From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Effect
of Acidic
and
Basic
Isoferritins
on In Vitro
Granulocyte-Monocyte
By Laura
Acidic
isoferritins
potent
have
inhibitors
trations
that
acidic
been
to
10_la
the
mol/L,
To
and
liver
ferritin
degree
fled
both
fernitin
from
did
sylated
preparation.
i0
ERRITIN
tions
and
to another
in
subunits
mate
are
most cells,
with
arranged
as hollow
whereas
cells,
not
including
Multiple
weight
of 500,000!
fashioned
These
may
isofernitins
found
the
observed
other
did
func-
to the
iron
isoferritins
S
A.
1986
Grune
p1)
and
and
L-subunit-rich
The
predominate
plasma,
acidic
in iron
whereas
p1) and
storage
organs
the
H-subunit-rich
occur
in tissues
are basic
(liver
acidic
progenitor
work,
we
and
spleen)
are
nonheme
iron
(heart,
erythroid
cells, monocytes,
lymphocytes)
and some malignant
tissues. This model for
and
tissue
ferritin
phenotypes
validated
based
by
the
on two distinct
demonstration
are encoded
by distinct
recent
evidence
for the
messenger
existence
subunit
species has
that the two subunits
RNAs
(mRNAs)5
and
of at least two ferritin
However,
the matter
could
be more
complex
a glycosylated
G subunit
(molecular
weight
about
has been isolated
from human serum.9 It is possible
genes.8
because
23,000)
to evaluate
basic
and
acidic
isoferritins
lymphocyte
has been
and
of myelopoiesis.
control
tory
there
migration’2
activity
monocyte
(LIA)
evidence
ofa role for ferritin
in
function’3
as well as in the
T cell
In fact,
directed
progenitors’4
a leukemia-associated
against
was identified
human
inhibigranulocyte-
by Broxmeyer
and
associates
as acidic isoferritins.’5
Acidic
isoferritin
activity
(AIFIA)
was later
found
in bone marrow
and
cells
monocyte-
Blood,
of normal
donors’6
Vol 67, No 3 (March),
and
a specific
1986:
pp 789-795
role
for
not
than
conditions
significance
of
regulaHow-
because
of certain
inconsistenof human
isoferritins.’7
have
studied
the
effects
and immunologic
of
different
granulocyte-macrothese
effects
characteristics
with
of the ferritin
tested.
MATERIALS
Purification
preparations
and
The
H.
Table
METHODS
heart,
and HeLa
made and
were
characteristics
coded
of these
Eight
ceilferritin.
as A,
B, C,
isoferritins
D,
E,
F, G,
are reported
in
The procedure for purification
of human
liver ferritin
(preparaA and B) was essentially
the same as previously
described
in
detail by Arosio et al.’8 It consisted
of heat extraction
at 75 #{176}C,
tions
ammonium
From
di
sulfate
precipitation
the C/mica
Medicina
Milano.
Medica
Interna
Dipariimento
(60%
saturation),
Medica.
e Tecnologie
Biomediche.
Pavia.
and
Universit#{225} di
Italy.
zione
June
Italiana
recipient
18, 1 985; accepted
per
the
Address
Ia Ricerca
sul
ofafellowshipfrom
Presented
Proteins
II.
at
Dipartimento
Universit#{225} di
Sept
27. 1985.
Supported
in part by Grants
84.00415.44
CNR,
Progetto
Finalizzato
Oncologia.
and
and
precipitation
A, Llniversit#{224} di Torino,
e Terapia
di Scienze
Submitted
his
blood
main
AND
liver,
1.
Experimental
inhibitory
ofhurnan
ferritin
using
separately
immunoassays
based
on monospecific
antibodies
raised
against
L-subunit-rich
and H-subunit-rich
molecules.’#{176}
Although
ferritin
function
is inextricably
linked
to iron
metabolism,”
normal
as feedback
was suggested.’6
cells
isoferritins
on the growth
of cultured
phage
precursor
cells
and
correlated
biochemical
are
higher
is uncertain.
isoferritins
ever, this has been disputed
cies with the present picture
this
this
are
Inc.
macrophage-derived
(high
isoferritins
of low
activity
of acidic
to concana-
for
under
is in
of colony
bind
physiologic
have
this
propor-
content
in fetal
been
(low
isoferritins
both
the
& Stratton.
tions of two distinct
subunit
classes, heavy (H) and light (L),
with molecular
weights
of approximately
2 1,000 and 19,000,
respectively.3
blood
At present,
inhibitory
by
and
concentrations
in human
and
preparations
responsible
effective
isoferri-
Inhibition
by some
the
blood.
