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TRANSPLANTATION
From the Center for Blood Research, the Department of
Pathology, and the Division of Clinical Genetics, Children's Hospital,
Harvard Medical School, Boston, MA; and the Howard Hughes Medical
Institute, University of Michigan, Ann Arbor.
Nonirradiated bone marrow (BM) venules and sinusoids in murine
skull support hematopoietic progenitor cell (HPC) rolling through constitutively expressed endothelial (P- and E-) selectins and VCAM-1.
Using intravital microscopy, we tested whether host conditioning with
total body irradiation (TBI) changes the molecular mechanisms by which
murine HPCs from fetal livers (FL) interact with BM endothelial cells.
Although a high dose of TBI did not affect the overall frequency of HPC
rolling in BM microvessels, the underlying molecular mechanisms
differed from those in nonirradiated BM. TBI induced VCAM-1
up-regulation in BM microvessels, whereas P-selectin expression was
reduced and the low baseline level of E-selectin remained unchanged.
Only the administration of anti-VCAM-1, but not anti-P- or
-E-selectin monoclonal antibodies, decreased FL HPC rolling. Rolling
was frequently followed by firm arrest (sticking), even in
nonirradiated BM microvessels in which sticking was entirely pertussis
toxin-insensitive Bone marrow transplantation (BMT) has been
increasingly used to replace the BM of patients with inborn errors of
metabolism, malignancies, and chronic viral infections with
hematopoietic progenitor cells (HPCs) from a healthy donor. BMT is also
used to induce immunologic tolerance to other transplanted
organs.1-3 In nearly all these clinical settings,
recipients of donor HPCs must be preconditioned by total body
irradiation (TBI) before receiving a BM graft.1 In
patients receiving allografts, preconditioning prevents alloresponse
against the graft by mature T and B cells. Animal studies also suggest
that engraftment is more complete in irradiated
recipients,4,5 presumably because of improved access for
transplanted HPCs to supportive niches within the BM.
Outside the BM, irradiation increases leukocyte interaction with
endothelium. This effect of TBI is caused by the induction of
endothelial cell (EC) adhesion molecules, similar to changes seen
during inflammation.6 However, HPC homing may not require the up-regulation of adhesion molecules above their constitutive levels7-9: some studies report a decrease in HPC
accumulation in the BM shortly after irradiation,10,11
even though others found increased expression of VCAM-1 on irradiated
BM ECs.7 Whether TBI affects the expression of other
endothelial surface adhesion molecules (eg, selectins) in BM has not
been determined.
P- and E-selectin are constitutively expressed in normal BM, where they
mediate HPCs rolling together with VCAM-1, which interacts with
Our study examines the effects of TBI on the expression of adhesion
molecules and chemoattractants on BM ECs, and it explores the
consequences of these effects for HPC adhesion in BM microvessels. We
have shown previously that fetal liver (FL) HPCs from 11-day-old embryos roll in normal BM microvessels using the same adhesion pathways
as murine HPC cell lines.8 FL cells, whose functional properties and adhesion molecules are largely similar to those of adult
HPCs,19,20 not only seed the BM during late gestation but
also efficiently repopulate the irradiated BM of adult recipients after
adoptive transfer.21 Thus, FL is a relevant source of HPCs. We show that TBI did not change the overall frequency of FL HPC
rolling in BM, but the molecular mechanisms mediating this process
differed from those in nonirradiated BM. VCAM-1, but not P- and
E-selectin, supported FL HPC rolling in irradiated BM. Indeed,
