Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells

Hoxb8 conditionally immortalised macrophage linesmodel inflammatory monocytic cells with importantsimilarity to dendritic cells

Marcela Rosas1, Fabiola Osorio2, Matthew J. Robinson2, Luke C. Davies1,

Nicola Dierkes1, Simon A. Jones1, Caetano Reis e Sousa2

and Philip R. Taylor1

1 Infection, Immunity and Biochemistry, Cardiff University School of Medicine, Heath Park,

Cardiff, Wales, UK2 Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn

Fields Laboratories, London England, UK

We have examined the potential to generate bona fide macrophages (MØ) from condi-

tionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8

conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or

GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF

bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen

phenotype and functional responses to microbial stimuli. In spite of this high similarity

between the two cell types and the ability of GM-MØP to effectively present antigen to a

T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-c

responses from naı̈ve CD41 T cells. The generation of MØP from transgenic or genetically

aberrant mice provides an excellent opportunity to study the inflammatory role of

GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation

of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of

inflammatory monocyte-like cells, with important differences from bone marrow-derived

dendritic cells, which will facilitate functional studies relating to the many ‘sub-pheno-

types’ of inflammatory monocytes.

Keywords: DC . Macrophage . Monocyte

Introduction

Some members of the Homeobox family of transcription factors

promote expansion of hematopoietic progenitors, and have been

implicated in both human and mouse myeloid leukaemia. Hoxb8

has been shown to arrest myeloid differentiation and in

conjunction with growth factor, e.g. stem cell factor (SCF) or

GM-CSF [1–3] leads to expansion of myeloid precursors. In

WEHI-3B acute myeloid leukemia cells, enhanced Hoxb8 activity

and enhanced IL-3 production prevents terminal differentiation

and allows proliferation of the undifferentiated cell [2, 3]. To

exploit the ability of Hoxb8 to expand myeloid precursors, Hoxb8

was fused to the binding domain of the oestrogen receptor as

engineered chimeric protein [1]. This oestrogen-regulated

strategy enabled the immortalisation and expansion of differently

committed MØ or neutrophil progenitors. The conditional nature

of the immortalisation also alleviated some of the problems

associated with aberrant constitutive transcription factor activity

present throughout differentiation making it one of the best

current approaches to the in vitro generation of MØ or

neutrophils [1]. Such systems are readily genetically manipulated

[1, 4] and hence represent a valuable experimental model, which

would consequently replace the need for animals for a large

number of experimental studies. However for this to be a viableCorrespondence: Dr. Philip R. Taylore-mail: [email protected]

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201040962 Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.356

approach, the immunological and functional potential of these

novel lines need to be fully explored.

Macrophages (MØ) and DC have been grown from mouse

bone marrow (BM) for several decades, using M-CSF and GM-

CSF respectively [5–7], and have become the major model for

analysis of mononuclear phagocyte cell function in vitro. The

study of BM-derived DC (BMDC) cultures as a model cell type has

been extensive and has been used to assign the functional char-

acteristics of DC. BMDC are now widely considered not to be

representative of most steady-state tissue DC [8]. This is consis-

tent with the lack of requirement for GM-CSF in generation of the

normal constitution of the majority [8], but not all [9], of

peripheral DC pool. In contrast, a strong dependence on Flt3

ligand for the population of adult animals with ‘steady state

conventional DC’ subsets indicates its more fundamental role in

this process [8, 10]. Instead, BMDC have been proposed to

represent an inflammatory monocyte-derived cell [11]. The

phenotype of inflammatory monocytes in vivo is quite varied,

which most likely reflects the diversity of stimuli encountered in

inflammatory lesions. These phenotypes range from pro-inflam-

matory, host-protective and immune-stimulatory to the ability to

suppress antigen specific T-cell responses [11–18].

Using the previously described oestrogen-regulated Hoxb8

system, we have performed a systematic analysis of cells derived

from GM-CSF differentiation of Hoxb8 immortalised MØP to

determine if they have DC-associated properties. Specifically, we

provide data relating to antigen presentation, anti-microbial

responsiveness, T-cell priming and effector cytokine production.

Based on these findings we conclude that this Hoxb8-regulated

system generates inflammatory monocyte-like cells that func-

tionally display both similarities and differences with more

traditional BMDC cultures. These cells are easily genetically

modified, therefore this approach provides an excellent model for

manipulating the biological responsiveness of monocytic cells and

represents a valuable model that may cast further light on, for

example, the requirements of antigen presenting cells (APC) for

efficient presentation of antigen to naı̈ve T cells.

Results

Differentiation of Hoxb8 conditionally immortalisedMØP in GM-CSF or M-CSF

MØPs were differentiated for 4 days in either GM-CSF or M-CSF

and were analysed by flow-cytometry for evidence of MØ

maturation. Undifferentiated MØP expressed the immature

myelomonocytic markers Ly-6B.2 (7/4 antigen [19]) and Gr-1.

