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*
Department of Chemical Engineering, Johns Hopkins University, Baltimore, MD 21218; and
Department of Medicine, Division of Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
,
and IFNs (1). However, several independent studies have
demonstrated that PMNs enhance the metastatic potential of tumor cells.
This concept is substantiated by experiments showing that PMNs
facilitate tumor cell extravasation in vitro (4, 5) and
promote the arrest and deposition of tumors in the microvasculature of
target organs in animal models (4, 6, 7). Detailed light
and electron microscopic studies have revealed that PMNs are often in
close association with metastatic tumor cells during the process of
tumor cell arrest and extravasation in vivo (8). To date, the literature on the molecular mechanisms that mediate PMN-tumor cell adhesive interactions is highly fragmentary. PMNs have been reported to bind to certain melanoma, neuroblastoma, and colon adenocarcinoma cells through the CD18 integrin receptor on the PMN surface in a selectin-independent manner (9, 10, 11). Alternatively, PMNs may attach to colon carcinomas via PMN CD62L (L-selectin) in the absence of any CD18 integrin contribution, presumably because these experiments were performed at 4°C, a temperature that renders integrins inactive (12). It is currently unknown whether L-selectin can function in concert with CD18 integrins to mediate optimal PMN-tumor cell interactions. The salient feature of selectins, not shared by integrins, is their ability to initiate cell binding under high shear conditions (13, 14). This is attributed to the fact that selectin-ligand bonds have fast on and off rates and an important ability to stretch before breaking under shear conditions (15).
A major limitation of our current knowledge stems from the fact that all previous studies aimed at investigating PMN-tumor cell interactions were performed exclusively under static conditions (2, 9, 10, 11, 12), which neglect the rheological parameters of fluid flow in the vasculature. It is now well established that the local fluid mechanical environment of the circulation critically affects the molecular pathways of cell-cell interactions. As has been appropriately argued in the literature, data obtained in vitro using static binding assays may not be relevant to the fluid dynamic environment encountered in the vasculature. Consequently, the present study was undertaken to systematically characterize the effects of hydrodynamic shear on PMN-tumor cell interactions at the molecular level using a human colon carcinoma cell model, because colon cancer is among those tumors with a propensity for hematogenous spread. The metastatic (16) sialyl Lewisx (sLex)-bearing LS174T human colon adenocarcinoma cell line, which expresses both selectin and CD18 integrin ligands on the cell surface, was used in this study because it has been extensively characterized and widely used in a number of diverse assays (10, 12, 17, 18). For comparison purposes, we also looked at the nonmetastatic (16, 19) HCT-8 colon adenocarcinoma cell line expressing minimal levels of sLex (sLex-low HCT-8 cells) (16, 19). Recent findings illustrate the presence of activated PMNs in the circulatory system of patients with metastatic adenocarcinomas of the colon, pancreas, and breast (20). The activation of PMNs could be induced by cytokines or chemokines produced by the tumor or an inflammatory response to bacterial or viral infection (20, 21). Hence, the interaction of PMNs activated by bacterial products with tumor cells could be physiologically important.
