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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 478-486
HEMATOPOIESIS
Relationship between selectin-mediated rolling of hematopoietic
stem and progenitor cells and progression in hematopoietic development
Adam W. Greenberg,
William G. Kerr, and
Daniel A. Hammer
From the Department of Bioengineering and the Department of
Molecular and Cellular Engineering, Institute for Human Gene Therapy,
and the Department of Chemical Engineering, Institute for Medicine and
Engineering, University of Pennsylvania, Philadelphia, PA.
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Abstract |
Current understanding of the adhesion molecules and mechanisms
regulating hematopoietic stem and progenitor cell (HSPC) homing to the
bone marrow is limited. In contrast, the process by which mature
leukocytes are able to home to and extravasate out of blood vessels at
sites of inflammation has been well characterized and invites
comparison. We studied the interaction of human HSPC from adult bone
marrow (ABM) and fetal liver (FL) with E-, P-, and L-selectin
immobilized in a flow chamber. CD34+ HSPC from both ABM
and FL rolled avidly on E-, P-, and L-selectin across a range of
physiologic shear rates, indicating the presence of ligands for all
three selectins on HSPC. Results indicate that CD34+ ABM
and FL cells roll more efficiently (to a greater extent and more
slowly) than more differentiated CD34 cells, especially
on P- and L-selectin. In a similar fashion, increased rolling
efficiency was seen with CD34+CD38 ABM
cells when compared with committed progenitor cells of the CD34+CD38+ phenotype. Rolling of
CD34+ ABM cells on P-selectin could be partially
inhibited by monoclonal antibody (mAb) against PSGL-1, and was not
inhibited by a mAb against CD34, suggesting that HSPC have unique
carbohydrate repertoires that facilitate selectin-mediated rolling. Our
results provide direct evidence of selectin ligands on HSPC under
physiologic flow conditions and are the first to show a correlation
between the maturity of HSPC during development and rolling efficiency on selectins, suggesting a mechanism by which HSPC subsets may differentially home to the extravascular spaces of the bone marrow.
(Blood. 2000;95:478-486)
© 2000 by The American Society of Hematology.
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Introduction |
The objective in bone marrow (BM) transplantation is to
seed a damaged or destroyed host BM with pluripotent hematopoietic stem
and progenitor cells (HSPC) to reestablish the host immune response and
erythroid and myeloid cell lineages. The mechanism of HSPC
transplantation, where donated HSPC are injected into the blood stream
and "home" to the BM, suggests HSPC readily traffic into tissues.
Further evidence exists from other physiologic contexts that HSPC
traffic avidly.1 During embryonic development, HSPC migrate
from the yolk sac to the fetal liver and spleen and then to the BM.
After birth and under normal physiologic conditions, HSPC can
intravasate into the blood circulation from the BM. This process can be
expanded by stem cell mobilization in which cytokines such as
granulocyte colony-stimulating factor (G-CSF) increase intravasation
from the BM.
Our current understanding about the adhesion molecules and mechanisms
regulating HSPC trafficking into and out of the BM is limited. This is
in stark contrast to the detailed characterization of leukocyte
trafficking in the inflammatory response, in which mature leukocytes
are able to home to and extravasate out of blood vessels at sites of
inflammation.2 Similarities in the trafficking ability of
leukocytes and HSPC to home to particular tissue sites in vivo suggest
that we examine whether HSPC trafficking shares common molecular
mechanisms with leukocyte trafficking. The selectin family of adhesion
molecules includes E-selectin (CD62E, endothelial selectin), P-selectin
(CD62P, platelet selectin), and L-selectin (CD62L, leukocyte selectin).
Selectins are involved in the initial steps of the inflammation process
by mediating the rolling of leukocytes on vascular endothelium that has
been activated as a result of inflammatory response. Subsequent steps
involve the up-regulation of integrins on the leukocyte surface by
chemoattractants that bind to cell surface receptors, integrin binding
to endothelial ligands, causing firm arrest of the rolling leukocyte,
and leukocyte migration through the blood vessel wall to enter the
extravascular tissue. It has not been established whether a similar
process occurs for HSPC that home to and extravasate out of BM microvessels.
Very few studies have looked at the interaction of HSPC with selectins
under physiologic flow conditions. CD34 isolated from KG1a cells, a
CD34+ human hematopoietic progenitor cell line, was shown
to support lymphocyte rolling and tethering through interactions with
L-selectin on the lymphocyte surface.3 Because of
anticipated differences between CD34+ BM cells and KG1a
cells, especially in their carbohydrate repertoires, it is not clear
whether these results fully translate to CD34+ BM cells.
