Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors

38 Pages·2000·0.818 MB·English

by  Oppenheim J.J., Howard O.M.Z., Goetzl E.

Chemotactic Factors,
Neuropeptides, and
Other Ligands for
Seven Transmembrane
Receptors
Joost J. Oppenheim1,*, O. M. Zack Howard2 and Edward Goetzl3
1Laboratory of Molecular Immunoregulation, Intramural Research Support Program,
Building 560, Room 21-89A, Frederick, MA 21702-1201, USA
2Frederick Cancer Research and Development Center, Frederick, MA 21702-1201, USA
3University of California San Francisco, U-B8B, 533 Parnassus, Box 0711, San Francisco,
CA 94143-0711, USA
*corresponding author tel: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2000.02011.
SUMMARY have adapted and learned to circumvent these medi-
ators of host defense by producing agonists or antag-
This introductory review serves to introduce the onistsor,asillustratedbyHIV-1,byusingchemokine
readertoseveralfamiliesofcellularchemoattractants receptors as entry sites for cellular invasion.
including cytokines known as chemokines, as neuro-
peptides, components of the complement cascade,
and cyclooxygenase pathways. These ligands share
INTRODUCTION
thecapacitytoutilizeseventransmembrane(STM)G
protein-coupled receptors. As is true of other cyto-
kines, these ligands are multifunctional and, in Well over a century ago histopathological studies
addition to mobilizing inflammatory cells to sites of revealed inflammatory sites to be heavily infiltrated
infection and injury, act on a wide spectrum of pro- by leukocytes (as reviewed by Gallin et al., 1992).
genitor cells and on nonleukocytes. Consequently, Phagocytic leukocytes present at sites of infection
someoftheseligandsparticipateinfetaldevelopment, engulfed bacteria. Metchnikoff was the first to
othersonhematopoiesisontraffickingandhomingof recognize that phagocytes engage in host defense by
cellstotissuesandorgans,ontheinductionofinflam- diapedesis from the circulation into inflamed tissues,
matory and immune responses, and on regulating and by internalizing and digesting the microorgan-
cell adhesion and angiogenesis. We will also review isms (Metchnikoff, 1901). This raised questions as to
reports that numerous viruses and microorganisms whether this directional migration of leukocytes was
986 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl
in response to exogenous bacterially derived signals concentrations these responses will be attenuated
ortoendogenousmediators.Wenowknowthatboth and the cells become desensitized to subsequent
contribute. Although scientists tried various in vitro stimulation by the ligand. This is associated with
approaches, it was not until the invention of the transient receptor phosphorylation, internalization,
Boyden chamber in the 1960s that it became possible and ligand degradation, and can be reversed by recy-
quantitatively to assay the directional in vitro migra- cling and reexpression of the receptor on the cell
tion of cells through intervening filters in response to surface.
a chemotactic attractant (Boyden, 1962). This led to A wide variety of ligands that use STM receptors
the identification of potent exogenous and endoge- and share signal transduction pathways will be con-
nous chemotactic factors for phagocytic cells such as sidered and compared in this overview. This includes
fMLPfragmentsofbacterialcellwalls,C5agenerated cell-derived chemokines, LTB , PAF, complement-
4
by activation ofthe complement cascade, cell-derived derived C5a, bacterial-derived fMLP, neuropeptides,
lipidmoietiessuchasplatelet-activatingfactor(PAF), and related ligands.
and leukotriene B (LTB ) generated by arachidonic Thecontributionoftheindividualligandsandtheir
4 4
acidmetabolism.Atthe endofthe1960sconditioned receptors to host defense will be discussed in greater
media of antigenically activated T cell cultures were detailintheindividualchapters.Inordertominimize
shown to contain lymphokines that were chemotactic redundancy, this overview will provide tables and
for monocytes (Ward et al., 1969) and neutrophils figures aimed at comparing the properties of these
(Ward et al., 1970). ligandsandreceptorstooneanotherandwillconsider
Duringthenext30years,awidevarietyofpeptides them in groups based on their shared pathophysio-
derived from bacteria, serum proteins, and various logicalroles.Wewillbeginbydiscussingchemokines,
cell types and tissues have been shown to have the followed by a brief introductory discussion of other
capacity to direct cell migration along gradients of chemotactic ligands that utilize STM.
increasing concentration to their site of origin.