The present study demonstrates that hydrodynamic shear applied by the use of a cone-and-plate rheometer regulates the dynamics and molecular constituents mediating adhesion between chemotactically stimulated PMNs and LS174T colon carcinoma cells. At low shear (100 s-1), CD11b alone is sufficient to mediate optimal PMN-LS174T heteroaggregation. In marked contrast, PMN adhesion to LS174T cells at high shear is a two-step, sequential process involving L-selectin-dependent tethering followed by CD11a and CD11b stable adhesion. Along these lines, the sLex-low HCT-8 colon carcinoma cells, which fail to bind to L-selectin, do not aggregate with PMNs at high shear conditions, despite their extensive heterotypic adhesive interactions at low shear.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IgG murine mAbs 6.7 (blocking anti-CD18), HI111 (blocking anti-CD11a), ICRF44(44) (blocking anti-CD11b), KPL-1 (blocking anti-CD162), Dreg-56 (anti-CD62L, purified as well as conjugated with either FITC or PE), 581 (anti-CD34), MOPC-21 (an irrelevant control Ab conjugated with either FITC or PE), and HI30 (anti-CD45 conjugated with PE) were purchased from BD PharMingen (San Diego, CA). The function-blocking anti-CD62L mAb LAM1-116 was generously provided by Dr. T. F. Tedder (Duke University Medical Center, Durham, NC) (22). Anti-sLex mAb KM93 (mouse IgM) was from Kiyama (Seattle, WA). The mAbs BU15 (anti-CD11c), 84H10 (anti-CD54), and 4B4 (anti-CD29) were obtained from Beckman Coulter (Fullerton, CA). The blocking F(ab')2 anti-CD102 mAb CBR-IC2/2 and anti-CD54 MHCD54F were from Caltag Laboratories (Burlingame, CA). Anti-CD50 mAb 76205.11 was purchased from R&D Systems (Minneapolis, MN). Anti-CD61-FITC mAb Y2/51, specific for platelet gpIIIa, was obtained from DAKO (Carpinteria, CA). Isotype-matched IgG and IgM mAbs were from Sigma-Aldrich (St. Louis, MO). Ro-31-9790, a synthetic hydroxamic acid-based metalloproteinase inhibitor that effectively blocks L-selectin shedding (23), was a kind gift by Dr. D. S. Walter (Roche Discovery Welwyn, Welwyn Garden City, Hertfordshire, U.K.). CellTracker CFSE and 5-(and 6-)4-chloromethyl-benzoyl-amino-tetra-methylrhodamine (CMTMR) were purchased from Molecular Probes (Eugene, OR). CFSE and CMTMR are excited efficiently at 488 nm by the argon laser of a flow cytometer, and their emission spectra are well separated (515 nm for CFSE and 570 nm for CMTMR), thereby allowing simultaneous two-color immunofluorescence measurements.
Tumor cell line culture and labeling
The LS174T and HCT-8 human colon adenocarcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured in the recommended medium. Both cell lines tested negative on a regular basis for mycoplasma contamination using a commercially available PCR kit (Maxim Biotech, San Francisco, CA). LS174T and HCT-8 cells were detached from culture flasks by mild trypsinization (0.25% trypsin/EDTA for 2 min at 37°C; Life Technologies, Gaithersburg, MD), resuspended in the appropriate medium, and then incubated for 2 h at 37°C to regenerate surface glycoproteins, as previously described (12, 19). During this period, the carcinoma cell suspensions (107 cells/ml) were also incubated with 1 µM CMTMR for 1 h at 37°C. Immediately thereafter, tumor cells were washed once to remove excess dye, resuspended in Dulbecco’s PBS (D-PBS) containing Ca2+/Mg2+/0.1% BSA (Sigma-Aldrich), and stored at 4°C for no longer than 3 h before use in aggregation assays or flow cytometry.
PMN preparation
Human PMNs were obtained from citrate phosphate dextrose
(Sigma-Aldrich) anticoagulated venous blood of healthy volunteers (1.4
ml citrate phosphate dextrose/10 ml blood) by centrifugation through a
PMN isolation medium (Robbins Scientific, Sunnyvale, CA). To minimize
erythrocyte contamination in the PMN preparations, a RBC agglutination
reagent (Red-Out; Robbins Scientific) was incubated with anticoagulated
blood for 
5 min at room temperature (RT) before the aforementioned
centrifugation step (24). Isolated PMNs were washed once,
resuspended in D-PBS lacking
Ca2+/Mg2+ at a
concentration of 107 cells/ml, and then incubated
with 0.1 µM CFSE for 1 h at 4°C. CFSE-stained PMNs were washed
once, resuspended in
Ca2+/Mg2+-free D-PBS/0.1%
BSA, and stored at 4°C for no longer than 3 h before use in
aggregation assays or flow cytometry. Neither the expression levels of
L-selectin on resting PMNs nor the extent of homotypic PMN aggregation
in response to hydrodynamic shear and chemotactic stimulation were
affected by CFSE (data not shown). Flow cytometric analysis using a
platelet-specific mAb, anti-CD61-FITC (25), revealed
that <1% of resting, unlabeled PMNs had surface-bound platelets. Near
background levels of PMN-platelet binding were also detected when
chemotactically stimulated PMNs were subjected to shear (data not
shown).
