Leukemia and Lymphoma, Vol. 18, pp.

4 2 9 4 3 3 0 1995 Harwood Academic Publishers GmbH

Reprints available directly from the publisher Printed in Singapore
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A Role for Ferritin in Hematopoiesis and the

Immune System
KEIKO MORIKAWA,* FUMIMARO OSEKO* and SHICERU MORlKAWA"
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The Departments of 'Internal Medicine and "Pathology, Shimane Medical University, 89- I , Enya-cho, Izumo, Shimane, 693, Japan

(Received July 16, 1994)

Elevated serum ferritin levels have been reported in a number of pathological states. These observa-
tions indicate that cells of the immune system can participate in the prevention of potential tissue ton:-
icity from iron accumulation, and iron and iron-binding protein have important effects on immune
systems.Ferritin is generally regarded as an iintracellular iron storageprotein. However, small amounls
of ferritin circulate in the serum of normal individuals, and the physiological role of serum ferritin
remains obscure. Although the function of fenitin is inevitably linked to iron metabolism, a role for
fenitin in hematopoiesis and the immune rsystem has drawn attention for years. Fenitin has an in-
For personal use only.

hibitory effect on the in vitro growth of human hematopoietic progenitor cells and on the prolifera-
tion of T lymphocytes in vitro. Recently we report that ferritin may directly suppress the differentiation
of human B lymphocytes maturing into antibody producing cells in vitro. In the present review, we
summarise this field of research.
KEY WORDS: fenitin hematopoiesis mmunosuppression
1 lymphocyte