They also expressed low levels of F4/80 and moderate levels of

CD11b (Fig. 1A). The majority of undifferentiated MØPs were

negative for expression of maturation-dependent mannose

receptor (MR) and dectin-2. Dectin-1 expression was evident

only on a subset of progenitors. Differentiation in M-CSF resulted

in an F4/80hiCD11bhiMRhiDectin-1lowDectin-2low surface pheno-

type (Fig. 1A). M-CSF-differentiated cells (M-MØ precursor

(MØP)) also consistently expressed CD40, but not CD11c, CD86

or MHCII, and lacked expression of the immature markers

Ly-6B.2 and Gr-1 (Fig. 1A). M-MØP resembled BM-derived MØ

and phenotypically grew as a uniform monolayer in culture

(Fig. 1B). Consistent with M-CSF treatment, differentiation of

MØP in GM-CSF (GM-MØP) triggered a loss of staining for

A+ GM-CSF + M-CSF

+ β-estradiol – β-estradiol+ GM-CSF

C

96

128

Ly-6B.2:

SS

FSC

0 32 64 96 128 0 32 64 96 128 0 32 64 96 1280

32

64

96

128

0

32

64

96

128

0

32

64

Dectin-1:

F4/80:

Gr-1:

CD11b:

MR/CD206:

Dectin-2:

CD40:

CD11c:

CD80:

CD86:

MHCII:

Cou

nts

Receptor

GM-MØP-S6 M-MØP-S6B

C Receptor

Figure 1. MØP can be differentiated in GM-CSF or M-CSF resulting incells that morphologically resemble BMDC and MØ respectively.(A) FACS analysis of MØP (1b-estradiol) and GM-CSF and M-CSF-differentiated cells (–b-estradiol). The top row shows gating and belowhistograms are representative of plots from two to three independentexperiments for each marker. Shaded histograms represent receptorspecific staining and bold lines denote isotype control staining.(B) Photomicrographs of MØP differentiated in GM-CSF (GM-MØP)and M-CSF (M-MØP) show that GM-MØP tend to grow in clusterswith distributed adhered cells, whereas M-MØP grow in uniformmonolayers.


Eur. J. Immunol. 2011. 41: 356–365 Cellular immune response 357

immature markers (Ly-6B.2 and Gr-1). However, GM-MØP

cells display an F4/80intCD11b1CD11chiMR1Dectin-1hiDectin-2hi

CD401CD80hiCD861MHCII1 phenotype (Fig. 1A). Furthermore,

GM-MØP grew in vitro with distinct cell clusters that resembled

BMDC cultures (Fig. 1B).

Cytokine response of GM-MØP to microbial stimuli

Given the apparent similarity of GM-MØP to BMDC, the cytokine

responses of the two cell types in response to microbial stimuli were

compared. Curdlan (a dectin-1 agonist [4, 20, 21]), zymosan

(dectin-1, dectin-2 and TLR2 agonist [22–25]), LPS and Pam3CSK4

(TLR4 and TLR2 agonists respectively) were incubated with

GM-MØP or BMDC for 16 h (Fig. 2A). All stimuli were capable of

inducing variable levels of IL-6, TNF and IL-12p40 from both cell

types, although, Two-way ANOVA analysis of the data indicated that

the GM-MØP produced slightly lower levels of these factors.

Curdlan, zymosan and to a lesser extent LPS and Pam3CSK4

induced IL-10 production from both cell types with no evident

difference in the amount produced by the two cell types. Notably,

stimulation with either curdlan or zymosan induced secretion of

IL-2 by BMDC that was not evident with GM-MØP (Fig. 2A).

Quantitative PCR confirmed that there was a markedly lower (�20-

fold) induction of IL-2 mRNA by GM-MØP in response to Curdlan

stimulation when compared to BMDC (Fig. 2B). Given the

selectivity of Curdlan and zymosan for the induction of IL-2 we

examined the expression of dectin-1 by both cell types. The

expression of dectin-1 exhibited a non-significant trend to reduced

levels in the GM-MØP cell line (mean7SDDMFI of 28.84720.14

and 31.84716.83 for adherent and non-adherent cells from GM-

CSF cultures respectively) when compared to the BMDC (mean

7SDDMFI of 67.24740.26 and 49.05724.16 for adherent and

non-adherent cells from GM-CSF cultures respectively). Data were

from three independent experiments and dectin-1 expression levels

were analysed by one-way ANOVA with Bonferonni post tests.

Nitric oxide (NO) production in response to endotoxin

A feature of BMDC is the induction of NO production in response to

endotoxin [11]. We compared the ability of GM-MØP and BMDC to

produce NO in response to increasing concentrations of LPS. As

previously reported [11] BMDC produced NO in a dose-dependent

response to LPS challenge (Fig. 3). GM-MØP appeared to be more

robust producers of NO in response to LPS challenge (Fig. 3).

