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-Mediated Chemotaxis of Human Cord Blood CD34+ Progenitor Cells1
Department of Microbiology/Immunology and The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; and The Walther Cancer Institute, Indianapolis, IN 46208
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF-1) at its
position two proline. CXCL12/SDF-1 induces migration of hemopoietic
stem and progenitor cells, and it is thought that CXCL12 plays a
crucial role in homing/mobilization of these cells to/from the bone
marrow. We found that CD26/DPPIV is expressed by a subpopulation of
CD34+ hemopoietic cells isolated from cord blood and that
these cells have DPPIV activity. The involvement of CD26/DPPIV in
CD34+ hemopoietic stem and progenitor cell migration has
not been previously examined. Functional studies show that the
N-terminal-truncated CXCL12/SDF-1 lacks the ability to induce the
migration of CD34+ cord blood cells and acts to inhibit
normal CXCL12/SDF-1-induced migration. Finally, inhibiting the
endogenous CD26/DPPIV activity on CD34+ cells enhances the
migratory response of these cells to CXCL12/SDF-1. This process of
CXCL12/SDF-1 cleavage by CD26/DPPIV on a subpopulation of
CD34+ cells may represent a novel regulatory mechanism in
hemopoietic stem and progenitor cells for the migration, homing, and
mobilization of these cells. Inhibition of the CD26/DPPIV peptidase
activity may therefore represent an innovative approach to increasing
homing and engraftment during cord blood
transplantation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD26/dipeptidylpeptidase IV (DPPIV)3 is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains after a proline or an alanine; under some conditions it is capable of cleaving after other amino acids (9). It was originally described as a T cell activation molecule, but is now regarded as a non-lineage-specific Ag whose expression in hemopoietic cells is regulated by differentiation and activation. For example, CD26/DPPIV is not expressed on the surface of resting B and NK cells, but it is induced upon stimulation (10). It is possible that many chemokines encounter CD26/DPPIV during their life span, since it is also expressed on activated T lymphocytes, endothelial cells, fibroblasts, and epithelial cells, which are all involved in cell migration (11, 12). In addition, CD26/DPPIV is present in a catalytically active soluble form in plasma (13). Several other natural substrates have been identified, including the pancreatic polypeptide family (neuropeptide Y and peptide YY) and the glucagon family (glucagons, glucagon-like peptide-1, and glucagon-like peptide-2) (14).
CD26/DPPIV has the ability to cleave only certain chemokines
(15, 16, 17, 18, 19, 20, 21, 22, 23, 24). This is due to the fact that only some
chemokines contain the essential N-terminal X-Pro or X-Ala motif
preferred by CD26/DPPIV, and even then some of these chemokines resist
cleavage. The in vitro ability of CD26/DPPIV to truncate chemokines,
and thereby affect function, is highly dependent on dosage and
incubation time. This would suggest that not all chemokines that can be
cleaved serve as in vivo substrates. A recent study suggests that
CD26/DPPIV has selectivity toward stromal cell-derived factor 1
(SDF-1)/CXCL12 and macrophage-derived chemokine/CCL22
compared with other chemokines able to be cleaved by CD26/DPPIV
(25). This study examined the steady state kinetics of
truncation between chemokines that had been previously reported to have
altered activity ability after treatment with CD26/DPPIV (CCL3b,
CCL5, CCL11, CCL22, CXCL9, CXCL10, CXCL11, and CXCL12). Truncation of
CXCL12/SDF-1 occurs after the proline at position 2 (Fig. 1
) (25).
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chemoattracts human T lymphocytes. CD26/DPPIV is
reported to reduce the ability of T lymphocyte cell lines with
endogenously expressed and transformed CD26/DPPIV to migrate in
response to CXCL12/SDF-1 and exhibit anti-HIV-1 activity
compared with non-CD26/DPPIV-expressing cells (19).
In addition, N-terminal-truncated CXCL12/SDF-1, lacking
the first two amino acids, was reported to lack chemotactic and
anti-HIV-1 activities. This suggests that CD26/DPPIV down-regulates
CXCL12/SDF-1 activation of the CXCR4 receptor in T lymphocytes by
cleaving the N-terminal dipeptide of CXCL12/SDF-1.
CXCL12/SDF-1 chemoattracts human CD34 + cells
and stem and progenitor cell populations within this phenotype
(26, 27, 28), and it is considered an important component of
the migration, homing, and mobilization of these important cells
(28). However, very little is known about CD26/DPPIV
expression in CD34+ hemopoietic cells. What is
known is that CD26/DPPIV is expressed in peripheral blood samples from
breast cancer patients after mobilization with G-CSF; some mobilized
CD34+ cells expressed CD26/DPPIV and possessed
peptidase activity (29). The expression of CD26/DPPIV in
nonmobilized CD34+ cells or other sources of
CD34+ cells is not known. More importantly, the
functional activity of CD26/DPPIV on CD34+ cells
for CXCL12/SDF-1 and its implications for migration and homing of
hemopoietic stem/progenitor cells has not yet been addressed.
