granulocyteâmacrophage colony-stimulating factor: not just another haematopoietic growth factor
TRANSCRIPT
REVIEW ARTICLE
Granulocyte–macrophage colony-stimulating factor: not justanother haematopoietic growth factor
Alejandro Francisco-Cruz • Miguel Aguilar-Santelises • Octavio Ramos-Espinosa •
Dulce Mata-Espinosa • Brenda Marquina-Castillo • Jorge Barrios-Payan •
Rogelio Hernandez-Pando
Received: 25 September 2013 / Accepted: 13 November 2013
� Springer Science+Business Media New York 2013
Abstract Granulocyte–macrophage colony-stimulating
factor (GM-CSF) is often used to treat leucopenia. Other
haematopoietins may increase the number of circulating
leucocytes with higher efficiency, but GM-CSF has addi-
tional effects that may be far more relevant than its hae-
matopoietic activity. GM-CSF induces differentiation,
proliferation and activation of macrophages and dendritic
cells which are necessary for the subsequent T helper cell
type 1 and cytotoxic T lymphocyte activation. GM-CSF
haematopoietic and non-haematopoietic functions have
pro-inflammatory and immune regulatory potential to treat
a variety of autoimmune diseases and tumours. On the
other hand, GM-CSF deficiency leads to various immune
dysfunctions and the current utilization of GM-CSF as
haematopoietic factor might be an accurate but very
incomplete indication for a cytokine with vast clinical
potential.
Keywords Haematopoietic growth factor �Granulocyte–macrophage colony-stimulating factor �Innate immunity � Adaptive immunity
Abbreviations
AML Acute myeloid leukaemia
AMØ Alveolar macrophage
BM Bone marrow
CISH Cytokine inducible SH2-domain
DC Dendritic cell
GRAP GM-CSFRa subunit-associated protein
iNKT Invariant natural killer T
IjK IjB kinase
MØ Macrophage
MAPK Mitogen-activated kinase-like protein
NFjB Nuclear factor kappa-light-chain-enhancer of
activated B cells
IkB Nuclear factor of kappa light polypeptide
gene enhancer in B cells inhibitor bPI3K Phosphatidylinositol 3 kinase
PKC Protein kinase C
PAP Pulmonary alveolar proteinosis
rhGM-CSF Recombinant human GM-CSF
rhG-CSF Recombinant human granulocyte colony-
stimulating factor
STAT Signal transducers and activators of
transcription
SLAP SRC-like adapter protein
TLR Toll-like receptor
Introduction
Colony-stimulating factors (CSF) are crucial for survival,
proliferation, differentiation, maturation and functional
activation of haematopoietic cells [1]. There are three types
of CSF, namely macrophage colony-stimulating factor (M-
CSF) or CSF1, granulocyte–macrophage colony-stimulating
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12032-013-0774-6) contains supplementarymaterial, which is available to authorized users.
A. Francisco-Cruz � O. Ramos-Espinosa � D. Mata-Espinosa �B. Marquina-Castillo � J. Barrios-Payan �R. Hernandez-Pando (&)
Department of Pathology, National Institute of Medical Sciences
and Nutrition ‘‘Salvador Zubiran’’, Vasco de Quiroga 15,
Tlalpan, 14000 Mexico City, Mexico
e-mail: [email protected]
A. Francisco-Cruz � M. Aguilar-Santelises
Department of Immunology, National School of Biological
Sciences, National Polytechnic Institute, Mexico City, Mexico
123
Med Oncol (2013) 30:774
DOI 10.1007/s12032-013-0774-6
factor (GM-CSF) or CSF2 and granulocyte colony-stimu-
lating factor (G-CSF) or CSF3 (Fig. 1). CSF were uninten-
tionally discovered when solid-state culture systems allowed
specific precursors of granulocytes and macrophages to
proliferate and form colonies of maturing but non-autono-
mous proliferating cells [1, 2]. The newly found cell product
that needed to be added to stimulate colony formation
received the operational name ‘‘colony-stimulating factor’’
[1, 2].
As part of the haematopoietin family, GM-CSF is a
four-helix packing cytokine (Fig. 2a). The GM-CSF gene
is located in the 5q31 region. Deletion of 5q is associated
with acute myeloid leukaemia (AML), but AML is not
always generated by deletion of 5q. The same chromo-
somal location is associated with other genes encoding
cytokines such as IL-4, IL-5 and IL-13 [2–4]. The stem cell
factor (SCF), leukaemia inhibitory factor, Flt3 ligand
(Flt3L), erythropoietin (EPO), thrombopoietin (TPO) and
IL-3, IL-5, IL-11 also have haematopoietic activity [1].
GM-CSF was first described for its ability to induce
mouse myelopoiesis, in addition to its capacity to stimulate
granulocyte, macrophage, eosinophil, megakaryocyte and
erythroid colony formation. However, there was not a clear
functional distinction between CSF and IL-3 until the
human GM-CSF was sequenced in 1985. The murine GM-
CSF was sequenced in 1994. Cloning techniques also
revealed in 1986 that the hypothetical human ‘‘pluripoie-
tin’’ was actually G-CSF which has since then been used as
the most important haematopoietic factor in humans [2–5].
