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Molecular Immunology 56 (2013) 675– 682

Contents lists available at ScienceDirect

Molecular Immunology

jo ur nal home p age: www.elsev ier .com/ locate /mol imm

eview

M-CSF as a therapeutic target in inflammatory diseases

nnemarie van Nieuwenhuijzea,b,c, Marije Koendersb, Debbie Roeleveldb,atthew A. Sleemand, Wim van den Bergb, Ian P. Wicksa,c,e,∗

Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, AustraliaDepartment of Rheumatology and Advanced Therapeutics, University Medical Centre Nijmegen, Nijmegen, The NetherlandsDepartment of Medical Biology, University of Melbourne, Parkville, Victoria, AustraliaMedImmune Ltd., Cambridge, United KingdomMelbourne Health, PO Royal Melbourne Hospital, Parkville, Victoria, Australia

r t i c l e i n f o

rticle history:eceived 14 March 2013eceived in revised form 29 April 2013ccepted 4 May 2013vailable online 8 August 2013

eywords:M-CSFutoimmunity

a b s t r a c t

GM-CSF is a well-known haemopoietic growth factor that is used in the clinic to correct neutropaenia,usually as a result of chemotherapy. GM-CSF also has many pro-inflammatory functions and recent dataimplicates GM-CSF as a key factor in Th17 driven autoimmune inflammatory conditions. In this reviewwe summarize the findings that have led to the development of GM-CSF antagonists for the treatmentof autoimmune diseases like rheumatoid arthritis (RA) and discuss some results of recent clinical trialsof these agents.

© 2013 Elsevier Ltd. All rights reserved.

nflammatory diseaseheumatoid arthritisytokinesutoimmune diseaseM-CSF receptorh17 pathway

cells

. Biology of GM-CSF

.1. Production of GM-CSF

Granulocyte macrophage colony stimulating factor (GM-CSF, orSF2) was first purified from LPS treated mouse lung-conditionededium. It was subsequently characterised as a haemopoietic

rowth factor that stimulates the proliferation of myeloid cellsrom bone-marrow progenitors (Burgess et al., 1977). Human and

ouse GM-CSF share a high level of amino acid homology (Gought al., 1984; Lee et al., 1985), but are species-specific in termsf receptor binding (Shanafelt et al., 1991). GM-CSF can be pro-uced by a wide variety of cell types, including activated T cells,-cells, macrophages, endothelial cells, fibroblasts and tumour cells

Fleetwood et al., 2005). The production of GM-CSF can be stimu-ated by a range of factors, including T cell receptor and co-receptortimulation (Fraser and Weiss, 1992; Shannon et al., 1995), toll like

∗ Corresponding author at: Reid Rheumatology Laboratory, Inflammation Divi-ion, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade,arkville, Victoria, Australia. Tel.: +61 3 9345 2466; fax: +61 3 9347 0852.

E-mail address: wicks@wehi.edu.au (I.P. Wicks).

161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.molimm.2013.05.002

receptor (TLR) agonists, TNF, IL-1 and prostaglandin E2 (Quill et al.,1989; Hamilton, 2008) (Fig. 1).

1.2. Action of GM-CSF on mature cells

Besides the production and differentiation of haemopoietic cellsfrom precursors, GM-CSF is now recognised to have a range ofother functions on mature haemopoietic cells, including enhancedantigen presentation, induction of complement- and antibody-mediated phagocytosis, and promotion of leukocyte chemotaxisand adhesion (Cook et al., 2004; Fleetwood et al., 2005; Gomez-Cambronero et al., 2003; Metcalf, 2008). GM-CSF, in combinationwith other inflammatory stimuli, can polarise macrophages into“M1-like” inflammatory macrophages (Fleetwood et al., 2007;Mantovani et al., 2002). M1-macrophages produce a range ofinflammatory cytokines, such as TNF, IL-6, IL-12p70 and IL-23. Incontrast, “M2-like” macrophages are maintained in a non-activatedstate by M-CSF, and produce anti-inflammatory factors IL-10 andCCL2 (Fleetwood et al., 2007; Mantovani et al., 2002). In the cen-

tral nervous system, GM-CSF can activate resident macrophage-likemicroglia and promote neuroinflammation through the upregula-tion of TLR4 and CD14 (Parajuli et al., 2012). In mature neutrophils,the integrin CD11b is upregulated by GM-CSF, which increases

676 A. van Nieuwenhuijze et al. / Molecular Immunology 56 (2013) 675– 682

Fig. 1. GM-CSF as a therapeutic target for inflammation and pain. The effects of inhibition of GM-CSF on inflammation and pain are shown. Inhibition of GM-CSF duringinflammation (e.g., joint inflammation during RA or neuroinflammation during MS) may inhibit differentiation, adhesion, chemotaxis and activation of multiple inflammatoryand immune cells such as monocytes, macrophages, neutrophils, DC, microglia and their respective precursors. GM-CSF inhibition may therefore reduce the production ofother inflammatory cytokines, such as IL-1�, TNF, IL-23 and IL-6. These cytokines are particularly important in shaping the responses of T cells, causing differentiation andp ion of

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roduction of inflammatory cytokines such as IL-17 and GM-CSF, through the activat; CNS – central nervous system.

dhesion to vascular endothelium and tissue entry (Hansen et al.,008). Priming of neutrophils by GM-CSF increases antimicro-ial functions, such as phagocytosis and oxidative burst (Condliffet al., 1998; Fleischmann et al., 1986). GM-CSF can have multi-le effects on mature DC, such as increased cross-presentationZhan et al., 2011) and increased uptake capacity (Daro et al.,000). GM-CSF also induced proliferation of CD103+ intestinalendritic cells in vivo in mice (Schulz et al., 2009). How GM-SF regulates the development and function of dendritic cellubsets is summarised in a recent review (Zhan et al., 2012).M-CSF can also activate non-haemopoietic cells, such as endothe-

ial cells and sensory neurons (Bussolino and Mantovani, 1991;

chweizerhof et al., 2009). Elevated tissue levels of GM-CSF haveeen detected in multiple inflammatory conditions, including RA,ultiple sclerosis (MS), obesity, lung disease and cancer (Hamilton,

008).

transcription factors ROR-�T and NF-�B. DC – dendritic cell; COX2 – cyclooxygenase

1.3. GM-CSF: a T cell cytokine

Several recent findings have sparked interest in the relationshipbetween GM-CSF and T cells. The discovery that GM-CSF promotesthe induction and survival of Th17 cells via IL-6 and IL-23 and thatGM-CSF is required for the pathogenicity of Th17 cells in exper-imental allergic encephalomyelitis (EAE), shows that GM-CSF isrelevant outside of the myeloid cell lineage (Codarri et al., 2011;El-Behi et al., 2011). In these studies, GM-CSF was shown to beinduced via ROR-�t, a lymphocyte-specific transcription factor thatwas initially described as the master regulator for Th17 develop-ment (Ivanov et al., 2007). Production of GM-CSF by T cells is also

regulated by the transcription factor NF-�B (Campbell et al., 2011a;Holloway et al., 2003; Osborne et al., 1995). It was shown that T cellsspecifically require the NF-�B1 subunit (p50, most likely as part ofthe active p50/p65 NF-�B heterodimer) for production of GM-CSF,

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nd that T cell-derived GM-CSF was crucial for the local differenti-tion of monocyte derived inflammatory dendritic cells in synovialissue during a model of inflammatory arthritis (Campbell et al.,011a).

