Human B cells express the orphan chemokine receptor CRAM-A/B in a maturation-stage-dependent and CCL5-modulated manner

Human B cells express the orphan chemokine receptor CRAM-A/Bin a maturation-stage-dependent and CCL5-modulated manner

Introduction

Chemokines are a family of growth factor-like proteins

that can be secreted by virtually all types of cells. They

work as cellular recruitment molecules, which induce

leucocytes to adhere to specific sites on blood vessels and

subsequently to extravasate into the tissue.1 Specific com-

binations of chemokines, chemokine receptors and adhe-

sion molecules determine which subgroups of leucocytes

migrate and what their destinations are. In addition,

chemokines not only facilitate leucocyte migration and

positioning, but are also involved in other processes such

as leucocyte degranulation, angiogenesis, cell differentia-

tion and transformation,2 cell invasion, integrin regula-

tion and metastasis.3 The chemokines are classified into

four highly conserved groups depending on whether they

express a C, CC, CXC, or CX3C amino acid motif at their

N termini.1 More than 50 chemokines have been discov-

ered so far, and there are at least 18 human chemokine

receptors.4 Normally, the chemokine receptors are G-pro-

tein coupled and seven-transmembrane-domain spanning.

The DRYLAR/IV motif in the second intracellular loop is

critical for G protein coupling in conventional chemokine

receptors,5 but besides these conventional receptors other

chemokine receptors with alterations in this motif have

been described, namely the Duffy antigen receptor for

chemokines (DARC),6 D67,8 and CCX CKR.9 As a result

of the lack of G protein coupling, there are no reports of

calcium signalling or of intrinsic migratory activity of

these chemokine receptors but there is emerging evidence

that these atypical chemokine receptors scavenge or alter

the localization of chemokines and so regulate the com-

plex chemotactic network.10 One orphan receptor

presenting structural similarities with known chemokine

receptors but bearing alterations in this DRYLAR/IV

motif is the chemokine (CC motif) receptor-like 2

Tanja N. Hartmann,1 Marion

Leick,1,2 Susann Ewers,1 Andrea

Diefenbacher,1 Ingrid

Schraufstatter,3 Marek

Honczarenko4 and Meike Burger1

1Department of Internal Medicine and2Developmental Biology Unit, Freiburg

University Clinic, Freiburg, Germany, 3Torrey

Pines Institute for Molecular Studies, San

Diego, CA, USA and 4BiogenIdec, Cambridge,

MA, USA

doi:10.1111/j.1365-2567.2008.02836.x

Received 23 February 2008; revised 25

February 2008; accepted 26 February 2008.

Correspondence: M. Burger, MD,

Department of Hematology and Oncology,

University Clinic of Freiburg, Hugstetter

Strasse 55, 79106 Freiburg, Germany.

Email: [email protected]

Senior author: Meike Burger

Summary

Chemokines orchestrate the organization of leucocyte recruitment during

inflammation and homeostasis. Despite growing knowledge of chemokine

receptors, some orphan chemokine receptors are still not characterized.

The gene CCRL2 encodes such a receptor that exists in two splice vari-

ants, CRAM-A and CRAM-B. Here, we report that CRAM is expressed by

human peripheral blood and bone marrow B cells, and by different B-cell

lines dependent on the B-cell maturation stage. Intriguingly, CRAM sur-

face expression on the pre-B-cell lines Nalm6 and G2 is specifically up-

regulated in response to the inflammatory chemokine CCL5 (RANTES), a

chemokine that is well known to play an important role in modulating

immune responses. Although Nalm6 cells do not express any of the

known CCL5 binding receptors, extracellular signal-regulated kinases 1

and 2 (ERK1/2) are phosphorylated upon CCL5 stimulation, suggesting a

direct effect of CCL5 through the CRAM receptor. However, no calcium

mobilization or migratory responses upon CCL5 stimulation are induced

in B-cell lines or in transfected cells. Also, ERK1/2 phosphorylation

cannot be inhibited by pertussis toxin, suggesting that CRAM does not

couple to Gi proteins. Our results describe the expression of a novel,

non-classical chemokine receptor on B cells that is potentially involved in

immunomodulatory functions together with CCL5.

Keywords: B cells; chemokine; GPCR; signal transduction

252 � 2008 The Authors Journal compilation � 2008 Blackwell Publishing Ltd, Immunology, 125, 252–262

I M M U N O L O G Y O R I G I N A L A R T I C L E

(CCRL2) molecule. The CCRL2 gene was initially mapped

by fluorescence in situ hybridization to the Xq13 region

of the human genome,11 but this was later corrected to

the main cluster of the CC-chemokine receptor genes in

the 3p21-23 region, together with CCR1 to CCR5, CCR8

to CCR10, XCR1 and CX3CR1.12 Northern blot analysis

revealed expression in lymphoid organs (spleen, lymph

node, fetal liver, bone marrow) as well as non-lymphoid

organs (heart, lung).11 The CCRL2 messenger RNA

(mRNA) has been found on almost all haematopoietic

cells, including monocytes, macrophages, polymorpho-

nuclear cells, T cells, dendritic cells, natural killer cells

and CD34+ progenitor cells.13,14 CCRL2 has been

reported as not expressed on B cells.13 CCRL2 exists in

two splice variants, one coding for a putative receptor of

345 amino acids, referred to in the literature as CCRL2B,

CRAM-B, CKRX and HCR, and another splice variant, 12

amino acids longer, which is referred to in the literature

as CCRL2A or CRAM-A. As a result of the high sequence

homology, CCRL2 has been suggested to be the human

homologue to the murine orphan chemokine receptor

L-CCR.15

Here we present data indicating that B cells express the

orphan chemokine receptor CRAM with a preferential

expression of the CRAM-A splice variant in pre-B cells.

We show that CRAM expression is modulated by CCL5

binding and suggest that this receptor might belong to

the group of atypical chemokine receptors.