Deletion of genes encoding for EPO, G-CSF, TPO or
M-CSF produces dramatic reductions in the amount of
cells that are normally stimulated by each one of these
cytokines. However, deletion of the GM-CSF gene only
reduces neutrophils function without significantly affecting
Multipotent stem cell
Primitive progenitor cells
Committed precursor cells
Linage commited cells
Neutrophils, eosinophils, basophilsMonocytes
Self-renewal
SCF TPO
GM-CSF
HSC
G-CSF, IL-5, SCF M-CSF
CMP
GM
MP GP
Fig. 1 GM–CSF relationship
with haematopoiesis. Formation
of blood cells progress from a
haematopoietic stem cell, which
can undergo either self-renewal
or differentiation into a
multilineage committed
progenitor cell named common
myeloid progenitor. This cell
gives rise to a more
differentiated progenitor by
stimulation with GM–CSF
committed to the granulocytes
and macrophages (GM) linage.
Finally, these cells give rise to
uni-lineage committed
progenitors for granulocytes or
monocytes. SCF stem cell
factor, TPO thrombopoietin
[115]
Page 2 of 14 Med Oncol (2013) 30:774
123
their number in circulation [1]. Recombinant human GM-
CSF (rhGM-CSF) has been available in USA for thera-
peutic use since the early nineties. Initially prescribed only
to enhance myeloid reconstitution in transplanted patients,
it is now also used as myeloproliferative agent [1, 5, 6].
Clinical trials revealed its pro-inflammatory effect and, in
consequence, its potential usefulness as a prophylactic or
adjuvant therapeutic agent for patients at high risk of
infection [7]. In 1996, the Food and Drug Administration
approved GM-CSF for treatment for leucopenia associated
with fungal infections [7]. GM-CSF applications will be
most probably further extended due to its ability to induce
inflammatory responses of differentiated eosinophils,
neutrophils, macrophages (MØ) and dendritic cells (DC)
[1, 5, 6].
GM-CSF expression and target cells
GM-CSF can be detected under physiological conditions at
serum concentrations ranging from 20 to 100 pg/ml [8].
Higher serum levels of GM-CSF require stimulation with
cytokines, antigens, microbial products or inflammatory
agents such as IL-1, TNF-a or lipopolysaccharide (LPS) [8].
In fact, GM-CSF was first purified from the conditioned
medium of lung tissue from mice after LPS administration
Fig. 2 Structure, cellular sources and cellular targets of GM–CSF.
a Type I cytokine or alpha short chain with four packing helix, native
GM–CSF is a monomeric glycosylated protein of 127 aa. rhGM-CSF
is associated with dimeric complexes. Structure obtained from Protein
Data Bank PDB: 2GMF. b GM-CSF production usually requires
stimulation with cytokines, antigens, microbial products or inflam-
matory agents (IL-1, TNF-a or LPS). Both in steady state and induced
conditions GM-CSF may be produced by CD4? T cells (TH1, TH2
and TH17), B lymphocytes, macrophages, keratinocytes, eosinophils,
neutrophils, endothelial cells, CD56hiCD16- NK cells, bone-forming
osteoblasts, mast cells, multipotent mesenchymal stromal cells and
Paneth’s cell. Upon appropriate stimulation, much of the production
and action of GM-CSF occurs locally at the site of inflammation.
c The major target cells and effects of GM-CSF are maturation in DC,
activation, survival and differentiation in granulocytes, steady-state
differentiation in alveolar MØ (AMØ) and invariant natural killer T
(iNKT) cells and proliferation in microglia and pneumocytes type I
Med Oncol (2013) 30:774 Page 3 of 14
123
[8]. GM-CSF is produced by a variety of cells, including
stromal cells, Paneth’s cells, MØ, DC, mast cells, endothelial
cells, smooth muscle cells, fibroblasts, chondrocytes, as well
as IL-23-stimulated TH17 cells and IL-1b-stimulated TH1
and TH17 cells (Fig. 2b) [8, 9]. Among these, T cell-derived
GM-CSF has a particularly important role in the crosstalk
between antigen-presenting cells and T cells [8, 9]. Although
the major role for GM-CSF seems to induce DC maturation,
GM-CSF also induces granulocytes activation, microglia
proliferation, steady-state differentiation in invariant natural
killer T (iNKT) cells and survival and differentiation of
alveolar MØ (AMØ; Fig. 2c). Moreover, lymphocytes and
endothelial cells have GM-CSF receptors (GM-CSFR)
which makes them susceptible to GM-CSF stimulation, but
the consequences of such stimulation are not clear [8–11].
GM-CSF knockout (KO) mice do not suffer abnormal-
ities in myelopoiesis, but they do have the characteristic
abnormalities of pulmonary alveolar proteinosis (PAP),
indicating that GM-CSF might have a significant role in
maintenance of the normal lung physiology [5, 10]. GM-
CSF deficient mice also have a deficient iNKT cells dif-
ferentiation during thymic ontogeny since GM-CSF acts at
the early thymic iNKT cell precursor stage, after lineage
commitment and positive selection by regulating the
acquisition of secretory function for immune synapse-
directed release of immunoregulatory cytokines [5, 10].