Another study found that rolling of peripheral blood CD34+
cells on primary and transformed human BM endothelial cells was E-selectin dependent.4
Recently, in vivo rolling of HSPC in murine BM has been observed using
intravital microscopy.5 Rolling was found not to involve
L-selectin, but was reduced in wild-type mice treated with antibodies
to E- and P- selectin and in knockout mice deficient in these
selectins. In vivo experiments also provide evidence of the role of
selectins in regulating the circulation and homing of HSPC. Knockout
mice deficient in E- and P-selectin were found to have abnormalities in
hematopoiesis6 and defective homing of HSPC to the
BM.7
HSPC are heterogeneous and it is possible that distinct subpopulations
of HSPC may have different homing activity. The presence or lack of
various cell surface markers and combinations of these markers on HSPC
have been shown to be associated with increased stem cell activity, as
measured by long-term multilineage hematopoietic engraftment
ability.8,9 CD34 is a surface antigen present on 1% to 3%
of human BM cells that serves as a marker for the identification and
separation of HSPC, because it is not found on fully differentiated, or
mature, hematopoietic cells.10,11 The cell population
expressing CD34 is heterogeneous, with CD34 antigen density highest on
early progenitors and its density progressively decreasing to
undetectable levels as cells mature.12 CD38 is a surface
glycoprotein that is absent from the most primitive adult and fetal BM
CD34+ HSPC but is expressed by their
differentiation-committed immediate progeny.13,14 The
CD34+CD38 population comprises
approximately 10% to 20% of the total CD34+ population
and is highly enriched for multiprogenitor and stem cell activity,
including engraftment ability.8,9,14
We studied the interaction of human HSPC from adult bone marrow (ABM)
and fetal liver (FL) with E-, P-, and L-selectin surfaces in a flow
chamber to gain insight into the mechanisms behind stem cell
trafficking. CD34+ HSPC from both ABM and FL rolled on E-,
P-, and L-selectin when subjected to physiologic shear rates,
indicating the presence of ligands for all the selectins on HSPC. Our
results indicate that CD34+ and
CD34+CD38ABM and FL cells roll more
efficiently (to a greater extent and more slowly) than the
corresponding more differentiated CD34 and
CD34+CD38+ cells. These results suggest the
existence and differential expression of selectin ligands on HSPC under
physiologic flow conditions and suggest a mechanism for the homing of
HSPC into the extravascular spaces of the BM, including the improved
ability of more primitive HSPC to engraft in the BM after transplantation.
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Materials and methods |
E-, P-, and L-selectin substrates
The E-, P-, and L-selectin-IgG chimeras used in this study were a
gift from Ray Camphausen (Genetics Institute, Cambridge, MA). The
chimeras consisted of the lectin, epidermal growth factor, and multiple
short consensus repeat domains for human E-, P-, or L- selectin linked
to the Fc region of human IgG1. Controls or selectin
chimeras were incubated on silanated glass microscope slides (Sigma,
St. Louis, MO) in modified flexiPERM wells (Sigma) at 2.0 µg/mL in
phosphate-buffered saline (PBS) overnight at 4°C. Slides were
washed with PBS, then incubated with blocking buffer for 1 hour at
22°C to block nonspecific adhesion. Blocking buffer is PBS
containing 2% bovine serum albumin (BSA) (Sigma) that is heated at
56°C for 30 minutes before blocking to denature the BSA. Control
slides were made by incubating only with blocking buffer or by
incubating with human IgG1 (Calbiochem, San Diego, CA).
Antibodies and reagents
All antibodies used were murine monoclonal antibodies (mAbs)
directed against the appropriate human molecule. Chimera-coated slides
were incubated for 1 hour in 20 µg/mL anti-E-selectin (68-5H11, IgG1, Pharmingen, San Diego, CA), anti-P-selectin (G1,
IgG1, Ancell, Bayport, MN), or anti-L-selectin (DREG-56,
IgG1, Caltag, Burlingame, CA) in PBS and then inserted into
the flow chamber. Cell suspensions at 1 × 106/mL
were incubated for 30 minutes at 4°C with 10 µg/mL anti-PSGL-1 (PL1, IgG1, Ancell) or 2 µg/mL anti-CD34 (QBEND/10,
IgG1, Biosource International, Camarillo, CA) in RPMI 1640 medium.
Isolation of cell populations
A single cell suspension of FL tissue (18 weeks gestation) was
minced, pipetted in complete RPMI medium several times, and passed
through a cell strainer. The cells were pelleted (5 minutes, 1100 rpm)
and the red blood cells lysed with NH4Cl lysis buffer. After lysis, the cell suspension was diluted 5-fold with complete RPMI
and pelleted. This cell pellet was resuspended in complete RPMI in
preparation for isolation of CD34+ cells as described
later. Post-Ficoll ABM mononuclear cells were obtained from Poietic
Technologies (Gaithersburg, MD). The mononuclear cells from ABM or FL
were then enriched for CD34 by immunomagnetic positive selection (CD34
progenitor cell isolation kit; Miltenyi Biotech, Inc, Auburn, CA).