Direct comparison of antigen phenotype betweenBMDC and GM-MØP

The expression of DC-associated antigens by GM-MØP and BMDC

was compared directly both on the non-adherent and adherent cell

fractions isolated from cell culture. The surface antigen phenotype

between adherent and non-adherent cells was essentially identical

30

40

50

I: n.s.

*

*

0

10

20

80

100

IL-6

(ng

/ml)

T: P=0.0144C: P<0.0001

I: P=0 0258

***

60

0

20

40

60

TN

F (

ng/m

l)

T: P=0.0003C: P=0.0137

300

0

20

40

IL-1

2p40

(ng

/ml)

I: n.s.T: n.s.C: P=0.0015

*

0

100

200

8

IL-2

(pg

/ml) I: n.s.

T: P=0.0193C: P=0.0132

0

2

4

6

IL-1

0 (n

g/m

l) I: n.s.T: P=0.0048C: n.s.

Cur

dlan

Zym

osanNon

e

LPS

Pam

3CS

K4

52.6

1053.5

GM-MØP

BMDC

P=0.0159

IL-2 fold-increase101 102 103 104

A

B

Figure 2. Cytokine responses of GM-MØP to microbial stimuli are similarto those of BMDC. (A) Production of select cytokines (as indicated) during24h after stimulation of BMDC (black bars) and GM-MØP (white bars) withthe indicated microbial stimuli (see the Materials and methods section). Dataare mean7SEM of data pooled from three (2 for IL-2) identical independentexperiments and are representative of four independent experiments intotal. Two-way ANOVA analysis was conducted and the p values for theeffects of an ‘Interaction’ (I), the Treatment (T) and the Cell type (C) areindicated (n.s., not significant). �po0.05, ��po0.001 (Bonferonni post tests).(B) SYBR Green quantitative PCR of IL-2 mRNA induction by curdlan (500mg/mL) in GM-MØP and BMDC. Data show mean795% confidence intervals ofdata from three independent experiments (the mean is indicated next tothe bar). The p value was determined using a paired two-tailed t-test.


Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.358

(biotinylated anti-MHCII antibodies were now used to increase

sensitivity) (Fig. 4A). One-way ANOVA and Bonferonni post-test

analysis of the expression of these markers (data from five

independent experiments) indicated that MHCII was significantly

lower in the GM-MØP, when compared to the BMDC (Fig. 4B).

Effective antigen presentation by GM-MØP

The presence of similar MHCII, CD11c and co-stimulatory molecule

expression by GM-MØP prompted us to examine the capacity of

these cells to function as professional APC. As an additional control,

M-CSF-differentiated M-MØP were included in the antigen

presentation assays as they were predicted to be poor APC by

analogy to BMMØ and because they expressed little MHCII. Both

adherent and non-adherent cells isolated from culture were co-

cultured with the 2G7.1 MHCII I-Ek-restricted hen egg lysozyme

(HEL)-specific T-cell hybridoma [26] (Fig. 4C). As expected from

previous studies, non-adherent BMDC were more effective than

adherent cells at presenting HEL to 2G7.1 as measured by IL-2

production (Fig. 4C, left panel). GM-MØP were at least as efficient

at presenting antigen to the 2G7.1 T-cell hybridoma as non-

adherent BMDC, whether or not they were from the non-adherent

fraction (Fig. 4C, right panel). M-MØP, as expected, failed to

stimulate IL-2 production by 2G7.1 cells (Fig. 4C, right panel).

Presentation of antigen by GM-MØP to naı̈ve CD41

T cells

The T-cell hybridoma experiments described above demonstrated

that GM-MØP were efficient APC. To assess the ability

14

BMDC-S6

GM-MØP-S6

2

4

6

8

10

12

NO

(μM

)

0.001 0.01 0.1 1 100

LPS (μg/ml)

Figure 3. NO production of GM-MØP in response to LPS. BMDC andGM-MØP were stimulated with increasing concentrations of LPS andthe production of NO was measured by the Griess reaction. GM-MØPproduced substantial amounts of NO in response to LPS in a dose-dependent manner. Dashed line denotes basal levels of NO fromunstimulated cells. Data show mean7SEM of triplicate samples andare representative of two independent experiments.

A

SS

C

FSC 64

128

192

256

Non-Ad:

Non-Ad:

Ad:GM

-MØ

P(C

BA

)

0 64 128 192 2560

MHCII CD11c CD80 CD86 CD40

MHCII CD11c CD80 CD86 CD40

Cou

nts

Receptor

Ad:BM

DC

(CB

A)

104B ******

102

103

ΔMF

I

100

101

Adherent BMDC

Adherent GM-MØP

Non-adherent BMDC

Non-adherent GM-MØP

0.8

1.2

1.6

0.8

1.2

1.6C BMDC (CBA) GM-MØP (CBA)

100 1000 10000 1000000.0

0.4

100 1000 10000 1000000.0

0.4

IL-2

(ng/

ml)