GM-CSF receptor
The GM-CSFR is a heterodimer formed by a ligand-spe-
cific a subunit (GM-CSFRa or CD116; 60–80 kDa) and a
b subunit (GM-CSFRbc or CD131; 120–140 kDa) shared
with IL-3 and IL-5 receptors [12]. Both subunits are type I
transmembrane glycoprotein structurally characterized by
the presence of cytokine-receptor homology modules,
consisting of two domains of fibronectin type III [13]. GM-
CSFRa binds its ligand with low affinity (KD =
0.2–100 nM), but the expression of GM-CSFRbc increases
GM-CSFRa affinity to KD = 100 pM [13]. Eight confor-
mational variants have been described for GM-CSFRa, but
only two are biologically relevant for its transductional
effect. The a-1 and a-2 isoforms contain transmembrane
and cytoplasmic regions with abundant serine and proline
residues. The importance of GM-CSFRa is illustrated by
the fact that the complete deletion of its cytoplasmic region
results in lack of cell growth and differentiation. GM-
CSFRbc is constitutively expressed on the cell surface
[14]. Similarly to IL-3R and IL-5R, GM-CSFR is expres-
sed at very low levels on the surface of haematopoietic
cells, with only 100–1,000 molecules per cell [13]. GM-
CSFR is also expressed on granulocytes and macrophages
progenitor cells, mature cells such as monocytes, MØ, DC,
megakaryocytes, neutrophils, plasma cells, T lymphocytes,
vascular endothelial cells, uterine cells, gastrointestinal
tract epithelial cells, astrocytes, oligodendrocytes and
microglia cells [9, 15, 16].
GM-CSF/GM-CSFR interactions
GM-CSF is able to bind GM-CSFRbc in the absence of
GM-CSFRa, but the heterodimerization with both subunits
is required for intracellular signal transduction. GM-CSFR
activation follows general rules regarding receptor dimer-
ization and transphosphorylation of tyrosine residues in the
receptor cytoplasmic domains. GM-CSFR does not have
intrinsic tyrosine kinase activity. Instead, it requires the
association of GM-CSFRbc with JAK-2 for GM-CSFRbc
transphosphorylation, involving two bc chains closely sit-
uated in the cytoplasmic region [17]. Crystallographic
studies have shown that GM-CSFRbc is in fact homodi-
meric, but its cytoplasmic regions are quite separated
(120 A), which makes transphosphorylation difficult to
occur [17–19].
A unique tertiary structure of the GM-CSF/GM-CSFR
complex has been described. It corresponds to a coordi-
nated dodecamer structure, which is necessary for receptor
activation (Fig. 3a). The association between GM-CSF and
GM-CSFR involves three sites of interaction. The first
interaction is between GM-CSF and GM-CSFRa, the sec-
ond is between GM-CSF and two domains of two different
GM-CSFRbc molecules and the third one is a stabilizing
site formed between GM-CSFRa and GM-CSFRbc. These
three combinations determine the formation of a higher-
order dodecamer complex composed by two hexameric
complexes linked by a fourth site of interaction. Antibodies
and mutations directed to the fourth site of interaction
significantly decrease GM-CSF transduction and initiate
the loss of the dodecamer complex [13]. These interactions,
which are only observed in GM-CSFR, explain the trans-
phosphorylation by GM-CSF/GM-CSFR in which JAK-2 is
associated with the beta chain. The dodecamer complex
product relocates closer two GM-CSFRbc, at a distance of
10 A permitting transphosphorylation and the activation of
subsequent signalling pathways [13, 19].
GM-CSF signalling
GM-CSFR transduction is similar to the activation carried
on by the interferon production regulator family. Binding
of GM-CSF with its receptor leads to the activation of
kinases of the JAK family which then phosphorylate signal
transducers and activators of transcription (STAT) that
migrate to the nucleus and bind specific DNA elements
directing the transcription of specific genes related to cell
differentiation. GM-CSF induces cell proliferation mainly
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by activation and signalling of protein kinase C, preventing
cell death by phosphatidylinositol 3 kinase and JAK/
STAT5-Bcl-2 signalling. GM-CSF induces proliferating
and inflammatory responses by mitogen-activated protein
kinases (MAPK) and NFjB activation (Fig. 3B) [14, 20].
GM-CSF increases AMØ and neutrophils function.
Proliferating AMØ increase their own expression of toll-
like receptor 4 (TLR4) and nuclear factor kappa-light-
chain-enhancer of activated B cells (NFjB) by direct GM-
CSFR and IjB kinase (IjK) interaction. GM-CSF regulates
the expression of TLR2 and TLR4 in neutrophils and TLR2
in monocytes. Additionally, GM-CSF induces the release
of IL-12 and TNF-a and enhances the expression of
monocyte chemoattractant protein (MCP)-1 by JAK2-
STAT5 signalling in monocytes [21, 22]. Recently, a spe-
cific signalling pathway has been described for inducing
classic MØ phenotype or M1 and inflammatory DC, which
is described below. SRC-like adapter protein (SLAP)
functions as a negative regulator for DC maturation
induced by GM-CSF. The need for SLAP action suggests a
specific threshold for correct stimulation by GM-CSF for
monocytic DC maturation [23].
GM-CSF binding to GM-CSFRbc triggers most of the
intracellular signals that induce inflammation, cell survival,
differentiation and proliferation in monocytes and granu-
locytes. c-Kit and GM-CSFRa are well-known markers of
haematopoietic cells differentiation. Haematopoietic stem
cells down-regulate c-Kit and up-regulate GM-CSFRawhen they enter to the granulocyte/macrophage lineage
[24, 25]. GM-CSFRa has two isoforms. Stimulation of the
GM-CSFRa-1 isoform induces overexpression of CD86
and F4/80 cell surface proteins, and it may also induce
intense phosphorylation of JAK-2 [26]. GM-CSFRa-2
isoform has a different cytoplasmic domain, but it associ-
ates with GM-CSFRbc in the same manner than GM-
CSFRa-1 does it [27]. There also exist soluble isoforms of
both a-subunits derived from alternative mRNA splicing,
but their functions are not known [27, 28].
