Interferon-a Overrides the Deficient Adhesion of

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Interferon-a Overrides the Deficient Adhesion of Chronic Myeloid Leukemia
Primitive Progenitor Cells to Bone Marrow Stromal Cells
By Charles Dowding, Ai-Pu Guo, Jurgen Osterholz, Martin Siczkowski, John Goldman, and Myrtle Gordon
Primitive blast colony-forming cells (BI-CFC) from chronic
myeloid leukemia (CML) patients are defective in their attachment t o bone marrow-derived stromal cells compared with
normal BI-CFC. We investigated the effect of recombinant
interferon-% (IFN-a) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells
by culturing normal stromal cells with IFN-a (50 t o 5,000
U/mL). At 50 U/mL we found that: (1)the capacity of stromal
cells to bind two types of CML primitive progenitor cells
(BI-CFC and long-term culture-initiating cells) was increased;
and (2)the amount of sulfated glycosaminoglycans (GAGS)in
the stromal layer was increased. However, sulfated GAGS
were not directly involved in binding CML BI-CFC, unlike
binding by normal BI-CFC, which is sulfated GAG-dependent.
R
ECOMBINANT interferon-a (IFN-a) has proved to
be useful in the treatment of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML). About
70% of patients show long-lasting hematologic control.’ In
some responding patients, IFN-a permits the re-emergence
of a significant degree of Ph-negative hematopoiesis,‘.’ but
the mechanism of its apparently selective action in CML is
unknown. IFN-a has a direct antiproliferative effect on
both normal and CML progenitor cells in vitro but no
selective anti-leukemic activity on progenitor cell proliferation has been dem~nstrated.”~
In contrast to IFN-a, recombinant IFN-y has proved to be ineffective in treating
or in combinapatients with CML either as a single agent5*6
tion with IFN-a.’
The bone marrow (BM) microenvironment is a possible
alternative target for IFN-a. The binding of CML progenitor cells to stromal cells in vitro is deficient,* in vivo this
deficiency could lead to release of progenitor cells into the
circulation and hematopoiesis in extramedullary sites. We
therefore speculated that IFN-a might act by modifying the
interaction between CML and stromal cells in a way that
allows normal microenvironmental control to be restored.
Accordingly, we investigated the binding of CML progenitor cells to stromal cells cultured with IFN-a or IFN-y and
the effect of IFN-a on the proliferation of Ph-positive and
Ph-negative long-term culture-initiating cells.
We also investigated the influence of IFN-a and IFN-y
on extracellular matrix (ECM) synthesis by stromal cells.
Heparan sulfate and chondroitin sulfate, both sulfated
glycosaminoglycan (GAG) molecules, are highly negatively
charged ECM components identified in cultures of BMderived stromal cells? Heparan sulfate has been implicated
in normal hematopoietic cell-stromal cell interactions” and
as a growth factor sequestrator within the ECM of stromal
cells.” Another negatively charged molecule, neuraminic
acid, is also involved in cell-to-cell interaction~.’~”~
Therefore, we investigated the effects of IFN-a and IFN-y on (1)
the synthesis of sulfated GAG by stromal cells, and ( 2 ) the
role of sulfated GAG and neuraminic acid molecules in the
attachment of primitive myeloid progenitor cells to BMderived stromal cells.
Blood, Vol78,
No 2 (July 15). 1991: pp 499-505
Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of
IFN-a, whereas the binding t o IFN-ortreated stromal cells
was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells t o
INE-ortreated stromal cells might be due t o qhanges in the
neuraminic acid composition in the stromal cell layer. Our in
vitro evidence may provide insights into the mechanism of
action of IFN-a in vivo. Prolonged administration may alter
the marrow microenvironment in some patients such that it
can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative
stem cells t o function normally.
o 1991by The American Society of Hematology.