Briefly, CD34+ HSPC were indirectly magnetically labeled
using a monoclonal hapten-conjugated CD34 antibody (QBEND/10, IgG1, 0.5 µg/106 cells) and colloidal superparamagnetic MACS
microbeads conjugated to an anti-hapten antibody. The magnetically
labeled cells were then enriched on positive selection columns in a
magnetic field. CD34 content was assessed by FACS and purity was
routinely > 90%.15 For experiments involving
CD34+CD38+/ fractions, the CD34+
fraction from the column was stained with murine mAbs against human
CD34 (581, FITC, Pharmingen, San Diego, CA) and CD38 (HIT2, R-PE,
Caltag, Burlingame, CA) for FACS sorting and analysis. Cells collected
from FACS sorting were always strongly positive for CD34+.
The percentage of CD34+ cells that were sorted as
CD38 ranged from 11% to 21% of the live cell gate.
The proportion of the live cell gate that was sorted as
CD38+ varied from 39% to 50%. The purity of FACS purified
cells was routinely higher than 95%.
Flow chamber
All experiments were conducted in a parallel plate flow chamber with
a tapered channel design that allows for a linear variation of shear
stress down the length of the flow channel at a single flow rate and
channel height. This design is ideal for these experiments as it allows
us to measure adhesion at many different shear stresses in a single
experiment, thus minimizing the necessary supply of ABM or FL cells,
which are expensive. The design is based on Hele-Shaw flow theory
between parallel plates and has been previously
described.16,17 The plates were separated by 250 µm
Duralastic sheeting (Allied Biomedical, Paso Robles, CA), which
compressed to 180 µm when the flow chamber was fully assembled and
tightened. During experiments, the chamber was secured on the stage of
a Nikon Diaphot inverted phase contrast microscope (Melville, NY)
connected to a monochrome CCD video camera (Cohu, Inc., San Diego, CA)
and an S-VHS videocassette recorder (Model SVO-9500MD; Sony
Electronics, Park Ridge, NJ). Buffer and cell suspensions were drawn
through the chamber by an infusion/withdrawal syringe pump (Harvard
Apparatus, South Natick, MA).
Adhesion experiments
E-, P-, or L-selectin coated slides were placed in the well of the
flow chamber that was assembled in PBS to prevent air bubbles and then
secured on the microscope stage. The perfusion buffer or cell
suspension flow rate was set to 0.004 21 mL/s (0.253 mL/min), which
for a channel height of 180 µm, gives an observable range in wall
shear rates from 77 s1 to 487 s1
down the length of the channel. The chamber was perfused with RPMI 1640 medium (Gibco BRL, Life Technologies, Rockville, MD) supplemented with
10 mmol/L HEPES for 15 minutes; then the cell suspension
(1 × 105/mL) was introduced. Data were collected by
stepping down the chamber from inlet to outlet in 5.0-mm steps,
allowing at least 1 minute between steps. Cell interaction with the
surface was recorded for future analysis at a total magnification of
300× (using a 10× objective). All experiments were
performed at room temperature (22°C). Perfusion buffer with 5 mmol/L EDTA (Sigma) or 10 µg/mL fucoidan (Sigma) was used for
inhibition experiments with these reagents. Fucoidan is a sialylated,
fucosylated plant polysaccharide that has been shown to block the
carbohydrate binding domains of L- and P- selectin, but not E-selectin,
in a specific, saturable fashion.18
Data analysis
To determine the rolling flux (cells rolling/min/mm2),
the number of rolling interactions in each field of view, consisting of
a 0.438 mm2 area, were manually counted for 1 minute at
each shear stress. Cells were counted if they rolled for > 10 cell
diameters while remaining in the field of view. Firmly attached cells
were not included in rolling calculations. Cells were considered firmly attached if they remained stationary for > 10 seconds. Velocity measurements were obtained from recorded data using National
Instruments' image acquisition (IMAQ) PCI-1408 frame grabber board
(Austin, TX), IMAQ software, and LabVIEW 4.0. Virtual instruments (VIs) used in LabVIEW were developed to determine rolling velocities. Briefly, VIs automatically advanced the VCR a specified number of
frames, grabbed a designated number of frames spaced equally apart,
converted each captured frame into a binary image according to user
supplied criteria, detected cells on each image according to user
inputted criteria, and recorded these cell positions as coordinates on
a 2-dimensional array. Another VI was then used to plot cell
trajectories by using the coordinate arrays from the images and
representing each detected cell from each grabbed frame as a point on a
background plot. Trajectories were then manually selected for and
instantaneous and average velocities in both the x and y directions,
along with the standard deviation of the average velocities, were
automatically calculated and sent to a tab delimited text file that
could be imported into spreadsheet and graphing programs. Instantaneous
velocity was calculated by dividing the displacement of a rolling cell
by the time between incremental captured frames. Average velocity was
calculated by averaging the instantaneous velocities for a given
trajectory. The time between incremental captured frames was set to 3 seconds, or 90 frames, for E- and P-selectin and 0.5 seconds, or 15 frames, for L-selectin. The total time for each analyzed trajectory was 30 seconds, or 10 iterations, for E- and P-selectin, and 10 seconds, or
20 iterations, for L-selectin. Rolling concentration, an index of
overall rolling efficiency, is calculated as rolling flux/mean velocity, giving units of concentration (cells per microliter). For
shear rates in which there were < 2 rolling cells, rolling concentration was set equal to zero because mean rolling velocity cannot be determined.
