been shown to cleave L-selectin from PMN surface (12). In
the low-shear regime (100-200 s
1; Fig.
3A), the extent of LS174T cell binding to
chymotrypsin-treated PMNs was similar to that to untreated PMNs. In
marked contrast, chymotrypsin treatment reduced PMN-LS174T
heteroaggregation by 50% at 400 s
1 and by
85% at 800 s
1. These data suggest that the L-selectin contribution
in mediating adhesion becomes progressively larger with increasing
shear. Fucoidan, which has been shown to block PMN binding to
L-selectin (8), abrogated PMN-LS174T heteroaggregation at
800 s
1 (Fig. 4), providing
further evidence for L-selectin involvement in this process under
high-shear conditions.

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Fig. 4.
Characterization of L-selectin ligand on LS174T cells.
CFDA-SE-labeled PMNs (1 × 106 cells/ml) were
incubated with CMTMR-labeled LS174T cells (2 × 106
cells/ml) for 2 min at 37°C, stimulated with 1 µM fMLP, and sheared
at 800 s 1 for 30 s. Sheared samples were immediately
fixed with 1% formaldehyde and analyzed with a FACSCalibur flow
cytometer. In selected experiments, LS174T cells were cultured in the
presence of either
benzyl-2-acetamido-2-deoxy- -D-galactopyranoside (Bzl
GalNAc; 2 mM) or tunicamycin (200 ng/ml) for 48 h or with
d,l-threo-1-phenyl-2-amino3-morpholino-1-propanol
hydrochloride (threo-PPPP; 5 µM) for 96 h. Some experiments were
conducted with LS174T cells treated with either trypsin (20 µg/ml for
30 min) or phosphatidylinositol-specific phospholipase C (PI-PLC; 1 U/ml for 1 h). Others were conducted in the presence of dextran
sulfate (100 µg/ml), heparin (100 U/ml), or fucoidan (10 µg/ml).
Values are means ± SE (n = 3-5).
*P < 0.05 with respect to control (untreated)
samples.
|
|
We have recently shown that the L-selectin ligand on LS174T cells is a
sialylated molecule, as evidenced by abrogation of PMN binding to
neuraminidase-treated LS174T cells (12). We wanted to
further characterize the L-selectin ligand and determine whether it is
a glycoprotein or a glycosphingolipid. Enzymatic treatment of LS174T
cells with trypsin abrogated PMN-LS174T cell aggregation at high (Fig.
4) but not low shear (data not shown). In distinct contrast, LS174T
cells cultured in the presence of an inhibitor of ceramide:UDP
glucose transferase, threo-PPPP (1), retained their
ability to aggregate effectively with PMNs under high shear (Fig. 4).
Cumulatively, these data suggest that the L-selectin ligand on LS174T
colon carcinoma cells is a protease-sensitive glycoprotein rather than
a glycosphingolipid. We next investigated whether the L-selectin ligand
is a GPI-linked molecule by treating LS174T cells with PI-PLC, an
enzyme that cleaves GPI-anchored glycoproteins (21).
However, this treatment failed to affect PMN-LS174T heteroaggregation
at 800 s
1 (Fig. 4).
Further studies aimed at testing whether critical L-selectin binding
determinants on LS174T colon carcinoma cells are presented on O-linked
and/or N-linked glycans. To this end, we cultured LS174T cells in the
presence of either Bzl GalNAc or tunicamycin, which are known to
inhibit O- and N-linked glycosylation, respectively (30).
PMN binding to LS174T treated with these inhibitors of glycosylation
was not significantly different from that to untreated LS174T cells at
low shear rates (data not shown). However, PMN-LS174T heteroaggregation
was drastically inhibited by Bzl GalNAc, but not by tunicamycin, at 800 s
1 (Fig. 4). Together, these data illustrate that the
L-selectin ligand is an O-linked, sialylated, protease-sensitive
structure and does not require N-glycans for binding.