Cone-and-plate rheometry assays
PMN and colon carcinoma cell suspensions, prelabeled with spectrally distinct fluorophores, were mixed in a microcentrifuge tube at final concentrations of 1 x 106 and 2 x 106 cells/ml, respectively, and allowed to equilibrate at 37°C for 2 min. Thereafter, the heterotypic cell suspension was placed onto the stationary plate of a cone-and-plate rheometer (RS150; Haake, Paramus, NJ), and stimulated with 1 µM fMLP (Sigma-Aldrich) 1 s before the application of shear. Exposure of cell suspensions to a linear velocity gradient resulted in collisions between the faster moving cells near the rotating cone and slower moving cells near the stationary plate (26). Stable aggregate formation occurred when the strength of adhesive bonds formed during collisional contact outweighed the tensile forces experienced by aggregates in the shear field. Shear rates varied from 100 s-1 to 1200 s-1 (typical for the microcirculation) (27) for prescribed periods of time ranging from 10 to 120 s. The 0.50 cone and plate of the rheometer were maintained at 37°C during the entire experiment. Upon termination of shear, aliquots were immediately fixed with 1% formaldehyde at RT, and subsequently analyzed in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) or under a light microscope (Nikon TE300 equipped with x10 and x20 phase objectives; Nikon, Melville, NY).
Cell treatment with mAbs and enzymes
For some inhibition studies, CFSE-labeled PMNs were pretreated for 10 min at 37°C with function-blocking mAbs (20 µg/ml unless otherwise stated), which were kept present during the aggregation assays. For others, the proteolytic enzyme chymotrypsin was used to cleave L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) from the PMN surface (28). Following a 20-min incubation at RT with chymotrypsin (1 U/106 cells; Sigma-Aldrich) (29), stained-PMNs were washed once and resuspended in buffer before use in aggregation assays or flow cytometry. In some experiments, fluorescently labeled PMNs were treated with a metalloproteinase inhibitor, Ro 31-9790 (30 µM), for 20 min at 37°C to prevent L-selectin shedding caused by fMLP stimulation (23). In other experiments, tumor cells (107/ml) were incubated with 0.1 U/ml Vibrio cholerae neuraminidase (Roche Molecular Biochemicals, Indianapolis, IN) for 30 min at 37°C to cleave terminal cell surface sialic acid residues (24). Following enzyme treatment, tumor cells were washed once and analyzed by flow cytometry or used in the aggregation assays. In parallel, control experiments were performed in which PMNs and tumor cells were treated exactly as stated above but in the absence of any function-blocking mAb or enzyme. PMN-colon carcinoma cell adhesive interactions in response to hydrodynamic shear and fMLP stimulation were unaltered by the presence or absence of control mAb (data not shown).
Quantitation of aggregation
The size distribution and cellular composition of aggregates
generated in the rheometric assay were determined by a dual-color flow
cytometric methodology. In brief, CFSE-labeled PMNs and CMTMR-stained
tumor cells were identified on the basis of their characteristic
forward-scatter, side-scatter, and fluorescence profiles in a
FACSCalibur flow cytometer (see Fig. 1
A). The mean fluorescence
intensity of single PMNs (P1) was recorded, and
aggregates were quantified as integral multiples of PMN singlet
fluorescence values (see Fig. 1A). Using this methodology,
PMN doublets (P2) and higher-order homotypic PMN
aggregates (P3+), as well as heterotypic
aggregates comprised of a single tumor cell with one
(P1T), two (P2T), or three
or more (P3+T) adherent PMNs were detected and
enumerated (see Fig. 1A). In accordance with previous
studies (26, 30), aggregates consisting of more than three
PMN singlets were rare events representing less than 10% of the total
PMN population and were grouped into the P3+T and
P3+ categories. The extent of aggregation was
quantified as the fraction of total PMNs in either homotypic or
heterotypic aggregates as previously described (26, 30):
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For direct single-color immunofluorescence assays, PMNs were fixed with 0.25% formaldehyde at 4°C and incubated with fluorophore-conjugated mAbs for 30 min at 4°C (30). Thereafter, specimens were diluted with fixative and analyzed in a FACSCalibur flow cytometer. For indirect immunofluorescence measurements, tumor cells were incubated with the primary Ab for 30 min at 4°C and then washed once with D-PBS/0.1% BSA (19). After an additional 30-min incubation with 15 µg/ml PE-labeled horse anti-mouse IgG or FITC-conjugated goat anti-mouse IgM (Vector Laboratories, Burlingame, CA), the specimens were washed again, fixed with 1% formaldehyde, and analyzed by flow cytometry (19). Isolated PMNs or tumor cells were distinguished from debris on the basis of their characteristic forward- and side-scatter profiles, and the geometric mean PE or FITC fluorescence of each specimen was recorded. Appropriate isotype-matched mAbs, either purified or fluorophore-conjugated, were also included for background fluorescence determination.