CFU-GM.
in H subunits
the
heart
basic
in human
evidence.
mechanism(s)
the
of
with
human
that
type
±
whereas
part
against
of human
rich
32%
.01).
significant
indicate
fernitin
tors of hematopoietic
In
are
findings
are
human
of
by preincubation
directed
clinical
preparation
from
(P<
A
2A4
A
inhibition
mol/L
be obtained
The
but
a mean
i0
growth
that
in leukemia.
The strucvary from
different
formation
growth.
removed
indirect
and
consisting
of 24
having
an approxi-
from
with
Aglietta
isoferritins
ineffective.
was
on the
those
of
at
present
glyco8%
produce
predominant
clear.
cell differentiation
forms of ferritin
all molecules
protein
shells
The
valin
major
only
was
cell
the
its
one
of two
fernitin.
and
related
of
±
control
effect
CFU-GM
acidic
antibody
keeping
pun-
5%
the
ie, the
no
and Massimo
of
to
activity
ferritin.
tins,
CFU-GM
of 26%
detoxification.”2
of this molecule
of heteropolymers
molecular
families
and
human
Hela
glycosylated
in all
to other
factors
transformation.3’4
but also
malignant
occur
no
iron
of
One
containing
.02).
<
storage
and
immunologic
properties
one tissue
and
contained
heart.
inhibition
(P
acidic
were
formation.
fernitin.
dif-
and
on
of
11%
effect
found
inhibitory
irrespective
human
a mean
is ubiquitous
are
mol/1,
colony
mel/I
which
effects
was
Levi,
glycosylated
monoclonal
tested
isoferritins
and
heart
showed
at
any
Acidic
in vitro
of human
fernitin,
tural
show
cells
affect
we
any
for
another
colony-forming
preparations
up to i0
Hela
not
control
status
not
of glycosylation.
preparations
F
did
at concentrations
the
the
Three
patho-
Sonia
show
heart
species
(1-subunit-rich)
isoferritins.
(basic)
growth
basic
Arosio,
enriched
in the
in the
Paolo
not
suggested
ferritin
human
highly
concen-
a role
also
the
of
be
been
plays
and
growth
of
to
of Human
Progenitors
Piacibello,
at
(CFU-GM),
preparations
(H-subunit-rich)
it has
activity
unit-granulocyte-macrophage
ferent
found
characterize
in vitro
Wanda
progenitors
hematopoiesis
leukemia.
affect
previously
inhibitory
of normal
of
Cazzola,
hematopoietic
isoferritin
regulation
genesis
that
of
of 1016
Mario
Dezza,
Growth
in part
oflron
14th
at
the
Annual
requests
International
Lille,
Meeting
Hematology,
of
to Dr
Dezza
was
the
Conference
France,
June
the
International
Jerusalem,
Policlinico
S. Maiteo,
27100
© I 986 by Grune
& Stratton,
Dr Laura
in /985.
Seventh
Metabolism,
reprint
Cancro.
AIRC
and 84.00505.44
from
a grant from
Associa-
Mario
Pavia,
Inc.
July
30 to July
on
5, 1985,
Society
for
14 to 18. 1985.
Cazzola,
C/mica
Medica
Italy.
0006-4971/86/6703-0037$03.OO/O
789
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
790
DEZZA
TabI e 1 . Biochemical
and Immunologic
Characteristi
cs of the Isof erritin
ET
AL
Preparations
Immunologic
Reactivity
Relevant
Purification
Name
Origin
Steps
Purity
Liver
Heart
(%)
Type 1%)
Type (%)
Binding
to Con A (%)
A
liver
crystallization
>97
100
0
B
liver
crystallization
>97
100
0
2
C
heart
passage
15
85
0
D
heart
preparative
>97
14
86
5
>97
9
91
0
through
Con A-
95
2
Sepharose
electrophoresis;
no passage through
Con
A-Sepharose
E
HeLa
asinD
cells
F
heart
no heat
extraction;
elution
from
Con A-Sepharose
with
0. 1 mol/L
<5
46
54
100
<5
49
51
100
-.1Q
98
a-methyl
mannoside
G
heart
tissueheatedat75
#{176}Cand
as in F; ultracentri-
then
fuged
H
liver
Abbreviation:
Ferritin
antibody
asinG
Con A, concanavalin
was evaluated
concentration
2A4
against
human
heart
with
ferritin;
immunoradiometric
each
value
was
assays
then
expressed
300,000
g for 90 minutes, gel filtration
on a Sepharose 4B column,
and crystallization
with 5% cadmium
sulfate.