Materials and methods

Cell culture

NIH 3T3, HEK 293 and COS cells were maintained in

Dulbeccos’s modified Eagle’s medium (DMEM) contain-

ing 10% calf serum in a humidified atmosphere (5%

CO2) at 37�. Stable transfectants were additionally pro-

vided with 800 lg/ml G418. For isolation of chronic lym-

phocytic leukaemia (CLL) cells, blood samples were

collected from patients who fulfilled the diagnostic and

immunophenotypic criteria for common B-cell CLL.

Peripheral blood mononuclear cells were isolated by den-

sity gradient centrifugation over Ficoll–Hypaque (Phar-

macia, Uppsala, Sweden). The CLL samples usually

contained more than 90% CLL B cells.16 For cytometrical

detection of CRAM surface expression, B cells of the CLL

samples were costained with the B-cell marker CD19. For

RNA extraction, > 99% pure B-cell fractions that had

been sorted by CD19 expression were used. The CLL cells

and the B-cell lines Reh, Nalm6, Ramos, Daudi and Raji

were maintained in RPMI-1640 containing 10% fetal calf

serum (FCS) and penicillin–streptomycin–glutamine. The

G2 cell line was kindly provided by Prof. Dr M. Freed-

man (Hospital for Sick Children, Toronto, Canada),17

and was cultured in Iscove’s modified Dulbecco’s medium

containing 10% FCS, penicillin–streptomycin–glutamine

and 25 mM HEPES.

Complementary DNA constructs

The CRAM-B coding sequence (Genbank/EMBL accession

number U97123) was amplified by polymerase chain reac-

tion (PCR) from human B-CLL complementary DNA

(cDNA) using 50-ATGGCCAATTACACGCTGGCACCA

GAG-30 and 50-TTACACTTCGGTGGAATGGTCAGGTTC

TTCCC-30 (for expression of native protein) or 50-CACT

TCGGTGGAATGGTCAGGTTCTTCCCTC-30 (for expres-

sion of protein with histag). The CRAM-A coding sequence

(Genbank/EMBL accession number AF015524) was ampli-

fied by PCR from human B-CLL cDNA using 50-AAA

AAAAAATGATCTACACCCGTTTCTTAAAAGGC-30 and

50-AAAAAAAATTACACTTCGGTGGAATGGCAGG-30

(for expression of native protein) or 50-GCCATGATCTAC

ACCCGTTTCTTAAAAGGCAGTCTG-30 and 50-CACTTC

GGTGGAATGGTCAGGTTCTTCCCTC-30 (for expression

of protein with histag). The CRAM-A and CRAM-B PCR

products were cloned in pcDNA3.1/V5-His TOPO TA und

subsequently BamHI/KspI subcloned in pIRES2-EGFP or

BstXI subcloned in pcDEF3. The plasmids were transfected

with PolyFect (Qiagen, Hilden, Germany) in NIH 3T3 and

COS cells according to the manufacturer’s instructions.

Stably transfected cells were selected with 800 lg/ml G418.

The cDNA of the receptors CCR1, CCR3 and CCR5 cloned

into the vector pcDNA.3.1 were purchased from the UMR

cDNA Resource Center (http://www.cdna.org). The plas-

mids were also transiently transfected into HEK 293 cells

using the method described above using PolyFect.

RNA-extraction and reverse transcription–PCR

Total RNA was isolated using the RNeasy RNA isolation

kit (Qiagen) according to the manufacturer’s instructions.

Residual DNA was removed by DNase I Digestion

(Ambion, Austin, TX). The cDNA synthesis was performed

using 1 lg RNA as a template for oligo-dT (12–18 mer)

primers and 50 Units SuperScript II reverse transcriptase

(Super Script first-strand synthesis system for reverse tran-

scription (RT-) PCR; Invitrogen, Karlsruhe, Germany).

The cDNA was amplified using Taq polymerase (Qiagen).

The following primer pairs were used for PCR: CRAM-B:

50-ATGGCCAATTACACGCTGGCACCAGAG-30 and the

corresponding antisense primer 50-CACTTCGGTGGAATG

GTCAGGTTCTTCCCTC-30; CRAM-A: 50-GCCATGATCT

ACACCCGTTTCTTAAAAGGCAGTCTG-30 with the same

antisense primer as for CRAM-B (annealing temperature

58�); CCR1: 50-AGTTTGACTATGGGGATGCAACTCCG

TGCCA-30 and 50-CGAAGGCGTAGATCACTGGGTTGA

CACAGCAGT-30; CCR3: 50-CTGATACCAGAGCACTGAT

GGCCCAGTTTGTGC-30 and 50-TGCATGAGCAAGTGCC

TGTGGAAGAAGTGG-30; CCR5: 50-AAATCAATGTGAAG

� 2008 The Authors Journal compilation � 2008 Blackwell Publishing Ltd, Immunology, 125, 252–262 253

CRAM-A/B is expressed by B cells

CAAATCGCAGCCCGC-30 and 50-TGATGGGGTTGATGC

AGCAGTGCGTCAT-30 (annealing temperatures 62�). To

test expression of the CD44 molecule, primers recognizing

all splice variants of CD44, 50-GACACATATTGCTTCAA

TGCTTCAGC-30 and 50-GATGCCAAGATGATCAGCCA

TTCTGGA-30, were used (annealing temperature 64�). For

GAPDH, the sense primer 50-GGAGTCCACTGGCGTCTT

CACC-30 and the antisense primer 50-ATTGCTGATG

ATCTTGAGGCTGTTGTC-30 (annealing temperature 58�)

were employed.

