From the
The classical symptoms of inflammation (redness, swelling, heat,
and pain) are the result of underlying biochemical events triggered by
infection, foreign material, or tissue damage. The hallmark of
inflammation is the infiltration of specific leukocyte subsets from the
blood into the affected tissue. These leukocytes function as the
primary line of host defense in the destruction of microorganisms and
initiation of tissue repair. A variety of chemotactic proteins and
their receptors orchestrate the directed migration of leukocytes to
inflammatory sites. A number of neutrophil and monocyte
chemoattractants such as fMLP,
The chemokine superfamily consists of a number of small
(8-10 kDa), inducible, proinflammatory proteins
(2, 3) that show 20-50% homology at the amino acid level.
This superfamily is divided into an
Recently, a T cell chemoattractant, named lymphotactin, was
identified that has sequence homology with members of both the
IL-8, the most extensively studied chemokine, was originally
identified as a neutrophil chemotactic factor produced by LPS-activated
peripheral blood monocytes
(6, 7) . IL-8 is also directly
chemotactic for T cells and basophils
(3) . Furthermore, IL-8 can
attract T cells by an indirect mechanism: it induces neutrophils to
degranulate and release potent T cell chemoattractants
The open reading frame of IL-8 codes
for a 99-amino acid protein, which is shortened by a 20-amino acid
signal sequence and by further cell type-dependent proteolysis of the
NH
Numerous exogenous
agents and endogenous proinflammatory stimulants, such as endotoxin,
lectins, hypoxia, viruses, bacteria, IL-1, and TNF-
Glucocorticoids, such as dexamethasone, are potent immunomodulating
anti-inflammatory agents that have a marked inhibitory effect on the
gene transcription of several proinflammatory cytokines including IL-1,
TNF-
Other
IP-10 is one of the few
MCP-1/MCAF was originally purified from human PBMC
supernatants (2, 3). MCP-1/MCAF is induced by a number of irritants and
endogenous stimuli in multiple non-lymphocytic cell types including
endothelial cells, epithelial cells, fibroblasts, smooth muscle cells,
and hematopoietic cells such as macrophages and mast
cells
(2, 3) . The
Even less is known about the regulation of C-C
chemokine gene expression than for IL-8. Potential binding sites for
NF-
The receptors for chemokines and other chemoattractants
(CCRs: Chemokine and Chemoattractant Receptors) belong to the
serpentine superfamily of G protein-coupled receptors
(GPRs)
(1, 32, 33, 34) . Many
similarities exist between CCRs and other members of the GPR family,
yet CCRs initiate unique and specific cellular activities. A number of
receptor cDNAs (from various species) have been cloned and functionally
expressed, including those for IL-8 (designated IL-8RA and IL-8RB),
MIP-1
Hydropathy analysis of CCRs shows that CCRs contain seven
hydrophobic putative transmembrane domains, separated by three
intracellular and three extracellular loops. They all have an
intracellular carboxyl terminus and an extracellular amino
terminus
(1, 33, 34) . Many CCRs share both
sequence and structural similarities. Several recently published
reviews discuss the characteristic structure-function relationships of
CCRs. These include: domains involved in ligand binding, models for the
ligand-binding site(s), and the potential role of cysteine residues and
N`-glycosylation sites
(1, 33, 34) .
Studies on IL-8RA and receptors for fMLP and C5a suggest that both the
amino terminus of CCRs and their extracellular loops are involved,
although to a different extent and in a different manner, in the
ligand-receptor interaction
(1, 34, 38) . Studies
on IL-8RA and IL-8RB show that although both receptors bind IL-8 with
similar affinities, the ligand-receptor interaction to each of the
receptors is mediated through different regions of IL-8. The ELR motif
and a sequence from amino acids 7 to 50 are important for IL-8 binding
to IL-8RA, whereas the ELR motif and the carboxyl terminus of IL-8
(amino acids 52-72) are important for binding to
IL-8RB
(39) .
The intracellular domains of CCRs that are
involved in G protein coupling consist of two regions in the case of
the human fMLPR, localized at the second intracellular loop and at a
domain of the carboxyl terminus that is proximal to the plasma
membrane
(40) . Studies from our laboratory have identified the
membrane proximal domain of the carboxyl terminus of IL-8RB to be
involved in IL-8 signal transduction (41). Another motif that may be
important for signal transduction in CCRs is the DRY (Asp-Arg-Tyr)
sequence located in the second intracellular loop. This motif was shown
to be highly conserved in many CCRs and in other GPRs and was
implicated in signaling
(1, 32, 34) .