Free stream velocity calculations
Free stream velocities were calculated using the theory of Goldman,
Cox, and Brenner,19,20 for a 10-µm-diameter sphere at
particle to surface separation of 50 nm and range from 195 µm/s at a
shear rate of 77 s1 to 1232 µm/s at a shear rate
of 487 s1. At this particle to surface separation,
it is assumed that a cell would be able to interact
with the selectin surface.
Free stream velocity gives an estimate of the velocity of a cell very close to, but not rolling on, the selectin surface.
Statistics
Experiments were performed in triplicate when possible. Comparisons
of mean values over specified ranges of shear rates were examined with
2-tailed paired Student t tests, with significance taken at
P < .05. Data are presented as mean ± SEM, except where indicated.
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Results |
In order to characterize the interaction of human HSPC with
selectins, we immobilized E-, P-, or L-selectin chimeras on the surface
of a parallel plate flow chamber. Selectin chimeras consisting of the
extracellular domains of the selectins fused to the Fc domain of human
IgG1 were adsorbed to silanated glass microscope slides.
Interactions of HSPC purified from ABM and FL with the selectin
surfaces were observed at wall shear rates ranging from 77 s1 to 487 s1 that are similar to
the wall shear rates ranging from 23.7 s1 to 167.9 s1 that have been observed in murine BM
microvessels.5 Rolling velocity, rolling flux, and rolling
concentration at each shear rate observed were determined and used to
compare populations of cells. Rolling flux (rolling cells
counted/min/mm2) gives an indication of the relative number
of cells rolling, whereas rolling concentration (=rolling flux/rolling
velocity) is a measure of the overall rolling efficiency of cells
(cells that roll to a greater extent and slower than other cells roll more efficiently).
We used the CD34 and CD38 cell surface markers to define and isolate
subpopulations of ABM and FL cells. Immunomagnetic separation and FACS
sorting and analysis were used. CD34+ ABM and FL cells were
compared with more differentiated CD34 cells.
Comparisons were also made between
CD34+CD38 ABM cells and committed
progenitor cells represented by CD34+CD38+
cells.
CD34+ ABM and FL cells rolled on surfaces to which E-, P-,
or L-selectin chimeras had been immobilized, under physiologic flow conditions (Figures 1 to 5). Control
substrates composed of adsorbed human IgG1 or adsorbed BSA
alone did not support any cell rolling or firm adhesion (data not
shown). Monoclonal antibodies specific for E-, P-, and L-selectin
greatly reduced rolling interactions of CD34+ FL on
corresponding E-, P-, and L-selectin surfaces (Figure 1 and data not
shown). CD34+ ABM cell rolling on P- and
L-selectin was calcium dependent as shown by inhibition with EDTA
(Figure 2). Rolling on P- and L-selectin was blocked by fucoidan (Figure 2). Also, previously bound and rolling
cells were released immediately on infusion of 5 mmol/L EDTA or 10 µg/mL fucoidan. These observations indicate that CD34+
ABM and FL cells express functional E-, P-, and L-selectin ligands and
that their interactions with the selectins are specific.
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| Fig 1.
Rolling flux of CD34+ and
CD34 FL cells on P- and L-selectin at a shear rate of
210 s1.
Cells were subjected to wall shear rates from 487 s1
to 77 s1 on P- or L-selectin chimeras adsorbed at 2 µg/mL on silanated glass. Rolling flux, rolling velocity, and rolling
concentration was determined at 10 shear rates on P- and L-selectin,
but only the rolling flux at a representative shear rate of 210 s1 is shown. In control experiments, chimera-coated
slides were incubated with mAb. (A) Rolling flux on P-selectin. Control
experiment used the anti-P selectin (CD62P) mAb, G1. (B) Rolling flux
on L-selectin. Control experiment used the anti-L selectin (CD62L) mAb,
DREG-56. Data presented are from single experiments.
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| Fig 2.
Rolling flux of CD34+ ABM cells on P- and
L-selectin at a shear rate of 210 s1.