irradiation caused the up-regulation of VCAM-1, but endothelial selectins were largely lost from the lumen of BM microvessels. However,
unlike rolling, sticking was increased after irradiation because of the
elevated expression of an unidentified G-protein-coupled signal
probably distinct from SDF-1 Antibodies and reagents
For covalent protein coupling to fluorescent microspheres, mAbs to
VCAM-1, P-selectin, E-selectin, and isotype controls were purchased
from PharMingen (San Diego, CA). The following flow cytometry reagents
were from PharMingen: anti-CD16/CD32 Fc block; phycoerythrin
(PE)-conjugated or biotinylated anti-c-Kit; PE-conjugated mAbs to
Ter119, Gr-1, B220, CD11b, Thy1.2, L-selectin, CD44, CD11a, CD11b,
Animals
Cells HPCs were isolated from FLs generated by timed matings of FucT / or wild-type (WT) mice. Females were killed 11 days after observation of a vaginal plug. FL cell suspensions were
prepared by 1-hour incubation at 37°C in 0.05% collagenase type 1 (Worthington Biochemical, Lakewood, NJ) followed by mechanical
dissipation. For intravital microscopy (IVM) experiments, cells were
labeled with 2',7'-bis-(carboxyethyl)-5(and-6) carboxyfluorescein
(BCECF; Molecular Probes, Eugene, OR). After staining for 30 minutes at
37°C (2.5 µg BCECF/107 cells), cells were washed and
resuspended to 5 × 106/mL in RPMI 1640 (BioWhittaker,
Walkersville, MD) containing 10% fetal calf serum (JRH Biosciences,
Lenexa, KS). Cell viability was determined by trypan blue exclusion. In
some experiments, FL HPCs were treated with pertussis toxin (PTX;
CalBiochem, La Jolla, CA). Cells were resuspended to 107/mL
in RPMI 1640 with 10% fetal calf serum (FCS) and 100 ng/mL PTX,
incubated at 37°C for 2 hours, washed, resuspended to
5 × 106/mL, and used for IVM.
Flow cytometry Surface expression of adhesion molecules on day 11 FL cells and adult BM HPCs was assessed by flow cytometry. Freshly harvested cells were resuspended to 0.5 × 106/mL in ice-cold PBS with 1% FCS. Anti-CD16/CD32 Fc block was added, and aliquots were labeled with specific or control mAbs using standard procedures. Stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson).Intravital microscopy Mice were prepared for IVM of skull BM as described.8 HPC behavior was analyzed by determining the rolling fraction (RF), sticking fraction (SF), and sticking efficiency. RF was the number of interacting cells in each vessel per 100 cells passing through the same vessel; SF was the number of cells that arrested for 30 seconds or more per 100 rolling cells; and sticking efficiency was the percentage of firmly adherent (30 seconds or longer) cells in the total flux. For in vivo inhibition, 100 µg/mouse mAbs toVCAM-1, P- or E-selectin, or SDF-1 was injected 10 minutes before
cell injection. Monoclonal antibodies to L-selectin, CD44, LFA-1, and Mac-1 were incubated with FL cells (100 µg/107 cells,
37°C) for 10 minutes before cell injection.
Covalent coupling of monoclonal antibodies to fluorescent microspheres Nile red (NR; excitation/emission, 535 nm/575 nm) and yellow green (YG; 505 nm/515 nm) carboxylate-modified microspheres (1.0 µm diameter; Molecular Probes) were covalently labeled with mAbs; 35 µg protein in 2 mL 50 mM MES buffer (Sigma Chemical, St Louis, MO), pH 6.0, was added to 12.5 × 108 beads and incubated at room temperature for 15 minutes, and 2.4 mg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Molecular Probes) was added. The mixture was incubated on a rocker at pH 6.5 for 4 hours at room temperature. Next, 100 mM glycine (Fisher Scientific, Pittsburgh, PA) was added for 30 minutes at room temperature. The suspension was centrifuged (5000g, 20 minutes), and the pellet was washed 3 times in phosphate-buffered saline (PBS) and was resuspended in 500 µL PBS containing 1% bovine serum albumin (BSA; Sigma). To control for differences in nonspecific bead properties, NR