The cellular responses to chemokines are strictly regulated,
mainly by a desensitization process that is characteristic of
GPRs
(33, 34) . Desensitization can be referred to as
either ``homologous'' or ``heterologous.''
Homologous desensitization occurs characteristically at high
concentrations of ligand, is relatively transient, and results in
diminished responsiveness specific for the original desensitizing
agent. Heterologous desensitization is a reversible loss of
responsiveness to multiple ligands. In many GPRs homologous
desensitization is the outcome of the activity of G protein-coupled
receptor kinases, resulting in uncoupling of the G protein from the
receptor. Heterologous desensitization involves phosphorylation by
protein kinase A and protein kinase C (PKC) and results in uncoupling
of the ligand-specific receptor and other receptors from the G
proteins
(34, 42) . Desensitization that results from
uncoupling of the receptor from the G proteins is usually mediated by
phosphorylation of sites located on the third cytoplasmic loop and/or
the carboxyl terminus of GPRs
(34, 42) . A recent report
shows that during homologous desensitization fMLPR and C5aR are
phosphorylated and that a PKC-mediated mechanism is involved in
heterologously desensitizing the C5aR
(43) . Additional evidence
for phosphorylation-mediated desensitization in CCRs comes from recent
data showing that the carboxyl terminus of PAF receptor is required for
signal attenuation, induced by PAF through phosphate
accepters
(44) .
As implied by the name of the receptor family, the activation
of the receptors by specific ligands results in coupling to G proteins,
followed by a cascade of events that leads to specific cellular
responses. The G proteins consist of a large gene family coding for at
least sixteen
The best characterized
signal transduction pathway of G protein-coupled receptors starts with
ligand binding, followed by activation of a heterotrimeric G protein.
An exchange occurs in the
In general, PLC activation as
well as stimulation of various second messengers and inositol
phosphates participate in the response to chemokines and other
chemoattractants, mainly IL-8, fMLP, C5a, PAF, and
LTB
PLD activation was also
shown to be induced by IL-8, fMLP, C5a, PAF, and
LTB
There are indications that the different cellular activities,
such as chemotaxis, degranulation, and respiratory burst, are mediated
by distinct pathways of signaling. Although there is evidence that
chemotaxis results from PLC activation and release of
[Ca
Recent information with regard to
second messengers involved in superoxide production and the respiratory
burst suggests that a respiratory burst with subsequent generation of
superoxide anion by NADPH oxidase does involve formation of IP
Consequently, the activity of chemokines and
other chemoattractants is the outcome of a complex cascade that depends
on the cell type, the ligand, the structure and configuration of the
receptor, the G proteins involved, and the different enzymes that are
available to be activated in a given cell type.
Many of the chemokines have been detected in multiple disease
states that have an inflammatory component
(3, 34) , and
antibodies that neutralize IL-8 have been shown to reduce the
self-destructive inflammation seen in reperfusion injury, acute
glomerulonephritis, and arthritis
(29) . Other evidence for the
role of CCRs in inflammation arises from the generation of
``knockout'' mice lacking the murine IL-8 receptor homologue.
In these mice neutrophils exhibited a markedly reduced capacity to
migrate in response to thioglycolate in vivo and to human IL-8
and murine MIP-2 in vitro, indicating that this receptor is a
major mediator of neutrophil migration. In addition, these mice
developed bone marrow hyperplasia, lymphadenopathy, and splenomegaly
based on excessive myelopoiesis and plasmacytopoiesis
(8) . These
findings suggest that this receptor normally participates in
down-regulating the development of neutrophils and B cells.
There are a number of general principles and concepts
concerning chemokines that highlight their pivotal roles, which for the
sake of brevity can only be alluded to as ``appetizers'' in
this minireview. 1) Chemokines have direct chemotactic effects
as well as indirect activities. Generally, their indirect action does
not result from induction of other cytokines but rather from the
induction of other effector molecules (such as histamine, oxygen
intermediates, or the release of defensins, CAP-37, and
enzymes)
(3) .