Sulfated polysaccharides such as heparin and dextran sulfate have been
shown to inhibit L-selectin binding to sLex
(25). Our data indicate that both heparin and dextran
sulfate significantly inhibited PMN binding to LS174T cells at 800 s
1 (Fig. 4), but not in the low-shear regime (data not
shown), presumably by interfering with L-selectin-sLex
binding. The inhibitory effects of heparin and dextran sulfate may be
suggestive of the potential presence of sulfated groups on the
L-selectin ligand (18). On the other hand, HCT-8 cells express near background levels of sLex (Table 1). The lack
of PMN recruitment by HCT-8 cells at high shear reveals the absence of
functional PMN L-selectin ligands on these colon carcinoma cells.
Effect of shear stress on PMN-colon carcinoma cell aggregate
formation.
Exposure of cell suspensions to increasing levels of shear rate
increases the frequency of cell-cell collisions but decreases the
intercellular contact duration. Concomitantly, there is an increase in
the magnitude of tensile forces acting on the adhesive contact region
to dissociate intercellular bonds. To differentiate the contributions
of contact duration and tensile forces on cell aggregation, we used 6%
Ficoll to double the viscosity of the suspending medium, thereby
increasing the shear stress at a constant shear rate or contact time
(10, 29, 37). PMN homotypic aggregation was unaffected by
the increase in viscosity in the presence of LS174T (Fig.
5A) as well as HCT-8 cells
(data not shown). Similarly, increasing the viscosity did not
appreciably affect PMN-LS174T binding over the entire range of shear
rates examined (Fig. 5B), suggesting that these interactions
are dependent on contact duration and that the aggregates are
sufficiently stable to resist the increased shear forces. However, in
the case of HCT-8 cells, heterotypic aggregation at 30 s remained
~45% at low shear rates (50-200 s
1) for 0.8-cP
suspensions, whereas heteroaggregation decreased sharply for 1.6-cP
suspensions from ~55% at 50 s
1 to 22% at 200 s
1 (Fig. 5C). The significant reduction in
PMN-HCT-8 heterotypic adhesion observed at 1.6 cP compared with that at
0.8 cP at a shear rate of 200 s
1 suggests that the
adhesive bonds are not strong enough to resist the increased shear
force acting on them. For shear rates >400 s
1, PMN-HCT-8
cell heteroaggregation was close to background levels in 0.8-cP as well
as 1.6-cP suspensions (Fig. 5C).

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Fig. 5.
Effect of shear stress on PMN-colon carcinoma cell aggregate
formation. Neuraminidase (0.1 U/ml)-treated or untreated CMTMR-labeled
colon carcinoma cells (LS174T or HCT-8, 2 × 106
cells/ml) were incubated with CFDA-SE-labeled PMNs (1 × 106 cells/ml) at 37°C for 2 min. In selected experiments,
the viscosity of the suspending medium was increased from 0.8 cP to 1.6 cP at 37°C with 6% Ficoll. The cell mixture was stimulated with 1 µM fMLP and sheared for 30 s at prescribed shear rates. Sheared
specimens were immediately fixed with 1% formaldehyde and analyzed
with a FACSCalibur flow cytometer. A and B:
percentage of PMNs in homotypic and heterotypic aggregates,
respectively, in the presence of untreated LS174T cells. C
and D: percentage of PMNs in heterotypic aggregates in
the presence of HCT-8 cells and neuraminidase-treated LS174T cells,
respectively. Values are means ± SE (n = 3-4). *P < 0.05 with respect to the aggregation
in 0.8-cP suspensions.
|
|
We next wanted to investigate whether the presence of L-selectin
ligands on LS174T cells is responsible for the differential effects
detected with LS174T and HCT-8 cells when the high viscosity (1.6 cP)
cell suspensions are subjected to shear. Treating LS174T cells with
neuraminidase cleaved cell surface sialic acid residues (12) and significantly impaired PMN-LS174T
heteroaggregation at
400 s
1 (Fig. 5, B and
D). We also observed that PMN binding to
neuraminidase-treated LS174T cells in 0.8-cP suspensions was similar to
that detected in 1.6-cP suspensions at shear rates of 50-100
s
1 (Fig. 5D). However, PMN-LS174T heterotypic
aggregation was significantly lower in 1.6-cP suspension than in 0.8-cP
buffer at 200 s
1 (Fig. 5D), which is in accord
with the results obtained with sLex-low HCT-8 cells.