Human heart (preparation
D) and HeLa (preparation
E) ferritins
were purified by similar
procedures
except
that the crystallization
step was omitted and replaced with diethyl aminoethyl
chromatography and preparative
gel electrophoresis
on a polyacrylamide
gra-
dient
(10%
to 30%)
Human
heart
preparation
C was purified
with
a similar
procedure
that the heated homogenate
was passed through a column of
Blue (Bio-Rad
Laboratories,
Richmond,
Calif);
equilibrated
in 20 mmol/L
Tris-HCI,
pH 7.4, and on a column of Con
A-Sepharose
(Pharmacia
Fine
Chemicals,
Uppsala, Sweden);
and
the break-through
peaks collected.
For this preparation,
the preparative gel electrophoresis
step was omitted.
Isoferritin
preparations
were analyzed
by means of isoelectrofosodium
dodecyl
blotting
as previously
appeared
whereas
pure by
preparation
protein
sulfate
(SDS)
electrophoresis,
and immuno-
described’0’5’9
Preparations
A,
native and SDS gel electrophoretic
C contained
5% contaminants.
concentration
was
on polyclonal
antibodies
as a percentage
heart
of the
ferritin
was
monoclonal
Binding
to
determined
by
the
B, D, and E
analyses,
The
method
ferritin
100
et
ferritin
prepared
human
liver ferritin
and the monoclonal
concentration.
as described
by Cavanna
This
determined
et a12’ using
the
2A4.
Con
A.
was
as
previously
detailed.’#{176}
CFU-GM
from
Normal
assayprocedures.
normal
volunteers
low-density
or from
diseases.
bone
Bone
heparin
(50
marrow
cells,
bone marrow
patients
marrow
to 100
chest
cells
were collected
U/mL).
separated
was obtained
undergoing
A quantity
as previously
surgery
in
of 1 x iO
described,23
were plated in a 1-mL mixture containing
Iscove’s modification
of
Dulbecco’s
medium (IMDM),
15% fetal
bovine
serum,
0.3% agar
(Difco Laboratories,
Detroit, Mich), and 10% conditioned
medium
from the 5637 cell line as a source
of colony-stimulating
activity
(CSA)
in the presence or absence of the various isoferritins.
Sodium
azide was removed from the isoferritin
preparations
by dialysis prior
to the cultures.
Preliminary
studies showed that ferritin concentrations
in the
bility
limit
incubated
of Lowry
against
total
antibody
preservative-free
gel
cusing,
based
for nonmalignant
gel slab.
except
Affi
2
A.
medium
5637-conditioned
of both the liver
were
below
the lower
detecta-
and 2A4 assays. Cultures
ferritin
at 37 #{176}C
in a humidified
atmosphere
of 5% CO2
were
in air.
were scored for colonies after seven days of incubation,
a time
has been shown previously
to be optimal
for the demonstration
of inhibition
of CFU-GM
from normal donors by AIFIA.’4”5
All
aggregates
containing
more
than 40 cells were counted as colonies.
To study
the neutralization
of a possible
isoferritin-associated
inhibitory
activity by a specific antibody, active isoferritin
preparations
were
preincubated
at 10_8 mol/L
with the monoclonal
antibody 2A4 prior to the culture. The final dilution of the ammonium
sulfate
precipitate
of the 2A4 ascitic fluid was calculated
so as to
Plates
that
Glycosylated
homogenates
ration
G) on a Con
glycosylated
side.
were obtained
ferritins
(preparations
A-Sepharose
proteins
These
by loading
F and H) or heated
column.
were eluted
preparations
were
nonheated
homogenates
After
extensive
with 0.1 mol/L
concentrated
tissue
(prepawashing,
a-methyl
either
on
an
glucoM
Amicon
membrane
(preparation
F and H) (Amicon
Corp.