-We thank Dr. E. Leonard, Dr. P. Murphy, Dr. D.
Taub, and Dr. D. Longo for critically reviewing the manuscript. The
secretarial assistance of R. Unger is gratefully acknowledged.
INTRODUCTION
Chemokines
Interleukin 8 and Other
-Chemokines (C-X-C
Chemokines)
-Chemokines (C-C Chemokines)
Receptors for Chemokines and Other Chemoattractants
(CCRs)
Structure-Function Relationships in CCRs
Desensitization of CCRs
Signal Transduction by CCRs
Signaling Pathways in CCR-mediated Effects
The in Vivo Role of CCRs in Inflammation
Prospects and Unresolved Issues
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
C5a,
LTB
, and PAF have been studied for several
decades
(1) . Since 1986, a superfamily of closely related and
conserved cytokines, which specialize in attracting a variety of
leukocytes to sites of injury, has been identified, cloned, sequenced,
and designated ``chemokines'' (short for
chemoattractant cytokines). We will cryptically
review the function and regulation of chemokines and focus on the
signaling pathways mediated by chemokine receptors in comparison with
receptors of other chemoattractants.
-chemokine subfamily based on
the presence of an intervening amino acid between the first and second
of four conserved cysteine residues (C-X-C) and a
-chemokine subfamily of proteins that has no intervening amino
acid between the first two cysteine residues (C-C). The first cysteine
forms a disulfide bond with the third cysteine, and the second with the
fourth, resulting in a similar tertiary structure for many of the
chemokines. So far at least 14 distinct
-chemokines and 12
-chemokines have been described at the protein and/or cDNA level.
Members of the human
-chemokine subfamily include IL-8, GRO,
IP-10, ENA-78, PF-4, MIP-2, GCP-2, and NAP-2; members of the human
-chemokine subfamily include RANTES, MCAF/MCP-1, MCP-2,
MCP-3/MARC, MIP-1
, MIP-1
, and I-309
(1, 3) .
The
- and
-chemokine subfamilies differ in their cell target
selectivity, with five of the
-chemokines (IL-8, GRO, NAP-2,
ENA-78, and GCP-2) acting primarily as potent chemoattractants and
activators of neutrophils
(3) . PF-4 and
-TG attract both
neutrophils and fibroblasts, whereas IP-10 attracts only monocytes, T
cells, and NK cells
(3) . An ELR motif (Glu-Leu-Arg) at positions
4, 5, and 6 preceding the first cysteine residue of
-chemokines
appears to be necessary, but not sufficient, for all
neutrophil-stimulating chemokines
(4) . In contrast to
-chemokines,
-chemokines primarily chemoattract monocytes and
T lymphocytes and do not have a common NH
-terminal motif
similar to the ELR sequence
(3) . In addition,
- and
-chemokines differ in their chromosomal location, with the
-chemokine genes colocalized at chromosome 4q12-q21 and the
-chemokine genes at chromosome 17q11-q21
(3) .
-
and
-chemokine subfamilies but has only two of the four conserved
cysteine residues
(5) . Lymphotactin may therefore be the
representative of a third subfamily of chemokines.
-Chemokines (C-X-C
Chemokines)
(
)
in the presence of secondary activators such as TNF-
or cytochalasin B, which enhance IL-8-stimulated neutrophil
degranulation
(9) . The resulting T cell chemoattractants were
recently identified as the defensins HNP-1 and HNP-2 and
CAP37/azurocidin.
terminus generating the two major natural forms: a
77-amino-acid form generated by tissue cells such as endothelial cells
and fibroblasts and a more active 72-amino-acid form generated by
monocytes and leukocytes
(2) . Similar to a number of
inflammatory cytokines, the mRNA for IL-8 contains a 3` AU-rich
sequence that causes the mRNA to be highly unstable and to be degraded
with a half-life of less than 1 h
(10) .