Flow cytometry and immunofluorescence

Murine monoclonal antibodies against CCRL2 (anti-

HCR/CRAM-A/B) were produced by R&D Systems (Min-

neapolis, MN). All experiments were performed with

clone 152254. Cells were adjusted to a concentration of

5 · 106 cells/ml in RPMI-1640 with 0�5% bovine serum

albumin (BSA). Bone marrow mononuclear cells were

obtained from Lonza Walkersville, Inc (Walkersville,

MD). Surface staining of bone marrow cells was per-

formed with the following antibodies (all from BDPharm-

ingen, San Diego, CA unless otherwise stated): fluorescein

isothiocyanate-labelled anti-immunoglobulin D (IgD),

phycoerythrin-labelled anti-j and anti-k light chain,

PE-Texas red (ECD)-labelled anti-CD23 (Beckman Coul-

ter-Immunotech, Marseille, France), phycoerythrin-Cy5-

labelled anti-CD27 (Beckman Coulter-Immunotech),

phycoerythrin-Cy7-labelled anti-CD34, allophycocyanin-

Cy5.5-labelled anti-CD38 (Ebioscience, San Diego, CA),

allophycocyanin-Cy7-labelled anti-CD19. CRAM was

detected with the unconjugated mouse monoclonal anti-

body mentioned above, followed by the secondary anti-

body, Cy5-labelled goat anti-mouse IgG (Jackson

Immunoresearch Laboratories, West Grove, PA). Cells

were resuspended in ice-cold staining buffer [1 · phos-

phate-buffered saline (PBS) with 2% fetal bovine serum]

and incubated for 30 min with the first-step reagent. Cells

were washed three times in staining buffer and incubated

for 30 min with the second-step reagents. Stages of bone

marrow B-cell differentiation were defined as previously

described.18 Briefly, the earliest B-cell population, desig-

nated here as pro-B cells, was j)/k) CD19+ CD34+. The

next developmental B-lineage populations were designated

as pre-B cells, identified as j)/k) CD19+ CD34); followed

by immature B cells identified as j+/k+ CD19+ IgD); and

mature B cells were identified as j+/k+ CD19+ IgD+. Sam-

ples were acquired using an LSRII cytometer (Becton

Dickinson Immunocytometry Systems, Franklin Lakes,

NJ) and analysed using FLOWJO software (Tree Star, Inc.,

Ashland, OR). Data are presented at a ‘logicle’ scale as

previously described.19

For immunofluorescent staining, Nalm6 on Bross adhe-

sion slides were fixed in 4% paraformaldehyde (PFA),

washed, permeabilized with 0�1% saponin, and stained

with anti-CCRL2 (5 lg/ml) and secondary antibodies

(Alexa488-conjugated anti-mouse immunoglobulin; Mole-

cular Probes, Eugene, OR). For visualization of the cyto-

plasm and nucleus, cells were stained with 2 lM

CellTracker� Orange (Molecular Probes) and 100 lM

DAPI (Sigma Aldrich, Taufkirchen, Germany). Slides were

mounted in ProLong Antifade (Molecular Probes) and

analysed using a confocal microsope (Leica TCS SP2

AOBS spectral confocal microscope).

Calcium

For calcium measurements, 1 · 107/ml cells were incu-

bated for 30 min in 2 lM fluo-3-AM (Molecular Probes)

in PBS. Afterwards, cells were washed twice in PBS and

resuspended in 0�1% BSA in PBS. Cells were diluted

1 : 10 in RPMI-1640 containing 1�5 mM CaCl2/0�1% BSA,

and fluorescence was cytometrically determined up to

120 seconds after addition of the chemokine.

Chemotaxis

Transfectants and Nalm6 cells were suspended in RPMI-

1640 with 0�5% BSA. A total of 100 ll, containing 5 · 105

cells, was added to the top chamber of transwell culture

inserts (Costar, Cambridge, MA) with a pore size of 5 lm

(HEK 293) or 8 lm (Nalm6 and NIH 3T3). Duplicates

were assayed for each condition. Filters were transferred to

wells containing medium with or without different concen-

trations of CXCL12, CCL2 and CCL5. The chambers were

incubated for 2 hr at 37� in 5% CO2. After this incubation,

the cells in the lower chamber were suspended and counted

with a FACSCalibur at high flow for 40 seconds.

Actin stress fibres

NIH 3T3 cells stably transfected with CRAM-A or CRAM-B

were seeded at low density on cover slips and grown in

DMEM containing 10% calf serum and G418. The cells

were starved of serum for 16–18 hr, and stimulated with

CCL2 or CCL5 for 1, 5, 15, or 30 min or left unstimulated.

All experiments were performed at 37� in a tissue culture

incubator. For F-actin localization, cells were fixed for

20 min in 3% paraformaldehyde in PBS, permeabilized for

5 min in 0�2% Triton X-100, incubated with 25 mU/ml of

Alexa 488-phalloidin (Molecular Probes) for 30 min,

washed three times with PBS and mounted with Antifade

(Molecular Probes). Fluorescence microscopy was

performed on a Zeiss Axiovision 2.0 microscope with the

AXIOPLAN 2.0 software to obtain digital images.

Western blotting

Transfectant cells were grown on 60-mm tissue culture

plates, and serum-starved for 16–18 hr, whereas Nalm6


T. N. Hartmann et al.

cells were starved for 2 hr only. The cells were then stim-

ulated with CCL2 or CCL5 for the indicated times at 37�.

Protein lysates were prepared with 100 ll lysis buffer.

Lysis buffer was 20 mM Tris–HCl pH 8�0, 150 mM KCl,

1 mM ethylenediaminetetraacetic acid, 0�2 mM Na3VO4,

1% Triton X, 0�5 mM phenylmethylsulphonyl fluoride,

protein inhibitor cocktail (complete�, Roche Applied Sci-

ence, Penzberg, Germany). Equal amounts of protein

were separated by 10% sodium dodceyl sulphate–poly-

acrylamide gel electrophoresis and transferred onto poly-

vinylidene fluoride membranes. Western blot analysis was

performed using the appropriate antibodies recognizing

the phosphorylated form of the proteins or the total

proteins. The antibodies for p44/42, p38 and Akt were

provided by Cell Signaling Technology, Beverly,

MA. Immunoreactive bands were visualized using horse-

radish peroxidase-conjugated secondary antibody and

the enhanced chemiluminescence system (Amersham

Biosciences, Freiburg, Germany).