APC number APC number

Adherent

Adherent + LPS

Non-adherent

Non-adherent + LPS

M-MØP

M-MØP + LPS

Figure 4. GM-MØP are effective at antigen presentation. (A) Separateanalysis of adherent (Ad) and non-adherent (Non-Ad) cells from GM-MØP and BMDC cultures indicates a high-similarity of antigenphenotype between both adherent and non-adherent cells and GM-MØP and BMDC. The top panel shows a representative gating of cellsfor analysis. Profiles are representative of results from five indepen-dent experiments. Shaded histograms represent receptor specificstaining and bold lines denote isotype control staining. (B) Quantifica-tion of receptor expression as difference in mean fluorescenceintensity between receptor specific and isotype control staining (DMFI)was represented as mean7SD for data from five independentexperiments. ��po0.001, Bonferonni post tests (One way ANOVA).(C) Antigen (HEL) presentation to the 2G7.1 MHCII-restricted T-cellhybridoma by GM-MØP and BMDC from CBA/ca mice resulting in IL-2production measured by ELISA. Data show mean7SEM of triplicatesamples and are representative of two independent experiments.



of GM-MØP to prime and expand T-cell responses, naı̈ve

CD41CD25–CD62L1CD44low T cells were derived from

OTII anti-ovalbumin (OVA) TCR transgenic mice and

maintained under conditions that polarise towards IFN-gproduction (LPS).

Naı̈ve T-cells were co-cultured with GM-MØP or BMDC for 5

days in the presence of OVA or OVA-derived peptide. T-cell

proliferation and effector function were recorded using carboxy-

fluorescein diacetate succinimidyl diester (CFSE) labelling and

intracellular IFN-g staining. When compared to BMDC cultures,

GM-MØP were consistently less efficient at inducing proliferation

and IFN-g production (Fig. 5A). Examination of the number of

CD41 T cells present at the end of the stimulation confirmed

markedly increased numbers of cells when BMDC were present

consistent with reduced priming and/or expansion in the

presence of GM-MØP (Fig. 5B).

Response of GM-MØP to fungal stimuli

BMDC exhibit robust cytokine responses to fungal particles such as

zymosan and purified b–glucan particles (curdlan). To examine

how effective GM-MØP were as a cell line model of DC function

under microbial stimulation we treated them with increasing doses

of zymosan or ‘TLR-agonist-depleted’ zymosan and examined the

mechanism of response (Fig. 6A). Dectin-1-deficient GM-MØP

(Clec7a�/�) only exhibited significant impairment of the response

to zymosan when dectin-2 was concurrently blocked. The response

of GM-MØP to depleted-zymosan was significantly more dependent

on dectin-1. Thus, GM-MØP used the same receptor mechanism to

recognise these particles as BMDC, namely the use of both dectin-1

and dectin-2 to recognise and respond to zymosan [24] and

primarily dectin-1 to respond to depleted-zymosan (Fig. 6A).

To demonstrate the usefulness of this cell line as a model of

GM-CSF BM culture, we retrovirally transduced dectin-1-deficient

MØP with the two short polymorphic isoforms of dectin-1, dectin-

1B.1 and dectin-1B.2 using the Moloney Murine Leukemia Virus

(MMLV)-derived vector pMXs-IZ, as previously described [4]. After

appropriate antibiotic selection we derived two stable cell lines

expressing comparable levels of the two dectin-1B variants

(Fig. 6B). To determine whether there were any differences

between these two polymorphic forms in their ability to respond to

dectin-1 stimuli, cells were treated with low doses of curdlan and

cytokine production assessed. No differences were seen in the levels

A Beads

10.7 9.1 11.5 1.6

No Ag2µg/ml

OVA prot10µg/ml OVA prot

10 nM OVA peptide

None:

SS

C

CD4

CD4+

11.0 22.5 39.4 22.9

2.5 2.4 4.5 3.3

52.6 72.159.259.1

79.3 97.177.367.0

LPS:

BMDC

GM-MØP5.2 3.2 5.3 2.0

51.6 35.538.419.4

58.9 54.141.128.2

FNγ

None:

LPS:

IF

CFSE

3

4

5

B

BMDC-B6

GM-MØP-B6

I: n.s.

0

1

2

5)

CD

4+ T

cel

ls (

x10

T: n.s.T: n.s.C: P=0.0134

+ LPS

Figure 5. IFN-g-production and expansion of naı̈ve CD41 T cells by GM-MØP. (A) BMDC or GM-MØP were cocultured for 5 days withCFSE-labelled naı̈ve CD41 OTII anti-OVA peptide-specific T cells in the absence or presence of LPS and OVA protein or peptide as indicated.IFN-g production was assessed by intracellular staining after PMA and ionomycin stimulation and proliferation was determined byCFSE dilution. CD41 cells were gated as shown in the upper panel alongside the counting beads. Percentages of expanded T cells producingIFN-g are indicated in the gated regions. Data are representative of four independent experiments. (B) Quantification of CD41 T-cellnumbers performed at the end of the experiments displayed in (A) above indicated that surviving cell numbers were substantially lower whenGM-MØP were used in place of BMDC. Data show mean7SEM of three independent experiments, was analysed by two-way ANOVA in the sameway as indicated in Fig. 2A.



of TNFa and IL-6 produced by the two different polymorphic

variants of dectin-1B (Fig. 6C).

