Transcript Role of Chemokines Genetic Polymorphisms in Diseases

Role of Chemokines
Genetic Polymorphisms
in Diseases
Ali H. Ad’hiah (Ph.D. Human Immunogenetics)
Tropical-Biological Research Unit
College of Science
University of Baghdad
Introductory Theme
 The interest into chemokine polymorphisms came
with the discovery of allelic variants of HIV coreceptors that confers protection against virus entry
into target cells.
 Since then, chemokines genetic background has been
deeply studied in order to find associations between
allelic variants and inflammation-related diseases as
well as infectious diseases.
 In addition, chemokines genetic variations have been
involved in other infectious diseases as HCV and
Malaria, and also in a variety of non-infectious
diseases such as cancer, autoimmune and
cardiovascular diseases.
 This seminar aims to present genetic variations in
chemokines encoding genes and discuss their role,
sometimes controversial, in a variety of diseases.
What are Chemokines?
 Chemokines are a large superfamily of small (~ 8–15 kDa)
structurally related chemotactic cytokines that regulate cell
trafficking of various types of leukocytes to areas of injury
and play different roles in both inflammatory and homeostatic
processes.
 They selectively, and often specifically, control the adhesion,
chemotaxis, and activation of many types of leukocyte
populations and subpopulations. Consequently, they are major
regulators of leukocyte traffic.
 Besides leukocyte migration, chemokines can also influence
leukocyte cell survival and effector functions such as
degranulation.
 Furthermore, chemokines act on other cell types and tumor
cells, thereby contributing to angiogenesis, hematopoiesis,
organogenesis and tumor growth and metastasis.
Chemokines recruit the cells to sites of infection
(a) The four sequential but
overlapping steps in
neutrophil extravasation.
(b) Cell-adhesion molecules and
chemokines involved in the
first three steps of
neutrophil extravasation.
1. Initial rolling is mediated by
binding of E-selectin
molecules on the vascular
endothelium to Mucinlike
CAMs.
2. A chemokine such as IL-8
then binds to a G-protein–
linked receptor on the
neutrophil, triggering an
activating signal.
3. This signal induces a
conformational change in
the integrin molecules,
enabling them to adhere
firmly to Ig-superfamily
molecules on the
endothelium.
CHEMOKINES: A DOUBLE-EDGED SWORD
Host defense
Angiogenesis
Inflammation
Hematopoiesis
chemokines
Autoimmune Disease
Neoplasia
Atherogenesis
Infection
Chemokines and their receptors are a system which has
evolved to protect the host and maintain homeostasis, but
disordered function of the system or exploitation by
pathogens can result in antagonistic negative effects.
Hence the concept of a double-edged sword
Functional Classification of Chemokines
 Chemokines can be either structurally or functionally
classified into subgroups.
 Based on their function, they are designated as
inflammatory and homeostatic chemokines.
 The inflammatory chemokines are inducible, expressed
in response to infection, tissue injury or stress factors.
They are responsible for the recruitment of effector
leukocytes to the site of inflammation.
 In contrast, the homeostatic chemokines are
constitutively produced by tissue cells and control
basal leukocyte trafficking, such as lymphocyte
homing to secondary lymphoid organs and lymphocyte
recirculation through peripheral tissues.
 Some chemokines exert both inflammatory and
homeostatic functions and are therefore called dualfunction chemokines.
CXC chemokines
Microbes
inflammatory cytokines
CXCL8/IL-8
CXCL1/GROα
CXCL4/PF4
leukocytes
endothelial cells
epithelial cells
fibroblasts
CC chemokines
CCL2/MCP-1
CCL3/MIP-1
CCL5/RANTES
CCL11/Eotaxin
CXCL10/IP-10
neutrophils
lymphocytes
Endothelial cells
lymphocytes
basophils
eosinophils
Mononuclear phagocytes
Under normal conditions, homeostatic chemokines regulate
cellular traffic by directing cells that express certain chemokine
receptors to specific locations where their chemokine ligands are
expressed
Structural Classification of Chemokines
 Approximately 50 chemokines (and 20 receptors) are
identified to date.
 Structurally, they are divided into four families on
the basis of the pattern of the first two of four
cysteine residues of the ligand:
I.
The large CC family (Most of them are clustered
on chromosome 17q11–21 and 9p13).
II.
The CXC family (Most of them are clustered on
chromosome 4q12–13 and 4q21).
III. The CX3C family (with only one member,
CX3CL1).
IV.
The XC family (consisting of two highly related
chemokines, both binding to the XCR1
receptor).