Statistics
Data are expressed as the mean ± SEM. Statistical significance of differences between the means was determined by either Student’s t test for comparisons between two groups or one-way ANOVA for multiple comparisons. Post-tests were performed using the Tukey method. Values of p < 0.05 were selected to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that human PMNs interact extensively
with a variety of cancerous cell lines, including human colon
adenocarcinoma LS174T cells, but these heterotypic cell adhesive
interactions were studied exclusively under static conditions (2, 9, 10, 11, 12). To simulate the fluid mechanical environment of the
vasculature, PMN and LS174T cell suspensions, prelabeled with distinct
fluorophores, were subjected to controlled, well-defined levels of
hydrodynamic shear in a cone-and-plate rheometer for various periods of
time. Application of shear in conjunction with fMLP stimulation induced
both PMN homotypic and heterotypic aggregation (Fig. 1
A). A
dual-color flow cytometric technique enabled the detection and
enumeration of single, noninteracting PMNs and LS174T cells, PMN
homotypic aggregates comprised of up to three or more PMN singlets, as
well as heterotypic aggregates composed of a single LS174T cell with up
to three or more adherent PMNs (Fig. 1A). The flow
cytometric detection of aggregates generated in the rheometric assay
was also confirmed by light microscopy (Fig. 1B). Under the
aforementioned experimental conditions, LS174T cells were not
incorporated into homotypic aggregates. Furthermore, exposure of
PMN-LS174T cell suspensions to hydrodynamic shear alone in the absence
of the chemotactic peptide fMLP failed to induce either homotypic or
heterotypic aggregation.
The kinetics of aggregation was measured over a wide range of fluid
flow conditions typically encountered within the circulatory system,
varying from 100 s-1 to 1200
s-1 (27). Fig. 2A shows that the extent of
PMN homotypic aggregation increased with increasing the shear, from a
minimum at a shear rate of 100 s-1 to a maximum
at 800 to 1200 s-1, at which 35% of PMNs
were incorporated into aggregates. In marked contrast, PMN-LS174T
heteroaggregation was maximal at 100 s-1 and
decreased with increasing the shear (see Fig. 2B). At low
shear rates, the extent of PMN homotypic aggregation diminished with
the shear exposure time, presumably due to the recruitment of these
particles into heterotypic aggregates (see Fig. 2). To the contrary,
PMN-LS174T heteroaggregation increased with time (up to 120 s) at
low levels of hydrodynamic shear (see Fig. 2B). However,
both PMN homotypic and heterotypic aggregation peaked at the 30- to
60-s time point under high shear conditions (see Fig. 2). Taken
together, these data indicate that hydrodynamic shear affects the
binding kinetics of PMN and LS174T cell adhesive interactions.
Furthermore, because CD62L (L-selectin) shedding from the PMN surface
occurs rapidly in response to chemotactic stimulation (see
below), these data are suggestive of the potential involvement
of L-selectin in heteroaggregate formation in the high but not low
shear regime.
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Ensuing experiments examined the potential contribution of PMN
L-selectin to PMN-LS174T cell adhesive interactions as a function of
the dynamic shear environment. L-selectin expression on the PMN surface
was modulated by two distinct agents, chymotrypsin and Ro-319790.
Chymotrypsin is a protease that cleaves L-selectin and CD162 (PSGL-1)
from the PMN surface (28), whereas Ro-319790 is a
metalloproteinase inhibitor that effectively blocks L-selectin
shedding induced by chemotactic factor
stimulation (23) (see Table I). Fig. 3A shows that modulation of
L-selectin expression by either agent did not significantly affect
PMN-LS174T heteroaggregation at a shear rate of 100
s-1. In marked contrast, L-selectin removal by
chymotrypsin abolished the formation of heterotypic aggregates at the
high shear regime of 800 s-1 (see Fig. 3B). Conversely, inhibition of the fMLP-induced PMN
L-selectin shedding by the use of Ro-319790 significantly potentiated
the extent of PMN-LS174T heteroaggregation at relatively long (120 s)
shear exposure times (see Fig. 3B).