GM-CSF clinical applications as haematopoietin
GM-CSF is the growth factor most extensively used as
hemopoietin in the clinical practice. rhGM-CSF is pro-
duced in S. cerevisiae, and it is named sargramostim or
Leukine� (Bayer HealthCare Pharmaceuticals, USA). Its
amino acid sequence is homologous to the endogenous
human GM-CSF, except for a leucine instead of a proline
at the 23rd position and different glycosylated motives. The
degree of glycosylation affects the biological activity,
antigenicity, toxicity and pharmacokinetics. When rhGM-
CSF is produced in E. coli is named molgramostim
(Zenotech Laboratories, India) and when it is produced in
Chinese hamster ovary cells is named regramostim (Lan-
ospharma Laboratories, China) [7]. Sargramostim may help
to prevent infections by enhancing the immune cell func-
tion. It is the only growth factor approved in USA for
treatment of older adults with AML, after induction che-
motherapy, to shorten the time to neutrophil recovery and
to reduce the incidence of life-threatening infections [7,
29]. Sargramostim is also approved in USA for myeloid
reconstitution following allogeneic bone marrow trans-
plantation (BMT), autologous BMT or peripheral blood
stem cell transplantation. Furthermore, sargramostim is
also approved for peripheral blood stem cell mobilization,
BMT failure and engraftment delay [29].
Pharmacokinetics and pharmacodynamics of rhGM-
CSF
When sargramostim is administered to the patients by
intravenous infusion during 2 h, it reaches a mean beta
half-life of approximately 60 min. The peak concentra-
tions of GM-CSF are observed in blood samples
obtained during or immediately after completion of the
sargramostim infusion, while minor concentrations are
still detected in blood 6 h after the beginning of the
infusion [7, 29]. GM-CSF is also detected in serum first
after 15 min of subcutaneous injection of sargramostim
to healthy volunteers. Then, the mean beta half-life is
approximately 162 min, and peak levels are reached after
one to 3 h post-injection and remain detectable for up to
6 h. Parenteral administration of rhGM-CSF alters the
kinetics of myeloid progenitor cells in bone marrow
accelerating the cell-cycle entry and reducing the cell-
cycle time. These effects are reversible once adminis-
tration is discontinued. However, the numbers of mobi-
lized progenitor cells that are released in peripheral
blood by GM-CSF are 10 times lower than those
released by G-CSF [6, 30].
rhGM-CSF and rhG-CSF in leucopenia
rhGM-CSF produces small increases in the number of
peripheral neutrophils and circulating monocytes. It is
currently indicated as second-line treatment for patients
with severe neutropenia [7, 31]. rhGM-CSF also induces a
mild increase in the number of circulating eosinophils and
basophils and enhances the phagocytic function of neu-
trophils. Filgrastim (rhG-CSF) and pegfilgrastim (pegylat-
ed rhG-CSF) are currently approved by FDA for prevention
of chemotherapy-induced neutropenia [1, 7].
The therapeutic application of rhGM-CSF for leuco-
penia could be overestimated. G-CSF recommendations
for neutropenia are supported by various results from
clinical trials and head-to-head comparative studies about
Med Oncol (2013) 30:774 Page 5 of 14
123
clinical benefits of G-CSF compared with GM-CSF are
lacking. Moreover, G-CSF has higher myeloproliferative
activity than GM-CSF [30]. CD34? mononuclear cells
mobilization into the peripheral blood by GM-CSF
administration is approximately 10 times smaller than
G-CSF (12.6 9 106 cells vs. 119 9 106 cells, respec-
tively). These effects are not synergistic when both CSF
are combined [6, 7, 30]. Besides, there are ample differ-
ences in therapeutic doses between G-CSF (5 lg/kg) and
GM-CSF (250 lg/m2), which are considered in the clin-
ical guidelines from the National Comprehensive Cancer
Network (NCCN) and the American Society of Clinical
Oncology (ASCO) recommending rhG-CSF as first-line
therapy for febrile neutropenia [31].
Toxicological profile of rhGM-CSF
Sargramostim is usually well tolerated by healthy subjects,
who have not shown clinical alterations in their clinical
analysis, as compared to placebo-treated individuals [29].
NCCN guidelines recommend sargramostim administration
Page 6 of 14 Med Oncol (2013) 30:774
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to 55 years old and older AML patients 24 h after induc-
tion chemotherapy [31]. Although sargramostim adminis-
tration increases cellularity in haematological, heart and
lung tissue on monkeys, these effects have not been serious
enough to fulfil toxicity standard criteria [29]. However,
sargramostim-treated patients with febrile neutropenia
secondary to chemotherapy, risk to develop fluid retention,
respiratory symptoms due to sequestration of granulocytes
in pulmonary circulation, cardiovascular symptoms, renal
and hepatic dysfunction, as well as adverse reactions
related to bone marrow transplant or peripheral blood
progenitor cell transplant [31]. Paediatric patients suffering
Crohn’s disease have been treated with subcutaneous doses
of 4 or 6 lg/kg sargramostim every day during 8 weeks,
and 90 % of them only had mild injection site reactions,
whereas the resting 10 % required dose reductions due to
elevated absolute neutrophil counts [32]. Syncope is the
worst side effect triggered thus far by sargramostim
administration to human subjects. Since it was attributed to
EDTA that was included in the liquid form, the FDA
prohibited the preparation of Leukine� in its liquid for-
mulation [33].
Non-haematopoietic functions of GM-CSF
GM-CSF has significant non-haematopoietic effects in
various physiological processes other than haematopoiesis.