Rolling flux, rolling velocity, and rolling concentration were
determined at 10 shear rates for P- and L-selectin, but only the
rolling flux at a representative shear rate of 210 s1 is shown. In control experiments, cells were
perfused with 5 mmol/L EDTA or 10 µg/mL fucoidan. (A) Rolling flux on
P-selectin chimera. (B) Rolling flux on L-selectin chimera. Data for
control experiments represents single experiments and is compared with
the mean rolling flux for 3 independent experiments ± SEM.
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CD34+ ABM cells rolled on E-, P-, and L-selectin at wall
shear rates ranging from 77 s1 to 487 s1 (Figures
3-5).
Free stream velocities for cells very close to, but not interacting
with, the selectin surfaces were calculated to range from 195 µm/s at
a shear rate of 77 s1 to 1232 µm/s at a shear rate
of 487 s1 (see "Materials and Methods"). Thus,
for all the shear rates observed, velocities of cells rolling along the
selectin surfaces (Figures 3B, 4B, and 5B) are much slower than free
stream velocities. Both CD34+ and CD34
cells rolled much faster on L-selectin than on E- or P-selectin. Rolling velocities were 1- to 2-fold higher on P-selectin than on
E-selectin. Rolling velocities of CD34+ ABM cells ranged
from 0.7 to 1.0 µm/s on E-selectin, 0.8-1.9 µm/s on P-selectin, and
24-118 µm/s on L-selectin. Increases in shear rate resulted in
increases in rolling velocities. However, a 6-fold increase in shear
rate produced only a 1.4-fold increase in rolling velocities on
E-selectin, a 2.4-fold increase on P-selectin, and a 4.8-fold increase
on L-selectin. Rolling flux (Figures 3A, 4A, and 5A) and rolling
concentration (Figures 3C, 4C, and 5C) generally decreased with
increases in shear rate on E-, P-, and L-selectin. An apparent shear
threshold effect21,22 was observed on L-selectin in which
rolling flux and concentration increased with increases in shear rate
up to 150 s1 and then decreased (Figure 5A, C) and
also on P-selectin in which rolling flux and concentration peaked at
approximately 200 s1 (Figure 4A and C).
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| Fig 3.
CD34+ and CD34 ABM cells
rolling on E-selectin.
Cells were subjected to wall shear rates from 487 s1
to 77 s1 on E-selectin chimera adsorbed at 2 µg/mL
on silanated glass. (A) Rolling flux. Rolling flux was determined at 10 shear rates for each experiment. (B) Rolling velocity. Rolling
velocities were determined for every rolling cell at each shear rate
for which there were 2 or more rolling cells and then averaged at each
shear rate for each experiment. At higher shear rates for which there
were < 2 rolling cells, mean rolling velocity is not presented. (C)
Rolling concentration. Rolling concentration ( = rolling flux/mean
rolling velocity) was determined at 10 shear rates for each experiment.
Each data point represents results from 2 independent experiments. Data
are presented as mean ± SEM.
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| Fig 4.
CD34+ and CD34 ABM cells
rolling on P-selectin.
Cells were subjected to wall shear rates from 487 s1
to 77 s1 on P-selectin chimera adsorbed at 2 µg/mL
on silanated glass. (A) Rolling flux. (B) Rolling velocity. (C) Rolling
concentration. Each data point represents results from 3 independent
experiments. Data are presented as mean ± SEM.
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| Fig 5.
CD34+ and CD34 ABM cells
rolling on L-selectin.
Cells were subjected to wall shear rates from 487 s1
to 77 s1 on L-selectin chimera adsorbed at 2 µg/mL
on silanated glass. (A) Rolling flux. (B) Rolling velocity. (C) Rolling
concentration. Each data point represents results from 3 independent
experiments. Data are presented as mean ± SEM.
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Significant differences in rolling properties were observed for
CD34+ cells compared with CD34cells.
CD34 cells are those cells that were not selected by
CD34+ magnetic separation. CD34+ ABM and FL
cells had a higher rolling flux than CD34 cells on
E-, P-, and L-selectin (Figures 1, 3A, 4A, and 5A). For ABM cells, this
effect was the most pronounced on L-selectin, but was significant
across a range of shear rates from 77 s1 to 339 s1 on E-selectin (P = .002), 77 s1 to 390 s1 on P-selectin
(P = .0005), and 77 s1 to 433 s1 on L-selectin (P = .0006). Differences
were greater when comparing CD34+ and
CD34 FL cells. CD34 FL cells
showed greatly reduced E-, P-, and L-selectin ligand activity compared
with CD34+ FL cells with little rolling of
CD34 FL cells at any shear rate observed (Figure 1
and data not shown). CD34+ ABM cells rolled more slowly
than CD34 ABM cells on P- and L-selectin (Figures 4B
and 5B), but not on E-selectin (Figure 3B). The reduction in rolling
velocities on P- and L-selectin was significant across a range of shear
rates from 77 s1 to 410 s1
(P = .001 and P = .003, respectively). Rolling
concentration was much lower for CD34 ABM cells than
CD34+ ABM cells on E-, P-, and L-selectin (Figures 3C, 4C,
and 5C). This effect was most pronounced on L-selectin, but was
significant at shear rates from 77 s1 to 339 s1 on E-selectin (P = .001) and 77 s1 to 390 s1 on P- and
L-selectin (P = .001 and P = .002, respectively).