Cord blood is a functional source of transplantable hemopoietic
stem/progenitor cells (30, 31, 32). Herein we present evidence
that CD26/DPPIV is expressed by a subpopulation of
CD34+ hemopoietic cells isolated from cord blood.
In addition, CD26+/CD34+
cord blood cells possess DPPIV peptidase activity. Functional studies
show that the N-terminal-truncated CXCL12/SDF-1 produced by
treatment with DPPIV lacks the ability to induce the migration of
CD34+ cord blood cells, and the
granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM)
progenitor cells within this population and acts to inhibit normal
CXCL12/SDF-1-induced migration. Finally, specifically inhibiting the
endogenous CD26/DPPIV activity on CD34+ cells, by
treatment with diprotin A, enhances the chemotactic response of these
cells to CXCL12/SDF-1, findings that may be of future clinical trial
utility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal human cord blood mononuclear cells were collected with institutional approval and were isolated by density gradient centrifugation over Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ). CD34+ cells were isolated from mononuclear cells using direct CD34+ magnetic bead separation over two sequential columns (Miltenyi Biotec, Auburn, CA). CD34+ cells (97.6 ± 0.76% pure) were then used immediately.
CD26/DPPIV expression in CD34+ hemopoietic stem cells
CD26/DPPIV cell surface expression was measured by multivariant flow cytometry. Isolated CD34+ cord blood cells were stained with fluorochrome-conjugated mAbs to CXCR4, CD26, and CD34 (BD PharMingen, San Diego, CA) in accordance with the manufacturer’s specifications and then counted by flow cytometric analysis. The conjugates used were CD26-FITC, CXCR4-PE, and CD34-PerCP-Cy5.5. The staining protocol was as follows. Cells were first washed in PBS/pennicillin/streptomycin/1% BSA and resuspended in 100 µl of PBS/pennicillin/streptomycin/1% BSA containing the appropriate Abs. Samples were mixed and incubated at 4°C in the dark for 40 min. The cells were then washed twice in PBS/pennicillin/streptomycin/1% BSA and fixed in PBS/1% paraformaldehyde for later flow cytometric analysis. One hundred thousand events were accumulated for each analysis. Six samples were analyzed separately, and then the data were averaged for statistical analysis. Data are presented as the mean ± SEM, and comparisons were made using two-tailed Student’s t test.
CD26/DPPIV activity in CD34+ hemopoietic stem cells
The DPPIV activity of CD26+/CD34+ sorted cord blood cells was measured in 96-well microplates using the chromogenic substrate Gly-Pro-p-nitoanilide (Gly-Pro-pNA; Sigma-Aldrich, St. Louis, MO) (33, 34). Proteolytic activity was determined by measurement of the amount of nitroanilide (pNA) formed in the supernatant at 405 nm. One thousand CD26+/CD34+ cord blood cells per well in the 96-well flat-bottom plate were incubated at 37°C with 4 mM Gly-Pro-pNA in 100 µl of PBS buffer (pH 7.4) containing 10 mg/ml of BSA. Absorbance was measured at 405 nm on a microplate spectrofluorometer (SpectraMax 190; Molecular Devices, Menlo Park, CA) every 2 min, and the picomoles of pNA formed was calculated by comparison with a pNA standard curve. The results were plotted as picomoles of pNA per minute, and the slope was calculated at the linear portion of the curve giving a measure of DPPIV activity expressed as picomoles per minute per 1000 cells. The activity of CD26+/CD34+ cells was compared with that of CD26+/CD3+ peripheral blood T lymphocytes. Tests were run using four separate CD26+/CD34+ cord samples (n = 2–7 for each sample) and two separate CD26+/CD3+ peripheral blood samples (n = 4 for each sample); cell-free blanks, substrate-free blanks, as well as CD26- cells were run in parallel. Data are presented as the mean ± SEM of all tests (total n = 15 for cord blood samples and total n = 8 for peripheral blood samples).
Migration of CD34+ hemopoietic stem cells
Chemotaxis assays were performed using 96-well chemotaxis
chambers (NeuroProbe, Gaithersburg, MD) in accordance with the
manufacturer’s instructions as described previously with minor
variations (35). Briefly, 0, 6.25, 12.5, 25, 50, 100, 200,
and 400 ng/ml CXCL12/SDF-1 were added to 300 µl of RPMI without
phenol medium supplemented with 10% FBS in the lower chamber. Twenty
thousand fluorescence-tagged (4 µg/ml of Calcein/AM; Molecular
Probes, Eugene, OR) isolated CD34+ cord blood
cells in 50 µl of medium were added to the upper side of the membrane
(5.7 mm diameter, 5 µm pore size, polycarbonate membrane).