 |
DISCUSSION |
The main findings of this work are as follows: 1) the
adhesion efficiency of PMN homotypic aggregation is not affected by the
presence of colon carcinoma cells over the entire range of shear rates
examined in this work; 2) the efficiency of PMN-colon carcinoma cell heterotypic aggregation decreases with increasing shear,
with PMN binding to CD54-bearing HCT-8 cells being more efficient than
that to CD54-negative LS174T cells at low shear; 3) under
these low-shear conditions, both CD11a and CD11b contribute to
PMN-HCT-8 cell aggegation, with CD54 on HCT-8 cells acting as a CD11a
ligand only at early time points; 4) CD11a involvement becomes progressively larger with increasing shear by binding to a yet
unidentified ligand distinct from CD54; and 5) in the high-shear regime, only PMN-LS174T cell aggregation occurs, which is
initiated by PMN L-selectin binding to a sialylated, O-linked, protease-sensitive ligand on LS174T cells. Thus we provide evidence that both fluid shear and shear exposure time modulate the molecular interactions between PMNs and colon carcinoma cells.
PMN homotypic adhesion efficiency is unaffected by the presence of
colon carcinoma cells.
PMN homotypic aggregation in PMN-colon carcinoma cell suspensions was
significantly lower than that in pure PMN suspensions whenever
appreciable PMN-colon carcinoma heteroaggregation occurred. We argue
that the reduction in PMN homotypic aggregation occurred because of
recruitment of PMNs by colon carcinoma cells and not because of the
release of any inhibitory enzyme. This concept is corroborated by the
fact that PMN homotypic adhesion efficiency estimated in the presence
of colon carcinoma cells did not differ significantly from that
calculated for pure PMN suspensions. PMN homotypic adhesion efficiency
decreased with increasing shear in the low-shear regime. This decrease
is attributed to the lower probability of CD18 integrin receptors to
mediate stable adhesion at increased shear rates and correspondingly
shorter intercellular contact durations in the absence of any selectin
contribution. Nevertheless, the efficiency is nearly unchanged at
higher shear rates (
200 s
1) because of the involvement
of L-selectin, which binds to its counterreceptor, PSGL-1, and mediates
transient tethering between PMNs, thereby increasing the collisional
contact duration and allowing the CD18 integrins to mediate stable
adhesion (37). The PMN homotypic adhesion efficiency in
our studies with pure suspensions was consistently lower than that
previously reported (37). This discrepancy may be ascribed
to three reasons. First, the coefficient for collision frequency used
by Taylor et al. (37) was in error by a factor of 2, as
acknowledged by the authors in a later publication (10).
Second, omission of (1 +
i,j
k,l) from the stoichiometric coefficients in the PBE (17)
results in a significant error, as shown in Table
2. Last, the extent of PMN homotypic
aggregation, which varies from donor to donor, was observed to be lower
in our study than that reported by Taylor et al. (37).
The molecular mechanisms of PMN-binding to LS174T and HCT-8 colon
carcinoma cells are shear and time dependent.