Furthermore, virtually all cell types, including T and
B lymphocytes, natural killer (NK) cells, eosinophils,
CHEMOKINES: HISTORICAL
basophils, dendritic cells (DC), and nonleukocytic
DISCOVERY
cells, such as endothelial cells, fibroblasts, smooth
muscle cells, have the capacity to respond to chemo-
tactic stimuli. Although it is difficult to demonstrate Based on the fact that interleukin-1 (IL-1) induces
that cells migrate along a gradient in vivo, in vitro migration of leukocytes to sites of injection, IL-1 was
assays of chemotaxis in general reflect in vivo inflam- thought to have chemotactic activity. In fact, leuko-
matory or cell-homing responses and are therefore cyte-derived chemoattractants for phagocytic cells
regarded as correlates of cellular immunity. werepresentinpartiallypurifiedpreparationsofIL-1
Even though widely different types of chemoat- (Luger et al., 1983; Sauder et al., 1984). However,
tractants have been identified, they have a similar when purified to homogeneity, IL-1 lost its chemo-
signaling mechanism,bind to specific members of the tactic activity. Further studies revealed that the par-
family STM receptors and activate G-type G pro- tially purified IL-1 preparations were contaminated
i
teins. These receptors are characterized by biphasic with both a chemotactic factor for monocytes and
doseresponsesandtheircapacitytoberapidlyturned a monocyte-derived neutrophil chemotactic factor,
on and off. At lower ligand concentrations, STM termed MDNCF (Yoshimura et al., 1987, 1989;
receptor engagement rapidly induces signal transduc- Matsushima et al., 1988, 1989). These paradoxical
tion,resultinginchangesincellshapewithpseudopod observationswereresolvedbydatashowingthatIL-1
formation at the leading edge, actin polymerization, was a potent inducer of these chemotactic factors.
redistribution and activation of cell surface adhesion Thesamemononuclearcell-derivedneutrophilattrac-
proteins leading to adhesion to endothelial cells, dia- tantswerealsoidentifiedbyseveralotherlaboratories
pedesis through the endothelium, and migration of (Schroder et al., 1987; Walz et al., 1987). Later,
along a gradient of increasing chemoattractant con- Larsen et al. (1989) reported that MDNCF was
centration present on cell surfaces and matrix pro- chemotacticforTlymphocytesaswellasneutrophils,
teins to the site of origin of the chemoattractant. At and,basedonthepresumedinvolvementofMDNCF
increased concentrations of about a log or more, the intheimmuneresponse,itwasrenamedIL-8(Larsen
chemoattractants induce cells to degranulate with et al., 1989; Oppenheim et al., 1991).
release of enzymes, histamine, antimicrobial peptides Analysis of DNA sequence data revealed that IL-8
and the production of oxygen radicals, all of which was a member of a cytokine gene family with related
have potential antimicrobial effects. At even higher structural features. IL-8 was the first member of this
Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 987
group to be shown to have substantial chemotactic presumably are used to circumvent host defenses
activity and this rapidly stimulated investigators to (Murphy, 1996, 1997).
identifyotherfamilymemberswithchemotacticeffects
on various cell types. The first international meeting
focusing on this group of chemoattractant cytokines CHEMOKINE CLASSIFICATION
wasorganizedbyJohnWestwickin1988attheRoyal
CollegeofSurgeonsinLondon,UK(Westwicketal., The chemokine subfamilies are delineated by the
1989). Enormous progress has been made, resulting numberofcysteinesandpresenceofinterveningamino
in the identification of over 50 ligands to date, acids,leadingtocommondesignationsofCXC(or(cid:11)),
using techniques embodied in bioinformatics, differ- CC ((cid:12)), C ((cid:13)), and CX3C ((cid:14)). All but two of the
ential nucleic acid and protein screens and protein chemokines belong to the CXC and CC subgroups,
purification. and there is only one each of the C and CX3C
Although the naming of the individual molecules chemokines. The linear amino acid alignment of the
has been based on the rather anarchistic proclivities human chemokine subfamilies is shown in Figure 1.