. The GM-CSF receptor

The GM-CSF receptor (CSF2R) is a heterodimer, composed of specific ligand-binding �-chain (CSF2R�), which binds GM-SF with low affinity and a signal-transducing �-chain (CSF2R�)Gearing et al., 1989; Hayashida et al., 1990). High affinity bind-ng follows dimerization of both chains. For signal transduction,he cytoplasmic domains of both the � and � chain are required,ut it is mainly the � chain that binds JAK2 (Hansen et al., 2008).ignalling from the CSF2R complex activates the Ras/MAPK andAK/STAT/SOCS pathways (Hansen et al., 2008; Jenkins et al., 1998;ato et al., 1993). Alternative signalling pathways, such as the-fps/fes pathway, have also been suggested (Hanazono et al.,993). The CSF2R is expressed on myeloid cells and on someon-haemopoietic cells, such as endothelial cells (Colotta et al.,993; Rosas et al., 2007), but interestingly, not on T cells (Hercust al., 2009). In both mice and humans, the �-subunit is sharedith the GM-CSF, IL-3, and IL-5 receptors, and is known as the

ommon � chain (�c) (Hayashida et al., 1990; Kitamura et al.,991; Tavernier et al., 1991). In the mouse, an additional IL-3-pecific �-chain exists, known as �IL-3 (Itoh et al., 1990), whichs used in preference to �c for signalling by IL-3 (Nicola et al.,996).

In addition to the membrane-bound form of the CSF2R�, mul-iple splice variants of soluble CSFR2 have been described, bothn vitro and in vivo (Brown et al., 1995; Pelley et al., 2007; Prevostt al., 2002). It is not clear whether these soluble receptors are ofunctional importance, but they may inhibit the ligand-membraneound CSFR2 interaction (Heaney and Golde, 1998).

. Too much or too little?

Abnormalities in GM-CSF production or CSF2R function haveeen implicated in multiple human pathologies such as RABell et al., 1995), juvenile myelomonocytic leukaemia (Birnbaumt al., 2000), chronic myelomonocytic leukaemia (Ramshawt al., 2002) and pulmonary alveolar proteinosis (PAP) (Dirksent al., 1998; Seymour et al., 1998). Many of the features ofhese human conditions have been studied in mice deficientor GM-CSF or the CSF2R and mice that overexpress GM-SF. In addition, therapeutic blockade of GM-CSF in animalodels using neutralising antibodies has yielded crucial informa-

ion.

.1. GM-CSF and CSF2R knock out mice

Mice have been generated that are deficient in GM-CSF (Stanleyt al., 1994). Surprisingly, these mice do not have a defect in myeloidell development, but display infiltration of the lungs with lympho-ytes and defective maturation of alveolar macrophages (Stanleyt al., 1994). As a consequence, there is accumulation of lung sur-actant, which results in the development of a lung phenotypequivalent to idiopathic PAP (Dranoff et al., 1994; Stanley et al.,994; Trapnell et al., 2003; Uchida et al., 2007). In humans, PAP

s a rare lung disease in which patients present with an accumu-ation of surfactant proteins in the lungs that interferes with gas

xchange. In some patients this can render them more suscepti-le to infections (Trapnell et al., 2003). Notably, GM-CSF deficientice have been reported to have a somewhat increased mortal-

ty from microbial infections (Dranoff et al., 1994; Stanley et al.,

Immunology 56 (2013) 675– 682 677

1994). This suggests that GM-CSF plays an important role in thehost response to infectious agents, particularly in the lungs, pos-sibly through regulating macrophage function (Fleetwood et al.,2005). More recently it was shown that GM-CSF deficient micealso have a defect in the maturation of invariant natural killer cells(Bezbradica et al., 2006), which could be important in innate immu-nity.

Mice lacking GM-CSF are protected from a wide range ofautoimmune disease models, such as methylated BSA(mBSA)/IL-1induced arthritis (Lawlor et al., 2005), collagen induced arthritis(CIA) (Campbell et al., 1998), EAE (McQualter et al., 2001) andexperimental autoimmune myocarditis (EAM) (Sonderegger et al.,2008). Intriguingly, these models all require T cells to developpathology.

Mice deficient for the �-chain (CSF2R�) of the GM-CSF recep-tor (Nishinakamura et al., 1995; Robb et al., 1995) have alsobeen generated. Although the phenotype of these mice cannot beattributed to perturbed signalling of GM-CSF alone, due to the acti-vation of this receptor subunit by IL-3 and IL-5 (Hayashida et al.,1990; Kitamura et al., 1991; Tavernier et al., 1991), the pheno-type was in many aspects similar to that of the GM-CSF deficientmice. CSF2R� gene knockout mice displayed a lung phenotyperesembling PAP, and had normal development of haemopoi-etic cells (Nishinakamura et al., 1996; Robb et al., 1995). Takentogether, the data from GM-CSF gene knockout animals clearlyillustrates that GM-CSF and the GM-CSF receptor are not essentialfor steady state haemopoiesis, but play more complex biologicalroles.

3.2. Overexpression of GM-CSF

Systemic administration of GM-CSF is used in the clinic for treat-ment of neutropenia after chemotherapy (Metcalf, 2008), and asan adjuvant in anti-tumour immunity (Disis et al., 1996; Slingluff,2003). However, when GM-CSF is administered to patients withFelty’s syndrome, a flare of RA can occur (Hazenberg et al.,1989). Likewise, administered GM-CSF also exacerbated arthri-tis in mice (Campbell et al., 1997). In addition, GM-CSF given asan adjuvant with a whole-cell melanoma vaccine, caused sys-temic recruitment of eosinophils and basophils and there was atrend towards worse survival (Faries et al., 2009). These findingsillustrate that not only the relative absence, but also the abun-dance, of GM-CSF can have significant effects on the immunesystem.

Besides exogenous administration of GM-CSF, there have beennumerous studies in which GM-CSF was overexpressed endoge-nously, either transiently using adenoviral vectors (Steinwede et al.,2011; Xing et al., 1996) or constitutively using transgenic mice(Biondo et al., 2001; Breuhahn et al., 2000; Lang et al., 1987;Paine et al., 2003; Van Nieuwenhuijze et al., unpublished obser-vations). A common feature of these transgenic animal models waslocal macrophage and DC recruitment/differentiation, with pheno-types including blindness due to accumulation of macrophages inthe eye, myelo-proliferation and inflammatory tissue destruction(Lang et al., 1987), the induction of autoimmune gastritis (Biondoet al., 2001), and the development of disseminated histiocytosis(Van Nieuwenhuijze et al., unpublished observations). These mod-els emphasize the importance of regulating the level of bioactiveGM-CSF to prevent exaggerated myelopoiesis or a break of immunetolerance.