Classification of chemokines
The chemokine wheel
Chemokine receptors and their ligand-specificity
Atypical Chemokine Receptors
 A subset of chemokine receptors, initially called
“silent” on the basis of their apparent failure to
activate conventional signalling events, has recently
attracted growing interest due to their ability to
internalize, degrade, or transport ligands and thus
modify gradients and create functional chemokine
patterns in tissues.
 These receptors recognize distinct and complementary
sets of ligands with high affinity, have evolved to fulfill
fundamentally different functions to the classical
signalling chemokine receptors.
 Based on these considerations, these receptors [D6,
Duffy antigen receptor for chemokines (DARC), CCXCKR1 and CXCR7] are now collectively considered as
an emerging class of ‘atypical’ chemokine receptors.
DUFFY BLOOD GROUP ANTIGEN
 The Duffy antigen receptor for chemokines (DARC) has recently
become the focus of studies investigating interactions of
inflammatory chemokines with erythrocytes during systemic
inflammatory responses, as well as, with venular endothelial
cells during chemokine-induced leukocyte adhesion and
emigration. These studies uncovered new functional scope of
this rather “old” molecule.
 DARC was first described in 1950 as the Duffy blood group
antigen. Three “Duffy-positive” phenotypes were described:
Fy(a+b−), Fy(a−b+), and Fy(a+b+), arising from combinations
of the co-dominant FYA and FYB genes. However, some
individuals, designated “Duffy-negative,” express neither Fya
nor Fyb antigens, Fy(a−b−).
 The Duffy-negative phenotype was first linked with resistance
to malaria when Fy(a−b−) volunteers exposed to the bites of P.
vivax-infected mosquitoes, in contrast to Duffy-positives, did
not develop malaria.
 Duffy blood group antigen was designated DARC after it was
shown to mediate the binding of inflammatory CC and CXC
chemokines to erythrocytes.
Viral chemokines
 Several virus [human herpesviruses (Kaposi sarcomaassociated herpes virus, HHV-8) and pox viruses (Molluscum
contagiosum virus, MCV) have been shown to encode CK-like
proteins.
 In HHV-8 two viral proteins with homology to CCL3– CCL4 (MIP
proteins), called vMIP-I and vMIP-II, are produced. Both
expressed proteins are angiogenic, showing a pathogenic role
in Kaposi sarcoma. In addition, vMIP-II has unique biological
activities in that it blocks infection by several different HIV-1
strains. This occurs because vMIP-II binds to a wide range of
CKRs, some of which are used by HIV to gain cell. The HHV-8
virus thus appears to have ‘hijacked’ a human MIP-like CK,
modifying it so that it can bind to more than one CKR and
thereby increasing the pathogenicity of the virus, helping it to
spread and proliferate.
 The poxvirus MCV CK proteins also closely resemble CCL3 and
appear to share the inhibitory effect that this CK has on human
haematopoietic progenitor cells. These proteins are potent
antagonists and can inhibit the chemotactic response to the
human CK. It is likely that their major function in the virus is to
aid it in immune evasion during infection.
Map of genomic organization of human chemokines
CC chemokines in red, CXC chemokines in green, CX3C
chemokine in yellow and C chemokines in blue.
Genetic Polymorphism of Chemokines
 In evolution, diversification through the generation of
multiple alleles is very common and the immune
system contains several groups of genes with
prominent allelic variations.
 The CK superfamily constitutes a very revealing case of
how, through evolution, a complex network of genes
has acquired a very diverse set of related functions.
 Most, if not all, CKs probably arose by gene duplication
of a single ancestral gene and, consequently, many
CKs (just as many CKRs) are clustered in defined
chromosomal locations.
 Two main clusters have been recognized, both of them
codifying the essential inflammatory CKs: the CXC
cluster, located in chromosome 4q12–21 and the CC
cluster, located in chromosome 17q11.2.
 Those CKs that map in the CXC and CC clusters seem to
maintain some similar functions: CXC cluster CKs
recruit mainly neutrophils, whereas CC cluster
members typically attract mononuclear cells.
Genetic Polymorphism of Chemokines
 Another important way by which CK variations
increased during CK evolution at the genomic level is
through the generation of polymorphisms, especially
single nucleotide polymorphisms (SNPs).
 Other types of polymorphisms such as deletion/
insertion polymorphisms (DIPs), copy number
polymorphisms (CNPs) or those due to repeated
elements (as minisatellites and microsatellites) also
contributed importantly to the CK genomic variation,
but their distribution is more restricted.
 Additionally, beyond the contribution of
polymorphisms to the overall variability in CK
superfamily, some CK genes that are polymorphic have
alleles that are found to be repeatedly associated with
disease.