and YG beads were conjugated to specific and control mAbs, and colors were swapped between experiments.IVM studies with fluorescent beads in skull BM Microspheres (5 × 107) of each color were sonicated and resuspended separately in 1 mL 0.01% Tween-20 (Fisher Scientific) in PBS. Nonspecific mAb-coated microspheres were injected intra-arterially, and microsphere accumulation in BM microvessels was videotaped 10 to 15 minutes later using rhodamine and FITC filters for NR and YG beads, respectively. After 15 minutes, when unbound microspheres were cleared from the circulation, specific mAb-conjugated microspheres of the second color were recorded in 8 to 14 fields of view in the same vascular bed. Specific binding was calculated as the number of bound specific mAb-conjugated microspheres divided by the number of bound nonspecific beads.Accumulation of fluorescent beads in BM of long bones NR and YG microspheres (7.5 × 107) coated with specific and control mAbs, respectively, were prepared as described above, mixed, and injected intra-arterially in anesthetized control or irradiated mice. An aliquot of the injection mixture was saved to control for differences in bead input using fluorescence-activated cell sorter (FACS) analysis. After 15 minutes, recipients were exsanguinated by cutting the abdominal vena cava and performing whole-body perfusion with ice-cold saline containing 1 U/mL heparin (Sigma). BM was harvested from femora and tibiae, single-cell suspensions were prepared, erythrocytes were lysed, and BM cells were used for duplicate cytospins. NR and YG microspheres were counted using epifluorescence microscopy. The bead accumulation ratio was calculated by dividing the number of specific mAb-coated beads by the number of control beads in the same field.Chemotaxis assay Chemotaxis assays were performed in 24-well plates with 5-µm pore size inserts (Costar, Cambridge, MA). Supernatant from the murine BM stroma cell line MS-5 with or without anti-SDF-1 mAb (50 µg/mL) or UltraCulture medium (BioWhittaker) with 1 to 500 ng/mL mouse SDF-1 (R&D Systems) was added to the lower chamber. FL cells or BM
mononuclear cells in UltraCulture medium (5 × 106/mL)
were loaded onto the inserts. Some cells were treated with PTX as
described above. Cells migrating to the bottom well were collected
after 3 hours and were counted by FACS after gating on
c-Kit+ FL HPCs or Lin CD34+ BM
HPCs. In some experiments, migrated cells were instead plated in 0.9%
methylcellulose (Sigma) with 15% FCS, 1% BSA, 0.13 mM  -mercaptoethanol (Sigma), 2.5 U/mL erythropoietin (Amgen, Thousand Oaks, CA), 35 ng/mL recombinant murine SCF (PeproTech, Rocky Hill, NJ),
and 35 ng/mL recombinant murine IL-3 (PeproTech). Cultures were
maintained at 37°C in 5% CO2. Colony-forming units were
counted after 8 days.
Statistics For comparison of 2 samples, a 2-tailed Student t test was used. Multiple comparisons were performed by one-way analysis of variance with Bonferroni correction. Significance was set at P < .05.
Effect of TBI on BM microvascular hemodynamics and barrier function We measured the diameters and hemodynamics of BM microvessels in normal and irradiated mice. Mean blood flow velocity, wall shear rates, and shear stresses (Table 1) were significantly reduced after irradiation. The most prominent differences were observed in BM sinusoids and intermediate venules (described in detail in Mazo et al8), which were typically surrounded by a thick extravascular layer of hematopoietic tissue, suggesting that edematous swelling of the irradiated tissue might have compressed these microvessels (data not shown). Subsequent experiments revealed a massive post-TBI breakdown of endothelial barrier function as evidenced by extravasation of FITC-dextran, which diffused rapidly into BM cavities (Figure 1).
