Monocyte - CiteSeerX

Monocyte

chemotactic

and signaling

protein-4:

through

CC chemokine

Ronald Godiska, David Chantry, and Patrick W. Gray Icos Corporation, Bothell, Washington

Abstract:

Chemokines

molecular-weight ety ofcell types,

constitute

proteins including

and fibroblasts. An Expressed Sequence

electronic Tags

tial eDNA sequence monocyte chemotactic the full-length clone mokine

MCP.4,

described

search database

Carol

family

of

or activate endothelial of the uncovered

an eosinopbil

GenBank a par-

chemoauractant

Exp

et al.

matography. Sequencing protein corroborated the binant MCP-4 produced calcium flux protein-coupled nizes MCP-1

lowa vanceII,

recently

Med.

183,

23 79-

Recombinant MCP-.4 was expressed cells and purified by hepanin-Sepharose

malian

J. Raport,

with homology to the chemokine protein-i (MCP-1). Isolation of revealed that it encodes the che-

by Ugucciom

23841.

a

that attract leukocytes,

the

amino

reported

in insect

in mamchro-

terminus of this sequence of recomcells. As shown by

assays, MCP-4 activated the cloned G receptor CCR-2, which also recogand MCP-3. Northern hybridization in-

dicated that MCP-4 is constitutively expressed at high levels in the small intestine, colon, and lung. This expression profile is consistent with its role as a chemo-. attractant for eosinophils, which can be rapidly mobilized to the lung or intestine in response to invading

pathogens. not

stimuli

such

a.

factor Key tion

In marked in cell lines

induced

.

contrast treated

to MCP-1, MCP-4 with pro-inflammatory

as lipopolysacchanide Leukoc.

J.

Words: chemotaxts eosinophil

cytokine

.

or tumor 61:

Biol.

was

1997.

inflammation

.

trarumigra-

a variety of agents, lipid metabolites,

into

inflammatory

sites

is mediated

including bacterial peptides, and chemotactic cytokines,

Chemokines with potent

Chemokines

MCP-3 tinct These

CC chemokines

are

localized

and

generally activate monoand eosinophils. proteins MCP-1, MCP-2. and

are 59-72% identical, representing part of a dissubgroup within the CC chemokine family (4, 5J. MCPs are produced by numerous cell types, includ-

ing fibroblasts, endothelial cells, and kocytes, in response to pro-inflammatory cytokines, MCPs are benefit

lipopolysaccharide, also synthesized

through

or by tumor

MCP-induced

mononuclear stimuli

infectious agents. cells, which may

angiogenesis

and

leuas

such

The either

metastasis

or be destroyed by the attracted monocytes 151. By virtue of their ability to attract monocytes, the MCPs play an important role in host defense; however, they may also mediate pathological inflammatory responses mulation of macrophages at atherosclerotic In addition to monocyte recruitment, the basophils

91

[8,

transendothelial Although tional

the

considerably to the fact

and

appear

an

important

their

efficacy

on

various

cell

in

1l. and func-

types

varies

due in part a particular

MCP-1 interacts with CCR-2 [14, MCP-3 interacts with CCR-1 114, 17,

18J, CCR-2

[12, 171, and

not interact Furthermore,

with

CCR-3

[19,

any of the known MCP-1 production

ofMCP-2

role

l0,

[12, 131. This disparity may be that each of the MCPs interacts with of receptors: CCR-4 [16J;

that

to play

such as acculesions 16, 71. MCPs activate

migration of T lymphocytes MCPs display much structural

similarity,

and

MCP-3

20J;

and

MCP-2

does

CC chemokine receptors. is about 10-fold higher

in most

cellular

systems

ana-

are

also

phosphotermed

are a family of structurally releukocyte activating properties

70-90

acids

in length

Abbreviations: mokine and

reaction; serum;

MIP,

ovary; DMEM, LPS,

tumor

Received

Journal

protein; activation

sequence modified

necrosis

factor

CCR, T

protein; tags;

Eagle’s

PCR,

CHO,

che-

Chinese

polymerase

medium;

a; PBS,

CC

cell-expressed

FCS,

chain fetal

calf

phosphate-buffered

sa-

lipopolysaccharide.

Avenue

cember

on

inflammatory

expressed

Dulbeccos

Correspondence: 20th

chemotactic

regulated

macrophage

EST,

TNF-a,

line;

monocyte

RANTES,

secreted;

generally can be subdivided into two groups based on the position of two of four conserved cysteine residues: CXC chemokines contain a single amino acid between the first whereas CC chemokines The CXC chemokines are 4 and primarily activate

MCP,

receptor;

hamster

cysteine residues, cysteine residues. human chromosome

amino

properties.

by

and

and second have adjacent encoded on

most

to human chromosome 17 cytes, lymphocytes, basophils, The monocyte chemotactic

tional

11-31.

In contrast,

Hai Le Trong,

lyzed 1211. Serum levels of the MCPs likewise vary independently of each other during sepsis 1221. Thus, despite their similarity, the MCPs have distinctly different func-

infiltration

chemokines. lated peptides

L. Schweickart,

neutrophils.

than

INTRODUCTION Leukocyte

expression

receptor-2

Vicki

repertoire 151 and

necrosis

353-360;

.

tissue-specific

SE, June

17,

Dr.

Patrick

W.

Bothell,

WA

98021.

3,

1996;

revised

Gray,

ICOS

December

Corporation,

16,

1996;

22021 accepted

De-

1996.

of

Leukocyte

Biology

Volume

61,

March

1997

353

Two recent additions kines are eotaxin 123,

56-61%

identical

motactic 3, the

to MCP-1,

for eosinophils, predominant

20J. Uguccioni cium

-2,

acting eosinophil

and

and

chemotaxis

-3.

that

of

also utilize CCR-3. and T lymphocytes

MCP-4

In addition, [251,

of functional hamster ovary

recombinant (CHO) cells.

MCP-4

CCR119,

induces

1251

cal-

and

MCP-4 suggesting

it

in that

interacts specifically In addition, the expression

with the MCP receptor CCR-2. pattern of MCP-4 is shown to

be

of MCP-1.

quite

distinct

from

that

Products,

and

Peripheral

a St.

and

for

Northern

amplification

Cloning

of the MCP-4

primers

coding

BLAST

region

service

similar

The

the

EST

“NCBI_ID

(PCR)

electronic

1271

EST

Tags

entries

were

program

are

tamed

sis and Nuclear, tiple

labeled Boston,

Tissue

washed

was

cDNA

complementary

was

obtained

selected

amplified

library

C-terminus additional

reaction ing

to

no termination

The

bases

was

tension

derived

The

gel cell

from

human

for

30

chemicals,

15

labeled

fragment

Test,

Inc.

cDNA

library

from

hybridizing

from

Promega,

Madison,

(Applied

Biosystems,

containing

the

coded Uguccioni

which

clone

et

was

at high

and

coding

sequence

is identical

cycles exBio-

isolated

were umns

was

automated CA).

An

recovered.

sequence

the

The

ing

70

sequencer 860-bp

of MCP-4

clone

protein

en-

described

by

Type

Culture

becco’s

cell

Collection

modified with

were

(Rockville,

Eagle’s

supplemented

lines

MD).

medium

10%

fetal

obtained Cells

(DMEM, calf

The

(I-HUVEC)

serum

(FCS;

and G418 St.

354

was

cultured

(Life Louis,

human

obtained in

from

RPMI

Technologies, MO),

Journal

and

of

sg/mL

Leukocyte

Jay

supplemented Grand

30

umbilical Dr.

Island, endothelial

Biology

the cultured

with NY), cell

Volume

in

25

penicillin,

vein

Nelson,

American

Gaithersburg, GIBCO),

acid,

immortalized was

were

GIBCO,

hydroxyethylpiperazine-N’-2-ethanesulfonic

tomycin.

from

mM

endothelial

10% 1 U/mL growth

61,

FCS, factor

March

probed

and rec-

1-4

days

at

lines

was

using to the

entire

digestion.

RNA

was

RNA

isolated

STAT-60

manufacturer’s

on

0.8%

and

(Telinstruc-

agarose

hybridized,

formalde-

washed

Autoradiograph

or 5 days

the

under

exposures

(MCP-4)

at

were

-80#{176}C with

inten-

of recombinant

MCP-4

9-383

ofthe

and

primers

was

were

cloned

the

the

the

dihydrofolate

For

electroporation,

were

pH

of

pH 8.

and

methotrexate to pH

CHO

For

sodium

columns

protein glycine

gel

apparent

Dul-

were

washed

kine

was

maining

molecular on

an

mass

automated

larger-scale with

0.35

eluted

with

on the

column

of 6.8

was

in 20

M NaCl eluted

contain-

in with

surviving CL-6B

mM 1.5

col-

purification

0.2

M

M NaC1 was

NaCl

CA)

from in

in 20

20

mM

mM

Tris,

fractionated

by

through

and

an

transferred

to

MCP-4

band

migrating

was

excised

for

N-terminal

(Applied

20

this

supernatants

The

kDa

from

M NaCl 0.7

hundred

electrophoresis

MA).

sequencer

purification

several

MCP-4

Diego,

no. UT)

medium

Culture

0.6

gel

San

(cat. Logan,

Colonies

with

with

eluted

Bedford,

DG44

a

small-scale

washed

the

medium

Heparin-Sepharose

For

was eluted

(NOVEX,

(Millipore.

MO).

onto

sequencing,

in

line

from in

expanded.

sulfate-polyacrylamide

membrane

For

and

were

amplification

(Hyclone,

replated

NJ).

of gene

of the

a-

in

FCS

Louis,

loaded

the chemokine

dodecyl

Tris

St.

for

cells

Cells

and

isolated and

growth

thymidine.

Piscataway,

medium,

8, and

by

a derivative

in 1 mL PBS, mixed with 25 to a 0.4-cm cuvette. The susGene Pulser at 290 volts, 960

dialyzed

(Sigma, 6.8

pDC1, gene

10

pooled

were

(Pharmacia, mL

which

sequence.

phosphotransferase

reductase

10%

were

of selection

Tris,

clone.

5’-AATCGGATCCGG-

vector

neomycin

selected

containing

hypoxanthine

brought

cDNA

of 3’ non-coding

as follows:

into

in which

with

nM

MCP-4

11 bp

300

mL

Tris, mM

Biosystems, pH

M NaCl

model

of medium,

Tris,

columns

8, and

the

pH

8.

Material

mM

Tris,

in 20

a

chemorepH

8.

strepcell

400

England Mul-

and 5’-CCATGAA’VFCGGTAGCAGAGVFCAA-

colonies

MD)

line

of Oregon,

heparin

5’-CAG-

by restriction above.

1291.

bases

PCR

fragment

GIBCO)

473A).

N-2-

and

University

cell

described

fractionated

of 5’ non-coding

cells.

20

at an IMR-90

were for

macrophages

as described

(InVitrogen)

sequencing and

of

according

p.tg) was

to amplify

lacking

18%

Cell culture A549

the

manufacturer’s

exposed

vector

to nitrocellulose,

Transformants

PVDF

The

TX)

ofthe

transformed

System,

an

as

(20

bp

replaced

and

electro-

was

on

used

12000,

round

City,

labeled cultured

and purification

67

The

tF.

macrophage

DNA

CA)

to the

the

washed in PBS, resuspended Mg of linearized plasmid, and transferred pension was electroporated with a BioRad

to the Purification

and from

8 h (MCP-1),

sequences

was

stringency

Foster

to the

follow-

by gel

was

pRc/CMV

1301,

sequenced 373,

the

Mannheim

DNA

by

using

by gel electrophore-

Alto,

were

1321 were

PCR

s at 60#{176}C, and

Plasmid

generated

clone

screens.

GIG.

protocols

Miniprep

Palo

from

conditions

mammalian

product.

by 30

isolated

was

cDNA

and

hybridization

CGGAACAGCCAGAGGAG

standard

amplified.

model

15

con-

The

using

tissue

MCP-4

according

excised

transferred

Expression

The

to append PCR

and

RNA

gels,

includes

1251.

al.

Total

hyde

sifying

reaction

type.

(Boehnnger

Following

it was

the bold

1291, for

product

(Wizard

WI)

in

ad-

macro-

based

fragment

at 94#{176}C,followed

priming

hybridized

entire

by this

PCR

random

colonies

designed

previously

mm

for 4

IN).

EST

was

into

screens.

Friendswood,

,

the

respectively, and 5’-TATUnderlined por-

the

onto

are

s at 94#{176}C, annealing by

was

Stop)

region

as described

Indianapolis,

labeled

Thr

this

s at 72#{176}C.The

and

Lys

primer

gra-

plastic

and f32PJTTP (DuPont-New priming (Boehringer Mannheim).

(Clontech,

Northern

purified

3 h (GAPDH),

macrophages

primers.

by

differentiation

was purified

autoradiographs

sequence

lines

fragment

conditions

intensifying

probe

from

blots

stringent

80#{176}Cwith

stringent

View,

study chain

6 h.

were

Mountain

for further

Because

reverse

(...Pro

incubation for

for

phoresis

and

of the

[32PJCTP by random

Northern

nu-

through

by polymerase

EST.

the

encoding

performed times:

of denaturation

the

codon,

of MCP-l

reaction

The

isolated

of whole

7-619 The

with MA)

under

PCR

fragment

a plasmid

the

database.

(Intelligenetics,

118741”

to the

to identify

([email protected])

I28J. The sequences of the forward and reverse were as follows: 5’-TATAAGClTVitAACATGAAAGTCTC TCTAGA1A’R’It’ITDGTGTGAAC1TI’CCGGCCC. tions

mail

MCP-1.

EST

from

these

GeneWorks to

above

via

Sequence

of GenBank

with

its similarity The

Expressed

of

sent

([email protected])

the

service

analyzed CA).

in

sequences

RETRIEVE

1261 was

MCP-l

of GenBank

sequences

cleotide

on

of

tumor for

histopaque

5’-AATCGGATCCGGCGGAACAGCCAGAGGAG

tions. The

70-80%

MN) on

were allow

hybridization

CAACCTACTTGCTCAAG.

It was

cDNA

purified

Monocytes to

to

Minneapolis,

were

6 days

of bases

ommendations.

METHODS

for

grown

of 10 ng/mL

blotting

probe

PCR

The

AND

cells MO).

cultured

MCP-4 cDNA

MATERIALS

R & D Systems,

Louis,

were

or absence

[31J.

The

-

Cells

presence

mononuclear

(Sigma,

herence

MA).

in the

(TNF-a;

blood

dients

Bedford,

cultured

factor

Northern

study we describe cDNA and char-

MCP-4 produced We demonstrate

Biomedical

confluency

phages

may

activates that

tive

necrosis

is che-

receptor receptor

eosinophils

binds to an additional receptor. In this the independent isolation of the MCP-4 acterization Chinese

Eotaxin

through the chemokine

et al. demonstrated

flux

therefore monocytes

to the MCP subgroup of chemo241 and MCP-4 1251, which are

tg/mL (Sigma,

(Collabora-

1997

Transmigration Transmigration scribed

T1B202).

assay was

by Casale Transwells

studied

et al. [33. were

by

use

THP-1 purchased

of a transwell cells from

were Costar

assay obtained (8-tm

system,

as

de-

from

ATCC

(no.

pore,

PVP

free.

Cambridge,

MA).

England added be

to the

tested

mm

at

upper

mM

fallen

Fullerton,

CA).

in

0.5

to the

mL

lower

adhering

the

in

labeled

to

with

0.5

the

lower

side

The

the

of

the

counter

to

of 60-90 were

washed

to the of these

(model

and

chemokine

filter

added

radioactivity

scintillation

medium

incubation

and

chamber.

a Beckman

plus

RESULTS

(DuPont-New

RPM!

After

lower

5tCr

mL

of RPM!

chamber.

ethylenediaminetetraacetate

into

measured

cells

resuspended

chamber;

added

37#{176}C, cells

0.5

106

were

were

off with had

Briefly,

Nuclear)

cells

that

cells

was

Gamma

55008,

Calcium flux assay Changes

in

CCR-2 in

intracellular

cell

HEK-293

115.

1 mL

(AM;

line

34J.

Cells

complete

cells/mL.

tinuously

2,

cated

and

resuspended milliliters cuvette

NY).

while

nm

every

from

the

were

added

MCP-i

0.5

relative to

and

was

mm

at room

were

fluorimeter

monitored

calcium

at a 510-nm

expressed

as

380-nm

excitation

concentration

from

the

emission

ratio

of the

nM

at the

Peprotech

mdiwave-

and

380

emissions

Chemokines indicated

(Rocky

times. Hill,

NJ).

of the

Protein

analysis

comparisons

GeneWorks

program

dendrogram

analysis

(Intelligenetics,

were

Mountain

View,

performed

with

the

amino

Tags

database

been Pairwise each ofthe identical identical

Figure

The

The

.

to that

encoded

four

and eotaxin chemokines

characteristic

other

amino

fragment

residues

These include protein, threonine

which

MCP-4 protein that it is 55-61%

residues

among

tyrosine at position at 32, phenylalanine

1.

of 23 ture

cDNA

and

predicted

The

leader

sequence

amino protein

of the the

MCP-4 sequence.

is in italics;

is in bold

amino

first

GenBank

acids acids

residue accession

type.

(see of the no.

text)

the

ma-

30% inthe 28 of at 37,

60

C

L

L

L

M

TA

T

C

180 C

240

SLQRLKSTVI

TT 300

AGCAGGTGTCCCCAGAAGGCTGTCATCTTCAGAACCAAACTGGGCAAGGAGATCTGTGCT SRC PQKAVI FRTKLGKE Fig.

to

in addition

TTCACATTTAGCAGTGAAGATCTCCTTGCAGAGGCTGAAGAGCTATGTGATCACCACC

protein

98

1251.

conserved

GCTTTCAACCCCCAGGGACTTGCTCAGCCAGATGCACTCAACGTCCCATCTACTTGCTGC AFNPQGLAQ PDALNVPS

I

of recently

120

CTCTTAACCTTCAACATGAAAGTCTCTGCAGTGCTTCTGTGCCTGCTGCTCATGACAGCA MK V SA VL L

SKK

was

same macrocDNA clone

and approximately (Fig. 2). Identities

cysteine

acid

were used fragment

AAAAGGCCGGCGGAACAGCCAGAGGAGCAGAGAGGCAAAGAAACATTGTGAAATCTCCAA

FTFS

with

protein

of MCP-4,

described by Uguccioni et al. comparisons of the predicted known CC chemokines indicate

CC chemokines. mature

library.

clones from the of a full-length 1

is identical

to the MCPs to the other the

from this EST corresponding

cDNA

hybridizing The sequence in

acids

has

the

CA).

Sequence

macrophage

to isolate library.

to several

and

Expressed

of recombinant

oligonucleotides derived reaction to amplify the

a human

used phage

dude

Computer

characterization

the BLAST sequence comparison program 1271 was used to identify sequences in the database that have homology to MCP-1. The EST sequence NCBLJD no. 118741 represented a previously undescribed sequence that was highly homologous but not identical to MCP-1, -2, -3, and eotaxin.

is presented

was

of 340

wavelengths.

of 25

purchased

at

in a con-

(AMINCO-Bowman

wavelengths

are

(PBS)

placed

of intracellular

A search

from

temperature. saline

cells

excitation

data

were

Fura-2/acetoxymethylester

30

and

Synthetic in a PCR

gene

incubated

for

a

level

to the

a final

MCP-3

37#{176}C in

The

between

s. The

340-

(GIBCO),

1 tM OR)

in an

receptor

in phosphate-buffered

which

switching

monitored

chemokine

versene

of suspended at

Rochester,

were

the

with

containing Eugene.

by fluorescence.

length

with

harvested

Two stirred

Series

were

Probes,

once,

106

concentrations

transfected

media

Molecular

washed

calcium

stably

Cloning MCP-4

GACCCAAAGGAGAAGTGGGTCCAGAATTATATGAAACACCTGGGCCGCAAAGCTCACACC P K E KWV Q N Y N K H

D

I

CA 360

L

G

R

KA

H

T

Numbering begins

mature

with

protein.

CTGAAGACTTGAACTCTGCTACCCCTACTGAAATCAAGCTGGAGTACGTGAAATGACTTT LKT *

420

TCCATTCTCCTCTGGCCTCCTCTTCTATGCTTTGGAATACTTCTACCATAATTTTCAAAT

480

AGGATGCATTCGGTTTTGTGATTCAAAATGTACTATGTGTTAAGTAATATTGGCTATTAT

540

TTGACTTGTTGCTGGTTTGGAGTTTATTTGAGTATTGCTGATCTTTTCTAAAGCAAGGCC

600

TTGAGCAAGTAGGTTGCTGTCTCTAAGCCCCCTTCCCTTCCACTATGAGCTGCTGGCAGT

660

GGGTTTGTATTCGGTTCCCAGGGGTTGAGAGCATGCCTGTGGGAGTCATGGACATGAAGG

720

GATGCTGCAATGTAGGAAGGAGAGCTCTTTGTGAATGTGAGCTGTTGCTAAATATGTTAT

780

TGTGGAAAGATGAATGCAATAGTAGGACTGCTGACATTTTGCAGAAAATACATTTTATTT

840

AAAATCTCCAAAAAAAAAAA

860

U59808.

Go&ska

et at.

Monocyte

chemotactic

protein.4

355

(100%) (55%) (58%)

MCP-4 MCP-2

MCP-3

Fig. the

2.

Dendrogram

analysis

CC chemokine

is indicated

family.

illustrating

Percent

the

identity

MCP-1

(6 1%)

Eotaxin

(56%)

1-309

(33%)

MIP-la

(36%)

M1P-1

(37%)

RANTES

(27%)

HCC-1

(37%)

sub-group

of each

of MCPs

chemokine

is similar pected

cine

a tryptophan-valine

pair

cessing

within

and

MCP-4

was

in mammalian

cells

by stable transfection with an expression plasmid ing the MCP-4 cDNA. It was partially purified from ture medium by heparin-Sepharose chromatography

containthe culand

fractionated representing

by polyacrylamide approximately

small-scale

preparations

produced

gel electrophoresis. 20% of the eluted

MCP-1

1141,

although their concentrations,

was

and absent from untransfected grated at a molecular mass

MCP-1

in this

gel

not

purity (Fig.

was obtained by increasing the 3, right). Automated sequencing

cleavage of a 23-residue sor protein. Cleavage

band (data

leader at this

-3, -1 model

shown).

Material

yielded the not shown),

sequence from site is consistent

of signal

sequence

the

transfected

into

HEK-293

[141.

with

response a series the The

the

receptor

CCR-2

not equivalent. At idenof CCR-2 with MCP-1 to subsequent with MCP-3

to MCP-1

(Fig.

of calcium

treatment only par-

[14J.

4)

mobilization

response of CCR-2 CCR-2 receptor cDNA

We

experto MCP-4, was stably

the human embryonic kidney cell line Recombinant MCP-4 derived from CHO stimulated a calcium flux measurable by spec-

trofluorimetry

(Fig. pletely Similar

(Fig.

4).

Furthermore,

4). However, blocked results

pretreatment subsequent were obtained

eral blood mononuclear tions, or the monocytic

amino tersuggesting

cleavage

interact

by the parthe pro-

using

an

equal

con-

centration of each chemokine (25 nM), exposure of CCR-2 transfectants to MCP-4 partially decreased the calcium flux induced by subsequent addition of either MCP-1 or MCP-3

of sig-

the

[25J.

potencies are pretreatment

to characterize and MCP-3.

transfectants

transfectants

(data

gel-fractionated MCP-4 sequence XPXALNVP

Heijne’s

to the

MCP-3

undertook

MCP-1,

controls (Fig. 3, left). It miof 6.8 kDa, similar to that of

system

nificantly greater scale of production of the minal

unique

and

diminishes

therefore

A band protein in

to pyroglutamic

was suggested and corroborates

cells

A ex-

of MCP-4

iments

at 61. Recombinant

MCP-3.

is generally

conversion

completely inhibits the response with MCP-3, whereas pretreatment

leu-

and

terminus

of MCP-4 signal

in insect

Both tical

of MCP-1

amino

spontaneous

observed

Activity

to MCP-4

at 53-54,

sites

at an

to undergo

tially at 57,

processing

residue

acid. Such modification ticularly weak sequencing

in parentheses.

alanine

to the

glutamine

precurwith von

shown). Culture transfected CHO fected HEK-293

1351

(data

and

not

with

MCP-1

or MCP-3

com-

activation of CCR-2 by MCP-4. with freshly isolated periph-

cells, tumor

monocyte cell line

enriched THP-1

popula(data not

supernatants similarly purified from uncells produced no signal, and untranscells did not respond to MCP-1 or MCP-4

shown).

43 29 I 8.8 16.5

6.4 3.4 2.3

Fig.

3.

Production

cultures gel NaCl

analysis

purification

of MCP-4

from

cells

and

expressing

of a larger-scale

elution),

molecular

356

and

of untransfected and weight

Journal

D (1.5 markers

of

Leukocyte

(CHO) purification.

M NaCI

cells Gel

elution).

is indicated

Biology

CHO

lanes

cells.

contain

MCP-4

Left

MCP-4

eluted

unfractionated in fraction

(kDa).

Volume

61,

March

panel

(MCP-4).

1997

shows

material

Arrow

indicates

culture C.

Fraction

supernatant B (0.35

purified

on

position

(Supe), M NaC1

heparin-Sepharose

of MCP-4. and

elution)

Right fractions is not

CL-6B panel

from

A (Flow-through), shown

small-scale

is a chromatogram on

this

gel.

and C (0.7

Migration

M of

/I .

4

MCP-4

MCP-1

4

( MCP-4

4

MCP-3

4

MCP-1

4

MCP-3 -r--

4

MCP-3

____

4

MCP-3

MCP-1

150

100

50

4.

Calcium

flux

chemokines

were

determined

by

It has

analyzing

the

assays

were with

both cells

been

with MCP-4

fected

by MCP-1,

The

also

interacts whether

induced

added.

the

final

and

reported

of the

that

-4 in HEK-293 ofeach

performed

on

CCR-1

cDNA.

intracellular

MCP-3,

MCP-4

HEK-293

but

induced

not

calcium

Fura-2

MCP-1,

stably

CCR-2

(220

(see

cDNA.

ng/mL).

Materials

tants

and

Arrows

Each and

sponse

nM,

trace

depict

time

represents

points

the

level

at which

the

indicated

of intracellular

calcium.

Methods).

in THP-1

to MCP-4,

relatively maximal

in these

in CCR-2

with

nM

cells,

its

predicted

ability

to induce

chemotaxis was tested in a transwell migration assay. 1 cells showed a distinct dose-dependent chemotactic

trans-

of 25

a response

flux

25

To determine calcium flux

cells

to elicit

transfected was

dye

At a concentration

failed

cells

chemokine

the receptor CCR-1 1181. interacts with this receptor,

MCP-4 and MCP-1 (data not shown).

Because

-3,

concentration

fluorescence

1 50

Tine(sec)

Tne(sec) Fig.

4

‘

--

100

50

MCP-4

transfec-

but

the

response

was

high concentrations of stimulation of chemotaxis

tion

of MCP-4

that

of

approximately

MCP-1.

Furthermore,

THPre-

observed

only

at

MCP-4 (Fig. 5). Halfrequired a concentra-

10-

to 20-fold

at

concentrations

higher

than

up

to

‘U

cn + +

b

b

0

E

0

E ‘a U

.0001

.001

.01

.1

1

Chemokine

Fig.

5.

The

lower

Chemotaxis chamber

available

MCP-1

the

chamber

lower

of THP-1 contained induced

cells

induced

various Note

by

response the

100

1000

.0001

10000

.001

.01

(nglml)

concentrations

a chemotactic

is plotted.

10

added

difference

.1

hemokine

MCP-1

and

MCP-4.

of recombinant similar in scale

to the

Cells MCP-4

CHO-derived

(y-axis)

between

labeled

with

(left)

or

MCP-1 the

two

5tCr

were

MCP-1 (data

not

to the

derived

shown).

10

added

added

(right)

1

The

from

100

1000

10000

(nglml)

upper CHO

radioactivity

chamber

of each

transfectants. of cells

transwell.

Commercially that

migrated

into

panels.

Godiska

et at.

Monocyte

chemotactic

protein-4

357

1

_G.

..

Fig.

6.

sues. was

Expression

A probe hybridized

to

-80#{176}C with molecular

ig/mL

(115 nM) the 3.1-fold above

4 was imal

increase

(6 nM). gration

highest chemotactic response background, compared with

of 7.1-fold

induced

potencies

were

Similar

of monocytes

and

Expression

of MCP-4

To determine of the cDNA

the was

and

lung

(Fig.

described

lymphocytes

pattern and

at 50 in the

ng/mL transmi-

and

Overexposure

cell

of MCP-4, hybridized

of the

film

(data

Northern

and

tiscDNA

for

1 day

migration

at

of RNA

(kb).

thymus,

No

human MCP-4

exposed The

is indicated

placenta,

normal

of the

screens.

standards

shown).

in

7-619

blots

low expression prostate, testis, not

mRNA

bases

intensifying

in the heart,

dition to very ney, pancreas, cytes

two

mass

or spleen. MCP-4 mRNA

lines

thelial

a fragment to a North-

from various normal human tissues. Sigwas observed in small intestine, colon, 6).

expression

MCP-4

and

uterus

in ad-

in liver, skeletal and peripheral

muscle, kidblood leuko-

expression

apparent

was

in

brain

[25J.

in tissues

expression radiolabeled

em blot of RNA nificant expression

by MCP-1

to MCPthe max-

of containing

revealed

lower

(A549), VEC) origin, cells following

could

trast, MCP-1 expression by TNF-a. Low levels phages

and

not

fibroblast nor could stimulation

be detected

in cell

lines

of epi-

(IMR-9O), or endothelial (HUexpression be induced in these with TNF-a (Fig. 7). In conwas readily induced in these cells of MCP-4 were detected in macro-

peripheral

blood

mononuclear

cells

(Fig.

7).

‘F 28S

18s

MCP-4

Fig. 7. Comparison human cell lines and 20

tg

ment

of total with

derived

from

plastic

GAPDH

358

Journal

of

Leukocyte

Biology

Volume

61,

March

1997

from

human

RNA

bands

exposed

for

(GAPDH).

MCP-1

RNA ng/mL

for 6 days.

somal were

10

of MCP-4 macrophages. cell

TNFa

and lines

for

peripheral The

positions are

5 days

indicated. (MCP-4),

MCP-1 expression Each lane contained with

6 h.

or without

of the The

treat-

Macrophages

monocytes 285

were

by plating and

in

185

on ribo-

autoradiographs

8 h (MCP-1),

or

3 h

DISCUSSION

to activate ling

The

human

(MCP-1,

MCP

group

contains

at least

five

-2, -3, -4, and

eotaxin) that share 59-72% identity, forming a discrete branch within the chemokine family. The MCPs share some receptors and activities, but they exhibit -4 were

distinct differences each able to elicit

transfected

with

ing that

they

the

in their a calcium

human

all interact

MCP

with

this

and MCP-3 induced greater cal concentration of MCP-4. desensitized

the

effects. MCP-1, -3, and flux in HEK-293 cells receptor

CCR-2,

receptor.

However,

calcium fluxes Furthermore,

receptor

to

indicatMCP-1

than an identithey completely

subsequent

treatment

with

MCP-4, whereas MCP-4 only partially desensitized the receptor to MCP-1 or MCP-3 (Fig. 4). Nearly identical results were obtained using the monocytic cell line THP-1 (data not shown) and freshly isolated human monocytes [25J. It is therefore possible that cytes to MCP-1, MCP-3,

the asymmetric and MCP-4

entirely

to their

metric affinities

desensitization of the MCPs

may be or different

desensitizing

events

stream

interaction

with

phenomenon

response of monomay be attributable

CCR-2.

The

due to abilities

in CCR-2

apparent

different binding to induce down(e.g.,

of asymmetric

asym-

phosphoryla-

tion).

The

desensitization

has

been kines results

reported for the interaction of several other chemowith their receptors [13, 14, 361. The calcium flux were also consistent with the chemotactic response

of THP-1 cells to MCP-1 and MCP-4. The relative efficacies of the molecules varied, with MCP-1 producing a much greater effect than MCP-4 over the range of concentrations studied (Fig. In contrast tants

MCP-4

5). to the

results

observed

with

(Fig. 4), eosinophils respond much than to MCP-3 [25J. This disparity

predominant MCP-4 receptor A likely alternate candidate

CCR-2

more strongly suggests that

on eosinophils is CCR-3, which

expressed

on eosinophils

and

[19, 20]. MCP-4

binds

has

pattern

transfecto the

is not CCR-2. is abundantly

to MCP-3

and

eotaxin

and

tissue-

of induction

expression ofmost by pro-inflammatory

CC chemomediators

such as lipopolysaccharide pression was not similarly

(LPS) and TNF-a, induced. The level

MCP-4 exof MCP-4

message was low in resting peripheral blood mononuclear cells or freshly isolated monocytes and could not be augmented by treatment with LPS or with phytohemagglutinin

MCP-4 (A549),

plus phorbol expression fibroblasts

by treatment tions MCP-1 types. normal

On the tissues,

parasites,

myristate could not (IMR 90),

acetate (pmA). Furthermore, be induced in epithelial cells or endothelial cells (HUVEC)

with TNF-a (Fig. 7). expression was readily other hand, particularly

(Fig. 6). Because for

are

essential

in control-

10 human

CC chemokines

have

been

reported

at this time, and lated to chemokine

many other novel human sequences genes are present in sequence

bases.

family

This

large

(and perhaps ulation about kines may recruitment

of proteins

functional) apparent

similarity redundancy.

be necessary of particular

effect types.

type-specific may be

structural

or preferential This selectivity

expression of chemokine of the chemokines, such expression imparted by

reas

of MCP-4. Adthe differential

of a given concentration of chemokine on various cell For example, low concentrations of MIP-1 a attract

B cells tions pear

notable

has led to much specThe variety of chemo-

in part for selective subsets of cells.

may be achieved by localized ceptors or localized expression the tissueand cell ditional selectivity

with

redata-

and

cytotoxic

T cells,

whereas

attract helper T cells instead f37. to have a similar concentration-dependent

higher

concentra-

MCP-4

would profile

apon

eosinophils and monocytes. Finally, chemokines may play a much broader role in cellular physiology than previously anticipated. Chemokines recently have been shown to be involved in such diverse processes as hematopoiesis 1381,

and

angiogenesis

inhibition

of

[4OJ. The cloning and characterization ily of genes will allow their role in both physiology

to be

more

ACKNOWLEDGM

fully

HIV of this normal

proliferation growing famand disease

understood.

ENTS

The authors wish to thank Dan Allison for construction of the plasmid pDC1 used for CHO transfection. We also wish to thank Dma Leviten and Christi Wood for DNA sequencing

and

oligonucleotide

synthesis.

REFERENCES a unique

specffic expression. Whereas kines is rapidly stimulated

(PMA)

which

infection.

At least

members

1251,

eosinophils

parasitic

these MCP-4

cytes to these sites Consistent with this

MCP-4 small tissues

may

play

Under the detectable

same condiin these cell

was expressed intestine, colon, are

primary

a role

as a defense against concept is the robust

2. 3.

4.

5.

6.

in several and lung

routes

of entry

in recruiting

leuko-

such ability

1.

pathogens. of MCP-4

7.

8.

Oppenheim. J. J. (1993) Overview of chemokines. Adv. Exp Med. Biol. 351. 183-186. Baggiolini, M., Dewald, B., Moser, B. (1994) lnterleukin-8 and related chemotactic cytokines-CXC and CC chemokines. Adv. immunol. 55. 97-179. Schall, 1. J. (1991) Biology of the RANTESISIS cytokine family. fyiokine 3. 165-183. Van Damme, J., Decock, R, Lenaerts, i-P.. Conings. R.. Bertini. R.. Mantovani, A.. Billiau, A. (1989) Identification by sequence analysis of chemotactic factors for monocytes produced by normal and transformed cells stimulated with virus, double-stranded RNA or cytokine. Eur. .1 Immunol. 19. 2367-2373. Van Damme, J.. Proost. P.. Lenaerts, i-P.. Opdenakker. G. (1992) Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J. Exp. Med. 176, 59-65. Yu, X., Dluz, S., Graves, D. T., Zhang. L.. Antoniades, H. N., Hollander, W., Prusty, S., Valente, A. J., Schwartz, C. J.. Sonenshein, G. E. (1992) Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hyperchoksterolemic primates. Proc. Nail. Acad. Sci. USA 89, 6953-6957. Yla-Herttuala, S., Lipton, B. A.. Rosenfeld, M. E., Sarkioja, T., Yoshimura. 1., Leonard, E. J., Witztum, J. L., Steinberg. D. (1991) Expression of monocyte chemoauractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc. NaiL Aced. Sci. USA 88, 5252-5256. Bisehoff, S. C.. Krieger. M., Brunner, T., Rot, A., Von Tscharner, V., Bag-

GodLska

ci

at.

Monocyte

chemotactic

protein-4

359

giolini,

M.,

Dahinden,

(1993)

C. A.

RANTES

tivate human basophil granulocytes through ceptors. Em-. J. Immunol. 23, 761-767.

9.

and

related

different

chemokines

G protein-coupled

ac-

J. C., Mackay.

re-

tant,

basophil

154. 10.

and

eosinophil

leukocytes

M., Clark-Lewis, MCP-2 activates to MCP-3. .1 Immunol.

similar

M. W., Roth,

cyte

chemoattractant

S. J., Luther,

E., Rose,

protein

1 acts

S. S., Springer,

as

a T-lymphocyte

T. A. (1994)

Nail.

protein-lO,

stimulate

transendotheial

chemotaxis

Immunol. 25, 3482-3488. Proost, P., Wuyts, A., Van Damme,

12.

proteins-2

26.

i4cad.

and

-3:

structural

and

of T lymphocytes.

but not inducible J. Eur.

27. 28.

J. (1996) Human monocyte chemotactic functional comparison with MCP-i. J. Leu-

Biol. 59, 67-74. Sozzani, S.. Zhou, D., Locati, M., Rieppi. M., Proost, P., Magazin, M.. Vita, N., Van Damme. J. , Mantovani, A. (1994) Receptors and transduction pathways for monocyte chemotactic protein-2 and monocyte chemotactic protein3. Similarities and differences with MCP-1. J. Immunol. 152, 3615-3622. Franci, C., Wong, L. M., Van Damme, J., Proost, P., Charo, I. F. (1995) koc.

13.

14.

Monocyte

15.

16.

17.

18.

19.

20.

21 22.

23.

24.

360

chemoattractant

protein-3,

but

not

monocyte

.

tiple

leukocyte

tory

protein-i

receptors.

C-C

CKR1.

a receptor

for

macrophage

inflamma-

a1phaIRANTES, is also a functional receptor for MCP3. J. Biol. Chem. 270, 22123-22128. Ponath, P. D., Qin, S., Post, T. W., Wang, J., Wu, L., Gerard, N. P., Newman, W., Gerard, C., Mackay, C. R. (1996) Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J. Exj.i. Med. 183, 2437-2448. Daugherty, R L., Siciliano, S. J., DeMartino, J. A., Malkowitz, L., Sirotina, A., Springer, M. S. (1996) Cloning, expression, and characterization of the human eosinophil eotaxin receptor. J. Exp. Med 183, 2349-2354. Van Damme, J. (1994) Interleukin-8 and related chemotactic cytokines. In Cytokine Handbook 3rd ed. (A. Thomson, ed.) London: Academic Press. Bossink, A. W., Paemen, L., Jansen, P. M., Hack, C. E., Thijs, L. G., Van Damme, J. (1995) Plasma levels of the chemokines monocyte chemotactic proteins-i and -2 are elevated in human sepsis. Blood 86, 3841-3847. Kitaura, M., Nakajima, T., Imai, T., Harada, T., Combadiere, C., Tiffany, H. L., Murphy, P. M., Yoshie, 0. (i996) Molecular cloning of human eotaxin, an eosinphil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. .1 RiO!. Chem. 271, 7725-7730. Ponath, P. D., Qui, S., Ringler, D. i., Qark-Lewis, I., Wang, J., Kassam, N., Smith, H., Shi, X., Gonzalo, J. A., Newman, W., Gutierrez-Ramos,

Journal

of

Leukocyte

Biology

Volume

61,

March

1997

Li,

eosinophil

and

recruitment

P., Forssmann,

Y., Kreider,

Monocyte

chemotactic

analogue

of

chemoattrac-

functional

properties

of eosinophils.

sug-

J. Clin.

Invest.

Yamada,

Y.,

B., Garotta,

U., Dewald, G., Thelen,

M.,

B.. Li, H., Baggiolini,

Hensehe M. (1996)

4 (MCP-4), a novel structural and functional J. Exp. Med. 183, 2379-2384. Shyy, Y. J., Li, Y. S.. Kolattukudy, P. E. (1990) Structure of human chemotactic protein gene and its regulation by TPA. Biochem. Biophys. Res. Commun. 169, 346-351. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. (i990) Basic local alignment search tool. J. Mol. BiOL 215, 403-4i2. Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M., Dietsch, G., Schimpf, a, Hooper, S., Le Trong, H., Cousens, L. S., Zimmerman, G. A., MCP-3

McIntyre,

inflammatory

properties

374, Godiska,

549-552. R., Chantry,

expression munol.

in murine

protein

and

eotaxin.

T.

M.,

Prescott,

S.

M.,

of a platelet-activating

D., Dietsch, experimental

Gray, factor

G. N., Gray, allergic

P. W.

(i995)

Anti-

Na-

acetylhydrolase.

P. W. (1995)

encephalomyelitis.

Chemokine J. New-oim-

36.

i67-176. J., Fritsch, E. F., Maniatis, ‘F. (1987) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Stafforini, D. M., Elstad, M. R., McIntyre, T. M., Zimmerman, G. A., Prescott, S. M. (1990) Human macrophages secrete platelet-activating factor acetylhydrolase. .1 Biol. C/tern. 265, 9682-9687. Urlaub, G., Mitchell, P. J., Kas, E., Chasm, L. A., Funanage, V. L., Myoda, T. T., Hamlin, J. (i986) Effect ofgamma rays at the dihydrofolate reductase locus: deletions and inversions. Somatic Cell Mol. Genet. 12, 555-566. Casale. T. B., Abbas, M. K., Carolan, E. J. (1992) Degree of neutrophil chemotaxis is dependent upon the chemoattractant and barrier. Am. J. Respir. Cell. Mo! BiOL 7, 112. Charo, I. F., Myers. S. J., Herman, A., Franci, C.. Connolly. A. J., Coughlin, S. R. (1994) Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Tht,c. NatL Acad. Sci. USA 91, 2752-2756. von Heijne. G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. Neote, K., DiGregorio, D., Mak. J. Y., Horuk, R., Schall, ‘F. J. (1993) Molec-

37.

chemokine receptor. Cell 72, 415-425. Schall, T., Bacon, K., Camp, R. D. R., Kaspari,

30.

chemoattractant

protein-2, is a functional ligand of the human monocyte chemoattractant protein-i receptor. J. Immunol. 154, 6511-6517. Myers, S. J., Wong, L. M., Charo, I. F. (1995) Signal transduction and ligand specificity of the human monocyte chemoattractant protein-i receptor in transfected embryonic kidney cells. J. Biol. C/tern. 270, 5786-5792. Power, C. A., Meyer, A., Nemeth, K., Bacon, K. R, Hoogewerf, A. J., Proudfoot, A. E., Wells, N. (1995) Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line. J. Biol. Chem. 270, 19495-19500. Combadiere, C., Ahuja, S. K., Van Damme, J., Tiffany. H. L., Gao, J. L., Murphy, P. M. (1995) Monocyte chemoattractant protein-3 is a functional ligand for CC chemokine receptors 1 and 2B. J. Biol. C/tern. 270, 29671-29675. Ben-Baruch, A., Xu, L., Young, P. R., Bengali, K., Oppenheim, J. J., Wang, J. M. (1995) Monocyte chemotactic protein-3 (MCP3) interacts with mul-

of the human binding,

for the selective

M., Loetscher,

S.,

ture

29.

Cloning receptor

604-612.

Uguccioni, Lima,

Mono-

chemoattractant.

Sci. USA 91, 3652-3656. Roth, S. J., Carr, M. W.. Springer, T. A. (1995) C-C chemokines, the C-X-C chemokines interleukin-8 and interferon-gamma Proc.

11.

97.

25.

4166-4172.

Carr,

Expression,

gest a mechanism

Weber, M., Uguccioni, M., Ochensherger. B.. Baggiolini, I., Dahinden, C. A. (1995) Monocyte chemotactic protein human

C. R. (1996)

eotaxin.

31.

32.

33.

34.

35.

ular

cloning,

Human

38.

40.

functional

macrophage

expression,

inflammatory

and

protein

signaling

characteristics

inflammatory

protein

family

of a C-C

J. W., Goeddel, D. V. (1993) (MIP-la) and MIP-i3 chemolymphocytes. J. Exp. Med. 177,

a

kines attract distinct populations and i82i-i825. Broxmeyer, H. E., Sherry, R, Cooper, S.. Lu, L., Maze, M. P., Cerami, A., Ralph, P. (1993) Comparative analysis macrophage

39.

58,

Sambrook,

of cytokines

R., Beckmann, of the human

(chemokines)

on

pro-

liferation of human myeloid progenitor cells. Interacting effects involving suppression, synergistic suppression, and blocking of suppression. .1 ImmunoL 150, 3448-3458. Stricter, R. M., Polverini, P. J., Kunkel, S. L., Arenberg, D. A.. Burdick, M. D., Kasper, J., Dzuiba, J., Van Damme, J., Walz, A., Marriott, D., Chan, S.-Y., Roczniak, S., Shanafelt, A. R (1995) The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J. BiOL Chem. 270, 27348-27357. Cocchi, F., DeVico A. L., Garzino-Demo, A., Arya, S. K., Gab, R. C., Lusso, P. (1995) Identification of RANTES, MIP-i alpha, and MIP-i beta as the major HIV-suppressive factors produced by CD8 T cells. Science 270, l8ii-i815.