, stimulate
IL-8 production in a wide variety of cell types
(3) by
stabilization of IL-8 mRNA
(11) as well as by activation of IL-8
gene transcription
(3) . The genomic sequence for IL-8 contains
putative binding sites for several transcription regulatory elements in
the 5`-flanking region preceding the first exon, including sites for
NF-
B, NF-IL-6-C/EBP, AP-1, glucocorticoid receptor, hepatocyte
nuclear factor-1, interferon regulatory factor-1, and an
octamer-binding motif
(12, 13) . Analysis of the IL-8
promoter showed that the sequences between positions -91 and
-71 contain the NF-
B-binding site and the NF-IL-6 (or C/EBP)
site and are sufficient for the induction of IL-8 gene transcription by
LPS, IL-1, TNF-
, phorbol 12-myristate 13-acetate, or hepatitis B
virus protein X. Studies on Jurkat and HeLa cells showed that the IL-8
gene is regulated by a cooperative binding to the DNA of the C/EBP
binding-protein (NF-IL-6) and the NF-
B-binding protein
(RelA)
(13, 14) . These nuclear factors form a tertiary
complex with the IL-8 promoter, resulting in a synergistic effect on
IL-8 expression
(14) . Overexpression of I
B, the NF-
B
inhibitor, abolished this RelA/NF-IL-6-dependent synergistic
effect
(14) . However, the transcription of the IL-8 gene appears
to be differentially regulated in various cell types, with cooperation
at AP-1, NF-IL-6, and NF-
B sites, possibly due to the availability
and activation of different nuclear factors. In gastric cancer cell
lines and monocytic cell lines only the NF-IL-6 site is indispensable
for IL-8 gene expression (i.e. either AP-1 + NF-IL-6 or
NF-
B + NF-IL-6 can stimulate). On the other hand, in human
glioblastoma cells the NF-
B is required for IL-8 gene expression
(stimulation at either AP-1 + NF-
B sites or NF-IL-6 +
NF-
B sites)
(15, 16, 17) .
, and IL-8. A glucocorticoid-responsive element, present at
positions -330 to -325 in the IL-8 promoter, may play a
role in a number of cell types in the inhibition of IL-8 expression
observed with dexamethasone
(12) . However, dexamethasone also
suppresses IL-1-induced IL-8 production through the NF-
B
site
(18) . Interferon-
also appears to regulate
inflammatory cytokine transcription as it inhibits TNF-
-induced
IL-8 gene expression at the transcriptional level via the NF-
B
site
(19) .
-chemokine genes appear to be under
similar regulatory controls. IL-1 and TNF-
stimulate the
expression of many of the
-chemokines including GRO
, GRO
(MIP-2
), and GRO
(MIP-2
) in monocytes, fibroblasts,
endothelial cells, and mammary epithelial cells through NF-
B sites
in their promoters
(20) . There is, however, a growth-related
serum-response pathway, not involving NF-
B, that preferentially
stimulates GRO
expression
(21) . ENA-78, one of the
neutrophil attractant chemokines, is produced and secreted by
epithelial cells in response to IL-1
or TNF-
, along with
IL-8, GRO
, and GRO
(3, 22, 23) .
-chemokines that is not active on
neutrophils but is a chemoattractant for monocytes and T cells and
promotes T cell-dependent antitumor activity. IP-10 was originally
identified as an IFN-
-inducible protein of 10 kDa from the human
U937 monocytic leukemia cell line
(24) . IFN-
induces IP-10
expression through an ISRE element present in the region flanking the
transcription start site
(25) . IL-4 can inhibit IFN-
induction by activation of a negative regulator that competes for the
ISRE site (26). LPS also stimulates IP-10 expression via the ISRE
through the intermediate expression of endogenous IFN-
/-
and
through two NF-
B sites
(27) . Similar to the IL-8 gene,
optimal expression of IP-10 by either IFN-
or LPS requires
cooperation between at least two of these sites.
-Chemokines (C-C Chemokines)
-chemokines RANTES, MIP-1
,
MIP-1
, and I-309 are largely produced by stimulated T lymphocytes.
More recently, RANTES has been isolated from platelets and endothelial
cells.
-Chemokines also undergo similar proteolytic processing as
many of the
-chemokines to mature forms
(2, 3) . In
addition to chemoattracting and activating monocytes and T lymphocytes,
eosinophils are also important targets for RANTES, MIP-1
, and
MCP-3
(3) . Several of the C-C chemokines, including MCP-1,
MCP-2, MCP-3, RANTES, and MIP-1
, induce histamine release and
chemotaxis of basophils
(28) . Furthermore, MCP-1 and RANTES also
attract resting mast cells.