Danvers,
Mass) or by precipitation
at 300,000 g for 90 minutes (preparation
G). The ferritin
concentration
was detected
by immunoassays.
In
ferritin preparations
F and G, ferritin did not account for more than
5% of the total protein, whereas preparation
H was somewhat more
than
10% pure.
300
Monoclonal
body
2A4
antibody
against
al,2’ and its
human
previously
assay
described.22
heari
heart
characteristics
Immunoassaysfor
noradiometric
to
ferritin
were
and
for
human
The
by Arosio
heariferritin.
two-site
monoclonal
was developed
reported
liver
The
ferritin.
liver
et
et al.’#{176}
The
ferritin
two-site
was
immunoradiometric
immu-
prepared
assay
an excess
The
bone
fashion.
(S.L.
as
for
of antibody
marrow
The various
and PA.)
working
anti-
by Cavanna
have
as compared
culture
isoferritin
and marked
with the CFU-GM
to ferritin
were
preparations
studies
with
different
molecules.
carried out in a blind
were made by two of us
letters
(A
to H).
assays did not know the content
People
of such
preparations.
Two
plates
were
scored
for each
sample
by two
independent
investigators,
and the mean value of the four scores was
considered
the result of that single experiment.
Colony
growth
in the
presence of different preparations
was expressed
as a percentage
of
the control, and the results of different
experiments
were expressed
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ISOFERRITINS
AND
GRANULOPOIESIS
791
....
guishable.
HLF
2O
C
.
;
\
IA.
.4
Fig 1 .
Isoferritin
analysis
of the heart ferritin
preparation
G
containing
only glycosylated
isoferritins
eluted
from a Con ASepharose
column
with 0.1 mol/L
a-methyl
mannoside.
In this
analysis.
isoferritins
were
separated
on polyacrylamide
gel isoelectrofocusing.’7
The bands containing
isoferritins
were cut and
eluted.
Then,
on each sample,
the pH was measured.
and the
ferritin
concentration
was evaluated
by means
of immunoradiometric
assays based on polyclonal
antibodies
against
human liver
ferritin
(HIF)
and the monoclonal
antibody
2A4 against
human
heart
ferritin.
Values
for pH (
) and concentrations
of
liver-type
(--)
and heart-type
(----)
ferritin
of different
fractions
are reported.
and
not
using the
i
I SD.
Differences
test for paired
tion of the original
between
means
observations
colony
after
scores (number
were
determined
logarithmic
by
transforma-
per plate).
RESULTS
Isoferritin
chemical
and
preparations
immunologic
used
preparations
ences
Table
1 summarizes
the biocharacteristics
of the ferritin
preparations.
A
in their
in the
and
following
experiments.
not
B did
show
any
profiles
isoelectrofocusing
Liver
2.
or subunit
Effects
ferritin
significant
differcomposi-
tion as judged
by SDS electrophoresis.
Biochemical
of heart preparations
C and D showed
that these
had about
85%
H subunits;
their
isoelectrofocusing
Table
portion
ferritin
to Con
of
ferritin
and the results
of these
major
addressed
question
studies
with
isoferritin
analyses
isoferritins
profiles
of Different
Isoferritin
the
bands
(preparation
100%
E)
of H subunits
Immunoassays
had
studies
are reported
in this
work
value
of 4.6 for pI;
metric
assay
liver
value
ferritin
of 5.2
based
(2) those
A
large
isoferritin
populations:
assay and
had a peak
by the immunoradio-
antibodies
rich
against
in L subunits
isoferritins
Molarity
HeLa
cell
formation.
on
tion
ferritin
(preparation
Of the two preparations
C, which
column
effect
to
had been
remove
CFU-GM
a peak
growth.