of investigators, the catchy term chemokine (short However,withinasubfamilythehomologycanrange
for chemotactic cytokines) was proposed by the from25%to95%.Intramolecularcysteinebondsare
Third International Symposium on Chemotactic formed between C1 and C3, C2 and C4, of the CXC
Cytokines held in Baden, Austria in 1992 (Kunkel and CC subfamilies, resulting in stably folded mole-
etal.,1995).TheterminologyfortheSTMGprotein- cules.AsubgroupoftheCCchemokinesconsistingof
coupled receptors for chemokines has been simplified I-309, HCC-2, MPIF1, and SLC, have six cysteines
and they are termed CXCR1-5, CCR1-8, XCR1, or (Miller et al., 1990; Schulz-Knappe et al., 1996;
CX3CR1 depending on structural features (as will Nagiraetal.,1997;Younetal.,1997,1998).InI-309,
be discussed) of the subfamily of the binding ligand HCC-2,andMPIF1oneadditionalcysteineislocated
(as proposed at the Second Gordon Conference withinthe(cid:12) sheetdomainandasecondoneislocated
on Chemotactic Cytokines held at Plymouth, NH in the C-terminal (cid:11) helix, suggesting that these cys-
in 1996). teines may form a third disulfide bond anchoring the
C-terminalhelixtothe(cid:12)sheet.SLChastwocysteines
ontheC-terminaldomain.Eotaxin2hasanunpaired
OVERVIEW OF CHEMOKINE fifth cysteine at the C-terminus (Nagira et al., 1997).
The effect of these additional cysteines on the
PATHOPHYSIOLOGY
secondary structure and on the functional activity of
these chemokines remains to be determined. A
Like most other cytokines, chemokines are multi- dendogram illustrating the relative genetic divergence
functional. In addition to mobilizing inflammatory of the human chemokines is shown in Figure 2;
cellstositesofinfectionsandinjury,theyalsoengage murine chemokines is shown in Figure 3. A dendo-
in regulation of cell adhesion, angiogenesis, fetal gram comparing both species with the virally derived
development, hematopoiesis, immune responses, and chemokines is shown on Figure 4.
the trafficking and homing of lymphocytes and den-
dritic cells to their appropriate locations in lymphoid
The C chemokine: lymphotactin
tissues(reviewedinBaggiolinietal.,1994,1997;Ben-
Baruch et al., 1995; Kunkel et al., 1995; Oppenheim
et al., 1996; Baggiolini, 1998; Krakauer et al., 1999; We will first discuss the C subfamily of chemokines
Zlotnik et al., 1999). since it has only one member. Although the C
In addition to their afore-mentioned beneficial subgroup just has one cysteine bond, it is functional.
effects, chemokines can participate in deleterious Lymphotactin is the name for the only characterized
reactions and at times are pre-empted by pathogens C chemokine (Kelner et al., 1994). Alignment of the
to the detriment of the host. Chemokines may also amino acid sequence shows that this chemokine is
have undesirable injurious side-effects by contri- missingoneofthetwoN-terminalcysteinesaswellas
buting to autoimmune diseases and participate in thecorrespondingthirdcysteine.Althoughitdoesnot
reperfusion injury. In addition, certain microbial have all the conserved cysteines, it does have
organisms, including Plasmodium vivax and HIV-1, chemotactic activity, indicating that the conserved
have been found to exploit chemokine receptors as amino acid substitutions in lymphotactin are able to
sites for host cell entry (Fauci, 1996). Furthermore, a maintain the functional conformation (Table 1). The
number of viral gene products are homologous lymphotactinreceptorhasrecentlybeencharacterized
to the chemokines or chemokine receptors and and named XCR1 (Yoshida et al., 1998).
9
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Figure 1 Pileup/ClustalX alignment of the CXC, CC, and C chemokines. The alignment begins at 10 amino acids from the second cysteine, not at the putative h
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initiation site. Alignment performed by Dr Robert Stephens of the Frederick Biomedical Super Computer Group SAIC, Frederick, NCI-FCRDC. im
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Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 989
Figure 2 Dendogram of human chemokines. Figure 4 Dendogram of human, murine, and viral
chemokines.