Few differences between ABM and FL cells were observed on E-, P-, or
L-selectin. Comparison of CD34+ versus
CD34 FL cells generally revealed the same trends in
rolling flux (Figure 1), rolling velocity (data not shown), and rolling
concentration (data not shown) as CD34+ versus
CD34 ABM cells.
Neither the CD34-specific mAb QBEND/10 nor PSGL-1- specific mAb PL1
significantly reduced the rolling flux of CD34+ ABM cells
on P-selectin (Figure 6A). However, PSGL-1
mAb PL1 greatly increased rolling velocities on P-selectin from 77 s1 to 410 s1
(P = .0005, Figure 6B), indicating at least partial blocking of the P-selectin ligands on CD34+ ABM cells with this mAb.
The CD34 mAb QBEND/10 had no effect on rolling velocities on
P-selectin. The addition of either CD34 or PSGL-1 mAbs reduced rolling
flux on L-selectin from 77 s1 to 410 s1 (P = .02 for each, Figure 6C) but had
no effect on rolling velocities (Figure 6D). These results indicate
that PSGL-1 acts as a ligand for P-selectin on the surface of
CD34+ ABM cells as shown by increased rolling velocities.
The epitope recognized by the QBEND/10 mAb on CD34 does not act as a
ligand for P-selectin. Less conclusive results were obtained on
L-selectin, but it appears that both PSGL-1 and CD34 on the surface of
CD34+ ABM cells may both play a modest role in mediating
interactions with L-selectin as shown by the effect of the mAbs on
rolling fluxes.
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| Fig 6.
Antibody blocking experiments for CD34+ ABM
cells on P- and L- selectin.
Cells were preincubated with mAb indicated before being perfused over
P- or L-selectin chimera surface. The CD34 mAb used was
QBEND/10.32,36 The PSGL-1 mAb used was
PL-1.31,32 (A) Rolling flux on P-selectin. (B) Rolling
velocity on P-selectin. (C) Rolling flux on L-selectin. (D) Rolling
velocity on L-selectin. Each data point represents results from a
single experiment.
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The CD34+ ABM population was further fractionated into the
CD38+ and CD38 subpopulations by FACS
sorting and analysis. The rolling efficiency of
CD34+CD38 ABM cells was greater than for
CD34+CD38+ ABM cells. Rolling flux was greater
for CD34+CD38 ABM cells than
CD34+CD38+ ABM cells on E-selectin from 77 s1 to 211 s1 (P = .01,
data not shown), on P-selectin from 77 s1 to 289 s1 (P = .005, Figure
7A), and on L-selectin from 211 s1 to 339 s1 (P = .02,
Figure 8A). This difference was most
evident on P-selectin. CD34+CD38 ABM
cells rolled slower than CD34+CD38+ ABM cells
on P-selectin from 211 s1 to 289 s1 (P = .007, Figure 7B) and on L-selectin
from 211 s1 to 410 s1
(P = .003, Figure 8B). The reduction in rolling velocity was most pronounced on L-selectin. Thus, the fast rolling of
CD34+ ABM cells on L-selectin could be resolved into faster
(CD34+CD38+) and slower
(CD34+CD38) rolling populations (Figure
8B). Rolling concentration was greater for
CD34+CD38 cells than
CD34+CD38+ cells on E-selectin at shear rates
from 77 s1 to 211 s1
(P = .01, data not shown), on P-selectin from 77 s1 to 289 s1 (P = .003,
Figure 7C), and on L-selectin from 77 s1 to 339 s1 (P = .04, Figure 8C). This difference
was the most evident on P-selectin. Interestingly, a maximum in rolling
flux and rolling concentration on L-selectin (Figure 8A, C) was seen
with CD34+CD38 ABM cells, suggesting
that the shear threshold effect on L-selectin21 is
preserved for this subpopulation.
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| Fig 7.
CD34+CD38 ABM and
CD34+CD38+ ABM cells rolling on P-selectin.
(A) Rolling flux. (B) Rolling velocity. (C) Rolling concentration. Each
data point represents results from 3 independent experiments. Data are
presented as mean ± SEM.
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| Fig 8.
CD34+CD38 ABM and
CD34+CD38+ ABM cells rolling on L-selectin.
(A) Rolling flux. (B) Rolling velocity. (C) Rolling concentration. Each
data point represents results from 3 independent experiments. Data are
presented as mean ± SEM.