Total cell migration was obtained by measuring fluorescence (excitation, 485 nm; emission, 530 nm) on a microplate spectrofluorometer and calculating cell number in the lower well by comparison with a cell number standard curve after 2 or 4 h of incubation at 37°C in 5% CO2. The percent migration was calculated by dividing the number of the cells in the lower well by the total cell input multiplied by 100 and subtracting random migration (always <5%) to the lower chamber without chemokine presence. Three samples were analyzed separately in triplicate, and then the data were averaged for statistical analysis. Data ire presented as the mean ± SEM, and comparisons were made using two-tailed Student’s t test.
Inhibition of endogenous CD26/DPPIV activity on
CD34+ cells was accomplished by pretreatment of
cells with 5 mM diprotin A (Peptides International, Louisville, KY) for
15 min at 37°C. Diprotin A was allowed to remain in the chemotaxis
chamber during the assay. Chemotaxis assays were performed with and
without diprotin A in conjunction with a CXCL12/SDF-1 dose response
between 0 and 400 ng/ml. In addition, treated and untreated sorted
CD26-/CD34+ cord blood
cells were examined and compared with the diprotin A-treated and
-untreated total CD34+ cord blood cells.
The N-terminal-truncated CXCL12/SDF-1
(CXCL12/SDF-13–68) was produced by treatment
with DPPIV (Enzyme Systems Products, Livermore, CA) for 18 h at
37°C in PBS, pH 7.4. Mass spectroscopy (matrix-assisted laser
desorption ionization after V8 digestion) was performed on
CXCL12/SDF-1 and CXCL12/SDF-13–68 and the
efficiency of the DPPIV digestion was 100% as determined by
a complete shift from a 1831.889 peak (corresponding to the
KPVSLSYRCPCRFFE fragment of CXCL12/SDF-1) to a 1636.730 peak
(corresponding to the VSLSYRCPCRFFE fragment of
CXCL12/SDF-13–68) after treatment with DPPIV.
The CXCL12/SDF-1 sample had no detectable peak at 1636.730, and the
CXCL12/SDF-13–68 sample had no detectable
peak at 1831.889. Chemotaxis assays using
CXCL12/SDF-13–68 were performed using 100
ng/ml chemokine. Chemotaxis assays examining the inhibitory effect of
CXCL12/SDF-13–68 used 100 ng/ml of
CXCL12/SDF-1 and a pretreatment of 100 ng/ml
CXCL12/SDF-13–68 for either 15 or 45
min. Fifteen minutes of pretreatment represents the minimum setup time
for the chemotaxis assay after addition of
CXCL12/SDF-13–68 to the system. An additional
30-min pretreatment (45 min total) was assayed to establish whether
CXCL12/SDF-13–68 had the ability to
competitively inhibit the function of normal CXCL12/SDF-1.
Myeloid progenitor cell colony assays
Additional chemotaxis assays for purposes of cell collection
following migration were performed using 24-well Transwell chambers
(Corning, Corning, NY) as previously described with minor variations
(36). Two-hundred thousand isolated
CD34+ cells in 200 µl of RPMI supplemented with
10% FBS were placed in the upper chamber above the membrane (6.5 mm
diameter, 5 µm pore size, polycarbonate membrane). Zero, 100, or 200
ng/ml CXCL12/SDF-1 was added to 1 ml of RPMI supplemented with 10%
FBS in the lower chamber. The total number of cells in the lower
chamber after 4 h of incubation at 37°C in 5%
CO2 was obtained by counting using a
hemocytometer.
Input and migrated cells were collected from 24-well chemotaxis assays, and 1000 cells were plated for colony formation by CFU-GM and CFU-GEMM (37). Cells were plated for colony formation in 1% methylcellulose culture medium with 30% FBS, 1 U/ml recombinant human erythropoietin, 100 U/ml recombinant human GM-CSF, 100 U/ml recombinant human IL-3, and 50 ng/ml recombinant human Steel factor (stem cell factor). Cells were scored after 14-day incubation at 5% CO2 and 5% O2. Since CFU-GM and CFU-GEMM, but not erythroid progenitor (BFU-E), are detected when this combination of growth factors is used to stimulate colony formation of cord blood progenitor cells (38), we did not assess BFU-E chemotaxis. The absolute number of cells in the lower chamber that give rise to a specific colony type was calculated by dividing the number of colonies formed in one plate by either 500 or 1000 and multiplying this number by the total number of cells in the lower chamber as determined previously.