The extent of PMN binding to HCT-8 cells was consistently higher than
that to LS174T cells at low shear (50-200 s
1) and
could be at least partially attributed to the presence of CD54 on the
HCT-8 carcinoma cell surface. Under these low shear conditions, CD11a
and CD11b are involved in PMN-HCT-8 binding at both short and long
shear exposure times. This finding is in contrast to PMN homotypic
aggregation (24) as well as PMN binding to
CD54-transfected mouse cells (23), where adhesion was
almost entirely CD11b dependent at long shear exposure times. However, this discrepancy may be attributed to the presence of distinct CD11a
ligands with markedly different binding kinetics in each of these cell
types. The lack of any additive inhibitory effect upon simultaneous use
of CD11a (on PMNs) and CD54 (on HCT-8 cells) suggests that PMN CD11a
binds to CD54 expressed on HCT-8 colon carcinoma cells at short shear
exposure times. However, at longer shear exposure, the inability of
anti-CD54 mAb alone to substantially reduce PMN-HCT-8 cell adhesion
suggests the presence of another ligand for CD11a with a higher
affinity than CD54. Furthermore, CD11b appears to interact with a yet
unidentified ligand on HCT-8 cells, rather than CD54 at all shear rates
and shear exposure times examined here. Along these lines, PMN CD11b
binds to CD54-negative LS174T colon carcinoma cells and mediates
heteroaggregation under shear (12). It is noteworthy that
at low shear (100 s
1), CD11a does not seem to be involved
in PMN binding to LS174T cells. This is in accord with previous studies
performed under static conditions showing that transmigration of
fMLP-activated PMNs across CD54-negative T84 human colon adenocarcinoma
cell layers is mediated by CD11b and is independent of CD11a
(26-28). However, the contribution of CD11a to
PMN-colon carcinoma heteroaggregation becomes evident (LS174T cells) or
even more pronounced (CD54-expressing HCT-8 cells) in the high-shear
regime (
400 s
1). In distinct contrast, a previous study
shows that PMN homotypic aggregation via CD11a alone is barely
detectable at 400 s
1 and becomes entirely
CD11a-independent/CD11b-dependent at even higher shear rates
(24). The partial disaggregation of PMN-colon carcinoma
cell aggregates observed at long shear exposure times may reflect a
decay in the avidity of PMN CD11a and CD11b (24) as well
as of their respective counterreceptors on the carcinoma cell surface.
L-selectin-mediated PMN-colon carcinoma cell binding depends on
intercellular contact duration and is shear stress resistant.
PMN homotypic and PMN-LS174T heterotypic aggregation were unaffected
when the shear stress was doubled independently of the shear rate by
increasing the viscosity of the medium from 0.8 to 1.6 cP. However, the
twofold increase in shear stress caused a significant reduction of PMN
binding to sLex-low HCT-8 cells and neuraminidase-treated
(sLex-low) LS174T cells at 200 s
1. These data
suggest that L-selectin-ligand interactions increase the contact
duration between PMNs and sLex-expressing LS174T cells,
thereby allowing a sufficient number of CD18 integrin bonds to form
that can withstand the increase in shear stress by elevating the
viscosity of the suspending medium. PMN L-selectin is not involved in
PMN-LS174T heteroaggregation at low shear rates, but its contribution
increases progressively with increasing shear rate, which is in
agreement with previously published data on PMN homotypic aggregation
(37). However, unlike PMN homotypic adhesion efficiency,
the PMN-LS174T cell heterotypic efficiency falls with increasing shear
(200-800 s
1), even though L-selectin is involved.
This decrease may occur because the L-selectin ligand on LS174T cells
may not be expressed in large numbers or may have a lower binding
affinity for L-selectin. On the other hand, PMNs expressing L-selectin
as well as its counterreceptor, PSGL-1, may preferentially bind other
PMNs, rather than LS174T cells, which express only the L-selectin
ligand and are devoid of L-selectin. The L-selectin ligand on LS174T
colon carcinoma cells appears to be a sialylated, O-glycosylated,
protease-sensitive molecule distinct from PSGL-1 (12).
Moreover, the lack of any inhibitory effects on PMN binding to LS174T
cells at high shear with the use of certain specific enzymes and
biosynthetic inhibitors suggests that the L-selectin ligand is neither
a glycosphingolipid nor a GPI-anchored molecule and does not require
N-glycans for binding to L-selectin.
