Leukemia and Lymphoma, Vol. 18, pp.

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

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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-
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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 defined. We review the studies camed out in the last sev-
eral years to clarify the possible role: for femtin in
The role for ferritin includes specialized functions such hematopoiesis and the immune reaction.
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- GENERAL FEATURES
tochromes, nitrogenase, ribonucleotide reduchse,
hemoglobin, myoglobin, etc, and possibly for detoxica-
Ferritin is a large protein formed from a spherical protein
tion, if excess iron enters the cells. Thus, ferritin is gen-
coat (apoferritin) with a molecular weight of about 500Kd,
erally regarded as an intracellular iron storage protein.
that surrounds a core of hydrous ferric oxide.' The pro-
Ferritins are also present in the extracellular fluids of nor-
tein coat is made up of 24 subunit polymers comprising
mal individuals. Serum femtin is elevated in a number of
any ratio of two major types, designated H(eart) and
pathological states, including acute inflammation, chronic
L(iver). Femtin is mainly localized intracellularly, where
infection, hepatocellular damage, and malignancy. The
biological role of this circulating ferritin i s obscure. it plays the major role of storing and detoxifying iron."
Other roles for ferritin, such as its involvement in The synthesis of ferritin in cells is regulated by iron, and
as with transferrin receptor, the process has been shown
immunosuppression and the regulation of hematopoiesis
have been reported for years, but have yet to be fully 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
Address for correspondence: Keiko Morikdwa, M. D., Department
of Internal Medicine, Shimane Medical University, 89- I , Enya-cho, aminoacids) and glycosylated (G, 23Kd). The cDNAs and
Izumo, Shimane 693, Japan. genes of the L and H peptides have been found to have
429
 430 K.MORIKAWA ET AL.

55% sequence homology.5 They are encoded in several hemagglutinin (PHA)-stimulated lymphocytes,22indicat-
different chromosomes,6 and have large immunologic ing that the distribution of femtin binding sites is not re-
differences.’ The G subunit, which is present only in the lated to cell lineage or function. Although ferritin binding
extracellular fluid appears to derive from a posttranscrip- sites are observed in lymphocytes at all cell cycle phases,
tional modification of the L chain.8 the expression of H-ferritin binding sites appears to be
Tissue ferritins are composed of variable proportions closely and positively linked to the proliferative status of
of H and L subunits. The L subunit-rich ferritins, named the lymphocytes, which is in accord with the findings
basic for their higher PI, are more abundant in iron-loaded shown in K562 cells. L-ferritin binding sites either do not
tissues such as liver and spleen, while the H subunit-rich exist on lymphocytes or are very different from H-ferritin
ferritins, named acidic for their lower PI, are found in binding sites, because they are undetectable with conven-
heart, kidney, hempatopoietic, and malignant cells. The tional methods. However, other studies demonstrate that
feature of function common to all ferritins is the storage ferritin rich in L subunits binds to both T and B lymphoid
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of iron in a soluble form. Therefore, the function offer- cell lines independently of the binding of H ferritin,zI im-
ritin is inevitably linked to iron rnetabolism.2.3However, plying that binding sites for both the H and L subunits of
small amounts of ferritin are present in serum and other ferritin are expressed on these cells.
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 ACTION OF FERRITIN ON HEMATOPOIETIC
or only low concentrations in serum. The origin and PROGENITORS
biologic significance of these extracellular proteins are
far from clear, but a number of functions have been attri- Acidic, H subunit-rich isofemtins have inhibitory activ-
buted to them, including negative regulation of ity on the in vitro growth of human hematopoietic prog-
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hematopoiesis~~~4 and the direction of lymphocyte mo- enitors, these molecules have been identified as having
bility and functionality.15-18 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
BINDING SITE FOR FERRITIN hematopoiesis assay showed that femtin suppression re-
sults from ferroxidase activity of the H-chain, because it
Receptor for ferritin have been reported in several normal disappears in the mutant in which this activity deleted.
tissues and on tumor cells in culture.1 Cell surface recep- However this suppression is reversed by iron supplemen-
tors are presumed to play an important role in mediating tation to cells in the form of hemin.23 Ferroxidase activity
the function of ferritin. A potential iron transport function has been suggested to interfere with cellular proliferation
for the protein has been suggested, but there is little direct by inducing iron starvation. The H-type isoferritins are
evidence for this as a general process. The ferritin recep- present in many cell types, and in particular are predom-
tor also can be considered as a potential scavenging sys- inant in monocytes24 and it has been suggested that these
tem for tissue fenitins found in the circulation. cells secrete acidic isoferritins which could act as nega-
The recognition that ferritin may have other functions tive feed-back regulators of hematopoiesis.11These results
unrelated to its role in iron storage has been closely linked suggest a specific role for monocyte-macrophage-derived
to an understanding of the subunit composition of the pro- acidic isoferritins as feedback regulators of hematopoietic
tein. Membrane binding sites specific for H-ferritin have progenitor cells.
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 IMMUNOSUPPRESSIVE ACTION OF FERRITIN
the growth of the cells reach the plateau phase, and the in-
duction of differentiation by hemin results in disappear- Immunosuppressive effect of ferritin has been shown in
ance of the binding sites.19 These results suggest that the the proliferation of T lymphocytes.~5-~8 H-rich, and not L-
expression of ferritin binding sites is modulated by cellu- rich, ferritins inhibit lymphocytes’ E rosette formation,
lar proliferation and differentiation. The binding sites for lymphocyte migration, and lymphocyte blastogenesis in-
ferritin have been demonstrated on lymphoid cell lines21 duced by PHA and concanavalin A (Con A) in vitro. The
and normal human lymphocytes.22In human lymphocyte clonal expansion and maturation of T precursor cells into
subsets, both CD4 and CD8 T lymphocytes, and CD19 B effector cells is also suppressed in ferritin-treated mice in
lymphocytes express H-ferritin binding sites in phyto- the delayed type hypersensitivityresponse.!*The presence
 IMMUNOSUPPRESSIVEACTIVITY OF FERRITIN 43 1

of ferritin binding sites on lymphocytes might explain why B cells may not be linked with the ferritin type (Fig. 1). It
ferritin interacts with the lymphocytes probably via these has been reported that human T lymphocytes synthesize
sites. This binding would not require internalization, sirice and secrete both types of ferritin molecules with a high
it may be aimed at cell-cell surface interactions. proportion of H subunits.26 In that case, T lymphocytes in-
Although ferritin partially inhibits the blastic transfior- terfere with B-cell Ig production through secretion of fer-
mation of T lymphocytes stimulated by Con A in vitro or ritin, which is distinct from T-T interaction phenomenon
in mixed-lymphocyte culture,l5+16this suppression could known as suppressor T cells.
be reversed by adding conditioned medium containing in- The effective concentration of ferritin in the inhibition
terleukin-2 (IL-2). 16 These results suggest that ferritin of Ig generation is 4 to 500 ng/ml ( I x 10-10 to 8 x 10-12
might induce impairment of IL-2 production by T lym- mol/L),25 which is very close to that involved in the inhi-
phocytes. Though the inhibitory activity of ferritin on lym- bition of human CFU-GM growth (10-9 to 10-12 moVL)
phocyte blastogenesis is predominantly associated with reported by Cazzoll2 and higher than that of the acidic iso-
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the H-chain,17 L-chain is also effective.15-17 Ferritin hiad ferritin inhibitory activity (IO-IO to lO-I5 mom) reported
no effect on E, EA, EAC rosette formation or pokeweed by Broxmeyer.10
mitogen (PWM)-induced lymphocyte blastogenesis.15 Small amounts of ferritin circulate in the sera of
Previous studies suggest that the immunosuppressive ef- normal individuals, although serum ferritin is predomi-
fect of ferritin appears to relate mainly to T cell rather thlan nantly basic. Very low concentrations of ferritin, which
B cell function. are at physiological levels, cause significant inhibition
Recently, we examined the effect of ferritin on humlan of marrow colony formation and Ig generation in the
B lymphocyte activation25 and found that ferritins do not in vitro system. Although the possible physiological
suppress the proliferation of resting B lymphocytes stim- significance of the inhibitory activity of ferritin remains
ulated by polyclonal B cell mitogen, or Staphylococcus unknown, it is assumed that serum ferritin does not have
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aureus Cowan strain I (SAC) (Table 1) nor do they inhibit a direct effect on the immune reaction and hematopoiesis
the proliferation of activated B cells, (Table I). However, in the in vivo states. Indeed it is probable that this
both L- and H-rich ferritins exert their inhibitory activity activity is blocked or overwhelmed by various factors
on immunoglobulin (Ig) generation by B lymphocytes in including cytokines, or that ferritin might interact with
a T-independent as well as a T-dependent way (Fig. 1). other active proteins. It has been shown in fact that human
The cytoplasmic Ig-containing cells decrease in propor- serum contains binding factors for acidic ferritins,
tion to the reduction of Ig secretion (Table 2). The action probably related to the complement proteins and alpha-2-
of ferritin on Ig generation by B cells appears to be regu- macroglobulin.27 Further studies on the mode of action of
lated at the transcriptional level. These results indicate that ferritins in the cytokine network will be investigated in the
ferritins directly suppress the differentiation of B lym- near future.
phocytes maturing into antibody producing cells. The sup- It is possible that the impairment of cell-mediated im-
pressive activity of L-rich ferritin on Ig production was munity classically observed in patients with hyperfer-
not different from that of the H-rich ferritin, implying that ritinemia is, at least, in part due to the mechanisms
the inhibitory activity of ferritin on the differentiation of 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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