GM-CSF KO mice do not suffer alterations in the steady
state of haematopoiesis. Instead, GM-CSF KO animals
acquire a particular lung injury phenotype characterized by
accumulation of lipids and surfactant proteins in alveolar
spaces, similar to the human condition described as PAP
[3, 4, 34].
Inflammation
The contribution of GM-CSF to the inflammatory response
has been evaluated in vitro. rhGM-CSF induces longer
survival in monocytes, MØ and neutrophils, augments the
amount of inflammatory mediators released by these cells
and contributes to the elimination of pathogens and
tumours [35, 36]. MØ are more efficiently stimulated by
secondary stimuli such as LPS and IFN-c, when they are
initially stimulated with GM-CSF. Intra-peritoneal admin-
istration of GM-CSF induces a strong recruitment of
human MØ, and both human and murine monocytes show
higher pro-inflammatory response when they are primary
stimulated with GM-CSF and then restimulated with LPS
[37, 38].
One of the most interesting features of GM-CSF was
observed in patients with Felty’s syndrome (rheumatoid
arthritis, splenomegaly and neutropenia) that received
rhGM-CSF and experienced worsening of arthritis [39].
Additionally, patients with rheumatoid arthritis (RA) also
experienced symptoms exacerbation when treated with
rhGM-CSF after chemotherapy [40]. Moreover, the exac-
erbation of RA symptoms may be reproduced with GM-
CSF administration in experimental models [41, 42]. On
the other hand, administration of mavrilimumab, a human
monoclonal antibody targeting GM-CSFRa, induces rapid
and significant responses in RA subjects [43].
Activation and regulation of the immune system
GM-CSF stimulates the expression of adhesion molecules,
IgGFcR and activated complemented receptors on neutro-
phils, enhancing their response to chemotactic factors,
phagocytosis, synthesis of leukotriene B4, arachidonic acid
release and superoxide anion generation. Moreover, GM-
CSF increases survival of neutrophils and induces its
MHC-II expression enabling them to activate T cells
response to superantigens [44, 45].
AMØ orchestrate structural and functional parenchyma
repair processes that are essential for homoeostasis [46].
Different extracellular signals are integrated to shape up
pulmonary MØ phenotypes, during lung inflammation
Fig. 3 Dodecameric complex of GM-CSFR activation and signalling
pathways induced by GM–CSF. a Schematic representation of GM-
CSFR and steps for dodecameric complex formation and activation.
Low-affinity binary complex consists of linked GM–CSF and GM-
CSFRa. Interaction with free dimeric forms of GM-CSFRbc chains
makes possible the formation of a hexameric complex formed by two
GM-CSFRbc chains. Each one of these two chains is joined to one
GM-CSFRa chain and carries a GM-CSF molecule (2:2:2). The
dodecameric complex, the highest order complex, is formed by the
lateral aggregation of two hexameric complexes thanks to a fourth site
of interaction that forms a signalling competent structure, diminishing
the distance between bc chains and allowing the transphosphorylation
of JAK-2. b GM–CSF exerts its biological functions by phosphory-
lating at least two domains in the b-chain of its receptor. Once GM-
CSFRbc is transphosphorylated, adapter proteins and kinases are
recruited for inducing activation of nuclear factors that up-regulate the
expression of specific genes related to inflammation, survival,
differentiation and cell proliferation. One of the GM-CSFRbc trans-
phosphorylated domains induces the activation of Ras and mitogen-
activated protein kinases (MAPK) with the consequent induction of
c-fos and c-jun and PI3 K/Akt/p21waf-1. The other phosphorylated
domain mediates the activation of Janus kinase 2 (JAK2)/signal
transducer and activator of transcription 5 (STAT5) signalling
pathway. Activated STAT5 migrates into the nucleus after mutual
phosphorylation and dimerization and binds to specific DNA-binding
sites, followed by the expression of STAT5 target genes. Jak kinase–
STAT5 transcription factor pathway induces the expression of
interferon-regulating factor 5 (IRF5). High expression of IRF5 results
in classical MØ activation (M1) or inflammatory MØ induction. GM–
CSF supports differentiation, survival and proliferation in part through
phosphorylated STAT5 (pSTAT5) signalling. In parallel, pSTAT
activity leads to an accumulation of cytoplasmic cytokine inducible
SH2-domain protein (CISH). CISH induces feedback inhibition of
STAT5 activation, triggering the differentiation of type 1 polarized
DC. GM-CSFRa subunit-associated protein (GRAP), a protein that
binds to the cytoplasmic region of the alpha chain, is highly expressed
in neoplastic cells
b
Med Oncol (2013) 30:774 Page 7 of 14
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TNF-a released from activated AMØ induces epithelial
GM-CSF expression, which initiates alveolar epithelial cell
proliferation and helps to restore the alveolar barrier
function [47]. Pneumocytes type II express GM-CSFRaand GM-CSFRbc. Proliferating type II pneumocytes may
then down-regulate GM-CSFR expression during its trans-
differentiation into type I pneumocytes occurring in
response to acute lesion [46, 47].
GM-CSF regulates the inflammatory response by acti-
vation of the JAK kinase–STAT5 transcription factor
pathway, inducing the expression of the interferon-regu-
lating factor 5 (IRF5). A high IRF5 expression by GM-CSF
or IFN-c results in M1 MØ activation. IRF5 acts together
with RelA (NFjB protein) inducing TNF-a gene expres-
sion and IRF5 inhibits the transcription of IL-10 and pro-
motes IFN-c expression [48]. In contrast, when M-CSF
predominates, IRF4 is overexpressed inducing a M2 MØ
phenotype. Several cell types, including fibroblasts, endo-
thelial cells, stromal cells and osteoblasts, constitutively
produce M-CSF. There is a steady-state production of
M-CSF that turns MØ towards the M2 phenotype trough
IRF4, probably balancing the pro-inflammatory microen-
vironment achieved by the expression of IL-12, IL-6 and
IL-23 that are produced by GM-CSF-stimulated MØ [46,
48].