Recent findings illustrate the presence of activated PMNs in the
circulatory system of patients with metastatic adenocarcinomas of the
colon, pancreas, and breast (31). The activation of PMNs could be induced by cytokines or chemokines produced by the tumor or an
inflammatory response to bacterial or viral infection (4, 31). Hence, the interaction of PMNs activated by bacterial
products with tumor cells could be physiologically important. Although this study does not address the effect of PMNs on blood-borne metastasis, which is currently controversial (4, 7), it provides a quantitative analysis of the dynamics and molecular mechanisms mediating PMN-neoplastic emboli formation that could potentially promote the hematogenous dissemination of tumor cells (3, 5, 35, 38). A recent study through the use of
L-selectin-deficient mice suggested that L-selectin facilitates the
metastatic spreading of tumor cells, thereby implicating leukocytes as
enhancers of blood-borne metastasis (3). Consequently,
this study provides a mechanistic interpretation of potential adhesion
events between PMNs and tumor cells occurring in vivo. It is noteworthy
that at the shear rate of 100 s
1, ~4 of 100 collisions
between PMNs and LS174T cells result in stable binding compared with
~2 of 1,000 collisions between thrombin-treated platelets and LS174T
cells (21a). Taking into account the physiological concentrations of platelets (2 × 108 per ml) and PMNs
(6 × 106 per ml), the probability of PMN-LS74T colon
carcinoma cell aggregate formation is of the same order of magnitude as
that of platelet-LS174T binding. Together, our data clearly show that
the hydrodynamic shear environment and shear exposure time influence
the kinetics, stability, and the molecular constituents of PMN-colon
carcinoma cell adhesion.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Ronald L. Schnaar (Johns Hopkins University) for
helpful discussions.
 |
FOOTNOTES |
This work was supported by National Science Foundation Grants BES
9978160 and BES 0093524.
Address for reprint requests and other correspondence: K. Konstantopoulos, Dept. of Chemical Engineering, The Johns Hopkins Univ., 3400 N. Charles St., Baltimore, MD 21218-2694 (E-mail: konst_k{at}jhu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 20, 2002;10.1152/ajpcell.00104.2002
Received 6 March 2002; accepted in final form 4 June 2002.
 |
REFERENCES |
1.
Abe, A,
Radin NS,
Shayman JA,
Wotring LL,
Zipkin RE,
Sivakumar R,
Ruggieri JM,
Carson KG,
and
Ganem B.
Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth.
J Lipid Res
36:
611-621,
1995[Abstract].
2.
Belegundu, AD,
and
Chandrupatla TR.
Optimization Concepts and Applications in Engineering. Upper Saddle River, NJ: Prentice-Hall, 1999.
3.
Borsig, L,
Wong R,
Hynes RO,
Varki NM,
and
Varki A.
Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis.
Proc Natl Acad Sci USA
99:
2193-2198,
2002[Abstract/Free Full Text].
4.
Coussens, LM,
and
Werb Z.
Inflammatory cells and cancer: think different!
J Exp Med
193:
F23-F26,
2001[ISI][Medline].
5.
Crissman, JD,
Hatfield J,
Schaldenbrand M,
Sloane BF,
and
Honn KV.
Arrest and extravasation of B16 amelanotic melanoma in murine lungs. A light and electron microscopic study.
Lab Invest
53:
470-478,
1985[ISI][Medline].
6.
Dallegri, F,
Ottonello L,
Ballestrero A,
Dapino P,
Ferrando F,
Patrone F,
and
Sacchetti C.
Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen 1.
Inflammation
15:
15-30,
1991[ISI][Medline].
7.
Di Carlo, E,
Forni G,
Lollini P,
Colombo MP,
Modesti A,
and
Musiani P.
The intriguing role of polymorphonuclear neutrophils in antitumor reactions.
Blood
97:
339-345,
2001[Free Full Text].
8.
Fuhlbrigge, RC,
Alon R,
Puri KD,
Lowe JB,
and
Springer TA.
Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions.
