Biological and transductional effects of allosteric antagonists on the activity of chemoattractant receptors

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1.INTRODUCTION 
 
 
1.1 The chemokine system 
 
Over 40 chemokines have been identified to date
1
. Chemokines are produced by a 
variety of cell types either constituvely or in response to inflammatory stimuli, the 
biological activities of chemokines range from the control of leukocyte trafficking in 
basal and inflammatory conditions to regulation of hematopoiesis, angiogenesis, 
tissue architecture and organogenesis. 
The basis for such diversified activities rests, on one hand, upon the ubiquitous nature 
of chemokine production and chemokine receptor expression. Indeed virtually every 
cell type can produce chemokines and expresses a unique combination of chemokine 
receptors. On the other hand, chemokine receptors make use of a flexible and 
complex network of intracellular signaling machineries that can regulate a variety of 
cellular functions ranging from cell migration, growth, differentiation and death
2
. 
 
1.2 Structure and biological function of chemokines  
Chemokines are small secreted proteins with the molecular weights in the range of 8-
12-KDa with 20 to 70 percent homology in amino acids sequences. They have been 
subdivided into families based on structural and genetic considerations; structurally 
they are similar, having at least three β-pleated sheets and a C-terminal α-helix, with 
disulfide bonds stabilizing overall topology (Figure 1)
1, 3
.  
Most chemokines have four characteristics cysteines (cys) in highly conserved 
positions and depending on the motif displayed by the first two cysteins, they have 
been classified into CXC or alpha (α)-chemokines, CC or beta (β), C or gamma (γ), 
and CX3C or delta (δ) chemokines classes
1, 3
. The only exception to the cysteins rule 
is lymphotactin, which has only two cysteins. In addition the CXC or alpha subfamily
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has been divided into two groups depending on the presence of the ELR motif 
(glutamate-leucine-arginine) preceding the first cys: the ELR-CXC chemokines and the 
no-ELR-CXC chemokines
4
 (Figure 2). 
After translation, most chemokines are secreted from the cell, with the exception of 
CX3CL1 and CXCL16, which are tethered to the extracellular surface. In the case of 
CXC3CL1 can act not only as chemoattractant but also as adhesion molecule
5
. 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Chemokines family. Chemokines are divided 
into families based on structural consideration. Structurally 
they are similar, having at least three β-pleated sheets 
(indicated as β1-3) and a C-terminal α-helix. Chemokines 
have at least four cysteins in conserved positions. Except 
lymphotactin C family. (Rollins, B.J. Blood, 1997).
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Important aspects concerning chemokines include their biological activities and the 
chromosomal location of the genes that encode them. They are best known from 
mammals, but chemokine genes have also been found in chicken, zebrafish, shark 
and jawless fish genomes, and possible homologs of chemokine receptor have been 
reported in nematodes. Careful analysis of the members of the superfamily and their 
receptors shows a logical order to its genomic organization and function, which in turn 
is the result of evolutionary pressures
6
. 
The chemokines have been divided into two major groups based on their expression 
patterns and functions. Those that are expressed by cells of the immune system 
(leukocytes) or other cells (epithelial and endothelial cells, fibroblasts and so on) only 
upon activation belong to the “inflammatory” class, whereas those that are expressed 
in discrete location in the absence of apparent activating stimuli have been classified 
as “homeostatic”. However, the genomic organization has allowed dividing into two 
alternative groups: those whose genes are located in large clusters at particular 
chromosomal location (the “major-clusters”, CC and CXC chemokines genes) and the 
“no cluster” or “mini-cluster”. This common evolutionary origin suggests that the 
cluster chemokines are a group of proteins sharing a common primary function. In the 
case of the chemokines encoded by CXC GRO cluster on chromosome 4, which in 
humans include CXCL1 - CXCL8, the primary function is the regulation of neutrophils 
recruitment to inflammatory sites mediated by through interaction with CXCR1 and 
CXCR2 receptors. Similarly, the main function of the cytokines encoded in the MIP 
(Macrophage Inflammatory Protein) and MCP (Monocyte Chemotactic Protein) clusters 
of CC chemokines in human chromosome 17, which includes CCL1-CCL16, CCL18 and 
CCL23 is the recruitment of monocytes, subset of T cells, and eosinophils, to sites 
where inflammation is developing, through their interaction with CCR1, CCR2, CCR3 
and/ or CCR5 (Figure 2).