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A). Similar results were also
obtained for Ro-319790-pretreated PMNs (Ro-treated specimens: 44.0
± 8.0% PMNs in heteroaggregates; Ro-treated plus
anti-L-selectin-treated specimens: 4.2 ± 0.5% PMNs in
heteroaggregates; n = 3; mean ± SEM).
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A). The lack of any inhibitory effect is not related
to the inability of the mAb to block PSGL-1 function, because it
effectively suppressed PMN homotypic aggregation under high shear
(control samples: 36.8 ± 5.7% PMNs in homotypic aggregates;
PSGL-1-treated samples: 16.1 ± 2.8% PMNs in homotypic
aggregates; n = 3; mean ± SEM), a finding which
is in agreement with previous work (31, 32).
Ensuing experiments aimed to characterize the counterreceptor for PMN
L-selectin on the LS174T cell surface. Earlier work has shown that
sialic acid and fucose residues are critical components of the
L-selectin ligand activity (33, 34). To examine whether
sialylated moieties present on LS174T cells mediate binding to PMN
L-selectin at high shear conditions, tumor cells were treated with
neuraminidase, an enzyme that cleaves sialic acid residues from cell
surfaces (see Table II). Fig. 4A shows that this enzyme treatment nearly abrogated
PMN-LS174T heteroaggregation at a shear rate of 800
s-1, suggesting that the tumor cell L-selectin
ligand is sialylated. In contrast, neuraminidase did not alter the
extent of PMN-LS174T heterotypic aggregation at 100
s-1 (see Fig. 4B), a finding which is
in accord with the lack of L-selectin involvement in the low shear
regime. To further validate that sialylated molecules on the colon
carcinoma cell surface are essential for binding PMNs at high levels of
hydrodynamic shear, we chose to look at the
sLex-low cell line HCT-8 (see Table II)
(19). The results indicate that HCT-8 cells failed to
aggregate with PMNs at high shear, despite their extensive adhesive
interactions in the low shear regime of 100 s-1
(see Fig. 5).
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Prior work has shown that PMNs interact with a variety of colon
carcinoma cell lines in a CD18 (2 integrin)-dependent manner under
static conditions (10, 11). Therefore, we explored the
potential involvement of CD18 in PMN-LS174T heteroaggregation under
hydrodynamic shear in conjunction with chemotactic stimulation.
Preincubation of PMNs with an anti-CD18 mAb (see Fig. 4) inhibited
heteroaggregation to essentially baseline levels at all shear rates
examined. We next wished to assess the relative contributions of the
2 integrin receptors, CD11a, CD11b, and CD11c, to this process.
Blocking CD11b function alone essentially abrogated heterotypic
aggregation at both low and high levels of shear (see Fig. 4). However,
use of an anti-CD11a mAb failed to reduce the extent of heterotypic
interactions at a shear rate of 100 s-1 (see
Fig. 4B), a finding which is in agreement with previous work
performed under stationary conditions (10, 11). In
distinct contrast, CD11a blockade alone dramatically inhibited
PMN-LS174T heteroaggregation at 800 s-1 (see
Fig. 4A). Under these conditions, an anti-CD11c mAb did
not alter the extent of PMN binding to LS174T cells (control samples:
29.4 ± 6.5% PMNs in heteroaggregates; CD11c-treated samples:
28.9 ± 1.3% PMNs in heteroaggregates; n = 2;
mean ± range). Similar results were also obtained at the low
shear level of 100 s-1 (data not shown),
suggesting that CD11c does not contribute to PMN-LS174T heterotypic
aggregation. Taken together, these data clearly suggest that
hydrodynamic shear regulates the receptor specificity of PMN-LS174T
colon carcinoma cell adhesive interactions.