Figure 3 Dendogram of murine chemokines.
The CX3C chemokine: fractalkine/
neurotactin
Human fractalkine is the only CX3C chemokine
identified to date, and is unique because there are
three amino acids between the first two cysteines and
990 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl
Table 1 Properties of human C, CX3C, and CXC ELR+ chemokine (cid:11) subfamilies
Chemokine Chromosome Cell sources Stimulants Major in vitro effects Major in vivo effects
location
Lymphotactin 1 CD8>CD4 Attracts thymocytes, Lymphocyte
(C) (LPTN) T cells, mast DC, T and NK cells trafficking
cells, NK1.1
Augments antitumor
CD4+ T cells
effects
Fractalkine 16q13 EC TNF(cid:11) Transmigration of Brain inflammation
(CX3C) endothelial cells by
(neurotactin) Microglial IL-1 mononuclear cells Adhesion to endothelial
cells cells
Attracts T, M, N,
M and NK cells Nerve repair
Acts on microglia
and astrocytes
IL-8 4 M LPS Activate neutrophil, Mobilize BM neutrophils
chemotaxis, adhesion,
N Mitogens shape changes, Antibacterial host
degranulation, defense
F Particulates enzyme release, and
respiratory burst Acute and chronic
EC Viruses inflammation
Endothelial cell
K Bacteria proliferation Angiogenesis
NK IL-1 Inhibits IL-4-mediated Modulates hematopoiesis
IgE production
T TNF Promotes viral
Inhibits interferon replication
SMC IL-3 antiviral effects
Ep IL-13
H O
2 2
IL-7
Hypoxia
UVB
GRO((cid:11),(cid:12),(cid:13))/ 4 M LPS Neutrophil activation Acute inflammation
MGSA
F IL-1 Melanoma cell Fibroplasia
proliferation
EC TNF Angiogenesis
Endothelial cell
Mesothelial proliferation
cells
Fibroblast
proliferation
Activates T cells
NAP-2 4 Plt Platelet Neutrophil activation Acute inflammation
M activation Clot resorption
Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 991
Table 1 (Continued)
Chemokine Chromosome Cell sources Stimulants Major in vitro effects Major in vivo effects
location
ENA-78 4 K LPS Endothelial cell Acute inflammation
proliferation
F IL-1 Angiogenesis
Neutrophil activation
M TNF(cid:11) Mobilize BM neutrophils
EC
SMC
GCP-2 4 Osteosarcoma cells Neutrophil Acute inflammation
gelatinase-(cid:12) release
EC,endothelialcells;M,monocytes;N,neutrophils;F,fibroblasts;Plt,platelets;SMC,smoothmusclecells;K,keratinocytes;
Ep,epithelialcells;T,Tcells;NK,naturalkillercells;DC,dendriticcells.
it is membrane-anchored (Bazan et al., 1997; Pan forms a sheet floor for the hydrophobic C-terminal (cid:11)
et al., 1997). Many of the other chemokines merely helix to lay across. The N-terminus up to the first
tether noncovalently to glycosaminoglycans (GAG), cysteine does not have an ordered structure in
as will be discussed. In addition to the chemokinex solution, with the exception of RANTES which has
domain at the N-terminus, fractalkine contains a asmallregionof(cid:12) strandjustbeforethefirstcysteine
mucin stalk, linked to transmembrane and cytoplas- (Skelton et al., 1995). The spacial relationship of the
mic domains. Although fractalkine is expressed as a N-terminus, (cid:12) sheet and(cid:11) helixis maintainedby two
tethered cytokine, a shed, soluble 95kDa form has disulfide bonds. The three structural domains have
also been detected.The soluble76 aminoacidrecom- distinct functions. The N-terminus is essential for
binant chemokine component of fractalkine is func- activation of G protein-coupled receptors (Gong and
tionalandcanactivatepertussistoxin(PTX)-sensitive Clark-Lewis, 1995). The N-terminus and the (cid:12) sheet
Ca2(cid:135) flux and induce chemotactic responses by cells provide both a scaffold for quaternary interactions
expressing the monospecific fractalkine receptor, resulting in high-affinity ligand bindingto G protein-
CX3CR1 (Table 1 and Table 2). coupled receptors and the formation of chemokine
Neurotactin, which was independent identified in dimers. The C-terminal (cid:11) helix of some chemokines
1998,isamurinehomologoffractalkine,andishighly has been shown to interact with low-affinity
expressed in both the central nervous system and in (K (cid:136)10(cid:255)5M) GAGs which are found on cell
d
peripheralneurons,whereasastrocytesandmicroglial surfaces and matrix proteins (Witt and Lander,
cells expressCX3CR1(Harrisonet al.,1998).Tether- 1994). The basic charge of these ligands accounts for
ing by the mucin domain appears indirectly to facili- their heparin-binding properties. Although not
tate activation of the chemokine domain (Imai et al., essential for function, the GAG-binding capabilities
1997). Fractalkine also directly induces adhesion of the chemokines are reported to facilitate receptor
of leukocytes to endothelial cells in a PTX-resistant interactions and the haptotactic migration of recep-
and-specificmanner.Consequently,theadhesionme- tor-bearing cells over matrix proteins and cellular
diated by cell-associated fractalkine appears to occur faces.