GM-CSF has an important role in the production of BM-
derived type 1 DC. BM-derived DC acquires potent anti-
gen-presenting capacity, representing a crucial reservoir of
professional antigen-presenting cells (APC). In type 1 DC
differentiation, the expression and activation of STAT5 is
gradually enhanced by GM-CSF during the early stage of
DC development. In parallel, pSTAT activity leads to the
accumulation of cytoplasmic cytokine inducible SH2-
domain (CISH) protein, at the later stage [49]. When a
threshold concentration of CISH is reached, CISH inhibits
the GM-CSF-mediated STAT5 phosphorylation, triggering
its differentiation to strong type 1 polarized DC, promoting
MHC class I expression. Thus, CISH can act as a molecular
switch from the DC progenitor stage to immature DC and a
control point for cytotoxic T lymphocytes activation [49].
GM-CSF is a DC-activating cytokine inducing a strong
differentiation of TH1 type cells. GM-CSF enhances the
oxidative metabolism, cytotoxicity and antibody-dependent
phagocytosis [7, 9]. Human monocytes and monocyte-
derived DC treated with GM-CSF exhibited higher
expression of MHC-II molecules, CD80, CD86 and CD40,
increasing the immune response against antimicrobial
agents [50, 51]. However, the endometrium originated
GM-CSF induces tolerance during early pregnancy [52],
GM-CSF directs DC from the central nervous system
(CNS) towards an inhibitory phenotype. In contrast, DC
recruited by GM-CSF from the peripheral circulation into
the CNS tissue has a pro-inflammatory phenotype, which
has been related to the pathogenesis of autoimmune
encephalomyelitis [53].
GM–CSF relationships with disease
The ability of GM-CSF to regulate the immune system has
been related to Felty’s syndrome, RA and various other
autoimmune, cardiovascular and metabolic diseases [43,
54]. Local over expression of GM-CSF in the stomach of
mice leads to autoimmune gastritis [55]. On the other hand,
GM-CSF deficient mice are less prone to develop experi-
mental allergic encephalitis, myocarditis and collagen-
induced arthritis [5, 8, 10]. GM-CSF promotes autoim-
munity by enhancing IL-6-dependent survival of antigen-
specific T CD4? cells. It has been described as the en-
cephalitogenic factor produced by IL-23-driven pathogenic
TH cells. GM-CSF is required for the response to IL-6 and
IL-23 by DC and subsequent TH17 cell generation [54].
Moreover, GM-CSF promotes autoimmunity by enhancing
IL-6-dependent survival of antigen-specific T CD4? cells
[54, 55].
GM-CSF is expressed by adipose tissue, but its role in
there remains unclear. GM-CSF KO mice are obese,
hyperphagic and show larger mesenteric fat adipocytes,
decreased number of MØ, lower transcription of pro-
inflammatory cytokines and enhanced peripheral uptake of
glucose when receive a high-fat diet [56]. iNKT cells
constitute up to 50 % of liver lymphocytes, and the relation
between GM-CSF, liver iNKT cells and metabolism has
not yet been investigated [57]. On the other hand, M-CSF
production in CD68? MØ found in human fatty streaks and
atherosclerotic plaques suggests that the ratio between
M-CSF and GM-CSF is important to determine MØ het-
erogeneity in atherosclerotic lesions [58]. The antagonism
between M-CSF and GM-CSF is also important to define
the functional polarization of M2 and M1 MØ during the
course of diverse inflammatory and metabolic disorders
[59, 60]. MØ migration to areas of myocardial infarction is
associated with tissue damage, whereas the treatment with
anti-GM-CSF antibodies decreases the amount of damaged
tissue after infarction [61]. A model of cerebral ischaemia
also shows that GM-CSF promotes collateral arteries
growth and reduces cerebral ischaemia [62].
GM–CSF relationship with tumour cells
The presence of GM-CSFR on tumour cells makes them
susceptible to GM-CSF stimulation. GM-CSFRa binding
may be particularly important for signal transduction in
tumour cells overexpressing the GM-CSFRa subunit-
associated protein [63]. The interaction between tumour
cells and sensory neurons seems to be part of the patho-
physiology of cancer-induced pain [64, 65]. The intense
Page 8 of 14 Med Oncol (2013) 30:774
123
bone pain occurring as side effect in rhGM-CSF-treated
patients that suffer myelodysplastic syndromes may be
caused by a two-way interaction between GM-CSFRabearing tumour cells and neurons [66]. GM-CSFRa is also
expressed on pancreatic nerves from healthy subjects and
in large hypertrophic nerves close to tumours in pancreatic
carcinoma patients [67]. GM-CSFR on nerve fibres may
contribute to tumour-induced pain through the JAK-
STAT3 pathway and ERK1/2 signalling involving both
GM-CSF and G-CSF (Fig. 4) [66, 67].
Myeloid-derived suppressive cells (MDSC) are a het-
erogeneous population of immature granulocytic and
monocytic cells, not expressing cell surface markers
associated with differentiated monocytes, MØ or DC.