MATERIALS AND METHODS
Samples. Peripheral blood cells from 13 patients with Ph
chromosome-positive CML in chronic phase were collected before
initiation of therapy with cytotoxic drugs. The median white blood
cell (WBC) count for the patients investigated was 198 x lOPLand
ranged from 124 to 287 x 109/L.Normal BM cells collected from
iliac crests of donors of marrow for allogeneic transplantion were
used as controls. Informed consent was given to use samples in
experimental procedures. For most experiments mononuclear cell
fractions (MNCs) from blood or marrow were prepared by centrifugation over Lymphoprep (density 1.077 gimL; Nycomed, Oslo,
Norway) according to the method of Boyum.14 For the long-term
culture experiments, MNCs were prepared by centrifugation (400g,
30 minutes) over iso-osmotic Percoll (density 1.065 g/mL; Pharmacia, Uppsala, Sweden). MNCs prepared by both techniques were
washed twice in Hanks’ balanced salt solution (HBSS; GIBCO,
Paisley, UK) before use.
Stromal cell cultures with IFN-a or IFN-y. Stromal layers were
grown by plating 5 x lo5 normal BM MNCs per milliliter in a
medium (GIBCO) containing fetal calf serum (FCS; 15%, GIBCO),
methylprednisolone (1.6 x
mol& Upjohn, Crawley, UK),
with (50 to 5,000 UImL) or without recombinant IFN-(y, (2 X 10’
Ulmg protein; Roferon, Hoffman La Roche, Basel, Switzerland) or
with (25 to 100 UImL) or without recombinant IFN-y (2 to 4 X lo8
U/mg protein; Biogen S.A., Basel, Switzerland) in 35”
petri
dishes or 25-cm’ flasks (GIBCO). Cultures were fed weekly by total
replacement of medium and additives. Stromal layers achieved
confluence after 3 to 5 weeks and were used in the procedures
described below. The effect of IFN-a (50 to 5,000 UImL) on
From the MRCILRF Leukaemia Unit, Royal Postgraduate Medical
School; and the Leukaemia Research Fund Centre, Chester Beatty
Laboratories, London, UlC
Submitted December 6, 1990; accepted March 21, 1991.
Supported by the Medical Research Council and the Leukaemia
Research Fund of Great Britain.
Address repnnt requests to Charles Dowding, MD, BSc, MRCILRF
Leukaemia Unit, Royal Postgraduate Medical School, Hammersmith
Hospital, Du Cane Rd, London W12 ONN, UK.
The publication costs of thls 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 I734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971I9117802-0034$3.0010
499
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500
stromal cell colony formation was assayed using a modification of a
standard CFU-F assay.” Stromal cultures were initiated and
colonies composed of 2 50 cells were scored on day 10. Colonies of
cells with fibroblastoid morphology were identified by staining with
hematoxylin for 2 minutes. In one representative experiment all
colonies stained positively with alkaline phosphatase, confirming a
fibroblastoid phenotype.’&
Attachment of progenitor cells to stromal layers. Adherent cells
were removed from normal BM or CML peripheral blood MNCs
by incubating 4 x loh cells in a medium (8 mL) containing FCS
(15%) for 2 hours in 25-cm’ tissue culture flasks. The nonadherent
MNCs were assayed for blast colony-forming cells (BI-CFC) using
the standard assay previously described.” In brief, the nonadherent
equivalent of 5 x 10’ MNCs was added to each petri dish culture of
stromal cells (three replicate dishes per experimental point) and
were incubated for 2 hours. Cells not attached to the stromal cells
were decanted and the stromal layers were washed vigorously three
times with HBSS, overlaid with a medium (1 mL) containing FCS
(15%) and agar (0.3%; Difco Laboratories, Detroit, MI), and
incubated at 37°C in 4% CO, in humidified air. Colonies were
counted 5 or 6 days later and dishes were desiccated and stained
with May-GrunwaldiGiemsa to identify colonies composed of blast
cells. In different experiments, stromal cells grown ? IFN-a, f
IFN-y, or f enzyme treatment (see below) were used.