J Cell Biol
135:
837-848,
1996[Abstract].
9.
Gupta, SK.
Numerical Methods for Engineers. New Delhi, India: Wiley Eastern, 1995.
10.
Hentzen, ER,
Neelamegham S,
Kansas GS,
Benanti JA,
McIntire LV,
Smith CW,
and
Simon SI.
Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule-1.
Blood
95:
911-920,
2000[Abstract/Free Full Text].
11.
Ishikawa, M,
Koga Y,
Hosokawa M,
and
Kobayashi H.
Augmentation of B16 melanoma lung colony formation in C57BL/6 mice having marked granulocytosis.
Int J Cancer
37:
919-924,
1986[ISI][Medline].
12.
Jadhav, S,
Bochner BS,
and
Konstantopoulos K.
Hydrodynamic shear regulates the kinetics and receptor specificity of polymorphonuclear leukocyte-colon carcinoma cell adhesive interactions.
J Immunol
167:
5986-5993,
2001[Abstract/Free Full Text].
13.
Konstantopoulos, K,
Kukreti S,
and
McIntire LV.
Biomechanics of cell interactions in shear fields.
Adv Drug Deliv Rev
33:
141-164,
1998[ISI][Medline].
14.
Konstantopoulos, K,
Kukreti S,
Smith CW,
and
McIntire LV.
Endothelial P-selectin and VCAM-1 each can function as primary adhesive mechanisms for T cells under conditions of flow.
J Leukoc Biol
61:
179-187,
1997[Abstract].
15.
Konstantopoulos, K,
Neelamegham S,
Burns AR,
Hentzen E,
Kansas GS,
Snapp KR,
Berg EL,
Hellums JD,
Smith CW,
McIntire LV,
and
Simon SI.
Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and
2-integrin.
Circulation
98:
873-882,
1998[Abstract/Free Full Text].
16.
Kushner, BH,
and
Cheung NKV
Absolute requirement of CD11/CD18 adhesion molecules, FcRII, and the Phosphatidylinositol-Linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity.
Blood
79:
1484-1490,
1992[Abstract].
17.
Laurenzi, IJ,
and
Diamond SL.
Monte Carlo simulation of the heterotypic aggregation kinetics of platelets and neutrophils.
Biophys J
77:
1733-1746,
1999[Abstract/Free Full Text].
18.
Ley, K,
Cerrito M,
and
Arfors KE.
Sulfated polysaccharides inhibit leukocyte rolling in rabbit mesentery venules.
Am J Physiol Heart Circ Physiol
260:
H1667-H1673,
1991[Abstract/Free Full Text].
19.
Lichtenstein, A.
Stimulation of the respiratory burst of murine peritoneal inflammatory neutrophils by conjugation with tumor cells.
Cancer Res
47:
2211-2217,
1987[Abstract].
20.
Mannori, G,
Crottet P,
Cecconi O,
Hanasaki K,
Aruffo A,
Nelson RM,
Varki A,
and
Bevilacqua MP.
Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.
Cancer Res
55:
4425-4431,
1995[Abstract].
21.
McCarty, OJ,
Mousa SA,
Bray PF,
and
Konstantopoulos K.
Immobilized platelets support human colon carcinoma cell tethering, rolling, and firm adhesion under dynamic flow conditions.
Blood
96:
1789-1797,
2000[Abstract/Free Full Text].
21a.
McCarty, OJ,
Jadhav S,
Burdick MB,
Bell WR,
and
Konstantopoulos K.
Fluid shear regulates the kinetics and molecular mechanisms of activation-dependent platelet binding to colon carcinoma cells.
Biophys J
83:
836-848,
2002[Abstract/Free Full Text].
22.
Miyata, R,
Iwabuchi K,
Watanabe S,
Sato N,
and
Nagaoka I.
Short exposure of intestinal epithelial cells to TNF-
and histamine induces Mac-1-mediated neutrophil adhesion independent of protein synthesis.
J Leukoc Biol
66:
437-446,
1999[Abstract].