MDSC exert an immunosuppressive effect of the immune
response against cancer [68]. Although the suppression
mechanism is not clear, it has been strongly associated with
the local presence of MDSC and M2 MØ in mice and
humans [68, 69]. In humans, MØ can be differentiated
either towards pro-inflammatory M1 MØ or anti-inflam-
matory M2 MØ. M1 MØ express IL-12high, IL- 23high, IL-
10low and have high bactericidal and tumoricidal capacity,
whereas M2 MØ expressing IL-12low, IL-23low, IL-10high
are involved with promotion of tissue remodelling, tumour
growth and immunoregulation [70]. The possibility exists
that tumour cells may be favoured by GM-CSF since they
can be stimulated to in vitro proliferation. However,
tumour cells proliferation may not be the predominant
result, due to the presence of GM-CSF and interaction with
other GM-CSF responder cells. If tumour growth promo-
tion occurs, it may be rather due to M-CSF stimulation,
leading to activation of M2 MØ and other tumour-associ-
ated macrophages that produce anti-inflammatory cyto-
kines which are linked to in vivo growth, migration and
spreading of a variety of cancers [48, 71].
Therapeutic applications of GM-CSF
GM-CSF KO mice are more susceptible than wild-type
mice to several infections and intestinal inflammation [72].
Whereas GM-CSF production is necessary to maintain a
fully functional intestinal innate immunity and to facilitate
recruitment and survival of intestinal DC, lack of GM-CSF
may be related to an immune deficiency associated with
Crohn’s disease [73]. Mutations in the intracellular pattern
recognition receptor NOD2, also known as inflammatory
bowel disease protein 1, have been identified in patients
with Crohn’s disease. These patients produce normal levels
of TNF-a, but they do not produce enough GM-CSF [74].
Multicentre randomized clinical trials have demonstrated
that subcutaneous administration of GM-CSF induces sig-
nificant remission of Crohn’s disease [75, 76]. PAP is
another disease in which GM-CSF production is relevant.
Tumor cells
Inflammatory cells
Sensory nerves
HyperalgesiaNerver emodelling
Release of proinflammatorycytokines
Proliferation
Fig. 4 Tumour–nerve interactions and cancer-induced pain. GM–
CSF may contribute to autocrine and paracrine mechanisms involving
proliferation of tumour cells. GM–CSF sensitizes nerves to mechan-
ical stimuli in vitro and in vivo, potentiates calcitonin gene-related
peptide (CGRP) release and causes sprouting of sensory nerve
endings in the skin mediating the sensitization of nociceptors
following exposure to CSF trough JAK-STAT3 and ERK1/2 signal-
ling. GM–CSF production can lead to recruitment and activation of
inflammatory MØs and monocytes and subsequent release of pro-
inflammatory cytokines such as TNF-a
Med Oncol (2013) 30:774 Page 9 of 14
123
PAP is a fatal lung disease characterized by respiratory
failure secondary to a deficient AMØ clearance, abnor-
malities in host defence and accumulation of surfactant
proteins [34]. Autoantibodies against GM-CSF and defi-
cient expression of the GM-CSFRa or bc chain have been
described in patients with PAP [34]. rhGM-CSF inhalation
is beneficial for PAP patients, and gene therapy with vec-
tors encoding GM-CSFRbc has shown efficacy in pre-
clinical results [77, 78].
Recent clinical data suggest that the expression of GM-
CSF may enhance the immune response to tumours. The
first FDA approved vaccine against tumours (Sipuleucel-T;
Provenge�) is an individually tailored DC-based vaccine
that incorporates the use of a key prostate cancer antigen
and GM-CSF to activate DC ex vivo, which upon DC
reconstitution recruits and stimulates T cells to destroy
prostate tumour cells [79]. Another therapy approach has
been to reduce the elevated levels of transforming growth
factor (TGF)-b2 that are frequently linked to immunosup-
pression in cancer patients by simultaneously administer-
ing a TGF-b2 antisense and GM-CSF plasmid and cancer
vaccines destined to enhance autologous tumour cells
recognition [80]. A third type of is currently in a phase III
clinical trial to evaluate the results of expressing GM-CSF
together with IL-12 in allogeneic cancer cells as treatment
for lung cancer [81, 82]. This kind of therapy may be also
effective against colorectal cancer, metastatic renal cell
carcinoma and pancreatic adenocarcinoma [83–85]. The
experimental administration of an adenovirus encoding
GM-CSF together with an adenovirus encoding p16 tumour
suppressor gene induces a potent antitumour response by
increasing the expression of MHC I molecules and the
cytotoxic activity of CD8 T cells [86]. Moreover, the
administration of therapeutic vaccines with plasmids
encoding GM-CSF and tumour antigens enhances the IgG
antibody response antitumour antigens, as well as the IFN-
c and IL-6 production [87, 88]. Another interesting strat-
egy, still in experimental phase, is the combined applica-
tion of armed oncolytic adenovirus expressing IL-12 and
GM-CSF with radiotherapy to suppress primary hepato-
carcinoma in mice [89].
In vitro and in vivo microbicidal activity of neutrophils
and MØ is enhanced by GM-CSF. GM-CSF also prevents
the immunosuppressant effect of dexamethasone, allowing
an effective response against Aspergillus fumigatus hyphae
[90]. Preincubation of neutrophils with GM-CSF increases
the fungicidal activity against Candida glabrata and His-
toplasma capsulatum [7, 91, 92]. In despite of this, there
are no current clinical trials that focus on GM-CSF effect
as an adjuvant for the treatment against invasive fungal
diseases. There are also reports about GM-CSF antibacte-
rial activity in immunodeficient patients. GM-CSF enhan-
ces ex vivo neutrophil bactericidal activity against
S. aureus in children with HIV infection [93]. The use of
GM-CSF in haematopoietic stem cell transplanted patients
reduces the incidence of bacterial infections and decreases
the infection-related hospitalization rates [94]. Local
administration of GM-CSF protects mice against lethal
pneumococcal pneumonia and Chlamydia trachomatis
[95–97].