independentofactivationofthechemokinedomain. A number of chemokines contain consensus sites
for N-linked and/or O-linked glycosylation. The nat-
urally produced chemokines are heavily glycosylated
STRUCTURE–FUNCTION but the bacterially produced recombinant proteins,
which lack glycosylation, retain receptor-binding
RELATIONSHIPS OF
activity and induce calcium flux and chemotaxis
CHEMOKINE LIGANDS
both in vitro and in vivo. The in vivo function of
glycosylation remains to be determined, but it may
A prominent secondary structural feature of chemo- playarolein prolongingthe chemokinehalf-life,and
kines is the triple-stranded antiparallel (cid:12) sheet that may improve GAG binding.
Table 2 Leukocytes targeted by chemokine–receptor interactions
Chemokine ligands Functional Neutrophils Resting Activated T cell NK B cells Monocytes Dendritic Basophils Eosinophils
chemokine T cells T cells subsets cells cells
receptors
IL-8>GCP-2(cid:29)NAP-2 CXCR1 X X
IL-8, GRO, ENA-78, CXCR2 X X
GCP-2, NAP-2
IP-10, MIG, I-TAC, (SLC+) CXCR3 X TH1(cid:29)TH2 X X X X
SDF-1(cid:11), SDF-1(cid:12) CXCR4 X X X X X Mature
BLC (BLR-1 ligand) CXCR5 X RO+, IL-2R(cid:11) X
Fractalkine (neurotactin) CX3CR1 X X CD8 X X
MIP-1(cid:11), RANTES, HCC-1, CCR1 X X X Immature X
HCC-2, MCP-2, MCP-3,
MCP-4, MPIF1
MCP-1, MCP-2, CCR2 X X X X
MCP-3, MCP-4
Eotaxin 1, eotaxin 2, CCR3 X TH2, IL-4 X X X X
RANTES, HCC-2, MCP-2, producers
MCP-3, MCP-4, MIP-1(cid:11)
TARC, MDC CCR4 X TH2, RO+ X
MIP-1(cid:12), MIP-1(cid:11), CCR5 X TH1>TH2 X X Immature
RANTES, MCP-2
LARC (MIP-3(cid:11)) CCR6 X CD4 X Immature
SLC, ELC (MIP-3(cid:12)) CCR7 X X TH1, CD4, CD8 X Mature
I-309, TARC, MIP-1(cid:12) CCR8 X TH2 X X Immature
Lymphotactin XCR1 X X
Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 993
Figure 5 Ribbon structure of human IL-8 exhibit the MIP-1(cid:12) dimer form and the CXC
overlainwithRANTES.Thesimilarregionsof chemokines produce the IL-8 dimer form. However,
IL-8(amino acids 12–26/41–53)and RANTES the CC chemokines MCP-1 and MCP-3 can form
(amino acids 14–28/41–53) are shown in blue.