(
)
MIP-1
suppresses the replication of hematopoietic stem cells
(3) .
Interestingly, RANTES has overlapping activity with many of the other
-chemokines, and it competes with MIP-1
and MCP-1 for the
same receptors on monocytes.
-Chemokines show the usual pattern
of cytokine responses with increases in mRNA on cell activation,
whereas constitutive expression is seen only in transformed cell lines.
Nevertheless, RANTES was shown to be constitutively produced by
unstimulated T cells, and its mRNA and protein expression may be
increased following T cell activation. The regulation of RANTES
expression is therefore unique and suggests a distinct physiological
role for RANTES.
B, NF-IL-6, AP-1, and AP-2 have been identified in the
5`-flanking region of the mouse MCP-1 (JE) gene
(30) . NF-IL-6
(C/EPB), NF-
B, and c-Ets sites have been identified in the
promoter for MIP-1
(31) , and it was shown that LPS and
IFN-
rapidly up-regulate MIP-1
mRNA in macrophages. Much
remains to be discovered about the activation elements regulating C-C
chemokine expression.
/RANTES, MCP-1, fMLP, C5a, PAF, and an ubiquitous chemokine
receptor on red blood cells, known as Duffy
antigen
(1, 32, 34, 35) . The Duffy
antigen was not yet shown to transduce signals and is thought to
promote clearance of chemokines from the
circulation
(1, 32) . The genomic localization and
organization of some of the genes for these receptors as well as of
inactive isoforms and a pseudogene have also been
established
(1, 3, 34) . The CCRs are expressed
on a number of responding cell types, and their expression is also
regulated by exogenous and endogenous stimuli. For example, the
transcription of IL-8RB gene in human T lymphocytes decreases with
in vitro incubation at 37 °C and is restored by incubating
T cells in the presence of monocytes
(36) . Studies of the
regulation of neutrophil expression of CCRs show that a 30-min
incubation of neutrophils with granulocyte-macrophage
colony-stimulating factor down-regulates the expression of IL-8R and
C5a receptor (C5aR) and up-regulates fMLP receptor (fMLPR)
expression
(37) . In addition, in vitro incubation of
neutrophils with granulocyte colony-stimulating factor enhances,
whereas LPS inhibits, the expression of IL-8R mRNA, IL-8 binding, and
chemotactic responses by neutrophils.
(
)
In
contrast, LPS up-regulates the expression of neutrophil fMLP receptors
by increasing gene transcription.
(
)
, four
, and multiple
subunits
(45) . Most of the reactions induced by CCRs are
pertussis toxin (PT)-sensitive, but some activities were shown to be
PT-resistant. Recent studies with transfected cells identified the
G
involved in signaling by CCRs. These studies showed that
G
and G
mediated the PT-sensitive
activities of IL-8, fMLP, and C5a
(46, 47) . The
PT-resistant effects of C5a were mediated by G
(48,
49), whereas those of IL-8 were induced by the activation of both
G
and G
(46) . PAF-induced
PT-resistant activities were proposed to be mediated by G
and/or G
(48) .
subunit of the G protein from a GDP- to
a GTP-bound state, resulting in a dissociation of the
subunit
from the
subunits. The free
subunit can activate both
phospholipase C (PLC)
1 and PLC
2, whereas the free
complex activates preferentially PLC
2. The activation of PLCs
results in hydrolysis of phosphatidylinositol 4,5-bisphosphate
(PIP
) to generate two second messengers: inositol
1,4,5-trisphosphate (IP
) and diacylglycerol (DG). IP
mobilizes Ca
from intracellular stores leading
to a transient rise in
[Ca
]
, whereas DG
stimulates PKC
(1, 33, 34, 45) .
Thereafter, a variety of effectors are phosphorylated and activated,
giving rise to diverse cellular responses. Moreover, activation of PKC
and elevation in cytosolic Ca
can thereafter induce
PLC and phospholipase D (PLD) to yield DG and phosphatidic acid,
respectively, resulting in a positive feedback loop
(50) .
However, two additional second messengers, inositol
1,3,4,5-tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate,
also were recently reported to have an important role in signaling by
GPRs and CCRs
(51, 52) .