The percent migration was then calculated by dividing the number of cells in the lower well that gave rise to a specific colony type by the total cell input of that specific colony type, multiplying by 100, and subtracting random migration (always <1.0%) to the lower chamber without chemokine presence. Three samples were analyzed separately in triplicate, and then the data were averaged for statistical analysis. Data are presented as the mean ± SEM, and comparisons were made using two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD26/DPPIV cell surface expression was measured by multivariant
flow cytometry using fluorochrome-conjugated mAbs to CXCR4, CD26, and
CD34. CD34+ cord blood cells are 8.6 ±
2.1% CD26+ (mean ± SEM; n
= 12; Fig. 2). Simultaneous examination
of CXCR4 expression in these cells reveals that, on the average,
82.8 ± 4.9% of
CD26+/CD34+ cells express
CXCR4 (n = 12; Fig. 3, A–C). Similarly, 81.9 ± 3.1% of
CD26-/CD34+ cells are
CXCR4+ (n = 12; Fig. 3, A–C). In addition, it was noted that
CD26+/CD34+ cells have a
higher level of CXCR4 expression than
CD26-/CD34+ cells, as
measured by changes in mean fluorescence intensity (MFI; MFI =
118.8 ± 16.2 vs MFI = 71.7 ± 5.8, respectively;
n = 12; p = 0.015; Fig. 3D).
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Having shown that a subpopulation of CD34+
cells existed in which CD26 was expressed, we then set out to show that
thispopulation of cells had DPPIV activity. Using the chromogenic
substrate Gly-Pro-pNA, we monitored the production of pNA at 405 nm
produced by DPPIV cleavage. The results of this assay were plotted as
picomoles of pNA produced per minute (Fig. 4A), and the slope was
calculated at the linear portion of the enzymatic curve giving a
measure of DPPIV activity expressed as units per 1000
CD26+/CD34+ cells, where 1
U = 1 pmol pNA/min.
CD26+/CD34+ cord blood
cells have DPPIV activity, and it was determined to be 43.35 U/1000
cells (n = 15; Fig. 4A). This is less than
the activity recorded for
CD26+/CD3+ peripheral blood
T lymphocytes (113.30 U/1000 cells; n = 8; Fig. 4C). The DPPIV activity of the total
CD34+ cord blood cell population (15.45 U/1000
cells; n = 8; Fig. 4B) was less than the
activity of CD26+/CD3+
peripheral blood T lymphocytes and less than the activity of
CD26+/CD34+ cord blood
cells. Neither CD26-/CD34+
cells nor CD26-/CD3+
peripheral blood T lymphocytes exhibited measurable DPPIV activity.
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Chemotaxis assays were performed to test the functional role of
CD26/DPPIV in CD34+ hemopoietic cell migration.
Normal CD34+ cell migration, after incubation at
37°C for 2 h (Fig. 5A)
or 4 h (Fig. 5B), shows a dose response to
CXCL12/SDF-1.
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at
both 2 h (n = 9; p < 0.01; Fig. 5
A) and 4 h (n = 6; p
< 0.01; Fig. 5B). This increase represents a 2-fold
increase in total CD34+ cord blood cell migration
in response to 200 and 400 ng/ml CXCL12/SDF-1 at both 2 h (Fig. 5C) and 4 h (Fig. 5D). When the
concentration of CXCL12/SDF-1 was lowered to 50 ng/ml, the increase
in migration was 5-fold (Fig. 5, C and D). Sorted
CD26-/CD34+ cells had a
greater migratory response than total CD34+ cells
at both 2 h (n = 6; p < 0.01;
Fig. 5A) and 4 h (n = 6;
p < 0.01; Fig. 5B).
CD26-/CD34+ cells were not
significantly different from CD34+ cells treated
with diprotin A (Fig. 5, A and B). Treatment of
CD26-/CD34+ cells with
diprotin A had no effect on their migration compared with untreated
CD26-/CD34+ cells (Fig. 5, A and B).
An increased 4-h migration of cord blood CFU-GM and CFU-GEMM was also
observed after treatment with diprotin A (n = 3;
p < 0.01; Fig. 6). When
comparing treated vs untreated CFU-GM, treatment resulted in 3.2
± 0.4- and 2.4 ± 0.3-fold increases in cell migration in
response to 200 and 400 ng/ml of CXCL12/SDF-1, respectively. When
comparing treated vs untreated CFU-GEMM, treatment with 5 mM diprotin A
resulted in 3.3 ± 0.5- and 2.7 ± 0.4-fold increases in cell
migration in response to 200 and 400 ng/ml CXCL12/SDF-1,
respectively.
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(CXCL12/SDF-13–68) produced by treatment with
DPPIV did no induce the migration of CD34+ cells
after 2 h (n = 9; p < 0.01; Fig. 7A) or 4 h
(n = 9; p < 0.01; Fig. 7B).
In addition, 15-min pretreatment with CXCL12/SDF-1 3–68(3–68) blocked
the migratory response to normal CXCL12/SDF-1 (n =
9, 2 h, p = 0.06, Fig. 7A; 4 h,
p = 0.07, Fig. 7B). A 15-min pretreatment of
CD34+ cord blood cells with 100 ng/ml of
CXCL12/SDF-13–68 followed by addition to a
chemotaxis chamber containing 100 ng/ml of CXCL12/SDF-1 reduced the
percent migration after 2 h from 5.6 ± 1.2 to 2.9 ±
0.9% and after 4 h from 13.3 ± 1.5 to 7.3 ± 0.7%,
representing 48 and 45% losses in migration, respectively.