Infectious diseases: HIV Infection
 The main relation between HIV and chemokines
resides in the virus’s need to bind to a co-receptor in
addition to CD4 at the surface of permissive cells to
enter the cell.
 The main co-receptors of HIV are CCR5, especially
during primary infection, and CXCR4 during the
asymptomatic/late stages of progression to AIDS.
 Although no obvious polymorphism has been identified
for CXCR4, several genetic variations in the CCR5 gene
have been clearly associated with HIV restriction, viral
control, and progression to AIDS.
CC-receptors and HIV infection
CCR5 Polymorphism and HIV Infection
 The strongest effect identified, which restricts HIV,
involves the CCR5Δ32 variant with 20 base pairs
missing in the coding sequence. This absence produces
an mRNA frame shift and the emergence of a stop
codon.
 In term of polymorphism, 13 single nucleotide
polymorphisms (SNPs) have been identified in the
CCR5 gene promoter: A29G, G208T, G303A, A612G,
C626A, C627T, C630T, C647A, A676G, T684C, C714G,
G811A, and C927T, that is, at least 10 promoter
haplotypes (numbered CCR5-P1 to P10). The CCR5Δ32
mutants is associated in linkage disequilibrium with
the CCR5-P1 haplotype.
Infectious diseases: HCV Infection
 The association study scanned for polymorphisms in
CCL5, CCR5, CCR2, CCL3, CCL2 and CCR3 and reported
four main findings:
i.
The CCR5Δ32 allele is associated with mild fibrosis
and reduced portal inflammation.
ii. Polymorphisms in a CCR5 promoter—the 2132-C
allele—are associated with increased risk of HCV
persistence and with better initial response to
interferon.
iii. The CCL5-403 promoter polymorphism is associated
with portal inflammation.
iv. The MCP-2 Q46K mutation is associated with more
severe fibrosis.
Transplantation

The major factors associated with transplant rejection episodes are HLA
mismatches, donor age, and delayed graft function. But, Some graft rejection
episodes cannot be explained by these factors, and the hypothesis of
Chemokine genetic polymorphisms responsible for these events has been an
attractive hypothesis according to the following observations:
i.
The outcome of kidney transplants showed that the CCR2-64I variant and the
CCR5 59029-A allele both protect against acute rejection.
ii.
CCL2-2518-G/G homozygotes are more susceptible to renal graft rejection
over the long term than either A/G heterozygotes or A/A homozygotes at this
position.
iii. The finding that neither the CCL2-2518 nor CCR264I polymorphisms are
involved in acute graft rejection suggested that the CCL2-2518-G allele, which
is correlated with higher CCL2 secretion levels in PBMCs isolated from
kidneys, plays a role only in the long-term survival of the transplant organ.
iv. Another chemokine ligand, CCL4L1, and more specifically, the number of its
copies, might influence lung transplantation outcome. Patients with more
than 2 copies of CCL4L1 had a greater risk of developing acute rejection.
Autoimmune diseases: SLE
 SLE is one of the most common autoimmune diseases and is due
mainly to genetic variations that can be enhanced by environmental
factors. Among the genetic factors associated with lupus is a
polymorphism in the CXCL8 gene, CXCL8-845C, which might
predispose African-Americans with SLE nephritis to more severe
renal damage, possibly by influencing CXCL-8 expression.
 The gene most often studied in association with SLE is CCL2. It has
been reported that A/G or G/G genotype at position 2518 might
predispose individuals to the development of SLE and further that
SLE patients with these genotypes might be at higher risk of
developing lupus nephritis (LN), a major cause of morbidity and
mortality in patients with SLE.
 A further finding revealed that the CCL2-2518-G allele is a significant
risk factor for SLE among Whites but not African-Americans suggests
that genetic differences in CCL2 expression play a role in SLE.
Autoimmune diseases: Sjogren syndrome
 Sjogren syndrome (SS), which is characterized mainly by CD4+ Tcell infiltration of exocrine glands, appears to be due to a
predisposition associated with multiple gene variations and
environmental triggers. Several chemokines, including CCL3, CCL4
and CCL5, may play a role in this disease.
 An initial study reported a significantly lower frequency among SS
patients, compared with healthy controls, of the CCR5Δ32 allele and
consequently of subjects heterozygous for it.
 Gene targeting in mice revealed that Ccr7-deficient animals are
severely impaired in the induction of central and peripheral
tolerance. Due to these defects, Ccr7-deficient mice spontaneously
develop multi-organ autoimmunity showing symptoms similar to
those observed in humans suffering from connective tissue
autoimmune diseases.