Surface expression of adhesion molecules on FL and BM HPC Previous experiments8 show that the minimal number of cells required for one IVM experiment in mouse skull BM is approximately 7 × 106. We obtained a yield of less than approximately 2 × 105 BM CD34+ HPCs (which contain only a small fraction of lin uncommitted
progenitors) from one adult mouse. Thus, it is not feasible to obtain a
sufficient number of adult BM HPCs for routine experiments in our
model. Therefore, we resorted to isolating FL day 11 of gestation as a
source of well-characterized, largely uncommitted
HPCs.19,20 At this stage, FLs contained approximately 50%
mononuclear cells expressing c-Kit (Figure
2), a marker for embryonic
HPCs.23 Up to 20% of c-Kit+ FL cells
coexpressed CD34 (Table 2). Erythroid
Ter119+ cells represented most of the c-Kit
CD34 population (not shown). Because it was difficult to
separate c-Kit+ from c-Kit FL cells without
losing a large fraction of HPCs in the process, we used unseparated
heterogeneous FL cells for in vivo experiments. Previous work has shown
that the Ter119+ fraction is poorly labeled by BCECF and
remains under the visualization threshold of our video camera.
Consequently, approximately 90% of cells detected in vivo under
fluorescence illumination were c-Kit+
HPC.8
Although FL HPCs have potent BM homing abilities,24,25
they may differ from adult BM HPCs, the standard source of HPCs in
clinical settings. Therefore, we compared the expression pattern of
adhesion molecules implicated in HPC trafficking on HPC subsets from
day 11 FL and adult BM (Table 2). The c-Kit+
CD34+ FL subset, containing most HPCs with long-term
repopulating activity,20 was similar to that of BM
HPCs, except the latter expressed lower levels of TBI does not alter the frequency of HPC rolling in BM microvessels but does change the underlying molecular mechanisms Skull BM microvessels constitutively support rolling of murine progenitor cell lines and day 11 FL HPC.8 Irradiation had no significant effect on the frequency of FL cell rolling irrespective of the time interval between irradiation and HPC injection (Table 3). We next studied the molecular mechanisms of FL HPC rolling after TBI. As shown previously, nonirradiated BM microvessels support HPC rolling through P- and E-selectin and VCAM-1. All 3 molecules contribute to the overall rolling frequency and support rolling independently. Thus, day 11 FL HPCs roll at significantly lower frequency in the BM of P- and E-selectin-deficient mice than they do in WT BM, and rolling is further reduced by antibodies to VCAM-1.8 Consistent with these findings, neutralization of P- and E-selectin in WT mice reduced FL HPC rolling by 45.5% ± 18.1% (data not shown). Thus, day 11 FL HPCs interact readily with vascular selectins in normal BM, despite lower levels of PSGL-1 than on adult HPCs.
In contrast, mAbs to P- or E-selectin alone or in combination had no
significant effect in irradiated BM 3 hours after TBI (Figure
3A), even though irradiation induces
selectin expression and leukocyte rolling in other
tissues.6 Because irradiation-induced transcriptional
effects may lead to E-selectin up-regulation after more than 3 hours,26 we also tested the roles of P- and E-selectin at
48 hours after irradiation. No significant effect of selectin inhibition was detected (Figure 3B). Anti-L-selectin treatment of FL
HPCs did not affect rolling either, consistent with our previous
findings8 and the fact that most FL HPCs were
L-selectin
To confirm that TBI results in the loss of selectin-mediated rolling in
the BM, we tested FL HPCs from FucT Thus, endothelial selectins lose their ability to mediate HPC rolling, whereas VCAM-1 continues to initiate and maintain this process. However, rolling was not completely abolished after anti-VCAM-1 treatment, indicating that additional unknown adhesion pathway(s) are involved. Up-regulation of VCAM-1, but not endothelial selectins, in irradiated BM microvessels To explore the unexpected finding that selectins do not contribute to HPC rolling in irradiated BM, we analyzed endothelial surface adhesion molecule expression in live BM by using 2 sets of fluorescent beads, visualized by epifluorescence through rhodamine (NR beads) and FITC (YG beads) filters (Figure 4). One set of beads was coated with mAbs to VCAM-1, E-selectin, or P-selectin, whereas the other was conjugated to control mAb to evaluate background binding. Equivalent numbers of both sets were injected into recipient mice, and bead accumulation in skull BM vessels was recorded by IVM.