3.3. Experimental blockade of GM-CSF

Genetic ablation of GM-CSF or CSF2R has provided manyinsights into the role of GM-CSF and GM-CSF signalling in steady

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tate and emergency haemopoiesis and autoimmunity. However,or the development of neutralising GM-CSF therapeutics fornflammatory disorders, it is paramount that inhibition is evaluateduring pathological processes. For the treatment of RA for instance,

t is anticipated that GM-CSF blockade might be an option foratients who do not respond adequately to conventional treatment,o these patients will have pre-existing inflammation. The thera-eutic effect of GM-CSF blockade after disease onset was shown

n CIA (Cook et al., 2000), streptococcal cell wall-induced arthritisSCW) (Plater-Zyberk et al., 2007), mBSA/IL-1 experimental mono-rthritis (Yang and Hamilton, 2001), Alzheimer’s disease (Manczakt al., 2009) and LPS-induced lung inflammation (Bozinovski et al.,004). In all of these studies, there was rapid improvement in estab-

ished disease features.An important issue for the development of GM-CSF inhibitors

s the possibility of adverse effects, in particular PAP and increasedusceptibility to microbial lung infections (Seymour et al., 1998).o date, these effects have not been observed in animals treatedith anti-GM-CSF. It is not clear if this is due to the duration

nd extent of GM-CSF deficiency, locally controlled regulation ofM-CSF during PAP, or because experimental animals are housednder controlled conditions and are exposed to a reduced infec-ious load. As many cytokine inhibitors have been trialled andpproved in a broad range of inflammatory conditions, physiciansnd regulatory authorities have a strong understanding of manag-ng the risk of opportunistic infections, however additional lung

onitoring is likely required in human clinical trials of GM-CSFnhibitors in which patients may present with a different side effectrofile.

. GM-CSF and Th17 cells

Perhaps surprisingly, GM-CSF is actually produced in high lev-ls by T cells during inflammation (Wada et al., 1997; Ponomarevt al., 2006; Shi et al., 2006) and it has been found recently that GM-SF is linked to the IL-23/IL-17 pathway (Campbell et al., 2011a;odarri et al., 2011; El-Behi et al., 2011; Poppensieker et al., 2012;an Nieuwenhuijze et al., unpublished observations). GM-CSF pro-oted the induction and survival of autoimmune Th17 effector

ells in a CD4 T cell-mediated model of myocarditis (Sondereggert al., 2008). During models of acute inflammatory arthritis or peri-onitis, GM-CSF production by T cells was dependent on NF-�B1 andas required for the local differentiation of inflammatory mono-

yte derived DCs (Campbell et al., 2011a). It was also shown thatM-CSF is essential for the pathogenicity of Th17 cells and for

he CCR4-dependent production of IL-23 by DC in EAE (Codarrit al., 2011; El-Behi et al., 2011; Poppensieker et al., 2012). Theroduction of GM-CSF was regulated not only by the transcriptionactor NF-�B1, but also by ROR-�t (Codarri et al., 2011). As notedn chapter 2, T cells lack GM-CSF receptors and so the effect of Tell derived GM-CSF on Th17 mediated inflammation occurs vianhancement of IL-6 and IL-23 production from antigen presentingells (APC) (El-Behi et al., 2011). Through binding to their respec-ive receptors on T cells, IL-6 and IL-23 enhance GM-CSF productiony T cells, creating a positive feedback loop (El-Behi et al., 2011).he close relationship between GM-CSF and IL-17 in inflamma-ory conditions suggests that therapeutic inhibition of one of these

olecules might have an effect on the other. It will be interest-ng to see what happens to the level of GM-CSF in patients treated

ith IL-17 inhibitors that are currently in clinical trials (Patel et al.,012). Since IL-17 inhibitors reduce the number of Th17 cells in

he circulation, one would assume there will also be a reduction inhe level of GM-CSF production (Patel et al., 2012). However, it islso possible that the effects on Th17-derived GM-CSF are mostlyocal, at the site of inflammation, and difficult to measure due to

Immunology 56 (2013) 675– 682

the generally low levels of GM-CSF in the circulation. Likewise,the levels of IL-17 and the number of Th17 cells are likely to beaffected in patients treated with GM-CSF inhibitors. The potentialof IL-17 and the IL-17 receptor as therapeutic targets in inflam-matory conditions has recently been reviewed (Roeleveld et al.,2013).

5. GM-CSF autoantibodies

Intriguingly, serum GM-CSF in both mice and humans is gen-erally present in a complex with GM-CSF autoantibodies (Uchidaet al., 2009). Free GM-CSF is maintained at very low, often unde-tectable levels in the serum and tissues (Carraway et al., 2000;Uchida et al., 2009), but is nevertheless essential for the main-tenance of some myeloid cell functions in the lungs. In humans,autoimmune PAP is characterized by high levels of auto-antibodiesto GM-CSF, or the defective expression of the CSF2R (Browneand Holland, 2010; Dirksen et al., 1998, 1997; Sakagami et al.,2009; Suzuki et al., 2008; Trapnell et al., 2003). As describedabove, the features of human PAP are similar to those seen inGM-CSF knockout mice (Stanley et al., 1994). Human GM-CSFautoantibodies inhibit GM-CSF bioactivity (Uchida et al., 2004),thereby reducing GM-CSF-dependent cell functions (Dranoff et al.,1994; Stanley et al., 1994; Trapnell et al., 2003; Uchida et al.,2007). GM-CSF autoantibodies are also found at low levels inhealthy individuals (Uchida et al., 2009). It is possible that theseautoantibodies, together with soluble CSFR2 molecules, functionas additional regulators of GM-CSF bioactivity in vivo (Heaneyand Golde, 1998; Uchida et al., 2009). To prevent autoimmunepathology and the development of PAP, anti-GM-CSF antibod-ies and the levels of free GM-CSF need to be maintained ina tightly controlled balance. This balance will need to be con-sidered with the introduction of anti-GM-CSF therapeutics forinflammatory conditions (Fleetwood et al., 2005; Uchida et al.,2009).

6. GM-CSF inhibitors in the clinic

Conventional disease-modifying anti-rheumatic drugs(DMARDS), such as methotrexate, are routinely used alone orin combination for the treatment of RA. However, despite thebeneficial effect on clinical symptoms and joint damage, DMARDSdo not yield the desired outcome for some patients (Smolen et al.,2007). In the past decade, new biological therapies have beenintroduced in the clinic or are currently in clinical trials for RA andother inflammatory conditions, which has significantly improvedclinical outcomes (Furst et al., 2012). These therapies include TNFinhibitors (infliximab, etanercept, adalimumab, certolizumab andgolimumab), a B-cell-depleting anti-CD20 antibody (rituximab),an inhibitor of costimulation (cytotoxic T-lymphocyte antigen-4fusion (CTLA-4) protein abatacept), an IL-1 receptor antagonist(anakinra) and an inhibitor of the IL-6 receptor (tocilizumab)(Campbell et al., 2011b; Smolen et al., 2007). However, about30% of patients treated with anti-TNF agents show no clinicalbenefit, and only a minority of patients achieve disease remission(Campbell et al., 2011b). The other four biologicals abatacept,rituximab, anakinra and tocilizumab have now been approvedfor use in RA patients, and have shown variable clinical efficacyin reducing disease activity in patients for whom TNF inhibitortherapy fails (Nam et al., 2010). There is great interest in tryingto understand the basis for clinical response, or lack of response,

to these agents in order to better tailor anti-cytokine therapy forindividual patients.