Autoimmune diseases: Kawasaki disease
 Kawasaki disease (KD) is characterized by acute systemic
vascularitis in young children and is the leading cause of acquired
heart disease in North America and Europe. Its incidence, however,
is 7–15 times higher in Japanese than American or European
children, which suggests that genetic variations promote its
occurrence.
 In a Dutch cohort of KD patients, the disease was associated with
common genetic variants of the chemokine receptor gene cluster
CCR3-CCR2-CCR5.
 Of note, the lower frequency of the CCR5Δ32 allele in KD subjects
than in the control group provides some confirmation of this allele’s
protective role in the context of autoimmune diseases, as seen in SS.
 Two additional studies found, in confirmation of the Dutch findings,
that genetic variations in CCR5/CCL5 genes are associated either
with KD or its coronary complications.
Autoimmune diseases: Arthritis
 Because of the important role chemokines play in pro-inflammatory
diseases, their contribution to rheumatoid arthritis (RA) has been
extensively evaluated. Early results showed that the CCR5Δ32 allele
might influence RA variables including IgM rheumatoid factor. These
data suggest that the CCR5 polymorphism has a specific effect on
the severity of RA .
 A recent genome-wide analysis study of about 7000 RA patients and
20,000 controls identified a SNP in CCR6 that is significantly
associated with RA. This SNP, rs3093024, is in strong LD with a triallelic dinucleotide polymorphism (CCR6DNP) and influences CCR6
transcription: the CCR6DNP-T allele induces stronger gene
expression than that seen with either the CCR6DNP-A or -C alleles.
Furthermore, the observed correlation of the CCR6DNP genotype
with IL-17 in the sera of RA patients suggests that CCR6 plays an
important role in IL-17-driven autoimmunity.
Allergy/Asthma
 Atopic dermatitis (AD): The first polymorphism identified was in the
CCL5 promoter (−401-A/G), and children homozygous for the −401-A
allele were present in the AD group only, not in the control group, but
no association was identified with asthma.

Also, this allele showed racial variation (the frequency of −401-A/A
genotype was 15% in Africans compared with about 2.1% in whites).
 Further study reported that the CCL2-2518-G allele (which is
associated with increased CCL2 transcription) might be associated
with asthma, and children carrying the −2518-G/G genotype were
significantly more susceptible to severe asthma, which is correlated
with increased levels of eosinophils.
 A similar subsequent work confirmed the role of the CCL2-2518-G
allele and extended it to allergic phenotypes in general, particularly by
showing an association with higher sensitization levels to allergens.
Chemokines & Breast Cancer Metastasis
 Metastasis is an orderly, multistep
process involving the movement
of cancer cells from the primary
tumor to specific organs under the
guidance of specific chemokines.
 First, cancerous mammary
epithelial cells undergo clonal
proliferation, invade local tissue,
induce angiogenesis, and CXCR4
on their surface. Then, cancer
cells detach from the primary
tumor, migrate across lymphatic
and vascular walls in the tumor,
and enter the systemic circulation.
 Cancer cells are arrested in
vascular beds in organs that
produce high levels of the CXCR4
ligand (CXCL12), which is
expressed on the surface of
vascular endothelial cells. Binding
of CXCL12 to CXCR4 induces the
migration of cancer cells into
normal tissue, where the cells
proliferate, induce angiogenesis,
and form metastatic tumors.
 Breast-cancer cells do not usually
metastasize to organs that
produce low levels of CXCL12,
such as the kidney.
Breast
Blood
Target Organ
Repertoire of chemokines and chemokine receptors
expressed in cancer tissues: Close interactions occur
between cancer cells and cells of the tumor
microenvironment
Cancer: Leukemia
 AML is characterized by uncontrolled proliferation of myeloid
progenitors in the bone marrow, and CXCR4 exerts a central
role in the trafficking of these malignant cells. The
CXCL12/CXCR4 axis has thus been screened for
polymorphisms related to blast dissemination.
 It has been shown that the 801-G/A gene polymorphism
(which leads to higher CXCL12 secretion) in CXCL12 is
associated with the number of peripheral blood blasts (PBB)
and the number of extra-medullary tumor sites. More
specifically, individuals with the 801-A/A genotype had more
PBB than 801-G/G carriers did, as well as almost triple the
likelihood of developing extra-medullary tumors.