INTRODUCTION
The role for ferritin includes specialized functions such
as recycling iron in macrophages, short- and long-term
iron storage as in hepatocytes and intracellular house-
keeping functions providing a reserve of iron for cy-
tochromes, nitrogenase, ribonucleotide reduchse,
hemoglobin, myoglobin, etc, and possibly for detoxica-
tion, if excess iron enters the cells. Thus, ferritin is gen-
erally regarded as an intracellular iron storage protein.
Ferritins are also present in the extracellular fluids of nor-
mal individuals. Serum femtin is elevated in a number of
pathological states, including acute inflammation, chronic
infection, hepatocellular damage, and malignancy. The
biological role of this circulating ferritin i s obscure.
Other roles for ferritin, such as its involvement in
immunosuppression and the regulation of hematopoiesis
have been reported for years, but have yet to be fully
Address for correspondence: Keiko Morikdwa, M. D., Department
of Internal Medicine, Shimane Medical University, 89- I , Enya-cho,
Izumo, Shimane 693, Japan.
429
defined. We review the studies camed out in the last sev-
eral years to clarify the possible role: for femtin in
hematopoiesis and the immune reaction.
GENERAL FEATURES
Ferritin is a large protein formed from a spherical protein
coat (apoferritin) with a molecular weight of about 500Kd,
that surrounds a core of hydrous ferric oxide.' The pro-
tein coat is made up of 24 subunit polymers comprising
any ratio of two major types, designated H(eart) and
L(iver). Femtin is mainly localized intracellularly, where
it plays the major role of storing and detoxifying iron."
The synthesis of ferritin in cells is regulated by iron, and
as with transferrin receptor, the process has been shown
to be translational and dependent on what is most likely
the same regulatory mRNA-binding protein (the iron-re-
sponse-element binding protein; IREBP).4
Three subunit types have been identified in human fer-
ritin: light (L; 19Kd, I74 residues), heaky (H; 21 Kd, 182
aminoacids) and glycosylated (G, 23Kd). The cDNAs and
genes of the L and H peptides have been found to have
 430 K.MORIKAWA ET AL.
55% sequence homology.5 They are encoded in several
different chromosomes,6 and have large immunologic
differences.’ The G subunit, which is present only in the
extracellular fluid appears to derive from a posttranscrip-
tional modification of the L chain.8
Tissue ferritins are composed of variable proportions
of H and L subunits. The L subunit-rich ferritins, named
basic for their higher PI, are more abundant in iron-loaded
tissues such as liver and spleen, while the H subunit-rich
ferritins, named acidic for their lower PI, are found in
heart, kidney, hempatopoietic, and malignant cells. The
feature of function common to all ferritins is the storage
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of iron in a soluble form. Therefore, the function offer-
ritin is inevitably linked to iron rnetabolism.2.3However,
small amounts of ferritin are present in serum and other
body fluids. Serum ferritin contains almost exclusively L
and G subunits and little or no iron.8.9 Acidic ferritins are
present in all biologic fluids, particularly in milk, but not
or only low concentrations in serum. The origin and
biologic significance of these extracellular proteins are
far from clear, but a number of functions have been attri-
buted to them, including negative regulation of
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hematopoiesis~~~4 and the direction of lymphocyte mo-
bility and functionality.15-18
BINDING SITE FOR FERRITIN
Receptor for ferritin have been reported in several normal
tissues and on tumor cells in culture.1 Cell surface recep-
tors are presumed to play an important role in mediating
the function of ferritin. A potential iron transport function
for the protein has been suggested, but there is little direct
evidence for this as a general process. The ferritin recep-
tor also can be considered as a potential scavenging sys-
tem for tissue fenitins found in the circulation.
The recognition that ferritin may have other functions
unrelated to its role in iron storage has been closely linked
to an understanding of the subunit composition of the pro-
tein. Membrane binding sites specific for H-ferritin have
been identified in various cell lines such as K562 and
HL60.1920 H-type ferritin binding sites expressed on K562
cells progressively tend to decrease and disappear when
the growth of the cells reach the plateau phase, and the in-
duction of differentiation by hemin results in disappear-
ance of the binding sites.19 These results suggest that the
expression of ferritin binding sites is modulated by cellu-
lar proliferation and differentiation. The binding sites for
ferritin have been demonstrated on lymphoid cell lines21
and normal human lymphocytes.22In human lymphocyte
subsets, both CD4 and CD8 T lymphocytes, and CD19 B
lymphocytes express H-ferritin binding sites in phyto-
hemagglutinin (PHA)-stimulated lymphocytes,22indicat-
ing that the distribution of femtin binding sites is not re-
lated to cell lineage or function. Although ferritin binding
sites are observed in lymphocytes at all cell cycle phases,
the expression of H-ferritin binding sites appears to be
closely and positively linked to the proliferative status of
the lymphocytes, which is in accord with the findings
shown in K562 cells. L-ferritin binding sites either do not
exist on lymphocytes or are very different from H-ferritin
binding sites, because they are undetectable with conven-
tional methods. However, other studies demonstrate that
ferritin rich in L subunits binds to both T and B lymphoid
cell lines independently of the binding of H ferritin,zI im-
plying that binding sites for both the H and L subunits of
ferritin are expressed on these cells.
ACTION OF FERRITIN ON HEMATOPOIETIC
PROGENITORS
Acidic, H subunit-rich isofemtins have inhibitory activ-
ity on the in vitro growth of human hematopoietic prog-
enitors, these molecules have been identified as having
leukemia-inhibitory activity.10 The mechanism responsi-
ble for the inhibitory activity of H-subunit-richisoferritins
remain to be clarified. A recent study of the in vitro
hematopoiesis assay showed that femtin suppression re-
sults from ferroxidase activity of the H-chain, because it
disappears in the mutant in which this activity deleted.
However this suppression is reversed by iron supplemen-
tation to cells in the form of hemin.23 Ferroxidase activity
has been suggested to interfere with cellular proliferation
by inducing iron starvation. The H-type isoferritins are
present in many cell types, and in particular are predom-
inant in monocytes24 and it has been suggested that these
cells secrete acidic isoferritins which could act as nega-
tive feed-back regulators of hematopoiesis.11These results
suggest a specific role for monocyte-macrophage-derived
acidic isoferritins as feedback regulators of hematopoietic
progenitor cells.
IMMUNOSUPPRESSIVE ACTION OF FERRITIN
Immunosuppressive effect of ferritin has been shown in
the proliferation of T lymphocytes.~5-~8 H-rich, and not L-
rich, ferritins inhibit lymphocytes’ E rosette formation,
lymphocyte migration, and lymphocyte blastogenesis in-
duced by PHA and concanavalin A (Con A) in vitro. The
clonal expansion and maturation of T precursor cells into
effector cells is also suppressed in ferritin-treated mice in
the delayed type hypersensitivityresponse.!*The presence
 IMMUNOSUPPRESSIVEACTIVITY OF FERRITIN 43 1

of ferritin binding sites on lymphocytes might explain why
ferritin interacts with the lymphocytes probably via these
sites. This binding would not require internalization, sirice
it may be aimed at cell-cell surface interactions.
Although ferritin partially inhibits the blastic transfior-
mation of T lymphocytes stimulated by Con A in vitro or
in mixed-lymphocyte culture,l5+16this suppression could
be reversed by adding conditioned medium containing in-
terleukin-2 (IL-2). 16 These results suggest that ferritin
might induce impairment of IL-2 production by T lym-
phocytes. Though the inhibitory activity of ferritin on lym-
phocyte blastogenesis is predominantly associated with
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B cells may not be linked with the ferritin type (Fig. 1). It
has been reported that human T lymphocytes synthesize
and secrete both types of ferritin molecules with a high
proportion of H subunits.26 In that case, T lymphocytes in-
terfere with B-cell Ig production through secretion of fer-
ritin, which is distinct from T-T interaction phenomenon
known as suppressor T cells.
The effective concentration of ferritin in the inhibition
of Ig generation is 4 to 500 ng/ml ( I x 10-10 to 8 x 10-12
mol/L),25 which is very close to that involved in the inhi-
bition of human CFU-GM growth (10-9 to 10-12 moVL)
reported by Cazzoll2 and higher than that of the acidic iso-

the H-chain,17 L-chain is also effective.15-17 Ferritin hiad
no effect on E, EA, EAC rosette formation or pokeweed
mitogen (PWM)-induced lymphocyte blastogenesis.15
Previous studies suggest that the immunosuppressive ef-
fect of ferritin appears to relate mainly to T cell rather thlan
B cell function.
Recently, we examined the effect of ferritin on humlan
B lymphocyte activation25 and found that ferritins do not
suppress the proliferation of resting B lymphocytes stim-
ulated by polyclonal B cell mitogen, or Staphylococcus
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ferritin inhibitory activity (IO-IO to lO-I5 mom) reported
by Broxmeyer.10
Small amounts of ferritin circulate in the sera of
normal individuals, although serum ferritin is predomi-
nantly basic. Very low concentrations of ferritin, which
are at physiological levels, cause significant inhibition
of marrow colony formation and Ig generation in the
in vitro system. Although the possible physiological
significance of the inhibitory activity of ferritin remains
unknown, it is assumed that serum ferritin does not have

aureus Cowan strain I (SAC) (Table 1) nor do they inhibit
the proliferation of activated B cells, (Table I). However,
both L- and H-rich ferritins exert their inhibitory activity
on immunoglobulin (Ig) generation by B lymphocytes in
a T-independent as well as a T-dependent way (Fig. 1).
The cytoplasmic Ig-containing cells decrease in propor-
tion to the reduction of Ig secretion (Table 2). The action
of ferritin on Ig generation by B cells appears to be regu-
lated at the transcriptional level. These results indicate that
ferritins directly suppress the differentiation of B lym-
phocytes maturing into antibody producing cells. The sup-
pressive activity of L-rich ferritin on Ig production was
not different from that of the H-rich ferritin, implying that
the inhibitory activity of ferritin on the differentiation of
a direct effect on the immune reaction and hematopoiesis
in the in vivo states. Indeed it is probable that this
activity is blocked or overwhelmed by various factors
including cytokines, or that ferritin might interact with
other active proteins. It has been shown in fact that human
serum contains binding factors for acidic ferritins,
probably related to the complement proteins and alpha-2-
macroglobulin.27 Further studies on the mode of action of
ferritins in the cytokine network will be investigated in the
near future.
It is possible that the impairment of cell-mediated im-
munity classically observed in patients with hyperfer-
ritinemia is, at least, in part due to the mechanisms
discussed in this review.

Table 1 Effect of basic and acidic femtins on the proliferative response of human B cells
Re,sting B Activated B
~_
Ferritin Concentration
(nghl) None SAC None IL-2
Basic 0.0 0.9~t0.1 18.6 i 0.3 6.6 * 0. I 37.5 * 0.2
0.8 0.9 f 0.2 19.8 i 0.6 6.8 f 0. I *
39.6 1.7
4.0 0.9 i 0. I 21 .0 f 0.9 6.5 i 0.1 28.9 i I .4
20.0 *
1 .o 0.0 20.0 f 0.6 6.5 * 0.7 36.4 * 1.3
100.0 1.3i0.1 15.0* 1.1 5.5 i 0.6 38.6 i I .O
Acidic 0.8 1.1 t0.1 17.5 f 0.3 *
5.9 0.2 *
37.3 0.3
4.0 1.3i0.1 *
20.4 0.2 5.0 i 0.6 33.2 f 0.9
20.0 0.9 f 0. I *
19.2 1.2 5.6 ~f:0. I 35.6 & I .O
IOO.O 1 . 1 +0.1 16.6 i 0.5 5.3 f 0. I 34.2 f 0. I
Re\ting or in vilro activated B cells from tonsillar sampler were cultured for 3 days in thc presence or nbhence of SAC (I.10’ v/v) or IL-2 (IIXJ Ulml). re.;pectively. Various concentrations of human
b a w ( ~ l c r i v c dfrom \plernJ or acidic (denvsd from heart) ferntin were added at the initiation afculturea. B cell rerponx to the \ttmuI.uors wah measured by the incorporation of ‘H-thymidine over the
la\( IXhr of culture\. The data was shown mean (cpm x 10 I) 01 triplicate culture t SE.
 432 K. MORIKAWA ETAL
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~~~~ ~

0 0.16 0.8 Y 10 100

Concentration of ferritins ( n g l m l )

Figure 1 Effect of femtins on Ig generation by PWM-driven system and by SAC-induced system. PBMC were cultured with PWM( 1:25 v/v) in the
presence of H ( 0 ) - and L(0)- fenitins for 7 days in PWM-driven system( (A) ). In SAC-induced system( (B) ), SAC-prestimulated B cells were incu-
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bated with IL-2( 100 Ulml) in the presence of H(0)- and L(0)- femtins for 7 days. Amounts of Ig secreted in the culture supernatants were measured
by ELISA. The results are expressed as mean of triplicate cultures. The representative results of IgG suppression by both ferritins in two systems from
six data were demonstrated in Figure 1.

Table 2 Cytoplasmic Ig positive (cig+) cell ratio and secreted Ig level in SAC-activated B cells in the presence of L-ferritin
Addition IR secretion
L-ferritin clg+ cells
fndmlJ f%J
0.0 17.5 91 1 1612
0.8 15.1 816 1367
4.0 10.5 422 968
20.0 7.9 288 456
100.0 3.7 101 189
SAC-activated B cells ( 2 x INIwell) were cultured with L-ferritin in the presence of rhlL-2 (lo0 Ulml) for 7 days. Then, the slides were prepared using a cytocentrifuge, fixed and were stained with
FITC-conjugated anti-human Ig Clg+cell? were detected by fluorexence microscopy. The percentage was determined by counting at least 400 cells. Amounts of I g secreted in the culture supernatants
were measured by ELISA. The values represent the mean of triplicate cultures. The values of control cultures are 3. I % ofclg'cells and I 13 ng/ml and 169 nglrnl of IgM and IgC recretion. respectively.

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