(1, 46, 48, 51, 52, 53, 54, 55, 56) .
The only receptor whose signaling mechanism seems to deviate from the
general scheme is MCP-1R; MCP-1 stimulation of human monocytes does not
result in PIP
turnover and production of
IP
(57) . A ligand-stimulated increase in
[Ca
]
was obtained in
native cells with most of the chemokines and with other
chemoattractants
(1, 49, 52, 55, 56, 57, 58, 59, 60, 61) .
Unlike many of the other ligands, the MCP-1- and MCP-3-induced rise in
[Ca
]
depends on
external Ca
(57, 58) . MCP-2 is an
exception since it does not induce an increase in
[Ca
]
when assayed at
chemotactic concentrations
(58) .
(62) . PLD activation resembles PLC activation in
terms of PT sensitivity. Yet, studies on fMLP-induced activation of
both PLC and PLD suggest that the two pathways are mediated by distinct
G proteins
(63) . Activation of PKC and a number of additional
serine/threonine kinases was demonstrated in response to IL-8, fMLP,
MCP-1, MCP-2, and MCP-3
(58, 64, 65) . Tyrosine
phosphorylation was also shown to be induced by almost all of the
chemokines and chemoattractants
(58, 66, 67) .
The substrates of the tyrosine phosphorylation may belong to another
pathway known as the MAP kinase cascade. The MAP kinase cascade
involves a series of enzymes with phosphorylating
activities
(68) . The involvement of this signaling pathway in
the action of IL-8, fMLP, C5a, PAF, and LTB
has recently
been implicated
(64, 69, 70) . Buhl et
al.(71) have proposed a model for the C5aR-induced signal
transduction network in human polymorphonuclear leukocytes, proceeding
from PLC
through PKC to stimulation of the MAP kinase pathway.
]
(1, 61) ,
other reports indicate that PKC activation or an increase in
intracellular Ca
is not always essential for a
migratory response or for actin polymerization (58, 59, 72). Studies in
polymorphonuclear leukocytes also indicate that PKC is probably not the
major mediator of degranulation and of oxidative
burst
(73, 74) .
and DG but can be either Ca
-dependent or
Ca
-independent
(73, 75, 76) .
Tyrosine phosphorylation as well as DG/PLD interaction were shown to be
involved in superoxide anion production and in the assembly or
activation of NADPH oxidase,
respectively
(66, 75, 77) . It is important to
note that PLD activation by IL-8 and fMLP occurs in the concentration
range needed for activation of respiratory burst, rather than the
10-100-fold lower concentrations that trigger
chemotaxis
(62) .
2) Although chemokines have
been proposed to contribute to the homing and migration of cells in
development, this hypothesis needs more data in order to be evaluated.
3) Chemokines generally appear to promote cell functions and
differentiation rather than cell growth
(3) . 4) The
observation that the deletion of the murine IL-8 receptor homologue
results in a phenotype with excessive myelopoiesis suggests that
chemokines may also be negative regulators of hematopoiesis
(8) .
5) There are reports that chemokines may be important positive
(e.g. IL-8) and negative (e.g. PF4) regulators of
angiogenesis (3). 6) Chemokines modulate leukocyte adhesion
and enhance the binding capacity of leukocytes
(3) . 7)
The angiogenic activities of chemokines and their ability to induce
adhesion proteins suggest that they may have a role in tumor growth and
metastatic spread. The expression of chemokines by tumor cells
(3) also suggests that chemokines may have a role in anti-tumor
defenses. 8) The extensive redundancy in the activities of
chemokines remains a mystery to be clarified. Obviously, further
elucidation of their ligand-receptor interactions and signal
transduction processes is needed to generate antagonists and inhibitors
with therapeutic promise.
, inositol 1,4,5-trisphosphate; LPS, lipopolysaccharide;
LTB
, leukotriene B
; MAP, mitogen-activated
protein; NF, nuclear factor; PAF, platelet-activating factor; PKC,
protein kinase C; PLC, phospholipase C; PLD, phospholipase D;
TNF-
, tumor necrosis factor-
; IFN, interferon; ISRE,
interferon stimulus response element; PBMC, peripheral blood
mononuclear cells; PIP
, phosphatidylinositol
4,5-bisphosphate; PT, pertussis toxin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.