Specific beads to all 3 endothelial antigens bound more frequently in
nonirradiated skull BM than beads coated with control mAb (Figure
5A). After TBI, anti-VCAM-1-coated bead
binding increased significantly (P < .05 vs
nonirradiated), whereas anti-P-selectin bead binding was abolished
(P < .01 vs nonirradiated). TBI made no difference in the
low-level specific binding of anti-E-selectin-coated beads. The
concentration of soluble serum P-selectin measured by enzyme-linked
immunosorbent assay (ELISA) was similar before and 48 hours after TBI
(not shown), excluding the possibility that high concentrations of
soluble antigen interfered with anti-P-selectin bead binding. Thus,
TBI causes the up-regulation of VCAM-1, little change in
E-selectin (at least within the sensitivity limits of our bead assay),
and, surprisingly, a dramatic reduction in P-selectin on the luminal
surfaces of ECs in skull BM.
To test whether BM ECs in long bones respond differently to TBI than those in the skull, we performed a modified experiment by injecting equivalent numbers of specific and nonspecific beads into normal and irradiated recipients. Bead accumulation in femoral and tibial BM was determined by fluorescence microscopy in BM cytospins (Figure 5B). As in the skull, anti-VCAM-1-coated beads accumulated more efficiently after TBI in long bones. No difference in accumulation was seen with anti-E-selectin beads. In 4 of 5 experiments, irradiation resulted in decreased anti-P-selectin bead accumulation, but this tendency did not reach statistical significance (P = .09). TBI increases HPC sticking, which is mostly mediated by VCAM-1 Although TBI did not affect rolling, it increased FL HPC sticking (Figure 6A). Administration of anti-VCAM-1 mAb had a similar and significant effect in nonirradiated and irradiated BM microvessels, reducing sticking fractions by 75% and 67%, respectively. Because rolling was also reduced by anti-VCAM-1 (Figure 3A-B), we conclude that most FL HPCs require VCAM-1 for sticking, even if they have alternative means to roll. Indeed, anti-VCAM-1 reduced the sticking efficiency (percentage of sticking HPCs in the total flux) in normal and irradiated BMs by 94.2% ± 3.2% and 89.4% ± 6.0%, respectively. Thus, VCAM-1 has a dual function in BM microvessels, supporting both rolling and firm arrest. Interestingly, there was no significant effect on HPC sticking by blocking2 integrins (CD11a and CD11b) or P- and
E-selectin function (Figure 6B).
TBI induces pertussis toxin-sensitive signals that stimulate FL HPC sticking Our studies indicate that FL HPC sticking is largely mediated by VCAM-1 counter-receptors (most likely41), which must be in a
high-affinity state to mediate firm arrest.12 This
suggests that irradiation causes BM to increase the production of an
integrin activating factor(s), presumably chemokines, which signal
through pertussis toxin (PTX)-sensitive Gi
protein-coupled receptors.31 To test whether FL HPC
sticking in BM depends on this mechanism, we compared HPC sticking
fraction before and after PTX treatment in normal and irradiated BM
(Figure 6C). Forty-eight hours after TBI, PTX treatment significantly
attenuated sticking, though most FL HPCs continued to stick in a
PTX-independent manner. Interestingly, HPC sticking in nonirradiated BM
was equal to sticking of PTX-treated cells in irradiated BM and was not
affected by PTX treatment.
FL HPCs respond poorly to SDF-1 i protein-coupled signal) by BM cells,
leading to increased PTX-sensitive sticking of FL HPCs in BM
microvessels. One possible pathway is SDF-1 and its receptor, CXCR4,
which have been implicated in the homing of human CD34+
HPCs to BM of NOD/SCID mice.16 To determine whether day 11 FL HPCs respond to SDF-1, we performed chemotaxis assays measuring the migration of FL HPCs to either recombinant SDF-1 or supernatant of MS5 BM stroma cells, which are known to secrete
SDF-1.13,32 Surprisingly, FL HPCs migrated poorly to
recombinant SDF-1, whereas adult BM-derived HPCs responded well to
this chemokine (Figure 7A). When plated
on methylcellulose, the few FL HPCs that migrated to SDF-1 failed to
form colonies (Figure 7B). Similarly, though FL HPCs migrated avidly to
the MS5 supernatant, the addition of a blocking anti-SDF-1 mAb did
not reduce this migration. Moreover, when irradiated mice were treated
with anti-SDF-1, the sticking fraction of PTX-treated FL HPCs was
105% ± 10% of untreated FL HPCs (not shown). Thus, it is unlikely
that SDF-1 triggered Gi-independent FL HPC
sticking.