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Discussion |
During BM and purified HSPC transplantation, as well as in normal
physiologic processes, HSPC must home to the BM. The similarities between HSPC and leukocyte trafficking invite comparison and suggest that an examination of selectin-mediated HSPC adhesion is warranted. In
vitro flow chambers have been an effective tool to elucidate leukocyte
rolling mechanisms23-25 and thus should be useful for elucidating the molecular mechanisms of HSPC rolling. The increased ability of more primitive HSPC populations to roll on selectins may be
an important feature that contributes to their ability to home to the
marrow spaces and subsequently engraft.8,9
Human HSPC isolated from ABM and FL rolled on E-, P-, and L-selectin
under physiologic flow conditions. Results indicated the presence of
ligands on HSPC that supported rolling and are calcium dependent, as
shown by inhibition of rolling with EDTA. Rolling was inhibited by
antibodies to each of the selectins and by fucoidan on P- and
L-selectin. These results give direct evidence of ligands for E-, P-,
and L-selectin on human HSPC.
Our results also show that rolling, characterized by rolling velocity,
rolling flux, and rolling concentration, differs during the progression
from primitive stem/progenitor hematopoietic cells to committed
progenitor cells to lineage committed cells. Our results indicate that
primitive ABM and FL stem/progenitor cells (CD34+ and
CD34+CD38) roll more efficiently than
more differentiated cells (CD34 and
CD34+CD38+, respectively) on E-, P-,
and L-selectin. This is the first study to show that HSPC demonstrate
different capacities for rolling on selectins, based on their
progression in hematopoietic development.
Both in vivo and in vitro studies, including this study, are limited
because of the rarity of the HSPC and the associated difficulty in
isolating a large enough number of cells for multiple experiments. As
such, there remains much to be done in studying the interactions of
HSPC with selectins. Cell lines, such as KG1a, a CD34+
human hematopoietic progenitor cell line, or animal HSPC were not used
because of the difficulty in extrapolating results to nontransformed
human HSPC. Animal or transformed human HSPC may express completely
different or slightly modified ligands and receptors than primary HSPC.
Fine differences in carbohydrate chemistry can greatly affect
interactions between the selectins and their ligands on the cell
surface.17,26 Analysis of primary HSPC isolated from human
ABM and FL cells provides the most direct test of the mechanism of
human HSPC transplantation.
There is considerable evidence for the expression of selectins and
their ligands by BM endothelial cells and HSPC. BM endothelial cells
express E- and P-selectin,4,27 as well as
CD34.4 HSPC express L-selectin,5,28-30
PSGL-1,5,27,31,32 and CD34.10-12 It should also
be noted that L-selectin has been reported to express sialyl
LewisX (sLeX) and to function as a
counter-receptor for E- and P-selectin.33 Because the
endothelium of BM microvessels represents a continuous barrier5 that homing HSPC must cross to nest in the BM
extravascular space, it is certainly plausible that a critical step in
this process is the adhesion of HSPC to E- and P-selectin expressed by
the endothelium of BM microvessels and/or L-selectin expressed by HSPC
adherent to BM microvessels, which could be presented to other HSPC in flow.
The HSPC cell surface molecules responsible for the homing of HSPC to
the BM are currently not known. However, our initial results suggest
roles for PSGL-1 and possibly CD34 as evidenced by antibody blocking
experiments on substrates of P- and L-selectin. The PSGL-1 mAb PL1
greatly increased rolling velocities of CD34+ ABM cells on
P-selectin, indicating that PSGL-1 acts as a ligand for P-selectin on
CD34+ ABM cells. Both the PSGL-1 mAb PL1 and the CD34 mAb
QBEND/10 modestly reduced the rolling flux of CD34+
ABM cells on L-selectin. One recognized difficulty with studying CD34
recognition with antibodies is that CD34 is a heterogeneous ligand that
is differentially glycosylated in different phenotypes (reviewed in
Krause et al34), and study of its activity would be greatly
aided by a more diverse repertoire of antibodies against CD34.
Further experiments are necessary to completely elucidate the selectin
ligands responsible for mediating the rolling on selectin surfaces.
Differential expression of selectin ligands may be a way of determining
which HSPC can home to the BM most effectively. The increased rolling
ability on E-, P-, and L-selectin for CD34+ cells compared
with CD34 cells suggests that differential
expression of selectin ligands on ABM and FL cells exists. These
results, in conjunction with a similar increase in rolling efficiency
on E-, P-, and L-selectin for CD34+CD38
ABM cells compared with CD34+CD38+ ABM cells,
indicate a correlation between selectin ligand expression on the
surface of HSPC and their progression in hematopoietic development.