 Chemokines are also used to assess drug efficacy in acute
lymphoblastic leukemia (ALL). The presence of minimal
residual disease (MRD) is a marker of this anti-leukemic
efficacy currently used to assess risk status in children with
ALL. A study has demonstrated the CCR5 243-A/A genotype
is associated with a less favorable MRD status
Lung cancer
 The first associations between chemokine polymorphisms
and lung cancer were reported in 2004, when Campa et al.
showed that an CXCL8 promoter polymorphism was
associated with a protective effect against lung cancer in
women: the risk of developing non-small-cell lung cancer
(NSCLC) was drastically reduced among women carrying the
CXCL8-251-A/A genotype.
 Analysis of the genotype frequencies for the CXCL12-3
polymorphism has indicated that individuals with genotypes
not carrying the A allele are nearly 3.5 times more likely to
develop a long-distance metastasis of epidermoid NSCLC and
thus suggests that the involvement of chemokine
polymorphisms is not limited to primary tumor sites only.
 Assessment of postoperative metastatic recurrence found
significantly higher values of CXCR4 and CXCR7 expression in
patients with these recurrences than in those without them.
The 5-year disease-free survival rate for patients with high
CXCR7 levels was significantly lower than that for patients
with low CXCR7 levels (63.2 vs. 84.8%)
Functions of glioma produced chemokines
Glioblastoma
 Patients homozygous for the allele CX3CR1-I249 survived
for a substantially longer period (mean: 23.5 vs. 14.1
months; P < 0.0001) after surgical operation.
 The common CX3CR1 allele was also associated with a
reduction in infiltration by microglial cells.
 Accordingly, the authors proposed that this polymorphism
might be useful in predicting survival.
Cardiovascular and cerebrovascular diseases
Hypertension
 A study showed significantly elevated levels of soluble CCL2
in hypertensive patients with diffuse atheroma, due to
overexpression of this gene by endothelial cells.
 More recently, a further study confirmed that CCL2
polymorphisms might play a role in blood pressure. The
authors showed that blood pressure values were associated
with the CCL2-2518-A/G polymorphism, and subjects with
the mutant G allele had higher levels of both systolic and
diastolic blood pressure than individuals with allele A; the
same was true among asymptomatic subjects.
Other cardiovascular and cerebrovascular
diseases: Atherosclerosis
 Atherosclerosis is a major public health problem, as a cause
of both myocardial infarction (MI) and brain infarction (BI).
The first robust study to associate chemokine polymorphisms
with these diseases found the CX3CR1-I249 and -M280
alleles to be associated with an increased risk of BI
independently of other established risk factors. In addition,
BI patients homozygous for the rare alleles were much more
frequent in the group with no previous cardiovascular
events.
 Finally, ex vivo monocyte adhesion was tested and found to
be highest in blood from individuals carrying the rare
CX3CR1 alleles, and such finding is consistent with the
mechanisms leading to stroke.
 It has also been suggested that eotaxin/CCL11, which is
known to promote cell migration, plays a role in MI. The
authors showed that the CCL11-23T allele was associated
with an increased risk of MI.
Central and peripheral nervous system
diseases: Alzheimer disease
 Alzheimer disease (AD) is the principal cause of dementia in
older people. The formation of β-amyloid plaques and
neurofibrillary tangles are the main events that lead to
neuron degeneration in the brain, but pro-inflammatory
processes promote disease progression.
 Until now, the only chemokines thought to contribute to AD
were CCL2 and CCL3. The A-2518G polymorphism of CCL2
was initially reported not to be a risk factor for AD
development, even though this genotype is correlated with
higher serum levels of CCL2, which can contribute to the AD
inflammatory process. Further study showed that the same
CCL2 gene genotype was associated with AD in a
homogenous Italian population.
 A Chinese study suggested that the CCL3-906
(TA)(6)/(TA)(6) genotype contributes to elevated serum
CCL3 levels in AD patients, which in turn play a role in the
inflammatory process in AD.
Conclusions
1. Chemokines and their receptors take part in the outcome of various
diseases, from viral infections to autoimmune syndromes.
2. They are major players in inflammatory events, which most often involve
the recruitment of leukocytes at the right sites to eliminate pathogens. In
some cases, however, impaired regulation of gene expression or a
structural mutation in the coding sequence can lead to chronic
inflammation-related diseases.
3. Two genetic polymorphisms of chemokine receptors have an almost
fully penetrant genetic effect for two pathogens: CCR5Δ32 for HIV, and
Duffy antigen for the P. vivax malaria parasite. These polymorphisms
confer almost complete protection from these pathogens on the people
bearing the homozygous genotype.
4. Strong evidence indicates that chemokines play a role in autoimmune
diseases, although controversy remains in some areas. The genetic
variations that can lead to chronic inflammation-related dysregulation
can be attributed, at least in part, to the genes encoding
chemokines/receptors.
5. Racial differences can also contribute to inconsistent observations.