BM stroma cells generate potent G-protein-coupled chemotactic and chemokinetic signals for FL HPC Although FL HPCs responded to a potent chemoattractant in MS5 supernatant distinct from SDF-1, this response was reduced after
PTX treatment, indicating Gi protein dependence (Figure 7C, left panel). In the absence of a chemotactic gradient, with undiluted MS5 supernatant added in both wells (Figure 7C, right panel),
FL HPC migration to the lower well was reduced (by 48%, on average).
Although this tendency did not reach statistical significance, these
results suggest that MS5 supernatant exerts chemotactic and
chemokinetic effects on FL HPCs. PTX treatment abolished the
chemokinetic component of FL HPC migration (not shown).
The data presented here show that a single lethal dose of TBI causes profound changes in BM microvessels and alters the molecular interactions between microvascular ECs and circulating HPCs. To uncover these changes, we used an IVM technique we had used previously to examine HPC rolling in normal BM.8 Here, we modified our approach to approximate the clinical situation during BMT, which requires ablation of the recipient's resident BM. Adhesion and extravasation of transplanted HPCs in BM vessels is a critical first step for the success of BMT. Thus, we characterized HPC interactions with BM endothelium during the first 2 days after TBI. This period was chosen because experimental BMT in mouse models is typically performed during this time interval.4,33 Because adult BM-derived HPCs are not obtainable at sufficient quantities for IVM studies, we used FL HPCs, which have similar marrow repopulating capabilities.34 Injection of day 11 FL cells efficiently rescues lethally irradiated recipients for at least 5 weeks after TBI (I.B.M., unpublished data, March/April 1998). Although one recent study on the competitive repopulation of recipient BM by donor HPCs from FLs and adult BM detected long-term repopulating ability in day 12 but not day 11 FL HPCs,35 others found a long-term repopulating capacity of the c-Kit+ CD34+ subset also in day 11 FLs.20 In our study, approximately 20% of c-Kit+ cells, which represent at least 90% of visualized FL cells,8 coexpressed CD34. Although this approach enabled us to study a relatively pure population
of primary murine HPCs, day 11 FL cells differ somewhat from postnatal
HPCs.36 Our FACS analysis revealed that FL HPCs are nearly
devoid of L-selectin and express less PSGL-1 but more By using this approach in irradiated mice, we made several novel
observations on the effects of TBI. First, we found that TBI altered
the composition of endothelial adhesion molecules mediating rolling;
though VCAM-1 expression was up-regulated, contributions by selectins
were greatly diminished. Second, TBI enhanced HPC arrest by triggering
G TBI also reduced blood flow and microvascular diameters in the BM and caused endothelial barrier breakdown. Several mechanisms might have contributed to these changes. Radiation-induced damage to ECs may lead to the exposure of subendothelial matrix and local platelet and leukocyte deposition, resulting in increased microvascular resistance. This effect may be exacerbated by vasoconstriction because of local cellular responses to radiation-induced and/or vasoactive mediators. In addition, the loss of vascular integrity and the ensuing edema likely increased the interstitial pressure, resulting in microvascular compression. Although these findings agree with microvascular changes in irradiated hamster cremaster muscle,37 studies in other tissues suggest that responses to irradiation vary. For example, irradiation increased rat pial and dermal vessel diameters and blood flow.38,39 Thus, microhemodynamic effects in any particular tissue cannot be extrapolated from observations in other organs. Nevertheless, if we assume that the observed effects in murine skull BM may occur in irradiated BM throughout a patient's body, this might result in significant hypoperfusion. Reduced blood flow to the BM may negatively influence the outcome of BMT because fewer blood-borne HPCs would reach that tissue. Indeed, short-term