both IL-8 and MIP-1(cid:12)-like dimers (Lubkowski et al.,
The N-terminal and C-terminal regions of
1997; Meunier et al., 1997). In addition, the crystal
IL-8 are shown in yellow. The N-terminal and
structure of stromal derived factor (SDF-1) reveals a
C- terminal regions of RANTES are shown in
monomer with both CXC and CC structural proper-
magenta. The data was taken from PDB
ties, supporting the notion that SDF-1 is a unique
(Abola et al., 1989) database numbers 1RTO
(Skelton et al., 1995) and 3IL8 (Rajarathnam primordialchemokine(Dealwisetal.,1998).Solution
et al., 1994). Graphic was generated by Dr studies of PF4 and a MIP-1(cid:12) mutant show that these
Nagarajan Pattabiraman of the Frederick chemokines from stable tetrameres (Mayo et al.,
Biomedical Super Computer Group SAIC, 1995; Dealwis et al., 1998; Laurence et al., 1998). In
Frederick, NCI-FCRDC using Insight 95, conclusion, all the chemokine monomers are very
Molecular Simulation Inc. (Full colour figure similar, with the exception of the N-terminal loop,
can be viewed online.)
and the CC and CXC subfamilies have the potential
to form both types of homodimers that can assemble
into tetramers.
The primary structural differences between the
chemokines account for the specificity of interaction.
Mutational analysis of IL-8 and MCP-1 has shown
that single amino acid substitutions can permit a
CXC chemokine to interact with a CC receptor and
the inverse is also true. For example, mutation of the
leucine at position 25 to tyrosine (L25Y) or mutant
variant V27Y in IL-8 results in decreased binding to
CXCR2 by 100-fold and diminished neutrophil che-
motaxis(Lusti-Narasimhanetal.,1995).Furthermore,
mutation of the corresponding large neutral residues
in MCP-1 to small neutral residues (Y28L and
N30V) means that they become chemotactic for
neutrophils, butare nolongerchemotactic formono-
cytes (Beall et al., 1992). These data demonstrate a
The solution and/or crystal structures of a few close association between primary structure and
chemokines have been determined. Several general- function.
izations can be made concerning the chemokine Chemokines have well-defined structural and func-
tertiary structure. The chemokine monomer confor- tional domains. Interaction between the chemokine
mation is globular. The spacing of the first two ligand and the chemokine receptor occurs at several
cysteines results in a difference in the orientation of sites. A high-affinity binding interaction occurs
theloopformedbetweentheN-terminusandthefirst between the N-terminus, before the first turn, of the
(cid:12) strand. In the CXC chemokines, this loop has a chemokine and the N-terminus (the so-called first
right-hand hook compared to the left-hand spiral extracellular domain) of the chemokine receptor
found in CC chemokines. This results in an increased (Samson et al., 1997). The activation of the receptor
spacingbetweentheN-terminusandthe(cid:12)sheetinthe is stimulated by amino acids located in the (cid:12) turns
CC chemokines (Abola et al., 1989), as shown in (Clark-Lewis et al., 1994). One structural feature of
Figure 5. Differences in electrostatic potential and the receptorsthatis essentialforproperfoldingisthe
hydrophobicity differences have been used to explain disulfide bond between extracellular domains. The
the different quaternary structures observed for CC residuesinvolvedintheactivationsiteonthereceptor
and CXC chemokines (Covell et al., 1994). The appear to vary among the chemokines. For example,
chemokines form dimers at micromolar concentra- the residues of CCR5 that are essential for activa-
tions, under conditions of low pH and high salt con- tion by RANTES do not appear to be identical to
centration. Two modes of dimerization have been those used by MIP-1(cid:12) (Samson et al., 1997). The
detected and these were called the IL-8 and MIP-1(cid:12) sites determining specificity appear to be located in
dimer forms because these were two of the first extracellular domains 1 and 2 of the receptor.
chemokines for which solution structures were deter- However, sites of activation appear to reside in do-
mined. It appears that the CC chemokines typically mains 3 and 4. Thus, it appears that the high-affinity
994 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl
chemokine-binding sites are separate and distinct activatingpeptide2(NAP-2).PBPisnotchemotactic,
from the activation sites on the extracellular domains but progressive proteolysis produces (cid:12)TG and NAP-
of the chemokine receptors. 2,whicharechemotactic(Walzetal.,1989).CTAPIII
has been reported to antagonize NAP-2 function.