Results

CCL5 induces p44/42 signalling in Nalm6 cells whichis independent of known CCL5-binding moleculesCCR1, CCR3, CCR5 and CD44

Studying the effects of different homeostatic and

inflammatory chemokines on B-cell lines, we detected

that CCL5 stimulation leads to phosphorylation of

p44/42 (ERK1/2) in cells of the pre-B acute lympho-

blastoid leukaemia cell line Nalm6 (Fig. 1a). CCL5 is

known to bind to the chemokine receptors CCR1,

CCR3 and CCR5 and to the adhesion molecule CD44.

Intriguingly, we could not attribute the observed CCL5-

induced p44/42 activation to any of these known

CCL5-binding molecules because Nalm6 cells do not

express CCR1, CCR3, CCR5 or CD44 (Fig. 1b). We

clearly excluded cell surface expression and transcripts

of these molecules in the Nalm6 cells by flow cytome-

try and RT-PCR. To confirm the quality of our experi-

mental settings, primers and antibodies, we performed

parallel RT-PCR and cytometric stainings of CCR1,

CCR3 and CCR5 transfectants, which resulted in strong

signals (Fig. 1b). As a positive control for CD44

RT-PCR and flow cytometry, the cell line HL60 was

used (Fig. 1b). The murine orphan receptor L-CCR was

reported to functionally respond to CCL5 and CRAM-

A/B is thought to be the human homologue of this

receptor15 so we tested if the CCL5-induced p44/42

phosphorylation in Nalm6 cells could be attributed to

this receptor. We found that, in contrast to the known

CCL5 binding receptors, the orphan chemokine receptor

CRAM was strongly expressed on the surface of Nalm6

cells and we observed mRNA of both known CRAM

splice variants, CRAM-A and CRAM-B, in these cells

(Fig. 1c).

0′CCL5(a)

(b)

(c)

Phospho-p44/42

p44/42

CCR1, CCR3, CCR5

GAPDH

GAPDH

GAPDH

Positive controlsPositivecontrol

NALM6NALM6

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CCR1-PE CCR3-PE CCR5-PE

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2′ 5′ 10′ 15′ 20′

Figure 1. p44/42 mitogen-activated protein

kinase activation by CCL5 in Nalm6 cells can-

not be attributed to surface expression of any

known CCL5 receptor. (a) Nalm6 cells were

stimulated with 250 ng/ml CCL5 for the indi-

cated times and lysates were prepared. Western

blots were performed with antibodies against

phospho-p44/42 and total p44/42. Data shown

are representative for three independent exper-

iments. (b) Expression of CCR1, CCR3, CCR5

or CD44 could neither be determined by

reverse transcription–polymerase chain reaction

(RT-PCR) nor by flow cytometry. GAPDH

expression is shown as a PCR control. The

fluorescence histograms show the relative fluo-

rescence intensity of the cells stained with

anti-CCR1, anti-CCR3, or anti-CCR5 anti-

body (black lines) compared to the corre-

sponding isotype control (tinted histograms).

(c) CRAM-A and CRAM-B expression in

Nalm6 cells as determined by RT-PCR and

flow cytometry. The fluorescence histograms

show the relative fluorescence intensity of the

cells stained with anti-CRAM antibody (black

line) compared to the corresponding isotype

control (tinted histogram).



CRAM-A and CRAM-B splice variants aredifferentially expressed dependent on the maturationstage of the B lymphocytes

It was previously reported that B cells do not express

CRAM.13 The unexpected detection of high CRAM sur-

face expression in the B-cell line Nalm6 therefore led us

to a comprehensive investigation of CRAM expression in

non-malignant and malignant primary B cells from

human blood and bone marrow as well as in different

B-cell lines. We studied the distribution of CRAM-A

and CRAM-B transcripts by RT-PCR and their surface

expression by flow cytometry in the cell lines Nalm6,

G2, Reh, Ramos, Daudi and Raji. These cell lines served

as models to cover different stages of B-cell maturation.

Reh is an acute lymphocytic leukaemia cell line of pro-B

stage cells. Nalm6 and G2 are pre-B cells from acute

lymphoblastoid leukaemia. Raji, Ramos and Daudi are B

cells from Burkitt’s lymphoma and represent B cells of

the germinal centre. The used antibody is highly specific

for CRAM-A/B but it does not differentiate between the

two splice variants. All reported surface expression of

the receptor includes CRAM-A and/or CRAM-B expres-

sion and is named CRAM expression. The cDNAs were

derived from B lymphocytes sorted from whole blood of

healthy patients, from B lymphocytes taken from blood

samples of patients with CLL, and from different

malignant and non-malignant B-cell lines (Fig. 2). We

detected transcripts of the shorter splice variant

CRAM-B in the CLL samples tested (n = 4) but could

not detect the longer splice variant CRAM-A (Fig. 2a).

Accordingly, we found only CRAM-B, but no CRAM-A,

transcripts in B lymphocytes from healthy donors. In

addition, surface expression of the receptor was found

in all six CLL samples as well as in the B lymphocytes

of healthy donors. Interestingly, only pre-B cells of

Nalm6 and G2 cell lines expressed transcripts of the

longer splice variant CRAM-A. CRAM-B transcripts

could be detected in all the investigated B-cell stages

with the exception of the IgM+ B-cell lines Ramos and

Daudi. There was a correlation between the existence of

CRAM-B mRNA transcripts and surface expression in all

the investigated B-cell types.