23.
Neelamegham, S,
Taylor AD,
Burns AR,
Smith CW,
and
Simon SI.
Hydrodynamic shear shows distinct roles for LFA-1 and Mac-1 in neutrophil adhesion to intercellular adhesion molecule-1.
Blood
92:
1626-1638,
1998[Abstract/Free Full Text].
24.
Neelamegham, S,
Taylor AD,
Shankaran H,
Smith CW,
and
Simon SI.
Shear and time-dependent changes in Mac-1, LFA-1, and ICAM-3 binding regulate neutrophil homotypic adhesion.
J Immunol
164:
3798-3805,
2000[Abstract/Free Full Text].
25.
Nelson, RM,
Cecconi O,
Roberts WG,
Aruffo A,
Linhardt RJ,
and
Bevilacqua MP.
Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation.
Blood
82:
3253-3258,
1993[Abstract].
26.
Parkos, CA,
Colgan SP,
Bacarra AE,
Nusrat A,
Delp-Archer C,
Carlson S,
Su DHC,
and
Madara JL.
Intestinal epithelia (T84) possess basolateral ligands for CD11b/CD18-mediated neutrophil adherence.
Am J Physiol Cell Physiol
268:
C472-C479,
1995[Abstract/Free Full Text].
27.
Parkos, CA,
Colgan SP,
Diamond SL,
Nusrat A,
Liang TW,
Springer TA,
and
Madara JL.
Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.
Mol Med
2:
489-505,
1996[ISI][Medline].
28.
Parkos, CA,
Delp C,
Arnaout MA,
and
Madara JL.
Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction.
J Clin Invest
88:
1605-1612,
1991[ISI][Medline].
29.
Rinker, KD,
Prabhakar V,
and
Truskey GA.
Effect of contact time and force on monocyte adhesion to vascular endothelium.
Biophys J
80:
1722-1732,
2001[Abstract/Free Full Text].
30.
Sawada, T,
Ho JJ,
Chung YS,
Sowa M,
and
Kim YS.
E-selectin binding by pancreatic tumor cells is inhibited by cancer sera.
Int J Cancer
57:
901-907,
1994[ISI][Medline].
31.
Schmielau, J,
and
Finn OJ.
Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients.
Cancer Res
61:
4756-4760,
2001[Abstract/Free Full Text].
32.
Skinner, MP,
Lucas CM,
Burns GF,
Chesterman CN,
and
Berndt MC.
GMP-140 binding to neutrophils is inhibited by sulfated glycans.
J Biol Chem
266:
5371-5374,
1991[Abstract/Free Full Text].
33.
Smoluchowski, MV.
Versuch einer mathematichen Theorie der koagulationskinetik Kolloider losungen.
Z Phys Chem
1992:
129-168,
1917.
34.
Springer, TA.
Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration.
Annu Rev Physiol
57:
827-872,
1995[ISI][Medline].
35.
Starkey, JR,
Liggitt HD,
Jones W,
and
Hosick HL.
Influence of migratory blood cells on the attachment of tumor cells to vascular endothelium.
Int J Cancer
34:
535-543,
1984[ISI][Medline].
36.
Tandon, P,
and
Diamond SL.
Kinetics of
2-integrin and L-selectin bonding during neutrophil aggregation in shear flow.
Biophys J
75:
3163-3175,
1998[Abstract/Free Full Text].
37.
Taylor, AD,
Neelamegham S,
Hellums JD,
Smith CW,
and
Simon SI.
Molecular dynamics of the transition from L-selectin- to
2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.
Biophys J
71:
3488-3500,
1996[Abstract].
38.
Wu, QD,
Wang JH,
Condron C,
Bouchier-Hayes D,
and
Redmond HP.
Human neutrophils facilitate tumor cell transendothelial migration.
Am J Physiol Cell Physiol
280:
C814-C822,
2001[Abstract/Free Full Text].
Am J Physiol Cell Physiol 283(4):C1133-C1143
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