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By contrast, the non cluster or mini-cluster chemokines are relatively conserved 
between species and tend not to act on multiple receptors (Figure 2). Indeed several 
of these have a single ligand-receptor relationship, such as CCL25-CCR9 or CXCL13-
CXCR5. These particular chemokine ligand-receptor pairs probably have a pivotal roles 
in the development of the organism or in the function of physiological systems 
necessary for the organism’ survival to reproductive age. For example, CXCR4 
deficient and CXCL12-deficient mice both have various defects in critical organs, such 
as the heart, brain and bone marrow
7
.   Therefore, throughout evolution, several non-
cluster chemokines have participated in organogenesis, and their critical functions 
must be conserved in order for the species to survive
6, 8
.  
Functionally, the CXC chemokines play a key role in acute inflammation
9
. The 
prototypic CXC chemokine is CXCL8, which was purified by several groups as a 
monocytes-derived factor that attracts neutrophils, but no monocytes in Boyden 
chamber assay. Several other CXC chemokines have been described as potent 
neutrophil chemoattractants, and structure/activity analyses show that this property 
depends on the presence of a three amino acids motif ELR between the N-terminus 
and the first cysteine. However, these amino acids must appear in position close to 
the protein’s N-termini. For example PBP (platelet basic protein) and two of its N-
terminally truncated derivatives, CTAPIII and β –thromboglobulin, have very weak 
neutrophil chemoattractant activity despite the presence of ELR. Only NAP-2, a further 
truncated product in which ELR appears close to the N-terminus, is an active PBP-
derived neutrophil attractant
8
. 
CXCL8 is produced by a variety of cells types including monocytes, T lymphocytes, 
neutrophils, fibroblasts, endothelial cells and epithelial cells. The most abundant form 
of  CXCL8 is a protein with 72 amino acids.  There is a variant form extended at the 
N-terminus occasionally called endothelial IL-8 because its synthesis by these cells. In
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vitro the longer protein is ~10- fold less potent than the shorter protein in attracting 
and activating neutrophils, but has similar potency, perhaps due to proteolytic 
processing to the short form. The 77 amino acid form may be involved in neutrophil 
adherence to the endothelium as a prelude to diapedesis. Other properties attributed 
to CXCL8 include chemoattraction of T lymphocytes and angiogenic activity even if the 
latter effect is controversial by reported absence of IL-8 receptors on endothelial 
cells
8
. Other members belonging to this family is CXCL1 also functionally identified as 
a neutrophil-specific chemoattractant secreted by activated mononuclear cells along 
with CXCL8 and having similar potency. CXCL2 and CXCL3 are closely related proteins 
that are also potent neutrophil attractants. CXCL5 places into the GRO proteins and 
like these chemokines it specifically attracts neutrophils. Similarly, CXCL6 is a 
neutrophil-specific chemoattractant and activator, but has a specific activity ~ 5-10 
folds lower than CXCL8. 
The ELR-chemokines have an apparent uniformity of function which it makes to think 
of them as a family of neutrophil chemoattractants and activators. In contrast,  the 
non-ELR CXC chemokines have a poor neutrophil atracttant activity. For example 
CXCL4 found in platelet α-granules along with PBP and its processed products , but 
unlike those proteins it has no ELR motif and is an extremely weak attractant for 
neutrophils. Another non-ELR CXC chemokine that has antiangiogenic properties is 
CXCL10, the product of an interferon-γ (IFN-γ)-inducible gen. CXCL10 is expressed by 
a variety of cells types including mononuclear cells, keratinocytes, fibroblasts, 
endothelial cells, and T lymphocytes. In mice, IFN-γ administration induces high levels 
of CXCL10 in liver and kidney with lower levels in the spleen. Similar to other non-ELR 
chemokines, CXCL10 is a poor neutrophil chemoattractant and activator. 
