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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 453-460
HEMATOPOIESIS
CD13/N-aminopeptidase is involved in the development of dendritic
cells and macrophages from cord blood CD34+ cells
Michelle Rosenzwajg,
Ludovic Tailleux, and
Jean
Claude Gluckman
From the Laboratoire de Biologie et Thérapeutique des
Pathologies Immunitaires, Université Paris 6-Centre National de
la Recherche Scientifique (ESA 7087), and the Laboratoire
d'Immunologie Cellulaire de l'Ecole Pratique des Hautes Etudes,
Hôpital Pitié-Salpêtrière, Paris, France.
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Abstract |
Expression of CD13/N-aminopeptidase may reflect cell activation and
growth. We examined its role regarding cell growth in cultures of cord
blood CD34+ cells with stem cell factor/Flt-3
ligand/granulocyte-macrophage colony-stimulating factor/tumor
necrosis factor- . Indeed, 82% ± 6% of cells from culture
day 5 were CD13hi, 25% ± 8% of which were still
Lin . About 50% of CD13hiLin cells, which comprise
progenitors of dendritic cells (DC), monocytes/macrophages and
granulocytes, and 30% of CD13loLin cells were
CD34+. Sorted CD34+CD13hiLin
cells, cultured further for 7 days with the same
cytokines, expanded 31-fold and
CD34-CD13hiLin cells 7-fold, but
CD34+CD13loLin and
CD34 CD13loLin cells did not grow. Thus,
cell growth correlated with CD13 expression, all the more so that cells
were CD34+. Actinonin, the most potent N-aminopeptidase
inhibitor, was used to engage CD13 on sorted CD13hiLin
cells and on culture day-7 bulk cells. In both cases, this resulted in
reversible cell growth arrest, with 30% to 60% fewer cells in the
G2/S-M phase than in controls. Interestingly, similar effects were
noted with CD13 monoclonal antibody TÜK1, which does not
inhibit N-aminopeptidase activity, but not with
N-aminopeptidase-blocking antibodies WM15 and F23. All cycling cells
appeared susceptible to actinonin, which induced cell apoptosis at the
same time as Bcl-2 was downregulated and caspase-3 activity increased,
but finally percentages and yields of DC and macrophage precursors were
affected more than those of granulocytic cells. Thus, through engagement of N-aminopeptidase enzymatic site but possibly also of an
independent determinant, CD13 plays a role in the growth of
DC/macrophage progenitors and precursors.
(Blood. 2000;95:453-460)
© 2000 by The American Society of Hematology.
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Introduction |
Cord blood CD34+ hematopoietic
progenitor cells (HPC) cultured with granulocyte/macrophage
colony-stimulating factor (GM-CSF), stem cell factor (SCF), Flt-3
ligand (FL), and tumor necrosis factor (TNF)- differentiate into
dendritic cells (DC) and monocytes/macrophages, and also into
granulocytes.1-6 The intermediate stages between the
CD34+ HPC and DC are not yet fully characterized, though it
has been shown that DC obtained in these cultures are heterogeneous and derive either from CD1a+CD14 unipotent
DC/Langerhans cell (LC) precursors or from bipotent CD1aCD14+ macrophage/DC precursors some
of which pass through a CD1a+CD14+
stage.4,6,7-10 We have identified a population of
CD13hiLin cells that appears during the first days
of culture of initially CD34+CD13lo HPC and
comprises either distinct or common progenitors of macrophages and DC
inasmuch as both cell types differentiate from this
population.4,11
CD13, a membrane-bound cell surface glycoprotein, was originally
recognized as a marker for subsets of normal and malignant myeloid
cells, but later found on different other cells.12 It is
identical to aminopeptidase N (APN; EC 3.4.11.2),13 a
Zn-dependent metalloprotease, which cleaves N-terminal neutral amino
acids from proteins. This ectoenzyme can cleave bioactive proteins on the cell surface, including cytokines, to either activate or inactivate them, and it is even involved in down-regulation of signal
peptides.12 Little is known about the mechanisms regulating
CD13 lineage-restricted expression and about its function on cells of
hematopoietic origin, apart that it is considered as a cell activation
marker the expression of which is associated with high capacity to
proliferate. Moreover, CD13 is involved in cell-surface antigen
processing through trimming of HLA class II- or class I-bound peptides
on antigen-presenting cells, and it is highly expressed on the most
potent of these, the DC,14-16 at all stages of their
differentiation/maturation be they of myeloid or lymphoid
origin.4,17-19
Here, we investigated whether CD13/APN expression level on
hematopoietic cells, particularly DC/macrophage progenitors and precursors, correlated with their capacity to expand in culture and, by
using known specific inhibitors of APN or monoclonal antibodies (mAb) to engage CD13, we examined its possible role during the growth
of these cells in cultures from human cord blood CD34+ HPC.
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Materials and methods |
Cells
Cord blood CD34+ HPC were obtained from normal cord
blood (Laboratoire Senders, Hopital Saint Vincent de Paul; Service de
Gynécologie-Obstétrique, Hôpital Saint Antoine,
Paris, France) and collected according to institutional guidelines.
After Ficoll-Paque (Pharmacia, Uppsala, Sweden) centrifugation,
mononuclear cells were enriched in light-density cells by
centrifugation over Percoll (d = 1.070, Pharmacia). CD34+
HPC were purified with CD34 mAb 561-coated M-450 Dynabeads (Dynal, Oslo, Norway), as described,4 yielding 87% ± 13%
viable CD34+ cells (n = 24). The latter cells (1 to
2 × 104/mL) were cultured in 6-well plates (Costar,
Cambridge, MA) at 37°C in humidified 5% CO2, in RPMI
1640, 10% fetal calf serum (FCS; Dutscher, Brumath, France), 1%
L-glutamine, 1% antibiotics (GIBCO BRL, Paisley, UK), with the
following recombinant human growth factors, as reported6:
GM-CSF, 5 ng/mL (gift of Schering Plough, Kenilworth, NJ); TNF-, 10 U/mL (Genzyme, Cambridge, MA); SCF, 50 ng/mL (R & D Systems,
Minneapolis, MN); FL, 50 ng/mL (gift of Immunex, Seattle, WA). From day
5, medium and cytokines were renewed every 2 days.
Cells of the lymphoid CEM line and monocytic U937 line, which were
CD13 and CD13+ (data not shown),
respectively, were also used in some experiments to assay peptidase inhibitors.
Flow cytometry analysis
Cells were incubated for 30 minutes at 4°C in phosphate-buffered
saline (PBS), 2% FCS, with 1:100 final fluorescein isothiocyante (FITC)-, phycoerythrin (PE)-, phycoerythrin-cyanin 5 (PCya5)- and/or
allophycocyanin (APC)-conjugated mAb: CD34 (581), CD1a (BL6), CD13
(SD1J1), CD95 (UB2), CD116 (SC06) (Immunotech, Marseille, France); CD1a
(OKT6; Ortho, Raritan, NJ); CD13 (Leu-M7), CD14 (Leu-M3), CD15
(Leu-M1), BrdU (Becton Dickinson, San Jose, CA; or PharMingen, San
Diego, CA); CD13 (WM15) and Fas-L (NOK1) (PharMingen); Bcl-2 (124) and
p53 (D0-7) (Dako, Golstrup, Denmark), and Bcl-x (SantaCruz, Santa Cruz,
CA). Other CD13 mAb were TÜK1 (Caltag, San Francisco, CA) and
F23 (gift from E. Stokert; Ludwig Institute for Cancer Research, New
York, NY). After washing, cells were analyzed with a FacsCalibur or a
FACScan (Becton Dickinson). Percentage of dead cells was determined by
propidium iodide (PI) staining (2.5 µg/mL; Sigma, Saint Louis,
MO) and FACS analysis.
Assay of cellular CD13/APN activity
CD13/APN activity was analyzed by spectrophotometry to assess
increased 405 nm absorbance due to release of free para-nitroaniline (pNA) from the cleavage of Leu-pNA derivatives in solution. Assays were
performed in duplicates in 96-well plates (Costar), using 5 × 104 cells/100 µL/well in PBS to which 1 mmol/L Leu-pNA and 10-3 to 10-6 mol/L peptidase
inhibitors arphamenine B, actinonin or bestatin (Sigma), or 0.1 to 50 µg/mL CD13 mAb WM15, TÜK1 or F23, or isotype control mAb
were added for up to 6 hours at 37°C.
Determination of cell growth, cell cycle, and apoptosis
On different days of culture, sorted or bulk cells
(5 × 105/mL) were cultured overnight under the
standard conditions in the presence or absence of peptidase inhibitors
or mAb. To assess proliferation, 5 × 104 cells were
seeded into 96-well plates (Costar) and cultured under the same
conditions but with 37 MBq/well
[3H]thymidine ([3H]dThd;
Amersham, Amersham, UK). Results are expressed as mean cpm of duplicates.
To determine DNA content in cells at the end of the overnight
incubation period, cells were washed in PBS and 75% (v/v) cold ethanol
was added dropwise to cell pellets. Fixed cells were incubated at
20°C for  2 hours and stored at 4°C in ethanol
until used. Just before staining, cells were washed with PBS and
incubated for 5 minutes on ice with cold PBS, 0.25% Triton-X100
(Sigma), after which they were resuspended in 300 µL PI (10 µg/mL)
and incubated on ice for at least 10 minutes before FACS analysis. Also, to evaluate cell cycle, bromodeoxyuridin (BrdU) incorporation was
assessed as reported21: BrdU (30 µg/mL) was added to
cultures for 2 hours; cells were washed, stained as usual for surface
markers, and fixed in 0.5% paraformaldehyde (100 µL/sample) at
4°C; after 4 hours, 25 µL PBS and 5% Tween 20 (Sigma) were added
and cells were allowed to permeabilize overnight at 4°C; they were
then washed in PBS and resuspended in 20 µL PBS, 10 µg/mL DNAse I
(Boehringer, Mannheim, Germany), 4 µL anti-BrdU-FITC or anti-BrdU-PE
mAb; after 1 hour, 250 µL PBS were added, and samples were FACS analyzed.
To assess apoptosis, cells were washed in PBS, cytospun onto glass
slides, dried, and fixed for 10 minutes in PBS, 1% formaldehyde, and
0.2% glutaraldehyde; they were then incubated for 10 minutes with 0.5 µg/mL Hoechst 33 342 (Sigma), dried at 37°C, washed 3 times in
PBS, mounted in glycerol, and examined under UV fluorescence microscopy. External translocation of inner membrane phosphatidylserine (PS) was determined with the Annexin V Apoptosis kit (PharMingen) according to the manufacturer's instructions, followed by FACS analysis.
Caspase-3 activity was determined by the Caspase-3 Cellular Activity
Assay kit (BIOMOL Research, Plymouth Meeting, PA) according to the
manufacturer's instructions. Briefly, cells were resuspended in
ice-cold lysis buffer and centrifuged, and cytosolic extracts (kept in
ice or stored at 70°C until use) were then incubated for up
to 10 hours at 37°C in microtiter plates with caspase-3 substrate
Ac-DEVD-pNa and with or without caspase-3 inhibitor Ac-DEVD-CHO, before
absorbance at 405 nm was determined.
Statistics
Results are presented as means ± SD of data from different
individual cultures. Statistical analysis used the paired (Excel 5, Microsoft, Redmond, WA), or 1 sample Student's t test
(GB-STAT, Dynamic Mycrosystems, Silver Spring, MD) when indicated.
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Results |
Cellular CD34 and CD13 expression and cell growth
On the basis of CD13 expression, 2 cell populations could be
distinguished after 5-day culture of the CD34+ HPC with
SCF/FL/GM-CSF/TNF-: 82% ± 6% of cells were
CD13hi, the rest being CD13lo (n = 7). Most
CD13hi cells already expressed DC, macrophage, or
granulocytic lineage markers CD1a, CD14, or CD15,4,20
respectively, whereas 25% ± 8% had no lineage markers and are
referred to as CD13hiLin cells (ie, ~20% of total
cells were CD13hiLin). All CD13lo cells
were Lin .20 Under our culture conditions,
53% ± 16% CD13hiLin and 30% ± 14%
CD13lo cells were still CD34+ (~11% and 6%
of total cells, respectively) at that time (Figure 1A). After sorting according to CD34 and
CD13 expression and further culture for 1 week with the same cytokines,
CD34+CD13hiLin cells expanded by
31- ± 9-fold and CD34CD13hi
Lin cells expanded 7- ± 3-fold, whereas
CD34+CD13loLin cells and
CD34CD13loLin cells did not grow
(Figure 1B).
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| Fig 1.
Cellular CD34 and CD13 expression and cell growth.
(A) Flow cytometry 2-color cytograms of culture day 5 cells: After
labeling with FITC-CD1a, FITC-CD14, FITC-CD15, PE-CD13, and PCya5-CD34
mAb (left panel), Lin cells were gated (R2) by excluding
FITC-labeled cells; CD13 and CD34 expression of Lin cells was
then evaluated (right panel, with cell percentages indicated);
fluorescence intensity is indicated along the 2 axes; data are from 1 experiment of 7. (B) Growth of day-5 sorted cells: after labeling as
described in A, cells were sorted according to CD34 and CD13 expression
after excluding FITC-labeled cells, and the sorted
CD34+CD13lo,
CD34CD13lo,
CD34+CD13hi, and
CD34CD13hi cells were cultured further
for 7 days; cell recovery is presented as fold increase cell numbers
over the number of initially seeded sorted cells; results are expressed
as means ± SD (n = 6; with only 2 points examined on day 10).
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At that time, the progeny of
CD34+CD13hiLin cells comprised
7% ± 3% CD1a+CD14 and
11% ± 8% CD1a+CD14+ DC,
38% ± 24% CD1aCD14+
monocytes/macrophages, and 28% ± 2% CD15+
granulocytes (n = 3), the corresponding figures being
22% ± 8%, 21% ± 5%, 14% ± 3%, and
23% ± 9%, respectively, for cells that derived from day 5 CD34CD13hiLin cells. Not enough
cells were usually recovered from cultures of the 2 CD13lo Lin cell populations to allow for
phenotyping (data not shown).
Thus, subsequent growth of culture day 5 cells in this system
correlated with expression of CD13, all the more so that they were
CD34+. Because CD13 may represent a proliferation marker of
HPC22 as well as of tumor cells or activated T
lymphocytes,23 we hypothesized that CD13 by itself could
influence the potential of progenitors and precursors of DC and
monocytes/macrophages to proliferate and differentiate in culture.
APN activity of cultured cells and its inhibition
To test this hypothesis, we used molecules known to engage CD13
and/or affect APN activity. Peptidase inhibitors bestatin24 and actinonin, the latter known as the most potent and specific inhibitor of CD13/APN activity,25 were chosen to this end;
arphamenine B, a specific inhibitor of aminopeptidase B,25
served as the control. In a first series of experiments, the compound
was added for up to 6 hours to culture day 7 bulk
cells 93% ± 4% (n = 7) of which were CD13hi
expressing or not lineage markers CD1a, CD14, or CD15 and APN activity was then determined. Incubation with actinonin had the strongest effect, reducing APN activity to about 23% ± 6%
(n = 4) that of cells incubated without inhibitor at 10-3
mol/L and to 57% ± 6% at 10-6 mol/L (Figure
2A). Although apparently incomplete, this
inhibiting activity level actually corresponds to that which is usually
noted under similar conditions when using APN.25-29
Bestatin was 10-fold less effective, and arphamenine B had a
noticeable effect only at the highest 10-3 mol/L
concentration used (Figure 2A). Therefore, only actinonin and control
arphamenine B, both at 10-4 mol/L, were used in subsequent
experiments.
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| Fig 2.
Assay of membrane APN activity of cultured cells.
(A) Determination of APN activity of cells incubated for 6 hours with
peptidase inhibitors arphamenine B (Arpha.B), bestatin, or actinonin.
(B) Determination of APN activity of cells incubated for 6 hours with
CD13 mAb F23, WM15, or TÜK1. Data, from 1 experiment out of 4 (A) or 3 (B), are presented as percentages of the APN activity (405 nm
absorbance) of peptidase inhibitor- or mAb-treated cells relative to
control cells incubated without inhibitor or mAb.
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CD13 mAb F23 and WM15, both known to neutralize APN
activity,24,30,31 and TÜK1 were also assayed:
indeed, F23 and WM15 mAb inhibited APN activity, if only by
39% ± 8% (n = 4) and 45% ± 8% (n = 3),
respectively, at 10 µg/mL or higher concentrations,32 whereas TÜK1 had no effect (Figure 2B; and data not
shown).29
Inhibition of cell growth by actinonin
Inasmuch as we found that cell growth correlated with CD13
expression on Lin cells and that actinonin potently inhibited CD13/APN activity on cultured cells, we next examined its effect on the
proliferative capacity of sorted culture day 5 CD13hiLin and CD13lo cells. Indeed, DNA
synthesis in cells of the 2 populations was reduced in the presence of
actinonin, but not of arphamenine B, albeit to a much greater extent as
regards CD13hiLin cells (Figure
3A). The same was noted when examining day 5 and day 7 bulk cells, > 90% of which were CD13hi as
already indicated4: [3H]dThd uptake by these
cells was also strongly reduced by actinonin (Figure 3B). When cells
were transiently treated for 1 to 5 hours with actinonin and washed and
recultured overnight without, instead of with the inhibitor, DNA
synthesis was then comparable to that in control cultures (data not
shown), indicating that this effect of actinonin was reversible and was
not the mere result of nonspecific toxicity.33
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| Fig 3.
Effect of the inhibition of APN activity on cultured cell
growth.
(A) Sorted CD13hi and CD13lo cells were
incubated overnight in the presence of 3[H]dThd, with or
without 104 mol/L arphamenine B (Arpha.B) or
actinonin; data are from 1 experiment of 2. (B) Bulk culture day 5 or
day 7 cells were treated in the same manner as in A; data are presented
as mean [3H]dThd incorporation ± SD (n = 6);
statistical significance of differences: P < .001 for
actinonin vs control and vs arphamenine B. (C) Culture day 7 cells were
incubated overnight with CD13 mAb F23, WM15 or TÜK1, or with
isotype control mAb IgG2a for F23 and IgG1 for WM15 and TÜK1
(all at 50 µg/mL) in the presence of [3H]dThd; data are
from 1 experiment of 2.
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To provide evidence that actinonin inhibited cell proliferation by
specifically interacting with CD13/APN, we used CD13+ U937
and CD13CEM cells. First, spectrophotometry analysis
of APN activity of these cells showed 30% lower absorbance with CEM
than with U937 cells (data not shown); arphamenine B had little (at the
highest 103 mol/L concentration used) or no effect
on this activity, whereas actinonin was 30-fold more efficient for U937
than for CEM cells (50% inhibiting concentration:
1 × 105 mol/L versus
3 × 103 mol/L; Figure
4A), confirming its specificity for the
CD13+ cells. Accordingly, only actinonin reduced in a
dose-dependent manner 3H]dThd uptake by U937 but not by
CEM cells, whereas arphamenine B affected neither cell type (Figure
4B).
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| Fig 4.
Inhibition of APN activity on CEM and U937 cells.
(A) Assay of APN activity of CEM or U937 cells incubated for 6 hours
with different concentrations of arphamenine B (CEM(arpha),
U937(arpha)) or actinonin (CEM(act), U937(act)); mean percentages ± SD (n = 2) of the APN activity of treated cells relative to control
cells without inhibitor. (B) CEM or U937 cells were cultured overnight
in the presence of [3H]dThd, with or without arphamenine
B or actinonin at the indicated concentrations; each bar corresponds to
the mean (+ SD) of 3 to 8 independent determinations; the significance
of differences between percentages of incorporated cpm and 100%
control values was assessed by the 1 sample t test: ***
P < .001.
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Finally, we examined the effect of incubating culture day 7 cells with
CD13 mAb. Surprisingly, only TÜK1 inhibited
3H]dThd incorporation by about 30% at 50 µg/mL, but
neither WM15 nor F23 had any effect in this respect (Figure 3C) though
both inhibited CD13/APN activity.
Actinonin affects the cell cycle and induces apoptosis of cultured
cells
Because actinonin inhibited cell growth to such extent, we examined
whether it affected the cell cycle. As shown previously,21 only a minority of viable cells were found to be in the S/G2-M phase
after 7 days of culture. The proportion of these cells decreased from
19% ± 6% in control cultures (19% ± 7% in arphamenine
B-treated cultures) to 11% ± 5% (44% ± 14% reduction
relative to controls; P = .001; n = 7) when culture day 7 cells were incubated overnight with actinonin,24 and the
percentage of G0/G1 cells increased in parallel (from 81% ± 6%
to 88% ± 5%), whereas no change occurred with arphamenine B
(Figure 5A). In addition, actinonin
increased cell mortality since the proportion of PI+ cells
reached 25% ± 12% of actinonin-treated cells relative to 6% ± 3% or 5% ± 2% when cells were incubated either in
the control condition or with arphamenine B (P = .005;
n = 8). This indicates that at least part of the actinonin effect was
to arrest cells in the G0/G1 phase and block passage to the S/G2-M
phase, which might then lead to cell death.
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| Fig 5.
Effect of the inhibition of APN activity on the cell
cycle and the induction of apoptosis.
Culture day 7 cells were incubated overnight with or without
104 mol/L arphamenine B or actinonin before being
examined. (A) DNA content was determined by flow cytometry after
ethanol fixation, permeabilization and PI staining: percentages of
cells in the G0/G1 or S/G2-M phase, assessed relative to viable cell
numbers, are indicated; data are from 1 representative experiment out
of 7. (B) Nuclear morphology was visualized by ultraviolet light
microscopy after Hoechst 33 342 staining; the arrow shows a typical
apoptotic cell; data are from 1 experiment of 3. (C) Surface expression
of inner membrane PS was evaluated by flow cytometry after Annexin-V
staining: the percentage of cells with increased PS expression is
indicated; data are from 1 experiment of 4.
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This led us to examine the mechanism of cell death. Ultraviolet light
microscopy after Hoechst 33 342 staining showed nuclear fragmentation
typical of apoptosis among actinonin-treated but not control cells
(Figure 5B). There was also an increased proportion of cells with
subdiploid fragmented DNA noted when analyzing the cell cycle, as
exemplified by the subG1 population (see Figure 5A) that increased to
18% ± 9% of all cells versus 2% ± 2% in arphamenine
B-treated and 2 ± 1% in control cultures (P = .002, n = 8). Actinonin-induced apoptosis was further confirmed by the increased surface expression of inner-membrane PS after Annexin-V staining (Figure 5C), indicating loss of membrane asymmetry that is an
early feature of apoptosis.
We also investigated cell expression of molecules known to be involved
in apoptosis34 such as Bcl-2 and CD95 (Fas), which were
both expressed by culture day 7 cells incubated under the control
condition, suggesting that Bcl-2 anti-apoptotic mechanisms were then
operative. In the presence of actinonin, Bcl-2 expression decreased and
that of CD95 was unchanged (Figure 6A),
whereas FasL, Bcl-xL, and p53 were not detected in actinonin-treated or in control cells (data not shown). Also, using an assay that assesses cleavage of a chromogenic substrate in the presence of cell lysates, caspase-3 activity was detected in actinonin-treated cells, whereas it
was at the same low level in arphamenine B-treated and in control cells
(Figure 6B).
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| Fig 6.
Cell expression of molecules known to be involved in
apoptosis.
Culture day 7 cells were incubated as indicated in the legend of Figure
5. (A) Flow cytometry analysis of Bcl-2 and Fas (CD95) expression; open
histograms: labeling with the irrelevant mAb; shaded histograms:
staining by the relevant mAb; data are from 1 experiment of 7 (Bcl-2)
or 2 (Fas). (B) Assay of caspase-3 activity in cells: cell lysates were
incubated for 10 hours with Ac-DEVD-pNa, the cleavage of which was
assessed at 405 nm; data are from 1 experiment of 3.
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Actinonin preferentially affects DC and macrophage precursors
Altogether, overnight treatment of culture day 7 cells with
actinonin led to decreased cell yields at the end of this period: for
example, starting from 5 × 105 cells seeded
initially, 7 ± 1.6 × 105 cells were recovered
under the control condition and
7.7 ± 1.6 × 105 after arphamenine B
treatment, but only 3.9 ± 0.9 × 105 cells
were obtained with actinonin (P = .001 and P = .002
vs controls and arphamenine B, respectively; n = 5). This decrease in
cell numbers under the influence of actinonin prompted us to examine
whether it affected distinct or all cell populations in cultures.
Indeed, on culture day 7, most cells are CD13hi4 and
represent mainly a mixture of
CD1a+CD14DC precursors and
differentiated DC, CD1a+CD14+ and
CD1aCD14+ bipotent macrophage/DC
precursors and more differentiated cells of the macrophage lineage, and
CD15+ granulocytic cells.8,10,21
We first analyzed the proportion of cycling BrdU+ cells
among these different types of cells. Under the control condition, there were about 50% more cycling CD1a+CD14+
than CD1a+CD14DC precursors
(24% ± 9% versus 16% ± 9%; P = .014;
n = 6); CD1aCD14+BrdU+
bipotent cell percentages (19% ± 7%) were not significantly
different from either, whereas 58% ± 6% of CD15+
granulocytic cells were BrdU+ (P = .010 to .044 vs the other cell types; n = 3). Arphamenine B did not affect these
percentages, but actinonin reduced by 75% to 90% percentages of
cycling cells, whatever their phenotype, but not those of
BrdU- cells (Figure 7). This
was associated with increase of PI+ cells over the control
condition in all populations, but with the
CD1aCD14+ cells, the cells that were
still CD13hiLin and
CD1a+CD14+ cells displaying the highest rates
(Figure 8).
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| Fig 7.
Effect of the inhibition of APN activity on the cell
cycle of the different populations of cultured cells.
Culture day 7 cells were incubated as indicated in the legend of Figure
5, and labeled with BrdU, and with CD1a, CD14, and CD15 mAb; cells were
gated according to CD1a and/or CD14 and CD15 expression. Results are
shown as flow cytometry 2-color cytograms of BrdU incorporation into
the indicated gated cells. Markers are indicated along the fluorescence
intensity horizontal and vertical axes. Percentages of
BrdU+ cells in each gated population are indicated in the
right corners of the cytograms. Data are from 1 experiment of 6.
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| Fig 8.
Effect of the inhibition of APN activity on the mortality
of the different populations of cultured cells.
Culture day 7 cells were incubated as indicated in the legend of Figure
5, labeled with CD1a, CD14, and CD15 mAb, and analyzed by FACS after PI
staining. Results are shown as the percentages of differently labeled
cells among IP+ cells under the different conditions (means
of 2 experiments).
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Nevertheless, analysis of the cells recovered at the end of the
overnight incubation showed that, relative to controls or arphamenine
B-treated cells, CD1a+ DC percentages in actinonin-treated
cultures were significantly reduced by about 30%, mostly due to the
40% reduction in the proportion of CD1a+CD14+
precursor cells, whereas percentages of the
CD1aCD14+ cells of the macrophage
lineageincluding bipotent DC precursorswere not modified, and
CD15+ cell percentages increased by about 60% (Table
1). Because of the reduction in overall
cell numbers, this resulted in reduced yields of cells of the DC and
macrophage lineages, from 3.9 ± 1.3 × 105 to
1.9 ± 0.7 × 105 (P = .01; n = 4)
and from 2.0 ± 0.3 × 105 to
0.7 + 0.2 × 105 (P = .006),
respectively, whereas granulocytic cell numbers were not significantly
affected (1.0 ± 0.3 × 105 vs
0.8 ± 0.3 × 105; P = .08).
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Table 1.
Percentages of CD1a+, CD14+,
and CD15+ cells among culture day 7 cells after overnight
incubation with or without 104 Mol arphamenine B or
actinonin
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Discussion |
We previously isolated a major CD13hi cell population
that appeared in  5 days in cultures of cord blood CD34+
HPC with SCF/GM-CSF/TNF-.4,20 This population comprised cells already expressing CD1a, CD14, and CD15 lineage markers as well
as CD13hiLin precursors thereof: CD15+
granulocytes appeared and plateaued earlier than CD1a+ DC
and CD14+ monocytes/macrophages, confirming early branching
off from the latter lineages.21,35 These findings allowed
us to delineate a differentiation pathway from CD34+ HPC to
CD13hiLin progenitors of either or both DC and
monocytes/macrophages,4,20 and it is now known that there
are in fact 2 intermediate precursors of DC differentiating from
CD13hiLin cells:
CD1a+CD14precursors of DC/LC and
CD1aCD14+ bipotent precursors of DC and
macrophages7-10 (and data not shown). In our initial
report, only a minority of CD13hiLin- cells were
CD34+,4,20 whereas here, presumably because of the
additional use of FL, which not only potentiates the growth of, but
also acts as survival factor for HPC,36 initially
CD34+CD13lo HPC differentiated into
CD13hiLin cells half of which were still
CD34+ on culture day 5. Even when they were
CD34, CD13hiLin cells had a
greater capacity than CD34+ or
CD34CD13lo cells to proliferate and
differentiate into DC and monocytes/macrophages under the culture
condition used here. Moreover, when their clonogenicity was examined in
a semisolid assay with the same cytokines as in liquid culture,
CD34+CD13hiLin cells yielded the highest
number of colonies, most of which were mixed CFU-M/DC or pure CFU-DC;
CD34-CD13hiLin cells yielded less
colonies of the same types. The capacity to generate colonies from
CD34+CD13loLin cells was lower and
limited to CFU-G, whereas
CD34CD13loLin cells had almost no
clonogenic capacity (M. Guigon, personal communication). Expansion and
engagement of CD13hiLin progenitors toward DC rather
than macrophages or granulocytes should then be enhanced by the
cytokine combination used here. At any rate, in liquid as in semisolid
culture, growth and differentiation of cells of the DC and
monocyte/macrophage lineages correlated foremost with CD13 expression level.
CD13 is expressed on stem cells and during most development stages of
myeloid cells and therefore it is generally considered as a
myelomonocytic marker.37 It is highly expressed on DC
precursors as well as on differentiated DC.4,17-19 However,
CD13 expression has also been found to correlate with growth and
activation of different cell types: (i) it is indeed preferentially
expressed on proliferating CFU-GM progenitors,22,38 on
malignant acute myeloblastic cells but also on lymphoblastic leukemia
cells,39,40 (ii) its expression is associated with
proliferation of activated T lymphocytes,41,42 and (iii)
inhibition of CD13 expression or of APN activity reduces proliferation
of different cell types.24,33,41-43 This led us to
hypothesize that CD13/APN could play a direct role in the development
of DC and/or macrophages from their progenitors.
Natural substrates of CD13 include vasoactive peptides, neuropeptides
such as leu- and metenkephalin, and the chemokine IL-8.37 Leu-enkephalin was first used to evaluate whether CD13 engagement influenced the growth of CD13hi cells, but its effect was
limited and only detectable at the highest 103 mol/L
concentration used (data not shown). Of note, bradykinin or substance P
is known to inhibit APN activity in the same concentration range.37 Therefore, we next used pseudopeptide inhibitors
of APN activity and CD13 mAb to this end. The naturally occurring derivative of L-prolinol considered as the most selective inhibitor of
APN,25,44 actinonin, strongly reduced APN activity on
cultured cells while APN-blocking CD13 mAb F23 and WM15 were less
potent. These findings may indicate that actinonin could also inhibit APN-like activities mediated by membrane ectopeptidases other than
CD13/APN,25,26,29 but the ability of CD13 mAb to inhibit APN activity has also been reported to vary depending on the cells used, which may be due to the occurrence of multiple CD13 species on
the same or different types of cell.27-29 For example, five CD13 species with different glycosylation patterns have been reported on U937 cells, and this may impinge on the epitopes recognized by
different mAb.45
We next found that actinonin inhibited growth of
CD13hiLin or CD13hiLin+ cells, which was
associated with an increase of cycling cells in the G0/G1 phase and
with an increased proportion of apoptotic cells. This is compatible
with arrest of cells in the G0/G1 phase and subsequent death, or with
actinonin inducing death selectively beyond G0/G1. Bcl-2, which
protects cells from apoptosis, is normally upregulated during DC
differentiation/maturation and, with CD95, it participates to the
regulation of apoptosis during distinct phases of DC
development.18 Here, CD95 expression was unchanged on
actinonin-treated cells, whereas that of Bcl-2 decreased concomitantly with the increase of caspase-3 activity, an enzyme that could then
cleave Bcl-2 and inactivate its antiapoptotic function.46 This suggests that this may be 1 of the pathways leading to apoptosis through engagement of CD13. All cycling cells in the cultures were
susceptible to actinonin, but finally the percentages and yields of DC
and macrophage precursors appeared to be affected to a greater extent
than cells of the granulocytic lineage. This observation is in line
with data indicating that CD13 expression level remains high on
differenciating DC and macrophages whereas it decreases as
granulocytes differentiate11 (other data not shown: J. Langner, Martin-Luther-Universität Halle/Saale, Germany, personal
communication). Thus, it is possible that CD13/APN
participates in regulating expansion and survival of DC and
monocyte/macrophage progenitors and precursors through cleavage of
molecules controlling hematopoiesis.
However, no physiologic substrate of CD13/APN has yet been implicated
in such a process and, of the cytokines tested so far, only IL-8 has
been shown to be degraded by APN.31,37 It may be relevant
here that CD13 reportedly plays a role in tumor cell migration47,48 through its function as an adhesion
molecule,37 inasmuch as DC precursors and differentiated DC
are highly motile and migrate in response to chemotactic
signals.49 Although there is no indication that IL-8 is
involved in expansion of DC precursors, one may not rule out that
CD13/APN could be involved in this process in vivo during migration of
DC precursors from the bone marrow in response to this or other
chemokines.31,49,50 Nevertheless, we examined whether
GM-CSFa critical myeloid growth factor, which also preserves
viability of immature as well as mature
DC51-53could be a target of APN. This enzyme
splits off the penultimate N-terminal amino acid of proteins,
with the exception of Pro.31 However, GM-CSF has a
penultimate N-terminal Pro and cannot thus be directly cleaved by
APN,54 though APN might well collaborate with other aminopeptidases (CD10, CD26, BP-1) in this regard.54 Using
an ELISA (Immunotech), we found increased GM-CSF levels in
actinonin-treated relative to control culture day 7 cell supernatants
(data not shown). This may merely reflect decreased comsumption by
cells blocked in the G0/G1 phase or undergoing apoptosis, but one may not rule out that actinonin could act by preventing uptake and/or consumption of GM-CSF by the cells, perhaps through a mechanism involving its inhibiting capacity of CD13/APN proteolytic activity. Finally, exposure to bestatin has been reported to modulate GM-CSF receptor surface expression on cells of the TF-1 and U937
lines55 but, here, this expression on actinonin-treated
cells was comparable to that on control cells (data not shown).
Thus, CD13/APN may well play a role in the growth of progenitors and
pure DC and bipotent DC/macrophage precursors through engagement of its
enzymatic site and processing of biologically active molecules.
However, when we used CD13 mAb instead of actinonin to confirm the CD13
specificity of its effect, only the nonblocking mAb, and not the
APN-blocking mAb, inhibited cell growth. The same type of findings was
reported by other investigators in experimental models with different
cell systems.24,56 This suggests that actinonin-induced
cell growth inhibition may be associated with either or both inhibition
of APN activity and/or binding of CD13 to a cell membrane ligand that
may then act as a signal-transducing molecule, as reported for
dipeptidyl peptidase IV (CD26).57,58
| |
Acknowledgments |
We gratefully acknowledge the help of the following colleagues and
companies: Pr. J. Milliez and his staff of the service de
Gynécologie-Obstétrique, Hôpital Saint-Antoine
(Paris, France) for the gift of cord blood samples; Drs E. Thomas and
E. Maraskovski (Immunex, Seattle, WA) for the gift of recombinant
Flt-3 ligand; Schering Plough (Kenilworth, NJ) for the gift of
recombinant GM-CSF; and Dr E. Stokert (Ludwig Institute for Cancer
Research, New York, NY) for the gift of mAb F23. We also thank M. Yagello for his invaluable contribution to cell sorting, Dr P. Auberger
(CJF INSERM 96-05, Nice, France) for his advice and fruitful
discussions, and Pr. Jürgen Langner and his colleagues
(Martin-Luther-Universität, Halle/Saale, Germany) for their
helpful interest in our work.
| |
Footnotes |
Submitted March 18, 1999; accepted September 10, 1999.
Supported by the Agence Nationale de Recherche sur le SIDA,
the Association de Recherche contre le Cancer, the Comité de Paris de la Ligue Nationale contre le Cancer, and the Association pour la Recherche sur les Déficits Immunitaires Viro-Induits (Paris, France).
Reprints: Jean Claude Gluckman, Laboratoire d'Immunologie,
CERVI, Hôpital de la Pitié-Salpêtrière, 83 Bld
de l'Hôpital, 75651 Paris Cedex 13, France.
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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M. Kruse, O. Rosorius, F. Kratzer, D. Bevec, C. Kuhnt, A. Steinkasserer, G. Schuler, and J. Hauber
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Top
Abstract
Introduction