Discussion

Hoxb8-conditional-immortalisation of MØ progenitors [1]

offers an approach for the in vitro generation of large numbers

of MØ from an array of genetically modified mice. Indeed,

the ease of manipulation makes this strategy an attractive

way of simplifying complex experiments involving mononuclear

phagocytes. This technology therefore has the capacity to

reduce the use of animals in research and cell lines generated

in this manner have the potential to be applied to high-

throughput screening assays. However, the phenotype of cells

derived from these progenitors in vitro has not been the subject of

extensive immunological characterisation. Studies outlined in

this current article have addressed this issue by examining the

functional properties of GM-CSF-differentiated MØP (GM-MØP)

cultures.

Here we demonstrate that GM-MØP cultures resemble BMDC,

but also display some notable functional differences. Specifically,

BMDC and GM-MØP display comparable surface antigen

20 BA

0

4

8

12

16

TN

F (

ng/m

l)

None SS

C

FSC

GM-MØP-S6.Clec7a –/– + IgG2aGM-MØP-S6.Clec7a –/– + α-Dectin-2

GM-MØP-S6 + IgG2aGM-MØP-S6 + α-Dectin-2

Zym d-Zym

pMXs-IZ: Dectin-1B.1

C 0

300

600

900

1200

256

256

192

192

128

128

64

640

0

0.0

0.5

1.0

1.5

2.0

2.5

0

2

4

6

8

10

pMXs-IZ: Dectin-1B.2

TN

F (

ng/m

l)

IL-6

(ng

/ml)

100

101

102

103

104

100

101

102

103

104

0

300

600

900

1200

Cou

nts

Dectin-1

50 µg/ml curdlan

untreated

pMX

s-IZ

pMX

s-IZ

Dec

tin-1

B.1

Dec

tin-1

B.1

Dec

tin-1

B.2

Dec

tin-1

B.2

Figure 6. GM-MØP share the receptor dependency of BMDC for fungal particle recognition and are useful to model minor functional differences.(A) GM-MØP from WT 129S6/SvEv (S6) and dectin-1-deficient (Clec7a�/�) mice were pretreated with blocking anti-dectin-2 (a-dectin-2) or rat IgG2aisotype control and TNF production was assessed after stimulation with the fungal particle zymosan (Zym). GM-MØP recognise zymosan usingboth dectin-1 and dectin-2, in a manner directly analgous to BMDC [24], whereas alkali-depleted zymosan (d-Zym), which is reported to lack theTLR agonist of zymosan, was more dectin-1-specific. Data show mean7SEM of triplicate samples and are representative of three independentexperiments. (B) Dectin-1-deficient MØP were reconstituted with polymorphic variants of dectin-1 isoforms (dectin-1B.1 and dectin-1B.2) [31] andthen differentiated to GM-MØP, where they displayed similar expression of both variants by flow cytometric analysis of surface expression (cellswere gated as shown in the upper panel to derive the histograms in the lower panels). Data are representative of two experiments. Shadedhistograms and bold lines represent receptor specific staining on reconstituted cells and empty vector control cells respectively. (C) Whenstimulated with low doses of the dectin-1-agonist curdlan, reconstituted, but not vector control (pMXs-IZ), cells produced TNF and IL-6. Data showmean7SEM of triplicate samples and are representative of two independent experiments.



expression, anti-microbial responses and the capacity to effec-

tively present antigen. Some of the antigens expressed by

these cells and the cytokine responses observed in response to

LPS were also consistent with the differences noted at the

RNA level in previous microarray analyses [1]. In addition,

both cell types mechanistically use dectin-1 and dectin-2 to

recognise fungal particles [24], which most likely reflects the

activity of inflammatory monocytes in vivo [27]. Whilst the

ability of GM-MØP to produce IL-6, IL-12p40 and TNF was

slightly reduced when directly compared to BMDC, this is unli-

kely to be solely because of slightly reduced dectin-1 expression,

since the additional dectin-1/Syk-induced cytokines IL-10 and

IL-2 were released at comparable and markedly lower levels

respectively, indicative of more complex signalling control.

Collectively though, this indicates that GM-MØP resemble the

phenotype of inflammatory monocyte-derived cells [11]. We

observed a couple of notable marked differences between GM-

MØP and bona fide BMDC cultures. First, whilst GM-MØP are

very effective at presenting antigen to a T-cell hybridoma, they

are relatively poor at priming the expansion of naı̈ve CD41 T cells

under conditions that promote IFN-g production. Second,

induction of IL-2 secretion by BMDC cultures following

stimulation with curdlan and zymosan was not observed in

GM-MØP cultures and a concurrent�20-fold lower induction of

IL-2 mRNA was observed in GM-MØP compared to BMDC

responding to curdlan. The factors that govern these differences

may themselves provide further insight into the in vitro and in

vivo requirements for efficient activation of naı̈ve T cells by

monocytic cells.

