Immunity
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Erythropoietin Surprises: An Immune Saga
Hal E. Broxmeyer1,*
1Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
*Correspondence: hbroxmey@iupui.edu
DOI 10.1016/j.immuni.2011.01.004
Erythropoietin (EPO) an erythropoietic stimulating agent also exerts effects on other cell systems. Nairz et al.
(2011) now link EPO and intracellular signaling through the EPO receptor (EPOR) to innate immune cell activity
via macrophages.
Erythropoietin (EPO) is a member of the
cytokine family of molecules and
a humoral regulator of erythropoiesis
(red blood cell production). EPO effects
on proliferation and survival of erythroid
progenitor cells are mediated via the
EPO receptor (EPOR) and downstream
intracellular signaling events (Shaheen
and Broxmeyer, 2009; Papayannopoulou
et al., 2009). EPO was the first cytokine
to be biochemically purified, a labor-
intensive task undertaken with massive
amounts of urine obtained from anemic
patients (Miyake et al., 1977). The amino
acid sequence of the purified EPO mole-
cule led to cloning of the EPO gene (Lin
et al., 1985; Jacobs et al., 1985). Subse-
quently, the gene for murine EPOR was
cloned. Having available large quantities
of purified recombinant EPO greatly
accelerated our understanding of EPO
activities. However, studies with recombi-
nant EPO at first only confirmed the then
prevailing belief that cytokines, to be
considered physiologically relevant,
would have great cell and tissue speci-
ficity of action, in this case for EPO on
cells of the early erythroid lineage and
selectively for erythropoiesis. It is only
recently that expression of the EPOR
has been reported on nonerythroid cells,
with stimulation of proliferation and antia-
poptotic effects noted on cells of the
nervous system and a number of other
tissues (reviewed in Shaheen and Brox-
meyer, 2009; Nairz et al., 2011). See
Figure 1 for a diagram of reported targets
and a growing number of possible targets
responsive to EPO.
Cloning of the EPO gene and expres-
sion and purification of recombinant
EPO set the stage for EPO to become
the first cytokine to demonstrate clinical
efficacy. EPO has been used in clinical
settings of disease- and treatment-
induced erythropoietic insufficiency.
6 Immunity 34, January 28, 2011 ª2011 Elsevier Inc.
EPO was first tested successfully in dial-
ysis patients suffering from severe anemia
whose only previous treatment at the time
was blood transfusions with inherent
possibilities of infectious episodes and
build-up of toxic levels of iron. EPO was
approved in 1989 by the United States
Food and Drug Administration for treat-
ment of the anemia of chronic kidney
disease to increase or maintain red blood
Proliferation or survival enhancing effects on:
Erythroid progenitor cells (BFU-E)
Neurons
Pancreatic beta cells
Retina cells
Cancer cells (?)
Kidney cells
Liver cells
+ attempts to modulate selective EPO
Erythropoietin
- from thioglycolate-elicited peritoneal
? exudates of mice, as well as a mouse
Suppressing effects on:
Macrophages
(by blocking NF-κB p65)
Unknown effects on:
Macrophage subsets
Other cell types
Cell-cell interactions
Figure 1. Diagram of Targets and Possible
Targets Responsive to Action(s) of
Erythropoietin
The originally described action of erythropoietin
was on enhancing proliferation and survival of cells
within the erythroid system, with one target for
erythropoiesis (production of red blood cells)
being the erythroid progenitor cell (defined func-
tionally as a burst forming unit-erythroid [BFU-E])
(Shaheen and Broxmeyer, 2009). Recently, other
targets of erythropoietin have been reported
(Shaheen and Broxmeyer, 2009; Nairz et al., 2011).
cell levels (Unger et al., 2010). EPO has
been used in a plethora of cases to
enhance erythropoiesis including during
treatment of patients with cancer. How-
ever, side effects of EPO treatment,
some life-threatening, have necessitated
revised EPO treatment guidelines (Rizzo
et al., 2010; Unger et al., 2010). These
side effects, in part, may relate to nonery-
thropoietic responses to EPO, especially
in treatment of patients with cancer.
Therefore, there is a timely and critically
important need to better understand the
totality of cell types that express EPORs,
what the functional outcomes of EPO
activities are on these nonerythroid cell
populations, and how these different
effects may be mechanistically mediated
within the cell. Once we know the range
of EPO actions, and how the different
actions are mechanistically mediated,
effects for clinical advantage become
possible.
Toward these important goals, Nairz
et al. (2011) have now used macrophages
macrophage-like cell line, RAW264.7, to
demonstrate EPO inhibition of a number
of proinflammatory genes in these acti-
vated cells, including tumor necrosis
factor-a (TNF-a) and inducible nitric oxide
synthetase (iNOS), effects apparently re-
sulting from EPO blockage of nuclear
factor (NF)-kB p65 activation. NF-kB is
a master controller of numerous cytokine
genes, so it is likely that there is yet
much more to uncover regarding EPO
effects on production of other cytokines,
and these effects may vary depending
on target EPOR-expressing cell types. In
this study, EPO inhibition of NF-kB activa-
tion was probably responsible for the
noted fact that in vivo administered EPO
reduced survival of mice and impaired
Immunity
Previews
clearance of systemic infection in mice in-
jected with Salmonella, because neutrali-
zation of endogenous EPO or genetic
ablation of EPOR allowed elimination of
the Salmonella infection in such mice,
confirming the effects of EPO in this
model of infection. Interestingly, the
authors also noted that this same
blockage of NF-kB-induced immune
mediator cytokines by EPO resulted in
limited tissue damage and amelioration
of disease severity in a different system,
one of a chemically induced mouse model
of colitis, thus highlighting positive and
negative effects of EPO depending on
the situation.
Exactly how EPO-EPOR engagement
works on macrophages and the complete
signaling cascades set forth are still to be
elucidated. Also, not clear is whether there
are similar or dissimilar intracellular
signals mediating EPO actions in different
cell types such as neurons or even normal
versus cancer cells. As with other cyto-
kines, there is a ‘‘double-edged sword’’
effect that requires finely balanced timing
and dosage for efficacious treatment. In
their paper, the authors provide a number
of relevant scenarios for correct timing of
treatment. One example mentions over-
coming the potential negative effects
associated with EPO enhancement of
bacterial infection by first treating patients
to kill these microbes so that other EPO
effects, such as enhanced erythropoiesis,
can manifest without compromising the
immune system. Moreover, the purported
effects of EPO on enhancing growth and
survival of cancer cells, which in part
necessitated changes in EPO clinical use
guidelines (Rizzo et al., 2010), may as the
authors suggest, also entail EPO impair-
ment of immune responses directed
against neoplastic cells. Two recent
papers (reviewed by Jelkmann, 2010)
found EPOR protein cell surface expres-
sion barely detectable on a large number
of human tumor cell lines, even though
EPOR mRNA expression was apparent.
Thus, it may be that not all cancer cells
have EPOR or respond to EPO, and that
the immune dampening effects of EPO
noted by Nairz et al., (2011) are as impor-
tant, or are more important, than direct
EPO-stimulating effects on proliferation
and survival of tumor cells.
More studies are needed with regards
to rigorously evaluating direct and indirect
effects of EPO on nonerythroid cells. Even
effects purported to be direct acting may
be indirect. The only way to truly prove
a direct cytokine-acting effect is at the
level of a single isolated cell (Lu et al.,
1998). Without such analysis, one cannot
rule out cross-talk between cells for
even an apparently homogenous popula-
tion of cells. Also, multiparameter cell
surface expression and intracellular flow
cytometry could be used to prove effects
at a single-cell level on intracellular
signaling and exact type of cytokine
expression within the specific individual
cells expressing EPOR.
In the context of the paper by Nairz et al.
(2011), it is of importance and interest to
know whether all primary thioglycolate-
induced peritoneal macrophages express
cell surface EPOR protein (because pres-
ence of EPOR mRNA does not a priori
equate with functional surface EPOR)
(Jelkmann, 2010), or whether it is only
within the EPOR-expressing subset of
these macrophages that NF-kB activity
and cytokine production is affected. If
not all these macrophages express
EPOR, cross-talk between cells could be
responsible for some effects. Subpopula-
tions of macrophages exist. Do different
macrophage subsets similarly express
EPOR protein and respond to EPO in
a similar manner? What about resident,
not thioglycolate-elicited, macrophages,
and monocytes, precursors of macro-
phages? Macrophages interact with other
cell types, such as T lymphocytes (see
Figure 1). What role, if any, do such
macrophage-other cell type interactions
play in the final outcome of EPO effects?
A better understanding of the above
questions could significantly advance
Immunity 34, January 28, 2011 ª2011 Elsevier Inc. 7
and enhance the efficacy of EPO treat-
ment, as well as the interesting and
provocative observations of Nairz et al.
(2011).
It will be of great interest to determine
the full repertoire of EPOR-expressing
and EPO-responding cell types in the
body, exactly what effects EPO has on
these EPOR-expressing cells, and physi-
ological and pathological aspects of
EPO-EPOR signaling.
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