Collectively, a wealth of pre-clinical studies now highlight GM-CSF as a key mediator of inflammatory and immune disorders,

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uggesting it might be an excellent candidate for therapeutic inter-ention (Cornish et al., 2009; Hamilton, 2008). The action of GM-CSFan be inhibited by at least two different approaches: inhibition ofM-CSF itself by neutralising antibodies, or the specific blockade ofM-CSF binding to its receptor by antibodies against the GM-CSF

eceptor � chain. These approaches have resulted in humanizedr fully human anti-GM-CSF and anti-CSF2R� therapeutics thatre currently being tested in clinical trials. Mavrilimumab, a fullyuman antibody that binds CSF2R�, thereby preventing signallinghrough this receptor, has now successfully been tested in Phase Ind Phase II clinical trials in RA patients (Burmester et al., 2011,012). In a recent study of patients with active RA in spite ofethotrexate, 55.7% of all participants treated with subcutaneous

njection of mavrilimumab (every two weeks for 12 weeks) methe primary end point of achieving ≥1.2 decrease from base-ine in the disease activity score (DAS28-CRP) at week 12. At theighest dose of mavrilimumab (100 mg), 66.7% of subjects methe primary endpoint compared with 34.7% of placebo subjects.reatment responses were often rapid, with differences observedetween placebo as early as two weeks, and were sustained dur-

ng 12 weeks of follow up. Throughout the duration of this studydverse events were reported as mild or moderate in intensity.f note is that there were no significant pulmonary complica-

ions reported during the study (Burmester et al., 2012). Likewise,OR103, another fully human high affinity anti-GM-CSF mono-

lonal antibody, has also recently completed evaluation in a Phaseb/2a trial in RA patients, with an acceptable safety profile and indi-ations of efficacy (Morphosys AG). Whilst both clinical studiesere different in size, route of administration and duration, it isoteworthy that both report a rapid onset of action and providevidence of clinical efficacy that support further clinical investi-ation. In addition to RA, MOR103 is currently being tested in ahase Ib trial for multiple sclerosis (MS) (Morphosys AG). A panelf other GM-CSF inhibitors are also under under investigation,owever results have not yet been reported. Of particular note

s KB003, which is a “humaneered” anti GM-CSF antibody thats currently undergoing clinical trials in severe asthma (KaloBiosharmaceuticals). Whilst there is compelling evidence that thisathway also plays a role in severe respiratory diseases suchs asthma and chronic obstructive pulmonary diseases (COPD)Botelho et al., 2011; Saha et al., 2009), a fine dosing balance mayave to be achieved so as not to promote additional pulmonaryomplications.

. GM-CSF and pain

Pain is one of the main symptoms of inflammatory diseases likeA, and rated as one of the highest priorities by RA patients (Lee,013; Wolfe and Michaud, 2007). The relatively recent and sur-rising finding that functional GM-CSF receptors are present onensory nerves has led to the investigation of GM-CSF as a mediatorf pain (Schweizerhof et al., 2009). In this study, GM-CSF secreted byumour cells was linked to signal transduction in nociceptors, andirectly mediated bone tumour-induced pain. It was suggested thathe locally produced GM-CSF at sites of inflammation or tumourould sensitise nociceptors. The high levels of GM-CSF found innflammatory synovitis could therefore be linked to joint pain expe-ienced by RA patients. This link has recently been examined innimal models of inflammatory arthritis and osteoarthritis (Cookt al., 2013, 2012), where it was shown that GM-CSF was requiredor arthritic pain. GM-CSF deficient mice were completely protected

rom pain associated with the induction of inflammatory arthri-is. Interestingly, GM-CSF mediated pain could be relieved by thedministration of the cyclooxygenase inhibitor indomethacin, buthis did not have any effect on joint inflammation (Cook et al., 2013).

Immunology 56 (2013) 675– 682 679

These results raise the possibility that GM-CSF inhibitors might beof particular value in relieving pain as well as inflammation.

8. Concluding remarks

There are many facets to GM-CSF biology – haemopoieticgrowth factor, inducer of myeloid and T cell differentiation, pro-inflammatory mediator and neuromodulator. The pre-clinical dataprovides a strong rationale for considering GM-CSF as a poten-tial therapeutic target for inflammatory diseases. Recent trialswith specific GM-CSF or CSF2R inhibitors promise improved treat-ment options for patients with inflammatory conditions. Carefulmonitoring for potential side effects, particularly PAP, remainsparamount, but we anticipate anti-GM-CSF therapeutics will beof value for many autoimmune and inflammatory conditions andperhaps tumour associated pain. Continued study of the in vivoconsequences of GM-CSF inhibition in patients with inflammatorydiseases will no doubt provide further insights into this fascinatingcytokine.

Acknowledgements

This work was supported by The Reid Charitable Trusts, NHMRCProgram Grant 1016647, NHMRC Clinical Practitioner Fellowship1023407 (IW), NHMRC Independent Research Institute Infra-structure Support Scheme Grant (361646), and a Victorian StateGovernment Operational Infrastructure Support grant.

References

Bell, A.L., Magill, M.K., McKane, W.R., Kirk, F., Irvine, A.E., 1995. Measurement ofcolony-stimulating factors in synovial fluid: potential clinical value. Rheuma-tology International 14, 177–182.

Bezbradica, J.S., Gordy, L.E., Stanic, A.K., Dragovic, S., Hill, T., Hawiger, J., Unutmaz, D.,Van Kaer, L., Joyce, S., 2006. Granulocyte-macrophage colony-stimulating factorregulates effector differentiation of invariant natural killer T cells during thymicontogeny. Immunity 25, 487–497.

Biondo, M.M., Nasa, Z.Z., Marshall, A.A., Toh, B.H.B., Alderuccio, F.F., 2001. Local trans-genic expression of granulocyte macrophage-colony stimulating factor initiatesautoimmunity. Journal of Immunology 166, 2090–2099.

Birnbaum, R.A., O’Marcaigh, A., Wardak, Z., Zhang, Y.Y., Dranoff, G., Jacks, T., Clapp,D.W., Shannon, K.M., 2000. Nf1 and Gmcsf interact in myeloid leukemogenesis.Molecular Cell 5, 189–195.

Botelho, F.M., Nikota, J.K., Bauer, C., Davis, N.H.E., Cohen, E.S., Anderson, I.K., Coyle,A.J., Kolbeck, R., Humbles, A.A., Stampfli, M.R., et al., 2011. A mouse GM-CSFreceptor antibody attenuates neutrophilia in mice exposed to cigarette smoke.European Respiratory Journal 38, 285–294.

Bozinovski, S., Jones, J., Beavitt, S.-J., Cook, A.D., Hamilton, J.A., Anderson, G.P., 2004.Innate immune responses to LPS in mouse lung are suppressed and reversed byneutralization of GM-CSF via repression of TLR-4. American Journal of Physiol-ogy – Lung Cellular and Molecular Physiology 286, L877–L885.

Breuhahn, K., Mann, A., Müller, G., Wilhelmi, A., Schirmacher, P., Enk, A.,Blessing, M., 2000. Epidermal overexpression of granulocyte-macrophagecolony-stimulating factor induces both keratinocyte proliferation and apoptosis.Cell Growth and Differentiation 11, 111–121.

Brown, C.B., Beaudry, P., Laing, T.D., Shoemaker, S., Kaushansky, K., 1995. In vitrocharacterization of the human recombinant soluble granulocyte-macrophagecolony-stimulating factor receptor. Blood 85, 1488–1495.

Browne, S.K., Holland, S.M., 2010. Anticytokine autoantibodies in infectious diseases:pathogenesis and mechanisms. Lancet Infectious Diseases 10, 875–885.