We further examined CRAM expression during B-cell

development on subsets of human B lymphocytes from

adult bone marrow (Fig. 2c). Consistent with the expres-

sion on B-cell lines, we detected robust CRAM expression

on a subpopulation of pro-B cells (j)/k) CD19+ CD34+)

and pre-B cells (j)/k) CD19+ CD34)). Among immature

B cells identified as j+/k+ CD19+ IgD), CRAM expression

disappeared and reappeared on a small subpopulation of

mature B cells identified as j+/k+ CD19+ IgD+. In sum-

mary, in contrast to previous expression reports,13,14

CRAM was clearly expressed on different B-cell types with

a preferential expression in the early stages of differentia-

tion and lack of CRAM-A or CRAM-B expression in

immature bone marrow B cells and IgM+-expressing

B-cell lines.

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(b)

(c)

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pro-B pre-B Immature Mature

B-CLL Reh Raji G2

CLL1 CLL2 CLL3 B cell NALM Reh RAMOSDAUDI Raji G2

Figure 2. Expression of CRAM-A and CRAM-B in human B cells. (a) CRAM-A and CRAM-B messenger RNA in malignant and non-malignant

peripheral blood B lymphocytes and different B-cell lines as detected by reverse transcription–polymerase chain reaction (RT-PCR). GAPDH

expression is shown as housekeeping control. (b) CRAM surface expression in different peripheral blood B lymphocytes and B-cell lines as

detected by flow cytometry. The fluorescence histograms depict the relative fluorescence intensity of the cells stained with anti-CRAM antibody

(black lines) compared to the corresponding isotype control (tinted histograms). The experiments were performed at least twice. (c) CRAM

surface expression of primary bone marrow B lymphocytes. Fluorescence-activated cell sorting histograms are presented at a ‘logicle’ scale (see

the Materials and methods section). Data shown are representative for four independent experiments.



CCL5 stimulation increases CRAM surface expressionin pre-B cells but does not promote calcium signallingor migration

To further investigate CRAM-mediated functional

responses in B cells, we focused on the pre-B-cell line

Nalm6, which showed the highest CRAM surface expres-

sion of all the investigated B cells. We investigated cal-

cium signalling and migratory responses upon both CCL2

and CCL5 stimulation in Nalm6 cells. In contrast to the

observations on L-CCR transfectants,15 we could not

detect any calcium release in Nalm6 cells in response to

CCL5, whereas Nalm6 cells easily mobilized calcium in

response to CXCL12 (Fig. 3a). In addition, we could not

detect any chemotactic response of Nalm6 cells towards

CCL5 in a broad range of concentrations from 50 to

800 ng/ml (Fig. 3b and data not shown). Chemotaxis of

Nalm6 cells towards CXCL12 served as a positive control

of our experimental settings and was up to 30% depend-

ing on CXCL12 concentration (Fig. 3b).

A further characteristic of classical chemokine receptors

is receptor internalization by endocytosis and so a desen-

sitization of the chemokine receptor to the ligand. To

investigate this issue, we first determined the subcellular

localization of the CRAM expression by immunofluores-

cence. Costaining with the cytosolic dye CellTracker and

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r

Rel

ativ

e ce

ll nu

mbe

r

Rel

ativ

e ce

ll nu

mbe

r

CCL3 CCL4

CRAM Colocalization

Time (seconds)90 120 0

0

0

20

40

60

80

100

120

140

160

0Incubation time (min)

Rel

ativ

e C

RA

M e

xpre

ssio

n (%

)

5 10 30 60 120

ng/mlChemokine

– 100CCL5 CXCL12200 500 10 50

–

Che

mot

axis

(%

of i

nput

)

10

20

30

30 60 90

CCL5CXCL12ControlControl

120

Figure 3. CRAM surface expression is increased after CCL5 stimulation in pre-B cells. (a) Nalm6 cells were stimulated with different CCL5

concentrations (ranging from 200 to 2000 ng/ml), all with similar results. For clarity reasons, only stimulation with 500 ng/ml CCL5 is shown.

Calcium transients were determined with flow cytometry using fluo-3 AM. For comparison, calcium transients after stimulation with 100 ng/ml

CXCL12 are shown. (b) Chemotaxis of Nalm6 cells towards different concentrations of CCL5 (100, 200 and 500 ng/ml) and CXCL12 (10 and

50 ng/ml). Results show the percentage of migrating cells and show the mean and range of double samples of one representative out of two inde-

pendent experiments. (c) Fixed and permeabilized cells were stained with 5 lg/ml anti-CRAM monoclonal antibody and anti-mouse Alexa488

secondary antibodies32 and additionally labelled with CellTracker�Orange (red) and DAPI (blue). (d) Time kinetics of the observed upregulation

of CRAM surface expression in Nalm6 cells after stimulation with 500 ng/ml CCL5 for the indicated times. Data show the mean and standard

error of three independent experiments. (e) CRAM surface expression after 10 min preincubation with 500 ng/ml CCL2, CCL3, CCL4 (black

lines) or medium alone (tinted light grey). (f) CRAM surface expression was cytometrically determined on the B-cell lines G2, Reh, Raji and

Nalm6 (for comparison) after 10 min preincubation with 500 and 800 ng/ml CCL5 (increasingly darker lines) or medium alone (tinted light

grey).



DAPI as a marker for the nucleus revealed a predominant

surface expression of CRAM without excluding minor

intracellular pools (Fig. 3c). For receptor internalization

experiments, we incubated Nalm6 cells with 500 or

800 ng/ml CCL5 and determined CRAM surface expres-

sion after 5–120 min of incubation (Fig. 3d). Treatment

with high levels of CCL5 did not change unspecific anti-

body binding to the cells, as determined for each concen-

tration using separate isotype controls (data not shown).

Intriguingly, upon CCL5 stimulation we did not find

desensitization of the receptor but instead there were sig-

nificant increases in surface expression of 62 ± 18%

(P = 0�004) when stimulated with 500 ng/ml CCL5 and

of 86 ± 11% (P = 0�023) when stimulated with 800 ng/ml

CCL5 (Fig. 3d, f). The increase in surface expression was

highest after 5 min of CCL5 incubation, was still robust

after 10 min and then decreased to normal levels after

about 60 min, which did not support the presence of

transcriptional modifications. CCL5 specificity of the

increase in surface expression was evaluated by incubating

the cells with CCL2, CCL3 or CCL4 instead of CCL5.