Pretreatment for 45 min with CXCL12/SDF-13–68
almost completely eliminated the chemotactic response to normal
CXCL12/SDF-1 (n = 9, 2 and 4 h,
p < 0.01, Fig. 7). The 45-min pretreatment of
CD34+ cord blood cells with 100 ng/ml of
CXCL12/SDF-13–68 followed by addition to a
chemotaxis chamber containing 100 ng/ml of CXCL12/SDF-1 reduced the
percent migration after 2 h to 0.4 ± 0.7% and after 4
h to 1.5 ± 0.8%, representing 93 and 89% losses in percent
migration, respectively.
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To examine the progenitor cell composition of
CD26+ and CD26- cells,
CD34+ cells were sorted based on CD26 and CXCR4
expressions. Colony formation assays were then performed on the
resulting populations of CD34+ cells, which were
comprised of CD26+/CXCR4-,
CD26+/CXCR4+,
CD26-/CXCR4+, and
CD26-CXCR4-
subpopulations. Significantly fewer CFU-GM (Fig. 8A) and CFU-GEMM (Fig. 8B) progenitors were observed in the
CD26+ population of cells compared with the
CD26- population regardless of CXCR4 expression
(n = 3, p < 0.01). Specifically,
2.6 ± 3.3, 3.7 ± 2.9, 60.8 ± 6.6, and 59.4 ±
8.6 CFU-GM progenitors/1000 cells were detected in the
CD26+/CXCR4-,
CD26+/CXCR4+,
CD26-/CXCR4+, and
CD26-CXCR4-
subpopulations, respectively (Fig. 8A). Some 2.8 ±
4.0, 1.7 ± 2.7, 43.7 ± 4.8, and 95.6 ± 9.9 CFU-GEMM
progenitors/1000 cells were detected in the
CD26+/CXCR4-,
CD26+/CXCR4+,
CD26-/CXCR4+, and
CD26-CXCR4-
subpopulations, respectively (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/CXCL12 after the
proline at position 2 (25). Removal of the N-terminal
amino acids results in significant changes in the functional activity
of many chemokines (1). Given that CD26/DPPIV reduces the
ability of T lymphocytes to migrate in response to CXCL12/SDF-1
compared with that of non-CD26/DPPIV-expressing cells
(19), the involvement of CD26/DPPIV in regulating the
migration of CD34+ cells induced by
CXCL12/SDF-1 was examined. We first tested the expression of
CD26/DPPIV in CD34+ hemopoietic cells isolated
from cord blood. Multivariant flow cytometry revealed that a low, but
highly significant, subpopulation of CD34+ cells
expresses CD26/DPPIV. It was also discovered that CXCR4, the receptor
for CXCL12/SDF-1, is expressed in both the
CD26+ and CD26-
populations of CD34+ cells. This data define two
populations of cells that exist within cord blood. The majority of
cells that are CD34+ are also
CXCR4+/CD26-. A much
smaller, but significant, population of CD34+
cells is CXCR4+/CD26+. This
suggests that either CD26/DPPIV regulates the activity of a small
portion of CD34+ hemopoietic cells or that, as we
believe, the small CD26+ subpopulation has the
ability to regulate the entire population of
CD34+ hemopoietic cells.
Having shown that a subpopulation of CD34+ cells
existed that expressed CD26, we then tested the DPPIV activity of this
population of cells using the chromogenic substrate Gly-Pro-pNA. Based
on the production of pNA by DPPIV cleavage it was shown that
CD26+/CD34+ cells possess
DPPIV activity equivalent to 43.3 U/1000 cells (1 U = 1 nM
pNA/min). The total CD34+ cell population has an
activity equal to 15.4 U/1000 cells, about one-third that of sorted
CD26+. DPPIV activity in T lymphocytes has
previously been established (39). We tested sorted
peripheral blood CD26+/CD3+
T lymphocytes and showed their DPPIV activity to be equivalent to 113.3
U/1000 cells. As a control we also tested CD26-
cell populations and were unable to detect DPPIV activity in this
assay. These data establish that not only do
CD34+ cord blood cells express the extracellular
peptidase CD26/DPPIV in an active form, but that the activity of
CD26+/CD34+ cells, on a per
cell basis, is a little more than one-third that of
CD26+ T lymphocytes. Lower levels of CD26/DPPIV
activity observed in
CD26+/CD34+ cells suggest
that they may have a decreased negative regulation of CXCL12/SDF-1
on a per cell basis compared with
CD26+/CD3+ T lymphocytes,
but probably have the ability to significantly negatively regulate
CXCL12/SDF-1 by N-terminal truncation.