CXCL11 is another IFN-γ-inducible protein isolated from macrophages. It has 
chemoattractant activity in vitro for tumor-infiltrating lymphocytes. Either CXCL11 or
13 
 
CXCL10 attract tumor-infiltrating lymphocytes and cross-desensitize in other 
measures of receptor activation, consistent with the fact that on tumor-infiltrating 
lymphocytes they share the same receptor CXCR3. 
Finally CXCL12, cloned from mouse bone marrow stromal cells. In humans CXCL12 
has a low-potency as chemoattractant for T lymphocytes in vitro. However, perhaps 
relevant to its provenance, CXCL12 is also a potent chemoattractant for CD34+ 
hematopoietic progenitors. In vivo, targeted gene disruption of murine CXCL12 
indicates that it’s required for normal B lymphocyte development and for normal 
cardiac organogenesis
8
.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. The chemokine and their receptors. Systematic nomenclature for each 
chemokine is given along with common old name or names. The select ligands are identified 
with old acronym and the new nomenclature, in which the first part of the name identifies the 
family and L stands for ligand, followed by a progressive number. Red indentifies predominantly 
“inflammatory  or “inducible” chemokines; green, “homeostatic agonists; yellow molecules 
belong to both realms. Ba, basophils; Eo, eosinophils; iDC, immature dendritic cells; MC, mast 
cells; mDC, mature dendritic cells; Mo, monocytes; MØ, macrophages, NK, natural killer cells; 
PMN, neutrophils; T act, activated T cells; T naïve, naïve T cells; T reg, regulatory T cells; T 
skin, skin homing T cells (Locati, M. Am J Clin Pathol, 2005).
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1.3 Receptors and chemokine interactions 
Chemokines interact with 22 G protein-coupled receptors possessing a seven 
transmembrane domain (7TM)organization
5
.  Approximately 20 signaling chemokine 
receptors have been reported 7 CXCRs, 10 CCRs, 1 CX3CR, and 1 XCR,
10, 11
 plus 3 
nonsignaling receptors with high structural  similarity to classic conventional signaling 
receptors, namely the Duffy antigen receptor for chemokines (DARC), D6, and CCX 
CKR. These molecules share the ability to bind chemokines with high affinity in the 
absence of any demonstrable signaling function and therefore are indicated as “silent” 
receptors.  “Silent receptors” have been suggested to favor transfer of chemokines 
across endothelial barriers and/or to act as decoy receptors which dampen 
inflammatory reaction by binding, internalizing and, in the case of D6, degrading 
chemokines
10, 12
.  
Chemokines receptors belong to the large family of 7TM receptors which couple to 
heterotrimeric GTP-binding proteins (G proteins). All chemokine receptors are single 
polypeptide chains with seven helical membrane- spanning regions connected by 
extramembranous loops, with an acidic N-terminal extracellular domain and three 
extracellular loops exposed outside the cell, whereas the serine-threonine- rich C-
terminus and three intracellular loops face to the cytoplasm. Two disulfide bonds 
between the N-terminal domain and the second extracellular loop and between the 
first and third extracellular loop normally are required for the molecule structure
3, 10
. 
The ability of chemokine receptors to signal upon ligand binding is due, at least in 
part, to the presence of a DRY motif in the second intracellular loop, which is missing 
in scavenger receptors. Despite a wealth of data related to GPCRs in general, many 
aspects of ligand binding and signaling are poorly understood at the molecular level. 
Structural understanding of GPCRs has benefited from a number or recent 
breakthrough, including the recent release of the first structure of a chemokine
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receptor
13
 in complex with a small- molecule antagonist and with a cyclic peptide 
inhibitor. Similarly to the previously determined high- resolution structures of the β
2
- 
adrenergic receptor (β
2
-AR) and A
2A
 adenosine receptor (A
2A
AR), the overall structure 
of CXCR4 bound to the small molecule IT1t consists of the canonical bundle 7TM α-
helices (Figure 3A) with some differences regarding to the disposition of the TM 
helices compared with β
2
-AR and A
2A
AR. Substantially both intracellular and 
extracellular tips of helix IV in CXCR4 deviate (~5 and ~3Å, respectively)from their 
consensus  positions in other GPCRs. The extracellular end of the helix V in CXCR4 is 
about one turn longer. Helix VI has a similar shape in all structures and is 
characterized by a sharp kink at the highly conserved residue,  Proline 254. Finally, 
the extracellular end of helix VII in CXCR4 is two helical turns longer that in other 
GPCR structures. It is comes as a surprise that in CXCR4 structure, helix VII is about 
one turn shorter at the intracellular side, ending just after the GPCR-conserved NPxxY 
motif, and that the structure lack the short α helix VIII
13
 (Fig 3B). 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3. Chemokine receptor structure. Overall fold of the CXCR4-IT1t complex and comparison with 
other GPCRs structures. (A) Overall fold of the CXCR4. The receptor is colored blue. The N terminus, 
ECL1, ECL2, and ECL3 are highlighted in brown, blue and red, respectively. The compound IT1t is shown 
in magenta stick representation. The disulfide bonds are yellow. Conserved  water molecules are shown as 
red spheres. (B) Comparison of TM helices for CXCR4 (blue); B
2
AR (yellow); A
2A
AR (green); and 
rhodopsin (pink) (Wu, B. Science, 2010 in press).
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Receptor expression is a crucial determinant of the spectrum of action of chemokines. 
Early studies have indicate that polarized T helper type 1/T cytotoxic type 1 (Th1/Tc1) 
and Th2/Tc2  populations show differential receptor expression and responsiveness to 
chemokines, and that activation is associated with differential regulation of receptor 
expression
5
  (Figure 4).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4. Chemokines expression in polarized type 1 and type 2 responses.
Chemokines in polarized type 1 and type2 T-cell responses. During type 1 (a) and type 
2 (b) immune responses, master cytokines regulate chemokine production by stromal 
and inflammatoru cells: chemokines then support selective recruitment of polarized T 
cells and specific type 1 and type 2 effector cells expressing distinct panels of 
chemokines receptors. Eo, eosinophils; Ba, basophils; DC, dendritic cells; IFNγ, 
interferon-γ; MC, mast cells; NK, natular killer cells; Tc, cytotoxic cells; Th, T helper 
cells (Mantovani, A. Immunol Today, 1999).
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Numerous chemokine receptors are highly promiscuous in their chemokine selectivity, 
and viceversa, numerous chemokines bind to more than one receptor. These 
redundancy in the chemokine system is most frequently associated with inflammation, 
and is in contrast with several monogamous chemokine systems involved in 
homeostatic leukocyte development and migration processes
11
.  
Chemokine receptors are expressed on different types of leukocytes. Some receptors 
are restricted to certain cells, (e.g., CXCR1 is predominantly restricted to neutrophils), 
whereas others are more widely expressed (e.g., CCR2 is expressed on monocytes, T 
cells, natural killer cells, dendritic cells, and basophils). In addition, chemokine 
receptors are constituvely expressed in some cells, whereas they are inducible on 
others. CCR1 and CCR2 are constituvely expressed on monocytes but are expressed 
on lymphocytes only after stimulation by IL-2
1
. In contrast, the expression of other 
chemokine receptors is restricted to a cell state of activation and differentiation. For 
example, CXCR3 is expressed on T lymphocytes of the T helper type 1 (Th1) 
phenotype, whereas CCR3, in addition to being expressed on eosinophils and 
basophils, is preferentially expressed on activated lymphocytes of the T helper type 2 
(Th2) (Figure 4). In this way, transient up-regulation of chemokine receptors on 
leukocytes allows for the selective amplification of either a cell mediated Th1-type 
immune response or a Th2-type response. Some chemokine receptors are also 
expressed on non hematopoietic cells, including neurons, astrocytes, epithelial cells 
and endothelial cells, suggesting that chemokine system has other roles in addition to 
leukocyte chemotaxis
1
.