With all these CC chemokines, we performed the same

time kinetics as shown for CCL5. In none of the cases did

the incubation with these chemokines affect CRAM sur-

face expression (Fig. 3e). To test if the increase in surface

expression by CCL5 preincubation was specific for certain

stages of B-cell differentiation or a common B-cell phe-

nomenon, we used the pro-B-cell line Reh, a second pre-

B-cell line, G2, and the germinal centre B-cell line Raji.

CRAM expression was significantly increased after CCL5

preincubation by 32 ± 7% (P = 0�048, 800 ng/ml) in Reh

and by 45 ± 14% (P = 0�023, 800 ng/ml) in G2 cells but

not in Raji cells, indicating that this increase in surface

expression was relevant in the early stages of B-cell differ-

entiation (Fig. 3f).

CCL5 induces stress fibres and p44/42phosphorylation but does not induce chemotaxis inCRAM-A/B transfectants

In addition to all the experiments with B-cell lines, we

decided to use CRAM-A-transfected and CRAM-B-trans-

fected cells, which gave us certain advantages over the cell

lines that naturally express CRAM. First, by using trans-

fectants we were able to compare the functional responses

of transfected cell lines compared to the corresponding

parental cell line, which did not express CRAM. This

allowed us to separate the CCL5-induced responses that

were mediated by glycosaminoglycan binding without

CRAM involvement from those responses mediated by

CCL5 binding to CRAM. Second, using transfectants gave

us the possibility to differentiate between the two CRAM

splice variants, CRAM-A and CRAM-B. We therefore

cloned both the existing splice variants of CRAM that dif-

fer by 12 amino acids at the N-terminal ends. The longer

variant is named CRAM-A or CCRL2A (accession num-

ber AF015524), the shorter version CRAM-B, CCRL2B,

HCR or CKRX (accession numbers AF015525, U97123).

To further characterize both splice variants of the recep-

tor separately, we cloned CRAM-A and CRAM-B from

total RNA from the lymphocyte fraction of blood samples

including B cells, T cells and monocytes using RT-PCR.

Sequencing of the CLL-derived PCR products confirmed

that CRAM-B possessed 99�6% nucleotide sequence iden-

tity and 100% amino acid sequence identity to the pub-

lished sequence (accession number U97123). CRAM-A

showed 99�8% nucleotide sequence identity to the pub-

lished sequence (accession number AF015524) with one

difference in the amino acid sequence: at position 255 we

found an isoleucine instead of the proposed valine resi-

due. At this position, the shorter sequence with the acces-

sion number U97123 also proposes an isoleucine residue,

indicating a database error in the sequence AF015524,

which we reported to LocuslinkProteom. To exclude the

possibility that the observed amino acid sequence differ-

ence was a result of PCR errors, several different clones

were sequenced. All sequenced clones showed the isoleu-

cine residue. PCR products were cloned in pcDNA3.1/V5-

His TOPO TA or pcDEF3 plasmids and transfectants

were established. We confirmed CRAM-B and CRAM-A

expression in the transfectants by RT-PCR and flow

cytometry (Fig. 4). We did not observe CRAM-B or

CRAM-A cDNA in untransfected or mock-transfected

NIH 3T3 cells, whereas the cDNA was found in the trans-

fectants (Fig. 4a). We furthermore determined that the

antibody used specifically recognized the binding of

NIH 3T3Untransf.

(a)

(b)

Untransf.CRAM-A CRAM-BControl

CRAM-A

CRAM-A

GAPDH

CRAM-B

CRAM-BMock

100

Rel

ativ

e ce

ll nu

mbe

r

80

60

40

20

0

CRAM-PE100 101 102 103 104

GAPDH

Figure 4. CRAM-A/B expression in transfectants. (a) Reverse tran-

scription–polymerase chain reaction (RT-PCR) for CRAM-A and

CRAM-B expression. (b) CRAM surface expression in CRAM-

A-transfected cells (black line) and CRAM-B-transfected cells (light

grey line) compared to mock-transfected cells (‘control’, grey dotted

line) and isotype control (tinted histogram).



CRAM to transfected cells but not to mock-transfected

cells (Fig. 4b).

Both CCL2 and CCL5 have been suggested to be agon-

ists of the CRAM-A/B mouse homologue L-CCR.20 We

therefore tested both chemokines and evaluated the ability

of CCL2 and CCL5 to induce changes in the reorganiza-

tion of filamentous actin (F-actin) in CRAM-B- or

CRAM-A-transfected NIH 3T3 cells. Cells were serum

starved and stimulated with the potential agonist CCL5

for up to 30 min. Polymerized F-actin was visualized with

phalloidin fluorophores as previously described,21 and

stress fibres were detected by fluorescence microscopy.

The cells revealed a disassembled cytoskeleton when they

had been serum starved for 16 hr (Fig. 5a,g,j). CCL2 did

not reproducibly induce stress fibres in these trans-

fectants, whereas CCL5 clearly did. After CCL5 stimula-

tion, the fluorescence intensity rapidly increased in the

CRAM-B- or CRAM-A-transfected cells. Stress fibre for-

mation could already be observed 1 min after the stimu-

lation, was clearly seen after 5 min, and reached a

maximum at 15 min after stimulation (CRAM-B: Fig. 5b,

c, d; CRAM-A: Fig. 6h, i). Afterwards the fluorescence

intensity declined; and 30 min after stimulation only a

few stress fibres were visible (Fig. 5e). The presence of the

receptor was definitely necessary for the CCL5-mediated

reorganization of actin. Untransfected NIH 3T3 cells did

not react to stimulation with CCL5 with actin fibre for-

mation (Fig. 5k, l). We therefore attribute these changes

in actin reorganization to CCL5-mediated CRAM cycling

from and to the surface. As a positive control, Fig. 6(f)

shows prominent stress fibre formation in CRAM-B-

transfected NIH 3T3 cells when the cells were cultured

with 10% fetal bovine serum.