Since the involvement of CD26/DPPIV in CD34+
hemopoietic cell migration had never been previously examined in
CD34+ cells from any source, chemotaxis assays
were performed at 2 and 4 h points using
CD34+ cord blood cells to test the functional
role of CD26/DPPIV. Treatment of CD34+ cord blood
cells with the CD26/DPPIV inhibitor, diprotin A, enhanced the migratory
response of these cells 2-fold at high concentrations (200 and 400
ng/ml) of CXCR4/SDF-1. This enhancement was roughly equivalent to
the migratory response to SDF-1 observed in
CD26-/CD34+ cells. This
suggests that treatment with the CD26/DPPIV inhibitor blocks the
majority of DPPIV activity expressed in the total
CD34+ population of cord blood cells. Treatment
of CD26-/CD34+ cells with
diprotin A did not affect the migration of these cells, suggesting that
the action of the inhibitor is CD26/DPPIV specific. By colony formation
assay we showed that the immature, highly proliferative subsets of
CFU-GM and CFU-GEMM within this population of
CD34+ cells (38) were also enhanced
in their migratory response to CXCL12/SDF-1 after treatment of cells
with diprotin A. Increases in migration of CFU-GM of 3.2- and 2.4-fold
in response to 200 and 400 ng/ml CXCL12/SDF-1, respectively, were
observed. Increases in migration of CFU-GEMM of 3.3- and 2.7-fold,
respectively, were also observed.
Comparison of CD34+ cell migration induced by the
normal CXCL12/SDF-1 to the truncated form,
CXCL12/SDF-13–68, produced by DPPIV treatment
showed an inability of CXCL12/SDF-13–68 to
induce migration. In addition,
CXCL12/SDF-13–68 acts as an antagonist,
resulting in the reduction of CD34+ cell
migratory response to normal CXCL12/SDF-1 after 15-min pretreatment,
representing the minimal setup time, and resulting in an almost
complete loss of migratory response to normal CXCL12/SDF-1 after
45-min pretreatment. The additional 30-min pretreatment (45 min total)
suggests that the inactive CXCL12/SDF-13–68
still has the ability to bind CXCR4 and competitively inhibit normal
activation by CXCL12/SDF-1. Similar studies using pretreatment of
cells with normal CXCL12/SDF-1 have shown that CXCR4 receptor can be
desensitized, reducing subsequent treatments with CXCL12/SDF-1
(27). These data suggest that the N-terminal-truncated
form of CXCL12/SDF-1 has no chemotactic activity toward
CD34+ cord blood cells, but still has the ability
to bind the CXCR4 receptor and block migration of cells induced by
normal CXCL12/SDF-1.
Importantly, this information shows that even though a small percentage
of CD34+ cells are expressing CD26/DPPIV on their
surface, the migratory response of the total population of
CD34+ cells to CXCL12/SDF-1 can be altered
greatly by inhibition of DPPIV activity. This suggests that CD26/DPPIV
indirectly has the ability to negatively regulate CXCL12/SDF-1
signaling through the CXCR4 receptor in CD34+
hemopoietic cells by cleaving local pools of CXCL12/SDF-1.
Therefore, CD26/DPPIV expressed on the surface of any one cell or
subpopulation of CD34+ hemopoietic
stem/progenitor cells collectively has the ability to self-regulate not
only their own activation/migration by CXCL12/SDF-1, but also the
activation/migration of surrounding CD34+
hemopoietic stem/progenitor cells.
To address the question of what portion of progenitor cells expresses
CD26/DPPIV, colony formation assays were performed on sorted
populations of CD34+ cord blood cells. The
CD26+/CXCR4- and
CD26+/CXCR4+ populations of
cells formed much fewer CFU-GM and CFU-GEMM progenitors compared with
the CD26-/CXCR4+ and
CD26-/CXCR4- populations
of cells. The majority of progenitors were within the
CD26- population of cells, and
CD26+ cells contained very few progenitors
regardless of their CXCR4 expression status. These data support the
concept that a small subpopulation of CD26+ cells
regulates the response of the entire population of hemopoietic
stem/progenitor cells to CXCL12/SDF-1.
CXCL12/SDF-1 is believed to be an important chemokine involved in
the homing of hemopoietic stem cells to the bone marrow (32, 40, 41). CD26/DPPIV cleavage of CXCL12/SDF-1 results in an
N-terminal-truncated CXCL12/SDF-1. This cleaved form of
CXCL12/SDF-1 lacks migratory ability and inhibits the migratory
ability of normal CXCL12/SDF-1. In this way it is possible for
CD26/DPPIV expressed on a small population of cells to inhibit the
migration of all CD34+ hemopoietic cells within a
local pool of cells. This process of CXCL12/SDF-1 cleavage by
CD26/DPPIV may represent a novel regulatory mechanism in hemopoietic
stem cells for the migration, homing, and mobilization of these cells.
Suppressing the inhibitory effects of CD26/DPPIV can be viewed as a
potential mechanism for increasing stem cell migration and therefore a
novel therapeutic approach for increasing the homing and engraftment of
hemopoietic stem and progenitor cells to the bone marrow.