In vitro studies demonstrated that human MØ stimula-
tion with GM-CSF enhances microbicidal response against
M. tuberculosis [98]. GM-CSF KO mice are highly sus-
ceptible to mycobacterial infections that produce intra-
bronchial and intra-alveolar lesions without formation of
granulomas that are usually produced in wild-type mice
[99]. GM-CSF protects alveolar structure and regulates
early recruitment of MØ and DC, which help to control
mycobacteria growth through granulomas formation [100].
In mice, overexpression of GM-CSF by gene therapy has
shown protection for the reactivation of latent tuberculosis
and avoids the transmissibility of hypervirulent M. tuber-
culosis [101].
The administration of GM-CSF to septic patients did not
produce favourable results. GM-CSF mildly increases the
number of circulating neutrophils in small-for-gestational
age babies but do not improve neonatal sepsis-free survival
[102]. Besides, production of GM-CSF negatively corre-
lates with survival to septic shock [103]. Extremely high
doses of GM-CSF may have a negative impact with
extensive lesions and production of cytokines and inflam-
matory mediators that failed to concentrate T cells and MØ
into sites of infection, MØ accumulation, blindness and
severe damages to various tissues [104, 105].
Prophylactic vaccines based on GM-CSF
The adjuvant role of GM-CSF in prophylactic vaccines
against infectious diseases has been evaluated in preclinical
trials. Vaccines based on recombinant vectors encoding
mycobacterial antigens or vaccines based on recombinant
BCG have shown effectiveness in experimental models of
TB. However, the use of GM-CSF as adjuvant improves
protection against disseminated infection increasing the
number of APC as well as the production of IL-12, IFN-c,
antibodies and the cytotoxic response [106–111]. Intra-
cerebral administration of recombinant rabies virus
encoding GM-CSF prevents the development of experi-
mental rabies after infection with the wild-type virus [112].
A similar DNA vaccine co-expressing GM-CSF against
Clostridium botulinum has shown protection [113]. GM-
CSF is also under evaluation as a new tool for the treatment
for TB, aspergillosis, candida, and influenza [5, 10, 114].
GM-CSF may induce tumour cell death by activation of
circulating monocytes. This and its other immunomodulatory
Page 10 of 14 Med Oncol (2013) 30:774
123
properties are currently exploited by means of its adminis-
tration as adjuvant of vaccines against prostate cancer, and it is
also under evaluation in clinical trials for treatment for mel-
anoma and haematological malignancies. Nine hundred sixty-
eight studies were found related to ‘‘sargramostim’’ or ‘‘GM-
CSF’’ in the clinicaltrials.gov page in October 2013. Most
current studies are analysing the usefulness of GM-CSF in
haematological disorders and cancer as adjuvant therapy.
Additionally, GM-CSF is also currently being evaluated as
adjuvant for prophylactic vaccines against HIV, sepsis, peri-
odontitis and Crohn’s disease. Table 1 (offered as Supple-
mental Material) shows some of the most interesting ongoing
clinical trials and the potential clinical applications of GM-
CSF that now motivate pharmaceutical companies to inves-
tigate the immunoregulatory and protective effects of this
cytokine in a variety of illnesses.
Conclusion
CSF has a long history of clinical use, but their entire
potential has not yet been fully exploited. They have been
almost exclusively applied to increase the absolute num-
bers of circulating innate immune effector cells which
greatly differ since every CSF has different effect on bone
marrow cells production. Furthermore, neither maturation,
chemotaxis, phagocytosis nor microbicidal functions have
been considered when analysing the effect of diverse CSF,
which has made researchers to miss the very important
value of GM-CSF as immunoregulatory cytokine. It is
quite intriguing how little overlap there is in biological
actions among lymphoid and haematopoietic regulatory
factors. One difference between haematopoietic and lym-
phoid regulatory factors is the circumscribed site in bone
marrow of their production and action confining its stim-
ulation to specific cell colony types. This is one of the most
important differences between G-CSF GM-CSF and
M-CSF, which should be considered as cytokines that
affect rather the quality than the quantity of leucocytes in
circulation. Such functional effect should be first consid-
ered in the clinical guidelines for therapeutic use.
The inclusion of biological products has changed and
expanded the possibilities of modern therapy. GM-CSF
effects on the inflammatory response influence the outcome
of multiple tissue insults in several systems and organs.
Virus-based immune agents such as GM-CSF armed vec-
tors are among the early efforts to reach a potent immu-
nostimulation therapy. GM-CSF has a wide range of
lymphoid regulatory actions, linking innate and adaptive
immunity, acting as a bridge between haematopoietic and
lymphoid factors and should be considered first-line treat-
ment for all kind of illnesses requiring a fully competent
immune response.
Acknowledgments A.F.C., O.R.E., and M.A.S., are scholarship
holders from the National Council for Science and Technology
(CONACYT), Mexico. Point of view and discussion from Leon Islas
is gratefully acknowledged. The graphic design expertise from Ari-
adna Mendez is gratefully acknowledged. The authors declare not to
have any conflict of interest related to publishing this article.
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