The fact that BMDC are now considered as a model of an

inflammatory monocyte-derived DC, such as the ‘TNF and iNOS

producing DC’ (Tip-DC) is very interesting in this context

[11, 17]. The phenotype of inflammatory monocyte-derived cells

in vivo is likely to be very heterogeneous. Some populations

produce TNF and/or iNOS during inflammatory response and are

considered immune stimulatory, while others possess immuno-

suppressive myeloid-derived suppressor cell (MDSC)-like activ-

ities or more ‘conventional’ MØ phenotypes [12–18]. The actual

phenotype would be logically dictated by the inflammatory

context in which the monocyte arrives. In many of the studies of

these in vivo inflammatory cells different antigen presentation

assays are used varying from allogeneic responses to stimulation

of T-cell hybridomas, but few have examined priming and

expansion of naı̈ve T cells. It has been suggested that an ability to

prime the T-cell response by these cells may not be a necessary

feature [16]. GM-MØP are effective at antigen presentation, but

have reduced key functionality in the priming and expansion of

naı̈ve T cells. Collectively, our observations indicate that

GM-MØP are a useful model for studying the function of

cells of the monocytic lineage and a potential model for the

spectrum of responsiveness of inflammatory monocytes. It also

exemplifies at its extreme conclusion the similarity between cells

categorised as DC and MØ [28] and that classification based on

markers is an inadequate replacement for functional character-

isation [29].

MØP can be easily generated from genetically modified mice

[1, 4], and we have shown here how they can be stably recon-

stituted with MMLV-derived vectors with subtle polymorphic

differences in a manner that would be more difficult to achieve

with retroviral transduction of primary BM cultures. In addition,

the stability of the genetic modification means that cells can be

subjected to thorough and repeated examination of these subtle

differences, although expression of transgenes from these types of

vectors should be monitored as they are prone to loss of

expression in hematopoietic cells, this system could no doubt be

improved by the use of specialised vectors [30]. In this context,

we used the dectin-1-deficient cells to demonstrate how easily

naturally occurring SNPs in murine dectin-1 [31] could be

examined reproducibly in vitro. In this case, the assays used

revealed no clear difference in the physiological response of the

cells when transduced with viral vectors encoding dectin-1B.1 or

dectin-1B.2. Whilst it is possible that more subtle assays of

dectin-1 function may determine differences, these studies do,

however, highlight the ease in which complex multiple modifi-

cations can be introduced in a stable manner. This approach

could easily be adapted and extended to the study of many other

molecular systems. In the context discussed above, the fact that

these cells are effective antigen presenters, but poor stimulators

of naı̈ve T cells, coupled with the ease with which they can be

genetically modified, could be exploited as part of cDNA

screening strategy designed to further probe the requirements of

an effective priming capacity.

In summary, Hoxb8 conditionally immortalised MØP are a

valuable genetically tractable model for the replacement of BM-

derived MØ and DC. When differentiated in GM-CSF the resultant

cell line shares significant similarities with GM-CSF derived

BMDC and will be a useful model in which to examine general

MØ and inflammatory monocyte biology.

Materials and methods

Mice and reagents

WT 129S6/SvEv or 129S6/SvEv.Clec7a�/� mice [32] were

obtained from our own breeding colonies, kept and handled in

accordance with institutional and UK Home Office guidelines.

C57BL/6 mice were obtained from Harlan Laboratories

and CBA/ca mice were from B&K Universal. Biotinylated

anti-dectin-1 (clone 2A11 [33]), biotinylated and purified anti-

dectin-2 (clone D2.11E4 [27]) and biotinylated anti-mannose

receptor/CD206 (clone 5D3 [34]) were produced in our

laboratory. Biotinylated-anti-CD40 (clone 3/23), biotinylated

anti-CD11c (clone N418), anti-dectin-1-AlexaFluor647 (clone

2A11), rat IgG2b-AlexaFlour647 and the anti-rat IgG2a

used as isotype control (MCA1212EL) for the anti-dectin-2

antibody were purchased from Serotec (Oxford, UK). Biotin-

labelled antibodies against MHCII (Clone 2G9), CD80 (clone

16-10A1), CD86 (clone PO3) (including their isotype controls)



and streptavidin-allophycocyanin were from BD Pharmingen

(Franklin Lakes, NJ). ELISA kits to measure the production of

TNF, IL-2, IL-6, IL-10 and IL-12p40 were also from BD

Pharmingen. Recombinant GM-CSF and M-CSF were purchased

from PeproTech (Rocky Hill, NJ). The Griess assay kit was from

Promega (Southampton, UK). LPS (Salmonella typhimurium) and

b-estradiol were obtained from Sigma-Aldrich. Alternatively, LPS

(Escherichia coli serotype R515) was obtained from Alexis

biochemicals. Zymosan was purchased from Invitrogen. Depleted

zymosan and Pam3CSK4 were from Invivogen (San Diego, CA).