Changes in the cytoskeletal reorganization, like the for-

mation of actin stress fibres, can occur with p44/42

(ERK1/2) activation.22 We tested CCL5-induced p44/42

phosphorylation in CRAM-B transfectants and used

mock-transfected cells as a negative control. The p44/42

mitogen-activated protein kinase (MAPK) was strongly

activated in CRAM-B transfectants upon CCL5 stimula-

tion and slightly activated in mock-transfectants (Fig. 6a).

To determine if the CCL5-induced p44/42 phosphoryla-

tion was Gi protein dependent, we used the Gi inhibitor

pertussis toxin. The p44/42 phosphorylation in response

to CCL5 was not sensitive to pertussis toxin (Fig. 6b). We

also tested activation of p38 MAPK and Akt (protein

kinase B) upon stimulation in CRAM-B-transfected cells,

but could not show any activation of these kinases upon

stimulation with CCL5 (data not shown).

Discussion

The human orphan chemokine receptor CRAM, which is

encoded by the gene CCRL2, is still awaiting characteriza-

tion. In this study we show for the first time CRAM sur-

face expression on human B cells. CRAM expression is

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

Figure 5. Stress fibre formation in CRAM-B-

and CRAM-A-transfected NIH 3T3 cells in

response to CCL5. NIH 3T3 cells expressing

CRAM-B (a–f), or CRAM-A (g–i) or untrans-

fected cells (j–l) were serum starved overnight

(all except f), stimulated with CCL5, and the

stress fibres were stained with Alexa 488 phal-

loidin. (a–e) CRAM-B-transfected cells, serum

starved (a), and stimulated with CCL5 for

(b) 1 min, (c) 5 min, (d) 15 min, (e) for

30 min; (f) CRAM-B-transfected cells without

serum starvation; (g–i) CRAM-A-transfected

cells, serum-starved (g), and stimulated with

CCL5 for (h) 5 min, (i) 15 min; (j–l) untrans-

fected cells, serum-starved (j), and stimulated

with CCL5 for (k) 5 min, (l) 15 min. The

images shown are representative for the

whole population and the experiment was per-

formed twice (differences in size of cells are

the result of different magnifications).



dependent on the maturation stage of the B lymphocytes

and is upregulated upon stimulation with the inflamma-

tory chemokine CCL5.

As suggested by the database LocusLink Proteom, the

gene CCRL2 encodes a seven-transmembrane G-protein

coupled receptor that is most probably a chemokine

receptor. It exists in two variants, the variant CRAM-B

(also known as HCR, CKRX, CCRL2B) and a longer

splice variant CRAM-A (also known as CCRL2A), which

differs by 12 amino acids at the N-terminal end. The

CCRL2 gene is located in the main cluster of CC-chemo-

kine receptor genes together with CCR1 to CCR5, CCR8

to CCR10, XCR1 and CX3CR112, and it shows consider-

able sequence homology to the CC chemokine receptor

genes CCR1, CCR2, CCR3 and CCR5.11 The location and

the sequence similarity suggest that CRAM-A/B belongs

to the CC chemokine receptor class of G-protein coupled

receptors (GPCRs). Distribution of the longer splice vari-

ant CRAM-A had not been investigated previously and

only expression data on the distribution of the shorter

splice variant, CRAM-B, was available. Migeotte and col-

leagues13 found abundant CRAM-B transcripts in thymus,

spleen, lymph nodes and lung. Furthermore, surface

expression of CRAM was found on T cells, neutrophils,

monocytes, monocyte-derived dendritic cells and macro-

phages as well as on CD34+ cells but not on B cells. In

contrast to this previous assumption that B cells do not

express CRAM-A/B, we detected CRAM-B mRNA and

surface expression in several B-cell types. The previous

failure to detect CRAM on B cells is most probably

because its expression on the B cells strongly depends on

their maturation stage. We tested primary B cells of dif-

ferent maturation stages derived from human bone mar-

row aspirates and found that CRAM expression was high

on pro- and pre-B cells. During further differentiation the

expression disappeared in immature B cells and reap-

peared on mature B cells. In addition, we confirmed these

data using several B-cell lines that represent different

B-cell maturation stages. The B-cell lines Reh, Nalm6 and

G2, which represent pro-B-cell and pre-B-cell stages,

expressed CRAM strongly, whereas the IgM+ cell lines

Ramos and Daudi, which represent B cells from the ger-

minal centre, were negative. Importantly, the longer splice

variant CRAM-A was restricted to pre-B cells. We focused

on CRAM surface expression and function in the pre-B

acute lymphoblastic cell lines Nalm6 and G2 and found

that CCL5 induced p44/42 MAP kinase activation and

upregulation of CRAM surface expression in these cells.

CCL5 (MCP2, RANTES, ‘regulated on activation, normal

T cell expressed and secreted’) is a CC chemokine, which

has been shown to be a chemoattractant for peripheral

blood monocytes, dendritic cells, T cells, natural killer

cells, eosinophils and basophils. Receptors for CCL5 are

CCR1, CCR3 and CCR5,23 all of which show considerable

sequence similarity to CRAM-A/B. Furthermore, CCL5

can bind to CD44 and to glycosaminoglycans presented

on the surface of cells. Nalm6 cells expressed none of the

CCL5 binding chemokine receptors or CD44, as we con-

firmed by flow cytometry and RT-PCR. Therefore, the

observed CCL5-induced responses could not be attributed

to any known CCL5 receptor. Moreover, CRAM-A/B on

Nalm6 cells exhibited unusual properties compared to

typical chemokine receptors. We could not detect any cal-

cium response upon CCL5 stimulation or any migration

towards CCL5 in Nalm6 cells, a pre-B-acute lymphoblastic

leukaemia cell line that has high endogenous CRAM-A/B,

and did not observe CRAM-A/B internalization or down-

regulation after incubating the cells with CCL5 but on the

contrary reported an active upregulation in cell surface

expression.