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1.4 Roles of chemokine and chemokine receptors in inflammation and 
immune surveillance  
To ensure immunity is necessary to control the homeostatic circulation of leukocytes 
through tissues. Different types of cells are addressed into inflamed tissue in an 
organized manner bringing naïve lymphocytes into the lymph nodes, where they 
encounter antigen and become in memory lymphocytes. This process is regulated by 
other bone marrow-derived cells such a macrophages, eosinophils, and mast cells all 
of them regulated by chemokines that ensures the movement of cells
1
. 
The dramatic increase in the secretion of chemokines results in the selective 
recruitment of leukocytes into inflamed tissues. Chemokines have been detected 
during inflammation in most organs, including the skin, brain, joints, meninges, lungs, 
blood vessels, kidneys, and gastrointestinal tract. They have also been identified in 
many types of cells during inflammation in these organs, indicating that most, if not 
all cells can secrete chemokines given the appropriate stimulus
1
. 
The role of chemokines in regulating movement of the cells into tissues began to be 
elucidated on the basis of studies in mice deficient in a particular chemokine.  
Several groups have developed models to provide insights into how chemokines work 
in vivo. For example CXCL12  is critical for the migration of myeloid precursor from 
the fetal liver to the bone marrow
14
. 
Mice expressing CXCL8 under the control of liver-specific promoter/enhancers did not 
develop neutrophil infiltrates in their livers
15
. Instead, they had high serum levels of 
IL-8 that were associated with L-selectin shedding from circulating neutrophils and an 
inability to induce neutrophil extravasation in response to local stimuli. This was 
similar to the observations that intravenous administration of CXCL8 in rabbits 
prevents local neutrophil accumulation
16, 17
. Along the same line, mice over expressing 
CCL2 under the control of the MMTV-LTR had high serum levels of CCL2 and no
19 
 
monocytic infiltration in expressing organs. This suggests that like, CXCL8 over 
expression, CCL2 over expression rendered circulating monocytes inacapable of 
responding to local physiological levels of CCL2
18
. 
In contrast to these models, expression of chemokines at low levels in anatomically 
restricted areas can produce leukocytic infiltration. Consistent with their in vitro 
properties, mice expressing murine CXCL1 in the thymus or brain had neutrophil-rich 
infiltrates in these organs but it was not observed tissue damage associated with the 
acute infiltrate
19
. These results all together provided at least two insights into how 
chemokines work in vivo. First, chemokines exert their attractant activity only when 
they are expressed locally at low levels; systemically administered actually antagonize 
the local effect. Second, chemokines appear to attract leukocytes without activating 
them, suggesting that chemokine function in leukocyte trafficking is restricted to 
attraction, and other signals are necessary for activation. 
 
1.4.1 Role and implications in leukocyte movement 
Cells migrating in a tissue encounter many different signals that can potentially direct 
their path. Cells of the immune system require very accurate positioning within tissues 
to perform their biological functions. However little is known about how leukocytes 
navigate through the complex environments
20
. Each leukocytes type has the capacity 
to respond to multiple different chemokines and/or classical attractants. Furthermore, 
many of the attractants that act on a given cell may be present together in a 
recruiting tissue. One classical example is a site of bacterial infection, in which a 
variety of neutrophil’s chemoattractant are produced by different sources: host 
endothelial, epithelial, and stromal cells produce arrays of attractants including 
CXCL8, CXCL1 and others; complement deposition on pathogens releases the