Thus, N-terminal proteolysis provides a level of
Biologically functional ligand unit
regulation of chemokine function.
The mature secreted chemokine may be further
Theidentityofthebiologicallyfunctionalformofthe cleaved extracellularly by a number of enzymes.
chemokines is quite controversial. For example, CD26 is a dipeptidylpeptidase, which
Bioassay observations indicate that chemokines recognizes the GP site expressed on the surface of
can act either as monomers or as multimers in vivo. activatedleukocytes.Recentworkhasshownthatthis
The functional chemokine form is likely to be the peptidase removes the first two amino acids from the
multimerasitbindstoGAG(StringerandGallagher, N-terminus of RANTES and GCP-2 (Proost et al.,
1997;Spillmannetal.,1998).BothPF4andIL-8have 1998a).TruncatedRANTESisinactiveinchemotaxis
beenshowntobindheparinsulfate;however,theonly assays and has a greatly attenuated ability to induce
structural model meeting the charge and position calcium flux. However, truncated RANTES main-
constraints of this interaction involves chemokine tainsahighaffinityforRANTESreceptors.Thus,the
multimerization.ItappearsthatGAGbindingrequires N-terminally deleted RANTES acts as a natural
thedimerformofIL-8andthetetramerformofPF4. antagonist for RANTES. Similar results have been
The PF4 concentration at sites of trauma has been observed with SDF-1; however, the CD26-truncated
determined to be 170mM, indicating that local con- form of GCP-2 has been found to retain activity. N-
centrations of PF4 could favor tetramer formation terminal truncated versions of MCP-1, MCP-2, and
and association with GAG molecules (Stringer and MCP-3 have been isolated from mononuclear cells
Gallagher,1997).Thisissueiscomplicatedevenfurther (Proost et al., 1998b). The MCP-2 deletion, missing
by the possibility that the chemokine receptors may the five N-terminal residues, is a functional inhibitor
dimerize. of chemotaxis. The peptidase that truncates the
Several lines of evidence favor the monomer as the MCPs remains to be identified.
functionalformofthechemokine.Structuralanalysis
conditions require very high concentrations of pro-
tein - levels 10- to 1000-fold greater than the protein
PATHOPHYSIOLOGICAL AND
concentrations needed for biological function, which
may not occur in vivo. Also, mutational changes can DEVELOPMENTAL ROLES OF
be made in the primary structure that prevent multi- CHEMOKINES
merization and do not significantly affect function
(Rajarathnam et al., 1994). Finally, the primary
Rather than discussing each chemokine individ-
structure of vMIP-II, a virally encoded (human her-
ually we will discuss them in groups based on
pesvirus 8 or HHV8) chemokine, resembles that
their structural relationships and major functional
of MIP-1(cid:11); however, vMIP-II fails to dimerize
activities.
regardless of the pH or protein concentration
(personal communication from Barry L. Schweitzer).
Role of CXC (ELR(cid:135)) chemokines
Posttranslational regulation in acute inflammation
Chemokines are produced as propeptides and are SomeoftheCXCchemokines,includingIL-8,NAP-2,
cleaved to yield the active secreted form. N-terminal ENA-78,GRO(cid:11),GRO(cid:12),GRO(cid:13),andGCP-2,contain
proteolysis is necessary to produce the active mature a tripeptide motif at N-terminal positions 4, 5, and 6
protein and further N-terminal proteolysis inacti- composed of glutamic acid, leucine, and arginine
vates mature chemokines. The peptidases that clip (Table 1). This ELR motif is essential for high-
the proform, yielding the functionally mature form, affinity binding to CXCR2 (Schraufstatter et al.,
have not been fully characterized. Perhaps one of 1993; Clark-Lewis et al., 1994) (Table 2). Only IL-8,
the most complicated posttranslational modifications andto alesserextentGCP-2, bind CXCR1with high
is the sequential processing of platelet basic protein affinity. These ELR(cid:135) CXC chemokines are produced
(PBP) to produce connective tissue-activating protein by a wide variety of cells in response to many stimu-
(CTAPIII), (cid:12)-thromboglobin ((cid:12)TG), and neutrophil lants, most notably proinflammatory cytokines such