Cord blood has been used as a transplantable source of hemopoietic stem and progenitor cells since our initial reports (30, 31, 32). However, to date its use in the majority of cases has been confined to children. The amount of cord blood collected appears more limiting for use in adults. To counter this problem, many laboratories have evaluated ex vivo stem cell expansion procedures, but the initial results are discouraging (32). Since not all stem cells home to the appropriate marrow niches necessary for engraftment, an alternative means to enhance the efficiency of cord blood transplantation for adults is to enhance the efficiency of stem cell homing (32). In this context, inactivation of CD26/DPPIV activity on CD26+/CD34+ cord blood cells may be one means to accomplish this clinically important functional scenario for more effective stem cell homing.
It has been proposed by others that direct degradation of
CXCL12/SDF-1 by proteolytic enzymes, including neutrophil elastase
and cathepsin G, may play a role in hemopoietic stem cell mobilization
(40). It is also possible that increasing the expression
and/or activity of endogenous CD26/DPPIV or augmenting the level of
CD26/DPPIV activity with exogenous peptidase could be a viewed as an
alternative method of enhancing hemopoietic stem cell mobilization.
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2 Address correspondence and reprint requests to Dr. Hal E. Broxmeyer, The Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202-5254. E-mail address: hbroxmey{at}iupui.edu
3 Abbreviations used in this paper: DPPIV, dipeptidylpeptidase IV; CFU-GEMM, multipotential progenitor cells; CFU-GM, granulocyte-macrophage progenitor cells; MFI, mean fluorescence intensity; pNA, nitroanilide; SDF-1, stromal cell-derived factor 1.
Received for publication July 5, 2002. Accepted for publication October 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chemokine MDC. Science 278:695. and GRO
from tumor cells and leukocytes reveals NH2-terminal heterogeneity. Functional comparison of different natural isoforms. Eur. J. Biochem. 260:421.[Medline]
-stimulated dermal fibroblasts. Eur. J. Biochem. 253:114.[Medline]
. FEBS Lett. 432:73.[Medline]
(SDF-1) and SDF-1 are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage. Proc. Natl. Acad. Sci. USA 95:6331.This article has been cited by other articles:
|
|
 |
|
  S. Struyf, S. Noppen, T. Loos, A. Mortier, M. Gouwy, H. Verbeke, D. Huskens, S. Luangsay, M. Parmentier, K. Geboes, et al. Citrullination of CXCL12 Differentially Reduces CXCR4 and CXCR7 Binding with Loss of Inflammatory and Anti-HIV-1 Activity via CXCR4 J. Immunol., January 1, 2009; 182(1): 666 - 674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Focosi, R. E. Kast, S. Galimberti, and M. Petrini Conditioning response to granulocyte colony-stimulating factor via the dipeptidyl peptidase IV-adenosine deaminase complex J. Leukoc. Biol., August 1, 2008; 84(2): 331 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Wang, W.-J. Cherng, N.-I Yang, C.-M. Hsu, C.-H. Yeh, Y.-J. Lan, J.-S. Wang, and S. Verma Cyclosporine increases ischemia-induced endothelial progenitor cell mobilization through manipulation of the CD26 system Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2008; 294(3): R811 - R818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, J. Yan, P. De, H.-C. Chang, A. Yamauchi, K. W. Christopherson II, N. C. Paranavitana, X. Peng, C. Kim, V. Munugulavadla, et al. Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins J. Immunol., December 15, 2007; 179(12): 8322 - 8331. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Drucker Dipeptidyl Peptidase-4 Inhibition and the Treatment of Type 2 Diabetes: Preclinical biology and mechanisms of action Diabetes Care, June 1, 2007; 30(6): 1335 - 1343. [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, M. Wittner, H. Bouamar, P. Jarrier, W. Vainchenker, and F. Louache Identification of CXCR4 as a New Nitric Oxide-Regulated Gene in Human CD34+ Cells Stem Cells, January 1, 2007; 25(1): 211 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kawai, U. Choi, L. Cardwell, S. S. DeRavin, N. Naumann, N. L. Whiting-Theobald, G. F. Linton, J. Moon, P. M. Murphy, and H. L. Malech WHIM syndrome myelokathexis reproduced in the NOD/SCID mouse xenotransplant model engrafted with healthy human stem cells transduced with C-terminus-truncated CXCR4 Blood, January 1, 2007; 109(1): 78 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. H. Peranteau, M. Endo, O. O. Adibe, A. Merchant, P. W. Zoltick, and A. W. Flake CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation Blood, December 15, 2006; 108(13): 4268 - 4274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. K. Kim, M. De La Luz Sierra, C. K. Williams, A. V. Gulino, and