Ensuing experiments aimed to identify the CD18 counterreceptor(s) on
LS174T cells. The most obvious candidate capable of binding CD11a and
CD11b is CD54 (ICAM-1) (13, 14). However, using indirect
immunofluorescence and flow cytometry, we were unable to detect
significant ICAM-1 expression levels on the LS174T cell surface (see
Table II). Blocking ICAM-1 function with a
F(ab')2 mAb fragment minimally reduced the extent
of PMN-LS174T heteroaggregation at 800 s-1
(control samples: 29.3 ± 1.5% PMNs in heteroaggregates;
ICAM-1-treated samples: 23.5 ± 1.7% PMNs in heteroaggregates;
n = 3; mean ± SEM). Several lines of evidence
have also shown that CD11a binds to CD102 (ICAM-2) and CD50 (ICAM-3)
(13, 14, 35). However, flow cytometric analysis of LS174T
adhesion receptor expression failed to detect ICAM-2 and ICAM-3 on the
tumor cell surface, and mAb blockade did not alter the extent of
heterotypic aggregate formation at 800 s-1
(control samples: 29.2 ± 1.5% PMNs in heteroaggregates;
ICAM-2-treated samples: 32.8 ± 0.3% PMNs in
heteroaggregates; ICAM-3-treated samples: 35.3 ± 1.2% PMNs in
heteroaggregates; n = 2; mean ± range).
Therefore, the identity of the CD18 ligand(s) on LS174T cells remains
unknown.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMN L-selectin is required for optimal PMN-LS174T heteroaggregation under high, but not low, shear conditions
A variety of colon carcinoma cell lines expressing sialylated and fucosylated glycans on their surfaces have been shown to interact with PMNs via an L-selectin-dependent, CD18-independent fashion under static conditions (12). Consequently, the contribution of L-selectin to PMN-LS174T heteroaggregation was thoroughly examined in our studies using agents that either abrogate L-selectin function (e.g., chymotrypsin or blocking mAbs) or preserve L-selectin surface expression by blocking its cleavage induced by chemotactic factor stimulation. Our data show that none of these interventions affected PMN binding to LS174T cells in the low shear regime, suggesting the lack of L-selectin involvement under these conditions. These results are in accord with other previously published data on PMN-colon carcinoma cell interactions under static conditions (10). In distinct contrast, L-selectin appears to be critical for PMN-LS174T heteroaggregation at high shear, as evidenced by the abrogation of these heterotypic interactions upon PMN treatment with chymotrypsin or a function-blocking anti-L-selectin mAb. Along these lines, blockade of L-selectin shedding in response to fMLP stimulation of PMNs potentiated their binding to LS174T cells at longer exposure times in the high shear regime, an adhesion process that was eliminated by an anti-L-selectin mAb.
Abundant evidence indicates that L-selectin ligands are carbohydrate
structures bearing sialylated and fucosylated moieties (33, 34). Indeed, treatment of LS174T cells with neuraminidase
essentially abolished PMN-LS174T heteroaggregation at 800
s-1, suggesting that sialylated moieties
represent essential components of the ligand structure. This is further
corroborated by observations showing that the
sLex-low HCT-8 colon carcinoma cells fail to form
heterotypic aggregates with PMNs under high shear conditions. The
sensitivity of LS174T L-selectin ligand to neuraminidase may
distinguish them from other proposed L-selectin ligands such as heparan
sulfate glycosaminoglycans and sulfo-Lex
(34). Furthermore, CD34, a sialomucin that bears a major
portion of the L-selectin ligand activity in peripheral node addressin
(36), is not expressed by LS174T cells (see Table II) and
thus is not likely to serve as the LS174T L-selectin
counterreceptor.
CD11b is sufficient to mediate optimal PMN-LS174T heteroaggregation at low shear
Prior work performed under static conditions indicated that PMNs
bind to cytokine-activated HT29 colon carcinoma cells in a
CD11b-dependent but CD11a- and L-selectin-independent manner
(10). Similarly, CD11b has been shown to mediate the
attachment of fMLP-stimulated PMNs to T84 colon adenocarcinoma cells in
the absence of any CD11a or CD11c contribution (11). Our
data on PMN-LS174T cell adhesive interactions induced by fMLP
stimulation at 100 s-1 are in concert with those
previously published results (10, 11). At this low level
of shear, our rheometric assay permits long intercellular contact times
(25 ms) that could only be observed under near static conditions in
a parallel-plate assay.
A recent study suggests that the capacity of CD11a to support adhesion decays with time at a much faster rate than that of CD11b, having a negligible role in PMN-PMN interactions after 5 min of fMLP stimulation (37). In light of these observations, we hypothesized that the lack of CD11a involvement in PMN-tumor cell interactions might be ascribed to the prolonged incubation times of the static adhesion assays (5- to 30-min incubation at 37°C) (10, 11). However, mAb blockade of CD11a function failed to affect the extent of PMN-LS174T heteroaggregation even at the 30-s time point (data not shown), suggesting that CD11a does not participate in this process in the low shear regime.