Burgess, A.W., Camakaris, J., metcalf, D., 1977. Purification and properties of colony-stimulating factor from mouse lung-conditioned medium. Journal of BiologicalChemistry 252, 1998–2003.

Burmester, G.R., Feist, E., Sleeman, M.A., Wang, B., White, B., Magrini, F.,2011. Mavrilimumab, a human monoclonal antibody targeting GM-CSFreceptor-, in subjects with rheumatoid arthritis: a randomised, double-blind,placebo-controlled, phase I, first-in-human study. Annals of the Rheumatic Dis-eases 70, 1542–1549.

Burmester, G.R., Weinblatt, M.E., McInnes, I.B., Porter, D., Barbarash, O., Vatutin, M.,Szombati, I., Esfandiari, E., Sleeman, M.A., Kane, C.D., et al., 2012. Efficacy andsafety of mavrilimumab in subjects with rheumatoid arthritis. Annals of theRheumatic Diseases, Online published December 2012.

Bussolino, F., Mantovani, A.A., 1991. Effect of granulocyte-macrophage colony-stimulating factor on endothelial cells. Blood 78, 2475–2476.

Campbell, I.K.I., Bendele, A.A., Smith, D.A.D., Hamilton, J.A.J., 1997. Granulocyte-macrophage colony stimulating factor exacerbates collagen induced arthritisin mice. Annals of the Rheumatic Diseases 56, 364–368.

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80 A. van Nieuwenhuijze et al. / Mole

ampbell, I.K.I., Rich, M.J.M., Bischof, R.J.R., Dunn, A.R.A., Grail, D.D., Hamilton,J.A.J., 1998. Protection from collagen-induced arthritis in granulocyte-macrophage colony-stimulating factor-deficient mice. Journal of Immunology161, 3639–3644.

ampbell, I.K., van Nieuwenhuijze, A., Segura, E., O’Donnell, K., Coghill, E., Hommel,M., Gerondakis, S., Villadangos, J.A., Wicks, I.P., 2011a. Differentiation of inflam-matory dendritic cells is mediated by NF-�B1-dependent GM-CSF production inCD4 T cells. Journal of Immunology 186, 5468–5477.

ampbell, J., Lowe, D., Sleeman, M.A., 2011b. Developing the next generation ofmonoclonal antibodies for the treatment of rheumatoid arthritis. British Journalof Pharmacology 162, 1470–1484.

arraway, M.S., Ghio, A.J., Carter, J.D., Piantadosi, C.A., 2000. Detection ofgranulocyte-macrophage colony-stimulating factor in patients with pulmonaryalveolar proteinosis. American Journal of Respiratory and Critical Care Medicine161, 1294–1299.

odarri, L., Gyülvészi, G., Tosevski, V., Hesske, L., Fontana, A., Magnenat, L., Suter, T.,Becher, B., 2011. ROR�t drives production of the cytokine GM-CSF in helper Tcells, which is essential for the effector phase of autoimmune neuroinflamma-tion. Nature Publishing Group 12, 560–567.

olotta, F.F., Bussolino, F.F., Polentarutti, N.N., Guglielmetti, A.A., Sironi, M.M.,Bocchietto, E.E., De Rossi, M.M., Mantovani, A.A., 1993. Differential expres-sion of the common beta and specific alpha chains of the receptors forGM-CSF, IL-3, and IL-5 in endothelial cells. Experimental Cell Research 206,311–317.

ondliffe, A.M., Kitchen, E., Chilvers, E.R., 1998. Neutrophil priming: pathophysio-logical consequences and underlying mechanisms. Clinical Science 94, 461–471.

ook, A.D., Braine, E.L., Campbell, I.K., Rich, M.J., Hamilton, J.A., 2000. Blockade ofcollagen-induced arthritis post-onset by antibody to granulocyte-macrophagecolony-stimulating factor (GM-CSF): requirement for GM-CSF in the effectorphase of disease. Arthritis Research 3, 293–298.

ook, A.D., Braine, E.L., Hamilton, J.A., 2004. Stimulus-dependent requirement forgranulocyte-macrophage colony-stimulating factor in inflammation. Journal ofImmunology 173, 4643–4651.

ook, A.D., Pobjoy, J., Sarros, S., Steidl, S., Dürr, M., Lacey, D.C., Hamilton, J.A.,2013. Granulocyte-macrophage colony-stimulating factor is a key mediatorin inflammatory and arthritic pain. Annals of the Rheumatic Diseases 72,265–270.

ook, A.D., Pobjoy, J., Steidl, S., Dürr, M., Braine, E.L., Turner, A.L., Lacey, D.C.,Hamilton, J.A., 2012. Granulocyte-macrophage colony-stimulating factor is a keymediator in experimental osteoarthritis pain and disease development. ArthritisResearch and Therapy 14, R199.

ornish, A.L., Campbell, I.K., McKenzie, B.S., Chatfield, S., Wicks, I.P., 2009. G-CSF andGM-CSF as therapeutic targets in rheumatoid arthritis. Nature Reviews Rheuma-tology 5, 554–559.

aro, E., Pulendran, B., Brasel, K., Teepe, M., Pettit, D., Lynch, D.H., Vremec, D., Robb,L., Shortman, K., McKenna, H.J., et al., 2000. Polyethylene glycol-modified GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high) murinedendritic cells in vivo: a comparative analysis with Flt3 ligand. Journal ofImmunology 165, 49–58.

irksen, U.U., Hattenhorst, U.U., Schneider, P.P., Schroten, H.H., Göbel,U.U., Böcking, A.A., Müller, K.M.K., Murray, R.R., Burdach, S.S., 1998.Defective expression of granulocyte-macrophage colony-stimulatingfactor/interleukin-3/interleukin-5 receptor common beta chain in chil-dren with acute myeloid leukemia associated with respiratory failure. Blood 92,1097–1103.

irksen, U., Nishinakamura, R., Groneck, P., Hattenhorst, U., Nogee, L., Murray, R.,Burdach, S., 1997. Human pulmonary alveolar proteinosis associated with adefect in GM-CSF/IL-3/IL-5 receptor common beta chain expression. Journal ofClinical Investigation 100, 2211–2217.

isis, M.L.M., Bernhard, H.H., Shiota, F.M.F., Hand, S.L.S., Gralow, J.R.J., Huseby, E.S.E.,Gillis, S.S., Cheever, M.A.M., 1996. Granulocyte-macrophage colony-stimulatingfactor: an effective adjuvant for protein and peptide-based vaccines. Blood 88,202–210.

ranoff, G., Crawford, A.D., Sadelain, M., Ream, B., Rashid, A., Bronson, R.T., Dickersin,G.R., Bachurski, C.J., Mark, E.L., Whitsett, J.A., 1994. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264,713–716.

l-Behi, M., Ciric, B., Dai, H., Yan, Y., Cullimore, M., Safavi, F., Zhang, G.-X., Dittel, B.N.,Rostami, A., 2011. The encephalitogenicity of TH17 cells is dependent on IL-1-and IL-23-induced production of the cytokine GM-CSF. Nature Publishing Group12, 568–575.

aries, M.B., Hsueh, E.C., Ye, X., Hoban, M., Morton, D.L., 2009. Effect ofgranulocyte/macrophage colony-stimulating factor on vaccination with anallogeneic whole-cell melanoma vaccine. Clinical Cancer Research 15,7029–7035.

leetwood, A.J.A., Lawrence, T.T., Hamilton, J.A.J., Cook, A.D.A., 2007. Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependentmacrophage phenotypes display differences in cytokine profiles andtranscription factor activities: implications for CSF blockade in inflammation.Journal of Immunology 178, 5245–5252.

leetwood, A.J., Cook, A.D., Hamilton, J.A., 2005. Functions of granulocyte-

macrophage colony-stimulating factor. Critical Reviews in Immunology 25,405–428.

leischmann, J., Golde, D.W., Weisbart, R.H., Gasson, J.C., 1986. Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria byhuman neutrophils. Blood 68, 708–711.