Curdlan was from Wako Chemicals GmbH (Neuss, Germany).

OVA peptide (residues 323–339) was synthesised and purified by

high performance liquid chromatography at Cancer Research UK.

OVA protein was from Calbiochem. Anti-CD3e (clone 145-2C11)

antibody was from BD pharmingen.

Generation of MØP from different mouse strains

MØPs were generated from several strains of mice, including

C57BL/6 and CBA/ca, as previously described [4]. MØP from

129S6/SvEv and 129S6/SvEv.Clec7a�/� mice have been

described elsewhere [4, 32]. In brief, following the basic

protocols of Wang et al. [1], we generated MMLV-derived

retroviral vector expressing an estrogen dependent Hoxb8

transcription factor [4]. CD117-enriched BM cells (using

CD117-biotin (BD) and anti-biotin-MACS, Miltenyi-Biotec) were

expanded for 2 days in IL-3 (10 ng/mL), IL-6 (20 ng/mL) and SCF

(25 ng/mL) (all from Peprotec) in Iscove’s-modified Dulbecco’s

medium (IMDM) (Invitrogen) supplemented with 15% heat-

inactivated FBS as described [1, 4]. The cells were then spin

infected with packaged retrovirus, which was generated as

previously described by transient (Fugene 6, Roche) transfection

of phoenix packaging cells [4, 27, 35]. After 2 days, the cells were

selected in puromycin (1.5 mg/mL; Sigma-Aldrich) for 10–14 days

in the presence of GM-CSF (10 ng/mL; Peprotec) and b-estradiol

(1 mM; Sigma), after which polyclonal, conditionally immorta-

lised MØP were stable in culture.

Growth and differentiation of MØP and BMDC

To differentiate MØP they were washed three times with RPMI

1640 medium containing 10% heat-inactivated FBS to

remove the b-estradiol and GM-CSF before differentiation in

bacterial dishes or multi-well plates, depending on the experi-

ment, and incubated at 371C for 4 days in the same medium

supplemented with either M-CSF (M-MØP) or GM-CSF (GM-

MØP) (both 20 ng/mL; Peprotec) for 4 days.

BMDC were produced by culture of BM cells from the femur

and tibia of mice in the presence of GM-CSF (Peprotec) for 7 days

as previously described [6]. In brief, BM cells were isolated by

flushing femurs and tibias of mice with RPMI. Red blood cells

were lysed with ACK buffer (150 mM NH4Cl, 10 mM KHCO3,

0.1 mM Na2EDTA pH 5 7.4) and the cells were washed twice with

RPMI. BMDC were produced by culture in RPMI 1640 supple-

mented with 10% heat-inactivated FBS, 2 mM L-glutamine,

penicillin/streptomycin, 50 mM b-mercaptoethanol and GM-CSF

(10 ng/mL). Cells were plated in bacterial petri dishes and

incubated at 371C for 7 days.

Stimulation of cells with microbial agonists

BMDC or GM-MØP cells derived from 129S6/SvEv or C57BL/6

mice were cultured in GM-CSF as indicated above. After

differentiation, cells were counted and added to 48-well plates

(2�105 cells per well) with various concentrations of

Curdlan (50, 500mg/mL), zymosan (5, 50 mg/mL), LPS (10,

100 ng/mL) and Pam3CSK4 (10, 100 ng/mL). After 16 h incuba-

tion at 371C, supernatants were collected. Production of IL-6,

TNF, IL-12p40, IL-2 and IL-10 was measured using ELISA kits (BD

Biosciences).

MØP cells derived from WT (129S6/SvEv) or 129S6/

SvEv.Clec7a�/� mice [4, 32] were cultured in 96-well U-bottom

plate (12� 103 cells per well) in differentiation medium with GM-

CSF for 4 days (see above). On the 4th day, medium was removed

from the wells (180mL) and replaced with 100mL of media

containing 10mg/mL anti- Dectin-2 antibody (D2.11E4) or a rat

IgG2a isotype control (Serotec, MCA1212EL). Cells were incubated

with the antibodies for 2 h at 371C. Then, increasing concentrations

of zymosan or depleted zymosan (0–30mg/mL) were added to a

final 200mL culture volume. After 6 h at 371C, supernatants were

collected and TNF production was measured using as above.

Quantitative RT-PCR

Total RNA was extracted from GM-MØP cells and BMDC using

the RNeasy kit with optional on column DNase digestion

(Qiagen). cDNA was synthesised using the qScript kit (Ambion)

and diluted to 5 ng/ml. Primers for IL-2 amplification

(50-GTAAAACTAAAGGGCTCTGACAAC-30 and 50-GGCTTGTTGA-

GATGATGCTTTG-30) and the housekeeping gene Ywhaz

(sequence undisclosed) were obtained from PrimerDesign.