CD11a and CD11b cooperate to support optimal PMN-LS174T heteroaggregation at high shear
In contrast to the data obtained at low shear, blockade of CD11a
function with a mAb dramatically inhibited PMN binding to LS174T cells
at 800 s-1. The molecular requirement of CD11a
in this process became evident at the shear rate of 400
s-1, at which use of an anti-CD11a mAb
reduced heteroaggregation by 50% (data not shown). However, CD11b
is requisite for PMN-LS174T stable aggregate formation over the entire
of range of shear rates examined in this work. In contrast, CD11c does
not contribute to this process under either low or high shear
conditions.
It is currently known that ICAM-1, ICAM-2 (13, 14), and ICAM-3 (35, 37) can serve as ligands for CD11a, whereas CD11b binds to ICAM-1 (14). However, the absence of ICAM-1, ICAM-2, and ICAM-3 expression on the LS174T cell surface coupled with the lack of any significant inhibitory effects upon use of their respective function-blocking mAbs in the rheometric assays eliminate their potential involvement in these heterotypic adhesive interactions. Further studies are needed to define the CD18 ligand.
Model of PMN-LS174T heteroaggregation
Altogether, our data suggest that CD11b alone is sufficient to
mediate PMN binding to LS174T cells at low shear conditions. However,
PMN L-selectin, CD11a, and CD11b are all requisite for optimal
PMN-LS174T heteroaggregation at high shear. Taking into consideration
that CD18 integrins require considerably more time for binding than
selectins under conditions of flow due to differences on their
respective kon rates, we propose the following
two-step model of PMN-LS174T aggregate formation (see Fig. 6): PMN L-selectin initiates LS174T cell
tethering by binding rapidly and transiently to the sialylated
counterstructure on the carcinoma cell surface in the high shear
regime. This receptor-ligand interaction increases the duration of
intercellular contact, thereby allowing CD11a and CD11b to mediate
stable aggregate formation (see Fig. 6). The involvement of CD11a in
the PMN binding to LS174T cells that is observed only under high shear
conditions made us speculate that CD11a and CD11b act sequentially in
mediating stable heteroaggregation under high shear. In particular, we
hypothesize that CD11a facilitates the transition from L-selectin
tethering to CD11b firm adhesion. A similar scenario has been
postulated for PMN binding to surface-anchored platelets and ICAM-1
transfectants (26, 38).
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In conclusion, this study addresses a very important question that has been recently posed in the literature: are selectins involved in tumor metastasis (41, 42)? Although prior work in this area focused primarily on E-selectin and P-selectin (19, 41, 42, 43), our data provide evidence for the potential involvement of L-selectin in adhesion events pertinent to the process of blood-borne metastasis. However, to demonstrate its role, the interplay of fluid mechanics and cell biology in the field of cancer research had to be considered. In this study, we show that chemotactically stimulated PMNs interact with the metastatic sLex-bearing LS174T cells (16) significantly more than with the nonmetastatic sLex-low HCT-8 cells (16) at high shear, via a mechanism that has an absolute requirement for PMN L-selectin. Several lines of evidence support the concept that progression and poor prognosis of carcinomas, including colon cancer, are associated with enhanced expression of sialylated, fucosylated glycans such as sLex and sLea (41, 44). Although this study does not address the effect of PMNs on blood-borne metastasis, which is currently controversial (1, 21), it elucidates the dynamics and molecular mechanisms mediating PMN-neoplastic emboli formation that could potentially promote the hematogenous dissemination of tumor cells (4, 5, 8, 45, 46). Our data clearly show that the fluid mechanical environment of the circulatory system regulates both the kinetics and receptor-specificity of PMN-tumor cell interactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Konstantinos Konstantopoulos, Department of Chemical Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2694. E-mail address: konst_k{at}jhu.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; sLex, sialyl Lewisx; CMTMR, 5-(and 6-)4-chloromethyl-benzoyl-amino-tetra-methylrhodamine; RT, room temperature; D-PBS, Dulbecco’s PBS; PSGL-1, P-selectin glycoprotein ligand-1.
Received for publication July 6, 2001. Accepted for publication September 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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