G. Tosato G-CSF down-regulation of CXCR4 expression identified as a mechanism for mobilization of myeloid cells Blood, August 1, 2006; 108(3): 812 - 820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kakinuma and S. T. Hwang Chemokines, chemokine receptors, and cancer metastasis J. Leukoc. Biol., April 1, 2006; 79(4): 639 - 651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Christmas, K. Tolentino, V. Primo, K. Z. Berry, R. C. Murphy, M. Chen, D. M. Lee, and R. J. Soberman Cytochrome P-450 4F18 Is the Leukotriene B4 {omega}-1/{omega}-2 Hydroxylase in Mouse Polymorphonuclear Leukocytes: IDENTIFICATION AS THE FUNCTIONAL ORTHOLOGUE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE CYP4F3A IN THE DOWN-REGULATION OF RESPONSES TO LTB4 J. Biol. Chem., March 17, 2006; 281(11): 7189 - 7196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Foudi, P. Jarrier, Y. Zhang, M. Wittner, J.-F. Geay, Y. Lecluse, T. Nagasawa, W. Vainchenker, and F. Louache Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4-/- chimeric mice Blood, March 15, 2006; 107(6): 2243 - 2251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Narducci, E. Scala, A. Bresin, E. Caprini, M. C. Picchio, D. Remotti, G. Ragone, F. Nasorri, M. Frontani, D. Arcelli, et al. Skin homing of Sezary cells involves SDF-1-CXCR4 signaling and down-regulation of CD26/dipeptidylpeptidase IV Blood, February 1, 2006; 107(3): 1108 - 1115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Segal, R. Shah, A. Afzal, C. M. Perrault, K. Chang, A. Schuler, E. Beem, L. C. Shaw, S. Li Calzi, J. K. Harrison, et al. Nitric Oxide Cytoskeletal-Induced Alterations Reverse the Endothelial Progenitor Cell Migratory Defect Associated With Diabetes Diabetes, January 1, 2006; 55(1): 102 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Lapidot, A. Dar, and O. Kollet How do stem cells find their way home? Blood, September 15, 2005; 106(6): 1901 - 1910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Guo, G. Hangoc, H. Bian, L. M. Pelus, and H. E. Broxmeyer SDF-1/CXCL12 Enhances Survival and Chemotaxis of Murine Embryonic Stem Cells and Production of Primitive and Definitive Hematopoietic Progenitor Cells Stem Cells, September 1, 2005; 23(9): 1324 - 1332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. B. Slayton, D. A. Wainman, X. M. Li, Z. Hu, A. Jotwani, C. R. Cogle, D. Walker, R. C. Fisher, J. R. Wingard, E. W. Scott, et al. Developmental Differences in Megakaryocyte Maturation Are Determined by the Microenvironment Stem Cells, September 1, 2005; 23(9): 1400 - 1408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kucia, R. Reca, K. Miekus, J. Wanzeck, W. Wojakowski, A. Janowska-Wieczorek, J. Ratajczak, and M. Z. Ratajczak Trafficking of Normal Stem Cells and Metastasis of Cancer Stem Cells Involve Similar Mechanisms: Pivotal Role of the SDF-1-CXCR4 Axis Stem Cells, August 1, 2005; 23(7): 879 - 894. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Berahovich, Z. Miao, Y. Wang, B. Premack, M. C. Howard, and T. J. Schall Proteolytic Activation of Alternative CCR1 Ligands in Inflammation J. Immunol., June 1, 2005; 174(11): 7341 - 7351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, A. Foudi, J.-F. Geay, M. Berthebaud, D. Buet, P. Jarrier, A. Jalil, W. Vainchenker, and F. Louache Intracellular Localization and Constitutive Endocytosis of CXCR4 in Human CD34+ Hematopoietic Progenitor Cells Stem Cells, November 1, 2004; 22(6): 1015 - 1029. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. W. Christopherson II, G. Hangoc, C. R. Mantel, and H. E. Broxmeyer Modulation of Hematopoietic Stem Cell Homing and Engraftment by CD26 Science, August 13, 2004; 305(5686): 1000 - 1003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. De La Luz Sierra, F. Yang, M. Narazaki, O. Salvucci, D. Davis, R. Yarchoan, H. H. Zhang, H. Fales, and G. Tosato Differential processing of stromal-derived factor-1{alpha} and stromal-derived factor-1{beta} explains functional diversity Blood, April 1, 2004; 103(7): 2452 - 2459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W. Christopherson II, S. Cooper, and H. E. Broxmeyer Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells Blood, June 15, 2003; 101(12): 4680 - 4686. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Cottler-Fox, T. Lapidot, I. Petit, O. Kollet, J. F. DiPersio, D. Link, and S. Devine Stem Cell Mobilization Hematology, January 1, 2003; 2003(1): 419 - 437. [Abstract] [Full Text] [PDF]
|
|
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) and untreated (
)
CD34+ cord blood cells. Normal CD34+ cell
migration (
) were observed to have
a greater migratory response than total CD34+ cells (
) cord
blood cells. Input and migrated cells underwent colony formation
assays. Normal cell migration (