Real-Time PCR amplification was performed with SYBR green

reagents (Primer Design) using the ABI-7900HT fast RT-PCR

system. IL-2 cycle threshold values were normalised against

Ywhaz and these values expressed as a fold increase over the

untreated control.

NO measurement

BMDC or GM-MØP cells derived from 129S6/SvEv mice were

counted and added into a 96-well U-bottom plate (2� 104 cells per

well) with increasing concentrations of LPS (0-1.5mg/mL). After

48 h incubation at 371C, supernatants were collected to assess NO

production by measurement of nitrate concentration using Griess

assay according to the manufacturer’s instructions (Promega).



Antigen presentation assays

T-cell hybridoma

BMDC or GM-MØP cells derived from CBA/ca mice were

differentiated in bacterial plates in GM-CSF as indicated above.

Additionally, MØP were also differentiated in M-CSF. Suspension

and adherent cells from the GM-CSF-differentiated cultures were

independently plated at increasing concentrations (0–32�103

per well) in 96-well U-bottom plates. M-CSF-differentiated cells

were plated at the same concentrations. All the APC were co-

cultured with 2G7.1 cells (3� 104 cells per well), a CBA-derived

MHCII I-Ek-restricted T-cell hybridoma specific for HEL [26]. HEL

was added at a final concentration of 50 mg/mL and some cells

were additionally treated with 100 ng/mL LPS. Co-cultures were

incubated for 48 h at 371C and supernatants were collected for

subsequent assay of IL-2 production by ELISA (BD Biosciences).

Naı̈ve OT-II CD41 T cells in vitro T-cell assays

CD41CD25�CD62LhiCD44lo (naı̈ve) T cells from OT-II mice were

purified from spleen by cell sorting using a MoFlo cell sorter

(Dako Cytomation) and labelled with 2mM CFSE for 12 min at

371C before culture. 1�104 BMDC or GM-MØP cells were

cultured for 5 days with 5� 104 naı̈ve T cells in the presence of

various doses of OVA323–339 peptide or OVA protein. 100 ng/mL

LPS (E. coli serotype R515) was used as innate stimulus. Cells

from cultures were re-stimulated on day 5 for 4 h with phorbol

12-myristate 13-acetate (10 ng/mL; Sigma), ionomycin (1 mg/

mL; Calbiochem) and brefeldin A (5mg/mL; Sigma). Intracellular

staining for IFN-g was analysed by flow cytometry. Alternatively,

half of the content of each well were re-stimulated on day 5 on

plate bound anti-CD3e (5 mg/mL) for 48 h before cytokines in the

supernatant were analysed by sandwich ELISA.

Flow cytometry

Flow cytometry was performed as previously described [36, 37].

BMDC or GM-MØP cells derived from CBA/ca or C57BL/6 mice

were cultured in GM-CSF as indicated above. The cells in

suspension were harvested by removing the medium from the dish

whereas the adherent cells were lifted with lidocaine. After

spinning down and removing the supernatant, cells were incubated

in blocking buffer (PBS containing 5% heat-inactivated rabbit

serum, 0.5% BSA, 5 mM EDTA, 2 mM NaN3, 10mg/mL 2.4G2) for

1 h at 41C. Biotin-labelled antibodies were added at 10mg/mL in a

final volume of 100mL of washing buffer (PBS containing 0.5%

BSA, 5 mM EDTA and 2 mM NaN3). After 1 h at 41C, cells were

washed twice with washing buffer and incubated with streptavidin-

allophycocyanin for a further 1 h at 41C. After this, cells were

washed three times with washing buffer and resuspended in 1%

formaldehyde (in PBS). Data were acquired on either a CyAn ADP

analyser (Beckman-Coulter) or a FACS Calibur and analysed in

either Summit (Beckman-Coulter) or FlowJo (Tree Star) software.

Statistics

Statistical analysis was accomplished using Graph Pad Prism. The

tests used are indicated where appropriate in the text and exact p

values or the following abbreviations are used: �po0.05;��po0.01; ��po0.001. Data were considered significant when

po0.05.

Acknowledgements: This study was funded by the UK Medical

Research Council (MRC) and P. R. T is an MRC Senior Non-

Clinical Research Fellow (G0601617). F. O. was supported by

GSRS and ORS studentships from University College London and

by a CRUK PhD studentship. The authors acknowledge the help of

their animal facility staff for the care of the animals used in this

study. All animal experiments were conducted in accordance with

institutional and UK Home Office guidelines.

Conflict of Interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: BMDC: bone marrow-derived dendritic cells � HEL: hen

egg lysozyme � MØ: macrophages � MØP: MØ precursor � MMLV:

moloney murine leukemia virus

Full correspondence: Dr. Philip R. Taylor, Infection, Immunity and

Biochemistry, Cardiff University School of Medicine, Tenovus Building,

Heath Park, Cardiff, Wales CF14 4XN, UK

Fax: 144-292068303

e-mail: [email protected]

Received: 17/8/2010

Revised: 1/11/2010

Accepted: 23/11/2010

Accepted article online: 6/12/2010



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Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells