Quimoquinas

Mensajeros en paz y en guerra: las quimoquinas
Inmunologa Bsica
Septiembre, 2012
Persio D. Lpez Loyo
Thursday, October4, 12
L as qui mo qui nas ( c i t o qui nas
qui mo tc t i c as ) s on pro te nas
e xt rac e l ul are s i nmunor re gul adoras
de f i ni das por homol o g a e n l a
s e c ue nc i a y una e s t r uc t u ra c omn.
Paul WE. Fundamental Immunology. 6th ed. Philadelphia:
Wolters Kluwer/Lippincott Williams & Wilkins; 2008. Figure
26.1, Chemokine classication and nomenclature; p. 805.
Clasicacin y nomenclatura
26.1, Chemokine classication and nomenclature; p. 805.
variable variable
24 AA 15 AA
8-12 kDa
Allen SJ, Crown SE, Handel TM. Chemokine: receptor
structure, interactions, and antagonism. Annu. Rev. Immunol.
2007;25:787820. Figure 1, Chemokine structures; p. 791.
ANRV306-IY25-26 ARI 11 February 2007 13:23
binding to GAGs (4648). The dimers fall
into two general classes (Figure 1). CC
chemokines (e.g. CCL2/MCP-1) associate
primarily through the formation of an an-
tiparallel -sheet involving residues near the
N terminus, including the rst two cysteines
(approximately residues 912 in CCL2). The
net result is an elongated structure with con-
siderable exibility in the disposition of the
two subunits with respect to each other. In
the CXC chemokines (e.g., CXCL8/IL-8),
residues in the rst strand of the -sheet
from one subunit hydrogen bond with the
same strand from a second subunit, forming
one extended six-stranded sheet. The dimer
is further stabilized by interactions between
the ends of the C-terminal -helices with the
-sheet of the opposing subunit. The overall
topology in these CXC dimers is effectively
a -sheet platform topped by two -helices
with a slight cavity between the helices. In
comparison to CC chemokines, CXC dimers
have a much more globular shape.
A few chemokines are known to form
tetrameric structures (Figure 2). Although
the structure of CCL2 was initially solved as a
dimer in solution by NMR, subsequent crys-
tallographic studies revealed the presence of
both dimers and tetramers from two differ-
ent crystal forms (49) (Figure 2ac). Interest-
ingly, the tetramer features bothCCand CXC
dimer interfaces, but since the CC dimer pre-
dominates in solution, it must be the more sta-
ble interface. Earlier studies of CXCL4/PF-
4 showed a similar tetrameric architecture as
the dominant form of the protein in solution
(50) (Figure 2e). In the case of CXCL10/IP-
10, two different tetramers were observed
by crystallography, one similar to the CCL2
and CXCL4 tetramers and the other consist-
ing of a novel 12-stranded -sheet structure
(43) (Figure 2d,e). Other chemokines such
as CCL5/RANTES, CCL3/MIP-1, and
CCL4/MIP-1 aggregate into even higher-
order oligomers (51). For this reason, struc-
tures of these chemokines were solved at ex-
ceptionally lowpH(<4.0), which destabilized
the aggregated formbut left the CCdimer in-
Figure 1
Chemokine structures. Ribbon diagrams of monomer (left) and dimer
(right) structures for (a) the CC-family chemokine, CCL2, and (b) the
CXC-family chemokine, CXCL8. The disulde bonds are shown in yellow
and red. The gure illustrates the similarity in the structures at the tertiary
level and the differences in CC versus CXC dimers.
tact. We therefore anticipate that the dimers
are the fundamental oligomeric subunits of
the higher-order aggregates. Tetramers are
likely to be the next level of organization, as
point mutants of both CCL5 and CCL3 have
been identied that form predominantly CC
dimers or tetramers of as yet unknown struc-
ture (51). Thus, the higher-order structures
are likely organized assemblies rather than
random precipitates.
Despite the prevalence of chemokines
that oligomerize, it is now accepted that
chemokines interact with receptors as
monomers, at least in the context of mi-
gration. This nding was demonstrated
with structure-based design of mutants that
are obligate monomers. Clark-Lewis (52)
www.annualreviews.org Chemokines and Chemokine Receptors 791
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Figure 1
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Figure 2
Chemokine tetramers. Structures of three chemokines have been solved by X-ray crystallography as
tetramers. Ribbon diagrams of CCL2 are shown for three views that highlight the presence of (a) the CC
dimerlike interface (blue and green, red and pink), (b) the CXC dimerlike interface (red and green, blue
and pink), and (c) a novel interface involving contacts between all four subunits. CXCL10 was solved in
two forms: (d ) the M-form, which resembles the CCL2 tetramer, and (e) the H-form, which is a novel
12-stranded -sheet structure. ( f ) CXCL4 forms a tetramer similar to the CCL2 tetramer.
made a synthetic variant of CXCL8/IL-8
containing a methyl group on the amide of
Leu25, which inhibits hydrogen bonding
between the central -strands of opposing
subunits in the dimer. The mutant does not
dimerize; nevertheless, the receptor binding
afnity of the variant and its ability to induce
cell migration and elastase release in vitro
are equivalent to the wild-type protein (52).
Subsequently, a similar chemical modication
of Thr8 in CCL5 produced a monomeric
variant with in vitro activity equivalent to the
wild-type protein (9). A different strategy was
used with CCL2 and CCL4. In recombinant
forms of these chemokines, mutation of Pro8,
an amino acid that anks the dimer interface
792 Allen
Crown
Handel
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2007;25:787820. Figure 2, Chemokine tetramers; p. 792.
Los guantes del queche
2007;25:787820. Table 1, Chemokine receptors and their
ligands; p. 789.
Table 1 Chemokine receptors and their ligands
Receptor Ligands
CCR1 CCL3,CCL5,CCL7,CCL13,CCL14,CCL15,CCL16,CCL23
CCR2 CCL2,CCL7,CCL8,CCL13,CCL16
CCR3 CCL5,CCL7,CCL8,CCL11,CCL13,CCL15,CCL16,CCL24,CCL26,CCL28
CCR4 CCL17,CCL22
CCR5 CCL3,CCL4,CCL5,CCL8,CCL11,CCL14,CCL16
CCR6 CCL20
CCR7 CCL19,CCL21
CCR8 CCL1
CCR9 CCL25
CCR10 CCL27,CCL28
CXCR1 CXCL6,CXCL7,CXCL8
CXCR2 CXCL1,CXCL2,CXCL3,CXCL5,CXCL6,CXCL7,CXCL8
CXCR3-A CXCL9,CXCL10,CXCL11
CXCR3-B CXCL4,CXCL9,CXCL10,CXCL11
CXCR4 CXCL12
CXCR5 CXCL13
CXCR6 CXCL16
CXCR7 CXCL12
XCR1 XCL1,XCL2
CX
3
CR1 CX
3
CL1
CCX-CKR CCL19,CCL21,CCL25
D6 CCL2,CCL3L1,CCL4,CCL5,CCL7,CCL8,CCL11,CCL13,CCL14,CCL17, CCL22
DARC/Duffy CCL2,CCL7,CCL8,CCL11,CCL13,CCL14,CCL16,CCL17,CXCL1,CXCL5,
CXCL6,CXCL7,CXCL8,CXCL9,CXCL11,CXCL13
Table 1 summarizes the chemokine recep-
tors and their known ligands and illustrates
the fact that many different ligands bind the
same receptor and many ligands bind multi-
ple receptors. Very little is known about the
functional consequences of this apparent re-
dundancy, except that it is reected in the
lack of signicant phenotypes of knockout
mice of most inammatory chemokine re-
ceptors unless faced with pathogenic or in-
ammatory challenge (15). Redundancy may
allow exceptional ne-tuning of immune re-
sponses (12, 16), which may be further en-
hanced by the possibilities that arise not only
fromthe combinatorial interactions of the lig-
ands and receptors, but also by GAGs and
adhesion molecules and, as described below,
the various oligomerization states of the lig-
ands and the receptors. However, tempo-
ral and spatial compartmentalization of these
molecules in vivo may attenuate what seems
to be an innitely complex system.
Despite their pivotal roles in the immune
system, chemokines and their receptors are
associated with an extraordinary number
of pathologies (1719). These include au-
toimmune disorders (20, 21) (e.g., psoriasis,
rheumatoid arthritis, and multiple sclerosis),
pulmonary diseases (asthma and chronic ob-
structive pulmonary disease), transplant re-
jection, cancer (2226), and vascular disease
(27, 28) (Table 2). Human immunodeciency
virus (HIV) also uses chemokine receptors
as essential cofactors during viral entry into
host cells (29, 30). Thus, chemokine recep-
tors are subjects of signicant medical im-
portance, and consequently there is intense
interest in obtaining structural information
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s
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l
y
.
Table 2 Chemokine receptors and links to disease
Target Likely indications
a
References
CCR1 MS, psoriasis, RA, cancer, transplant rejection, kidney
disease
198204
CCR2 MS, RA, diabetes, obesity, atherosclerosis, cancer,
transplant, asthma
27, 28, 198, 201, 205211
CCR3 Asthma, allergic rhinitis 212
CCR4 Asthma, skin disease 213217
CCR5 HIV, transplant, cancer 25, 179181, 200204, 211
CCR6 Asthma 218, 219
CCR7 Cancer 220, 221
CCR9 IBD 222, 223
CXCR1/2 Sepsis, atherosclerosis, RA, COPD 198
CXCR3 Transplant, psoriasis, MS, RA, cancer 22, 198
CXCR4 HIV, cancer 24, 25, 179181, 220, 224, 225
CX3CR1 Atherosclerosis 226
a
Abbreviations: RA, rheumatoid arthritis; MS, multiple sclerosis; COPD, chronic obstructive pulmonary disease; IBD,
irritable bowel disease.
about these proteins to aid the develop-
ment and optimization of therapeutics to in-
hibit their function(3134). Extensive reviews
have been published on the links between
chemokines/receptors and disease, and these
topics are not reiteratedhere. Instead, we limit
our discussiontotherapeutic strategies target-
ing chemokine receptors CXCR4 and CCR5
with respect to their roles in HIV, and CCR1,
a recent example of a chemokine receptor for
whicha small molecule antagonist bindingsite
has been extensively mapped.
TERTIARY AND QUATERNARY
STRUCTURE OF CHEMOKINES
Tertiary Structure
For the most part, the chemokine ligands are
812 kDa proteins that contain 13 (usually 2)
disuldes. Their sequence homology is highly
variable, ranging from less than 20% to over
90%, but as described below, all share very
similar tertiary structures. After translation,
most chemokines are secreted from the cell,
with the exception of CX
3
CL1/fractalkine
and CXCL16, which are tethered to the ex-
tracellular surface. These proteins consist of
a chemokine domain at the N terminus fol-
lowed by an approximately 110 amino acid
mucin-like stalk rich in Ser and Thr residues,
a transmembrane domain, and a cytoplasmic
tail. CXCL1 and CXCL16 are produced in
bothmembrane-bound and soluble forms (35,
36) and, at least in the case of CX
3
CL1, can
act not only as chemoattractants, but also as
adhesion molecules.
Structures of many chemokines have been
solved by NMR and X-ray crystallography
(3745). These studies revealed that despite
low sequence homology, chemokines adopt a
remarkably conserved tertiary structure con-
sisting of a disordered N terminus of 610
amino acids, which functions as a key signal-
ing domain in all chemokines characterized
to date. This region is followed by a long
loop (the N-loop) that ends in a 3
10
helix
and invariably contains important binding de-
terminants, a three-stranded -sheet, and a
C-terminal helix. Disulde bonds stabilize the
overall topology (Figure 1).
Oligomerization
Many chemokines form dimers and higher-
order oligomers alone in solution or upon
790 Allen
Crown
Handel
A
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n
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.

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.

2
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:
7
8
7
-
8
2
0
.

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o
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.
2007;25:787820. Table 2, Chemokine receptors and links to
disease; p. 790.
Table 2 Chemokine receptors and links to disease
Target Likely indications
a
References
CCR1 MS, psoriasis, RA, cancer, transplant rejection, kidney
disease
198204
CCR2 MS, RA, diabetes, obesity, atherosclerosis, cancer,
transplant, asthma
27, 28, 198, 201, 205211
CCR3 Asthma, allergic rhinitis 212
CCR4 Asthma, skin disease 213217
CCR5 HIV, transplant, cancer 25, 179181, 200204, 211
CCR6 Asthma 218, 219
CCR7 Cancer 220, 221
CCR9 IBD 222, 223
CXCR1/2 Sepsis, atherosclerosis, RA, COPD 198
CXCR3 Transplant, psoriasis, MS, RA, cancer 22, 198
CXCR4 HIV, cancer 24, 25, 179181, 220, 224, 225
CX3CR1 Atherosclerosis 226
a
Abbreviations: RA, rheumatoid arthritis; MS, multiple sclerosis; COPD, chronic obstructive pulmonary disease; IBD,
irritable bowel disease.
about these proteins to aid the develop-
ment and optimization of therapeutics to in-
hibit their function(3134). Extensive reviews
have been published on the links between
chemokines/receptors and disease, and these
topics are not reiteratedhere. Instead, we limit
our discussiontotherapeutic strategies target-
ing chemokine receptors CXCR4 and CCR5
with respect to their roles in HIV, and CCR1,
a recent example of a chemokine receptor for
whicha small molecule antagonist bindingsite
has been extensively mapped.
TERTIARY AND QUATERNARY
STRUCTURE OF CHEMOKINES
Tertiary Structure
For the most part, the chemokine ligands are
812 kDa proteins that contain 13 (usually 2)
disuldes. Their sequence homology is highly
variable, ranging from less than 20% to over
90%, but as described below, all share very
similar tertiary structures. After translation,
most chemokines are secreted from the cell,
with the exception of CX
3
CL1/fractalkine
and CXCL16, which are tethered to the ex-
tracellular surface. These proteins consist of
a chemokine domain at the N terminus fol-
lowed by an approximately 110 amino acid
mucin-like stalk rich in Ser and Thr residues,
a transmembrane domain, and a cytoplasmic
tail. CXCL1 and CXCL16 are produced in
bothmembrane-bound and soluble forms (35,
36) and, at least in the case of CX
3
CL1, can
act not only as chemoattractants, but also as
adhesion molecules.
Structures of many chemokines have been
solved by NMR and X-ray crystallography
(3745). These studies revealed that despite
low sequence homology, chemokines adopt a
remarkably conserved tertiary structure con-
sisting of a disordered N terminus of 610
amino acids, which functions as a key signal-
ing domain in all chemokines characterized
to date. This region is followed by a long
loop (the N-loop) that ends in a 3
10
helix
and invariably contains important binding de-
terminants, a three-stranded -sheet, and a
C-terminal helix. Disulde bonds stabilize the
overall topology (Figure 1).
Oligomerization
Many chemokines form dimers and higher-
order oligomers alone in solution or upon
790 Allen
Crown
Handel
A
n
n
u
.

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e
v
.

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.

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0
0
7
.
2
5
:
7
8
7
-
8
2
0
.

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.
2007;25:787820. Table 2, Chemokine receptors and links to
disease; p. 790.
2007;25:787820. Figure 5, Hypothetical model of the
interaction of a chemokine with its receptor; p. 800.
receptor, CXCR4, and unlike the full-length
proteins, cannot inhibit CXCR4-dependent
entry of HIV-1 into CD4
+
cells. In addi-
tion, injection of CXCL12 (567) into the
basal ganglia in mice causes neurodegener-
ation and dementia (111). This result sug-
gests a survival role for CXCL12, similar to
its role in many cancers, such as chronic lym-
phocytic leukemia (112). Although the most
well-studied consequences of proteolysis are
direct effects on signaling, MMP cleavage of
chemokines may also have functional con-
sequences that are less obvious. These con-
Figure 5
Hypothetical model of the interaction of a chemokine (pink) with its
receptor (blue) based on the structure of a chemokine and the structure of
bovine rhodopsin. The model illustrates two hypothetical interactions:
the interaction of the N-terminal domain of the ligand with the receptor
helical bundle, and the interaction of the core domain of the ligand with
the ECLs of the receptor (only the N-terminal extracellular domain of
the receptor is shown).
sequences include release of the membrane-
bound chemokine CX
3
CL1 from the mem-
brane (113) and changes in the ability to form
oligomers and/or bind GAGs.
The relevance of MMP-mediated cleav-
age of chemokines in vivo is demonstrated
by studies showing the presence of truncated
forms of chemokines in cell culture (114117)
and the specic detection of the 576 form of
CCL7 in the rheumatoid synovial uid of an
arthritis patient (108). MMPs andchemokines
also regulate one another in vivo (109, 118
120), indicating a form of reciprocal regula-
tion during the course of the inammatory re-
sponse. As a note of caution, the discovery that
chemokines can be modied by proteolytic
processing in vivo should be taken into ac-
count when interpreting expression patterns
to understand the importance of chemokines
in inammation and disease. Otherwise, the
presence of a chemokine or its perceived ab-
sence due to the inability to detect a modi-
ed version of the protein may be incorrectly
interpreted.
The N-Loop and Core Domain
In addition to the role of the N terminus, the
N-loop between the rst two cysteines and
the 3
10
helix invariably contains residues in-
volved in receptor binding and is often por-
trayed as the primary docking site in the
two-step model of receptor activation (121,
122). In CXCL12, a RFFRESH motif in
this region provides a great deal of binding
afnity (123). However, in other chemokines,
residues distributed along the entire face of
the chemokine, as shown in Figure 2, par-
ticipate in receptor binding. Mutagenesis of
CCL2 and CXCL10 revealed that the loops
between the -strands of the chemokines also
contact the receptor and contribute to high-
afnity binding (89, 94). A particularly inter-
esting nding is that the binding determinants
within a ligand may differ for each receptor
that it binds (124). However, there is almost
nothing known about where these regions of
the ligands contact the receptor. Figure 5
800 Allen
Crown
Handel
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:
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8
7
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8
2
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.

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.
expected to yield information that cannot be
obtained by any other method and therefore
is worth the effort to prepare the requisite
libraries.
Even with heparin, some differences in the
chemokine:octasaccharide interactions have
been observed. As described above, CCL2
and CCL8 bind heparin octasaccharides as
dimers, whereas CCL7, CCL11, and CCL13
bind as monomers. CCL7, CCL11, and
CCL13 also show slightly more promiscuous
behavior by binding to heparin octasaccha-
rides with 10 sulfates in addition to those with
11 and 12 sulfates (79). These results indicate
that chemokines that share a common recep-
tor (in this case CCR2) can have unique prop-
erties. Despite the apparent redundancy, these
chemokines can be distinguished with respect
to their oligomeric state, the stoichiometry
of the chemokine:GAG complexes they form,
and the specicity of GAGs bound by indi-
vidual chemokines. However, whether these
Figure 4
Left: Ribbon diagram of CCL2 with the disordered N-terminal signaling
domain labeled. Right: Receptor binding epitopes of CCL2 for CCR2 are
highlighted on a surface topology model of the CCL2 monomer in the
same orientations as the ribbon diagram (61, 89). The N-terminal domain
( green) and Y13 ( pink) are required to induce signaling of CCR2; deletion
of the N terminus or mutation of Tyr13 to Ala results in a CCR2
antagonist. Other basic hotspots (blue) likely bind acidic residues in the
extracellular loops (ECLs) of the receptor. Reprinted from Reference 228
by permission.
observations translate into differences in vivo
remains to be demonstrated.
INTERACTION OF
CHEMOKINES WITH
RECEPTORS: DETERMINANTS
OF BINDING AND SIGNALING
The N-Terminal Signaling Domain
Mutagenesis studies of several chemokines
coupled with assays of receptor binding and
activation have been used to dene binding
and signaling epitopes on the ligands (61,
89, 90). The primary epitopes on CCL2, the
ligand for the receptor CCR2, are summa-
rized in Figure 4. Since the earliest studies
of CXCL8 by Clark-Lewis (92), the general
concept has emerged that the N termini of
chemokines are key signaling domains and
that deletion or modication of the N ter-
mini results in variants that in some cases
do not induce signaling but frequently re-
tain high-afnity interactions with their re-
ceptors (61, 89, 91, 92). Studies of CCL2,
CCL5, CCL9, CCL19, and CXCL10 all re-
vealed the importance of the N terminus of
the ligand for inducing signaling by their re-
spective receptors (61, 93, 94). For exam-
ple, deletion of seven residues from the N
terminus of CCL2 results in a CCR2 an-
tagonist with an afnity of 1.4 nM (61, 91,
92). Other modications also modulate the
functional properties of chemokines. Reten-
tion of the N-terminal Met in CCL5 and
CCL2 produces antagonists, and addition
of amino-oxypentane to the N terminus of
CCL5 results in an exceptional antagonist
of HIV entry (95, 96). Certain motifs in
the N terminus of chemokines also corre-
late with function. CXC chemokines con-
taining N-terminal Glu-Leu-Arg (ELR) se-
quences are angiogenic (e.g., CXCL18),
whereas most non-ELR CXC chemokines
are angiostatic (e.g., CXCL911, CXCL4,
CXCL13), with one notable exception (SDF-
1/CXCL12) (97).
798 Allen
Crown
Handel
A
n
n
u
.

R
e
v
.

I
m
m
u
n
o
l
.

2
0
0
7
.
2
5
:
7
8
7
-
8
2
0
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

w
w
w
.
a
n
n
u
a
l
r
e
v
i
e
w
s
.
o
r
g
b
y

H
I
N
A
R
I

o
n

0
9
/
1
8
/
1
2
.

F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
2007;25:787820. Figure 4; p. 798.
receptor, CXCR4, and unlike the full-length
proteins, cannot inhibit CXCR4-dependent
entry of HIV-1 into CD4
+
cells. In addi-
tion, injection of CXCL12 (567) into the
basal ganglia in mice causes neurodegener-
ation and dementia (111). This result sug-
gests a survival role for CXCL12, similar to
its role in many cancers, such as chronic lym-
phocytic leukemia (112). Although the most
well-studied consequences of proteolysis are
direct effects on signaling, MMP cleavage of
chemokines may also have functional con-
sequences that are less obvious. These con-
Figure 5
Hypothetical model of the interaction of a chemokine (pink) with its
receptor (blue) based on the structure of a chemokine and the structure of
bovine rhodopsin. The model illustrates two hypothetical interactions:
the interaction of the N-terminal domain of the ligand with the receptor
helical bundle, and the interaction of the core domain of the ligand with
the ECLs of the receptor (only the N-terminal extracellular domain of
the receptor is shown).
sequences include release of the membrane-
bound chemokine CX
3
CL1 from the mem-
brane (113) and changes in the ability to form
oligomers and/or bind GAGs.
The relevance of MMP-mediated cleav-
age of chemokines in vivo is demonstrated
by studies showing the presence of truncated
forms of chemokines in cell culture (114117)
and the specic detection of the 576 form of
CCL7 in the rheumatoid synovial uid of an
arthritis patient (108). MMPs andchemokines
also regulate one another in vivo (109, 118
120), indicating a form of reciprocal regula-
tion during the course of the inammatory re-
sponse. As a note of caution, the discovery that
chemokines can be modied by proteolytic
processing in vivo should be taken into ac-
count when interpreting expression patterns
to understand the importance of chemokines
in inammation and disease. Otherwise, the
presence of a chemokine or its perceived ab-
sence due to the inability to detect a modi-
ed version of the protein may be incorrectly
interpreted.
The N-Loop and Core Domain
In addition to the role of the N terminus, the
N-loop between the rst two cysteines and
the 3
10
helix invariably contains residues in-
volved in receptor binding and is often por-
trayed as the primary docking site in the
two-step model of receptor activation (121,
122). In CXCL12, a RFFRESH motif in
this region provides a great deal of binding
afnity (123). However, in other chemokines,
residues distributed along the entire face of
the chemokine, as shown in Figure 2, par-
ticipate in receptor binding. Mutagenesis of
CCL2 and CXCL10 revealed that the loops
between the -strands of the chemokines also
contact the receptor and contribute to high-
afnity binding (89, 94). A particularly inter-
esting nding is that the binding determinants
within a ligand may differ for each receptor
that it binds (124). However, there is almost
nothing known about where these regions of
the ligands contact the receptor. Figure 5
800 Allen
Crown
Handel
A
n
n
u
.

R
e
v
.

I
m
m
u
n
o
l
.

2
0
0
7
.
2
5
:
7
8
7
-
8
2
0
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

w
w
w
.
a
n
n
u
a
l
r
e
v
i
e
w
s
.
o
r
g
b
y

H
I
N
A
R
I

o
n

0
9
/
1
8
/
1
2
.

F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
2007;25:787820. Figure 4; p. 798.
expected to yield information that cannot be
obtained by any other method and therefore
is worth the effort to prepare the requisite
libraries.
Even with heparin, some differences in the
chemokine:octasaccharide interactions have
been observed. As described above, CCL2
and CCL8 bind heparin octasaccharides as
dimers, whereas CCL7, CCL11, and CCL13
bind as monomers. CCL7, CCL11, and
CCL13 also show slightly more promiscuous
behavior by binding to heparin octasaccha-
rides with 10 sulfates in addition to those with
11 and 12 sulfates (79). These results indicate
that chemokines that share a common recep-
tor (in this case CCR2) can have unique prop-
erties. Despite the apparent redundancy, these
chemokines can be distinguished with respect
to their oligomeric state, the stoichiometry
of the chemokine:GAG complexes they form,
and the specicity of GAGs bound by indi-
vidual chemokines. However, whether these
Figure 4
Left: Ribbon diagram of CCL2 with the disordered N-terminal signaling
domain labeled. Right: Receptor binding epitopes of CCL2 for CCR2 are
highlighted on a surface topology model of the CCL2 monomer in the
same orientations as the ribbon diagram (61, 89). The N-terminal domain
( green) and Y13 ( pink) are required to induce signaling of CCR2; deletion
of the N terminus or mutation of Tyr13 to Ala results in a CCR2
antagonist. Other basic hotspots (blue) likely bind acidic residues in the
extracellular loops (ECLs) of the receptor. Reprinted from Reference 228
by permission.
observations translate into differences in vivo
remains to be demonstrated.
INTERACTION OF
CHEMOKINES WITH
RECEPTORS: DETERMINANTS
OF BINDING AND SIGNALING
The N-Terminal Signaling Domain
Mutagenesis studies of several chemokines
coupled with assays of receptor binding and
activation have been used to dene binding
and signaling epitopes on the ligands (61,
89, 90). The primary epitopes on CCL2, the
ligand for the receptor CCR2, are summa-
rized in Figure 4. Since the earliest studies
of CXCL8 by Clark-Lewis (92), the general
concept has emerged that the N termini of
chemokines are key signaling domains and
that deletion or modication of the N ter-
mini results in variants that in some cases
do not induce signaling but frequently re-
tain high-afnity interactions with their re-
ceptors (61, 89, 91, 92). Studies of CCL2,
CCL5, CCL9, CCL19, and CXCL10 all re-
vealed the importance of the N terminus of
the ligand for inducing signaling by their re-
spective receptors (61, 93, 94). For exam-
ple, deletion of seven residues from the N
terminus of CCL2 results in a CCR2 an-
tagonist with an afnity of 1.4 nM (61, 91,
92). Other modications also modulate the
functional properties of chemokines. Reten-
tion of the N-terminal Met in CCL5 and
CCL2 produces antagonists, and addition
of amino-oxypentane to the N terminus of
CCL5 results in an exceptional antagonist
of HIV entry (95, 96). Certain motifs in
the N terminus of chemokines also corre-
late with function. CXC chemokines con-
taining N-terminal Glu-Leu-Arg (ELR) se-
quences are angiogenic (e.g., CXCL18),
whereas most non-ELR CXC chemokines
are angiostatic (e.g., CXCL911, CXCL4,
CXCL13), with one notable exception (SDF-
1/CXCL12) (97).
798 Allen
Crown
Handel
A
n
n
u
.

R
e
v
.

I
m
m
u
n
o
l
.

2
0
0
7
.
2
5
:
7
8
7
-
8
2
0
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

w
w
w
.
a
n
n
u
a
l
r
e
v
i
e
w
s
.
o
r
g
b
y

H
I
N
A
R
I

o
n

0
9
/
1
8
/
1
2
.

F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
26.2, Chemokine signal transduction in chemotaxis; p. 805.
Bennett LD, Fox JM, Signoret N. Mechanisms regulating
chemokine receptor activity. Immunology. 2011 Nov;134(3):
24656. Figure 1, Agonist-dependent and independent
chemokine receptor desensitization; p. 250.
dem. Figure 3, Dierent tracking routes proposed for
agonist-treated CCR5; p. 251.
whereby clusters of dimers are packed at the cell surface,
with the potential for allosteric cross-talk between neigh-
bouring dimers to affect more distant receptors in a dom-
ino effect.
54
The physiological relevance of chemokine receptor olig-
omerization was highlighted initially with CCR5, when a
naturally occurring truncation (D32) of this receptor lead-
ing to retention of wild-type CCR5/CCR5D32 heterodi-
mers in the endoplasmic reticulum was found to confer
resistance to HIV-1 infection.
78,79
More recently, it was
shown using blood cells from CCR5D32-expressing indi-
viduals that CCR2/CXCR4/CCR5 heteromers accounted
for a negative ligand-binding co-operativity, which inhib-
ited leucocyte recruitment in vitro and in vivo.
73
Overall,
multimerization is emerging as an additional level of reg-
ulation providing cell and tissue specicity to ne-tune
chemokine receptor activity in vivo.
Chemokine receptor desensitization
Chemokine receptors are coupled to heterotrimeric G
proteins and undergo conformational changes following
ligand binding. The G protein dissociates into guanosine
triphosphate (GTP)-bound Ga and the Gb/c complex,
which activate second messengers and stimulate effector
proteins leading to intracellular signalling.
80
It has
emerged that GPCRs can also elicit G protein-indepen-
dent signals through interaction with the scaffolding pro-
teins b-arrestins, linking activated receptors to various
signalling pathways that act independently of, in synergy
with or in opposition to, G protein-mediated signals.
81
However, b-arrestins are best known for their pivotal role
in the regulation of GPCR signals via the process of
desensitization, a feedback mechanism protecting cells
from overstimulation. In this section we consider what is
called homologous desensitization only affecting agonist-
activated receptors (Fig. 1).
82
Briey, following agonist
binding, signalling receptors become rapidly phophorylat-
ed on their cytoplasmic tail, usually by one member of
the G protein receptor kinase (GRK) family, which
uncouples the G protein from the receptor and prevents
further activation. Phosphorylated receptors interact with
one of the b-arrestins acting as a scaffold targeting recep-
tors for internalization, leading to a permanent or tran-
sient loss of cell surface receptors due to degradation or
subsequent recycling of internalized molecules, respec-
tively.
5
The ability of a chemokine receptor to interact
with b-arrestins can inuence its fate in multiple ways.
First, the strength and stability of receptor/b-arrestins
interactions seem critical in determining whether or not
an agonist-activated chemokine receptor is internalized,
as described for CCR7 and CCR2.
8385
Secondly, the
afnity of these interactions can inuence the destiny of
receptors once internalized. Indeed, GPCRs that rapidly
recycle (Class A) preferentially bind b-arrestin 2 with low
afnity and dissociate from it upon internalization,
whereas those that slowly recycle or are degraded (Class
B) bind both b-arrestins with high afnity and remain b-
arrestin-bound inside the cell.
86
To date, only class B
chemokine receptors have been described, with evidence
for b-arrestins binding to agonist-treated CXCR4, CCR2
and CCR5 in internal compartments
8789
(see Fig. 2).
Chemokine receptors can be internalized via clathrin-
or caveolin-dependent endocytosis, although other inde-
pendent pathways have also been reported.
5
Interestingly,
CCR2 and CCR5 have been shown to follow both clath-
rin-dependent and caveolin-mediated pathways and the
route of endocytosis could be cell-type dependent.
42,9093
The intracellular path followed by a chemokine receptor
determines the fate of this receptor, i.e. being sent for
degradation (down-regulation) or being sequestered intra-
cellularly before returning to the cell surface (resensitiza-
tion). Receptors can follow one path exclusively, such as
Internalization
(a)
(b)
Internalization
Degradation
Degradation
Cross-
phosphorylation
P
x
P
P
P
P P
P
P
P
P
P P
P
P
G
R
K
?
Recycling
Recycling
a
a
g b
a
g b
b
b
g
b
-
a
r
r
b-arr
b-arr
b
-
a
r
r
g
Figure 1. Agonist-dependent (a) and independent (b, heterologous)
chemokine receptor desensitization. (a) Following agonist binding
and G protein mediated signalling, the chemokine receptor cytoplas-
mic tail is rapidly phosphorylated, usually by a G protein receptor
kinase (GRK); this uncouples the G protein, which dissociates into
guanosine triphosphate (GTP)-bound Ga and the Gbc complex, and
enables interaction with a b-arrestin, which acts as a scaffold target-
ing the receptor for internalization. Once internalized, the receptor
follows recycling or degradation pathways. (b) Receptor X mediates
cross-phosphorylation of the chemokine receptor, which may involve
protein kinase C (PKC), leading to inhibition of chemokine-induced
signalling and in some cases internalization of the receptor.
250 2011 The Authors. Immunology 2011 Blackwell Publishing Ltd, Immunology, 134, 246256
L. D. Bennett et al.
CCR5 or CXCR3 sent for recycling or degradation,
respectively.
9498
Alternatively, they can enter either path-
way depending on the cell-type and duration of ligand
treatment, as reported for CXCR2 and CXCR4.
99101
Note
that the agonist itself can impact upon the fate of a recep-
tor. For instance, with CCR5, any agonist-stimulated
receptors seem to follow the recycling route but the dis-
tribution of receptors along the pathway could be ago-
nist-specic (Fig. 3). Following internalization, CCR5
receptors treated with the natural chemokine CCL5 [regu-
lated upon activation normal T cell expressed and
secreted (RANTES)] are located in recycling endosomes
(RE) before re-accumulating in the plasma membrane.
95
In contrast, they keep cycling back from the cell surface
to the RE after exposure to the chemically modied
aminooxypentane (AOP)-RANTES,
95
become trapped in
the trans-Golgi network (TGN) after passage through RE
with Na-(n-nonanoyl)-des-Ser1-[l-thioproline2, l-a-cyclo-
hexyl-glycine3] PSC-RANTES,
102
and appear to bypass
the RE to accumulate in the TGN with methionine MET-
RANTES.
103
Sorting of internalized chemokine receptors to the recy-
cling or degradative pathways requires complex interac-
tions with the machinery mediating movement of
molecules between intracellular compartments. Endocytic
adaptors recognize specic determinants in the cytoplas-
mic domains of the receptors, mainly small sorting motifs
and post-translational modications.
5,104
Two of these
determinants, the PDZ ligand motif and ubiquitination,
have received much interest recently, and were shown to
support recycling or degradation of chemokine receptors,
respectively. At least 12 chemokine receptors have been
identied as containing potential PDZ ligand motifs in
their extreme C-terminal cytoplasmic tail.
5
The PDZ
ligand motifs are presumed to interact with PDZ domain
containing proteins of the sorting machinery, but only a
few of these interactions have been unveiled. CCR5 post-
endocytic sorting to the recycling pathway is dependent
on its PDZ ligand motif,
94
which has been shown to
interact with a protein implicated in receptor recycling
called EBP50/NHERF-1.
105
For CXCR2 that can be both
recycled following short ligand exposure and degraded
following more prolonged ligand treatment,
99
the PDZ
ligand motif serves to delay degradation by preventing
lysosomal sorting, due probably to interaction with an as
yet unknown PDZ-containing protein.
106
Ubiquitination
has emerged as an important modication for sending
the chemokine receptor CXCR4
107
and other GPCRs
104
to
degradation. For CXCR4, CXCL12 stimulation leads to
ubiquitination of cell surface receptors as well as ubiqu-
itin-dependent endocytosis and trafcking of ubiquitinat-
ed CXCR4 to lysosomes.
108,109
However, ubiquitination
does not seem to be required for the degradation of all
chemokine receptors.
98,106
Cross-talk and heterologous regulation
In addition to co-operativity within chemokine receptor
multimers, various examples for regulation by indirect
T0 3
60
Figure 2. Intracellular transport of b-arrestin-bound CCR5 receptors
following CCL5-treatment. Isolated human blood monocytes were
treated with 100 nm CCL5 for the indicated time-period. Cells were
xed and permeabilized before labelling for CCR5 (red) and b-arres-
tins (green), as described previously.
88
Scale bar 5 lm.
CCR5
All
Golgi
Late endosomes
& lysosomes
Nucleus
CCL5 (RANTES)
95
AOP-RANTES
95
PSC-RANTES
102
MET-RANTES
103
TGN
Recycling
endosomes endosomes
Early sorting
Figure 3. Different trafcking routes proposed for agonist-treated
CCR5. Following agonist-stimulation, internalized CCR5 receptors
are transported through the early endocytic pathway towards recy-
cling and avoiding degradation. However, there are suggestions that
the route followed by CCR5 may be ligand-dependent, as summa-
rized here for the chemokine CCL5 and three of its derivatives.
2011 The Authors. Immunology 2011 Blackwell Publishing Ltd, Immunology, 134, 246256 251
Chemokine receptor regulation
Cmo funcionan
in vivo?
Zlotnik A, Burkhardt AM, Homey B. Homeostatic chemokine
receptors and organ-specic metastasis. Nature Publishing
Group. Nature Publishing Group; 2011Sep.1;11(9):597606.
Figure 1, The human body contains cellular highways
through which cells travel to reach dierent sites or organs in
the body; p. 598.
Nature keviews Immuno|ogy
Sloon
Smull inosino
Lunq
Livor
Lymb
nodos
5kin
5econdary |ymphoid tissues
5ma|| intestine
one marrow
Liboliul
coll
CCL2S
CCL2S
8ruin
CXCL12
CXCL12
CCP0
41
T coll

CCL21
CCL21
CCL10
CCL21
CXCL13
CXCL12
Dormul
coll
CCP10
CLA
T coll
Sociulizod
sromul colls
Lndoboliul
coll
8lood
CXCP4
CD34
HSC
HFV
T coll
8 coll
CCP1
T coll
CCL21
CCL10
one
marrow
niche
1 ce|| rone
CXCL12
CXCP4
unuqonis
CXCPS
CXCL13
CXCPS
CCL10
CCL21
CXCL13
liquro 1 The human body contains cellular highways through which cells travel to reach different sites or
organs in the body. Undor normul condiions, bomoosuic cbomolinos roquluo collulur rullic by dirocinq colls bu
oxross coruin cbomolino rocoors o socilic locuions vboro boir cbomolino liqunds uro oxrossod. Somo collulur
biqbvuys uro sbovn boro. ln bo slin, CC-cbomolino liqund21 (CCL21) rocruis cuunoous loulocyo uniqon (CLA)
+
T colls
bu oxross CC-cbomolino rocoor10 (CCP10). ln bo smull inosino, CCL2S rocruis T colls bu oxross 41 inoqrin
und CCP0. ln bo lymb nodos, CCL21 und CCL10 rocrui CCP1
+
T colls, und CXC-cbomolino liqund13 (CXCL13)
rocruis CXC-cbomolino rocoorS (CXCPS)
+
8 und T colls. ln bo bono murrov, CXCL12 rocruis CXCP4
+
buomuooioic
som colls (HSCs) und crouos u 'nicbo' lor boso colls. lnbibiors ol bo CXCL12-CXCP4 inorucion 'liboruo' HSCs lrom
boir bono murrov nicbo, und boso colls cun bon onor bo circuluion. Tboso collulur biqbvuys cun ulso bo usod by
cuncor colls durinq moususis, lor oxumlo vbon CCP1-oxrossinq umour colls miqruo ino lymb nodos or vbon
CCP0-oxrossinq molunomu colls miqruo o bo smull inosino. Obor imorun biqbvuys includo boso uccossod by
umour colls oxrossinq CXCP4, vbicb modiuos rocruimon ol umour colls o sios vboro CXCL12 is biqbly oxrossod,
sucb us bo lunq, livor, bono murrov und bruin. HLV, biqb ondoboliul vonulo.
they are involved in the recruitment of specific popu-
lations of lymphoid cells that have prominent roles in
either the innate or acquired immune responses that
develop in certain tissues. Some examples include the
CCL25CCR9 axis, which is crucial for the initial seed-
ing of progenitor Tcells to the thymus and for intra-
thymic thymocyte development
18,19
. CCL25 is also
highly expressed in the small intestine and promotes the
recruitment of CCR9
+
Tcells that express 47 integrin
to the gut
20
. These cells are believed to have an important
role in inflammatory intestinal diseases
21
. Other exam-
ples include the CCL27CCR10 axis, which specifically
recruits cutaneous lymphocyte antigen (CLA)
+
Tcells to
the skin
22
, and the CCL28CCR10 axis, which recruits
IgA
+
plasmablasts to the mammary gland at the onset of
lactation
23
(FIG.1).
The model that emerges from these observations is
one in which homeostatic chemokines and their recep-
tors play a key part in the genesis and organization of
the immune system by recruiting specific precursor
cells and promoting the formation of lymphoid tissues,
or by recruiting discrete lymphoid populations to spe-
cific organs to promote regional immune responses. A
suitable metaphor to understand these concepts is that
mammalian bodies have cellular highways (FIG.1) that
are regulated by chemokines and their receptors, the
function of which is to control the migration of various
cell types to specific locations within the body. Although
immune cells are the best examples to illustrate these
mechanisms, the migration of other cell types can also be
controlled by chemokines, as long as these cells express
the appropriate chemokine receptors.
REVI EWS
598 | SEPTEMBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
2011 Macmillan Publishers Limited. All rights reserved
Cmo funcionan
in vivo?
2006 Nature Publishing Group

Plasma cell
Pre-pro-
B cell
HSC
Medullary
vascular sinus
Pro-B cell
Pre-B cell
Endothelial
cell
Immature
B cell
CXCL12
IL-7
Osteoblast
IL-7-
expressing
cell
CXCL12
hi
reticular
cell
Bone
function in vivo. Recently, several cell types have been
implicated in providing the specific cellular niches for
B-cell development (FIG. 3), as described next.
Osteoblasts. Osteoblasts are highly specialized cells that
are responsible for synthesis, deposition and mineraliza-
tion of the extracellular matrix of bone. They are thought
to arise from mesenchymal stem cells (MSCs) and have
an important role in the development of all bones. Both
in vitro and in vivo studies indicate that osteoblasts are
also essential regulators of the development of all blood
cells, including B cells in the bone marrow, and such
studies indicate that osteoblasts might function as niches
for HSCs
7,8,98100
. In agreement with these observations,
a recent study has shown that primary mouse osteo-
blasts can support early B-cell lymphopoiesis in in vitro
cultures
101
. Furthermore, the observations that human
osteoblasts express CXCL12 (REF. 102) and that primary
mouse osteoblasts express RANKL
94
, indicate that
osteoblasts might participate in B-cell lymphopoiesis.
CXCL12
hi
reticular cells. The observation that CXCL12
is required for the generation of the earliest identifi-
able B-cell precursors, pre-pro-B cells
33
, indicates that
CXCL12 might attract and/or tether these precursors
immediately after commitment to the B-cell lineage in
an appropriate microenvironment for their develop-
ment. Therefore, the localization of cells expressing
CXCL12 in the bone marrow has been analysed using
mice in which the gene encoding green fluorescent
protein (GFP) was knocked into the Cxcl12 locus
(Cxcl12GFP knock-in mice)
10
. High levels of expression
of CXCL12GFP was seen in a small population of stro-
mal cells (termed CXCL12
hi
reticular cells). Lower levels
of CXCL12GFP expression were found in some other
fibroblast-like cells. CXCL12
hi
reticular cells occurred
singly and were uniformly scattered throughout the
bone marrow
10
. Within the bone marrow, reticular cells
as well as sinusoid endothelial cells have been shown to
express vascular cell-adhesion molecule 1 (VCAM1)
103
.
Almost all of the CXCL12
hi
reticular cells expressed
VCAM1, and constituted ~20% of the total VCAM1
+

cells
10
, supporting the idea that CXCL12
hi
reticular cells
are a subset of bone-marrow reticular cells. In addition,
almost all of the CXCL12
hi
reticular cells lacked expres-
sion of platelet/endothelial cell-adhesion molecule 1
(PECAM1) and phenotypic markers of osteoblasts,
including osteopontin and osteocalcin, indicating that
CXCL12
hi
reticular cells are different from endothelial
cells and osteoblasts. Consistent with this observation,
CXCL12
hi
reticular cells are located away from the
bone surface. The molecular mechanisms that control
CXCL12 expression in CXCL12
hi
reticular cells remain
unclear. It has however been shown that the cytokine
granulocyte colony-stimulating factor (G-CSF) induces
a reduction of CXCL12 expression in the bone mar-
row
104,105
. Further studies investigating the effect of
environmental factors, including G-CSF, on CXCL12
hi

reticular cells are needed.
IL-7-expressing cells. Cells that express IL-7 in the bone
marrow have been examined by immunohistochemi-
cal analysis. Staining with an IL-7-specific antibody
was observed for some fibroblast-like cells, which also
expressed VCAM1 (REF. 10). These IL-7-expressing cells
were scattered throughout bone marrow
10,106
. Because both
CXCL12 and IL-7 are required for B-cell lympho poiesis
and they have synergistic or additive functions on B-cell
precursors, expression of both these factors by stromal
cells would be expected
33,36,37,65
. However, analysis of bone
marrow from Cxcl12GFP knock-in mice showed that
CXCL12
hi
reticular cells did not stain with IL-7-specific
antibody and that the IL-7-expressing cells were located
away from the CXCL12
hi
reticular cells
10
, indicating that
these cells might represent distinct stromal-cell subsets.
It has previously been shown that IL-7 expression by
a stromal-cell line is induced by stimuli, including
several cytokines, and B-cell precursors
107
. How IL-7
expression is regulated in cellular niches comprised of
IL-7-expressing cells, for example, is an important issue
for the future.
B-cell-precursorniche interactions
To clarify the functions of niches for B-cell development,
it is important to characterize the association of these
niches with B cells and their progenitors. To achieve this,
the association between multipotential haematopoietic
progenitors and CXCL12
hi
reticular cells was analysed
10
.
Immunohistochemical staining analysis using antibod-
ies against KIT and SCA1 was carried out to visualize
multipotential haematopoietic progenitors, including
Figure 3 | Candidates for cellular niches for B-cell development and a model of the
movement of B cells and their precursors in the bone marrow. In this model, the
intermediate precursor cells between haematopoeitic stem cells (HSCS) which are
located near the osteoblasts
7,8
, endothelial cells
113
or CXC-chemokine ligand 12
hi

(CXCL12
hi
) reticular cells
10
and pre-pro-B cells would move towards CXCL12
hi

reticular cells. Pre-pro-B cells associate with CXCL12
hi
reticular cells, whereas
pro-B cells move away and instead adjoin interleukin-7 (IL-7)-expressing cells
10
.
Subsequently, pre-B cells leave IL-7-expressing cells
10
. B cells expressing cell-surface
IgM exit the bone marrow and enter the blood to reach the spleen, where they mature
into peripheral mature B cells. End-stage B cells (plasma cells) again home to CXCL12
hi

reticular cells in the bone marrow
10
.
REVI EWS
NATURE REVIEWS | IMMUNOLOGY VOLUME 6 | FEBRUARY 2006 | 113
FOCUS ON EARLY LYMPHOCYTE DEVELOPMENT
Nagasawa T. Microenvironmental niches in the bone marrow
requi red for B-cel l devel opment. Nature Revi ews
Immunology. 2006Feb.;6(2):10716. Figure 3, Candidates for
cellular niches for B-cell development and a model of the
movement of B cells and their precursos in the bone marrow.
Cmo funcionan
in vivo?
Chemokines and thymocyte development
Table 1. Expression of chemokines and chemokine receptors in the thymus
Receptor Expression pattern Ligand Expression pattern
CXC family
CXCR3 DN TCR
+
MIG (CXCL9) Stroma
stimulated DN TCR
IP-10 (CXCL10, CRG-2) Stroma, stimulated thymocytes

ITAC (CXCL11) NS
CXCR4 DN > DP > SP SDF-1/ (CXCL12) Fibroblasts, outer cortex
CC family
CCR3 DP, SP Eotaxin (CCL11) Hassals corpuscles, DC-like cells
CCR4 CD69
+
DP TARC (CCL17) DC
CD69
+
CD62L
lo
CD4SP MDC (CCL22, STCP-1/h, ABCD-1) Medullary epithelial cells
CCR5 DP, SP MIP-1 (CCL3) DN
MIP-1 (CCL4) NS
RANTES (CCL5) NS
CCR6 Thymic B cells, CD4SP (low) LARC (CCL20, MIP-3, Exodus-1) NS
CCR7 SP ELC (CCL19, MIP-3, Exodus-3) NS
SLC (CCL21, 6Ckine, Exodus-2, Medullary stromal cells,
TCA-4/m) vessels at CM junction
CCR8 DP (low), CD4SP TCA-3/h, I-309/m (CCL1) NS
CCR9 DP, SP TECK (CCL25) Cortical > medullary epithelial cells,
medullary DC
Unknown PARC (CCL18, DC-CK1) NS
XCR family
XCR1 DN and/or CD8SP Lymphotactin (XCL-1, SCM-1, ATAC) DN, activated CD8SP
The expression pattern is listed for the principal chemokines and their receptors present in the thymus. Expression data
for some chemokine receptors is inferred from chemotaxis assay results. Alternate names of chemokines are indicated in
parentheses, some of which are specic human (h) or murine (m) forms. Abbreviations include: NS (non-specied), CM
(corticomedullary).
markers to trace thymic reconstitution by puried
DN thymocytes.
2527
The rst cycling cells to enter
the thymus were present in vessels of the inner cortex,
corticomedullary junction, and medulla. Cortical
capillaries are surrounded by an impermeable ep-
ithelial layer and are thought to be sequestered from
developing thymocytes. In contrast, post-capillary
venules in the medulla and corticomedullary junc-
tion lie within a perivascular space in close proximity
to thymocytes, making them the likely site for entry of
thymocyte progenitors (Figure 1).
2830
Once inside
the thymic parenchyma, early DN cells make their
way to the cortical subcapsular region, apparently
doing so in the face of more mature DN and DP cells
that are moving in the opposite direction.
While a number of chemokines are expressed at
high levels in the fetal or adult thymus,
31, 32
the traf-
cking of early DN progenitors has been best studied
for the CXC chemokine SDF-1. DN thymocytes in
humans and mice express CXCR4 and efciently
respond to SDF-1 in chemotaxis assays.
13, 3335
While
SDF-1 is expressed at high levels throughout the fetal
thymus, expression in newborn and adult mice ap-
pears to be restricted to the outer cortex, specically
in thymic broblasts by RT-PCR.
35, 36
DN cells mature
through four stages dened by expression of CD44
(a proteoglycan adhesion molecule) and CD25,
proceeding in the following order: CD44
+
CD25
to
CD44
+
CD25
+
to CD44
CD25
+
to CD44
CD25
.
37
SDF-1/CXCR4 have been implicated in the hom-
ing of progenitors to the thymus as some groups
have observed that very early CD44
+
CD25
DN
thymocytes migrate to SDF-1. For example, murine
CD44
+
CD25
thymocytes
13
or human CD34
+
cord
blood cells
38
are reported to migrate to SDF-1.
However, other data suggest that SDF-1/CXCR4 may
direct recent immigrants to the outer cortex, or
promote the association of DN cells with subcorti-
cal stromal elements after arrival.
35, 39
This would
be more consistent with the proposed function
447
Norment AM, Bevan MJ. Role of chemokines in thymocyte
development. Semin. Immunol. 2000Oct.;12(5):44555.
Table1, Expression of chemokines and chemokine receptors
in the thymus; p. 447.
Cmo funcionan
in vivo?
Andrian von UH, Mackay CR. T-Cell Function and Migration:
Two Sides of the Same Coin. Mackay IR, Rosen FS, editors.
N Engl J Med. 2000Oct.5;343(14):102034. Table 2, Role of
chemokine receptors and their ligands in the migration of T
cells; p. 1026.
1026 October 5, 2000
The New Engl and Jour nal of Medi ci ne
*CCR denotes receptor for CC chemokine, CXCR receptor for CXC chemokine, SLC secondary lymphoid-tissue che-
mokine, TCA-4 thymus-derived chemotactic agent 4, ELC EpsteinBarr virusinduced gene 1 ligand chemokine, MIP
macrophage inflammatory protein, SDF-1a stroma-derived factor 1a, TARC thymus- and activation-regulated chemokine,
MDC-1 macrophage-derived chemokine 1, TECK thymus-expressed chemokine, MCP monocyte chemotactic protein,
RANTES regulated on activation normal T cell expressed and secreted, Th1 type 1 helper T cells, Th2 type 2 helper
T cells, IP-10 inducible protein of 10 kd, Mig monokine induced by interferon-g, I-TAC interferon-inducible T cell alpha
chemoattractant, HCC human CC chemokine, BLC B-lymphocyte chemoattractant, BCA-1 B-cellattracting chemokine
1, LARC liver- and activation-regulated chemokine, MPIF-1 myeloid progenitor inhibitory factor 1, GCP-2 granulocyte
chemotactic protein 2, Gro growth-related activity, Nap-2 neutrophil-activating protein 2, ENA-78 epithelial-cellderived
neutrophil attractant 78, SCM-1b single C motif 1b, GPR-2 G-proteincoupled receptor 2, CTACK cutaneous T cell
attracting chemokine, and ILC interleukin-11 receptor alpha-locus chemokine.
The physiologic relevance of several chemokine receptors varies. For instance, CCR9 functions in the homing of pro-
thymocytes to the thymus and in the migration of T cells to the gut. CXCR4 is widely expressed and appears to have
multiple roles.
The most common names that are currently in use for human chemokines are given here, with frequently used alter-
native names shown in parentheses. Recently, a more systematic classification for chemokines has been proposed that is
based on the nomenclature for the corresponding chemokine genes.
22
TABLE 2. ROLE OF CHEMOKINE RECEPTORS AND THEIR LIGANDS IN THE MIGRATION OF T CELLS.*
BIOLOGIC ACTIVITY
CHEMOKINE
RECEPTORS PREDOMINANT CHEMOKINE AGONISTS
Migration of naive T cells to lymph nodes
and Peyers patches
Migration of naive T cells within
lymphoid tissues
CCR7
CXCR4
SLC (also called TCA-4, 6C-kine, exodus-2),
ELC (also called MIP-3b)
SDF-1a
Migration of memory T cells to
lymphoid tissues
Migration of memory T cells to the skin
Migration of memory T cells to the gut
Migration of memory T cells to
sites of inflammation
CCR7
CCR4
CCR9
CCR2
CCR5
TARC, MDC-1
TECK
MCP-1, 3, and 4
RANTES, MIP-1a and 1b
Migration of effector T cells (Th1)
Migration of effector T cells (Th2)
CCR2
CCR5
CXCR3
CCR3
CCR4
CCR8
CXCR4
MCP-1, 3, and 4
1P-10, Mig, I-TAC
Eotaxin-1, 2, and 3; RANTES; MCP-2, 3, and 4; HCC-2
TARC, MDC-1
I-309
SDF-1a
Migration of B cells CCR7
CXCR4
CXCR5
SDF-1a
BLC (also called BCA-1)
Migration of dendritic cells to
lymphoid tissues
Migration of dendritic cells to normal skin
Migration of dendritic cells to
sites of inflammation
CCR7
CCR6
CCR1
CCR2
CCR5
CXCR1
MIP-3a (also called LARC, exodus-1)
RANTES; MIP-1a; MCP-3; HCC-1, 2, and 4; MPIF-1
MCP-1, 3, and 4
Interleukin-8, GCP-2
Recruitment of monocytes CCR1
CCR2
CCR5
CCR8
CXCR1
CX
3
CR1
RANTES; MIP-1a; MCP-3; HCC-1, 2, and 4; MPIF-1
MCP-1, 3, and 4
I-309
Fraktalkine (also called neurotactin)
Recruitment of neutrophils CXCR1
CXCR2
Interleukin-8, Groa, b, and g; Nap-2; GCP-2; ENA-78
Recruitment of eosinophils CCR3 Eotaxin-1, 2 and 3; RANTES; MCP-2, 3, and 4; HCC-2
Migration of hematopoietic progenitor
cells and B-cell development
CXCR4 SDF-1a
Function unknown XCR1
CCX CKR
GPR-2
D6
Lymphotactin, SCM-1b
ELC (also called MIP-3b), TECK
CTACK (also called ILC)
Multiple CC chemokines
Downloaded from www.nejm.org on April 26, 2010 . Copyright 2000 Massachusetts Medical Society. All rights reserved.
Cmo funcionan
in vivo?
L. Ohl et al. / Seminars in Immunology 15 (2003) 249255 251
5. Lymph nodes
Lymph nodes are placed at strategic locations within the
body to lter lymph from skin and solid organs for the
presence of foreign material. They show a characteristic
segregation of B cells to the subcapusular area and the T
cell-rich zone situated beneath in the cortex. The majority
of dendritic cells locate to the T cell areas, while follicular
dendritic cells (FDC) are situated within the B cell follicles.
Although soluble antigen can be transported passively along
the lymph drainage, recent evidence demonstrates that con-
siderable amounts of foreign material is taken up and pro-
cessed by antigen presenting cells (APC) in the periphery.
Subsequently, these cells enter the subcapsular sinus of the
draining lymph node via afferent lymphatics. From there
APC migrate to the T cell-rich area where they present
the processed material to nave T cells. Data derived from
gene targeted mice demonstrate that the chemokine receptor
CCR7 is essentially required for antigen-triggered mobiliza-
tion and migration of Langerhans cells residing within of
the epidermis [20]. While APC and memory T cells reach
the lymph node by afferent lymphatics, the majority of
nave B and T cells enter the lymph node T cell area through
venules decorated with a single layer of cubic, highly spe-
cialized endothelium (high endothelia venules, HEV). Two
ligands for CCR7, CCL19 and CCL21 have been identi-
ed on the luminal site of HEV [21,22], both of which are
missing at this place in a naturally occurring mouse mutant,
paucity of lymph node T cells (plt) [23]. These animals as
well as CCR7-decient mice exert a profound defect in the
migration (homing) of T cells into LN demonstrating the
importance of CCR7 and its ligands for emigration of lym-
phocytes from the blood stream into secondary lymphoid
organs [20,23]. Since CCL19 and CCL21 are also ex-
pressed by various cell populations including APCs within
lymph nodes, it seems that CCR7 signaling also mediates
the contact of T cells to dendritic cells within the T cell
area. This interaction is a prerequisite to specically ac-
tivate those T cells carrying a T cell receptor tting to
the presented foreign material. Recent data demonstrate
that CXCR4 contributes to B cell but not T cell homing
through HEV [24]. This nding might help to explain why
CCR7-decient mice show normal B cell counts despite the
observation that CCR7-decient B cells display impaired
homing to LN following short-term adoptive transfer [20].
Once within the lymph node B cell take advantage of an-
other chemokine receptor, CXCR5, to access the B cell
follicles [25]. There FDC express high levels of CXCL13
the only known activator of CXCR5 [26,27]. Data obtained
from CXCR5- as well as CXCL13-decient mice corrobo-
rate the importance of this chemokine/chemokine receptor
pair, since lymph nodes of either animal lack FDC-rich
B cell follicles [25,28]. CXCR5 is also expressed on a
small sub-population of activated CD4
+
T cells [29], which
primarily localize to the B cell rich areas and support im-
munoglobulin secretion [22,30]. Based on the characteristic
localization and functional properties, these cells were
termed Follicular B helper T cells (T
FH
). In peripheral
blood, T
FH
cells co-express CCR7 which permits entry
into secondary lymphoid organs. Once there, they loose
expression of CCR7, which allows them to localize to B
cell follicles and germinal centers where they express high
levels of CD40L, a co-stimulatory molecule required for B
cell activation, and ICOS, a co-stimulatory molecule of the
CD28 family [22,30]. The expression of CXCR5 on T cells
is a striking example illustrating how chemokine receptors
determine the localization and the concomitant function
of T cells. Therefore, chemokine receptors are markers to
characterize dened functional stages of T cells within this
extremely heterogeneous cell population [31].
6. Spleen
While antigens reach the lymph nodes by afferent lym-
phatics, the spleen is specialized for uptake of blood born
antigens and pathogens. The two major compartments of the
spleen are the red pulp and the white pulp cords (Fig. 1).
The red but not the white pulp is permeated by a reticular
meshwork consisting of bers and reticular cells. Red and
white pulp are separated by marginal sinuses surrounding
the white pulp cords and marginal zones locating adjacent
to the marginal sinus [32].
Blood enters the spleen through afferent arterial vessels,
the central arterioles embedded in the white pulp. Whereas
some of the radial branches of this arteri divert into cap-
illaries within the follicles, others extend to the marginal
Fig. 1. Cell migration within the spleen: the migration events within the
spleen are dependent on chemokines expressed in dened compartments
and the corresponding chemokine receptors found on traveling target
cells. Chemokine receptors expressed on the migrating cells are indicated
by the receptor name on the dotted line with the arrow heading to the
compartment to which the cells migrate. RP, red pulp; MZ, marginal zone;
MS, marginal sinus; MZBC, marginal zone bridging channels; B, B cell
rich follicle; GC, germinal center; T, T cell-rich peri arteriolar lymphoid
sheath; V, venous sinuses.
L. Ohl et al. / Seminars in Immunology 15 (2003) 249255 253
Fig. 2. Chemokine receptors expressed on dened leukocyte populations
of Peyers patches believed to participate in guiding these cells to the
various compartments. High endothelial venules (HEV) in the T cell
rich area (T) express CCL19 and CCL21 on their luminal surface, while
HEV in the B cell rich follicle (B) express CXCL13. MC, microfold
cells; SED, sub-epithelial dome; GC, germinal center; IEL, intraepithelial
lymphocytes; LPC, lamina propria cells.
and migration of T cells into the T cell zone [20,25,28].
However, unlike in lymph nodes, the entry of B cells into PP
is also facilitated via interaction of CXCR5 on B cells and
CXCL13 expressed on endothelial cells [24]. Besides HEV
locating to the T cell zone PP harbour CXCL13-positive
HEV. These are positioned in the follicular region and
seem to facilitate the migration of B cells into this organ
[24]. In addition to the organized lymphoid structures in
the gut, single immune cells are dispersed throughout this
tissue. These cells are either found associated with the
epithelium (intraepithelial lympocytesIEL) or inside the
lamina propria (lamina propria cellsLPC). Most IEL are
CD8
+
T cells that express CCR9 and, in humans, have
also been shown to express CXCR3 and CCR5 [24,43,44].
Inactivation of CCR9 revealed a decrease in a subset of
intestinal T cells [3,45]. Additionally, CCL25/CCR9
has been suggested to be crucial for the migration of
re-circulating CD8
+
cells from the periphery into the
gut upon antigenic activation [46]. These ndings show that
CCR9 is important for the homeostasis of IEL although
the precise mechanism underlying this phenomenon is still
unknown.
Within the lamina propria a large fraction of IgA-secreting
plasma cells is present that express CCR10 and CCR9.
IgA-secreting plasma cells have been shown to migrate to-
wards to CCL25 [47,48]. Taken together these data sug-
gest that in addition to organized GALT, isolated leukocytes
are also largely dependent on dened chemokine receptors
and differential expression of chemokines by epithelial cells.
This may allow the high degree of diversication between
different sub-compartments as observed in the small and
large intestine.
The second largest MALT is found in the lung and orga-
nized lymphoid follicles have been described in the bronchi
of at least some species. As described for the gut, im-
mune cells in the lung can be found dispersed in the lamina
propria and between epithelial cells. However, signicant
sub-populations also reside in the interstitium as well as in
the intravascular and in the bronchoalveolar space [49]. Up-
take of inhaled antigens in the airways is achieved by imma-
ture DC residing in the broncheoalveolar cavity [50]. Upon
antigen encounter, airway DC seem to up-regulate CCR7
and migrate to the draining mediastinal lymph node [51]
suggesting that airway DC use the same chemokine recep-
tors as skin DC. Additionally, CCR6 decient mice which
display a reduced responsiveness to oral antigens also exert
a reduced allergic airway response although CCL20 is not
constitutively expressed in the lung [52].
Lung T cells share the expression of some chemokine re-
ceptors such as CXCR3 and CCR5 with intestinal T cells but
do not express others such as CCR9. However, it should be
noted that it is currently unknown whether these receptors
actually contribute to normal lung homeostasis [53]. Thus,
although aspects of chemokine triggered cell behaviour are
shared among the different MALT systems the differential
combination of chemokines and adhesion factors such as in-
tegrins allow for a high degree of specication and diversi-
cation in leukocyte populations of MALT.
8. Extralymphatic tissue (the lighthouse-effect)
In some autoimmune disorders, the establishment of the
chronic inammatory syndrome is frequently accompanied
by the formation of lymphoid-like structures in the diseased
tissue, e.g. in synovium in the case of Rheumatoid Arthritis,
thyroid gland in Hashimotos thyreoiditis, or salivary gland
in Sjogrens syndrome [54]. The cause of these extralym-
phatic tissues is thought to be the sustained expression of
lymphotoxins or/and chemokines such as CCL21, CXCL12,
and CXCL13, at the sites of inammation. Glandular fea-
tures of the affected organs may facilitate this process: the
transgenic expression of CCL21 in pancreas but not in skin
provokes the formation of lymph node-like compartments
[55], revealing that the presence of a single homeostatic
chemokine in an ambient non-lymphoid parenchyma already
sufces to create neo-lymphoid structures resembling those
observed in lymphoid organs. Is there any better proof to
substantiate the importance of chemokines as organizers of
lymphoid organs?
Acknowledgements
We apologize to all our colleagues whose work could not
be cited due to space limitations. This work was supported
in part by the Deutsche Forschungsgemeinschaft Grant Fo
334/1-1 to RF.
Ohl L, Bernhardt G, Pabst O, Frster R. Chemokines as
organizers of primary and secondary lymphoid organs.
Semin. Immunol. 2003Oct.;15(5):24955. Figure 1, Cell
migration within the spleen; p. 252.
dem. Figure 2, Chemokine receptors expressed on dened
leukocyte populations; p. 253.
Y la clnica?
Garin A, Proudfoot AEI. Chemokines as targets for therapy.
Experimental Cell Research. Elsevier Inc; 2011Mar.10;317(5):
60212. Figure 1, The association of chemokines and their
receptors to disease; p. 603.
Introduction
Chemokines are the key drivers in cellular recruitment, being at the
heart of both innate and adaptive immunity and playing a significant
physiological role in lymphoid tissue ontogenesis, organogenesis,
vasculogenesis and tissue repair. The fine tuning of the regulation of
the chemokine system is essential and aberrant expression is often
associated with pathological processes. Indeed, genetic mutations
leading to impaired chemokine function (such as CCR532 or
CX3CR1-I249 M280) are reported to cause an impairment of
pathogen clearance [13] or tumor regression [4]. On the contrary,
reduced resolution of inflammation or increased chemokine expres-
sion is associated with chronic tissue damage [5], auto-immunity
[6,7] and tumor development [8,9]. Validation of the role of this
family indiseasehas beenprovidedbypharmacological studies using
genetically modified animals or neutralization of chemokines and/or
chemokine receptors. Yet, despite all the evidence of the importance
of this family in diseases, trials to date have shown a high attrition
rate, which interestingly has not stopped industry fromstarting new
programs and launching clinical trials. In this review, we will discuss
thevalue andrationale of targetingthechemokinesystemfor specific
diseases to provide novel effective medicines.
As the first mediators of cell recruitment, chemokines are major
players ininflammation. The inflammatory response is necessary to
eradicate pathogens but a lack of resolution of this response will
result in pathological inflammation and diseases. Evidence linking
chemokine receptors and ligands to these diseases has been drawn
from several sources and experimental approaches, and is
summarized in Fig. 1. The majority of disease association is derived
from animal models, but in parallel to the animal data, human
genetic associations and correlation with expression levels from
human disease samples have confirmed the animal data. We will
address some of these disease associations in this review, although
we will not be able to cover all disease conditions in which the
chemokine system is involved, to support our postulate for the
rationale of targeting the chemokine system to treat diseases.
Inflammatory diseases
Atherosclerosis
Cardiovascular disease is the leading cause of death in the
developed world, and atherosclerosis is one of the most exten-
sively studied inflammatory disorders for which the involvement
Fig. 1 The association of chemokines and their receptors to disease. A selection of disease association obtained fromanimal models
using gene deletions, neutralizing antibodies and receptor antagonists, as well as expression data in human samples and positive
results from clinical trials.
a
CP 481,715 showed positive results in a PhIb trial, and MDX-1100 (anti-CXCL10) met its primary
endpoints in a Ph II trial;
b
MLN1202 (anti-CCR2) and showed a trend in a small Ph II trial abbreviations: Sep, Sepsis; RA, Rheumatoid
arthritis; T, Transplant; IBD, Inflammatory Bowel Disease; Onc, Oncology; SLE, Systemic Lupus; MS, Multiple Sclerosis; Ath Scl,
Atherosclerosis; COPD: Chronic Obstructive Pulmonary Disease; AMD, Acute macular degeneration; NP, Neuropathic pain; Asth,
Asthma; At. Derm, Atopic dermatitis; Hep, Hepatitis; Panc, Pancreatitis; Pso, Psoriasis; GVHD, Graft vs Host disease.
603 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 6 0 2 6 1 2
Next class: Sistema inmune innato humoral
Paul WE. Fundamental Immunology. 6th ed. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins; 2008.
Captulo 33
Roitt IM, Brostoff J, Male DK. Immunology. 7th ed. Edinburgh ; New York: Mosby; 2009.
Captulo 4
Kindt TJ, Goldsby RA, Osborne BA, Kuby J. Kuby immunology. 6th ed. New York: W.H. Freeman; 2007.
Captulo 13
Bellanti JA, Escobar-Gutirrez A, Tsokos GC. Immunology IV: clinical applications in health and disease.
Bethesda, Md.: I Care Press; 2012.
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