Immunology 56 (2013) 675– 682

Fraser, J.D., Weiss, A., 1992. Regulation of T-cell lymphokine gene transcription bythe accessory molecule CD28. Molecular and Cellular Biology 12, 4357–4363.

Furst, D.E.D., Keystone, E.C.E., Braun, J.J., Breedveld, F.C.F., Burmester, G.R.G., DeBenedetti, F.F., Dörner, T.T., Emery, P.P., Fleischmann, R.R., Gibofsky, A.A., et al.,2012. Updated consensus statement on biological agents for the treatmentof rheumatic diseases, 2011. Annals of the Rheumatic Diseases 71 (Suppl. 2),i2–i45.

Gearing, D.P., King, J.A., Gough, N.M., Nicola, N.A., 1989. Expression cloning of a recep-tor for human granulocyte-macrophage colony-stimulating factor. The EMBOJournal 8 (12), 3667–3676.

Gomez-Cambronero, J., Horn, J., Paul, C.C., Baumann, M.A., 2003. Granulocyte-macrophage colony-stimulating factor is a chemoattractant cytokine for humanneutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway.Journal of Immunology 171, 6846–6855.

Gough, N.M., Gough, J., metcalf, D., Kelso, A., Grail, D., Nicola, N.A., Burgess, A.W.,Dunn, A.R., 1984. Molecular cloning of cDNA encoding a murine haematopoieticgrowth regulator, granulocyte-macrophage colony stimulating factor. Nature309, 763–767.

Hamilton, J.A., 2008. Colony-stimulating factors in inflammation and autoimmunity.Nature Reviews Immunology 8, 533–544.

Hanazono, Y., Chiba, S., Sasaki, K., Mano, H., Miyajima, A., Arai, K., Yazaki, Y., Hirai, H.,1993. c-fps/fes protein-tyrosine kinase is implicated in a signaling pathway trig-gered by granulocyte-macrophage colony-stimulating factor and interleukin-3.The EMBO Journal 12, 1641–1646.

Hansen, G., Hercus, T.R., McClure, B.J., Stomski, F.C., Dottore, M., Powell, J., Ramshaw,H., Woodcock, J.M., Xu, Y., Guthridge, M., et al., 2008. The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.Cell 134, 496–507.

Hayashida, K.K., Kitamura, T.T., Gorman, D.M.D., Arai, K.K., Yokota, T.T., Miyajima,A.A., 1990. Molecular cloning of a second subunit of the receptor for humangranulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution ofa high-affinity GM-CSF receptor. Proceedings of the National Academy of Sci-ences of the United States of America 87, 9655–9659.

Hazenberg, B.P., Van Leeuwen, M.A., Van Rijswijk, M.H., Stern, A.C., Vellenga, E., 1989.Correction of granulocytopenia in Felty’s syndrome by granulocyte-macrophagecolony-stimulating factor, Simultaneous induction of interleukin-6 release andflare-up of the arthritis. Blood 74, 2769–2770.

Heaney, M.L., Golde, D.W., 1998. Soluble receptors in human disease. Journal ofLeukocyte Biology 64, 135–146.

Hercus, T.R., Thomas, D., Guthridge, M.A., Ekert, P.G., King-Scott, J., Parker, M.W.,Lopez, A.F., 2009. The granulocyte-macrophage colony-stimulating factor recep-tor: linking its structure to cell signaling and its role in disease. Blood 114,1289–1298.

Holloway, A.F., Rao, S., Chen, X., Shannon, M.F., 2003. Changes in chromatin acces-sibility across the GM-CSF promoter upon T cell activation are dependenton nuclear factor kappaB proteins. Journal of Experimental Medicine 197,413–423.

Itoh, N.N., Yonehara, S.S., Schreurs, J.J., Gorman, D.M.D., Maruyama, K.K., Ishii,A.A., Yahara, I.I., Arai, K.K., Miyajima, A.A., 1990. Cloning of an interleukin-3 receptor gene: a member of a distinct receptor gene family. Science 247,324–327.

Ivanov, I.I., Zhou, L., Littman, D.R., 2007. Transcriptional regulation of Th17 cell dif-ferentiation. Seminars in Immunology 19, 409–417.

Jenkins, B.J.B., Blake, T.J.T., Gonda, T.J.T., 1998. Saturation mutagenesis of thebeta subunit of the human granulocyte-macrophage colony-stimulating fac-tor receptor shows clustering of constitutive mutations, activation of ERK MAPkinase and STAT pathways, and differential beta subunit tyrosine phosphoryla-tion. Blood 92, 1989–2002.

KaloBios Pharmaceuticals. Effect of KB003 in subjects with asthma inadequatelycontrolled by corticosteroids (KB003-04). ClinicalTrials.Gov (Internet).

Kitamura, T., Sato, N., Arai, K., Miyajima, A., 1991. Expression cloning of the humanIL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSFreceptors. Cell 66, 1165–1174.

Lang, R.A., metcalf, D., Cuthbertson, R.A., Lyons, I., Stanley, E., Kelso, A., Kan-nourakis, G., Williamson, D.J., Klintworth, G.K., Gonda, T.J., 1987. Transgenicmice expressing a hemopoietic growth factor gene (GM-CSF) develop accumu-lations of macrophages, blindness, and a fatal syndrome of tissue damage. Cell51, 675–686.

Lawlor, K.E., Wong, P.K.K., Campbell, I.K., van Rooijen, N., Wicks, I.P., 2005.Acute CD4+T lymphocyte-dependent interleukin-1-driven arthritis selectivelyrequires interleukin-2 and interleukin-4, joint macrophages, granulocyte-macrophage colony-stimulating factor, interleukin-6, and leukemia inhibitoryfactor. Arthritis and Rheumatism 52, 3749–3754.

Lee, F., Yokota, T., Otsuka, T., Gemmell, L., Larson, N., Luh, J., Arai, K., Rennick, D., 1985.Isolation of cDNA for a human granulocyte-macrophage colony-stimulating fac-tor by functional expression in mammalian cells. Proceedings of the NationalAcademy of Sciences of the United States of America 82, 4360–4364.

Lee, Y.C., 2013. Effect and treatment of chronic pain in inflammatory arthritis. Cur-rent Rheumatology Reports 15, 300.

Manczak, M., Mao, P., Nakamura, K., Bebbington, C., Park, B., Reddy, P.H., 2009.Neutralization of granulocyte macrophage colony-stimulating factor decreases

amyloid beta 1-42 and suppresses microglial activity in a transgenic mousemodel of Alzheimer’s disease. Human Molecular Genetics 18, 3876–3893.

Mantovani, A., Sozzani, S., Locati, M., Allavena, P., Sica, A., 2002. Macrophagepolarization: tumor-associated macrophages as a paradigm for polarized M2mononuclear phagocytes. Trends in Immunology 23, 549–555.

cular

M

MM

N

N

N

N

O

P

P

P

P

P

P

P

P

Q

R

R

R

R

S

A. van Nieuwenhuijze et al. / Mole

cQualter, J.L.J., Darwiche, R.R., Ewing, C.C., Onuki, M.M., Kay, T.W.T., Hamilton, J.A.J.,Reid, H.H.H., Bernard, C.C.C., 2001. Granulocyte macrophage colony-stimulatingfactor: a new putative therapeutic target in multiple sclerosis. Journal of Exper-imental Medicine 194, 873–882.

etcalf, D., 2008. Hematopoietic cytokines. Blood 111, 485–491.orphosys, A.G., A study of the safety and preliminary efficacy of MOR103, a

human antibody to granulocyte macrophage colony-stimulating factor (GM-CSF), in patients with active rheumatoid arthritis (NCT01023256); Phase Ib studyto evaluate MOR103 in multiple sclerosis (NCT01517282). ClinicalTrials.Gov(Internet).

am, J.L., Winthrop, K.L., van Vollenhoven, R.F., Pavelka, K., Valesini, G., Hensor,E.M.A., Worthy, G., Landewé, R., Smolen, J.S., Emery, P., et al., 2010. Currentevidence for the management of rheumatoid arthritis with biological disease-modifying antirheumatic drugs: a systematic literature review informing theEULAR recommendations for the management of RA. Annals of the RheumaticDiseases 69, 976–986.

icola, N.A.N., Robb, L.L., Metcalf, D.D., Cary, D.D., Drinkwater, C.C.C., Begley, C.G.C.,1996. Functional inactivation in mice of the gene for the interleukin-3 (IL-3)-specific receptor beta-chain: implications for IL-3 function and the mechanismof receptor transmodulation in hematopoietic cells. Blood 87, 2665–2674.

ishinakamura, R.R., Miyajima, A.A., Mee, P.J.P., Tybulewicz, V.L.V., Murray, R.R.,1996. Hematopoiesis in mice lacking the entire granulocyte-macrophagecolony-stimulating factor/interleukin-3/interleukin-5 functions. Blood 88,2458–2464.

ishinakamura, R., Nakayama, N., Hirabayashi, Y., Inoue, T., Aud, D., McNeil, T.,Azuma, S., Yoshida, S., Toyoda, Y., Arai, K., 1995. Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response,while beta IL3 receptor-deficient mice are normal. Immunity 2, 211–222.

sborne, C.S., Vadas, M.A., Cockerill, P.N., 1995. Transcriptional regulation ofmouse granulocyte-macrophage colony-stimulating factor/IL-3 locus. Journalof Immunology 155, 226–235.

aine, R., Wilcoxen, S.E., Morris, S.B., Sartori, C., Baleeiro, C.E.O., Matthay, M.A., Chris-tensen, P.J., 2003. Transgenic overexpression of granulocyte macrophage-colonystimulating factor in the lung prevents hyperoxic lung injury. American Journalof Pathology 163, 2397–2406.

arajuli, B., Sonobe, Y., Kawanokuchi, J., Doi, Y., Noda, M., Takeuchi, H., Mizuno,T., Suzumura, A., 2012. GM-CSF increases LPS-induced production of proin-flammatory mediators via upregulation of TLR4 and CD14 in murine microglia.Journal of Neuroinflammation 9, 268.

atel, D.D., Lee, D.M., Kolbinger, F., Antoni, C., 2012. Effect of IL-17A blockade withsecukinumab in autoimmune diseases. Annals of the Rheumatic Diseases, Onlinepublished December 2012.

elley, J.L., Nicholls, C.D., Beattie, T.L., Brown, C.B., 2007. Discovery and characteriza-tion of a novel splice variant of the GM-CSF receptor alpha subunit. ExperimentalHematology 35, 1483–1494.

later-Zyberk, C., Joosten, L.A.B., Helsen, M.M.A., Hepp, J., Baeuerle, P.A., van den Berg,W.B., 2007. GM-CSF neutralisation suppresses inflammation and protects carti-lage in acute streptococcal cell wall arthritis of mice. Annals of the RheumaticDiseases 66, 452–457.

onomarev, E.D.E., Shriver, L.P.L., Maresz, K.K., Pedras-Vasconcelos, J.J., Verthelyi,D.D., Dittel, B.N.B., 2006. GM-CSF production by autoreactive T cells is requiredfor the activation of microglial cells and the onset of experimental autoimmuneencephalomyelitis. Journal of Immunology 178, 39–48.

oppensieker, K., Otte, D.-M., Schuermann, B., Limmer, A., Dresing, P., Drews,E., Schumak, B., Klotz, L., Raasch, J., Mildner, A., et al., 2012. CC chemokinereceptor 4 is required for experimental autoimmune encephalomyelitis byregulating GM-CSF and IL-23 production in dendritic cells. Proceedings ofthe National Academy of Sciences of the United States of America 109,3897–3902.

revost, J.M.J., Pelley, J.L.J., Zhu, W.W., D’Egidio, G.E.G., Beaudry, P.P.P., Pihl, C.C.,Neely, G.G.G., Claret, E.E., Wijdenes, J.J., Brown, C.B.C., 2002. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuliup-regulate secretion of the soluble GM-CSF receptor in human monocytes:evidence for ectodomain shedding of the cell surface GM-CSF receptor alphasubunit. Journal of Immunology 169, 5679–5688.

uill, H., Gaur, A., Phipps, R.P., 1989. Prostaglandin E2-dependent induction ofgranulocyte-macrophage colony-stimulating factor secretion by cloned murinehelper T cells. Journal of Immunology 142, 813–818.

amshaw, H.S.H., Bardy, P.G.P., Lee, M.A.M., Lopez, A.F.A., 2002. Chronic myelomono-cytic leukemia requires granulocyte-macrophage colony-stimulating factor forgrowth in vitro and in vivo. Experimental Hematology 30, 1124–1131.

obb, L.L., Drinkwater, C.C.C., Metcalf, D.D., Li, R.R., Köntgen, F.F., Nicola, N.A.N.,Begley, C.G.C., 1995. Hematopoietic and lung abnormalities in mice with anull mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5. Proceedings ofthe National Academy of Sciences of the United States of America 92, 9565–9569.

oeleveld, D.M., van Nieuwenhuijze, A.E.M., van den Berg, W.B., Koenders, M.I., 2013.The Th17 pathway as a therapeutic target in rheumatoid arthritis and otherautoimmune and inflammatory disorders. BioDrugs, April 2013.

osas, M., Gordon, S., Taylor, P.R., 2007. Characterisation of the expression and func-tion of the GM-CSF receptor �-chain in mice. European Journal of Immunology

37, 2518–2528.

aha, S., Doe, C., Mistry, V., Siddiqui, S., Parker, D., Sleeman, M., Cohen, E.S., Brightling,C.E., 2009. Granulocyte-macrophage colony-stimulating factor expression ininduced sputum and bronchial mucosa in asthma and COPD. Thorax 64,671–676.

Immunology 56 (2013) 675– 682 681

Sakagami, T., Uchida, K., Suzuki, T., Carey, B.C., Wood, R.E., Wert, S.E., Whitsett, J.A.,Trapnell, B.C., Luisetti, M., 2009. Human GM-CSF autoantibodies and reproduc-tion of pulmonary alveolar proteinosis. New England Journal of Medicine 361,2679–2681.

Sato, N., Sakamaki, K., Terada, N., Arai, K., Miyajima, A., 1993. Signal transductionby the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of thecommon beta subunit responsible for different signaling. The EMBO Journal 12,4181–4189.

Schulz, O., Jaensson, E., Persson, E.K., Liu, X., Worbs, T., Agace, W.W., Pabst, O., 2009.Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymphand serve classical dendritic cell functions. Journal of Experimental Medicine206, 3101–3114.

Schweizerhof, M., Stösser, S., Kurejova, M., Njoo, C., Gangadharan, V., Agarwal, N.,Schmelz, M., Bali, K.K., Michalski, C.W., Brugger, S., et al., 2009. Hematopoieticcolony-stimulating factors mediate tumor-nerve interactions and bone cancerpain. Nature Medicine 15, 802–807.

Seymour, J.F.J., Begley, C.G.C., Dirksen, U.U., Presneill, J.J.J., Nicola, N.A.N., Moore,P.E.P., Schoch, O.D.O., van Asperen, P.P., Roth, B.B., Burdach, S.S., et al.,1998. Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating factor in patients with acquired pulmonary alveolar proteinosis.Blood 92, 2657–2667.

Shanafelt, A.B., Johnson, K.E., Kastelein, R.A., 1991. Identification of critical aminoacid residues in human and mouse granulocyte-macrophage colony-stimulatingfactor and their involvement in species specificity. Journal of Biological Chem-istry 266, 13804–13810.

Shannon, M.F.M., Himes, S.R.S., Coles, L.S.L., 1995. GM-CSF and IL-2 share commoncontrol mechanisms in response to costimulatory signals in T cells. Journal ofLeukocyte Biology 57, 767–773.

Shi, Y., Liu, C.H., Roberts, A.I., Das, J., Xu, G., Ren, G., Zhang, Y., Zhang, L., Yuan,Z.R., Tan, H.S.W., et al., 2006. Granulocyte-macrophage colony-stimulating fac-tor (GM-CSF) and T-cell responses: what we do and don’t know. Cell Research16, 126–133.

Slingluff, C.L., 2003. Clinical and immunologic results of a randomized phase II trial ofvaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells.Journal of Clinical Oncology 21, 4016–4026.

Smolen, J.S., Aletaha, D., Koeller, M., Weisman, M.H., Emery, P., 2007. New therapiesfor treatment of rheumatoid arthritis. Lancet 370, 1861–1874.

Sonderegger, I., Iezzi, G., Maier, R., Schmitz, N., Kurrer, M., Kopf, M.,2008. GM-CSF mediates autoimmunity by enhancing IL-6-dependent Th17cell development and survival. Journal of Experimental Medicine 205,2281–2294.

Stanley, E.E., Lieschke, G.J.G., Grail, D.D., Metcalf, D.D., Hodgson, G.G., Gall,J.A.J., Maher, D.W.D., Cebon, J.J., Sinickas, V.V., Dunn, A.R.A., 1994. Granu-locyte/macrophage colony-stimulating factor-deficient mice show no majorperturbation of hematopoiesis but develop a characteristic pulmonary pathol-ogy. Proceedings of the National Academy of Sciences of the United States ofAmerica 91, 5592–5596.

Steinwede, K., Tempelhof, O., Bolte, K., Maus, R., Bohling, J., Ueberberg, B., Länger, F.,Christman, J.W., Paton, J.C., Ask, K., et al., 2011. Local delivery of GM-CSF protectsmice from lethal pneumococcal pneumonia. The Journal of Immunology 187,5346–5356.

Suzuki, T., Sakagami, T., Rubin, B.K., Nogee, L.M., Wood, R.E., Zimmerman, S.L., Smo-larek, T., Dishop, M.K., Wert, S.E., Whitsett, J.A., et al., 2008. Familial pulmonaryalveolar proteinosis caused by mutations in CSF2RA. Journal of ExperimentalMedicine 205, 2703–2710.

Tavernier, J.J., Devos, R.R., Cornelis, S.S., Tuypens, T.T., Van der Heyden, J.J., Fiers,W.W., Plaetinck, G.G., 1991. A human high affinity interleukin-5 receptor (IL5R)is composed of an IL5-specific alpha chain and a beta chain shared with thereceptor for GM-CSF. Cell 66, 1175–1184.

Trapnell, B.C., Whitsett, J.A., Nakata, K., 2003. Pulmonary alveolar proteinosis. NewEngland Journal of Medicine 349, 2527–2539.

Uchida, K.K., Nakata, K.K., Trapnell, B.C.B., Terakawa, T.T., Hamano, E.E., Mikami,A.A., Matsushita, I.I., Seymour, J.F.J., Oh-Eda, M.M., Ishige, I.I., et al., 2004. High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonaryalveolar proteinosis. Blood 103, 1089–1098.

Uchida, K., Beck, D.C., Yamamoto, T., Berclaz, P.-Y., Abe, S., Staudt, M.K., Carey, B.C.,Filippi, M.-D., Wert, S.E., Denson, L.A., et al., 2007. GM-CSF autoantibodies andneutrophil dysfunction in pulmonary alveolar proteinosis. New England Journalof Medicine 356, 567–579.

Uchida, K., Nakata, K., Suzuki, T., Luisetti, M., Watanabe, M., Koch, D.E., Stevens,C.A., Beck, D.C., Denson, L.A., Carey, B.C., et al., 2009. Granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions inhealthy subjects. Blood 113, 2547–2556.

Wada, H., Noguchi, Y., Marino, M.W., Dunn, A.R., Old, L.J., 1997. T cell functions ingranulocyte/macrophage colony-stimulating factor deficient mice. Proceedingsof the National Academy of Sciences of the United States of America 94,12557–12561.

Wolfe, F., Michaud, K., 2007. Assessment of pain in rheumatoid arthritis: minimalclinically significant difference, predictors, and the effect of anti-tumor necrosis

factor therapy. Journal of Rheumatology 34, 1674–1683.

Xing, Z., Braciak, T., Ohkawara, Y., Sallenave, J.M., Foley, R., Sime, P.J., Jordana, M.,Graham, F.L., Gauldie, J., 1996. Gene transfer for cytokine functional studies in thelung: the multifunctional role of GM-CSF in pulmonary inflammation. Journalof Leukocyte Biology 59, 481–488.

6 cular

Y

Z

82 A. van Nieuwenhuijze et al. / Mole

ang, Y.H., Hamilton, J.A., 2001. Dependence of interleukin-1-induced arthritis ongranulocyte-macrophage colony-stimulating factor. Arthritis and Rheumatism44, 111–119.

han, Y., Carrington, E.M., van Nieuwenhuijze, A., Bedoui, S., Seah, S., Xu, Y., Wang,N., Mintern, J.D., Villadangos, J.A., Wicks, I.P., et al., 2011. GM-CSF increases

Immunology 56 (2013) 675– 682

cross-presentation and CD103 expression by mouse CD8+ spleen dendritic cells.European Journal of Immunology 41, 2585–2595.

Zhan, Y., Xu, Y., Lew, A.M., 2012. The regulation of the development and functionof dendritic cell subsets by GM-CSF: more than a hematopoietic growth factor.Molecular Immunology 52, 30–37.