To gain more insight into direct interactions between

CCL5 and CRAM, we decided to use CRAM-A-transfect-

ed and CRAM-B-transfected cells; this made it possible to

differentiate between the two CRAM splice variants,

CRAM-A and CRAM-B. CCL5 induced higher actin poly-

merization and p44/42 MAPK stimulation in both

CRAM-A- and CRAM-B-transfected cells than in mock

transfectants. Moreover, higher binding of labelled CCL5

to CRAM-transfected cells was detected compared to

mock-transfected cells but competitive binding is still

under assessment (data not shown). We could not inhibit

the functional responses upon CCL5 stimulation by per-

tussis toxin, suggesting that either this receptor is not cou-

pled to G proteins or is coupled to G proteins other than

Gi. For example, pertussis-toxin-insensitive heterotrimeric

CCL5MOCK

(a)

(b)

Phospho-p44/42

p44/42

Phospho-p44/42

Phospho-p44/42

p44/42

p44/42

Pertussis toxin –

0′ 10′ 0′ 10′

– + +

CCL5

CRAM-B

CRAM-B

0′ 2′ 5′ 10′ 15′ 20′

Figure 6. p44/42 mitogen-activated protein kinase activation by

CCL5 in CRAM-B transfected NIH 3T3 cells. (a) Cells were stimu-

lated with CCL5 for the indicated times and lysates were prepared.

Western blots were performed with antibodies against phospho-p44/

42 and total p44/42. (b) Western blots of transfected cells preincu-

bated with pertussis toxin and stimulated with CCL5. Data shown

are representative for five independent experiments.



G proteins include members of the G12 and Gq families.

Moreover, CRAM-A/B transfectants did not migrate

towards CCL5 in chemotaxis assays (data not shown).

Previously it was suggested that CRAM-A- or CRAM-

B-transfected HEK 293 cells move chemotactically

towards rheumatoid arthritis synovial fluid.24 We could

not reproduce these results using transfected HEK 293

cells with high CRAM-A or CRAM-B expression and

rheumatoid arthritis synovial fluid from several patients

as chemoattractant. Moreover, the unusual behaviour of

the receptor in the Nalm6 cells and the failure of pertussis

toxin to inhibit the responses in the transfectants indicate

that CRAM-A/B might rather modulate chemokine-

induced immune responses than acting as a migratory

receptor itself. Besides the classical chemokine receptors, a

group of chemokine-binding molecules with high struc-

tural similarity to chemokine receptors that are not cou-

pled to G proteins exists. Examples are the Duffy antigen

receptor for chemokines (DARC),6 D67,8 and CCX CKR.9

All members of this chemokine receptor subfamily are

unable to couple to major signalling pathways used by

typical chemokine receptors, or to mediate chemotaxis.

They all share alterations in the DRYLAR/IV motif in the

second intracellular loop, which is indispensable for G

protein coupling in conventional receptors,5,25 and are

reported to be regulators of chemokine networks by inter-

nalizing their ligands. Since CRAM-A/B displays a QRY

instead of a DRY motif and the CCL5-induced MAPK

activation was insensitive to pertussis toxin, we propose

that this receptor could be an atypical chemokine recep-

tor and that actin polymerization and p44/42 MAPK

activation upon CCL5 binding to the cells might be a

prominent sign for receptor cycling events upon chemo-

kine binding rather than the sign of the typical activa-

tion of a chemokine receptor. Recycling of ligands occurs

through actin filaments and is regulated by actin-related

kinases as shown for example for the chemokine receptors

CXCR1 and CXCR2.22 The activation of p44/42 can also

be related to b-arrestin-dependent chemokine receptor

recycling. Interestingly, PAR2, a class A GPCR which also

has the QRY modification, uses a b-arrestin-dependent

mechanism to sequester activated p44/42 in the pseudo-

podia.26 Moreover, PAR2 is shown to activate p44/42

through a pertussis-toxin-insensitive pathway, which is

similar to our findings for CRAM-A and CRAM-B.

The function of atypical chemokine receptors in general

might be to regulate chemokine networks and so a possi-

ble CRAM function could be hypothesized in regulating

chemokine networks in connection to CCL5. CCL5 is a

prototypic inflammatory chemokine and plays a role in

the functional relationship between inflammation and

haematological malignancies.27,28 A number of cancer

types, e.g. melanoma, prostate cancer and ovarian cancer,

have been shown to express CCL5,29 and to enhance

tumour growth of melanoma cells.30 Furthermore, CCL5

induces the migration of leucocytes into the tumour

site.31 Moreover, expression of CRAM receptors might

lead to differential regulation of other chemokines or

chemokine receptors. CCL5 is already known to induce a

heterologous desensitization of the CXCR4 on progenitor

B cells in the bone marrow,18 thereby possibly influencing

the retention of B-cell precursors in the bone marrow.

The functional characterization of the role of the high

CRAM expression on early B-cell stages needs to be

addressed in future studies.

In summary, we identified CRAM as a novel chemo-

kine receptor that is presented on the surface of B cells

dependent on their maturation stage. Surface presentation

of the receptor was modulated by CCL5 in pre-B cells.

Our results suggest a role of CRAM in modulating

chemokine-triggered immune responses rather than in

inducing direct migratory responses.

Acknowledgements

We are deeply grateful to Knut Biber for fruitful discus-

sion, and for provision of antibodies and DNA constructs

and to M. Freedman for providing the G2 cell line.

This study was supported by by the Deutsche Fors-

chungsgemeinschaft grant no. BU/1159/3-2 (to M.B.).

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Documents

Human B cells express the orphan chemokine receptor CRAM-A/B in a maturation-stage-dependent and CCL5-modulated manner