The binding efficiency of CML BI-CFC was determined by
measuring the recovery of BI-CFC following incubation of cell
suspensions with stromal layers. Adherent cell-depleted CML
MNCs were incubated (‘panned’) on stromal cells (primary) for 2
hours. Unattached cells were removed by sucking the medium up
and down 10 times with a Pasteur pipette and were then transferred to a second dish of control stromal cells (secondary) and
incubated for a further 2 hours. Blast cell colonies were scored in
both primary and secondary cultures and the attachment of CML
BI-CFC to primary stromal cells as a proportion of the primary +
secondary total was calculated.18
To determine the recovery of normal or CML burst-forming
unit-erythroid (BFU-E) and granulocyte-macrophage colonyforming cells (GM-CFC) from stromal cells cultured ? IFN-a (50
U/mL), the nonadherent equivalent of 5 x 16‘ normal or CML
MNCs were assayed directly using standard clonogenic techn i q u e ~ ’or~ ‘panned’ for 2 hours on stromal cells before assay.
Unattached cells were decanted from the stromal layers and the
number of BFU-E and GM-CFC was calculated.
Attachment to and culture of long-term culture (LTC)-initiating
cells with stromal cells grown f IFN-oL In replicate experiments,
MNCs (4 x lob, of density I1.065 g/mL) from four newly diagnosed CML patients were suspended in a medium (8 mL)
containing FCS (15%) and were then added to flasks (25 cm2) of
irradiated (30 Gy, I3’Cs source) normal allogeneic stromal cells
grown f 50 U/mL IFN-a from culture initiation. After 2 hours the
flasks were rocked ten times and the nonadherent cells (‘2h
non-adh’) were removed, and the remaining cells attached to
stromal cells were cultured in LTC medium (8 mL) consisting of a
medium containing horse serum (12.5%), FCS (12.5%),methylprednisolone (2 x
mol/L) and ? IFN-a (50 U/mL). The ‘2h
non-adh’ cells were pelleted, resuspended in LTC medium, and
added to identical (secondary) flasks of irradiated stromal cells
matched with the first according to exposure to IFN-a. LTCs set up
using IFN-a-treated stromal cells were maintained in the presence
of IFN-a (50 U/mL), whereas LTCs set up with control stromal
cells were maintained without IFN-CY.
Cultures were incubated at
33°C in 4% CO, in humidified air. On days 7, 17, and 27 the
cultures were demi-depopulated and re-fed with fresh medium
plus additives. In addition, cultures were harvested by trypsinization (1:20 in EDTA, 15 to 30 minutes at 37°C; GIBCO) of the
DOWDING ET AL
adherent layer and the numbers of GM-CFC per flask calculated.
The numbers of GM-CFC on day 27 were presumed to reflect the
numbers of LTC-initiating cells seeded because at this time few of
the original GM-CFC seeded remain.”
Generation of Ph-positivehegative GM-CFC. Individual granulocyte-macrophage colonies derived from the 27-day-old LTCs from
the same four CML patients studied above were analyzed cytogenetically using a standard technique with minor modifications.*’In
brief, on days 7 to 9 of culture a mixture of recombinant
granulocyte-macrophage colony-stimulating factor (GM-CSF; 1,000
UlmL; Genetics Institute, Cambridge, MA) and recombinant
interleukin-3 (IL-3; 1,000 UlmL; Genetics Institute) in a 50 p L
volume was added to a 35-mm dish containing the colonies. After
24 hours incubation at 37”C, colcemid (50 pL, 5 pg/mL; GIBCO)
was added dropwise to the culture and was incubated for 2 to 4
hours at 37°C. Individual colonies were then plucked from the
culture and resuspended in a droplet of KCI (0.075 mol/L) placed
on a poly-L-lysine-treated slide. After 30 minutes the colony was
fixed in methanol-acetic acid (3:1)2’and stained with Giemsa stain
and the chromosomes were analyzed. For ‘direct’ cytogenetic
analysis of cells from LTC, colcemid (40 pL, 50 pg/mL) was added
to the culture, and after 4 hours the cells were pelleted and
resuspended in KCI (0.085 mol/L). After 30 minutes of incubation
at 37”C, the cells were pelleted, fixed in methanokacetic acid (3:1),
and stained with Giemsa stain.
Sulfated GAG in stromal layers. To estimate the amount of
sulfated GAG in stromal layers grown ? IFN-a or ? IFN-y,
confluent cultures (in 25-cmZflasks or 35-mm dishes) were incubated for 72 hours in fresh culture medium f IFN-a (50 to 500
UlmL) or IFN-y (25 to 100 UlmL) containing [”SI sulfate (50
pCi/mL. specific activity 20 to 40 Ciimmol; Amersham, Aylesbury,
UK). The medium was then decanted and the cell layer was rinsed
three times in a medium and incubated in fresh medium for a
further 4 hours at 37°C to chase free [’’SI. The stromal cells were
washed three times, air dried, and the sides were cut off the culture
vessel. The surface to which the stromal cells were attached was
then placed against an X-ray film (X-Omat; Kodak, Rochester,
NY) within a cassette. The autoradiographs were developed after 4
to 12 hours. The intensity of the film blackening gave an indication
of the [”SI sulfate incorporation by GAGs in the stromal layer
because 90% of sulfate is used in sulfation of GAG molecules2*
with only a small proportion incorporated into glycoprotein molecules. ’’Autoradiographs were scanned using a light densitometer
(Chromoscan 3; Joyce-Loebl, Gateshead, UK). Quantitation of
[”SI sulfate incorporation into the stromal cell layer was also
measured using G25 gel filtration. Fractions were counted on an
LKB 1217 Rackbeta liquid scintillation counter (Pharmacia, Uppsala, Sweden) . The amount of incorporated isotope was determined from the isotope in the first of the two peaks.
Role of GAGs or neuraminic acid in binding CML BI-CFC.
Stromal layers (grown f 50 UimL IFN-a) were incubated with
neuraminidase (isolated from Clostridium perfringens, 0.1 U/mL;
Sigma), heparanase ( 5 UlmL Sigma), or chondroitinase ABC (0.1
U/mL; Sigma, St Louis, MO) dissolved in a medium, for 30 minutes
at 37°C. Control cultures were incubated with CY medium alone.
The stromal cells were rinsed three times in a medium and used in
the B1-CFC assay (above).
Statistics. Dr R. Szydlo (MRC/LRF Leukaemia Unit) performed the statistical analyses. A Student’s t-test was used to
compare differences between colony numbers under various culture conditions. A paired t-test was used to evaluate the effect of
IFN-a on the binding of LTC-initiating cells to stromal cells. The
IFN-a dose-response curve was evaluated using an analysis of
variance with repeated measures. A xz test was used to compare
the effect of IFN-a on generation of Ph-negative GM-CFC in LTC.
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IFN-a ON STROMNPROGENITOR INTERACTIONS
501
RESULTS
Effect of IFN-a and IFN-y on stromal cell capacity to bind
progenitor cells. The formation of blast cell colonies on
IFN-a-treated (50 U/mL) stromal cells by CML
(169.5% ? 21.5% of the control, mean ? SEM, P < .05;
Fig lA), but not by normal, Bl-CFC (104.7% ? 10.2%) was
increased. At higher IFN-a concentrations, there was a
non-significant increase in colony formation by both normal
and CML Bl-CFC (data not shown). We sought to confirm
that the peak in CML blast colony numbers detected on
IFN-a (50 U/mL)-treated stromal cells resulted from increased numbers of Bl-CFC attaching rather than an
increased ability of stromal cells to support colony formation. In 'panning' experiments comparing binding to IFN-atreated and -untreated stromal cells, the proportion of
CML BI-CFC attaching to IFN-a-treated stromal cells in 2
hours was 81% compared with 40% attachment to untreated stromal cells (Fig 1B). In contrast, blast colony
formation by CML Bl-CFC on IFN-y-treated (25 to 100
U/mL) stromal cells showed a dose-dependent decrease
(Fig 1C). At 100 U/mL of IFN-y, blast colony numbers were
reduced to 37.1% of the control (P < .05, n = 3) compared
with the twofold increase found in the presence of IFN-a.
The effects of IFN-a on progenitor cell binding were
further tested in the LTC system. The presence of IFN-a
"1
A
i
Normal
CML
- IFN-a + IFN-a
B
f
C
D
0
Days
Fig 2. Numbers of GM-CFC/flask in four LTCs of CML peripheral
blood cells of density less than 1.065 g/mL seeded onto irradiated
normal BM stromal cells. See Materials and Methods for culture
details. ( 0 )Control (no IFN-a). ( 0 ) IFN-a (50 U/mL).
+
(50 U/mL) during the period of LTC had no significant
effect on the relative numbers of LTC-initiating cells
detected for any of the four patients studied (Fig 2 and see
below). Using cells from these four CML patients, the mean
proportion of LTC-initiating cells (d I1.065 g/mL) that
bound to IFN-a-treated stromal cells (50 U/mL) during 2
hours of incubation was 0.59 compared with 0.38 on control
stromal cells (P < .OS, n = 4; Fig 1D). Stromal cells cultured with IFN-a (50 U/mL) slightly increased the attachment, though not significantly,of CML GM-CFC or BFU-E
during a 2-hour 'panning' procedure (89.0% rt 12.1% and
85.8% f 10.3% of numbers recovered from control stromal
cells, respectively; mean rt SEM, n = 5 for both).
Effect of IFN-a (50 UImL) on proliferation of CML cells in
LTC. The total numbers of GM-CFC, in the 2h-adherent
and -nonadherent fractions combined, after 27 days of LTC
of four CML samples are shown in Fig 2. There was little or
no effect on the total numbers of GM-CFC detected after
27 days of LTC despite the altered distribution of LTCinitiating cells between the 2h-adherent and -nonadherent
cultures. The generation of Ph-negative GM-CFC colonies
in 27-day-old LTCs grown ? IFN-a (50 U/mL) is shown in
Table 1. In three of four patients, only Ph-positive GMCFC were detected before LTC. In the fourth patient a
small proportion of Ph-negative GM-CFC were detected
1
-IFN-u +IFNU
Fig 1. Binding of primitive progenitor cells to stromal cells
grown f IFN-a or 2 IFN-y during 2 hours of incubation. (A) Blast
colony formation by normal BM MNCs ( 0 )(mean f SEM, range 22 to
49/1@ MNCs on control stromal cells, n = 7) or CML peripheral blood
MNCs (0) (mean f SEM, range 44 to 121/106 MNCs on control
stromal cells, n = 13) on stromal cells grown with IFN-a(50 U/mL). (B)
Recovery of CML blast colony forming cells from stromal cells grown
f IFN-a (mean f SEM, range 51 to 131/106 MNCs on control stromal
cells, n = 3). (C) CML blast colony formation on stromal cells grown
with IFN-y (mean f SEM, range 37 to 120/10* MNCs on control
stromal cells, n = 3). (D) Relative number of LTC-initiating cells
binding to stromal cells grown with IFN-a (50 U/mL). (*)P < .05 level
of significance compared with control.
Table 1. The Effect of IFN-a on the Generation of Ph-Negative
GM-CFC in LTC of CML Peripheral Blood
% Ph-Negative GM-CFC (coloniesanalyzed)
Patient
No.
1
2
3
4
PreCulture
0
4.2
0
0
(78)
(24)
(30)
(46)
Post-Culture*
- IFN-a
+ IFN-a
4.1 (73)
6.3 (206)t
0 (47)
0 (52)
6.0 (50)
7.5 (187)t
See Materials and Methods for culture details.
"Day 27 of LTC.
tOnly direct metaphases analyzed.
0 (64)
5.3 (75)
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502
DOWDING ET AL
I
II
111
Fig 3. Effect of IFN-a or IFN-y on stromal cells. (A)
Colony formation by normal B M stromal cells
grown r IFN-a (mean f SEM, range 20 to 42 colonies/
lo6 B M MNCs, n = 3). (6) [35S]sulfate incorporation
by stromal cells grown f IFN-a in 25-cmZflasks. I, no
IFN-a; II, 50 U/mL; 111, 100 U/mL; IV, 500 UlmL. Film
exposed for 8 hours. (C) ['5S] sulfate incorporation by
stromal cells grown f IFN-y in 35-mm petri dishes. 1,
no IFN-y; II, 25 U/mL; 111.50 U/mL; IV, 100 U/mL. Film
exposed for 4 hours. See Materials and Methods for
culture and metabolic labeling details.
IV
IFN-N (UlmL)
A
B
C
before LTC. After LTC without IFN-a, a small proportion
of Ph-negative GM-CFC (one patient) and Ph-negative
metaphases in an experiment where colony analysis failed
(one patient) were identified. In two patients, no Phnegative GM-CFC were found. In cultures seeded on
IFN-a-treated stromal cells and grown in the presence of
IFN-a, the proportion of Ph-negative GM-CFC or
metaphases detected was increased over that in control
cultures. In patient 4, whose preculture cells and postcontrol culture cells contained no Ph-negative GM-CFC,
there were 5.3% Ph-negative GM-CFC fotlowing LTC with
IFN-a. Overall, the differences did not achieve statistical
significance, but in no case was the number of Ph-negative
GM-CFC lower in cultures grown on IFN-a-treated stromal cells.
Effect of IFN-a and IFN-y on stromal cell proliferation and
GAG synthesis. Normal BM-derived stromal cell colony
formation was unaffected by culture with IFN-a at concentrations I100 U/mL (Fig 3A). At concentrations of 2 100
U/mL there was a dose-dependent reduction in stromal cell
colony numbers (P < .02).
Representative autoradiographs showing ["SI sulfate
incorporation by stromal layers cultured with IFN-a or
IFN-y are shown in Fig 3B and C. The intensity of film
blackening corresponded to the amount of sulfate incorporated into GAG. Figure 3B shows that at 50 to 100 U/mL,
IFN-a increased the amount of sulfated GAG in the
stromal layer compared with controls, whereas at higher
concentrations the amount of sulfated GAG was reduced in
parallel with inhibition of proliferation (Fig 3C). Densitometric scanning of the autoradiograph at 50 U/mL IFN-a
showed a 39% increase in intensity compared with the
control. Quantitation using G25 chromatography indicated
a mean 25% increase in sulfated GAG incorporation into
the stromal layer (Table 2). Incubation of stromal cells with
IFN-y (25 to 100 U/mL) did not appear to alter the amount
of sulfated GAG in the stromal layer (Fig 3C).
Involvement of stromal cell-derived GAGs and neuraminic
acid on attachment by CML Bl-CFC. Stromal layers
grown 2 IFN-a (50 U/mL) were stripped of sulfated GAGs
and neuraminic acid to see if there was any relationship
between the effects of IFN-a on these matrix components
and its effect on CML BI-CFC binding. The results of these
experiments are shown in Fig 4. Neither heparanase nor
chondroitinase ABC treatment of stromal cells had a
significant effect on attachment of CML Bl-CFC to stromal
cells grown 2 IFN-a, but neuraminidase treatment of
control stromal cells (ie, without IFN-a) increased blast
colony formation by CML cells (P < .05; Fig 4). Thus, the
enhancing effect of IFN-a on CML binding to stromal cells
was reproduced by treating control stromal cells with
neuraminidase. Consequently, the results are consistent
with IFN-a acting via modulation of the neuraminic acid
content in the hematopoietic microenvironment.
Table 2. p5S]Sulfate Incorporation Into Stromal Layers
Grown f IFN-a
Counts Per Minute
Experiment
No.
Control
(no IFN-N)
IFN-N
(50 UlmL)
1
13,910
2
77,967
3
54.91 7
4
15,884
18,079
(130.0%)'
80,871
(103.7%)
68,519
(124.8%)
22,492
(141.6%)
Background counts less than 30. See Materials and Methods for
labeling details.
*Percentage of control (no IFN-a) counts.
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IFN-CXON STROMNPROGENITOR INTERACTIONS
*
*
T,*
T *
cho
503
***
neu
Fig 4. Blast colony formation by CML peripheral blood MNCs on
stromal cells grown 2 IFN-a (50 UlmL) and 2 treatment: hep,
heparanase (n = 3); cho. chondroitinase (n = 2); neu, neuraminidase
(n = 4). Mean values k 1 standard deviation. Mean and range in
control colony numbers were 82 and 66 to 118/14 MNCs, respectively. ("I P < .05 level of significance compared with 'no IFN-a'
control. See Materials and Methods for conditions for enzyme treatment. (Dl no IFN-a; (8)no IFN-a treatment;).(
+ IFN-a;) . (
IFN-a + treatment.
+
+
DISCUSSION
A selective anti-leukemic activity of IFN-a has been
identified in a significant proportion of patients with CML','
but there is no evidence that this is due to a direct
cytostatic/cytotoxiceffect on the leukemic clone. We considered, therefore, that in addition to exerting a nonspecific
antiproliferative effect on normal and malignant cells,
IFN-a might act by modifying the BM microenvironment.
The microenvironment consists of a complex network of
cells with their associated extracellular matrix and growth
factors and is thought to play an active role in the regulation
of normal hematopoiesis. However, the Ph-positive stem
cell population in CML escapes this control.% We have
previously shown that a population of primitive progenitor
cells (Bl-CFC) found in the BM and blood of patients with
CML is deficient in its ability to bind to BM-derived stromal
cells. Moreover, CML Bl-CFC are also less discriminatory
than normal Bl-CFC because normal cell binding is restricted to stromal cells grown with methylprednisolone,
whereas CML cells bind, albeit inefficiently, to stromal cells
grown with or without methylprednisolone.' This abnormal
attachment of CML primitive progenitor cells could underlie some of the pathologic features such as: (1) lack of
responsiveness to microenvironment-derived control factors,% (2) inappropriate release from the BM, and (3)
extramedullary hematopoiesis. It follows that any maneuver that tends to normalize the adhesive interaction between CML progenitor cells and microenvironmental cells
might reinstate a more physiologic regulation of leukemic
hematopoiesis.
In this study we showed that IFN-a, but not IFN-y,
increased the attachment of two different types of CML
primitive progenitor cells (Bl-CFC and LTC-initiating cells)
to stromal cells. The increased efficiency in binding by CML
BI-CFC approached the efficiency reported for normal
Bl-CFC of which greater than 90% bind to stromal cells.' In
this previous study designed to determine the rate of
attachment of B1-CFC to stromal cells using serial 2-hour
panning steps, approximately 90% of normal BM-derived
BI-CFC attached to stromal cells during the first 2-hour
incubation step. However, apparently only about 40% of
the total CML B1-CFC adhered to stromal cells during the
first 2-hour panning step because progressively decreasing
numbers of B1-CFC could be detected by re-panning the
nonadherent cell fraction on stromal cells for further
2-hour incubations. Blast colony-forming cells from CML
patients did not have a different binding rate to stromal
cells because maximal binding occurred after 2
Instead, once bound, a time-dependent increasing proportion of CML B1-CFC detached upon further incubation in
liquid culture at 37"C, unlike normal Bl-CFC, which remained bound. This result suggests that a kinetic equilibrium between adhesion and detachment favors detachment
for CML Bl-CFC and attachment for normal Bl-CFC.
The enhanced binding was most marked at an IFN-a
concentration of 50 U/mL, which would fall within the
range of concentrations achieved in CML patients treated
with IFN-a. This concentration had no inhibitory effect on
fibroblast colony formation and little or no significant effect
on the proliferation of CML LTC-initiating cells or other
hematopoietic cells? In contrast to primitive progenitor
cells, the attachment of CML GM-CFC and BFU-E to
IFN-a-treated stromal cells during a similar 2-hour 'panning' step (previously shown to be <15% of normal
progenitor cells") was not significantly increased. However,
we have also recently found that IFN-a (100 U/mL) greatly
increased the number of adherent GM-CFC in a 10-day
liquid co-culture of CML BM cells with confluent normal
stromal cells,26suggesting that under different conditions
the attachment or release of lineage-committed progenitor
cells might also be enhanced by IFN-a.
In two of four patients, between 4% to 8% of the
circulating LTC-initiating cells were Ph-negative as previously reportedz4 and in three of four patients IFN-a
increased, though not significantly, the proportion of Phnegative GM-CFC detected on day 27. These IFN-amediated increases in Ph-negative GM-CFC detected could
reflect differences in the responses of Ph-positive and
Ph-negative LTC-initiating cells to the direct effect of
IFN-a or could reflect an effect through modification of the
interaction with stromal cells. If the modification of primitive progenitor cell-microenvironmental cell interactions by
IFN-a plays a role in the anti-leukemic process, microenvironment-derived negative regulators of hematopoiesis,
which might include stem cell inhibitory factor," transforming growth factor+,= or tumor necrosis factor-a,29might
then annul the proliferative advantage usually expressed by
leukemic cells.
The cell adhesion molecules (CAMS) and their receptors
involved in attachment of primitive progenitor cells to BM
microenvironmental cells are poorly characterized. He-
From www.bloodjournal.org by guest on October 25, 2016. For personal use only.
504
DOWDING ET AL
m~nectin,~'
fibronectin," thrombospondin;' and ad@,
integrin33may have a role in binding primitive progenitor cells.
Heparan sulfate, found in the ECM of stromal cell cultures," has been implicated in binding normal BI-CFC to
stromal cells."' Chondroitin sulfate is the major GAG in
r ~ d e n t ~and
'.~~
human' stromal cell-derived ECM and has
been identified in the membrane of murine factordependent cloned progenitor cells?' Roles for chondroitin
sulfate in the regulation of proliferation38s39
and the process
of detachment of progenitor cells from stromal cells" have
been proposed. We therefore investigated whether (1)
stromal cell-derived heparan sulfate or chondroitin sulfate
was involved in the attachment by CML BI-CFC to stromal
cells, and (2) whether the increased attachment of CML
BI-CFC to IFN-a-treated stromal cells was due to an
alteration in the synthesis or availability of sulfated GAG.
We found that IFN-a increased the total amount of sulfated
GAG (heparan sulfate + chondroitin sulfate) but enzymatic removal of either GAG from stromal cells did not
significantly affect CML BI-CFC attachment, suggesting
that sulfated GAGS do not have a primary role in the
attachment of CML BI-CFC to stromal cells.
Neuraminic acid (sialic acid) is a negatively charged
terminal nonreducing sugar important in some hematopoietic cell-stromal cell intera~tions.''~'~
Stromal cells of LTCs
can bind neuraminic acid: but its function within the
stromal layer is unknown. A role for neuraminic acid in the
IFN-a-induced changes in binding was suggested by results
of enzymatic stripping of neuraminic acid from control
stromal cells. This resulted in a significant increase in CML
attachment but had no significant effect on the ability of
IFN-a-treated stromal cells to bind CML BI-CFC. Whether
IFN-a qualitatively modifies or reduces the neuraminic acid
content in stromal cell ECM remains to be determined. The
influence of neuraminic acid and the lack of involvement of
heparan sulfate in the improved binding by CML cells to
stromal cells indicates that IFN-a probably does not restore
the normal cell adhesion mechanism because this would be
heparan sulfate-dependent. Instead, IFN-a might facilitate
the operation of a different mechanism of adhesion because
cells express a variety of cell adhesion molecules with
apparently overlapping functions."' Also, we have recently
demonstrated that CML BI-CFC lack a phosphatidylinositolanchored CAM43that is an essential requisite for efficient
and selective binding by normal BI-CFC. It is possible that
expression of CAMS by hematopoietic progenitor cells
controls their precise location in the marrow microenvironment so that they are properly regulated." The operation of
a different cell adhesion mechanism by CML Bl-CFC, in the
context of stromal cells exposed to IFN-a, raises the
possibility that the immobilized leukemic cells could be
placed at a proliferative disadvantage because they are
subjected to inappropriate rather than normal control
mechanisms.
In summary, we have presented evidence for a functional
difference in the effect of IFN-a on normal and CML
progenitor cells that may prove to be associated with the
finding of IFN-a-induced normalization of nuclear proteinDNA complexes observed in IFN-a-sensitive leukemic
cells.@ To a great extent IFN-a overcame the deficient
attachment of CML BI-CFC to stromal cells and increased
the proportion of LTC-initiating cells that attached to
stromal cells. This effect was associated with changes in the
neuraminic acid content of the stromal layer. It is possible
that the mechanism of action of IFN-a in CML patients
might include modification of BM microenvironmental cells
such that the interaction between hematopoietic cells and
stromal cells is enhanced; this could lead to suppression of
the leukemic clone and thus allow re-emergence of normal
hematopoiesis.
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1991 78: 499-505
Interferon-alpha overrides the deficient adhesion of chronic myeloid
leukemia primitive progenitor cells to bone marrow stromal cells
C Dowding, AP Guo, J Osterholz, M Siczkowski, J Goldman and M Gordon
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