Re-
on CFU-GM
growth,
ing 5% glycosylated
effect,
mainly
at i0
through
ferritin,
mol/L.
a Con
proteins,
whereas
did
A-Sepharose
not
preparation
show
showed a significant
inhibitory
Of the two heart
preparations
to produce
on In Vitro
CFU-GM
Growth
Formation
Control
With
Medium
t
P
1012
liver
±
8
0.01
NS
A(4)
io-9
liver
98
±
9
0.44
NS
B(4)
1012
liver
99
± 4
0.31
NS
B(4)
iO
liver
93
±
8
1.82
NS
C(4)
1012
heart
98
±
10
0.43
NS
C(4)
i-
heart
87
±
18
1.47
NS
D(4)
1O12
heart
88
±
6
3.99
<.05
D(4)
i0
heart
E(4)
1O12
HeLa cells
E(4)
i-
F(4)
100
74
±
8
5.52
<.02
102
±
12
0.32
NS
HeLacells
96
±
17
0.95
NS
1012
heart
99
±
8
0.27
NS
F(4)
i-
heart
95
±
14
0.79
NS
G(4)
1O12
heart
91
±
11
1.54
NS
G(4)
i-
heart
68
±
11
5.01
<.01
H(4)
10’s
liver
101
±
15
0.12
NS
iO
liver
96
±
8
1.06
NS
“Comparison
medium
was carried
out by applying
ranged from 59 to 499
the
per plate.
t test
for
paired
observations
on colony
any
D, contain-
glycosylated
isoferritins
(F and G), G was found
a mean inhibition
of 32% ± 1 1% of the control
at
containing
P reparations
passed
A(4)
H(4)
Abbreviation:
NS, not significant.
control
human
had
also did not affect colony
of heart ferritin,
prepara-
E)
glycosylated
as Percentage
of Control
Medium (Mean ± 1 SD)
Origin
of
5.8 to 4.2.
sults of these
studies
are reported
in Table
2. Irrespective
of
the degree of glycosylation,
the three preparations
of human
liver ferritin
(A, B, H) did not show any effect on CFU-GM
growth
at concentrations
equal to l0
and l0_2 mol/L.
Colony
(No. of
number
in p1 from
detected
on polyclonal
I . One
relationship
reactivity),
glycoof isoelectrofocus-
G.
ranging
two different
and therefore
for p1.
ofdifferent
ferritin
in Table
was the
preparation
seen,
revealed
approxi-
only immupurification,
detected
by the 2A4 immunoradiometric
containing
more than 60% H subunits
those
therefore
(1)
bound
nonglyco-
as heart
Comparison
Preparation
Experiments)
D
A.
heart
were
C and
ferritin
C was
among subunit
composition
(immunologic
sylation,
and p1. Figure
1 shows the results
ing
not distin-
preparations
(5%)
proportion
bind
and were
between
A whereas
cell
HeLa
the same
Effect
±
a minor
mately
did
of isoferritins
difference
Preparations
F and H could be characterized
nologically
because
of the low level of ferritin
j:\
Fractions
as means
only
to Con
sylated.
C
1o
that
specifically
2A4
spectrum
The
was
D
.6
\.:.
E
a large
covered
.7
,
30J
scores
after
logarithmic
transformation.
Colony
scores
of the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
792
DEZZA
l0
mol/L,
between
extraction
As
whereas
these
had
F was ineffective.
two
been
reported
preparations
omitted
with
in Table
The
major
was that
preparation
3, a significant
the
difference
step
2A4
directed
against
human
(t
5.14,
=
P
tions as low as I0_18
mately
six molecules
With
target
macrophage
progenitors
ducibility
of our studies,
heart
ferritin
according
ferritin.28
Although
inconsistencies
preparations
D and G. The
at iO
mol/L
significant
(n
an average
inhibition
of24%
=
4, i = 4.80, P < .02). Only
were
out
carried
with
the
new
and colony
formation
medium,
respectively.
mol/L,
control
was
are entirely
valid.
I . Experimental
G,,
and
at
68%
ferritin
subunits
the membranes
l0
of
the
ritins
control
in the
Broxmeyer
and
acidic isoferritins
multipotential,
genitor
cells
molecular
to 4.9;
a-methyl
antibodies
subunits
the
main
According
characteristics
to
of the
in vitro
they
are
the
ofabout
bind
mannoside;
prepared
from heart
following26:
550,000
to Con
(1)
they
and a p1 ranging
A and
are
specifically
have
4.6
eluted
by
ferritin.
A
Jacobs
and
(3) they are inactivated
by monoclonal
against
human
heart
ferritin
and H
ferritin,
but not by antibodies
against
his associates
in the data
Table
3.
Effects
have
of the
noted
Broxmeyer
of the Monoclonal
a number
group
and
Antibody
,yains
.-
and
ferritin
formation.
raised
Against
Human
Heart
Granulocyte-Macrophage
Ferritin
Monoclonal
and
within
Medium
with
the
ability
68
5
2A4
Inhibitory
Cells
93
5
none
100
5
2A4
±
±
1
67
5
2A4
With
both
and
t
preparations,
-
colony
1.29, respectively.
85
5
ln all experiments,
the ferritin
formation
after
100
preincubation
P > .05 in both cases).
concentration
was iO
Colony
mol/L.
of the feri’itin
scores
and
that
this
in the
fucose,
spleen
mannoside.
This
ferritin
and
with
of Acidic
to
inhibit
colony
Isoferritins
±
Test for Paired Observations)
(t
11
t
-
5.47,
12
t
-
±
5
t
=
±
11
t
=
±
13
t
-
±
5
t
=
the monoclonal
of the control
medium
2A4
ranged
P
<
control
P
4.37,
compared
medium
<
.02 when
compared
P
>
.05 when
compared
D + 2A4
4.96,
P
<
control
6.9 1 . P
.01 when
compared
medium
<
.0 1 when
compared
.05 when
compared
G alone
2.35,
P
<
G + 2A4
did not significantly
from
.0 1 when
D alone
1 .70,
with
2.30
of
Comparison
SD)
with
none
20%
attached
to
This is the
tissue
extracts
of Control
with
G
some
the ferritin
a129 reported
in crystalline
total
Activity
with
G
criticisms
that
a-methyl
of cell
with
none
or serum
Formation
(Mean
5
2A4
spleen,
vesicles
acidic
than
Shinjyo
et
hexosamine
with
D
has
in the data of Broxmeyer
suggests
eluted
Ferritin
on the
Progenitor
as Percentage
Experiments
none
D
of
do exist
group
granulocyte-
is generally
richer in H subunits
than
is the total
ferritin
of
the tissue extract.’#{176}
2. A variety
of products
from leukemic
cells can affect in
vitro granulopoiesis.3#{176} Consequently,
there can be no simple
relationship
between
immunologic
characteristics
of cell
Colony
No.
of
heart,
for 2% to 5% of the
accounts
of inconsis-
2A4
Jacobs
not all the aforementioned
is specifically
have
H.imn
the
mean approxicell within
the
studies in our laboratories’0
and some data
work show that in human
liver, spleen, and
component
of ferritin
exists that binds to Con
Previous
fraction
liver ferritin;
and (4) they are present
in normal
monocytes
and macrophages
and, in greater
amounts,
in leukemic
cells.
tencies
by either
assembled
is
of the present
heart a minor
a
from
not normally
contain
against
H-subunit-rich
to be active at concentra-
inhibition
evidence
the
do
important,
ferritin
could
be more
surrounding
cytoplasm.’
mannose,
galactose,
and
that manifest
suppressive
activity
upon the
granulocyte-macrophage,
and erythroid
pro-
weight
(2)
of hematopoiesis.”7’226
his associates,
isofer-
between
CFU-GM
growth
isoferritins;
(3)
are made in the polyribosomes
of the endoplasmic
reticulum.’
polyribosomes
the role of acidic
including
site at which export proteins
secreted
from cells
and glycosylation
takes place.
It has also been
that
some
ferritin
made
by membrane-bound
intracellular
are made
surrounding
more
demonstrate
his associates,’5”6
suggested
is controversy
to
which was
two experiments
DISCUSSION
There
and (5)
able
and
G,
79%
been
D, D1, produced
± 9%,
preparation
system;
not
of
with
preparations
followed
new preparation
no correlation
mol/L,
and this would
interacting
with one
preparation
G, colony
formation
was 68% ± 10% of the
control
medium,
and the difference
was again
significant
(t = 7.40, P < .001 ). In an attempt
to investigate
the reprowe remade
two
to the procedures
shown
biologic
fluids,
including
serum,
ferritins
that react with antibodies
isoferritins;
(4) AIFIA
is reported
medium,
.001).
<
have
leukemic
10% of the control
±
lines
ferritins,
ability
of their extracts
to inhibit
human
and their content
in acidic,
H-subunit-rich
were carried
out with heart
G at lO
mol/L.
With
ferritin
colony formation
was 70%
difference
being significant
D,
the
tissue
AL
cells, are nonglycosylated
A; (2) studies
on human
inhibitory
ferritin.
heart
Overall,
nine experiments
ferritin
preparations
D and
cell
(1)
and leukemic
bind to Con
formation
antibody
of the
activity
of preparations
D and G on in vitro colony
was removed
by preincubation
with the monoclonal
criticisms’”7’27:
those in monocytes
or at least do not
F.
part
following
the
of heat
ET
57 to 474
differ
per plate.
from
the control
(t
-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ISOFERRITINS
AND
3. Normal
spleen
serum
ferritin,
part
GRANULOPOIESIS
ferritin
has
a very
glycosylated.2
storage
by
iron,
the
and
rough
cells
and
reacts
with
iron
content,
low
Its
793
concentration
it is believed
endoplasmic
this
Acidic
the
of
amount
is synthesized
protein
of reticuloendothelial
are present
in many
reticulum
hepatocytes.
or
and is for the most
reflects
that
to liver
antibodies
ferritins
active
purified
portion
(5%)
one
was
fully
(F and
the active
human
probably
ferritins,
action
of
serum contains
binding
related to complement
H-subunit-rich
may
be responsible
from
circulation
with
ferritin
these
for the rapid clearance
and the low levels of acidic
of normal
plasma
this
in local
marrow.
In
there
subjects.
However,
rule out the possibility
not
does
isoferritins
fact,
would
factors
proteins.
cell-to-cell
if
acidic
therefore
mechanisms
in plasma
with
of tissue
isoferritins
ferritin
in the
ciated
the
a local
bone
function,
sure
that they
fluids.35 Alter-
to make
and other
the
strongest
within
had
body
cell growth
in addition,
low.36 There
tors
factors
act at very
need
to
be occupied
to
elicit
example,
as few as ten molecules
bound
to erythroid
progenitors.36
lopoiesis,
lactoferrin
colony
ing
i0’
has been
stimulatory
mol/L
In
this
work,
we
activity
have
consistently
affect
This is at variance
found
that
inhibitory
ever,
with
a
and
in inhibit-
at concentrations
as low
tried
concentrations
range from
the
aforementioned
human
liver
of human
CFU-GM.
of Broxmeyer
et al,’5
and spleen
ferritins
had
to l0’
mol/L.
Our data, howare in keeping
with
indirect
clinical
evidence.
Patients
thalassemia
major
who are not regularly
treated
with
desferoxamine
high as 20,000
circulating
lished
at 1010
have serum
ferritin
(4 x 108 mol/L),
may
tg/L
protein
binding
observation).
Their
total
tial
is either
normal
or may
nia
may
observed
only
be
to Con
A (M.
white
cell
be slightly
as
concentrations
as
about 50% of the
Cazzola,
count
and
of
the
differen-
picture
of
The results
preparations
suppressive
any
obtained
intriguing.
effect.
Only
with
heart
and HeLa
one of the two
severe
cell ferritins
purified
and one of the two glycosylated
effect,
The
whereas
HeLa
biochemical
and
cell
ferritin
immunologic
not show
preparations.
major
differences
between
active
Only the differences
in purification
and degree
of glycosylation
part,
the different
effects
(Table
on colony
heart
had a
and inactive
procedures
at least
in vitro.
2A4
that
inhibitory
the
The
been
added
as a
(Table
of
3) is one of the
activity
was
asso-
molecules.
proportion
of H subunits
AIFIA,’6
inhibitory
and glycosyla-
and its physiologic
significance
activity
was detected
by us at
to 10_12 mol/L,
and this
zg/L, assuming
a molecular
found that most
of acidic
ferritin
means
weight
normal
lower
a
of
subjects
than I .5
limit
of detection)33
and values are generally
5 tg/L
in patients
with
leukemia.#{176} As previously
out, however,
the low serum concentrations
could be
with
the
hypothesis
mechanism(s)
activity
are
preparations
CFU-GM
completely,
suppress
CFU-GM.4’
does not have
but interacts
evidence
that
serum.33’34
interaction
M.
sized
not
formation
not
only
effect
an
of
possibility
(V.
liver
but
also
exists
interaction
of
isoferritins
of myeloid
that
has been
binders
in
an in vitro
and the third
Bellotti,
P. Arosio,
C3
by
Ia-like
cycling
progenitors
observed
isoferritins
C3
of
about
act on a
progenitors
has been
by
observations).
as is the C3 proactivator,
growth
growth
on myeloid
have
complement,
unpublished
acidic
vitro
their
active
being
active
molecules.
There
proteins
may be ferritin
monocytes,
in vitro
in
the
a direct
by the
vitro
subpopulation
with other
complement
ofhuman
in
The
inhibition
Alternatively,
Cazzola,
exert
present.
block
More
recently,
we
between
H-subunit-rich
component
at
that acidic
isoferritins
granulocyte-monocyte
For example,
AIFIA
colony
ferritin
function.
isoferritins
maximum
antigen-positive
human
and
clear
did
the
local
acidic
not
50%. This
could
mean
subpopulation
of human
having
specific
receptors.
to
ofa
by which
isoferritin
tors.43
activity
did not have
analyses
did
1) may explain,
formation
are
ferritin
fractions
and
not.
is synthe-
and
macrophages
factor B.42 Whether
and C3 can result
the
in a
progenitors
to be
remains
established.
Furthermore,
it has been recently
shown
that
lactoferrin
can bind to ferritin,
the ferritin-lactoferrin
complex retaining
its capacity
of binding
to lactoferrin
recep-
hypersplenism.39
more
heated
was
(lower
below
pointed
reduced
and leukope-
elevated,
a part
unpub-
was
(F)
had
antibody
of 10
to 500
0.5
(HLA-DR)
the pure
is able to
one
ferritin
of the suppressive
activity
D and G by preincubation
500,000.
We have recently
have serum concentrations
found
to clarify
clearly
show that neither
fraction
of liver ferritin
normal
as
groups.37’38
one (G)
fluoride
isoferritin
a high
inhibitory
need to be
of granu-
acidic
previously
reported
is uncertain.
The
The
for
effect,
to be effective
the in vitro growth
with the findings
activity
biologic
of erythropoietin
Within
inhibitors
found
by two different
controversy.
Our findings
form nor the glycosylated
who
low concentrations,
the receptor
numbers
per cell are extremely
is also intriguing
evidence
that very few recep-
the
Although
in keeping
will
in the
ferritin
heart
tion appeared
to be prerequisites
for the observed
inhibitory
effect,
this activity
appears
to be much less potent
than the
poietic
as
a role
monoclonal
with
tg/L
play
inactive
HeLa
glycosylated
inactive
Inactivation
preparations
indications
be discussed,
ferritin-binding
proteins
may
interaction
of acidic isoferritins
with cells.
4. The extremely
low effective
concentrations
of AIFIA
(l0l6
to 10_8
mol/L)
should
not be considered
biologic
nonsense
per se. It has indeed been found that the hematonatively,
the
the
a minor
the
preparation,
which
is known
to contain
active
could have been partially
damaged
upon storage,
inhibitor.
isoferritin
factors
interactions
two
if phenylmethylsulfonyl
protease
acidic
binding
it should be noted that
of a role of acidic
isoferritins
be
do not accumulate
for acidic
The inter-
G),
whereas
whereas
as was
preparations
even
that
nonglycosylated,
ultracentrifuged,
contained
ferritin,
the
possible
explanation
for this
Covell
and her colleagues34’35
fact
preparation
As regards
nonheated
proteases,’t
by
in
ferritin
preparation.
tissues, cells, and some biologic
fluids, but cannot be detected
in most sera or are present only in low concentrations.3133
A
has been recently
advanced
and us.33 It has been shown
heart
of glycosylated
Since
lactoferrin
macrophages,37’38
in
the inhibitory
in the in vitro system could
interacting
with lactoferrin.
It is hoped
The
is able
to specific
by binding
that
a deeper
to inhibit
colony
receptors
on monocytes
activity
of acidic
be explained
by their
insight
into
isoferritins
on in vitro growth
of human
will be obtained
by using recombinant
mers”
and
investigating
the
aforementioned
the
effects
myeloid
human
stimulatory
and
isoferritins
capacity
of acidic
progenitors
homopoly-
mechanisms.
of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
794
DEZZA
ET
AL
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1986 67: 789-795
Effect of acidic and basic isoferritins on in vitro growth of human
granulocyte-monocyte progenitors
L Dezza, M Cazzola, W Piacibello, P Arosio, S Levi and M Aglietta
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