homing studies indicate that fewer HPCs accumulate in irradiated BM than in normal BM.10,11 Thus, modifications in treatment protocols may improve HPC engraftment and survival by minimizing the effects of TBI on blood flow in a patient's BM. Microvascular responses to irradiation are commonly viewed as a form of inflammation associated with increased selectin-mediated rolling.6 However, P-selectin up-regulation was found in microvessels of irradiated gliomas, but not in surrounding brain vessels.40 Conversely, in irradiated skin, leukocyte rolling was only enhanced in normal microvessels but not in adjacent adenocarcinomas, indicating that irradiation has divergent effects on ECs in different tissues.39 The present findings in irradiated BM microvessels show that HPC rolling remains unchanged. Inflammatory responses may depend on the treatment regimen because the same dose of irradiation increased leukocyte rolling in rat mesentery only when given at a high dose rate.41 Thus, different treatment regimens with TBI might also exert distinct effects on BM microvessels. Because a single high dose of TBI used here induced severe microvascular dysfunction, it is unlikely that the unresponsiveness of rolling was caused by a suboptimal stimulus. Our studies show that TBI profoundly altered the composition of
adhesion molecules on BM ECs. Although it abolished P- and E-selectin-mediated rolling interactions, the contribution of VCAM-1
to HPC rolling was increased. Interestingly, although the anti-VCAM-1
blocking mAb MK2.7 used in this study efficiently blocks Perhaps the most unexpected finding was the loss of selectin activity in irradiated BM. To confirm this, we evaluated the expression of luminal surface molecules on BM ECs. As expected,7-9 we found constitutive expression of P- and E-selectin and VCAM-1 in nonirradiated BM vessels using mAb-coated fluorescent beads. After TBI, specific binding of anti-P-selectin beads was lost, whereas anti-VCAM-1 bead accumulation increased. These findings are consistent with our functional studies on the post-TBI role of P-selectin and VCAM-1 and with previous reports that irradiation enhances VCAM-1.7 On the other hand, anti-E-selectin bead binding was equal before and after irradiation. Although this observation confirms a report that TBI does not alter E-selectin expression in the BM,44 it contrasts with our IVM studies, which failed to detect a significant contribution by E-selectin to HPC rolling after TBI, even though anti-E-selectin caused a significant reduction in rolling in normal BM.8 This discrepancy between E-selectin detectability and function after TBI remains unexplained. Subsequent bead homing experiments revealed that BM ECs in the skull and in long bones react similarly to TBI. Only VCAM-1 was significantly up-regulated in long bones, whereas E-selectin remained unchanged. Anti-P-selectin beads accumulated less in long bones in 4 of 5 irradiated animals, though this tendency did not reach statistical significance. Taken together, these findings indicate that TBI alters BM microvessels throughout the body, resulting in reduced emphasis on selectins and enhanced contribution by VCAM-1 to HPC trafficking. Because ELISA experiments showed that anti-P-selectin bead binding was not blocked by increased soluble P-selectin in the serum of irradiated mice, P-selectin loss after TBI could have been due to 3 (nonexclusive) reasons: reduction in overall expression, redistribution away from the lumen, or altered accessibility of the mAb epitope from microenvironmental changes (eg, glycosylation). All these mechanisms would be associated with a de facto loss of functional P-selectin in the vascular lumen. This notion is also supported by experiments with FucT These findings necessitate a reconsideration of recent work in P- and
E-selectin TBI increased HPC arrest, which was inhibited by treatment with PTX.
Thus, sticking was mediated by a TBI-induced G-protein-coupled signal
that led to integrin activation. FL HPCs did not use The mechanism of TBI-induced |
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Abstract
Introduction
that is, G