More immature HSPC roll at a higher concentration on E-, P-, and
L-selectin than more mature HSPC. As mentioned earlier, immature HSPC,
as defined by the presence of CD34 and lack of CD38 cell surface
antigens, are enriched for stem cell activity, as measured by long-term
multilineage hematopoietic engraftment ability, relative to other more
differentiated hematopoietic precursor populations
(CD34+CD38 or
CD34).8,9 This suggests a link between
selectin ligand expression and stem cell activity, including propensity
for engraftment. We propose that the increased rolling efficiency of
more primitive HSPC allows them to be retained in greater numbers by
the endothelium of BM microvessels, allowing more of these cells to
extravasate into the BM stromal matrix. Thus, primitive HSPC are able
to home more effectively than their more differentiated progeny.
These results suggest a role for the selectins in the homing of HSPC to
the BM. We hypothesize that HSPC may home to the BM as a result of
binding to E-, P-, or L-selectin or combinations of these. This process
might mimic the known multistep process for extravasation of mature
leukocytes. In the initial steps, HSPC would roll on E- and P-selectins
expressed by BM microvessel endothelial cells or on L-selectin
expressed by HSPC already adherent to the BM endothelium. Subsequent
steps would involve stronger binding of integrin molecules on HSPC to
their ligands on the BM endothelium resulting in firm adhesion of the
HSPC to the vessel walls. HSPC may then transverse through the
endothelial layer, adhere to the BM extracellular stromal matrix, and
undergo proliferation and maturation in the BM extravascular space.
Our results generally agree well with and may provide advantages over
in vivo studies that show a role for selectins in the rolling of HSPC
in murine BM microvessels5 and also in the homing of HSPC
to the BM of lethally irradiated knockout mice.7 In studies
with knockout mice deficient in E- and P-selectin,5-7 there
may exist compensation mechanisms, such as elevated cytokine levels,6 that can affect both results and conclusions about molecular mechanisms. Similarly, in normal animals, many molecular and
cellular interactions may play a role in homing, making it difficult to
test hypotheses. Such complications do not exist in a reconstituted
system such as ours. Although a previous study utilizing intravital
microscopy in mice found that rolling of an injected cell line in
normal adult BM microvessels did not involve L-selectin,5
we do not rule out a role for L-selectin in the homing of HSPC to the
BM. As mentioned earlier, cell lines may possess different or modified
adhesion molecules than primary cells. Also, interactions involving
L-selectin may play a more important role in BM affected by
chemotherapy or radiation or in developing fetal BM. In vitro
experiments have shown that neutrophils use L-selectin to
accumulate near already adherent neutrophils.21,35,36 We hypothesize that a similar process may occur for HSPC that would
allow accumulation of HSPC at sites of extravasation in BM microvessels.
Our results do not exclude a role for other adhesion molecules or
chemoattractants that may be critical for firm arrest of rolling cells
or subsequent extravasation. Also, because the selectins are expressed
on many tissues, additional mechanisms to ensure specific homing of
HSPC to the BM must be required. Perhaps, specificity is provided by
other adhesion molecules or unique chemoattractants secreted within the
BM microenvironment. Also, the selectins could be inducibly upregulated
or constitutively expressed on BM endothelial cells. Evidence of
constitutive expression of E- and P-selectin on murine BM venules and
sinusoids, in the absence of inflammation, has been
found.5 This same study also found that bone venules, in
immediate vicinity to BM venules and sinusoids, did not support rolling
of HSPC, suggesting that BM microvessels express the selectins selectively as well.
Because the success of BM and HSPC transplantation depends on the
efficient seeding of grafted cells in the recipient's BM, the further
characterization of HSPC trafficking is of great importance. The
evidence that more primitive subpopulations of HSPC that engraft better
in BM during transplantation and that bind preferentially to selectin
substrates invites examination of additional subpopulations of HSPC.
Results have shown that the subfraction of
CD34+CD38 ABM cells expressing c-mpl,
the thrombopoietin receptor, engrafts significantly better in a severe
combined immunodeficient-human bone model than the subfraction lacking
c-mpl expression,15 inviting investigation of whether this
subpopulation is further endowed for preferential homing to the BM.
These preferential homing abilities of the most primitive HSPC suggest
that evolution has developed a natural method for retention of stem
cells in the rich BM environment, a mechanism that can be further
exploited in BM transplantation through additional fractionation of
donor BM before transplantation.
| |
Acknowledgments |
We thank Heather McIntosh and John Ninos for the purification of the
human stem/progenitor cell populations used in this study.
| |
Footnotes |
Drs Kerr and Hammer contributed equally to this article.
Submitted May 19, 1999; accepted September 17, 1999.
Supported by a Whitaker Foundation fellowship to A.W.G. and
National Institute of Health (NIH) grants RO1 DK54767-01 to W.G.K. and HL18208 to D.A.H.
Reprints: Daniel A. Hammer, Department of Chemical
Engineering, 311A Towne Building, 220 S 33rd St, Philadelphia, PA 19104.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction