|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1359-1371
Calcium Ionophore-Treated Myeloid Cells Acquire Many Dendritic Cell
Characteristics Independent of Prior Differentiation State,
Transformation Status, or Sensitivity to Biologic Agents
By
Gary K. Koski,
Gretchen N. Schwartz,
David E. Weng,
Ronald E. Gress,
Friederike H.C. Engels,
Maria Tsokos,
Brian J. Czerniecki, and
Peter A. Cohen
From the Medicine Branch, National Cancer Institute, Laboratory of
Pathology, National Cancer Institute, Bethesda, MD; and the Department
of Surgery, University of Pennsylvania Medical Center, Philadelphia,
PA.
 |
ABSTRACT |
We previously reported that treatment of human peripheral blood
monocytes or dendritic cells (DC) with calcium ionophore (CI) led to
the rapid (18 hour) acquisition of many characteristics of mature DC,
including CD83 expression. We therefore investigated whether
less-mature myeloid cells were similarly susceptible to rapid CI
activation. Although the promyelocytic leukemia line HL-60 was
refractory to cytokine differentiation, CI treatment induced
near-uniform overnight expression of CD83, CD80 (B7.1), and CD86
(B7.2), as well as additional characteristics of mature DC. Several
cytokines that alone had restricted impact on HL-60 could enhance
CI-induced differentiation and resultant T-cell sensitizing capacity.
In parallel studies, CD34pos cells cultured from normal
donor bone marrow developed marked DC-like morphology after overnight
treatment with either rhCD40L or CI, but only CI simultaneously induced
upregulation of CD83, CD80, and CD86. This contrasted to peripheral
blood monocytes, in which such upregulation could be induced with
either CI or rhCD40L treatment. We conclude that normal and transformed
myeloid cells at many stages of ontogeny possess the capacity to
rapidly acquire many properties of mature DC in response to CI
treatment. This apparent ability to respond to calcium mobilization,
even when putative signal-transducing agents are inoperative, suggests strategies for implementing host antileukemic immune responses.
This is a US government work. There are no restrictions on its use.
| |
INTRODUCTION |
A NUMBER OF RECENT STUDIES have
suggested that human myeloid leukemias express antigens that can enable
their recognition by T cells, with attendant immune-mediated modulation
of tumor growth.1,2 Because tumors of myeloid and lymphoid
lineage share the ontogeny of professional antigen-presenting cells
(APC),3 the capacity of such malignant cells to present
endogenously expressed tumor-associated antigen (Ag) directly to T
cells has been the focus of intense recent interest. Studies of
myelogenous leukemias as well as lymphocytic leukemias, lymphomas, and
multiple myelomas indicate that native malignant cells typically have
heterogenous or absent expression of costimulatory molecules essential
for efficient T-cell sensitization.4-9 However,
costimulatory molecule expression can frequently be induced by agents
or combinations of agents identical to those that enhance APC function
in these cells' nonmalignant counterparts. For example, cytokine
combinations such as recombinant granulocyte-macrophage
colony-stimulating factor (rGM-CSF), tumor necrosis factor-alpha
(rTNF- ) and interleukin-4 (rIL-4), when added to cultured bone
marrow or peripheral blood mononuclear cells (PBMC) obtained from
chronic myelogenous leukemia (CML) patients, also can induce dendritic
cell (DC)-like differentiation in the Philadelphia
chromosome-expressing (Phpos) malignant cell subpopulation,
thereby enhancing APC function.10,11 Similar results have
been obtained when peripheral blasts from patients with acute
myelogenous leukemia (AML) were cultured with rGM-CSF, rTNF-, stem
cell factor, and IL-6.2 These results indicate
that CML and AML cells from at least some individuals remain
susceptible to normal signal transductive stimuli that promote elements
of DC-like differentiation and enhanced APC function. Similarly,
culture of B-cell-derived lymphoma, leukemia, and multiple myeloma
cells with CD40 ligand (CD40L) can induce marked expression of
costimulatory molecules such as B7.1, B7.2, and ICAM-1 on the tumor
cells, similar to the upregulation that occurs after CD40L-stimulation of normal DC and B cells.7-9,12-14
Efforts to enhance APC function in leukemia cells via cytokine or
ligand treatments are dependent on the malignant cells' expression of
specific receptors for the stimulating agent(s) as well as a capacity
for normal membrane signal transduction. One strategy for bypassing
such dependence is to transduce malignant cells with expression
constructs that contain the genes encoding costimulatory molecules such
as B7.1. Whereas this approach has successfully enhanced tumor cell
immunogenicity in several studies,15-17 strategies that
target activation of endogenous differentiation pathways may remain
clinically advantageous when they can induce a wider panoply of APC
functional characteristics.
We recently showed that pharmacologic agents that mobilize
intracellular calcium can be used to enhance APC function in both human
peripheral blood monocytes and immature DC, apparently bypassing initial receptor/ligand requirements involving cytokines or ligands previously shown to influence such development.18 We found
that each of these myeloid subpopulations responded to calcium
ionophore (CI) treatment by developing the morphologic characteristics
and functions of highly activated, mature DC. Whereas full morphologic differentiation during CI treatment required 72 to 96 hours of culture,
many immunophenotypic alterations occurred promptly within the first 20 hours of culture, including decreased expression of the
monocyte/macrophage-associated molecule CD14 (LPS binding protein
receptor), increased expression of major histocompatibility complex
(MHC) molecules, upregulation of B7.2, CD40, and ICAM-1 expression, and de novo expression of both B7.1 and the DC-associated activation marker CD83.19 In addition, enhanced T-cell
sensitizing efficiency was evident within the first 20 to 40 hours of
treatment. Such rapid activation kinetics contrasted to the much slower
activation observed when peripheral blood monocytes are treated with
cytokine combinations such as rGM-CSF, rIL-4, and rTNF-. For
example, monocyte CD83 expression appeared within 4 hours and peaked at 20 hours of CI treatment, whereas with combination cytokine treatment CD83 expression is seldom observed before 5 to 7 days of culture. These
contrasting kinetics do not preclude the possibility that cytokine
treatment and CI treatment induce DC differentiation and/or activation
through ultimately convergent molecular mechanisms. However,
pharmacologic calcium-mobilizing agents have the potential clinical
advantage of bypassing initial surface receptor/signal transduction requirements.
Our previous studies of peripheral blood monocytes and immature DC
raised the possibility that calcium-mobilizing agents might also
trigger elements of differentiation/activation in relatively undifferentiated myeloid leukemia cells as well as in normal myeloid bone marrow progenitor cells, rendering such treatment a means to
induce numerous DC characteristics in these populations. The present
report shows that, in addition to peripheral blood monocytes and DC,
both transformed and untransformed myeloid cells at earlier stages of
maturation possess the ability to respond rapidly to CI treatment.
Although the kinetics of morphologic responses vary among the
individual studied myeloid populations, a common observation for each
studied population is marked upregulation of the activation marker
CD8319 as well as the costimulatory molecules B7.1 and B7.2
within 20 hours of initiation of CI treatment, regardless of the
myeloid cells' prior differentiation status.
| |
MATERIALS AND METHODS |
Reagents and Ab.
CI A23187 (Sigma, St. Louis, MO) was used as described
previously.18 In some experiments, cultures were
supplemented with the following cytokines: human interferon-gamma
(rIFN- ) (Biogen, Cambridge MA; specific activity 1.6 × 107 U/mg); rh-c-kit ligand (sp act 2.7 × 105 U/mg), rhTNF- (sp act 1.1 × 108
IU/mg), and rhIL-4 (sp act 2.9 × 107 IU/mg) (all R&D
Systems, Minneapolis, MN); and rhGM-CSF (sp act 5.6 × 106 IU/mg (Amgen, Thousand Oaks, CA). Human recombinant
soluble trimeric CD40-ligand (rhCD40L) was a generous gift from
Immunex, Seattle, WA.
Myelogenous human cell line cultures.
The acute monocytic leukemia-derived THP-1 (TIB 202), the acute
myelogenous leukemia-derived KG-1 (CCL 246), the chronic myelogenous leukemia line K562 (CCL 243), the macrophage-like histocytic lymphoma U937 (CRL 1593), and the promyelocytic leukemia-derived HL-60 (CCL 240)
were obtained from American Type Culture Collection (Rockville, MD);
BV173 was a kind gift from Dr Alan Gevirtz at the University of
Pennsylvania (Philadelphia, PA). Cell lines were maintained in
exponential growth by serial passage in 162 cm2 tissue
culture flasks (Costar, Cambridge, MA) in culture medium (CM)
consisting of RPMI-1640 with 10% heat-deactivated fetal calf serum
(FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL gentamicin sulfate, .05 mmol/L 2-ME, 1 mmol/L sodium pyruvate, and 1 mmol/L nonessential amino acids (all reagents Biofluids, Rockville, MD)
at 37o C, 5% CO2. Cell lines used in all
experiments were split 5:1 every 2 to 3 days, and were carried no more
than 2 to 3 months in culture from the original stocks. To assess the
effects of treatments with CI and/or cytokines, cells were washed and
seeded in fresh medium at 1 × 106 or 5 × 105 per well in 24-well tissue culture plates (Costar),
then cultured for 18 hours before CI and/or cytokine addition.
CD34pos human myeloid cultures.
Normal bone marrow cells enriched for CD34pos cells (purity
91% to 98%) were obtained from three healthy donors (Poietic
Technologies, Inc, Gaithersburg, MD) and in one instance from cadaveric
vertebral bodies by positive immunomagnetic selection using a CD34
isolation kit (Miltenyi Biotech Inc, Auburn CA). To promote enrichment
and expansion of intermediate DC precursors,20,21
CD34pos cells were resuspended to 4 × 104
cells/mL and cultured for 6 days in an enriched Iscove's Modified Dulbecco's Medium22 supplemented with 10% FCS (Hyclone,
Logan, UT), 20 ng/mL rh-c-kit ligand, 10 ng/mL rhGM-CSF, and 10 ng/mL rhTNF-; fresh medium and factors were added on day 3. After the 6-day expansion and enrichment phase, cells were harvested, washed, and
replated in 48-well cluster plates (Costar) at 5 × 105 cells per well in fresh medium containing rhGM-CSF and
rhTNF- but not rh-c-kit ligand. Various doses of CI or rhCD40L were
added 45 minutes later, and wells were harvested at various subsequent timepoints for analysis as described in Results.
Human peripheral blood monocyte cultures.
Human monocytes were prepared from four healthy volunteers using
leukapheresis and countercurrent centrifugal elutriation as previously
described.18 A serum-free modification of our prior culture
method was used (Nguyen et al, manuscript in preparation); monocytes
were washed and resuspended in serum-free medium (Macrophage-SFM, Gibco, Grand Island, NY) with 50 ng/mL added rhGM-CSF, and plated at 5 × 106 cells per well in 24-well cluster plates
(Costar) overnight, after which various doses of CI or rhCD40L were
added as described in Results.
Ab and fluorescence-activated cell sorter (FACS) analysis.
Multicolor FACS analysis was used as previously developed to analyze
normal human monocytes and DC.18 PE-conjugated MoAb against
human B7.1, monomorphic HLA-DR, and CD33 were obtained from Becton
Dickinson (Mountain View, CA), B7.2, ICAM-1, and CD1a from Pharmingen
(San Diego, CA), monomorphic HLA-ABC from Sigma (St. Louis, MO), and
CD40 from Harlan (Oxford, UK); subclass-matched, conjugated control Ab
were obtained from Becton Dickinson and Caltag Laboratories (San
Francisco, CA). Unconjugated HB15a, a mouse MoAb recognizing human CD83
was a kind gift of Thomas Tedder (Duke University, Durham, NC).
PE-conjugated goat antimouse IgG (GAMPE) as well as unconjugated mouse
immunoglobulin (Ig) subclass MoAb controls were acquired from Caltag.
Cultured cell lines were harvested, distributed into 12 × 15 mm
polystyrene FACS tubes (Falcon, Lincoln Park, NJ), centrifuged, and
supernatants removed. Cells were FcR blocked at 4°C for 20 min with
unconjugated 1 mg/mL human IgG (Sigma) in FACS buffer
(Ca++/Mg++-free Hanks' Buffered Saline
Solution [HBSS] containing 0.1% bovine serum albumin [BSA] and
.02% sodium azide). Cells were then washed in FACS buffer,
centrifuged, and stained with various Ab under identical conditions for
20 min. Cells were then washed and centrifuged as before and
resuspended in 0.5 mL FACS buffer. In some experiments cells were
counterstained with FITC-conjugated anti-CD14 or subclass matched MoAb
(Becton Dickinson). Samples stained with CD83 or isotype-matched
control Ab were washed, then counterstained with GAMPE. In
later experiments PE-conjugated anti-CD83 antibody (Coulter) was
substituted for indirect staining of CD83.
Cells were then washed and resuspended in 0.5 mL FACS buffer and
subjected to three- or two-color analysis on a Becton Dickinson FACSort
using CellQuest analysis software; 0.8 µg/mL propidium iodide (PI)
was added to samples to enable exclusion of nonviable cells from analysis.
Microscopy.
Culture medium was carefully removed from cells cultured in 24- or
48-well plates, and fresh medium supplied. Cells were then resuspended
and transferred onto Lab-Tek 8-glass chamber slides (Nunc, Naperville,
IL) previously coated with 1% polylysine (Sigma, St Louis, Mo) and
incubated for 30 min at 37oC, 5%
CO2.18 Slides were then examined and
photographed with an Olympus IX-70 inverted microscope (Melville, NY)
using Nomarski differential interference contrast (DIC)
optics. Cells were either photographed while viable, or first fixed in
absolute ethanol and stained with Wright's solution.18
Allosensitization studies.
Human T lymphocytes obtained from lymphocyte-rich elutriation fractions
were purified using T-cell isolation columns (R&D, Minneapolis,
MN).18 In some experiments non-naive T cells were depleted
by incubating lymphocytes with 10 µg/mL anti-CD45RO (Caltag) before
column application.23 HL-60 cells or cultured
CD34pos bone marrow cells were subjected to various CI or
CI/cytokine combination treatments, were harvested, washed 3 times in
CM, -irradiated to 30 Gy, then cocultured with T lymphocytes in
96-well flat-bottomed plates (Costar). The T-cell number was held
constant at 1 × 105 cells/well while the irradiated
HL-60 cells were added to each well in graded numbers. Cocultures were
incubated for 96 hours, then pulsed with 1 µCi/well
[3H]-TdR, and harvested and counted 18 hours later.
| |
RESULTS |
Acute promyelocytic leukemia line HL-60 displays profound early and
delayed responses to CI treatment.
Previous studies of human monocytes indicated that the optimal dose of
CI for enhancing DC differentiation varied among culture media, with
relatively higher CI concentrations required in media with higher
protein content (not shown). Our preliminary screenings with human
transformed myeloid cell lines, each cultured in RPMI-1640 medium
containing 10% FCS, indicated that phenotypic responses to CI
treatment varied among lines, but were maximal for each cell line
tested in the same CI dose range (not shown). CI-reactive cell lines
displayed varied upregulation of surface costimulatory molecules B7.1,
B7.2, and/or ICAM-1 after CI treatment (HL-60>K562>THP-1>KG-1; BV173 and U937 nonreactive, Fig 1 and not
shown); HL-60 additionally displayed marked upregulation of CD83,
similarly to normal peripheral blood monocytes and immature DC (see
below).18 The optimally activating A23187 dose selected for
HL-60 in all subsequent experiments (conducted in 10% FCS) was 180 ng/mL, a concentration 2 to 5 times lower than that previously used by
others to induce apoptosis of HL-60 in similar media.24,25
This optimum A23187 concentration had a pronounced antiproliferative
effect on HL-60 (not shown) but was accompanied by only modest
(typically 15% to 20%) cell death (compared with untreated controls)
during periods of observation (see below).
View larger version (54K):
[in this window]
[in a new window]
| Fig 1.
Time-course analysis of A23187-inducible surface Ag
expression. HL-60 cells (5 × 105) were cultured in a
final volume of 2 mL with or without an optimal dose (180 ng/mL) of
A23187 as described in Materials and Methods. Cells were harvested at
20, 48, and 96 hours post addition of A23187 and stained with
PE-conjugated mouse-antihuman CD83, B7.1, B7.2, ICAM-1, CD40, CD1a,
CD33, or IgG subclass-matched control Ab, and analyzed by FACS as
described in Materials and Methods. Results displayed are at 20 hours
without A23187 treatment (UNTREATED), 20 hours with A23187 treatment
(20H TREATMENT), 48 hours with A23187 treatment (48H TREATMENT), and 96 hours with A23187 treatment (96H TREATMENT). Cultures not treated with
A23187 showed an identical immunophenotypic profile at 20, 48, and 96 hours (not shown). Histograms display staining intensity of viable
(PI-excluding) cells, comparing control Ab (light lines) to specific Ab
(heavy lines).
|
|
HL-60 cells were cultured during a 96-hour period with or without 180 ng/mL A23187 and evaluated periodically by FACS analysis. In the
absence of CI treatment during this culture period a subpopulation of
untreated HL-60 cells constitutively expressed B7.2 and/or HLA-DR, but
no expression of CD83, B7.1, ICAM-1, CD40, CD1a, or CD14 was observed
(Fig 1 and not shown). In addition, untreated cells uniformly expressed
both MHC Class-I molecules and the myeloid marker CD33 (see below).
After initiation of A23187 treatment HL-60 cells rapidly developed de
novo expression of CD83, a cell surface protein typically expressed
during DC activation.19 Although already detectable within
4 hours of A23187 treatment (not shown) CD83 expression became maximal
within 20 hours, declined after 48 hours, and was essentially
undetectable by 96 hours (Fig 1). This early and transient expression
of CD83 was similar to that reported previously for ionophore-treated
peripheral blood monocytes.18 Marked de novo expression of
cell surface B7.1 and ICAM-1, and markedly upregulated expression of
B7.2 also developed within 20 hours of A23187 treatment. Although
nearly uniform high expression of ICAM-1 was attained, B7.1 and B7.2
expression remained somewhat more heterogeneous, with a minority of
cells remaining negative for expression of these molecules (Fig 1).
However, in contrast to CD83 expression, B7.1, B7.2, and ICAM-1
continued to be maximally expressed on a majority of cells throughout
the culture period (Fig 1).
Expression of CD40 and CD1a did not become prominent until 48 to 96 hours of CI treatment (Fig 1). Although A23187 treatment alone was
insufficient to induce uniform upregulation of CD40, the proportion of
cells expressing CD40 increased in magnitude after 24 hours and
maintained this expression through the culture period (Fig 1). The de
novo appearance of CD1a expression on a subset of cells occurred even
later, being detectable only after 72 to 96 hours of A23187 treatment
(Fig 1).
In contrast to the above modulations, levels of the constitutively
expressed myeloid marker CD33 were not increased by A23187 treatment,
but instead were slightly attenuated at 48 and 96 hours (Fig 1). In
contrast to CI's effect on normal monocytes and immature DC, A23187
treatment alone did not enhance HL-60 HLA-DR expression, and
paradoxically downregulated HLA-ABC expression (see below).
In synchronous morphologic studies, treatment of HL-60 cells for 24 hours with CI A23187 (180 ng/mL) caused many cells to develop motile
dendritic processes when transferred to polylysine-coated glass slides
(Fig 2A-C). Continued incubation with
A23187 for 72 to 96 hours resulted in expression of dendritic processes
on nearly all treated cells (Fig 2E), which were absent on untreated cells (Fig 2D).
View larger version (116K):
[in this window]
[in a new window]
| Fig 2.
A23187-treated HL-60 cells display progressive
acquisition of dendritic processes. HL-60 cells (1 × 106)
were cultured in a final volume of 2 mL with or without an optimal dose
of A23187 (180 ng/mL) for up to 96 hours. Cells were harvested at 24 or
96 hours, resuspended in fresh medium, transferred to polylysine-coated
glass chamber slides, and incubated an additional 20 minutes at
37°C. Cells were either immediately examined microscopically
(600×) and photographed using DIC (Nomarski) optics (A, B, and C), or
were fixed in 100% ethanol, stained in Wright's solution, and then
photographed (D and E). Photomicrographs A, B, and C were taken at
consecutive 30-second intervals and depict living HL-60 cells treated
with A23187 for 20 hours. Note motility of dendritic processes.
Photomicrographs D and E depict fixed, stained, HL-60 cells previously
left untreated (D) or treated with ionophore for 96 hours (E).
|
|
Effect of cytokine treatments on HL-60 cells alone or in combination
with CI.
Because several cytokines exert marked differentiating effects on
human monocytes and DC,18,19,26-31 we investigated whether such cytokines could modulate the differentiating effect of CI on HL-60
cells. Granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (20 ng/mL), rhIL-4 (1000 U/mL), and rhTNF- (100 U/mL) were found to have
no detectable effect on studied HL-60 cell surface markers during 7 days of observation when added singly or together in the absence of CI
(data not shown). In contrast, single-agent rhIFN- (1000 U/mL), as
previously reported by others,32 induced a majority of
HL-60 cells to express HLA-DR at 72 hours (Fig 3), with such upregulation already
apparent in some cells after 24 hours of treatment (not shown).
Nonetheless, the upregulation of HLA-DR by rhIFN- was not completely
uniform, and rhIFN- caused negligible modulations of the other
studied surface molecules (Fig 3).
View larger version (40K):
[in this window]
[in a new window]
| Fig 3.
Effects of rhIFN- and rhGM-CSF on A23187-induced HL-60
surface Ag expression. HL-60 cells (5 × 105) were
cultured in a final volume of 2 mL with or without an optimal dose (180 ng/mL) of CI A23187, rhIFN- (1000 U/mL), or rhGM-CSF (20 ng/mL).
Cells were harvested 72 hours later, stained with PE-conjugated mouse
antihuman B7.2, HLA-DR, HLA-ABC, CD40, CD1a, CD83, or IgG
subclass-matched control Ab, and analyzed by FACS as described in
Materials and Methods. Cell viabilities (percent) at time of analysis
(72 hours of treatment) are indicated below histograms for each
treatment group. Histograms display staining intensity of viable
(PI-excluding) cells, comparing control Ab (light lines) to specific Ab
(heavy lines).
|
|
More striking modulatory effects were observed, however, when either
rhIFN- or rhGM-CSF was added to HL-60 cultures in conjunction with
CI treatment. Adjunct rhGM-CSF prevented the appearance of a
subpopulation of cells negative for B7.2 expression that became apparent in CI-treated cells by 72 hours (Fig 3), and had a similar effect on B7.1 expression (not shown). Although CI alone had no effect
on HLA-DR expression, CI plus rhIFN- modestly enhanced upregulation
of HLA-DR compared with rhIFN- treatment alone (Fig 3). Although
rhGM-CSF and rhIFN- each modestly enhanced CI-induced CD40
expression, CD1a expression was enhanced only by rhGM-CSF, with
rhIFN- displaying a dominant suppressive activity for the expression
of this surface molecule (Fig 3). Cytokines in conjunction with CI
displayed no profound effects on the transient expression of CD83.
As noted above, an unanticipated finding was that treatment of HL-60
cells with CI alone induced a drop in MHC Class-I expression that
approached background levels by 72 to 96 hours (Fig 3). Whereas rhGM-CSF did not prevent CI-induced downregulation of MHC Class I, the
addition of rhIFN--restored MHC Class-I expression in CI-treated
cells almost to untreated control levels (Fig 3).
To determine whether CI or CI plus adjunct cytokines enhanced APC
function in HL-60 cells, treated or untreated cells were irradiated and
cocultured with MHC-unmatched T lymphocytes in an allosensitization
assay. Neither untreated HL-60 cells nor HL-60 cells treated with
rhGM-CSF alone proved effective for allosensitizing T cells
(Fig 4). As previously reported by
others,32 treatment with rhIFN- alone (or rhIFN- plus
rhGM-CSF) enhanced HL-60's allosensitizing capacity (Fig 4), but only
two- to threefold. In contrast, marked enhancement of T-cell
allosensitization capacity was observed when HL-60 cells were treated
with CI alone (more than 25-fold increased T-cell
[3H]-TdR uptake v untreated group at a 5:1
T-cell:APC ratio). Still further augmentation of allosensitization
capacity was observed when HL-60 cells were treated both with CI and
adjunct cytokines. Combination treatment with CI, rhGM-CSF, and
rhIFN- was most effective (74-fold [3H]-TdR uptake
v untreated group at a 5:1 T-cell:APC ratio) followed by CI
plus rhIFN- (50-fold) and CI plus rhGM-CSF (39-fold).
View larger version (20K):
[in this window]
[in a new window]
| Fig 4.
Treatment with CI A23187 or A23187 with adjunct cytokines
enhances T-cell allosensitizing capacity of HL-60 cells. HL-60 cells (5 × 105/well) were cultured in 24-well plates in 2 mL CM
(Untreated) or CM supplemented with rhGM-CSF (20 ng/mL, GM-CSF),
rhIFN- (1000 U/mL, IFN-) or combined rhGM-CSF plus rhIFN-,
each of these conditions with or without CI A23187 (180 ng/mL). HL-60
cells were harvested 72 hours later, washed three times in CM,
-irradiated (30 Gy), and cocultured at various HL-60:T-cell ratios
with freshly prepared allogeneic human T lymphocytes (1 × 105/well) in triplicate 96-well tissue culture plate wells
in 200 µL final volume CM. Cells were maintained in culture for 96 hours, pulsed with 1 µCi [3H]-TdR, harvested 18 hours
later, and [3H]-TdR incorporation assessed by liquid
scintillation spectrometry. Ordinate displays [3H]-TdR
incorporation as thousands of CPM per well during the final 18-hour
culture period; abscissa displays ratio of APC:T cells. Not shown:
irradiated HL-60 cells alone, regardless of treatment, generated < 750 cpm when seeded at 20,000 cells per well; T cells alone generated < 250 cpm. Bars indicate SEM from triplicate wells.
|
|
Similar results were obtained in four separate experiments, and were
comparable whether or not CD45R0pos (ie, non-naive) T cells
were depleted before culture (data not shown). The maximal T-cell
alloproliferation induced by treated HL-60 cells, however, always
remained lower than that stimulated by normal peripheral blood
DC.18
CI treatment rapidly induces many DC characteristics in cultured
CD34pos cells that are not identical to the effects of
rhCD40L treatment.
Highly enriched CD34pos cells obtained from four normal
bone marrow donors were initially cultured for 6 days in rh-c-kit
ligand, rhGM-CSF, and rhTNF- to promote enrichment and expansion of
DC precursors. Cells were then centrifuged and suspended in identical fresh medium except for the absence of rh-c-kit ligand. At this time,
individual replated groups were also treated with varying doses of CI,
and each group was harvested for analysis 20 or 40 hours after
replating. We also studied in two of these experiments the effects of
rhCD40L, a ligand that has been reported to have differentiating
effects on CD34pos myeloid progenitors as well as on
incompletely differentiated peripheral DC.13,14
At the time of initial replating, the day six cultured
CD34pos cells were always highly heterogeneous in their
surface protein expression, with a majority of cells expressing modest
amounts of B7.2, a minority expressing B7.1 or CD83, and only a
relatively small fraction manifesting DC-like morphology (not shown);
this heterogeneous and predominantly nonactivated profile persisted without alteration when these cells were replated for 20 or 40 hours in
medium containing only rhGM-CSF and rhTNF-
(Figs 5 and 6).
However, including either rhCD40L (2.5 to 40 µg/mL) or CI (180 to 750 ng/mL) in culture caused the great majority of cells to acquire
dendritiform morphologic characteristics within 20 hours of replating
(Fig 5). Despite this profound morphologic effect of rhCD40L, the
latter caused negligible immunophenotypic activation of the replated
cells during this culture period at each of the tested concentrations
(Fig 6). In contrast, similar to previous observations in peripheral
blood monocytes, CI treatment of day 6 cultured
CD34poscells resulted in a dose-dependent marked and rapid
upregulation of B7.1, B7.2, and CD83 and a marked downregulation of
CD14 in the CD14pos subpopulation. In addition, modest
and/or more variable upregulation of ICAM-1, HLA-DR, CD1a, CD40, and
HLA-ABC were observed (Figs 6 and 7 and not
shown). CI's effect was essentially complete in three of four donors
within 20 hours of replating, but required an additional 20 hours to
generalize in the fourth donor. The allosensitizing capacity of
CD34pos cultured cells was modestly enhanced by such CI
treatment, though even without CI treatment, cells displayed
considerable allostimulatory potential at this time in culture
(Fig 8), consistent with the marked
phenotypic activation observed in a significant subpopulation of the
cells (Fig 6). Prolonging CI treatment for an additional 5 days
resulted in sustainedly high CD80 and CD86 expression and low CD14
expression, but as similarly observed in monocytes and HL-60, a decline
in CD83 expression (not shown).
View larger version (89K):
[in this window]
[in a new window]
| Fig 5.
Cultured CD34pos bone marrow cells acquire
the capacity to form dendritic processes in response to treatment with
A23187 or rhCD40L. Cultures of CD34pos bone marrow cells
initially expanded for 6 days in the presence of rh-c-kit ligand,
rhGM-CSF, and rhTNF- to induce numerical expansion. Cells were then
centrifuged and replated in identical fresh medium except for the
absence of rh-c-kit ligand, with individual wells also receiving CI
A23187 or rhCD40L. Twenty hours after replating, cells were harvested
and centrifuged, resuspended in fresh medium and transferrred onto
polylysine-coated glass chamber slides, incubated for an additional 20 minutes at 37oC, and photomicrographed (600×) using DIC
(Nomarski) optics. Photomicrographs are representative of multiple CI
and CD40L doses tested in two separate experiments: untreated cells
(A); cells treated for 20 hours with 375 ng/mL CI A23187 (B); cells
treated for 20 hours with 10 µg/mL rhCD40L (C). Note dendritic
processes exhibited on cells in panels B and C that are largely absent
in A. Similar results were observed after CI treatment at 180 ng or 750 ng/mL or rhCD40L at 2.5 or 40 µg/mL (not shown).
|
|
View larger version (43K):
[in this window]
[in a new window]
| Fig 6.
Alterations in immunophenotype of cultured human
CD34pos bone marrow cells and peripheral blood monocytes in
the presence of CI A23187 or rhCD40L. Cultures of CD34pos
bone marrow cells previously expanded for 6 days in the presence of
cytokines (see Fig 5) were replated and cultured for an additional 20 hours in the same medium without rh-c-kit ligand with various doses of
CI A23187 or rhCD40L added. In synchronous experiments, elutriated
monocytes were washed and plated overnight as described in Materials
and Methods, then cultured for an additional 20 hours with no additive
or CI A23187 or rhCD40L. After the 20-hour treatment bone marrow
progenitors and monocytes were harvested and stained with PE-conjugated
antibodies against human B7.1, B7.2, and CD83 and analyzed by FACS (see
Materials and Methods). Representative displayed data depict untreated
cells, cells treated with an optimal dose of CI (375 ng/mL for
CD34pos cells and 225 ng/mL for monocytes), or cells
treated with 10 µg/mL rhCD40L. Similar results were observed in
either cultured CD34pos progenitors or peripheral blood
monocytes treated with 2.5 or 40 µg/mL doses of rhCD40L (not
shown).
|
|
View larger version (45K):
[in this window]
[in a new window]
| Fig 7.
Dose-response analyses of immunophenotypic modulations of
cultured CD34pos bone marrow cells during CI A23187
treatment. Six-day cultured CD34pos myeloid cells enriched
from normal volunteer bone marrow were cultured in medium containing
rhGM-CSF and rhTNF- with graded doses of CI A23187 (0 to 750 ng/mL).
Cells were harvested and analyzed by flow cytometry with added
propidium iodide (PI) as described in Fig 6 and Methods. Abscissa
displays ng/mL of A23187 present during CI treatment. Two or three
separate dose-response experiments were performed and plotted for each
cell surface marker analyzed (B7.1, B7.2, CD83, ICAM-1, HLA-DR, CD1a,
and CD14). Also portrayed is the viable cell recovery relative to
control for three dose-response experiments (0 ng/mL group = 100%).
Cell surface Ag expression data are portrayed as Specific Mean
Fluorescence quotients (mean fluorescent intensity of cells stained
with specific Ab/mean fluorescent intensity of cells stained with
subclass-matched control Ab); a minimum of 5000 cells with
background-level PI staining was assayed per determination.
|
|
View larger version (12K):
[in this window]
[in a new window]
| Fig 8.
Treatment with CI A23187 enhances T-cell allosensitizing
capacity of cultured CD34pos bone marrow cells. The latter
were treated and harvested as in Fig 6, then cocultured at various
APC:T lymphocyte ratios with fixed numbers of freshly prepared
allogeneic human T lymphocytes as in Fig 4. Ordinate displays
CPM/105 input T cells during the last 18 hours in culture.
Groups are "Untreated" (continued culture in rhGM-CSF and
rhTNF-), "CD40L" (10 µg/mL rhCD40L) and "A23187" (375 ng/mL). Abscissa displays ratio of APC: T cells. Not shown:
CD34pos cells alone, regardless of treatment, generated < 250 cpm when seeded at 20,000 cells per well; T cells alone generated < 250 cpm. Bars indicate SEM from triplicate wells.
|
|
Despite the contrasting effects of CI and rhCD40L on CD80, CD86, and
CD83 expression in cultured CD34pos bone marrow cells, both
agents were capable of inducing upregulated expression of these
molecules by peripheral blood monocytes within a 20-hour exposure
period (Fig 6).
| |
DISCUSSION |
Our data are consistent with the hypothesis that agents known to
mobilize intracellular calcium can affect myeloid cells at apparently
differing stages of ontogeny and can serve as a decisive differentiation stimulus, leading to rapid acquisition of many characteristics often associated with mature DC. In certain cases such
calcium mobilization appears to result in true DC
differentiation,18 whereas in other cases (such as the
established leukemic cell line HL-60) it induces a somewhat more
circumscribed set of characteristics associated with mature DC. In
either case, treatments with such calcium mobilizing agents may rapidly
preempt alternate courses of differentiation that would have occurred
in its absence. In addition to our previous demonstrations of CI's
effects in peripheral blood monocytes and immature DC,18
the present report describes commensurate effects, including striking
rapid upregulation of CD83 and B7 costimulatory molecule expression and
acquisition of dendritic processes, when CD34pos bone
marrow cells and the leukemic cell line HL-60 are treated with CI. We
have recently found, in addition, that CI treatment of freshly obtained
chronic myelogenous leukemia (CML) cells from 10 out of 10 patients
induced many similar responses (Engels et al, manuscript submitted).
Although membrane signal transduction pathways may not be operative at
every stage of myeloid ontogeny to physiologically trigger sufficient
calcium mobilization to achieve differentiation, the myeloid cell's
inherent potential for such differentiation is readily demonstrable
with CI treatment. Although CI treatment appears to supply signals
leading to the acquisition of DC characteristics by bypassing at least
the initial requirements for receptor/ligand-mediated membrane signal
transduction, it remains a formal possibility that CI acts indirectly,
through induction of secretion of biologic agents by the treated
myeloid cells. These agents (presumably cytokines or chemokines) could in turn act in an autocrine fashion, by binding to specific receptors on the cell surface, and thus supply such signals that induce DC
differentiation/activation.
The cell line HL-60's response to CI treatment is of considerable
interest for several reasons. Originally isolated from a 36-year-old
female patient with acute promyelocytic leukemia,33,34 HL-60 has the demonstrated capacity to differentiate in vitro into
cells expressing many characteristics of different myeloid-lineage cell
types, including granulocytes in the presence of dimethyl sulfoxide
(DMSO) or retinoic acid,35,36 monocytes in the presence of
1,25 dihydroxy vitamin D3 or rhIFN-,37-40
macrophages in the presence of phorbol myristate acetate
(PMA),41 or eosinophils in the presence of
alkalinity.42 Because of the extraordinary multipotency of
HL-60 cells compared with other culture-adapted human myelogenous
leukemia lines, it appeared possible that appropriate signals delivered
to these cells would induce differentiation along a myeloid DC pathway.
However, previous studies that have attempted to use cytokines alone to
promote DC differentiation in HL-60 have not been
successful,2 even though such strategies successfully
promoted DC development in CML and AML cells from individual
patients.2,10,11 Our laboratory also has been unable to
induce differentiation in HL-60 with such cytokines (see above) or with
rhCD40L (data not shown). Nonetheless, calcium mobilization treatment
of HL-60 results in DC-like differentiation, showing the cell line's
inherent potential for such differentiation even when it cannot be
induced with putative biologic agents.
The phenotypic alterations induced by CI in HL-60 cells in many ways
paralleled previously shown effects in human peripheral blood monocytes
and immature DC,18 including rapid upregulation of B7.1,
B7.2, ICAM-1, and early peaking expression of CD83. Like peripheral
blood monocytes, HL-60 cells treated with CI acquired the tendency to
elaborate motile cellular processes that caused HL-60 cells to
morphologically resemble myeloid DC; as also observed in monocytes this
effect was maximal after 72 to 96 hours in culture. In addition, as in
monocyte studies (reference 18 and manuscript in preparation),
cytokines that had limited impact when added alone to HL-60 cultures
had more substantial impact when included in the context of CI treatment.
Several characteristics of HL-60 cells and their response to CI
treatment differed markedly, however, from normal (nontransformed) myeloid-lineage cells. HL-60 cells largely failed to express HLA-DR constitutively, and in contrast to normal peripheral blood monocytes or
immature DC failed to upregulate HLA-DR expression after CI treatment
unless IFN- was also present in the culture. CI also had the
anomalous action of downregulating HL-60's MHC Class-I expression,
which was in contrast to the modest enhancement of MHC Class-I
expression typically observed when normal blood monocytes or immature
DC are treated with CI.18 Aberrant MHC Class-II expression
is a frequently reported characteristic of acute promyelocytic leukemias such as HL-60.43-45 There also exist both
physiologic and pathologic precedents for somatic cell downregulation
of MHC Class I: for example, thyroid-stimulating hormone downregulates MHC Class-I expression in normal thyroid cells,46 and
adenovirus transformation downregulates MHC Class-I expression in a
variety of infectable somatic targets.47 Paralleling such
precedents, HL-60's MHC Class-I downregulation during CI treatment
could largely be countered by rhIFN- treatment (Fig 3).
In contrast to HL-60, CI treatment of fresh CML cells from 10 patients
has resulted in upregulation of both MHC Class-I and -II expression in
addition to costimulatory molecule, CD40 and CD83 upregulation; in no
case was paradoxical downregulation of MHC Class I induced by CI
(Engels et al, manuscript submitted). It remains to be determined
whether HL-60's anomalous MHC Class-I and -II regulation will be
observed among freshly obtained AML samples.
Similarly to peripheral blood myeloid cells and to HL-60 cells, 6-day
cultured, CD34pos bone marrow cells displayed prompt
responses to CI treatment, including rapid acquisition of CD83, B7.1,
and B7.2 expression. In contrast to peripheral monocytes and HL-60,
however, cultured CD34pos cells treated with CI displayed
much more rapid acquisition of dendritic processes; these differences
may reflect an artifact of the CD34pos cells' sustained
exposure to exogenous cytokines both before and after CI treatment.
Because CI treatment promotes rapid and near-uniform DC-like
differentiation of 6-day cultured CD34pos cells, it
preempts the subsequent heterogenous differentiation, predominantly to
neutrophils and monocytes, which is typically observed in such cells
between days 6 and 12 of culture.20
Given that myeloid cells respond to calcium mobilization at various
stages of ontogeny by acquiring elements of DC-like differentiation, it
is of great interest to determine which factors and/or ligands present
in normal bone marrow and peripheral tissues can induce such calcium
mobilization and differentiation via physiologic signal transduction.
Such calcium-mobilizing signals may vary during ontogeny with the
myeloid cell's expression (or lack) of receptors for individual
agents, and may include CD40L and/or IL-4, because other investigators
have reported that exposure of B lymphocytes to these agents can result
in significant calcium mobilization, and, at least in the case of
CD40L, apparent calcineurin-mediated cell activation.48,49
Because many of CI's activating effects on myeloid cells can be
blocked by calmodulin and calcineurin antagonists,50 we are
presently assessing the role of these enzymes in the capacity of CD40L,
IL-4, and other agents to induce myeloid cell differentiation.
However, it is apparent that most IL-4, GM-CSF, and/or TNF--based
myeloid differentiating regimens require many more days than CI
treatment (typically 1 week or longer) to induce uniform DC-like
activation, as evidenced by the late appearance of upregulated costimulatory molecule expression, CD83 expression, and/or enhanced T-cell sensitizing efficiency during such
treatments.2,10,11,19,29,30,51-57 It is possible that such
cytokine treatments may not operate through a calcium-dependent
differentiation pathway; alternatively, their activation of the
calcium-dependent pathway may be indirect and less efficient than CI
treatment, due to complex signal transduction issues. This is well
illustrated by CD40L. Although previously published reports indicate
that prolonged (6 day) CD40L exposure can induce immunophenotypic
activation of cultured CD34pos myeloid cells,14
it is apparent from the present studies that shorter (20 hour)
treatments with rhCD40L or CI do not have the same effects on cultured
CD34pos cells: rhCD40L induces profound morphologic effects
without immunophenotypic activation within the tested 20-hour window of
exposure (Figs 5 and 6), whereas CI induces both effects simultaneously
(Figs 5, 6, and 7). In contrast, 20-hour treatment of peripheral blood monocytes with either CI or rhCD40L can promote equivalent
immunophenotypic activation (Fig 6). We are investigating the
possibility that CD40L signal transduction displays variable coupling
to calcium-dependent signaling pathways during myeloid maturation.
In these studies, we showed that the HL-60 myeloid leukemia line had
the capacity to respond to CI in ways similar to those that we have
observed in normal myeloid cells,18 resulting in the
development of a mature, activated APC phenotype that in many regards
resembled activated myeloid DC. Anomalies in its response to CI
compared with normal myeloid cells could be normalized with rhIFN-
treatment, and cytokines (rhIFN- and rhGM-CSF) that independently had modest effects on HL-60's antigen-presenting efficiency had more
potent effects in the context of concomitant CI treatment. These data
suggest that CI treatment can serve as an alternative means to enhance
the immunogenicity of myeloid leukemias in vaccine strategies in which
leukemias are unresponsive to cytokine treatment alone, but in which an
endogenous calcium-dependent activation pathway is pharmacologically
inducible. We are currently analyzing the effects of CI and adjunct
cytokine treatments on leukemic cells isolated freshly from patients
with various classes of myelogenous leukemia. Efforts are directed to
using CI-treated fresh leukemia cells to efficiently sensitize
autologous T cells to leukemia-associated antigens.
| |
ACKNOWLEDGMENT |
The authors are indebted to Drs Nirbhay Kumar, Alan Scott, and Frances
Hakim for helpful suggestions and encouragement, as well as to Charles
Carter and Drs Susan Leitman, E.J. Read, and Harvey Kleinman of the NIH
Department of Transfusion Medicine. We also wish to thank Dr Kathy
Picha at Immunex for her helpful support and discussions in regards to
rhCD40L, as well as Cathy Bare for excellent technical support.
| |
FOOTNOTES |
Submitted August 31, 1998; accepted April 14, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests and correspondence to Peter A. Cohen, MD,
Center for Surgery Research, FF-50, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195; email: cohenp{at}ccf.org.
| |
REFERENCES |
1.
Lim SH, Coleman S:
Chronic myeloid leukemia as an immunological target.
Am J Hematol
54:61, 1997[Medline]
[Order article via Infotrieve]
2.
Santiago-Schwarz F, Coppock DL, Hindenburg AA, Kern J:
Identification of a malignant counterpart of the monocyte-dendritic cell progenitor in an acute myeloid leukemia.
Blood
84:3054, 1994[Abstract/Free Full Text]
3.
Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ:
The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand.
J Exp Med
185:1101, 1997[Abstract/Free Full Text]
4.
Munro JM, Freedman AS, Aster JC, Gribben JG, Lee NC, Rhynhart KK, Banchereau J, Nadler LM:
In vivo expression of the B7 costimulatory molecule by subsets of antigen-presenting cells and the malignant cells of Hodgkin's disease.
Blood
83:793, 1994[Abstract/Free Full Text]
5.
Hirano N, Takahashi T, Ohtake S, Hirashima K, Emi N, Saito K, Hirano M, Shinohara K, Takeuchi M, Taketazu F, Tsunoda S, Ogura M, Omine M, Saito T, Yazaki Y, Ueda R, Hirai H:
Expression of costimulatory molecules in human leukemias.
Leukemia
10:1168, 1996[Medline]
[Order article via Infotrieve]
6.
Tsukada N, Aoki S, Maruyama S, Kishi K, Takahashi M, Aizawa Y:
The heterogeneous expression of CD80, CD86 and other adhesion molecules on leukemia and lymphoma cells and their induction by interferon.
J Exp Clin Cancer Res
16:171, 1997[Medline]
[Order article via Infotrieve]
7.
Cardoso AA, Schultze JL, Boussiotis VA, Freeman GJ, Seamon MJ, Laszlo S, Billet A, Sallan SE, Gribben JG, Nadler LM:
Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen.
Blood
88:41, 1996[Abstract/Free Full Text]
8.
Schultze JL, Cardoso AA, Freeman GJ, Seamon MJ, Daley J, Pinkus GS, Gribben JG, Nadler LM:
Follicular lymphomas can be induced to present alloantigen efficiently: A conceptual model to improve their tumor immunogenicity (published erratum appears in Proc Natl Acad Sci USA 92:10818, 1995).
Proc Natl Acad Sci USA
92:8200, 1995[Abstract/Free Full Text]
9.
Urashima M, Chauhan D, Uchiyama H, Freeman GJ, Anderson KC:
CD40 ligand triggered interleukin-6 secretion in multiple myeloma.
Blood
85:1903, 1995[Abstract/Free Full Text]
10.
Smit WM, Rijnbeek M, van Bergen CA, de Paus RA, Vervenne HA, van de Keur M, Willemze R, Falkenburg JH:
Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells.
Hum Immunol
53:216, 1997[Medline]
[Order article via Infotrieve]
11.
Choudhury A, Gajewski JL, Liang JC, Popat U, Claxton DF, Kliche KO, Andreeff M, Champlin RE:
Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia.
Blood
89:1133, 1997[Abstract/Free Full Text]
12.
Caux C, Burdin N, Galibert L, Hermann P, Renard N, Servet-Delprat C, Banchereau J:
Functional CD40 on B lymphocytes and dendritic cells.
Res Immunol
145:235, 1994[Medline]
[Order article via Infotrieve]
13.
Caux C, Massacrier C, Vanbervliet B, Dubois B, Van Kooten C, Durand I, Banchereau J:
Activation of human dendritic cells through CD40 cross-linking.
J Exp Med
180:1263, 1994[Abstract/Free Full Text]
14.
Flores-Romo L, Bjorck P, Duvert V, Van Kooten C, Saeland S, Banchereau J:
CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells.
J Exp Med
185:341, 1997[Abstract/Free Full Text]
15.
Matulonis UA, Dosiou C, Lamont C, Freeman GJ, Mauch P, Nadler LM, Griffin JD:
Role of B7-1 in mediating an immune response to myeloid leukemia cells.
Blood
85:2507, 1995[Abstract/Free Full Text]
16.
Dunussi-Joannopoulos K, Weinstein HJ, Nickerson PW, Strom TB, Burakoff SJ, Croop JM, Arceci RJ:
Irradiated B7-1 transduced primary acute myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML.
Blood
87:2938, 1996[Abstract/Free Full Text]
17.
Hirano N, Takahashi T, Azuma M, Okumura K, Yazaki Y, Yagita H, Hirai H:
Protective and therapeutic immunity against leukemia induced by irradiated B7-1 (CD80)-transduced leukemic cells.
Hum Gene Ther
8:1375, 1997[Medline]
[Order article via Infotrieve]
18.
Czerniecki BJ, Carter C, Rivoltini L, Koski GK, Kim HI, Weng DE, Rorors JG, Hijazi YM, Shuwen X, Rosenberg SA, Cohen PA:
Calcium ionophore treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells.
J Immunol
159:3823, 1997[Abstract]
19.
Zhou LJ, Tedder TF:
CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.
Proc Natl Acad Sci USA
93:2588, 1996[Abstract/Free Full Text]
20.
Szabolcs P, Avigan D, Gezelter S, Ciocon DH, Moore MA, Steinman RM, Young JW:
Dendritic cells and macrophages can mature independently from a human bone marrow-derived, post-colony-forming unit intermediate.
Blood
87:4520, 1996[Abstract/Free Full Text]
21.
Lardon F, Snoeck HW, Berneman ZN, Van Tendeloo VF, Nijs G, Lenjou M, Henckaerts E, Boeckxtaens CJ, Vandenabeele P, Kestens LL, VanBockstaele DR, Vanham GL:
Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: Antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor-1.
Immunology
91:553, 1997[Medline]
[Order article via Infotrieve]
22.
Schwartz GN:
Use of gel-well culture chambers as a liquid culture system to measure responses of hematopoietic colony-forming cells to growth factors.
Int J Cell Cloning
7:360, 1989[Medline]
[Order article via Infotrieve]
23.
Mehta-Damani A, Markowicz S, Engleman EG:
Generation of antigen-specific CD4+ T cell lines from naive precursors.
Eur J Immunol
25:1206, 1995[Medline]
[Order article via Infotrieve]
24.
Kataoka A, Kubota M, Wakazono Y, Okuda A, Bessho R, Lin YW, Usami I, Akiyama Y, Furusho K:
Association of high molecular weight DNA fragmentation with apoptotic or non-apoptotic cell death induced by calcium ionophore.
FEBS Lett
364:264, 1995[Medline]
[Order article via Infotrieve]
25.
Naora H:
Differential expression patterns of beta-actin mRNA in cells undergoing apoptosis.
Biochem Biophys Res Commun
211:491, 1995[Medline]
[Order article via Infotrieve]
26.
Triozzi PL, Aldrich W:
Phenotypic and functional differences between human dendritic cells derived in vitro from hematopoietic progenitors and from monocytes/macrophages.
J Leukoc Biol
61:600, 1997[Abstract]
27.
Peters JH, Xu H, Ruppert J, Ostermeier D, Friedrichs D, Gieseler RK:
Signals required for differentiating dendritic cells from human monocytes in vitro.
Adv Exp Med Biol
329:275, 1993[Medline]
[Order article via Infotrieve]
28.
Kiertscher SM, Roth MD:
Human CD14+ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4.
J Leukoc Biol
59:208, 1996[Abstract]
29.
Pickl WF, Majdic O, Kohl P, Stockl J, Riedl E, Scheinecker C, Bellow-Fernandez C, Knapp W:
Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes.
J Immunol
157:3850, 1996[Abstract]
30.
Romani N, Gruner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, Konwalinka G, Fritsch PO, Steinman RM, Schuler G:
Proliferating dendritic cell progenitors in human blood.
J Exp Med
180:83, 1994[Abstract/Free Full Text]
31.
Sallusto F, Lanzavecchia A:
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.
J Exp Med
179:1109, 1994[Abstract/Free Full Text]
32.
Steeg PS, Sztein MB, Mann DL, Strong DM, Oppenheim JJ:
Interferon regulation of DR antigen expression and alloantigen-presenting capabilities of the promyelocytic cell line HL60.
Scand J Immunol
21:425, 1985[Medline]
[Order article via Infotrieve]
33.
Collins SJ, Gallo RC, Gallagher RE:
Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture.
Nature
270:347, 1977[Medline]
[Order article via Infotrieve]
34.
Collins SJ:
The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression.
Blood
70:1233, 1987[Abstract/Free Full Text]
35.
Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC:
Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.
Proc Natl Acad Sci USA
75:2458, 1978[Abstract/Free Full Text]
36.
Geary SM, Ashman LK:
HL-60 myeloid leukaemia cells acquire immunostimulatory capability upon treatment with retinoic acid: Analysis of the responding population and mechanism of cytotoxic lymphocyte activation.
Immunology
88:428, 1996[Medline]
[Order article via Infotrieve]
37.
Miyaura C, Abe E, Kuribayashi T, Tanaka H, Konno K, Nishii Y, Suda T:
1 alpha,25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells.
Biochem Biophys Res Commun
102:937, 1981[Medline]
[Order article via Infotrieve]
38.
Tanaka H, Abe E, Miyaura C, Kuribayashi T, Konno K, Nishii Y, Suda T:
1 alpha,25-Dihydroxycholecalciferol and a human myeloid leukaemia cell line (HL-60).
Biochem J
204:713, 1982[Medline]
[Order article via Infotrieve]
39.
McCarthy DM, San Miguel JF, Freake HC, Green PM, Zola H, Catovsky D, Goldman JM:
1,25-dihydroxyvitamin D3 inhibits proliferation of human promyelocytic leukaemia (HL60) cells and induces monocyte-macrophage differentiation in HL60 and normal human bone marrow cells.
Leuk Res
7:51, 1983[Medline]
[Order article via Infotrieve]
40.
Ball ED, Guyre PM, Shen L, Glynn JM, Maliszewski CR, Baker PE, Fanger MW:
Gamma interferon induces monocytoid differentiation in the HL-60 cell line.
J Clin Invest
73:1072, 1984
41.
Rovera G, Santoli D, Damsky C:
Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester.
Proc Natl Acad Sci USA
76:2779, 1979[Abstract/Free Full Text]
42.
Fischkoff SA, Pollak A, Gleich GJ, Testa JR, Misawa S, Reber TJ:
Eosinophilic differentiation of the human promyelocytic leukemia cell line, HL-60.
J Exp Med
160:179, 1984[Abstract/Free Full Text]
43.
Guglielmi C, Martelli MP, Diverio D, Fenu S, Vegna ML, Cantu-Rajnoldi A, Biondi A, Cocito MG, Del Vecchio L, Tabilio A, Avvisati G, Basso G, Lo Coco F:
Immunophenotype of adult and childhood acute promyelocytic leukaemia: Correlation with morphology, type of PML gene breakpoint and clinical outcome. A cooperative Italian study on 196 cases.
Br J Haematol
102:1035, 1998[Medline]
[Order article via Infotrieve]
44.
Paietta E, Andersen J, Gallagher R, Bennett J, Yunis J, Cassileth P, Rowe J, Wiernik PJ:
The immunophenotype of acute promyelocytic leukemia (APL): An ECOG study.
Leukemia
8:1108, 1995
45.
Stone RM, Mayer RJ:
The unique aspects of acute promyelocytic leukemia.
J Clin Oncol
8:1913, 1990[Abstract]
46.
Saji M, Shong M, Napolitano G, Palmer LA, Taniguchi SI, Ohmori M, Ohta M, Suzuki K, Kirshner SL, Giuliani C, Singer DS, Kohn LD:
Regulation of major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP response element-like sequence.
J Biol Chem
272:20096, 1997[Abstract/Free Full Text]
47.
Rotem-Yehudar R, Winograd S, Sela S, Coligan JE, Ehrlich R:
Downregulation of peptide transporter genes in cell lines transformed with the highly oncogenic adenovirus 12.
J Exp Med
180:477, 1994[Abstract/Free Full Text]
48.
Klaus GG, Choi MS, Holman M:
Properties of mouse CD40. Ligation of CD40 activates B cells via a Ca(++)-dependent, FK506-sensitive pathway.
Eur J Immunol
24:3229, 1994[Medline]
[Order article via Infotrieve]
49.
Finney M, Guy GR, Michell RH, Gordon J, Dugas B, Rigley KP, Callard RE:
Interleukin 4 activates human B lymphocytes via transient inositol lipid hydrolysis and delayed cyclic adenosine monophosphate generation.
Eur J Immunol
20:151, 1990[Medline]
[Order article via Infotrieve]
50. Koski GK, Schwartz GN, Weng DE, Czerniecki BJ, Carter C, Gress
RE, Cohen PA: Calcium mobilization in human myeloid cells results in
acquisition of individual dendritic cell-like characteristics through
discrete signaling pathways. J Immunol (in press)
51.
Lauener RP, Goyert SM, Geha RS, Vercelli D:
Interleukin 4 down-regulates the expression of CD14 in normal human monocytes.
Eur J Immunol
20:2375, 1990[Medline]
[Order article via Infotrieve]
52.
Ruppert J, Friedrichs D, Xu H, Peters JH:
IL-4 decreases the expression of the monocyte differentiation marker CD14, paralleled by an increasing accessory potency.
Immunobiology
182:449, 1991[Medline]
[Order article via Infotrieve]
53.
Ruppert J, Schutt C, Ostermeier D, Peters JH:
Down-regulation and release of CD14 on human monocytes by IL-4 depends on the presence of serum or GM-CSF.
Adv Exp Med Biol
329:281, 1993[Medline]
[Order article via Infotrieve]
54.
Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J:
GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.
Nature
360:258, 1992[Medline]
[Order article via Infotrieve]
55.
Santiago-Schwarz F, Belilos E, Diamond B, Carsons SE:
TNF in combination with GM-CSF enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages.
J Leukoc Biol
52:274, 1992[Abstract]
56.
Reid CD, Stackpoole A, Meager A, Tikerpae J:
Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow.
J Immunol
149:2681, 1992[Abstract]
57.
Szabolcs P, Moore MA, Young JW:
Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34+ bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-alpha.
J Immunol
154:5851, 1995[Abstract]
This is a US government work. There are no restrictions on its use.
0006-4971/99/9404-0026$0.00/0
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. J. A. M. Santegoets, A. J. M. van den Eertwegh, A. A. van de Loosdrecht, R. J. Scheper, and T. D. de Gruijl
Human dendritic cell line models for DC differentiation and clinical DC vaccination studies
J. Leukoc. Biol.,
December 1, 2008;
84(6):
1364 - 1373.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Illario, M. L. Giardino-Torchia, U. Sankar, T. J. Ribar, M. Galgani, L. Vitiello, A. M. Masci, F. R. Bertani, E. Ciaglia, D. Astone, et al.
Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells
Blood,
January 15, 2008;
111(2):
723 - 731.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Tanijiri, T. Shimizu, K. Uehira, T. Yokoi, H. Amuro, H. Sugimoto, Y. Torii, K. Tajima, T. Ito, R. Amakawa, et al.
Hodgkin's Reed-Sternberg cell line (KM-H2) promotes a bidirectional differentiation of CD4+CD25+Foxp3+ T cells and CD4+ cytotoxic T lymphocytes from CD4+ naive T cells
J. Leukoc. Biol.,
September 1, 2007;
82(3):
576 - 584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. A. Lyakh, M. Sanford, S. Chekol, H. A. Young, and A. B. Roberts
TGF-{beta} and Vitamin D3 Utilize Distinct Pathways to Suppress IL-12 Production and Modulate Rapid Differentiation of Human Monocytes into CD83+ Dendritic Cells
J. Immunol.,
February 15, 2005;
174(4):
2061 - 2070.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Lindner, M. A. Kharfan-Dabaja, E. Ayala, D. Kolonias, L. M. Carlson, Y. Beazer-Barclay, U. Scherf, J. H. Hnatyszyn, and K. P. Lee
Induced Dendritic Cell Differentiation of Chronic Myeloid Leukemia Blasts Is Associated with Down-Regulation of BCR-ABL
J. Immunol.,
August 15, 2003;
171(4):
1780 - 1791.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Masterson, C. C. Sombroek, T. D. de Gruijl, Y. M. F. Graus, H. J. J. van der Vliet, S. M. Lougheed, A. J. M. van den Eertwegh, H. M. Pinedo, and R. J. Scheper
MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors
Blood,
June 28, 2002;
100(2):
701 - 703.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. A. Lyakh, G. K. Koski, H. A. Young, S. E. Spence, P. A. Cohen, and N. R. Rice
Adenovirus type 5 vectors induce dendritic cell differentiation in human CD14+ monocytes cultured under serum-free conditions
Blood,
January 15, 2002;
99(2):
600 - 608.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. B. Faries, I. Bedrosian, S. Xu, G. Koski, J. G. Roros, M. A. Moise, H. Q. Nguyen, F. H. C. Engels, P. A. Cohen, and B. J. Czerniecki
Calcium signaling inhibits interleukin-12 production and activates CD83+ dendritic cells that induce Th2 cell development
Blood,
October 15, 2001;
98(8):
2489 - 2497.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. A. Lyakh, G. K. Koski, W. Telford, R. E. Gress, P. A. Cohen, and N. R. Rice
Bacterial Lipopolysaccharide, TNF-{alpha}, and Calcium Ionophore Under Serum-Free Conditions Promote Rapid Dendritic Cell-Like Differentiation in CD14+ Monocytes Through Distinct Pathways That Activate NF-{kappa}B
J. Immunol.,
October 1, 2000;
165(7):
3647 - 3655.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION